WO1991019797A1 - Heterohybridomes produisant des anticorps monoclonaux humains contre le vih-1 - Google Patents

Heterohybridomes produisant des anticorps monoclonaux humains contre le vih-1 Download PDF

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WO1991019797A1
WO1991019797A1 PCT/US1991/004304 US9104304W WO9119797A1 WO 1991019797 A1 WO1991019797 A1 WO 1991019797A1 US 9104304 W US9104304 W US 9104304W WO 9119797 A1 WO9119797 A1 WO 9119797A1
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hiv
epitope
antibody
human
cell line
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PCT/US1991/004304
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Susan Zolla-Pazner
Miroslaw K. Gorny
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New York University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/166—Animal cells resulting from interspecies fusion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • HIV-l 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.
  • AZT azidothymidine
  • ARC AIDS Related Complex— a prodrome of the disease
  • AZT also decreases the mortality rate and frequency of oppor ⁇ tunistic infections.
  • 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.
  • preliminary results of early clinical trials are not promising. Patients often suffer serious side effects, including low blood pressure, nausea, and diarrhea.
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • 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.
  • 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.
  • 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;
  • EBV Epstein-Barr virus
  • 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)) .
  • 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.
  • Putney et al. 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 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. 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.
  • oligopep ⁇ tides comprise the ⁇ -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 ⁇ -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. 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.. discloses 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.. 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.
  • 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:
  • the method may further comprise:
  • preferred heteromyelomas are mouse x human hybrids, preferably the cell line desig ⁇ nated SHM-D33 (ATCC #CRL1668) .
  • the neutralizing epitope preferably comprises a peptide of at least 6 amino acids, more preferably, of the sequence
  • 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;
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g/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.
  • Cell lines making human IgG mAb to a neutral ⁇ izing epitope of HIV-l 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.
  • heteromyeloma is intended a hybrid cell produced by fusion of a non-human myeloma cell line and a human myeloma cell line.
  • 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).
  • hetero- myeloma cells are used as fusion partners for selected EBV-transformed human cells to produce the hetero- hybridomas of this invention.
  • heteromyeloma SHM-D33 is used as a fusion partner.
  • This cell line is available from the ATCC, under accession number ATCC CRL1668.
  • heterohybridoma refers to a hybrid cell line produced by fusion of an antibody-producing cell of one species with a hetero- myeloma.
  • 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.
  • the term as used herein is more narrowly defined.
  • a human antibody-producing cell is fused with a mouse-human heteromyeloma.
  • 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.
  • the human-mouse hetero ⁇ myeloma is the cell line designated as SHM-D33.
  • neutralizing epitope 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • a preferred immunoassay is an Enzyme Linked Immunosorbent Assay, or ELISA.
  • ELISA Enzyme Linked Immunosorbent Assay
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • compositions comprising the antibodies of the invention include all compositions wherein the antibody is contained in an amount effective to achieve its intended purpose.
  • 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.
  • 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.
  • immunotoxins 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.
  • 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.
  • 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.
  • the antibody is conju ⁇ gated to a toxin such as ricin that, alone, is toxic to HIV-infected as well as uninfected cells.
  • a toxin such as ricin that, alone, is toxic to HIV-infected as well as uninfected cells.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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 ⁇ g/ml) (complete medium) . Periodically, hetero- myeloma cells were cultured with the antibiotic G418 at 200 ⁇ g/ml to eliminate neomycin-sensitive variants.
  • the SHM-D33 cells were cultured at a concentration of 1-2 x 10 5 cells/ml (log phase growth) .
  • 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.
  • 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 10 4 cells/100 ⁇ l/well.
  • 1 x 10 4 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 ⁇ M amino- pterin, 16 ⁇ M thymidine (HAT) and 1 ⁇ M ouabain (Sigma Chemicals) . Feeding was repeated twice weekly with fresh complete medium supplemented with HAT.
  • 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.
  • RIP radioimmunoprecipitation
  • Antibody isotypes were determined by ELISA. Immulon 2 plates were coated with 1 ⁇ g/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.
  • 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
  • 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 ⁇ quiz antibodies were further cloned three times at 100 to 10 cells/well.
  • both lymphoblastoid cell lines (257-2, 268-11) were fused with heteromyeloma SHM-D33. All wells showed growth of hybrid cells.
  • 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.
  • lymphoblastoid cell lines 257-2 (ATCC #CRL10483) and 268-11 (ATCC #CRL10482) produced 6.4 and 3.8 ⁇ g IgG/ml/10 6 cells/24 hr, respectively
  • the related heterohybridomas, 257-2D (ATCC #HB 10480) and 268-11D (ATCC #10481) produced 20.5 and 11.3 ⁇ g IgG/ml/10 6 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 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
  • 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 ⁇ g/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 ⁇ g/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
  • 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.
  • 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.
  • 96-well plates with EBV- transformed cells were screened for the presence of an antibody to HIV-l, 3-4 weeks after culture initiation.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-
  • Antigen 1.0 . . . 0.1 (K d in ⁇ M) MN 412 ⁇ 311 ⁇ 419 ⁇ 391 ⁇ 268 447 ⁇ 257 ⁇ 386 SF-2 311 ⁇ 391 ⁇ 268 ⁇ 386 ⁇ 447 «419 «257
  • EBV transformed human lymphocyte line producing a human IgGl, lambda antibody (ATCC accession #CRL 10483)
  • EBV transformed human lymphocyte line producing a human IgGl, lambda antibody (ATCC accession #CRL 10482)

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Abstract

On prépare des hétérohybridomes produisant des anticorps monoclonaux humains contre un épitope neutralisant le VIH-1 par transformation de lymphocytes de sang périphérique à l'aide du virus d'Epstein-Barr, par sélection de cellules produisant un anticorps contre un peptide synthétique représentant une partie de l'épitope neutralisant, et par fusion de celle-ci avec une cellule d'hétéromélome. L'invention concerne également un procédé de production d'un anticorps monoclonal humain spécifique à un épitope de neutralisation du VIH-1 à partir de l'hétérohybridome, ainsi que des anticorps monoclonaux humains spécifiques à des épitopes peptidiques particuliers de la boucle V3 de la glycoprotéine Gp120 du VIH.
PCT/US1991/004304 1990-06-15 1991-06-14 Heterohybridomes produisant des anticorps monoclonaux humains contre le vih-1 WO1991019797A1 (fr)

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EP0577243A2 (fr) * 1992-04-01 1994-01-05 Merck & Co. Inc. Anticorps monoclonaux recombinants, neutralisant le HIV pour la prévention et traitement des infections du HIV
EP0588750A2 (fr) * 1992-08-05 1994-03-23 Centro De Ingenieria Genetica Y Biotecnologia Procédé pour la production de polypeptides recombinants contenant des épitopes dérivées d'isolées d'HIV différentes, leur utilisations comme immunigènes et pour la détection d'anticorps contre le HIV
WO1995006119A1 (fr) * 1993-08-24 1995-03-02 Scotgen Limited Anticorps anti-vih humanise recombine
EP0693121A1 (fr) * 1994-02-04 1996-01-24 Akzo Nobel N.V. Anticorps monoclonal 88bv59, subclones et procede de production
EP0724651A1 (fr) * 1993-10-19 1996-08-07 The Scripps Research Institute Anticorps monoclonaux humains synthetiques diriges contre et neutralisant le virus de l'immunodeficience
US5558865A (en) * 1991-08-22 1996-09-24 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
US5618922A (en) * 1994-07-25 1997-04-08 Nissin Shokuhin Kabushiki Kaisha NM03 antibody materials and methods
US6057294A (en) * 1995-01-16 2000-05-02 Northern Sydney Area Health Service Of Pacific Highway Peptide

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NL8702403A (nl) * 1987-10-09 1989-05-01 Stichting Centr Diergeneeskund Oligopeptiden en gebruik daarvan voor diagnostische en vaccinatiedoeleinden voor aids en arc.
DD274448A1 (de) * 1988-07-29 1989-12-20 Univ Berlin Humboldt Verfahren zur herstellung humaner monoklonaler antikoerper gegen strukturproteine des hiv

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AIDS Research and Human Retrovirus, Volume 6, No. 5, issued May 1990, J.E. ROBINSON et al., "Identification of conserved and Variant Epitopes of Human Immunodeficiency Virus Type 1 (HIV-1) gp120 by Human Monoclonal Antibodies produced by EBV-transformed cell lines", pages 567-579, see entire article. *
Immunology, Volume 68, issued 1989, M. OHLIN et al., "Human monoclonal Antibodies against a recombinant HIV envelope antigen produce by primary in vitro immunization. Characterization and epitope mapping", pages 325-331, see entire article. *
Proceedings National Academy of Science, Volume 85, issued May 1988, J.R. RUSCHE et al., "Antibodies that inhibit fusion of human immunodeficiency virus infected cells bind a 24-amino acid sequence of the viral envelope, gp 120", pages 3198-3202, see entire article. *
Proceedings National Academy Sciences, Volume 80, issued December 1983, N.H.-H. TENG et al., "Construction and testing of more human heteromylomas for human monoclonal antibody production", pages 3708-3712, see entire article. *
See also references of EP0535154A4 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5558865A (en) * 1991-08-22 1996-09-24 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
US5665569A (en) * 1991-08-22 1997-09-09 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
EP0577243A3 (fr) * 1992-04-01 1994-11-02 Merck & Co Inc Anticorps monoclonaux recombinants, neutralisant le HIV pour la prévention et traitement des infections du HIV.
EP0577243A2 (fr) * 1992-04-01 1994-01-05 Merck & Co. Inc. Anticorps monoclonaux recombinants, neutralisant le HIV pour la prévention et traitement des infections du HIV
EP0588750A3 (fr) * 1992-08-05 1995-01-11 Cigb Procédé pour la production de polypeptides recombinants contenant des épitopes dérivées d'isolées d'HIV différentes, leur utilisations comme immunigènes et pour la détection d'anticorps contre le HIV.
EP0588750A2 (fr) * 1992-08-05 1994-03-23 Centro De Ingenieria Genetica Y Biotecnologia Procédé pour la production de polypeptides recombinants contenant des épitopes dérivées d'isolées d'HIV différentes, leur utilisations comme immunigènes et pour la détection d'anticorps contre le HIV
WO1995006119A1 (fr) * 1993-08-24 1995-03-02 Scotgen Limited Anticorps anti-vih humanise recombine
EP0724651A1 (fr) * 1993-10-19 1996-08-07 The Scripps Research Institute Anticorps monoclonaux humains synthetiques diriges contre et neutralisant le virus de l'immunodeficience
EP0724651A4 (fr) * 1993-10-19 1998-11-04 Scripps Research Inst Anticorps monoclonaux humains synthetiques diriges contre et neutralisant le virus de l'immunodeficience
US6395275B1 (en) 1993-10-19 2002-05-28 The Scripps Research Institute Synthetic human neutralizing monoclonal antibodies to human immunodeficiency virus
EP0693121A1 (fr) * 1994-02-04 1996-01-24 Akzo Nobel N.V. Anticorps monoclonal 88bv59, subclones et procede de production
EP0693121A4 (fr) * 1994-02-04 1999-11-03 Akzo Nobel Nv Anticorps monoclonal 88bv59, subclones et procede de production
US5618922A (en) * 1994-07-25 1997-04-08 Nissin Shokuhin Kabushiki Kaisha NM03 antibody materials and methods
EP0848013A1 (fr) 1994-07-25 1998-06-17 Nissin Shokuhin Kabushiki Kaisha NM03, un anticorps monoclonal dirige contre la VIH-1 gp120 protéine
US6057294A (en) * 1995-01-16 2000-05-02 Northern Sydney Area Health Service Of Pacific Highway Peptide

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