CA2028121A1 - Human monoclonal antibodies to human immunodeficiency virus - Google Patents

Abstract

(57) Abstract Disclosed herein are eleven human lymphoblastoid cell lines producing monoclonal antibodies directed against human im-munodeficiency virus (HIV) proteins gp41 and p24. Also provided are methods for treating HIV-infected individuals using the human monoclonal antibodies and pharmaceutical formulations comprising effective amounts of the human monoclonal antib-odies.

Description

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~90~09805 PCT/US90/01132 ~UMAN MONO~QNA~ ANTI~ODIES TO HUMAN ~MMUNODEFICIENCY VIRUS
BACKGROy~D OF~ INVENTION
The goverr~ent has rights to this invention by virtue of funding from grant AI-72658 from the National Institutes of ~ealth.
The human immunodeficiency virus (aIv) has been implicated as the causative agent of acquired immune deficiency syndrome (AIDS). Two different serotype~ of the virus have been identified to date: ~IV-l and ~IV-2. It i~ currently believed that the majority of individuals that become infected with ~IV eventually will develop AIDS and are likely to succumb to fatal infections and/or malignancies. At this time it is estimated that approxi~tely l.5 million individuals have been infected by ~IV in the United States alone~
Several avenues have been explored to treat individu~
afflicted with AIDS or ~IV infections. The antiviral ~t ~
azidothymidine (AZT) has been found to produce both clinic.ll and immunological improvements upon short term administratio to patients afflicted with AIDS and ARC (AIDS Related Comp1e~-a prodrome of the disease) and iecrease the mortality r~
and frequency of opportunistic ~ctions. Although clinical benefitfi are achieved with A it is costly. A furtheL
d~8~b~ that s~ifica~t drug toxi~ity oft~n accompanies administration of AZT. This may necessitate blood transfusions and/or reduction of the AZT dosage, or in some instances, discontinuance of AZT therapy altogether. Nonetheless, AZT is 'O90/09805 PCTtUS90/01l32 the only drug currently authorized for the treatment of AIDS.
An alternative treatment that is currently under evaluation involves a~inistration 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. ~owever, the prelimlnary 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 ~IV proteins expressed in infected ind~viduals as therapeutic agents. These proteins are part of the virus particles and are expressed by ~IV
infected cells and are designated inter alia as p24 and gp41.
The identification and isolation of gp41 is described in ~.S.
Patent No. 4,725,66~ 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 ~IY infections has been hampered because only a limited number of murine and rat monoclonal antibodies to ~IV proteins are available. In addition, none of the currently available monoclonal antibodies directed against ~IV proteins are of human origin. Administra-tion of murine antibodies to humans can cause dangerous lifethreatening immunologic reactions, and such antibodies may not be effective in binding to the target ~IV proteins in humans.
Stable hum~n cell lines which produce monoclonal antibodies directed against ~IV would be useful for treating and/or diagnosing individuals infected with the virus.
~owever, human monoclonal antibodies and particularly those directed against ~IV have proven to be far more difficult to produce than those of either rat or mouse origin. Amongst the explanations for this problem are: (a) the most available source of lymphocytes from humans, the peripheral blood, normally contains few antibody producing cells and in some instances, none at all; (b) transformation of antibody produc-'090/09805 PCT/US90/01132 ing cells can be achieved using Epstein-Barr virus (E~V), but the level of antibody production hy these transformed cells is often low and unstable; ~c) stability of antibody production can be enhanced, as can levels of antibody production, by fusion of E~V-transformed lines to mouse myeloma cells but, these 'heterohybridomas' readily delete human chromosomes and immunoglobulin production i6 often lost; and (d) fusion of normal or transformed B cell5 to human lymphoblastoid lines or to heteromyelomas stabilizes antibody production but, until recently, few satisfactory parent lines of this cell type have been available.

OBJECTS OF THE INVENTION
It is an object of the present invention to provide human lymphoblastoid cell lines producing monoclonal antibodies directed against protein components of HIV.
Another object of the present invention is to provide diagnostic and therapeutic agents comprising human monoclonal antibodies directed against ~IV proteins which have a low non-specific toxicity for use in the diagnosis and treatment of in-dividuals infected with ~IV.
A further object of the present invention is to provide a method for treating individuals uffering from ~IV infections by administering human monoclonal antibodies directed against protein components of ~IV to said individual~
A still further object of the present invention is to provide pharmaceutical formulations for treating individuals suffering from ~IV infections.
These and other objects of the present invention will be apparent to those of ordinary skill in the art in light of the following specification.

SUMMARY OF THE INVENTION
The present inventors have discovered new monoclonal antibodies for the treatment, prophylaxis and diagnosis of human immunodeficiency virus (~IV) infections. These are human monoclonal antibodies directed against ~IV proteins gp4l and 6~ . d -- ~ ~
`'O90/09805 PCT/US90/01132 p24 which are expressed by infected cells. The human monoclo-nal antibodies of the present in~ention 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-~IV 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 ~IV proteins gp4l and p24. The invention also provides human monoclonal antibodies directed against ~IV proteins p24 and gp4l.
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 f ormulations comprising an effective amount of a human monoclonal antibody to ~IV proteins.

~RIEF_DESCRIPTION OF TBE DRAWINGS
Figure 1 is a radio-immunoprecipitation assay of l25tI]-labelled ~IV lysate with serum from an HIV-infected subject or with antibodies from a sub~et 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 ~IV.
Figure 4 is a graph showing the inhibition testing of monoclonal antibodies 120-l6 and 71-3l.

~090J09X~5 PCT/USgO/01132 DETAILED DESCRIPTION OF TH~ INVENT~ON
All literature references and patents cited in this specification are hereby incorporated by reference in their entirety.
The pre6ent inventor~ have isolated fourteen novel stable human lymphoblastoid cell lines producing monoclonal antibodies directed against ~IV encoded proteins. The an-tibodies of the invention are useful in the prophylaxis of ~IV
and in the diagnosis, and treatment of humans suffering from ~IV infections. These human monoclonal antibodie~ are directed against ~IV protein gp41 and protein p24 (and the precursors and decomposition products of such E~roteins).
gp41 is a viral membrane glycoprotein expressed on the surface of infected cells and is a product of the env gene of ~IV (as described in E~sex, M., U.S. Patent No. 4,725,669 i~sued February 16, 1988). p24 is a viral core protein and is a product of the HIV aag gene (as described in Allan, J.S. et al., supra~).
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 a patient has been exposed to, or infected with, ~IV. Example 6 below illustrate~ the use of the antibodies of the invention in a diagnostie as~ay. Admini~tered to human~, the antibodies can provide passive immunization of HIV-infected individuals. In addition, the antibodies of the invention can serve prophylac-tically for administration to non-infected, high-risk in-dividuals tsuch as health care workers who have been exposed via a needle stick to ~IV). The anti~odies of the invention also can serve as research tools for epitope mapping of ~IV
proteins gp41 and p24.
A particularly important use of the human monoclonal antibodies of the present invention is for administration to ~IV infected expectant mothers. All of the antibodies of the present invention are of the IgG serotype (see below)-. Since IgG's can pass through the placenta and reach the fetus in utero, passive a~;nistration of the antibodies of the present ~ ~ 3 ~
-`'090~09805 PCT/US90/01t32 invention to ~IV-infected pregnant women would provide effec-tive therapy for the fetus.
The human monoclonal antibodies may be conjugated to cytotoxic agents and used as immunotoxins (as described in Vitetta, E.S. et al., Science 238: 1098-1104, 1987) or incor-porated onto the surface of liposomes containing anti-~IV drugs or toxins to specifically target such drugs or toxins to 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 cytotoxic agents that may be conjugated to the antibodies of the present invention are ricin, diphtheria toxin and radionuclides. ~icin is an extremely po-tent toxin produced by the beans of the plant Ricinus communis. In a typical treatment employing the human monoclonal antibodies of the present invention as immunotoxins, the antibody (which binds to a protein that is expressed by HIV-infected cells) is conjugated to a toxin (e.g. ricin) that is toxic to the ~IV-infected cell (and $o non-infected cells as well). By coupling the cytotoxic agent to the antibody, a high level of toxic efficacy can be achieved against the target cell with a markedly lower level of non-specific toxicity. The use of the toxic agent is possible because the human monoclonal antibodies to which the agent is coupled will carry the agent directly to the target (in this case, HIV-infected cells), thereby sparing non-infected cells from the toxin. Techniques that may be employed to conjugate human monoclonal antibodies, including those of the present invention, to cytotoxic agents are described in detail in Vitetta et al., supra and in European Patent Application Serial No. 279,668, published August 24, 1988 of Genentech, Inc The human lymphoblastoid cell lines (which produce the monoclonal antibodies of the present invention) were formed by immortalizing lymphocytes obtained from ~IV-seropositive patients by infecting such lymphocytes with Epstein Barr Virus (EBV) in vitro. Initially, blood was obtained from 58 ~IV-seropositive individuals, peripheral blood mononuclear cells were obtained and incubated overnight with EBV. The EBV

O90/09805 PCT/US90/01l32 infected cells were cultured at 80,Q00 cells per well in microtiter wells for 3-4 weeks and assayed for anti-~IV
antibody production using a non-commercial ELISA (see below) and a commercial ELISA employing HIV-coated beads. The specificity of each positive reaction obtained by the ELISA was confirmed by te6ting for their non-reactivity on identical beads coated with bovine serum albumin (BSA).
Approximately 9% of the lymphoblastoid cell cultures te~ted positively in the non-commercial ~LISA. After expansion the positive well6 were cultured for two more weeks. It was found that 2.4~ tested positively for ~IV proteins by ELISA and 0.67% proved to be specific for HIV by virtue of their non-reactivity on the BSA beads. The anti-~IV antibodies produced were found to be directed again~t gp41 or p24 and had suffi-cient avidity to show reactivity by ELISA, Western blot, radio-immunoprecipitation and~or i~munofluorescence. Therefore/ all of these monoclonal antibodies would be useful in diagnostic assays for HIV. The stable clones were then ~ubcultured 1 to 2 times at 10 or 100 cells per well with irradiated human lymphoblastoid feeder cells and expanded into tissue culture flasks.
In a ~econd round of immortalization/selection, peripheral blood mononuclear cells from another 36 HIV-seropositive individuals were obtained, immortalized by EBV
infection and as~ayed for anti-~IV production as above. Four stable lymphoblastoid cell lines were obtained producing monoclonal antibodies: 120-16, 126-6, 126-50, 167-7 and 191-3 against gp41, and 134-F6 against p24.
In addition, one cell line, 98-4.3, producing monoclonal antibodies against p24, was obtained from positive cultures derived from the first group of 58 patients de~cribed above. The culture was subcultured twice after several unsuccessful trials and is presently stable in culture.
The characteristics of the human monoclonal antibodies produced by the lymphoblastoid cell lines of the present invention are described in Table III of Example 5 below.
The present inventors have performed some epitope ~O90/09805 PCT/US90/01l32 mapping of the human monoclonal antibodies of the present invention. For example, it can be seen from the data presented in Example 5, Table III below that monoclonal antibodies 50-69 and 98-43 bind to the same epitope cluster (i.e. amino acids falling between residues 579 and 613) Whereas 98-6 binds to to a different re~ion (amino acids falling between residues 642 and 692~. ~owever, monoclonal antibodies 50-69 and 98-q3 differ in their epitope ~pecificity a5 demonstrated by the fact that 50-69 binds to peptide 599-613 whereas 98-43 binds to pepticle 579-604. Three of the anti-p24 antibodies have been tested (i.e. 71-31, 91-5 and 91-6). All bind to the same region of p24 (131-198).
All of the human monoclonal antibodies of the present 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 dependent cellular cytotoxicity (ADCC) as shown in Table III below. This is a most important finding in that serotherapy (e.g. passive immunization) and seroprophylaxis in animal retroviral models have been shown to be mediated by ADCC (Plata, F. et al., Cell 48: 231, 1987; Weinhold, K.J. et al, J. Natl. Cancer Inst. 75:
717, 1985). The ADCC immune response is directed by specific antibodies and involves mobilization of effector cells (cytotoxic T-cells, monocytes, and killer cells against speci~ic targets. It i8 believed that the ability to mount an ADCC response will be important for serotherapy and seroprophylaxis in HIV infections also.
The fourteen lymphoblastoid cell lines thus obtained are stable in culture and produce human monoclonal antibodies directed against targets on BIV which are expressed in vivo in infected patients.
The human monoclonal antibodies of the present inven-tion are all of-the IgG serotype and may be recovered from the ~upernatants of monoclonal antibody producing lymphoblastoid cell cultures and purified by conventional methods known in the 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, or 'O90/09805 PCT/US90/01132 ~igh Performance Liquid Chromatography (~PLC).
The eleven stable lymphoblastoid cell lines described in Examples 1-6 below produce human monoclonal antibodies which are directed against unique epitopes which are expressed in ~IV-infected patients. Although some epitope mapping has been performed (see Table III in Example 4 below), further epitope mapping will determine the exact specificity of each of the monoclonal antibodies and may reveal targets on the ~IV gp41 and p24 protein molecules which can be candidates for vaccine production.
The human monoclonal antibodies of the present inven-tion are directed against either immunodominant (common) or non-dominant epitopes of the gp41 and p24 viral proteins. As employed in this specification, the term "immunodominant"
refers to an antigenic determinant that most patients respond by the production of antibodies. Antibodies 50-69 and 120-16, directed against gp41, are to immunodominant epitopes. These two antibodies may be employed for passive immunizations and/or diagnostic reagents. Antibodies 71-31 and 91-5, directed ~0 against p24, are to non-dominant epitopes.
Lymphoblastoid cell lines 91-5 and 120-16 (producing human monoclonal antibodies directed against p24 and gp41, respectively) have been deposited with the American Type Culture (ATCC, Rockville, MD) and have received Accession Numbers CRL lgO38 and CRL 10037, respectively.
The classification of the antibodies of the invention into an immunodominant or non-dominant grouping was ac-complished by inhibition testing as describe~ in Example 6 below. In this assay, sera collected from HIV seropositive individual~ were used to inhibit the binding of biotinylated monoclonal antibodies directed against ~IV proteins to their re~pective antigens.
Of the monocional antibodies to p24 and gp41, only those to gp41 mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro cytotoxicity studies revealed that the most active induced significant levels of killing was at minimum concentrations of 15-250 ng Ab/ml. ADCC was assayed using `~090t09805 PCT~US90/01132 peripheral blood mononuclear cells (MNC) as effectors and the CEM.NKR cell line, infected with ~TLV-IIIB, MN and RF (viral strains), as targets.
Two monoclonal antibodies to gp4l 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 ug Ab/ml. IT made with monoclonal antibody to gp4l, however, reduced protein synthesis in infected ~9 cells by so%
(IC50) at concentrations of 500 ng/ml. The IC50 of anti-gp41 IT for infected U937 cells was lO00 ng/ml. In the presence of chloroquine, the IC50 of these immunotoxins (ITs) was 5-lO
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 l gram. The antibodies o~ 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 wee~ 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 treatment~, 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 `'090/09805 PCT/US90/01132 with ~IV or for prophylaxis in individuals at risk for such infections. The pharmaceutical formulations of the invention comprising an anti-~IV effective amount, range between about 200 mg and about 15 grams, of the human monoclonal antibodies of the present invention identified in Examples 1-4 below. The quantity of effective dose applied by each injection is relatively unimportant since the total dosage can be reached by 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-known in the art. Isotonic saline, sterile water, 10% maltose, human serum albumin, glycine or other pharmaceutically-acceptable materials may be u~ed as diluents, carriers or solvents in preparing the pharmaceutical formulations comprising the human monoclonal antibodies of the present invention.
The present invention is described below in specific working examples which are intended to illustrate the invention without limiting its scope.

EXAMPLE 1: IMMORTALIZATION OF UUMAN ~-CELLS
Blood was obtained from 58 HIV-seropositive individuals who were intravenous drug users or homosexuals. The presence of antibody to ~IV in the blood was determined using a commer-cial enzyme-linked immunosorbent assay (ELISA) (Organon-Teknika Bio-Enzabead UTLV-III ELISA, Durham, NC) and confirmed by Western blot (Novapath I~1~unoblot Assay, Bio-Rad, ~ercules, CA
and Biotech/DuPont, DuPont, Wilmington, DE). The disease status of patients was established on the basis of an im-munologic staging system as described by Zolla-Pazner et al.
(Proc. Nat. Acad. Sci. USA 84: 5404, 1987):
Scale Score T4/T8 ratio #T~lmm #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 the 58 patients were obtained by centrifugation of heparinized ~O90/09805 PCTtUS90/01132 blood, diluted 1:1 with RPMI-1640 and centrifuged on Histopa~ue (Sigma, St. Louis, MO) at 300 x g for 30 minutes. Cells at the medium/Histopaque interface were recovered, washed three times and incubated overnight at a density of 2 x 106 cells/ml with the filtered supernatant from the EBV-transformed marmoset cell line B95-8 (Proc. Nat. Acad. Sci. ~a 70: 190, 1973, available under Accession Number CRL 1612 from the American Type Culture Collection, ATCC, Rockville, MD). Lymphocytes were then washed once and cultured in RPMI-1640 medi~m ~M.A. Bioproducts, Walkersville, MD) supplemented with 10% fetal calf serum (~yclone Labs, Logan, UT) 2mM L-glutamine, 100U/ml penicillin, and 100 micrograms/ml streptomycin (complete medium) for four weeks in 96-well plates (Costar, Cambridge, MA) at 80,000 cells per well.

EXAMPLE 2: ISOLATION AND SCREENING OF LYMPHOBLASTOID
CELL LINES FOR ANTI-~IV ANTIBODY PRODUCTION
After screening for anti-HIV antibody production by a non-commercial ELISA (see below), positive cultures were expanded into wells of 24-well tissue culture plates (Costar) and cultured for two more weeks. All initial and expanded cultures were fed at weekly intervals with complete medium.
Cultures with supernatants showing specific reactivity to ~IV
were then subcultured one to two times at doubling dilution on feeder layers of irradiated GK-5 human lymphoblastoid cells (derived from a variant of GM1500; Satoh, J. et al., N. Engl.
J. Med. 309: 217, 1983) which had been exposed to 3000 Rads of gamma-radiation. Stable clones were then subcultured one to three times at 10 to 100 cells per well on feeder cells and then expanded into flasks.
Thus, initial cultures of immortalized B-cells (hereinafter referred to as lymphoblastoid cell lines) were established and further characterized as described below.
The screening of the initial cultures in 96-well plates was performed using a non-commercial ELISA. Immulon 2 plates 2 ~
O90/09805 PCTtUS90/01132 (Dynatech, Chantilly, VI) were coated overnight at 4C with 4 micrograms/ml of ~TLV-IIIg lysate (purchased from Electro-Nucleonics, Inc., Silver Spring, MD) diluted in carbonate buffer, pH 9.8. Plates were washed three times with phosphate buffered saline, p~ 7.2, containing 0.05% Tween 20 (PBS-Tween).
The culture supernatants to be assayed (0.1 ml per well) were then added and incubated for 90 minutes at 37C. Subsequently, plates were washed with PBS-Tween and incubated with goat anti-human immunoglobulin conjugated to alkaline phosphatase (Organon Teknika-Cappel, Malvern, PA) for another 90 minutes at 37C. The plates were washed again with PBS-Tween and the substrate, p-nitrophenyl phosphate in 10% diethanolamine, was added for 30 minutes. The reaction was terminated with 25 microliters of lN NaO~ and the ab~orbance was read at 405 nm in an automated ELISA reader (MR 600 Microplate Reader, Dynatech).
The specificity of the antibody bindinq was assessed by testing the supernatants for reacti~ity against ~IV-coated beads (Bio-Enza~ead) and against uncoated beads (obtained from Organon Teknika Cappel~ which were then coated with bovine serum albumin (BSA, Sigma Chemical Co.) by incubating the beads in 1.25% BSA diluted in PBS for 1 hour at room temperature.
Reactivity with ~IV-coated beads, but not with BSA-coated beads, was used as a criterion f or specificity. Further analysis of the specificity o~ the monoclonal antibodies was then carried out by Western blot using a commercially-available kit ~Aio-Rad, Richmond, CA) and by radioimmunoprecipitation (RIP). RIP assay~ were carried out using the method of Pinter and ~onnen (J. Immunol. Methods, in press). Briefly, 30 micrograms of ~TLV-IIIB lysate (purchased from Organon-Teknika), was labeled with 125[I] using the E3Olton-~unter reagent (New England Nuclear, Boston, MA). Bound label was separated from free label on a Bio~Gel P-4 column (Bio-Rad).
Fif ty microliters of culture supernatant were incubated with 5 x 106 cpm of the labeled lysate for 1 hour at 37~C, then 50 microliters of 10% fixed Staphlococcus aureus (Pansorbin, Calbiochem, La Jolla, CA) was added~ The immunoprecipitate was washed three times by centrifugation and the air-dried pellet YO90/~9805 PCTJUS90/01l32 was resuspended in buffer, boiled for 3 minutes and electrophoresed on a 10% SDS polyacrylamide gel. The gels were dried and exposed for one to three days to X-Omat S film (Kodak, Rochester, NY).
The class and light chain type of anti-~IV antibody was determined by ELIS~. For these assays, microtiter plates (Immulon 2) were coated with 4 micrograms/ml of HIV lysate tElectro-Nucleonics~ and then incubated with culture super-natants. The type of antibody binding to ~IV was determined using the following alkaline phosphatase-coupled antibodies:
goat anti-human IgG (gamma specific), goat anti-human kappa chain and goat anti-human lambda chain (Organon Teknika-Cappel, Malvern, PA). The subtype of the monoclonal antibody was also analyzed by ELISA using alkaline phosphatase-labeled mouse monoclonal antibodies against the four subclasses of human IgG
(Zymed, San Francisco, CA).
Immunoglobulin quantitation was also performed by ELISA. Immulon 2 plates were coated with goat anti-human IgG
(gamma specific) and incubated with culture supernatants.
Bound IgG was detected with alkaline phosphatase-labeled goat anti-human IgG (gamm~ specific). Affinity-purified human IgG
~Cappel) was used to produce standard curves.
A total of 14,329 culturec in microtitre wells were established using cells derived from the 58 subjects. Ap-proximately half of these cultures were derived from three serial bleeds from a single subject (with a scale score of 1) over a period of three months. The remaining wells were established using cells derived from 57 subjects whose scale scores ranged from O to 3. The results of this procedure are shown in Table I below.

'~ $ ?~ 3J d 'O90/09805 PCT/US90/01132 TABLE I
QUANTITATIVE RESULTS OF T~E PROCEDURE VSED TO
PRODUCE ~UNAN MONOCLONAL ANTIBODIES TO HIV

No. of % positive wells wells l. Infection of PBMC with EBV14,329 (l00) 1 4 weeks Screen for anti-~IV by Non-commercial ELISA 1,290 9.O
2. Expand positive wells ¦ 2 weeks ~r 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 RIP57 0.40 3. Subculture positive wells by doubling dilution (l0,000-l0 cells/well) 4-6 weeks r Screen by commercial ELISA16* 0.ll*
4. Subculture positive wells at (l00 and l0 cells/well) 1 4-6 weeks Screen by commercial ELISA 7* 0.05*

* No. and ~ of positive plates from ~ubcultures of individual wells which contain at lea~t one antibody positive well.

~ 3f~ .

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 ~IV.
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 statuR using the m~unologic staging sy~tem of Zolla-Pazner et al. (supra) and the results are shown in Table II
below.

2-32li3~ 2 ~
`vo90/0980s PCT/US90/01132 TABLE II

C~ARACTERISTICS OF CELL CULTURES DERIVED FROM
PATIENTS AT DIFFERENT STAGBS OF ~IV 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) antibody per well+
_ BIV-seronegative patients:
3 637 0 0 7.3 ~IV-~eropositive patients:
0 4725 18 ~2.4%) 6 (0.8%) 0.92 1 138,789 180 (2%)66 (0.7%) 0.88 2 202,792 54 (1.9%~ 16 (0.5%) 0.65 3 212,023 88 ~4.3%) 9 (0.4%) 0.22 5814,329 340 2.4%97 0.67% 0.66 * Specificity of anti-HIV antibodies were assessed by commer-cial ELISA using BIV-coated and BSA-coated beads.
~ B cells transformed by EBV stick together and create characteristic clumps of cells which were quantitated microscopically.

2 ~

The results, shown in Table II, revealed that cultures obtained from patients with a scale score of 3 (severe im-munodeficiency) gave a higher percentage of antibody producing wells than patients with lesser scale scores. However, only 10% (9/88) of reactive wells from the cells of stage 3 patients were specifically reactive with HIV whereas 30-37% of wells from cells of patients with scale scores of 0-2 were specifi-cally reactive with BIV. Thus, after six weeks of culture, cells from patient~ with lower scale scores produced a higher percentage of wells containing ~IV-specific antibody.
Analysis of antibodies from ELISA-positive expanded cultures was carried out by RIP. Only 59~ of supernatants from these cultures were also positive on RIP analysis~ RIP
analysis demonstrated that, out of 57 supernatants, 44 showed reactivity to env-e~coded proteins, ll to 9~ proteins and 2 to reverse transcriptase.
Therefore, specific lymphoblastoid cell lines were isolated and further cloned as described below.

EXAMPLE 3: SPBCIFICITY AND REACTIVITY OF THE ~UMAN MONOCLONAL
ANTIBODIES OF A SUBSET OF T~E PRESENT INVENTION
The 57 cell lines mentioned above were then cloned by doubling dilution from lO,OOO to lO cells per well. Wells with the lowest cell concentration which were producing antibodies were then picked and cloned at lOO or lO cells per well. Using this procedure, seven cell lines, 3 producing anti-gp4l antibodies and 4 producing anti-p24 antibodies were established which have been cloned from one to three times at lOO or lO
cells per well. The reactivities of the antibodies from these lines are shown in Figures l and 2.
All seven of the cell lines of this Example produced antibodies of the IgG subtype as shown in Figure l. In Fig1~re l, all lines received [l25I]-labeled ~IV lysate reacted Wi~ll lane l, serum from an HIV-infected subject, lane 2, with antibody from cell line 50-69, lane 3, with antibody from cell 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 from cell 91-5, lane 7, with antibody from cell and line 91-6 and lane 8, with antibody from 98-4.9. The molecular weights of the major viral proteins are shown on the left in kilodaltons.
Antibody from three of the cell lines bound to env-encoded protein gp41 (lines 50-69, 98-6 and 98-43, lanes 2-4 respec-tively). Antibodies from four of the cell lines bound to qag encoded protein p24 (lines 71-31, 91-5, 91-6 and 98-4.9, lanes 5-7 respectively). By RIP, antibodies from cell lines 71-31, 91-5 and 91-6 reacted with p24. Antibodies from 98-4.9 were 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 also tested by Western blot ~Figure 2).
In Figure 2, Western ~lot strips were used to show the reactivity o~ serum from a normal control (lane 1), an HIV-infected subject (lane 2), and of supernatant from cell lines 71-31 (lane 3), 91-51 (lane 4), 91-6 (lane 5), and 98-4.9 (lane 6j. Western blot analysis showed all 3 monoclonal antibodies reacted with p24 and with known aag precursors p55 and p40.
All four antibodies also reacted with a breakdown product of p24 which migrated with a mobility of approximately 22 kilodal-ton~ (kd); three of the four anti-~ monoclonal antibodies also reacted with additional intermediate precursors which were noted with a mouse monoclonal antibody and which had mobility of approximately 37, 31 and 28 kd.
Studies of the growth characteristics and level of antibody production o~ each line was performed. Each cell line was initially cultured in replicate wells at 0.5 x 106 cells/ml for 1-8 days. The number of cells and the amount of ;~
munoglobulin produced is shown in Figure 3. In Figure 3, each time point, separate wells were used to test for viahl-cell number (closed cir~les) l for secreted human IgG (o~-~n circles). Cell lines ~u~ied ~re 50-69(a), 98.6(b), sn ~ d~, g~-5~e~, 9~-~(f) and 9~-4 9(g)-Peak cell density was noted at 4 days with maximum densities of 1.0 to 2.4 x 106 cells/ml. The doubling time of cells in log phase ranged from 40-61 hours. The concentration 2 ~

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 LYMP~OBLASTOID CELL LINES
PRODUCING ~UMAN MONOCLONAL ANTIBODIES TO HIV
Peripheral blood mononuclear cells were obtained from another 39 ~IV-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 6upernatants were reactive with HIV-coated beads and unreactive with BSA-coated beads),~ by radioLmmunoprecipitation and by Western Blot as in Example 3 above. The characteristics of these human monoclonal antibodies are shown in Table III
below.

EXAMPLE 5: C~ARACTERIZATION OF T~E ~UMAN MONOCLONAL
ANTIBODIES OF T~E PRESENT INVENTION
The specificity and biological activities of the human monoclonal antibodie~ of the present invention are summarized in Table III below.

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-"O90/09805 PCT/US90/01132 In Table III, epitope mapping was performed using recombinant antigens using a Western slOt format. viral Neutralization and enhancement assa~s were performed as in J.
Ciin. Micro. 26: 231, 1988. ADCC assays were performed according to Lyerly, H.K. et al. AIDS and Human Retroviruses 3: 409-422, 1987. Western Blot analysis was performed using 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 761 (obtained from DuPont, Wilmington, DE). PE3 is a 286 amino acid sequence from gpl20 (obtained from DuPont, Wilmington, DE~. pl21 contains residues 561-649 of gp41 (obtained from Centocor, Malvern, PA).
As can be ~een in Table III above, all of the anti-gp41 human monoclonal antibodies of the present invention were o~
the IgG serotype and mediated antibody dependent cellular cytotoxicity (ADCC).
In addition, epitope mapping showed that five of the anti-gp41 antibodies bound to different epitopes on the viral protein. Monoclonal antibody 50-69 bound to residues 599-613;
monoclonal antibody 98-6 was directed against a peptide encompassing residuec 642-692; monoclonal antibody 98-43 bound to a peptide encompassing gp41 residues 579-604; monoclonal antibody 120-16 bound residues 644-663; and monoclonal antibody 116-7 bound to residues 661-683. The numberinq system for the gp41 peptide is according to J. Virol. 61: 570, 1987.
All three of the anti-p24 monoclonal antibodies tested (71-~1, 91-5 and 91-6) bound to a HIV p24 peptide encompassing 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 the present invention were able to neutralize the infectivity of aIV and one (120-16) enhanced viral pathoqenicity.

EXAMPLE 6: INHIBITION TESTING OF T~E MONOCLONAL
ANTIBODIES_OF T~E PRESENT INVENTION
Presented below is an example of the use of the `'O~0/09805 PCT/US90/01132 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 37C, and overnight at 4C.
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 l:10 to 1:1000. The plates were incubated at room temperature over-night and washed three times with PBS-Tween. A predetermined dilution of biotinylated monoclonal anti~HIV antibodies (see below) was then added in a volume of lO0 microliters and the plates incubated for 2 hours at 37C. The wells were washed three times with PBS-Tween and the reaction developed by adding an avidin-biotinylated-horseradish peroxidase complex (Vector Labs) and incubated for 2 hours at 37C. After washing 5 times with PBS-Tween, 2,2'-aæino-di-[3-ethylbenzthiazoline sulfonate]
(ABTS) was added as substrate and incuhated for 30 minutes at room temperature. The optical density of each well was read in an ELISA reader at 410nm.
~iotinylation of the monoclonal antibodies to ~IV was performed as follows. Each monoclonal antibody was partially purified by ammonium ~ulfate precipitation and/or chromatog-raphy on Protein A-Sepharose. After dialysis against O.lM
sodium bicarbonate 75 microliters of N-hydroxyl-suc-cini m; dobiotin (5 mg in 1 ml of DMSO~ was added to 1 ml of the antibody at a concentration of 5 mg/ml. The reaction was allowed to proceed at room temperature with haking for 3 hours and then dialyzed against phosphate buffered saline, pH 7.4.
The biotinylated monoclonal antibodies were stored at -25C in 50% glycerine before use.
The results of the inhibition tests are shown in Figure 4. In Figure 4, the seronegative sera is designated t-) and the seropositive (+).
The data presented in Figure 4 shows that antibodies 120-16 and 71-31 are directed against immunodominant epitopes 2 ~
~090~09805 PCT/US90/0113 and non-dominant epitopes respectively. Thus, seropositive sera are a~le to compete for the binding of labeled antibody 120-16 to the ~IV 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 thè ~IV lysate.

Claims (21)

WHAT IS CLAIMED IS:

1. A human monoclonal antibody directed against human immunodeficiency virus protein p24.

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

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

4. The human lymphoblastoid cell line of claim 3 derived from a human infected with human immunodeficiency virus.

5. 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 p24 and a phar-maceutically-acceptable carrier.

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

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

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

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

10. A human monoclonal antibody directed against human immunodeficiency virus protein gp41, said monoclonal antibody 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 Accession No. 10038.

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