WO2017133639A1 - Anticorps neutralisants à large spectre contre le vih-1 et leur utilisation - Google Patents

Anticorps neutralisants à large spectre contre le vih-1 et leur utilisation Download PDF

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WO2017133639A1
WO2017133639A1 PCT/CN2017/072749 CN2017072749W WO2017133639A1 WO 2017133639 A1 WO2017133639 A1 WO 2017133639A1 CN 2017072749 W CN2017072749 W CN 2017072749W WO 2017133639 A1 WO2017133639 A1 WO 2017133639A1
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antibody
seq
hiv
fragment
amino acid
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Yiming Shao
Jiang Zhu
Yuxing Li
Ian A. Wilson
Leopold KONG
Bin JU
Linling HE
Li Ren
Yajing Chen
Jiandong Liu
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National Center For Aids/Std Control And Prevention, Chinese Center For Disease Control And Prevention
The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • 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
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    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/624—Disulfide-stabilized antibody (dsFv)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2

Definitions

  • the present invention relates to broadly neutralizing antibodies against HIV-1 which specifically bind to gp120 of HIV-1.
  • the present invention also relates to the methods for preparing such antibodies and the use thereof.
  • NAbs HIV-1 neutralizing antibodies
  • Monoclonal antibody b12 was isolated from a phage display library, which neutralizes about 40%known HIV-1 isolates [1] . With the improvement of techniques including single B cell sorting and deep sequencing, a number of monoclonal broadly neutralizing antibodies have been isolated from HIV-1-infected donors which recognize a range of epitopes on HIV-1 viral spike since 2010.
  • These sites include the CD4-binding site on gp120 (VRC01) [2] , the glycan-containing regions of V1V2 on gp120 (PG9 and PG16) [3] , the V3 region centered on the N332 glycan of gp120 (PGT121) [4] and the membrane-proximal external region (MPER) on gp41 (10E8) [5] , and a conserved face on contiguous areas of gp41 and gp120 (35O22) [6] .
  • the present invention provides human monoclonal antibodies and functional fragments thereof which are capable of specifically binding to gp120 of HIV-1 and blocking the entry of HIV-1 into target cells.
  • the present invention also provides nucleic acid molecules encoding the above described antibodies or antibody fragments as well as an expression vector comprising at least one nucleic acid molecule as described above.
  • the present invention also provides a host cell transformed with at least one nucleic acid molecule or expression vector as described above.
  • the present invention also provides a method of producing an antibody by using at least one nucleic acid molecule or expression vector or host cell as described above.
  • the present invention also provides a pharmaceutical composition comprising at least one antibody or antibody fragment as described above.
  • the heavy chain variable domain (VH) of the antibody of the invention comprises VHCDR1, VHCDR2, and VHCDR3 respectively corresponding to amino acids 31-35, 50-66, and 99-109 of SEQ ID NO: 2.
  • the light chain variable domain (VL) of the antibody of the invention comprises VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 24-34, 50-56, and 89-93 of SEQ ID NO: 4.
  • the VL of the antibody of the invention comprises VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 22-32, 48-54, and 87-91 of SEQ ID NO: 6.
  • the VL of the antibody of the invention comprises VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 24-34, 50-56, and 89-93 of SEQ ID NO: 8.
  • the antibodies of the invention are broadly neutralizing antibodies which specifically bind to gp120 of HIV-1.
  • the present invention also provides method of detecting an HIV-l infection in a human subject comprising contacting a biological sample from the subject with an antibody or antibody fragment of the present invention, and determining the presence of an immune complex formed by the antibody or the antibody fragment in said sample, wherein the presence of said immune complex indicates that the subject has an HIV-1 infection.
  • the sample prior to the contacting, is immobilized on a solid substrate.
  • the antibody or antibody fragment prior to the contacting, is immobilized on a solid substrate.
  • the antibody or antibody fragment is labeled with a fluorescent, enzymatic, or radioactive label.
  • the immune complex is detected by using a second antibody capable of specifically binding to the antibody or antibody fragment.
  • the immune complex is detected by using a second antibody capable of specifically binding to an antigen of HIV-1.
  • the present invention also provides a kit for detecting an HIV-l infection in a human subject, comprising an antibody or antibody fragment of the present invention.
  • the present invention further provides a method of preventing or treating an HIV-l infection in a human subject, comprising administering to the subject an effective amount of at least one antibody or antibody fragment of the invention or pharmaceutical composition of the invention.
  • the subject has acquired immune deficiency syndrome (AIDS) .
  • the method further comprises administering to the subject at least one anti-viral agent against HIV-1.
  • the present invention also relates to use of the antibody or antibody fragment of the invention in manufacture of a kit for detecting an HIV-l infection in a human subject or in manufacture of a pharmaceutical composition for preventing or treating an HIV-l infection in a human subject.
  • Fig. 1 shows the binding capacity of the monoclonal antibodies of the invention to HIV-1 antigens.
  • A The binding capacity of DRVIA7 to gp140.
  • B The binding capacity of antibody to gp120.
  • C The binding capacity of antibody DRVIA7H+gDRVI01-L57 to gp140.
  • D The binding capacity of antibody DRVIA7H+gDRVI01-L40 to gp140.
  • Fig. 2 shows the ribbon diagrams of the crystal structure of DRVIA7 bound to gp120.
  • Fig. 3 shows the residue-based energy evaluation of gp120 in complex with DRVIA7.
  • Fig. 4 shows the crystal structure of N-terminus and CDRL1 of DRVIA7 bound to gp120.
  • Fig. 5 shows the sequence analysis of antibody DRVIA7 and VRC01 heavy and light chains with alignment to respective germline genes.
  • Fig. 6 shows the crystal structure of the DRVIA7 compared with VRC01-like antibodies.
  • Fig. 7 shows the identity/divergence 2D analysis of the light chain repertoires from the HIV-1 infected donor.
  • SEQ ID NO: 1 is the sequence encoding the DRVIA7 VH.
  • SEQ ID NO: 2 is the amino acid sequence of the DRVIA7 VH.
  • SEQ ID NO: 3 is the sequence encoding the DRVIA7 VL.
  • SEQ ID NO: 4 is the amino acid sequence of the DRVIA7 VL.
  • SEQ ID NO: 5 is the sequence encoding the VL gDRVI01-L57.
  • SEQ ID NO: 6 is the amino acid sequence of the VL gDRVI01-L57.
  • SEQ ID NO: 7 is the sequence encoding the VL gDRVI01-L40.
  • SEQ ID NO: 8 is the amino acid sequence of the VL gDRVI01-L40.
  • Escherichia coli strain carrying the expression vector containing the heavy chain gene of DRVIA7 was deposited at CGMCC on December 14, 2015 under the Deposit Number CGMCC No. 11879.
  • Escherichia coli strain carrying the expression vector containing the light chain gene of DRVIA7 was deposited at CGMCC on December 14, 2015 under the Deposit Number CGMCC No. 11880.
  • Escherichia coli strain carrying the expression vector containing the light chain gene encoding the light chain variable domain gDRVI01-L57 was deposited at CGMCC on December 14, 2015 under the Deposit Number CGMCC No. 11881.
  • Escherichia coli strain carrying the expression vector containing the light chain gene encoding the light chain variable domain gDRVI01-L40 was deposited at CGMCC on December 14, 2015 under the Deposit Number CGMCC No. 11882.
  • antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin.
  • an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site) .
  • Antibodies include antibody fragments, such as anti-HIV-1 antibody fragments.
  • the term antibody thus, includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies) , human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab'fragments, F (ab’) 2 fragments, Fv fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fd’fragments, single-chain Fvs (scFv) , single-chain Fabs (scFab) , diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above.
  • Fab fragments fragments, Fab'fragments, F (ab’) 2 fragments, Fv fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fd’fragments, single-chain Fvs (scFv) , single-chain
  • Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY) , any class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (e.g., IgG2a and IgG2b) .
  • immunoglobulin type e.g., IgG, IgM, IgD, IgE, IgA and IgY
  • any class e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • subclass e.g., IgG2a and IgG2b
  • an “antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody that binds antigen (e.g. one or more CDRs and/or one or more antibody combining sites) and thus retains the binding specificity, and at least a portion of the specific binding ability of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically, e.g.
  • An antibody fragment is included among antibodies.
  • antibody fragments include, but are not limited to, Fab, Fab', F (ab’) 2 , single-chain Fv (scFv) , Fv, dsFv, diabody, Fd and Fd’fragments and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003) ; Chapter 1; p 3-25, Kipriyanov) .
  • the fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers.
  • An antibody fragment generally contains at least or about 50 amino acids and typically at least or about 200 amino acids.
  • An antigen-binding fragment includes any antibody fragment that when inserted into an antibody framework (such as by replacing a corresponding region) results in an antibody that immunospecifically binds (i.e. exhibits Ka of at least or at least about 10 7 -10 8 M -1 ) to the antigen.
  • neutralizing antibody is any antibody or antigen-binding fragment thereof that binds to a pathogen and interferes with the ability of the pathogen to infect a cell and/or cause disease in a subject.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This property is in contrast to that of a polyclonal population of antibodies, which contains antibodies having a plurality of different sequences. Monoclonal antibodies can be produced by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000) , 53, 111-117) .
  • monoclonal antibodies can be produced by immortalization of a B cell, for example through fusion with a myeloma cell to generate a hybridoma cell line or by infection of B cells with virus such as EBV.
  • Recombinant technology also can be used to produce antibodies in vitro from clonal populations of host cells by transforming the host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.
  • a “conventional antibody” refers to an antibody that contains two heavy chains (which can be denoted H and H’) and two light chains (which can be denoted L and L’) and two antibody combining sites, where each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding capability (e.g. heavy chains include, but are not limited to, V H , chains V H -C H 1 chains and V H -C H 1-C H 2-C H 3 chains) , and each light chain can be a full-length light chain or any functional region of (e.g. light chains include, but are not limited to, V L chains and V L -C L chains) .
  • Each heavy chain (H and H’) pairs with one light chain (L and L’, respectively)
  • a full-length antibody is an antibody having two full-length heavy chains (e.g. V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L ) and hinge regions, such as human antibodies produced naturally by antibody secreting B cells and antibodies with the same domains that are synthetically produced.
  • a dsFv refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the V H -V L pair.
  • a Fab fragment is an antibody fragment that results from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g. by recombinant methods.
  • a Fab fragment contains a light chain (containing a V L and C L ) and another chain containing a variable domain of a heavy chain (V H ) and one constant region domain of the heavy chain (C H 1) .
  • a F (ab’) 2 fragment is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure that is produced synthetically, e.g. by recombinant methods.
  • the F (ab’) 2 fragment essentially contains two Fab fragments where each heavy chain portion contains an additional few amino acids, including cysteine residues that form disulfide linkages joining the two fragments.
  • a Fab’fragment is a fragment containing one half (one heavy chain and one light chain) of the F (ab’) 2 fragment.
  • an scFv fragment refers to an antibody fragment that contains a variable light chain (V L ) and variable heavy chain (V H ) , covalently connected by a polypeptide linker in any order.
  • the linker is of a length such that the two variable domains are bridged without substantial interference.
  • Exemplary linkers are (Gly-Ser) n residues with some Glu or Lys residues dispersed throughout to increase solubility.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants typically contain chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies.
  • Each light chain and each heavy chain has one variable region domain, V L and V H , respectively.
  • the variable domains provide antigen specificity, and thus are responsible for antigen recognition.
  • Each variable region contains CDRs that are part of the antigen-binding site domain and framework regions (FRs) .
  • antigen-binding domain As used herein, “antigen-binding domain, ” “antigen-binding site, ” “antigen combining site” and “antibody combining site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with cognate antigen.
  • a native conventional full-length antibody molecule has two conventional antigen-binding sites, each containing portions of a heavy chain variable region and portions of a light chain variable region.
  • a conventional antigen-binding site contains the loops that connect the anti-parallel beta strands within the variable region domains.
  • the antigen combining sites can contain other portions of the variable region domains.
  • Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions also are called complementarity-determining regions (CDRs) .
  • CDRs complementarity-determining regions
  • variable region domain contains three CDRs, named CDR1, CDR2 and CDR3.
  • the three CDRs are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide.
  • the CDRs are located within the loops that join the parallel strands of the beta sheets of the variable domain.
  • framework regions are the domains within the antibody variable region domains that are located within the beta sheets; the FR regions are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
  • a “constant region” domain is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved than that of the variable region domain.
  • each light chain has a single light chain constant region (C L ) domain and each heavy chain contains one or more heavy chain constant region (C H ) domains, which include, C H 1, C H 2, C H 3 and C H 4.
  • C L constant region
  • C H heavy chain constant region
  • Full-length IgA, IgD and IgG isotypes contain C H 1, C H 2, C H 3 and a hinge region, while IgE and IgM contain C H 1, C H 2, C H 3 and C H 4.
  • C H 1 and C L domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms.
  • Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g., through interactions with various cells, biomolecules and tissues.
  • a functional region of an antibody is a portion of the antibody that contains at least a V H , V L , C H (e.g. C H 1, C H 2 or C H 3) , C L or hinge region domain of the antibody, or at least a functional region thereof.
  • a functional region of a V H domain is at least a portion of the full V H domain that retains at least a portion of the binding specificity of the full V H domain (e.g. by retaining one or more CDR of the full V H domain) , such that the functional region of the V H domain, either alone or in combination with another antibody domain (e.g. V L domain) or region thereof, binds to antigen.
  • exemplary functional regions of V H domains are regions containing the CDR1, CDR2 and/or CDR3 of the V H domain.
  • a functional region of a V L domain is at least a portion of the full V L domain that retains at least a portion of the binding specificity of the full V L domain (e.g. by retaining one or more CDRs of the full V L domain) , such that the function region of the V L domain, either alone or in combination with another antibody domain (e.g. V H domain) or region thereof, binds to antigen.
  • exemplary functional regions of V L domains are regions containing the CDR1, CDR2 and/or CDR3 of the V L domain.
  • “specifically bind” or “immunospecifically bind” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site (s) of the antibody and the antigen.
  • the antigen can be an isolated antigen or presented in a virus.
  • an antibody that immunospecifically binds (or that specifically binds) to a virus antigen or virus is one that binds to the virus antigen (or to the antigen in the virus or to the virus) with an affinity constant Ka of about or 1 ⁇ 10 7 M -1 or 1x 10 8 M -1 or greater (or a dissociation constant (K d ) of 1x 10 -7 M or 1 ⁇ 10 -8 M or less) .
  • Ka affinity constant
  • K d dissociation constant
  • Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11: 54; Englebienne (1998) Analyst.
  • ITC isothermal titration calorimetry
  • other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989) ; see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of antibodies) .
  • Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27: 335) .
  • polypeptide refers to two or more amino acids covalently joined.
  • polypeptide and protein are used interchangeably herein.
  • Suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224) .
  • nucleic acid refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA) , joined together, typically by phosphodiester linkages.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Sequence "identity” has an art-recognized meaning and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule.
  • identity is well known to skilled artisans (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988) ) .
  • operably linked with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.
  • expression refers to the process by which polypeptides are produced by transcription and translation of polynucleotides.
  • the level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.
  • a “host cell” is a cell that is used in to receive, maintain, reproduce and amplify a vector.
  • a host cell also can be used to express the polypeptide encoded by the vector.
  • the nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.
  • a “vector” is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation.
  • Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression/display of the polypeptide encoded by the nucleic acid.
  • the vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome.
  • vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
  • a vector also includes “virus vectors” or “viral vectors. ”
  • Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
  • an “expression vector” includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • treating means that the subject’s symptoms are partially or totally alleviated, or remain static following treatment.
  • treatment encompasses prophylaxis, therapy and/or cure.
  • Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease.
  • Treatment also encompasses any pharmaceutical use of any antibody or antigen-binding fragment thereof provided or compositions provided herein.
  • a “therapeutic effect” means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition.
  • a “therapeutically effective amount” or a “therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
  • a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses.
  • a prophylactically effective amount can be administered in one or more administrations.
  • the inventors isolated a monoclonal antibody from an HIV-1-infected Chinese donor sample and found that it has broadly neutralizing activities on a number of HIV-1 isolates.
  • This monoclonal antibody is named DRVIA7.
  • DRVIA7 Based on the heavy chain of DRVIA7 and through light chain modification technique and light chain gene repertoire screening, additional antibodies with increased potency and broadly neutralizing activities were obtained.
  • the antibodies of the invention are capable of blocking the entry of HIV-1 into target cells.
  • the present invention provides an isolated human monoclonal antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises VHCDR1, VHCDR2, and VHCDR3 respectively corresponding to VHCDR1, VHCDR2, and VHCDR3 contained in the amino acid sequence of SEQ ID NO: 2, wherein the antibody is a neutralizing antibody which specifically binds to gp120 of HIV-1.
  • the heavy chain variable domain comprises VHCDR1, VHCDR2, and VHCDR3 respectively corresponding to amino acids 31-35, 50-66, and 99-109 of SEQ ID NO: 2.
  • the expression “the heavy chain variable domain comprises VHCDR1 corresponding to VHCDR1 contained in the amino acid sequence of SEQ ID NO: 2” means VHCDR1 in the heavy chain variable domain of said antibody has the same amino acid sequence with that of VHCDR1 contained in the amino acid sequence of SEQ ID NO: 2.
  • VHCDR1 of antibody DRVIA7 consists of amino acids 31-35 (SSFIH) of the heavy chain variable domain as shown in SEQ ID NO: 2, thus, the above expression means VHCDR1 of the heavy chain variable domain of the antibody of the invention also consists of SSFIH.
  • VHCDR As used herein, VHCDR, HCDR, and CDRH have the same meaning and can be used interchangeably to refer to the complementary determining region of the antibody heavy chain variable domain.
  • the heavy chain variable domain comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%or even higher sequence identity with SEQ ID NO: 2.
  • the heavy chain variable domain consists of an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%or even higher sequence identity with SEQ ID NO: 2.
  • the heavy chain variable domain comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO: 2.
  • the heavy chain variable domain comprises VHCDR1, VHCDR2, and VHCDR3 as set out in Table 1.
  • the light chain variable domain comprises:
  • VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to VLCDR1, VLCDR2, and VLCDR3 contained in the amino acid sequence of SEQ ID NO: 4,
  • VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to VLCDR1, VLCDR2, and VLCDR3 contained in the amino acid sequence of SEQ ID NO: 6, or
  • VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to VLCDR1, VLCDR2, and VLCDR3 contained in the amino acid sequence of SEQ ID NO: 8.
  • the light chain variable domain comprises:
  • VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 24-34, 50-56, and 89-93 of SEQ ID NO: 4,
  • VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 22-32, 48-54, and 87-91 of SEQ ID NO: 6, or
  • VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 24-34, 50-56, and 89-93 of SEQ ID NO: 8.
  • the expression “the light chain variable domain comprises VLCDR1 corresponding to VLCDR1 contained in the amino acid sequence of SEQ ID NO: 4” means VLCDR1 in the light chain variable domain of said antibody has the same amino acid sequence with that of VLCDR1 contained in the amino acid sequence of SEQ ID NO: 4.
  • VLCDR1 of antibody DRVIA7 consists of amino acids 22-32 (RASQRIDNWVA) of the light chain variable domain as shown in SEQ ID NO: 4, thus, the above expression means VLCDR1 of the light chain variable domain of the antibody of the invention also consists of RASQRIDNWVA.
  • VLCDR LCDR
  • CDRL CDRL
  • the light chain variable domain comprises or consists of an amino acid sequence selected from a group consisting of (i) an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 85%, at least 90%, at least 95%or even higher sequence identity with SEQ ID NO: 4, (ii) an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%or even higher sequence identity with SEQ ID NO: 6, and (iii) an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 90%, at least 95%or even higher sequence identity with SEQ ID NO: 8.
  • the light chain variable domain comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO: 4, 6, or 8.
  • the light chain variable domain comprises VLCDR1, VLCDR2, and VLCDR3 as set out in Table 1.
  • the present invention also provides an isolated antibody capable of specifically binding to an epitope on HIV-1 gp120 which is recognized by an antibody of the invention as described above.
  • the present invention also provides an isolated antibody capable of competing with an antibody of the invention as described above to bind to an epitope on HIV-1 gp120.
  • the ability of two antibodies to bind to the same epitope can be determined by known assays in the art such as, for example, surface plasmon resonance assays and antibody competition assays.
  • antibodies that immunospecifically bind to the same epitope can compete for binding to the epitope, which can be measured, for example, by an in vitro binding competition assay (e.g. competition ELISA) , using techniques known the art.
  • the antibody of the invention is IgG. In some other embodiments, the antibody of the invention is IgM. In some other embodiments, the antibody of the invention is IgA.
  • the present invention further provides an isolated antibody fragment, which is a functional fragment of the above described antibody of the invention and capable of specifically binding to gp120 of HIV-1.
  • the antibody fragment of the invention is selected from the group consisting of a Fab fragment, a Fab’fragment, a F (ab) ’2 fragment, a single chain Fv protein (scFv) , and a disulfide stabilized Fv protein (dsFv) .
  • the antibody fragment of the invention has broadly neutralizing activities against a number of HIV-1 isolates.
  • the present invention also provides an isolated polypeptide which is the heavy or light chain variable domain of immunoglobulin.
  • the polypeptide of the invention can be used to construct an antibody capable of specifically binding to gp120 of HIV-1 and having broadly neutralizing activities on HIV-1.
  • the polypeptide is a heavy chain variable domain of immunoglobulin and comprises VHCDR1, VHCDR2, and VHCDR3 respectively corresponding to amino acids 31-35, 50-66, and 99-109 of SEQ ID NO: 2.
  • the polypeptide is a heavy chain variable domain of immunoglobulin and comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO: 2.
  • the polypeptide is a light chain variable domain of immunoglobulin and comprises VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 24-34, 50-56, and 89-93 of SEQ ID NO: 4.
  • the polypeptide is a light chain variable domain of immunoglobulin and comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO: 4.
  • the polypeptide is a light chain variable domain of immunoglobulin and comprises VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 22-32, 48-54, and 87-91 of SEQ ID NO: 6.
  • the polypeptide is a light chain variable domain of immunoglobulin and comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO: 6.
  • the polypeptide is a light chain variable domain of immunoglobulin and comprises VLCDR1, VLCDR2, and VLCDR3 respectively corresponding to amino acids 24-34, 50-56, and 89-93 of SEQ ID NO: 8.
  • the polypeptide is a light chain variable domain of immunoglobulin and comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO: 8.
  • the present invention provides an isolated nucleic acid molecule encoding an antibody or antibody fragment or polypeptide of the invention.
  • the nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 1 encoding a heavy chain variable domain.
  • the nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 3, 5, or 7 encoding a light chain variable domain.
  • the nucleic acid molecule of the invention is operatively linked to a promoter.
  • the present invention also provides an expression vector comprising at least one nucleic acid molecule of the invention as described above.
  • the present invention also provides an isolated host cell which has been transformed with at least one nucleic acid molecule or expression vector of the invention as described above.
  • the present invention provides a method of producing an antibody, comprising:
  • the present invention also relates to an isolated antibody or antibody fragment obtained by performing the method of the invention as described above, which is capable of specifically binding to HIV-1 gp120.
  • the isolated antibody or antibody fragment obtained by the method of the invention has broadly neutralizing activities on various HIV-1 isolates.
  • the present invention provides a method of detecting an HIV-l infection in a human subject, comprising:
  • step (i) the sample is immobilized on a solid substrate prior to the contacting, and the contacting comprises applying the antibody or antibody fragment to the solid substrate on which the sample is immobilized.
  • the antibody or antibody fragment is labeled with a fluorescent, enzymatic, or radioactive label.
  • step (ii) the solid substrate is brought into contact with a first binding partner capable of specifically binding to the antibody or antibody fragment.
  • the first binding partner is a second antibody capable of specifically binding to the antibody or antibody fragment.
  • step (i) the antibody or antibody fragment is immobilized on a solid substrate prior to the contacting, and the contacting comprises applying the sample to the solid substrate on which the antibody or antibody fragment is immobilized.
  • the solid substrate is brought into contact with a second binding partner capable of specifically binding to an antigen of HIV-1.
  • the second binding partner is a second antibody capable of specifically binding to an antigen of HIV-1.
  • the second antibody is capable of specifically binding to gp120 of HIV-1.
  • the antibody or antibody fragment and the second antibody capable of specifically binding to an antigen of HIV-1 bind to different epitopes on HIV-1.
  • the biological sample from the subject is a blood, plasma, serum, blood cell, or blood cell lysate sample.
  • the biological sample from the subject contains blood cells, and the method further comprises, prior to, during, or after step (i) , contacting the biological sample with a third binding partner capable of specifically binding to the blood cells.
  • the third binding partner is an antibody capable of specifically binding to the blood cells.
  • the blood cells are lymphocytes, for example T cells, such as CD4+ T cells.
  • the blood cells are monocytes.
  • the third binding partner is an antibody capable of specifically binding to a marker on the blood cells which is characteristic for the blood cells.
  • the present invention relates to use of the antibody or antibody fragment of the invention in manufacture of a kit for detecting an HIV-l infection in a human subject.
  • the present invention also provides a kit for detecting an HIV-l infection in a human subject, comprising an antibody or antibody fragment of the invention.
  • the present invention also provides a method of preventing or treating an HIV-l infection in a human subject, comprising administering to the subject an effective amount of an antibody or antibody fragment or a pharmaceutical composition of the invention.
  • the subject has acquired immune deficiency syndrome (AIDS) .
  • the method further comprises administering to the subject at least one anti-viral agent against HIV-1.
  • a subject or candidate for treatment with an antibody or antigen-binding fragment thereof provided herein includes, but is not limited to, a human subject that has been exposed to a HIV-1 virus, a human subject who exhibits one or more symptoms of a HIV-1 infection and a human subject who is at risk of a HIV-1 infection.
  • the present invention also relates to use of the antibody or antibody fragment of the invention in manufacture of a pharmaceutical composition for preventing or treating an HIV-l infection in a human subject.
  • the subject has acquired immune deficiency syndrome (AIDS) .
  • AIDS acquired immune deficiency syndrome
  • the present invention provides a pharmaceutical composition, which comprises at least one antibody or antibody fragment of the invention as described above, and a pharmaceutically acceptable carrier.
  • an effective amount of antibody or antigen-binding fragment thereof to be administered therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient.
  • the attending physician takes into consideration various factors known to modify the action of drugs, including severity and type of disease, patient’s health, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Accordingly, it will be necessary for the therapist to titer the dosage of the antibody or antigen-binding fragment thereof and modify the route of administration as required to obtain the optimal therapeutic effect.
  • the clinician will administer the antibody or antigen-binding fragment thereof until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays. Exemplary assays for monitoring treatment of a viral infection are know in the art and include for example, viral titer assays.
  • the dosage ranges for the administration of the antibodies or antigen-binding fragments thereof provided herein are those large enough to produce the desired effect in which the symptom (s) of the pathogen-mediated disease (e.g. viral disease) are ameliorated or the likelihood of virus infection is decreased.
  • the antibodies or antigen-binding fragments thereof provided herein are administered in an amount effective for inducing an immune response in the subject.
  • the dosage is not so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema or congestive heart failure.
  • the dosage will vary with the age, condition, sex and the extent of the disease in the patient and can be determined by one of skill in the art.
  • Exemplary dosages for the prevention or treatment of a HIV-1 infection include, but are not limited to, about or 0.01 mg/kg to about or 300 mg/kg, such as for example, about or 0.01 mg/kg, about or 0.1 mg/kg, about or 0.5 mg/kg, about or 1 mg/kg, about or 5 mg/kg, about or 10 mg/kg, about or 15 mg/kg, about or 20 mg/kg, about or 25 mg/kg, about or 30 mg/kg, about or 35 mg/kg, about or 40 mg/kg, about or 45 mg/kg, about or 50 mg/kg, about or 100 mg/kg, about or 150 mg/kg, about or 200 mg/kg, about or 250 mg/kg, or about or 300 mg/kg.
  • the dosage of the antibodies or antigen-binding fragments thereof of the invention can vary depending on the type and severity of the disease.
  • the antibodies or antigen-binding fragments thereof can be administered single dose, in multiple separate administrations, or by continuous infusion.
  • the treatment can be repeated until a desired suppression of disease symptoms occurs or the desired improvement in the patient's condition is achieved.
  • Repeated administrations can include increased or decreased amounts of the antibody or antigen-binding fragment thereof depending on the progress of the treatment.
  • Other dosage regimens also are contemplated.
  • the antibodies or antigen-binding fragments thereof provided herein can be administered to a subject by any method known in the art for the administration of polypeptides, including for example systemic or local administration.
  • the antibodies or antigen-binding fragments thereof can be administered by routes, such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavity) , topical, epidural, or mucosal (e.g. intranasal or oral) .
  • the antibodies or antigen-binding fragments thereof can be administered externally to a subject, at the site of the disease for exertion of local or transdermal action.
  • compositions containing the antibodies or antigen-binding fragments thereof can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa) .
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa
  • compositions provided herein can be in various forms, e.g., in solid, semi-solid, liquid, powder, aqueous, or lyophilized form.
  • suitable pharmaceutically acceptable carriers include but are not limited to water, buffering agents, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, powders, among others.
  • compositions provided herein can contain other additives including, for example, antioxidants, preservatives, antimicrobial agents, analgesic agents, binders, disintegrants, coloring, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents, flavoring agents, emulsions, such as oil/water emulsions, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth
  • Such carriers and/or additives can be formulated by conventional methods and can be administered to the subject at a suitable dose.
  • Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can preserve the compositions from degradation within the body.
  • a monoclonal antibody DRVIA7 was isolated from a sample from an HIV-1-infected Chinese donor and has been identified as having broadly neutralizing activities against HIV-1 isolates.
  • Two Escherichia coli strains carrying the heavy chain gene expression vector (DRVIA7H) or light chain gene expression vector (DRVIA7L) were deposited at China General Microbiological Culture Collection Center (CGMCC) (NO. 1 West Beichen Road, Chaoyang District, Beijing, China) under the accession number of CGMCC No. 11879 and CGMCC No. 11880, respectively.
  • CGMCC General Microbiological Culture Collection Center
  • the sequence encoding the heavy chain variable region contained in DRVIA7H is SEQ ID NO: 1
  • the encoded amino acid sequence of the heavy chain variable region is SEQ ID NO: 2.
  • the sequence encoding the light chain variable region contained in DRVIA7L is SEQ ID NO: 3
  • the encoded amino acid sequence of the light chain variable region is SEQ ID NO: 4.
  • Escherichia coli strains carrying the heavy chain gene expression vector DRVIA7H or the light chain gene expression vector DRVIA7L were separately inoculated in 100ml LB culture medium (Amersham) containing kanamycin at a final concentration of 50 ⁇ g/ml, and then cultured at 37°C for 16 hours with shaking at 200rpm.
  • the expression vector plasmids were extracted from the cultures using Plasmid Midi Kit (Omega) following the manufacturer’s instructions. 293F cells were co-transfected with equal amount of heavy and light chain plasmids using PEI transfection reagent (Polysciences) , and then cultured at 8%CO 2 and 37°C for 6 days.
  • Monoclonal antibody DRVIA7 was purified from the culture supernatant using Protein-Acolumns (GE health) , and the concentration of antibody was determined by NanoDrop2000 (Thermo) . The purified antibody samples were stored at 4°Cfor further detection.
  • ELISA Enzyme Linked Immunosorbent Assay
  • monoclonal antibody DRVIA7 can specifically bind with CN54 GP140 protein (Fig. 1A) as well as wild-type and mutant gp120 proteins (Fig. 1B) .
  • the neutralizing activity of antibody DRVIA7 was measured using TZM-bl/pseudovirus neutralizing assay [7] .
  • antibody DRVIA7 was serially diluted in DMEM growth medium (Hyclone) .
  • the diluted antibody (100 ⁇ l/well) and the pseudovirus (50 ⁇ l/well, containing pseudovirus of 200 TCID 50 ) were added to each well of the 96-well flat bottom plates. The plates were then incubated at 37°C, 5%CO 2 for 1 hour.
  • TZM-bl cells were added at 1 ⁇ 10 4 /well to the plates in DMEM growth medium containing 11 ⁇ g/ml DEAE-dextran (Sigma) .
  • DRVIA7 can neutralize different subtypes of HIV-1 viruses, and thus is a broadly neutralizing antibody.
  • Crystallographic characterization of unliganded DRVIA7 and DRVIA7+gp120 complex were determined using X-ray crystallography (Table 3) . Ribbon diagrams of the crystal structure of DRVIA7 bound to gp120 was displayed in Fig. 2.
  • a Numbers in parentheses refer to the highest resolution shell.
  • R sym ⁇ hkl ⁇ i
  • R pim is a redundancy-independent measure of the quality of intensity measurements.
  • R pim ⁇ hkl (1/ (n-1) ) 1/2 ⁇ i
  • e R free was calculated as for R cryst , but on a test set comprising 5%of the data excluded from refinement.
  • Residue-based energy evaluation [8] of the complex formed between DRVIA7 and gp120 revealed that there might be a likelihood of structural interference between the N-terminus, CDRL1 and CDRL3 of DRVIA7 light chain and gp120 which may affect the binding thereof (Fig. 3) .
  • modified DRVIA7 antibodies with mutations on the light chain were generated. Antibodies with modifications on CDRL1 failed to show an improved neutralizing activity. However, a modified DRVIA7 antibody obtained by deleting two residues from the N-terminus of DRVIA7 light chain variable region significantly increased the binding capacity and neutralizing breadth.
  • the modified light chain variable region is named as gDRVI01-L57. Escherichia coli strain carrying the expression vector (gDRVI01-L57) containing the light chain gene encoding light chain variable region gDRVI01-L57 was deposited at CGMCC under the accession number of CGMCC No. 11881.
  • the sequence encoding the light chain variable region contained in the expression vector gDRVI01-L57 is SEQ ID NO: 5
  • the encoded amino acid sequence of the light chain variable region is SEQ ID NO: 6.
  • Antibody DRVIA7H+gDRVI01-L57 was obtained by an antibody expression and purification process similar to that described in Example 1 by using Escherichia coli strains carrying the heavy chain expression vector DRVIA7H and the light chain expression vector gDRVI01-L57.
  • the binding capacity of DRVIA7H+gDRVI01-L57 was measured by ELISA as described in Example 1. As shown in Fig. 1C, the binding capacity of DRVIA7H+gDRVI01-L57 to CN54 GP140 antigen was markedly improved as compared with DRVIA7.
  • the neutralizing activity of DRVIA7H+gDRVI01-L57 was measured using TZM-bl/pseudovirus neutralizing assay as described in Example 1. As shown in Tables 2A and 2B, the neutralizing breadth of DRVIA7H+gDRVI01-L57 was increased significantly in both the tested Global Panel and DRVI Panel as compared with that of DRVIA7.
  • the mechanism of improved neutralizing activity of DRVIA7H+gDRVI01-L57 was preliminarily discussed by analyzing the crystal structure of N-terminus (LC-Nt) and CDRL1 of DRVIA7 light chain bound to gp120. As shown in Fig. 4, the N-terminus of wild-type DRVIA7 light chain was very adjacent to the N461 glycosylation site on gp120. As it is believed that N-linked glycosylation sites may protect the virus against neutralizing activity of antibody, it is possible that the deletion of two N-terminal residues from the light chain variable region may help to avoid the interference of the N461 glycosylation site on the binding of the antibody with gp120 and thereby facilitate the neutralizing of virus by the antibody.
  • Example 3 Antibody DRVIA7H+gDRVI01-L40 obtained by antibody light chain gene repertoire screening
  • the CDR3 length was determined based on the Kabat numbering. As shown in Fig. 5, DRVIA7 heavy chain is derived from IgHV1-02*02 allele, and CDRH3 contains 11 amino acids. DRVIA7 light chain is derived from IgKV1-5*03 allele, and CDRL3 contains 5 amino acids. DRVIA7 heavy chain is derived from the same allele as VRC01 and CDRL3 of light chain has the same length as VRC01 [1] , suggesting that DRVIA7 may be a VRC01-like antibody.
  • DRVIA7 The crystal structure of DRVIA7 was determined in example 1, which strongly resembles some published VRC01-like antibodies such as VRC01 (PDBID: 3NGB) , VRC03 (PDBID: 3SE8) , PG04 (PDBID: 3SE9) and 12A21 (PDBID: 4JPW) (Fig. 6) .
  • VRC01 PDBID: 3NGB
  • VRC03 VRC03
  • PG04 PDBID: 3SE9
  • 12A21 PBID: 4JPW
  • the antibody light chain gene repertoire was established using deep sequencing technology from the blood sample of the HIV-1 infected donor from whom DRVIA7 was isolated, and a large number of DRVIA7-like light chain genes were found in this repertoire. Based on the sequence analysis of published VRC01-like antibodies, some common characteristics were identified.
  • the CDRL3 signatures (5 amino acids in length, hydrophobic residue at position 3 and Q/E at position 4) among VRC01-like antibody light chains were summarized and used to select candidate light chain genes from the antibody light chain repertoire. As shown in Fig. 7, 703 VRC01-like light chains (blue dots) were identified and 22 candidate light chains (red dots) were synthesized and the binding capacity and neutralizing activity were tested.
  • Escherichia coli strain carrying the expression vector (gDRVI01-L40) containing the light chain gene encoding light chain variable region gDRVI01-L40 was deposited at CGMCC under the accession number of CGMCC No. 11882.
  • the sequence encoding the light chain variable region contained in the expression vector gDRVI01-L40 is SEQ ID NO: 7, and the encoded amino acid sequence of the light chain variable region is SEQ ID NO: 8.

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  • Communicable Diseases (AREA)
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Abstract

L'invention concerne des anticorps neutralisants à large spectre contre le VIH-1, qui se lient spécifiquement à la gp120 du VIH-1, ainsi qu'un procédé de préparation desdits anticorps et l'utilisation de ceux-ci.
PCT/CN2017/072749 2016-02-02 2017-01-26 Anticorps neutralisants à large spectre contre le vih-1 et leur utilisation WO2017133639A1 (fr)

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WO2020010107A1 (fr) 2018-07-03 2020-01-09 Gilead Sciences, Inc. Anticorps se liant spécifiquement à la gp120 du vih pd-1 et leurs methodes d'utilisation
WO2020263830A1 (fr) 2019-06-25 2020-12-30 Gilead Sciences, Inc. Protéines de fusion flt3l-fc et procédés d'utilisation
WO2021011544A1 (fr) 2019-07-16 2021-01-21 Gilead Sciences, Inc. Vaccins contre le vih et leurs procédés de fabrication et d'utilisation
WO2021011891A1 (fr) 2019-07-18 2021-01-21 Gilead Sciences, Inc. Formulations à action prolongée de ténofovir alafénamide
WO2021130638A1 (fr) 2019-12-24 2021-07-01 Carna Biosciences, Inc. Composés modulant la diacylglycérol kinase
WO2021188959A1 (fr) 2020-03-20 2021-09-23 Gilead Sciences, Inc. Promédicaments de nucléosides de 4'-c-substitué-2-halo-2'-désoxyadénosine et leurs procédés de fabrication et d'utilisation
WO2021236944A1 (fr) 2020-05-21 2021-11-25 Gilead Sciences, Inc. Compositions pharmaceutiques contenant du bictégravir
WO2022031894A1 (fr) 2020-08-07 2022-02-10 Gilead Sciences, Inc. Promédicaments d'analogues nucléotidiques de phosphonamide et leur utilisation pharmaceutique
WO2022046644A1 (fr) 2020-08-25 2022-03-03 Gilead Sciences, Inc. Molécules de liaison à un antigène multi-spécifiques ciblant le vih et méthodes d'utilisation
EP3830108A4 (fr) * 2018-07-27 2022-08-10 International AIDS Vaccine Initiative Anticorps modifiés dirigés contre les env du vih
WO2022271677A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2022271659A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
WO2022271684A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
WO2022271650A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2023102523A1 (fr) 2021-12-03 2023-06-08 Gilead Sciences, Inc. Composés thérapeutiques pour l'infection par le virus du vih
WO2023102529A1 (fr) 2021-12-03 2023-06-08 Gilead Sciences, Inc. Composés thérapeutiques pour l'infection par le virus du vih
WO2023102239A1 (fr) 2021-12-03 2023-06-08 Gilead Sciences, Inc. Composés thérapeutiques contre l'infection par le virus du vih
WO2023196875A1 (fr) 2022-04-06 2023-10-12 Gilead Sciences, Inc. Composés de carbamoylpyridone tricycliques pontés et leurs utilisations
WO2024006982A1 (fr) 2022-07-01 2024-01-04 Gilead Sciences, Inc. Composés thérapeutiques utiles pour le traitement prophylactique ou thérapeutique d'une infection par le virus du vih
WO2024015741A1 (fr) 2022-07-12 2024-01-18 Gilead Sciences, Inc. Polypeptides immunogènes du vih et vaccins et utilisations de ceux-ci
WO2024076915A1 (fr) 2022-10-04 2024-04-11 Gilead Sciences, Inc. Analogues de 4'-thionucléosides et leur utilisation pharmaceutique

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WO2020010107A1 (fr) 2018-07-03 2020-01-09 Gilead Sciences, Inc. Anticorps se liant spécifiquement à la gp120 du vih pd-1 et leurs methodes d'utilisation
EP4257600A2 (fr) 2018-07-03 2023-10-11 Gilead Sciences, Inc. Anticorps se liant spécifiquement à la gp120 du vih pd-1 et leurs methodes d'utilisation
EP3830108A4 (fr) * 2018-07-27 2022-08-10 International AIDS Vaccine Initiative Anticorps modifiés dirigés contre les env du vih
WO2020263830A1 (fr) 2019-06-25 2020-12-30 Gilead Sciences, Inc. Protéines de fusion flt3l-fc et procédés d'utilisation
WO2021011544A1 (fr) 2019-07-16 2021-01-21 Gilead Sciences, Inc. Vaccins contre le vih et leurs procédés de fabrication et d'utilisation
WO2021011891A1 (fr) 2019-07-18 2021-01-21 Gilead Sciences, Inc. Formulations à action prolongée de ténofovir alafénamide
WO2021130638A1 (fr) 2019-12-24 2021-07-01 Carna Biosciences, Inc. Composés modulant la diacylglycérol kinase
EP4445902A2 (fr) 2019-12-24 2024-10-16 Carna Biosciences, Inc. Composés modulant la diacylglycérol kinase
WO2021188959A1 (fr) 2020-03-20 2021-09-23 Gilead Sciences, Inc. Promédicaments de nucléosides de 4'-c-substitué-2-halo-2'-désoxyadénosine et leurs procédés de fabrication et d'utilisation
WO2021236944A1 (fr) 2020-05-21 2021-11-25 Gilead Sciences, Inc. Compositions pharmaceutiques contenant du bictégravir
WO2022031894A1 (fr) 2020-08-07 2022-02-10 Gilead Sciences, Inc. Promédicaments d'analogues nucléotidiques de phosphonamide et leur utilisation pharmaceutique
WO2022046644A1 (fr) 2020-08-25 2022-03-03 Gilead Sciences, Inc. Molécules de liaison à un antigène multi-spécifiques ciblant le vih et méthodes d'utilisation
WO2022271684A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
WO2022271659A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
WO2022271650A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2022271677A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2023102523A1 (fr) 2021-12-03 2023-06-08 Gilead Sciences, Inc. Composés thérapeutiques pour l'infection par le virus du vih
WO2023102529A1 (fr) 2021-12-03 2023-06-08 Gilead Sciences, Inc. Composés thérapeutiques pour l'infection par le virus du vih
WO2023102239A1 (fr) 2021-12-03 2023-06-08 Gilead Sciences, Inc. Composés thérapeutiques contre l'infection par le virus du vih
EP4445900A2 (fr) 2021-12-03 2024-10-16 Gilead Sciences, Inc. Composés thérapeutiques pour une infection par le virus vih
EP4438123A2 (fr) 2022-04-06 2024-10-02 Gilead Sciences, Inc. Composés de carbamoylpyridone tricyclique pontés et leurs utilisations
WO2023196875A1 (fr) 2022-04-06 2023-10-12 Gilead Sciences, Inc. Composés de carbamoylpyridone tricycliques pontés et leurs utilisations
EP4310087A1 (fr) 2022-04-06 2024-01-24 Gilead Sciences, Inc. Composés de carbamoylpyridone tricycliques pontés et leurs utilisations
WO2024006982A1 (fr) 2022-07-01 2024-01-04 Gilead Sciences, Inc. Composés thérapeutiques utiles pour le traitement prophylactique ou thérapeutique d'une infection par le virus du vih
WO2024015741A1 (fr) 2022-07-12 2024-01-18 Gilead Sciences, Inc. Polypeptides immunogènes du vih et vaccins et utilisations de ceux-ci
WO2024076915A1 (fr) 2022-10-04 2024-04-11 Gilead Sciences, Inc. Analogues de 4'-thionucléosides et leur utilisation pharmaceutique

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