WO1989004370A1 - Human monoclonal anti-hiv-i-antibodies - Google Patents

Human monoclonal anti-hiv-i-antibodies Download PDF

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Publication number
WO1989004370A1
WO1989004370A1 PCT/EP1988/001072 EP8801072W WO8904370A1 WO 1989004370 A1 WO1989004370 A1 WO 1989004370A1 EP 8801072 W EP8801072 W EP 8801072W WO 8904370 A1 WO8904370 A1 WO 8904370A1
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Prior art keywords
hiv
human
immunotoxin
cells
lnfected
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PCT/EP1988/001072
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French (fr)
Inventor
Hermann Katinger
Rüdiger VON BAEHR
Alois Jungbauer
Tomas Porstmann
Franz J. Steindl
Roland Grunow
Andrea Buchacher
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Cl-Pharma Aktiengesellschaft
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Priority to EP89900809A priority Critical patent/EP0355140B1/en
Priority to DE3855134T priority patent/DE3855134T2/en
Publication of WO1989004370A1 publication Critical patent/WO1989004370A1/en

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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
    • C07K16/1063—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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A61K47/6827—Ricin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • 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
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • This invention is in the field of immunology specially AIDS therapy and in vivo imaging of HIV-lnfected cells. More particulary it concerns human mono ⁇ clonal anti-HIV-1-antibodies and immunochemicals made from these antibodies and therapeutic methods that use these immunochemicals.
  • the production of human monoclonal antibodies may present a new source of antibodies to be used in immunotherapy for infections and other diseases.
  • the application of mAb of human origin instead of murine origin avoids the induction of antibody response in humans.
  • the Fc-part of murine antibodies has to be removed prior to injetion in humans to minimize antibody response against heterogenous administered proteins. Human monoclonal antibodies however present a part of the homologous proteins of human individuals.
  • HIV human immunodeficin ⁇ y virus
  • AIDS aquired immunodeficiency syndrome
  • Human anti-HIV-monoclonal antibodies Human anti-HIV-1-mAb
  • the hu anti-HIV-1-mAb used is of IgG 1 subclass produced according to known hybridization procedures described by R. Kennet et al. in "Monoclonal antibodies and functional cell lines; progress and applications”. Plenum Press (New York), 1984. Samples of hybridoma 3D6 were deposited in ECACC, Porton Down, Salisbury SP4 OJG, UK. 3D6 was the only deposited one of the six hybridomas that produce antibodies recognizing HIV-envelope proteins. The ECACC accession number and deposit date for the deposited hybridoma is: 87110301; November 3 rd 1987.
  • HIV I serum positive donors were washed 3 times with serum free cell culture medium.
  • the cells were fused immediately or stimulated with 5 ng pokeweed mitogen (PWM) for three days prior to fusion.
  • PWM pokeweed mitogen
  • the cells were mixed at a ratio of 5:1 with HAT-sensitive fusion cells.
  • the cells were fused with PWM
  • IgG and IgM isotypes were analyzed from the culture supernatant by a sandwich enzyme linked immunosorbant assay (ELISA) with peroxidase conjugated anti human IgG, IgM (heavy chain specific) antibodies in a known manner. Screening for specific antibodies was performed by a sand ⁇ wich ELISA using HIV in a concentration of 10 microgram/ml. The virus isolated from tissue culture was coated in flat bottom microtiterplates and the unbound material was washed out. The culture supernatants were incubated overnight at 4°C. After washing out unbound material the specific antibodies were detected by anti human-Ig-heavy-chain specific antibodies labelled with peroxidase.
  • ELISA sandwich enzyme linked immunosorbant assay
  • Immunoblot using HIV envelope proteins prepared from native virons Purified virus was denaturated and reduced with 2.5 % SDS and 5.0 % 2-mercaptoethanol at 90° C for 5 min and applied to a 10 % polyacrylamide slab gel. After separation protein bands were electro-transferred into a nitrocellulose sheet (Schleicher & Schüll, FRG). After blocking the sheet with 5 % dry milk, strips were cut and immersed in 5 ml 1:2 diluted culture supernatant. Bound antibodies were detected by reacting with anti-human IgG antibodies labelled with HRP and staining with diaminobenzidine containing 0.1 % NiCl 2 .
  • SDS-gelelectrophoresis was performed according to Laemmli, (Nature 227, 1970; 680-685).
  • the electrophoretically separated envelope or core proteins resp. derived from genetically engineered E. coli (L.H. Gosting, et al., Journal of Clinical Microbiology 52 1987 (845-848)). were electroblotted to nitrocellulose. after blocking with 3 % bovine serum albumin (BSA dissolved in PBS) the strips were immersed in samples diluted in PBS buffer (containing 0.1 %
  • peptides were used for the evaluation of the antigen binding sites.
  • the peptides A and B were synthesized according to literature.
  • C and D were a gift from Biochrom (FRG). 4 differnt peptides were tested.
  • the hybridization probe was the 32 P-labelled plasmid Blur8 which contains a cloned Alu-sequence that is specific for human DNA and repeated about 300.000 times in the genome (Schmid et al., Science 216, 1982; 1065).
  • the membrane was washed 3 x in 100 ml 2 x SSC, 0.1 % SDS for 5' at room temperature and 2 x in 200 ml 1 x SSC, 0.1 % SDS for
  • H9 cells Fixed HIV-1 infected cells (H9 cells) were incubated with the hu anti-HIV-mAb. Binding of the mAb was demonstrated by a second incubation after washing out unbound mAb with FITC labelled anti human IgG.
  • the 3A6 antibody was purified by cation exchange chromatography using CM-Sepharose fast flow and Protein-A superose. Preparation of Ricin-A-chain Toxin
  • Ricin is extracted from castor beans (Ricinus Communis) by known methods. Ricin is extracted either from grinded whole castor beans (Ricinus Communis) or from castor bean cake, which is a byproduct of castor bean processing. The castor bean cake is defatted by extraction 3 times with 5 volumes of 40 % - 60 % (v/w) petroleum ether. The air-dried material is then extracted over night in phosphate buffered saline (PBS; 0.15 M NaCl, 0.01 M phosphate, pH 7.2). The extract is cleared by filtration through a nylon gauze followed by centrifugation at 1500 g for 1 hour. The clear super ⁇ natant is precipitated at 4° C with saturated ammonium sulfate.
  • PBS phosphate buffered saline
  • the precipitate is collected and harvested by centrifugation (1500 g, 1 hour) redissolved in a minimum amount of PBS and dialyzed against PBS until the extract is ammonium sulfate free.
  • the clarified and dialyzed toxin extract is further purified by lectin affinity chromatography.
  • the gel (Sepharose 4B, Fa. Pharmacia) is pretreated with 1 M propionic acid at room temperature for at least 4 weeks to enhance its binding capacity for lectins.
  • the column chromatography should be operated at temperatures lower than 10° C to optimize lectin binding.
  • the clarified and dialyzed extract is applied to a PBS equilibrated acid pretreated Sepharose 4B-column. After sample application the column is washed with PBS until the UV-absorbance return to the baseline.
  • the toxin is eluted together with other lectins with 100 mM galactose in PBS. This mixture is further separated by gelchromatography on Sephacryl S200 HR. The sample volume should not exceed 3-4 % of the bed total volume. The toxin is resolved completely from the other lectins under these conditions.
  • the toxin recovered from the affinity column is concentrated to 10 mg/ml by ultrafiltration (Milipore PTGC membrane) prior to application on the Sephacryl S200 HR column.
  • the toxin peak was collected and filter sterilized.
  • the sterile toxin solution was stored deep frozen at -30°C until cleavage into
  • the affinity purified and concentrated toxin is cleaved into A- and B-chain by a 5 % beta-mercaptoethanol solution.
  • the toxin is incubated at a concentration of 5 mg/ml in 0.1 M Tris-HCl pH 8.5 buffer completed with betamercaptoethanol (5 % end concentration) and galactose (0.5 M final concentration) overnight at room temperature, followed by 2-3 hours at 37° C.
  • the toxin is transferred from the starting buffer (PBS) into the incubation buffer (Tris buffer) by gelchromatography on Sephadex G25 columns equilibrated with the incubation buffer.
  • the sample is applied to a DEAE-Sepharose fast flow equilibrated with 0.1 M Tris/HCl buffer pH 8.5.
  • the column is washed with 0.1 M Tris/HCl buffer pH 8.5 until all unbound material is eluted.
  • the unbound material essentially pure A-chain, is collected and passed down an asialo-fetuin-Sepharose 4B column to remove contaminating toxin.
  • the asialo fetuin-Sepharose 4B was prepared according to the recommendation of Pharmacia company. The unbound material is collected and filter sterilized and stored at 4° C.
  • the DEAE-Sepharose fast flow is regenerated with a 0.1 M Tris-HCl buffer pH 8.5 containing 0.1 M Galactose and 1 M NaCl.
  • the isoelectric point of the hu anti-HIV-1-mAb was determined by a described method. Pharmalytes were used as carrier ampholytes. The whole procedure was carried out according the recommendation of the Pharmacia company
  • Light chains and subclass were determined by ELISA. Specific anti human KAPPA-chain antibodies labelled with alkaline phosphatase or specific anti human Gl, G2, G3 and G4 antibodies labelled with Peroxidase were used.
  • the mixture was rocked for 1 hour at 4°C and then diafiltrated at 4°C against 0.01 M sodium phosphate buffer containing 2.0 M sodium chloride.
  • the bulk of the unconjugated A-chain was separated from the immunotoxin by gelchromatography on Sephacryl S200 HR.
  • the column was equilibrated in 0.02 M sodium phosphate containing 3 M sodium chloride.
  • the first peak, which contained the immunotoxin was pooled and affinity purified on an anti-human IgG-Sepharose 4B.
  • the immunotoxin was eluted from the affinity column with 3.5 M ⁇ magnesium chloride and extensively dialyzed against sodium chloride and then against PBS.
  • Immunotoxins (0.2-2 mg/ml) were concentrated by ultrafiltration using PTGC membranes to the end concentration necessary for testing. The final preparation was sterile filtered and stored in aliquots at -20°C. The immunotoxins were analyzed by SDS-PAGE under both reducing and nonreducing conditions. The substitution of the immunotoxin was determined by radioimmunoassay. An average substitution of 1-2 moles A-chain per mole antibody was observed.
  • a dilution series of hu anti-HIV-mAb (in PBS 1 % BSA and 0.1 % Triton X-100) was dispensed in flat bottomed microtiter wells coated with purified HIV envelope proteins incubated overnight at 4°C. After washing out unbound material the bound conjugate was determined by reaction with 1.4 phenylendiamine. The developed colour was measured at 492 nm. A calibration curve was blotted optical density versus dilution. The dilution of the half saturation is calculated from this calibration curve.
  • Hu anti-HIV-mAb at a dilution corresponding to the half maximum saturation was mixed with sera from patients suffering from AIDS or with sera from probands who were seropositiv determined by a conventional screening (ELISA) followed by a confirmation test (westernblot).
  • the mixture of the conjugate (at half maximal dilution) and the serum from the probands were incu ⁇ bated in HIV envelope protein coated microtiter wells, washed and stained.
  • optical density of these samples is compared to the anti HIV mAb at half maximum saturation. A value below the half maximum saturation indicates a competition between the human sera and the human monoclonal anti-HIV antibody.
  • the assay for neutralizing activity of the hu anti-HIV-1-mAb was performed according to a known method (J. Virology, 61,2024-2028, 1987), Nature 316, 72-74, 1985; Biotechnology 5, 940-946, 1987).
  • An HIV-1 dose equivalent to twenty times the amount to cause infection in 50 % inoculated cultures on day 12 (20 x TICD 20) was used to infect the H9-cell line. Cells were collected 3,6,9 and 12 days after virus inoculation and percentage of infection was determined by immunofluorescence. 100 % infection was observed between day 6 and 9.
  • the neutralizing effect was also tested in the presence of the monoclonal antibody at variable dilutions.
  • the neutralizing activity of the 3D6 antibody was evaluated by addition of a single dose of antibody (final concentration l ⁇ g/ml) immediately after virus inoculation. The infection rate was expressed as percent of infected cells.
  • the test cell line used in the cytotoxicity tests were HIV1 infected H9. cells .
  • HIV-1 infected H9 cells were prepared as described under Neutralizing activity on the infectivity of HIV-1 virions. Uninfected H9 cells were used as negative control. Cells harvested after a period of 6-9 days after virus inoculation were suspended in 1 nil medium. Various dilutions of the hu mAb-toxin conjugate were added to the infected cells and the negative control. After incuba ⁇ tion for 24 hours at 37°C cells were washed with PBS and methionine free medium supplemented with 35 S-methionine was added.
  • the hybridoma No. 3D6 is deposited in the ECACC, Porton Down, Salisbury, UK under the deposition No. 87110301 (3 rd November 1987).
  • the hu mAb anti HIV-1 produced by this hybridoma line (3D6) is used as example to illustrate the present invention.
  • Immunoblots of the 3D6 antibody are shown in Fig. la,b and c.
  • Immunoblots of the 3A6, 19H2, 23H7, 37G12 and 42B9 antibodies are shown in fig. Id:
  • Fig. la Immunoblot of the human mAb produced by Hybridoma No 3D6. HIV envelope proteins were prepared from native virons by SDS and 2-mercaptoethanol treatment.
  • the human origin of the monoclonal antibody is shown by the hybridization of 32 P-labelled plasmid D Blur8. Autoradiographs are shown in fig. 2 with the DNA of the hybridoma cells. Also the reaction with anti human IgG and human IgM antisera and the more specific sera for subclass determination verifies the human origin of the monoclonal antibodies.
  • Fig. lb Immunoblot of the human anti HIV-mAb produced by Hybridoma 3D6 HIV envelope proteins were prepared from genetically engineered E. coli (according to a known procedure, L.H. Costing et al.: Journal of Clinical Microbiology 25,1987 (845-848)).
  • Fig. 1c Immunoblot of the human anti HIV mAb produced by Hybridoma 3D6. A commercially available blot strip (Du Pont Lot. : 70 44 128) was used.
  • Fig. Id Immunoblot of anti HIV mab produced by the hybridoma 3A6(1), 19H2(2), 23H7 (3), 37G(12) and 42B9(4). A purified virus were electrophoretically separated and blotted.
  • Fig. 2 Detection of human DNA by hybridization, a) 3D5; b) 3D9; c) 24G3; d) BJA-B (human lymphoid cell line positive control; e) P3X63Ag/653 (murine myeloma negative control; f) BUBTU (human bladder carcinoma positive control.
  • Fig. 3 Immunoflourescence of fixed HIV-1 infected H9- cells imaged with a sandwich of 3D6-antibody and anti human IgG conjugated with FITC. The clone 3D6 and 3D5 are shown.
  • Fig. 4 Neutralizing activity of 3D6-antibody expressed as percent of infected cells. Infected cells were shown by immunflourescence using 3D6 antibody.
  • the monoclonal antibody (3D6) covalently linked with the Ricin-A-chain was used to demonstrate the cytotoxicity of the antibody.
  • the cytotoxicity was measured by 35 s- methionine uptake.
  • the immunotoxin consisting of a conjugate between 3D6 antibody and Ricin A-chain, prepared as described under materials and methods killed specifically HIV-1 infected H9 cells.
  • the 35 s-methionine uptake is expressed in percent of control (Fig. 6).
  • the TCID 50 of the immunotoxin (3D6) -Ricin-A-chain) is less than 10 nm. Comparison of the hu anti-HIV-mAb with naturally occuring antibodies from seropositives and patients suffering from AIDS or pre AIDS
  • Table II shows the values from competitive EIA using a 3D6-antibody peroxidase conjugate and different sera. Table II: Comparision between 3D6 antibody and sera from seropositives and patients suffering from AIDS or pre AIDS.
  • FIG.7 Comparison between 3D6 antibody and sera from seropositives and patients suffering from AIDS or pre AIDS. The results according table II are shown in this figure

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Abstract

A human monoclonal antibody which binds selectively to envelope and/or core proteins of HIV 1 and also selectively to HIV-1 infected cells. When conjugated to a ricin A-chain it exhibits a TCID 50% against HIV-1 infected H-9 cells of less than 10 uM.

Description

HUMAN MONOCLONAL ANTI-HIV-I-ANTIBODIES
This invention is in the field of immunology specially AIDS therapy and in vivo imaging of HIV-lnfected cells. More particulary it concerns human mono▴clonal anti-HIV-1-antibodies and immunochemicals made from these antibodies and therapeutic methods that use these immunochemicals. The production of human monoclonal antibodies (hu mAbs) may present a new source of antibodies to be used in immunotherapy for infections and other diseases. The application of mAb of human origin instead of murine origin avoids the induction of antibody response in humans. The Fc-part of murine antibodies has to be removed prior to injetion in humans to minimize antibody response against heterogenous administered proteins. Human monoclonal antibodies however present a part of the homologous proteins of human individuals. In the sera of human immunodeficinσy virus (HIV) infected men anti-virus antibodies could be detected over a certain period after infection without any clinical manifestations of the aquired immunodeficiency syndrome (AIDS). At this state of active immune response high numbers of antigen-speci▴fic B cells were expected in the circulation. These B-cells were used as fusion partners for the generation of human monoclonal anti HIV antibodies. We describe here:
- the generation of human mAb to HIV antigens
- the substantial inhibition of growth of HIV-lnfected cells
- the preparation of immunotoxins conjugated with the described human anti-HIV-1-mAb and an A-chain toxin - the selective killing of HIV-1 infected cells with the immunotoxin
- the selective killing of HIV-1 infected cells by antibody dependent cellular cytotoxicity
- the selective prevention of infection of cells with the human monoclonal anti-HIV-1 antibody
-The selective determination of HIV-1 antigen in human sera
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
Human anti-HIV-monoclonal antibodies (Hu anti-HIV-1-mAb)
In the presently preferred embodiment of the invention the hu anti-HIV-1-mAb used is of IgG1 subclass produced according to known hybridization procedures described by R. Kennet et al. in "Monoclonal antibodies and functional cell lines; progress and applications". Plenum Press (New York), 1984. Samples of hybridoma 3D6 were deposited in ECACC, Porton Down, Salisbury SP4 OJG, UK. 3D6 was the only deposited one of the six hybridomas that produce antibodies recognizing HIV-envelope proteins. The ECACC accession number and deposit date for the deposited hybridoma is: 87110301; November 3rd 1987.
Fusion
Fusion as performed in a described manner (Köhler et al.,Europ.Journal of Immunology, 6 (1976):292). Prior to cell fusion with PEG 1500 peripheral blood lymphocytes from
HIV I serum positive donors were washed 3 times with serum free cell culture medium. The cells were fused immediately or stimulated with 5 ng pokeweed mitogen (PWM) for three days prior to fusion.The cells were mixed at a ratio of 5:1 with HAT-sensitive fusion cells. The cells were fused with
42 % PEG 1500 (Ferak,FRG). in the presence of 7.5 % DMSO
(Serva,FRG). Cells were cloned and subcloned by limiting dilution. Screening of the Hybridomas
IgG and IgM isotypes were analyzed from the culture supernatant by a sandwich enzyme linked immunosorbant assay (ELISA) with peroxidase conjugated anti human IgG, IgM (heavy chain specific) antibodies in a known manner. Screening for specific antibodies was performed by a sand▲wich ELISA using HIV in a concentration of 10 microgram/ml. The virus isolated from tissue culture was coated in flat bottom microtiterplates and the unbound material was washed out. The culture supernatants were incubated overnight at 4°C. After washing out unbound material the specific antibodies were detected by anti human-Ig-heavy-chain specific antibodies labelled with peroxidase.
Immunoblotting
Immunoblot using HIV envelope proteins prepared from native virons: Purified virus was denaturated and reduced with 2.5 % SDS and 5.0 % 2-mercaptoethanol at 90° C for 5 min and applied to a 10 % polyacrylamide slab gel. After separation protein bands were electro-transferred into a nitrocellulose sheet (Schleicher & Schüll, FRG). After blocking the sheet with 5 % dry milk, strips were cut and immersed in 5 ml 1:2 diluted culture supernatant. Bound antibodies were detected by reacting with anti-human IgG antibodies labelled with HRP and staining with diaminobenzidine containing 0.1 % NiCl2.
Immunoblot using HIV envelope proteins prepared from cloned material
SDS-gelelectrophoresis was performed according to Laemmli, (Nature 227, 1970; 680-685). The electrophoretically separated envelope or core proteins resp. derived from genetically engineered E. coli (L.H. Gosting, et al., Journal of Clinical Microbiology 52 1987 (845-848)). were electroblotted to nitrocellulose. after blocking with 3 % bovine serum albumin (BSA dissolved in PBS) the strips were immersed in samples diluted in PBS buffer (containing 0.1 %
Triton X-100, 1 % BSA, 0.5 % gelatine) overnight,washed 3 times and incubated with goat anti human GAMMA-chain labelled with peroxidase.
Immunoblots using commercially available blot strips:
Blot strips from Du Pont Lot 7044128 were used according to recommendations of the company
Commercial available EIA
Commercial available EIA were used to check the specifity of monoclonal antibodies. Both, catch-elisa and competitive EIA were used. Assays from Labsystems (Finland), Organon (Belgium), Pasteur (France), Biochrom (FRG), Abott (USA) and a ccompetitv EIA from Organon were used.
Peptide mapping
Different peptides were used for the evaluation of the antigen binding sites.The peptides A and B were synthesized according to literature. C and D were a gift from Biochrom (FRG). 4 differnt peptides were tested.
Peptid A: Wang et al. PNAS 83 6159-6163 (1986
RILAVERYLKDQLLGIWGCS
Peptide B: Gnann et al. J. Virol.61 2639-2641 (1987)
LGIWGCSGKLIC
Peptide C: own results
KDQQLLGIWGCSGKLIC
Peptide D: obtained from Biochrom
KDQQLLGIWGCSGKLICVPWNAS The peptides C and D reacted with the monoclonal antibody produced by the hybridoma no. 3D6.
Hybridization detection of human DNA
106 cells of each cell line were spotted on a nitrocellulose membrane. The hybridization probe was the 32P-labelled plasmid Blur8 which contains a cloned Alu-sequence that is specific for human DNA and repeated about 300.000 times in the genome (Schmid et al., Science 216, 1982; 1065).
After prehybridization and hybridization overnight the membrane was washed 3 x in 100 ml 2 x SSC, 0.1 % SDS for 5' at room temperature and 2 x in 200 ml 1 x SSC, 0.1 % SDS for
30' at. 60°C and autoradiographed for 80 hours.
Immunofluorescence
Fixed HIV-1 infected cells (H9 cells) were incubated with the hu anti-HIV-mAb. Binding of the mAb was demonstrated by a second incubation after washing out unbound mAb with FITC labelled anti human IgG.
purification of the 3D6 and 3A6-antibodies
Purification was performed in a known manner. The clarified culture supernatant was desalted using gelfiltration. on Sephadex G-25 column (Pharmacia) and further chromatographed on a CM-Sepharose fast flow column (Pharmacia). The eluate of the CM-Sepharose fast flow column was concentrated by ultrafiltration and rechromatographed using Phenyl-Superose (Pharmacia). The chromatographic steps were performed according to the recommendations of the Pharmacia company. Prior to the loading of the solution on the Phenyl-superose column the crude mAb solution was diluted by a 2 M-ammonium sulfate solution.
The 3A6 antibody was purified by cation exchange chromatography using CM-Sepharose fast flow and Protein-A superose. Preparation of Ricin-A-chain Toxin
Ricin is extracted from castor beans (Ricinus Communis) by known methods. Ricin is extracted either from grinded whole castor beans (Ricinus Communis) or from castor bean cake, which is a byproduct of castor bean processing. The castor bean cake is defatted by extraction 3 times with 5 volumes of 40 % - 60 % (v/w) petroleum ether. The air-dried material is then extracted over night in phosphate buffered saline (PBS; 0.15 M NaCl, 0.01 M phosphate, pH 7.2).The extract is cleared by filtration through a nylon gauze followed by centrifugation at 1500 g for 1 hour. The clear super▴natant is precipitated at 4° C with saturated ammonium sulfate. At a final concentration of 60 % ammonium sulfate the precipitate is collected and harvested by centrifugation (1500 g, 1 hour) redissolved in a minimum amount of PBS and dialyzed against PBS until the extract is ammonium sulfate free.
Affinity chromatography
The clarified and dialyzed toxin extract is further purified by lectin affinity chromatography. The gel (Sepharose 4B, Fa. Pharmacia) is pretreated with 1 M propionic acid at room temperature for at least 4 weeks to enhance its binding capacity for lectins.
The column chromatography should be operated at temperatures lower than 10° C to optimize lectin binding. The clarified and dialyzed extract is applied to a PBS equilibrated acid pretreated Sepharose 4B-column. After sample application the column is washed with PBS until the UV-absorbance return to the baseline. The toxin is eluted together with other lectins with 100 mM galactose in PBS. This mixture is further separated by gelchromatography on Sephacryl S200 HR. The sample volume should not exceed 3-4 % of the bed total volume. The toxin is resolved completely from the other lectins under these conditions. The toxin recovered from the affinity column is concentrated to 10 mg/ml by ultrafiltration (Milipore PTGC membrane) prior to application on the Sephacryl S200 HR column. The toxin peak was collected and filter sterilized. The sterile toxin solution was stored deep frozen at -30°C until cleavage into
A and B-chain.
Cleaveage of Ricin into A- and B-chain
The affinity purified and concentrated toxin is cleaved into A- and B-chain by a 5 % beta-mercaptoethanol solution. The toxin is incubated at a concentration of 5 mg/ml in 0.1 M Tris-HCl pH 8.5 buffer completed with betamercaptoethanol (5 % end concentration) and galactose (0.5 M final concentration) overnight at room temperature, followed by 2-3 hours at 37° C. The toxin is transferred from the starting buffer (PBS) into the incubation buffer (Tris buffer) by gelchromatography on Sephadex G25 columns equilibrated with the incubation buffer.
After incubation the sample is applied to a DEAE-Sepharose fast flow equilibrated with 0.1 M Tris/HCl buffer pH 8.5. The column is washed with 0.1 M Tris/HCl buffer pH 8.5 until all unbound material is eluted. The unbound material, essentially pure A-chain, is collected and passed down an asialo-fetuin-Sepharose 4B column to remove contaminating toxin. The asialo fetuin-Sepharose 4B was prepared according to the recommendation of Pharmacia company. The unbound material is collected and filter sterilized and stored at 4° C.
The DEAE-Sepharose fast flow is regenerated with a 0.1 M Tris-HCl buffer pH 8.5 containing 0.1 M Galactose and 1 M NaCl.
Determination of the isoelectric point
The isoelectric point of the hu anti-HIV-1-mAb was determined by a described method. Pharmalytes were used as carrier ampholytes. The whole procedure was carried out according the recommendation of the Pharmacia company
(booklet: Isoelectric focusing, Pharmacia fine chemicals). Determination of subclass and light chains
Light chains and subclass were determined by ELISA. Specific anti human KAPPA-chain antibodies labelled with alkaline phosphatase or specific anti human Gl, G2, G3 and G4 antibodies labelled with Peroxidase were used.
Quality control of Ricin A-chain
Quality control tests are performed on the purified Ricin-A chain. Gradient SDS-polyacrylamide gel electrophoresis under reduced conditions shows the absence of any contaminating material. Only one band at 33 and one at 30 kilo dalton respectively can be detected.
Conjugation of A-chain toxin with monoclonal antibody
5 mg of purified monoclonal antibody (1-2 mg/ml in PBS) were reacted with a 10-fold molar excess of SPDP (Pharmacia), dissolved at 1 mg/ml in dimethylformamide for 30 min at room temperature. PDP-substituted antibody was desalted by gelfiltration using Sephadex G-25. The protein peak as determined continuously at 280 nm was collected and placed on ice. Five mg of ricin A chain were reduced with 5 mM DTT for 1 hour at room tempera▲ture and desalted on a column of Sephadex G-25. The column was equilibrated with PBS. The protein peak was collected and was immediately mixed with the cold PDP-substituted antibody. The mixture was rocked for 1 hour at 4°C and then diafiltrated at 4°C against 0.01 M sodium phosphate buffer containing 2.0 M sodium chloride. The bulk of the unconjugated A-chain was separated from the immunotoxin by gelchromatography on Sephacryl S200 HR. The column was equilibrated in 0.02 M sodium phosphate containing 3 M sodium chloride. The first peak, which contained the immunotoxin was pooled and affinity purified on an anti-human IgG-Sepharose 4B. The immunotoxin was eluted from the affinity column with 3.5 M ▴ magnesium chloride and extensively dialyzed against sodium chloride and then against PBS.
Immunotoxins (0.2-2 mg/ml) were concentrated by ultrafiltration using PTGC membranes to the end concentration necessary for testing. The final preparation was sterile filtered and stored in aliquots at -20°C. The immunotoxins were analyzed by SDS-PAGE under both reducing and nonreducing conditions. The substitution of the immunotoxin was determined by radioimmunoassay. An average substitution of 1-2 moles A-chain per mole antibody was observed.
Competitive EIA
Purified hu anti-HIV-mAb were conjugated with Peroxidase by a known method according to M.B. Wilson and P.K. Nakane
(1978) in "Immunoflourescence and related techniques", ed.
W. Knapp et al. Elsevier Netherlands.
A dilution series of hu anti-HIV-mAb (in PBS 1 % BSA and 0.1 % Triton X-100) was dispensed in flat bottomed microtiter wells coated with purified HIV envelope proteins incubated overnight at 4°C. After washing out unbound material the bound conjugate was determined by reaction with 1.4 phenylendiamine. The developed colour was measured at 492 nm. A calibration curve was blotted optical density versus dilution. The dilution of the half saturation is calculated from this calibration curve.
Hu anti-HIV-mAb at a dilution corresponding to the half maximum saturation was mixed with sera from patients suffering from AIDS or with sera from probands who were seropositiv determined by a conventional screening (ELISA) followed by a confirmation test (westernblot). The mixture of the conjugate (at half maximal dilution) and the serum from the probands were incu▴bated in HIV envelope protein coated microtiter wells, washed and stained.
The optical density of these samples is compared to the anti HIV mAb at half maximum saturation. A value below the half maximum saturation indicates a competition between the human sera and the human monoclonal anti-HIV antibody.
SUBSTITUTE SHEET Neutralizing activity on the infectivity of HIV-1 virions
The assay for neutralizing activity of the hu anti-HIV-1-mAb was performed according to a known method (J. Virology, 61,2024-2028, 1987), Nature 316, 72-74, 1985; Biotechnology 5, 940-946, 1987). An HIV-1 dose equivalent to twenty times the amount to cause infection in 50 % inoculated cultures on day 12 (20 x TICD 20) was used to infect the H9-cell line. Cells were collected 3,6,9 and 12 days after virus inoculation and percentage of infection was determined by immunofluorescence. 100 % infection was observed between day 6 and 9. The neutralizing effect was also tested in the presence of the monoclonal antibody at variable dilutions. The neutralizing activity of the 3D6 antibody was evaluated by addition of a single dose of antibody (final concentration lμg/ml) immediately after virus inoculation. The infection rate was expressed as percent of infected cells.
Cytotoxicity tests
The test cell line used in the cytotoxicity tests were HIV1 infected H9. cells . HIV-1 infected H9 cells were prepared as described under Neutralizing activity on the infectivity of HIV-1 virions. Uninfected H9 cells were used as negative control. Cells harvested after a period of 6-9 days after virus inoculation were suspended in 1 nil medium. Various dilutions of the hu mAb-toxin conjugate were added to the infected cells and the negative control. After incuba▴tion for 24 hours at 37°C cells were washed with PBS and methionine free medium supplemented with 35S-methionine was added. Cells were incubated for 2 hours at 37°C, then the medium was removed by centrifugation and the cells transferred to a glassfiber filter and washed three times with 10 % trichloracetic acid, containing 1 mg/ml methionine. The cells were air dried on the filter and then transferred into a scintillation fluid. Radioactivity was counted in a scintillation counter. Cytotoxicity was expressed as the tissue culture inhibitory dose of the conjugate that resulted in 50 % of control untreated protein synthesis (TCID50).
EXPERIMENTAL RESULTS
A variety of experiments are shown to serve as examples for illustrating the present invention and its utilization in the following embodiments.
The human origin, the biochemical properties, the immunochemical characterization, the in vitro and in vivo behaviour are demonstrated by representative results.
Generation of clones and human origin of the monoclonal antibodies
A variety of clones was obtained by the fusion of peripheral blood lymphocytes and the fusion cell line. The specificity of 11 hybridomas is shown in table I.
Tab. I: Characterization of 11 hybridomas
Hybridoma Specifity of mAb isotype light No. test by immunoblotting and subclass chain - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
3D6 gp41, gp120 G1 KAPPA
3D9 gp41 G n.d.
24G3 gp160 G n.d.
25C2 gp120 G n.d.
54E7 gp41, gp160 M n.d.
81C7 gp160 M n.d.
3A6 p24 G n.d.
19H2 p24 G n.d.
23H7 p24 G n.d.
37G12 p55 G n.d.
42B9 p55, p65 G n.d. n.d. = not determined
The hybridoma No. 3D6 is deposited in the ECACC, Porton Down, Salisbury, UK under the deposition No. 87110301 (3rd November 1987). The hu mAb anti HIV-1 produced by this hybridoma line (3D6) is used as example to illustrate the present invention. Immunoblots of the 3D6 antibody are shown in Fig. la,b and c. Immunoblots of the 3A6, 19H2, 23H7, 37G12 and 42B9 antibodies are shown in fig. Id:
Figure imgf000014_0001
Fig. la: Immunoblot of the human mAb produced by Hybridoma No 3D6. HIV envelope proteins were prepared from native virons by SDS and 2-mercaptoethanol treatment.
The human origin of the monoclonal antibody is shown by the hybridization of 32P-labelled plasmid D Blur8. Autoradiographs are shown in fig. 2 with the DNA of the hybridoma cells. Also the reaction with anti human IgG and human IgM antisera and the more specific sera for subclass determination verifies the human origin of the monoclonal antibodies.
Figure imgf000015_0001
Fig. lb: Immunoblot of the human anti HIV-mAb produced by Hybridoma 3D6 HIV envelope proteins were prepared from genetically engineered E. coli (according to a known procedure, L.H. Costing et al.: Journal of Clinical Microbiology 25,1987 (845-848)).
Figure imgf000016_0001
Fig. 1c: Immunoblot of the human anti HIV mAb produced by Hybridoma 3D6. A commercially available blot strip (Du Pont Lot. : 70 44 128) was used.
Figure imgf000016_0002
Fig. Id: Immunoblot of anti HIV mab produced by the hybridoma 3A6(1), 19H2(2), 23H7 (3), 37G(12) and 42B9(4). A purified virus were electrophoretically separated and blotted.
Figure imgf000017_0001
Fig. 2: Detection of human DNA by hybridization, a) 3D5; b) 3D9; c) 24G3; d) BJA-B (human lymphoid cell line positive control; e) P3X63Ag/653 (murine myeloma negative control; f) BUBTU (human bladder carcinoma positive control.
Biochemical properties and immunochemical characterization of the antibody The isotype of the monoclonal antibody produced by Hybridoma No. 3D6 is G. The subclass determined by ELISA is G1. The isoelectric point, determined by isoelectric focusing using the Pharmacia the immobiline system is in the range of pH 8.6. A summary of the results is also shown in table I Immunoflourescence
Fixed HIV infected cells were incubated with the 3D6 antibody. Specific flourescence caused by the marker conjugate anti human IgG-FITC is shown in Fig. 3.
Figure imgf000018_0001
Figure imgf000018_0002
Fig. 3: Immunoflourescence of fixed HIV-1 infected H9- cells imaged with a sandwich of 3D6-antibody and anti human IgG conjugated with FITC. The clone 3D6 and 3D5 are shown.
In vitro behaviour of the monoclonal antibodies and related immunotoxins
Neutralizing activity
In the presence of up to 1 μg/ml 3D6 antibody H9 cells could not be infected by 20 times TCID50 of HIV virons. The neutralizing activity is shown in Fig. 4.
Figure imgf000019_0001
Fig. 4: Neutralizing activity of 3D6-antibody expressed as percent of infected cells. Infected cells were shown by immunflourescence using 3D6 antibody.
9 days after virus inoculation with 20 time TCID50 0.1 μg/ml 3D6 antibody could prevent HIV infection in H9 cells. The neutralizing activity of the 3A6 antibody is shown in fig.5: A 6 day delay could be observed.The infection rate was determined by immunofluorescence
Figure imgf000020_0001
Cytotoxicity
The monoclonal antibody (3D6) covalently linked with the Ricin-A-chain was used to demonstrate the cytotoxicity of the antibody. The cytotoxicity was measured by 35s- methionine uptake.The immunotoxin consisting of a conjugate between 3D6 antibody and Ricin A-chain, prepared as described under materials and methods killed specifically HIV-1 infected H9 cells. The 35s-methionine uptake is expressed in percent of control (Fig. 6).
Figure imgf000021_0001
Fig. 6: Inhibition of proteinsynthesis measured by 35smethionine uptake was taken as measure for cytotoxicity On the basis of assuming a molecular weight of 180000 dalton the molar concentration of the immunotoxin was calculated.
The TCID50 of the immunotoxin (3D6) -Ricin-A-chain) is less than 10 nm. Comparison of the hu anti-HIV-mAb with naturally occuring antibodies from seropositives and patients suffering from AIDS or pre AIDS
For the comparison of the claimed monoclonal antibodies with naturally occuring antibodies in men after a HIV infection a competitive EIA (Enzyme immunoassay) was choosen. The 3D6 monoclonal antibody was conjugated with peroxidase and the dilution of the half maximal saturation was determined. The 3D6 antibody at a dilution of half maximal saturation was mixed with different sera from seropositives without clinical manifestation of AIDS and with- sera from patients suffering from AIDS or pre AIDS in a ratio of 1:1.The competition between the 3D6 antibody and the natural occuring antibodies in blood are expressed in % of half maximal saturation.
The half maximal saturation by the 3D6 antibody is taken as 100 %. Table II shows the values from competitive EIA using a 3D6-antibody peroxidase conjugate and different sera. Table II: Comparision between 3D6 antibody and sera from seropositives and patients suffering from AIDS or pre AIDS.
------------------------------------------------------------ Patient No % Percentage of Clinical manifestation or propand half maximal of AIDS (1) pre AIDS
(2) saturation seropositive proband
(3) ------------------------------------------------------------
1 96 3
2 91 3
3 84 3
4 88 3 5 92 3
6 50 3
7 94 3
8 98 3
9 96 3 Table II continued
10 85 2
11 85 3
12 88 3
13 95 2
14 97 2
15 88 3
16 88 3 17 94 3
18 92 3
19 96 1
20 25 3
21 96 3 22 94 3
23 94 3
24 90 1
25 82 2
26 88 3 27 94 3
29 94 3
30 96 1
31 82 3
32 86 2 33 80 3
34 48 3
35 84 3
36 92 3
37 94 2 38 98 3
39 98 3
40 96 3
41 88 3
42 90 3 43 94 2
44 92 3
45 86 3
46 84 3
47 92 1 Table II continued
48 94 3 49 87 3 50 96 2
Figure imgf000024_0001
Figure.7: Comparison between 3D6 antibody and sera from seropositives and patients suffering from AIDS or pre AIDS. The results according table II are shown in this figure
Peptide analysis
Peptide C and D reacted with the 3D6 antibody. A similar repetitive sequence is also found in gp 120. This is the reason for reaction with gp 41 and gp 120
commercially available EIA
The results of commercially available EIA are shown in figure 8. In all EIA a positive result was obtained, except using the Abott ELISA. The Organon (2) assay is a competitive EIA.
Figure imgf000025_0001

Claims

We claim:
1) A human monoclonal antibody characterized in that it
a) binds selectively to envelope proteins of HIV 1 b) binds selectively to HIV 1 infected cells
2) A human monoclonal antibody characterized in that it
a) binds selectively to core proteins of HIV-1 b) Binds selectively to HIV-1 infected cells
3) A human monoclonal antibody characterized in that it
a) binds selectively to envelope proteins of HIV-1 (HTLV III/LAV), b) binds selectively to HIV-1 (HTLV III/LAV) infected cells, c) when conjugated to ricin A-chain, exhibits a TCID 50 % against HIV-1 infected H-9 cells of less then 10 nM, d) has a G or M isotype e) shows neutralizing activity on HIV-1 virons.
4) A human monoclonal antibody characterized in that it
a) binds selectively to core proteins of HIV-1 b) binds selectively to HIV-1 (HTLV III/LAV) infected cells, c) Binds selectively to p24 or p55 or p65 d) when conjugated to ricin A-chain, exhibits a TCID50 against HIV-1 infected H-9 cells of less then 10 nM, e) has a G or M isotype f) shows neutralizing activity on HIV-1 virons.
5) A human monoclonal antibody according to claim 1 characterized in that it is one of the following antibodies produced by hybridoma
a) 3D6 e) 54E7 b) 3D9 f) 81C7 c) 24G3 d) 25C2 2 g) monoclonal antibodies that are functionally equivalent to a member of said group.
6) A human monoclonal antibody according to claim 2 and 4 characterized in that it is one of the following antibodies produced by hybridoma a) 3A6 b) 19H2 c) 23H7 d) 37G12 e) 42B9 g) monoclonal antibodies that are functionally equivalent to a member of said group.
7) A monoclonal antibody according to claim 1 characterized in that it binds to an envelope protein of HIV-1
a) gp 160, b) gp 120 and c) gp 41 and d) HIV-1 infected cells.
8) A monoclonal antibody according to claim 2 characterized in that it binds to a core protein of HIV-1 a) p24 b) p55 c) p65 d) HIV-1 infected cells
9) A mouse x human hybridoma characterized in that it produces a human monoclonal antibody according to claim 1 and progeny of said hybridomas.
10) A mouse x human hybridoma characterized in that it produces a human monoclonal antibody according to claim 1 and 3 and progeny of said hybridomas. 11) A mouse x human hybridoma characterized in that it produces a human monoclonal antibody according to claim 2 and progeny of said hybridomas.
12) A mouse x human hybridoma characterized in that it produces a human monoclonal antibody according to claim 2 and 4 and progeny of said hybridomas.
13) An immunotoxin according characterized in that it is a conjugate of the antibody acorrding claim 1 or3 and a cytotoxic moiety
14) An immunotoxin according to claim 13 characterized in that the cytotoxic moiety of said immunotoxin is the A -chain of ricin.
15) An immunotoxin according to claim 13 characterized in that the cytotoxic moiety of said immunotoxin is the A -chain of abrin.
16) An immunotoxin according to claim 13 characterized in that the cytotoxic moiety of said immunotoxin is the A-chain of the diphteria toxin.
17) An immunotoxin characterized in that it is a conjugate of an antibody according to claim 7 and a cytotoxic moiety.
18) A cytotoxic cocktail comprising immunotoxin according to claim 14 and claim 15.
19) A method for preventing the HIV-1 infection of cells in human hosts by administering a prophylactic dose of the human monoclonal antibody according to claim 1.
20) A method for preventing the HIV-1 infection of cells in human hosts by administering a cockail of at least two different human antibodies according to claim 1 and 2. 21) A method for the treatment of HIV-lnfected cells comprisingadministering a therapeutic dose of the antibody to claim 1 to a human HIV-lnfected cell host.
22) A method for the treatment of HIV-lnfected cells comprising administering a therapeutic dose of an immunotoxin according to claim 14 ,15,16, and 17 to a human HIV-lnfected cell host.
23) A method for the treatment of HIV-lnfected cells by adiministering a therapeutic dose of said cytotoxic cocktail according to claim 18 to a human HIV-lnfected cell host.
24) A method for the treatment of human HIV-1 infected cells comprising administering approximately 0.01 to 20 milligrams of said immunotoxin according to claim 14,15,16, and 17 to a human HIV-lnfected cell host per kilogram of host body weight.
25) A method for the treatment of human HIV-1 infected cells comprising administering of said immunotoxin according to claim 17 to a HIV-1 infected cell host wherein the total dose of all immunotoxins is in the approximate range of 0.01 to 20 milligram per kilogram of host body weight.
26) An immunotoxin according characterized in that it is a conjugate of the antibody acorrding claim 2 or 4 and a cytotoxic moiety
27) An immunotoxin according to claim 26 characterized in that the cytotoxic moiety of said immunotoxin is the A
-chain of ricin.
28) An immunotoxin according to claim 26 characterized in that the cytotoxic moiety of said immunotoxin is the A -chain of abrin.
29) An immunotoxin according to claim 26 characterized in that the cytotoxic moiety of said immunotoxin is the A-chain of the diphteria toxin.
30) An immunotoxin characterized in that it is a conjugate of an antibody according to claim 8 and a cytotoxic moiety.
31) A cytotoxic cocktail comprising immunotoxin according to claim 27 and claim 28.
32) A method for preventing the HIV-l infection of cells in human hosts by administering a prophylactic dose of the human monoclonal antibody according to claim 2.
33) A method for preventing the HIV-l infection of cells in human hosts by administering a cockail of at least two different human antibodies according to claim 2 and
4.
34) A method for the treatment of HIV-lnfected cells comprising administering a therapeutic dose of the antibody to claim 2 to a human HIV-lnfected cell host.
35) A method for the treatment of HIV-lnfected cells comprising administering a therapeutic dose of an immunotoxin according to claim 27,28, 29,and 30 to a human HIV-lnfected cell host.
36) A method for the treatment of HIV-lnfected cells by adiministering a therapeutic dose of said cytotoxic cocktail according to claim 31 to a human HIV-lnfected cell host.
37) A method for the treatment of human HIV-1 infected cells comprising administering approximately 0.01 to 20 milligrams of said immunotoxin according to claim 27,28, 29,and 30 to a human HIV-lnfected cell host per kilogram of host body weight.
38) A method for the treatment of human HIV-1 infected cells comprising administering of said immunotoxin according to claim 31 to a HIV-1 infected cell host wherein the total dose of all immunotoxins is in the approximate range of 0.01 to 20 milligram per kilogram of host body weight.
39) A method for specific detection of HIV-1 infected cells in human blood or tissue culture fluid comprising at least one of said antibodies according to claim 1
40) A method for specific detection of HIV-1 infected cells in human blood or tissue culture fluid comprising at least one of said antibodies according to claim 2
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO1993000367A1 (en) * 1991-06-21 1993-01-07 Hafslund Nycomed As Abrin variants and immunnotoxins
US5558865A (en) * 1991-08-22 1996-09-24 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
WO1996033219A1 (en) * 1995-04-19 1996-10-24 Polymun Scientific Immunbiologische Forschung Gmbh Monoclonal antibodies against hiv-1 and vaccines made thereof
US5935579A (en) * 1990-09-25 1999-08-10 Retroscreen Limited AIDS therapy and vaccine
US7744887B2 (en) 2004-06-01 2010-06-29 Merck & Co., Inc. Human antibodies interacting with HIV gp41

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0251612A2 (en) * 1986-06-23 1988-01-07 Bristol-Myers Squibb Company Human monoclonal antibody to lymphadenopathy-associated virus

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5087557A (en) * 1986-06-23 1992-02-11 Genetic Systems Corporation Human monoclonal antibody to lymphadenopathy-associated virus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0251612A2 (en) * 1986-06-23 1988-01-07 Bristol-Myers Squibb Company Human monoclonal antibody to lymphadenopathy-associated virus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biochemical and Biophysical Research Communications, vol. 155, no. 3, 30 September 1988, Academic Press (US)T. Sugano et al.: "Human monoclonal antibody against glycoproteins of human immunodeficiency virus", pages 1105-1112 *
Interferon Y Biotecnologia", vol. 4, no. 3, 1987; R.Grunow et al.:"Obtencion de un heteromieloma de cultivo (CB-F7) con alta eficiencia para la inmortalizacion de cèlulas B." pages 261-264 *
Journal of Immunological Methods, vol. 106, 10 February 1988 Elsevier Science Publishers B.V. (NL) R. Grunow et al.: "The high efficiency, human B cell immortalizing heteromyeloma CB-F7", pages 257-265 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0517815A1 (en) * 1990-02-26 1992-12-16 Roger Williams General Hospital AN IgG-1 HUMAN MONOCLONAL ANTIBODY REACTIVE WITH AN HIV-1 GLYCOPROTEIN AND METHOD OF USE
EP0517815A4 (en) * 1990-02-26 1993-09-22 Roger Williams General Hospital An igg-1 human monoclonal antibody reactive with an hiv-1 glycoprotein and method of use
WO1991018983A1 (en) * 1990-05-29 1991-12-12 Alois Jungbauer Recombinant protein which binds to a complex viral antigen of hiv-1
AT396939B (en) * 1990-05-29 1993-12-27 Alois Dipl Ing Dr Jungbauer COMPLEX VIRAL ANTIQUE OF HIV-1 BINDING RECOMBINANT PROTEIN
US5935579A (en) * 1990-09-25 1999-08-10 Retroscreen Limited AIDS therapy and vaccine
WO1993000367A1 (en) * 1991-06-21 1993-01-07 Hafslund Nycomed As Abrin variants and immunnotoxins
US5558865A (en) * 1991-08-22 1996-09-24 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
WO1996033219A1 (en) * 1995-04-19 1996-10-24 Polymun Scientific Immunbiologische Forschung Gmbh Monoclonal antibodies against hiv-1 and vaccines made thereof
US7744887B2 (en) 2004-06-01 2010-06-29 Merck & Co., Inc. Human antibodies interacting with HIV gp41

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ATE135743T1 (en) 1996-04-15
JPH02502251A (en) 1990-07-26
EP0355140A1 (en) 1990-02-28
EP0355140B1 (en) 1996-03-20
DE3855134D1 (en) 1996-04-25
DE3855134T2 (en) 1996-10-02
US5753503A (en) 1998-05-19

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