WO2023004197A1 - Heavy chain antibodies binding to hepatitis b surface antigen - Google Patents

Heavy chain antibodies binding to hepatitis b surface antigen Download PDF

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Publication number
WO2023004197A1
WO2023004197A1 PCT/US2022/038228 US2022038228W WO2023004197A1 WO 2023004197 A1 WO2023004197 A1 WO 2023004197A1 US 2022038228 W US2022038228 W US 2022038228W WO 2023004197 A1 WO2023004197 A1 WO 2023004197A1
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seq
sequence
antibody
heavy chain
human
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PCT/US2022/038228
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French (fr)
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Nicole ALLEN
Priya Ganesan
Wenchao Sun
Willem VAN SCHOOTEN
Harish MEDLARI
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Teneoten, Inc.
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Publication of WO2023004197A1 publication Critical patent/WO2023004197A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention concerns human heavy chain antibodies (e.g., UniAbsTM) binding to Hepatitis B surface antigen (HBsAg).
  • UniAbsTM Hepatitis B surface antigen
  • the invention further concerns methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders that are characterized by the expression of HBsAg.
  • CHB Chronic Hepatitis B infection
  • HBsAg serum hepatitis B surface antigen
  • Curative treatments have remained elusive mainly due to the persistence of the viral covalently closed circular DNA (cccDNA) in infected hepatocytes, which provides the template for further viral replications and exhausted HBV specific humoral and cellular immunity.
  • the UniAbsTM of Camelidae ( Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) have a unique structure consisting of a single variable domain (VHH), a hinge region and two constant domains (CH2 and CH3), which are highly homologous to the CH2 and CH3 domains of classical antibodies.
  • VHH variable domain
  • CH2 and CH3 constant domains
  • These UniAbsTM lack the first domain of the constant region (CHI) which is present in the genome, but is spliced out during mRNA processing.
  • the absence of the CHI domain explains the absence of the light chain in the UniAbsTM, since this domain is the anchoring place for the constant domain of the light chain.
  • IgNAR immunoglobulin
  • IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).
  • Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBSLett. 414, 521-526 (1997)).
  • mice in which the l (lambda) light (L) chain locus and/or the l and k (kappa) L chain loci have been functionally silenced and antibodies produced by such mice are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been reported, for example, in W02006008548; U.S. Application Publication No. 20100122358; Nguyen et al., 2003, Immunology, 109(1), 93-101; Briiggemann et al., Crit. Rev.
  • CAR-T structures comprising single-domain antibodies as binding (targeting) domain are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.
  • aspects of the invention relate to heavy chain antibodies, including, but not limited to, UniAbsTM, with binding affinity to HBsAg. Further aspects of the invention relate to methods of making such antibodies, compositions comprising such antibodies, and their use in the treatment of disorders that are characterized by the presence and/or expression of HBsAg.
  • aspects of the invention include antibodies that bind to HBsAg, comprising a first heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 1-15; and/or (b) a CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 16-34; and/or (c) a CDR3 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 35-54.
  • an antibody further comprises a second heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 1-15; and/or (b) a CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 16-34; and/or (c) a CDR3 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 35-54.
  • said CDR1, CDR2, and CDR3 sequences are present in a human framework.
  • an antibody further comprises a heavy chain constant region sequence in the absence of a CHI sequence.
  • the first heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
  • the second heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
  • the first heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
  • the second heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
  • an antibody comprises a heavy chain variable region comprising: (a) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and aCDR3 sequence of SEQ ID NO: 37; (b) a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44; (c) a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49; (d) a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52; or (e) a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53.
  • an antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 55-77.
  • an antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 55-77.
  • the heavy chain variable region sequence is selected from the group consisting of: SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 75 and SEQ ID NO: 76.
  • the heavy chain variable region sequence is SEQ ID NO: 59.
  • the heavy chain variable region sequence is SEQ ID NO: 66.
  • the heavy chain variable region sequence is SEQ ID NO: 72.
  • the heavy chain variable region sequence is SEQ ID NO: 75.
  • the heavy chain variable region sequence is SEQ ID NO: 76.
  • aspects of the invention include antibodies that bind to HBsAg, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences comprise a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 1-54.
  • an antibody comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-54.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework, in a monovalent or bivalent configuration.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework, in a monovalent or bivalent configuration.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework, in a monovalent or bivalent configuration.
  • aspects of the invention include antibodies that bind to to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework, in a monovalent or bivalent configuration.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
  • aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework, in a monovalent or bivalent configuration.
  • an antibody is monospecific. In some embodiments, an antibody is multi-specific. In some embodiments, an antibody is bispecific. In some embodiments, an antibody has binding affinity to a CD3 protein and an HBsAg protein. In some embodiments, an antibody has binding affinity to two different epitopes on the same HBsAg protein. In some embodiments, an antibody has binding affinity to an effector cell. In some embodiments, an antibody has binding affinity to a T-cell antigen. In some embodiments, an antibody has binding affinity to CD3. In some embodiments, an antibody is in a CAR-T format.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
  • bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
  • aspects of the invention include bispecific three-chain antibody like molecules that binds to human CD3 and HBsAg, comprising: (i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 142; (ii) a second polypeptide subunit comprising the amino acid sequence selected from the group consistin of SEQ ID NOs: 143-145, wherein the first and second polypeptide subunits together form a first binding moiety that binds to human CD3; and (iii) a third polypeptide subunit that binds to HBsAg, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-118.
  • aspects of the invention include multispecific antibodies that binds to CD3 and HBsAg, comprising: (i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 142; (ii) a second polypeptide subunit comprising an amino acid sequence selected from the group consistin of SEQ ID NOs: 143-145, wherein the first and second polypeptide subunits together form a first binding moiety that binds to human CD3; and (iii) a third polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 147.
  • the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 143.
  • the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 145. [00049] Aspects of the invention include pharmaceutical compositions comprising an antibody as described herein.
  • aspects of the invention include methods for the treatment of a disorder characterized by the presence of HBsAg, comprising administering to a subject with said disorder an antibody or pharmaceutical composition as described herein.
  • aspects of the invention include use of an antibody as described herein, in the preparation of a medicament for the treatment of a disorder characterized by the presence of HBsAg.
  • aspects of the invention include an antibody as described herein for use in the treatment of a disorder characterized by the presence of HBsAg.
  • the disorder is selected from the group consisting of: acute hepatitis B infection, chronic hepatitis B infection, liver cirrhosis and hepatocellular carcinoma.
  • aspects of the invention include polynucleotides encoding an antibody as described herein, vectors comprising such polypeptides, and cells comprising such vectors.
  • aspects of the invention include methods of producing an antibody as described herein, comprising growing a cell as described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell.
  • aspects of the invention include methods of making an antibody as described herein, comprising immunizing a UniRat animal with an HBsAg protein and identifying HBsAg binding antibody sequences.
  • aspects of the invention include methods of treatment, comprising administering to an individual in need an effective dose of the antibody as described herein, or a pharmaceutical composition as described herein.
  • FIG. 1, panels A-F provide schematic illustrations of: a UniAb (panel A); an anti-CD3 x monovalent, monospecific anti-HBsAg antibody (panel B); an anti-CD3 x bivalent, monospecific anti- HBsAg antibody (panel C); an anti-CD3 x bivalent, biparatopic anti-HBsAg antibody (panel D); an anti-CD3 x trivalent, triparatopic anti-HBsAg antibody in which all three anti-HBsAg variable region sequences are positioned adjancent to the N-terminus (panel E); and an anti-CD3 x trivalent, triparatopic anti-HBsAg antibody in which two anti-HBsAg variable region sequences are positioned adjancent to the N-terminus, and one anti-HBsAg variable region sequence is positioned adjacent to the C-terminus (panel F), in accordance with embodiments of the invention.
  • FIG. 2 provides a graph showing results from anti-HBsAg HCAb binding to HB
  • FIG. 3 provides a graph showing results from anti-CD3, anti-HBsAg bispecific antibodies binding to HBsAg expressing HepG2-LMS cells.
  • FIG. 4 provides a graph showing results from monovalent bispecific antibody -mediated killing of HepG-LMS cells expressing HBsAg.
  • FIG. 5 provides an epitope bin chart of anti-HBsAg UniAbs. Solid line indicates mutual blocking. Dashed line with an arrowhead indicates one-directional blocking. 5 bins are identified.
  • FIG. 6 is a table showing binding affinity and epitope binning data for the indicated anti-HBsAg antibodies.
  • FIG. 7 is a graph showing % cytotoxicity as a function of antibody concentration for the indicated antibody constructs.
  • FIG. 8 is a graph showing % neutralization of HBV as a function of increasing antibody concentration for the indicated antibody constructs.
  • composition/method/kit By “comprising” it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim.
  • Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • the “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies mean residue numbering by the EU numbering system.
  • Antibodies also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VL) domain.
  • VH variable heavy
  • VL variable light
  • the two domains conventionally associate to form a specific binding region, although as will be discussed here, specific binding can also be obtained with heavy chain-only variable sequences, and a variety of non-natural configurations of antibodies are known and used in the art.
  • a “functional” or “biologically active” antibody or antigen-binding molecule is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events.
  • a functional antibody or other binding molecule e.g., a TCA
  • a TCA may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signal transduction or enzymatic activity.
  • a functional antibody or other binding molecule may also block ligand activation of a receptor or act as an agonist or antagonist.
  • the capability of an antibody or other binding molecule, e.g., a TCA, to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three chain antibodies, TCAs, single chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.
  • antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that comprises an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
  • the immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity.
  • Light chains of the subject antibodies can be kappa light chains (Vkappa) or lambda light chains (Vlambda).
  • the immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies in accordance with the present invention can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be made via recombinant protein production methods (see, e.g., U.S. Patent No. 4,816,567), for example.
  • variable refers to the fact that certain portions of the antibody variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a b-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the b-sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Rabat et al, Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Rabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
  • CDR complementarity determining region
  • CDR means a complementary determining region of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999).
  • Framework Region or “FR” residues are those variable domain residues other than the hypervariable region/CDR residues as herein defined.
  • CDR designations are shown herein, however one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Rabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used.
  • the Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989; 342:877-883).
  • CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes.” J Immunol. 2008;181:6230-6235; Almagro “Identification of differences in the specificity -determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.
  • heavy chain-only antibody and “heavy chain antibody” are used interchangeably herein and refer, in the broadest sense, to antibodies, or more or more portions of an antibody, e.g., one or more arms of an antibody, lacking the light chain of a conventional antibody.
  • the terms specifically include, without limitation, homodimeric antibodies comprising the VH antigen-binding domain and the CH2 and CH3 constant domains, in the absence of the CHI domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NAR comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR) and functional fragments thereof; and soluble single domain antibodies (sUniDabsTM).
  • a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4.
  • a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains. In another embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH2 domain. In a further embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain. Heavy chain-only antibodies in which the CH2 and/or CH3 domain is truncated are also included herein. In a further embodiment, a heavy chain is composed of an antigen binding domain, and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region.
  • the heavy chain-only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded or otherwise, covalently or non- covalently, attached with each other.
  • the heavy chain-only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein.
  • a heavy chain antibody is of the IgGl, IgG2, IgG3, or IgG4 subtype, in particular the IgGl or IgG4 subtype.
  • a heavy-chain antibody is of the IgG4 subtype, wherein one or more of the CH domains is modified to alter an effector function of the antibody.
  • the heavy -chain antibody is of the IgGl or IgG4 subtype, wherein one or more of the CH domains is modified to alter an effector function of the antibody. Modifications of CH domains that alter effector function are further described herein. Non-limiting examples of heavy-chain antibodies are described, for example, in W02018/039180, the disclosure of which is incorporated herein by reference in its entirety.
  • the heavy chain-only antibodies herein are used as a binding (targeting) domain of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the definition specifically includes human heavy chain- only antibodies produced by human immunoglobulin transgenic rats (UniRatTM), called UniAbsTM.
  • the variable regions (VH) of UniAbsTM are called UniDabsTM, and are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multi-specificity, increased potency and extended half-life. Since the homodimeric UniAbsTM lack a light chain and thus a VL domain, the antigen is recognized by one single domain, i.e., the variable domain of the heavy chain of a heavy -chain antibody (VH or VHH).
  • an “intact antibody chain” as used herein is one comprising a full length variable region and a full length constant region (Fc).
  • An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, hinge, CH2 and CH3 for secreted IgG.
  • CL light chain constant domain
  • Other isotypes, such as IgM or IgA may have different CH domains.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody.
  • effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody -dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors.
  • Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.
  • antibodies and various antigen-binding proteins can be provided as different classes.
  • the Fc constant domains that correspond to the different classes of antibodies may be referenced as a, d, e, g, and m, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310).
  • the light chains of antibodies from any vertebrate species can be assigned to one of two types, called k (kappa) and l (lambda), based on the amino acid sequences of their constant domains.
  • Antibodies in accordance with embodiments of the invention can comprise kappa light chain sequences or lambda light chain sequences.
  • a “functional Fc region” possesses an “effector function” of a native-sequence Fc region.
  • Non limiting examples of effector functions include Clq binding; CDC; Fc-receptor binding; ADCC; ADCP; down-regulation of cell-surface receptors (e.g., B-cell receptor), etc.
  • Such effector functions generally require the Fc region to interact with a receptor, e.g., the FcyRI; FcyRIIA; FcyRIIBl; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors, and the low affinity FcRn receptor; and can be assessed using various assays known in the art.
  • a “dead” or “silenced” Fc is one that has been mutated to retain activity with respect to, for example, prolonging serum half-life, but which does not activate a high affinity Fc receptor, or which has a reduced affinity to an Fc receptor.
  • a “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native-sequence human Fc regions include, for example, a native-sequence human IgGl Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof.
  • a “variant Fc region” comprises an amino acid sequence that differs from that of a native- sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s).
  • the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.
  • Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563).
  • Two amino acid substitutions in the complement Clq binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)).
  • Substitution into human IgGl or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL.
  • Fc variants are possible, including, without limitation, one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N- terminal end of a native Fc, or a methionine residue is added thereto.
  • one or more Fc portions of an antibody can comprise one or more mutations in the hinge region to eliminate disulfide bonding.
  • the hinge region of an Fc can be removed entirely.
  • an antibody can comprise an Fc variant.
  • an Fc variant can be constructed to remove or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding.
  • a deletion may occur in a complement-binding site, such as a Clq-binding site.
  • Techniques for preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478.
  • the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
  • an antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which can optionally be referred to herein as an IgG4 CH3 knob sequence.
  • an antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which can optionally be referred to herein as an IgG4 CH3 hole sequence.
  • the IgG4 CH3 mutations described herein can be utilized in any suitable manner so as to place a “knob” on a first heavy chain constant region of a first monomer in an antibody dimer, and a “hole” on a second heavy chain constant region of a second monomer in an antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.
  • an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235 A mutation, and a T366W mutation (knob).
  • and antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, and a Y407V mutation (hole).
  • Fc-region-comprising antibody refers to an antibody that comprises an Fc region.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody.
  • an antibody having an Fc region according to this invention can comprise an antibody with or without K447.
  • any of SEQ ID NOs. 78-118, 143, 144 and 145 can optionally comprise a K447 residue.
  • aspects of the invention include antibodies comprising a heavy chain-only variable region in a monovalent or bivalent configuration.
  • the term “monovalent configuration” as used in reference to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain is present, having a single binding site (see FIG. 1, Panel B, right arm of antibody).
  • the term “bivalent configuration” as used in reference to a heavy chain-only variable region domain means that two heavy chain-only variable region domains are present (each having a single binding target), and are connected by a linker sequence (see FIG. 1, Panel C, right arm of antibody).
  • Non-limiting examples of linker sequences are discussed further herein, and include, without limitation, GS linker sequences of various lengths.
  • each of the two heavy chain-only variable region domains can have binding affinity to the same antigen, or to different antigens (e.g., binding to different epitopes on the same protein; to two different proteins (biparatopic), etc.).
  • a heavy chain-only variable region denoted as being in a “bivalent configuration” is understood to contain two identical heavy chain-only variable region domains, connected by a linker sequence, wherein each of the two identical heavy chain-only variable region domains have binding affinity to the same target antigen.
  • aspects of the invention include antibodies having multi-specific configurations, which include, without limitation, bispecific, trispecific, etc.
  • a large variety of methods and protein configurations are known and used in bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, etc.
  • a first and a second antigen-binding domain on a polypeptide are connected by a polypeptide linker.
  • a polypeptide linker is a GS linker, having an amino acid sequence of four glycine residues, followed by one serine residue, and wherein the sequence is repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9.
  • Other suitable linkers can also be used, and are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357-69, the disclosure of which is incorporated herein by reference in its entirety.
  • three-chain antibody like molecule or “TCA” is used herein to refer to antibody- like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen binding region and at least one CH domain.
  • This heavy chain/light chain pair has binding specificity for a first antigen.
  • the third polypeptide subunit comprises, consists essentially of, or consists of a heavy-chain only antibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and one or more antigen binding domains (e.g., two antigen binding domains) that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain.
  • Parts of such variable region may be encoded by V H and/or V L gene segments, D and J H gene segments, or J L gene segments.
  • the variable region may be encoded by rearranged V H DJ h , V L DJ H , V H J L , or V L J L gene segments.
  • a TCA binding compound makes use of a “heavy chain only antibody” or “heavy chain antibody” or “heavy chain polypeptide” which, as used herein, mean a single chain antibody comprising heavy chain constant regions CH2 and/or CH3 and/or CH4 but no CHI domain.
  • the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains.
  • the heavy chain antibody is composed of an antigen binding domain, at least part of a hinge region and a CH2 domain.
  • the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain.
  • Heavy chain antibodies in which the CH2 and/or CH3 domain is truncated are also included herein.
  • the heavy chain is composed of an antigen binding domain, and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region.
  • the heavy chain only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded other otherwise covalently or non-covalently attached with each other, and can optionally include an asymmetric interface between one or more of the CH domains to facilitate proper pairing between polypeptide chains.
  • the heavy- chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein.
  • the heavy chain antibody is of the IgGl, IgG2, IgG3, orIgG4 subtype, in particular the IgGl subtype or the IgG4 subtype.
  • TCA binding compound are described in, for example, WO2017/223111 and W02018/052503, the disclosures of which are incorporated herein by reference in their entirety.
  • Heavy-chain antibodies constitute about one fourth of the IgG antibodies produced by the camelids, e.g., camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but are devoid of light chains. As a consequence, the variable antigen binding part is referred to as the VHH domain and it represents the smallest naturally occurring, intact, antigen-binding site, being only around 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)).
  • Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)).
  • HBsAg or “Hepatitis B surface antigen” as used herein refers to a surface protein of Hepatitis B Virus (“HBV” or “Hep B”).
  • HBsAg is encoded by a Gene S, which consists of one long open reading frame, but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-Sl, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes, called large, middle, and small (pre-Sl+pre-S2+S, pre-S2+S, or S) are produced (Beck et al., World J. Gastroenterol. (2007) 13; 48-64.
  • HBsAg includes an HBsAg surface protein from a Hepatitis B virus that is capable of infecting a human or non-human animal, and includes HBsAg on a Hepatitis B virus itself, and/or HBsAg that is generated by a cell in a human or non-human animal that is infected with a Hepatitis B virus.
  • HBsAg includes an HBsAg protein from a Hepatitis B virus (e.g., on the surface of a Hepatitis B virus), and also includes HBsAg produced in the cells of human and non-human mammals that are infected with a Hepatitis B virus, or that are transfected with one or more genes encoding HBsAg.
  • HBsAg includes any serotypes, variants, isoforms and species homologs of HBsAg (e.g.,UniProt Q81158), regardless of its source or mode of preparation.
  • HBsAg includes native HBsAg that is naturally expressed by human or animal cells infected by a Hepatitis B virus, as well as HBsAg expressed on cells transfected with an HBsAg gene.
  • native HBsAg as used herein includes HBsAg isolated from human or animal blood or tissues.
  • recombinant HBsAg as used herein includes HBsAg expressed by cells transfected with an HBsAg gene.
  • anti-HBsAg heavy chain-only antibody HBsAg heavy chain-only antibody
  • anti-HBsAg heavy chain antibody HBsAg heavy chain antibody
  • HBsAg heavy chain antibody HBsAg heavy chain antibody
  • HBsAg heavy chain antibody HBsAg heavy chain antibody
  • HBsAg heavy chain antibody HBsAg heavy chain antibody
  • HBsAg heavy chain antibody HBsAg heavy chain antibody
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • Antibodies of the invention include multi-specific antibodies.
  • Multi-specific antibodies have more than one binding specificity.
  • the term “multi-specific” specifically includes “bispecific” and “trispecific,” as well as higher-order independent specific binding affinities, such as higher-order polyepitopic specificity, as well as tetravalent antibodies and antibody fragments.
  • the terms “multi specific antibody,” “multi-specific heavy chain-only antibody,” “multi-specific heavy chain antibody,” and “multi-specific UniAbTM” are used herein in the broadest sense and cover all antibodies with more than one binding specificity.
  • the multi-specific heavy chain anti-HBsAg antibodies of the present invention specifically include antibodies immunospecifically binding to two or more non-overlapping epitopes on an HBsAg protein, such as a native HBsAg (i.e., bivalent and biparatopic).
  • the multi specific heavy chain anti-HBsAg antibodies of the present invention also specifically include antibodies immunospecifically binding to an epitope on an HBsAg protein, such as native HBsAg and to an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 (i.e., bivalent and biparatopic).
  • the multi-specific heavy chain anti-HBsAg antibodies of the present invention also specifically include antibodies immunospecifically binding to two or more non-overlapping or partially overlapping epitopes on an HBsAg protein, such as a native HBsAg protein, and to an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 protein (i.e., trivalent and biparatopic).
  • an HBsAg protein such as a native HBsAg protein
  • a different protein such as, for example, a CD3 protein, such as human CD3 protein (i.e., trivalent and biparatopic).
  • Antibodies of the invention include monospecific antibodies, having one binding specificity.
  • Monospecific antibodies specifically include antibodies comprising a single binding specificity, as well as antibodies comprising more than one binding unit having the same binding specificity.
  • the terms “monospecific antibody,” “monospecific heavy chain-only antibody,” “monospecific heavy chain antibody,” and “monospecific UniAbTM” are used herein in the broadest sense and cover all antibodies with one binding specificity.
  • the monospecific heavy chain anti-HBsAg antibodies of the present invention specifically include antibodies immunospecifically binding to one epitope on an HBsAg protein, such as a native HBsAg (monovalent and monospecific).
  • the monospecific heavy chain anti- HBsAg antibodies of the present invention also specifically include antibodies having more than one binding unit (e.g., multivalent antibodies) immunospecifically binding to an epitope on an HBsAg protein, such as native HBsAg.
  • a monospecific antibody in accordance with embodiments of the invention can include a heavy chain variable region comprising two antigen-binding domains, wherein each antigen-binding domain binds to the same epitope on an HBsAg protein (i.e., bivalent and monospecific).
  • An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.
  • Epitope mapping is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens.
  • Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.
  • “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).
  • the present invention specifically includes anti-HBsAg heavy chain antibodies with polyepitopic specificities, i.e., anti-HBsAg heavy chain antibodies binding to one or more non-overlapping epitopes on an HBsAg protein, such as a native HBsAg; and anti-HBsAg heavy chain antibodies binding to one or more epitopes on an HBsAg protein and to an epitope on a different protein, such as, for example, a CD3 protein.
  • non-overlapping epitope(s) or “non-competitive epitope(s)” of an antigen is defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies but not the other member. Pairs of antibodies, or antigen-binding regions targeting the same antigen on a multi-specific antibody, recognizing non-overlapping epitopes, do not compete for binding to that antigen and are able to bind that antigen simultaneously.
  • An antibody binds “essentially the same epitope” as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes.
  • the most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody.
  • the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.
  • the term “valent” as used herein refers to a specified number of binding sites in an antibody molecule.
  • a “monovalent” antibody has one binding site. Thus, a monovalent antibody is also monospecific.
  • a “multi-valent” antibody has two or more binding sites.
  • the terms “bivalent”, “trivalent”, and “tetravalent” refer to the presence of two binding sites, three binding sites, and four binding sites, respectively.
  • a bispecific antibody according to the invention is at least bivalent and may be trivalent, tetravalent, or otherwise multi-valent.
  • a bivalent antibody in accordance with embodiments of the invention may have two binding sites to the same epitope (i.e., bivalent, monoparatopic), or to two different epitopes (i.e., bivalent, biparatopic).
  • BsMAB bispecific monoclonal antibodies
  • tri-specific antibodies tri-specific antibodies
  • three-chain antibody like molecule or “TCA” is used herein to refer to antibody- like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen binding region and at least one CH domain.
  • This heavy chain/light chain pair has binding specificity for a first antigen.
  • the third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain.
  • Parts of such variable region may be encoded by V H and/or V L gene segments, D and J H gene segments, or J L gene segments.
  • the variable region may be encoded by rearranged V H DJ H , V L DJ H , V H J L , or V L J L gene segments.
  • a TCA protein makes use of a heavy chain-only antibody as hereinabove defined.
  • chimeric antigen receptor or “CAR” is used herein in the broadest sense to refer to an engineered receptor, which grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to membrane-spanning and intracellular-signaling domains.
  • a desired binding specificity e.g., the antigen-binding region of a monoclonal antibody or other ligand
  • the receptor is used to graft the specificity of a monoclonal antibody onto a T-cell to create a chimeric antigen receptors (CAR).
  • CAR-T cells are T-cells that have been genetically engineered to produce an artificial T-cell receptor for use in immunotherapy.
  • CAR-T cell means a therapeutic T-cell expressing a transgene encoding one or more chimeric antigen receptors comprised minimally of an extracellular domain, a transmembrane domain, and at least one cytosolic domain.
  • human antibody is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies herein may include amino acid residues not encoded by human germline immuno globulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo.
  • the term “human antibody” specifically includes heavy chain-only antibodies having human heavy chain variable region sequences, produced by transgenic animals, such as transgenic rats or mice, in particular UniAbsTM produced by UniRatsTM, as defined above.
  • a “chimeric antibody” or a “chimeric immunoglobulin” is meant an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, e.g., a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus.
  • Chimeric antibodies include transgenic antibodies with non-human Fc-regions or artificial Fc-regions, and human idiotypes.
  • Such immunoglobulins can be isolated from animals of the invention that have been engineered to produce such chimeric antibodies.
  • effector cell refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and carry out specific immune functions.
  • an effector cell such as a natural killer cell is capable of inducing antibody- dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcR, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens.
  • an effector cell may phagocytose a target antigen or target cell.
  • Human effector cells are leukocytes which express receptors such as T-cell receptors or FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T-cells and neutrophils; with NK cells being preferred.
  • the effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.
  • lymphocytes such as B-cells and T-cells including cytolytic T-cells (CTLs)
  • CTLs cytolytic T-cells
  • NK natural killer cells
  • macrophages macrophages
  • monocytes monocytes
  • eosinophils polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody.
  • Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody -dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptor; BCR), etc.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in US Patent No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.
  • a CDC assay e.g., as described in Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.
  • Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound.
  • Kd dissociation constant
  • the “Kd” or “Kd value” refers to a dissociation constant determined by BioLayer Interferometry, using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode.
  • anti-mouse Fc sensors are loaded with mouse-Fc fused antigen and then dipped into antibody-containing wells to measure concentration dependent association rates (kon).
  • Antibody dissociation rates (koff) are measured in the final step, where the sensors are dipped into wells containing buffer only.
  • the Kd is the ratio of koff/kon.
  • treatment covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • a “therapeutically effective amount” is intended for an amount of active agent which is necessary to impart therapeutic benefit to a subject.
  • a “therapeutically effective amount” is an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with a disease or which improves resistance to a disorder.
  • HBsAg characterized by the presence of HBsAg
  • characterized by expression of HBsAg broadly refer to any disease or disorder in which HBsAg presence and/or expression are associated with or involved with one or more pathological processes that are characteristic of the disease or disorder.
  • disorders include, but are not limited to, liver or hepatic disease (e.g., infection), acute or chronic hepatitis B infection, liver cirrhosis and hepatocellular carcinoma.
  • subject is used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated.
  • the mammal is a human.
  • subject encompass, without limitation, individuals having cancer, individuals with autoimmune diseases, with pathogen infections, and the like.
  • Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mouse, rat, etc.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
  • a “sterile” formulation is aseptic or free or essentially free from all living microorganisms and their spores.
  • a “frozen” formulation is one at a temperature below 0 °C.
  • a “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example. Stability can be measured at a selected temperature for a selected time period.
  • Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy -terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc.
  • aggregate formation for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection
  • icIEF image capillary isoelectric focusing
  • capillary zone electrophoresis amino-terminal or carboxy -terminal sequence analysis
  • mass spectrometric analysis SDS-PAGE analysis to compare reduced and intact antibody
  • peptide map for example
  • Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.
  • deamidation e.g., Asn deamidation
  • oxidation e.g., Met oxidation
  • isomerization e.g., Asp isomerization
  • clipping/hydrolysis/fragmentation e.g., hinge region fragmentation
  • succinimide formation unpaired cysteine(s)
  • N-terminal extension e.g., N-terminal extension, C-terminal processing, glycosylation differences, etc.
  • the present invention provides families of closely related antibodies that bind to HBsAg (e.g., HBsAg that is expressed in human cells infected with Hepatitis B virus (HBV)).
  • the antibodies of these families comprise sets of CDR sequences as defined herein and shown in Tables 1-2, and are exemplified by the provided heavy chain variable region (VH) sequences of SEQ ID NOs: 55 to 100 set forth in Tables 3 and 4.
  • VH heavy chain variable region
  • These families of antibodies provide a number of benefits that contribute to utility as clinically therapeutic agent(s).
  • the antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity.
  • Table 1 Anti-HBsAg heavy chain antibody unique CDR amino acid sequences.
  • Table 2 Anti-HBsAg heavy chain antibody unique CDR amino acid sequences.
  • a suitable antibody may be selected from those provided herein for development and therapeutic or other use, including, without limitation, use as a bispecific antibody, e.g., as shown in FIG. 1, panels B-D, or as part of a CAR-T structure.
  • FIG. 1, panel B provides an illustration of an anti- CD3 x anti-HBsAg multi-specific antibody, where the anti-HBsAg domain is monovalent and monospecific.
  • the anti-CD3 domain contains a CHI domain and pairs with a light chain, while the anti- HBsAg domain is derived from a heavy chain-only antibody and does not contain a CHI domain or interact with a light chain.
  • the two heavy chains are pared using, e.g., knobs- into-holes technology.
  • FIG. 1, panel C provides an illustration of an anti-CD3 x anti-HBsAg multi specific antibody, where the anti-HBsAg domain is bivalent and monospecific.
  • the anti-CD3 domain contains a CHI domain and pairs with a light chain, while the anti-HBsAg domain is derived from a heavy chain-only antibody and does not contain a CHI domain or interact with a light chain.
  • the two heavy chains are pared using, e.g., knobs-into-holes technology.
  • the antibody depicted in FIG. 1, panel B is an anti-CD3 x anti-HBsAg bispecific antibody wherein the anti-HBsAg binding arm is monovalent and monospecific, and the antigen-binding domain of the anti-HBsAg arm is in a monovalent configuration, meaning only one antigen-binding domain is present.
  • panel C is an anti-CD3 x anti-HBsAg bispecific antibody wherein the anti-HBsAg binding arm is bivalent and monospecific, and the antigen-binding domain of the anti-HBsAg arm is in a bivalent configuration, meaning there are two identical antigen binding domains placed in tandem.
  • an antibody can be bivalent and biparatopic, meaning that there are two antigen binding domains present on the anti-HBsAg arm of the antibody, and each of these antigen-binding domains contains a different sequence, and binds to a different epitope on an HBsAg protein (see FIG. 1, panel D).
  • Determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements.
  • Members of the antibody family may have an affinity for HBsAg with a Kd of from about 10 6 to around about 10 n , including without limitation: from about 10 6 to around about 10 10 ; from about 10 6 to around about 10 9 ; from about 10 6 to around about 10 8 ; from about 10 8 to around about 10 n ; from about 10 8 to around about 10 10 ; from about 10 8 to around about 10 9 ; from about 10 9 to around about 10 n ; from about 10 9 to around about 10 10 ; or any value within these ranges.
  • the affinity selection may be confirmed with a biological assessment for modulating, e.g., blocking, an HBsAg biological activity, including in vitro assays, pre-clinical models, and clinical trials, as well as assessment of potential toxicity.
  • the HBsAg-specific antibodies herein comprise a VH domain, comprising CDR1, CDR2 and CDR3 sequences in a human VH framework.
  • the CDR sequences may be situated, as an example, in the region of around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequences set forth in SEQ ID NOs: 23 to 74. It will be understood by one of ordinary skill in the art that the CDR sequences may be in different positions if a different framework sequence is selected, although generally the order of the sequences will remain the same.
  • an anti-HBsAg antibody comprises a CDR1 sequence of any one of SEQ ID NOs: 1-15.
  • an anti-HBsAg antibody comprises a CDR1 sequence of any one of SEQ ID NOs: 2, 5, 8, 12, and 14.
  • an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 2.
  • an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 5.
  • an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 8.
  • an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 12.
  • an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 14.
  • an anti-HBsAg antibody comprises a CDR2 sequence of any one of SEQ ID NOs: 16-34. In some embodiments, an anti-HBsAg antibody comprises a CDR2 sequence of any one of SEQ ID NOs: 18, 24, 29, 32, and 33. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 18. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 24. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 29. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 32. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 33.
  • an anti-HBsAg antibody comprises a CDR3 sequence of any one of SEQ ID NOs: 32-54. In some embodiments, an anti-HBsAg antibody comprises a CDR3 sequence of any one of SEQ ID NOs: 37, 44, 49, 52, and 53. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 37. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 44. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 49. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 52. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 53.
  • an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 2; a CDR2 sequence comprising the sequence of SEQ ID NO: 18; and a CDR3 sequence comprising the sequence of SEQ ID NO: 37.
  • an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 8; a CDR2 sequence comprising the sequence of SEQ ID NO: 24; and a CDR3 sequence comprising the sequence of SEQ ID NO: 44.
  • an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 5; a CDR2 sequence comprising the sequence of SEQ ID NO: 29; and a CDR3 sequence comprising the sequence of SEQ ID NO: 49.
  • an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 12; a CDR2 sequence comprising the sequence of SEQ ID NO: 32; and a CDR3 sequence comprising the sequence of SEQ ID NO: 52.
  • an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 14; a CDR2 sequence comprising the sequence of SEQ ID NO: 33; and a CDR3 sequence comprising the sequence of SEQ ID NO: 53.
  • an anti-HBsAg antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 55-77 (Table 3).
  • an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 59.
  • an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 66.
  • an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 72.
  • an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 75.
  • an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 76.
  • a CDR sequence in an anti-HBsAg antibody of the invention comprises one or two amino acid substitutions relative to a CDR1, CDR2 and/or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs: 1-54 (Table 1).
  • an anti-HBsAg antibody preferably comprises a heavy chain variable domain (VH) in which the CDR3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Tables 1 and 2, and binds to HBsAg.
  • VH heavy chain variable domain
  • an anti-HBsAg antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty- five percent (85%) sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Tables 1 and 2, and binds to HBsAg.
  • VH heavy chain variable domain
  • an anti-HBsAg antibody comprises a heavy chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identify, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 55-77 (shown in Table 3), and binds to HBsAg.
  • an anti-HBsAg antibody can comprise a plurality of heavy chain variable regions positioned at any suitable location on any one or more of the polypeptide subunits of the antibody.
  • an anti-HBsAg antibody can comprise a heavy chain variable region that is positioned at an N-terminus and/or a C-terminus of one or more of the polypeptide subunits (either a light chain or a heavy chain) that make up the antibody.
  • an anti-HBsAg antibody can comprise 1, 2, 3, 4, 5, 6, 7, or 8 or more heavy chain variable regions that are positioned at various locations on one or more of the polypeptide subunits of the antibody.
  • anti-HBsAg heavy chain variable regions can comprise the same sequence, or they can comprise a plurarlity of different sequences, e.g., such as 2, 3, 4, 5, 6, 7, or 8 different sequences, such that the resulting antibody is monoparatopic, biparatopic, triparatopic, tetraparatopic, pentaparatopic, hexaparatopic, heptaparatopic, or octaparatopic.
  • one or more heavy chain variable regions can be positioned, e.g., at an N-terminus of a light chain polypeptide subunit, at a C- terminus of a light chain polypeptide subunit, at an N-terminus of a heavy chain polypeptide subunit, or at a C-terminus of a heavy chain polypeptide subunit.
  • a single heavy chain variable region can be present, or a plurality of heavy chain variable regions can be present (e.g., a tandem configuration of two heavy chain variable regions, a tandem configuration of three heavy chain variable regions, etc.), each having the same or different binding sequences.
  • bispecific or multi-specific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific three-chain antibody like molecule (TCA).
  • a multi-specific antibody can comprise at least one heavy chain variable region having binding specificity for HBsAg, and at least one heavy chain variable region having binding specificity for a protein other than HBsAg.
  • a multi-specific antibody can comprise a heavy chain variable region comprising at least two antigen binding domains, wherein each of the antigen-binding domains has binding specificity for HBsAg.
  • a multi-specific antibody can comprise a heavy chain/light chain pair that has binding specificity for a first antigen (e.g., CD3), and a heavy chain from a heavy chain-only antibody that has binding specificity to a second antigen (e.g., HBsAb).
  • the heavy chain from the heavy chain-only antibody comprises an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain.
  • a bispecific antibody comprises a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (e.g., a CD3 protein on a T-cell), and a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain that has binding specificity for HBsAg.
  • an effector cell e.g., a CD3 protein on a T-cell
  • a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain that has binding specificity for HBsAg.
  • bispecific or multispecific anti-HBsAg antibodies in accordance with embodiments of the invention can also comprise a plurality of anti-HBsAg heavy chain variable regions positioned at any suitable location on any one or more of the polypeptide subunits of the antibody.
  • a multispecific anti-HBsAg antibody can comprise an anti-HBsAg heavy chain variable region that is positioned at an N-terminus and/or a C-terminus of one or more of the polypeptide subunits (either a light chain or a heavy chain) that make up the antibody, and which also contain one or more variable regions that bind to an antigen other than HBsAg, e.g., CD3.
  • a multispecific antibody comprises an anti-CD3 binding arm comprising a light chain polypeptide subunit and a heavy chain polypeptide subunit, and comprises one or more anti-HBsAg heavy chain variable regions that are positioned at an N-terminus and/or a C- terminus of one or more of the polypeptide subunits of the CD3 binding arm.
  • bispecifc or multispecific anti-HBsAg antibodies can comprise 1, 2, 3, 4, 5, 6, 7, or 8 or more anti-HBsAg heavy chain variable regions that are positioned at various locations on one or more of the polypeptide subunits of the antibody.
  • anti-HBsAg heavy chain variable regions can comprise the same sequence, or they can comprise a plurarlity of different sequences, e.g., such as 2, 3, 4, 5, 6, 7, or 8 different sequences, such that the resulting antibody is monoparatopic, biparatopic, triparatopic, tetraparatopic, pentaparatopic, hexaparatopic, heptaparatopic, or octaparatopic for HBsAg.
  • one or more heavy chain variable regions can be positioned, e.g., at an N-terminus of a light chain polypeptide subunit, at a C-terminus of a light chain polypeptide subunit, at an N-terminus of a heavy chain polypeptide subunit, or at a C-terminus of a heavy chain polypeptide subunit.
  • a single heavy chain variable region can be present, or a plurality of heavy chain variable regions can be present (e.g., a tandem configuration of two heavy chain variable regions, a tandem configuration of three heavy chain variable regions, etc.), each having the same or different binding sequences.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N-terminus, which are connected to one another by linker sequences.
  • a triparatopic anti-HBsAg heavy chain polypeptide subunit comprises SEQ ID NO: 146.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions, and is paired with a heavy chain light chain pair that binds to CD3, as depicted in FIG. IE.
  • a multispecific antibody comprises a first heavy chain polypeptide subunit that comprises SEQ ID NO: 147, a second heavy chain polypeptide subunit that comprises SEQ ID NO: 143, and a light chain polypeptide subunit that comprises SEQ ID NO: 142.
  • a multispecific antibody comprises a first heavy chain polypeptide subunit that comprises SEQ ID NO: 147, a second heavy chain polypeptide subunit that comprises SEQ ID NO: 144, and a light chain polypeptide subunit that comprises SEQ ID NO: 142.
  • a multispecific antibody comprises a first heavy chain polypeptide subunit that comprises SEQ ID NO: 147, a second heavy chain polypeptide subunit that comprises SEQ ID NO: 145, and a light chain polypeptide subunit that comprises SEQ ID NO: 142.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises two variable regions positioned adjacent to the N-terminus, which are connected to one another by a linker sequence, and a third variable region positioned adjacent ot the C-terminus.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises two variable regions at the N-terminus and 1 variable region at the C-terminus, and is paired with a heavy chain/light chain pair that binds to CD3, as depicted in FIG. IF.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F04A VH sequence, an F05B VH sequence, and an F03B VH sequence, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-55).
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, and an F04A VH sequence, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-59).
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-71).
  • linker sequences comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-71).
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T 10-83).
  • linker sequences comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T 10-83).
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-87).
  • linker sequences comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-87).
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F04A VH sequence, an F05B VH sequence, and an F03B VH sequence, paired with an F2F CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, and an F04A VH sequence, paired with an F2F CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2F CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2F CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2F CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F04A VH sequence, an F05B VH sequence, and an F03B VH sequence, paired with an F2B CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, and an F04A VH sequence, paired with an F2B CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2B CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2B CD3 arm.
  • a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2B CD3 arm.
  • a multi-specific antibody comprises a CD3-binding VH domain that is paired with a light chain variable domain.
  • the light chain is a fixed light chain.
  • the CD3-binding VH domain comprises a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and a CDR3 sequence of SEQ ID NO: 121, in a human VH framework.
  • the CD3 -binding VH domain comprises a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and a CDR3 sequence of SEQ ID NO: 124, in a human VH framework.
  • the CD3-binding VH domain comprises a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and a CDR3 sequence of SEQ ID NO: 127, in a human VH framework.
  • the fixed light chain comprises a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and a CDR3 sequence of SEQ ID NO: 130, in a human VL framework.
  • the CD3-binding VH domain and the light chain variable domain have binding affinity for CD3.
  • a CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 131.
  • a CD3- binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 131.
  • a CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 132.
  • a CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 132.
  • a CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 133. In some embodiments, a CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 133. In some embodiments, a fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 134. In some embodiments, a fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 134.
  • Multi-specific antibodies comprising the above -described CD3-binding VH domain and light chain variable domain have advantageous properties, for example, as described in published PCT application publication number W02018/052503, the disclosure of which is incorporated by reference herein in its entirety.
  • any of the multi-specific antibodies and antigen-binding domains described herein, having binding affinity to HBsAg, can be combined with any of the CD3 -binding domains and fixed light chain domains described herein (see, e.g., Table 6 and Table 7) and in published PCT application publication numbers W02018/052503 and WO2017/223111, the disclosures of which are incorporated by reference herein in their entireties, as well as additional sequences, such as those provided in Table 5, Table 8, and Table 9, to generate multi-specific antibodies having binding affinity to one or more HBsAg epitopes, as well as CD3.
  • Table 8 Human IgGl and IgG4 Fc region sequences.
  • bispecific or multi-specific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific three-chain antibody like molecule (TCA).
  • a bispecific antibody can comprise at least one heavy chain variable region having binding specificity for HBsAg, and at least one heavy chain variable region having binding specificity for a protein other than HBsAg.
  • a bispecific antibody can comprise a heavy chain/light chain pair that has binding specificity for a first antigen, and a heavy chain from a heavy chain-only antibody, comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen.
  • a bispecific antibody comprises a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (e.g., a CD3 protein on a T-cell), and a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain that has binding specificity for HBsAg.
  • an effector cell e.g., a CD3 protein on a T-cell
  • a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain that has binding specificity for HBsAg.
  • an antibody of the invention is a bispecific antibody
  • one arm of the antibody is specific for HBsAg
  • the other arm may be specific for target cells, tumor-associated antigens, targeting antigens, e.g., integrins, etc., pathogen antigens, checkpoint proteins, and the like.
  • one arm of the antibody is specific for HBsAg
  • the other arm is specific for CD3.
  • a multispecific (e.g., bispecific) antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 142, an anti-CD3 heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 143, 144 or 145, and an anti-HBsAg heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 101-118.
  • the anti-HBsAg heavy chain polypeptide can comprise an anti-HBsAg heavy chain variable region in a monovalent or bivalent configuration, as described herein.
  • a multispecific (e.g., bispecific) antibody comprises a first anti-HBsAg heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 78-100, paired with a second anti-HBsAg heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 78- 100
  • a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising any one of SEQ ID NOs: 1-15, a CDR2 sequence comprising any one of SEQ ID NOs: 16-34, and a CDR3 sequence comprising any one of SEQ ID NOs: 35-54.
  • CAR chimeric antigen receptor
  • a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 2, a CDR2 sequence comprising SEQ ID NO: 18, and a CDR3 sequence comprising SEQ ID NO: 37.
  • CAR chimeric antigen receptor
  • a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 24, and a CDR3 sequence comprising SEQ ID NO: 44.
  • CAR chimeric antigen receptor
  • a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 5, a CDR2 sequence comprising SEQ ID NO: 29, and a CDR3 sequence comprising SEQ ID NO: 49.
  • CAR chimeric antigen receptor
  • a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 12, a CDR2 sequence comprising SEQ ID NO: 32, and a CDR3 sequence comprising SEQ ID NO: 52.
  • CAR chimeric antigen receptor
  • a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 14, a CDR2 sequence comprising SEQ ID NO: 33, and a CDR3 sequence comprising SEQ ID NO: 53.
  • CAR chimeric antigen receptor
  • a CAR-T cell comprises an extracellular antigen-binding domain that binds to HBsAg and comprises a heavy chain variable region having at least 95% identity to any one of SEQ ID NOs. 55-77. In some embodiments, a CAR-T cell comprises an extracellular antigen-binding domain that binds to HBsAg and comprises a heavy chain variable region comprising any one of SEQ ID NOs. 55-77.
  • a CAR-T cell comprises an extracellular antigen-binding domain that binds to HBsAg and comprises a heavy chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 75, and SEQ ID NO: 76.
  • Aspects of the invention include pharmaceutical compositions comprising a CAR-T cell as described herein, as well as methods of treatment that comprise administering a therapeutically effective amount of a CAR-T cell as described herein.
  • multi-specific antibodies are within the ambit of the invention, including, without limitation, single chain polypeptides, two chain polypeptides, three chain polypeptides, four chain polypeptides, and multiples thereof.
  • the multi-specific antibodies herein specifically include T- cell multi-specific (e.g., bispecific) antibodies binding to HBsAg and CD3 (anti-HBsAg x anti-CD3 antibodies). Such antibodies induce potent T-cell mediated killing of cells expressing HBsAg.
  • the antibodies of the present invention can be prepared by methods known in the art.
  • the antibodies herein are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which the endogenous immunoglobulin genes are knocked out or disabled.
  • the heavy chain antibodies herein are produced in UniRatTM. UniRatTM have their endogenous immunoglobulin genes silenced and use a human immunoglobulin heavy-chain translocus to express a diverse, naturally optimized repertoire of fully human HCAbs.
  • Non- homologous end joining to silence a gene or locus via deletions up to several kb can also provide a target site for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67).
  • Human heavy chain antibodies produced in UniRatTM are called UniAbsTM and can bind epitopes that cannot be attacked with conventional antibodies. Their high specificity, affinity, and small size make them ideal for mono- and poly-specific applications.
  • heavy chain-only antibodies lacking the camelid VHH framework and mutations, and their functional VH regions.
  • Such heavy chain-only antibodies can, for example, be produced in transgenic rats or mice which comprise fully human heavy chain-only gene loci as described, e.g., in W02006/008548, but other transgenic mammals, such as rabbit, guinea pig, rat can also be used, rats and mice being preferred.
  • Heavy chain-only antibodies including their VHH or VH functional fragments, can also be produced by recombinant DNA technology, by expression of the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), E. coli or yeast.
  • a suitable eukaryotic or prokaryotic host including, for example, mammalian cells (e.g., CHO cells), E. coli or yeast.
  • Domains of heavy chain-only antibodies combine advantages of antibodies and small molecule drugs: can be mono- or multi-valent; have low toxicity; and are cost-effective to manufacture. Due to their small size, these domains are easy to administer, including oral or topical administration, are characterized by high stability, including gastrointestinal stability; and their half-life can be tailored to the desired use or indication. In addition, VH and VHH domains of HCAbs can be manufactured in a cost-effective manner.
  • the heavy chain antibodies of the present invention including UniAbsTM, have the native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Rabat numbering system), substituted by another amino acid residue, which is capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position.
  • Such hydrophobic patches are normally buried in the interface with the antibody light chain constant region but become surface exposed in HCAbs and are, at least partially, for the unwanted aggregation and light chain association of HCAbs.
  • the substituted amino acid residue preferably is charged, and more preferably is positively charged, such as lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R).
  • the heavy chain-only antibodies derived from the transgenic animals contain a Trp to Arg mutation at position 101.
  • the resultant HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.
  • VH variable region
  • Heavy chain antibodies binding to non-overlapping epitopes on an HBsAg protein can be identified by competition binding assays, such as enzyme-linked immunoassays (ELISA assays) or flow cytometric competitive binding assays. For example, one can use competition between known antibodies binding to the target antigen and the antibody of interest. By using this approach, one can divide a set of antibodies into those that compete with the reference antibody and those that do not. The non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody.
  • one antibody is immobilized, the antigen is bound, and a second, labeled (e.g., biotinylated) antibody is tested in an ELISA assay for ability to bind the captured antigen.
  • SPR surface plasmon resonance
  • This can be performed also by using surface plasmon resonance (SPR) platforms, including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR imager (Ibis technologies B.V.), as well as on biolayer interferometry platforms, such as Octet Red384 and Octet HTX (ForteBio, Pall Inc).
  • SPR surface plasmon resonance
  • an antibody “competes” with a reference antibody if it causes about 15-100% reduction in the binding of the reference antibody to the target antigen, as determined by standard techniques, such as by the competition binding assays described above.
  • the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher.
  • compositions comprising one or more antibodies of the present invention in admixture with a suitable pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.
  • a pharmaceutical composition comprises a heavy chain antibody (e.g., UniAbTM) that binds to HBsAg.
  • a pharmaceutical composition comprises a multi-specific (including bispecific) heavy chain antibody (e.g., UniAbTM) with binding specificity for two or more non-overlapping epitopes on an HBsAg protein.
  • a pharmaceutical composition comprises a multi-specific (including bispecific and TCA) heavy chain antibody (e.g., UniAbTM) with binding specificity to HBsAg and with binding specificity to a binding target on an effector cell (e.g., a binding target on a T-cell, such as, e.g., a CD3 protein on a T-cell).
  • a multi-specific (including bispecific and TCA) heavy chain antibody e.g., UniAbTM
  • a binding target on an effector cell e.g., a binding target on a T-cell, such as, e.g., a CD3 protein on a T-cell.
  • compositions of the antibodies used in accordance with the present invention are prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g. Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). The formulation depends on the route of administration chosen.
  • the antibodies herein can be administered by intravenous injection or infusion or subcutaneously.
  • the antibodies herein can be formulated in aqueous solutions, preferably in physiologically -compatible buffers to reduce discomfort at the site of injection.
  • the solution can contain carriers, excipients, or stabilizers as discussed above.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies, including UniAbsTM, of the present invention. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.
  • the anti-HBsAg antibodies and pharmaceutical compositions described herein can be used for the treatment of diseases and conditions characterized by the presence and/or expression of HBsAg, including, without limitation, the conditions and diseases described further herein.
  • the anti-HBsAg antibodies and pharmaceutical compositions described herein can be used for the treatment of infection with hepatitis B virus (HBV) and/or a disease or condition caused thereby.
  • HBV hepatitis B virus
  • HBsAg (UniProt Q81158) is the major antigen of the viral envelop and essential for viral attachment to heparan sulfate proteoglycans and a bile receptor — the sodium taurocholate co transporting polypeptide (NTCP) on the surface of hepatocytes.
  • NTCP sodium taurocholate co transporting polypeptide
  • HBsAgS Three related HBsAgs-small (HBsAgS), middle (HBsAgM) and large (HBsAgL) are encoded by a single viral open reading fame (S ORF) and translated from 2 subgenomic mRNAs (HBsAgL from the 2.4 kb subgenomic mRNA, and HBsAgS and HBsAgM from the 2.1 kb subgenomic mRNA). All three envelope proteins share the common S domain with a size of 226 amino acids (aa).
  • HBsAgM has an additional pre-S2 domain (55 aa) at the N-terminus of HBsAgS, and HBsAgL has an additional preSl-domain (108 or 119 aa depending on the genotype) extended from the N-terminus of HBsAgM.
  • the core region of the S domain comprising aa 99 to 169 and referred to as the major hydrophilic region (MHR), contains important epitopes recognized by neutralizing antibodies and T cells.
  • HBsAg Based on the antigenic heterogeneity of HBsAg, four serotypes were initially identified: adw, adr, ayw, and ayr, with a common “a” determinant and mutually exclusive “d/y” and “w/r”. Additional subdeterminants further divide HBsAg into a total of 10 serotypes.
  • HBsAgs are also secreted by infected cells as spherical or filamentous non-infectious subviral particles (SVPs) without the viral capsid.
  • SVPs non-infectious subviral particles
  • HBsAgS recombinantly produced in yeast and CHO cells also form SVPs and have been developed into HBV vaccines. Development of human derived monoclonal and bispecific neutralizing antibodies against HBsAg with therapeutic potentials have been described. Cellular confocal imaging studies and immunohistochemical tissue staining provide evidence of localization of HBsAg on the surface of infected hepatocytes. This and the importance of T cell immunity in viral clearance prompted the development of anti-HBsAg chimeric antigen receptor (CAR) T cells.
  • CAR antigen receptor
  • the anti-HBsAg antibodies (e.g., UniAbsTM) and pharmaceutical compositions herein can be used to treat disorders characterized by the presence and/or expression of HBsAg, including, without limitation, chronic Hepatitis B infection (CHB), liver cirrhosis and hepatocellular carcinoma.
  • CHB chronic Hepatitis B infection
  • the anti-HBsAg antibodies (e.g., UniAbsTM) and pharmaceutical compositions herein can be used in connection with liver transplantation procedures, wherein a liver from a first patient is transplanted into a second patient.
  • the first patient is currently suffering from, or has previously been infected with, Hepatitis B virus, and is receiving a liver from a patient who is Hepatitis B virus-negative.
  • the anti-HBsAg antibodies e.g., UniAbsTM
  • the anti-HBsAg antibodies and pharmaceutical compositions herein can be used to prevent the Hepatitis B virus infection from spreading to the transplanted liver.
  • the first patient has not previously been infected with Hepatitis B virus, but is receiving a liver from a patient that is currently suffering from, or has previously been infected with, Hepatitis B virus.
  • the anti-HBsAg antibodies (e.g., UniAbsTM) and pharmaceutical compositions herein can be used to prevent the Hepatitis B virus infection from spreading to the transplant recipient patient.
  • compositions of the present invention for the treatment of disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human, but non-human mammals may also be treated, e.g., companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.
  • Dosage levels can be readily determined by the ordinarily skilled clinician, and can be modified as required, e.g., as required to modify a subject's response to therapy.
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
  • the therapeutic dosage the agent may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once every two weeks or once a month or once every 3 to 6 months.
  • Therapeutic entities of the present invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient.
  • therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution of solid (e.g., lyophilized) compositions.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • Toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in humans.
  • the dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • compositions for administration will commonly comprise an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et ak, eds., 1996)).
  • kits comprising the active agents and formulations thereof, of the invention and instructions for use.
  • the kit can further contain a least one additional reagent, e.g. a chemotherapeutic drug, etc.
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • label as used herein includes any writing, or recorded material supplied on or with a kit, or which otherwise accompanies a kit.
  • Example 1 Flow cytometry analysis of binding to HBsAg expressing cells by anti-HBsAg UniAbsTM and monovalent HepBxCD3 bispecific antibodies.
  • Binding to HBsAg-positive cells was assessed by flow cytometry (BD FACSCelestaTM, BD Biosciences) using a HepG2-LMS cell line. Briefly, 50,000 target cells were stained with a dilution series of purified UniAbs or HepBxCD3 bispecific antibodies for 30 minutes at 4°C. Following incubation, the cells were washed twice with flow cytometry buffer (IX PBS, 1% BSA, 0.1% NaN3) and stained with goat F(ab’)2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, Cat No.: 2042-09) to detect cell-bound antibodies.
  • flow cytometry buffer IX PBS, 1% BSA, 0.1% NaN3
  • MFI mean fluorescence intensity
  • Table 10 summarizes the target binding activity of the anti-HBsAg UniAbs and HepBxCD3 bispecific antibodies. Column 1 indicates the clone ID while Column 2 indicates the family ID. Columns 3 and 4 show the EC50 values in nM and the max binding to HepG2-LMS cells measured as maximum fold over the background MFI signal.
  • HBsAg SVPs The binding affinity of anti-HBsAg UniAbs to HBsAg SVPs of different serotypes was measured by BLI using the Octet HTX (Sartorius). Since HBsAg SVPs are multivalent, they were immobilized onto the biosensors as the ligand while the UniAbs were in solution as the analyte. Two methods were employed to immobilize HBsAg SVPs onto streptavidin (SA) biosensors (Sartorius,
  • the VIR biosimilar HBC34v35 (WO2020132346A1) was used as the capture antibody.
  • HBC34v35 was first biotinylated by incubating with the EZ-Link NHS- PEG4-Biotin (Thermo Scientific, Cat No. : 21329) at a molar ratio of 1 : 10 (antibody :biotin) at room temperature for 30 min. After removal of unreacted biotin using a Zeba Spin Desalting Columns 7K MWCO (Thermo Scientific, Cat No.: 89882), biotinylated HBC34v35 was immobilized onto SA biosensors at 5 pg/mL for 300 secs.
  • the sensors were dipped into wells containing recombinant HBsAgs adw from yeast (abeam, Cat No.: ab91276), adr from CHO cells (ProSpec, Cat No.: hbs-875-b) or ayw from yeast (Fitzgerald, Cat No.: 30R-AH018), or native HBsAgs from human blood ad (Fitzgerald, Cat No.: 30-1815) or ay (Fitzgerald, Cat No.: 30- 1816) at 5 pg/mL for 900-1800 secs.
  • the sensors were washed in Kinetic Buffer for 300 secs followed by 120 secs of baseline readings in a separate set of wells containing Kinetic Buffer. Then the association with the anti-HBsAg UniAbs were measured for 300 secs at an antibody concentration of 250 nM or 100 nM followed by a dissociation phase of 300 secs in Kinetic Buffer. Dissociation rate constant (kofl) in 1/s and response in nm were analyzed by the ForteBio Data Analysis software vl 1.1.1.8.
  • recombinant HBsAg ayw from yeast were biotinylated and directly immobilized onto SA biosensors.
  • the same reagent EZ- Link NHS-PEG4-Biotin
  • HBsAg:biotin a molar ratio of 1 :2
  • incubation time 60 min.
  • biotinylated HBsAg ayw was loaded onto SA biosensors at 5 pg/mL for 900 secs.
  • the sensors were washed in Kinetic Buffer for 300 secs followed by 60 secs of baseline readings in a separate set of wells containing Kinetic Buffer. Then the association with the anti-HBsAg UniAbs were measured for 180 secs followed by a dissociation phase of 240 secs in Kinetic Buffer. After the first cycle, the sensors were regenerated in 10 mM glycine pH 1.7 to the biotinylated HBsAg level and reused for additional cycles of association and dissociation. For each UniAb, a series of 7 concentrations were measured starting from 250 or 500 nM with a 1 ⁇ 2 serial dilution.
  • biotinylated HBsAg ayw (as described in Example 2) was loaded onto SA biosensors at 5 pg/mL for 900 secs. After baseline readings, sensors were dipped into solutions containing Antibody 1 for 600 secs to saturate the sensors followed by another 60 secs baseline. Then the sensors were dipped into wells containing Antibody 2 for 300 secs. After the first cycle, the sensors were regenerated in 10 mM glycine pH 1.7 to the biotinylated HBsAg level and reused for additional cycles. An all-by-all binning study was conducted and the data was analyzed with ForteBio Data Analysis HT vl 1.1.1.39.
  • Example 4 Monovalent bispecific antibody mediated killing of HBsAg expressing hepatocellular carcinoma (HCC) cells through T-cell redirection
  • HepG2-LMS target cells were seeded at 10,000 cells per well in a 96-well plate and grown overnight at 37°C. Following incubation, increasing amounts of HepBxCD3 bispecific antibody were added together with resting human T-cells at a 10: 1 effector-to-target cell ratio and incubated for an additional 48 hours at 37°C. Cell death was measured using the cell proliferation reagent WST-1 (Sigma Cat No.: 11644807001). A small sample of each supernatant was collected after incubation, but prior to analysis of target cell viability, and saved for analysis of cytokine production.
  • Example 5 HepG2 cell killing data using anti-HBsAg antibody constructs comprising a trivalent HBsAb-binding arm
  • HepG2-LMS target cells were seeded at 10,000 cells per well in a 96-well plate and grown overnight at 37°C. Following incubation, increasing amounts of HepBxCD3 multispecific antibody constructs comprising a trivalent anti-HBsAg heavy chain polypeptide subunit as indicated in the legend of the graph in FIG. 7 were added together with resting human T-cells at a 10: 1 effector-to- target cell ratio and incubated for an additional 48 hours at 37°C. Cell death was measured using the cell proliferation reagent WST-1 (Sigma Cat No.: 11644807001).

Abstract

Hepatitis B surface antigen (HBsAg) heavy chain antibodies are disclosed, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders that are characterized by the presence and/or expression of Hepatitis B surface antigen (HBsAg).

Description

HEAVY CHAIN ANTIBODIES BINDING TO HEPATITIS B SURFACE ANTIGEN
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of the filing date of U.S. Provisional Patent Application No. 63/225,213, filed on July 23, 2021, the disclosure of which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns human heavy chain antibodies (e.g., UniAbs™) binding to Hepatitis B surface antigen (HBsAg). The invention further concerns methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders that are characterized by the expression of HBsAg.
BACKGROUND OF THE INVENTION
Chronic Hepatitis B infection
[0003] Chronic Hepatitis B infection (CHB) is the world’s most common liver infection and is the main cause of liver cirrhosis and hepatocellular carcinoma. While less than 5% of adults who acquired HBV develop a persistent infection, the risks of progression from acute to CHB significantly increase if the infection occurs in early childhood (20-30%) and during the perinatal period (95%). It is estimated that CHB affects 248 million individuals worldwide, causing 887,000 deaths annually. While a prophylactic vaccine is available, 10-15% of individuals do not respond to it adequately and are not protected. Although existing therapies including interferons and nucleoside analogs reduce morbidity and mortality, clearance of serum hepatitis B surface antigen (HBsAg), which is a favorable clinical treatment outcome, is only achieved in less than 10% of patients. Curative treatments have remained elusive mainly due to the persistence of the viral covalently closed circular DNA (cccDNA) in infected hepatocytes, which provides the template for further viral replications and exhausted HBV specific humoral and cellular immunity. Hehle et al. J Exp Med. 2020 Oct 5;217(10):e20200840.
Heavy Chain Antibodies
[0004] In a conventional IgG antibody, the association of the heavy chain and light chain is due in part to a hydrophobic interaction between the light chain constant region and the CHI constant domain of the heavy chain. There are additional residues in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions that also contribute to this hydrophobic interaction between the heavy and light chains. [0005] It is known, however, that sera of camelids (sub-order Tylopoda which includes camels, dromedaries and llamas) contain a major type of antibodies composed solely of paired H-chains (heavy- chain only antibodies or UniAbs™). The UniAbs™ of Camelidae ( Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) have a unique structure consisting of a single variable domain (VHH), a hinge region and two constant domains (CH2 and CH3), which are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain of the constant region (CHI) which is present in the genome, but is spliced out during mRNA processing. The absence of the CHI domain explains the absence of the light chain in the UniAbs™, since this domain is the anchoring place for the constant domain of the light chain. Such UniAbs™ naturally evolved to confer antigen-binding specificity and high affinity by three CDRs from conventional antibodies or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et ak, 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish, such as sharks, have also evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).
[0006] The ability of heavy chain-only antibodies devoid of light chain to bind antigen was established in the 1960s (Jaton et al. (1968) Biochemistry , 7, 4185-4195). Heavy chain immuno globulin physically separated from light chain retained 80% of antigen-binding activity relative to the tetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that removal of the CHI domain from a rearranged mouse m gene results in the production of a heavy chain-only antibody, devoid of light chain, in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.
[0007] Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBSLett. 414, 521-526 (1997)).
[0008] Mice in which the l (lambda) light (L) chain locus and/or the l and k (kappa) L chain loci have been functionally silenced and antibodies produced by such mice are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been reported, for example, in W02006008548; U.S. Application Publication No. 20100122358; Nguyen et al., 2003, Immunology, 109(1), 93-101; Briiggemann et al., Crit. Rev. Immunol., 2006, 26(5):377-90; and Zou et al., 2007 , J Exp Med, 204(13): 3271-3283. The production of knockout rats via embryo microinjections of zinc-finger nucleases is described in Geurts et ak, 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents comprising a heterologous heavy chain locus producing such antibodies are described in U. S. Patent Nos. 8,883,150 and 9,365,655. CAR-T structures comprising single-domain antibodies as binding (targeting) domain are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.
SUMMARY OF THE INVENTION
[0009] Aspects of the invention relate to heavy chain antibodies, including, but not limited to, UniAbs™, with binding affinity to HBsAg. Further aspects of the invention relate to methods of making such antibodies, compositions comprising such antibodies, and their use in the treatment of disorders that are characterized by the presence and/or expression of HBsAg.
[00010] Aspects of the invention include antibodies that bind to HBsAg, comprising a first heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 1-15; and/or (b) a CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 16-34; and/or (c) a CDR3 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 35-54.
[00011] In some embodiments, an antibody further comprises a second heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 1-15; and/or (b) a CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 16-34; and/or (c) a CDR3 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 35-54.
[00012] In some embodiments, said CDR1, CDR2, and CDR3 sequences are present in a human framework. In some embodiments, an antibody further comprises a heavy chain constant region sequence in the absence of a CHI sequence.
[00013] In some embodiments, the first heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
[00014] In some embodiments, the second heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
[00015] In some embodiments, the first heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
[00016] In some embodiments, the second heavy chain variable region comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
[00017] In some embodiments, an antibody comprises a heavy chain variable region comprising: (a) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and aCDR3 sequence of SEQ ID NO: 37; (b) a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44; (c) a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49; (d) a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52; or (e) a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53.
[00018] In some embodiments, an antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 55-77. In some embodiments, an antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 55-77. In some embodiments, the heavy chain variable region sequence is selected from the group consisting of: SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 75 and SEQ ID NO: 76. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 59. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 66. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 72. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 75. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 76.
[00019] Aspects of the invention include antibodies that bind to HBsAg, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences comprise a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 1-54.
[00020] In some embodiments, an antibody comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-54.
[00021] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
[00022] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework, in a monovalent or bivalent configuration.
[00023] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
[00024] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework, in a monovalent or bivalent configuration.
[00025] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
[00026] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework, in a monovalent or bivalent configuration.
[00027] Aspects of the invention include antibodies that bind to to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
[00028] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework, in a monovalent or bivalent configuration.
[00029] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
[00030] Aspects of the invention include antibodies that bind to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework, in a monovalent or bivalent configuration.
[00031] In some embodiments, an antibody is monospecific. In some embodiments, an antibody is multi-specific. In some embodiments, an antibody is bispecific. In some embodiments, an antibody has binding affinity to a CD3 protein and an HBsAg protein. In some embodiments, an antibody has binding affinity to two different epitopes on the same HBsAg protein. In some embodiments, an antibody has binding affinity to an effector cell. In some embodiments, an antibody has binding affinity to a T-cell antigen. In some embodiments, an antibody has binding affinity to CD3. In some embodiments, an antibody is in a CAR-T format.
[00032] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
[00033] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
[00034] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
[00035] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework. [00036] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
[00037] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
[00038] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
[00039] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
[00040] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
[00041] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
[00042] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
[00043] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
[00044] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
[00045] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
[00046] Aspects of the invention include bispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework; (ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
[00047] Aspects of the invention include bispecific three-chain antibody like molecules that binds to human CD3 and HBsAg, comprising: (i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 142; (ii) a second polypeptide subunit comprising the amino acid sequence selected from the group consistin of SEQ ID NOs: 143-145, wherein the first and second polypeptide subunits together form a first binding moiety that binds to human CD3; and (iii) a third polypeptide subunit that binds to HBsAg, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-118.
[00048] Aspects of the invention include multispecific antibodies that binds to CD3 and HBsAg, comprising: (i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 142; (ii) a second polypeptide subunit comprising an amino acid sequence selected from the group consistin of SEQ ID NOs: 143-145, wherein the first and second polypeptide subunits together form a first binding moiety that binds to human CD3; and (iii) a third polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 147. In some embodiments, the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 145. [00049] Aspects of the invention include pharmaceutical compositions comprising an antibody as described herein.
[00050] Aspects of the invention include methods for the treatment of a disorder characterized by the presence of HBsAg, comprising administering to a subject with said disorder an antibody or pharmaceutical composition as described herein.
[00051] Aspects of the invention include use of an antibody as described herein, in the preparation of a medicament for the treatment of a disorder characterized by the presence of HBsAg.
[00052] Aspects of the invention include an antibody as described herein for use in the treatment of a disorder characterized by the presence of HBsAg.
[00053] In some embodiments, the disorder is selected from the group consisting of: acute hepatitis B infection, chronic hepatitis B infection, liver cirrhosis and hepatocellular carcinoma.
[00054] Aspects of the invention include polynucleotides encoding an antibody as described herein, vectors comprising such polypeptides, and cells comprising such vectors.
[00055] Aspects of the invention include methods of producing an antibody as described herein, comprising growing a cell as described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell.
[00056] Aspects of the invention include methods of making an antibody as described herein, comprising immunizing a UniRat animal with an HBsAg protein and identifying HBsAg binding antibody sequences.
[00057] Aspects of the invention include methods of treatment, comprising administering to an individual in need an effective dose of the antibody as described herein, or a pharmaceutical composition as described herein.
[00058] These and further aspects will be further explained in the rest of the disclosure, including the Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[00059] FIG. 1, panels A-F, provide schematic illustrations of: a UniAb (panel A); an anti-CD3 x monovalent, monospecific anti-HBsAg antibody (panel B); an anti-CD3 x bivalent, monospecific anti- HBsAg antibody (panel C); an anti-CD3 x bivalent, biparatopic anti-HBsAg antibody (panel D); an anti-CD3 x trivalent, triparatopic anti-HBsAg antibody in which all three anti-HBsAg variable region sequences are positioned adjancent to the N-terminus (panel E); and an anti-CD3 x trivalent, triparatopic anti-HBsAg antibody in which two anti-HBsAg variable region sequences are positioned adjancent to the N-terminus, and one anti-HBsAg variable region sequence is positioned adjacent to the C-terminus (panel F), in accordance with embodiments of the invention. [00060] FIG. 2 provides a graph showing results from anti-HBsAg HCAb binding to HBsAg expressing HepG2-LMS cells.
[00061] FIG. 3 provides a graph showing results from anti-CD3, anti-HBsAg bispecific antibodies binding to HBsAg expressing HepG2-LMS cells.
[00062] FIG. 4 provides a graph showing results from monovalent bispecific antibody -mediated killing of HepG-LMS cells expressing HBsAg.
[00063] FIG. 5 provides an epitope bin chart of anti-HBsAg UniAbs. Solid line indicates mutual blocking. Dashed line with an arrowhead indicates one-directional blocking. 5 bins are identified.
[00064] FIG. 6 is a table showing binding affinity and epitope binning data for the indicated anti-HBsAg antibodies.
[00065] FIG. 7 is a graph showing % cytotoxicity as a function of antibody concentration for the indicated antibody constructs.
[00066] FIG. 8 is a graph showing % neutralization of HBV as a function of increasing antibody concentration for the indicated antibody constructs.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[00067] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et ak, 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et ak, eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et ak, ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et ak, 2001); kiarlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).
[00068] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [00069] Unless indicated otherwise, antibody residues herein are numbered according to the Kabat numbering system ( e.g ., Kabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
[00070] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.
[00071] All references cited throughout the disclosure, including patent applications and publications, are incorporated by reference herein in their entirety.
I. Definitions
[00072] By “comprising” it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim.
[00073] By “consisting essentially of’, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention.
[00074] By “consisting of’, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim.
[00075] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies mean residue numbering by the EU numbering system.
[00076] Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VL) domain. The two domains conventionally associate to form a specific binding region, although as will be discussed here, specific binding can also be obtained with heavy chain-only variable sequences, and a variety of non-natural configurations of antibodies are known and used in the art. [00077] A “functional” or “biologically active” antibody or antigen-binding molecule (including heavy chain-only antibodies and multi-specific (e.g., bispecific) three-chain antibody-like molecules (TCAs, described herein) is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, may also block ligand activation of a receptor or act as an agonist or antagonist. The capability of an antibody or other binding molecule, e.g., a TCA, to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.
[00078] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three chain antibodies, TCAs, single chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.
[00079] The term antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that comprises an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. Light chains of the subject antibodies can be kappa light chains (Vkappa) or lambda light chains (Vlambda). The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.
[00080] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies in accordance with the present invention can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be made via recombinant protein production methods (see, e.g., U.S. Patent No. 4,816,567), for example.
[00081] The term “variable”, as used in connection with antibodies, refers to the fact that certain portions of the antibody variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a b-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the b-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Rabat et al, Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
[00082] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Rabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a “hypervariable loop” residues 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, “CDR” means a complementary determining region of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region/CDR residues as herein defined.
[00083] Exemplary CDR designations are shown herein, however one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Rabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989; 342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes.” J Immunol. 2008;181:6230-6235; Almagro “Identification of differences in the specificity -determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.
[00084] The terms “heavy chain-only antibody,” and “heavy chain antibody” are used interchangeably herein and refer, in the broadest sense, to antibodies, or more or more portions of an antibody, e.g., one or more arms of an antibody, lacking the light chain of a conventional antibody. The terms specifically include, without limitation, homodimeric antibodies comprising the VH antigen-binding domain and the CH2 and CH3 constant domains, in the absence of the CHI domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NAR comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR) and functional fragments thereof; and soluble single domain antibodies (sUniDabs™). In one embodiment, a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains. In another embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH2 domain. In a further embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain. Heavy chain-only antibodies in which the CH2 and/or CH3 domain is truncated are also included herein. In a further embodiment, a heavy chain is composed of an antigen binding domain, and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region. The heavy chain-only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded or otherwise, covalently or non- covalently, attached with each other. The heavy chain-only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein. In a particular embodiment, a heavy chain antibody is of the IgGl, IgG2, IgG3, or IgG4 subtype, in particular the IgGl or IgG4 subtype. In one embodiment, a heavy-chain antibody is of the IgG4 subtype, wherein one or more of the CH domains is modified to alter an effector function of the antibody. In one embodiment, the heavy -chain antibody is of the IgGl or IgG4 subtype, wherein one or more of the CH domains is modified to alter an effector function of the antibody. Modifications of CH domains that alter effector function are further described herein. Non-limiting examples of heavy-chain antibodies are described, for example, in W02018/039180, the disclosure of which is incorporated herein by reference in its entirety.
[00085] In some embodiments, the heavy chain-only antibodies herein are used as a binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes human heavy chain- only antibodies produced by human immunoglobulin transgenic rats (UniRat™), called UniAbs™. The variable regions (VH) of UniAbs™ are called UniDabs™, and are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multi-specificity, increased potency and extended half-life. Since the homodimeric UniAbs™ lack a light chain and thus a VL domain, the antigen is recognized by one single domain, i.e., the variable domain of the heavy chain of a heavy -chain antibody (VH or VHH).
[00086] An “intact antibody chain” as used herein is one comprising a full length variable region and a full length constant region (Fc). An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody -dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.
[00087] Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be provided as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains that correspond to the different classes of antibodies may be referenced as a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called k (kappa) and l (lambda), based on the amino acid sequences of their constant domains. Antibodies in accordance with embodiments of the invention can comprise kappa light chain sequences or lambda light chain sequences. [00088] A “functional Fc region” possesses an “effector function” of a native-sequence Fc region. Non limiting examples of effector functions include Clq binding; CDC; Fc-receptor binding; ADCC; ADCP; down-regulation of cell-surface receptors (e.g., B-cell receptor), etc. Such effector functions generally require the Fc region to interact with a receptor, e.g., the FcyRI; FcyRIIA; FcyRIIBl; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors, and the low affinity FcRn receptor; and can be assessed using various assays known in the art. A “dead” or “silenced” Fc is one that has been mutated to retain activity with respect to, for example, prolonging serum half-life, but which does not activate a high affinity Fc receptor, or which has a reduced affinity to an Fc receptor.
[00089] A “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include, for example, a native-sequence human IgGl Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof.
[00090] A “variant Fc region” comprises an amino acid sequence that differs from that of a native- sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.
[00091] Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement Clq binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution into human IgGl or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. etal., 1999 Eur J Immunol 29(8):2613-24; and Shields RL. etal., 2001. J Biol Chem. 276(9):6591- 604). The human IgG4 Fc amino acid sequence (UniProtKB No. P01861) is provided herein as SEQ ID NO: 92. Silenced IgGl is described, for example, in Boesch, A.W., et al., “Highly parallel characterization of IgG Fc binding interactions.” MAbs, 2014. 6(4): p. 915-27, the disclosure of which is incorporated herein by reference in its entirety.
[00092] Other Fc variants are possible, including, without limitation, one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N- terminal end of a native Fc, or a methionine residue is added thereto. Thus, in some embodiments, one or more Fc portions of an antibody can comprise one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, an antibody can comprise an Fc variant.
[00093] Further, an Fc variant can be constructed to remove or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, and not limitation, a deletion may occur in a complement-binding site, such as a Clq-binding site. Techniques for preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
[00094] In some embodiments, an antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which can optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, an antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which can optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner so as to place a “knob” on a first heavy chain constant region of a first monomer in an antibody dimer, and a “hole” on a second heavy chain constant region of a second monomer in an antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.
[00095] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235 A mutation, and a T366W mutation (knob). In some embodiments, and antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, and a Y407V mutation (hole).
[00096] The term “Fc-region-comprising antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to this invention can comprise an antibody with or without K447. For example, any of SEQ ID NOs. 78-118, 143, 144 and 145 can optionally comprise a K447 residue.
[00097] Aspects of the invention include antibodies comprising a heavy chain-only variable region in a monovalent or bivalent configuration. As used herein, the term “monovalent configuration” as used in reference to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain is present, having a single binding site (see FIG. 1, Panel B, right arm of antibody). In contrast, the term “bivalent configuration” as used in reference to a heavy chain-only variable region domain means that two heavy chain-only variable region domains are present (each having a single binding target), and are connected by a linker sequence (see FIG. 1, Panel C, right arm of antibody). Non-limiting examples of linker sequences are discussed further herein, and include, without limitation, GS linker sequences of various lengths. When a heavy chain-only variable region is in a bivalent configuration, each of the two heavy chain-only variable region domains can have binding affinity to the same antigen, or to different antigens (e.g., binding to different epitopes on the same protein; to two different proteins (biparatopic), etc.). However, unless specifically noted otherwise, a heavy chain-only variable region denoted as being in a “bivalent configuration” is understood to contain two identical heavy chain-only variable region domains, connected by a linker sequence, wherein each of the two identical heavy chain-only variable region domains have binding affinity to the same target antigen.
[00098] Aspects of the invention include antibodies having multi-specific configurations, which include, without limitation, bispecific, trispecific, etc. A large variety of methods and protein configurations are known and used in bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, etc.
[00099] Various methods for the production of multivalent artificial antibodies have been developed by recombinantly fusing variable domains of two or more antibodies. In some embodiments, a first and a second antigen-binding domain on a polypeptide are connected by a polypeptide linker. One non- limiting example of such a polypeptide linker is a GS linker, having an amino acid sequence of four glycine residues, followed by one serine residue, and wherein the sequence is repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 81) (n=l) and GGGGSGGGGS (SEQ ID NO: 82) (n=2). Other suitable linkers can also be used, and are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357-69, the disclosure of which is incorporated herein by reference in its entirety.
[000100] The term “three-chain antibody like molecule” or “TCA” is used herein to refer to antibody- like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy-chain only antibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and one or more antigen binding domains (e.g., two antigen binding domains) that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain. Parts of such variable region may be encoded by VH and/or VL gene segments, D and JH gene segments, or JL gene segments. The variable region may be encoded by rearranged VHDJh, VLDJH, VHJL, or VLJL gene segments.
[000101] A TCA binding compound makes use of a “heavy chain only antibody” or “heavy chain antibody” or “heavy chain polypeptide” which, as used herein, mean a single chain antibody comprising heavy chain constant regions CH2 and/or CH3 and/or CH4 but no CHI domain. In one embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains. In another embodiment, the heavy chain antibody is composed of an antigen binding domain, at least part of a hinge region and a CH2 domain. In a further embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain. Heavy chain antibodies in which the CH2 and/or CH3 domain is truncated are also included herein. In a further embodiment, the heavy chain is composed of an antigen binding domain, and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region. The heavy chain only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded other otherwise covalently or non-covalently attached with each other, and can optionally include an asymmetric interface between one or more of the CH domains to facilitate proper pairing between polypeptide chains. The heavy- chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein. In a particular embodiment, the heavy chain antibody is of the IgGl, IgG2, IgG3, orIgG4 subtype, in particular the IgGl subtype or the IgG4 subtype. Non-limiting examples of a TCA binding compound are described in, for example, WO2017/223111 and W02018/052503, the disclosures of which are incorporated herein by reference in their entirety.
[000102] Heavy-chain antibodies constitute about one fourth of the IgG antibodies produced by the camelids, e.g., camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but are devoid of light chains. As a consequence, the variable antigen binding part is referred to as the VHH domain and it represents the smallest naturally occurring, intact, antigen-binding site, being only around 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, termed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)). [000103] The term “HBsAg” or “Hepatitis B surface antigen” as used herein refers to a surface protein of Hepatitis B Virus (“HBV” or “Hep B”). HBsAg is encoded by a Gene S, which consists of one long open reading frame, but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-Sl, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes, called large, middle, and small (pre-Sl+pre-S2+S, pre-S2+S, or S) are produced (Beck et al., World J. Gastroenterol. (2007) 13; 48-64. The term “HBsAg” includes an HBsAg surface protein from a Hepatitis B virus that is capable of infecting a human or non-human animal, and includes HBsAg on a Hepatitis B virus itself, and/or HBsAg that is generated by a cell in a human or non-human animal that is infected with a Hepatitis B virus. The term “HBsAg” includes an HBsAg protein from a Hepatitis B virus (e.g., on the surface of a Hepatitis B virus), and also includes HBsAg produced in the cells of human and non-human mammals that are infected with a Hepatitis B virus, or that are transfected with one or more genes encoding HBsAg.
[000104] The term “HBsAg” as used herein includes any serotypes, variants, isoforms and species homologs of HBsAg (e.g.,UniProt Q81158), regardless of its source or mode of preparation. Thus, “HBsAg” includes native HBsAg that is naturally expressed by human or animal cells infected by a Hepatitis B virus, as well as HBsAg expressed on cells transfected with an HBsAg gene. The term “native HBsAg” as used herein includes HBsAg isolated from human or animal blood or tissues. The term “recombinant HBsAg” as used herein includes HBsAg expressed by cells transfected with an HBsAg gene.
[000105] The terms “anti-HBsAg heavy chain-only antibody,” “HBsAg heavy chain-only antibody,” “anti-HBsAg heavy chain antibody” and “HBsAg heavy chain antibody” are used herein interchangeably to refer to a heavy chain-only antibody as hereinabove defined, immunospecifically binding to HBsAg, including native HBsAg, as hereinabove defined. The definition includes, without limitation, human heavy chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immuno globulin, including UniRats™ producing human anti- HBsAg UniAb™ antibodies, as hereinabove defined.
[000106] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
[000107] An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[000108] Antibodies of the invention include multi-specific antibodies. Multi-specific antibodies have more than one binding specificity. The term “multi-specific” specifically includes “bispecific” and “trispecific,” as well as higher-order independent specific binding affinities, such as higher-order polyepitopic specificity, as well as tetravalent antibodies and antibody fragments. The terms “multi specific antibody,” “multi-specific heavy chain-only antibody,” “multi-specific heavy chain antibody,” and “multi-specific UniAb™” are used herein in the broadest sense and cover all antibodies with more than one binding specificity. The multi-specific heavy chain anti-HBsAg antibodies of the present invention specifically include antibodies immunospecifically binding to two or more non-overlapping epitopes on an HBsAg protein, such as a native HBsAg (i.e., bivalent and biparatopic). The multi specific heavy chain anti-HBsAg antibodies of the present invention also specifically include antibodies immunospecifically binding to an epitope on an HBsAg protein, such as native HBsAg and to an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 (i.e., bivalent and biparatopic). The multi-specific heavy chain anti-HBsAg antibodies of the present invention also specifically include antibodies immunospecifically binding to two or more non-overlapping or partially overlapping epitopes on an HBsAg protein, such as a native HBsAg protein, and to an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 protein (i.e., trivalent and biparatopic).
[000109] Antibodies of the invention include monospecific antibodies, having one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity, as well as antibodies comprising more than one binding unit having the same binding specificity. The terms “monospecific antibody,” “monospecific heavy chain-only antibody,” “monospecific heavy chain antibody,” and “monospecific UniAb™” are used herein in the broadest sense and cover all antibodies with one binding specificity. The monospecific heavy chain anti-HBsAg antibodies of the present invention specifically include antibodies immunospecifically binding to one epitope on an HBsAg protein, such as a native HBsAg (monovalent and monospecific). The monospecific heavy chain anti- HBsAg antibodies of the present invention also specifically include antibodies having more than one binding unit (e.g., multivalent antibodies) immunospecifically binding to an epitope on an HBsAg protein, such as native HBsAg. For example, a monospecific antibody in accordance with embodiments of the invention can include a heavy chain variable region comprising two antigen-binding domains, wherein each antigen-binding domain binds to the same epitope on an HBsAg protein (i.e., bivalent and monospecific).
[000110] An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.
[000111] “Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.
[000112] “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As noted above, the present invention specifically includes anti-HBsAg heavy chain antibodies with polyepitopic specificities, i.e., anti-HBsAg heavy chain antibodies binding to one or more non-overlapping epitopes on an HBsAg protein, such as a native HBsAg; and anti-HBsAg heavy chain antibodies binding to one or more epitopes on an HBsAg protein and to an epitope on a different protein, such as, for example, a CD3 protein. The term “non-overlapping epitope(s)” or “non-competitive epitope(s)” of an antigen is defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies but not the other member. Pairs of antibodies, or antigen-binding regions targeting the same antigen on a multi-specific antibody, recognizing non-overlapping epitopes, do not compete for binding to that antigen and are able to bind that antigen simultaneously.
[000113] An antibody binds “essentially the same epitope” as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody. Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels. [000114] The term “valent” as used herein refers to a specified number of binding sites in an antibody molecule.
[000115] A “monovalent” antibody has one binding site. Thus, a monovalent antibody is also monospecific.
[000116] A “multi-valent” antibody has two or more binding sites. Thus, the terms “bivalent”, “trivalent”, and “tetravalent” refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, a bispecific antibody according to the invention is at least bivalent and may be trivalent, tetravalent, or otherwise multi-valent. A bivalent antibody in accordance with embodiments of the invention may have two binding sites to the same epitope (i.e., bivalent, monoparatopic), or to two different epitopes (i.e., bivalent, biparatopic).
[000117] A large variety of methods and protein configurations are known and used for the preparation of bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, and the like.
[000118] The term “three-chain antibody like molecule” or “TCA” is used herein to refer to antibody- like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain. Parts of such variable region may be encoded by VH and/or VL gene segments, D and JH gene segments, or JL gene segments. The variable region may be encoded by rearranged VHDJH, VLDJH, VHJL, or VLJL gene segments. A TCA protein makes use of a heavy chain-only antibody as hereinabove defined.
[000119] The term “chimeric antigen receptor” or “CAR” is used herein in the broadest sense to refer to an engineered receptor, which grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to membrane-spanning and intracellular-signaling domains. Typically, the receptor is used to graft the specificity of a monoclonal antibody onto a T-cell to create a chimeric antigen receptors (CAR). ( J Natl Cancer Inst, 2015; 108(7):dvj439; and Jackson et ak, Nature Reviews Clinical Oncology, 2016; 13:370-383). CAR-T cells are T-cells that have been genetically engineered to produce an artificial T-cell receptor for use in immunotherapy. In one embodiment, “CAR-T cell” means a therapeutic T-cell expressing a transgene encoding one or more chimeric antigen receptors comprised minimally of an extracellular domain, a transmembrane domain, and at least one cytosolic domain. [000120] The term “human antibody” is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies herein may include amino acid residues not encoded by human germline immuno globulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term “human antibody” specifically includes heavy chain-only antibodies having human heavy chain variable region sequences, produced by transgenic animals, such as transgenic rats or mice, in particular UniAbs™ produced by UniRats™, as defined above.
[000121] By a “chimeric antibody” or a “chimeric immunoglobulin” is meant an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, e.g., a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human Fc-regions or artificial Fc-regions, and human idiotypes. Such immunoglobulins can be isolated from animals of the invention that have been engineered to produce such chimeric antibodies.
[000122] As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and carry out specific immune functions. In some embodiments, an effector cell such as a natural killer cell is capable of inducing antibody- dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcR, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, an effector cell may phagocytose a target antigen or target cell.
[000123] “Human effector cells” are leukocytes which express receptors such as T-cell receptors or FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T-cells and neutrophils; with NK cells being preferred. The effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.
[000124] The term “immune cell” is used herein in the broadest sense, including, without limitation, cells of myeloid or lymphoid origin, for instance lymphocytes (such as B-cells and T-cells including cytolytic T-cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.
[000125] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody -dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptor; BCR), etc. [000126] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI. FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
[000127] “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.
[000128] “Binding affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound.
[000129] As used herein, the “Kd” or “Kd value” refers to a dissociation constant determined by BioLayer Interferometry, using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, anti-mouse Fc sensors are loaded with mouse-Fc fused antigen and then dipped into antibody-containing wells to measure concentration dependent association rates (kon). Antibody dissociation rates (koff) are measured in the final step, where the sensors are dipped into wells containing buffer only. The Kd is the ratio of koff/kon. (For further details see, Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).
[000130] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
A “therapeutically effective amount” is intended for an amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a “therapeutically effective amount” is an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with a disease or which improves resistance to a disorder.
[000131] The terms “characterized by the presence of HBsAg” and “characterized by expression of HBsAg”, as used interchangeably herein, broadly refer to any disease or disorder in which HBsAg presence and/or expression are associated with or involved with one or more pathological processes that are characteristic of the disease or disorder. Such disorders include, but are not limited to, liver or hepatic disease (e.g., infection), acute or chronic hepatitis B infection, liver cirrhosis and hepatocellular carcinoma.
[000132] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer, individuals with autoimmune diseases, with pathogen infections, and the like. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mouse, rat, etc.
[000133] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
[000134] A “sterile” formulation is aseptic or free or essentially free from all living microorganisms and their spores. A “frozen” formulation is one at a temperature below 0 °C.
[000135] A “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example. Stability can be measured at a selected temperature for a selected time period. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy -terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.
II. Detailed Description
Anti-HBsAs Antibodies
[000136] The present invention provides families of closely related antibodies that bind to HBsAg (e.g., HBsAg that is expressed in human cells infected with Hepatitis B virus (HBV)). The antibodies of these families comprise sets of CDR sequences as defined herein and shown in Tables 1-2, and are exemplified by the provided heavy chain variable region (VH) sequences of SEQ ID NOs: 55 to 100 set forth in Tables 3 and 4. These families of antibodies provide a number of benefits that contribute to utility as clinically therapeutic agent(s). The antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity.
[000137] Table 1: Anti-HBsAg heavy chain antibody unique CDR amino acid sequences.
Figure imgf000029_0001
Figure imgf000030_0001
[000138] Table 2: Anti-HBsAg heavy chain antibody unique CDR amino acid sequences.
Figure imgf000030_0002
Figure imgf000031_0001
[000139] Table 3. Anti-HBsAg heavy chain antibody variable domain amino acid sequences.
Figure imgf000031_0002
Figure imgf000032_0001
Figure imgf000033_0001
[000140] Table 4: Anti-HBsAg UniAb consensus full orf sequences:
Figure imgf000033_0002
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
[000141] Table 5: Anti-HBsAg bispecific consensus full orf sequences:
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
[000142] A suitable antibody may be selected from those provided herein for development and therapeutic or other use, including, without limitation, use as a bispecific antibody, e.g., as shown in FIG. 1, panels B-D, or as part of a CAR-T structure. FIG. 1, panel B provides an illustration of an anti- CD3 x anti-HBsAg multi-specific antibody, where the anti-HBsAg domain is monovalent and monospecific. The anti-CD3 domain contains a CHI domain and pairs with a light chain, while the anti- HBsAg domain is derived from a heavy chain-only antibody and does not contain a CHI domain or interact with a light chain. In some embodiments, the two heavy chains are pared using, e.g., knobs- into-holes technology. FIG. 1, panel C provides an illustration of an anti-CD3 x anti-HBsAg multi specific antibody, where the anti-HBsAg domain is bivalent and monospecific. The anti-CD3 domain contains a CHI domain and pairs with a light chain, while the anti-HBsAg domain is derived from a heavy chain-only antibody and does not contain a CHI domain or interact with a light chain. In some embodiments, the two heavy chains are pared using, e.g., knobs-into-holes technology.
[000143] The antibody depicted in FIG. 1, panel B is an anti-CD3 x anti-HBsAg bispecific antibody wherein the anti-HBsAg binding arm is monovalent and monospecific, and the antigen-binding domain of the anti-HBsAg arm is in a monovalent configuration, meaning only one antigen-binding domain is present. The antibody depicted in FIG. 1, panel C is an anti-CD3 x anti-HBsAg bispecific antibody wherein the anti-HBsAg binding arm is bivalent and monospecific, and the antigen-binding domain of the anti-HBsAg arm is in a bivalent configuration, meaning there are two identical antigen binding domains placed in tandem. In some embodiments, an antibody can be bivalent and biparatopic, meaning that there are two antigen binding domains present on the anti-HBsAg arm of the antibody, and each of these antigen-binding domains contains a different sequence, and binds to a different epitope on an HBsAg protein (see FIG. 1, panel D).
[000144] Determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family may have an affinity for HBsAg with a Kd of from about 106 to around about 10 n, including without limitation: from about 106 to around about 1010; from about 106 to around about 109; from about 106 to around about 108; from about 108 to around about 10 n; from about 108 to around about 1010; from about 108 to around about 109; from about 109 to around about 10 n; from about 109 to around about 1010; or any value within these ranges. The affinity selection may be confirmed with a biological assessment for modulating, e.g., blocking, an HBsAg biological activity, including in vitro assays, pre-clinical models, and clinical trials, as well as assessment of potential toxicity.
[000145] The HBsAg-specific antibodies herein comprise a VH domain, comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequences may be situated, as an example, in the region of around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequences set forth in SEQ ID NOs: 23 to 74. It will be understood by one of ordinary skill in the art that the CDR sequences may be in different positions if a different framework sequence is selected, although generally the order of the sequences will remain the same.
[000146] Representative CDR1, CDR2 and CDR3 sequences are shown in Tables 1 and 2. [000147] In some embodiments, an anti-HBsAg antibody comprises a CDR1 sequence of any one of SEQ ID NOs: 1-15. In some embodiments, an anti-HBsAg antibody comprises a CDR1 sequence of any one of SEQ ID NOs: 2, 5, 8, 12, and 14. In a particular embodiment, an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 2. In a particular embodiment, an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 5. In a particular embodiment, an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 8. In a particular embodiment, an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 12. In a particular embodiment, an anti-HBsAg antibody comprises a CDR1 sequence of SEQ ID NO: 14.
[000148] In some embodiments, an anti-HBsAg antibody comprises a CDR2 sequence of any one of SEQ ID NOs: 16-34. In some embodiments, an anti-HBsAg antibody comprises a CDR2 sequence of any one of SEQ ID NOs: 18, 24, 29, 32, and 33. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 18. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 24. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 29. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 32. In a particular embodiment, an anti-HBsAg antibody comprises a CDR2 sequence of SEQ ID NO: 33.
[000149] In some embodiments, an anti-HBsAg antibody comprises a CDR3 sequence of any one of SEQ ID NOs: 32-54. In some embodiments, an anti-HBsAg antibody comprises a CDR3 sequence of any one of SEQ ID NOs: 37, 44, 49, 52, and 53. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 37. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 44. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 49. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 52. In a particular embodiment, an anti-HBsAg antibody comprises a CDR3 sequence of SEQ ID NO: 53.
[000150] In a further embodiment, an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 2; a CDR2 sequence comprising the sequence of SEQ ID NO: 18; and a CDR3 sequence comprising the sequence of SEQ ID NO: 37.
[000151] In a further embodiment, an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 8; a CDR2 sequence comprising the sequence of SEQ ID NO: 24; and a CDR3 sequence comprising the sequence of SEQ ID NO: 44.
[000152] In a further embodiment, an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 5; a CDR2 sequence comprising the sequence of SEQ ID NO: 29; and a CDR3 sequence comprising the sequence of SEQ ID NO: 49. [000153] In a further embodiment, an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 12; a CDR2 sequence comprising the sequence of SEQ ID NO: 32; and a CDR3 sequence comprising the sequence of SEQ ID NO: 52.
[000154] In a further embodiment, an anti-HBsAg antibody comprises a CDR1 sequence comprising the sequence of SEQ ID NO: 14; a CDR2 sequence comprising the sequence of SEQ ID NO: 33; and a CDR3 sequence comprising the sequence of SEQ ID NO: 53.
[000155] In a further embodiment, an anti-HBsAg antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 55-77 (Table 3).
[000156] In a still further embodiment, an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 59. In a still further embodiment, an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 66. In a still further embodiment, an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 72. In a still further embodiment, an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 75. In a still further embodiment, an anti-HBsAg antibody comprises a heavy chain variable region sequence of SEQ ID NO: 76.
[000157] In some embodiments, a CDR sequence in an anti-HBsAg antibody of the invention comprises one or two amino acid substitutions relative to a CDR1, CDR2 and/or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs: 1-54 (Table 1).
[000158] In some embodiments, an anti-HBsAg antibody preferably comprises a heavy chain variable domain (VH) in which the CDR3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Tables 1 and 2, and binds to HBsAg.
[000159] In some embodiments, an anti-HBsAg antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty- five percent (85%) sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Tables 1 and 2, and binds to HBsAg.
[000160] In some embodiments, an anti-HBsAg antibody comprises a heavy chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identify, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 55-77 (shown in Table 3), and binds to HBsAg.
[000161] In some embodiments, an anti-HBsAg antibody can comprise a plurality of heavy chain variable regions positioned at any suitable location on any one or more of the polypeptide subunits of the antibody. For example, in some embodiments, an anti-HBsAg antibody can comprise a heavy chain variable region that is positioned at an N-terminus and/or a C-terminus of one or more of the polypeptide subunits (either a light chain or a heavy chain) that make up the antibody. In some embodiments, an anti-HBsAg antibody can comprise 1, 2, 3, 4, 5, 6, 7, or 8 or more heavy chain variable regions that are positioned at various locations on one or more of the polypeptide subunits of the antibody. Any or all of these anti-HBsAg heavy chain variable regions can comprise the same sequence, or they can comprise a plurarlity of different sequences, e.g., such as 2, 3, 4, 5, 6, 7, or 8 different sequences, such that the resulting antibody is monoparatopic, biparatopic, triparatopic, tetraparatopic, pentaparatopic, hexaparatopic, heptaparatopic, or octaparatopic. In some embodiments, one or more heavy chain variable regions can be positioned, e.g., at an N-terminus of a light chain polypeptide subunit, at a C- terminus of a light chain polypeptide subunit, at an N-terminus of a heavy chain polypeptide subunit, or at a C-terminus of a heavy chain polypeptide subunit. Moreover, at any of these positions, a single heavy chain variable region can be present, or a plurality of heavy chain variable regions can be present (e.g., a tandem configuration of two heavy chain variable regions, a tandem configuration of three heavy chain variable regions, etc.), each having the same or different binding sequences.
[000162] In some embodiments, bispecific or multi-specific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific three-chain antibody like molecule (TCA). In some embodiments, a multi-specific antibody can comprise at least one heavy chain variable region having binding specificity for HBsAg, and at least one heavy chain variable region having binding specificity for a protein other than HBsAg. In some embodiments, a multi-specific antibody can comprise a heavy chain variable region comprising at least two antigen binding domains, wherein each of the antigen-binding domains has binding specificity for HBsAg. In some embodiments, a multi-specific antibody can comprise a heavy chain/light chain pair that has binding specificity for a first antigen (e.g., CD3), and a heavy chain from a heavy chain-only antibody that has binding specificity to a second antigen (e.g., HBsAb). In certain embodiments, the heavy chain from the heavy chain-only antibody comprises an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain. In one particular embodiment, a bispecific antibody comprises a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (e.g., a CD3 protein on a T-cell), and a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain that has binding specificity for HBsAg.
[000163] Furthermore, bispecific or multispecific anti-HBsAg antibodies in accordance with embodiments of the invention can also comprise a plurality of anti-HBsAg heavy chain variable regions positioned at any suitable location on any one or more of the polypeptide subunits of the antibody. For example, in some embodiments, a multispecific anti-HBsAg antibody can comprise an anti-HBsAg heavy chain variable region that is positioned at an N-terminus and/or a C-terminus of one or more of the polypeptide subunits (either a light chain or a heavy chain) that make up the antibody, and which also contain one or more variable regions that bind to an antigen other than HBsAg, e.g., CD3. For example, in some embodiments, a multispecific antibody comprises an anti-CD3 binding arm comprising a light chain polypeptide subunit and a heavy chain polypeptide subunit, and comprises one or more anti-HBsAg heavy chain variable regions that are positioned at an N-terminus and/or a C- terminus of one or more of the polypeptide subunits of the CD3 binding arm. As reviewed above, in some embodiments, bispecifc or multispecific anti-HBsAg antibodies can comprise 1, 2, 3, 4, 5, 6, 7, or 8 or more anti-HBsAg heavy chain variable regions that are positioned at various locations on one or more of the polypeptide subunits of the antibody. Any or all of these anti-HBsAg heavy chain variable regions can comprise the same sequence, or they can comprise a plurarlity of different sequences, e.g., such as 2, 3, 4, 5, 6, 7, or 8 different sequences, such that the resulting antibody is monoparatopic, biparatopic, triparatopic, tetraparatopic, pentaparatopic, hexaparatopic, heptaparatopic, or octaparatopic for HBsAg. In some embodiments, one or more heavy chain variable regions can be positioned, e.g., at an N-terminus of a light chain polypeptide subunit, at a C-terminus of a light chain polypeptide subunit, at an N-terminus of a heavy chain polypeptide subunit, or at a C-terminus of a heavy chain polypeptide subunit. Moreover, at any of these positions, a single heavy chain variable region can be present, or a plurality of heavy chain variable regions can be present (e.g., a tandem configuration of two heavy chain variable regions, a tandem configuration of three heavy chain variable regions, etc.), each having the same or different binding sequences.
[000164] In one embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N-terminus, which are connected to one another by linker sequences. In one embodiment, a triparatopic anti-HBsAg heavy chain polypeptide subunit comprises SEQ ID NO: 146. In one embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions, and is paired with a heavy chain light chain pair that binds to CD3, as depicted in FIG. IE. In one preferred embodiment, a multispecific antibody comprises a first heavy chain polypeptide subunit that comprises SEQ ID NO: 147, a second heavy chain polypeptide subunit that comprises SEQ ID NO: 143, and a light chain polypeptide subunit that comprises SEQ ID NO: 142. In one preferred embodiment, a multispecific antibody comprises a first heavy chain polypeptide subunit that comprises SEQ ID NO: 147, a second heavy chain polypeptide subunit that comprises SEQ ID NO: 144, and a light chain polypeptide subunit that comprises SEQ ID NO: 142. In one preferred embodiment, a multispecific antibody comprises a first heavy chain polypeptide subunit that comprises SEQ ID NO: 147, a second heavy chain polypeptide subunit that comprises SEQ ID NO: 145, and a light chain polypeptide subunit that comprises SEQ ID NO: 142.
[000165] In one embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises two variable regions positioned adjacent to the N-terminus, which are connected to one another by a linker sequence, and a third variable region positioned adjacent ot the C-terminus. In one embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises two variable regions at the N-terminus and 1 variable region at the C-terminus, and is paired with a heavy chain/light chain pair that binds to CD3, as depicted in FIG. IF.
[000166] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F04A VH sequence, an F05B VH sequence, and an F03B VH sequence, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-55).
[000167] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, and an F04A VH sequence, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-59).
[000168] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-71).
[000169] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T 10-83).
[000170] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F1F CD3 arm (also referred to in FIG. 8 as T10-87).
[000171] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F04A VH sequence, an F05B VH sequence, and an F03B VH sequence, paired with an F2F CD3 arm.
[000172] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, and an F04A VH sequence, paired with an F2F CD3 arm.
[000173] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2F CD3 arm.
[000174] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2F CD3 arm.
[000175] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2F CD3 arm.
[000176] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F04A VH sequence, an F05B VH sequence, and an F03B VH sequence, paired with an F2B CD3 arm.
[000177] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, and an F04A VH sequence, paired with an F2B CD3 arm.
[000178] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F03B VH sequence, an F05B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2B CD3 arm.
[000179] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F03B VH sequence, an Fc region, and an F04A VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2B CD3 arm.
[000180] In one preferred embodiment, a multispecific antibody comprises a triparatopic anti-HBsAg heavy chain polypeptide subunit that comprises three variable regions positioned adjacent to the N- terminus, which are connected to one another by linker sequences, comprising, in N-term to C-term orientation, an F05B VH sequence, an F04A VH sequence, an Fc region, and an F03B VH sequence at the C-terminal end of the heavy chain subunit, paired with an F2B CD3 arm.
[000181] In some embodiments, a multi-specific antibody comprises a CD3-binding VH domain that is paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and a CDR3 sequence of SEQ ID NO: 121, in a human VH framework. In some embodiments, the CD3 -binding VH domain comprises a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and a CDR3 sequence of SEQ ID NO: 124, in a human VH framework. In some embodiments, the CD3-binding VH domain comprises a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and a CDR3 sequence of SEQ ID NO: 127, in a human VH framework. In some embodiments, the fixed light chain comprises a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and a CDR3 sequence of SEQ ID NO: 130, in a human VL framework. Together, the CD3-binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, a CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 131. In some embodiments, a CD3- binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 131. In some embodiments, a CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 132. In some embodiments, a CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 132. In some embodiments, a CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 133. In some embodiments, a CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 133. In some embodiments, a fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 134. In some embodiments, a fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 134.
[000182] Multi-specific antibodies comprising the above -described CD3-binding VH domain and light chain variable domain have advantageous properties, for example, as described in published PCT application publication number W02018/052503, the disclosure of which is incorporated by reference herein in its entirety. Any of the multi-specific antibodies and antigen-binding domains described herein, having binding affinity to HBsAg, can be combined with any of the CD3 -binding domains and fixed light chain domains described herein (see, e.g., Table 6 and Table 7) and in published PCT application publication numbers W02018/052503 and WO2017/223111, the disclosures of which are incorporated by reference herein in their entireties, as well as additional sequences, such as those provided in Table 5, Table 8, and Table 9, to generate multi-specific antibodies having binding affinity to one or more HBsAg epitopes, as well as CD3.
[000183] Table 6. Anti-CD3 Heavy and Light Chain CDR1, CDR2, CDR3 amino acid sequences.
Figure imgf000050_0001
[000184] Table 7. Anti-CD3 heavy and light chain variable region amino acid sequences.
Figure imgf000050_0002
Figure imgf000051_0001
[000185] Table 8: Human IgGl and IgG4 Fc region sequences.
Figure imgf000051_0002
Figure imgf000052_0002
[000186] Table 9: additional sequences.
Figure imgf000052_0001
Figure imgf000053_0001
[000187] In some embodiments, bispecific or multi-specific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific three-chain antibody like molecule (TCA). In some embodiments, a bispecific antibody can comprise at least one heavy chain variable region having binding specificity for HBsAg, and at least one heavy chain variable region having binding specificity for a protein other than HBsAg. In some embodiments, a bispecific antibody can comprise a heavy chain/light chain pair that has binding specificity for a first antigen, and a heavy chain from a heavy chain-only antibody, comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen. In one particular embodiment, a bispecific antibody comprises a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (e.g., a CD3 protein on a T-cell), and a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain that has binding specificity for HBsAg.
[000188] In some embodiments, where an antibody of the invention is a bispecific antibody, one arm of the antibody (one binding moiety, or one binding unit) is specific for HBsAg, while the other arm may be specific for target cells, tumor-associated antigens, targeting antigens, e.g., integrins, etc., pathogen antigens, checkpoint proteins, and the like. In some embodiments, one arm of the antibody (one binding moiety, or one binding unit) is specific for HBsAg, while the other arm is specific for CD3.
[000189] In some embodiments, a multispecific (e.g., bispecific) antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 142, an anti-CD3 heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 143, 144 or 145, and an anti-HBsAg heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 101-118. In some embodiments, the anti-HBsAg heavy chain polypeptide can comprise an anti-HBsAg heavy chain variable region in a monovalent or bivalent configuration, as described herein.
[000190] In some embodiments, a multispecific (e.g., bispecific) antibody comprises a first anti-HBsAg heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 78-100, paired with a second anti-HBsAg heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 78- 100
[000191] Aspects of the invention include one or more antibody sequences, as described herein, which are in a CAR-T format, for use as one or more binding domains that provide antigen specificity to a CAR-T cell. In certain embodiments, a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising any one of SEQ ID NOs: 1-15, a CDR2 sequence comprising any one of SEQ ID NOs: 16-34, and a CDR3 sequence comprising any one of SEQ ID NOs: 35-54. In certain embodiments, a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 2, a CDR2 sequence comprising SEQ ID NO: 18, and a CDR3 sequence comprising SEQ ID NO: 37. In certain embodiments, a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 24, and a CDR3 sequence comprising SEQ ID NO: 44. In certain embodiments, a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 5, a CDR2 sequence comprising SEQ ID NO: 29, and a CDR3 sequence comprising SEQ ID NO: 49. In certain embodiments, a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 12, a CDR2 sequence comprising SEQ ID NO: 32, and a CDR3 sequence comprising SEQ ID NO: 52. In certain embodiments, a CAR-T cell comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to HBsAg, and comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 14, a CDR2 sequence comprising SEQ ID NO: 33, and a CDR3 sequence comprising SEQ ID NO: 53.
[000192] In some embodiments, a CAR-T cell comprises an extracellular antigen-binding domain that binds to HBsAg and comprises a heavy chain variable region having at least 95% identity to any one of SEQ ID NOs. 55-77. In some embodiments, a CAR-T cell comprises an extracellular antigen-binding domain that binds to HBsAg and comprises a heavy chain variable region comprising any one of SEQ ID NOs. 55-77. In some embodiments, a CAR-T cell comprises an extracellular antigen-binding domain that binds to HBsAg and comprises a heavy chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 75, and SEQ ID NO: 76. Aspects of the invention include pharmaceutical compositions comprising a CAR-T cell as described herein, as well as methods of treatment that comprise administering a therapeutically effective amount of a CAR-T cell as described herein.
[000193] Various formats of multi-specific antibodies are within the ambit of the invention, including, without limitation, single chain polypeptides, two chain polypeptides, three chain polypeptides, four chain polypeptides, and multiples thereof. The multi-specific antibodies herein specifically include T- cell multi-specific (e.g., bispecific) antibodies binding to HBsAg and CD3 (anti-HBsAg x anti-CD3 antibodies). Such antibodies induce potent T-cell mediated killing of cells expressing HBsAg.
Preparation ofHBsAe antibodies
[000194] The antibodies of the present invention can be prepared by methods known in the art. In a preferred embodiment, the antibodies herein are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which the endogenous immunoglobulin genes are knocked out or disabled. In a preferred embodiment, the heavy chain antibodies herein are produced in UniRat™. UniRat™ have their endogenous immunoglobulin genes silenced and use a human immunoglobulin heavy-chain translocus to express a diverse, naturally optimized repertoire of fully human HCAbs. While endogenous immunoglobulin loci in rats can be knocked out or silenced using a variety of technologies, in UniRat™ the zinc-finger (endo)nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J-locus, light chain CK locus and light chain Cl locus. ZNF constructs for microinjection into oocytes can produce IgH and IgL knock out (KO) lines. For details see, e.g., Geurts et ak, 2009, Science 325:433. Characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. Advantages of the ZNF technology are that non- homologous end joining to silence a gene or locus via deletions up to several kb can also provide a target site for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind epitopes that cannot be attacked with conventional antibodies. Their high specificity, affinity, and small size make them ideal for mono- and poly-specific applications.
[000195] In addition to UniAbs™, specifically included herein are heavy chain-only antibodies lacking the camelid VHH framework and mutations, and their functional VH regions. Such heavy chain-only antibodies can, for example, be produced in transgenic rats or mice which comprise fully human heavy chain-only gene loci as described, e.g., in W02006/008548, but other transgenic mammals, such as rabbit, guinea pig, rat can also be used, rats and mice being preferred. Heavy chain-only antibodies, including their VHH or VH functional fragments, can also be produced by recombinant DNA technology, by expression of the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), E. coli or yeast.
[000196] Domains of heavy chain-only antibodies combine advantages of antibodies and small molecule drugs: can be mono- or multi-valent; have low toxicity; and are cost-effective to manufacture. Due to their small size, these domains are easy to administer, including oral or topical administration, are characterized by high stability, including gastrointestinal stability; and their half-life can be tailored to the desired use or indication. In addition, VH and VHH domains of HCAbs can be manufactured in a cost-effective manner.
[000197] In a particular embodiment, the heavy chain antibodies of the present invention, including UniAbs™, have the native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Rabat numbering system), substituted by another amino acid residue, which is capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position. Such hydrophobic patches are normally buried in the interface with the antibody light chain constant region but become surface exposed in HCAbs and are, at least partially, for the unwanted aggregation and light chain association of HCAbs. The substituted amino acid residue preferably is charged, and more preferably is positively charged, such as lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment the heavy chain-only antibodies derived from the transgenic animals contain a Trp to Arg mutation at position 101. The resultant HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.
[000198] As part of the present invention, human IgG anti-HBsAg heavy chain antibodies with unique sequences from UniRat™ animals (UniAb™) were identified that bind to native HBsAg in ELISA protein and cell-binding assays. The identified heavy chain variable region (VH) sequences are positive for HBsAg binding and/or for binding to HBsAg+ cells, and are all negative for binding to cells that do not express HBsAg. See, e.g., Table 10.
[000199] Heavy chain antibodies binding to non-overlapping epitopes on an HBsAg protein, e.g., UniAbs™ can be identified by competition binding assays, such as enzyme-linked immunoassays (ELISA assays) or flow cytometric competitive binding assays. For example, one can use competition between known antibodies binding to the target antigen and the antibody of interest. By using this approach, one can divide a set of antibodies into those that compete with the reference antibody and those that do not. The non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized, the antigen is bound, and a second, labeled (e.g., biotinylated) antibody is tested in an ELISA assay for ability to bind the captured antigen. This can be performed also by using surface plasmon resonance (SPR) platforms, including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR imager (Ibis technologies B.V.), as well as on biolayer interferometry platforms, such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details see the examples herein.
[000200] Typically, an antibody “competes” with a reference antibody if it causes about 15-100% reduction in the binding of the reference antibody to the target antigen, as determined by standard techniques, such as by the competition binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher.
Pharmaceutical Compositions, Uses and Methods of Treatment
[000201] It is another aspect of the present invention to provide pharmaceutical compositions comprising one or more antibodies of the present invention in admixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.
[000202] In one embodiment, a pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to HBsAg. In another embodiment, a pharmaceutical composition comprises a multi-specific (including bispecific) heavy chain antibody (e.g., UniAb™) with binding specificity for two or more non-overlapping epitopes on an HBsAg protein. In a preferred embodiment, a pharmaceutical composition comprises a multi-specific (including bispecific and TCA) heavy chain antibody (e.g., UniAb™) with binding specificity to HBsAg and with binding specificity to a binding target on an effector cell (e.g., a binding target on a T-cell, such as, e.g., a CD3 protein on a T-cell).
[000203] Pharmaceutical compositions of the antibodies used in accordance with the present invention are prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g. Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; saltforming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
[000204] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). The formulation depends on the route of administration chosen. The antibodies herein can be administered by intravenous injection or infusion or subcutaneously. For injection administration, the antibodies herein can be formulated in aqueous solutions, preferably in physiologically -compatible buffers to reduce discomfort at the site of injection. The solution can contain carriers, excipients, or stabilizers as discussed above. Alternatively, antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[000205] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies, including UniAbs™, of the present invention. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.
Methods of Use
[000206] The anti-HBsAg antibodies and pharmaceutical compositions described herein can be used for the treatment of diseases and conditions characterized by the presence and/or expression of HBsAg, including, without limitation, the conditions and diseases described further herein. The anti-HBsAg antibodies and pharmaceutical compositions described herein can be used for the treatment of infection with hepatitis B virus (HBV) and/or a disease or condition caused thereby.
[000207] HBsAg, (UniProt Q81158) is the major antigen of the viral envelop and essential for viral attachment to heparan sulfate proteoglycans and a bile receptor — the sodium taurocholate co transporting polypeptide (NTCP) on the surface of hepatocytes. Three related HBsAgs-small (HBsAgS), middle (HBsAgM) and large (HBsAgL) are encoded by a single viral open reading fame (S ORF) and translated from 2 subgenomic mRNAs (HBsAgL from the 2.4 kb subgenomic mRNA, and HBsAgS and HBsAgM from the 2.1 kb subgenomic mRNA). All three envelope proteins share the common S domain with a size of 226 amino acids (aa). HBsAgM has an additional pre-S2 domain (55 aa) at the N-terminus of HBsAgS, and HBsAgL has an additional preSl-domain (108 or 119 aa depending on the genotype) extended from the N-terminus of HBsAgM. The core region of the S domain, comprising aa 99 to 169 and referred to as the major hydrophilic region (MHR), contains important epitopes recognized by neutralizing antibodies and T cells. Based on the antigenic heterogeneity of HBsAg, four serotypes were initially identified: adw, adr, ayw, and ayr, with a common “a” determinant and mutually exclusive “d/y” and “w/r”. Additional subdeterminants further divide HBsAg into a total of 10 serotypes. In addition to forming the envelops of infectious virions (Dane particles), HBsAgs are also secreted by infected cells as spherical or filamentous non-infectious subviral particles (SVPs) without the viral capsid. In patients’ circulation, SVPs are present in 100 to 10,000 fold excess over virions which are believed to allow the absorption of neutralizing antibodies and exhaustion of the host immune system. HBsAgS recombinantly produced in yeast and CHO cells also form SVPs and have been developed into HBV vaccines. Development of human derived monoclonal and bispecific neutralizing antibodies against HBsAg with therapeutic potentials have been described. Cellular confocal imaging studies and immunohistochemical tissue staining provide evidence of localization of HBsAg on the surface of infected hepatocytes. This and the importance of T cell immunity in viral clearance prompted the development of anti-HBsAg chimeric antigen receptor (CAR) T cells. Iannacone and Guidotti, Nat Rev Immunol. 2021 May 17 doi: 10.1038/s41577-021-00549-4. Epub ahead of print. PMID: 34002067, Lazarevic et ak, Viruses 2019, 11, 778., and Ho, J.K.-T. et ak, Viruses. 2020; 12(2): 126.
[000208] In one aspect, the anti-HBsAg antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to treat disorders characterized by the presence and/or expression of HBsAg, including, without limitation, chronic Hepatitis B infection (CHB), liver cirrhosis and hepatocellular carcinoma. In some embodiments, the anti-HBsAg antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used in connection with liver transplantation procedures, wherein a liver from a first patient is transplanted into a second patient. In some embodiments, the first patient is currently suffering from, or has previously been infected with, Hepatitis B virus, and is receiving a liver from a patient who is Hepatitis B virus-negative. In such embodiments, the anti-HBsAg antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to prevent the Hepatitis B virus infection from spreading to the transplanted liver. In some embodiments, the first patient has not previously been infected with Hepatitis B virus, but is receiving a liver from a patient that is currently suffering from, or has previously been infected with, Hepatitis B virus. In such embodiments, the anti-HBsAg antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to prevent the Hepatitis B virus infection from spreading to the transplant recipient patient.
[000209] Effective doses of the compositions of the present invention for the treatment of disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals may also be treated, e.g., companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.
[000210] Dosage levels can be readily determined by the ordinarily skilled clinician, and can be modified as required, e.g., as required to modify a subject's response to therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
[000211] In some embodiments, the therapeutic dosage the agent may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once every two weeks or once a month or once every 3 to 6 months. Therapeutic entities of the present invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
[000212] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution of solid (e.g., lyophilized) compositions. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
[000213] Toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
[000214] The compositions for administration will commonly comprise an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et ak, eds., 1996)).
[000215] Also within the scope of the invention are kits comprising the active agents and formulations thereof, of the invention and instructions for use. The kit can further contain a least one additional reagent, e.g. a chemotherapeutic drug, etc. Kits typically include a label indicating the intended use of the contents of the kit. The term “label” as used herein includes any writing, or recorded material supplied on or with a kit, or which otherwise accompanies a kit. [000216] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.
EXAMPLES
Example 1 : Flow cytometry analysis of binding to HBsAg expressing cells by anti-HBsAg UniAbs™ and monovalent HepBxCD3 bispecific antibodies.
[000217] Binding to HBsAg-positive cells was assessed by flow cytometry (BD FACSCelesta™, BD Biosciences) using a HepG2-LMS cell line. Briefly, 50,000 target cells were stained with a dilution series of purified UniAbs or HepBxCD3 bispecific antibodies for 30 minutes at 4°C. Following incubation, the cells were washed twice with flow cytometry buffer (IX PBS, 1% BSA, 0.1% NaN3) and stained with goat F(ab’)2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, Cat No.: 2042-09) to detect cell-bound antibodies. After a 20-minute incubation at 4°C, the cells were washed twice with flow cytometry buffer and the mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI of cells stained with secondary antibody alone were used for determination of background signal and binding of each antibody was converted to fold over background. EC50 values were calculated using GraphPad Prism 9.
[000218] Table 10 summarizes the target binding activity of the anti-HBsAg UniAbs and HepBxCD3 bispecific antibodies. Column 1 indicates the clone ID while Column 2 indicates the family ID. Columns 3 and 4 show the EC50 values in nM and the max binding to HepG2-LMS cells measured as maximum fold over the background MFI signal.
Table 10: Cell binding
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Example 2: Binding affinity to HBsAg measured by biolayer Interferometry (BLI)
[000219] The binding affinity of anti-HBsAg UniAbs to HBsAg SVPs of different serotypes was measured by BLI using the Octet HTX (Sartorius). Since HBsAg SVPs are multivalent, they were immobilized onto the biosensors as the ligand while the UniAbs were in solution as the analyte. Two methods were employed to immobilize HBsAg SVPs onto streptavidin (SA) biosensors (Sartorius,
Cat No.: 18-5020). In the first method, the VIR biosimilar HBC34v35 (WO2020132346A1) was used as the capture antibody. HBC34v35 was first biotinylated by incubating with the EZ-Link NHS- PEG4-Biotin (Thermo Scientific, Cat No. : 21329) at a molar ratio of 1 : 10 (antibody :biotin) at room temperature for 30 min. After removal of unreacted biotin using a Zeba Spin Desalting Columns 7K MWCO (Thermo Scientific, Cat No.: 89882), biotinylated HBC34v35 was immobilized onto SA biosensors at 5 pg/mL for 300 secs. After washing in Kinetic Buffer for 120 secs, the sensors were dipped into wells containing recombinant HBsAgs adw from yeast (abeam, Cat No.: ab91276), adr from CHO cells (ProSpec, Cat No.: hbs-875-b) or ayw from yeast (Fitzgerald, Cat No.: 30R-AH018), or native HBsAgs from human blood ad (Fitzgerald, Cat No.: 30-1815) or ay (Fitzgerald, Cat No.: 30- 1816) at 5 pg/mL for 900-1800 secs. After loading of the HBsAgs onto the biosensors through the capture antibody, the sensors were washed in Kinetic Buffer for 300 secs followed by 120 secs of baseline readings in a separate set of wells containing Kinetic Buffer. Then the association with the anti-HBsAg UniAbs were measured for 300 secs at an antibody concentration of 250 nM or 100 nM followed by a dissociation phase of 300 secs in Kinetic Buffer. Dissociation rate constant (kofl) in 1/s and response in nm were analyzed by the ForteBio Data Analysis software vl 1.1.1.8. In the second method, recombinant HBsAg ayw from yeast (Fitzgerald, Cat No.: 30R-AH018) were biotinylated and directly immobilized onto SA biosensors. For the biotinylation reaction, the same reagent (EZ- Link NHS-PEG4-Biotin) was used with a molar ratio of 1 :2 (HBsAg:biotin) and an incubation time of 60 min. After removal of unreacted biotin, biotinylated HBsAg ayw was loaded onto SA biosensors at 5 pg/mL for 900 secs. After loading, the sensors were washed in Kinetic Buffer for 300 secs followed by 60 secs of baseline readings in a separate set of wells containing Kinetic Buffer. Then the association with the anti-HBsAg UniAbs were measured for 180 secs followed by a dissociation phase of 240 secs in Kinetic Buffer. After the first cycle, the sensors were regenerated in 10 mM glycine pH 1.7 to the biotinylated HBsAg level and reused for additional cycles of association and dissociation. For each UniAb, a series of 7 concentrations were measured starting from 250 or 500 nM with a ½ serial dilution. Equilibrium dissociation constant (Kd) were calculated using the ForteBio Data Analysis software vl 1.1.1.8. As shown in FIG. 6, anti-HBsAg UniAbs that belong to F04 and F13 have overall good affinity to all the serotypes tested while other UniAbs show differential affinities to different serotypes.
Example 3: Epitope binning by BLI
[000220] Six anti-HBsAg UniAbs (HepB_F02A, F03B, F04A, F05A, F12A and F13A) with small kdis and large response were selected for the epitope binning study. They represent 6 distinct antibody families. VIR biosimilar HBC34v35 (WO2020132346A1) and a commercial mouse anti-HBsAg antibody clone A10F1 (Biolegend, Cat No.: 932302) were also included. Epitope binning analysis was performed on the Octet HTX (Sartorius). Briefly, biotinylated HBsAg ayw (as described in Example 2) was loaded onto SA biosensors at 5 pg/mL for 900 secs. After baseline readings, sensors were dipped into solutions containing Antibody 1 for 600 secs to saturate the sensors followed by another 60 secs baseline. Then the sensors were dipped into wells containing Antibody 2 for 300 secs. After the first cycle, the sensors were regenerated in 10 mM glycine pH 1.7 to the biotinylated HBsAg level and reused for additional cycles. An all-by-all binning study was conducted and the data was analyzed with ForteBio Data Analysis HT vl 1.1.1.39. Since the binding of UniAb HepB_F05A and F12A to HBsAg were affected by antigen biotinylation and/or regeneration, the binning study was repeated for these 2 UniAbs (2-by-all) using the capture antibody immobilization method (as described in Example 2) without sensor regeneration. As shown in FIG. 5, five separate bins were identified. The epitope recognized by HepB_F13A partially overlaps with both HepB_F02A (Bin 1) and HepB_F04A (Bin 2). Bin 3 and Bin 5 show one-directional blocking of Bin 4.
Example 4: Monovalent bispecific antibody mediated killing of HBsAg expressing hepatocellular carcinoma (HCC) cells through T-cell redirection
[000221] HepG2-LMS target cells were seeded at 10,000 cells per well in a 96-well plate and grown overnight at 37°C. Following incubation, increasing amounts of HepBxCD3 bispecific antibody were added together with resting human T-cells at a 10: 1 effector-to-target cell ratio and incubated for an additional 48 hours at 37°C. Cell death was measured using the cell proliferation reagent WST-1 (Sigma Cat No.: 11644807001). A small sample of each supernatant was collected after incubation, but prior to analysis of target cell viability, and saved for analysis of cytokine production. When cell viability was analyzed with WST-1 reagent, the reagent stock was added to each well at a 1:10 dilution and incubated for 120 minutes at 37°C. The absorbance was then measured at 450 nm (reference 690 nm), and the percent specific lysis was calculated. Wells containing untreated target cells were used to normalize for spontaneous cell death. The results are provided in Table 11.
Table 11: Cytotoxicity
Figure imgf000066_0001
Example 5: HepG2 cell killing data using anti-HBsAg antibody constructs comprising a trivalent HBsAb-binding arm
[000222] HepG2-LMS target cells were seeded at 10,000 cells per well in a 96-well plate and grown overnight at 37°C. Following incubation, increasing amounts of HepBxCD3 multispecific antibody constructs comprising a trivalent anti-HBsAg heavy chain polypeptide subunit as indicated in the legend of the graph in FIG. 7 were added together with resting human T-cells at a 10: 1 effector-to- target cell ratio and incubated for an additional 48 hours at 37°C. Cell death was measured using the cell proliferation reagent WST-1 (Sigma Cat No.: 11644807001). When cell viability was analyzed with WST-1 reagent, the reagent stock was added to each well at a 1:10 dilution and incubated for 120 minutes at 37°C. The absorbance was then measured at 450 nm (reference 690 nm), and the percent specific lysis was calculated. Wells containing untreated target cells were used to normalize for spontaneous cell death. The results are provided in FIG. 7.
Example 6: HBV Neutralization
[000223] An HBV genotype D reporter virus assay utilizing secretable NanoLuc was used to determine the neutralizing activity of several antibody constructs. Each antibody construct was co-incubated in the assay with reporter virus using 3-fold dilutions to calculate the titer at which 50% of infectivity was suppressed. The results are provided in FIG. 8. T10-55 corresponds to XX; T10-59 corresponds to XX; T10-71 corresponds to XX; T10-83 corresponds to XX; T10-87 corresponds to XX.
[000224] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS:
1. An antibody that binds to HBsAg, comprising a first heavy chain variable region comprising:
(a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and/or
(b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and/or
(c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
2. The antibody of claim 1, further comprising a second heavy chain variable region comprising:
(a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and/or
(b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and/or
(c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
3. The antibody of any one of claims 1 or 2, wherein said CDR1, CDR2, and CDR3 sequences are present in a human framework.
4. The antibody of any one of claims 1-3, further comprising a heavy chain constant region sequence in the absence of a CHI sequence.
5. The antibody of any one of claims 1-4, wherein the first heavy chain variable region comprises:
(a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and
(b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and
(c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
6. The antibody of any one of claims 2-5, wherein the second heavy chain variable region comprises:
(a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-15; and
(b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 16-34; and
(c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 35-54.
7. The antibody of any one of claims 1-6, comprising a heavy chain variable region comprising:
(a) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37;
(b) a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44; (c) a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49;
(d) a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52; or
(e) a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53.
8. The antibody of any one of claims 1-7, comprising a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 55-77.
9. The antibody of any one of claims 1-8, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 55-77.
10. The antibody of claim 9, wherein the heavy chain variable region sequence is selected from the group consisting of: SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 75 and SEQ ID NO: 76.
11. An antibody that binds to HBsAg, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-54.
12. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
13. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework, in a monovalent or bivalent configuration.
14. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
15. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework, in a monovalent or bivalent configuration.
16. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
17. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework, in a monovalent or bivalent configuration.
18. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
19. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework, in a monovalent or bivalent configuration.
20. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
21. An antibody that binds to HBsAg, comprising: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework, in a monovalent or bivalent configuration.
22. The antibody of any one of claims 1-21, which is monospecific.
23. The antibody of any one of claims 1-21, which is multi-specific.
24. The antibody of claim 23, which is bispecific.
25. The antibody of claim 23 or 24, which has binding affinity to a CD3 protein and an HBsAg protein.
26. The antibody of claim 23 or 24, which has binding affinity to two different epitopes on the same HBsAg protein.
27. The antibody of claim 23 or 24, having binding affinity to an effector cell.
28. The antibody of claim 27, having binding affinity to a T-cell antigen.
29. The antibody of claim 28, having binding affinity to CD3.
30. The antibody of any one of claims 1 to 29, which is in a CAR-T format.
31. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
32. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
33. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
34. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
35. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 119, a CDR2 sequence of SEQ ID NO: 120, and CDR3 sequence of SEQ ID NO: 121, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
36. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
37. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
38. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
39. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
40. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 122, a CDR2 sequence of SEQ ID NO: 123, and CDR3 sequence of SEQ ID NO: 124, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
41. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 37, in a human VH framework.
42. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 24, and a CDR3 sequence of SEQ ID NO: 44, in a human VH framework.
43. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 49, in a human VH framework.
44. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and (iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 32, and a CDR3 sequence of SEQ ID NO: 52, in a human VH framework.
45. A bispecific antibody comprising:
(i) a heavy chain variable region having binding affinity to CD3, comprising a CDR1 sequence of SEQ ID NO: 125, a CDR2 sequence of SEQ ID NO: 126, and CDR3 sequence of SEQ ID NO: 127, in a human VH framework;
(ii) a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 128, a CDR2 sequence of SEQ ID NO: 129, and CDR3 sequences of SEQ ID NO: 130, in a human VL framework; and
(iii) an antigen-binding domain of an anti-HBsAg heavy chain antibody, comprising a CDR1 sequence of SEQ ID NO: 14, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 53, in a human VH framework.
46. A bispecific three-chain antibody like molecule that binds to human CD3 and HBsAg, comprising:
(i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 142;
(ii) a second polypeptide subunit comprising an amino acid sequence selected from the group consistin of SEQ ID NOs: 143-145, wherein the first and second polypeptide subunits together form a first binding moiety that binds to human CD3; and
(iii) a third polypeptide subunit that binds to HBsAg, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-118.
47. A multispecific antibody that binds to CD3 and HBsAg, comprising:
(i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 142;
(ii) a second polypeptide subunit comprising an amino acid sequence selected from the group consistin of SEQ ID NOs: 143-145, wherein the first and second polypeptide subunits together form a first binding moiety that binds to human CD3; and
(iii) a third polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 147.
48. The multispecific antibody of claim 47, wherein the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 143.
49. The multispecific antibody of claim 47, wherein the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 144.
50. The multispecific antibody of claim 47, wherein the second polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 145.
51. A pharmaceutical composition comprising the antibody of any one of claims 1 to 50.
52. A method for the treatment of a disorder characterized by the presence of HBsAg, comprising administering to a subject with said disorder an antibody of any one of claims 1 to 50, or the pharmaceutical composition of claim 51.
53. Use of an antibody of any one of claims 1 to 50, in the preparation of a medicament for the treatment of a disorder characterized by the presence of HBsAg.
54. An antibody of any one of claims 1 to 50, for use in the treatment of a disorder characterized by the presence of HBsAg.
55. The method, use, or antibody of any one of claims 52 to 54, wherein the disorder is selected from the group consisting of: acute hepatitis B infection, chronic hepatitis B infection, liver cirrhosis and hepatocellular carcinoma.
56. A polynucleotide encoding an antibody of any one of claims 1 to 50.
57. A vector comprising the polynucleotide of claim 56.
58. A cell comprising the vector of claim 57.
59. A method of producing an antibody of any one of claims 1 to 50, comprising growing a cell according to claim 58 under conditions permissive for expression of the antibody, and isolating the antibody from the cell.
60. A method of making an antibody of any one of claims 1 to 50, comprising immunizing a UniRat animal with an HBsAg protein and identifying HBsAg binding antibody sequences.
61. A method of treatment, comprising administering to an individual in need an effective dose of the antibody of any one of claims 1 to 50, or the pharmaceutical composition of claim 51.
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