WO2005097822A1 - Identification de la sequence precise d'acides amines de l'epitope identifie par l'anticorps monoclonal igg1b12 puissant de neutralisation anti-vih 1 humain - Google Patents

Identification de la sequence precise d'acides amines de l'epitope identifie par l'anticorps monoclonal igg1b12 puissant de neutralisation anti-vih 1 humain Download PDF

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WO2005097822A1
WO2005097822A1 PCT/CA2005/000547 CA2005000547W WO2005097822A1 WO 2005097822 A1 WO2005097822 A1 WO 2005097822A1 CA 2005000547 W CA2005000547 W CA 2005000547W WO 2005097822 A1 WO2005097822 A1 WO 2005097822A1
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hiv
amino acid
acid sequence
seq
peptide
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PCT/CA2005/000547
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English (en)
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Jillian L.M. Waruk
Jody D. Berry
T. Blake Ball
Francis A. Plummer
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University Of Manitoba
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Priority to US10/599,734 priority Critical patent/US20080146499A1/en
Priority to CA002562385A priority patent/CA2562385A1/fr
Publication of WO2005097822A1 publication Critical patent/WO2005097822A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/625—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier binding through the biotin-streptavidin system or similar
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site

Definitions

  • the present invention relates generally to the field of medical treatments.
  • Gp120 amino acids involved in gp120-CD4 binding include Asp 368 (fig 3. D362), GIu 370 (G364) and Trp 427 (W484) (8).
  • Gp120 amino acids determined to be involved in gp120-lgG1b12 binding are, according to the sequence in figure 3, G371 , D373, P374, 1376 and Y389.
  • the neutralizing capabilities of lgG1b12 have been described by many groups for many strains of HIV-1, and under many conditions (refs. 6,9-24).
  • lgG1b12 is an invaluable tool for vaccine research and development.
  • the abilities of a vaccine to elicit immune responses that block viral infection of target cells and/or replication within these cells are critical to its success.
  • Antibodies are capable of combating invading virus in many ways. When HIV-1 exits an infected cell, it acquires its envelope from that cell's membrane. Gp120 is therefore expressed on the surface of infected cells containing replicating virus. Gp120 may also exist on the surface as a result of HIV fusion to the cell membrane.
  • Antibody can bind to the gp120 and mediate antibody dependant cellular cytotoxicity (ADCC), or compliment-dependant cytotoxicity (CDC) of the infected cell, or potentially block viral release.
  • ADCC antibody dependant cellular cytotoxicity
  • CDC compliment-dependant cytotoxicity
  • Antibodies can bind to surface proteins on the virus and specifically block virus particles required for cell invasion. Higher affinity antibodies will remain tightly attached to the viral surface, out-competing for binding by the target cell receptor molecule(s) and are in general more powerfully neutralizing. Antibody-bound viruses can also trigger complement-mediated virolysis or phagocytosis (25). Virus that has entered the body is unable to infect target cells because neutralizing antibody mops up free virus which is then cleared by normal mechanisms. Infection cannot be established, and the host remains healthy. In contrast, cytotoxic T cells can only be specifically activated after an infection has been established and cells begin presenting antigen.
  • Sterilizing immunity does not result in this case, and it is important to remember that once HIV has infected cells, it can begin to mutate and evolve to escape the immune response.
  • Antibody-focused vaccine researchers strive to create a vaccine that will elicit a sterilizing antibody response. It is also important to induce cross clade- specific antibody responses so that the vaccine recipient is protected from infection by HIV of any clade.
  • One of the most important HIV-related phenomena to have been discovered in the last ten years is the existence of individuals who are resistant to HIV infection. This model of protection may hold the secret to the specific and/or innate immune responses required to successfully block HIV infection.
  • Our group has identified a group of Kenyan female sex workers who, despite repeated exposure to HIV, remain uninfected (26).
  • HIV-1 gp120-specific IgA has been isolated from the cervix of these women.
  • the cervical IgA not only neutralizes HIV, but it can also inhibit the transcytosis of HIV across human epithelial cells (28).
  • These women are exposed to HIV through heterosexual contact, therefore HIV initially comes into contact with cells of the genital tract. The virus must pass through epithelial cells via transcytosis in order to establish an infection. It is therefore plausible that neutralizing, transcytosis-inhibiting antibody may play a crucial role in HIV resistance in these women. Any vaccine that could educe such antibodies may provide sterilizing immunity to its recipients.
  • the lgG1b12 antibody was cloned from an HIV+ donor who had been HIV+ for over 6 years.
  • the lgG1b12 epitope specificity may provide information, in the form of a marker, about those individuals who will not progress quickly to AIDS. Knowledge- of what comprises a neutralizing epitope for antibodies may be applicable to clinical settings as well. For instance, if antibodies from patient 'X' recognized a specific sequence in the HIV envelope protein, they have an increased chance of being a long-term non-progressor. Doctors could use this information to tailor drug regimens specifically for each patient.
  • a purified polypeptide the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6.
  • a method of immunizing an individual against HIV infection comprising administering to an individual a purified polypeptide, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6.
  • a purified polypeptide as a vaccine, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6.
  • a purified polypeptide as a medicament, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6.
  • a method of preparing an immune globulin effective against Human Immunodeficiency virus comprising: vaccinating a plurality of donors with a purified polypeptide, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6; isolating plasma from each of said donors after a period of time sufficient to allow production of antibodies against said polypeptide; pooling the plasma; and preparing an immune globulin from the pooled plasma.
  • a method of determining a course of treatment for an individual infected with human immunodeficiency virus comprising: screening a sample from an individual infected with human immunodeficiency virus for antibodies binding to a purified polypeptide, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6, wherein presence of antibodies against said polypeptide indicates that a less aggressive treatment is needed.
  • a method of treating an individual infected or suspected of being infected by human immunodeficiency virus comprising administering to said individual a therapeutically effective amount of a purified polypeptide, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6.
  • a method of treating an individual infected or suspected of being infected by human immunodeficiency virus comprising administering to said individual a purified polypeptide, the amino acid sequence of which comprises at least 6 contiguous residues of any one of SEQ ID No. 1-6.
  • the second peak corresponds to the sequence KLWVTVYYGVPVWK.
  • the third peak, 2097 corresponds to the sequence TEKLWVTVYYGVPVWKE.
  • Figure 2. MALDI QqTOF mass spectrometry results of gp120 epitope mapping. IIIB (a) or MN (b) gp120 was bound tb lgG1b12 antibody and was digested overnight with trypsin endopeptidase. Protein fragments not bound by igG1b12 were washed away. Antibody-bound fragments protected from digestion were analyzed by mass spectrometry. The size of the peak 1357 corresponds to the N-terminal sequence EATTTLFCASDAK.
  • Gp120 MN was incubated with either lgG1 b12 (A) or KZ52 control (B) antibodies linked to Sepharose beads.
  • the antigen-antibody complex was digested with the endoprotease glu-C, washed and tested by MALDI QqTOF mass spectrometry for bound epitopes.
  • Figure 9 Epitope excision mapping by trypsin digestion of gp120 confirms that the lgG1 b12 epitope recognition by gp120 is variable region-specific.
  • Gp120 MN was incubated with either (a) lgG1 b12 or (b) KZ52 control antibodies linked to Sepharose beads.
  • lane 3 shows b12 binding to BSA (negative control).
  • Figure 11 lgG1b12 recognizes synthetic peptide sequence from the amino- terminus of gp120, and not a scrambled version of the same peptide. ELISA plates were coated with lgG1 b12, biotinylated N-term and scrambled peptides were added and tested for binding. Representative data from one of three experiments is shown.
  • Figure 12. The binding of N terminal peptide to lgG1 b12 can be blocked by soluble gp120. ELISA plates were coated with lgG1 b12. Soluble gp120 was added then biotinylated peptide was added and tested for binding.
  • FIG. 13 Representative data from one of 3 experiments is shown.
  • Figure 13 Antigen recognized by differentially immunized mice. Four groups of 4 mice were immunized on five separate occasions with adjuvant plus PBS alone, gp120, N terminal peptide, or scrambled peptide. Serum was collected and tested for antibody recognition of gp120 (A), N terminal peptide (B), and scrambled peptide (C) by indirect ELISA. Representative data from one of 3 experiments is shown.
  • an amount of a given compound that achieves the desired effect.
  • purified does not require absolute purity but is instead intended as a relative definition. For example, purification of starting material or natural material to at least one order of magnitude, preferably two or three orders of magnitude is expressly contemplated as falling within the definition of "purified”.
  • isolated requires that the material be removed from its original environment.
  • the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causitive agent other abnormal condition.
  • the region recognized by lgG1b12 which is the most potent antibody yet described which is capable of neutralizing HIV-1, has been identified.
  • the neutralizing ability of lgG1b12 is likely involved in protective immune responses to HIV-1 and this can be induced in others to generate protective HIV-1 specific responses.
  • the sequence of this particular epitope could be used in blocking HIV infection. Knowledge of when/how responses to this epitope develop may also be useful in tailoring alternate therapeutic interventions, as discussed below.
  • the minimal epitopes as defined by Glu-C digestion are:
  • LWVTVYYGVPVWKE and ATTTLFCASDAK while the minimal epitopes as defined by Trypsin digestion are: LWVTVYYGVPVWK and EATTTLFCASDAK
  • LWVTVYYGVPVWK and EATTTLFCASDAK This leads to .
  • a consensus sequence for lgG1b12 binding of LWVTVYYGVPVWKEATTTLFCASDAK SEQ ID No. 1, shown in Figure 7
  • GVPVWKEATTTL SEQ ID No. 2
  • the sequence of this region varies somewhat in different strains and clades.
  • an isolated- and/or purified polypeptide the amino acid sequence of the polypeptide comprised of or consisting essentially of 6 or more consecutive residues of SEQ ID No. 1 or SEQ ID No. 2, that is, LWVTVYYGVPVWKEATTTLFCASDAK or GVPVWKEATTTL or a variant thereof, for example, as shown in Figure 7.
  • the polypeptide may consist of 7 or more consecutive residues, 8 or more consecutive residues, 9 or more consecutive residues or 10 or more consecutive residues of SEQ ID No. 1 or SEQ ID No. 2, that is, LWVTVYYGVPVWKEATTTLFCASDAK or GVPVWKEATTTL or a variant thereof.
  • variant thereof refers to peptides derived from or based op the amino acid sequence from the same region of gp120 from a different clade or isolate of HIV that act as a neutralizing peptide, as discussed below. Examples of such variants are shown in Figure 7. Other potential variants can readily be determined using means known in the art and any suitable database containing gp120 sequences.
  • an isolated and/or purified polypeptide the amino acid sequence of the polypeptide comprised of or consisting essentially of 6 or more consecutive residues of SEQ ID No. 3 or SEQ ID No. 4, that is,
  • the polypeptide may consist of 7 or more consecutive residues, 8 or more consecutive residues, 9 or more consecutive residues or 10 or more consecutive residues of SEQ ID No. 3 or SEQ ID No. 4, that is, LWVTVYYGVPVW(E/K/R)(E/D)A(E/T/N/K/D/A)(T/P)(T/P/V)LFCASDAK or
  • an isolated and/or purified polypeptide the amino acid sequence of the polypeptide comprised of or consisting essentially of 6 or more consecutive residues of SEQ ID No. 5 or SEQ ID No. 6, that is,
  • the polypeptide may consist of 7 or more consecutive residues, 8 or more consecutive residues, 9 or more consecutive residues or 10 or more consecutive residues of SEQ ID No. 5 or SEQ ID No.
  • the above-described peptides may include peptides that differ by conservative amino acid substitutions.
  • the peptides of the present invention also extend to biologically equivalent peptides that differ by conservative amino acid substitutions.
  • conservative amino acid substitutions refers to the substitution of one amino acid for another at a given. location in the peptide, where the substitution can be made without substantial loss of the relevant function, in this case, the folding of the epitope.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. It is of note, that one of skill i ⁇ the art would anticipate that unconserved or not highly conserved amino acids are more likely candidates for substitution without loss of function.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6)s are assigned to amino acid residues (as detailed in United States Patent No.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0).
  • each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: He (+4.5); Val (+4.2); Len (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Set (- 0.8); Trp (-0.9); Tyr (-.1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Am (-3.5); Lys (-3.9); and Arg (-4.5).
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Len, lie, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr.
  • non-polar Ala, Val, Len, lie, Phe, Trp, Pro, Met
  • acidic Asp, Glu
  • basic Lys, Arg, His
  • neutral Gly, Ser, Thr, Cys, Asn, Gin, Tyr.
  • the b12 antibodies are recognizing a conformational site within the above-described sequence, for example, a conformational epitope formed by non-adjacent, that is, non-contiguous residues.
  • the peptide may comprise 2 or more contiguous amino acids from a first region of SEQ ID No. 1 separated by a linker of variable sequence to 2 or more contiguous amino acids from a second region of SEQ ID No. 2.
  • the linker does not necessarily need to correspond verbatim to the intervening residues between the two regions but should be such that the confirmation of the residues within the conformational epitope is approximated. That is, in other embodiments, there is provided a peptide comprising 2 or more amino acids from the N-terminus region of any one of the peptides according to SEQ ID No.
  • a method of immunizing an individual against HIV infection comprising administering to an individual an effective amount the isolated or purified polypeptide described above.
  • the effective amount is an amount sufficient to induce an immune response within the individual.
  • the preparation may include at least one suitable excipient.
  • administration of the polypeptide to an individual results in the individual obtaining sterilizing immunity against the human immunodeficiency virus, as discussed below.
  • immunization will result in the production of neutralizing antibodies against the above-described polypeptide under subsequent challenge. That is, on subsequent exposure to gp120, antibodies will be produced which will bind to gp120 at the site required for binding to CD4, thereby preventing viral infection of T-cells and will also target the viral particles for removal by antibody dependant cellular cytotoxicity or complement dependent cytotoxicity.
  • any one of the above-described peptides is administered to an individual infected with or suspected of being infected with Human Immunodeficiency virus.
  • immunization will promote the production of neutralizing antibodies, which will in turn bind to gp120, thereby preventing viral infection of T-cells.
  • immunization in these embodiments will slow disease progression by decreasing the rate of viral infection.
  • the immunization may be combined with other anti-HIV compounds, for example, azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddi), dideoxycytidine (ddc) and ritonavir, as well as other reverse transcriptase and protease inhibitors.
  • an immune globulin effective against Human Immunodeficiency virus may be prepared by vaccinating a plurality of donors with any one of the above-described isolated or purified polypeptides; isolating plasma from each of said donors after a period of time sufficient to allow production of antibodies against said polypeptide; pooling the plasma; and preparing an immune globulin from the pooled, plasma using means known in the art.
  • the immune globulin preparation may be used as a treatment for individuals having been recently infected or suspected of having been infected with human immunodeficiency virus.
  • antibodies within the immune globulin preparation will bind to gp120, preventing binding to CD4 and targeting the viral particles for removal as discussed above.
  • a method of treating an individual infected or suspected of being infected by human immunodeficiency virus comprising administering to said individual a therapeutically effective amount of any one of the above-described purified polypeptides.
  • the above-described polypeptide interacts with CD4, effectively acting as a decoy substrate and preventing or greatly reducing gp120 binding to CD4 by occupying CD4 binding sites.
  • this treatment may be combined with other treatments known in the art as well as for example the immune globulin preparation described above.
  • a method of determining a course of treatment for an individual infected with human immunodeficiency virus comprising screening a sample from an individual infected with human immunodeficiency virus for antibodies binding to any one of the above- described purified polypeptides, wherein presence of antibodies against said polypeptide indicates that a less aggressive treatment is needed.
  • the presence of antibodies against this region of gp120 has been shown to result in non-progressio ⁇ of the disease.
  • individuals having natural immunity against this specific region of gp120 may not need to be treated aggressively, as discussed below.
  • any one of the above-described polypeptides may be combined with a suitable carrier peptide known in the art.
  • the purified polypeptide or immune globulin may be combined with other compounds or compositions known in the art such that the is a pharmaceutical composition in the form of, for example, a pill, tablet, liquid, film or coating using means known in the art and as discussed below.
  • the purified polypeptide or immune globulin discussed above may be prepared to be administered in a variety of ways, for example, topically, orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally or by local or systemic intravascular infusion using means known in the art and as discussed below.
  • the above-described pharmaceutical composition may be combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable.
  • a pharmaceutically or pharmacologically acceptable carrier include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, mathaerylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like.
  • Standard excipients include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, .
  • the carrier may be pH-sensitive, thermo-sensitive, thermo-gelling, arranged for sustained release or a quick burst.
  • carriers of different classes may be used in combination for multiple effects, for example, a quick burst followed by sustained release.
  • the above-described pharmaceutical composition at concentrations or dosages described above may be encapsulated for delivery.
  • the pharmaceutical composition may be encapsulated in biodegradable microspheres, microcapsules, microparticles, or nanospheres.
  • the delivery vehicles may be composed of, for example, hyaluronic acid, polyethylene glycol, poly(lactic acid), gelatin, poly(E-caprolactone), or a poly(lactic-glycolic) acid polymer. Combinations may also be used, as, for example, gelatin nanospheres may be coated with a polymer of poly(lactic-glycolic) acid.
  • these and other suitable delivery vehicles may be prepared according to protocols known in the art and utilized for delivery of the.
  • the delivery vehicle may be suspended in saline and used as a nanospray for aerosol dispersion.
  • the above-described pharmaceutical compounds at therapeutically effective dosages would therefore reduce the spread of an HIV infection by accomplishing at least one of the following: decreasing viral load, preventing or limiting the rate of viral infection and preventing further infection by the virus.
  • kits of the invention comprise one or more containers comprising a purified polypeptide or immune globulin as described above, a suitable excipient as described herein and a set of instructions, generally written instructions although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable, relating to the use and dosage of the for the intended treatment.
  • the instructions included with the kit generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers of the glandular kallikrein may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • lgG1b12 antibody Full length, purified lgG1b12 antibody was contributed by Carlos Barbas, III (The Scripps Research Institute, La Jolla, CA, USA). The antibody was linked to cyanogen-bromide activated sepharose beads (Sigma-Aldrich, Oakville, Ontario). Epitope excision was performed to include potential conformational epitopes. The antibody-bead mixture was incubated with either MN or IIIB HIV-1 gp120 (ImmunoDiagnostics, Inc. Woburn, MA.) or synthetic HIV-1 gp120 peptides (United Biochemical Research, Inc, Seattle, Washington, USA) for 2 hours under physiological conditions.
  • MN or IIIB HIV-1 gp120 ImmunoDiagnostics, Inc. Woburn, MA.
  • synthetic HIV-1 gp120 peptides United Biochemical Research, Inc, Seattle, Washington, USA
  • trypsin Calbiochem-Novabiochem Corporation, San Diego, California, USA
  • Glu-C Roche Diagnostics Canada. Laval, Quebec
  • enzyme digests were performed. Unbound, digested material was washed away. Antibody-bound fragments protected from digestion were analyzed by mass spectrometry. Bead + antibody + peptide complex were spotted on a gold plate along with DHB matrix (Sigma-Aldrich, Oakville, Ontario) (3,4). The sample was analyzed on the prototypic QqTOF mass spectrometer by matrix-assisted laser desorption/ionization. Theoretical MN and IIIB trypsin and Glu-C digests were performed on
  • ProMac which provided molecular masses for each possible fragment created by enzyme digestion.
  • IIIB (a) or MN (b) gp120 was bound to lgG1b12 antibody and was digested overnight with Glu-C endopeptidase. Protein fragments not bound by lgG1b12 were washed away. Antibody-bound fragments protected from digestion were analyzed by mass spectrometry.
  • 1867, the second peak corresponds to the sequence KLWVTVYYGVPVWK.
  • the third peak, 2097 corresponds to the sequence TEKLWVTVYYGVPVWKE.
  • This is the first experiment describing a region other than the CD4-binding site as the lgG1b12 epitope.
  • the CD4-binding site epitope expected peak size is 2067.01 m/z with the sequence QFGNNKTIIFKQSSGGDPE.
  • This epitope is clearly missing, implying that it may not be involved in the specific interactions between lgG1b12 and gp120.
  • IIIB (a) or MN (b) gp120 was bound to lgG1b12 antibody and was digested overnight with trypsin endopeptidase.
  • Protein fragments not bound by lgG1b12 were washed away. Antibody-bound fragments protected from digestion were analyzed by mass spectrometry.
  • the size of the peak 1357 corresponds to the N-terminal sequence EATTTLFCASDAK.
  • the peak at 1609 corresponds to the N-terminal sequence LWVTVYYGVPVWK. This indicates that the amino terminus is the source of the lgG1b12 epitope.
  • the mass spectrometer has unequivocally identified the amino terminus as the epitope.
  • the trypsin digest mass spectrometry results strengthens the results seen in the first figure.
  • Figure 3 shows the amino acid sequence of HIV-1 gp120 IIIB (Immunodiagnostics, Inc.).
  • Amino acids identified by mass spectrometry are underlined.
  • Figure 4 shows the amino acid sequence of HIV-1 gp120 MN (Immunodiagnostics, Inc.). Amino acids identified by mass spectrometry are underlined.
  • Bold G glycine
  • G- ⁇ T threonine
  • these amino acids correspond to Ser 370 , Asp 373 , lie 376 , and likely Val 435 for the IIIB sequence, and Ser 341 , Asp 344 , lie 347 , and likely Val 403 in the MN sequence.
  • Saphire et al then confirmed their results by alanine mutation studies.
  • These amino acids are near the CD4-binding site, and therefore it is assumed that lgG1b12 physically blocks the CD4 binding site.
  • LWVTVYYGVPVWKEATTTLFCASDAK as the sequence containing the amino acids involved in lgG1b12 binding. According to the ImmunoDiagnostics IIIB gp120 sequence, these amino acids fall between Leu 34 and Lys 59 , and the amino acids Leu 6 and Lys 31 in the MN sequence. Because, as discussed above, previous experiments have suggested that residues near the CD4 binding site may be important for lgG1b12-gp120 interactions, further studies are required. It has been proposed that lgG1b12 may interact with an isoleucine residue that would otherwise be cleaved off our trypsin digested CD4 binding site peptide (...SSGGDPE/7) (7).
  • N-term amino terminus
  • the N-term sequence is as follows: KLWVTVYYGVPVWKEATTTLFCASDAKAYDTE.
  • a second peptide covered the CD4 binding site, including the isoleucine (I) residue.
  • the sequence QFGNNKTIIFKQSSGGDPEIVTHSFNCGGE was tested for antibody binding, and these results were measured by mass spectrometry, as shown in Figure 5. With the peptides as starting material, lgG1b12 still specifically recognizes the amino terminus and not the CD4 binding site.
  • a 2-hour trypsin digest confirmed the identity of our peptide (figure 6).
  • the 1737 peak corresponds to the digested fragment amino acid sequence KLWVTVYYGVPVWK.
  • tandem mass spectrometry MS/MS
  • MS/MS tandem mass spectrometry
  • One ⁇ g of soluble gp120 MN (ImmunoDiagnostics, Inc., Woburn, MA.) was digested with either trypsin or glu-C and subjected to matrix-assisted laser desorption/ionization quadrupole time of flight mass spectrometry (MALDI QqTOF).
  • Antigen capture ELISA was carried out by first coating 96 well plates (NUNC, Mississauga, ON) with 2.5 ⁇ g/ml lgG1 b12 at 4°C overnight. Plates were washed with phosphate buffered saline (PBS) 0.05% Tween 20 and blocked with PBS containing 0.17% bovine serum albumin.
  • PBS phosphate buffered saline
  • gp120 or BSA was run on a 7.5% SDS-PAGE minigel.
  • the gel was blotted onto a nitrocellulose membrane by Transblot semi-dry transfer cell (Bio-Rad, Mississauga, ON.), blocked, and lgG1 b12 was added at 0.25 ⁇ g/ml and incubated for 2 hours at 37°C.
  • the blot was washed with PBS Tween 20 and HRP-sheep anti-human antibody (The Binding Site, San Diego, CA.) was incubated on the blots. Blots were washed and detected by ECL Advance western blot detection system (Amersham Bioscience, Baie d'Urfe, PQ.).
  • mice 4 groups of 4 BALB/c mice (16 total) were immunized intraperitoneally (i.p.) 5 times over 2 months.
  • the first group of mice received PBS plus Freund's adjuvant in each immunization, and the second group received 10-50 ⁇ g gp120.
  • the third and fourth groups received 10 ⁇ g biotinylated peptide (group 3 received N-term, group 4, scrambled) linked to avidin.
  • Biotinylated peptides were linked to avidin (Zymed Laboratories, Inc., San Francisco, CA) by incubating them at a 1:1 molecular ratio for 30 minutes at 37°C before inoculation.
  • ELISAs to detect Ab responses in mice were carried out by coating 96 well plates with 2.5 ⁇ g/ml gp120 or 5 ⁇ g/ml biotinylated peptide overnight at 4°C. Blood was obtained by venous tail puncture, serum was obtained by collecting the supernatant of blood that had been incubated at 4°C for one hour and centrifuged in an Eppendorf microcentrifuge (Centrifuge 5417C, Brinkmann Instruments, Ltd. Mississauga, ON) twice for 30 minutes at maximum speed.
  • Serum was diluted at 1/50 down in doubling dilutions and incubated on plates for 2 hours at 37°C, washed, and HRP-goat anti-mouse secondary antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) was added. ABTS substrate (Roche Diagnostics Canada, Laval, PQ) was used for detection. Plates were read at 405nm on the Spectramax Plus. In order to confirm that the mass spectrometry epitope mapping experiments specifically revealed peptides involved in interactions with the variable region of lgG1 b12, control experiments were performed. A non-HIV specific anti- Ebola isotype matched antibody KZ52 was used for such experiments.
  • the KZ52 antibody shares the same constant region and framework region as lgG1 b12, but differs at the variable region (Fv) responsible for binding antigen.
  • This first set of experiments was performed using glu-C endoprotease to digest whole gp120 bound to antibodies.
  • Figure 8 (a) shows epitope excision mapping results of lgG1 b12 and KZ52 revealing the N-terminal specificity of lgG1 b12 (m/z value 1806.8, 1866.0 and 2096.1); whereas KZ52 (b) lacks these peaks.
  • the masses of the 3 peaks correspond to the earlier identified linear sequence TEKLWVTVYYGVPVWKEATTTLFCASDAK located near the amino terminus of gp120.
  • Figure 9 confirms the results observed in Figure 8.
  • Epitope excision mapping using the endoprotease trypsin shows specific peaks for lgG1 b12 (a) at 1357 and 1609 (roughly), whereas the KZ52 antibody (b) revealed no specific gp120 peptide epitopes. These peaks correspond to the earlier identified linear sequence KLWVTVYYGVPVWKEATTTLFCASDAK, again demonstrating the recognition of lgG1 b12 to the amino terminus of gp120.
  • MALDI QqTOF mass spectrometry epitope mapping identifies peptide masses that can then be assigned an amino acid sequence based on a theoretical digest of gp120.
  • digested soluble gp120 was subjected to MS/MS sequencing. Soluble gp120, and not antibody-bound gp120 fragments, were used for confirmation because MALDI QqTOF epitope excision mapping yields peptide quantities high enough to be detected, but too low to sequence.
  • MALDI QqTOF epitope excision mapping yields peptide quantities high enough to be detected, but too low to sequence.
  • tandem mass spectrometry the gp120 peak masses 1357, 1609 (trypsin digest) and 2097 (glu-C digest) sequences were confirmed. This confirmation came by matching more than 50% of the theoretical peaks generated by MS/MS to the actual ones.
  • Results show that the gp120 peptide bound lgG1 b12, and this binding was dose dependant. The binding of the scrambled peptide was significantly lower, and did show dose-dependant binding as did the N-term peptide.
  • This experiment strengthens the evidence that lgG1 b12 recognizes N-term sequence on gp120.
  • To substantiate the evidence that lgG1 b12 targets the amino terminus of gp120 we assessed the ability of whole gp120 to compete with peptide for binding to lgG1 b12 by ELISA. We incubated plate-bound lgG1 b12 with soluble gp120 before the addition of biotinylated peptide.
  • the mass spectrometry control experiments validate the mass spectrometry results that lgG1 b12 specifically recognizes gp120 at a sequence located near the amino terminus of the whole gp120 molecule. Since the control KZ52 antibody and lgG1 b12 share the same constant region (Fc), but differ in their variable regions (Fv), the differences in their recognition of gp120 by mass spectrometry are attributable only to specific interactions occurring at the antibody Fv. In this set of experiments, the control antibody used, lgG1 KZ52 recognizes specifically Ebola glycoprotein, and not the HIV glycoprotein gp120.
  • the negative lgG1 KZ52 mass spectrometry results indicate that the nature of the interaction between lgG1 b12 and gp120 are highly specific and localized at the antibody variable region. These results solidify and confirm the original mass spectrometry results.
  • MALDI QqTOF epitope mapping yields peptide peaks that large enough to detect, but too small to sequence. We therefore confirmed the sequence through MS/MS of soluble gp120, a commonly utilized practice.
  • the mass spectrometry mapping uncovered an amino acid sequence that was, in fact, linear and located at the amino terminus of gp120. It has yet to be shown whether lgG1 b12 binds denatured gp120. The binding of lgG1 b12 to denatured gp120 suggests that the previously described IgGI b12-CD4 binding site interactions are incorrect. While the mass spectrometry results do not prove that gp120 conformation is irrelevant to IgGI b12 binding, it is suggested.
  • mice immunizations were to test the immunogenicity of the peptide in an animal model - that is to determine if the peptide can elicit antibodies in an in vivo situation. Mice are commonly and easily used as a first step for testing the capability of an antigen to elicit responses in a live animal model. Our mouse data shows that amino terminal peptide was able to elicit antibodies that were able to bind both N terminal peptide and whole gp120. Background-level responses only were seen on the scrambled peptide-coated plates.
  • the N terminal peptide is immunogenic.
  • the positive mouse data also suggests that the Nterm peptide can be exploited as a vaccinogen.
  • the amino terminus is the binding site of a powerfully neutralizing anti-HIV antibody, it follows that immunization with that amino-terminal sequence will elicit antibody production, and that these antibodies will be HIV-neutralizing.
  • IgGI b12-like antibodies should block HIV infection of cells in neutralization assays. A future direction will be to perform neutralization assays with the mouse serum generated as above. Briefly, cells will be incubated with dilutions of HIV-1 and mouse serum and infection will be measured by p24 and ⁇ gal production.
  • the mouse serum will block HIV-1 infection in vitro.
  • the next step will be to adapt the N-term peptide for human testing and eventually human vaccine phase trials.
  • the serum generated above will be tested for potency of neutralization of live HIV-1 in vitro cell culture assays. Basically, serum generated above will be incubated with laboratory strains and primary isolates of HIV-1 before the virus is used to infect a variety of susceptible cell lines. The ability to block HIV-1 infection will be determined via HIV-1 p24 protein production and compared to non-specific control antibody preparations. We can also test for the ability of antibodies specific for the described epitope to inhibit transcytosis (the ability of HIV-1 viruses to pass across stratified cell layers) as previously described (27).
  • the ability of antibodies to block this process will be compared to non-specific controls antibodies. If antibody responses against the described epitope appear to be capable of generating protective responses in vitro (tested above), the ability of the peptide to protect against HIV infection will be assessed in one of two in vivo animal models currently used in HIV-1 challenge studies (14, 15). Basically animals will be vaccinated with the appropriate adjuvant a number of times. They will then be challenged with live HIV-1 and their susceptibility to infection will be assessed at the appropriate time for the animal model involved.
  • TZM-b1 cells a cell line derivative expressing CD4, CXCR4, and CCR5, and firefly luciferase upon infection with HIV were seeded at a density of 3 x 10 3 cells/96-well plate (J. Biol. Chem. 2005; 280: 4095-4101).
  • next day cells were treated with mouse serum at different concentrations, and one thousand infectious units/well (as determined on TZM-b1 cells) of HIV-1 strains IIIB, SF162, and QH0692 were used to challenge the cells, and 2 days later the cells were lysed and the activity of firefly luciferase activity was determined (Steady-Glo luciferase system, Promega). Because of the induction of firefly luciferase upon infection, the reduction of the relative light units detectable correlates with the inhibition of infection by mouse serum. The viability of the ceils was not affected by the addition of serum. The serum dilutions are listed in table 2.
  • Binding of synthetic peptides to b12 will assessed by ELISA and other EIA based methods as described and will be done to confirm that this epitope binds to the b12 Mab under approachable physiologic conditions in vivo. This information will be used to design a panel of synthetic peptides that can be used to assess the minimal inhibitory peptide epitope, or peptide sequence that will inhibit binding of b12 to the described epitope. These minimal epitopes will be assessed for their ability to interfere with b12/gp120 binding using ELISA based methods.
  • the ability of the minimal inhibitory peptide epitope (identified above) to block HIV-1 infection will be determined essentially as previously described, but using peptides corresponding to the identified epitope rather than serum, or MAb's to inhibit HIV-1 infection of the target cells.
  • the ability of the minimal inhibitory peptide epitope (identified above) to block HIV-1 transcytosis will be determined essentially as previously described, but using peptides corresponding to the identified epitope rather than serum, or MAb's to inhibit HIV-1 transcytosis across target cells.
  • One further application of the invention is detecting the presence of Ab capable of recognizing described epitope as a means of correlating or analyzing HIV disease progression and/or resistance to infection by HIV-1.
  • Kessler II J. A., McKenna, P. M., Emini, E. A., Chan, C. P., Patel, M. D., Gupta, S. K., Mark III, G. E., Barbas III, C. F., Burton, D. R., and A. J. Conley. 1997.
  • Recombinant human monoclonal antibody lgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res. Hum. Retro. 13(7):575-82.
  • Tandem mass spectrometry sequencing confirms 3 of the 5 sequences derived from epitope mapping. Peaks 1357, 1609, 1807, 1867 and 2097 were selected from digested gp120 complete spectra for MS/MS on the MALDI QqTOF. The a, b. and y ionic fragmentation peptide masses were measured and percent match was calculated. Tabulated are the y ion fragment matches, which are representative of the three forms of fragmentation.
  • mice immunized with the N terminal epitope Serum was isolated and from 4 groups of 4 mice immunized with PBS (P), gp120(G), N terminal peptide (N), or Scrambled peptide (S), then pooled by group. Neutralizing ability was tested for both serum collected from a pre- immunization bleed (PO, GO, NO, SO), and post immunization (P1, G1, N1 , S1). Neutralization ability was tested against 3 strains of HIV-1 (IIIB, SF162.LS and QH0692.4) in TZM-b1 cells.
  • RLUs relative luminescence units

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Abstract

La présente invention a trait à l'anticorps monoclonal humain IgG1b12 de forte liaison à la gp120, constituant l'anticorps le plus puissant connu de neutralisation du VIH. La séquence exacte d'acides aminés de ce site de liaison n'est pas connue. On a identifié les parties minimales de la protéine d'enveloppe gp 120 du VIH 1, auxquelles se lie l'anticorps IgG1b12. Cela a été réalisé par la spectrométrie de masse de temps de vol quadrupolaire (QqTOF) par mise en oeuvre de l'excision d'épitopes. L'excision d'épitopes permet la détermination d'épitopes conformationnels. A cet effet, on a réalisé la liaison de la gp120 à l'anticorps IgG1b12, la digestion de toutes les portions non liée de gp120, suivie de l'exécution de la spectrométrie de masse du complexe peptidique IgG1b12-gp120 obtenu. Les résultats ont permis la détermination de la séquence exacte d'acides aminés impliquée dans l'interaction IgG1b12-gp120.
PCT/CA2005/000547 2004-04-09 2005-04-11 Identification de la sequence precise d'acides amines de l'epitope identifie par l'anticorps monoclonal igg1b12 puissant de neutralisation anti-vih 1 humain WO2005097822A1 (fr)

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EP2928492A4 (fr) * 2012-09-11 2017-09-13 The Regents of The University of California Protéines d'enveloppe du vih-1 et leurs fragments qui possèdent des épitopes reconnus par des anticorps largement neutralisants
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