WO2009137632A2 - Immunogène du vih et son procédé de fabrication et d'utilisation - Google Patents

Immunogène du vih et son procédé de fabrication et d'utilisation Download PDF

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WO2009137632A2
WO2009137632A2 PCT/US2009/043054 US2009043054W WO2009137632A2 WO 2009137632 A2 WO2009137632 A2 WO 2009137632A2 US 2009043054 W US2009043054 W US 2009043054W WO 2009137632 A2 WO2009137632 A2 WO 2009137632A2
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Prior art keywords
hiv
immunogen
antibody
antibodies
human
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PCT/US2009/043054
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WO2009137632A3 (fr
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Dimiter S. Dimitrov
Mei-Yun Zhang
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Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services
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Publication of WO2009137632A2 publication Critical patent/WO2009137632A2/fr
Publication of WO2009137632A3 publication Critical patent/WO2009137632A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates generally to the field of vaccination, treatment and diagnosis of human immunodeficiency virus (HIV) infections.
  • HIV human immunodeficiency virus
  • the invention provides recombinant HIV immunogens and corresponding antibodies that are effective for the vaccination and/or immunization and/or treatment of HIV.
  • the invention further provides methods for making and using the immunogens and antibodies of the invention to vaccinate and/or treat and/or diagnose HIV infections.
  • HIV human immunodeficiency virus
  • Vaccine development has been elusive and made difficult due to genetic diversity of HIV-I and due to the fact that the virus rapidly mutates and "hides" conserved epitopes of its envelope glycoprotein by using variable loops, heavy glycosylation, oligomerization and conformational masking.
  • Key challenges in vaccine development have been to identify antigens that are capable of eliciting broadly cross-reactive neutralizing antibodies and to understand the epitope landscape providing for such antibodies, such as the epitope landscape occurring during the process of viral entry.
  • Enveloped viruses enter cells by a two-step process.
  • the first step involves the binding of a viral surface protein to receptors on the plasma membrane of a host cell. After receptor binding, a membrane fusion reaction takes place between the lipid bilayer of the viral envelope and host cell membranes. Viral proteins embedded in the lipid bilayer of the viral envelope catalyze receptor binding and membrane fusion reactions.
  • the Env glycoprotein performs the functions of viral entry. Env is synthesized as a polyprotein precursor molecule which is proteolytically processed by a host protease to generate the surface (gpl20) and transmembrane subunits (gp41) of the mature Env glycoprotein complex.
  • the unprocessed Env precursor is known as gpl60, reflecting its apparent molecular mass, which is further processed to form a transmembrane subunit, gp41, and an extracellular subunit, gpl20, which are assembled on the native virion surface as trimers of gpl20/gp41 noncovalently associated heterodimers. Both subunits are involved in the viral entry process and the eliciting of an immune response.
  • the initial step in HIV infection involves the binding of gpl20 to the cell surface molecule CD4, which serves as the major receptor for HIV-I and HIV-2.
  • the membrane fusion process is initiated by the interaction of gpl20 with a G protein-coupled co-receptor, either the CCR5 or the CXCR4 chemokine receptor, generally after prior contact of gpl20 with CD4.
  • Gp41 is involved in the fusion process; however its exact role in membrane fusion is not fully understood.
  • gp41 first engages contact with the target cell membrane by its amino- terminal hydrophobic domain, termed the fusion peptide, and then undergoes conformational changes in order to bring the viral and cellular lipid bilayers into proximity, allowing their external leaflets to merge, thereby forming a hemifusion intermediate.
  • an aqueous connection termed a fusion pore, must open across the internal leaflets of the merged membranes and expand to leave open passage to the nucleocapsid.
  • Gp41 like other retroviral transmembrane proteins, consists of an amino- terminal extracellular domain (or ectodomain), a membrane-spanning domain, and a carboxy-terminal cytoplasmic (or intraviral) domain. Mutations in the cytoplasmic domain can modify the efficiency of membrane fusion, but the ectodomain can continue to function despite the cytoplasmic domain's complete deletion, at least in syncytium-formation assays. Fusion activity is also observed when the gp41 ectodomain is anchored by the membrane-spanning domain of an unrelated protein such as CD4. Therefore, only the ectodomain of gp41 seems to have a direct role in the membrane fusion process.
  • gpl20 and gp41 of Env are involved in eliciting an immune response during an HIV infection.
  • the antibody response to Env is robust.
  • the antibodies elicted against HIV and Env usually have a narrow spectrum of neutralization activity.
  • some HIV-I -positive individuals induce broadly cross-reactive neutralizing antibodies that are believed to account for the containment of the virus.
  • Monomeric gpl20s can elicit neutralizing antibodies in vivo, but the neutralization activity is restricted to autologous viruses or the spectrum of neutralization activity is narrow. Recent findings, however, suggest that the induction of broadly cross-reactive antibodies is possible. For example, oligomeric forms of Env ectodomain from HIV-I, strain R2 (gpl4U R2 ), which contain both gpl20 and a truncated gp41 that lacks both transmembrane domains and cytoplasmic tails, induced better antibody response than gpl2U R2 alone.
  • the present invention provides HIV antigens or derivatives and/or fragments thereof and nucleic acid molecules encoding same, as well as methods of using same for vaccinating against an HIV infection and which are capable of eliciting broadly cross-reactive neutralizing antibodies against HIV, and which overcome the deficiencies in the art regarding known anti-HIV antibodies and HIV antigens, e.g., anti-gp41 antibodies and/or gp41 -based antigens.
  • the invention also provides diagnostic methods for detecting HIV infections using the HIV antigens or derivatives and/or fragments thereof or the anti-HIV antibodies of the invention, e.g., the recombinant anti-gp41 antibodies of the invention.
  • the present invention further provides methods for obtaining anti-HIV antibodies which are broadly cross- reactive neutralizing antibodies against HIV, e.g., the anti-gp41 antibodies of the invention. Also provided by the present invention are broadly cross-reactive neutralizing anti-HIV antibodies or fragments thereof and pharmaceutical compositions comprising such antibodies or fragments thereof, such as, the anti- gp41 antibodies described herein.
  • the invention also provides methods of utilizing the antigens and antibodies of the invention for immunizing against and/or treating HIV infections.
  • the invention further provides pharmaceutical and/or diagnostic packages comprising the HIV antigens and/or the anti-HIV antibodies of the invention for use in immunizing against or treating HIV infections or for detecting HIV in a subject or a biological sample.
  • the present invention provides an immunogen for vaccinating against and/or treating an HIV infection, comprising an HIV gp41 polypeptide or fragment thereof translationally linked to an Fc receptor ligand, optionally through a flexible linker.
  • the HIV gp41 polypeptide of the immunogen of the invention comprises at least the ectodomain of gp41. In other embodiments, the HIV gp41 polypeptide of the immunogen of the invention comprises a gp41 that lacks the cytoplasmic tail region, the transmembrane region or the fusion domain region, or any combination thereof.
  • the gp41 polypeptide of the immunogen of the invention is translationally fused to the amino-terminal end of the Fc receptor ligand. In other embodiments, the gp41 polypeptide of the immunogen of the invention is translationally fused to at its amino-terminal end to the carboxy-terminal end of a Fc receptor ligand.
  • the Fc receptor ligand can be an antibody Fc region or fragment thereof.
  • the Fc region is the Fc region of a human IgG.
  • the Fc region is the Fc region of any IgG, IgA, IgM, or IgE antibody.
  • the immunogen of the invention comprises a flexible linker that couples the gp41 portion of the immunogen with the Fc receptor ligand component of the immunogen.
  • the flexible linker can be a polypeptide having between 1 and 50 amino acids in length.
  • the flexible linker is a hinge region of an antibody, such as the hinge region of IgG or any other antibody.
  • the gp41 component of the immunogen of the invention corresponds with the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the flexible linker component of the immunogen of the invention is the hinge region of a human IgG having the amino acid sequence of SEQ ID NO: 6 or or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the Fc receptor ligand component of the immunogen of the invention is the CH2-CH3 region of a human IgG having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the gp41-Fc fusion protein immunogen of the invention is capable of eliciting broadly-cross reactive neutralizing antibodies against HIV, which are effective in vitro and in vivo.
  • the present invention provides a method of vaccinating a subject against an HIV infection comprising administering a therapeutically effective amount of a gp41-Fc fusion protein immunogen of of the invention.
  • the present invention provides a method of treating a subject having an HIV infection comprising administering a therapeutically effective amount of a gp41-Fc fusion protein immunogen and/or antibodies specific for or raised against the gp41-Fc immunogens of the invention.
  • the present invention provides isolated nucleic acid molecules which encode the gp41-Fc fusion protein immunogens and/or antibodies of the invention, as well as methods for preparing the nucleic acid molecules, and methods for using the nucleic acid molecule to make the antigens/immunogens and antibodies of the invention.
  • the present invention provides a recombinant HIV antigen comprising a human immunodeficiency virus envelope protein gp41 or fragment thereof, e.g., the ectodomain of gp41, which is coupled to an Fc (fragment crystallizable region) receptor ligand optionally by a flexible linker.
  • a human immunodeficiency virus envelope protein gp41 or fragment thereof e.g., the ectodomain of gp41, which is coupled to an Fc (fragment crystallizable region) receptor ligand optionally by a flexible linker.
  • the gp41 is a full length gp41 polypeptide from any known strain of HIV-I or HIV-2, e.g., or from any isolate obtained from an infected individual.
  • the gp41 is a fragment of gp41, wherein the fragment comprises at least the ectodomain of gp41 or a fragment of the ectodomain.
  • the gp41 is a gp41 fragment comprising the ectodomain or fragment thereof and at least one or both of the membrane-spanning domain and the carboxy-terminal cytoplasmic domain.
  • the recombinant HIV antigen comprises a human HIV gp41 ectodomain sequence defined by SEQ ID NO: 4 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto, and an Fc receptor ligand optionally coupled to the gp41 sequence or component through a flexible linker.
  • the Fc receptor ligand can be an Fc region or fragment thereof of an antibody, e.g., an IgG, or a polypeptide having an amino acid sequence that has at least about 80%, 85%, 90%, 95% or 99% sequence identity to the Fc region of an antibody.
  • the Fc receptor ligand can be an Fc region or fragment thereof from an IgG, IgA, IgD, IgM or an IgE antibody, or a polypeptide having an amino acid sequence with at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the Fc receptor ligand corresponds with the CH2-CH3 region of human IgG as defined by SEQ ID NO: 5, or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the recombinant HIV antigen is defined by SEQ ID NO: 3, which comprises an ectodomain of HIV-I gp41 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a human IgG Fc region, joined to the gp41 region via a flexible linker human IgG hinge region.
  • the recombinant HIV antigen comprises an human immunodeficiency virus envelope protein gp41 or fragment thereof which is coupled at its carboxy-terminal end to the amino-terminal end of an Fc receptor ligand optionally through a flexible linker, e.g., a human IgG hinge region.
  • thegp41 is defined by SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the flexible linker is not required, and the recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 or fragment thereof (e.g., gp41 ectodomain) is coupled directly to an Fc receptor ligand or fragment thereof, e.g., an IgG Fc region or fragment thereof.
  • the antigen comprises a gp41 protein or fragment thereof coupled at its carboxy-terminal end to the amino-terminal end of an Fc receptor ligand or fragment thereof, optionally through a flexible linker.
  • the antigen comprises an Fc receptor ligand or fragment thereof coupled at its carboxy-terminal end to the amino-terminal end of a gp41 protein or fragment thereof, optionally through a flexible linker.
  • the flexible linker can be any suitable molecule, such as, for example, a polypeptide of a suitable length and composition of amino acids.
  • the flexible linker can be single amino acid residue, or even a polypeptide having at least 2 amino acids in length, or to at least about 5 amino acids in length, or from about 5 to about 10 amino acids in length, or about 5 to about 25 amino acids in length, or about 5 to about 50 amino acids in length or even about 5 to 100 amino acids or more in length.
  • the linker corresponds to the hinge region of human IgG as defined by SEQ ID NO: 6, or an amino acid sequence or fragment thereof having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the hinge region of any antibody can be use as the flexible linker.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a recombinant gp41 antigen of the invention, and at least one pharmaceutically acceptable carrier or excipient.
  • the present invention provides a method of immunizing a subject against an HIV infection, comprising administering a therapeutically effective amount of a recombinant gp41 antigen of the invention.
  • the present invention provides a method of making a broadly cross-reactive neutralizing anti-HIV antibody, comprising raising the antibody against a recombinant HIV antigen of the invention.
  • the invention provides a method of making a broadly cross-reactive neutralizing anti-HIV antibody by screening an antibody library for antibodies that specifically bind to an antigen of the invention.
  • the invention also provides a method of making a broadly cross-reactive neutralizing anti-HIV antibody against HIV, comprising immunizing an animal with an antigen of the invention to elicit the production of anti-HIV antibodies in the animal, obtaining the sera of the animal, and isolating the anti-HIV antibodies from the sera.
  • the invention provides an isolated broadly cross- reactive neutralizing anti-HIV antibody against HIV isolated according to the methods of the invention.
  • the present invention provides a method of treating an HIV infection in a subject in need thereof, comprising: obtaining a broadly cross- reactive neutralizing anti-HIV antibody prepared by the methods of the invention; and administering a therapeutically effective amount of the anti-HIV antibody to the subject, thereby neutralizing the HIV infection and treating the subject.
  • the administration can be local, topical, or systemic (e.g., injection), or any other suitable mode of administration.
  • the present invention additionally provides a vaccine for immunizing a subject, e.g., a human, against an HIV infection, wherein the vaccine comprises a recombinant gp41 antigen of the invention.
  • vaccinating a human with a vaccine of the invention or administering an antigen or immunogen of the invention can further include the step of co-administering a suitable co-therapeutic agent, e.g., an anti- viral drug or an agent that modulates an immune response.
  • a suitable co-therapeutic agent e.g., an anti- viral drug or an agent that modulates an immune response.
  • the anti- viral drug can be, for example, an anti-HIV agent.
  • the present invention provides a packaged pharmaceutical comprising a recombinant antigen of the invention and instructions for use in accordance with a therapeutic method of the invention.
  • the instant invention provides for nucleic acid molecules encoding the recombinant antigens and antibodies of the invention.
  • the nucleic acid molecule is defined by the nucleic acid sequence of SEQ ID NO: 1, which encodes a gp41-Fc antigen of the invention, or a nucleic acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • the invention provides an expression vector comprising a nucleic acid molecule encoding a recombinant antigen of the invention, and host cells comprising the expression vectors of the invention.
  • the host cells can be capable of expressing the antigens and/or antibodies of the invention, i.e., the products encoded by the expression vectors of the invention, to allow a means for making the antigens and/or antibodies of the invention.
  • the present invention provides a method of making a human recombinant antigen of the invention comprising, coupling the 5 ' end of a nucleic acid molecule encoding the Fc receptor ligand to the 3' end of the nucleic acid molecule encoding gp41 or fragment thereof, optionally via a nucleic acid molecule encoding a flexible linker to form a gp41-Fc fusion gene encoding a recombinant gp41-Fc antigen; operably linking the gp41-Fc fusion gene to an expression vector; introducing the expression vector into a host cell; permitting the host cell to express the recombinant antigen; and isolating the recombinant antigen from the cell.
  • the invention still further provides diagnostic methods for detecting an HIV infection, including detecting HIV antigens and/or anti-HIV antibodies, utilizing the antigens and antibodies of the invention.
  • Figure 1 provides a schematic depicting the general structure of an embodiment of the HIV antigen of the invention, gp41Fc.
  • Figure 2 shows the results of an antibody- antigen assay to evaluate binding of anti-gp41 antibodies to a gp41Fc antigen of the invention.
  • Three-fold serially diluted anti-gp41 antibodies (Abs) were added to 96- well plates coated with 5 ⁇ g/ml of a gp41Fc of the invention.
  • Bound Abs were detected using HRP-conjugated anti- human IgG, F(ab')2 (for human Abs) or anti-mouse IgG (for NC-I and T3) and
  • Figure 3 depicts the titration of rabbit serum with a gp41Fc of the invention
  • Figure 4 provides a table showing the percentage HIV- 1 neutralization by purified IgG from immunized rabbit serum in a cell line/pseudovirus assay. All four HIV isolates, BaL (B), GXC-44(C), 92UG037.8(A), and CM243(E), were tested against 50 ⁇ g/ml of purified IgG sample. Pre-1214/1215: pre-immunization serum;
  • PB1-1214/1215 1 st test bleeding serum
  • PB2- 1214/1215 2 nd test bleeding serum
  • FT1214/1215 final bleeding serum.
  • Figure 5 provides an SDS-PAGE of a gp41Fc embodiment of the invention under non-denaturing and denaturing conditions.
  • M rainbow protein markers
  • lane 1 IgGl ml6, reduced
  • 2 gp41Fc, reduced
  • 3-4 empty 5: IgGl ml6 (non-reduced);
  • 6 gp41Fc (non-reduced).
  • Figure 6 shows the DNA coding (sense) strand sequence of gp41g 9 6 in vector pEAKIO (indicated as SEQ ID NO: 1). The single underlined portion at the 5' end of the molecule corresponds to the CD5ss signal peptide.
  • the double underlined portion at the 5' end corresponds to the Nhel restriction site.
  • the double underlined portion at the 3' end corresponds to the BamHI site.
  • the italicized region corresponds to the complete 89.6 gp41 region without the N-terminal fusion peptide-encoding region.
  • Figure 7 shows the sense (SEQ ID NO: 1) and antisense (SEQ ID NO. 2) strands of the DNA of SEQ ID NO: 1 of Figure 6.
  • the figure shows the predicted amino acid sequence as defined by the sense strand or coding strand (i.e., the top strand of DNA).
  • Figure 8 shows the amino sequence of a gp41Fc embodiment of the invention (SEQ ID NO: 3), which lacks the fusion domain, transmembrane domain and cytoplasmic tail of 89.6 gp41.
  • 89.6 gp41Fc in pEAKIO has 151 amino acids (22-172) and does not contain the fusion peptide (i.e., the first 20 amino acids at the N-terminus of gp41), or the transmembrane domain and cytoplasmic tail.
  • the complete gp41 extracellular portion has 171 amino acids in length.
  • the flexible linker region is shown in bold, which is formed by the hinge region of human IgG (EPKSCD KTHTCPPCP).
  • the Fc receptor ligand is CH2 and CH3 regions derived from human IgG (see Figure 10, SEQ ID NO: 5).
  • the DPE sequence is derived from the restriction site BamHI and becomes a part of the linker between gp41 and human Fc.
  • Figure 9 shows the amino sequence (SEQ ID NO: 4) of 89.6 gp41, which lacks the fusion domain, transmembrane domain and cytoplasmic tail, and which corresponds to the italicized portion of SEQ ID NO: 1.
  • Figure 10 shows the amino acid sequence of the CH2-CH3 region (Fc region) of human IgG, which corresponds to SEQ ID NO: 5.
  • Figure 11 shows the amino acid sequence of the hinge region of human IgG, which corresponds to SEQ ID NO: 6.
  • Figure 12 shows the amino acid sequence of an embodiment of the gp41Fc fusion protein of the invention which corresponds to SEQ ID NO: 7, including the gp41 sequence, flexible linker and IgG Fc region.
  • Figure 13 shows the nucleotide sequence (SEQ ID NO: 8) of an embodiment of the gp41Fc fusion of the invention in pEAKIO vector.
  • Underlined is the CD5ss signal peptide; bolded is the Nhel (gctagc) and BamHI (ggatcc) sites; in italics is the hinge region; highlighted is human CH2; highlighted and underlined is human CH3; bold and underlined region is the complete 89.6gp41 region without the N-terminal fusion peptideATG at the end is stop codon (TGA).
  • Figure 14 shows the binding of anti-HIV-1 Env mAbs to an gp41Fc fusion protein of the invention (SEQ ID NO: 7).
  • Three-fold serially diluted anti-gp41 mAbs were added to 96-well plates coated with 5 ⁇ g/mL of purified gp41Fc.
  • Bound Abs were detected using HRP conjugated to anti-human IgG, F(ab')2 (for human Abs) or anti-mouse IgG (H+L) (for mAbs NC-I and T3) and ABTS as substrate.
  • Figure 15 shows titration of rabbit serum.
  • One ⁇ g/mL 89.6gpl40 linker protein (left panel) or lug/mL of an gp41Fc fusion protein (right panel) of the invention (SEQ ID NO: 7) was coated on microwell plates. After blocking the wells, 2-fold serially diluted rabbit serum was added to the plates. Bound Abs were detected using HRP- anti-rabbit IgG, F(ab') 2 and ABTS as substrate.
  • Figure 16 shows that neutralization activity of immune rabbit IgG was decreased in the presence of increased concentration of a gp41Fc fusion protein of the invention (SEQ ID NO: 7) in neutralizing pseudo-typed HIV-I (BaI).
  • SEQ ID NO: 7 a gp41Fc fusion protein of the invention
  • Fifty ⁇ g/mL FT1214 IgG was tested in cell line-based pseudovirus assay against HIV-I isolate BaI in the presence of serially diluted gp41Fc. Gp41Fc alone was also tested in the assay.
  • Figure 17 shows that binding of immune rabbit IgG to non-denatured and denatured gpl4U 89 6 and a gp41Fc fusion protein of the invention (SEQ ID NO: 7).
  • Two ⁇ g/mL non-denatured and denatured gp 1408 9 6 and gp41Fc were coated on micro well plates.
  • Three-fold serially diluted rabbit IgG were added to the wells. Bound antibodies were detected using HRP conjugated to anti-rabbit IgG (H+L) and ABTS as substrate.
  • Figure 18 shows binding of rabbit IgGs to peptides derived from gp41 immunodominant loop region. Five ⁇ g/mL of each peptide was coated on high- binding microwell plates.
  • Figure 19 shows the results of capture ELISA of a gp41Fc fusion protein (SEQ ID NO: 7) of the invention in immune rabbit IgG.
  • Gp41Fc in the rabbit IgGs purified from immune rabbit sera collected from different time of bleed was captured by mouse mAb T3 coated on 96-well microplates.
  • Bound gp41Fc was detected by using biotinylated mouse mAb D61 and HRP-conjugate to streptavidin. The optical density was measured 30min after addition of ABTS (top left and top right panels).
  • the concentration of gp41Fc in purified rabbit IgG was calculated using purified recombinant gp41Fc as a standard and then converted to the concentration of gp41Fc in rabbit serum (middle panel and corresponding bottom table).
  • the present inventors have for the first time developed gp41 -based HIV antigens which advantageously elicit antibodies having broad neutralization activity against different HIV isolates, are highly immunogenic, exhibit high stability and enhanced half-life, and exhibit in vivo effectiveness, while lacking the disadvantages of prior gp41 -based HIV antigens, including problems of eliciting self-reacting antibodies, having low immunogenicity and exhibiting in vivo ineffectiveness.
  • the gp41 -based antigens provided by the invention fuse HIV gp41 or a fragment thereof (e.g., gp41 ectodomain) with an Fc receptor ligand, e.g., the Fc region of a human IgG), optionally through a flexible linker (e.g., the hinge region of human IgG), wherein the gp41 fusion protein stabilizes the structure of gp41 in the absence of gpl20, and exhibits enhanced immunogenicity by enabling it to bind to immune cells having Fc receptors, such as, macrophages or dendritic cells.
  • the invention further provides methods and compositions for vaccinating and/or immunizing a subject against an HIV infection by administering a therapeutically effective amount of the gp41 -based HIV antigen of the invention or a fragment thereof, or a composition thereof.
  • the present invention also provides antibodies specific for and/or raised against the gp41 -based antigen of the invention, methods for obtaining the antibodies, and compositions comprising the antibodies of the invention.
  • the antibodies of the invention are advantageously provided with broad cross-reactive neutralization activity against a wide spectrum of HIV isolates and/or clades and can be utilized in therapeutic methods for treating an HIV infection.
  • Compositions comprising the antibodies can be prepared for systemic, e.g., parenteral administration, or local administration, e.g., topical creams, ointments, or suaves for applying to bodily fluids that may comprise HIV viruses, or for administering by any other suitable means.
  • the instant invention further provides diagnostic methods and compositions comprising the antigens and/or antibodies of the invention for clinical and/or research purposes for detecting HIV viruses, antigens and/or anti-HIV antibodies.
  • the present invention also provides pharmaceutical and/or diagnostic packages or kits comprising the antigens and/or antibodies of the invention, instructions for use, and additional materials and components suitable for a particularly desired diagnostic or therapeutic use.
  • the instant invention provides for nucleic acid molecules encoding the recombinant antigens and antibodies of the invention, as well as expression vectors comprising the nucleic acid molecules and host cells for expressing the encoded proteins.
  • the nucleic acid molecule is defined by the nucleic acid sequence of SEQ ID NO: 1, which encodes a gp41-Fc antigen of the invention, or a nucleic acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • antibody is meant to refer to immunoglobulin molecules (e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or any class, including, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) isolated from nature or prepared by recombinant means or chemically synthesized.
  • immunoglobulin molecules e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or any class, including, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • immunoglobulin molecules e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or any class, including, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • antibody fragment is meant to refer to a portion of a whole antibody which retains the ability to exhibit antigen binding activity or immunogenicity. Examples include, but are not limited to, Fv, disulphide-linked Fv, single-chain Fv, Fab, variable heavy region (V H ), variable light region (V L ), and fragments of any of the above antibody fragments which retain the ability to exhibit antigen binding activity, e.g., a fragment of the variable heavy region V H retains its ability to bind its antigen.
  • any technique known in the art can be used (see, e.g., Kohler and Milstein, Nature 256:495497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in
  • immunoassay is meant to refer to an assay that uses an antibody to specifically bind to and detect an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • biological sample or "patient sample” as used herein, is meant to refer to a sample obtained from an organism or from components (e.g., cells) of an organism.
  • the sample can be of any biological tissue or fluid.
  • the sample may be a clinical sample which is a sample derived from a patient.
  • Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue samples, biopsy samples, urine, peritoneal fluid, and pleural fluid, saliva, semen, breast exudate, cerebrospinal fluid, tears, mucous, lymph, cytosols, ascites, amniotic fluid, bladder washes, and bronchioalveolar lavages or cells therefrom, among other body fluid samples.
  • the patient samples may be fresh or frozen, and may be treated, e.g. with heparin, citrate, or EDTA, or other suitable treatment known in the art.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Samples can be infected with HIV.
  • epitope is meant to refer to any antigenic determinant on an antigen, e.g., a gpl20 or gp41 protein, to which an antibody binds through an antigenic binding site.
  • Determinants or antigenic determinants on an antigen usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the epitope can be an inducible epitope, i.e., an epitope that becomes available or accessible only once exposed by a change in conformation, e.g., the CD4-inducible epitope of gp41.
  • antibody that "specifically (or selectively) binds to" or is “specific for” or is “specifically (or selectively) immunoreactive with” a particular antigen, e.g., a polypeptide, or a particular epitope on a particular antigen, e.g., a polypeptide epitope, is one that binds to that particular antigen or epitope without substantially binding to any other antigen or epitope.
  • Antibody affinity for antigens and epitopes can be measured by enzyme linked immunosorbent assay (ELISA) or other suitable affinity tests.
  • An antibody that specifically binds to an antigen refers to the binding of an antigen by an antibody or fragment thereof with a dissociation constant (K d ) of 1 ⁇ M or lower, as measured by surface plasmon resonance analysis using, for example, a BIACORE surface plasmon resonance system and BIACORE kinetic evaluation software (eg. version 2.1).
  • K d dissociation constant
  • the affinity or dissociation constant (K d ) for a specific binding interaction is preferably about 500 nM or lower, more preferably about 300 nM or lower and preferably at least 300 nM to 50 pM, 200 nM to 50 pM, and more preferably at least 100 nM to 50 pM, 75 nM to 50 pM, 10 nM to 50 pM.
  • antigen refers to a substance, e.g., a polypeptide or polysaccharide or nucleic acid molecule, which can specifically bind to or is specifically recognized by an antibody. Antigens can, but may not necessarily, prompt an immune response.
  • immunogen refers to a subclass of antigen which are capable of eliciting a response by the immune system, i.e., the eliciting of antibodies that specifically bind to the immunogen.
  • HIV antigen refers to a substance that is capable of eliciting antibodies that specifically recognize HIV or a component thereof, e.g., an HIV polypeptide or nucleic acid molecule.
  • the HIV antigen can be obtained from HIV or prepared synthetically and can include any suitable modification (e.g., chemical or translational fusion to heterologous polypeptide to form fusion protein, including gp41-Fc fusion protein of the invention.
  • gpl60 refers to the human immunodeficiency virus-1 envelope glycoprotein gpl60 kDa (or its corresponding gene), which is processed to form the 120 kDa (gpl20) subunit and the 41 kDa (gp41) subunit.
  • gpl20 or "gpl20 subunit”, as used herein, is meant to refer to the human immunodeficiency virus-1 envelope glycoprotein gpl20.
  • gpl20 variant or "gpl20 derivative” refers to a protein which is characterized by: (1) having an amino acid subsequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably
  • nucleic acid and amino acid sequences of HIV gp-120 are readily available to the public through the HIV sequence database on the world wide web at hiv.lanl.gov/content/sequence/HIV/mainpage.html; (2) binding to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of HIV-I gpl20; (3) specifically hybridizing under stringent hybridization conditions to a nucleic acid sequence encoding HIV-I gpl20 and (4) having a nucleic acid sequence that has greater than about 85%, preferably greater than about 90%, 95%, 98%, 99%, or higher nucleotide sequence identity to the nucleic acid sequence encoding HIV-I gpl20.
  • gp41 or "gp41 subunit”, as used herein is meant to refer to the human immunodeficiency virus-1 envelope glycoprotein gp41.
  • gp41 variant or "gp41 mutant” refers to a protein which is characterized by: (1) having an amino acid subsequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably
  • nucleic acid and amino acid sequences of HIV gp-41 are readily available to the public through the HIV sequence database on the world wide web at hiv.lanl.gov/content/sequence/HIV/mainpage.html; (2) binding to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of HIV-I gp41; (3) specifically hybridizing under stringent hybridization conditions to a nucleic acid sequence encoding HIV-I gp41 and (4) having a nucleic acid sequence that has greater than about 85%, preferably greater than about 90%, 95%, 98%, 99%, or higher nucleotide sequence identity to the nucleic acid sequence encoding HIV-I gp41.
  • the "fusion domain" of HIV-I gp41 is meant to refer to the domain comprising the first 20 amino acids at the N-terminus of gp41.
  • the fusion domain of gp-41 corresponds to position 512-532 according to HXB2 numbering.
  • extracellular domain or “ectodomain” or HIV-I gp41 is meant to refer to the part of the protein that is on the outside of the cell.
  • the extracellular domain of gp41 consists of position 512 to 683 according to HXB2 numbering.
  • transmembrane domain of HIV-I gp41 is meant to refer to the region corresponding to the portion of the protein that spans the cell. In certain examples, the transmembrane domain of gp-41 corresponds to the position 684 to 703 according to HXB2 numbering.
  • cytoplasmic tail of HIV-I gp 41 is meant to refer to the region corresponding to the portion of the protein that interacts with the interior of the cell or organelle, n certain examples, the cytoplasmic tail of gp-41 corresponds to position 704 to 846 according to HXB2 numbering.
  • the term “flexible linker” refers to any heterologous polypeptide of at least 1, 2, 3, 4 or 5 or more amino acids in length, which when inserted between the carboxy-terminal end of gp41 and the amino-terminal end of a Fc receptor ligand yields a functional linkage joining the gp41 region with the Fc receptor ligand.
  • a flexible linker is not required, and the gp41 and the Fc receptor ligand are joined without a linker.
  • Fc or “Fc region,” as used herein, refers to the fragment crystallizable region ("Fc region”) or an antibody, which is known as the tail region of an antibody. It will be appreciated that the Fc region of an antibody interacts with or function as ligands for cell surface receptors called “Fc receptors,” which are protein receptor molecules found on the surface of certain cells, including macrophages and neutrophils, and which play a role in the immune system.
  • Fc receptors are activated upon binding to a cognate Fc region of an antibody, which in turn stimulates the activities of cytotoxic and phagocytic cells against invading pathogens or unwanted cells or substances, thereby mediating various physiological effects of antibodies, such as opsonization, cellular lysis, and cell degranulation.
  • the Fc regions or fragments thereof can include those Fc regions from any type of antibody, including IgG, IgA, IgD, IgM and IgE antibody types.
  • Fc receptors are classified based on the type of antibody that they recognize, and include, for example, Fc receptors that recognize IgG Fc regions ("Fc-gamma receptors"), IgA Fc regions ("Fc-alpha receptors”) and IgE Fc regions ("Fc-epsilon receptors").
  • Fc-gamma receptors IgG Fc regions
  • Fc-alpha receptors IgA Fc regions
  • Fc-epsilon receptors IgE Fc regions
  • Fc receptor ligand refers to any Fc region (or fragment thereof) capable of being specifically recognized and/or bound by an Fc receptor and which causes the Fc receptor to become activated, thereby triggering the downstream physiological responses associated with the binding of an Fc receptor with an Fc region.
  • regulatory sequences refers to those sequences, both 5' and 3' to a structural gene, that are required for the transcription and translation of the structural gene in the target host organism. Regulatory sequences include a promoter, ribosome binding site, optional inducible elements and sequence elements required for efficient 3' processing, including polyadenylation. When the structural gene has been isolated from genomic DNA, the regulatory sequences also include those intronic sequences required for splicing of the introns as part of mRNA formation in the target host.
  • gp41 fusion protein refers to a polypeptide comprising both a gp41 protein or fragment thereof (e.g., the ectodomain of gp41) translationally fused to an Fc receptor ligand.
  • the order of the polypeptides of the fusion protein is not limited.
  • the gp41 fusion protein can be a fusion of the C- terminal end of gp41 (or fragment thereof) with the N-terminal end of an Fc receptor ligand.
  • the gp41 protein can be a fusion of the C-terminal end of the Fc receptor ligand with the N-terminal end of the gp41 protein or fragment thereof.
  • the gp41 fusion protein may comprise a flexible linker between the gp41 and Fc receptor ligand portions; however, the flexible linker is not absolutely required.
  • ligand-receptor complexes refers to a specific association between a fusion protein and the extracellular subunit of HIV receptors or with an Fc receptor on an effector immune cell.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.
  • purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide - nucleic acids (PNAs).
  • PNAs peptide - nucleic acids
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides likewise, may be referred to by their commonly accepted single-letter codes.
  • a "nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • the probes can be directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • an "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • host cell is meant a cell that contains an expression vector and supports the replication or expression of the expression vector.
  • Host cells can be mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo, or bacterial host cells.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, 65%, 70%, 75%, 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence or a nucleotide sequence when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • sequences are then said to be “substantially identical.”
  • This definition also refers to the compliment of a test sequence.
  • the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of HIV envelope glycoproteins fusion proteins comprising envelope glycoproteins and nucleic acid sequences encoding the same, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoI. Biol.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nln.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10C° lower than the thermal melting point (Tm for the specific sequence at a defined ionic strength pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T.sub.m 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30C for short probes (e.g., 10 to 50 nucleotides) and at least about 6OC for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5xSSC and 1% SDS incubated at 42C or 5xSSC and 1% SDS incubated at 65C, with a wash in 0.2xSSC and 0.1% SDS at 65C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37C, and a wash- in 1. times. SSC at 45C A positive hybridization is at least twice background.
  • gp41 -based HIV antigen refers any antigen comprising gp41 or a fragment or derivative thereof.
  • vaccine is meant to encompass any immunogenic composition that is capable of inducing an immune response in a subject which establishes or enhances immunity to a particular disease, such as HIV, thereby either vaccinating against a future infection or treating an existing infection.
  • immune response is meant to include responses that result in at least some level of immunity in the treated subject thereby treating an existing infection or vaccinating against a future infection.
  • the nucleic acid sequences encoding HIV envelope glycoproteins may be obtained by recombinant DNA methods, such as screening reverse transcripts of mRNA, or screening genomic libraries from any HIV-infected cell or HIV isolate.
  • the DNA may also be obtained by synthesizing the DNA from published sequences using commonly available techniques such as solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984).
  • Synthesis may be advantageous because unique restriction sites may be introduced at the time of preparing the DNA, thereby facilitating the use of the gene in vectors containing restriction sites not otherwise present in the native source. Furthermore, any desired site modification in the DNA may be introduced by synthesis, without the need to further modify the DNA by mutagenesis.
  • Purification of oligonucleotides is by either native acrylamide gel electrophoresis, agarose electrophoresis or by anion-exchange HPLC as described in Pearson and Reanier, J. Chrom. 255:137-149 (1983), depending upon the size of the oligonucleotide and other characteristics of the preparation. The sequence of cloned genes and synthetic oligonucleotides can be verified using, e.g., the chain termination method for sequencing double- stranded templates as described by Wallace et al., Gene 16:21-26 (1981).
  • DNA encoding the envelope glycoproteins described herein can be obtained by constructing a cDNA library from mRNA recovered from field or laboratory isolates and (1) screening with labeled DNA probes encoding portions of the envelope glycoprotein sought in order to detect clones in the cDNA library that contain homologous sequences or (2) amplifying the cDNA using polymerase chain- reaction (PCR) and subcloning and screening with labeled DNA probes.
  • PCR polymerase chain- reaction
  • Clones can then be analyzed by restriction enzyme analysis, agarose gel electrophoresis sizing and nucleic acid sequencing so as to identify full-length clones and, if full-length clones are not present in the library, recovering appropriate fragments from the various clones and ligating them at restriction sites common to the clones to assemble a clone encoding a full-length molecule.
  • DNA probes for envelope glycoproteins are common in the art and can be prepared from the genetic material set forth in SEQ ID NO: 1.
  • sequences missing from the 5' end of the cDNA may be obtained by the 3' extension of the synthetic oligonucleotides complementary to sequences encoding the protein using mRNA as a template (so- called primer extension), or homologous sequences may be supplied from known cDNAs.
  • Polynucleic acid sizes are given in either kilobases (Kb) or base pairs (bp). These sizes are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • Amplification techniques using primers can also be used to isolate HIV envelope glycoproteins from DNA or RNA.
  • Suitable primers are commonly available in the art, or can be derived from SEQ ID NO: 1, then synthesized by conventional solid-phase techniques common in the art and described.
  • Primers can be used, e.g., to amplify either the full length sequence or a probe of one to several hundred nucleotides, which is then used to screen a library for full-length HIV envelope glycoproteins.
  • Nucleic acids encoding HIV envelope glycoproteins can also be isolated from expression libraries using antibodies as probes. Such polyclonal or monoclonal antibodies can be raised using the sequence of SEQ ID NO:3, or any immunogenic portion thereof. HIV envelope glycoprotein strain variants and orthologs can be isolated using corresponding nucleic acid probes known in the art to screen libraries under stringent hybridization conditions. Alternatively, expression libraries can be used to clone sequences encoding HIV envelope glycoprotein strain variants and orthologs by detecting expressed proteins immunologically with commercially available antisera or antibodies, or antibodies made against SEQ ID NO:3, or portions thereof, which also recognize and selectively bind to the HIV envelope glycoprotein strain variants and orthologs.
  • a source that is rich in the HIV envelope glycoprotein(s) of interest such as the primary R5X4 HIV-I isolate 89.6 described in Collman, R, et al. "An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1", J. Virol., 66, 7517-7521 (1992).
  • the mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of HIV envelope glycoprotein-encoding mRNA in physiological samples, for nucleic acid sequencing, or for other purposes.
  • Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • HIV envelope glycoprotein gene expression can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A.sup.+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, high density polynucleotide array technology and the like.
  • Synthetic oligonucleotides can be used to construct recombinant HIV envelope glycoprotein genes for use as probes or for expression of protein. This method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and non-sense (antisense) strands of the gene. These DNA fragments are then annealed, ligated and cloned. Alternatively, amplification techniques can be used with precise primers to amplify a specific gene subsequences for HIV envelope glycoproteins. The specific subsequence is then ligated into a suitable eukaryotic expression vector.
  • DNA encoding HIV envelope glycoprotein strain variants and orthologs typically show at least 70% sequence identity between strains, as defined supra, and are capable of selectively cross-hybridizing when annealed under stringent hybridization conditions. Coding regions for field isolates of gpl60/140, gpl20 or gp41 will typically not vary in length by more than 6 base pairs.
  • HIV envelope glycoprotein genes can also be identified by reference to the proteins produced when expressed in a eukaryotic system.
  • a nucleic acid sequence or a restriction fragment putatively encoding gpl60 can be inserted into a vector capable of transfecting a eukaryotic cell, providing a recombinant vector.
  • the vector can then be used to transfect a eukaryotic cell capable of expressing the gpl60 human immunodeficiency virus envelope protein.
  • the cell preparation can be tested for the presence of the HIV envelope using one of the protein-specific assays described infra.
  • the invention provides a gp41 -based HIV antigen which advantageously exhibits broad cross-reactivity to HIV antibodies, elicits antibodies having broad neutralization activity against different HIV isolates, is highly immunogenic, exhibits high stability and enhanced half-life, and exhibits in vivo effectiveness, while lacking the disadvantages of prior art gp41 -based HIV antigens, including problems of eliciting self -reacting antibodies, having low immunogenicity and exhibiting in vivo ineffectiveness.
  • the gp41-based antigen provided by the invention fuses HIV gp41, or a fragment thereof, with an Fc receptor ligand, e.g., the Fc portion of a human antibody, which stabilizes the structure of gp41 in the absence of gpl20, and enhances the immunogenecity of the antigen by enabling it to bind to immune cells having Fc receptors, such as, macrophages or dendritic cells.
  • an Fc receptor ligand e.g., the Fc portion of a human antibody
  • the invention provides a gp41 -based antigen that fuses a portion of HIV-I gp41 with the Fc portion of human IgG, which forms a highly immunogenic and stable antigen that is broadly cross-reactive with anti-HIV antibodies and which can be utilized in a variety of therapeutic and diagnostic methods, including as an immunogen for immunizing a subject against a broad range of HIV infections.
  • This gp41 fusion protein can also be utilized diagnostically to detect anti-HIV antibodies in a sample.
  • the present invention provides an HIV antigen comprising a 151 amino acid portion of the extracellular domain of gp41 from HIV- 1 strain 89.6 (i.e., excluding the fusion domain, transmembrane domain and cytoplasmic tail domains) which is fused at its C-terminus to the N-terminus of the Fc domain (CH2-CH3 region) of human IgG joined through the IgG hinge (H) domain.
  • This antigen is broadly cross-reactive against antibodies specific for a wide range of HIV isolates and features enhanced stability and half- life.
  • the Fc moiety is thought to provide the antigen with enhanced immunogenicity by enabling its binding to immune effector cells having Fc receptors, such as macrophages and dendritic cells.
  • the gp41Fc antigen of the invention is highly immunogenic.
  • antibodies raised in response to immunization with gp41Fc in test animals were shown to have broad HIV neutralization activity against several HIV isolates from different clades.
  • gp41Fc was found to elicit broadly cross reactive antibodies which did not react with self antigens - both desirable characteristics for an HIV vaccine.
  • linker Any linker envisioned by one of skill in the art can be used in the instant invention. In certain embodiments, a linker is not required, and the gp-41 and the Fc receptor ligand are joined without a linker.
  • the invention also provides for neutralization antibodies that are specifically immunoreactive against the gp41-based antigens of the invention, e.g., gp41Fc, and which are broadly cross reactive against a wide spectrum of HIV isolates.
  • a cell line/ pseudovirus assay is used as a neutralization assay.
  • Such assays are well-known in the art and easily performed by the skilled practicioner.
  • Montefiore et al. describe neutralizing antibody assays as tools for assessing humoral immunity in AIDS virus infection and vaccine development.
  • This reference describes two assays utilizing a genetically engineered cell lines that are susceptible to infection by most strains of HIV-I, SIV, and SHIV.
  • One assay is designed for optimal performance with uncloned viruses produced in either PBMC or CD4(+) T cell lines.
  • a second assay is designed for single-cycle infection with molecularly cloned pseudoviruses produced by transfection in 293T cells. Both assays are performed in a 96-well format and use tat-responsive luciferase reporter gene expression as readout.
  • PBMC peripheral blood mononuclear cells
  • assays are well-known in the art and easily performed by the skilled practicioner. For example, Montefiore et al. (J. Virol., 03 1997, 2512-2517, VoI 71, No.
  • HIV-I human immunodeficiency virus type 1
  • PBMC peripheral blood mononuclear cells
  • various host animals may be immunized by injection with a protein, or a portion thereof (e.g. any one of the SEQ ID Nos 1 - 7 of the invention or fragments thereof).
  • Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a cystatin gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection with a cystatin gene product supplemented with adjuvants as also described above.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein (1975) Nature 256:495-497; and U.S. Pat. No. 4,376,110, the human B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today 4:72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • chimeric antibodies or “humanized antibodies” may be utilized to modify mouse monoclonal antibodies to reduce immunogenicity of non-human antibodies.
  • Such antibodies are generated by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments may include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed (Huse et al. (1989) Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the method according to an embodiment of the present invention may further comprise screening an antibody for neutralization antibodies that are specifically immunoreactive against the gp41 -based antigens of the invention, e.g., gp41Fc, and which are broadly cross reactive against a wide spectrum of HIV isolates.
  • an antibody for neutralization antibodies that are specifically immunoreactive against the gp41 -based antigens of the invention, e.g., gp41Fc, and which are broadly cross reactive against a wide spectrum of HIV isolates.
  • the screening may be performed using column chromatography filled with, for example, peptides having amino acid sequences of SEQ ID NOs. 1 - 7, or fragments thereof.
  • the neutralizing anti-gp41 antibodies of the invention can be further modified by methods known in the art. The modifications may be genetic modifications to the nucleic acid encoding the antibodies of the invention or they may be chemical, structural, or physical modifications made directly to an isolated antibody of the invention to impart additional advantageous properties to an antibody of the invention regarding, for example, the level of expression, stability, solubility, epitope affinity, antigen neutralization activity, or penetration characteristics, etc.
  • the present invention contemplates introducing genetic modifications into one or more CDRs or to the framework sequence of the antibodies of the invention which are identified by library screening methods.
  • Such genetic modifications can confer additional advantageous characterics, i.e. genetic optimization, of the antibodies identified from library screening, including, for example, enhanced solubility, enhanced affinity, and enhanced stability.
  • Any type of genetic modification is contemplated by the present invention, including, for example, site-directed mutagenesis, random mutagenesis, insertions, deletions, and CDR grafting (i.e. genetic replacement of one CDR for another CDR). All of these techniques are well known to those skilled in the art.
  • modifications contemplated by the present invention relate to chemical modifications of the antibodies of the invention to confer additional advantageous features, such as enhanced stability and/or solubility and/or half-life.
  • the antibodies of the present invention can be
  • PEGylated or coupled to polymers of similar structure, function and purpose (“PEG or PEG-like polymers"), to confer enhanced stability and half- life.
  • PEGylation can provide increased half- life and resistance to degradation without a loss in activity (e.g. binding affinity) relative to non-PEGylated antibody polypeptides.
  • PEGylation may not be advantageous with respect to some targets, in particular, those epitopes which are sterically-obstructed.
  • the antibody should be minimally PEGylated so as not to negatively impact the accessibility of the antibody to the size -restricted antigen.
  • this general principle should be applied to any modifications made to the antibodies of the invention.
  • PEG or PEG-like moieties which can be utilized in the invention can be synthetic or naturally occurring and include, but are not limited to, straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymers, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.
  • Preferred examples of synthetic polymers which can be used in the invention include straight or branched chain poly(ethylene glycol) (PEG), poly(propylene glycol), or poly(vinyl alcohol) and derivatives or substituted forms thereof.
  • Substituted polymers for linkage to the antibodies of the invention can also particularly include substituted PEG, including methoxy(polyethylene glycol).
  • Naturally occurring polymer moieties which can be used in addition to or in place of PEG include, for example, lactose, amylose, dextran, or glycogen, as well as derivatives thereof which would be recognized by persons skilled in the art.
  • PEGylation of the antibodies of the invention may be accomplished by any number of means (see for example Kozlowski-A & Harris -J M (2001) Journal of Controlled Release 72:217).
  • PEG may be attached to an antibody construct either directly or by an intervening linker.
  • the first step in the attachment of PEG or other polymer moieties to the antibody construct of the invention typically is the substitution of the hydroxyl end-groups of the PEG polymer by electrophile-containing functional groups.
  • PEG polymers are attached to either cysteine or lysine residues present in the antibody construct monomers or multimers. The cysteine and lysine residues can be naturally occurring, or can be engineered into the antibody molecule.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monomethoxy polyethylene glycol (MPEG) using tresylchloride. Following reaction of amino acid residues with tresylated MPEG, polyethylene glycol is directly attached to the amine groups.
  • MPEG monomethoxy polyethylene glycol
  • the invention includes protein-polyethyleneglycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
  • U.S. Pat. No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
  • Protein- polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'- carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG- succinate derivatives.
  • MPEG-succinimidylsuccinate MPEG activated with 1,1'- carbonyldiimidazole
  • MPEG-2,4,5-trichloropenylcarbonate MPEG-p- nitrophenolcarbonate
  • MPEG- succinate derivatives A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are
  • polymer molecules include, for example, derivatives which have additional moieties or reactive groups present therein to permit interaction with amino acid residues of the antibodies described herein.
  • derivatives include N-hydroxylsuccinimide (NHS) active esters, succinimidyl propionate polymers, and sulfhydryl- selective reactive agents such as maleimide, vinyl sulfone, and thiol.
  • the reactive group e.g., MAL, NHS, SPA, VS, or Thiol
  • the size of polymers useful in the invention can be in the range of 500 Da to
  • 60 kDa for example, between 1000 Da and 60 kDa, 10 kDa and 60 kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40 kDa and 60 kDa, and up to between 50 kDa and 60 kDa.
  • the polymers used in the invention, particularly PEG, can be straight chain polymers or may possess a branched conformation.
  • the present invention also contemplates the coupling of adduct molecules, which can be various polypeptides or fragments thereof which occur naturally in vivo and which resist degradation or removal by endogenous mechanisms.
  • Molecules which increase half life may be selected from the following: (a) proteins from the extracellular matrix, eg. collagen, laminin, integrin and fibronectin; (b) proteins found in blood, e.g., serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobin, protein, ubiquitin, uteroglobulin, ⁇ -2 microglobulin, plasminogen, lysozyme, cystatin C, alpha- 1 -antitrypsin and pancreatic kypsin inhibitor; (c) immune serum proteins, e.g. IgE, IgG, IgM and their fragments e.g.
  • Fc transport proteins, e.g. retinol binding protein;
  • defensins e.g. beta- defensin 1, neutrophil defensins 1, 2 and 3;
  • proteins found at the blood brain barrier or in neural tissues e.g. melanocortin receptor, myelin, ascorbate transporter;
  • transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin- like growth factor 1 (IGF 1) receptor, insulin- like growth factor 2 (IGF 2) receptor, insulin receptor;
  • proteins localised to the kidney e.g.
  • polycystin, type IV collagen, organic anion transporter Kl, Heymann's antigen proteins localized to the liver, e.g. alcohol dehydrogenase, G250;
  • blood coagulation factor X blood coagulation factor X;
  • ⁇ - 1 antitrypsin (1) HNF 1 ⁇ .
  • proteins localised to the lung e.g. secretory component (binds IgA);
  • proteins localised to the heart eg. HSP 27;
  • proteins localised to the skin eg, keratin;
  • bone specific proteins such as bone morphogenic proteins (BMPs) e.g.
  • BMP-2, -4, -5, -6, -7 also referred to as osteogenic protein (OP-I) and -8 (OP-2);
  • tumour specific proteins eg. human trophoblast antigen, herceptin receptor, oestrogen receptor, cathepsins eg cathepsin B (found in liver and spleen);
  • disease-specific proteins eg.
  • LAG-3 lymphocyte activation gene
  • osteoprotegerin ligand OPGL
  • OX40 a member of the TNF receptor family, expressed on activated T cells and the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukaemia virus type-I (HTLV-I)-producing cells-see Pankow R et al J. Immunol. (2000) JuI.
  • angiogenic growth factors including acidic fibroblast growth factor (FGF-I), basic fibroblast growth factor (FGF- 2), Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor- ⁇ (TGF- ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet derived endothelial growth factor (PD-ECGF), placental growth factor (PIGF), midkine platelet-derived growth factor-BB (PDGF), fractalkine; (s) stress proteins (heat shock proteins); and (t) proteins involved in Fc transport.
  • FGF-I acidic fibroblast growth factor
  • FGF- 2 basic fibroblast growth factor
  • VEGF/VPF Vascular endothelial growth factor/vascular permeability factor
  • TGF- ⁇ tumor necrosis factor-alpha
  • TGF- ⁇ tumor necrosis factor-alpha
  • IL-8
  • the antibodies of the invention may be multimerized, as for example, hetero- or homodimers, hetero- or homotrimers, hetero- or homotetramers, or higher order hetero- or homomultimers. Multimerisation can increase the strength of antigen binding, wherein the strength of binding is related to the sum of the binding affinities of the multiple binding sites.
  • the antibodies can be multimerized in another aspect by binding to an additional one, two, three or more polypeptide which function to stabilize the dAb against degradation.
  • polypeptides may include common blood proteins, such as, albumin, or fragments thereof.
  • modifications relating to enhancing or modifying antibody activity are contemplated by the present invention.
  • the antibody of the invention may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating a condition, infection or disorder.
  • cysteine residue(s) may be introduced in the antibody polypeptide, thereby allowing interchain disulfide bond formation in a multimerized form of the inventive antibodies.
  • the homodimeric or heterodimeric (or multimeric) antibodies may include combinations of the same antibody polypeptide chains or different antibody polypeptide chains, such that more than one epitope is targeted at a time by the same construct. Such epitopes can be proximally located in the target (e.g.
  • the invention also contemplates modifying the antibodies of the invention to form immunoconjugates comprising the antibodies of the invention conjugated to cytotoxic agents, such as a chemotherapeutic agents, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), radioactive isotopes (i.e., a radioconjugate), or antiviral compounds (e.g. anti-HIV compounds).
  • cytotoxic agents such as a chemotherapeutic agents, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), radioactive isotopes (i.e., a radioconjugate), or antiviral compounds (e.g. anti-HIV compounds).
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term can include radioactive isotopes (e.g., I 13I , I 125 , Y 9 0 and Reig ⁇ ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • a “chemotherapeutic agent” is a type of cytotoxic agent useful in the treatment of cancer.
  • chemotherapeutic agents include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins, Melphalan and other related nitrogen mustards.
  • the invention also contemplates immunoconjugation with enzymatically active toxins or fragments thereof.
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • inventive antibodies are intended to target HIV infections that might also involve infection by other viruses, bacteria or other pathogens
  • the invention also contemplates immunoconjugation of the antibodies with anti- viral, anti-bacterial or other chemicals and/or compounds that might improve or increase the effectiveness of the antibodies of the invention against intended targets, such as, for example, HIV.
  • inventive antibodies can be immunoconjugated, or in the alternative, co-administered with, an antibacterial compound, such as, for example, a macrolide (e.g., tobramycin (TOBI®)), a cephalosporin (e.g., cephalexin (KEFLEX®), cephradine (VELOSEF®), cefuroxime (CEFTIN®), cefprozil (CEFZIL®), cefaclor (CECLOR®), cefixime (SUPRAX®) or cefadroxil (DURICEF®), a clarithromycin (e.g., clarithromycin (BIAXIN®)), an erythromycin (e.g., erythromycin (EMYCIN®)), a penicillin (e.g., penicillin V (V-CILLIN K® or PEN VEE K®)) or a quinolone (e.g., ofloxacin (FLOXIN®), ciproflox
  • inventive antibodies can be immunoconjugated, or in the alternative, co-administered with, an antiviral compound, such as, for example, a zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir, adefovir, clevadine, entecavir, and pleconaril.
  • an antiviral compound such as, for example, a zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopina
  • immunoconjugates of the antibody and cytotoxic agents can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- di
  • bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987).
  • Carbon- 14-labeled 1-isothiocyanatobenzyl- 3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibodies can also be modified with useful detectable agents, such as, for example, fluorescent compounds.
  • useful detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l- napthalenesulfonyl chloride, phycoerythrin and the like.
  • the antibody construct may also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When the antibody construct is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product.
  • the antibody construct may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • biotin and detected through indirect measurement of avidin or streptavidin binding.
  • linkers such as flexible polypeptide chains.
  • Such linkers may be required to avoid a loss in activity of the antibodies, or to avoid sterically restricting the antibodies such that they lose their effectiveness in binding to cognate epitopes, in particular, those epitopes which themselves may be sterically restricted.
  • the linkers can be the same or different as the linkers described herein elsewhere which are used to fuse the gp41 subunit (or fragment or derivative thereof) with the Fc receptor ligand.
  • Another type of covalent modification contemplated by the present invention involves chemically or enzymatically coupling glycosides to the antibodies of the invention. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation.
  • the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • Removal of any carbohydrate moieties present on the antibodies of the invention may be accomplished chemically or enzymatically.
  • Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact.
  • Chemical deglycosylation is described by Hakimuddin, et al. Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al. Anal. Biochem., 118:131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987).
  • Analytical/preparative methods for antigens and/or antibodies of invention Once an antigen or antibody in accordance with the invention is identified or obtained, for example, by any of the methods herein described, it may be preferable to carry out further steps to characterize and/or purify and/or modify the antigen or antibody. For example, it may be desirable to prepare a purified, high-titer composition of the desirable antibody or to test the immunoreactivity of the identified antibody.
  • the present invention contemplates any known and suitable methods for characterizing, purifying, or assaying the antigens and/or antibodies of the present invention and it is expected the any person of ordinary skill in the art to which the invention pertains will have the requisite level of technical know-how and resources, e.g. technical manuals or treatises, to accomplish any further characterization, purification and/or assaying of the antigens and/or antibodies of the invention without undue experimentation.
  • any useful means to describe the strength of binding (or affinity) between a antibody of the invention and an antigen of the invention can be used.
  • Affinity can be assessed and/or measured by a variety of known techniques and immunoassays, including, for example, enzyme-linked immunospecific assay (ELISA), Bimolecular Interaction Analysis (BIA) (e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345, 1991; Szabo, et al., Curr. Opin. Struct. Biol. 5:699-705, 1995, each incorporated herein by reference), and fluorescence- activated cell sorting (FACS) for quantification of antibody binding to cells that express antigen.
  • BIA is a technology for analyzing biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORETM).
  • BIAcore is based on determining changes in the optical phenomenon surface plasmon resonance (SPR) in real-time reactions between biological molecules, such as, an antibody of the invention and an antigen of interest, e.g. CD4L References relating to BIAcore technology can be further found in U.S. Published Application Nos: 2006/0223113, 2006/0134800, 2006/0094060, 2006/0072115, 2006/0019313, 2006/0014232, and 2005/0199076, each of which are incorporated herein in their entireties by reference.
  • SPR optical phenomenon surface plasmon resonance
  • antigens and antibodies of the invention may be assayed for immunospecific binding by any suitable method known in the art.
  • Assays involving an antibody and an antigen are known as "immunoassays," which can be employed in the present invention to characterize both the antibodies and the antigens of the invention.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8% 20% SDS- PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or nonfat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer; blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 12 sl) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen.
  • ELISAs typically comprise preparing antigen (e.g., gp41Fc), coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1, which is incorporated herein by reference.
  • any suitable method for purifying antigens and/or antibodies of the invention is contemplated herein.
  • chromatographic methods such as, for example, immuno-affinity chromatography (immobilized ligand to bind and trap antibody of interest), affinity chromatography, protein precipitation, ion exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, as well as electrophoresis, can be found described in the technical literature, for example, in Methods in Enzymology, Volume 182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1 st Edition, 1990, which is incorporated herein by reference.
  • suitable materials for performing such purification steps, such as chromatographic steps are known to those skilled in the art.
  • Such methods are suitable for purification of any of the antibodies, antigens or any fragments thereof that are in accordance with the invention as described herein. Certain embodiments may require the purification or isolation of expressed antigens or antibodies or fragments thereof from a host cell or a portion thereof.
  • Conventional procedures for isolating recombinant proteins from transformed host cells are contemplated by the present invention.
  • Such methods include, for example, isolation of the protein or fragments of interest by initial extraction from cell pellets or from cell culture medium, followed by salting-out, and one or more chromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, high performance liquid chromatography (HPLC), and affinity chromatography may be used to isolate the recombinant protein or fragment.
  • the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of the antigens and/or antibodies of the invention, together with a pharmaceutically acceptable carrier.
  • the present invention provides a method for vaccinating against an HIV infection by administering a therapeutically effective amount of an antigen of the invention (e.g., gp41Fc), together with a pharmaceutically acceptable carrier or diluent. Administration can occur before or after HIV infection.
  • the present invention provides a method for treating an HIV infection by administering a therapeutically effective amount of an antibody of the invention (e.g., anti-gp41Fc), together with a pharmaceutically acceptable carrier or diluent. Administration can occur before or after HIV infection.
  • an antibody of the invention e.g., anti-gp41Fc
  • treatment includes any process, action, application, therapy, or the like, wherein a subject (or patient), including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject, or ameliorating at least one symptom of the disease or disorder under treatment.
  • combination therapy means the administration of two or more therapeutic agents to treat a disease, condition, and/or disorder.
  • administration encompasses co-administration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each inhibitor agent.
  • administration encompasses use of each type of therapeutic agent in a sequential manner. The order of administration of two or more sequentially co- administered therapeutic agents is not limited.
  • terapéuticaally effective amount means the amount of each agent administered that will achieve the goal of improvement in a disease, condition, and/or disorder severity, and/or symptom thereof, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment.
  • pharmaceutically acceptable means that the subject item is appropriate for use in a pharmaceutical product.
  • an embodiment of this invention includes a method of treating and/or preventing a particular condition (e.g. HIV infection) in a patient which comprises administering to said patient a composition containing an amount of an antibody of the invention that is effective in treating the target condition, e.g., HIV infection.
  • a particular condition e.g. HIV infection
  • a composition containing an amount of an antibody of the invention that is effective in treating the target condition, e.g., HIV infection.
  • an embodiment of this invention includes a method of vaccinating against HIV infections in a subject comprising administering to said subject a pharmaceutical composition containing an amount of an antigen of the invention that is effective in immunizing (at least partially) against HIV infection.
  • the antigens and/or antibodies of the present invention may be administered alone or in combination with one or more additional therapeutic agents.
  • Combination therapy includes administration of a single pharmaceutical dosage formulation which contains an antibody of the present invention and one or more additional therapeutic agents, as well as administration of the antibody of the present invention and each additional therapeutic agents in its own separate pharmaceutical dosage formulation.
  • an antibody of the present invention and a therapeutic agent may be administered to the patient together in a single oral dosage composition or each agent may be administered in separate oral dosage formulations.
  • the antibody of the present invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).
  • the order of administration of the agents is not limited.
  • co-administration of an antibody or antibody fragment of the invention together with one or more anti-HIV agents to potentiate the effect of either the antibody or the anti-HIV agent(s) or both is contemplated for use in treating HIV infections.
  • anti-HIV agents include, but are not limited to AGENERASE (ampreavir), APTIVUS (tipranavir), ATRIPLA, COMBIVIR, RETROVIR, EPIVIR, CRIXIVAN (indinavir), EMTRIVA (emtricitabine), EPZICOM, FORTOVASE (saquinavir), FUZEON (enfuvirtide), HIVID (ddc /zalcitabine), INTELENCE (Etravirine), ISENTRESS (raltegravir), INVIRASE (saquinavir), KAETRA (lopinavir), LEXIVA (Fosamprenavir), NORVIR (ritonavir), PREZISTA (darunavir),
  • the one or more anti-cancer agents can include any known and suitable compound in the art, such as, for example, chemoagents, other immunotherapeutics, cancer vaccines, anti-angiogenic agents, cytokines, hormone therapies, gene therapies, and radiotherapies.
  • a chemoagent or "anti-cancer agent” or “anti-tumor agent” or “cancer therapeutic” refers to any molecule or compound that assists in the treatment of a cancer.
  • chemoagents contemplated by the present invention include, but are not limited to, cytosine arabinoside, taxoids (e.g., paclitaxel, docetaxel), anti-tubulin agents (e.g., paclitaxel, docetaxel, epothilone B, or its analogues), macrolides (e.g., rhizoxin) cisplatin, carboplatin, adriamycin, tenoposide, mitozantron, discodermolide, eleutherobine, 2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and c
  • compositions comprising one or more chemoagents (e.g., FLAG, CHOP) are also contemplated by the present invention.
  • FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF.
  • CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • the chemoagent can be an anti-angiogenic agent, such as, for example, angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin, canstatin, maspin, anti-VEGF antibodies or peptides, anti-placental growth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Flt-1 antibodies or peptides, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, IP-IO, Gro- ⁇ , thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CMlOl, Marimastat, pentosan polysulphate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K pro
  • an anti-angiogenic agent advantageously may lead to the increase in MN expression in a tumor, thereby making the tumor more susceptible to the antibodies and antibody conjugates of the invention.
  • said chemoagent is gemcitabine at a dose ranging from 100 to
  • said chemoagent is dacarbazine at a dose ranging from 200 to 4000 mg/m 2 cycle. In another aspect, said dose ranges from 700 to 1000 mg/m 2 /cycle. In yet another aspect, said chemoagent is fludarabine at a dose ranging from 25 to 50 mg/m 2 /cycle. In another aspect, said chemoagent is cytosine arabinoside (Ara-C) at a dose ranging from 200 to 2000 mg/ m 2 /cycle. In still another aspect, said chemoagent is docetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle.
  • said chemoagent is paclitaxel at a dose ranging from 5 to 15 mg/kg/cycle.
  • said chemoagent is cisplatin at a dose ranging from 5 to 20 mg/kg/cycle.
  • said chemoagent is 5- fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle.
  • said chemoagent is doxorubicin at a dose ranging from 2 to 8 mg/kg/cycle.
  • said chemoagent is epipodophyllotoxin at a dose ranging from 40 to 160 mg/kg/cycle.
  • said chemoagent is cyclophosphamide at a dose ranging from 50 to 200 mg/kg/cycle.
  • said chemoagent is irinotecan at a dose ranging from 50 to 150 mg/m /cycle.
  • said chemoagent is vinblastine at a dose ranging from 3.7 to 18.5 mg/m 2 /cycle.
  • said chemoagent is vincristine at a dose ranging from 0.7 to 2 mg/m /cycle.
  • said chemoagent is methotrexate at a dose ranging from 3.3 to 1000 mg/m 2 /cycle.
  • the antigens and/or antibodies of the present invention are administered in combination with one or more immuno therapeutic agents, such as antibodies or immunomodulators, which include, but are not limited to,
  • HERCEPTIN® RETUXAN®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Campath IZH, Smart MI95, LymphoCide, Smart I DlO, and Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.
  • the invention also contemplates administering the antigens and/or antibodies of the present invention with one or more anti- angiogenic agents, which include, but are not limited to, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor) anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a ⁇ -amino acid peptide corresponding to a fragment of platelet factor-4 (Maione et al., 1990, Cancer Res.
  • anti- angiogenic agents include, but are not limited to, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor) anti-thrombin, 29 kDa N-terminal and a 40
  • the antigens and/or antibodies of the present invention can also be administered in combination with one or more cytokines, which includes, but is not limited to, lymphokines, tumor necrosis factors, tumor necrosis factor-like cytokines, lymphotoxin- ⁇ , lymphotoxin- ⁇ , interferon- ⁇ , macrophage inflammatory proteins, granulocyte monocyte colony stimulating factor, interleukins (including, but not limited to, interleukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CD137 ligands, Fas- Pas ligand, 4- IBBL, endothelial monocyte activating protein or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable salts thereof.
  • cytokines which includes, but is not limited to, lymphokines, tumor necrosis factors, tumor necrosis factor-like cytokines, lymphotoxin-
  • the antigens and/or antibodies of the present invention can also be administered in combination with a cancer vaccine, examples of which include, but are not limited to, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins (e.g., gplOO, MART- 1/MelanA, TRP-I (gp75), tyrosinase, widely shared tumor-associated, including tumor-specific, antigens (e.g., BAGE, GAGE-I, GAGE-2, MAGE-I, MAGE-3, N- acetylglucosaminyltransferase-V, pl5), mutated antigens that are tumor-associated ( ⁇ -catenin, MUM-I, CDK4), nonmelanoma antigens (e.g., HER-2/neu (breast and ovarian carcinoma), human
  • the antigens and/or antibodies of the present invention are used in association with a hormonal treatment.
  • Hormonal therapeutic treatments comprise hormonal agonists, hormonal antagonists (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), and antiandrogens (e.g., cyproterone acetate).
  • hormonal antagonists e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON), LH-RH antagonists
  • inhibitors of hormone biosynthesis and processing e.
  • the antigens and/or antibodies described herein may be provided in a pharmaceutical composition
  • a pharmaceutically acceptable carrier may be non-pyrogenic.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • aqueous carriers may be employed including, but not limited to saline, glycine, or the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well- known sterilization techniques (e.g., filtration).
  • the phrase "pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the antibody compositions of the invention.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and the like.
  • concentration of the antibody of the invention in such pharmaceutical formulation may vary widely, and may be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected. If desired, more than one type of antibody may be included in a pharmaceutical composition (e.g., an antibody with different IQ for MN binding).
  • compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which may be used pharmaceutically.
  • Pharmaceutical compositions of the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • compositions of the invention additionally contemplate suitable immunocarriers, such as, proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solid phases.
  • suitable immunocarriers such as, proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solid phases.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Other protein-derived or non-protein derived substances are known to those skilled in the art.
  • Formulations suitable for parenteral, subcutaneous, intravenous, intramuscular, and the like; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and e
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the determination of a therapeutically effective dose is well within the capability of those skilled in the art.
  • a therapeutically effective dose refers to the amount of an antibody that may be used to effectively treat a disease (e.g., cancer) compared with the efficacy that is evident in the absence of the therapeutically effective dose.
  • the therapeutically effective dose may be estimated initially in animal models (e.g., rats, mice, rabbits, dogs, or pigs). The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity (e.g., ED 50 - the dose therapeutically effective in 50% of the population and LD 50 - the dose lethal to 50% of the population) of an antibody may be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD 50 /ED 50 .
  • the data obtained from animal studies may used in formulating a range of dosage for human use.
  • the dosage contained in such compositions may be within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage may be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration may be adjusted to provide sufficient levels of the antibody or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • the antigens and/or antibodies of the invention may also be administered by introducing genetically engineered bacteria which express and release the expressed antigens and/or antibodies of the invention once the bacteria are present in the patient. This format might be suitable for treating HIV infections.
  • the antigen and/or antibody-expressing bacteria can be introduced into mucus membranes of the throat, for example, or in other mucosal regions in which HIV might be found. Methods for constructing and/or engineering such recombinant bacteria are well known in the art.
  • Polynucleotides encoding the antigens and/or antibodies of the invention may be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antigen and/or antibody are in the range of about 5 ⁇ g to about 500 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 500 ⁇ g of DNA.
  • the antigens and/or antibodies of the present invention can also be delivered in a microsphere or microsome bodies.
  • the mode of administration of antigen- and/or antibody-containing pharmaceutical compositions of the present invention may be any suitable route which delivers the antibody to the host.
  • pharmaceutical compositions of the invention may be useful for parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or intranasal administration, or microsomal or lipid microsome bodies).
  • parenteral administration e.g., subcutaneous, intramuscular, intravenous, or intranasal administration, or microsomal or lipid microsome bodies.
  • Example 1 gp41 fused to Fc as HIV vaccine immunogen
  • the HIV envelope glycoprotein (Env) is composed of two subunits - gpl20 and gp41, of which gp41 is more conserved but unstable in the absence of gpl20.
  • this Example describes the construction of a fusion protein where a truncated gp41 lacking the fusion peptide, the transmembrane domain and the cytoplasmic tail was joined to the Fc region of human IgG by a long flexible linker.
  • gp41Fc This molecule, designated gp41Fc, exhibited binding to recently identified and characterized broadly cross- reactive HIV-I -neutralizing human monoclonal antibodies m43, m44, m46, m47 and m48, which recognize conformational epitopes on gp41.
  • the fusion antigen, gp41Fc also exhibited binding to known broadly cross-reactive gp41-specific antibodies, 2F5, 4E10, and Z13, each of which target the membrane proximal external region (MPER).
  • gp41Fc if used as vaccine immunogen, could potentially elicit the same or similar human monoclonal antibodies that could bind to these two major groups of neutralizing epitopes and neutralize primary HIV-I isolates from different clades.
  • the inventors have found that such a possibility appears to be confirmed based on the results of the immunization of rabbits with purified gp41Fc fusion protein.
  • the gp41Fc is highly immunogenic - the serum titer was 102,400 for the gp41Fc fusion protein and 5,120 for a recombinant Env ectodomain (gpl40) containing both gpl20 and gp41 from the same HIV-I isolate (isolate 89.6).
  • purified rabbit IgG from one of the four immunized rabbits showed broad neutralization activity against several HIV isolates from different clades in a cell line/pseudovirus assay.
  • Viruses pseudotyped with Envs from HIV- 1 primary isolates representing HIV-I group M, clades A-F, and laboratory adapted HIV-I isolates and JRCSF were used in this Example. Cloning of HIV- 1 envelope genes and preparation of pseudoviruses have been previously described. Briefly, pseudotyped viruses were prepared by cotransfection of 70% to 80% confluent HEK 293T cells with pNL4- 3.1uc.E-R- and HIV-I Env plasmid using the calcium phosphate/HEPES buffer, according to manufacturer's instruction (Promega, Madison, WI). Eighteen hours after the transfection, medium was replaced with medium supplemented with 0.1 mM sodium butyrate (Sigma, St. Louis, USA).
  • gp4 IFc fusion protein Preparation of gp4 IFc fusion protein
  • the gp41 89 6 gene without the fusion peptide was PCR amplified, digested with Nhe I and BamHI and cloned to pEAKIO digested with the same enzymes. Recombinant DNA was confirmed by DNA sequencing and used for transient transfection of free-style 293 cells (Invitrogen). The culture supernatant four days post transfection was collected and gp41Fc fusion protein purified from the supernatant by protein A affinity purification.
  • the 89.6 virus was a gift of Dr. Robert Doms, University of Pennsylvania, Philadelphia, PA. 89.6gp41 was amplified from 89.6 construct. Results
  • the gp41Fc antigen of the invention is capable of eliciting antibodies that do not react with self antigens. This feature also makes the fusion protein an attractive vaccine immunogen based on the possibility that it may elicit antibodies which are not regulated by tolerance mechanisms.
  • the HIV-I envelope glycoprotein (Env) is known to exist as trimers of heterodimers on native virions that is composed of a non-covalently associated extracellular subunit gpl20 and a transmembrane subunit gp41.
  • Env induces a robust antibody response, but these antibodies usually have a narrow spectrum of neutralization activity.
  • Env-based vaccines have been extensively studied. Monomeric gpl20s can elicit neutralizing antibodies in vivo, but the neutralization activity is restricted to autologous viruses or the spectrum of neutralization activity is narrow.
  • Oligomeric forms of Env ectodomain from HIV-I, strain R2 (gpl4U R2 ), which contain both gpl20 and a truncated gp41 that lacks both transmembrane domains and cytoplasmic tails, induced better antibody response than gpl2U R2 alone.
  • Rabbit sera immunized with gpl4U R2 neutralized not only the autologous virus, but also heterologous viruses from diverse HIV-I subtypes, suggesting that induction of broad spectrum neutralizing antibodies is an achievable goal in HIV- 1 vaccine development.
  • This study utilizes gp41 derived from a dual tropic HIV-I primary isolate of 89.6.
  • Gp41 is relatively conserved compared to gpl20, but unstable in the absence of gpl20.
  • gp41Fc a fusion protein, designated as gp41Fc, as outlined in Example 1, which was hypothesized to increase its half life in vivo and enhance its binding to immune cells like macrophages, dendritic cells, and B cells that express receptors specific for Fc, thereby enhancing the elicitation of broadly cross-reactive neutralizing antibodies.
  • gp41Fc was used to localize the epitopes of a panel of gp41-specific neutralizing antibodies by alanine scanning mutagenesis. It was found that gp41Fc not only bound to recently identified mAbs conformational epitopes, m44, m46 and m48, but also to known broadly neutralizing human mAbs that include 2F5, 4E10, Zl 3 that recognize linear epitopes in the membrane proximal region (MPER), suggesting that gp41Fc retains critical conformation required for presenting neutralization epitopes and may induce neutralizing antibodies against HIV-I in vivo.
  • MPER membrane proximal region
  • This Example investigates the possibility of developing the gp41Fc fusion protein as HIV-I vaccine immunogen by immunizing rabbits with purified recombinant gp41Fc.
  • This Example reports the results from the characterization of immune rabbit sera and purified IgGs from two rabbits that had different immune response to gp41Fc. The following Methods and Materials were used in this Example.
  • 293T cells were purchased from ATCC. Other cell lines and HIV-I isolates were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP). Recombinant gpl20 and gpl40 Envs from primary isolates were produced as described previously (Zhang MY et al, 2003, J. Immunol. Methods, Dec; 283(1-2): 17-25, which is incorporated herein by reference). Human mAbs 2F5 and 4E10 were obtained from the NIH-ARRRP. Mouse mAbs NC-I was generously provided by Dr. Shibo Jiang (New York Blood Center). Mouse mAbs T3 and D61 were obtained from Dr. Christopher Broder (Uniformed Services University of the Health Sciences).
  • Fab Z13 and other gp41-specific mAbs m44, m46, m48 were produced by the inventors.
  • Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors.
  • HRP-conjugated streptavidin was purchased from Zymed Laboratories Inc., San Francisco, CA.
  • HIV- 1 MN Env (15-mer) peptides (complete set, cat# 6451, Lot# 12) were obtained from NIH-ARRRP.
  • the gp41Fc fusion protein was prepared by cloning a gp41g 9 6 ectodomain gene lacking the fusion peptide region in a pEAKIO plasmid upstream of a human Fc region.
  • the recombinant plasmid DNA for transient transfection of free-style 293 cells (Invitrogen) was produced in E. coli.
  • the culture supernatant was collected four days post transfection and gp41Fc fusion protein purified from the culture supernatant using a protein A affinity purification.
  • FCA Freund's Complete Adjuvant
  • FIA Freund's Incomplete Adjuvant
  • Fig. 15 Titration of immune rabbit serum was done by coating one ⁇ g/mL gp41Fc or gpl4U 89 6 on maxisorp 96- well plates, then washing and blocking with 3% BSA in PBS followed by the addition of two-fold serially diluted rabbit immune sera. Bound antibodies were detected using horse radish peroxidase (HRP) conjugated to goat anti-rabbit IgG (H+L) (1:10,000) as a second antibody and ABTS as a substrate. The optical density at 405 nm was measured after a 20 minute color development.
  • HRP horse radish peroxidase
  • Binding of gp41-specific mAbs to recombinant gp41Fc was performed by coating 5 ⁇ g/mL gp41Fc on 96-well plates followed by the addition of 3-fold serially diluted biotinylated mAbs. The bound mAbs was detected using HRP conjugated to streptavidin (1:10,000) as a second antibody and ABTS as a substrate.
  • Binding of immune rabbit IgG to denatured gpl40 and gp41Fc was carried out by diluting purified gpl40 and gp41Fc to 20 ⁇ g/mL in PBS containing
  • ELISA assay with HIV-I MN gp41-derived peptides (6341 to 6377) (Fig. 18) was carried out by coating 5 ⁇ g/mL of each peptide on Easy- wash microplates followed by the addition of 3 -fold serially diluted immune rabbit IgG. The bound rabbit IgG was detected as described above.
  • Capture ELISA was used to measure gp41Fc concentration in immune rabbit
  • IgG sera utilizing the following procedure: gp41Fc was captured by conformation- dependent mouse mAb T3 coated on 96-well plates. Bound gp41Fc was detected by using biotinylated mouse mAb D61 and HRP conjugated to streptavidin. The optical density was measured 30min after addition of ABTS. The concentration of gp41Fc in purified rabbit IgG was calculated using purified recombinant gp41Fc for making a standard curve and then converted to the concentration of gp41Fc in rabbit serum.
  • PBMC-based HIV-I neutralization assay The PB MC -based assay was carried out as previously described [10]. Briefly, Fresh human PBMCs, seronegative for HIV and HBV, were isolated from blood of screened donors (Biological Specialty Corporation; Colmar, PA) using Lymphocyte Separation Medium (LSM; Cellgro® by Mediatech, Inc.; density 1.078+/-0.002 g/mL) following the manufacturer's instructions. Cells were stimulated by incubation in 4 ⁇ g/mL Phytohemagglutinin (PHA; Sigma) for 48-72 hours. Mitogenic stimulation was maintained by the addition of 20 U/mL recombinant human IL-2 (R&D Systems, Inc) to the culture medium.
  • LSM Lymphocyte Separation Medium
  • PHA Phytohemagglutinin
  • PHA- stimulated PBMCs from at least two donors were pooled, diluted in fresh medium and added to 96-well plates at 5xlO 4 cells/well. Cells were infected (final MOI ⁇ 0.1) in the presence of 9 different concentrations of antibody (triplicate wells/concentration; 50 ⁇ g/mL high-test concentration) and incubated for 7 days. To determine the level of virus inhibition, cell-free supernatant samples were collected for analysis of reverse transcriptase activity [H].
  • cytotoxicity was measured by the addition of 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; CellTiter 96 Reagent, Promega) following the manufacturer's instructions.
  • MTS 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
  • the cell line-based assay was carried out in triplicate using an ⁇ IV- 1 Env pseudotyping system and ⁇ OS CD4 + CCR5 + or ⁇ OS CD4 + CXCR4 + target cells containing a tat-inducible luciferase reporter that express CD4, CCR5 or CXCR4.
  • the degree of virus neutralization by antibody was achieved by measuring luciferase activity as described previously (Zhang et al., J. Immunol. Methods, 2003, 283(1-2): 17-25).
  • Recombinant gp41Fc retains its antigenic structure.
  • Recombinant gp41Fc retains its antigenic structure as measured by ELISA (Fig. 13). Recombinant gp41Fc binds to all gp41 -specific mAbs tested including human mAbs m44, m46 and m48, and mouse NC-I and T3 that recognize the conformational epitopes of gp41, and the best characterized human mAbs, 2F5,
  • BSA and anti- gpl20 human mAb ml4 were used as controls. BSA did not bind to gp41Fc, but ml4 showed weak binding at high concentrations likely due to non-specific interactions.
  • Recombinant gp41Fc is highly immunogenic.
  • Gp41 -specific rabbit IgG accounts for neutralization activity of the immune serum IgG.
  • Rabbit IgG was purified from the antisera using a protein A affinity column and tested the rabbit IgG with a PBMC assay against a panel of HIV-I primary isolates from group M and O, and HIV-2 (Table 1, below).
  • TC 50 S of both FT1214 and FT1215 IgGs were over 50 ⁇ g/mL for all isolates tested.
  • ICso s were converted to the serum dilutions based on the amount of FT1214, 1215 IgG isolated from the corresponding serum, 6.7 mg and 6.0 mg per ml serum, respectively. Isolates neutralized by the rabbit IgG were highlighted in grey.
  • IgG IC 50 IgG IC 50 IC 50 serum serum type ( ⁇ g/mL) ( ⁇ g/mL) (nM) dilution dilution
  • rabbit FT 1214EX IgG neutralized half of the HIV-I primary isolates tested from group M (clades A, B, C, D, B/F and G), one out of three isolates from group O and two HIV-2 isolates with IC 50 values lower than 50 ⁇ g/mL, the highest concentration tested.
  • Rabbit FT1215EX IgG did not neutralize the HIV-I isolates tested, but did neutralize one of the two HIV-2 isolates, CSC310342. It was further confirmed that the neutralization activity of FT1214EX IgG was attributed to the gp41-specific IgG of the immune rabbit IgG (Fig. 16).
  • Immune rabbit IgG recognizes conformation-independent epitopes on gp41 and preferentially binds to peptides derived from the immunodominant loop region. Because each rabbit IgG immune serum had similar titres to gp41Fc and gpl4U8 9 6, but showed very different neutralization activity against HIV-I isolates, we investigated the composition of the rabbit IgG by measuring their binding to denatured gp41Fc and gpl40 89 6 (Fig- 17). Binding profiles were strikingly similar, suggesting that rabbit IgG recognizes mainly conformation-independent epitopes on gp41 (Fig. 17).
  • the neutralizing FT1214EX IgG bound to peptides 6357 (LLGFWGCSGKLICTT) (SEQ ID NO: 9), 6358 (WGCS GKLICTTT VP W) (SEQ ID NO: 10) and 6359 (GKLICTTTVPWNASW) (SEQ ID NO: 11) that are located in the C-terminal immunodominant loop, while the weak-neutralizing FT1215EX IgG bound to peptides 6354 (VLA VER YLKD QQLLG) (SEQ ID NO: 12) and 6355 (ERYLKDQQLLGFWGC) (SEQ ID NO: 13), as well as 6357, 6358 and 6359.
  • VLA VER YLKD QQLLG VLA VER YLKD QQLLG
  • 6355 ERYLKDQQLLGFWGC
  • FT1214EX IgG bound more strongly to peptides 6358 and 6359 than FT1215EX IgG. Both bound well to peptide 6357 (Fig. 18). No binding was observed to peptides derived from MPER and other regions of gp41 (data not shown). Prolonged existence of the immunogen was detected in the neutralizing rabbit IgG. In an effort to explain why two rabbit IgG sera with similar titers had different neutralization activity, gp41Fc level in rabbit IgG (Fig. 19) was measured.
  • the FT1214EX IgG at 87 days post immunization contained a much higher gp41Fc level (about 46 ng/mg rabbit IgG, equivalent to 305 ng/mL rabbit serum) than the FT1215EX IgG ( ⁇ 10 ng/mg rabbit IgG, or ⁇ 60 ng/mL rabbit serum).
  • the gp41Fc level in the FT1214PB 1 and FT1214PB2 rabbit IgG purified from the production bleed after the 3 rd and 4 th booster was comparable to that in FT1215PB1 and FT1215PB2.
  • This Example describes the results from two rabbits immunized with a gp41Fc fusion protein.
  • the major observation of this Example is that prolonged existence of the immunogen in rabbit serum correlates with the neutralization activity of rabbit IgG. These results suggest that prolonging the existence of the immunogen in vivo may enhance elicitation of broadly cross-reactive HIV-I neutralizing Abs.
  • FcRn neonatal Fc receptor
  • mouse FcRn binds to all IgG from these species (Ober et al., Int. Immunol, 2001, 13: 1551-9). Further study showed that bovine FcRn binds stronger to human IgG than to bovine IgG, and human IgG has two times longer serum half life in normal and transchromosomic calves than its bovine counterpart (Kacskovics et al., Int. Immunol. 2006, 18:525-36). Presently, there is no study reported on the interaction of human IgG with rabbit FcRn, but the results from this Example suggest that rabbit FcRn may bind to human Fc and mediate elongated human Fc serum half life in rabbits.
  • the current Example used human Fc fused to gp41 ectodomain as immunogen to immunize rabbits.
  • the immunogen level of the two rabbits during the course of immunization was comparable, but dropped quickly in rabbit FT1215 after the last booster, indicating a difference in the half-life of the immunogen in different host individuals. Further studies will be required to elucidate the mechanism for what leads to a fast drop of the immunogen level in vivo and how to extend the in vivo half- life of the immunogen.
  • the percentage of Fc-specific antibodies in the rabbit IgG by absorption of the rabbit IgG with human Fc-conjugated sepharose 4B column was determined (data not shown). Rabbit anti-Fc antibodies accounts for 30% of total rabbit IgG.
  • Human Fc-specific antibodies in rabbits may shorten the half life of gp41Fc in rabbits.
  • Administration of gp41Fc in humans may result in a better immune response than in rabbits because human Fc is non-immunogenic in human and it can bind to human FcRn on immune cells including macrophages and dendritic cells thus contributing to a potentially longer half-life.
  • the prolonged serum half-life of gp41Fc in rabbit FT1214 may contribute to the high level of cross- reactive HIV-neutralizing antibodies, but other mechanisms should not be excluded that may explain the difference in the level of cross-reactive HIV-neutralizing antibodies in the two rabbits used in this study.
  • Lin G Nara PL. Designing immunogens to elicit broadly neutralizing antibodies to the HIV-I envelope glycoprotein. Curr HIV Res 2007 Nov;5(6):514-41.
  • MHC class I- related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells. J Immunol 2001 Mar l;166(5):3266-76.

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Abstract

L'invention concerne un antigène du VIH à base de gp41, comprenant un polypeptide gp41 ou un fragment immunogène de celui-ci, fusionné à un complexe ligand-récepteur Fc. Ledit antigène se révèle hautement immunogène, très stable et présente une bonne efficacité in vivo. Il est également à l'origine de la production d'anticorps neutralisants anti-VIH de réaction croisée et a pour intérêt de ne pas présenter les inconvénients des antigènes à base de gp41 déjà connus. L'invention concerne également des procédés de vaccination d'un sujet contre une infection par le VIH, procédés impliquant l'administration de l'antigène de l'invention ou d'une composition en contenant.
PCT/US2009/043054 2008-05-06 2009-05-06 Immunogène du vih et son procédé de fabrication et d'utilisation WO2009137632A2 (fr)

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CN107383190A (zh) * 2017-08-03 2017-11-24 深圳市慢性病防治中心 人源抗HIVgp41特异性抗体及其应用
WO2019097466A1 (fr) * 2017-11-17 2019-05-23 Grifols Diagnostic Solutions Inc. Nouveaux antigènes protéiques d'enveloppe du virus de l'immunodéficience humaine exprimés chez les mammifères

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160331830A1 (en) * 2014-01-08 2016-11-17 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Ras pathways as markers of protection against hiv and methods to improve vaccine efficacy
US10398772B2 (en) * 2014-01-08 2019-09-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Ras pathways as markers of protection against HIV and methods to improve vaccine efficacy
CN107383190A (zh) * 2017-08-03 2017-11-24 深圳市慢性病防治中心 人源抗HIVgp41特异性抗体及其应用
CN107383190B (zh) * 2017-08-03 2020-09-25 深圳市慢性病防治中心 人源抗HIV gp41特异性抗体及其应用
WO2019097466A1 (fr) * 2017-11-17 2019-05-23 Grifols Diagnostic Solutions Inc. Nouveaux antigènes protéiques d'enveloppe du virus de l'immunodéficience humaine exprimés chez les mammifères
CN110049995A (zh) * 2017-11-17 2019-07-23 盖立复诊断解决方案公司 哺乳动物表达的新型人类免疫缺陷病毒包膜蛋白抗原
JP2020503839A (ja) * 2017-11-17 2020-02-06 グリフォルス ダイアグノステック ソリューションズ インコーポレーテッド 哺乳類で発現した新規なヒト免疫不全ウイルスエンベロープタンパク質抗原
JP2022022308A (ja) * 2017-11-17 2022-02-03 グリフォルス ダイアグノステック ソリューションズ インコーポレーテッド 哺乳類で発現した新規なヒト免疫不全ウイルスエンベロープタンパク質抗原
AU2018366480B2 (en) * 2017-11-17 2023-06-15 Grifols Diagnostic Solutions Inc. Novel mammalian expressed human immunodeficiency virus envelope protein antigens
AU2018366480C1 (en) * 2017-11-17 2024-02-01 Grifols Diagnostic Solutions Inc. Novel mammalian expressed human immunodeficiency virus envelope protein antigens

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