EP0418347A1

EP0418347A1 - Human monoclonal antibodies to human immunodeficiency virus

Info

Publication number: EP0418347A1
Application number: EP90904509A
Authority: EP
Inventors: Susan Zolla-Pazner; Miroslaw K. Gorny
Original assignee: New York University NYU
Current assignee: New York University NYU
Priority date: 1989-02-28
Filing date: 1990-02-28
Publication date: 1991-03-27
Also published as: JPH04505099A; CA2028121A1; AU5225693A; AU5188590A; AU680836B2; EP0418347A4; WO1990009805A1; AU651794B2

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.The invention relates to eleven human lymphoblastoid cell lines producing monoclonal antibodies to the gp41 and p24 proteins of the human immunodeficiency virus (HIV). The invention also provides methods of treating individuals with HIV using human monoclonal antibodies, as well as pharmaceutical preparations comprising the human monoclonal antibodies in effective amounts.

Description

HUMAN MONOCLONAL ANTIBODIES TO HUMAN IMMUNODEFICIENCY VIRUS BACKGROUND OF THE INVENTION

The government has rights to this invention by virtu of funding from grant AI-72658 from the National Institutes o Health.

The human immunodeficiency virus (HIV) has bee implicated as the causative agent of acquired immune deficienc syndrome (AIDS). 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. ,

Several avenues have been explored to treat individua afflicted with AIDS or HIV infections. The antiviral dr azidothymidine (AZT) has been found to produce both clinic and ixαmunological improvements upon short term administrati to patients afflicted with AIDS and ARC (AIDS Related Comple a ,prodrome of the disease) and to decrease the mortality ra and frequency of opportunistic infections. Although 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.

An alternative treatment that is currently under evaluation involves administration of one or more lymphokines. Interferon (particularly gamma-interferons) and interleukin-2 are currently being studied for possible use in the treatment of HIV infections. However, 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. It has been proposed to use 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. Essex, issued February 16, 1988 as is its use in the treatment and diagnosis of AIDS. The identification of p24 has been described in Allan, J.S. et al.. Science 228; 1091, 1985. However, the use of monoclonal antibodies for the treatment of HIV infections has been hampered because only a limited number of murine and rat monoclonal antibodies to HIV proteins are available. In addition, none of the currently available monoclonal antibodies directed against HIV proteins are of human origin. Administra- tion of murine antibodies to humans can cause dangerous life threatening immunologic reactions, and such antibodies may not be effective in binding to the target HIV proteins in humans.

Stable human cell lines which produce monoclonal antibodies directed against HIV would be useful for treatin and/or diagnosing individuals infected with the virus. However, 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. Amongst th explanations for this problem are: (a) the most availabl source of lymphocytes from humans, the peripheral blood, normally contains few antibody producing cells and in som instances, none at all; (b) transformation of antibody produc ing cells can be achieved using Epstein-Barr virus (EBV) , bu the level of antibody production by these transformed cells i often low and unstable; (c) stability of antibody productio can be enhanced, as can levels of antibody production, b fusion of EBV-transformed lines to mouse myeloma cells but these 'heterohybridomas' readily delete human chromosomes an immunoglobulin production is often lost; and (d) fusion o normal or transformed B cells to human lymphoblastoid lines o to heteromyelomas stabilizes antibody production but, unti recently, few satisfactory parent lines of this cell type hav been available.

OBJECTS OF THE INVENTION

It is an object of the present invention to provid human lymphoblastoid cell lines producing monoclonal antibodie directed against protein components of HIV.

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.

These and other objects of the present invention wil be apparent to those of ordinary skill in the art in light the following specification.

SUMMARY OF THE INVENTION

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.

In a further aspect, the present invention comprises pharmaceutical formulations comprising an effective amount of a human monoclonal antibody to HIV proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

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 (a-g) 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

All literature references and patents cited in thi specification are hereby incorporated by reference in thei entirety. The present inventors have isolated fourteen nove stable human lymphoblastoid cell lines producing monoclona antibodies directed against HIV encoded proteins. The an tibodies of the invention are useful in the prophylaxis of HI and in the diagnosis, and treatment of humans suffering fro HIV infections. These human monoclonal antibodies are directe against HIV protein gp41 and protein p24 (and the precursor and decomposition products of such proteins) . 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 σaσ 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. Administered to humans, the antibodies ca provide passive immunization of HIV-infected individuals. I addition, 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 imπtunotoxins (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. As employed herein the term "immunotoxin" refers to a conjugate of an antibody with one or more toxins, drugs, radionuclides or cytotoxic agents. Among the 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. In a typical treatment employing the huma monoclonal antibodies of the present invention as immunotoxins, the antibody (which binds to a protein that is expressed b HIV-infected cells) is conjugated to a toxin (e.g. ricin) tha is toxic to the HIV-infected cell (and to non-infected cells a well). By coupling the cytotoxic agent to the antibody, a hig level of toxic efficacy can be achieved against the target cel with a markedly lower level of non-specific toxicity. The us of the toxic agent is possible because 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. Initially, 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) .

Approximately 9% of the 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.

In a second round of immortalization/selection 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.

In addition, 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 characteristics of the human monoclonal antibodie produced by the lymphoblastoid cell lines of the presen invention are described in Table III of Example 5 below.

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).

All of the human monoclonal antibodies of the presen invention directed against gp41 (i.e. 50-69, 98-6, 98-43, 120 16, 126-6, 126-50, 167-7 and 191-3) mediated antibody dependen cellular cytotoxicity (ADCC) as shown in Table III below. Thi is a most important finding in that serotherapy (e.g. passiv immunization) and seroprophylaxis in animal retroviral model have been shown to be mediated by ADCC (Plata, F. et al.. Cel .48.* 231, 1987; Weinhold, K.J. et al, i. Natl. Cancer Inst. 75 717, 1985). 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).

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. employed in this specification, the term "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 (produci human monoclonal antibodies directed against p24 and gp4 respectively) have been deposited with the American Ty Culture (ATCC, Rockville, MD) and have received Accessi Numbers CRL 10038 and CRL 10037, respectively.

The classification of the antibodies of the inventi into an immunodominant or non-dominant grouping was a complished by inhibition testing as described in Example below. In this assay, sera collected from HIV serqpositi individuals were used to inhibit the binding of biotinylat monoclonal antibodies directed against HIV proteins to the respective antigens.

Of the monoclonal antibodies to p24 and gp41, on those to gp41 mediated antibody-dependent cellular cytotoxici (ADCC). In vitro cytotoxicity studies revealed that the mo active induced significant levels of killing was at minim concentrations of 15-250 ng Ab/ml. ADCC was assayed usi peripheral blood mononuclear cells (MNC) as effectors and the CEM.NKR cell line, infected with HTLV-IIIB, MN and RF (viral strains) , as targets.

Two monoclonal antibodies to gp41 and one monoclonal antibody to p24 were purified and coupled to the deglycosylated A chain of ricin. 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.

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. The pharmaceutical formulations 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. In addi tion, 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.

The present invention is described below in specifi working examples which are intended to illustrate the inventio without limiting its scope.

EXAMPLE 1: IMMORTALIZATION OF HUMAN B-CELLS

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). The diseas status of patients was established on the basis of an i munologic staging system as described by Zolla-Pazner et a (Proc. Nat. Acad. Sci. USA 84: 5404, 1987):

Scale Score T4/T8 ratio #T4/mm #lymphocytes/mm

0 > 1.0 > 500 > 1500

1 < 1.0 > 500 > 1500

2 < 1.0 < 500 > 1500

3 < 1.0 < 500 < 1500

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.

EXAMPLE 2: ISOLATION AND SCREENING OF LYMPHOBLASTOID

CELL LINES FOR ANTI-HIV ANTIBODY PRODUCTION After screening for anti-HIV antibody production by non-commercial ELISA (see below), positive cultures wer expanded into wells of 24-well tissue culture plates (Costar) and cultured for two more weeks. All initial and expande cultures were fed at weekly intervals with complete medium. Cultures with supernatants showing specific reactivity to HI were then subcultured one to two times at doubling dilution o feeder layers of irradiated GK-5 human lymphoblastoid cell

(derived from a variant of GM1500; Satoh, J. et al., _j_ Engl

J. Med. 309: 217, 1983) which had been exposed to 3000 Rads o gamma-radiation. Stable clones were then subcultured one t three times at 10 to 100 cells per well on feeder cells an then expanded into flasks.

Thus, initial cultures of immortalized B-cell (hereinafter referred to as lymphoblastoid cell lines) wer 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) wer then added and incubated for 90 minutes at 37°C. Subsequently plates were washed with PBS-Tween and incubated with goat anti human immunoglobulin conjugated to alkaline phosphatas (Organon Teknika-Cappel, Malvern, PA) for another 90 minutes a 37°C. The plates were washed again with PBS-Tween and th substrate, p-nitrophenyl phosphate in 10% diethanolamine, wa added for 30 minutes. The reaction was terminated with 2 microliters of IN NaOH and the absorbance was read at 405 nm i an automated ELISA reader (MR 600 Microplate Reader, Dynatech)

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

Organon Teknika Cappel) which were then coated with bovin serum albumin (BSA, Sigma Chemical Co. ) by incubating the bead in 1.25% BSA diluted in PBS for 1 hour at room temperature Reactivity with HIV-coated beads, but not with BSA-coate beads, was used as a criterion for specificity. Furthe analysis of the specificity of the monoclonal antibodies wa then carried out by Western blot using a commercially-availabl kit (Bio-Rad, Richmond, CA) and by radioimmunoprecipitatio (RIP). RIP assays were carried out using the method of Pinte and Honnen {d _ Immunol. Methods, in press). Briefly, 3 micrograms of HTLV-III_B lysate (purchased from Organon

125 Teknika), was labeled with [I] using the Bolton-Hunte reagent (New England Nuclear, Boston, MA) . Bound label wa separated from free label on a Bio-Gel P-4 column (Bio-Rad)

Fifty microliters of culture supernatant were incubated with x 10^ cpm of the labeled lysate for 1 hour at 37°C, then 5 microliters of 10% fixed Staphlococcus aureus (Pansorbi

Calbiochem, La Jolla, CA) was added. The immunoprecipitate w washed three times by centrifugation and the air-dried pell was resuspended in buffer, boiled for 3 minutes an electrophoresed on a 10% SDS polyacrylamide gel. The gels wer dried and exposed for one to three days to X-Omat S fil (Kodak, Rochester, NY) . The class and light chain type of anti-HIV antibody was determined by ELISA. For these assays, microtiter plates (Immulon 2) were coated with 4 micrograms/ml of HIV lysate (Electro-Nucleonics) and then incubated with culture super¬ natants. 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 wer 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 wer 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

QUANTITATIVE RESULTS OF THE PROCEDURE USED TO

PRODUCE HUMAN MONOCLONAL ANTIBODIES TO HIV

No. of % positiv wells wells

Infection of PBMC with EBV 14,329 (100)

4 weeks

V Screen for anti-HIV by Non-commercial ELISA 1,290 9.0

Expand positive wells

2 weeks

V Screen for anti-HIV by: Non-commercial ELISA 573 4 . 0

Commercial ELISA 340 2 .4

Screen for specificity (reactive HIV, unreactive with BSA) 97 0.67

Screen for reactivity by RIP 57 0.40

Subculture positive wells by doubling dilution (10,000-10 cells/well)

4-6 weeks

V

Screen by commercial ELISA 16* 0.11*

Subculture positive wells at (100 and 10 cells/well)

4-6 weeks

V

Screen by commercial ELISA 0.05*

No. and % of positive plates from subcultures of individu wells which contain at least one antibody positive well. After four weeks, 9% of the wells displayed antibody production as revealed by the non-commercial ELISA (Table II). After expansion, only 2.4% of the original cultures continued to produce antibody reactive with a commercial ELISA kit and only one-quarter of these (0.67% of the original 14,329 cultures) were producing antibodies which reacted specifically with HIV.

To determine whether the severity of disease in the cell donor affected the number of cultures able to produce antibodies and the specificity of antibody produced, 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.

TABLE II

CHARACTERISTICS OF CELL CULTURES DERIVED FROM PATIENTS AT DIFFERENT STAGES OF HIV INFECTION

No. of positive wells No. of

(Includes wells HIV-specific with HIV- No. of

Scale No. of No. of & non-specific specific clumps

Score patients wells reactivity) antibody⁵ per well

HIV-seronegative patients:

3 637 0 0 7.3

HIV-seropositive patients:

0 4 725 18 (2.4%) 6 (0.8%) 0.92

1 13 8,789 180 (2%) 66 (0.7%) 0.88

2 20 2,792 54 (1.9%) 16 (0.5%) 0.65

3 21 2,023 88 (4.3%) 9 (0.4%) 0.22

58 14,329 340 2.4% 97 0.67% 0.66

* Specificity of anti-HIV antibodies were assessed by commer cial ELISA using HIV-coated and BSA-coated beads.^' + B cells transformed by EBV stick together and creat characteristic clumps of cells which were quantitate microscopically.

The results, shown in Table II, revealed that culture obtained from patients with a scale score of 3 (severe im munodeficiency) gave a higher percentage of antibody producin wells than patients with lesser scale scores. However, onl 10% (9/88) of reactive wells from the cells of stage 3 patient were specifically reactive with HIV whereas 30-37% of well from cells of patients with scale scores of 0-2 were specifi cally reactive with HIV. Thus, after six weeks of culture, cells from patients with lower scale scores produced a highe percentage of wells containing HIV-specific antibody.

Analysis of antibodies from ELISA-positive expande cultures was carried out by RIP. Only 59% of supernatants fro these cultures were also positive on RIP analysis. RI analysis demonstrated that, out of 57 supernatants, 44 showe reactivity to env-encoded proteins, 11 to gag proteins and 2 t reverse transcriptase.

Therefore, specific lymphoblastoid cell lines wer isolated and further cloned as described below.

EXAMPLE 3: SPECIFICITY AND REACTIVITY OF THE HUMAN MONOCLONA

ANTIBODIES OF A SUBSET OF THE PRESENT INVENTION

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. In Figur 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 ejήv-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). By RIP, antibodies from cell lines 71-31, 91-5 and 91-6 reacted with p24. Antibodies from 98-4.9 wer 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).

In Figure 2, Western Blot strips were used to show th reactivity of serum from a normal control (lane 1) , an HIV infected subject (lane 2), and of supernatant from cell line 71-31 (lane 3), 91-51 (lane 4), 91-6 (lane 5), and 98-4.9 (lan 6) . Western blot analysis showed all 3 monoclonal antibodie reacted with p24 and with known gag precursors p55 and p40 All four antibodies also reacted with a breakdown product o p24 which migrated with a mobility of approximately 22 kilodal tons (kd) ; three of the four anti-gag monoclonal antibodie also reacted with additional intermediate precursors which wer noted with a mouse monoclonal antibody and which had mobilit of approximately 37, 31 and 28 kd.

Studies of the growth characteristics and level o antibody production of each line was performed. Each cell lin was initially cultured in replicate wells at 0.5 x 10^ cells/m for 1-8 days. The number of cells and the amount of im munoglobulin produced is shown in Figure 3. In Figure 3, a each time point, separate wells were used to test for viabl cell number (closed circles) and for secreted human IgG (ope circles). Cell lines studied were 50-69(a), 98.6(b), 98-43(c) 71-31(d), 91-5(e), 91-6(f) and 98-4.9(g). Peak cell density was noted at 4 days with maximu densities of 1.0 to 2.4 x 10° cells/ml. The doubling time o cells in log phase ranged from 40-61 hours. The concentratio of immunoglobulin produced varied widely, generally peaking at day 5 of culture and ranging from 9-112 micrograms/ml.

It should be noted that cell line 98-4.9 recently lost the ability to produce human monoclonal antibodies.

EXAMPLE 4: GENERATION OF ADDITIONAL LYMPHOBLASTOID CELL LINES

PRODUCING HUMAN MONOCLONAL ANTIBODIES TO HIV

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.

Each of the monoclonal antibodies of this Example were tested for their specificities by means of commercial ELISA (the supernatants were reactive with HIV-coated beads and unreactive with BSA-coated beads), by radioimmunoprecipitation and by Western Blot as in Example 3 above. The characteristics of these human monoclonal antibodies are shown in Table III below.

EXAMPLE 5: CHARACTERIZATION OF THE HUMAN MONOCLONAL

ANTIBODIES OF THE PRESENT INVENTION The specificity and biological activities of the human monoclonal antibodies of the present invention are summarized in Table III below. TABLE III Characteristics of human monoclonal antibodies against HIV

Cell line Isotype, RIP ELISA Western Blot Function Approx.

Subclass ENV9 PE3 pl21 r-gp41 r-p24 Neutr. Enhanc. ADCC Epitope Region and Light

8 Chain Type

50-69 IgG₂k¹ gp41 + + - _ - + 599 - 613²,^a

98-6 IgG₂k gp41 + + - - - + 642 - 692

98-43 IgG₂k gp41 + + - - - + 579 - 604² 120-16 IgG₂k gp41 + NT³ - - + + NT 126-6 IgG₂k gp41 + NT - - - + 567 - 649 126-50 IgG₂k gp41 + NT - - - + 567 - 649 167-7 igGii gp41 NT NT NT NT NT NT NT NT 661 - 683 194-1 IgG_jl gp41 NT NT NT NT NT NT NT NT NT

71-31 gGii⁴ p24 NT + - - - 131 - 198⁵

91-5 igGxi p24 NT + - - - 131 - 198

91-6 IgG_jl p24 NT + - - - 131 - 198

CM 98-4. ≠ IgG₃l p24 NT NT - - - NT

98-4.3 igGii p24 NT NT NT NT NT NT NT NT NT 134-F6 IgG₃k p24 NT NT NT NT NT NT NT NT NT

* k = Kappa

² The Modrow numbering system according to i. Virol. 61: 570, 1987.

³ NT = Not tested

⁴ 1 = Lambda

⁵ The numbering system according to Wain-Hobson, et al., Cell 40; 9, 1985

O 00 ON 6 This lymphoblastoid cell line has not been stabilized with respect to the production of monoclonal antibodies e ^a Tested for reactivity on LABSYSTEMS kit

I

In Table III, epitope mapping was performed usin recombinant antigens using a Western Blot format. Vira Neutralization and enhancement assays were performed as in J Clin. Micro. 26: 231, 1988. ADCC assays were performe according to Lyerly, H.K. et al. AIDS and Human Retroviruse 3.: 409-422, 1987. Western Blot analysis was performed usin recombinant p24 or gp41 (supplied by Organon-Teknica) .

Elisa's were performed using cloned gp41 antigens 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) .

As can be seen in Table III above, all of the anti-gp4 human monoclonal antibodies of the present invention were o the IgG serotype and mediated antibody dependent cellula cytotoxicity (ADCC).

In addition, epitope mapping showed that five of th anti-gp41 antibodies bound to different epitopes on the vira protein. Monoclonal antibody 50-69 bound to residues 599-613 monoclonal antibody 98-6 was directed against a peptid encompassing residues 642-692; monoclonal antibody 98-43 boun to a peptide encompassing gp41 residues 579-604; monoclona antibody 120-16 bound residues 644-663; and monoclonal antibod 116-7 bound to residues 661-683. The numbering system for th gp41 peptide is according to J . Virol. 61: 570, 1987.

All three of the anti-p24 monoclonal antibodies teste (71-31, 91-5 and 91-6) bound to a HIV p24 peptide encompassin residues (131-198 (the numbering system according to Wain Hobson et al. Cell 40: 9, 1987) (Table III).

Finally, none of the monoclonal antibodies of th present invention were able to neutralize the infectivity o HIV and one (120-16) enhanced viral pathogenicity.

EXAMPLE 6: INHIBITION TESTING OF THE MONOCLONAL

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^βC, 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. A predetermine dilution of biotinylated monoclonal anti-HIV antibodies (see below) was then added in a volume of 100 microliters and the plates incubated for 2 hours at 37°C. The wells were washe three times with PBS-Tween and the reaction developed by addin an avidin-biotinylated-horseradish peroxidase complex (Vecto Labs) and incubated for 2 hours at 37^βC. After washing 5 time with PBS-Tween, 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) was added as substrate and incubated for 30 minutes a room temperature. The optical density of each well was read i an ELISA reader at 410nm.

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.

The results of the inhibition tests are shown in Figur 4. In Figure 4, the seronegative sera is designated (-) an the seropositive (+).

The data presented in Figure 4 shows that antibodie 120-16 and 71-31 are directed against immunodominant epitope and non-dominant epitopes respectively. Thus, seropositive sera are able to compete for the binding of labeled antibody 120-16 to the HIV lysate defining the epitope of this monoclonal antibody as immunodominant. Non-dominance was established for the epitope with which 71-31 reacts as a result of the inability of most seropositive sera to compete with this antibody for binding to the HIV lysate.

Claims

WHAT IS CLAIMED IS: 1. A human monoclonal antibody directed against human immunodeficiency virus protein p24.

2. The human monoclonal antibody of claim 1, wherei said antibody is of the IgG serotype.

3. A human lymphoblastoid cell line producing a huma monoclonal antibody directed against human immunodeficienc virus protein p24.

4. The human lymphoblastoid cell line of claim derived from a human infected with human immunodeficienc virus.

5. A method for treating a mammal infected with huma immunodeficiency virus comprising administering to a mammal i need of such treatment an effective amount of a human monoclo nal antibody directed against HIV protein p24 and a phar maceutically-acceptable carrier.

6. The method of claim 5 wherein - said effectiv amount ranges between about 200 mg and 15 grams

7. The method of claim 5 comprising administerin said monoclonal antibody parenterally.

8. A pharmaceutical formulation for treating mammal infected with human immunodeficiency virus comprising a effective amount of a human monoclonal antibody directe against HIV protein p24.

9. The pharmaceutical formulation of claim 8 furthe comprising a pharmaceutically-acceptable carrier.

10. A human monoclonal antibody directed against huma immunodeficiency virus protein gp41, said monoclonal antibod having the ability to mediate antibody dependent cellular cytotoxicity.

11. The monoclonal antibody of claim 10 wherein said monoclonal antibody is of the IgG serotype.

12. A human lymphoblastoid cell line producing a human monoclonal antibody directed against human immunodeficiency virus protein gp41.

13. The human lymphoblastoid cell line of claim 12 derived from a human infected with human immunodeficiency virus.

14. A method for treating a mammal infected with human immunodeficiency virus comprising administering to a mammal in need of such treatment an effective amount of a human monoclo- nal antibody directed against HIV protein gp41, said monoclonal antibody having the ability to mediate antibody dependent cellular cytotoxicity.

15. The method of claim 14 wherein said antibody is of the IgG serotype.

16. The method of claim 14 wherein said effective amount ranges between about 200 mg and 15 grams

17. The method of claim 16 comprising administering said monoclonal antibody parenterally.

18. A pharmaceutical formulation for treating mammals infected with human immunodeficiency virus comprising an effective amount of a human monoclonal antibody directed against HIV protein gp41, said human monoclonal antibody having the ability to mediate antibody dependent cellular toxicity.

19. The pharmaceutical formulation of claim 18 further comprising a pharmaceutically-acceptable carrier.

20. A human monoclonal antibody having the charac- teristics of the monoclonal antibody produced by ATCC Accessio No. 10038.

21. A human monoclonal antibody having the charac- teristics of the monoclonal antibody produced by ATCC Accessio No. 10037.