[go: up one dir, main page]

WO1993019786A1 - Anticorps monoclonaux fortement neutralisants et possedant une affinite importante, diriges contre le site de liaison cd-4 de la glycoproteine gp120 du virus immunodeficitaire humain - Google Patents

Anticorps monoclonaux fortement neutralisants et possedant une affinite importante, diriges contre le site de liaison cd-4 de la glycoproteine gp120 du virus immunodeficitaire humain Download PDF

Info

Publication number
WO1993019786A1
WO1993019786A1 PCT/US1993/003010 US9303010W WO9319786A1 WO 1993019786 A1 WO1993019786 A1 WO 1993019786A1 US 9303010 W US9303010 W US 9303010W WO 9319786 A1 WO9319786 A1 WO 9319786A1
Authority
WO
WIPO (PCT)
Prior art keywords
mab
hiv
mabs
neutralization
cells
Prior art date
Application number
PCT/US1993/003010
Other languages
English (en)
Inventor
Shermaine Tilley
Abraham Pinter
Original Assignee
The Public Health Research Institute Of The City Of New York, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Public Health Research Institute Of The City Of New York, Inc. filed Critical The Public Health Research Institute Of The City Of New York, Inc.
Priority to AU49591/93A priority Critical patent/AU4959193A/en
Publication of WO1993019786A1 publication Critical patent/WO1993019786A1/fr

Links

Classifications

    • 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

Definitions

  • This invention relates to monoclonal antibodies ("mAbs”) that strongly recognize the CD-4 binding site of human immunodeficiency virus, type 1 ("HIV-l”) .
  • 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 gp41 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
  • This* is an immunodominant epitope cluster eliciting potent neutralizing Abs in man and experimental animals (summarized in (Javaherian et al. 1990) ) .
  • 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 __ _ _ _
  • 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 of the invention are specific for HIV- envelope glycoprotein gpl20, and achieve at least about 50% neutralization in vitro of about 2 x 10* infectious units of the RF HIV-l strain at a concentration of about 1 ⁇ g/ l. This reflects a higher binding and neutralization ability with respect to this strain than has been seen with previous anti-CD-4 binding site mAbs.
  • the mAb is specific for an epitope that is partially or completely within the CD-4 binding site of gpl20.
  • certain anti-gpl20 Abs are combined to obtain a synergistic result.
  • the invention includes a combination of mAbs, the first being one of the novel anti-CD-4 binding site mAbs of the invention, and the second being an Ab that is specific for the V3 region of HIV-l envelope glycoprotein gpl20, the combination being effective at synergistically binding or neutralizing HIV-l. -4-
  • Figure 1 shows the results of a radioimmunoprecipitation/SDS gel analysis discussed in the examples.
  • FIG. 2 shows the results of an im unoblot experiment described in the examples.
  • Figure 3 is a graph of the results of a competitive inhibition experiment described in the examples.
  • Figures 4-6 are graphs of neutralization experiments described in the examples.
  • Figure 7 is a graph of combination index values calculated from the results shown in figures 5 and 6, and discussed in the examples.
  • the mAbs of the invention can be used in many ways, including immunoassays, affinity chromatography and any other procedures that use anti-HIV mAbs.
  • the strong recognition of HIV makes the mAbs especially useful in detection, either indirectly through a competitive assay for HIV antibody, or directly in an assay for HIV antigen.
  • the mAbs described below are especially useful in affinity chromatography procedures for purifying a wide variety of HIV strains.
  • the mAbs of this invention are useful in a conventional format competitive immunoassay, such as described below, to determine antibodies, such as human antibodies, to the HIV-l epitopes recognized by the mAbs.
  • the present invention also includes test kits to measure the presence of HIV antigens or of human Abs against the epitopes recognized by the novel mAbs.
  • a kit for a competitive ELISA contains mAbs of the invention, a solid phase on which is coated an antigen which the mAbs are -5-
  • 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.
  • a typical direct assay the mAb is reacted with a sample and the formation of a complex between the mAb and antigen from the sample is determined using well known conventional methods.
  • An assay for determining the presence of an antigen which the mAb of the invention binds to can be performed using, for example, a sandwich format wherein a solid phase is coated with antibody to HIV envelope, the sample is added, and then labeled mAb of the invention (such as with biotin) is added. Following a wash, enzyme labeled avidin can be added as well as enzyme substrate.
  • enzyme labeled avidin can be added as well as enzyme substrate.
  • Kits for determining an antigen for which mAbs of the invention are specific may comprise a mAb (or mAbs) of the invention, a solid phase on which is coated an antibody specific for HIV-l env, and means for detecting the formation of a complex among the mAb of the invention, the antibody specific for HIV-l env, and an HIV antigen for which the mAbs are specific.
  • the mAb of the invention has high neutralization ability with respect to the RF strain, achieving at least about 50% neutralization in vitro of about 2 x 10 4 infectious units of the RF HIV-l strain at a mAb concentration of about 1 ⁇ g/ml.
  • the mAb achieves that degree of neutralization at a concentration of about 0.5 ⁇ g/ml.
  • the mAb of the invention achieves at least about 50% neutralization in vitro of the RF strain in a concentration of about 0.5 ⁇ g/ml under conditions where another highly neutralizing anti-CD- 4 binding site mAb, mAb 1125H, achieves 50% neutralization in a mAb concentration of about 5 ⁇ g/ml.
  • the amounts of neutralization is as determined using a standard 24 hr. neutralization assay described in the examples. Other assays may arrive at different results. It is not necessary for a mAb of the invention to have in vivo the neutralization activity measured in the in vitro test described.
  • the mAb of the invention is against the CD-4 binding site region of gpl20 of HIV-l, it does not necessarily bind only within the CD-4 binding site.
  • the CD- 4 binding site is a conformational region that encompasses many disparate areas of gpl20. It has been found that addition of soluble CD4 at about a 1000 fold excess over mAb inhibits binding to HIV-l ⁇ B of the antibody of the invention to its epitope, by about 90%.
  • the mAb of the invention is specific for a highly conserved gpl20 epitope.
  • the epitope is conserved among strains MN, IIIB, SF-2 and RF.
  • Human mAbs are also preferred.
  • the mAb of the invention substantially has the epitope specificity of human mAb 5145A.
  • mAb 5145A is produced by an EBV-transformed human 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 10982. Methods for obtaining 5145A and its further characterization are described below.
  • mAb 5145A is specific for an epitope overlapping the CD-4 binding site that is destroyed by reduction of disulfide bonds. It does not react with LAV-2. 5145A recognizes a wide variety of strains, including almost all African strains tested.
  • a preferred mAb neutralizes in vitro about 2 x 10 4 infectious units of virus of all four strains to a level of 50% at concentrations of antibody that do not differ by more than about 3X.
  • binding of a preferred mAb of the invention to gpl20 is markedly decreased by the following amino acid changes from HXB2 wild type: 368 D/R, 368 D/T and 370 E/R, while the following amino acid changes did not markedly decrease binding: 257 T/R, 257 T/G, 381 E/P and 427 W/S.
  • an immortalized cell line of the invention such as the deposited cell line CRL 10982
  • 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.
  • the mAb substantially has the complementarity determining regions of mAb 5145A.
  • mAb 5145A is also useful in screening to determine whether cultures obtained of anti gpl20 mAbs (human or otherwise) are producing mAbs to an epitope closely related to one recognized by antibody 5145A, such as by using a competitive ELISA assay with labeled 5145A.
  • a competitive ELISA assay determines if mAbs from the culture being screened compete with 5145A 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.
  • a suitable competitive assay is described below, as is methodology for obtaining mAb-producing cells for screening. 3 1
  • the invention also includes a synergistically neutralizing combination of mAbs.
  • New or already known antibodies against the V3 region can be screened for use in the synergistic combination of the invention.
  • a peptide consisting of amino acids 305-328 of the MN strain (or the corresponding sequence in other strains) can be substituted for recombinant gpl60 in screening using the procedures of the Examples.
  • That (MN) sequence is NYNKRKRIHIGPGRAFYTTKNIIGC, described in Gurgo et al. 1988.
  • the anti-V3 antibody of the combination can be specific for any part of the V3 region, so long as it synergistically neutralizes with the anti-CD4 binding site antibody (as determined in the test for synergistic neutralization described herein) . It preferably competitively inhibits the binding to gpl60»» ⁇ , or V m of antibodies produced by the cell line deposited under accession no. CRL 10770, the inhibition preferable being more than about 80%, most preferably more than about 95%.
  • mAb 4117C is characterized by its reactivity with the V3 peptide of the following strains: MN, SF-2, NY-5, CD-451, WMJ-1, WMJ-3, Z- 3, Z-321, and SC; and by its lack of reactivity with the following strains: WMJ-2, LAV-MA, BR, LAV-IIIB, PV-22, ELI, Z-6, NX3-3, JY-1, HXB-2 and MAL.
  • mAb 4117 is produced by EBV transformed human B-cells deposited on June 14, 1991 at the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852 USA and assigned accession No. CRL 10770.
  • the mAb specific for the V3 region has the epitope specificity of mAb 4117c.
  • a mAb can be produced, for example, by cells derived from the deposited cell line. -9-
  • the anti-CD4 mAb of the combination is the novel mAb that is described herein, in all its different aspects.
  • the human mAb specific for the CD-4 binding site used in the combination can be one that achieves at least about 50% neutralization of about 2 x 10 4 infectious units of RF HIV-l strain at a concentration of about 1 ⁇ g/ml.
  • the preferred mAb specific for the CD-4 binding site of the combination has the epitope specificity of the 5145A, or results from the manipulation of the genes obtained from the cell line producing 5145A (such as described herein) .
  • a given combination of a mAb against the V-3 region and for example, CRL 10982 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 antibody combination to synergize will be 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 guantitated by calculating the Combination Index using known statistical methods (Chou et al. 1984; Chou et al. 1989; Chou 1991).
  • the combination index value at greater than 20% neutralization preferably is less than 0.8 to indicate significant synergy.
  • Polyclonal compositions can also be obtained which are specific for V3 and which synergize with the anti-CD-4 binding site mAb of the invention.
  • Immunization and screening of polyclonal sera from, for example chimpanzees, rabbits or sheep can be accomplished using the immunogens described herein for generating mAbs, and by using the screening techniques described herein.
  • Antibodies from different sources may be employed to obtain antibodies for use in 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 gpl60 (Berman et al., 1990). 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-transfor ation procedure described herein.
  • Antibodies can be purified by immunoaffinity chromatography.
  • AH-Sepharose beads are activated by treatment with glutaraldehyde, and conjugated either to purified V3 peptide or gpl20.
  • Antibodies against V3 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 and 0.5M NaCI solutions.
  • V3-specific antibodies can be eluted from these columns by washing with tris-glycine buffer, pH2.7 or by passing through excess V3 peptide.
  • Antibodies of the invention can be used, if desired, in methods of neutralizing infection of T-cells by HIV.
  • the antibodies are used in the manner described to reduce the possibility of infection.
  • passive administration of the human mAbs of the invention can be performed to decrease the chance of HIV-l infection in cases of acute exposure to HIV.
  • the HIV-l neutralizing mAbs of this invention could be passively -11-
  • mAbs can be used to prevent HIV-l infection by administering them to individuals near the time of their exposure to HIV-l, such as following a needle stick.
  • Human, or other primate, mAbs have distinct advantages over, for example, rodent mAbs, in administration to humans for prevention, or therapy, of viral infection. They have increased stability and very low immunogenicity in humans. Thus, human 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 human mAbs in humans.
  • a neutralizing mAb is desired to be used for treating HIV-infected individuals or preventing infection by HIV, the 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 desirable 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 synergizing mAbs of the invention make possible a reduction in the concentration of mAb required when used for this purpose.
  • the anti-CD-4 binding site mAb of the invention can also be used in combination with other anti-HIV-1 mAbs, in order to attain an additive neutralization effect, if desired.
  • the mAb 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. For example, a 1:1 ratio of 5145A and an anti-V3 antibody with the neutralization ability of 4117C can be used.
  • concentration of each should be determined to give very roughly comparable levels of neutralization to each other (i.e. an equipotent ratio is preferably used) .
  • 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.
  • the reagent can consist of as few as one type of antibody, although it is believed that the most efficient composition will contain a large number of different antibodies directed against major antigenic sites, preferably including synergizing antibodies. This is in order to increase the affinities and cross-reactivity of the antibodies to different HIV variants which may exist and to decrease the likelihood of a deleterious anti-idiotype response.
  • 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.
  • Excellent properties of the preferred mAb of this invention are: 1) its demonstrated HIV-l neutralizing activity in vitro at low mAb concentrations, 2) its high affinity for antigen (HIV-l gp 120), 3) its broad HIV-l strain specificity, 4) the fact that it is of human origin and will, therefore, elicit few, if any, deleterious immune reactions when administered to humans, and 5) the heavy chain isotype of the preferred mAb is IgG, which is significant because human IgG Abs 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
  • the mAbs of this invention may be modified 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 dapable 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 mAbs are preferably obtained for the various in vitro or in vivo uses possible. 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 3/1
  • Antibodies are preferred that result from cloning of variable region genes from 5145A, and thus substantially have the complementarity determining regions of that mAb.
  • the invention also includes moieties having the same function as mAbs, 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 mAbs 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 such as the EBV transformed cell line described herein, or cell lines producing mAbs of the invention that result from the cloning of immunoglobulin genes or portions thereof using immunoglobulin expression vectors into an appropriate antibody producing cell line.
  • the preferred reagent 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.
  • HumAbs Human monoclonal antibodies
  • PBMC peripheral blood mononuclear 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 4 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 fro the 4117C cell line 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 1 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 human cultures for production of anti-env Ab was done using recombinant gpl60 BRU (Kieny et al. 1988) or V3 MN to coat PVC ELISA plates (Flow/ICN) .
  • This version of gpl60 supplied by Pasteur Merieux, lacks the site which is normally cleaved to form gpl20 and gp41.
  • a variety of other HIV-l proteins or peptides can be used to determine the specificity of the mAbs.
  • 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. 1990) synthetic V3 peptides from a variety of strains (strain specificity is described above) ; pl21, a gp41 peptide (amino acids 565-646) sold commercially by Dupont or described in Chang, et al. , European Patent Application 0199438 published October 29, 1986, or other peptides described below. 4117C is negative for gpl20 of the IIIB strain and for pl21. 5145A is negative for V3 peptides and gp41.
  • 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 human 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 human serum, and bound Ab was detected (Pinter et al. 1989) .
  • Human mAbs were purified from cell supernatants on MASS protein A filters (Nygene Corp., Yonkers, N.Y.) and concentrated using CentriCell ultrafilters (Polysciences, Inc.) (Tilley et al 1991). The mAbs were guantitated by ELISA using affinity-purified human IgG (Cappel) of known concentration as standard.
  • HIV-l strains IIIB Popovic et al. 1984; Ratner et al. 1985
  • SF2 Levy et al. 1984; Sanchez-Pescador et al. 1985
  • strains MN Gaallo et al. 1984; Shaw et al. 1984
  • RF Popovic et al. 1984; Starcich et al. 1986
  • the identities of 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 provided by Dr. Robert Neurath, New York Blood Center.
  • An HIV-2 strain, LAV-2 (Clavel et al. 1986) was obtained from Luc Montagnier, Pasteur Institute via Dr. Alvin Friedman-Kien, New York University School of Medicine. African strains were isolated by Dr. Ellen Murphy at PHRI from blood samples obtained from Bangui (Central Africa) .
  • the Haitian isolate, AL was a gift from Dr. David Ho, Aaron Diamond AIDS Research Center, New York.
  • 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
  • the slides were washed IX in distilled water and then incubated in 100-200 ml of acetone or methanol for 8 mins. The slides were then removed from the fixative and allowed to air dry.
  • the heaving chain class was selected to be IgG by the use of goat anti-human IgG in the primary screening. Heavy chain subclass was determined for 4117C using a variation of the -immunofluorescence assay. Human mAb-producing cells were attached to slides and fixed with acetone. The slides -22-
  • Light chain isotype was determined by a variation of the ELISA assay discussed above. Following incubation of supernatant from mAb-producing human 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.
  • 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 0.1 to 20 ⁇ g/ml in a total volume of lOO ⁇ l. Included in this volume was approximately 2 x 10 4 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. Following a 24 hr.
  • 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) or serum from a seropositive individual (1/200) followed by a species specific anti-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 immunofluorescent cells versus total counterstained cells under the fluorescence microscope. Under the conditions discussed above, approximately 3% of the cells (3000 cells) are infected at the end of the assay in the absence of neutralizing antibody.
  • the affinity of mAb for gpl60 was determined by diluting mAbs of known concentration and assaying the -24-
  • panel of gp 120 mutants used in these experiments was derived from HXB2 (a III B clone) and was a subset of those reported by others (Thali et al 1992, Olshevsky et al. 1992, Thali et al. 1991). Specifically, all of the mutants reported in Thali et al. 1992 were used except 76 P/Y, 256 S/Y, 257 T/A, 297-329, 368 D/P, 368 D/N, 368 D/K, 368 D/E, 370 E/D, 380-381 GE/YV, 386 N/Q, 457 D/N, 457 D/E, and 470 P/G.
  • mutants described by Olshevsky et al. 1992 were used — 36 V/L 45 W/S, 429 K/L, 470 P/L, AND 500-501KA/KGIPKA — and mutants described by Thali et al. — 356 N/I, 386 N/R, AND 450 T/N.
  • the wells were washed 2 times with TBS.
  • TMSS 2% milk powder, 20% sheep serum in TBS
  • the recognition index for each human mAb against a given gpl20 mutant was calculated as: OD (mAb on mutant gpl20)/OD (mAb on wild type gpl20) x OD (seropositive serum on wild type gpl20/OD (seropositive serum on mutant gpl20) . This is similar to the calculation of recognition index used by Thali et al.
  • 4117C was found to recognize a variety of divergent HIV strains, including MN, SF-2, FV (New York), 11699 (Central Africa) , and the JR-CSF primary isolate (Los Angeles) (Koyanagi et al. 1987) . 4117C human mAb is less strain specific than other anti-V3 human mAbs that have been described (Scott et al. 1990, Zolla-Pazner et al. 1990). Comparison of the V3 sequences of the isolates recognized by 4117C reveals that the sequence GPGR at the tip of the loop is shared by all of them. In addition, the sequence IXI just to the left of the GPGR is highly conserved among these isolates.
  • the apparent affinity constant, K, of 5145A was measured against recombinant gp 160 from either BRU or MN strains and found to be approximately 2 x 10 9 L/mole. This is comparable to the apparent affinity constants of two of other potent neutralizing HuMAbs, 1125H (anti-CD4 binding site), 1.3 x 10 9 L/mole, and 4117C (anti-V3 loop), 0.9 x 10 9 L/mole.
  • mAb 1125H is described herein for comparative purposes. It is produced by EBV- transformed human B-cells and was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. , 20852, USA, on October 23, 1990, and has been assigned accession number CRL 10582.
  • Fig. 1 demonstrates the specificity of the 5145A (and 1125H) for gpl20 by radioimmunoprecipitation/SDS gel analysis.
  • Fig. 2 shows, by immunoblot analysis, that the epitope of 5145A (and the epitope of 1125H) is destroyed by reduction of gpl20 disulfide bonds. The latter feature is common to all HuMAbs against the CD4-binding site that we are aware of.
  • Fig. 3 is a graph of the results of the study of competitive inhibition of 5145A binding to gpl60 by soluble CD-4. The figure shows that 5145A binding to its epitope is inhibited in a concentration-dependent manner by soluble CD-4.
  • the V3 loop of each of these Central African strains has been sequenced.
  • the Central African Republic HIV- 1 strains are representatives of African subtype A, African subtype D, and Northern Thailand sub-type E based on their V3 region sequences.
  • Table l shows that both 5145A and 1125H reacted with all of the North American (MN, SF-2, IIIB) and Haitian (RF, AL) strains tested.
  • 5145A also recognized 9 of 10 Central African Republic strains tested, while 1125H reacted with only 4 of these 10 strains.
  • the group of African strains recognized by 1125H includes African subtype A and Northern Thailand subtype E strains, while the group of strains not recognized by 1125H includes subtypes A and E as well as a single African subtype D strain.
  • Fig. 4 shows that 5145A has potent neutralizing activity against the four North American HIV-l isolates that it reacts with by immunofluorescence assay. Fifty percent of approximately 2 x 10 4 infectious units of virus in this assay is neutralized by approximately 0.2-0.5 ⁇ g/ml of 5145A. Furthermore, 90-100% of each of these viral strains is neutralized by 10 ⁇ g/ml of 5145A. These neutralization results contrast with those seen using other anti-CD4 binding site HuMAbs known to us. 1125H HuMAb has similar neutralizing activity against MN and IIIB strains to 5145A, but significantly less neutralizing activity against SF-2 and RF strains than 5145A (Tilley et al. 1991) . Specifically, a 5-10 fold greater concentration (5 ⁇ g/ml) of 1125H is required to neutralize 50% of the SF-2 and RF virus than was required for this level of neutralization of the -29-
  • 1125H neutralizes a maximum of only about 75% of the SF-2 and RF virus at a concentration of 20 ⁇ g/ml.
  • Three other anti-CD4 binding site HuMAbs (Ho et al. 1991; Posner et al. 1991; Lake et al.) exhibit more narrow strain specificity and/or lower neutralizing activity than either 1125H or 5145A.
  • the 1.5e HuMAb does not recognize nor neutralize the RF strain (Ho et al. 1991) and the Sl-1 HuMAb neutralizes RF only in the presence of complement (Lake et al.), whereas both 1125H and 5145A neutralize RF significantly in the absence of complement (Tilley et al. 1991).
  • the F105 HuMAb has orders of magnitude lower neutralizing activity against the MN strain than does either 1125H or 5145A (Posner et al.). These differences among other anti-CD4 binding site HuMAbs in strain specificity and neutralizing activity indicate that each of these other HuMAbs is directed against different epitopes than that of 5145A that are within or overlapping the CD4-binding site.
  • Mutant or wild-type gpl20 molecules present in the supernatant of COS-1 cells in which they were expressed were captured by antibody against their C-terminus onto ELISA plates.
  • Anti-gpl20 mAbs were then reacted with the bound gpl20 molecules, and binding of the mAbs was detected with enzyme-conjugated anti-Ig antibodies via standard techniques. This assay yields very similar results to those obtained by the radioimmunoprecipitation/SDS gel technique. -30-
  • Results for 5145A represent the mean of three independent experiments.
  • 5145A did not lose substantial reactivity with gpl20 and gpl60 upon removal of N-linked carbohydrates from these glycoproteins under non-reducing conditions.
  • This experiment was carried out by isolating virus from cells grown in the presence of 1-MNN, a substance that inhibits the processing of high mannose sugars to complex carbohydrates. The high mannose sugars on viral glycoproteins obtained from these 1-MNN-treated cells were then digested with endoglycosidase I (EH) under non-reducing conditions, resulting in the removal of all but one N- linked sugar residue (N-acetylglucosamine) from each glycan attachment site on the proteins.
  • EH endoglycosidase I
  • N-linked glycans are not essential components of the 5145A epitope nor are they required for maintaining its conformation.
  • Figs. 5 and 6 show neutralization curves demonstrating that 5145A and 4117C (anti-V3 loop HuMAb) synergistically neutralize the MN and SF-2 strains, respectively.
  • the plots of combination index vs. F. (% neutralization / 100) for the two synergistic neutralization experiments shown in Figs. 5 and 6 are given in Figs. 7A and 7B, respectively.
  • Other parameters calculated from these two experiments using a computer program developed by Chou and Chou (Chou and Chou 1989), are shown in Table 3.
  • the linear correlation coefficient, r, for the data shown in Figs. 5 and 6 is shown in Table 3 below. /19786
  • 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.
  • Thali M. , C Furman, D.D. Ho, J. Robinson, S.A. Tilley, A. Pinter, and J. Sodroski. (1992) J. Virol. 66:5635-5641.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • AIDS & HIV (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Anticorps monoclonaux présentant une affinité pour le VIH-1 et neutralisant fortement ce dernier. Ces anticorps monoclonaux sont spécifiques contre un déterminant antigénique (épitope) qui est situé partiellement ou entièrement dans le site de liaison CD-4 de la glycoprotéine gp120.
PCT/US1993/003010 1992-03-31 1993-03-31 Anticorps monoclonaux fortement neutralisants et possedant une affinite importante, diriges contre le site de liaison cd-4 de la glycoproteine gp120 du virus immunodeficitaire humain WO1993019786A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49591/93A AU4959193A (en) 1992-03-31 1993-03-31 High affinity, strongly neutralizing monoclonal antibodies against the CD-4 binding site of GP120 of human immunodeficiency virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US86110892A 1992-03-31 1992-03-31
US07/861,108 1992-03-31

Publications (1)

Publication Number Publication Date
WO1993019786A1 true WO1993019786A1 (fr) 1993-10-14

Family

ID=25334900

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/003010 WO1993019786A1 (fr) 1992-03-31 1993-03-31 Anticorps monoclonaux fortement neutralisants et possedant une affinite importante, diriges contre le site de liaison cd-4 de la glycoproteine gp120 du virus immunodeficitaire humain

Country Status (2)

Country Link
AU (1) AU4959193A (fr)
WO (1) WO1993019786A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474894A (en) * 1994-01-21 1995-12-12 Quality Biological, Inc. Immunodeficiency virus neutralization assay
WO2013016468A3 (fr) * 2011-07-25 2013-05-02 California Institute Of Technology Compositions et procédés permettant d'améliorer la puissance et la portée d'anticorps anti-vih
WO2013090644A3 (fr) * 2011-12-13 2013-10-10 California Institute Of Technology Anticorps anti-vih ayant des puissance et étendue accrues
WO2013192589A1 (fr) * 2012-06-21 2013-12-27 California Institute Of Technology Anticorps ciblant des mutants échappés du vih
US8722861B2 (en) 2007-11-19 2014-05-13 Kumamoto University Monoclonal antibodies that bind to the V3 loop of HIV-1 gp120
US9493549B2 (en) 2011-07-25 2016-11-15 The Rockefeller University Antibodies directed toward the HIV-1 GP120 CD4 binding site with increased potency and breadth

Non-Patent Citations (6)

* 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 Monoclonol Antibodies Against the V3 Lo9op 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 (HuMaAb) Against the CD4-Binding Site of HIV-1 gp120". *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474894A (en) * 1994-01-21 1995-12-12 Quality Biological, Inc. Immunodeficiency virus neutralization assay
US8722861B2 (en) 2007-11-19 2014-05-13 Kumamoto University Monoclonal antibodies that bind to the V3 loop of HIV-1 gp120
EP2230250B1 (fr) * 2007-11-19 2015-11-04 Kumamoto University Anticorps monoclonal anti-vih
US10590187B2 (en) 2011-07-25 2020-03-17 California Institute Of Technology Highly active agonistic CD4 binding site anti-HIV antibodies (HAADS) comprising modified CDRH2 regions that improve contact with gp120
US9493549B2 (en) 2011-07-25 2016-11-15 The Rockefeller University Antibodies directed toward the HIV-1 GP120 CD4 binding site with increased potency and breadth
US9890207B2 (en) 2011-07-25 2018-02-13 California Institute Of Technology Highly active agonistic CD4 binding site anti-HIV antibodies (HAADS) comprising modified CDRH2 regions that improve contact with GP120
WO2013016468A3 (fr) * 2011-07-25 2013-05-02 California Institute Of Technology Compositions et procédés permettant d'améliorer la puissance et la portée d'anticorps anti-vih
US11472868B2 (en) 2011-07-25 2022-10-18 California Institute Of Technology Highly active agonistic CD4 binding site anti-HIV antibodies (HAADS) comprising modified CDRH2 regions that improve contact with GP120
US12054538B2 (en) 2011-07-25 2024-08-06 California Institute Of Technology Highly active agonistic CD4 binding site anti-HIV antibodies (HAADS) comprising modified CDRH2 regions that improve contact with GP120
WO2013090644A3 (fr) * 2011-12-13 2013-10-10 California Institute Of Technology Anticorps anti-vih ayant des puissance et étendue accrues
WO2013192589A1 (fr) * 2012-06-21 2013-12-27 California Institute Of Technology Anticorps ciblant des mutants échappés du vih
US9879068B2 (en) 2012-06-21 2018-01-30 California Institute Of Technology Antibodies targeting HIV escape mutants
US11149081B2 (en) 2012-06-21 2021-10-19 The Rockefeller University HIV-1 GP120 CD4 binding site antibodies targeting HIV escape mutants
US12173052B2 (en) 2012-06-21 2024-12-24 The Rockefeller University HIV-1 gp120 CD4 binding site antibodies targeting HIV escape mutants

Also Published As

Publication number Publication date
AU4959193A (en) 1993-11-08

Similar Documents

Publication Publication Date Title
US5922325A (en) Synergistic neutralization of HIV-1 by human monoclonal antibodies and other antibodies directed against the v3 loop and the CD-4 binding site of GP-120,and the use for immunotherapy of HIV-1 infection
Tilley et al. A human monoclonal antibody against the CD4-binding site of HIV1 gp120 exhibits potent, broadly neutralizing activity
JP2520464B2 (ja) Ｈｉｖ―１を中和するモノクロ―ナル抗体
Thali et al. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding
Bou-Habib et al. Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization
US5911989A (en) HIV-vaccines
Tilley et al. Synergistic neutralization of HIV-1 by human monoclonal antibodies against the V3 loop and the CD4-binding site of gp120
Sun et al. Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gp120
Ugolini et al. Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line–adapted HIV-1 neutralization
US5854400A (en) Monoclonal antibodies which neutralize HIV-1 infection
AU632475B2 (en) Human immunodeficiency virus (hiv) env-coded peptide capable of eliciting hiv-inhibiting antibodies in mammals
RU2128222C1 (ru) Мышиное моноклональное антитело nm01, гибридомная клеточная линия, фрагмент мышиного могоклонального антитела (варианты)
EP0492560B1 (fr) Anticorps monoclonaux humains contre la glycoprotéine transmembranaire (gp41) de HIV-1, et peptides associés
US6241986B1 (en) Human monoclonal antibodies to the CD4-binding domain of HIV, uses thereof and synergistic neutralization of HIV
JPH0768276B2 (ja) Ｈｉｖのｃｄ４結合領域に特異的な抗体
US6268484B1 (en) HIV-vaccines
Pinter et al. A potent, neutralizing human monoclonal antibody against a unique epitope overlapping the CD4-binding site of HIV-1 gp120 that is broadly conserved across North American and African virus isolates
Thomas et al. Anti-idiotypic antibody to the V3 domain of gp120 binds to vimentin: a possible role of intermediate filaments in the early steps of HIV-1 infection cycle
US5981278A (en) Chimeric monoclonal antibodies which neutralize HIV-1 infection and their applications in therapy and prevention for AIDS
WO1991009625A1 (fr) Anticorps monoclonaux qui neutralisent l'infection par hiv-1 et leurs anti-idiotypes
Corre et al. Anti‐idiotypic antibodies to human anti‐gp120 antibodies bind recombinant and cellular human CD4
US20070292390A1 (en) Broadly Cross-Reactive Hiv-1 Neutralizing Human Monoclonal Antibodies
Boudet et al. Antibodies of HIV-1 positive subjects and experimentally immunized primates and rodents bind to sequence divergent regions of the third variable domain (V3) of gp120
WO1993019786A1 (fr) Anticorps monoclonaux fortement neutralisants et possedant une affinite importante, diriges contre le site de liaison cd-4 de la glycoproteine gp120 du virus immunodeficitaire humain
Matsushita et al. Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

COP Corrected version of pamphlet

Free format text: FORMS PCT/RO/134,INDICATIONS RELATING TO A DEPOSITED MICROORGANISM,ADDED

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i
121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase