AU8228791A - Heterohybridomas producing human monoclonal antibodies to hiv-1 - Google Patents

Description

HETEROHYBRIDOMAS PRODUCING HUMAN MONOCLONAL ANTIBODIES TO

This invention was funded in part by a research contract from the National Institute of Allergy and Infectious Disease, National Institutes of Health, No. AI72658, which provides to the United States Government certain rights in this invention.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention in the fields of immunolo- gy and virology relates to a method for producing lympho- blastoid cell lines and heterohybridomas which make human monoclonal antibodies specifically directed to HIV-l neutralizing antigens, a method for producing the human monoclonal antibodies, the lymphoblastoid and heterohybri- doma cell lines and their monoclonal antibody products.

DESCRIPTION OF THE BACKGROUND ART

The human immunodeficiency virus (HIV) has been implicated as the causative agent of acquired immune deficiency syndrome (AIDS) . Two different HIV serotypes have been identified to date: HIV-l and HIV-2. It is currently believed that the majority of individuals that become infected with HIV eventually will develop AIDS and are likely to succumb to fatal infections and/or malignan- cies. Currently, it is estimated that approximately 1.5 million persons have been infected by HIV in the United States alone.

Several avenues have been explored to treat patients afflicted with AIDS or HIV infections. The antiviral drug, azidothymidine (AZT) , produces both clinical and immunological improvements upon short term administration to patients afflicted with AIDS and ARC (AIDS Related Complex— a prodrome of the disease) . AZT also decreases the mortality rate and frequency of oppor¬ tunistic infections. Although AZT has clinical benefit, it is expensive and not without untoward side effects. AZT toxicity often requires blood transfusion and/or reduction in dosage, and, in some instances, total cessa¬ tion of drug therapy. Nevertheless, AZT is the only drug currently authorized for the treatment of AIDS. Most of the drugs currently being tested in AIDS therapy exhibit unforeseen side effects and thus may not be suitable for administration to all AIDS patients.

Lymphokine therapy of AIDS with one or more lymphokines is currently under evaluation. Interferons, particularly ga ma-interferon, and interleukin-2 are being tested for the treatment of HIV infections. However, preliminary results of early clinical trials are not promising. Patients often suffer serious side effects, including low blood pressure, nausea, and diarrhea.

One approach to the treatment of AIDS is the use of monoclonal antibodies (mAbs) of defined specificity directed against HIV-l proteins expressed in infected patients. These proteins are constituents of the HIV-l virions, and are expressed by HIV infected cells. These proteins are designated inter alia as p24, gp41, gpl20, etc. Essex (U.S. Patent No. 4,725,6569) describes the identification and isolation of gp41, as well as its use in the treatment and diagnosis of AIDS.

However, the use of mAbs for treatment of HIV-l infections has been hampered because most mAbs directed against HIV-l proteins currently available in therapeutic quantities are of rodent origin. Administration of non- human antibodies to humans can cause dangerous and even life-threatening immunologic reactions. In addition, such rodent mAbs may not be as effective in interacting with human effector cells or effector molecules (such as the complement system) . Stable human cell lines producing HIV-1-specific mAbs, and the mAb products directed against HIV-l compo¬ nents, are useful for treating and/or diagnosing individu¬ als infected with this virus. However, human mAbs in general, and those directed against HIV in particular, have proven to be extremely difficult to produce.

Among the explanations for this difficulty are:

(a) The most readily 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-producing cells can be achieved using Epstein-Barr virus (EBV) , but production is often unstable and the level of antibody produced is often low;

(c) whereas the level and the stability of antibody production can be enhanced by fusion of EBV- transformed human B lymphocyte lines to mouse myeloma cells, such hybrids (called "heteromyelomas") readily lose human chromosomes, and ultimately antibody production as well; and

(d) whereas fusion of normal or transformed B cells to human lymphoblastoid lines or to heteromyelomas can stabilize antibody production, few satisfactory parent lines of this cell type have been available.

Neutralizing antibodies are considered to be essential for protection against viral infection. For this reason, any synthetic vaccine against HIV-l must include epitopes which induce neutralizing antibodies. Analysis of the reactivity patterns of sera of HIV- infected subjects, and of rodent anti-HIV mAbs, has revealed the existence of several HIV-l protein epitopes that elicit neutralizing antibodies. Most of the epitopes are localized in the envelope glycoprotein, gpl20, and the transmembrane protein, gp41. One report has identified a neutralizing epitope in the pl7 core protein (Sarin, P.S. et al.. Science 232:1135 (1986)) . Some of these neutralizing epitopes are located in conserved regions of the proteins (Ho, D.D. et al.. Science 239: 1021 (1988)). However, they do not seem to elicit particularly strong responses nor to represent sensitive principal neutralizing domains since (a) only low serum titers of antibodies recognizing these epitopes are demonstrable, and (b) high concentrations of murine mAbs to these epitopes are required to neutralize the virus. Several groups have shown that the principal immunodominant neutralizing epitope of HIV-l appears to reside in the V3 domain of gpl20 (Goudsmit, J. et al. , Proc. Natl. Acad. Sci. USA 85:4478-4482 (1988); Goudsmit, J. AIDS 2:S41 (1988); Matsushita, S.M. et al.. J. Virol. 62.:2107 (1988); Javaherian, K. et al.. Proc. Natl. Acad.

Sci. USA JL6.6768 (1989); Kowalski et al. , Science 237:1351 (1987); Palker et al.. Proc. Natl. Acad. Sci. USA 5:1758-1762 (1988)). This particular epitope is located between two cysteine residues which participate in an intrachain disulfide bond which is predicted to form a hairpin loop structure in the protein. The "tip" of the loop consists of a sequence of four amino acids, Gly-Pro- Gly-Arg (G-P-G-R) that is essentially conserved and is flanked by amino acids which differ between various HIV-l isolates.

The V3 loop of gpl20 has been shown to be important for biological activity of the virus including infectivity. Proteolytic cleavage in this region, between amino acids 315 and 316, appears essential for infectivity (Stephens et al.. Nature 343:219 (1990); Hattori et al.. FEBS Lett. 248:48 (1989)). Neutralizing antibodies binding to this region may therefore prevent infection by inhibiting such cleavage.

Therapeutic use of mAbs of defined specificity directed against HIV-l proteins p24 and gp4i, which are expressed in infected individuals, has been proposed (Jackson, G. G, Lancet ii:647 (1988); Chanh, T.C. et al. , EMBO J. 5:3065 (1986); Karpas, A. et al. , Proc. Natl. Acad. Sci. USA 85:9234 (1988)).

A number of approaches have been taken to pre¬ paring antibodies against HIV. For example, Putney et al. (European Patent Publication EP311228) disclose proteins which can be used in assays for detecting and quantifying antibody against HIV, as well as DNA encoding these proteins.

Putney et al. (European Patent Publication EP255190) disclose recombinant DNA transfer vectors which comprise all or part of the nucleotide sequence, the translated regions of which encode the RIO, PB1, 590, or KH1 fragments of the HTLV-III (HIV-l) envelope protein. These protein fragments are said to be useful in immuno- assays for detection of HIV antibodies, as antigenic components of AIDS vaccines, and for stimulation of lymph¬ ocyte proliferative responses in infected individuals.

Wang (European Patent Publication EP328403) discloses peptides which have specific immunoreactivity to antibodies to HIV-l, and which neutralize antibodies to HIV-gpl20. The disclosed peptides comprise 15-40 amino acids in a sequence corresponding to a region in HIV gpl20 which are peptide 126, peptide 127, and analogues thereof. These peptides are used as solid phase immunoadsorbents for detection of antibodies to HIV gpl20, including neutralizing antibodies.

Goudsmit et al. (European Patent Publication EP311219) disclose oligopeptides composed of 8-17 amino acids in a sequence corresponding to a sequence occurring in the variable region (V3) in the gpl20 protein of an AIDS- or ARC-causing or related virus. These oligopep¬ tides comprise the β-turn amino acid sequence G-P-G or G- P-G-R at positions 312-314 or 312-315 in the amino acid numbering of HTLV-IIIB (BH10) and flanking amino acids sequences having a length of at least 1 and preferably at least 2 amino acids and variations in which the GPG or GPGR sequence has been replaced by a different β-turn sequence, and variations in which the free amino group of the N-terminal amino acid and/or the free carboxyl group of the C-terminal amino acid has been blocked or otherwise modified. This document also discusses antibodies to these oligopeptides.

Rusche et al. (European Patent Publication EP306219) disclose HIV-l proteins or peptides as well as DNA sequences coding for the proteins or peptides and DNA transfer vectors containing the DNA. These proteins and peptides are said to be useful in the diagnosis, prophy¬ laxis, and therapy of AIDS, in the preparation of HIV vaccines, and for stimulation of lymphocyte proliferative responses in HIV-infected humans.

Petteway et al.. (PCT Publication WO8805824) , disclose a method for producing and selecting a hybridoma cell which produces a mAb to a viral glycoprotein, such as an HIV glycoprotein. The method is said to be useful for obtaining mAbs to HIV proteins such as gpl20 and gp41. Kennedy et al.. (European Patent Publication EP245362) disclose a synthetic peptide for producing an immune response to the viral causative agents of AIDS and ARC. This peptide has a sequence homologous to a portion of the amino acid sequence of the gpl20 or gp41 envelope glycoproteins of HIV. These proteins are said to be useful to vaccinate against viral causative agents of AIDS and in diagnostic assays for AIDS.

Grunow et al.. (J. I muno1. Meth. 106:257-265 (1988) describe the construction a heteromyeloma cell line designated CB- 57 by fusing normal human B lymphocytes and a urine myeloma. This heteromyeloma was then used for fusion with EBV-transformed B cells. Three cloned hetero- hybridomas were obtained which showed about 30-fold enhanced secretion of IgM antibodies relative to the EBV- transformed parent line. CB-57 cells were also fused with PEL from individuals having anti-HIV antibodies, resulting in 4 heterohybridomas producing IgM anti-p25 antibodies or IgGl antibodies to p25 or gp41. No anti-gpl20 hetero- hybridomas were reported. A later report from the same group (Berzow, D. et al. , Proc. IVth Int'l. AIDS Conf., Montreal, 1989, abstr. T.C.P. 150) indicated that one clone capable of continuous production of antibodies to gpl20 was obtained but no data were available on the activity of this antibody.

SUMMARY OF THE INVENTION it is an object of the present invention to overcome the aforementioned deficiencies in the prior art.

The invention is directed to a method for pro¬ ducing a heterohybridoma cell line which produces a human mAb against a neutralizing epitope of HIV-l comprising:

(a) transforming human peripheral blood lymphocytes in vitro with Epstein-Barr virus;

(b) selecting those cells transformed by Epstein-Barr virus which produce an epitope-specific human anti- body specific for the HIV-l epitope;

(c) fusing the transformed cells with a heteromyeloma, thereby producing the heterohybridoma cell line.

The method may further comprise:

(d) selecting heterohybridoma cells which produce the above antibody; and

(e) cloning the heterohybridoma cell line.

For the above method, preferred heteromyelomas are mouse x human hybrids, preferably the cell line desig¬ nated SHM-D33 (ATCC #CRL1668) . in the above method, the neutralizing epitope preferably comprises a peptide of at least 6 amino acids, more preferably, of the sequence

Y-N-K-R-K-R-I-H-I-G-P-G-R-A-F-Y-T-T-K-N-I-I-G.

In the above method, the heterohybridoma of this invention preferably produces a human mAb specific for an epitope of the gpl20 glycoprotein of HIV-l, more prefera¬ bly from the V3 loop. This invention is also directed to a method for producing a human mAb against a neutralizing epitope of HIV-l, preferably an epitope of the V3 loop of HIV gpl20, comprising: (a) producing a heterohybridoma according to the method described above;

(b) culturing the heterohybridoma; and

(c) recovering the mAb antibody from the culture supernatant. According to the present invention, EBV trans¬ formation of peripheral blood lymphocytes followed by fusion to a heteromyeloma results in a heterohybridoma which is capable of producing larger quantities of human mAb to HIV-l, than are human antibody-producing cells obtained by EBV transformation alone.

The present invention is further directed to a human monoclonal antibody specific for a peptide having the amino acid sequence Y-N-K-R-K-R-I-H-I-G-P-G-R-A-F-Y-T-T-K-N-I-I-G, the anti- body having HIV-l neutralizing activity.

Also intended within the scope of the present invention is a human mAb produced by the above method, the antibody being specific for a neutralizing epitope of the V3 loop of HIV-l glycoprotein gpl20, wherein the epitope has an amino acid sequence selected from the group con¬ sisting Of R-K-R-I-H-I-G, H-I-G-P-G-R, K-R-I-H-I, G-P-G-R, and H-I-G-P. Preferably, the antibody of the present invention is one that is produced by the cell lines described below. The present invention is further directed to a heterohybridoma cell line which produces a human mono¬ clonal antibody specific for a neutralizing epitope of HIV-l, preferably wherein the the neutralizing epitope is from the gpl20 glycoprotein of HIV-l, most preferably from the V3

Preferred cells lines according to the present invention are selected from the group consisting of the cell lines 257-2D (ATCC #HB 10480) , 268-11D (ATCC #HB 10481), 447-52D (ATCC #HB 10725), 386-D, 477-D, 311-11-D, 391-95-D, 419-D and 412-D, most preferably 257-2D (ATCC #HB 10480) , 268-11D (ATCC #HB 10481) or 447-52D (ATCC# HB 10725) .

In the EBV transformation technique of the present invention, unfractionated peripheral blood mono- nuclear cells, not just B cells, are infected with EBV.

An important advantage of the present invention is the screening technique for selecting an EBV- transformed lymphocyte line which makes the antibody of desired specificity. First, the screening method utilizes synthetic peptides representing the epitope or epitopes of interest, rather than crude viral preparations. Further- more, this intense screening procedure is performed at an earlier stage of the development process than is common in the prior art. This combination of early and highly rigorous screening of antibody-producing EBV-transformed lymphocytes avoids disadvantages accompanying the unneces- sary production and testing of many cell lines which make undesired antibodies of broad or questionable specificity. The invention is also directed to methods in which these selected EBV-transformed, antibody-producing, cell lines are fused with mouse-human heteromyelomas followed at an early time by cloning, resulting in selection of hetero¬ hybridoma cell lines which are more stable and produce greater amounts of antibody than known heretofore.

The present invention is therefore also directed to an Epstein-Barr virus-transformed lymphocyte cell line which produces a human monoclonal antibody specific for a neutralizing epitope of HIV-l, preferebly wherein the neutralizing epitope is from the gpl20 glycoprotein of HIV-l, most preferably from the V3 loop.

A preferrred Epstein-Barr virus-transformed cells line is selected from the group consisting of the cell lines 257-2 (ATCC #CRL 10483) , 268-11 (ATCC #CRL 10482), 386, 447-52, 477, 311-11, 391-95, 419 and 412, most preferably, 257-2 (ATCC #CRL 10483) or 268-11 (ATCC #CRL 10482) .

The invention is directed to human mAbs produced by these EBV-transformed cell lines, the mAbs having specificity to a neutralizing epitope of the gpl20 mole¬ cule of HIV-l.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the immune reactivi- ty of 257-2D (o) and 268-11D (•) to the 23-mer HIV-l synthetic peptide in an antigen-limited ELISA.

Figure 2 is a gel pattern from a radioim uno- precipitation assay of human mAbs with HIV lysates. Lanes 1 and 2 show the reactivity of a serum specimen from an HIV-infected individual. Lanes 3 and 4: reactivity of supernatant 257-2D. Lanes 5 and 6: reactivity of super¬ natant 268-11D. Lanes 7 and 8: reactivity of supernatant 280-2 (which is unreactive with HIV antigens) . Lanes 1, 3, 5, and 7 represent the reactivity of specimens to HIV_{M N} lysate. Lanes 2, 4, 6 and 8 represent reactivity of specimens to HTLV-IIIB lysate.

Figure 3 is a graph showing the results of a scan of serum and mAb reactivities by ELISA with over¬ lapping hexapeptides homologous with the 23-mer of the HIV_HN V3 loop. The reactivity of each hexapeptide with seronegative (0 ) or seropositive (Q) sera (panel A) or with supernatants from heterohybridomas 257-2D (o) or 268-11D (■) (panel B) is shown on the ordinate and each hexapeptide is designated by the single letter code of its N-terminal residue and the subsequent five amino acids. Thus, the sequence appearing on the abscissa is the sequence of the 23-mer.

Figure 4 is a graph showing HIV neutralization by serial dilutions of mAb 257-2D (starting concentration: 14.0 μg/ml) . Panel A: Neutralization of HIV_HN incubated with 257-2D in the absence of complement for 1 hr. Panel B: Neutralization of HIV_HN incubated with 257-2D in the presence of complement for 18 hrs. The data are normal¬ ized as the percentage of the mean plaque count in 12 or 24 replicate control wells (o) for experiments shown in panels A and B, respectively. The number of control plaques in the experiment shown in panel A was 9.6 ± 1.4 (Mean ± SEM) and, in panel B, 9.1 ± 1.1. Error bars represent the standard error (SEM) .

Figure 5 is a graph showing the correlation between the 50% neutralizing concentrations and the disso- ciation constants (Kd) of five human anti-HIV mAbs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cell lines making human IgG mAb to a neutral¬ izing epitope of HIV-l, such as epitopes associated with the gpl20 glycoprotein, are produced by EBV transformation of human peripheral blood mononuclear cells followed by selection of cell lines making antibody of the desired specificity, followed by fusion of the selected EBV- transformed cells to a heteromyeloma cell line. The resultant heterohybridoma cells each makes a human mAb having the epitope-specificity (e.g. for the gpl20 epitope) of the antibodies produced by the selected parent EBV-transformed cells.

The gpl20 glycoprotein, which contains one or more neutralizing epitopes recognized by the cells and antibodies of the present invention, may be derived from any of the known HIV-l strains, such as the relatively common MN strain.

By the term "heteromyeloma" is intended a hybrid cell produced by fusion of a non-human myeloma cell line and a human myeloma cell line. Typically, a mouse myeloma or plasmacytoma cell is the fusion partner of the human myeloma cell. Such non-human and human myeloma and heteromyeloma cell lines are well-known in the art and are exemplified by cell lines reported in Teng, N.N. et al. , Proc. Natl. Acad. Sci. USA 80:7308 (1983); Kozbor, D. et al., Hybridoma 2:7 (1983); and Grunow, R. et al. , J.Immunol. Meth. 106:257-265 (1988).

As intended in the present invention, hetero- myeloma cells are used as fusion partners for selected EBV-transformed human cells to produce the hetero- hybridomas of this invention.

In a preferred embodiment, the heteromyeloma SHM-D33 is used as a fusion partner. This cell line is available from the ATCC, under accession number ATCC CRL1668.

The term "heterohybridoma", as used herein, refers to a hybrid cell line produced by fusion of an antibody-producing cell of one species with a hetero- myeloma. The term "heterohybridoma" has also been used elsewhere to refer to any interspecies hybridoma, such as one resulting from the fusion of an antibody-producing human lymphocytoid cell line cell and a murine myeloma cell. However, the term as used herein is more narrowly defined.

In one embodiment of this invention, a human antibody-producing cell is fused with a mouse-human heteromyeloma. In a preferred embodiment, the hetero¬ hybridoma is the result of fusing an EBV-transformed human lymphocyte which is producing an antibody to a neutral¬ izing epitope of HIV, with a human-mouse heteromyeloma. In a more preferred embodiment, the human-mouse hetero¬ myeloma is the cell line designated as SHM-D33.

By the term "neutralizing epitope" is intended an epitope which, when bound by an antibody specific for this epitope, results in neutralization of the virus. Neutralization of any biological activity of the virus, such as, for example, syncytium formation, falls within the scope of "neutralization", as used herein. To generate human mAbs against a neutralizing epitope of HIV-l gpl20, human peripheral blood lymphocytes are transformed by EBV, as described, for example in Gorny, M.K. et al., Proc. Nat'l. Acad. Sci. USA 86:1624- 1628 (1989) , which is hereby incorporated by reference.

Preferably, the cells to be transformed are derived from the blood of an individual producing anti- HIV-l antibodies.

The cultures of EBV-transformed cells are screened for antibody to the epitope of interest. In one embodiment the epitope is a neutralizing epitope of the gpl20 protein and the screening is performed using puri- fied gpl20, a fragment thereof, or a synthetic peptide representing a portion thereof. In a preferred embodi¬ ment, cultures are screened for antibody to an epitope of the V3 loop of gpl20 using a synthetic 23-mer peptide from the V3 loop representing amino acids 306-328 (see below for sequence) . In addition to such a peptide, additional peptides having at least 6 amino acids are useful for screening the EBV-transformed cells in order to identify antibody producing cells of the desired epitope specificity. Any of a number of immunoassays well known in the art can be used for this screening process. A preferred immunoassay is an Enzyme Linked Immunosorbent Assay, or ELISA. Using such an assay, the culture super- natants are tested for the presence of antibodies of desired specificity and isotype.

Positive EBV-transformed cultures are cloned repeatedly by any of a number of cloning methods known in the art, such as, for example, by doubling dilution. Cells from cultures found to be positive for the desired antibody specificity are also fused with cells of the heteromyeloma line to produce a heterohybridoma. Fused cells are subsequently cloned by culturing at a density of about 1-100 cells per well.

Specificity of the antibody produced by the heterohybridoma is determined by immunoassay methods which are well known in the art. In a preferred embodiment, ELISA and radioimmunoprecipitation (RIP) procedures are used. The antigen preparation comprises HIV-l virions (such as strain MN) , lysates of viruses or of infected cells, such as MN and HTLV-IIIB lysates, viral proteins such as gpl20, or recombinant or synthetic viral peptides such as the 23-mer described above.

The mAbs of the present invention are of the IgG isotypes and may be recovered from the supernatants of the heterohybridoma cell cultures and purified by conventional methods known in the art for purification of IgG. Such methods include, but are not limited to, protein-A Sephar- ose affinity chromatography, a combination of Affigel Blue (BioRad, Richmond, CA) and Protein-A Sepharose chromatog¬ raphy, or High Performance Liquid Chromatography.

When administered to humans infected with HIV-l, or at risk for HIV infections, the antibodies of the present invention can provide therapeutic or prophylactic benefits. Such individuals particularly at risk are known in the art and include health care workers who have been exposed via a needle stick to HIV-l. The antibodies of the present invention are also useful in diagnostic assays of the type used to determine if a patient has been exposed to, or infected with, HIV-l. The antibodies are also useful for analyzing the expression of HIV proteins for which they are specific. The HIV-specific human mAb of the present inven¬ tion can be used to treat individuals infected by HIV or suffering from AIDS. The antibodies according to the invention are administered parenterally or enterally by any of a number of known routes. For example, administra- tion may be subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or intrathecal. Alterna¬ tively, or concurrently, administration may be by the oral or rectal route. The antibodies may also be administered into the amniotic cavity for in utero treatment. The preferred routes are intravenous and intramuscular. The dosage of antibody administered will be dependent upon the age, health, and weight of the recipi¬ ent, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Effec- tive amounts of the mAbs are from about 0.1 to about 500 mg per day, and preferably from about 3 to about 30 mg per. day. Treatment may require infusion or injection of the antibody over a period of days, weeks, months, or even years, as would be readily ascertained by one of skill in the art.

A typical treatment regimen comprises adminis¬ tration of an effective amount of antibody administered over between one week and about six months. Duration of treatment required to achieve a therapeutic result will vary from patient to patient, depending upon the severity and stage of the illness and the individual characteris¬ tics of each patient.

The total dose required for each treatment may be administered by multiple doses or in a single dose. The mAbs may be administered alone or in conjunction with other therapeutics directed to HIV-l infection, such as AZT, or directed to other disease symptoms.

The mAbs of the present invention can be admin¬ istered to HIV-infected expectant mothers. Since the antibodies of the present invention are of the IgG isotype, they can cross the placenta and reach the fetus. This may prevent infection of the fetus or, alternatively, provide effective therapy for an infected fetus.

Pharmaceutical compositions comprising the antibodies of the invention include all compositions wherein the antibody is contained in an amount effective to achieve its intended purpose. In addition to the anti¬ body, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceuti¬ cally. An additional pharmaceutical composition within the scope of the present invention is a combination of the antibody of the invention with an intravenous immunoglobu- lin preparation as is known in the art.

Pharmaceutical compositions include suitable solutions for administration by injection or orally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e. the antibody) together with the excipient. Pharmaceutical compositions for oral administration include tablets and capsules. Compositions which can be administered rec- tally, include suppositories.

The mAbs of the present invention can be conju¬ gated to cytotoxic agents and used as immunotoxins (see, for example, Vitetta et al.. Science 238:1098-1104 (1987)), or incorporated onto the surface of liposomes containing anti-HIV drugs or toxins to specifically target such drugs or toxins to infected cells. As used herein, the term "immunotoxin" refers to a conjugate of an anti¬ body with one or more toxins, drugs, radionuclides, or cytotoxic agents. A toxic moiety can either be chemically conjugated to the antibody of the invention, or alterna¬ tively, can be ligated through recombinant DNA technology. In such a ligation, the DNA encoding the toxic protein or an active fragment thereof is ligated to the DNA encoding the entire, or a portion of, the mAb heavy chain, light chain, or both. Such genetic constructs and method for making them are known in the art. Among the toxins that may be conjugated to the antibodies of the present inven¬ tion are ricin, diphtheria toxin, Pseudomonas toxin, tumor necrosis factor-alpha, and others known in the art.

In a typical treatment using the mAbs of the present invention as immunotoxins, the antibody is conju¬ gated to a toxin such as ricin that, alone, is toxic to HIV-infected as well as uninfected cells. By coupling the cytotoxic agent to the antibody, a high level of toxic efficacy can be achieved in a highly localized manner, against the target cell to which the antibody has deliv¬ ered the toxin, with a sparing of neighboring infected cells to which the antibody did not bind.

EXAMPLE I

OPTIMIZING PRODUCTION OF HUMAN MONOCLONAL ANTIBODIES TO

HIV-l

The objective of this study was to examine the optimal conditions for establishing cell lines producing human mAbs against HIV-l. METHODS

Peripheral blood lymphocytes derived from 74 HIV seropositive individuals were transformed with EBV.

Cultures producing antibodies to HIV were expanded and cloned several times on irradiated GK5 feeder cells by doubling dilution (5000 to 10 cells/well) . Five of the 74 specimens could be processed by both cloning and fusions. Simultaneous with the first cloning (i.e. 5 to 7 weeks after initiation of culture) , the lymphoblastoid cells from expanded cultures were fused with heteromyeloma SHM- D33 cells. Anti-HIV positive hybrids were cloned at 100 to 1 cell/well. The specificity of the mAb was tested by ELISA, Western blot and RIP.

RESULTS

The immortalization of PBL by EBV alone gave rise to 2 cell lines synthesizing human mAbs. However when EBV-transformed cells were fused to SHM-D33, 5 hybrid lines making human mAb were obtained. All cell lines have been in culture for 6-12 months.

Conclusion

EBV transformation of blood cells followed by fusion to a heteromyeloma appears to be the most effective method for the generation of human mAb to HIV-l and is more efficient than EBV transformation alone.

EXAMPLE II

Subjects group of 41 asymptomatic HIV-seropositive individuals participated in the study. The presence of serum antibodies to HIV-l was tested by commercial ELISA (Genetic Systems) and confirmed by Western blot using Novapath Immunoblot Assay (Bio-Rad) . The CD4 and CD8 phenotype of lymphocytes from each subject was determined using Leu 3a and Leu2a antibodies (supplied by Becton- Dickinson) by flow cytometry using a Cytofluorograf II (Ortho) . Peripheral blood white blood cell counts were processed by a Coulter Counter and differential counts were performed manually.

Patients were classified as to disease progres¬ sion using an immunologic staging system such that patients were divided into four categories on the basis of the following, previously described criteria (Zolla- Pazner, S. et al.. Proc. Natl. Acad. Sci. USA 84:5404 (1987) ) : Scale Score CD4:CD8 Ratio CD4 cells mm³ L m hoc tes mm³

A peptide which spans 23 amino acids of the gpl20 V3 loop of the MN strain of HIV-l (23-mer peptide) was synthesized by solid-phase methodology (Peninsula Laboratories, Inc. Belmont, CA) . The peptide has the following sequence:

Y N K R K R I H I G P G R A F Y T T K N I I G This peptide was used in an ELISA assay to screen for antibodies reactive to it. Establishment of EBV-Transformed Cell Lines

The method for producing human cell lines synthesizing mAbs to HIV-l was described by Gorny et al. , 1989, supra. Peripheral blood mononuclear cells were incubated with Epstein-Barr virus (EBV) and cultured for 3-4 weeks in 96-well microplates. After screening for antibodies in the culture supernatants using the 23-mer peptide in an ELISA, cells from cultures having super- natants positive for antibodies were expanded, subcultured several times, and finally further expanded into flasks. These cells are called lymphoblastoid cells. Cell Fusion

The heteromyeloma (mouse-human hybrid) SHM-D33 (Teng, N.H. et al. , Proc. Natl. Acad. Sci. USA, 80:7308 (1983)) was grown in Iscove's modified Dulbecco's medium supplemented with 15% fetal bovine serum, 2 mM L- glutamine, penicillin (100 units/ml) , and streptomycin (100 μg/ml) (complete medium) . Periodically, hetero- myeloma cells were cultured with the antibiotic G418 at 200 μg/ml to eliminate neomycin-sensitive variants. Two days prior to fusion, the SHM-D33 cells were cultured at a concentration of 1-2 x 10⁵ cells/ml (log phase growth) . The viability of the cells, as determined by erythrosin B dye exclusion, exceeded 95%. The SHM-D33 cells were washed twice in phosphate-buffered saline and then mixed with the lympho- blastoid cells which had been expanded from initial cul¬ ture but had not yet been cloned. The cells were mixed at a ratio of 1:3 and centrifuged. Then, 1 ml of 50% poly- ethylene glycol 1300-1600 (Sigma Chemicals) was added dropwise to the pellet over a period of one minute with constant agitation that was continued for another one minute. During the next five minutes, the cells were slowly diluted with Iscove's medium and, after pelleting by centrifugation at 200 Xg, the cells were gently resus- pended in complete medium and plated in 96-well micro- plates at a concentration of 8 x 10⁴ cells/100 μl/well. The next day, 1 x 10⁴ mouse peritoneal cells were added per well as feeder cells, and the cultures were continued in the presence of 0.5 mM hypoxanthine, 0.2 μM amino- pterin, 16 μM thymidine (HAT) and 1 μM ouabain (Sigma Chemicals) . Feeding was repeated twice weekly with fresh complete medium supplemented with HAT. After two to three weeks all culture wells were screened for antibody produc-^* tion against the aforementioned peptide and heterohybrids producing antibodies reactive with the 23-mer peptide were expanded in 24-well plates. Hybrids that produced the highest level of antibodies (and IgG) measured by ELISA were cloned at concentrations of 100, 25, and (at least twice at) 1 cell per well.

Antibody Detection and Characterization

Culture supernatants were screened against the 23-mer by ELISA. Immulon 2 plates (Dynatech) were coated overnight at 4^aC with the synthetic peptide (1 μg/ml) , diluted in sodium carbonate buffer, pH 9.6. Plates were washed three times and culture supernatants were added to each well and incubated for 90 minutes at 37^aC, then washed. Goat anti-human IgG (gamma chain-specific) conju¬ gated to alkaline phosphatase (Zymed Laboratories) was added and incubated for another 90 minutes at 37^aC and washed as above. The substrate, p-nitrophenyl phosphate (Sigma Chemicals) , was added for 30 min and the absorbance was read at 405 n o MR 700 Microplate Reader (Dynatech) .

The specificity of antibody binding was assessed by radioimmunoprecipitation (RIP) . RIP assays were carried out by the method of Pinter et al. (J. Immunol. Meth. 112:735 (1988)) with 30 μg of HTLV-IIIB lysate (Organon Teknika) and/or MN lysate (Advanced Biotechnolo¬ gies, Inc.) labelled with ¹²⁵I using the Bolton-Hunter reagent (New England Nuclear) . Culture supernatants were incubated with viral lysate and further processed as described by Gorny et al. , supra. and Pinter et al. , supra. Analysis of Human Antibodies

Antibody isotypes were determined by ELISA. Immulon 2 plates were coated with 1 μg/ml of the 23-mer and incubated with culture supernatants. The subtype of the IgG mAb was detected by alkaline phosphatase-labelled mouse mAbs against the four subclasses of human IgG (Zymed Laboratories) . The light chain of mAb was analyzed by ELISA using microplates coated with rabbit antibodies to human kappa chain or lambda chain (Dakopatts) . The developing antibodies used were alkaline phosphatase-coupled goat anti-human kappa chain and goat anti-human lambda chain (Sigma Chemicals) , respectively.

IgG quantitation was also performed by ELISA. Plates were coated with goat anti-human IgG (gamma chain- specific) and incubated with serially diluted culture supernatants. Bound IgG was detected with alkaline phosphatase-labelled goat anti-human IgG (gamma chain- specific) . Affinity-purified human IgG (Organon Teknika- Cappel) was used as a standard. Plates were read and standard curves were generated using an automated MR-700 Microplate Reader (Dynatech Laboratories) . Epitope Mapping

The fine specificity of the mAb was determined using the Epitope Mapping Kit (Cambridge Research Biochem- icals, Valley Stream, New York) which utilizes the method developed by Geysen et al. (Geysen, H. M. et al. Proc. Natl. Acad. Sci. (USA) 81:3998-4002 (1984)) to synthesize hexapeptides on plastic pins. Eighteen sequential, over- lapping hexapeptides which spanned the 23-mer were synthe¬ sized in situ on plastic pins with two additional control peptides. The peptides were deprotected, then washed and dried according to the manufacturer's instructions. Because the configuration of pins fit to 96-well micro- plate, the ELISA assays were carried out in standard microplates as recommended by the manufacturer. Thus, all peptide-containing pins were allowed to react with culture supernatants from the cell lines being tested at a 1:10 dilution in 0.1% Tween-20 in PBS containing 1% ovalbumin and 1% bovine serum albumin. Thereafter, the pins were washed and reacted with horseradish peroxidase-conjugated goat anti-human IgG. The color reaction was read in a Dynatech MR-700 plate reader as absorbance at 405 nM. RESULTS total of 46 blood specimens derived from 41

HlV-seropositive individuals were processed and trans¬ formed with EBV. After 3 to 4 weeks of culture, an aver¬ age of 2.9% of the wells were positive for antibody against the 23-mer of the V3 loop as revealed by ELISA. Table II shows that the percentage of positive wells was slightly increased in the group of subjects with a scale score of 1, but that there was no significant difference in the yield of positive cultures from patients with different levels of severity of the disease. TABLE II

PRODUCTION OF ANTIBODIES SPECIFIC FOR A 23-mer OF THE MN STRAIN gpl20 V3 LOOP BY EBV-TRANSFORMED HUMAN

LYMPHOCYTES

Lymphoblastoid cells from positive wells were further expanded in 24-well plates and, once per week, fresh culture supernatants were tested for antibody speci- ficity by ELISA using the 23-mer peptide. Two lymphoblas¬ toid cell lines, 257-2 (ATCC #CRL10483) and 268-11 (ATCC #CRL10482) , that were producing high levels of specific antibody against the 23-mer were cloned by doubling dilu¬ tion (from 10,000 to 10 cells per well). Cells from wells plated at the lowest cell density that continued to pro¬ duce antibodies were further cloned three times at 100 to 10 cells/well.

Simultaneously with the original cloning, both lymphoblastoid cell lines (257-2, 268-11) were fused with heteromyeloma SHM-D33. All wells showed growth of hybrid cells. Three weeks after fusion, 50 of 183 wells (29%) plated with 257-2 heterohybrids and 43 out of 48 wells (90%) plated with 268-11 heterohybrids were found to contain antibody against the 23-mer. From each fusion, the eighteen clones producing the highest concentration of antibody (based on absorbance in ELISA) , were expanded in 24-well plates. The production of antibodies was moni¬ tored weekly, and cells producing supernatant yielding the highest specific antibody and IgG concentrations were selected for cloning. The heterohybridomas were cloned at 100 and 25 cells/well and subsequently twice at 1 cell/well.

While the lymphoblastoid cell lines 257-2 (ATCC #CRL10483) and 268-11 (ATCC #CRL10482) produced 6.4 and 3.8 μg IgG/ml/10⁶ cells/24 hr, respectively, the related heterohybridomas, 257-2D (ATCC #HB 10480) and 268-11D (ATCC #10481) produced 20.5 and 11.3 μg IgG/ml/10⁶ cells/24 hr, respectively. The mAbs were shown to react in ELISA with the 23-mer when the latter was bound to the wells of microtiter plates at concentrations as low as 1 ng/ml (Figure 1) .

The specificity of these mAbs was further defined by RIP, the results of which are shown in Figure 2. Both mAbs react with the env-encoded protein gpl20 of HIV_MN but not with the gpl20 derived from HTLV-III_B , revealing the type-specificity of these mAbs.

The mAbs were found to be of the IgG isotype with lambda light chains. Table III shows some character¬ istics of the two EBV- transformed parent lines and the two related heterohybridomas. The heterohybridomas produce three times as much IgG in 24 hours as the EBV- transformed lines, even though the EBV-transformed lines produce considerably more than most EBV-transformed lines described in the literature (Kozbor, D. et al., Immunol. Today 4:72 (1983) ; Casali, P. et al. , Science 234:476 (1986) ; and Steinitz, M. et al.. Nature 269:420 (1977)) . TABLE III

CHARACTERISTICS OF CELL LINES PRODUCING HUMAN MONOCLONAL ANTIBODIES SPECIFIC FOR A 23-mer OF THE MN gpl20 V3 LOOP

μg/10⁶ cells/ml/24h)

In order to define the fine specificity for each antibody, epitope mapping was performed using overlapping hexapeptides which represent sequential hexapeptides overlapping by five amino acids. Each peptide was synthe¬ sized in quadruplicate, so that is was possible to test four samples on one microplate simultaneously. The over¬ lapping antigenic regions and the results of these experi¬ ments are shown in Table IV and Figure 3, panels A and B.

A pool of seronegative sera was not reactive. A seropositive serum sample (serum from HIV-seropositive individual at a dilution of 1:1000) reacted above back¬ ground levels with all pins, giving peak reactions with three pins spanning the region P G R A F Y T T at the tip and right side of the V3 loop.

MAb 257-2D, at a dilution of 1:10 (3.7 μg/ml), bound strongly to two adjacent hexapeptides representing amino acid 309-315 to the left of the top of the loop (R- K-R-I-H-I-G) . MAb 268-11D (5.4 μg/ml) bound to one hexa¬ peptide covering the amino acid sequence H-I-G-P-G-R. Table IV shows the overlapping antigenic regions recog¬ nized by the two mAbs. TABLE IV

REACTIVITY OF HUMAN SERA AND HUMAN MONOCLONAL ANTIBODIES WITH HEXAPEPTIDES OF THE MN gpl20 V3 LOOP

ELISA REACTIVITY (Absorbance units)

These results indicate that the smallest reactive peptide (core of the epitope) that 257-2D recognizes is K R I H I, located to the left of the conserved tip of the V3 loop. The flanking N-and C-terminal arginine and glycine resi¬ dues may also contribute to the binding of this mAb. The mAb 268-llD bound to a single hexapeptide consisting of H I G P G R which spans the tip of the loop and the two adjacent N-terminal amino acids.

EXAMPLE III HUMAN HETEROHYBRIDOMA PRODUCTION WITHOUT PROLONGED EXPANSION OF EBV-TRANSFORMED LYMPHOCYTES

Method

The fusion of EBV-transformed cells and hetero- myeloma SHM-D33 is usually performed after 2-3 weeks of expansion of EBV immortalized cells in 24-well micro- plates. This is equivalent to 5-7 weeks after culture initiation. However, the expansion period is very criti¬ cal for production of the mAb because the majority (at least 90%) of culture wells become negative for mAb pro¬ duction during this period. We therefore tested an alter¬ native method in which the expansion period was excluded and fusion took place 3-4 weeks after culture initiation. Thus, 96-well plates with EBV- transformed cells were screened for the presence of an antibody to HIV-l, 3-4 weeks after culture initiation. Cells from all wells producing anti-HIV were pooled and immediately fused with heteromyeloma SHM-D33 as described in Example II. Results PBL derived from 4 HIV-seropositive individuals produced 8 cell lines secreting mAB to HIV-l after EBV transformation followed by early fusion. Six mAB were directed against p24 and 2 mAbs to gp41 of HIV-l as deter¬ mined by RIP and ELISA. To date, we have obtained 21 stable lines from 300 patients by EBV- transformation or by EBV-transformation and fusion as described in Examples I and II. This is equivalent to 6-7 lines per 100 patient specimens when 20-40 ml of patient's blood are used. Using the "early fusion method" described in this example, 8 lines were obtained from 4 patient specimens, a signifi¬ cant increase in the efficiency of obtaining stable antibody-producing lines. Conclusion

The early fusion of EBV-transformed cells without expansion of positive wells is a more efficient method than our previous techniques for the generation of human mAbs to HIV-l.

EXAMPLE IV

DETERMINATION OF MONOCLONAL ANTIBODY AFFINITY

Determination of the dissociation constants (K_d ) of human mAbs was performed using an ELISA methodology as described by B. Friguet et al.. J. Immunol. Methods 27:305-319 (1985) . Briefly, the culture supernatants of 257-2D and 268-llD were tested at concentrations of 0.5 and 0.6 μg/ml, respectively. The 20-mer (MW=2702 Da), described above, was dissolved in distilled water to a concentration of 1 mg/ml (3.7 x 10" ⁴ M) , diluted in phosphate-buffered saline, pH 7.2, and used at concentra¬ tions ranging from 10' ⁵ to 10" ⁸ M. Supernatant and pep¬ tide were mixed in equal volumes and after 16 hr, the mixture was added to plates coated with the 23-mer (1 μg/ml) and the amount of unbound mAb was measured by ELISA. Data were plotted according to the Friguet modifi¬ cation of the Klotz method (Friguet et al.. supra) to determine the K_d .

The K_d of mAbs 257-2D and 268-llD were found to be 2.3 x 10" ⁷ and 5.9 x 10" ⁷ M, respectively. These values are in the range of those reported by others for IgG mAbs (Friguet et al. , supra; Larsson, A. et al. , Molec. Immunol. .2_4:569-576 (1987)). The K_d of the mAbs produced by the human lymphoblastoid cell lines (257-2 and 268-11) were similar to the those of the mAbs produced by the related heterohybridomas (257-2D and 268-llD) . The K_d described above are for the binding of the mAbs to the 20- mer peptide. The K_d of these mAbs for native gpl20 mole¬ cules may be lower due to the contributions of the confor¬ mation of the whole protein molecule to the epitopes to which the mAbs react. EXAMPLE V

NEUTRALIZATION OF HIV INFECTIVITY A plaque assay which measures the inhibition of HIV infection of MT-2 cells was used to detect the neutralizing activity of the mAbs of the present invention in the presence or absence of human complement (C.V. Hanson et al.. J. Clin. Micro. 128: (1990)). Thus, mAbs were serially diluted in 50% assay medium (Hanson et al. , supra) and 50% of a normal human plasma pool. The plasma pool served as the source of human complement; for studies in the presence of complement, mAb and virus were incu¬ bated for 18 hr at 37oC. For tests in the absence of complement, the plasma pool was heat- inactivated and the mAb and virus were incubated under these conditions for 1 hr at 37@C. The dilution at which 50% of the input virus was neutralized on the basis of plaque counts was calcu¬ lated by interpolation using third order regression analy¬ sis of the mean plaque count at each dilution. RESULTS

When supernatant fluids from 257-2D and 268-llD were incubated with HIV_MN for 1 hr (no complement) prior to addition to permissive MT-2 cells, 50% neutralization was achieved at dilutions of 1:4,700 and 1:2,000, corre- sponding to mAb concentrations of 3.0 and 23.0 ng/ml, respectively (Table 5) . No neutralization of HTLV-IIIB was observed.

When the mAbs from 257-2D and 268-llD were tested in a more sensitive assay, wherein antibodies were incubated with virus for 18 hours in the presence of human complement, neutralization was achieved at dilutions of 1:44,000 and 1:41,000, corresponding to mAb concentrations of 0.3 and 1.1 ng/ml, respectively. Again, no neutraliza¬ tion of HTLV-IIIB occurred under these conditions. A representative dose-response curve for the activity of mAb 257-2D against HIV_{H N} in the absence and presence of complement, is shown in Figure 4. Human mAb 50-69, specific for the HIV transme brane protein, gp41, and mAb 71-31, specific for the core protein, p24, previ¬ ously described by Gorny, M.K. et al.. Proc. Natl. Acad. Sci. (USA) 8£:1624-1628 (1989)), were tested in parallel and displayed essentially no neutralizing activity for either strain of HIV.

TABLE 5

NEUTRALIZING ACTIVITY OF HUMAN MONOCLONAL ANTIBODIES AGAINST HIV

Neutralizing Antibody Titers (ng/ml) 1 hr. no C 18 hr + C

MN IIIB 1:44000 (0.3) neg

268-llD gpl20 1:2000 (23) neg 1:41000 (1.1) neg

50-69 gp41 1:3 neg 1:3 neg

71-31 p24 neg neg neg neg

EXAMPLE VI

HUMAN mABS TO HIV-l V3 LOOP REACT WITH DIVERGENT VIRUS

STRAINS Using the methods described above, additional

EBV-transformed cell lines and heterohybridomas producing human mAbs specific for the V3 loop of HIV_{M N} gpl20 were produced. Several of the heteromyelomas were designated 386-D, 391-D, 419-D, 447-52D, 477-D, 311-11D, 391-95D and 412-D. The reactivity patterns of some of these mAbs are compared to those or 257-2D and 268-llD (described above) are shown in Table 6, below.

TABLE 6

HIV-Type Specificity and Neutralization Activity of Six Human Monoclonal Antibodies Specific for gpl20

Reactive in ELISA to synthetic V3 Neutralization

mAb Core E ito e IIIB MN SF-2 RF IIIB MN SF-2 RF

* Not determined

These results lead to the following conclusions.

(1) The tip of the V3 loop constitutes a cluster of epitopes recognized by human mAbs; (2) the human mAbs cross-react in ELISA with some or all synthetic V3 peptides from divergent HIV-l strains; (3) all tested anti-V3 (MN) human mAbs neutralize the MN virus, including one mAb (257-2D) directed primarily to a region N-terminal to the most conserved region of the loop; (4) cross- neutralization can occur even when 2 out of 5 amino acids in the core epitope are changed (e.g., 257-2D reacting with MN and SF-2) , but some changes in the core epitope abrogate neutralizing activity (e.g., 268-llD reacting with MN and not with IIIB) ; and (5) cross-reactivity as detected by ELISA is much less stringent than cross- reactivity measured in a biologic assay.

EXAMPLE VTI

RELATION BETWEEN CROSS-REACTIVITY AND AFFINITY OF HUMAN ABS TO SYNTHETIC PEPTIDES OF THE V3 LOOP OF DIVERGENT HIV

STRAINS

The reactivity of human mAbs, described above, was measured by direct ELISA against diverse synthetic peptides (19-mers through 23-mers) which had been coated on plates at concentrations of 1 μg/ml. Antibody affini¬ ty, measured as dissociation constants (Kd) of binding to these peptides, were determined as described in Example IV, above.

The results are shown in Table 7. Anti-V3 human mAbs at 2-4 μg/ml cross-reacted with synthetic peptides of the V3 region of HIV-l strains MN, SF-2 and RF, in an ELISA. No reaction was detected with the V3 peptide derived from HIV-l strain IIIB. Kd's for binding to these peptides ranged from >10"⁶M (268-llD binding to RF) to 10^{' 7}M (268-llD binding to MN) . TABLE 7

ELISA Reactivity to V3 from:

Absorbance units at 410 nm

These results lead to the following conclusions:

(1) Human mAbs specific for the HIV-l V3 region display cross-reactivity to V3 regions of divergent virus strains, as measured by ELISA or as antibody affinity; (2) affini¬ ties of human mAbs to different V3 peptides may vary by about one order of magnitude or more; (3) affinity differ¬ ences cannot be explained simply by the amino acid sequence in the relevant epitope; and (4) human mAbs with identical epitope-specificity vary in their affinities for different V3 peptides.

EXAMPLE VIII

A number of additional human mAbs were produced and tested using the methods described above. These antibodies and their specificity and affinity characteris¬ tics are described in Table 8, below.

The correlation between the dissociation con¬ stants and the neutralization capacity of five human mAbs is shown in Figure 5. The results indicate a direct relationship between these two properties, suggesting that the affinity of binding to the 23-mer peptide from the V3 loop is a good predictor of efficacy for virus neutralization.

TABLE 8 Characteristics of Human Monoclonal Antibodies Specific for HIV-l gpl20 Epitopes

IgG 50

ELISA Reactivity^* Affinity (Kd in uM) Titer MN SF-2 RF NY5 IIIB ELI

+ + + +

+ + + +

+ + + + + + + + + +

311-11D IgGl, lambda ** + + + 391-95D IgGl, appa ** + + + 419-D IgGl, lambda ** + + +

412-D IgGl, lambda ** 27 NT NT NT Neg

NT not tested * tested with mAbs at 10 μg/ml except for 477-D which was tested at <5 μg/ml *^* : not determined by Epitope Mapping Kit but shown to be reactive with the peptide

RKRIHIGPGRAFYTT IgG 50% Titer refers to the neutralization titer, indicating the concentration in ng/ml at which the antibody gave 50% neutralization at 18 hr with no complement (except for 257-

2D and 268-1D, which were tested at lhr without complement.

HIV Relative affinity of human mAbs (from low to high)

Antigen 1.0 . . . . 0.1 (K_d in μM) MN 412 < 311 < 419 < 391 < 268=447 < 257 < 386 SF-2 311 < 391 < 268 < 386 < 447«419«257

The references cited above are all incorporated by reference herein, whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

DEPOSITS

The following illustrative cell lines secreting human monoclonal antibodies specific for HIV-l gpl20 neutralizing epitopes were deposited at the ATCC, Rock- ville, Maryland.

1. 257-2

EBV transformed human lymphocyte line producing a human IgGl, lambda antibody (ATCC accession #CRL 10483)

2. 268-11

EBV transformed human lymphocyte line producing a human IgGl, lambda antibody (ATCC accession #CRL 10482)

3. 257-2D Human x Human x Mouse Heterohybridoma cell line producing a human IgGl, lambda antibody (ATCC accession #HB 10480)

4. 268-llD

Human x Human x Mouse Heterohybridoma cell line producing a human IgGl, lambda antibody (ATCC accession #HB 10481)

5. 447-52D

Human x Human x Mouse Heterohybridoma cell line producing a human IgG3 , lambda antibody (ATCC accession #HB 10725)

Claims (34)

WHAT IS CLAIMED IS:

1. A method for producing a heterohybridoma cell which makes a human monoclonal antibody against a neutralizing epitope of HIV-l comprising:

(a) transforming human peripheral blood lymphocytes in vitro with Epstein-Barr virus;

(b) selecting a cell transformed by Epstein- Barr virus which produces an epitope-specific human antibody specific for said HIV-l epitope;

(c) fusing said transformed cell with a heteromyeloma, thereby producing said heterohybridoma cell.

2. The method of claim 1, additionally comprising after step (c) :

(d) selecting said heterohybridoma cell which produces said antibody; and

(e) cloning said heterohybridoma cell.

3. The method of claim 1 wherein said neutralizing epitope is from the gpl20 glycoprotein of HIV-l.

4. The method of claims 3 wherein said neutralizing epitope is from the V3 loop of said gpl20 glycoprotein.

5. The method of claim 1 wherein said selecting is performed using as antigen a synthetic peptide comprising at least part of said neutralizing epitope.

6. The method of claim 5 wherein said neutralizing epitope is from the gpl20 glycoprotein of said HIV.

7. The method of claim 6 wherein said neutralizing epitope is from the V3 loop of said gpl20 glycoprotein.

8. The method of claim 7 wherein said neutralizing epitope comprises a peptide of at least 6 amino acids.

9. The method of claim 8 wherein said peptide has the sequence:

Y N K R K R I H I G P G R A F Y T T K N I I G.

10. The method of claim 1 wherein said heteromyeloma is a murine-human hybrid.

11. The method of claim 10 wherein said heteromyeloma is the cell line designated SHM-D33 (ATCC #CRL1668) .

12. A method for producing a human monoclonal antibody against a neutralizing epitope of HIV-l comprising:

(a) producing a heterohybridoma according to the method of claim 1; (b) culturing said heterohybridoma; and

(c) recovering said monoclonal antibody from said culture.

13. A human monoclonal antibody specific for a peptide having the amino acid sequence:

Y N K R K R I H I G P G R A F Y T T K N I I G.

14. A human monoclonal antibody produced by the method of claim 12, said antibody being specific for a neutralizing epitope of the V3 loop of HIV-l glycoprotein gpl20, wherein said epitope has an amino acid sequence selected from the group consisting of R-K-R-I-H-I-G, H-I-G-P-G-R, K-R-I-H-I, G-P-G-R, and H-I-G-P.

15. The antibody of claim 14 wherein said epitope has the amino acid sequence R-K-R-I-H-I-G.

16. The antibody of claim 14 wherein said epitope has the amino acid sequence H-I-G-P-G-R.

17. The antibody of claim 14 wherein said epitope has the amino acid sequence G-P-G-R.

18. The antibody of claim 14 having HIV-l neutralizing activity.

19. A heterohybridoma cell line which produces a human monoclonal antibody specific for a neutralizing epitope of HIV-l.

20. A cell line according to claim 19 wherein said neutralizing epitope is from the gpl20 glycoprotein of HIV-l.

21. A cell line according to claim 20 wherein said neutralizing epitope is from the V3 loop of said gpl20 glycoprotein.

22. A cell line according to claim 21 which is selected from the group consisting of the cell lines 257-2D (ATCC #HB 10480) , 268-llD (ATCC #HB 10481) , 447-52D (ATCC #HB 10725) , 386-D, 477-D, 311-11-D, 391-95-D, 419-D and 412-D.

23. A cell line according to claim 22 which is 257-2D (ATCC #HB 10480) .

24. A cell line according to claim 22 which is 268-llD (ATCC #HB 10481) .

25. A cell line according to claim 22 which is 447-52D (ATCC# HB 10725) .

26. An antibody produced by a cell line according to claim 19.

27. An antibody produced by a cell line according to claim 20.

28. An antibody produced by a cell line according to claim 21.

29. An Epstein-Barr virus-transformed lymphocyte cell line which produces a human monoclonal antibody specific for a neutralizing epitope of HIV-l.

30. A cell line according to claim 29 wherein said neutralizing epitope is from the gpl20 glycoprotein Of HIV-l.

31. A cell line according to claim 29 wherein said neutralizing epitope is from the V3 loop of said gpl20 glycoprotein.

32. A cell line according to claim 29 which is selected from the group consisting of the cell lines 257-2 (ATCC #CRL 10483), 268-11 (ATCC #CRL 10482), 386, 447-52, 477, 311-11, 391-95, 419 and 412.

33. A cell line according to claim 32 which is 257-2 (ATCC #CRL 10483) .

34. A cell line according to claim 32 which is -11 (ATCC #CRL 10482) .