WO2012106578A1 - HIV NEUTRALIZING ANTIBODIES HAVING MUTATIONS IN CONSTANT DOMAIN (Fc) - Google Patents

HIV NEUTRALIZING ANTIBODIES HAVING MUTATIONS IN CONSTANT DOMAIN (Fc) Download PDF

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Publication number
WO2012106578A1
WO2012106578A1 PCT/US2012/023737 US2012023737W WO2012106578A1 WO 2012106578 A1 WO2012106578 A1 WO 2012106578A1 US 2012023737 W US2012023737 W US 2012023737W WO 2012106578 A1 WO2012106578 A1 WO 2012106578A1
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Prior art keywords
antibody
seq
hiv
heavy chain
isolated
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PCT/US2012/023737
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French (fr)
Inventor
Gary J. Nabel
Sung-Youl Ko
Zhi-Yong Yang
Richard Blumberg
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Brigham And Women's Hospital
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Publication of WO2012106578A1 publication Critical patent/WO2012106578A1/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
    • C07K16/10—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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This relates to monoclonal neutralizing antibodies that bind to the CD4 binding site of HIV-1 gpl20 having mutations in the constant domain (Fc), and methods for their use.
  • HIV-1 vaccine will likely need to induce neutralizing antibodies (NAbs) that block HIV-1 entry into human cells.
  • NAbs neutralizing antibodies
  • vaccine induced antibodies will have to be active against most circulating strains of HIV-1.
  • HIV-1 vaccines are unable to induce potent and broadly reactive NAbs.
  • antibodies with increased half-life are desirable.
  • HIV-1 neutralizing mAb can bind to a site on gpl20 that is required for viral attachment to its primary cellular receptor, CD4.
  • Another previously characterized HIV-1 neutralizing mAb called 2F5
  • mAb bl2 was derived from a phage display library, a process which makes it impossible to know if the antibody was naturally present in an infected person, or was the result of a laboratory combination of antibody heavy and light chains.
  • bl2 can neutralize about 75% of clade B strains of HIV-1 (those most common in North America), but it neutralizes less than 50% of other strains of HIV-1 found worldwide.
  • Prior attempts to design a vaccine that induces NAbs similar to bl2 have been unsuccessful. Therefore, there is a need to develop Nabs for HIV-1, including those that have an increased half-life.
  • Isolated human monoclonal neutralizing antibodies that specifically bind HIV-l gpl20 and have one or more mutations in the constant region (Fc) are provided herein. In some examples such antibodies have an increased half-life relative to the same antibodies with native Fc sequences. Also disclosed herein are compositions including these antibodies, nucleic acids encoding these antibodies, expression vectors comprising the nucleic acids, and isolated host cells that express the nucleic acids.
  • any IgG antibody can be generated that has equivalent mutations in its Fc region.
  • isolated IgG antibodies wherein a heavy chain of the IgG includes one or more amino acid substitutions in the Fc region, wherein the one or more amino acid substitutions comprise: T250Q/M428L; M428L/N434S; N434A;
  • T307A/E380A/N434A M252Y/S254T/T256E; S239D/I332E;
  • M428L/N434S S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
  • an isolated IgG antibody can include a S239D/I332E or S239D/A330L/I332E substitution, and in some examples can further include one or more of
  • IgG molecules have increased FcRn binding relative to the native IgG molecule, such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
  • IgG molecules can include IgGl, IgG2, IgG3, or IgG4.
  • the heavy chain of the isolated human monoclonal antibody (mAb) having one or more mutations in Fc has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and wherein the antibody comprises a light chain, and wherein the mAb specifically binds gpl20 of HIV-1, and wherein the antibody is neutralizing.
  • the heavy chain of the isolated human mAb having one or more mutations in Fc has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the antibody comprises a light chain, wherein the mAb specifically binds gpl20 of HIV-1, wherein the antibody is neutralizing, and wherein the mAb includes the particular mutation provided by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (for example, a sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 includes the T250Q/M428L mutation and a sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 includes the M428L/N434S
  • the light chain of the mAb comprises amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO: 36.
  • the heavy chain of the mAb includes one or more of the following amino acid substitutions:
  • the heavy chain of the mAb includes a sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 38 or 42, and wherein the antibody comprises a light chain (such as a sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 40 or 44), and wherein the antibody specifically binds gpl20 of HIV-1, wherein the antibody is neutralizing, and wherein the heavy chain of the antibody has one or more of the following amino acid substitutions:
  • the isolated mAb has a heavy chain as shown in SEQ ID NO: 38 comprising one or more of the amino acid substitutions shown above and a light chain as shown in SEQ ID NO: 40. In other examples, the isolated mAb has a heavy chain as shown in SEQ ID NO: 42 comprising one or more of the amino acid substitutions shown above and a light chain as shown in SEQ ID NO: 44.
  • the disclosed neutralizing hmAbs having one or more Fc mutations and have the ability to bind gpl20 have increased FcRn binding and/or increase antibody-dependent cellular cytotoxicity (ADCC) activity relative to the native IgG molecule (such as VRCOl, VRCOl, or VRC03), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
  • ADCC antibody-dependent cellular cytotoxicity
  • the disclosed neutralizing hmAbs having one or more Fc mutations and have the ability to bind gpl20 have increased half-life relative to the native IgG molecule (such as VRCOl, VRCOl, or VRC03), such as an increase of at least 1.5-fold, 1.8-fold, 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5 fold, or at least 5-fold.
  • the native IgG molecule such as VRCOl, VRCOl, or VRC03
  • isolated functional fragments of the disclosed mAbs specific for gpl20 that have one or more mutations in the Fc.
  • Exemplary fragments include a Fab fragment, a Fab' fragment, a F(ab)' 2 fragment, a single chain Fv protein (scFv), or a disulfide stabilized Fv protein (dsFv).
  • the disclosed antibodies and fragments thereof can be labeled, for example with a fluorescent, enzymatic, or radioactive label.
  • compositions that include the disclosed antibodies are provided, for example those that include a pharmaceutically acceptable carrier, such as water or saline.
  • nucleic acid molecules that encode the human mAbs disclosed herein (or a functional fragment thereof).
  • a nucleic acid molecule includes a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98, or at least 99% sequence identity to any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41, as well as sequences that encode a sequence having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 38 or 42 which have one or more of the following amino acid substitutions: T250Q/M428L;
  • the mAbs and compositions disclosed herein can be used for a variety of purposes, such as for detecting an HIV-1 infection or diagnosing AIDS in a subject. These methods can include contacting a sample from the subject (such as one suspected of having or who has been diagnosed with HIV-1 or AIDS) with a human monoclonal antibody that specifically binds gpl20 and has one or more mutations in the Fc region, and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample relative to binding of the antibody to a control sample confirms that the subject has an HIV-1 infection and/or AIDS.
  • the methods further include contacting a second antibody that specifically binds gpl20 or gp41 with the sample, and detecting binding of the second antibody.
  • a second antibody that specifically binds gpl20 or gp41 with the sample, and detecting binding of the second antibody.
  • an increase in binding of the antibody to the sample relative to a control sample detects HIV-1 in the subject.
  • the antibody specifically binds soluble gpl20 in the sample.
  • the methods further include contacting a second antibody that specifically recognizes the gpl20- or gp41-specific antibody with the sample and detecting binding of the second antibody.
  • a method for treating a subject with an HIV infection, such as, but not limited to, a subject with AIDS.
  • the methods include administering a therapeutically effective amount of a human gpl20 specific monoclonal antibody having one or more mutations in the Fc region disclosed herein to the subject.
  • Also provided is a method for testing a potential vaccine wherein the method includes contacting the potential vaccine with a human mAb disclosed herein (or a functional fragment thereof) and detecting the binding of the antibody to an immunogen in the potential vaccine.
  • FIG. 1 is a table showing mutations made to the Fc region of the heavy chain of the VRCOl antibody.
  • FIG. 2 is a schematic drawing showing the structures of the VRCOl antibody and its Fc mutants. Blue (those at the top) and red spheres (those closer to the bottom) represent ADCC enhancing mutation and hFcRn-binding enhancing mutation, respectively.
  • FIG. 3 is a pair of graphs showing binding of the VRCOl antibody and its Fc mutants to hFcRn at pH 6 (left) and pH 7.4 (right).
  • FIG. 4 is a pair of graphs showing binding of the VRCOl antibody and its Fc mutants to FcyRIIIa (left) and RSC3 (right).
  • FIG. 5 shows a pair of graphs showing antibody-dependent cellular cytotoxicity (ADCC) of the VRCOl antibody and its Fc mutants.
  • ADCC antibody-dependent cellular cytotoxicity
  • FIG. 6 is a schematic drawing showing the binding of IgG to hFcRn (left) and FcyRIIIa (right).
  • FIG. 7 shows an alignment of the heavy chain amino acid sequences for mAbs VRCOl (SEQ ID NO: 46), VRC02 (SEQ ID NO: 38) and VRC03 (SEQ ID NO: 42), and a consensus sequence (SEQ ID NO: 47).
  • Specific Fc mutations are disclosed herein that were made to VRCOl (see FIG. 1).
  • One skilled in the art will appreciate that equivalent mutations can be made to VRC02 and 03 at the equivalent positions (underlined).
  • FIG. 8 is a bar graph showing that the LS mutant antibodies were transcytosed across MDCK cells expressing hFcRn more than other mutant groups.
  • FIG. 9 is a graph showing that the hFcRn-binding enhancing mutant antibody, LS, had a half-life about 2 times longer than native VRCOl in the hFcRn Tg/Tg mouse model.
  • FIG. 10 is a graph showing that the hFcRn-binding enhancing mutant antibody, LS, had a half-life about 3 times longer than native VRCOl in the Rhesus macaques model.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NOS: 1 and 2 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 3 and 4 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 5 and 6 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a N434A (A) mutation in its Fc.
  • SEQ ID NOS: 7 and 8 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 9 and 10 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 11 and 12 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 13 and 14 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 15 and 16 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 17 and 18 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 19 and 20 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 21 and 22 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 23 and 24 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 25 and 26 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 27 and 28 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 29 and 30 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 31 and 32 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 33 and 34 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
  • SEQ ID NOS: 35 and 36 are the nucleic acid and corresponding amino acid sequence of the light chain of gp 120- specific antibody VRCOl.
  • SEQ ID NOS: 37 and 38 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRC02.
  • SEQ ID NOS: 39 and 40 are the nucleic acid and corresponding amino acid sequence of the light chain of gp 120- specific antibody VRC02. CDRs are shown in amino acids 27-30 (CDRl), 48-50 (CDR2), and 87-91(CDR3) of SEQ ID NO: 40.
  • SEQ ID NOS: 41 and 42 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRC03.
  • SEQ ID NOS: 43 and 44 are the nucleic acid and corresponding amino acid sequence of the light chain of gp 120- specific antibody VRC03. CDRs are shown in amino acids 24-33 (CDRl), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO: 44.
  • SEQ ID NOS: 45 and 46 are the nucleic acid and corresponding amino acid sequence of the native sequence of the heavy chain of gp 120- specific antibody VRCOl.
  • SEQ ID NO: 47 is a VRC heavy chain consensus sequence.
  • compositions such as the antibodies disclosed herein, into a subject by a chosen route, for example topically, orally, intravascularly such as intravenously, intramuscularly, intraperitoneally,
  • Administration can be local or systemic, such as intravenous or intramuscular.
  • the composition is administered by introducing the composition into a vein of the subject.
  • a disclosed antibody specific for an HIV protein is administered to a subject at an effective dose.
  • Amino acid substitution The replacement of one amino acid in peptide with a different amino acid.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non- human mammals, such as primates.
  • subject includes both human and veterinary subjects.
  • Antibody A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or antigen binding fragments thereof, which specifically binds and recognizes an analyte (antigen), such as gpl20 or an antigenic fragment of gpl20.
  • Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Exemplary neutralizing gpl20 rriAbs are provided herein that have mutations in the Fc region, which in some examples increases their half-life and/or antibody-dependent cellular cytotoxicity (ADCC) activity.
  • Antibodies exist, for example as intact immunoglobulins and as a number of well characterized fragments produced by digestion with various peptidases. For instance, Fabs, Fvs, and single-chain Fvs (scFvs) that specifically bind to gpl20 or fragments of gpl20 would be gp 120 -specific binding agents.
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies), heteroconjugate antibodies such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
  • Antibody fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab') 2 , the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab') 2 , a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
  • a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • H heavy chain
  • L light chain
  • ⁇ lambda
  • ⁇ kappa
  • IgM immunoglobulin heavy chain classes
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
  • the heavy and the light chain variable regions specifically bind the antigen.
  • Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs.” The extent of the framework region and CDRs have been defined (see, Kabat et ah, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a V L CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • Light chain CDRs are sometimes referred to as CDR LI, CDR L2, and CDR L3.
  • Heavy chain CDRs are sometimes referred to as CDR HI, CDR H2, and CDR H3.
  • immunoglobulin heavy chain including that of an antibody fragment, such as Fv, scFv, dsFv or Fab.
  • references to "V L " or "VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas.”
  • Monoclonal antibodies include humanized monoclonal antibodies. In some examples monoclonal antibodies are isolated from a subject. The amino acid sequences of such isolated monoclonal antibodies can be determined.
  • a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a "donor,” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they are substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
  • a humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (for example, see U.S. Patent No. 5,585,089).
  • Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.
  • "Epitope” or “antigenic determinant” refers to the region of an antigen to which B and/or T cells respond.
  • T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.
  • Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents .
  • An epitope can include at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.
  • antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell.
  • antigens include peptides derived from HIV, such as a gpl20 polypeptide or antigenic fragment thereof, such as a gpl20 outer domain or fragment thereof.
  • a “target epitope” is a specific epitope on an antigen that specifically binds an antibody of interest, such as a monoclonal antibody.
  • a target epitope includes the amino acid residues that contact the antibody of interest, such that the target epitope can be selected by the amino acid residues determined to be in contact with the antibody of interest.
  • Antigenic surface A surface of a molecule, for example a protein such as a gpl20 protein or portion thereof, capable of eliciting an immune response.
  • An antigenic surface includes the defining features of that surface, for example the three-dimensional shape and the surface charge.
  • An antigenic surface includes both surfaces that occur on gpl20 polypeptides as well as surfaces of compounds that mimic the surface of a gpl20 polypeptide (mimetics).
  • an antigenic surface includes all or part of the surface of gpl20 that binds to the CD4 receptor.
  • Binding affinity Affinity of an antibody or antigen binding fragment thereof for an antigen.
  • an antibody that binds preferentially to a particular target protein such as gpl20
  • affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay. In several examples, a high binding affinity is at least about
  • a high binding affinity is at least about 1.5 x 10 - " 8 , at least about 2.0 x 10 "8 , at least about 2.5 x 10 "8 , at least about 3.0 x 10 "8 , at least about 3.5 x 10 - “ 8 , at least about 4.0 x 10 - “ 8 , at least about 4.5 x 10 - " 8 , or at least about 5.0 x 10 "8 M.
  • CD4 Cluster of differentiation factor 4 polypeptide; a T-cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV on T-cells during HIV-I infection. CD4 binds to gpl20 from HIV.
  • the sequence of the CD4 precursor has a hydrophobic signal peptide, an extracellular region of approximately 370 amino acids, a highly hydrophobic stretch with significant identity to the membrane-spanning domain of the class II MHC beta chain, and a highly charged intracellular sequence of 40 resides (Maddon, Cell 42:93, 1985).
  • the term "CD4" includes polypeptide molecules that are derived from CD4 include fragments of CD4, generated either by chemical (for example enzymatic) digestion or genetic engineering means.
  • Such a fragment may be one or more entire CD4 protein domains.
  • the extracellular domain of CD4 consists of four contiguous immunoglobulin-like regions (Dl, D2, D3, and D4, see Sakihama et ah, Proc. Natl. Acad. Sci. 92:6444, 1995; U.S. Patent No. 6,117,655), and amino acids 1 to 183 have been shown to be involved in gpl20 binding.
  • a binding molecule or binding domain derived from CD4 would comprise a sufficient portion of the CD4 protein to mediate specific and functional interaction between the binding fragment and a native or viral binding site of CD4.
  • One such binding fragment includes both the Dl and D2 extracellular domains of CD4 (D1D2 is also a fragment of soluble CD4 or sCD4 which is comprised of Dl D2 D3 and D4), although smaller fragments may also provide specific and functional CD4-like binding.
  • the gpl20- binding site has been mapped to Dl of CD4.
  • Placement in direct physical association includes both in solid and liquid form, which can take place either in vivo or in vitro.
  • Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody.
  • Contacting can also include contacting a cell, for example by placing an antibody in direct physical association with a cell.
  • Framework Region Amino acid sequences interposed between CDRs. Includes variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.
  • Fc region The protein sequence that includes the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM.
  • An Fc region may also include part or all of the flexible hinge N-terminal to these domains.
  • an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain.
  • the Fc region comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the lower part of the hinge between Cgammal (Cyl) and C 2.
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • the Fc region comprises
  • gpl20 An envelope protein from Human Immunodeficiency Virus (HIV). This envelope protein is initially synthesized as a longer precursor protein of 845- 870 amino acids in size, designated gpl60. gpl60 is cleaved by a cellular protease into gpl20 and gp41. gpl20 contains most of the external, surface-exposed, domains of the HIV envelope glycoprotein complex, and it is gpl20 which binds both to cellular CD4 receptors and to cellular chemokine receptors (such as CCR5).
  • HIV Human Immunodeficiency Virus
  • the mature gpl20 wild-type proteins have about 500 amino acids in the primary sequence. gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD.
  • the protein is comprised of five conserved regions (C1-C5) and five regions of high variability (V1-V5).
  • Exemplary sequence of wt gpl20 proteins are shown on GENBANK®, for example accession numbers AAB05604 and AAD12142 (as available on February 4, 2011), incorporated by reference herein. It is understood that there are numerous variation in the sequence of gpl20 from what is given in GENBANK®, for example accession numbers AAB05604 and AAD12142, and that these variants are skill recognized in the art as gpl20.
  • the numbering used in gpl20 polypeptides disclosed herein is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al, Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM which is incorporated by reference herein in its entirety.
  • Host cells Cells in which a vector can be propagated and its DNA expressed, for example a disclosed antibody can be expressed in a host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.
  • Immunoadhesin A molecular fusion of a protein with the Fc region of an immunoglobulin, wherein the immunogloblin retains specific properties, such as Fc receptor binding and increased half-life.
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein, polypeptide, peptide, or small molecule.
  • immunoadhesin includes the hinge, CH 2 , and CH 3 domains of the immunoglobulin gamma 1 heavy chain constant region.
  • the immunoadhesin includes the CH 2 , and CH 3 domains of an IgG.
  • Immunologically reactive conditions Includes reference to conditions which allow an antibody raised against a particular epitope (such as an HIV gpl20 epitope) to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes.
  • a particular epitope such as an HIV gpl20 epitope
  • Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions.
  • the immunologically reactive conditions employed in the methods are "physiological conditions" which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.
  • IgA A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype includes IgAi and IgA 2 .
  • IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA.
  • the constant chain of wild- type IgA contains an 18-amino-acid extension at its C- terminus called the tail piece (tp).
  • Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.
  • IgG A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG 1; IgG 2 , IgG 3 , and IgG > in mice IgG 1; IgG 2a , IgG 2b , IgG .
  • Inhibiting or treating a disease Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as acquired immunodeficiency syndrome (AIDS).
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
  • a "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • Isolated An "isolated" biological component (such as a cell, for example a
  • B cell a nucleic acid, protein or antibody
  • B cell has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as, other
  • nucleic acids and proteins which have been "isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • an antibody such as an antibody specific for gpl20 can be isolated, for example isolated from a subject infected with HIV or from a cell that expresses the antibody.
  • Ka The dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction.
  • a polypeptide ligand interaction such as a polypeptide ligand interaction or an antibody antigen interaction.
  • an antibody such as VRCOl, VRC02, or VRC03 or a mutant thereof
  • an antigen such as gpl20
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • molecule such as an antibody or a protein
  • labels include fluorescent tags, enzymatic linkages, quantum dots, and radioactive isotopes.
  • a disclosed antibody is labeled.
  • Neutralizing antibody An antibody which reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent.
  • the infectious agent is a virus.
  • an antibody that is specific for gpl20 neutralizes the infectious titer of HIV.
  • HIV-l resists neutralization by most antibodies.
  • the disclosed hmAbs having one or more Fc mutations successfully neutralize at least 50% of current circulating HIV-l isolates, such as at least 75%, at least 80%, or at least 90% of current circulating HIV-l isolates.
  • compositions of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes
  • compositions and formulations suitable for pharmaceutical delivery of the antibodies herein disclosed are provided.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions ⁇ e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the protein (such as an antibody) is more enriched than the protein is in its natural environment within a cell.
  • a preparation is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • a recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by methods known in the art, such as chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Cells that express such molecules are referred to as recombinant or transgenic cells.
  • Sequence identity The similarity between amino acid or nucleic acid sequences are expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Variants of the disclosed mAbs that specifically bind gpl20 and have one or more mutations in the Fc region are encompassed by this disclosure typically characterized by possession of at least about 75%, for example at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid or nucleic acid sequence of interest, such as any of SEQ ID NOS: 1-47.
  • Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • Therapeutically effective amount A quantity of a specific substance, such as a disclosed mAb (or combinations of mAbs), sufficient to achieve a desired effect in a subject administered the mAb. For instance, this can be the amount necessary to inhibit HIV replication or treat AIDS. In several embodiments, a therapeutically effective amount is the amount necessary to reduce a sign or symptom of AIDS, and/or to decrease viral titer in a subject (such as a decrease of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, or at least 95% as compared to an absence of the mAb). When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect.
  • T Cell A white blood cell critical to the immune response.
  • T cells include, but are not limited to, CD4 + T cells and CD8 + T cells.
  • a CD4 + T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • CD8 + T cells carry the "cluster of differentiation 8" (CD8) marker.
  • a CD8 T cells is a cytotoxic T lymphocytes.
  • a CD8 cell is a suppressor T cell.
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • a virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.
  • RNA viruses wherein the viral genome is RNA.
  • the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells.
  • the integrated DNA intermediate is referred to as a pro virus.
  • the term "lentivirus” is used in its conventional sense to describe a genus of viruses containing reverse transcriptase.
  • the lentiviruses include the "immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), and feline
  • FMV immunodeficiency virus
  • HIV-I is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS).
  • HIV disease refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, for example, as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T cells.
  • Isolated human monoclonal antibodies that specifically bind gpl20 are disclosed herein, which include one or more mutations in their Fc region.
  • mutations include one or more of the following: T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E;
  • hmAbs that specifically bind gpl20 and have a S239D/I332E or S239D/A330L/I332E substitution in combination with one or more of T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; or M252Y/S254T/T256E, have greater FcRn binding, and/or half-life, than any of these mutations alone.
  • mAbs that specifically bind gpl20 and have a M428L/N434S substitution have a greater half-life and increased transcytosis activity, as compared to the native VRCOl antibody, and that the combination M428L/N434S and S239D/I332E substitutions provide a mutant antibody with better transcytosis and a similar half-life relative to the native VRCOl antibody.
  • hFcRn-binding enhancing mutants (hFcRn mutant) have lower FcgRIIIa-binding affinity and ADCC activity
  • FcgRIIIa-binding mutants have lower hFcRn-binding affinity than wild type.
  • an FcgR mutation When an FcgR mutation is introduced into an hFcRn mutnat, the binding affinity of the combined mutant to FcgRIIIa and ADCC activity becomes similar to wild type and vice versa, n some examples, combining an hFcRn mutant with an FcgR-binding enhancing mutation increases ADCC activity relative to a wildtype antibody (e.g., VRCOl).
  • a wildtype antibody e.g., VRCOl
  • Such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased FcRn binding relative to the native hmAb (such as VRCOl), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
  • such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased FcyRIIIa binding relative to the native hmAb (such as VRCOl), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
  • native hmAb such as VRCOl
  • such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased antibody-dependent cellular cytotoxicity (ADCC) relative to the native hmAb (such as VRCOl), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
  • ADCC antibody-dependent cellular cytotoxicity
  • such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased half-life relative to the native hmAb (such as VRCOl), such as an increase of at least 1.5-fold, at least 1.8 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold.
  • the native hmAb such as VRCOl
  • any IgG antibody can be generated that has equivalent mutations in its Fc region.
  • isolated IgG antibodies wherein a heavy chain of the IgG includes one or more amino acid substitutions in the Fc region, wherein the one or more amino acid substitutions comprise: T250Q/M428L; M428L/N434S; N434A;
  • T307A/E380A/N434A M252Y/S254T/T256E; S239D/I332E;
  • M428L/N434S S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
  • an isolated IgG antibody can include a S239D/I332E or
  • S239D/A330L/I332E substitution can further include one or more of T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; and M252Y/S254T/T256E substitutions.
  • such variant IgG molecules have increased FcRn binding relative to the native IgG molecule (such as IgGl, IgG2, IgG3, or IgG4), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
  • compositions including these variant IgGs or hmAbs and a pharmaceutically acceptable carrier.
  • Nucleic acids encoding these antibodies, expression vectors comprising these nucleic acids, and isolated host cells that express the nucleic acids are also provided.
  • compositions comprising the disclosed hmAbs specific for gpl20 and having one or more Fc mutations can be used for research, diagnostic and
  • the hmAbs disclosed herein can be used to diagnose or treat a subject having an HIV-1 infection and/or AIDS.
  • the hmAbs can be used to determine HIV-1 titer in a subject.
  • the hmAbs disclosed herein also can be used to study the biology of HIV.
  • an isolated hmAb specifically binds gpl20, and includes one or more (such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10) of the following mutations (relative to the native Ab Fc sequence): T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E;
  • M428L/N434S S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
  • the isolated hmAbs specifically bind gpl20 (and may neutralize HIV), and include a heavy chain having one or more of the mutations listed above (relative to the native Ab Fc sequence) and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • the heavy chain of the hmAb has at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least 80%, at least
  • the antibody can include a heavy chain that comprises or consists of any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • the light chain of the hmAb includes amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO: 36, for example, a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 36, or a sequence comprising or consisting of SEQ ID NO: 36.
  • the light chain of the hmAb includes amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91(CDR3) of SEQ ID NO: 40, for example, a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40, or a sequence comprising or consisting of SEQ ID NO: 40.
  • the light chain of the hmAb includes amino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO: 44, for example, a sequence having at least 80%, at least
  • the isolated hmAb has a heavy chain with a sequence that includes or consists of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and the light chain of the antibody includes or consists of or consists of SEQ ID NO: 36.
  • the isolated human monoclonal antibody of has a heavy chain comprising SEQ ID NO: 4 or 18.
  • substitutions include a variant of a native heavy chain of the VRCOl hmAb (SEQ ID NO: 46) having one or more mutations in the Fc disclosed herein.
  • FIG. 1 shows several amino acid substitutions made to a native VRCOl sequence.
  • the equivalent mutation can be made to other antibodies, such as VRC02 and VRC03.
  • an amino acid alignment of the heavy chain is provided for VRCOl, VRC02 and VRC03.
  • the amino acid in the Fc region mutated in VRCOl is underlined, as is the equivalent amino acid in VRC02 and VRC03. Based on this alignment and the mutations provided herein, equivalent mutations are provided for VRC02 and VRC03.
  • hmAbs wherein a heavy chain of the antibody includes a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 38 or 42, wherein the heavy chain of the antibody has one or more of the following amino acid substitutions: T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E; S239D/A330L/I332E; S239D/I332E + T250Q/M428L; S239D/I332E + M428L/N434S; S239D/I332E + N434A;
  • the light chain of such hmAbs include a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40 or 44.
  • the disclosed hmAbs can include a heavy chain that includes SEQ ID NO: 38 with one or more of the amino acid substitutions listed above and a light chain that includes SEQ ID NO: 40.
  • a disclosed hmAb has a heavy chain comprising SEQ ID NO: 42 with one or more of the amino acid substitutions listed above and a light chain that includes SEQ ID NO: 44.
  • the isolated human monoclonal antibody is isolated human monoclonal antibody
  • these antibodies retain the binding affinity of the parental antibody (VRCOl or VRC02 or VRC03) for the antigenic epitope.
  • these antibodies have a KD of ⁇ 3 nM for the antigenic epitope of gpl20.
  • Fully human monoclonal antibodies include human framework regions.
  • any of the antibodies that specifically bind gpl20 and have a mutated Fc region provided herein can include the human framework region and can include the framework regions of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or SEQ ID NO: 38 or 42 with one or more of the mutations provided herein).
  • the framework regions can be from another source. Additional examples of framework sequences that can be used include the amino acid framework sequences of the heavy and light chains disclosed in PCT Publication No. WO 2006/074071 (see, for example, SEQ ID NOs: 1-16), herein incorporated by reference.
  • the monoclonal antibody can be of any isotype.
  • the monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgGiOr an IgG 2 .
  • the class of an antibody that specifically binds gpl20 can be switched with another.
  • a nucleic acid molecule encoding V L or V H is isolated using methods well- known in the art, such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively.
  • the nucleic acid molecule encoding V L or V H can be operatively linked to a nucleic acid sequence encoding a C L or C H from a different class of immunoglobulin molecule.
  • a hmAbs disclosed herein that was originally IgM may be class switched to an IgG. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG 2 .
  • the disclosed hmAbs are oligomers of antibodies, such as dimers trimers, tetramers, pentamers, hexamers, septamers, octomers and so on. In some examples, the hmAbs are pentamers.
  • the CDRs of the light chain are bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded by the residues at positions 31 and 35b (H- CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using the numbering convention delineated by Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5 Edition, U.S. Department of Health and Human
  • Antibody fragments of the disclosed neutralizing hmAbs that specifically bind gpl20 and have one or more mutations in the Fc region are encompassed by the present disclosure, such as Fab, F(ab') 2 , and Fv which include a heavy chain and light chain variable region . These antibody fragments retain the ability to selectively bind with the antigen. These fragments include:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • Fv a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • a dimer of a single chain antibody (scFV 2 ), defined as a dimer of a scFV. This has also been termed a "miniantibody.”
  • the antibodies are Fv antibodies, which are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain.
  • the V H and the V L can be expressed from two individual nucleic acid constructs in a host cell.
  • the V H amino acid sequence includes the CDRs from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or SEQ ID NO: 38 or 42 with one or more of the mutations provided herein).
  • the V L amino acid sequence includes the CDRs from SEQ ID NO: 36, 40 or 44.
  • the chains of the Fv antibody are typically held together by noncovalent interactions.
  • these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker.
  • the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • conservative variants of the disclosed antibodies can be produced.
  • Such conservative variants employed in antibody fragments such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V H and the V L regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules.
  • Amino acid substitutions (such as at most one, at most two, at most three, at most four, at most five, or at most 10 amino acid substitutions, such as 1 to 10 or 1 to 5 conservative substitutions) can be made in the V H and the V L regions to increase yield.
  • the V H sequence is from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or SEQ ID NO: 38 or 42 with one or more of the mutations provided herein).
  • the V L sequence is from one of SEQ ID NOs: 36, 40 or 44.
  • the antibodies disclosed herein can be isolated using cloaked antigens, as described in PCT Publication No. WO 2009/100376. Briefly, antigens are cloaked to target antigenicity of the antigen to a specific epitope that specifically bound by the antibody of interest, such as a neutralizing antibody.
  • the amino acid substitutions result in the antigen not being bound by antibodies in a polyclonal serum that specifically bind surface exposed amino acid residues of the wild- type antigen located exterior of the target epitope.
  • the amino acid substitutions alter antigenicity of the antigen in vivo as compared to the wild-type antigen (unsubstituted antigen) but do not introduce additional glycosylation sites as compared to the wild-type antigen.
  • that antigen is glycosylated.
  • the cloaked antigen is modified to substitute one or more residues recognized by the antibody of interest to abolish antigen recognition.
  • a biotinylation peptide can be fused to the cloaked antigen. Biotinylated cloaked antigen can then be used to stain and thus identify cells, such as PBMC, expressing an antibody of interest.
  • the hmAbs or antibody fragments disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein).
  • the antibody or portion thereof is derivatized such that the binding to gpl20 is not affected adversely by the derivatization or labeling.
  • the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m- maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).
  • Such linkers are available from Pierce Chemical Company (Rockford, IL).
  • An antibody that specifically binds gpl20 and has an Fc mutation (or a functional fragment thereof) can be labeled with a detectable moiety.
  • Useful detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, Green fluorescent protein, Yellow fluorescent protein.
  • An antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • enzymes that are useful for detection
  • an antibody When an antibody is labeled with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable.
  • An antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label. In one example, an antibody or fragment thereof disclosed herein can be labeled with a quantum dot.
  • An antibody or fragment thereof disclosed herein can be labeled with a magnetic agent, such as gadolinium.
  • Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese.
  • Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels.
  • An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • an antibody or fragment thereof disclosed herein can also be labeled with a radiolabeled amino acid.
  • the radiolabel may be used for both diagnostic and therapeutic purposes.
  • Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleo tides: 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, U1 ln, 125 I, 131 I.
  • An antibody or fragment thereof disclosed herein can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.
  • PEG polyethylene glycol
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • Nucleic acid molecules encoding the peptides provided herein can readily be produced by one of skill in the art.
  • these nucleic acids can be produced using the amino acid sequences provided herein (such as the CDR sequences, heavy chain and light chain
  • sequences are sequences, sequences available in the art (such as framework sequences), and the genetic code.
  • nucleic acid molecules that encoding any of the neutralizing hmAbs disclosed herein (or a functional fragment thereof).
  • a nucleic acid molecule comprises a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41.
  • the nucleic acid molecule comprises or consists of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41.
  • the isolated nucleic acid molecule encodes a heavy chain and a light chain of an antibody, wherein (a) the heavy chain of the antibody comprises a sequence having at least 80%, at least 90%, at least 95% or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and having one or more of the following
  • T307A/E380A/N434A M252Y/S254T/T256E; S239D/I332E;
  • the light chain of the antibody comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 36;
  • the heavy chain of the antibody comprises the heavy chain of the antibody comprises any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and wherein the light chain of the antibody comprises SEQ ID NO: 36;
  • the heavy chain of the antibody comprises SEQ ID NO: 36 or 42 having one or more of the following substitutions/mutations:
  • the light chain of the antibody comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 40 or 44; (d) the heavy chain of the antibody comprises SEQ ID NO: 36 having one or more of the following substitutions/mutations: T250Q/M428L; M
  • T307A/E380A/N434A M252Y/S254T/T256E; S239D/I332E;
  • the light chain of the antibody comprises SEQ ID NO: 40; (e) the heavy chain of the antibody comprises SEQ ID NO: 42 having one or more of the following substitutions/mutations: T250Q/M428L;
  • Such isolated nucleic acid molecules can be operably linked to a promoter.
  • the disclosed isolated nucleic acid molecules can be part of an expression vector, such as a plasmid.
  • the expression vector includes a promoter and an enhancer, such as a cytomegalovirus promoter or cytomegalovirus enhancer.
  • the expression vector can include RNA splicing donor sites (such as HTLV-1 or CMV RNA splicing donor sites), RNA splicing acceptor sites (such as HTLV-1 or CMV RNA splicing acceptor sites) and/or internal ribosomal binding sequences.
  • RNA splicing donor sites such as HTLV-1 or CMV RNA splicing donor sites
  • RNA splicing acceptor sites such as HTLV-1 or CMV RNA splicing acceptor sites
  • internal ribosomal binding sequences can be used to express the heavy chain of the antibody and the light chain of the antibody as a fusion polypeptide following the introduction of the expression vector in a host cell.
  • the expression vector can include a nucleic acid sequence encoding a furin cleavage site between the heavy chain and the light chain of the antibody.
  • the expression vector also encodes a selectable marker. Isolated host cells transformed with the disclosed nucleic acid molecules or vectors are also provided.
  • nucleic acid sequences encoding the neutralizing hmAbs that specifically bind gpl20 and have one or more Fc mutations can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et ah, Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et ah, Meth. Enzymol.
  • Exemplary nucleic acids can be prepared by routine cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et ah, supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN),
  • Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include but are not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and the self- sustained sequence replication system (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self- sustained sequence replication system
  • nucleic acids encoding any of the antibodies, V H and/or V L , disclosed herein (or fragment thereof) can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. These antibodies can be expressed as individual V H and/or V L chain, or can be expressed as a fusion protein. An immunoadhesin can also be expressed . Thus, nucleic acids encoding a V H and V L , and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.
  • the V H - and V L -encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H domains joined by the flexible linker (see, e.g., Bird et al., Science 242:423-426, 1988;
  • cleavage site can be included in a linker, such as a furin cleavage site.
  • the nucleic acid encoding the V H and/or the V L optionally can encode an Fc domain (immunoadhesin).
  • the Fc domain can be an IgA, IgM or IgG Fc domain.
  • the Fc domain can be an optimized Fc domain, as described in U.S. Published Patent Application No. 20100/093979.
  • the immunoadhesin is an IgG! Fc.
  • the single chain antibody may be monovalent, if only a single V H and V L are used, bivalent, if two V H and V L are used, or polyvalent, if more than two V H and V L are used. Bispecific or polyvalent antibodies may be generated that bind specifically to gpl20 and to another molecule, such as gp41.
  • the encoded V H and V L optionally can include a furin cleavage site between the V H and V L domains.
  • the host cell can be a gram positive bacteria including, but are not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,
  • Lactobacillus Lactococcus, Clostridium, Geobacillus, and Oceanobacillus.
  • Gram negative bacteria include, but not limited to, E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.
  • One or more DNA sequences encoding the antibody or fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the mutant mAbs provided herein are also encompassed by this disclosure.
  • nucleic acids encoding the isolated proteins described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette.
  • the promoter can be any promoter, including a cytomegalovirus promoter and a human T cell lymphotrophic virus promoter (HTLV)-l.
  • an enhancer such as a cytomegalovirus enhancer, is included in the construct.
  • the cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein.
  • the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • the vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
  • expression cassettes can include a strong promoter to direct transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a
  • transcription/translation terminator For E. coli, this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal.
  • the control sequences can include a promoter and/or an enhancer derived from, for example, an
  • the cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
  • Eukaryotic cells can also be cotransformed with
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps. In addition to recombinant methods, the
  • immunoconjugates, effector moieties, and antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis.
  • the recombinant immunoconjugates, antibodies, and/or effector molecules can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column
  • the antibodies, immunoconjugates and effector molecules need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.
  • a reducing agent is present to separate disulfide bonds.
  • An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9:5015-21, 1970, and by Buchner et al., supra.
  • Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer.
  • An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
  • the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution.
  • An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5-fold molar excess of one protein over the other is not exceeded.
  • Excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution after the redox-shuffling is completed.
  • the antibodies, labeled antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis.
  • Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence.
  • the methods include prevention or inhibition of infection with HIV-1.
  • the methods include contacting a cell with an effective amount of one or more of the hmAbs disclosed herein that specifically bind gpl20 and have one or more Fc mutations, or a functional fragment thereof.
  • the method can also include administering to a subject a therapeutically effective amount of the hmAbs to a subject.
  • the method includes selecting a subject infected with HIV, such as HIV-1, or who is at risk for such infection (such as medical personnel).
  • the subject is an HIV-positive pregnant woman, or child recently born to such a woman. Studies have shown that the rate of HIV
  • hmAbs that can decrease HIV-transmission from mother to infant.
  • a therapeutically effective amount one or more of the disclosed hmAbs specific for gpl20- is administered in order to prevent transmission of HIV, or decrease the risk of transmission of HIV, from a mother to an infant.
  • a therapeutically effective amount of the antibody is administered to mother and/or to the child at childbirth. In other examples, a therapeutically effective amount of the antibody is administered to the mother and/or infant prior to breast feeding in order to prevent viral transmission to the infant or decrease the risk of viral transmission to the infant. In some
  • both a therapeutically effective amount of the antibody and a therapeutically effective amount of another agent, such as zidovudine, is a therapeutically effective amount of the antibody and a therapeutically effective amount of another agent, such as zidovudine, is
  • Methods to assay for neutralization activity include, but are not limited to, a single-cycle infection assay as described in Martin et al. (2003) Nature Biotechnology 21:71-76.
  • the level of viral activity is measured via a selectable marker whose activity is reflective of the amount of viable virus in the sample, and the IC 50 is determined.
  • acute infection can be monitored in the PM1 cell line or in primary cells (normal PBMC).
  • the level of viral activity can be monitored by determining the p24 concentrations using ELISA. See, for example, Martin et al. (2003) Nature
  • the cell is also contacted with an effective amount of an additional agent, such as anti-viral agent.
  • an additional agent such as anti-viral agent.
  • the cell can be in vivo or in vitro.
  • the disclosed mAbs can be combined with anti-retroviral therapy.
  • Antiretro viral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits.
  • the disclosed antibodies can be administered in conjunction with nucleoside and nucleotide reverse transcriptase inhibitors (nRTI), non- nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors, entry inhibitors (or fusion inhibitors), maturation inhibitors, or a broad spectrum inhibitors, such as natural antivirals.
  • Exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine and efavirenz.
  • the mAbs and the anti-retroviral therapy is administered simultaneously or contemporaneously. In other examples the therapies are administered separately.
  • compositions are provided that include one or more of the hmAbs or functional fragments thereof disclosed herein in a carrier.
  • the compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes.
  • the antibody can be formulated for systemic or local
  • the hmAbs or functional fragments thereof disclosed herein is formulated for parenteral administration, such as intravenous
  • compositions for administration can include a solution of the hmAbs or functional fragments thereof in a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • a pharmaceutically acceptable carrier such as an aqueous carrier.
  • aqueous carriers can be used, for example, 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, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the concentration of antibody 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 subject's needs.
  • Controlled-release parenteral formulations containing the therapeutic mAb can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic mAb as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 ⁇ are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ so that only nanoparticles are administered intravenously. Microparticles are typically around 100 ⁇ in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
  • Polymers can be used for ion-controlled release of the mAb compositions disclosed herein.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et ah, Pharm. Res. 9:425-434, 1992; and Pec et al, J. Parent. Sci. Tech. 44(2):58-65, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)).
  • Numerous additional systems for controlled delivery of therapeutic proteins, such as mAbs, are known (see U.S.
  • a typical pharmaceutical composition for intravenous administration includes at least 1 mg/kg (such as at least 5 mg/kg, at least 10 mg/kg, at least 20 mg/kg or at least 40 mg/kg, such as 1 to 100 mg/kg, 1 to 50 mg/kg, 5 to 50 mg/kg, or 10-40 mg/kg) administered every two to four weeks.
  • the dosage for a small rodent is about 50 to 500 ⁇ g mAb, such as 200 ⁇ g mAb (e.g., 10 mg/kg).
  • the dosage for a large mammal, such as a human or non-human primate is about 1 to 100 mg mAb /kg subject, such as 1 to 50, 10 to 24 or 20 mg/kg.
  • Dosages from 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ.
  • Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's
  • Antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 75 mg/kg of body weight (such as 5 to 50 or 10 to 40 mg/kg).
  • Antibodies can be administered by slow infusion, rather than in an
  • a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
  • an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
  • the Abs are administered at least once a week, at least once a month, at least twice a month, or at least once every two months, or In one example, at least 1 mg mAb/kg (such as at least 5 mg/kg, at least 10 mg/kg, at least 20 mg/kg or at least 40 mg/kg, such as 1 to 100 mg/kg, 1 to 50 mg/kg, 5 to 50 mg/kg, or 10-40 mg/kg) is administered to the patient every two to four weeks. In some examples, the mAbs are administered iv, subcutaneously or im.
  • a therapeutically effective amount of a mAb disclosed herein will depend upon the severity of the disease and/or infection and the general state of the patient's health.
  • a therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • These compositions can be administered in conjunction with another therapeutic agent, either simultaneously or sequentially.
  • administration of the antibody results in a reduction in the establishment of HIV infection and/or reducing subsequent HIV disease progression in a subject.
  • a reduction in the establishment of HIV infection and/or a reduction in subsequent HIV disease progression encompass any statistically significant reduction in HIV activity.
  • methods are disclosed for treating a subject with an HIV-1 infection. These methods include administering to the subject a therapeutically effective amount of an antibody, or a nucleic acid encoding the antibody, thereby preventing or treating the HIV-1 infection.
  • a composition can decrease HIV infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV infected cells), as compared to HIV infection in the absence of the composition.
  • HIV replication can be reduced or inhibited by use of the disclosed mAbs. HIV replication does not need to be completely eliminated for the composition to be effective.
  • a composition can decrease HIV replication by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV), as compared to HIV replication in the absence of the composition.
  • Single or multiple administrations of the compositions including the mAbs disclosed herein are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient.
  • the dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • a dose of the antibody is infused for thirty minutes every other day.
  • about one to about ten doses can be administered, such as three or six doses can be administered every other day.
  • a continuous infusion is administered for about five to about ten days.
  • the subject can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
  • gpl20 is detected in a biological sample, and can be used to detect HIV-1 infection.
  • the sample can be any sample, including, but not limited to, tissue from biopsies or check swabs, autopsies and pathology specimens.
  • Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes.
  • Biological samples further include body fluids, such as blood, serum, plasma, semen, sputum, breast milk, spinal fluid or urine.
  • a method for detecting AIDS and/or an HIV- 1 infection in a subject provides a method for detecting HIV-1 in a biological sample, wherein the method includes contacting a biological sample with a hmAb or functional fragment thereof disclosed herein under conditions conducive to the formation of an immune complex, and detecting the immune complex to detect the gpl20 in the biological sample.
  • the detection of gpl20 in the sample indicates that the subject has an HIV infection.
  • the detection of gpl20 in the sample indicates that the subject has AIDS.
  • detection of gpl20 in the sample confirms a diagnosis of AIDS and/or an HIV-1 infection in a subject.
  • the method includes comparing the gpl20-hmAb complex detected to an amount of such complex detected in a control, such as a sample known to not contain HIV or gpl20, or known to be infected with HIV or contain gpl20. In some examples the method includes comparing an absolute or relative amount of the gpl20-hmAb complex detected to a reference value or range of values expected of such complex expected if the sample does not contain gpl20, or expected if the sample does contain gpl20.
  • the disclosed antibodies are used to test vaccines, for example to determine if a vaccine composition assumes the same conformation as a native gpl20 peptide.
  • a method for detecting testing a vaccine wherein the method includes contacting a sample containing the test vaccine, such as a gpl20 immunogen, with an antibody disclosed herein under conditions conducive to the formation of an immune complex, and detecting the immune complex, to detect the vaccine in the sample.
  • the detection of the immune complex in the sample indicates that vaccine component, such as such as a gpl20 immunogen assumes a conformation capable of binding the antibody.
  • the anti-gpl20 antibody is directly labeled with a detectable label.
  • the anti-gpl20 antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the anti-gpl20 antibody is utilized.
  • a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody.
  • the first antibody is a human IgG
  • the secondary antibody may be an anti-human-lgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
  • Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, quantum dots, magnetic agents and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • a non-limiting exemplary exemplary enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include
  • luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I, 131 I, 35 S or 3 H.
  • Kits for detecting a polypeptide will typically include one or more hmAbs or functional fragments thereof disclosed herein.
  • an antibody fragment such as an Fv fragment or a Fab is included in the kit.
  • the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).
  • the disclosure provides a kit that includes one more of the mAbs provided herein, for example in separate containers.
  • the kit includes an antibody having a heavy chain comprising the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and can further include a light chain comprising the sequence of SEQ ID NO: 36.
  • Such antibodies may include a label.
  • such a kit further includes a labeled secondary antibody in a separate container, which permits detection of the primary antibody.
  • such a kit further includes additional therapeutic agents, such as one or more antiretro viral compounds, in separate containers.
  • kits includes instructional materials disclosing means of use.
  • the instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • the diagnostic kit comprises an immunoassay.
  • the method of detecting gpl20 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to gpl20.
  • the antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
  • This example describes the mutation of the Fc region of VRCOl, a neutralizing human monoclonal antibody that specifically binds to gpl20.
  • VRC02 SEQ ID NOS: 37-38
  • VRC03 SEQ ID NOS: 41-42
  • the neutralization blocking effect of the proteins was calculated as the percent reduction in the IC50 value of the antibody in the presence of protein compared to PBS.
  • Synergistic or additive neutralization was assessed by mixing a fixed concentration (10 ⁇ g/ml) of the test antibody with serial dilutions of sCD4, CD4-Ig or VRCOl for 15 min prior to the addition of virus. The baseline of viral entry at each concentration of sCD4, CD4-Ig or VRCOl was used to calculate the adjusted percent neutralization.
  • Neutralization was also assessed using Env-pseudoviruses generated by 293T transfection using the pNL4-3 ⁇ HIV-1 backbone containing a lucif erase reporter gene to infect activated PBMC.
  • PBMC-derived HIV- 1 primary isolates were performed by single-round infection of either TZM-bl cells using luciferase as readout, or activated PBMC using flow cytometry staining for HIV- 1 p24 antigen.
  • CD4-facilitated virus entry was performed in the CCR5+/CD4- cell line Cf2Th/syn CCR5 with Env-pseudoviruses 5 containing the luciferase pNL4-3 ⁇ HIV- 1 backbone.
  • a mixture of 40 ⁇ of viral stock and 10 ⁇ of serial dilutions of sCD4, CD4-Ig or VRCOl was incubated at 37°C for 30 min before adding 1 x 10 4 Cf2Th/syn CCR5 cells. Virus entry was measured 2 days later by luciferase activity in cell lysates.
  • the VRCOl Fc mutants have similar neutralization 10 activity as the wild- type VRCOl antibody.
  • Table 2 provides a comparison of the
  • IC 50 between wild type VRCOl and the mutant VRCOl antibodies Table 3 provides a comparison of ICso between wild type and the mutants.
  • Tables 4 and 5 show the fold increase/decrease of IC 50 and ICso of the Fc mutants comparing with wild type.
  • hFcRn human MHC class I-related Fcy-receptor
  • Anti-human IgG-HRP diluted with 2.5%-BSA in PBS, pH6.0 or PBS, pH7.4 were incubated for one hour and washed with PBS, pH6.0 + 0.05 % Tween20 or PBS, pH7.4 + 0.05 % Tween20.
  • HRP substrate TMB was added and the color development was stopped by 0. IN H2S04.
  • Optical density at 450 nm was measured by ELISA reader.
  • Antibody binding to FcyRIIIa provides an indication of ADCC activity.
  • RSC3 is the resurfaced stabilized core from gpl20, and is the preserved antigenic structure of the neutralizing surface of the CD4 binding site but does not include other antigenic regions of HIV-1
  • Antibody binding to RSC3 provides an indication of the ability of the antibody to specifically bind gpl20.
  • Nickel-coated plates were incubated with His tagged recombinant FcyRIIIa for one hour, and washed with PBS, pH7.4 + 0.05 % Tween20 (washing buffer) (left).
  • ELISA plates were coated with RSC3 for overnight, blocked with 5 % BSA in PBS, pH7.4, and washed with washing buffer (right).
  • VRCOl and its Fc mutants diluted with 2.5%-BSA in PBS, pH7.4 were incubated for two hours and washed.
  • Anti-human IgG-HRP in PBS, pH7.4 were incubated for one hour and washed.
  • HRP substrate TMB was added and the color development was stopped by 0.1N H2S04. Optical density at 450 nm was measured by ELISA reader.
  • This example describes the methods used to measure the antibody-dependent cellular cytotoxicity (ADCC) of the VRCOl antibody mutants as compared to the native VRCOl antibody.
  • ADCC antibody-dependent cellular cytotoxicity
  • HIV-infected CD4+CEM-NKr cells were double- stained with PKH26 and CFSE, and suspended in RPMI with 10%-FBS (Target cells).
  • Human PBMC was prepared from human buffy coat by Ficoll gradient centrifugation, and suspended in RPMI with 10%-FBS (Effector cells).
  • Target cells 10,000 cells/well
  • Effector cells 500,000 cells/well
  • Percent killing was calculated from % CFSE negative within PKH26hi population.
  • ADCC was lower for the hFcRn-binding enhancing mutations, while the combined LS, A and AAA mutants had stronger ADCC activities.
  • the combined LS and A mutants showed higher ADCC activities and higher binding to FcyRIIIa. That is, DE or DLE mutants combined with LS or A mutations have higher affinity to FcyRIIIa and functional ADCC activities. As shown in FIG. 2, LS and A mutants have mutations only at CH3, whereas DE and DLE mutations are made in CH2 region close to hinge.
  • FIG. 6 shows the binding of IgG to hFcRn and FcyRIIIa.
  • hFcRn binds to the CH2 and CH3 region of IgG, while FcyRIIIa bind to the hinge and CH2 regions of IgG Therefore, hFcRn-binding enhancing mutations did not affect (or affected less) the enhanced binding to FcyRIIIa by LS and A mutants. This may the reason why combined LS and A mutants showed higher ADCC activities than other mutants.
  • This example describes methods used to measure transcytosis of the antibody mutants as compared to the native antibody.
  • hFcRn is expressed in the epithelial cells in mucosa and mediates bidirectional transcytosis of human IgG across cellular epithelium.
  • hFcRn-binding enhancing mutants of VRCOl can increase transcytosis of the HIV neutralizing antibody, VRCOl
  • a monolayer of Madin Darby canine kidney (MDCK) cells expressing hFcRn was grown in transwell plates.
  • the native VRCOl antibody or the Fc mutants were added to the basolateral side of the cells at 1.0 mg/mL of and incubated for 2 hours. The concentrations of the antibodies were then measured on the apical side by ELISA coated with RSC3 to compare the levels of transcytosis.
  • the LS mutant groups (LS, DE-LS, and DLE-LS) showed a higher level of transcyotis than VRCOl or other mutant antibodies.
  • This example describes methods used to measure the half-life of the VRCOl antibody mutants as compared to the native VRCOl antibody.
  • each mAb (native VRCOl, DE, LS, or DE-LS) was injected into a hFcRn Tg/Tg mouse by i.v.
  • concentrations of the mAbs in the sera were examined by indirect ELISA with RSC3-coated plates over time, and their half-life was calculated.
  • the hFcRn-binding enhancing mutant antibody, LS had a half-life about 2 times longer than VRCOl. Table 6.
  • the disclosed mutant antibodies such as the LS antibodies, have a half-life that is greater than the native antibody (such as native VRCOl), such as an at least 1.5 fold, at least 1.8 fold, at least 2-fold, at least 2.5- fold, or at least 3-fold.
  • native antibody such as native VRCOl
  • This example describes methods used to measure the half-life and the clearance of the VRCOl antibody mutants as compared to the native VRCOl antibody.
  • the disclosed mutant antibodies such as the LS antibodies, have a half-life that is greater than the native antibody (such as native VRCOl), such as an at least 1.5 fold, at least 1.8 fold, at least 2-fold, at least 2.5- fold, or at least 3-fold.
  • native antibody such as native VRCOl
  • This example describes methods used to compare the protection immunity against SHIV challenge after the passive immunization with the mutant mAbs.
  • each mAb (native VRCOl, DE, LS, or DE-LS) was injected into rhesus macaques by i.v.
  • the passively immunized monkeys were challenged with repetitive, intrarectal SHIV BaL two days later. The number of challenges required to establish infection and plasma viral load over time was examined.
  • This example describes methods that can be used to measure ADCC activity of the antibody mutants as compared to the native antibody in vivo. It is expected that the ADCC activity of the mutant antibodies (e.g., mutant VRCOl antibodies) will be equal to or better than the native antibody (such as native VRCOl).
  • mutant antibodies e.g., mutant VRCOl antibodies
  • hFcRn transgenic SCID mouse and monkey infected with SIV can be administered the disclosed hmAbs, and ADCC activity measured (for example as in Dall'Acqua et al. (J. Biol. Chem. 281:23514-24, 2006, herein incorporated by reference). Mutant and native hmAb ADCC activity can be compared.
  • mice are administered 200 ⁇ g Ab or 2 mg/kg and monkeys receive 20 mg/kg via i.v. administration.
  • This example describes the use of the disclosed HIV-1 monoclonal neutralizing antibodies specific to gpl20 having one or more Fc mutations for the detection of HIV-1 in a subject. This example further describes the use of these antibodies to confirm the diagnosis of HIV-1 in a subject.
  • a biological sample such as a blood sample is obtained from the patient diagnosed with, or suspected of having an HIV-1 infection.
  • a blood sample taken from a patient who is not infected can be used as a negative control.
  • An ELISA is performed to detect the presence of HIV-1 in the blood sample.
  • Proteins present in the blood samples are immobilized on a solid support, such as a 96-well plate, according to methods well known in the art (see, for example, Robinson et ah, Lancet 362: 1612-1616, 2003).
  • a solid support such as a 96-well plate
  • one or more mutant mAbs disclosed herein that is directly labeled with a fluorescent marker or quantum dot is applied to the protein-immobilized plate.
  • the plate is washed in an appropriate buffer, such as PBS, to remove any unbound antibody and to minimize non-specific binding of antibody. Fluorescence can be detected using a fluorometric plate reader according to standard methods.
  • An increase in fluorescence intensity of the patient sample, relative to the negative control sample, indicates the anti-gpl20 antibody with an Fc mutation specifically bound proteins from the blood sample, thus detecting the presence of HIV- 1 protein in the sample. Detection of HIV-1 protein in the patient sample indicates the patient has HIV-1, or confirms diagnosis of HIV-1 in the subject.
  • This example describes a particular method that can be used to treat HIV in a human subject by administration of one or more of the disclosed Fc mutant gpl20 specific human neutralizing mAbs. Although particular methods, dosages, and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.
  • HIV-1 can be treated by administering a therapeutically effective amount of one or more of the neutralizing mAbs described herein, thereby reducing or eliminating HIV infection. Screening subjects
  • the subject is first screened to determine if they have an HIV infection.
  • methods that can be used to screen for HIV include a combination of measuring a subject' s CD4+ T cell count and the level of HIV in serum blood levels. Additional methods using the gp 120- specific mAbs described herein can also be used to screen for HIV (e.g., see Example 10).
  • HIV testing consists of initial screening with an enzyme- linked immunosorbent assay (ELISA) to detect antibodies to HIV, such as to HIV-1.
  • ELISA enzyme- linked immunosorbent assay
  • Specimens with a nonreactive result from the initial ELISA are considered HIV- negative unless new exposure to an infected partner or partner of unknown HIV status has occurred.
  • Specimens with a reactive ELISA result are retested in duplicate. If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (e.g., Western blot or an immunofluorescence assay (IFA)).
  • IFA immunofluorescence assay
  • Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection.
  • indeterminate Western blot result which may be either an incomplete antibody response to HIV in an infected person, or nonspecific reactions in an uninfected person.
  • IFA can be used to confirm infection in these ambiguous cases.
  • a second specimen will be collected more than a month later and retested for subjects with indeterminate Western blot results.
  • nucleic acid testing e.g. , viral RNA or proviral DNA amplification method
  • the detection of HIV in a subject's blood is indicative that the subject has
  • HIV and is a candidate for receiving the therapeutic compositions disclosed herein. Moreover, detection of a CD4+ T cell count below 350 per microliter, such as 200 cells per microliter, is also indicative that the subject is likely to have HIV.
  • the subject is treated prior to administration of a therapeutic agent that includes one or more antiretroviral therapies known to those of skill in the art.
  • a therapeutic agent that includes one or more antiretroviral therapies known to those of skill in the art.
  • pre-treatment is not required, and can be determined by a skilled clinician.
  • a therapeutically effective dose of a gpl20 specific neutralizing mAb described herein is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV or known to be infected with HIV).
  • Additional agents such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular,
  • the amount of the composition administered to prevent, reduce, inhibit, and/or treat HIV or a condition associated with it depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition.
  • a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV) in a subject without causing a substantial cytotoxic effect in the subject.
  • An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves.
  • these compositions may be formulated with an inert diluent or with a pharmaceutically acceptable carrier.
  • antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the particular stage of HIV. In an example, the antibodies are administered continuously. In another example, antibodies or antibody fragments are administered at 50 ⁇ g per kg given twice a week for 2 to 3 weeks.
  • Administration of the therapeutic compositions can be taken long term (for example over a period of months or years). Assessment
  • subjects having HIV can be monitored for reductions in HIV levels, increases in a subjects CD4+ T cell count, or reductions in one or more clinical symptoms associated with HIV.
  • subjects are analyzed one or more times, starting 7 days following treatment.
  • Subjects can be monitored using any method known in the art . For example, biological samples from the subject, including blood, can be obtained and alterations in HIV or CD4+ T cell levels evaluated. Additional treatments
  • a partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70% in HIV infection, HIV replication or combination thereof.
  • a partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter.

Abstract

IgG antibodies having one or more mutations in the Fc domain are disclosed. Monoclonal neutralizing antibodies having one or more mutations in the constant domain are disclosed that specifically bind to the CD4 binding site of HIV-1 gpl20. In some examples, such antibodies have a longer half-life than the corresponding native antibodies. The use of these antibodies to diagnose HIV infection or treat an HIV infection are also disclosed.

Description

HIV NEUTRALIZING ANTIBODIES HAVING MUTATIONS IN
CONSTANT DOMAIN (Fc)
CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to US Provisional Application No.
61/439,678 filed February 4, 2011, herein incorporated by reference.
FIELD OF THE DISCLOSURE
This relates to monoclonal neutralizing antibodies that bind to the CD4 binding site of HIV-1 gpl20 having mutations in the constant domain (Fc), and methods for their use.
BACKGROUND
An effective HIV-1 vaccine will likely need to induce neutralizing antibodies (NAbs) that block HIV-1 entry into human cells. To be effective, vaccine induced antibodies will have to be active against most circulating strains of HIV-1.
Unfortunately, current HIV-1 vaccines are unable to induce potent and broadly reactive NAbs. In addition, antibodies with increased half-life are desirable.
One previously characterized HIV-1 neutralizing mAb, called bl2, can bind to a site on gpl20 that is required for viral attachment to its primary cellular receptor, CD4. Another previously characterized HIV-1 neutralizing mAb, called 2F5, can bind to a site on gp41. mAb bl2 was derived from a phage display library, a process which makes it impossible to know if the antibody was naturally present in an infected person, or was the result of a laboratory combination of antibody heavy and light chains. bl2 can neutralize about 75% of clade B strains of HIV-1 (those most common in North America), but it neutralizes less than 50% of other strains of HIV-1 found worldwide. Prior attempts to design a vaccine that induces NAbs similar to bl2 have been unsuccessful. Therefore, there is a need to develop Nabs for HIV-1, including those that have an increased half-life. SUMMARY OF THE DISCLOSURE
Isolated human monoclonal neutralizing antibodies that specifically bind HIV-l gpl20 and have one or more mutations in the constant region (Fc) are provided herein. In some examples such antibodies have an increased half-life relative to the same antibodies with native Fc sequences. Also disclosed herein are compositions including these antibodies, nucleic acids encoding these antibodies, expression vectors comprising the nucleic acids, and isolated host cells that express the nucleic acids.
One skilled in the art will appreciate based on this discovery, that any IgG antibody can be generated that has equivalent mutations in its Fc region. Thus, also provided are isolated IgG antibodies, wherein a heavy chain of the IgG includes one or more amino acid substitutions in the Fc region, wherein the one or more amino acid substitutions comprise: T250Q/M428L; M428L/N434S; N434A;
T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E;
S239D/A330L/I332E; S239D/I332E + T250Q/M428L; S239D/I332E +
M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; and S239D/A330L/I332E +
M252Y/S254T/T256E. The numbering of these mutations is based on the VRC antibodies (see FIGS. 1 and 7), and one skilled in the art will appreciate how to make equivalent mutations in other IgG molecules, for example by aligning the Fc region of the IgG with the mutated Fc regions shown in FIG. 7. For example, an isolated IgG antibody can include a S239D/I332E or S239D/A330L/I332E substitution, and in some examples can further include one or more of
T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; and
M252Y/S254T/T256E substitutions. In some examples such variant IgG molecules have increased FcRn binding relative to the native IgG molecule, such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%. IgG molecules can include IgGl, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain of the isolated human monoclonal antibody (mAb) having one or more mutations in Fc has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and wherein the antibody comprises a light chain, and wherein the mAb specifically binds gpl20 of HIV-1, and wherein the antibody is neutralizing. In some embodiments, the heavy chain of the isolated human mAb having one or more mutations in Fc has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the antibody comprises a light chain, wherein the mAb specifically binds gpl20 of HIV-1, wherein the antibody is neutralizing, and wherein the mAb includes the particular mutation provided by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (for example, a sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 includes the T250Q/M428L mutation and a sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 includes the M428L/N434S mutation, and so forth).
In some examples, the light chain of the mAb comprises amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO: 36. In some examples, the heavy chain of the mAb includes one or more of the following amino acid substitutions:
T250Q/M428L;
M428L/N434S;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E.
In some examples, the heavy chain of the mAb includes a sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 38 or 42, and wherein the antibody comprises a light chain (such as a sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 40 or 44), and wherein the antibody specifically binds gpl20 of HIV-1, wherein the antibody is neutralizing, and wherein the heavy chain of the antibody has one or more of the following amino acid substitutions:
T250Q/M428L;
M428L/N434S;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E. In some examples, the isolated mAb has a heavy chain as shown in SEQ ID NO: 38 comprising one or more of the amino acid substitutions shown above and a light chain as shown in SEQ ID NO: 40. In other examples, the isolated mAb has a heavy chain as shown in SEQ ID NO: 42 comprising one or more of the amino acid substitutions shown above and a light chain as shown in SEQ ID NO: 44.
In some examples, the disclosed neutralizing hmAbs having one or more Fc mutations and have the ability to bind gpl20 have increased FcRn binding and/or increase antibody-dependent cellular cytotoxicity (ADCC) activity relative to the native IgG molecule (such as VRCOl, VRCOl, or VRC03), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%. In some examples, the disclosed neutralizing hmAbs having one or more Fc mutations and have the ability to bind gpl20 have increased half-life relative to the native IgG molecule (such as VRCOl, VRCOl, or VRC03), such as an increase of at least 1.5-fold, 1.8-fold, 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5 fold, or at least 5-fold.
Also provided are isolated functional fragments of the disclosed mAbs specific for gpl20 that have one or more mutations in the Fc. Exemplary fragments include a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single chain Fv protein (scFv), or a disulfide stabilized Fv protein (dsFv).
The disclosed antibodies and fragments thereof can be labeled, for example with a fluorescent, enzymatic, or radioactive label.
Compositions that include the disclosed antibodies are provided, for example those that include a pharmaceutically acceptable carrier, such as water or saline.
Also provided are isolated nucleic acid molecules that encode the human mAbs disclosed herein (or a functional fragment thereof). In some examples, such a nucleic acid molecule includes a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98, or at least 99% sequence identity to any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41, as well as sequences that encode a sequence having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 38 or 42 which have one or more of the following amino acid substitutions: T250Q/M428L;
M428L/N434S;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E.
The mAbs and compositions disclosed herein can be used for a variety of purposes, such as for detecting an HIV-1 infection or diagnosing AIDS in a subject. These methods can include contacting a sample from the subject (such as one suspected of having or who has been diagnosed with HIV-1 or AIDS) with a human monoclonal antibody that specifically binds gpl20 and has one or more mutations in the Fc region, and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample relative to binding of the antibody to a control sample confirms that the subject has an HIV-1 infection and/or AIDS. In some embodiments, the methods further include contacting a second antibody that specifically binds gpl20 or gp41 with the sample, and detecting binding of the second antibody. In some non-limiting examples an increase in binding of the antibody to the sample relative to a control sample detects HIV-1 in the subject. In some non-limiting examples, the antibody specifically binds soluble gpl20 in the sample. In some embodiments, the methods further include contacting a second antibody that specifically recognizes the gpl20- or gp41-specific antibody with the sample and detecting binding of the second antibody.
In additional embodiments, a method is disclosed for treating a subject with an HIV infection, such as, but not limited to, a subject with AIDS. The methods include administering a therapeutically effective amount of a human gpl20 specific monoclonal antibody having one or more mutations in the Fc region disclosed herein to the subject.
Also provided is a method for testing a potential vaccine, wherein the method includes contacting the potential vaccine with a human mAb disclosed herein (or a functional fragment thereof) and detecting the binding of the antibody to an immunogen in the potential vaccine.
The foregoing and other features of this disclosure will become more apparent from the following detailed description of a several embodiments which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a table showing mutations made to the Fc region of the heavy chain of the VRCOl antibody.
FIG. 2 is a schematic drawing showing the structures of the VRCOl antibody and its Fc mutants. Blue (those at the top) and red spheres (those closer to the bottom) represent ADCC enhancing mutation and hFcRn-binding enhancing mutation, respectively.
FIG. 3 is a pair of graphs showing binding of the VRCOl antibody and its Fc mutants to hFcRn at pH 6 (left) and pH 7.4 (right).
FIG. 4 is a pair of graphs showing binding of the VRCOl antibody and its Fc mutants to FcyRIIIa (left) and RSC3 (right).
FIG. 5 shows a pair of graphs showing antibody-dependent cellular cytotoxicity (ADCC) of the VRCOl antibody and its Fc mutants.
FIG. 6 is a schematic drawing showing the binding of IgG to hFcRn (left) and FcyRIIIa (right).
FIG. 7 shows an alignment of the heavy chain amino acid sequences for mAbs VRCOl (SEQ ID NO: 46), VRC02 (SEQ ID NO: 38) and VRC03 (SEQ ID NO: 42), and a consensus sequence (SEQ ID NO: 47). Specific Fc mutations are disclosed herein that were made to VRCOl (see FIG. 1). One skilled in the art will appreciate that equivalent mutations can be made to VRC02 and 03 at the equivalent positions (underlined).
FIG. 8 is a bar graph showing that the LS mutant antibodies were transcytosed across MDCK cells expressing hFcRn more than other mutant groups.
FIG. 9 is a graph showing that the hFcRn-binding enhancing mutant antibody, LS, had a half-life about 2 times longer than native VRCOl in the hFcRn Tg/Tg mouse model.
FIG. 10 is a graph showing that the hFcRn-binding enhancing mutant antibody, LS, had a half-life about 3 times longer than native VRCOl in the Rhesus macaques model.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOS: 1 and 2 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
T250Q/M428L (QL) mutation in its Fc.
SEQ ID NOS: 3 and 4 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
M428L/N434S (LS) mutation in its Fc.
SEQ ID NOS: 5 and 6 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a N434A (A) mutation in its Fc.
SEQ ID NOS: 7 and 8 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
T307A/E380A/N434A (AAA) mutation in its Fc. SEQ ID NOS: 9 and 10 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
M252Y/S254T/T256E (YTE) mutation in its Fc.
SEQ ID NOS: 11 and 12 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/I332E (DE) mutation in its Fc.
SEQ ID NOS: 13 and 14 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/A330L/I332E (DLE) mutation in its Fc.
SEQ ID NOS: 15 and 16 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/I332E + T250Q/M428L (DE - QL) mutation in its Fc.
SEQ ID NOS: 17 and 18 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/I332E + M428L/N434S (DE - LS) mutation in its Fc.
SEQ ID NOS: 19 and 20 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/I332E + N434A (DE - A) mutation in its Fc.
SEQ ID NOS: 21 and 22 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/I332E + T307A/E380A/N434A (DE - AAA) mutation in its Fc.
SEQ ID NOS: 23 and 24 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/I332E + M252Y/S254T/T256E (DE -YTE) mutation in its Fc.
SEQ ID NOS: 25 and 26 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/A330L/I332E + T307A/E380A/N434A (DLE - AAA) mutation in its Fc.
SEQ ID NOS: 27 and 28 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/A330L/I332E + M252Y/S254T/T256E (DLE - YTE) mutation in its Fc. SEQ ID NOS: 29 and 30 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/A330L/I332E + T250Q/M428L (DLE - QL) mutation in its Fc.
SEQ ID NOS: 31 and 32 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/A330L/I332E + M428L/N434S (DLE - LS) mutation in its Fc.
SEQ ID NOS: 33 and 34 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRCOl having a
S239D/A330L/I332E + N434A (DLE - A) mutation in its Fc.
SEQ ID NOS: 35 and 36 are the nucleic acid and corresponding amino acid sequence of the light chain of gp 120- specific antibody VRCOl.
SEQ ID NOS: 37 and 38 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRC02.
SEQ ID NOS: 39 and 40 are the nucleic acid and corresponding amino acid sequence of the light chain of gp 120- specific antibody VRC02. CDRs are shown in amino acids 27-30 (CDRl), 48-50 (CDR2), and 87-91(CDR3) of SEQ ID NO: 40.
SEQ ID NOS: 41 and 42 are the nucleic acid and corresponding amino acid sequence of the heavy chain of gpl20-specific antibody VRC03.
SEQ ID NOS: 43 and 44 are the nucleic acid and corresponding amino acid sequence of the light chain of gp 120- specific antibody VRC03. CDRs are shown in amino acids 24-33 (CDRl), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO: 44.
SEQ ID NOS: 45 and 46 are the nucleic acid and corresponding amino acid sequence of the native sequence of the heavy chain of gp 120- specific antibody VRCOl.
SEQ ID NO: 47 is a VRC heavy chain consensus sequence.
DETAILED DESCRIPTION
/. Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287- 9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Terms describing protein structure and structural elements of proteins can be found in Creighton, Proteins, Structures and Molecular Properties, W.H. Freeman & Co., New York, 1993 (ISBN 0-717-7030).
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.
It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All references cited herein are incorporated by reference.
To facilitate review of the various embodiments of this disclosure, the following explanations of terms are provided:
Administration: The introduction of a composition, such as the antibodies disclosed herein, into a subject by a chosen route, for example topically, orally, intravascularly such as intravenously, intramuscularly, intraperitoneally,
intranasally, intradermally, transdermally, intrathecally, subcutaneously, via inhalation or via suppository. Administration can be local or systemic, such as intravenous or intramuscular. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. In some examples a disclosed antibody specific for an HIV protein is administered to a subject at an effective dose. Amino acid substitution: The replacement of one amino acid in peptide with a different amino acid.
Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non- human mammals, such as primates. Similarly, the term "subject" includes both human and veterinary subjects.
Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or antigen binding fragments thereof, which specifically binds and recognizes an analyte (antigen), such as gpl20 or an antigenic fragment of gpl20. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Exemplary neutralizing gpl20 rriAbs are provided herein that have mutations in the Fc region, which in some examples increases their half-life and/or antibody-dependent cellular cytotoxicity (ADCC) activity.
Antibodies exist, for example as intact immunoglobulins and as a number of well characterized fragments produced by digestion with various peptidases. For instance, Fabs, Fvs, and single-chain Fvs (scFvs) that specifically bind to gpl20 or fragments of gpl20 would be gp 120 -specific binding agents. A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies), heteroconjugate antibodies such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
Antibody fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. The term
"antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs have been defined (see, Kabat et ah, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human
Services, 1991, hereby incorporated by reference in its entirety). Thus one of ordinary skill in the art will recognize the numbering of the residues in the disclosed antibodies is made with reference to the Kabat convention. The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as CDR LI, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR HI, CDR H2, and CDR H3.
References to "VH" or "VH" refer to the variable region of an
immunoglobulin heavy chain, including that of an antibody fragment, such as Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
A "monoclonal antibody" (mAb) is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies. In some examples monoclonal antibodies are isolated from a subject. The amino acid sequences of such isolated monoclonal antibodies can be determined.
A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they are substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (for example, see U.S. Patent No. 5,585,089).
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. "Epitope" or "antigenic determinant" refers to the region of an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents . An epitope can include at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial
conformation. Methods of determining spatial conformation of epitopes are known in the art and include, for example, x-ray crystallography and nuclear magnetic resonance.
Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. In some examples, antigens include peptides derived from HIV, such as a gpl20 polypeptide or antigenic fragment thereof, such as a gpl20 outer domain or fragment thereof.
A "target epitope" is a specific epitope on an antigen that specifically binds an antibody of interest, such as a monoclonal antibody. In some examples, a target epitope includes the amino acid residues that contact the antibody of interest, such that the target epitope can be selected by the amino acid residues determined to be in contact with the antibody of interest.
Antigenic surface: A surface of a molecule, for example a protein such as a gpl20 protein or portion thereof, capable of eliciting an immune response. An antigenic surface includes the defining features of that surface, for example the three-dimensional shape and the surface charge. An antigenic surface includes both surfaces that occur on gpl20 polypeptides as well as surfaces of compounds that mimic the surface of a gpl20 polypeptide (mimetics). In some examples, an antigenic surface includes all or part of the surface of gpl20 that binds to the CD4 receptor.
Binding affinity: Affinity of an antibody or antigen binding fragment thereof for an antigen. For example, under designated conditions, an antibody that binds preferentially to a particular target protein (such as gpl20) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject, is referred to an antibody that specifically binds to its target. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In several examples, a high binding affinity is at least about
1 x 10 -"8 M. In other embodiments, a high binding affinity is at least about 1.5 x 10 -"8 , at least about 2.0 x 10"8, at least about 2.5 x 10"8, at least about 3.0 x 10"8, at least about 3.5 x 10 -"8 , at least about 4.0 x 10 -"8 , at least about 4.5 x 10 -"8 , or at least about 5.0 x 10"8 M.
CD4: Cluster of differentiation factor 4 polypeptide; a T-cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV on T-cells during HIV-I infection. CD4 binds to gpl20 from HIV. The sequence of the CD4 precursor has a hydrophobic signal peptide, an extracellular region of approximately 370 amino acids, a highly hydrophobic stretch with significant identity to the membrane-spanning domain of the class II MHC beta chain, and a highly charged intracellular sequence of 40 resides (Maddon, Cell 42:93, 1985). The term "CD4" includes polypeptide molecules that are derived from CD4 include fragments of CD4, generated either by chemical (for example enzymatic) digestion or genetic engineering means. Such a fragment may be one or more entire CD4 protein domains. The extracellular domain of CD4 consists of four contiguous immunoglobulin-like regions (Dl, D2, D3, and D4, see Sakihama et ah, Proc. Natl. Acad. Sci. 92:6444, 1995; U.S. Patent No. 6,117,655), and amino acids 1 to 183 have been shown to be involved in gpl20 binding. For instance, a binding molecule or binding domain derived from CD4 would comprise a sufficient portion of the CD4 protein to mediate specific and functional interaction between the binding fragment and a native or viral binding site of CD4. One such binding fragment includes both the Dl and D2 extracellular domains of CD4 (D1D2 is also a fragment of soluble CD4 or sCD4 which is comprised of Dl D2 D3 and D4), although smaller fragments may also provide specific and functional CD4-like binding. The gpl20- binding site has been mapped to Dl of CD4.
Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell, for example by placing an antibody in direct physical association with a cell.
Framework Region: Amino acid sequences interposed between CDRs. Includes variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.
Fc region: The protein sequence that includes the constant region of an antibody excluding the first constant region immunoglobulin domain. Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the lower part of the hinge between Cgammal (Cyl) and C 2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. For IgA, the Fc region comprises
immunoglobulin domains Calpha2 and Calpha3 (Ca2 and Ca3) and the lower part of the hinge between Calphal (Cal) and Ca2.
gpl20: An envelope protein from Human Immunodeficiency Virus (HIV). This envelope protein is initially synthesized as a longer precursor protein of 845- 870 amino acids in size, designated gpl60. gpl60 is cleaved by a cellular protease into gpl20 and gp41. gpl20 contains most of the external, surface-exposed, domains of the HIV envelope glycoprotein complex, and it is gpl20 which binds both to cellular CD4 receptors and to cellular chemokine receptors (such as CCR5).
The mature gpl20 wild-type proteins have about 500 amino acids in the primary sequence. gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD. The protein is comprised of five conserved regions (C1-C5) and five regions of high variability (V1-V5). Exemplary sequence of wt gpl20 proteins are shown on GENBANK®, for example accession numbers AAB05604 and AAD12142 (as available on February 4, 2011), incorporated by reference herein. It is understood that there are numerous variation in the sequence of gpl20 from what is given in GENBANK®, for example accession numbers AAB05604 and AAD12142, and that these variants are skill recognized in the art as gpl20.
The numbering used in gpl20 polypeptides disclosed herein is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al, Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM which is incorporated by reference herein in its entirety.
Host cells: Cells in which a vector can be propagated and its DNA expressed, for example a disclosed antibody can be expressed in a host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.
Immunoadhesin: A molecular fusion of a protein with the Fc region of an immunoglobulin, wherein the immunogloblin retains specific properties, such as Fc receptor binding and increased half-life. An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein, polypeptide, peptide, or small molecule. In one example, and immunoadhesin includes the hinge, CH2, and CH3 domains of the immunoglobulin gamma 1 heavy chain constant region. In another example, the immunoadhesin includes the CH2, and CH3 domains of an IgG.
Immunologically reactive conditions: Includes reference to conditions which allow an antibody raised against a particular epitope (such as an HIV gpl20 epitope) to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes.
Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions. The immunologically reactive conditions employed in the methods are "physiological conditions" which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.
IgA: A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype includes IgAi and IgA2. IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild- type IgA contains an 18-amino-acid extension at its C- terminus called the tail piece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.
IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG1; IgG2, IgG3, and IgG > in mice IgG1; IgG2a, IgG2b, IgG .
Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as acquired immunodeficiency syndrome (AIDS). "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
Isolated: An "isolated" biological component (such as a cell, for example a
B cell, a nucleic acid, protein or antibody) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as, other
chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. In some examples an antibody, such as an antibody specific for gpl20 can be isolated, for example isolated from a subject infected with HIV or from a cell that expresses the antibody.
Ka: The dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody (such as VRCOl, VRC02, or VRC03 or a mutant thereof) and an antigen (such as gpl20) it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.
Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, quantum dots, and radioactive isotopes. In some examples, a disclosed antibody is labeled.
Neutralizing antibody: An antibody which reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent. In some examples the infectious agent is a virus. In some examples, an antibody that is specific for gpl20 neutralizes the infectious titer of HIV. In specific examples, HIV-l resists neutralization by most antibodies. However, the disclosed hmAbs having one or more Fc mutations successfully neutralize at least 50% of current circulating HIV-l isolates, such as at least 75%, at least 80%, or at least 90% of current circulating HIV-l isolates.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes
compositions and formulations suitable for pharmaceutical delivery of the antibodies herein disclosed.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions {e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the protein (such as an antibody) is more enriched than the protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein represents at least 50% of the total protein content of the preparation.
Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by methods known in the art, such as chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Cells that express such molecules are referred to as recombinant or transgenic cells.
Sequence identity: The similarity between amino acid or nucleic acid sequences are expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
Methods of alignment of sequences for comparison are well known in the art.
Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5: 151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
Variants of the disclosed mAbs that specifically bind gpl20 and have one or more mutations in the Fc region are encompassed by this disclosure typically characterized by possession of at least about 75%, for example at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid or nucleic acid sequence of interest, such as any of SEQ ID NOS: 1-47. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
Therapeutically effective amount: A quantity of a specific substance, such as a disclosed mAb (or combinations of mAbs), sufficient to achieve a desired effect in a subject administered the mAb. For instance, this can be the amount necessary to inhibit HIV replication or treat AIDS. In several embodiments, a therapeutically effective amount is the amount necessary to reduce a sign or symptom of AIDS, and/or to decrease viral titer in a subject (such as a decrease of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, or at least 95% as compared to an absence of the mAb). When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect.
T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8 T cells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell is a suppressor T cell.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.
Virus: A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.
"Retroviruses" are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a pro virus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the "immunodeficiency viruses" which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), and feline
immunodeficiency virus (FIV).
HIV-I is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). "HIV disease" refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, for example, as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T cells.
//. Description of Several Embodiments
A. Neutralizing Monoclonal Antibodies
Isolated human monoclonal antibodies (hmAbs) that specifically bind gpl20 are disclosed herein, which include one or more mutations in their Fc region. In one example, such mutations include one or more of the following: T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E;
S239D/I332E; S239D/A330L/I332E; S239D/I332E + T250Q/M428L; S239D/I332E + M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or S239D/A330L/I332E +
M252Y/S254T/T256E. The numbering of amino acid residue in IgGl follows the Eu system (Edelman et al., PNAS 63:78-85, 1969).
It has been unexpectedly found that hmAbs that specifically bind gpl20 and have a S239D/I332E or S239D/A330L/I332E substitution, in combination with one or more of T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; or M252Y/S254T/T256E, have greater FcRn binding, and/or half-life, than any of these mutations alone. It has also been unexpectedly found that mAbs that specifically bind gpl20 and have a M428L/N434S substitution, have a greater half-life and increased transcytosis activity, as compared to the native VRCOl antibody, and that the combination M428L/N434S and S239D/I332E substitutions provide a mutant antibody with better transcytosis and a similar half-life relative to the native VRCOl antibody. In some examples, hFcRn-binding enhancing mutants (hFcRn mutant) have lower FcgRIIIa-binding affinity and ADCC activity, and FcgRIIIa-binding mutants (FcgR mutants) have lower hFcRn-binding affinity than wild type. When an FcgR mutation is introduced into an hFcRn mutnat, the binding affinity of the combined mutant to FcgRIIIa and ADCC activity becomes similar to wild type and vice versa, n some examples, combining an hFcRn mutant with an FcgR-binding enhancing mutation increases ADCC activity relative to a wildtype antibody (e.g., VRCOl).
Methods of introducing such mutations into the Fc region are routine. In some examples such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased FcRn binding relative to the native hmAb (such as VRCOl), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%. In some examples such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased FcyRIIIa binding relative to the native hmAb (such as VRCOl), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%. In some examples such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased antibody-dependent cellular cytotoxicity (ADCC) relative to the native hmAb (such as VRCOl), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%. In some examples such hmAbs that specifically bind gpl20 that include one or more mutations in their Fc region have increased half-life relative to the native hmAb (such as VRCOl), such as an increase of at least 1.5-fold, at least 1.8 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold.
One skilled in the art will appreciate based on this discovery, that any IgG antibody can be generated that has equivalent mutations in its Fc region. Thus, also provided are isolated IgG antibodies, wherein a heavy chain of the IgG includes one or more amino acid substitutions in the Fc region, wherein the one or more amino acid substitutions comprise: T250Q/M428L; M428L/N434S; N434A;
T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E;
S239D/A330L/I332E; S239D/I332E + T250Q/M428L; S239D/I332E +
M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; S239D/A330L/I332E +
M252Y/S254T/T256E, or combinations thereof. The numbering of these mutations is based on the VRC antibodies (see FIGS. 1 and 7), and one skilled in the art will appreciate how to make equivalent mutations in other IgG molecules, for example by aligning the Fc region of the IgG with the mutated Fc regions shown in FIG. 7. For example, an isolated IgG antibody can include a S239D/I332E or
S239D/A330L/I332E substitution, and in some examples can further include one or more of T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; and M252Y/S254T/T256E substitutions. In some examples such variant IgG molecules have increased FcRn binding relative to the native IgG molecule (such as IgGl, IgG2, IgG3, or IgG4), such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%.
Also disclosed herein are compositions including these variant IgGs or hmAbs and a pharmaceutically acceptable carrier. Nucleic acids encoding these antibodies, expression vectors comprising these nucleic acids, and isolated host cells that express the nucleic acids are also provided.
Compositions comprising the disclosed hmAbs specific for gpl20 and having one or more Fc mutations can be used for research, diagnostic and
therapeutic purposes. For example, the hmAbs disclosed herein can be used to diagnose or treat a subject having an HIV-1 infection and/or AIDS. For example, the hmAbs can be used to determine HIV-1 titer in a subject. The hmAbs disclosed herein also can be used to study the biology of HIV.
In some embodiments, an isolated hmAb specifically binds gpl20, and includes one or more (such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10) of the following mutations (relative to the native Ab Fc sequence): T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E;
S239D/A330L/I332E; S239D/I332E + T250Q/M428L; S239D/I332E +
M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or S239D/A330L/I332E +
M252Y/S254T/T256E. In some embodiments, the isolated hmAbs specifically bind gpl20 (and may neutralize HIV), and include a heavy chain having one or more of the mutations listed above (relative to the native Ab Fc sequence) and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
In specific examples, the heavy chain of the hmAb has at least 80%, at least
85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and specifically binds gpl20 of HIV-1 and is neutralizing. For example, the antibody can include a heavy chain that comprises or consists of any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
In some examples, the light chain of the hmAb includes amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO: 36, for example, a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 36, or a sequence comprising or consisting of SEQ ID NO: 36. In some examples, the light chain of the hmAb includes amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91(CDR3) of SEQ ID NO: 40, for example, a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40, or a sequence comprising or consisting of SEQ ID NO: 40. In some examples, the light chain of the hmAb includes amino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO: 44, for example, a sequence having at least 80%, at least
90%, or at least 95% sequence identity to SEQ ID NO: 44, or a sequence comprising or consisting of SEQ ID NO: 44.
In one example, the isolated hmAb has a heavy chain with a sequence that includes or consists of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and the light chain of the antibody includes or consists of or consists of SEQ ID NO: 36. In a specific example, the isolated human monoclonal antibody of has a heavy chain comprising SEQ ID NO: 4 or 18.
In some examples the disclosed antibodies having Fc amino acid
substitutions (mutations) include a variant of a native heavy chain of the VRCOl hmAb (SEQ ID NO: 46) having one or more mutations in the Fc disclosed herein. For example, FIG. 1 shows several amino acid substitutions made to a native VRCOl sequence. One skilled in the art will appreciate that the equivalent mutation can be made to other antibodies, such as VRC02 and VRC03. As shown in FIG. 7, an amino acid alignment of the heavy chain is provided for VRCOl, VRC02 and VRC03. The amino acid in the Fc region mutated in VRCOl is underlined, as is the equivalent amino acid in VRC02 and VRC03. Based on this alignment and the mutations provided herein, equivalent mutations are provided for VRC02 and VRC03. For example, provided herein are hmAbs, wherein a heavy chain of the antibody includes a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 38 or 42, wherein the heavy chain of the antibody has one or more of the following amino acid substitutions: T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E; S239D/A330L/I332E; S239D/I332E + T250Q/M428L; S239D/I332E + M428L/N434S; S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A; S239D/A330L/I332E+ T307A/E380A/N434A; S239D/A330L/I332E + M252Y/S254T/T256E, or combinations thereof, wherein the antibody comprises a light chain, and wherein the antibody specifically binds gpl20 of HIV-1 and is neutralizing. In some examples, the light chain of such hmAbs include a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40 or 44. For example, the disclosed hmAbs can include a heavy chain that includes SEQ ID NO: 38 with one or more of the amino acid substitutions listed above and a light chain that includes SEQ ID NO: 40. In another example, a disclosed hmAb has a heavy chain comprising SEQ ID NO: 42 with one or more of the amino acid substitutions listed above and a light chain that includes SEQ ID NO: 44.
In further embodiments, the isolated human monoclonal antibody
specifically binds gpl20, and includes a heavy chain with at most one, at most two, at most three or at most four amino acid substitutions in the CDR1, CDR2, or CDR3 of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or
SEQ ID NO: 38 or 42 with one or more of the mutations provided herein) and a light chain. In some examples, these antibodies retain the binding affinity of the parental antibody (VRCOl or VRC02 or VRC03) for the antigenic epitope. Thus, in some examples, these antibodies have a KD of < 3 nM for the antigenic epitope of gpl20.
Fully human monoclonal antibodies include human framework regions. Thus, any of the antibodies that specifically bind gpl20 and have a mutated Fc region provided herein can include the human framework region and can include the framework regions of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or SEQ ID NO: 38 or 42 with one or more of the mutations provided herein). However, the framework regions can be from another source. Additional examples of framework sequences that can be used include the amino acid framework sequences of the heavy and light chains disclosed in PCT Publication No. WO 2006/074071 (see, for example, SEQ ID NOs: 1-16), herein incorporated by reference.
The monoclonal antibody can be of any isotype. The monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgGiOr an IgG2. The class of an antibody that specifically binds gpl20 can be switched with another. In one aspect, a nucleic acid molecule encoding VL or VH is isolated using methods well- known in the art, such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. The nucleic acid molecule encoding VL or VH can be operatively linked to a nucleic acid sequence encoding a CL or CH from a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule that comprises a CL or CH chain, as known in the art. For example, a hmAbs disclosed herein that was originally IgM may be class switched to an IgG. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG2.
In some examples, the disclosed hmAbs are oligomers of antibodies, such as dimers trimers, tetramers, pentamers, hexamers, septamers, octomers and so on. In some examples, the hmAbs are pentamers.
By definition, the CDRs of the light chain are bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded by the residues at positions 31 and 35b (H- CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using the numbering convention delineated by Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5 Edition, U.S. Department of Health and Human
Services, Public Health Service, National Institutes of Health, Bethesda, MD (NIH Publication No. 91-3242, incorporated herein by reference in its entirety).
Antibody fragments of the disclosed neutralizing hmAbs that specifically bind gpl20 and have one or more mutations in the Fc region are encompassed by the present disclosure, such as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region . These antibody fragments retain the ability to selectively bind with the antigen. These fragments include:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
(6) A dimer of a single chain antibody (scFV2), defined as a dimer of a scFV. This has also been termed a "miniantibody."
Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York, 1988). In further embodiments, the antibodies are Fv antibodies, which are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce these antibodies, the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. In particular examples, the VH amino acid sequence includes the CDRs from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or SEQ ID NO: 38 or 42 with one or more of the mutations provided herein). In other examples, the VL amino acid sequence includes the CDRs from SEQ ID NO: 36, 40 or 44.
If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
In an additional example, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in
Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al, Bio/Technology 11: 1271, 1993; and Sandhu, supra). Dimers of a single chain antibody (scFV2), are also contemplated.
Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et ah, Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et ah, Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
One of skill will realize that conservative variants of the disclosed antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, at most five, or at most 10 amino acid substitutions, such as 1 to 10 or 1 to 5 conservative substitutions) can be made in the VH and the VL regions to increase yield. In particular examples, the VH sequence is from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 (or SEQ ID NO: 38 or 42 with one or more of the mutations provided herein). In other examples, the VL sequence is from one of SEQ ID NOs: 36, 40 or 44.
Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
The antibodies disclosed herein can be isolated using cloaked antigens, as described in PCT Publication No. WO 2009/100376. Briefly, antigens are cloaked to target antigenicity of the antigen to a specific epitope that specifically bound by the antibody of interest, such as a neutralizing antibody.
In some examples, the amino acid substitutions result in the antigen not being bound by antibodies in a polyclonal serum that specifically bind surface exposed amino acid residues of the wild- type antigen located exterior of the target epitope. In some embodiments, the amino acid substitutions alter antigenicity of the antigen in vivo as compared to the wild-type antigen (unsubstituted antigen) but do not introduce additional glycosylation sites as compared to the wild-type antigen. I n some embodiments, that antigen is glycosylated. In some embodiments the cloaked antigen is modified to substitute one or more residues recognized by the antibody of interest to abolish antigen recognition. In some examples, a biotinylation peptide can be fused to the cloaked antigen. Biotinylated cloaked antigen can then be used to stain and thus identify cells, such as PBMC, expressing an antibody of interest.
The hmAbs or antibody fragments disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibody or portion thereof is derivatized such that the binding to gpl20 is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m- maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company (Rockford, IL).
An antibody that specifically binds gpl20 and has an Fc mutation (or a functional fragment thereof) can be labeled with a detectable moiety. Useful detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, Green fluorescent protein, Yellow fluorescent protein. An antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, β- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labeled with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label. In one example, an antibody or fragment thereof disclosed herein can be labeled with a quantum dot.
An antibody or fragment thereof disclosed herein can be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
An antibody or fragment thereof disclosed herein can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleo tides: 3H, 14C, 15N, 35S, 90Y, "Tc, U1ln, 125I, 131I.
An antibody or fragment thereof disclosed herein can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.
Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
B. Polynucleotides and Expression
Nucleic acid molecules encoding the peptides provided herein (including, but not limited to antibodies) can readily be produced by one of skill in the art. For example, these nucleic acids can be produced using the amino acid sequences provided herein (such as the CDR sequences, heavy chain and light chain
sequences), sequences available in the art (such as framework sequences), and the genetic code.
Thus, the disclosure provides isolated nucleic acid molecules that encoding any of the neutralizing hmAbs disclosed herein (or a functional fragment thereof). In one example, such a nucleic acid molecule comprises a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41. In some examples the nucleic acid molecule comprises or consists of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41.
In one example, the isolated nucleic acid molecule encodes a heavy chain and a light chain of an antibody, wherein (a) the heavy chain of the antibody comprises a sequence having at least 80%, at least 90%, at least 95% or at least 99% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and having one or more of the following
substitutions/mutations: T250Q/M428L; M428L/N434S; N434A;
T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E;
S239D/A330L/I332E; S239D/I332E + T250Q/M428L;S239D/I332E +
M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+
N434A;S239D/A330L/I332E+ T307A/E380A/N434A; or S239D/A330L/I332E + M252Y/S254T/T256E, and wherein the light chain of the antibody comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 36; (b) the heavy chain of the antibody comprises the heavy chain of the antibody comprises any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and wherein the light chain of the antibody comprises SEQ ID NO: 36; (c) the heavy chain of the antibody comprises SEQ ID NO: 36 or 42 having one or more of the following substitutions/mutations:
T250Q/M428L; M428L/N434S; N434A; T307A/E380A/N434A;
M252Y/S254T/T256E; S239D/I332E; S239D/A330L/I332E; S239D/I332E + T250Q/M428L;S239D/I332E + M428L/N434S; S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+ N434A;S239D/A330L/I332E+ T307A/E380A/N434A; or S239D/A330L/I332E + M252Y/S254T/T256E, and wherein the light chain of the antibody comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 40 or 44; (d) the heavy chain of the antibody comprises SEQ ID NO: 36 having one or more of the following substitutions/mutations: T250Q/M428L; M428L/N434S; N434A;
T307A/E380A/N434A; M252Y/S254T/T256E; S239D/I332E;
S239D/A330L/I332E; S239D/I332E + T250Q/M428L;S239D/I332E +
M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+
N434A;S239D/A330L/I332E+ T307A/E380A/N434A; or S239D/A330L/I332E + M252Y/S254T/T256E, and wherein the light chain of the antibody comprises SEQ ID NO: 40; (e) the heavy chain of the antibody comprises SEQ ID NO: 42 having one or more of the following substitutions/mutations: T250Q/M428L;
M428L/N434S; N434A; T307A/E380A/N434A; M252Y/S254T/T256E;
S239D/I332E; S239D/A330L/I332E; S239D/I332E + T250Q/M428L;S239D/I332E + M428L/N434S; S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A; S239D/I332E + M252Y/S254T/T256E; S239D/A330L/I332E+ T250Q/M428L; S239D/A330L/I332E+ M428L/N434S; S239D/A330L/I332E+
N434A;S239D/A330L/I332E+ T307A/E380A/N434A; or S239D/A330L/I332E + M252Y/S254T/T256E, and wherein the light chain of the antibody comprises SEQ ID NO: 44, wherein the encoded antibody specifically binds gpl20 of HIV- 1, and wherein the encoded antibody is neutralizing.
Such isolated nucleic acid molecules can be operably linked to a promoter.
The disclosed isolated nucleic acid molecules can be part of an expression vector, such as a plasmid. In one example, the expression vector includes a promoter and an enhancer, such as a cytomegalovirus promoter or cytomegalovirus enhancer. The expression vector can include RNA splicing donor sites (such as HTLV-1 or CMV RNA splicing donor sites), RNA splicing acceptor sites (such as HTLV-1 or CMV RNA splicing acceptor sites) and/or internal ribosomal binding sequences. Such expression vectors can be used to express the heavy chain of the antibody and the light chain of the antibody as a fusion polypeptide following the introduction of the expression vector in a host cell. For example, the expression vector can include a nucleic acid sequence encoding a furin cleavage site between the heavy chain and the light chain of the antibody. In some examples, the expression vector also encodes a selectable marker. Isolated host cells transformed with the disclosed nucleic acid molecules or vectors are also provided.
One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the heavy or light chain nucleic acid sequence. Nucleic acid sequences encoding the neutralizing hmAbs that specifically bind gpl20 and have one or more Fc mutations can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et ah, Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et ah, Meth. Enzymol. 68: 109-151, 1979; the diethylphosphoramidite method of Beaucage et ah, Tetra. Lett. 22: 1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20): 1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham- VanDevanter et ah, Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that longer sequences may be obtained by the ligation of shorter sequences.
Exemplary nucleic acids can be prepared by routine cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et ah, supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN),
Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources.
Nucleic acids can also be prepared by amplification methods. Amplification methods include but are not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and the self- sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known.
Any of the nucleic acids encoding any of the antibodies, VH and/or VL, disclosed herein (or fragment thereof) can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. These antibodies can be expressed as individual VH and/or VL chain, or can be expressed as a fusion protein. An immunoadhesin can also be expressed . Thus, nucleic acids encoding a VH and VL, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.
To create a single chain antibody, (scFv) the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker (see, e.g., Bird et al., Science 242:423-426, 1988;
Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al, Nature 348:552-554, 1990). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.
The nucleic acid encoding the VH and/or the VL optionally can encode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fc domain. The Fc domain can be an optimized Fc domain, as described in U.S. Published Patent Application No. 20100/093979. In one example, the immunoadhesin is an IgG! Fc.
The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to gpl20 and to another molecule, such as gp41. The encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains.
It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. The host cell can be a gram positive bacteria including, but are not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,
Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus.
Methods for expressing protein in gram positive bacteria, such as Lactobaccillus are well known in the art, see for example, U.S. Published Patent Application No.
20100/080774. Expression vectors for lactobacillus are described, for example in U.S. Pat. Nos. 6,100,388 and 5,728,571. Leader sequences can be included for expression in Lactobacillus. Gram negative bacteria include, but not limited to, E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.
One or more DNA sequences encoding the antibody or fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the mutant mAbs provided herein are also encompassed by this disclosure.
The expression of nucleic acids encoding the isolated proteins described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter, including a cytomegalovirus promoter and a human T cell lymphotrophic virus promoter (HTLV)-l. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
To obtain high level expression of a cloned gene, expression cassettes can include a strong promoter to direct transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a
transcription/translation terminator. For E. coli, this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an
immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with
polynucleotide sequences encoding the antibody, labeled antibody, or functional fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One can readily use an expression system, such as plasmids and vectors, to produce proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa, and myeloma cell lines.
Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps. In addition to recombinant methods, the
immunoconjugates, effector moieties, and antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis.
Once expressed, the recombinant immunoconjugates, antibodies, and/or effector molecules can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column
chromatography, and the like (see, generally, R. Scopes, PROTEIN
PURIFICATION, Springer- Verlag, N.Y., 1982). The antibodies, immunoconjugates and effector molecules need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.
Methods for expression of antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies disclosed herein. See, Buchner et ah, Anal. Biochem. 205:263-270, 1992; Pluckthun,
Biotechnology 9:545, 1991; Huse et al, Science 246: 1275, 1989 and Ward et al, Nature 341:544, 1989.
Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, a reducing agent is present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9:5015-21, 1970, and by Buchner et al., supra.
Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5-fold molar excess of one protein over the other is not exceeded. Excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution after the redox-shuffling is completed.
In addition to recombinant methods, the antibodies, labeled antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence.
Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al, J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et ah, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, 111., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide) are well known. C. Compositions and Therapeutic Methods
Methods are disclosed herein for the treatment of an HIV infection, such as an HIV-1 infection. In one example, the methods include prevention or inhibition of infection with HIV-1. The methods include contacting a cell with an effective amount of one or more of the hmAbs disclosed herein that specifically bind gpl20 and have one or more Fc mutations, or a functional fragment thereof. The method can also include administering to a subject a therapeutically effective amount of the hmAbs to a subject. In some examples, the method includes selecting a subject infected with HIV, such as HIV-1, or who is at risk for such infection (such as medical personnel).
In one example the subject is an HIV-positive pregnant woman, or child recently born to such a woman. Studies have shown that the rate of HIV
transmission from mother to infant is reduced significantly when zidovudine is administered to HIV-infected women during pregnancy and delivery and to the offspring after birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541). Several studies of mother- to-infant transmission of HIV have demonstrated a correlation between the maternal virus load at delivery and risk of HIV transmission to the child. The present disclosure provides isolated hmAbs that can decrease HIV-transmission from mother to infant. Thus, in some examples a therapeutically effective amount one or more of the disclosed hmAbs specific for gpl20- is administered in order to prevent transmission of HIV, or decrease the risk of transmission of HIV, from a mother to an infant. In some examples, a therapeutically effective amount of the antibody is administered to mother and/or to the child at childbirth. In other examples, a therapeutically effective amount of the antibody is administered to the mother and/or infant prior to breast feeding in order to prevent viral transmission to the infant or decrease the risk of viral transmission to the infant. In some
embodiments, both a therapeutically effective amount of the antibody and a therapeutically effective amount of another agent, such as zidovudine, is
administered to the mother and/or infant.
Methods to assay for neutralization activity, such as HIV neutralization, include, but are not limited to, a single-cycle infection assay as described in Martin et al. (2003) Nature Biotechnology 21:71-76. In this assay, the level of viral activity is measured via a selectable marker whose activity is reflective of the amount of viable virus in the sample, and the IC50 is determined. In other assays, acute infection can be monitored in the PM1 cell line or in primary cells (normal PBMC). In this assay, the level of viral activity can be monitored by determining the p24 concentrations using ELISA. See, for example, Martin et al. (2003) Nature
Biotechnology 21:71-76.
In one example, the cell is also contacted with an effective amount of an additional agent, such as anti-viral agent. The cell can be in vivo or in vitro. For example, the disclosed mAbs can be combined with anti-retroviral therapy.
Antiretro viral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. The disclosed antibodies can be administered in conjunction with nucleoside and nucleotide reverse transcriptase inhibitors (nRTI), non- nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors, entry inhibitors (or fusion inhibitors), maturation inhibitors, or a broad spectrum inhibitors, such as natural antivirals. Exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine and efavirenz. In some examples the mAbs and the anti-retroviral therapy is administered simultaneously or contemporaneously. In other examples the therapies are administered separately.
Compositions are provided that include one or more of the hmAbs or functional fragments thereof disclosed herein in a carrier. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes. The antibody can be formulated for systemic or local
administration. In one example, the hmAbs or functional fragments thereof disclosed herein is formulated for parenteral administration, such as intravenous
administration.
The compositions for administration can include a solution of the hmAbs or functional fragments thereof in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, 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, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody 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 subject's needs. Controlled-release parenteral formulations containing the therapeutic mAb can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
Microcapsules contain the therapeutic mAb as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μιη are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μιη so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μιη in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
Polymers can be used for ion-controlled release of the mAb compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et ah, Pharm. Res. 9:425-434, 1992; and Pec et al, J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins, such as mAbs, are known (see U.S.
Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).
A typical pharmaceutical composition for intravenous administration includes at least 1 mg/kg (such as at least 5 mg/kg, at least 10 mg/kg, at least 20 mg/kg or at least 40 mg/kg, such as 1 to 100 mg/kg, 1 to 50 mg/kg, 5 to 50 mg/kg, or 10-40 mg/kg) administered every two to four weeks. In one example, the dosage for a small rodent is about 50 to 500 μg mAb, such as 200 μg mAb (e.g., 10 mg/kg). In another example, the dosage for a large mammal, such as a human or non-human primate is about 1 to 100 mg mAb /kg subject, such as 1 to 50, 10 to 24 or 20 mg/kg. Dosages from 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's
Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).
Antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 75 mg/kg of body weight (such as 5 to 50 or 10 to 40 mg/kg).
Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RITUXAN® in 1997. Antibodies can be administered by slow infusion, rather than in an
intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated. In some examples the Abs are administered at least once a week, at least once a month, at least twice a month, or at least once every two months, or In one example, at least 1 mg mAb/kg (such as at least 5 mg/kg, at least 10 mg/kg, at least 20 mg/kg or at least 40 mg/kg, such as 1 to 100 mg/kg, 1 to 50 mg/kg, 5 to 50 mg/kg, or 10-40 mg/kg) is administered to the patient every two to four weeks. In some examples, the mAbs are administered iv, subcutaneously or im.
A therapeutically effective amount of a mAb disclosed herein will depend upon the severity of the disease and/or infection and the general state of the patient's health. A therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with another therapeutic agent, either simultaneously or sequentially.
In one embodiment, administration of the antibody results in a reduction in the establishment of HIV infection and/or reducing subsequent HIV disease progression in a subject. A reduction in the establishment of HIV infection and/or a reduction in subsequent HIV disease progression encompass any statistically significant reduction in HIV activity. In some embodiments, methods are disclosed for treating a subject with an HIV-1 infection. These methods include administering to the subject a therapeutically effective amount of an antibody, or a nucleic acid encoding the antibody, thereby preventing or treating the HIV-1 infection.
HIV infection does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV infected cells), as compared to HIV infection in the absence of the composition.
In additional examples, HIV replication can be reduced or inhibited by use of the disclosed mAbs. HIV replication does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV replication by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV), as compared to HIV replication in the absence of the composition. Single or multiple administrations of the compositions including the mAbs disclosed herein are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. In one example, a dose of the antibody is infused for thirty minutes every other day. In this example, about one to about ten doses can be administered, such as three or six doses can be administered every other day. In a further example, a continuous infusion is administered for about five to about ten days. The subject can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient. D. Diagnostic Methods and Kits
Methods are provided herein for the detection of gpl20 or in vitro or in vivo. In one example, gpl20 is detected in a biological sample, and can be used to detect HIV-1 infection. The sample can be any sample, including, but not limited to, tissue from biopsies or check swabs, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, semen, sputum, breast milk, spinal fluid or urine.
In one embodiment, a method is provided for detecting AIDS and/or an HIV- 1 infection in a subject. The disclosure provides a method for detecting HIV-1 in a biological sample, wherein the method includes contacting a biological sample with a hmAb or functional fragment thereof disclosed herein under conditions conducive to the formation of an immune complex, and detecting the immune complex to detect the gpl20 in the biological sample. In one example, the detection of gpl20 in the sample (for example above a background level) indicates that the subject has an HIV infection. In another example, the detection of gpl20 in the sample indicates that the subject has AIDS. In another example, detection of gpl20 in the sample confirms a diagnosis of AIDS and/or an HIV-1 infection in a subject. In some examples the method includes comparing the gpl20-hmAb complex detected to an amount of such complex detected in a control, such as a sample known to not contain HIV or gpl20, or known to be infected with HIV or contain gpl20. In some examples the method includes comparing an absolute or relative amount of the gpl20-hmAb complex detected to a reference value or range of values expected of such complex expected if the sample does not contain gpl20, or expected if the sample does contain gpl20.
In some embodiments, the disclosed antibodies are used to test vaccines, for example to determine if a vaccine composition assumes the same conformation as a native gpl20 peptide. Thus provided herein is a method for detecting testing a vaccine, wherein the method includes contacting a sample containing the test vaccine, such as a gpl20 immunogen, with an antibody disclosed herein under conditions conducive to the formation of an immune complex, and detecting the immune complex, to detect the vaccine in the sample. In one example, the detection of the immune complex in the sample indicates that vaccine component, such as such as a gpl20 immunogen assumes a conformation capable of binding the antibody.
In one embodiment, the anti-gpl20 antibody is directly labeled with a detectable label. In another embodiment, the anti-gpl20 antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the anti-gpl20 antibody is utilized. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-lgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
Suitable labels for the antibody or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, quantum dots, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non- limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary
luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I, 131 I, 35 S or 3H.
The immunoassays and method disclosed herein can be used for a number of purposes. Kits for detecting a polypeptide will typically include one or more hmAbs or functional fragments thereof disclosed herein. In some embodiments, an antibody fragment, such as an Fv fragment or a Fab is included in the kit. In a further embodiment, the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).
Thus, the disclosure provides a kit that includes one more of the mAbs provided herein, for example in separate containers. In one example the kit includes an antibody having a heavy chain comprising the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and can further include a light chain comprising the sequence of SEQ ID NO: 36. Such antibodies may include a label. In some examples such a kit further includes a labeled secondary antibody in a separate container, which permits detection of the primary antibody. In some examples such a kit further includes additional therapeutic agents, such as one or more antiretro viral compounds, in separate containers.
In one embodiment, a kit includes instructional materials disclosing means of use. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art. In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting gpl20 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to gpl20. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
EXAMPLES
Example 1
Mutation of the Fc Region of VRCOl
This example describes the mutation of the Fc region of VRCOl, a neutralizing human monoclonal antibody that specifically binds to gpl20. One skilled in the art will appreciate that similar mutations can be made to the heavy chains of VRC02 (SEQ ID NOS: 37-38) or VRC03 (SEQ ID NOS: 41-42), for example based on the alignment shown in FIG. 7.
Mutations were made in the Fc region of VRCOl using site-directed mutagenesis PCR in the heavy chain of VRCOl. The resulting VRCOl mutant heavy chain and native light chain (SEQ ID NOS: 35 and 36) were transiently transfected to 293F cells and the supernatant collected six days later. The resulting VRCOl antibodies having one or more Fc mutations were purified by Protein A from the supernatant. The mutations made are shown in SEQ ID NOS: 1-34 and in the Table below and FIGS. 1 and 2. Table 1. VRCOl Fc mutations
Figure imgf000055_0001
S254T / T256E at position 353, E at position
355, Y at position 275, T at position 277, E at position 279, of SEQ ID NO: 28)
DLE-QL S239D / A330L / I332E + T250Q / 29 and 30 (D at position 262, L
M428L at position 353, E at position
355, Q at position 273, L at position 451 of SEQ ID NO:
30)
DLE-LS S239D / A330L / I332E + M428L / 31 and 32 (D at position 262, L
N434S at position 353, E at position
355, L at position 451, S at position 457, of SEQ ID NO:
32)
DLE-A S239D / A330L / I332E + N434A 33 and 34 (D at position 262, L at position 353, E at position 355, A at position 457, of SEQ
ID NO: 34)
Viral entry, neutralization and protein competition assays. Neutralization was measured using single round infection by HIV-1 Env-pseudoviruses and TZM- bl target cells. Neutralization curves were fit by nonlinear regression using a 5- parameter hill slope equation. The 50% and 80% inhibitory concentrations (IC50 and IC80) were reported as the antibody concentrations required to inhibit infection by 50% and 80% respectively. Competition of serum or mAb neutralization was assessed by adding a fixed concentration (25 μg/ml) of the RSC3 or ARSC3 glycoprotein to serial dilutions of antibody for 15 min prior to the addition of virus. The resulting IC50 values were compared to the control with mock protein added. The neutralization blocking effect of the proteins was calculated as the percent reduction in the IC50 value of the antibody in the presence of protein compared to PBS. Synergistic or additive neutralization was assessed by mixing a fixed concentration (10 μg/ml) of the test antibody with serial dilutions of sCD4, CD4-Ig or VRCOl for 15 min prior to the addition of virus. The baseline of viral entry at each concentration of sCD4, CD4-Ig or VRCOl was used to calculate the adjusted percent neutralization. Neutralization was also assessed using Env-pseudoviruses generated by 293T transfection using the pNL4-3 ΔΕην HIV-1 backbone containing a lucif erase reporter gene to infect activated PBMC. Neutralizations using uncloned PBMC-derived HIV- 1 primary isolates were performed by single-round infection of either TZM-bl cells using luciferase as readout, or activated PBMC using flow cytometry staining for HIV- 1 p24 antigen. CD4-facilitated virus entry was performed in the CCR5+/CD4- cell line Cf2Th/syn CCR5 with Env-pseudoviruses 5 containing the luciferase pNL4-3 ΔΕην HIV- 1 backbone. A mixture of 40 μΐ of viral stock and 10 μΐ of serial dilutions of sCD4, CD4-Ig or VRCOl was incubated at 37°C for 30 min before adding 1 x 104 Cf2Th/syn CCR5 cells. Virus entry was measured 2 days later by luciferase activity in cell lysates.
As shown in Tables 2-5, the VRCOl Fc mutants have similar neutralization 10 activity as the wild- type VRCOl antibody. Table 2 provides a comparison of the
IC50 between wild type VRCOl and the mutant VRCOl antibodies. Table 3 provides a comparison of ICso between wild type and the mutants. Tables 4 and 5 show the fold increase/decrease of IC50 and ICso of the Fc mutants comparing with wild type.
Table 2: IC50 values (mg/mL)
Figure imgf000057_0001
DLE-A CVL1062.002 0.061 0.025 0.141 0.029 >50 0.094 0.132 >50 >50
DLE-
CVL1062.002 0.079 0.026 0.138 0.031 >50 0.094 0.132 >50 >50 AAA
DLE-
CVL1062.002 0.063 0.030 0.139 0.030 >50 0.085 0.121 >50 >50 YTE
Table 3: ICso values (mg/mL)
Figure imgf000058_0001
5 Table 4: IC50 values (fold-difference)
Figure imgf000059_0001
Table 5: ICso values (fold-difference)
Figure imgf000060_0001
Example 2
Binding of the VRCOl Mutants to hFcRn
5 This example describes the methods used to measure the binding of the
VRCOl antibody mutants to the human MHC class I-related Fcy-receptor (hFcRn) as compared to the native VRCOl antibody. Binding to hFcRn provides an indication as to the likely degradation of the antibody; antibodies while boudn to hFcRn avoid degradation.
0 Nickel-coated plates were incubated with His tagged recombinant hFcRn for one hour, and washed with PBS, pH7.4 + 0.05 % Tween20. VRCOl and its Fc mutants were diluted with 2.5%-BSA in PBS, pH 6.0 or PBS, pH 7.4 were incubated for two hours and washed with PBS, pH6.0 + 0.05 % Tween20 or PBS, pH7.4 + 0.05 % Tween20. Anti-human IgG-HRP diluted with 2.5%-BSA in PBS, pH6.0 or PBS, pH7.4 were incubated for one hour and washed with PBS, pH6.0 + 0.05 % Tween20 or PBS, pH7.4 + 0.05 % Tween20. HRP substrate TMB was added and the color development was stopped by 0. IN H2S04. Optical density at 450 nm was measured by ELISA reader.
As shown in FIG. 3, all of the VRCOl Fc mutants have a higher binding to hFcRN at pH 6 and 7.4 relative to VRCOl, except for the combined A mutants. Thus, these mutants are expected to have an increase half-life relative to the native VRCOl antibody (FIG. 1).
Example 3
Binding of the VRCOl Mutants to FcyRIIIa and RSC3 This example describes the methods used to measure the binding of the
VRCOl antibody mutants to the Fc fragment of IgG low affinity Ilia receptor (FcyRIIIa) and resurfaced stabilized core 3 (RSC3) as compared to the native VRCOl antibody. Antibody binding to FcyRIIIa provides an indication of ADCC activity. RSC3 is the resurfaced stabilized core from gpl20, and is the preserved antigenic structure of the neutralizing surface of the CD4 binding site but does not include other antigenic regions of HIV-1 Antibody binding to RSC3 provides an indication of the ability of the antibody to specifically bind gpl20.
Nickel-coated plates were incubated with His tagged recombinant FcyRIIIa for one hour, and washed with PBS, pH7.4 + 0.05 % Tween20 (washing buffer) (left). ELISA plates were coated with RSC3 for overnight, blocked with 5 % BSA in PBS, pH7.4, and washed with washing buffer (right). VRCOl and its Fc mutants diluted with 2.5%-BSA in PBS, pH7.4 were incubated for two hours and washed. Anti-human IgG-HRP in PBS, pH7.4 were incubated for one hour and washed. HRP substrate TMB was added and the color development was stopped by 0.1N H2S04. Optical density at 450 nm was measured by ELISA reader.
As shown in FIG. 4, all of the combined VRCOl antibody Fc mutants had higher binding to FcyRIIIa than the native VRCOl antibody, while all VRCOl antibody Fc mutants had higher binding to RSC3 than the native VRCOl. Thus, the combined mutants are indicated to have higher ADCC activity than the native VRCOl antibody, and all mutants bind to the gpl20 target the same as the native VRCOl antibody.
Example 4
ADCC Activity of VRCOl Mutants
This example describes the methods used to measure the antibody-dependent cellular cytotoxicity (ADCC) of the VRCOl antibody mutants as compared to the native VRCOl antibody.
HIV-infected CD4+CEM-NKr cells were double- stained with PKH26 and CFSE, and suspended in RPMI with 10%-FBS (Target cells). Human PBMC was prepared from human buffy coat by Ficoll gradient centrifugation, and suspended in RPMI with 10%-FBS (Effector cells). Target cells (10,000 cells/well) and serial dilutions of VRCOl Fc mutants were incubated together at R.T. for 15 min. Effector cells (500,000 cells/well) were added and incubated at 37 °C, C02 incubator for four hours. Percent killing was calculated from % CFSE negative within PKH26hi population.
As shown in FIG. 5, ADCC was lower for the hFcRn-binding enhancing mutations, while the combined LS, A and AAA mutants had stronger ADCC activities.
In summary, in the ADCC assay and ELISA binding assay, the combined LS and A mutants showed higher ADCC activities and higher binding to FcyRIIIa. That is, DE or DLE mutants combined with LS or A mutations have higher affinity to FcyRIIIa and functional ADCC activities. As shown in FIG. 2, LS and A mutants have mutations only at CH3, whereas DE and DLE mutations are made in CH2 region close to hinge. FIG. 6 shows the binding of IgG to hFcRn and FcyRIIIa. hFcRn binds to the CH2 and CH3 region of IgG, while FcyRIIIa bind to the hinge and CH2 regions of IgG Therefore, hFcRn-binding enhancing mutations did not affect (or affected less) the enhanced binding to FcyRIIIa by LS and A mutants. This may the reason why combined LS and A mutants showed higher ADCC activities than other mutants.
Example 5
Transcytosis of the Mutant mAbs
This example describes methods used to measure transcytosis of the antibody mutants as compared to the native antibody.
hFcRn is expressed in the epithelial cells in mucosa and mediates bidirectional transcytosis of human IgG across cellular epithelium. To demonstrate that hFcRn-binding enhancing mutants of VRCOl can increase transcytosis of the HIV neutralizing antibody, VRCOl, a monolayer of Madin Darby canine kidney (MDCK) cells expressing hFcRn was grown in transwell plates. The native VRCOl antibody or the Fc mutants were added to the basolateral side of the cells at 1.0 mg/mL of and incubated for 2 hours. The concentrations of the antibodies were then measured on the apical side by ELISA coated with RSC3 to compare the levels of transcytosis.
As shown in FIG. 8, the LS mutant groups (LS, DE-LS, and DLE-LS) showed a higher level of transcyotis than VRCOl or other mutant antibodies. Example 6
Half- Life of the Mutant mAbs
This example describes methods used to measure the half-life of the VRCOl antibody mutants as compared to the native VRCOl antibody.
2 mg/Kg of each mAb (native VRCOl, DE, LS, or DE-LS) was injected into a hFcRn Tg/Tg mouse by i.v. The concentrations of the mAbs in the sera were examined by indirect ELISA with RSC3-coated plates over time, and their half-life was calculated.
As shown in FIG. 9 and Table 6, the hFcRn-binding enhancing mutant antibody, LS, had a half-life about 2 times longer than VRCOl. Table 6. Half-life of antibodies in vivo
Half-life (days)
WT 2.65 + 0.28
DE 2.20 + 0.08
LS 4.47 + 0.52
DE-LS 2.15 + 0.11
Thus, in some examples the disclosed mutant antibodies, such as the LS antibodies, have a half-life that is greater than the native antibody (such as native VRCOl), such as an at least 1.5 fold, at least 1.8 fold, at least 2-fold, at least 2.5- fold, or at least 3-fold.
Example 7
Half-Life of the Mutant mAbs in Non-Human Primates
This example describes methods used to measure the half-life and the clearance of the VRCOl antibody mutants as compared to the native VRCOl antibody.
10 mg/Kg of each mAb (native VRCOl, DE, LS, or DE-LS) was injected into rhesus macaques by i.v. The concentrations of the mAbs in the sera were examined by indirect ELISA with RSC3-coated plates over time, and their half-life was calculated by WinNonlin software.
As shown in FIG. 10 and Table 7, the hFcRn-binding enhancing mutant antibody, LS had a half-life about 3 times longer than VRCOl, and the clearance rate of LS is about 2 times slower than VRCOl. Table 7. Half-life of antibodies in vivo
Half-life (days) Clearance (mL/day/Kg)
WT 4.88 + 0.71 13.34 + 1.03
LS 14.32 + 6.15 5.79 + 1.61
DE 3.40 + 0.41 24.27 + 4.35
DE-LS 5.17 + 1.08 12.51 + 2.75
Thus, in some examples the disclosed mutant antibodies, such as the LS antibodies, have a half-life that is greater than the native antibody (such as native VRCOl), such as an at least 1.5 fold, at least 1.8 fold, at least 2-fold, at least 2.5- fold, or at least 3-fold.
Example 8
SHIV challenge after the passive immunization with the Mutant mAbs
This example describes methods used to compare the protection immunity against SHIV challenge after the passive immunization with the mutant mAbs.
0.3 mg/Kg of each mAb (native VRCOl, DE, LS, or DE-LS) was injected into rhesus macaques by i.v. The passively immunized monkeys were challenged with repetitive, intrarectal SHIV BaL two days later. The number of challenges required to establish infection and plasma viral load over time was examined.
Example 9
In vivo ADCC activity
This example describes methods that can be used to measure ADCC activity of the antibody mutants as compared to the native antibody in vivo. It is expected that the ADCC activity of the mutant antibodies (e.g., mutant VRCOl antibodies) will be equal to or better than the native antibody (such as native VRCOl).
For example, hFcRn transgenic SCID mouse and monkey infected with SIV can be administered the disclosed hmAbs, and ADCC activity measured (for example as in Dall'Acqua et al. (J. Biol. Chem. 281:23514-24, 2006, herein incorporated by reference). Mutant and native hmAb ADCC activity can be compared. In one example mice are administered 200 μg Ab or 2 mg/kg and monkeys receive 20 mg/kg via i.v. administration.
Example 10
Fc mutant HIV-1 monoclonal neutralizing antibodies specific to gpl20 for detecting HIV-1 in a subject
This example describes the use of the disclosed HIV-1 monoclonal neutralizing antibodies specific to gpl20 having one or more Fc mutations for the detection of HIV-1 in a subject. This example further describes the use of these antibodies to confirm the diagnosis of HIV-1 in a subject. A biological sample, such as a blood sample is obtained from the patient diagnosed with, or suspected of having an HIV-1 infection. A blood sample taken from a patient who is not infected can be used as a negative control. An ELISA is performed to detect the presence of HIV-1 in the blood sample. Proteins present in the blood samples (the patient sample and control sample) are immobilized on a solid support, such as a 96-well plate, according to methods well known in the art (see, for example, Robinson et ah, Lancet 362: 1612-1616, 2003). Following immobilization, one or more mutant mAbs disclosed herein that is directly labeled with a fluorescent marker or quantum dot is applied to the protein-immobilized plate. The plate is washed in an appropriate buffer, such as PBS, to remove any unbound antibody and to minimize non-specific binding of antibody. Fluorescence can be detected using a fluorometric plate reader according to standard methods. An increase in fluorescence intensity of the patient sample, relative to the negative control sample, indicates the anti-gpl20 antibody with an Fc mutation specifically bound proteins from the blood sample, thus detecting the presence of HIV- 1 protein in the sample. Detection of HIV-1 protein in the patient sample indicates the patient has HIV-1, or confirms diagnosis of HIV-1 in the subject.
Example 11
Fc mutant HIV-1 monoclonal neutralizing antibodies specific to gpl20 for Treating HIV-1
This example describes a particular method that can be used to treat HIV in a human subject by administration of one or more of the disclosed Fc mutant gpl20 specific human neutralizing mAbs. Although particular methods, dosages, and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.
Based upon the teaching disclosed herein HIV-1 can be treated by administering a therapeutically effective amount of one or more of the neutralizing mAbs described herein, thereby reducing or eliminating HIV infection. Screening subjects
In particular examples, the subject is first screened to determine if they have an HIV infection. Examples of methods that can be used to screen for HIV include a combination of measuring a subject' s CD4+ T cell count and the level of HIV in serum blood levels. Additional methods using the gp 120- specific mAbs described herein can also be used to screen for HIV (e.g., see Example 10).
In some examples, HIV testing consists of initial screening with an enzyme- linked immunosorbent assay (ELISA) to detect antibodies to HIV, such as to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV- negative unless new exposure to an infected partner or partner of unknown HIV status has occurred. Specimens with a reactive ELISA result are retested in duplicate. If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (e.g., Western blot or an immunofluorescence assay (IFA)).
Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection.
Specimens that are repeatedly ELISA-reactive occasionally provide an
indeterminate Western blot result, which may be either an incomplete antibody response to HIV in an infected person, or nonspecific reactions in an uninfected person. IFA can be used to confirm infection in these ambiguous cases. In some instances, a second specimen will be collected more than a month later and retested for subjects with indeterminate Western blot results. In additional examples, nucleic acid testing (e.g. , viral RNA or proviral DNA amplification method) can also help diagnosis.
The detection of HIV in a subject's blood is indicative that the subject has
HIV and is a candidate for receiving the therapeutic compositions disclosed herein. Moreover, detection of a CD4+ T cell count below 350 per microliter, such as 200 cells per microliter, is also indicative that the subject is likely to have HIV.
Pre- screening is not required prior to administration of the therapeutic compositions disclosed herein F 're-treatment of subjects
In particular examples, the subject is treated prior to administration of a therapeutic agent that includes one or more antiretroviral therapies known to those of skill in the art. However, such pre-treatment is not required, and can be determined by a skilled clinician.
Administration of therapeutic compositions
Following subject selection, a therapeutically effective dose of a gpl20 specific neutralizing mAb described herein is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV or known to be infected with HIV). Additional agents, such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular,
intraperitoneal, or subcutaneous.
The amount of the composition administered to prevent, reduce, inhibit, and/or treat HIV or a condition associated with it depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV) in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. As such, these compositions may be formulated with an inert diluent or with a pharmaceutically acceptable carrier.
In one specific example, antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the particular stage of HIV. In an example, the antibodies are administered continuously. In another example, antibodies or antibody fragments are administered at 50 μg per kg given twice a week for 2 to 3 weeks.
Administration of the therapeutic compositions can be taken long term (for example over a period of months or years). Assessment
Following the administration of one or more therapies, subjects having HIV can be monitored for reductions in HIV levels, increases in a subjects CD4+ T cell count, or reductions in one or more clinical symptoms associated with HIV. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art . For example, biological samples from the subject, including blood, can be obtained and alterations in HIV or CD4+ T cell levels evaluated. Additional treatments
In particular examples, if subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same schedule and preparation of agents that they previously received for the desired amount of time, including the duration of a subject's lifetime. A partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70% in HIV infection, HIV replication or combination thereof. A partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter. In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:
1. An isolated antibody, wherein a heavy chain of the antibody comprises one or more amino acid substitutions in the Fc region, wherein the one or more amino acid substitutions comprise:
M428L/N434S;
T250Q/M428L;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A;
S239D/A330L/I332E + M252Y/S254T/T256E; or combinations thereof.
2. The isolated antibody of claim 1, wherein the one or more amino acid substitutions comprise M428L/N434S, S239D/I332E, or S239D/I332E +
M428L/N434S.
3. The isolated antibody of claim 2, wherein the one or more amino acid substitutions further comprise one or more of T250Q/M428L; N434A;
T307A/E380A/N434A; and M252Y/S254T/T256E.
4. The isolated antibody of any of claims 1-3, wherein the antibody comprises:
a light chain; and
a heavy chain comprising,
a sequence having at least 80%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, or
any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34,
wherein the antibody specifically binds gpl20 of HIV-1, and wherein the antibody is neutralizing.
5. The isolated antibody of any of claims 1-4, wherein the antibody comprises a light chain comprising:
amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID
NO: 36, or
a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 36, 40 or 44. 6. The isolated antibody of any of claims 1-5, wherein the antibody comprises a heavy chain and a light chain, wherein heavy chain of the antibody comprises any of SEQ ID NOS: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and the light chain of the antibody comprises SEQ ID NO: 36.
7. The isolated antibody of claim 4 or 5, wherein the heavy chain of the antibody comprises one or more of the following amino acid substitutions:
M428L/N434S;
T250Q/M428L;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E; S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A
S239D/A330L/I332E + M252Y/S254T/T256E; or
combinations thereof.
8 The isolated antibody of any of claims 1-7, wherein the antibody is a human monoclonal antibody.
9. An isolated human monoclonal antibody, comprising:
a light chain; and
a heavy chain, wherein the heavy chain comprises:
a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 38 or 42, and
one or more of the following amino acid substitutions:
M428L/N434S;
T250Q/M428L;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A; S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E,
wherein the antibody specifically binds gpl20 of HIV-1, and wherein the antibody is neutralizing.
10. The isolated human monoclonal antibody of claim 9, wherein: the heavy chain of the antibody comprises SEQ ID NO: 38 and one or more of the amino acid substitutions, and the light chain of the antibody comprises SEQ ID NO: 40; or
the heavy chain of the antibody comprises SEQ ID NO: 42 and one or more of the amino acid substitutions and the light chain of the antibody comprises SEQ ID NO: 44.
11. The isolated antibody of any of claims 1-10, wherein the antibody is an IgG, IgM or IgA.
12. An isolated functional fragment of the isolated antibody of any of claims 1-11.
13. The isolated antibody of any of claims 1-11, or the functional fragment of claim 12, wherein the antibody or functional fragment is labeled.
14. A composition comprising the antibody of any of claims 1-11, or the functional fragment of claim 12, and a pharmaceutically acceptable carrier.
15. An isolated nucleic acid molecule encoding the antibody of any of claims 1-11, or the functional fragment of claim 12.
16. The isolated nucleic acid molecule of claim 15, wherein the molecule comprises a nucleic acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37 or 41.
17. The isolated nucleic acid molecule of claim 16, encoding a heavy chain and a light chain of an antibody, wherein
(a) the heavy chain of the antibody comprises a sequence having at least 80%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and comprising one or more of the following mutations:
M428L/N434S;
T250Q/M428L;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E,
and wherein the light chain of the antibody comprises a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 36; (b) the heavy chain of the antibody comprises the heavy chain of the antibody comprises any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and wherein the light chain of the antibody comprises SEQ ID NO: 36;
(c) the heavy chain of the antibody comprises SEQ ID NO: 36 or 42 comprising one or more of the following mutations:
T250Q/M428L;
M428L/N434S;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E,
and wherein the light chain of the antibody comprises a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40 or 44;
(d) the heavy chain of the antibody comprises SEQ ID NO: 36 having one or more of the following mutations:
T250Q/M428L;
M428L/N434S;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E; S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E,
and wherein the light chain of the antibody comprises SEQ ID NO: 40; (e) the heavy chain of the antibody comprises SEQ ID NO: 42 having one or more of the following mutations:
T250Q/M428L;
M428L/N434S;
N434A;
T307A/E380A/N434A;
M252Y/S254T/T256E;
S239D/I332E;
S239D/A330L/I332E;
S239D/I332E + T250Q/M428L;
S239D/I332E + M428L/N434S;
S239D/I332E + N434A;
S239D/I332E + T307A/E380A/N434A;
S239D/I332E + M252Y/S254T/T256E;
S239D/A330L/I332E+ T250Q/M428L;
S239D/A330L/I332E+ M428L/N434S;
S239D/A330L/I332E+ N434A;
S239D/A330L/I332E+ T307A/E380A/N434A; or
S239D/A330L/I332E + M252Y/S254T/T256E, and wherein the light chain of the antibody comprises SEQ ID NO: 44, wherein the encoded antibody specifically binds gpl20 of HIV- 1, and wherein the encoded antibody is neutralizing.
18. The isolated nucleic acid molecule of any of claims 15-17, operably linked to a promoter.
19. An expression vector comprising the isolated nucleic acid molecule of any one of claims 15-18.
20. The expression vector of claim 19, wherein the antibody is an IgA or an IgG.
21. The expression vector of claim 19 or 20 comprising RNA splicing donor sites, RNA splicing acceptor sites and/or internal ribosomal binding sequences.
22. The expression vector of any of claims 19-21, wherein the heavy chain of the antibody and the light chain of the antibody are expressed as a fusion polypeptide following the introduction of the expression vector in a host cell.
23. The expression vector of claim 22, comprising a nucleic acid sequence encoding a furin cleavage site between the heavy chain and the light chain of the antibody.
24. The expression vector of any of claims 19-22, encoding a selectable marker.
25. An isolated host cell transformed with the nucleic acid molecule of any of claims 15-218 or the vector of any of claims 29-24.
26. A method of detecting a human immunodeficiency virus (HIV)-l infection in a subject comprising:
contacting a biological sample from the subject with at least one isolated antibody of any claims 1-11, or 13 or the functional fragment of claim 12;
detecting antibody bound to the sample; and
determining that the subject has an HIV-1 infection when the antibody is detected bound to the sample.
27. The method of claim 26, wherein the isolated antibody is directly labeled.
28. The method of claim 26 or 27, further comprising:
contacting the sample with a second antibody that specifically binds the isolated antibody;
detecting the binding of the second antibody; and
determining the presence of an HIV-1 infection in the subject when an increase in binding of the second antibody to the sample is detected as compared to binding of the second antibody to a control sample.
29. A method for treating an human immunodeficiency virus (HIV)-l infection in a subject, comprising:
administering to the subject a therapeutically effective amount of at least one antibody of any one of claims 1-11, or 13 or the functional fragment of claim 12, thereby preventing or treating the HIV-1 infection.
30. The method of claim 29, wherein the method is a method for treating an HIV-1 infection, and wherein the subject has acquired immune deficiency syndrome (AIDS).
31. The method of claim 29 or 30, further comprising administering to the subject an anti-viral agent.
32. The method of any one of claims 29-31, further comprising measuring HIV-1 viral titer in the subject.
33. A method for testing a potential vaccine, comprising:
contacting the potential vaccine with an antibody of any one of claims 1-11 , or 15 or the functional fragment of claim 13; and
detecting the binding of the antibody to an immunogen in the potential vaccine.
PCT/US2012/023737 2011-02-04 2012-02-03 HIV NEUTRALIZING ANTIBODIES HAVING MUTATIONS IN CONSTANT DOMAIN (Fc) WO2012106578A1 (en)

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