WO1990009805A1 - Anticorps monoclonaux humains contre le virus d'immunodeficience humaine - Google Patents

Anticorps monoclonaux humains contre le virus d'immunodeficience humaine Download PDF

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Publication number
WO1990009805A1
WO1990009805A1 PCT/US1990/001132 US9001132W WO9009805A1 WO 1990009805 A1 WO1990009805 A1 WO 1990009805A1 US 9001132 W US9001132 W US 9001132W WO 9009805 A1 WO9009805 A1 WO 9009805A1
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Prior art keywords
human
monoclonal antibody
hiv
antibody
directed against
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PCT/US1990/001132
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English (en)
Inventor
Susan Zolla-Pazner
Miroslaw K. Gorny
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New York University
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Priority to AU51885/90A priority Critical patent/AU651794B2/en
Publication of WO1990009805A1 publication Critical patent/WO1990009805A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • HIV human immunodeficiency virus
  • AIDS acquired immune deficienc syndrome
  • HIV-1 and HIV-2 Two different serotypes of the virus ha been identified to date: HIV-1 and HIV-2. It is currentl believed that the majority of individuals that become infect with HIV eventually will develop AIDS and are likely to succu to fatal infections and/or malignancies. At this time it estimated that approximately 1.5 million individuals .have be infected by HIV in the United States alone.
  • AZT antiviral dr azidothymidine
  • clinic benefits are achieved with AZT, it is costly.
  • a furth drawback is that significant drug toxicity often, accompani administration of AZT. This may necessitate blood transfusio and/or reduction of the AZT dosage, or in some instance discontinuance of AZT therapy altogether. Nonetheless, AZT the only drug currently authorized for the treatment of AIDS.
  • Interferon particularly gamma-interferons
  • interleukin-2 are currently being studied for possible use in the treatment of HIV infections.
  • the preliminary results of early clinical trials are not promising.
  • Patients receiving lym- phokine therapy often suffer serious side effects including low blood pressure, nausea and diarrhea.
  • monoclonal antibodies of defined specificities directed against HIV proteins expressed in infected individuals as therapeutic agents. These proteins are part of the virus particles and are expressed by HIV infected cells and are designated inter alia as p24 and gp41. The identification and isolation of gp41 is described in U.S. Patent No. 4,725,669 of M.
  • Stable human cell lines which produce monoclonal antibodies directed against HIV would be useful for treatin and/or diagnosing individuals infected with the virus.
  • human monoclonal antibodies and particularly thos directed against HIV have proven to be far more difficult t produce than those of either rat or mouse origin.
  • Another object of the present invention is to provid diagnostic and therapeutic agents comprising human monoclona antibodies directed against HIV proteins which have a low non specific toxicity for use in the diagnosis and treatment of in dividuals infected with HIV.
  • a further object of the present invention is to provid a method for treating individuals suffering from HIV infection by administering human monoclonal antibodies directed agains protein components of HIV to said individual.
  • a still further object of the present invention is t provide pharmaceutical formulations for treating individual suffering from HIV infections.
  • the present inventors have discovered new monoclon antibodies for the treatment, prophylaxis and diagnosis human immunodeficiency virus (HIV) infections. These are hum monoclonal antibodies directed against HIV proteins gp41 a p24 which are expressed by infected cells.
  • the human monoclo ⁇ nal antibodies of the present invention may be used as diagnos ⁇ tic agents, directly as therapeutic agents, as the basis for vaccines or to form conjugates by covalent coupling with cytotoxic agents, specific anti-HIV drugs or radionuclides (the antibody/toxin conjugates are alternatively referred to herein as immunotoxins) for use in the diagnosis and treatment of in ⁇ dividuals that have been exposed to or infected with HIV.
  • the present invention provides stable human lymphoblastoid cell lines which secrete human monoclonal antibodies directed against HIV proteins gp41 and p24.
  • the invention also provides human monoclonal antibodies directed against HIV proteins p24 and gp41.
  • Another aspect of the present invention comprises a method for treating a mammal infected with HIV comprising administering to a mammal in need of such treatment an effec ⁇ tive amount of a human monoclonal antibody directed against HIV.
  • the present invention comprises pharmaceutical formulations comprising an effective amount of a human monoclonal antibody to HIV proteins.
  • Figure 1 is a radio-immunoprecipitation assay of 125 [l]-labelled HIV lysate with serum from an HIV-infected subject or with antibodies from a subset of the human monoclonal antibody producing cell lines of the present invention.
  • Figure 2 is a Western blot analysis of human sera and an anti-p24 monoclonal antibody of the present invention.
  • Figure 3 are graphs of the growth kinetics and immunoglobulin production of a subset of the human lymphoblas ⁇ toid cell lines of the present invention which produce monoclonal antibodies directed against HIV.
  • Figure 4 is a graph showing the inhibition testing of monoclonal antibodies 120-16 and 71-31. DETAILED DESCRIPTION OF THE INVENTION
  • gp41 is a viral membrane glycoprotein expressed on th surface of infected cells and is a product of the env gene o HIV (as described in Essex, M., U.S. Patent No. 4,725,66 issued February 16, 1988).
  • p24 is a viral core protein and i a product of the HIV ⁇ a ⁇ gene (as described in Allan, J.S. e al., supr ) .
  • the human monoclonal antibodies of the present inven tion may be employed as the antibody component in the conven tional diagnostic assays of the type used to determine if patient has been exposed to, or infected with, HIV.
  • Example below illustrates the use of the antibodies of the invention i a diagnostic assay.
  • the antibodies Administered to humans, the antibodies ca provide passive immunization of HIV-infected individuals.
  • the antibodies of the invention can serve prophylac tically for administration to non-infected, high-risk in dividuals (such as health care workers who have been expose via a needle stick to HIV) .
  • the antibodies of the inventi also can serve as research tools for epitope mapping of HI proteins gp41 and p24.
  • a particularly important use of the human monoclon antibodies of the present invention is for administration HIV infected expectant mothers.
  • All of the antibodies of t present invention are of the IgG serotype (see below). Sin IgG's can pass through the placenta and reach the fetus utero, passive administration of the antibodies of the prese invention to HIV-infected pregnant women would provide effec tive therapy for the fetus.
  • the human monoclonal antibodies may be conjugated t cytotoxic agents and used as im ⁇ tunotoxins (as described i Vitetta, E.S. et al.. Science 238: 1098-1104, 1987) or incor porated onto the surface of liposomes containing anti-HIV drug or toxins to specifically target such drugs or toxins t infected cells.
  • im ⁇ tunotoxins as described i Vitetta, E.S. et al.. Science 238: 1098-1104, 1987
  • incor porated onto the surface of liposomes containing anti-HIV drug or toxins to specifically target such drugs or toxins t infected cells.
  • the term "immunotoxin” refers to a conjugate of an antibody with one or more toxins, drugs, radionuclides or cytotoxic agents.
  • cytotoxi agents that may be conjugated to the antibodies of the presen invention are ricin, diphtheria toxin and radionuclides.
  • Rici is an extremely potent toxin produced by the beans of the plan Ricinus communis.
  • the antibody which binds to a protein that is expressed b HIV-infected cells
  • a toxin e.g. ricin
  • tha is toxic to the HIV-infected cell (and to non-infected cells a well).
  • the human monoclona antibodies to which the agent is coupled will carry the agen directly to the target (in this case, HIV-infected cells) thereby sparing non-infected cells from the toxin.
  • Technique that may be employed to conjugate human monoclonal antibodies including those of the present invention, to cytotoxic agent are described in detail in Vitetta et al., supra and i European Patent Application Serial No. 279,668, publishe August 24, 1988 of Genentech, Inc..
  • the human lymphoblastoid cell lines (which produce th monoclonal antibodies of the present invention) were formed b immortalizing lymphocytes obtained from HIV-seropositiv patients by infecting such lymphocytes with Epstein Barr Viru (EBV) in vitro.
  • EBV Epstein Barr Viru
  • blood was obtained from 58 HIV seropositive individuals, peripheral blood mononuclear cell were obtained and incubated overnight with EBV.
  • the EB infected cells were cultured at 80,000 cells per well i microtiter wells for 3-4 weeks and assayed for anti-HI antibody production using a non-commercial ELISA (see below and a commercial ELISA employing HIV-coated beads.
  • Th specificity of each positive reaction obtained by the ELISA wa confirmed by testing for their non-reactivity on identica beads coated with bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • lymphoblastoid cell culture tested positively in the non-commercial ELISA. After expansio the positive wells were cultured for two more weeks. It wa found that 2.4% tested positively for HIV proteins by ELISA an 0.67% proved to be specific for HIV by virtue of their non reactivity on the BSA beads.
  • the anti-HIV antibodies produce were found to be directed against gp41 or p24 and had suffi cient avidity to show reactivity by ELISA, Western blot, radio immunoprecipitation and/or immunofluorescence. Therefore, al of these monoclonal antibodies would be useful in diagnosti assays for HIV.
  • the stable clones were then subcultured 1 to times at 10 or 100 cells per well with irradiated huma lymphoblastoid feeder cells and expanded into tissue cultur flasks.
  • peripheral blood mononuclear cells from another 36 HIV seropositive individuals were obtained, immortalized by EB infection and assayed for anti-HIV production as above.
  • Fou stable lymphoblastoid cell lines were obtained producin monoclonal antibodies: 120-16, 126-6, 126-50, 167-7 and 191- against gp41, and 134-F6 against p24.
  • one cell line, 98-4.3, producin monoclonal antibodies against p24 was obtained from positiv cultures derived from the first group of 58 patients describe above. The culture was subcultured twice after severa unsuccessful trials and is presently stable in culture.
  • the present inventors have performed some epito mapping of the human monoclonal antibodies of the presen invention. For example, it can be seen from the data presente in Example 5, Table III below that monoclonal antibodies 50-6 and 98-43 bind to the same epitope cluster (i.e. amino acid falling between residues 579 and 613) Whereas 98-6 binds to t a different region (amino acids falling between residues 64 and 692). However, monoclonal antibodies 50-69 and 98-4 differ in their epitope specificity as demonstrated by the fac that 50-69 binds to peptide 599-613 whereas 98-43 binds t pepticle 579-604. Three of the anti-p24 antibodies have bee tested (i.e. 71-31, 91-5 and 91-6). All bind to the sam region of p24 (131-198).
  • ADCC cellular cytotoxicity
  • the ADCC immune response is directed by specifi antibodies and involves mobilization of effector cell (cytotoxic T-cells, monocytes, and killer cells agains specific targets. It is believed that the ability to mount a ADCC response will be important for serotherapy an seroprophylaxis in HIV infections also.
  • the fourteen lymphoblastoid cell lines thus obtaine are stable in culture and produce human monoclonal antibodie directed against targets on HIV which are expressed in vivo i infected patients.
  • the human monoclonal antibodies of the present inven tion are all of the IgG serotype and may be recovered from th supernatants of monoclonal antibody producing lymphoblastoi cell cultures and purified by conventional methods known in th art for the purification of IgG. Such methods include Protein A Sepharose chromatography, a combination of Affigel Blue (Bio Rad, Richmond, CA) and Protein-A Sepharose chromatography, o High Performance Liquid Chromatography (HPLC).
  • HPLC High Performance Liquid Chromatography
  • the eleven stable lymphoblastoid cell lines describe in Examples 1-6 below produce human monoclonal antibodies whic are directed against unique epitopes which are expressed i HIV-infected patients. Although some epitope mapping has bee performed (see Table III in Example 4 below), further epitop mapping will determine the exact specificity of each of th monoclonal antibodies and may reveal targets on the HIV gp4 and p24 protein molecules which can be candidates for vaccin production.
  • the human monoclonal antibodies of the present inven tion are directed against either immunodominant (common) o non-dominant epitopes of the gp41 and p24 viral proteins.
  • immunodominant refers to an antigenic determinant that most patients respon by the production of antibodies.
  • Antibodies 50-69 and 120-16 directed against gp41 are to immunodominant epitopes.
  • Thes two antibodies may be employed for passive immunizations and/ diagnostic reagents.
  • Antibodies 71-31 and 91-5, directe against p24, are to non-dominant epitopes.
  • Lymphoblastoid cell lines 91-5 and 120-16 have been deposited with the American Ty Culture (ATCC, Rockville, MD) and have received Accessi Numbers CRL 10038 and CRL 10037, respectively.
  • ADCC antibody-dependent cellular cytotoxici
  • the anti-p24 immunotoxin (IT) did not kill infected or uninfected H9 or U937 cells at concentrations up to 20 ug Ab/ml. IT made with monoclonal antibody to gp41, however, reduced protein synthesis in infected H9 cells by 50% (IC50) at concentrations of 500 ng/ml. The IC50 of anti-gp41 IT for infected U937 cells was 1000 ng/ml. In the presence of chloroquine, the IC50 of these immunotoxins (ITs) was 5-10 ng/ml.
  • the human monoclonal antibodies of the present invention When employed to treat individuals infected by HIV or suffering from AIDS, the human monoclonal antibodies of the present invention (having a specificity and a binding affinity for HIV proteins p24 and gp41) may be administered as passive immunization agents in effective amounts broadly ranging between about 200 mg and about 15 grams and preferably between 50 mg and 1 gram.
  • the antibodies of the invention are adminis ⁇ tered parenterally, and preferably via the intravenous route.
  • a typical treatment regimen would comprise administration of an effective amount of antibody administered over between about one week and about 6 months.
  • the number of treatments required to control a patient's disease will vary from individual to individual, depending upon the severity and stage of the illness and the individual characteristics of each patient being treated.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the human monoclonal antibodies may be administered alone or in conjunction with other HIV treatments, such as AZT, in order to control a patient's disease.
  • the anti-HIV treatment may be administered one or two times a week or more as determined by the patient's condition and the stage of the patient's disease.
  • the human monoclonal antibodies of the present inven ⁇ tion can be incorporated into conventional pharmaceutical formulations for use in treating individuals that are afflicted with HIV or for prophylaxis in individuals at risk for suc infections.
  • compositions of the inventio comprising an anti-HIV effective amount, range between abou 200 mg and about 15 grams, of the human monoclonal antibodie of the present invention identified in Examples 1-4 below. Th quantity of effective dose applied by each injection i relatively unimportant since the total dosage can be reached b administration of one or a plurality of injections.
  • such formulations may comprise pharmaceutically-accep table carriers, diluents, salts and other materials well-know in the art.
  • Isotonic saline, sterile water, 10% maltose, huma serum albumin, glycine or other pharmaceutically-acceptabl materials may be used as diluents, carriers or solvents i preparing the pharmaceutical formulations comprising the huma monoclonal antibodies of the present invention.
  • Blood was obtained from 58 HIV-seropositive individual who were intravenous drug users or homosexuals.
  • the presenc of antibody to HIV in the blood was determined using a commer cial enzyme-linked i munosorbent assay (ELISA) (Organon-Teknik Bio-Enzabead HTLV-III ELISA, Durham, NC) and confirmed b Western blot (Novapath Immunoblot Assay, Bio-Rad, Hercules, C and Biotech/DuPont, DuPont, Wilmington, DE).
  • ELISA commer cial enzyme-linked i munosorbent assay
  • ELISA Organon-Teknik Bio-Enzabead HTLV-III ELISA, Durham, NC
  • Western blot Novapath Immunoblot Assay, Bio-Rad, Hercules, C and Biotech/DuPont, DuPont, Wilmington, DE.
  • the diseas status of patients was established on the basis of an i munologic staging system as described
  • Peripheral blood mononuclear cells collected from t 58 patients were obtained by centrifugation of hepariniz blood, diluted 1:1 with RPMI-1640 and centrifuged on Histopaqu (Sigma, St. Louis, MO) at 300 x g for 30 minutes. Cells at th medium/Histopaque interface were recovered, washed three time and incubated overnight at a density of 2 x 10 ⁇ cells/ml wit the filtered supernatant from the EBV-transformed marmoset cel line B95-8 (Proc. Nat. Acad. Sci. USA 70: 190, 1973, availabl under Accession Number CRL 1612 from the American Type Cultur Collection, ATCC, Rockville, MD).
  • Lymphocytes were then washe once and cultured in RPMI-1640 medium (M.A. Bioproducts, Walkersville, MD) supplemented with 10% fetal calf seru (Hyclone Labs, Logan, UT) 2mM L-glutamine, lOOU/ml penicillin, and 100 micrograms/ml streptomycin (complete medium) for fou weeks in 96-well plates (Costar, Cambridge, MA) at 80,000 cell per well.
  • lymphoblastoid cell lines initial cultures of immortalized B-cell (hereinafter referred to as lymphoblastoid cell lines) were established and further characterized as described below.
  • the screening of the initial cultures in 96-well plate was performed using a non-commercial ELISA.
  • Immulon 2 plate (Dynatech, Chantilly, VI) were coated overnight at 4°C with micrograms/ml of HTLV-III B lysate (purchased from Electro Nucleonics, Inc., Silver Spring, MD) diluted in carbonat buffer, pH 9.8. Plates were washed three times with phosphat buffered saline, pH 7.2, containing 0.05% Tween 20 (PBS-Tween) The culture supernatants to be assayed (0.1 ml per well) were then added and incubated for 90 minutes at 37°C.
  • the specificity of the antibody binding was assessed b testing the supernatants for reactivity against HIV-coate beads (Bio-EnzaBead) and against uncoated beads (obtained fro
  • the type of antibody binding to HIV was determine using the following alkaline phosphatase-coupled antibodies: goat anti-human IgG (gamma specific), goat anti-human kapp chain and goat anti-human lambda chain (Organon Teknika-Cappel, Malvern, PA) .
  • the subtype of the monoclonal antibody was als analyzed by ELISA using alkaline phosphatase-labeled mouse monoclonal antibodies against the four subclasses of human Ig (Zymed, San Francisco, CA) .
  • Immunoglobulin quantitation was also performed b ELISA. Immulon 2 plates were coated with goat anti-human Ig (gamma specific) and incubated with culture supernatants. Bound IgG was detected with alkaline phosphatase-labeled goa anti-human IgG (gamma specific). Affinity-purified human Ig (Cappel) was used to produce standard curves.
  • a total of 14,329 cultures in microtitre wells were established using cells derived from the 58 subjects. Ap proximately half of these cultures were derived from thre serial bleeds from a single subject (with a scale score of 1) over a period of three months. The remaining wells were established using cells derived from 57 subjects whose scal scores ranged from 0 to 3. The results of this procedure ar shown in Table I below. TABLE I
  • the seropositive cell donors were categorized with respect to disease status using the immunologic staging system of Zolla- Pazner et al. (supra) and the results are shown in Table II below.
  • HIV-seronegative patients
  • lymphoblastoid cell lines were isolated and further cloned as described below.
  • EXAMPLE 3 SPECIFICITY AND REACTIVITY OF THE HUMAN MONOCLONA
  • the 57 cell lines mentioned above were then cloned b doubling dilution from 10,000 to 10 cells per well. Wells wit the lowest cell concentration which were producing antibodie were then picked and cloned at 100 or 10 cells per well. Usin this procedure, seven cell lines, 3 producing anti-gp4 antibodies and 4 producing anti-p24 antibodies were establishe which have been cloned from one to three times at 100 or 1 cells per well. The reactivities of the antibodies from thes lines are shown in Figures 1 and 2.
  • All seven of the cell lines of this Example produce antibodies of the IgG subtype as shown in Figure 1.
  • all lines received [ 125I]-labeled HIV lysate reacted wit lane 1, serum from an HIV-infected subject, lane 2, wit antibody from cell line 50-69, lane 3, with antibody from cel line 98-6, lane 4, with antibody from cell line 98-43, lane 5 with antibody from cell line 71-31 lane 6, with antibody fro cell 91-5, lane 7, with antibody from cell and line 91-6 an lane 8, with antibody from 98-4.9.
  • the molecular weights o the major viral proteins are shown on the left in kilodaltons.
  • Antibody from three of the cell lines bound to ej ⁇ v-encode protein gp41 (lines 50-69, 98-6 and 98-43, lanes 2-4 respec tively) .
  • Antibodies from four of the cell lines bound to ga encoded protein p24 (lines 71-31, 91-5, 91-6 and 98-4.9, lane 5-7 respectively).
  • RIP antibodies from cell lines 71-31, 91-5 and 91-6 reacted with p24.
  • Antibodies from 98-4.9 was unreactive by RIP since IgG3, the subtype of this antibody, does not bind to Protein A and is therefore not precipitated.
  • Antibody from all 3 of these anti-gag cell lines were als tested by Western blot ( Figure 2).
  • cell line 98-4.9 recently lost the ability to produce human monoclonal antibodies.
  • Peripheral blood mononuclear cells were obtained from another 39 HIV-seropositive individuals, the cells immortalized by EBV infection, screened and selected as in Examples 1-3 above. Positive cultures were expanded, subcultured by doubling dilution and again subcultured one to three times at 10 to 100 cells per well.
  • Four stable lymphoblastoid cell lines producing human monoclonal antibodies were obtained as follows: 120-16, 126-6 and 126-50 directed against gp41; and 134-F6 directed against p24.
  • ENV9 is a cloned gp41 protein encompassing residues 461 to 76 (obtained from DuPont, Wilmington, DE).
  • PE3 is a 286 amin acid sequence from gpl20 (obtained from DuPont, Wilmington DE).
  • pl21 contains residues 561-649 of gp41 (obtained fro Centocor, Malvern, PA) .
  • ANTIBODIES OF THE PRESENT INVENTION Presented below is an example of the use of th monoclonal antibodies of the present invention in a diagnostic assay for HIV.
  • Immulon 2 plates (Dynatech) were coated with 0.5 micrograms/well of an HIV lysate diluted in 0.05M bicarbonate buffer, pH 9.6 for 2 hours at 37 ⁇ C, and overnight at 4°C. After washing the plates with phosphate buffered saline, pH 7.4, containing 0.05% Tween (PBS-Tween), samples of human sera, obtained from HIV seropositive or seronegative individuals were added to each well at 0.5 micrograms/well, diluted 1:10 to 1:1000. The plates were incubated at room temperature over ⁇ night and washed three times with PBS-Tween.
  • PBS-Tween phosphate buffered saline
  • Biotinylation of the monoclonal antibodies to HIV wa performed as follows. Each monoclonal antibody was partiall purified by ammonium sulfate precipitation and/or chromatog raphy on Protein A-Sepharose. After dialysis against 0.1 sodium bicarbonate 75 microliters of N-hydroxyl-suc cinimidobiotin (5 mg in 1 ml of DMSO) was added to 1 ml of th antibody at a concentration of 5 mg/ml. The reaction wa allowed to proceed at room temperature with shaking for 3 hour and then dialyzed against phosphate buffered saline, pH 7.4 The biotinylated monoclonal antibodies were stored at -25°C i 50% glycerine before use.

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Abstract

L'invention concerne onze lignées cellulaires lymphoblastoïdes humaines produisant des anticorps monoclonaux dirigés contre les protéines gp41 et p24 du virus d'immunodéficience humaine (VIH). L'invention concerne également des procédés de traitement d'individus atteints du VIH à l'aide des anticorps monoclonaux humains, ainsi que des préparations pharmaceutiques comprenant les anticorps monoclonaux humains en quantités efficaces.
PCT/US1990/001132 1989-02-28 1990-02-28 Anticorps monoclonaux humains contre le virus d'immunodeficience humaine WO1990009805A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19809785A1 (de) * 1998-03-08 1999-09-09 Bergter Radioimmunpharmakon zur Behandlung der HIV-1-Infektion
FR2777285A1 (fr) * 1998-04-10 1999-10-15 Bio Merieux Ligand peptidique presentant une affinite specifique vis-a-vis de la proteine p24 du retrovirus hiv-1
US6413515B1 (en) 1996-03-12 2002-07-02 Ovogenix Immunpharma Gmbh Avian, vitelline antibodies directed against HIV antigens
US7744887B2 (en) 2004-06-01 2010-06-29 Merck & Co., Inc. Human antibodies interacting with HIV gp41
US10730933B2 (en) 2015-12-05 2020-08-04 Centre Hospitalier Universitaire Vaudois HIV binding agents

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0251612A2 (fr) * 1986-06-23 1988-01-07 Bristol-Myers Squibb Company Anticorps monoclonal humain contre le virus associé à la lymphadénopathie

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE86636T1 (de) * 1984-10-18 1993-03-15 Pasteur Institut F antigene vom menschlichen immunodefizienz-virus und deren verwendungen.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0251612A2 (fr) * 1986-06-23 1988-01-07 Bristol-Myers Squibb Company Anticorps monoclonal humain contre le virus associé à la lymphadénopathie

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Journal of Immunological Methods, Volume 106, 1988, GRUNOW et al., "The High Efficiency, Human B Cell Immortalizing Heteromyeloma CB-F7", pages 257-265. see pages 257, 258 and 263. *
Journal of Immunology, Volume 140, 01 February 1988 EVANS, "Human Monoclonal Antibody Directed Against Gag Gene Products of the Human Immunodeficiency Virus", pages 941-943. see Abstract. *
Proceedings National Academy Sciences, Volume 86 March 1989, GORNY et al, "Generation of Human Monoclonal Antibodies to Human Immunodeficiency Virus", pages 1624-1628. see Abstract. *
See also references of EP0418347A4 *
The Lancet 23 April 1988, DESGRANGES et al, Monoclonal Antibodies to HIV in a Non-Infected, Immunized Volunteer", pages 935-936. see entire article. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6413515B1 (en) 1996-03-12 2002-07-02 Ovogenix Immunpharma Gmbh Avian, vitelline antibodies directed against HIV antigens
DE19809785A1 (de) * 1998-03-08 1999-09-09 Bergter Radioimmunpharmakon zur Behandlung der HIV-1-Infektion
DE19809785C2 (de) * 1998-03-08 2000-02-10 Wolfgang Bergter Radioimmunpharmakon zur Behandlung der HIV-1-Infektion
FR2777285A1 (fr) * 1998-04-10 1999-10-15 Bio Merieux Ligand peptidique presentant une affinite specifique vis-a-vis de la proteine p24 du retrovirus hiv-1
WO1999053063A1 (fr) * 1998-04-10 1999-10-21 Bio Merieux LIGAND PEPTIDIQUE PRESENTANT UNE AFFINITE SPECIFIQUE VIS-A-VIS DE LA PROTEINE p24 DU RETROVIRUS HIV-1
US7744887B2 (en) 2004-06-01 2010-06-29 Merck & Co., Inc. Human antibodies interacting with HIV gp41
US10730933B2 (en) 2015-12-05 2020-08-04 Centre Hospitalier Universitaire Vaudois HIV binding agents

Also Published As

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EP0418347A4 (en) 1994-12-14
JPH04505099A (ja) 1992-09-10
AU5225693A (en) 1994-03-03
EP0418347A1 (fr) 1991-03-27
AU651794B2 (en) 1994-08-04
AU680836B2 (en) 1997-08-14
AU5188590A (en) 1990-09-26
CA2028121A1 (fr) 1990-08-29

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