WO1993020104A1 - Anticorps monoclonaux agissant contre un determinant antigenique dependant de glucides correspondant a la region v2 de gp120 de hiv-1 - Google Patents

Anticorps monoclonaux agissant contre un determinant antigenique dependant de glucides correspondant a la region v2 de gp120 de hiv-1 Download PDF

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WO1993020104A1
WO1993020104A1 PCT/US1993/003044 US9303044W WO9320104A1 WO 1993020104 A1 WO1993020104 A1 WO 1993020104A1 US 9303044 W US9303044 W US 9303044W WO 9320104 A1 WO9320104 A1 WO 9320104A1
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hiv
mab
gpl20
mabs
antibodies
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PCT/US1993/003044
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WO1993020104A9 (fr
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Shermain Tilley
Abraham Pinter
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The Public Health Research Institute Of The City Of New York, Inc.
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Priority to AU56952/94A priority Critical patent/AU5695294A/en
Publication of WO1993020104A1 publication Critical patent/WO1993020104A1/fr
Publication of WO1993020104A9 publication Critical patent/WO1993020104A9/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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

Definitions

  • This invention relates to monoclonal antibodies ("mAbs”) against a carbohydrate-dependent, V2 related epitope of HIV-1 and to certain glycosylated peptides which effectively present that epitope.
  • mAbs monoclonal antibodies
  • These mAbs and peptides are useful for example in immunoassays, affinity chromatography, in immunogenic compositions, or neutralizing compositions.
  • the HIV-l envelope is composed of two glycoproteins , gpl20 and gp41. These glycoproteins are initially synthesized in virus-infected cells as a precursor called gpl60. This molecule is cleaved into gpl20 and gp4l prior to assembly of virions. The latter two glycoproteins are non-covalently associated with each other and are anchored to the viral membrane via gp41, a transmembrane protein (reviewed in (Olshevsky et al. 1990)).
  • V3 region hypervariable loop hvl-V3
  • gpl20 amino acids 307-330
  • CD-4 binding site Another epitope cluster of HIV-l envelope that has been shown to elicit neutralizing antibodies is the CD-4 binding site of gpl20.
  • the CD-4 binding site is believed to be formed by non-contiguous protein loops from multiple regions of gpl20 (Olshevsky et al. 1990) .
  • the precise structure of the CD-4 binding site and its contact residues have yet to be defined.
  • mouse derived mAbs which only weakly bind and are only weakly neutralizing have been obtained by Fung et al. and Ho et al. which are specific for epitopes in or near the second variable region of HIV-l (the V2 region) , (Ho et al. 1991) .
  • a preliminary report indicates that human serum antibodies raised during HIV-l infection against an SF-2 strain- derived V2 peptide can neutralize virus in vitro (Stephens et al. 1991) .
  • chimpanzees were challenged with a stock of the IIIB strain of HIV-l that had previously been incubated with neutralizing serum Ab from an HIV-1-seropositive chimpanzee.
  • the challenged animals were protected against viral infection, as assessed by lack of serum Ab response to virus and attempts at viral isolation (Emini et al. 1990) .
  • Successful long term protection of two chimpanzees against HIV-l infection has been demonstrated by immunization with recombinant gpl60 followed by a V3 loop peptide (Girard et al. 1991) .
  • the mAbs are specific for an amino acid seguence included in about amino acid 160 to about amino acid 180 of gp 120 of the HXB2 strain or in the homologous sequences of other strains.
  • the binding of the mAbs to gpl20 is carbohydrate dependent, that binding being substantially reduced by complete removal of N-linked carbohydrate residues of gpl20.
  • glycosylated peptide lacking the CD-4 binding site, and also lacking the V3 region, and which contains at least part of the V2 region, is responsible for eliciting high affinity mAbs that are also very strongly neutralizing. This response is quite surprising in view of the weakly neutralizing mAbs known against unglycosylated V2 peptides.
  • the glycosylated peptide is useful in an immunoassay for detecting HIV antigen or antibodies, or as a component of an i munogenic composition.
  • the invention also includes a mAb against gpl20 whose binding to gp 120 is competitively inhibited by mAbs against V2 region peptides, the mAb having an affinity for gpl20 of at least about lxl0 9 L/mole, and preferably about lxlO 10 L/mole.
  • V2 peptides can synergistically neutralize HIV-l infection in vitro in combination either with anti-V3 antibodies, or in combinations with anti-CD-4 binding site antibodies.
  • cell lines producing the mAbs of the invention are included in the invention.
  • the invention includes using the mAbs of the invention in im unoassays for determining HIV antigen or a competing antibody, for prevention of HIV-l infection or for HIV- 1 therapy.
  • Immunoassay kits including the mAbs are also included.
  • Figure 1 shows the results of a RIPA analysis described in the examples.
  • FIG. 2 shows the results of an immunoblot experiment described in the examples.
  • Figure 3 shows the results of an experiment described in the examples related to an immunoblot.
  • Figure 4 is a graph of the results of a competitive inhibition experiment described in the examples involving competitive inhibition by soluble CD4 of the binding of certain mAbs to gpl ⁇ O.
  • Figure 5 is a graph of the results of a competitive inhibition experiment described in the examples involving competitive inhibition by certain mAbs of the binding of C108G to gpl ⁇ O.
  • Figure 6A-D are graphs of the results of competitive inhibition experiments described in the examples.
  • Figure 7 shows peptide mapping results of the epitope of C108G. The amino acid sequence of gpl20 is also shown with high mannose sugar and complex sugar glycosylation sites indicated.
  • Figure 8 shows the amino acid sequences homologous to the C108G epitope in six HIV-l strains, indicating the reactivity of mAb C108G with each of those strains.
  • Figures 9-11 are graphs of the results of neutralization experiments described in the examples.
  • Figure 12 is a graph of combination index values calculated from the neutralization results graphed in Figures 10 and 11.
  • the glycosylated peptide of the invention can produce a very strong neutralizing response even though it does not contain either of the two strong neutralization sites already known, i.e. the CD-4 binding site and the V3 loop.
  • the glycosylated peptide in one embodiment, includes part or all of the gpl20 sequence from about amino acids nos. 160-180 of HXB2 or the corresponding (i.e. homologous) sequence in other HIV-l strains.
  • the V2 region extends from amino acids 126-196.
  • the glycosylated peptide can include an immunologically effective part of amino acids 160-180 of gpl20 of the HXB2 strain, or the corresponding amino acids of another HIV-l strain.
  • a strongly neutralizing antibody against a new epitope or epitopes, in or near the V2 region achieves at least about 50% neutralization in vitro of about 1 x 10 5 infectious units of HIV-l strain at a concentration of about 0.05 ⁇ g/ml.
  • the mAb is very strongly neutralizing, neutralizing in vitro about 50% of about 10 5 infectious units of HIV-l at a concentration of about 0.03 ⁇ g/ml.
  • a particular mAb discovered by us has been found to be about 6000 times more effective at neutralizing than previously described neutralizing antibodies against the V2 region, and about twenty five times more effective than a previously described strongly neutralizing antibody against the CD4 binding site.
  • the neutralization values are those obtained using a standard 24 hr. neutralization assay described herein. Other assays may arrive at different values.
  • Binding to an epitope, or epitope cluster, by the antibodies of the invention is substantially reduced by removal of all N- linked carbohydrate residues in gpl20, such as by treatment with N-glycanase.
  • mAbs of the invention include those whose binding to gpl20 is inhibited at least about 75% by addition of C108G at the concentration of C108G required to substantially inhibit (i.e. 90% or more inhibition) its own binding to gpl20. Preferably the inhibition is more than about 90%.
  • the binding of C108G to the IIIB strain of HIV-l has been established and it is anticipated that C108G may bind to other strains as well though, as further described below, it is not believed to bind to several common strains.
  • a particular mAb of the invention may react with the strongly neutralizing V-2 related epitope in one strain, but not with the corresponding epitope in another.
  • C108G has been found to strongly neutralize the IIIB strain, but has not been found to significantly neutralize the MN strain. It also does not bind the SF-2, or the RF strains of the virus.
  • the epitope of the mAb of the invention need not be variable; i.e. it may be conserved.
  • the discovery of the new neutralization epitope(s) characterized herein allows the homologous (i.e. corresponding) epitopes in different strains to be easily identified by one skilled in the art. It also allows mAbs to those epitopes to be obtained using standard mAb production and screening procedures.
  • amino acid sequences of other strains can be aligned to obtain maximum homology by one skilled in the art.
  • Epitopes in homologous sequences need not be immunologically cross-reactive. For example, a homologous sequence of the MN strain would be about: NITTSIRDKMQKEYALLYKLD.
  • the mAbs may bind with an antigenic determining amino acid seguence contained in a smaller part of HXB2 gpl20, such as 160-170, or a homologous sequence. Binding to peptides which have such sequences is shown in the Examples.
  • mAb C108G is produced by an EBV-transformed chimpanzee B- cell line deposited on March 10, 1992 at the American Type Culture Collection, (ATCC) , located at 12301 Parklawn Dr. , Rockville MD, 20852, United States of America, and assigned accession number CRL 10983. Methods for obtaining C108G and its further characterization are described below.
  • C108G is of primate origin, which has distinct advantages over, for example, rodent mAbs.
  • Primate mAbs have increased stability and very low immunogenicity in humans.
  • primate mAbs are much less likely to create deleterious anti-immunoglobulin responses than are non-primate mAbs and it should be possible to obtain stable levels of therapeutic doses of the primate mAbs in humans.
  • Human mAbs are obtained following the same procedures described herein for obtaining chimp antibodies except that peripheral blood ononuclear cells of a patient infected with HIV-l, can be used as a source of antibody in the screening procedures.
  • an immortalized cell line of the invention such as the deposited cell line CRL 10983
  • genes coding for the mAb expressed by the cells may be altered and resulting mAbs screened to obtain mAbs with even greater affinity for antigen.
  • the genes may also be altered to change the isotype, idiotype, or effector functions of the mAbs.
  • Expression systems have been developed to allow expression and secretion of genetically engineered non-murine mAbs, such as human mAbs, in mouse cells. Using the genes, one may conventionally develop mAbs having the complementarity determining regions of C108G.
  • the immunogenic response of humans to chimp mAbs is believed to be no greater than to human mAbs as the immunoglobulin variable (V) regions of chimpanzees and humans do not appear to differ any more than human V regions differ from each other (Ehrlich et al., 1990; Ueda et al. 1988; Sakoyama et al. 1987) .
  • studies designed to test the i munogenicity of human or chimpanzee mAbs in rhesus monkeys indicate that antibodies from these three species have little, if any, immunogenicity when administered to the other two related species in the group.
  • the mAbs of the invention can be used in any conventional manner including immunoassays, affinity chromatography and any other procedures that use anti-HIV mAbs.
  • Their high affinity makes the mAbs patentability useful in detection of HIV, either indirectly through a competitive assay, or directly in any assay for HIV antigen.
  • the same property makes the mAbs especially useful in standard affinity chromatography procedures for purifying HIV.
  • the antibodies of this invention are used in a competitive immunoassay, such as described below, to determine antibodies to the HIV-l epitopes recognized by the mAb.
  • the present invention also includes test kits to measure the presence of HIV antigens or of Abs against the epitopes of the mAbs of this invention.
  • a kit for a competitive ELISA to determine antibodies contains mAbs of the invention, a solid phase on which is coated an antigen which the mAbs are specific for, and means for detecting the formation of a complex between the monoclonal antibodies and the antigen.
  • An ELISA using biotin labeled Abs can be performed similarly to the assay described below. Such an assay determines whether the sample has any antibody competing with the antibody of the invention.
  • kits for determining an antigen for which antibodies of the invention are specific may comprise a mAb of the invention, a solid phase on which is coated an antibody for HIV-l env, and an HIV antigen for which the mAbs are specific.
  • the invention allows antibodies to be obtained which are specific for the new strongly neutralizing V-2 related epitope(s) discovered by us.
  • Standard Kohler Millstein protocols can be used to obtain mAbs of the invention, immunizing a rodent with any HIV antigen (of any strain) that presents the epitope described herein.
  • Hybrido as obtained that produce mAbs against gpl20 can be further screened for binding to V2 peptide, preferably about amino acids 160-180 of HXB2 or the corresponding sequence of other HIV-l strains, and for N-glycan dependency.
  • Other screening procedures described below with respect to primate mAbs can be used to screen rodent mAbs as well.
  • PBMC peripheral blood mononuclear cells
  • EBV transformation a conventional immortalization process
  • Supernatants of cultures of the EBV-transformed PBMC can be screened for reactivity with gpl60, or gpl20.
  • Further screening to obtain cell lines of the invention can be accomplished by eliminating cell lines producing antibodies which do not bind to peptides of about amino acids 160-170 of the HXB2 strain or corresponding sequence from a different strain and are non N- glycan dependant.
  • peripheral blood mononuclear cells from HIV-l infected individuals chimpanzees can be immortalized by transformation with Epstein-Barr virus using a modification described below of the procedure of Gorny et al. (Gorny et al. 1989) .
  • the mAb cultures can be obtained by screening immortalized cultures for production of anti-env antibody using recombinant gpl60 coated ELISA plates.
  • Recombinant gpl ⁇ O obtained from higher eukaryotic transformed hosts is probably required, for example gpl60 expressed by transformed baby hamster kidney cells as per Kieny et al. (Kieny et al.
  • gpl60 supplied by Pasteur Merieux, lacks the site which is normally cleaved to form gpl20 and gp41. The deletion of this cleavage site, however, is not believed to have any effect on the screening process and to be distant from the epitope[s] which the antibodies of this invention are specific for.
  • Alternative preferred sources include gpl60 or gpl20 obtained from other transformed higher eukaryotic hosts. For example, recombinant gpl20 per Leonard et al. (Leonard et al. 1990) available through the AIDS Research and Reference Reagent Program (NIH) is also believed effective in screening cultures for mAbs of the invention.
  • An additional means to screen for the strongly neutralizing mAbs is to isolate a glycosylated peptide that contains V2 and adjacent peptide sequences from gpl20 or gpl60 of any desired strain, after treatment of the gpl20 or gpl60 with trypsin or other proteases. Procedures for doing this have been described by Leonard et al. (Leonard et al. 1990) . (Using the method of Leonard et al., peptides with disulfide linkages can be obtained.) The identity of the peptide can be determined by conventional sequencing, or by reactivity with anti-V2 antibodies having broad specificity (such as described by Ho et al) .
  • glycosylated peptide which can be used to directly screen for antibodies of the invention in ELISA assays.
  • An equivalent approach is to synthesize the peptide by standard methods, and then to add the relevant glycans at appropriate glycosylation sites. Preferably, however, screening is done against a linear peptide containing amino acids about 160-180 of HXB2 or a corresponding sequence and N-glycan dependence determined separately (see examples below) . Since the mAb of the invention is N-glycan dependent binding to the peptide will be low.
  • glycopeptides described above from the desired strain can be used in combination with a pharmaceutically acceptable carrier as an immunogenic composition for eliciting an immune response for obtaining mAbs, or for inducing a neutralizing response in an individual.
  • Glycopeptides including about amino acids 160-180 of HXB2 or a corresponding sequence of another strain, (but lacking a functional CD-4 binding site and the V3 region) , can be screened for effectiveness in generating strongly neutralizing antibodies in conventional immunization experiments in animals, such as chimpanzees, rodents or rabbits. Sera from immunized animals in such experiments are drawn and tested for neutralization ability. Vaccine trials in chimpanzees can then be carried out, the chimps being challenged with diverse strains.
  • Screening to determine whether cultures are producing mAbs to an epitope substantially overlapping the one recognized by antibody C108G can be done with a competitive ELISA assay.
  • Such an assay determines if mAbs from the culture being screened compete with C108G in binding to an epitope presented on, for example, recombinant gpl60 coated ELISA plates. MAbs which compete highly would be specific for the same or adjacent epitopes.
  • Other mAbs of the invention, obtained as described above, can be used to screen for mAbs that are not cross-reactive with C108G.
  • a suitable competitive assay is described below.
  • the invention includes synergistic combinations of antibodies against the V2 region with antibodies against either the CD-4 binding site or with antibodies against the V-3 region.
  • antibodies can be screened from those regions in order to find those that best synergize with each other.
  • antibodies produced by any method against each of these epitope clusters are screened for synergistic activity.
  • a given combination of a mAb against the V-2 region and against the CD-4 binding site can be screened in a standard neutralization assay for synergistic neutralizing activity. The individual neutralizing activity of each antibody individually is compared with the neutralization activity of the antibodies combined. A suitable neutralization assay is described below.
  • the ability of the given antibody combination to synergize is evidenced by a significant increase in neutralization activity over that obtained in the presence of equivalent concentrations of the individual antibodies.
  • the extent of synergy can be quantitated by calculating the Combination Index using known statistical methods such as those described by Chou (Chou and Talalay 1984; Chou et al. 1989; Chou 1991).
  • anti CD-4 binding site antibodies such as human monoclonal antibodies or other antibodies
  • anti V-3 antibodies can be screened for synergistic activity in combination with the C108G antibody of the invention.
  • the antibodies can be known antibodies or newly obtained.
  • Antibodies which competitively inhibit the binding of C108G to gpl20 to a high degree can also be screened for synergistic activity in combination with anti-CD4 binding site antibodies or anti-V3 antibodies.
  • Polyclonal antibodies from different sources may be employed to obtain antibodies for use in the synergistic combinations of the invention. Methods have been described in the literature for inducing neutralizing antibodies against different epitopes of HIV-l gpl20 in both rodents and chimpanzees. Antibodies against the V3 loop have been induced in both rodents (Javaherian et al, 1990) and chimps (Girard et al., 1991) by immunizing animals with synthetic V3 peptides either in free form, or conjugated to KLH. Anti-V3 antibodies have also been induced by immunizing chimps with purified gpl20 and gpl ⁇ O (Berman et al., 1990).
  • Antibodies against both regions can also be produced in chimpanzees which have been infected with HIV, although the V3 region is immunodominant, and anti-V3 antibodies will predominate over anti-CD-4 binding site antibodies.
  • Monoclonal antibodies against these gpl20 epitopes can be prepared from immunized mice by standard techniques, and monoclonal antibodies can be prepared from chimps by following the EBV-transformation procedure described herein.
  • Antibodies against the various regions can be purified by immunoaffinity chromatography.
  • AH-Sepharose beads are activated by treatment with glutaraldehyde, and conjugated either to purified V2 peptide or glycopeptide, or to gpl20.
  • Antibodies against V2 peptide or glycopeptide can be obtained by passing 10-fold diluted hyperimmune serum through the columns to allow the antibodies to bind, and washing off unbound antibodies with saline solution.
  • V2-specific antibodies can be eluted from a V2 column by washing with tris-glycine buffer, pH2.7 while V2 specific antibodies can be eluted form a gpl20 column by passing through excess V2 peptide or glycopeptide.
  • synergistic combinations of Abs can be employed for any of the various uses of anti-HIV antibodies described herein. High neutralization ability orrelates with high affinity. Thus, tests described for measuring neutralization ability are useful in determining high affinity as well. Affinity can of course be directly measured using known techniques (see Examples) . Thus, synergizing antibodies are useful in the immunoassays and affinity separation mentioned above, or for administration to an individual.
  • mAbs of the invention may be used to inhibit HIV-l infection in cases of acute exposure to HIV.
  • the HIV-l neutralizing mAbs of this invention could be passively administered to pregnant seropositive women to prevent their fetuses from becoming HIV-l infected.
  • mAbs can be used to prevent HIV-l infection by administering them to individuals near the time of their exposure to HIV-l.
  • the neutralizing mAb should be extremely potent, so that neutralizing concentrations can be attained in vivo following administration of milligram amounts of the mAb. It has been estimated that between 0.03 to 3 mg/ml of a neutralizing Ab with similar affinity to that of CD-4 for gpl20 would be required to eliminate HIV infection in vivo (Layne et al. 1989) . This would necessitate administration of approximately 0.15 to 15 g of Ab per patient, the higher ranges of which are not feasible because of the side-effects associated with administering such high protein doses and the difficulties and cost of producing such large amounts of purified antibodies.
  • the high affinity and neutralization ability of the mAbs of the invention make possible a reduction in the concentration of mAb required. Also, because, as further described below, the mAbs of the invention can be used synergistically with mAbs against the CD-4 binding site or in a synergistic combination with mAbs against the V3 region, less total mAb can be used. The mAbs of the invention can also be used in combination with other anti-HIV-1 mAbs, in order to attain an additive (i.e. non-synergistic) effect.
  • the antibodies can be adjusted to 5% solution in sterile saline, yielding a concentration of 50 mg/ml.
  • synergizing antibodies of the invention the best ratio of the synergizing antibodies is determined experimentally, using the 24 hour fluorescent focus assay described below.
  • a 1:25 ratio of C108G and a strongly neutralizing anti-CD4 binding site antibody can be used; i.e. a concentration of each which has been determined to give very roughly comparable levels of neutralization to each other.
  • the amount of this solution required for protection can be determined in animal experiments, performed first in Hu-SCID mice (Mosier et al. 1988, McCune et al. 1988) and subsequently in chimpanzees.
  • the reagent can consist of as few as one antibody, although it is believed that the most efficient composition will contain a larger number of different antibodies directed against major antigenic sites. This is in order to increase the cross-reactivity of the antibodies to different HIV variants.
  • engineered antibodies of different isotypes including IgGs, IgMs, and IgAs, in order to increase the affinities and effector activities of the antibodies. It is also believed to be beneficial to include antibodies conjugated to toxins, mentioned below, to increase the killing of infected cells, and engineered bispecific antibodies, to increase targeting of infected cells to immune cell-mediated cytotoxic mechanisms.
  • the preferred mAb of this invention which make it excellent for these applications are: 1) its demonstrated HIV- 1 neutralizing activity in vitro at low mAb concentrations, 2) it has high affinity for antigen (HIV-l gp 120) , 3) it is of primate origin and will, therefore, elicit few, if any, deleterious immune reactions when administered to humans, and 4) the heavy chain isotype of the preferred mAb is IgGl, which is significant because human IgGAbs are the only class of Ab able to cross the placenta, and Abs of the IgGl subclass can potentially kill HIV- 1-infected cells in vivo via Ab- and complement-dependent cytotoxicity (ACC) and/or Ab-dependent cellular cytotoxicity (ADCC) .
  • ACC complement-dependent cytotoxicity
  • ADCC Ab-dependent cellular cytotoxicity
  • mAbs can be used by themselves to prevent HIV-l infection, they may be modified to enhance their anti-viral activity by covalent attachment of a toxin such as ricin A, pokeweed antiviral protein, poisonous lectins, abrin, diphtheria toxin, or other toxins to the mAbs. It has been demonstrated that such anti-HIV-1 mAbs-toxins are capable of specifically killing HIV-l infected cells in vitro.
  • a toxin such as ricin A, pokeweed antiviral protein, poisonous lectins, abrin, diphtheria toxin, or other toxins.
  • the killing of HIV-l infected cells via ACC, ADCC, or following mAb conjugation with a toxin could complement the neutralizing activity of our mAbs by eliminating a very small percentage of HIV-l infected cells which might result if 100% neutralization of HIV-l by the mAbs is not obtained.
  • Gram quantities of this invention's mAbs are preferably obtained for the various uses described. These amounts can be obtained by growth of cell lines producing the mAbs of the invention in a mini-bioreactor. Additionally, cost-effective methods to increase mAb production are: 1) fusion of EBV- transformed lines with a human/mouse heteromyeloma (Teng et al. 1983; Kazbor et al. 1982) and 2) PCR amplification of expressed immunoglobulin V H and V L genes from cell lines using published primer sequences (Larrick et al. 1989) , followed by cloning of these genes into available eukaryotic expression vectors containing constant region genes (Orlandi et al. 1989) .
  • genes could be directly cloned from cDNAs or genomic DNA of antibody producing cells.
  • the latter constructs can then be expressed as mAbs at high levels in mouse myeloma cell lines.
  • Transformed cell lines producing recombinant monoclonal antibodies are included within the scope of the invention, as are the antibodies produced thereby.
  • Such antibodies may contain the complementarity determining regions of C108G or other mAbs of the invention.
  • the invention also includes moieties having the same function as monoclonal antibodies, such as Fab fragments, F(ab') 2 , Fd or other fragments, modified proteins such as chimeras with altered Fc regions, or having mutagenized idiotypic regions, or the heavy or light chains alone, so long as they bind to the same epitopes as the monoclonal antibodies of the invention.
  • Techniques for producing such fragments or modified antibodies are known to one skilled in the art (e.g., Parham 1986) .
  • the invention also includes cell lines producing the mAbs of the invention.
  • Those cell lines can be, for example, conventionally immortalized cell lines of any type obtained using the immunization and screening materials described herein, or other types of mAb producing cells such as described herein.
  • the preferred reagent for administration of this invention consists of mixtures of engineered antibodies of different isotypes, including IgGs, IgMs, and IgAs, in order to increase the affinities and effector activities of the antibodies.
  • an adjuvant can be included with the glycosylated peptide in the immunogenic composition of the invention in order to enhance the immune response.
  • adjuvants include Freunds complete adjuvant, saponin, aluminum hydroxide, or other available adjuvants or combinations of adjuvants.
  • a carrier should also be included in the immunogenic composition. Such carriers are well known to one skilled in the art and include, for example, saline solution.
  • the immunogenic composition can contain, for example, from about 0.2 to 5 mg of glycosylated peptide in a one ml dose including an adjuvant and carrier.
  • C108G ChmAb (chimpanzee antibody) was derived from chimpanzee #087 (“Owen”) that was part of HIV-l vaccine studies conducted by Dr. Marc Girard, Pasteur Institute, at L.E.M.S.I.P., New York Univ. School of Medicine. Chimpanzee #087 was naive for HIV-l and its components as well as for antibodies against HIV-l at the beginning of the study. On 1/23/90, #087 was infected with HIV-l IIIB strain (100 TCID 50 ) as a control animal (Girard et al. 1991) . Chimpanzee #087 remains HIV-l infected and healthy. On three dates (11/13/90, 12/10/90. 1/8/91) following the HIV-l infection of #087, the animal received injections of 150 ⁇ g of gpl ⁇ O M ,.
  • PBMC peripheral blood ononuclear cells
  • the cells were gently resuspended, diluted approximately 10-fold with RPMI 1640 medium, and spun down. The pellet was resuspended at a final density of 10* cells/ml in complete medium. The cells were then plated in U bottom 96-well plates at lOO ⁇ l (1000 cells) per well onto lOO ⁇ l of irradiated (3500 rads) rat embryo fibroblasts in complete medium. The cultures were fed weekly for 4 weeks at which time approximately 45% of the wells exhibited growth. Then their supernatants were assayed for anti-env Ab production (see below) .
  • DNA was isolated from C108G followed by restriction enzyme digestion, agarose gel electrophoresis, blotting to nitrocellulose, and hybridization to 32 P-labeled nick-translated probe (Eckhardt et al. 1982) .
  • the DNA was cut with Hind III, which allows visualization of rearrangements due to V-D-J joining upon hybridization with an immunoglobulin J H region probe (Ravetch et al. 1981) .
  • the J H probe used was a EcoRI-Hindlll fragment approximately 3.3 kilobases in length from the germ line J H locus; the Hindlll site at its 3' end is present in the germ line DNA [Ravetch, J. V., U. Siebenlist, S. Korsmeyer, T. Waldmann, and P. Leder. (1981) Cell 27:583-591], whereas the EcoRI site at its 5' end was created upon cloning. The monoclonality of the cell line was confirmed.
  • ELISA assays were used to detect HIV-l env-specific Abs.
  • the initial screening of EBV-transformed chimp cultures for production of anti-env Ab was done using recombinant gpl60 BRU (Kieny et al.) to coat PVC ELISA plates (Flow/ICN) .
  • gpl60 BRU Kieny et al.
  • Flow/ICN PVC ELISA plates
  • a variety of other HIV-l proteins or peptides can be used to determine the specificity of the mAbs. These include recombinant gpl20 of the IIIB strain produced by Celltech, Inc. and available through the AIDS Research and Reference Reagent Program (NIH) or described by (Leonard et al.
  • C108G The specificity of C108G was confirmed by both RIPA and immunoblot analyses (Figs. 1-3) .
  • glycoproteins in HIV-1- infected cells at 5-7 x 10 5 cells/ml were labeled with 3 H- glucosamine (lOO ⁇ Ci/ml) (Pinter et al. 1989) .
  • the cells were then lysed and immunoprecipitated as previously described (Pinter et al. 1988) . Briefly, the cell pellet was brought up in lysis buffer at a concentration of 5 x 10 6 cells per ml.
  • the lysate was then precleared with fixed, killed staphylococcus aureus cells (Staph A) , and 70 ⁇ l of pre-cleared lysate was added to 70 ⁇ l of supernatant from chimp Ab-producing cell lines or 1/400 dilution of human sera. Following an incubation and precipitation by Staph A, the pellet was brought up in Laemmli sample buffer containing 1% DTT and run on an 11% polyacrylamide gel as described (Laemmli 1970) . Fluorography (Bonner et al. 1974) then allowed detection of radiolabeled, immunoprecipitated glycoproteins in the gel.
  • Western blot analysis was performed using strips prepared with HIV-l lysate essentially as described by Pinter et al. (Pinter et al. 1989) .
  • the lysate was diluted in buffer composed of 0.01M Tris hydrochloride (pH 7.4) ,10% glycerol, 0.01% bromophenol blue, either 0 or 1% DTT, and 1% SDS.
  • the Western blot strips were incubated with a 1/2 dilution of supernatant from human Ab-producing cell lines or a 1/100 dilution of chimp serum, and bound Ab was detected (Pinter et al. 1989) .
  • HIV-l strains IIIB Popovic et al. 1984; Ratner et al. 1985
  • SF2 Levy et al. 1984; Sanchez-Pescador et al. 1985
  • strains IIIB, MN, and RF were confirmed by us using strain-specific antisera against the hypervariable V3 loop (hvl-v3) of each strain in an immunofluorescence assay.
  • the IIIB-specific chimpanzee antiserum was obtained through a collaboration with Dr. Marc Girard, Pasteur Institute, whereas the MN- and RF-specific rabbit antisera were generously provided by Dr. Robert Neurath, New York Blood Center.
  • the slides Prior to attachment of cells to Multi-spot microscope slides (Shandon) for immunofluorescence analysis, the slides were treated with poly-L-lysine (lOO ⁇ g/ml in PBS, 50ml per well) for 30 min at room temperature. The slides were then washed with distilled water and dried. Cells that were 100% HIV-1-infected or uninfected were then washed in sterile PBS, resuspended in PBS at a density of 1-2 x 10 6 cells/ml, and incubated on the poly-L lysine-coated slides (50 ⁇ l cell suspension/well) at 37°C for 30 min. The slides were then washed 2X in 100-200 ml PBS, using a slide-holder and trays.
  • poly-L-lysine lOO ⁇ g/ml in PBS, 50ml per well
  • Heavy chain subclass was determined using a variation of the immunofluorescence assay. Chimp mAb-producing cells were attached to slides and fixed with acetone. The slides were blocked with bovine gamma globulin and washed as discussed above. Next, a 1/5000 dilution of human IgG subclass-specific mouse monoclonal Ab (Zymed) (specifically, anti-IgGl and anti-IgG2 were used in these experiments) was added and incubated for 1 hr. at 37° C. Following washing and drying of the slides, biotinylated goat antimouse IgG (Zymed) , 1/200 dilution, was added and incubated for 1 hr. , 37° C.
  • Light chain isotype was determined by a variation of the ELISA assay discussed above. Following incubation of supernatant from mAb-producing chimp cells with gpl60 in duplicate ELISA wells, the mAb isotype was determined by development of one well with goat anti-human kappa Ab conjugated to alkaline phosphatase and the other well with goat anti-human lambda Ab conjugated to alkaline phosphatase. Both of the latter reagents (Tago) were used at 1/3250 dilution. mAb C108G was found to have a light chain kappa isotype and a heavy chain IgGl isotype.
  • the mAbs Prior to conducting neutralization assays, the mAbs were purified on recombinant protein A Sepharose columns essentially as described (Harlow et al. 1988) .
  • the column fractions containing mAb (as determined by ELISA assay of fraction aliquots) were concentrated in an AMICON centriprep 30 column and dialyzed against PBS.
  • the neutralization assay was carried out as follows. Purified Abs, or combinations of Abs, were diluted in complete media containing 10% FCS to obtain concentrations ranging from 0.01 to 25 ⁇ g/ml in a total volume of lOO ⁇ l. Included in this volume was approximately 10 A -10 5 tissue culture infectious units of HIV-l. After a 30 min. preincubation of virus and mAb at room temperature, the mixtures were each added to 1 x 10 5 H9 cells in a final volume of 200 ⁇ l.
  • the cells in each well were plated onto separate wells of poly L-lysine-coated slides and stained sequentially with a rat anti-nef serum (1/200) followed by a rabbit anti-rat IgG Ab conjugated to FITC (1/50) (Zymed) .
  • the latter two antibodies were diluted in 1 mg/ml bovine gamma globulin in PBS.
  • the cells were counterstained with Evan's Blue, and the percentage of infected cells from each culture relative to the control (no mAb added) was assessed by counting i munofluorescent cells versus total counterstained cells under the fluorescence microscope.
  • the affinity of mAb for gpl60 was determined by diluting mAbs of known concentration and assaying the various dilutions on gpl60 coated plates by ELISA as discussed above. It has been demonstrated that the concentration at which half-maximal Ab binding is observed is a rough value of 1/ ⁇ (van Heyningen et al. 1987) . C108G was found to have an affinity of about 1 x l ⁇ " L/mole.
  • C108G is specific for a new neutralization epitope in HIV-l gpl20 that is carbohydrate-dependent and near the V2 region.
  • C108G possesses approximately 25-fold greater neutralizing activity against IIIB strain of virus than previously described HumAb 1125H against the CD4-binding site (Tilley et al. 1991) or 0.5/3, a potent neutralizing mouse mAb against the V3 loop (Matsushita et al. 1988) .
  • C108G synergistically neutralizes the IIIB strain of virus when combined with either 1125H (anti-CD4 binding site human) or 0.5
  • C108G The C108G supernatant was found to react with recombinant gpl20 IIIB as well as gpl60 BRU indicating specificity of C108G for gpl20 rather than gp41.
  • C108G has no reactivity with recombinant gpl ⁇ Ojo,, V3 loop peptides from either the IIIB or MN strains, a V2 IIIB peptide (V15P) described by Fung et al. (sequence: VQKEYAFFYKLDIIP) , nor PB1, a recombinant gpl60 IIIB peptide expressed in E. coli (Putney et al. 1986) .
  • Figure 1 show results of RIPA analysis of 35 S-labelled HIV lysates of infected H9 cells, and shows that C108G reacts with gpl20.
  • C108G reacts strongly with the IIIB viral strain as well as with HXB-2, a virus molecularly cloned from IIIB (Ratner et al. 1987) .
  • C108G has no reactivity with NL43 (Adachi et al. 1986) , a virus with an env gene that is closely related to HXB-2.
  • C108G does not have significant reactivity with the MN, SF-2, nor RF strains of virus.
  • FIG. 2A shows immunoblot strips prepared with IIIB viral lysate.
  • Fig. 2B shows strips prepared with recombinant gpl60 BRU . They were reacted with antibodies as labeled.
  • EG is a control rat antiserum against gpl20.
  • FIG. 4 shows a binding competition assay in which biotinylated antibodies 1125H, 0.53, and C108G were competed with excess unlabeled CD-4.
  • the results show that soluble CD4 partially inhibited binding of C108G to its epitope at CD4 concentrations where HumAb 1125H (Tilley et al. 1991) is completely inhibited by soluble CD4.
  • the anti-V3 mouse mAb, 0.5/3, is not inhibited by CD4, as expected.
  • Fig. 5 shows the results of an experiment in which biotinylated C108G was competed by excess unlabeled mAbs to various gpl20 epitopes.
  • the figure shows that, at unlabeled mAb concentrations where biotinylated C108G is completely inhibited in binding to its epitope by unlabeled C108G, no inhibition of C108G binding is observed with 1125H, a human mAb against a conformational epitope overlapping the CD4-binding site nor by 0.5
  • Mouse mAb G3-4 recognizes an epitope that is destroyed by reduction of disulfide bonds and is also apparently dependent on N-linked sugars (Ho et al. 1991) .
  • mAb G3-4 is inhibited in binding to its epitope by mouse mAbs G3-136 and BAT085 that bind to a linear peptide (V15P) from the V2 region (Fung et al.).
  • V15P linear peptide
  • Reactivity of mAb G3-136 for its epitope is reduced, but not abolished, by either reduction of disulfide bonds or de- glycosylation of gpl20, whereas BAT085 reactivity is unaffected by either of these treatments.
  • mAbs G3-4 and G3-136 are partially inhibited by soluble CD4, whereas BAT085 is not (Fung et al.) .
  • the low background C108G reactivity with bovine serum albumin (BSA) which is used to "block" the ELISA plates, and with peptides 5 and 9 is shown for comparison (results with other peptides not shown) .
  • BSA bovine serum albumin
  • the three peptides with which C108G reacts share a region of 8 amino acids in common. This is probably the minimal epitope (of the amino acid sequence) that is recognized by C108G.
  • Fig. 7 is the location of this epitope within gpl20; it lies near the N-terminal border of the V2 region. This mapping of the C108G epitope (in terms of amino acid sequence) is in excellent agreement with the previously observed HIV-l strain specificity.
  • Figure 8 shows that there is no difference in the sequences of the HXB2 and BRU strains that are both recognized by C108G within the region spanned by peptides 6-8.
  • the NL43 strain that is highly related to HXB2 and BRU strains but is not recognized by C108G has a single nonconservative amino acid change from the latter strains within this region of a G to D (glycine to aspartic acid) ; this change lies within the minimal epitope of C108G (boxed in Fig. 8) , and apparently disrupts ClO ⁇ G's ability to bind to its epitope.
  • the MN, SF-2, and RF strains also carrying this G to D substitution as well as other mostly conservative amino acid changes within the minimal epitope with respect to HXB2 and BRU are not recognized by C108G.
  • Figure 9 shows the potent neutralizing activity of C108G against the IIIB strain of HIV-l. This experiment was done using approximately 10 5 infectious virions in our immunofluorescent focus assay. ClO ⁇ G's neutralizing activity, as assessed by the concentration of mAb required to achieve 50% neutralization, is approximately 25-fold greater than either that of 1125H or 0.5/3. For comparison, we assessed the neutralizing activity of mouse mAb G3-4 in this assay; this is the most potent neutralizing mouse mAb of the group of three discussed above (G3-4, G3-136, BAT085) that bind epitopes in or near the V2 region.
  • Figures 10 and 11 show results of combining C108G with 1125H or 0.5 / 3, respectively, at a 1:25 (C108G:1125H or C10 ⁇ G:0.5/3) ratio upon neutralization of the IIIB strain of HIV-l.
  • CI values below 1 indicate synergism, and CI values are inversely related to the level of synergism observed.
  • Kieny, M.P. Lathe, R. , Riviere, Y. , Dott, K. , Schmitt, D. , Girard, M. , Montagnier, L. , and Lecocq, J. (1988) Protein Engineering. 2:219-225.
  • Ratner, L. A. Fisher, L.L. Jagodzinski, H. Mitsuya, R.S. Liou, R.C. Gallo, and F. Wong-Staal. (1987) AIDS Res. Human Retroviruses 3:57-69.
  • variable region of HIV-l external envelope glycoprotein contains a neutralizing epitope, Vllth Intl. Conf. on AIDS, Florence, Italy Abstract:TH.A.66 (1991).

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Abstract

L'invention décrit de nouveaux anticorps monoclonaux à affinité élevée agissant contre gp120 de HIV-1 et spécifiques à un groupement de déterminants antigéniques de neutralisation élevée. Lesdits anticorps monoclonaux sont spécifiques à une séquence aminoacide comprise entre la position 160 aminoacide et la position 180 aminoacide de gp120 de la souche de HXB2 ou dans les séquences homologues d'autres souches. La fixation des anticorps monoclonaux à gp120 est dépendante de glucides.
PCT/US1993/003044 1992-03-31 1993-03-31 Anticorps monoclonaux agissant contre un determinant antigenique dependant de glucides correspondant a la region v2 de gp120 de hiv-1 WO1993020104A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US5674984A (en) * 1990-04-03 1997-10-07 Genentech, Inc. Method for isolation of unclipped HIV envelope protein
US5864027A (en) * 1993-06-07 1999-01-26 Genentech, Inc. HIV envelope polypeptides
US6585979B1 (en) 1996-07-08 2003-07-01 Genentech, Inc. HIV envelope polypeptides and immunogenic composition
US7041293B1 (en) 1990-04-03 2006-05-09 Genentech, Inc. HIV env antibodies

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* Cited by examiner, † Cited by third party
Title
AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol. 7, No. 12, issued December 1991, KENNEDY et al., "Analysis of Synergism/Antagonism Between HIV-1 Antibody-Positive Human Sera and Soluble CD4 in Blocking HIV-1 Binding and Infectivity", pages 975-981. *
AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol. 8, No. 4, issued April 1992, BUCHBINDER et al., "Synergy Between Human Monoclonal Antibodies to HIV Extends their Effective Biologic Activity Against Homologous and Divergent Strains", pages 425-427. *
AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol. 8, No. 4, issued April 1992, TILLEY et al., "Synergistic Neutralization of HIV-1 by Human Monoclonal Antibodies Against the V3 Loop and the CD4-Binding Site of gp120", pages 461-467. *
SIXIEME COLLOQUE DES CENT GARDES, issued 1991, TILLEY et al., "Potent Neutralization of HIV-1 by Human and Chimpanzee Monoclonal Antibodies Directed Against Three Distinct Epitope Clusters of gp120", pages 211-216. *
VII INTERNATIONAL CONFERENCE ON AIDS, issued 16 June 1991, TILLEY et al., "Human Monoclonal Antibodies Against the Putative CD4-Binding Site and the V3 Loop of HIV gp120 Act in Concert to Neutralize Virus", Abstract M A.70. *
VIII INTERNATIONAL CONFERENCE ON AIDS/III STD WORLD CONGRESS, issued 19 July 1992, TILLEY et al., "Very Broadly Neutralizing Human Monoclonal Antibody (HuMAb) Against the CD4-Binding Site of HIV-1 gp120". *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674984A (en) * 1990-04-03 1997-10-07 Genentech, Inc. Method for isolation of unclipped HIV envelope protein
US5849533A (en) * 1990-04-03 1998-12-15 Genetech, Inc. Method for making unclipped HIV envelope protein
US7041293B1 (en) 1990-04-03 2006-05-09 Genentech, Inc. HIV env antibodies
US5864027A (en) * 1993-06-07 1999-01-26 Genentech, Inc. HIV envelope polypeptides
US6042836A (en) * 1993-06-07 2000-03-28 Genentech, Inc. HIV envelope polypeptides
US6331404B1 (en) 1993-06-07 2001-12-18 Genentech, Inc. HIV envelope polypeptides
US6806055B2 (en) 1993-06-07 2004-10-19 Genentech, Inc. HIV envelopolype peptides
US6585979B1 (en) 1996-07-08 2003-07-01 Genentech, Inc. HIV envelope polypeptides and immunogenic composition
US7071322B2 (en) 1996-07-08 2006-07-04 Genentech, Inc. HIV envelope polynucleotides and immunogenic composition

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