WO1994028933A1 - Bispecific human monoclonal antibodies specific for human immunodeficiency virus - Google Patents

Bispecific human monoclonal antibodies specific for human immunodeficiency virus Download PDF

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Publication number
WO1994028933A1
WO1994028933A1 PCT/US1994/006170 US9406170W WO9428933A1 WO 1994028933 A1 WO1994028933 A1 WO 1994028933A1 US 9406170 W US9406170 W US 9406170W WO 9428933 A1 WO9428933 A1 WO 9428933A1
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hiv
human monoclonal
mabs
monoclonal antibody
mab
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PCT/US1994/006170
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French (fr)
Inventor
Susan Zolla-Pazner
Miroslaw K. Gorny
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New York University
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Priority to AU72031/94A priority Critical patent/AU7203194A/en
Publication of WO1994028933A1 publication Critical patent/WO1994028933A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel bispecific human monoclonal antibodies (bs-mAbs) directed against two neutralizing epitopes of human immunodeficiency virus (HIV) .
  • bs-mAbs bispecific human monoclonal antibodies
  • HIV human immunodeficiency virus
  • Such bs-mAbs are effective in neutralizing HIV, can be designed to have a broader range of activity against laboratory and primary HIV isolates than standard mAbs, and are useful in treating subjects with HIV infection and AIDS.
  • Monoclonal antibodies which are homogenous with respect to both physical characteristics and immunochemical reactivity provide the promise of absolute specific activity.
  • Murine mAbs which are easily generated against various human pathogens, have limited utility for treating humans due to the rapid induction of strong immune responses in the human recipient against the foreign mouse antibody which abrogates the murine mAb's efficacy (Schroff, R. et al . , 1985, Cane. Res . 45 : 879 ) .
  • Chimeric (human-mouse) and humanized mAbs are much less immunogenic, but nevertheless, result in the production of antibodies to mouse variable regions after repeated doses in some subjects (LoBuglio, A. F. et al . , 1989, Proc.
  • HIV-1 infection induces neutralizing Abs which are specific for gpl20 and gp4l, the envelope and transmembrane glycoproteins of the virus.
  • Abs specifically directed to several regions of gpl20 can neutralize HIV (Olshevsky, U. et al . , 1990, J. Virol . 64:5701)
  • the anti-gpl20 Abs which possess the most clearly defined neutralizing functions recognize two major regions of the protein: (a) the third variable domain or "V3 loop"; and (b) the domain which binds to the CD4 glycoprotein of the T lymphocytes, called the CD4-binding domain (CD4-bd) (Rusche, J.R. et al . , 1988, Proc. Natl .
  • Anti-gp41 neutralizing Abs appear to recognize a conserved region in the extracellular domain (Buchacher, A. et al . , 1992. In: VACCINES 92 : MODERN APPROACHES TO NEW VACCINES INCLUDING PREVENTION OF AIDS, F. Brown et al . . eds. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York p. 191) .
  • Neutralizing Abs are not present at high levels in the sera of HIV-1 infected individuals and might therefore not be sufficient to control HIV infection.
  • Human mAb 447-52-D is the most broadly reactive anti-V3 antibody, with a specificity directed towards the tetrapeptide GPGR, a very conserved sequence in the V3 loop.
  • enzyme linked immunosorbent assays ELISA
  • this human mAb reacts with eight out of eight peptides derived from American and European HIV strains, including such divergent strains as MN, IIIB and RF (Gorny et al . , supra) .
  • this 447- 52-D mAb neutralizes eight different HIV-1 laboratory strains (Gorny, M. K. et al . , 1992, J " . Virol . 66:7538) .
  • bispecific antibodies which are structurally bivalent, recognizing two different epitopes, but functionally monovalent for each antigen- binding site.
  • Bs-Abs can be produced by chemically linking two mAbs, by fusion of two different hybridoma cell lines, or by genetic engineering.
  • Biological methods result in production of intact molecules, both bispecific and parental, as well as some nonfunctional combinations of light and heavy chains of the parent Abs (Milstein, C. et al . , 1983. Nature 305 : 537) .
  • Two heterodimer- forming sequences derived from the leucine zipper regions of the transcription factors Fos and Jun were genetically fused with the Fab fragments of two different murine mAbs, anti-CD3 and anti-Tac (which binds to the human interleukin-2 receptor) .
  • Leucine zippers are specific amino acid sequences about 30 residues along with leucines occurring at every seventh residue. These sequences form amphipathic anti-helices, with the leucine residues lined up on the hydrophobic side for dimer formation.
  • the leucine zipper peptides of Fos and Jun have a much greater tendency to form heterodimers rather than homodimers (O'Shea, E. K. , 1989, Science 245:646) .
  • the F(ab' -zipper) 2 homodimers were individually expressed in the murine myeloma cell line Sp2/0 and purified by affinity chromatography from culture supernatant. Homodimeric proteins Fos and Jun were reduced at the hinge region using 2-mercaptoethylamine to form Fab' -zipper monomers. At this point, the Fos and Jun monomers were mixed together at a 1:1 ratio and reoxidized, and the resulting products were purified on an FPLC system. The heterodimers constituted 82% of the eluted F (ab' -zipper) 2 materials.
  • Bispecific anti-CD3 x anti-Tac F (ab' -zipper) 2 heterodimers were highly effective in recruiting cytotoxic T cells to lyse target cells in vi tro, whereas the homodimers in combination were totally ineffective.
  • this method appears to provide an effective means for producing bispecific antibodies with little side-product formation.
  • all known bs-mAbs are composed of murine mAbs or humanized murine mAbs.
  • their use in human therapy carries the threat of inducing an immune response against the murine portions of the therapeutic molecule (Kwak et al . , supra) .
  • HIV-specific human bs-mAbs and methods for their production and use have not been disclosed. The invention disclosed herein is directed to such antibodies.
  • the present invention relates to a novel bispecific mAb (bs-mAb) specific for two neutralizing epitopes of HIV, and the clinical use of such a bs-mAbs for immunoprophylaxis or immunotherapy of HIV infection.
  • bs-mAb bispecific mAb
  • the present invention is directed to a human bs-mAb in which one variable region is specific for an epitope of the V3 loop of an HIV glycoprotein gpl20 and which antibody is capable of neutralizing HIV.
  • the antibody may be specific for two different epitopes of the V3 loop of the gpl20 or for an epitope of the V3 loop and an epitope of the CD4-binding domain of the gpl20.
  • human bs-mAb as above, wherein the V3 epitope is a peptide of at least 4 amino acids from the amino acid sequence YNKRKRIHIGPGRAFYTTKNIIG (SEQ ID NO:l) .
  • the V3 epitope for which the human bs-mAb is specific is preferably an epitope included in SEQ ID N0:1, selected from the group consisting of HIGPGR, HIGPGRA, IGPGRAF, GPGRAFY, PGRAFYT, GRAFYTT, GPGRAF, RKRIHIG, KRIHI, KRIHIGP, IHIGPGR, IHIGP, IGPGR, GPGR, and GRAF.
  • RKRIHIGPGRAFYTR SEQ ID NO:2
  • HIGPIHIGP SEQ ID NO:3
  • GPGRVI SEQ ID NO:4
  • GPGRTL SEQ ID NO:5
  • GPGRVW SEQ ID NO:6
  • the above human bs-mAb is specific for an epitope having the amino acid sequence GPXR (SEQ ID NO:7) , where X may be any amino acid residue.
  • the human bs-mAb is preferably one in which the one variable region specific for a V3 loop epitope is derived from a human mAb selected from the group consisting of 257-2D, 268-10D, 311-11D, 386-10D, 391/95-D, 412-D, 418- D, 419-D, 447-52D, 453-D, 504-10-D, 537-D, 694/98-D, 782-D, 838-D and 908-D.
  • a human bs-mAb specific for an epitope of the V3 loop and an epitope of the CD4-binding domain of the gpl20 wherein:
  • one variable region is derived from a human mAb selected from the group consisting of 257-2D,
  • a human bs-mAb having one variable region specific for an epitope of the CD4-binding domain of HIV glycoprotein gpl20 and capable of neutralizing HIV.
  • the one variable region is derived from a human mAb selected from the group consisting of 448-D, 559/64-D, 588-D, 654-D, 728/29-D, 729-D, 855-D and 860-D.
  • a preferred human bs-mAb is selected from the group consisting of the human bs-mAbs 729/447D, 654D/447D, 694D/447D and 782D/447D.
  • Also provided is a method for neutralizing HIV comprising contacting the HIV with an effective amount of a human bs-mAb as described above.
  • the present invention is further directed to a cell line producing a human bs-mAb as above.
  • the cell line is a hybrid hybridoma.
  • the cell line may also be a mammalian or bacterial cell transfected with DNA encoding heavy and light chains of a bs- mAb as above.
  • the present invention also provides a composition useful for neutralizing HIV, preventing infection with HIV or treating a subject infected with HIV, comprising an effective amount of one or more bs- human monoclonal antibodies as above.
  • the composition may further comprise a drug useful in the treatment of HIV infection or AIDS.
  • a pharmaceutical composition comprising the bs- mAb as above and a pharmaceutically acceptable excipient.
  • the present invention is also directed to a method for preventing infection of a subject by HIV-1, or for treating a subject infected with HIV-1, comprising administering to the subject an effective amount of a human bs-mAb as above, or administering an effective amount of a composition or pharmaceutical composition, as above.
  • the present invention also provides a method for producing a hybrid hybridoma cell which makes a human bs- mAb specific for two neutralizing epitopes of the gpl20 protein of HIV, comprising:
  • one of the neutralizing epitopes recognized by the bs-mAb is from the V3 loop of the gpl20 glycoprotein, both of the neutralizing epitopes are from the V3 loop of the gpl20 glycoprotein, or one of the neutralizing epitopes is from the V3 loop of the gpl20 glycoprotein and the other of the neutralizing epitopes is from the CD4-binding domain of the gpl20 glycoprotein.
  • a method for producing a human bs-mAb specific for two neutralizing epitopes of the gpl20 protein of human immunodeficiency virus comprising: (a) producing a cell line using a method as described above ;
  • Figure 1 shows elution profiles of two HIV- specific bs-mAbs purified by HPLC.
  • Panel A shows the HPLC purification of supernatant of the hybrid hybridoma designated 729/447D.
  • Panel B shows the HPLC purification of supernatant of the hybrid hybridoma designated 71- 41/447D.
  • Supernatants were first purified on Protein A columns and applied to a Bakerbond Abx HPLC column.
  • the elution gradient was 10 mM MES, pH 5.6 -60% of 500 mM sodium acetate, pH 7.0, run at 1 ml/min over 1 hour.
  • the elution of protein was monitored by measuring absorbance at a wavelength of 280 nm.
  • Peak 1 Proteins not binding to the column eluted in the early fraction (peak 1) .
  • Parental mAbs 729-D and 71-31 eluted in the second fraction (peak 2)
  • parental mAb 447-52-D eluted in the fourth fraction (peak 4)
  • Peak 3 contains the bs-mAbs.
  • Figure 2 is a SDS-polyacrylamide gel electrophoresis pattern (under reducing conditions) of samples obtained from an HPLC column.
  • Single heavy (H) chain bands in Fractions 2 and 4 indicate the presence of parental mAbs, 729-D and 447-D, respectively.
  • Double H chain bands in fraction 3 represent the bs-mAbs 729/447D.
  • L indicates the presence of light chains.
  • the present invention relates to a bispecific human monoclonal antibody (bs-mAb) specific to two different neutralizing epitopes of HIV and pharmaceutical compositions comprising such an antibody, methods of producing such bs-mAb and methods of using such bs-mAb in - li ⁇ the prophylaxis or treatment of HIV infection and AIDS
  • bs-mAb bispecific human monoclonal antibody
  • the present invention provides a human bs-mAb specific for different epitopes of the V3 region and the CD4-bd of the viral gpl20 protein which antibodies neutralize HIV-1.
  • the bs-mAbs are typically generated by the fusion of two parent lines each producing a human mAb.
  • Preferred parent cells lines are human hybridomas or mouse- human heterohybridomas, or human lymphoblastoid cell lines which produce a human mAb specific for HIV.
  • Preferred cell lines are those shown in Table I, below, produced by the present inventors, each of which produces a human mAb which neutralizes HIV-1. Sixteen of these mAbs mediate the
  • any one of three different classes of bs-mAbs is preferred.
  • the first class is a bs-mAb directed against two different epitopes of the gpl20 V3 region.
  • Such a bs-mAb may be specific for two V3 epitopes of the most common HIV strain, HIV MN .
  • the bs-mAb is made by fusing two cell lines, the first of which produces a mAb which reacts strongly with the V3 region of HTLV UIB or HIVpj- and the second of which makes a mAb highly specific for the V3 region of HIV MN .
  • This second type of doubly V3-specific bs- mAb has a broader range of reactivity against different HIV strains than does the first type described above.
  • Bs-mAbs of the second class are directed against two different epitopes of the CD4-bd of gpl20.
  • the parent lines producing human mAbs are selected on the basis of the specificity of their anti-CD4bd mAbs such that the parent mAbs react with different sites within the CD4bd.
  • a third class of bs-mAbs is specific for two different neutralizing domains of gpl20: the V3 loop and the CD4-bd.
  • heterohybridomas making mAbs which act synergistically in the neutralization of HTLV IIIB and/or HIV MN are fused to make bs-mAbs specific for both a V3 epitope and a CD4-bd epitope.
  • a bs-mAb of the present invention is tested for its ability to bind to HIV-infected cells and to mediate neutralization of viral infection.
  • the neutralization is preferably tested using laboratory strains of HIV as well as various primary clinical HIV isolates.
  • the neutralizing activity of the bs-mAb is generally compared with that of the two parental mAbs.
  • Parent anti-V3 mAbs are selected for producing a bs-mAb on the basis of their HIV neutralizing activity, as well as their cross-reactivity between different HIV strains.
  • the mAb 447-D is a preferred choice as a parent supplying one of the antigen binding portions of a bs-mAb due to its unique specificity for the most conserved region of the V3 loop and due to its broad HIV strain reactivity.
  • Another unusual feature of the 447-D mAb is its IgG3 isotype.
  • the bs- mAb of the present invention is purified from parental type mAbs produced by hybrid hybridoma cells using standard purification techniques well-known in the art, including high performance liquid chromatography (see below) .
  • Another criteria for selecting parental mAbs for production of a bs-mAb is their binding activity to primary HIV isolates.
  • the present inventors' group recently showed that all 14 anti-V3 human mAbs which bound to gpl20 V3 peptides neutralized HIV MN (Gorny, M.K. et al., 1993, J " . Immunol . 150 : 635 ) .
  • the V3-specific mAb used to produce a bs-mAb of the present invention is preferably one which is specific for an epitope of at least 4 contiguous amino acids from the amino acid sequence YNKRKRIHIGPGRAFYTTKNIIG (SEQ ID NO:l) .
  • Other preferred mAbs are those specific for a peptide from within SEQ ID NO:l selected from the following group: KRIHI, KRIHIGP, IHIGPGR, HIGPGR, IGPGR, HIGPGRA, GPGR, GRAF, and RKRIHIGPGRAFYTR.
  • V3- specific mAb is specific for the GPGR epitope and is characterized by a broad cross-reactivity among HIV strains, such as the human mAb designated 447-D (or 447- 52D) . Also preferred is a mAb specific for GPXR (SEQ ID NO:7) , wherein X is any amino acid residue
  • the preferred binding assay for testing and selecting a mAb for bs-mAb production is based on a previously used antigen-capture ELISA.
  • the stock virus which is derived from supernatants of primary isolate cultures, is treated with Empigen ® and then used as a source of gpl20.
  • the gpl20 antigen is immobilized onto a solid phase carrier, preferably the surface of the wells of a 96-well microplate, with an antibody specific for a C- terminal portion of gpl20.
  • the binding of an anti-V3 mAb to the immobilized gpl20 is scored as the ratio of "positive" to "negative" wells (the P/N ratio) .
  • the level of gpl20 in a given virus stock is measured by another antigen-capture ELISA in order to normalize the concentration of gpl20 from different stocks.
  • the reactive mAbs are preferably also tested by flow cytometry for binding to cells infected with the HIV of the primary isolates.
  • Known staining procedures are used (Tyler, D. S. et al., 1990, J " . Immunol . 145 : 3276) .
  • cells from the primary cocultures are incubated with mAbs and then stained with enzyme-labeled anti-human immunoglobulins.
  • Flow cytometric analysis is performed using, for example, a FACScan ® flow cytometer. In this way, it is possible to select an anti-V3 mAb with the desired characteristics to serve as a "partner" for 447-D in the production of a bs-mAb.
  • the human mAbs which bind to gpl20 and block attachment of virus to the CD4 molecule on the surface of target cells recognize a conformational epitope.
  • the CD4- bd is does not act as a linear epitope but rather involves several constant domains including the C2, C3 and C4 domains of CD4.
  • Target epitopes for an anti-CD4-bd mAb which is a preferred parent for producing a bs-mAb can be determined with some approximation using the "Sodroski mutants" of HIV which have single amino acid changes in the conserved residues of gpl20 (Olshevsky, U. et al . , 1990, J " . Virol . 64:5701) .
  • the present inventors' group mapped the target epitopes of three anti-CD4-bd mAbs, termed 448-D, 559-D and 588-D.
  • Recognition of the envelope glycoproteins by 448-D was affected by different changes in the gpl20 amino acid residues to a greater extent than was recognition by mAbs 559-D and 588-D (McKeating, J. A. et al . , 1992, Virology 150:134) .
  • mAbs 559-D and 588-D McKeating, J. A. et al . , 1992, Virology 150:134
  • three more mAbs have been mapped more recently.
  • the mAb 654-D appeared to be unique among the anti-CD4-bd mAbs in terms of its binding to mutants.
  • the ones which bind strongly to gpl20 of primary isolates and/or to infected cells are used in combination with either 654-D or 448-D for producing a bs-mAb. Since all the anti-CD4-bd mAbs described above are of the IgGl isotype, the bs-mAbs are distinguishable from the parental mAbs on the basis of their different light chains: K versus ⁇ .
  • the mAbs 654-D (IgGl, ⁇ ) or 448-D (IgGl, ⁇ ) can be used for making bs-mAbs with a selected mAb from the group having K light chains (559-D, 588-D or 729-D.
  • K light chains 559-D, 588-D or 729-D.
  • the molecular genetic approaches described herein utilizing leucine zipper peptides for the production of bs- mAbs should allow the use of different combinations of parental mAbs which do not have distinct H chain or L chain isotypes. 5.1.3. BS-mAbs SPECIFIC FOR A V3 LOOP
  • Preferred mAbs for constructing a bs-mAb which recognizes a V3 loop epitope and a CD4-bd epitope are those which (a) bind strongly to primary HIV isolates, (b) are broadly reactive with various V3 peptides and (c) have potent virus neutralization activity against laboratory strains of HIV.
  • the present inventors' group and Tilley's laboratory provided the first descriptions of synergistic activity between two neutralizing anti-HIV mAbs, one specific for V3 and one for the CD4-bd, in the neutralization of HIV IIIB (Buchbinder, A. et al . , 1992, AIDS Res . Hum . Retrovir. 8:425; Tilley, S.A. et al . , 1992, AIDS Res . Hum. Retrovir. 8:461) .
  • Synergistic activity against two other strains of HIV-1, HIV MN and HIV Ala may also be tested to determine if the effective mixtures synergize in the neutralization of both virus strains or against only one. Results are analyzed for synergistic, additive or antagonistic activity using the computer program of Chou and Chou (DOSE-EFFECT ANALYSIS WITH MICROCOMPUTERS, Biosoft, Cambridge, 1987) .
  • Synergistic neutralizing activity between two mAbs is attributable to the ability of one neutralizing mAb to enhance the binding, and therefore the activity, of the other.
  • a mAb to the CD4-bd may cause a stereochemical change in the gpl20 molecule so that the V3 loop is better exposed for binding of an anti-V3 mAb (Mckeating, J. A. et al., 1992, Virology 191 : 732 ) .
  • the antibodies may act in the opposite sequence. (Such synergistic activities are described in co-pending commonly assigned U.S. patent application Serial Number 07/776,772, filed October 17, 1991 and incorporated by reference herein . )
  • Hybrid- hybridomas are preferably produced by a fusion technique developed in the present inventors' laboratory (Gorny, M.K. et al . , 1989, Proc . Natl . Acad. Sci . USA 86:1624; Gorny, M.K. et al . , 1991, Proc . Na tl . Acad. Sci . USA 88 : 3238 ) .
  • hybridomas which produce bs-mAbs For selecting hybridomas which produce bs-mAbs, one "parent" hybridoma is back-selected for hypoxanthine, aminopterin and thymidine (HAT) -sensitivity prior to fusion. This procedure is carried out by growing cells in the presence of 8-azaguanine (30 ⁇ g/ml) which induces an enzyme deficiency for hypoxanthine guanine phosphoribosyl transferase (HGPRT) . The hybridoma cells lacking HGPRT fail to grow in medium containing HAT, since both the DNA and RNA biosynthetic pathways are blocked. The other
  • hybridoma or lymphoblastoid cell line
  • a lethal dose 5 mM
  • the irreversible biochemical inhibitor iodoacetamide
  • Cells treated in this way are rendered non-viable unless "rescued” by fusion.
  • the selecting medium contains HAT and ouabain. Lymphoblastoid cells that result from Epstein Barr virus transformation of peripheral blood lymphocytes are ouabain-sensitive and are killed when unfused.
  • Both parental cell lines are preferably mixed at a ratio of about 1:1, and cell fusion is induced using polyethylene glycol with dimethyl sulfoxide (DMSO) .
  • the fused cells are plated at a density of about 8 x 10 4 cells/well on a feeder layer of about 1 x 10 4 murine peritoneal cells.
  • two to three weeks post-fusion all culture wells are screened for production of bs-mAbs in the supernatant using one or more techniques (described below) and the positive cultures are expanded in 24-well plates.
  • Hybrid-hybridomas that produce the highest level of antibodies as measured in ELISA are cloned at 100 to 1 cell/well. After cloning, the positive wells are expanded into flasks.
  • hybrid hybridomas are grown in the peritoneum of SCID mice. Mice are primed by intraperitoneal (i.p.) injection of 0.5 ml pristane (2,6,10,14 tetramethyldecanoic acid; Sigma Chemicals) . After 7-14 days, about 1-5 x 10 6 hybridoma cells are injected i.p. and ascites fluid is collected a few weeks later. A single mouse may yield 2-10 ml of ascites fluid. Antibody concentrations are generally between 1 and 10 mg/ml.
  • the presence of antibodies specific for peptides or recombinant proteins of HIV is determined using a direct ELISA.
  • mAbs specific for the V3 loop are detected by ELISA using 20-23-mer peptides that represent the V3 regions of HIV-strains MN, RF, and HXB2 (American Biotechnologies, Cambridge, MA) .
  • Antibodies to the CD4-bd which are specific for a discontinuous epitope, are identified with recombinant gpl20 from HIV IIIB (rgpl20 IIIB ) prepared in baculovirus-infected insect cells (Repligen, Cambridge, MA) .
  • antigens are coated onto plastic microplates at a concentration ranging from about 0.2 to 1.0 ⁇ g/ml.
  • the culture supernatants or purified mAbs are tested for reactivity to these antigens by a standard direct ELISA, such as that previously described by the present inventors' group (Gorny, M.K. et al . , 1989, Proc . Natl . Acad. Sci . USA 86:1624; Gorny, M.K. et al . , 1991, Proc. Natl . Acad. Sci . USA 88:3238) .
  • the hybrid hybridoma cells producing bs-mAbs are screened preferably by either a single antigen ELISA, a double isotype ELISA, or by an antigen-isotype ELISA.
  • a single antigen ELISA the culture supernatant is tested separately for its binding to plates coated with one of two different antigens.
  • the double isotype ELISA is used when bs-mAbs consist of two different isotypes, such as IgGl and IgG3.
  • an antibody to one of the isotypes e . g. , anti-IgGl
  • the antibody for the second isotype e . g.
  • anti-IgG3 is enzyme-labeled.
  • a positive well indicates the presence of a bs-mAb that binds to both the anti-IgGl and anti-IgG3 reagents .
  • the antigen-isotype ELISA is employed when the bs-mAbs consist of two different isotypes with two specificities that recognize two different peptides.
  • a bs-mAb may consist of a portion having the IgGl isotype and specificity for an epitope of the CD4-bd and a portion having the IgG3 isotype and specificity for an epitope of the V3 loop.
  • the V3 peptide is coated onto the plate and the binding of the bs- mAb is detected using an enzyme-labeled anti-IgGl reagent.
  • rgpl20 IIIB peptide is coated onto the plastic and the reactivity of the bs-mAb is detected using an enzyme-labeled anti-IgG3 reagent.
  • Binding of human mAbs to primary isolates of HIV-1 Preferred bs-mAbs are made using parental mAbs selected for their binding to the majority of HIV-1 primary isolates.
  • the present inventors have found that antibodies specific for antigens present in viral lysates at low concentrations are more readily detected using antigen - capture ELISAs than in direct ELISAs . Therefore, to detect binding activity of a human mAb to the gpl20 of a primary isolate of HIV-1, wells of microtiter plates (preferably Immulon-2) are coated with a commercially available sheep antibody specific for the C-terminus of gpl20.
  • the supernatants from primary cultures (see below) treated with 1% Empigen ® detergent and containing known levels of gpl20, are added to coated plates and incubated. After washing, human mAbs are added, followed by rabbit anti-human IgG coupled to alkaline phosphatase (or any of a large number of enzymes useful in ELISAs) and then a chromogenic substrate for the alkaline phosphatase (or for the other enzyme) .
  • the reaction can be further enhanced using the ELAST ® Amplification system.
  • This assay has been developed by the present inventors and is used for screening supernatants from primary cultures or as a read-out for a microtiter neutralization assay.
  • the assay has a limit of sensitivity of 150 pg/ml .
  • One human mAb specific for p24 (Gorny et al . , 1989, supra) is used to coat a microtiter plate, which is then blocked and washed.
  • Supernatants from primary cultures or from a neutralization assay are then added to the wells and the plate is incubated for about 3 hours.
  • the color reaction is developed using an enzyme-conjugated rabbit anti-p24 antibody. Diluted recombinant p24 in the range of about 0.2 to 20 ng/ml is used to determine the standard curve.
  • This assay was established in the present inventors' laboratory to determine the concentration of gpl20 in stocks of primary HIV isolates treated with the Empigen ® detergent .
  • the wells of microtiter plates are coated with a sheep antiserum specific for the C-terminus of gpl20. After washing and blocking, the plates are incubated with various dilutions of lysed virus.
  • An aliquot of an affinity purified pool of human mAbs directed to the CD4-bd is added, followed by an enzyme- (e.g., alkaline phosphatase) labeled anti-human IgG.
  • a standard curve is constructed using rgpl20 IIIB (Repligen) as a reference reagent in a range of about 0.1-20 ng/ml. The standard curve is used to estimate the concentration of gpl20 in a sample.
  • Antibodies are purified from culture supernatants using well-known methods.
  • the antibodies are preferably first by precipitated with 50% saturated ammonium sulfate and the resulting precipitate dissolved in phosphate buffered saline (PBS) and dialyzed against several changes of PBS.
  • Antibodies are further purified, first by Protein A affinity chromatography (Pharmacia) and subsequently by ion-exchange HPLC, for example, using a 7.75 x 100 mm Bakerbond Abx column (J.T.Baker, Phillipsburg, NJ) .
  • Samples are then preferably dialyzed against 10 mM 2- (N-morpholine) ethanol sulfonic acid (MES) and chromatographed in a linear gradient of 10 mM MES, pH 5.6, 500 mM sodium acetate (NaAc), pH 7.0, over a 60 minute period.
  • Immunoglobulins are collected in separate fractions every minute and then tested for antigen binding or binding to an isotype-specific antibody in the double isotype or antigen-isotype ELISA described above.
  • Samples of purified mAbs and bs-mAbs are diluted into a buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.002% bromphenol blue, 10 mM Tris HCl, pH 6.8, '• and are boiled for 3 minutes.
  • a 20 ⁇ l volume is electrophoresed on a 10% polyacrylamide gel according to standard methods (Laemmli, E.K., 1970, Nature 227:680) . Proteins are stained preferably using Coomassie blue.
  • Friguet et al is preferably employed (Friguet, B. et al . , 1985, J. Immun . Meth . 77:305) . Briefly, purified bs-mAbs and parental mAbs are diluted to concentrations ranging from 0.5 to 2.5 ⁇ g/ml . To establish the equilibrium phase in solution, the antibodies are incubated 16 hr at room temperature with antigen (V3 loop peptides or rgpl20 mB ) at various concentrations ranging between about 10 nM and 10 ⁇ M.
  • antigen V3 loop peptides or rgpl20 mB
  • Each mixture is added to microwells coated directly with the homologous antigen at a concentration of about 1 ⁇ g/ml .
  • the excess of antibodies not complexed with antigen in solution is measured by ELISA.
  • Absorbance and antigen concentration values are plotted according to the Friguet modification of Klotz (Friguet et al. , supra) to determine the K d .
  • the K d is generally taken as the average value of three or more determinations.
  • Three different methods for the production of primary HIV isolates may be used to generate viral proteins which are optimal for testing the binding activity of human mAbs to gpl20.
  • the binding capacity of the antibody serves as an indicator of the suitability of a mAb for bs-mAb production.
  • PBLs Peripheral blood lymphocytes derived from seropositive patients and from uninfected subjects are first stimulated independently for three days with phytohemagglutinin (PHA) and are then cocultured at a 1:3 ratio in the presence of medium supplemented with interleukin-2 (IL-2) . The cells are fed about twice a week with fresh medium.
  • PHA phytohemagglutinin
  • the culture supernatants are collected prior to feeding and used either as stock virus for neutralization assays (after titration) or treated with 1% Empigen ® detergent.
  • Supernatants with the highest p24 levels are be pooled and used as a source of viral proteins.
  • the second method is designed to avoid possible selection of variants which may occur during coculture.
  • Donors' PBLs and CD4+ T cells from infected patients are stimulated directly to enhance HIV-1 production (Hausner, M.A. et al . , 1993. J. Immunol Meth . 157:181) .
  • the patient PBLs are incubated for 1 hour at room temperature in an activated AIS MicroCELLector ® CD4 capture flask (Applied Immune Sciences, Menlo Park, CA) in which anti-CD4 mAbs are covalently attached to the plastic surface.
  • non-adherent cells are removed by rinsing the flask and the adherent CD4+ cells are cultured in the flask in medium supplemented with human IL-2 and an anti-CD3 antibody, such as the mAb OKT3, for 5 days.
  • the supernatant is collected and treated as previously described.
  • Another source of primary HIV isolates is patient plasma.
  • Cell-free virus in the plasma is expected to reasonably reflect the virus actually replicating in the host (although passage through PBLs derived from healthy donors may again cause some unwanted selection) . Therefore, about 1 ml of plasma from a patient with high circulating p24 levels is incubated with about 2.5 x 10 6 PHA-stimulated normal donor PBLs overnight. After washing, the cultures are maintained in medium supplemented with IL- 2 and fed bi-weekly. Supernatants are collected before feeding and tested for p24 antigen. These supernatants can be used either as a source of primary isolates for neutralization or as viral proteins after treatment with Empigen ® detergent.
  • the present inventors have established the neutralizing activity of over 20 human mAbs specific for HIV-1
  • various serial two-fold dilutions of a bs- mAb and, separately, its parental mAbs are incubated for 1 hr at room temperature with culture supernatants of HIV MN or HIV mB which have a titer of about 100-200 syncytium-forming units/ml.
  • the virus/antibody mixture is added to cells of the CEM-SS line previously adhered to the wells of a poly-L- lysine-coated 96-well plate, and the plate is incubated for 1 hr at 37°C.
  • the washed cells are then cultivated for 3 days and the amount of p24 produced in culture supernatants is assayed by antigen-capture ELISA.
  • PBLs of HIV-seropositive subjects Stocks of primary isolates from the supernatants of PBL cocultures are first titered using a p24-based assay system. Antibodies (bs-mAbs, parental mAbs and control mAbs specific for unrelated antigens) at different concentrations are added to 100 TCID 50 of primary HIV isolates in a volume of 100 ⁇ l and incubated for 1 h at room temperature. PBL (10 6 ) derived from seronegative donors, which cells have been stimulated with PHA for 3 days, are then exposed to the virus/mAb mixture or to medium alone after which the cells are incubated for 1 h at 37°C.
  • the resultant mixtures are transferred to 24-well plates, washed once and then cultured for 7 days.
  • the culture supernatants are tested for p24 levels by antigen-capture ELISA.
  • Neutralization is defined by the method used for neutralization of laboratory strains.
  • Another neutralization assay is preferably utilized to study the effect of bs-mAbs on viruses which are derived from primary isolates which have been maintained in culture for prolonged periods of time (versus a short period, as above) .
  • Antibodies bs-mAbs, parental mAbs or control mAbs
  • Antibody-containing cocultures of uninfected and HIV MN -infected PHA-blasts serve as positive virus controls.
  • the cultures are maintained for 21 days, and fed every 3 days.
  • the collected supernatants are assayed for p24.antigen.
  • the results are expressed as the antibody concentration needed for 50% neutralization.
  • Each bs-mAb of the present invention, produced as described herein, is preferably also tested for its ability to bind to virus-infected cells using flow cytometric methods described herein (see, Tyler et al . , supra) .
  • a biotinylated form of each bs-mAb is preferably used together with fluoresceinated - avidin.
  • Controls consist of staining uninfected cells or staining infected cells with biotinylated human mAbs specific for an unrelated antigen, such as a cytomegalovirus (CMV) antigen.
  • Functional studies include neutralization assays with (1) various laboratory isolates ( e . g. , IIIB and Ala) and with (2) primary HIV isolates from infected adults.
  • the mAbs described in Table I, above were selected for their specificities to various laboratory strains of HIV (Gorny, M.K. et al . , 1993, J. Immunol . 150 : 635 ; Gorny, M.K. et al . , 1992, J. Virol . 66: 7538 ; Karwowska, S. et al . , 1992, AIDS Res . Hum. Retrovir. 8:1099) .
  • Bs-mAbs are preferably tested against the same laboratory virus strains to prove that neutralizing activity is a property of the bs-mAb molecule. This activity is then compared to the parental mAb activity, to test whether incorporation of two different specificities into one antibody molecule results in greater virus neutralization capacity.
  • Use of laboratory isolates allows better standardization of the neutralization assay, and easier repetition and adaptation to different combinations of control Abs.
  • this assay alone is not sufficiently informative about the clinical utility of a given bs-mAb of this invention, because a laboratory strain of HIV represents one unique genome and phenotype of virus and does not reflect the complexity of the HIV-1 variants present in a single infected individual.
  • testing of the bs-mAb on primary HIV isolates is important for evaluation of natural immunity against HIV-1 infection, especially when mAbs or bs- mAbs are considered . as candidates for passive administration in the immunoprophylaxis or therapy of HIV infection.
  • Primary isolates comprise a mixture of closely related viral genomes which result from a high misincorporation rate caused by reverse transcriptase errors (Nara, P. L. et al . , 1991, FASEB J. 5:2437; Wain-Hobson, S., 1989, AIDS 3:sl3) . Variation among HIV isolates occurs within a given individual during the course of infection, as well as between individuals (Epstein, L.G. et al . , 1991, Virology 180 : 583 ; Meyerhans, A. et al. ,
  • virus variation occurs in the course of its amplification during in vi tro culture. Selection of virus variants adapted to tissue culture conditions induces and stabilizes alterations that are not necessarily representative of those which exist in an infected subject in vivo (Wain-Hobson, supra; Cheynier, R. et al . ,
  • the primary culture of HIV with IL-2 treated PBLs may preferentially select for lymphocytotropic viruses, at the expense of macrophage-tropic viruses.
  • primary isolates remain the most representative virus population available for testing the neutralizing activity of different anti-HIV antibodies in vi tro.
  • the virus is exposed to the bs-mAb for a brief period of time while in the second assay the bs-mAbs remain in the culture throughout the test.
  • a stock of primary HIV isolates which has been titered using a p24-based assay is incubated with the test bs-mAbs for about 1 hour.
  • Cultures without bs- mAbs or with non-neutralizing mAbs can serve as negative antibody controls, while cultures containing dilutions of known polyclonal serum pools can be used as positive antibody controls.
  • the mixture of virus and antibody is added to PHA- stimulated normal PBLs, the cells are then washed to remove antibody, and the cells then maintained in culture for 7 days . Effective virus neutralization is indicated by lack of p24 antigen in cultures treated with antibodies versus the presence of virus in cultures not treated or treated with a non-neutralizing mAb. Results may be expressed as the amount of IgG necessary for 50 and 90% neutralization.
  • the second assay is designed to mimic the situation in vivo under the most optimal conditions.
  • a bs-mAb and control antibody are added to the co-cultures of PHA-blasts from infected and uninfected donors.
  • the cultures are maintained for 21 days and the supernatants collected every 3 days and assayed for p24 antigen.
  • the cultures are fed with media supplemented with the appropriate antibody to maintain a constant antibody level .
  • Decreased levels of p24 antigen in culture supernatants may be explained as follows: (1) Cell-free virus is bound and neutralized by antibodies; (2) virus binding to CD4+ T cells is blocked; (3) the fusion or penetration of the virus into susceptible cells is inhibited; and/or (4) cell-to-cell transmission of the virus is prevented by the bs-mAb.
  • bispecific F(ab') 2 heterodimeric antibody fragments are produced in transfected cells using leucine zipper peptides from the Fos and Jun proteins.
  • the biological function of the new constructs including binding affinity to HIV antigens and the ability to neutralize laboratory strains and primary isolates of HIV, are compared with intact bs-mAbs.
  • bs-F(ab') 2 in large quantity is preferably carried out in mammalian cells.
  • An E. coli expression system is useful for further enhancement of production, and for producing bs-mAbs which can be utilized in animal studies and in the clinic.
  • mRNA is purified from cells producing human mAb, such as heterohybridoma cells, for example, 729-D, by lysis of the cells in a buffer containing SDS and proteinase K, followed by oligo-dT chromatography of the lysate. DNA and mRNA are removed by high and low salt washes, respectively, and polyA+ RNA is eluted in TE buffer (no salt) . The polyA+ RNA is then precipitated with ethanol and used directly in a reverse transcription reaction to convert it to first strand cDNA. First strand cDNA is used to amplify the specific light chain and Fd regions using the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Primers specific for the signal sequences of the immunoglobulin H or L chain is used in conjunction with primers in the hinge region or the region 3' to the L chain coding sequence.
  • cDNA fragments encoding the L chain and or the Fd fragment are cloned into pUC18 and several clones of each are sequenced using dideoxy technology and modified T7 DNA polymerase (Sequenase, US Biochemical) . This technology is well established and has been used by the present inventors to clone and sequence seven different human mAb H and L chain genes, including 447-D.
  • the leucine zipper regions of the Fos and Jun proteins are assembled from oligonucleotides. Since each of these segments is approximately 140 bp in length, four oligonucleotides from 70-75 bases long are required. Each 75mer is phosphorylated using T4 polynucleotide kinase and annealed to the appropriate 70-mer. The annealed pairs of oligonucleotides are then mixed and ligated together using T4 DNA ligase. The 140bp fragment is then purified using agarose gel electrophoresis. The assembled and ligated oligonucleotides are then joined to the Fd antibody fragment by recombinant PCR and cloned into mammalian expression vectors using the Ncol and Notl restriction sites introduced during PCR.
  • the respective Fd-zipper and L chain genes are inserted, as an Ncol-Notl fragment, into an expression vector, preferably pSJ301.
  • the pSJ301 vector contains the cytomegalovirus immediate early gene ( CMVie) promoter/enhancer for high level transcription of the immunoglobulin genes, a linker region containing a consensus translation initiation signal ("Kozak” sequence) as well as cloning sites for inserting genes, and the bovine growth hormone (bGH) polyadenylation signal for efficient RNA polyadenylation.
  • CMVie cytomegalovirus immediate early gene
  • Kozak consensus translation initiation signal
  • bGH bovine growth hormone
  • the expression cassettes for the L chain genes from two heterohybridomas, for example from 729-D and 447-D, and the Fd zipper gene are combined into a single plasmid using a strategy based on the compatible cohesive ends produced by the restriction enzymes BamHl and Bglll which flank the CMV promoter - polyA-i- region. This is accomplished by combining a Pvul-BamHl fragment from the L chain expression vector with a Bglll-Pvul fragment from the Fd expression vector.
  • Stable expression of the bivalent molecules is accomplished in a mammalian cell line, for example, the hfr-Chinese hamster ovary (CHO) cell line DG44.
  • the combined expression vectors for example the 447-D F(ab'- zipper) and 729-D F (ab' -zipper) expression vectors, are transfected separately into CHO cells by calcium phosphate coprecipitation and selected in nucleoside-free medium. Expression levels are measured after each subcloning step in methotrexate and only the highest producing colonies are carried forward. In this manner, the present inventors have routinely created cell lines stably producing, in under six months, antibody in quantities greater than 100 mg/liter/4 days.
  • the F(ab) ' 2 homodimers are purified by HPLC and/or affinity chromatography using anti-L chain antibody (anti- ⁇ for 447-D; anti- ⁇ for 729-D) immobilized on agarose.
  • Bivalent F(ab') 2 fragments are generated by mild reduction, mixing and reoxidation of the purified preparation.
  • F(ab' -zipper) The ability of transformed E. coli cells to secrete F(ab' -zipper) either to the periplasm or to the medium depends on several factors, most importantly the strength of the promoter used, the temperature, and the ability to tightly control expression.
  • the pelB leader is preferably used to secrete the individual chains expressed from the lac promoter.
  • Combined vectors are constructed using a similar strategy to that described above for the mammalian expression vectors.
  • Expression of the F (ab' -zipper) is induced by an appropriate inducer for the lac promoter, for example isopropylthio-jS-D-galactoside (IPTG) , at 25°C.
  • IPTG isopropylthio-jS-D-galactoside
  • Bacterially produced F (ab' -zipper) can be reoxidized directly after purification by dialyzing against redox buffer.
  • the resulting product should contain mostly heterodimers due to the preferential dimerization of proteins to which Fos and Jun are genetically fused.
  • the heterodimer 729D x 447D is purified using an HPLC system.
  • a combination of ELISA and SDS-PAGE are used to identify the peaks. 5.4. IN VIVO SCREENING OF bs-mAbs
  • the mAbs of the present invention may be screened for anti-HIV activity using a recently developed mouse model (CITE) .
  • CITE mouse model
  • SCID mice lacking T lymphocytes are infused with about 2 x 10 7 human peripheral blood lymphocytes i.p.
  • the mice are injected i.p. with the bs-mAbs being tested.
  • Other animals may receive control antibodies, the parental mAbs or a mixture of the two parent mAbs.
  • the animals are challenged with a preparation of HIV. At various time points thereafter, in particular about 6 weeks, the animals are examined for virus growth.
  • Neutralizing mAbs that are useful for therapy of humans are expected to neutralize the virus and limit infection in this SCID mouse model.
  • Bs-mAbs which incorporate two anti-viral specificities into a single molecule and neutralize HIV-1 with augmented potency over the parental mAbs are particularly useful in prophylaxis of HIV-1 infection.
  • Passive immunotherapy with the bs-mAbs of the present invention is useful in the prevention of transmission of the virus from an infected mother to a newborn, by treating the mother during pregnancy, and in particular, prior to delivery.
  • Another application for bs-mAbs of the present invention is in the prophylaxis of health care workers exposed to the virus through needle sticks. Needle injury is a common problem for medical staff in hospitals.
  • treating is intended the administering to subjects of a bs-mAb, or an HIV antigen-binding fragment or derivative thereof, such as the bs-F(ab') 2 fragment described above, for purposes which may include prevention, amelioration, or cure of HIV infection or AIDS.
  • Bs-mAb compositions within the scope of this invention include all compositions wherein the bs-mAb or derivative is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.01 to 100 mg/kg/body wt. The preferred dosages comprise 1 to 100 mg/kg/body wt. The most preferred dosages comprise 10 to 100 mg/kg/body wt. Administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • a pharmaceutical composition according to the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations comprise suitable solutions for administration by injection, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active bs-mAb together with the excipient.
  • suitable formulations for parenteral administration include aqueous solutions of the bs-mAb in water-soluble form, for example, as water-soluble salts.
  • suspensions of the bs-mAb as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions that may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
  • Suitable injectable solutions include intravenous subcutaneous and intramuscular injectable solutions.
  • the active compound may also be administered in the form of an infusion solution or as a nasal inhalation or spray.
  • the bs-mAb is preferably administered by drip infusion in a buffered aqueous solution.
  • the bs-mAb may be administered in single or divided doses.
  • the pharmaceutical composition of the present invention may be comprised of a bs-mAb as described herein in combination with another agent or medicament useful in treating HIV infection or symptoms associated with HIV infection or AIDS.
  • Medicaments are considered to be provided "in combination" with one another if they are provided to the patient concurrently or if the time between the administration of each medicament is such as to permit an overlap of biological activity.
  • the bs-mAbs of the present invention may be administered in combination with known anti-retroviral agents, for example, AZT, ddA, ddT, ddl, and ddC.
  • known anti-retroviral agents for example, AZT, ddA, ddT, ddl, and ddC.
  • Many additional anti-retroviral drugs known in the art which inhibit the activity of various HIV genes are included within the scope of this invention.
  • anti-retroviral peptides include anti-retroviral peptides, soluble CD4 peptides, or immunotoxins (Vitetta, E. et al., Science 215:644-650 (1983) ; Pastan, I. et al . , Cell 47:641-648 (1986) ; Olsnes, S. et al., Immunol . Today 10 : 291-295 (1989) ; Oeltmann, T.N. et al . , FASEB J. 5:2334-2337 (1991)) comprising such antibodies or CD4 peptides.
  • Anti-bacterial or anti- parasitic drugs such as drugs directed to Pneumocystis carinii disease, and drugs useful for alleviating any symptoms associated with AIDS, are included within the scope of the combination treatment methods and bs-mAb compositions of the present invention.
  • the bs-mAbs of the present invention may be used diagnostically to detect HIV in cells or body fluids, or to characterize the viral strain infecting a subject.
  • the bs-mAbs are used in the same manner in which mAbs or polyclonal anti-HIV sera are used in immunodiagnosis.
  • an HIV-1 population is not a homogeneous virus population but is rather a mixture of closely related variants (Wain-Hobson, S., 1992, Curr. Top. Micro . Immunol . 176:181) . These variants are subjected to selection by the host immune system resulting in the emergence of variants which can evade immune defenses. Studies performed in chimpanzees, and in vi tro using neutralizing antibodies or patient sera have demonstrated the immune selection of neutralization- resistant mutants (Reitz et al . , supra; Cotropia, J. et al .
  • VACCINES 92 MODERN APPROACHES TO NEW VACCINES INCLUDING PREVENTION OF AIDS, F. Brown et al . . eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 157) .
  • Escape mutants selected by neutralizing antibodies exhibited sequence changes in proteins which affected the antibody binding sites.
  • the bs-mAbs of the present invention with specificity against the V3 loop and/or the CD4-bd can be used for the selection of HIV escape mutants in vi tro .
  • a single neutralizing mAb may select a resistant HIV variant through a change or changes which can affect the epitope and shroud it from the mAbs.
  • more than one set of changes may be necessary to select a neutralization-resistant mutant.
  • the rate of mutations in the HIV-1 genome is relatively constant, approximately one mutation per genome per replication cycle (Dougherty, J.P. et al . , 1988, J. Virol . 62 : 2817) . While the frequency of appearance of escape mutants in the presence of one mAb will be as high as 10 "5 , in the presence of bs-mAb, the frequency will be in the order of 10 "10 .
  • bs-mAbs are expected to be advantageous over single mAbs and induce the emergence of fewer variants during the same time frame.
  • the present invention allows one to establish the frequency of appearance and the selection of HIV-1 escape mutants propagated in the presence of bs-mAbs as compared to the two parental mAbs.
  • Bs-mAbs may select escape mutants differently than parental mAbs.
  • This comparative analysis is based on: (1) the quantitative difference in the appearance of escape mutants as measured by the number of wells in a 96-well plate where p24 antigen is present, (2) the time period during which the escape mutants appear, and (3) the pattern of sequence changes in gpl20.
  • a quantitative assay was developed by the present inventors in which CEM-SS cells are infected with the molecularly characterized viral inoculum of HXB2D.
  • a virus stock is prepared for standardizing the virus input for selecting escape mutants.
  • the viral clone of HXB2D virus is obtained by transfecting H9 cells with the complete molecular proviral clone, pHXB2D, using a lipofection transfection procedure according to the manufacturer's instructions (GIBCO BRL, Gaithersburg, MD) .
  • the transfected H9 cells are monitored for productive viral infection by the detection of p24 using an antigen-capture assay.
  • the supernatant is harvested, filtered and used as a stock of cell-free virus.
  • the infectious titer of the stock is determined by end-point titration using 10-fold serial dilutions of virus (50 ⁇ l) incubated with 100 ⁇ l of CEM-SS cells at 2 x 10 5 cells per well in microtiter plates at 37°C for 7 days. The plates are scored for the presence of p24 antigen and the 50% TCID 50 is calculated.
  • CEM-SS cells at a density of 10 5 cells/well are exposed to 100 and 1000 TCID 50 of HXB2D virus stock for 1 hr at 37°C.
  • the unbound virus is removed by washing, and the cells are then cultured in the presence of various dilutions of either neutralizing bs-mAbs, parental mAbs or non-neutralizing irrelevant (control) human mAb at concentrations of 10 and 100 ⁇ g/ml.
  • the cultures are fed periodically (preferably biweekly) with medium containing the appropriate antibody and the supernatant is monitored once a week for the presence of p24 antigen. While the first escape mutants may appear after only 2 weeks, the cultures are preferably monitored for six weeks.
  • the percentage of wells containing p24 antigen indicates the frequency of appearance of escape mutants selected by either the bs-mAbs or the parental mAbs at their respective concentrations.
  • the time period during which the escape mutants appear may also indicate a difference in the selection effect between neutralizing bs- Abs and parental mAbs.
  • Virus is recovered from those wells with a high p24 level as cell-free supernatant and the progeny viruses are propagated in CEM-SS cells in the absence of antibodies. The virus stock is then tested for resistance to neutralization by the selecting mAbs. Recently, the present inventors' group characterized a human serum-selected HIV-1 escape mutant which was resistant to neutralization by CD4-bd mAbs. Virus was recovered after two weeks from the cultures containing the lowest serum dilutions (1/160 and 1/320) . Over a 7 week period no evidence of viral replication was observed at the highest concentration of selecting serum (1/40) .
  • Determination of a bs-mAb concentration which blocks the selection of escape mutants provides useful information for selecting bs-mAbs and their doses for use in a passive immunotherapy approach.
  • the escape mutants are subjected to nucleic acid sequencing for comparative analysis of mutations which occurred during culture in the presence of either the bs-mAb or the parental mAbs.
  • DNA is prepared by standard methods from cultures containing neutralization escape mutants.
  • the polymerase chain reaction (PCR) assay is performed according to the supplier's specifications. All oligonucleotide primers are chosen from HIV-HXB2CG GenBank sequence files.
  • Gpl20 gene amplifications are carried out in two steps by using a model 480 thermal cycler (Perkin Elmer/Cetus) .
  • the first amplification step consists of 30 cycles of PCR using oligonucleotide primers REC-07 and REC-09 (Smith, M.S. et al . , 1993, J. Infec . Dis . 167:445.) .
  • the second amplification step of 30 cycles uses 5% of the product from the first amplification, together with primers REC-27 and REC-12.
  • the 1550-base-pair gpl20 PCR products are purified by agarose gel electrophoresis and are subcloned in an expression vector, preferably M13mpl8. Pure single stranded DNA is prepared from this vector multiplied in infected E. coli cells and is sequenced directly using the Sequenase kit version 2.0 (U.S. Biochemical) .
  • the mAb 447-52-D which is the most broadly reactive with diverse HIV-1 strains (Gorny et al . , 1992, supra) , was tested for its ability to interact with other mAbs specific for the CD4-bd or with other V3-specific mAbs in neutralizing HIV.
  • Combination Index was calculated according to Chou et al . ( supra) .
  • a CI value which is less than 1.0 indicates synergy; a CI value of around 1.0 demonstrates additivity.
  • hybridoma cell line 447- 52-D was back selected for HAT-sensitivity and was fused separately with two hybridomas producing mAbs to the CD4-bd (654-D and 729-D) .
  • the 447-52-D cell line was also fused with hybridomas 694/98-D and 782-D making mAbs to the V3 loop.
  • 694/98-D and 782-D were chosen for the production of bs-mAbs on the basis of their strong reactivity and high affinity to HIV IUB and HIV RF , respectively, whereas 447-52-D had the most prominent activity against HIV MN .
  • bs-mAbs made with 447- 52-D and 694/98D or 782-D would have broadened specificity and an ability to neutralize a broader array of HIV-1 variants.
  • control cell line producing a non- neutralizing human mAb to p24 designated 71-31 was fused to the cell line producing 447-52-D to construct a "control bs-mAb” .
  • Table III describes the production of (a) hybrid hybridomas ("quadromas”) derived from the fusion of two hybridomas, and (b) one trioma (71-31/447D) obtained by fusion of an EBV transformed cell line with a hybridoma.
  • the frequency of wells containing hybrid hybridomas ranged from 8% to 65%.
  • the percentage of wells containing hybridomas which produced bs-mAbs ranged between 3% and 57%.
  • the hybrid hybridomas and trioma were cloned at 100, 10 and 1 cell/well to stabilize the production of bs- mAbs. After cloning, the positive wells were expanded into flasks.
  • Hybrid- Wells Hybrid- Wells with specific Abs Hybridoma Plated Hybridomas IgGl IgG3 bs-mAbs a
  • Peak 4 contained the second parental mAb, 447-52-D based on binding to the V3 MN peptide.
  • the 71-31/447D bs-mAb ( Figure 1, panel B) fractions from peak 4 contained antibody which bound to the V3 MN peptide and reacted with both anti-IgG3 (the isotype of 447-52D) and anti-IgGl (the isotype of 71-31) . This indicated the presence of some bs-mAb in this peak.
  • Peak 3 contained bs-mAbs only. This conclusion was based on the strong reactivity of fractions included in this peak in an antigen-isotype ELISA in which the antibodies bound V3 MN peptide while the of the IgGl isotype was associated with antibody to p24 but not V3 MN .
  • fractions comprising peaks 3 and 4 of bs-mAb 729/447-D were re-chromatographed by HPLC and, along with fractions from peak 2, were reduced and examined by SDS- PAGE ( Figure 2) .
  • the peak 3 fractions had showed bands representing the two H chains (an IgGl H chain and an IgG3 H chain) of the bs-mAb.
  • fractions from peak 2 and peak 4 each showed a single band, each of different mobility, indicative of H chains of the parental mAbs.
  • the CI for this mixture was 0.1, though the CI for the bs-mAb purified by HPLC was 10- fold lower. Furthermore, the final concentration of bs-mAb needed to achieve the same activity (50% neutralization) was 9-fold lower than the mixture of two parental mAbs.
  • mAbs For optimal selection of mAbs to produce bs-mAbs recognizing both the V3 loop and the CD4-bd, a group of mAbs was tested in a binding ELISA using primary HIV isolates as antigen. Five primary viral isolates derived from different HIV-positive individuals were used in an antigen capture ELISA. The solid phase was created using a capture antibody specific for the C-terminus of gpl20, and envelope glycoproteins were exposed for binding with the test mAbs.
  • the p24 level in primary isolate stocks was between 40 and 75 ng/ l, while the level of gpl20 ranged between 2.4 and 4.8 ng/ml.
  • Table V presents the results expressed as a ratio of optical density of bound mAb to the average background.
  • a gpl20 of primary HIV isolates was captured on an ELISA microplate using an anti-gpl20 antibody specific for the C- terminus and incubated with the mAbs as shown.
  • All mAbs were used at an IgG concentration of 10 ⁇ g/ml except in the case of 558/64D which was used at 4.2 ⁇ g/ ⁇ l.
  • a second partner is preferably 419-D, 412-D or 391/95-D. Additional studies with other primary isolates may be used to determine which of these three mAbs is a more preferred parent for a bs-mAb. Previous analysis of these three mAbs indicated that while they were restricted in their cross- reactivity to V3 peptides, they were quite cross-reactive in binding to various primary isolates. This result demonstrates the importance of evaluating reactivity of antibodies with primary HIV isolates for selecting partners for production of bs-mAbs of preferred specificity. All four of the anti-CD4-bd mAbs examined recognized each of the five primary isolates. Thus, other criteria, including epitope fine specificity and isotype are utilized for selecting parent mAbs for the production of bs-mAbs which recognize CD4-bd.
  • bispecific F(ab') 2 heterodimer of 729D x 447D will be compared with the 729/447D bs-mAb, described herein, in neutralization assays against laboratory and primary isolates of HIV. If such bispecific heterodimers produced recombinantly show similar functional activity, the molecular genetic approach will be utilized for large- scale production.
  • F(ab'- zipper) 2 bispecific heterodimer may have a shorter half-life in vivo than an intact antibody. This may be overcome by further modifications, for example, by alteration of charge properties or the addition of polyethylene glycol to form a complex, or by the additional through recombinant techniques of an Fc portion.

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Abstract

Novel bispecific human monoclonal antibodies (bs-mAbs) are directed against two neutralizing epitopes of human immunodeficiency virus (HIV). Such bs-mAbs, specific for two V3 loop epitopes, or specific for a V3 loop epitope and a CD4-binding domain epitope, are effective in neutralizing HIV and can be used for prophylactic or therapeutic administration to subjects with HIV infection and AIDS. Also provided are pharmaceutical compositions comprising one or more bs-mAbs, cell lines producing such bs-mAbs, methods for producing such cell lines and methods for producing the bs-mAbs.

Description

BISPECIFIC HUMAN MONOCLONAL ANTIBODIES SPECIFIC FOR HUMAN IMMUNODEFICIENCY VIRUS
1. INTRODUCTION
The present invention relates to novel bispecific human monoclonal antibodies (bs-mAbs) directed against two neutralizing epitopes of human immunodeficiency virus (HIV) . Such bs-mAbs are effective in neutralizing HIV, can be designed to have a broader range of activity against laboratory and primary HIV isolates than standard mAbs, and are useful in treating subjects with HIV infection and AIDS.
2. BACKGROUND OF THE INVENTION
Passive immunization with antibodies (Abs) is a recognized method of prophylaxis and treatment of infectious diseases. Traditionally, this approach involved preparing human immunoglobulins from donors which recovered from an infectious disease and utilizing such preparations, containing Abs specific for the infectious organism, to protect a recipient against the same disease. Such immunoglobulin preparations, containing a high titer of Abs derived from recovered individuals, are currently used for the prevention of hepatitis B virus and cytomegalovirus infections (Condie, R. M. et al . , 1984, Birth Defects 10:327; Theppisai, U. et al . , 1988, J. Med. Assoc . 1:413) . However, even preparations consisting of high concentrations of human immunoglobulin contain a very low proportion of protective Abs (Masuho, Y., 1988, Serodlag . Immunother . Inf . Dis . 2:319) .
Monoclonal antibodies (mAbs) which are homogenous with respect to both physical characteristics and immunochemical reactivity provide the promise of absolute specific activity. Murine mAbs, which are easily generated against various human pathogens, have limited utility for treating humans due to the rapid induction of strong immune responses in the human recipient against the foreign mouse antibody which abrogates the murine mAb's efficacy (Schroff, R. et al . , 1985, Cane. Res . 45 : 879 ) . Chimeric (human-mouse) and humanized mAbs are much less immunogenic, but nevertheless, result in the production of antibodies to mouse variable regions after repeated doses in some subjects (LoBuglio, A. F. et al . , 1989, Proc. Natl . Acad. Sci . USA 56:4220; Kwa , L. . et al . , 1992, N. Engl . J. Med. 327:1209.) . Thus, human mAbs remain the optimal reagent for passive immunization of humans.
Based on results of several experiments in chimpanzees, the use of passive immunotherapy for protection of humans against HIV infection has been proposed. The administration of high-titered human anti- HIV immunoglobulins to chimpanzees proved protective against viral challenge (Prince, A.M. et al . , 1991, AIDS Res . Hum. Retrovir. 7:971) . Notably, both the amount of Ab administered and the amount of virus used for challenge were critical for protection. In other primate studies
(Emini, E. et al . , 1992, Nature 355:728) , a chimeric mouse- human mAb (Cpl) specific for the V3 loop of gpl20, protected chimpanzees when administered before or after virus challenge. These experiments convincingly support the hypothesis that antibodies are capable of protecting against infection with HIV-1 in the absence of any specific cellular immunity to the virus.
Furthermore, clinical trials of passive immunization showed some effectiveness in the treatment of HIV-1 infected humans. Administration of plasma with virus neutralizing activity derived either from HIV-1 positive human donors or hyperimmune porcine immunoglobulins to HIV- 1 infected patients improved both the clinical and serologic parameters of infection (Karpas, A. et al . , 1988, Proc. Natl . Acad. Sci . USA 85:9234; Karpas, A. et al . ,
1990, Proc . Natl . Acad. Sci . USA 87:1; Vittecoq, D. et al . , 1992. J". Infect . Dis. 26"5:364; Osther, K. et al . , 1992. AIDS 6:1457) . Although clinical trials were limited and the immunoglobulin preparations had not been selected for neutralization of clinical viral isolates, these results suggest the beneficial effect of elimination of cell-free virus. While protection as well as treatment of HIV-1 infection could be attributable mainly to antibody-mediated neutralization of the virus, virolysis mediated by the antibody and complement or antibody-dependent cellular cytotoxicity (ADCC) could also have contributed to the outcome. HIV-1 infection induces neutralizing Abs which are specific for gpl20 and gp4l, the envelope and transmembrane glycoproteins of the virus. Although Abs specifically directed to several regions of gpl20 can neutralize HIV (Olshevsky, U. et al . , 1990, J. Virol . 64:5701), the anti-gpl20 Abs which possess the most clearly defined neutralizing functions recognize two major regions of the protein: (a) the third variable domain or "V3 loop"; and (b) the domain which binds to the CD4 glycoprotein of the T lymphocytes, called the CD4-binding domain (CD4-bd) (Rusche, J.R. et al . , 1988, Proc. Natl . Acad. Sci . USA 85 : 3198 ; Sterner, K. S. et al., 1991, Science 254:105) . Anti-gp41 neutralizing Abs appear to recognize a conserved region in the extracellular domain (Buchacher, A. et al . , 1992. In: VACCINES 92 : MODERN APPROACHES TO NEW VACCINES INCLUDING PREVENTION OF AIDS, F. Brown et al . . eds. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York p. 191) . Neutralizing Abs are not present at high levels in the sera of HIV-1 infected individuals and might therefore not be sufficient to control HIV infection. However, since the titer of neutralizing Abs is so critical for protection, as proven experimentally in chimpanzees (Prince et al . , supra) , passive immunization may deliver a level of Ab that is protective and/or therapeutic. Anti-V3 Abs are considered to be "type-specific," whereas anti-CD4bd Abs are considered "group-specific" (Palker, T.J. et al., 1988, Proc . Natl . Acad. Sci . USA 85:1932; Kang, C. et al . , 1991, Proc . Natl . Acad . Sci . USA 88:6171) . See, also, co-pending, commonly assigned United States Patent Application, Serial No. 07/776,772, filed October 17, 1991. However, some anti-V3 human mAbs developed in the present inventors' laboratory, are not narrowly type-specific and broadly cross-react with peptides of several different HIV-1 strains (Karwowska, S. et al., In: VACCINES 92 : MODERN APPROACHES TO NEW VACCINES INCLUDING PREVENTION OF AIDS, F. Brown et al . . eds . , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1992, p. 171; Gorny, M. K. et al . , 1993, J". Immunol . 150 : 635 ) . Human mAb 447-52-D, developed by the present inventors, is the most broadly reactive anti-V3 antibody, with a specificity directed towards the tetrapeptide GPGR, a very conserved sequence in the V3 loop. In enzyme linked immunosorbent assays (ELISA) , this human mAb reacts with eight out of eight peptides derived from American and European HIV strains, including such divergent strains as MN, IIIB and RF (Gorny et al . , supra) . Moreover, this 447- 52-D mAb neutralizes eight different HIV-1 laboratory strains (Gorny, M. K. et al . , 1992, J". Virol . 66:7538) .
Although the absolute specificity of mAbs gives them advantages over other immunoglobulin preparations, their specificity for only a single epitope may be a disadvantage in the clinical setting. This inherent limitation could potentially be overcome by using "cocktails" of more than one mAb in a preparation. Concomitant binding of Abs to multiple sites or to a cluster of epitopes may be more biologically effective in removing or neutralizing a virus. Animals studies with rubella, cytomegalovirus and Dengue virus demonstrated that combinations of mAbs to different viral epitopes can result in synergistic neutralizing activity (Gerna, G. et al . , 1987, J. Gen . Virol . 68 : 2007 ; Lussenhop, N. 0. et al . , 1988, Virology 164 : 362 ; Henchal, E. A. et al . , 1988, J. Gen . Virol . 69 : 2101 ) . A similar phenomenon has been observed with human mAbs to HIV-1 (Buchbinder, A. et al . , 1992, AIDS Res . Hum . Retrovir. 8:425; Tilley, S. A. et al., 1992, AIDS Res . Hum. Retrovir. 8:461) . Viruses can escape the neutralizing action of an antibody by mutating to a form which is no longer recognized by the antibody, also termed antibody-resistant. Such escape is known with rabies (Lafon, M. et al . , 1990, J. Gen . Virol . 71:1689) , equine infectious anemia virus (Hussain, K. et al . , 1987, J. Virol . 61:2956) and HIV (Nara, P. L. et al., 1990, J. Virol . 64 : 3779 ; Reitz, M. S. et al . , 1988. Cell 54-57 ; Mckeating, J.A. et al . , 1989. AIDS 3 : 777) . Thus, passive immunization with a single mAb may have limited value, whereas a "cocktail" of mAbs may increase the extent of viral variation required for escape by several orders of magnitude (Lafon et al . , supra) .
An alternative to administering a combination of antibodies is the use of bispecific antibodies (bs-Abs) which are structurally bivalent, recognizing two different epitopes, but functionally monovalent for each antigen- binding site. Bs-Abs can be produced by chemically linking two mAbs, by fusion of two different hybridoma cell lines, or by genetic engineering. Each of these methods has advantages and limitations. Biological methods result in production of intact molecules, both bispecific and parental, as well as some nonfunctional combinations of light and heavy chains of the parent Abs (Milstein, C. et al . , 1983. Nature 305 : 537) . Separation of bs-mAbs from other Abs in such preparations is a challenge because even in the best situation, only about 30% of hybrid-hybridoma (or "quadroma") products consist of bs-mAbs. Purification of bs-mAbs by high performance liquid chromatography (HPLC) techniques is possible, but economical large-scale production has yet to be achieved.
Chemical linkage appears to be the most straightforward approach but frequently results in heterogenous and ill-defined products. Large scale production also remains a problem. Molecular genetic techniques have some potential because they can be adapted to economically effective expression systems. Several approaches have been used to produce single-chain antibody-like proteins consisting of immunoglobulin VL and VH domains, and have affinities and specificities similar to those of the parent Abs (Bird, R. E. et al . , 1991, Trends Biotech . 9 : 132 ) . Single-chain fusion proteins have been shown to target cytotoxic lymphocytes to HIV- infected cells through hybrid CD4-FC7 or CD4-anti-CD3 constructs (Traunecker, A. et al . , EMBO J. 10 : 3655 ; Bryn, R. A. et al . , 1990, Na ture 344 : 667 ) .
Recent advances in the production of genetically engineered antibodies may provide a basis for applying molecular approaches in the production of bs-mAbs (Wright, A. et al . , 1992, Crit. Rev. Immunol. 12:125) . Kostelny et al . (Kostelny, S. et al . , 1992, J. Immunol . 148:1547) described a new method for producing bispecific antibody fragments using a leucine zipper peptide. Two heterodimer- forming sequences derived from the leucine zipper regions of the transcription factors Fos and Jun were genetically fused with the Fab fragments of two different murine mAbs, anti-CD3 and anti-Tac (which binds to the human interleukin-2 receptor) . Leucine zippers are specific amino acid sequences about 30 residues along with leucines occurring at every seventh residue. These sequences form amphipathic anti-helices, with the leucine residues lined up on the hydrophobic side for dimer formation. The leucine zipper peptides of Fos and Jun have a much greater tendency to form heterodimers rather than homodimers (O'Shea, E. K. , 1989, Science 245:646) .
In the Kostelny study ( supra) , the F(ab' -zipper)2 homodimers were individually expressed in the murine myeloma cell line Sp2/0 and purified by affinity chromatography from culture supernatant. Homodimeric proteins Fos and Jun were reduced at the hinge region using 2-mercaptoethylamine to form Fab' -zipper monomers. At this point, the Fos and Jun monomers were mixed together at a 1:1 ratio and reoxidized, and the resulting products were purified on an FPLC system. The heterodimers constituted 82% of the eluted F (ab' -zipper)2 materials. Bispecific anti-CD3 x anti-Tac F (ab' -zipper)2 heterodimers were highly effective in recruiting cytotoxic T cells to lyse target cells in vi tro, whereas the homodimers in combination were totally ineffective. Thus, this method appears to provide an effective means for producing bispecific antibodies with little side-product formation. At present, all known bs-mAbs are composed of murine mAbs or humanized murine mAbs. Hence, their use in human therapy carries the threat of inducing an immune response against the murine portions of the therapeutic molecule (Kwak et al . , supra) . HIV-specific human bs-mAbs and methods for their production and use have not been disclosed. The invention disclosed herein is directed to such antibodies.
3. SUMMARY OF THE INVENTION
The present invention relates to a novel bispecific mAb (bs-mAb) specific for two neutralizing epitopes of HIV, and the clinical use of such a bs-mAbs for immunoprophylaxis or immunotherapy of HIV infection.
In one embodiment, the present invention is directed to a human bs-mAb in which one variable region is specific for an epitope of the V3 loop of an HIV glycoprotein gpl20 and which antibody is capable of neutralizing HIV. The antibody may be specific for two different epitopes of the V3 loop of the gpl20 or for an epitope of the V3 loop and an epitope of the CD4-binding domain of the gpl20.
Also provided is human bs-mAb as above, wherein the V3 epitope is a peptide of at least 4 amino acids from the amino acid sequence YNKRKRIHIGPGRAFYTTKNIIG (SEQ ID NO:l) . The V3 epitope for which the human bs-mAb is specific is preferably an epitope included in SEQ ID N0:1, selected from the group consisting of HIGPGR, HIGPGRA, IGPGRAF, GPGRAFY, PGRAFYT, GRAFYTT, GPGRAF, RKRIHIG, KRIHI, KRIHIGP, IHIGPGR, IHIGP, IGPGR, GPGR, and GRAF. Other preferred epitopes include RKRIHIGPGRAFYTR (SEQ ID NO:2) , HIGPIHIGP (SEQ ID NO:3) , GPGRVI (SEQ ID NO:4), GPGRTL (SEQ ID NO:5) and GPGRVW (SEQ ID NO:6) .
In another embodiment, the above human bs-mAb is specific for an epitope having the amino acid sequence GPXR (SEQ ID NO:7) , where X may be any amino acid residue. The human bs-mAb is preferably one in which the one variable region specific for a V3 loop epitope is derived from a human mAb selected from the group consisting of 257-2D, 268-10D, 311-11D, 386-10D, 391/95-D, 412-D, 418- D, 419-D, 447-52D, 453-D, 504-10-D, 537-D, 694/98-D, 782-D, 838-D and 908-D.
Also provided is a human bs-mAb specific for an epitope of the V3 loop and an epitope of the CD4-binding domain of the gpl20, wherein:
(a) one variable region is derived from a human mAb selected from the group consisting of 257-2D,
268-10D, 311-11D, 386-10D, 391/95-D, 412-D, 418- D, 419-D, 447-52D, 453-D, 504-10-D, 537-D, 694/98-D, 782-D, 838-D and 908-D; and
(b) the other variable region derived from a human mAb selected from the group consisting of 448-D,
559/64-D, 588-D, 654-D, 728/29-D, 729-D, 855-D and 860-D.
Also provided is a human bs-mAb having one variable region specific for an epitope of the CD4-binding domain of HIV glycoprotein gpl20 and capable of neutralizing HIV. Preferably, the one variable region is derived from a human mAb selected from the group consisting of 448-D, 559/64-D, 588-D, 654-D, 728/29-D, 729-D, 855-D and 860-D. A preferred human bs-mAb is selected from the group consisting of the human bs-mAbs 729/447D, 654D/447D, 694D/447D and 782D/447D.
Also provided is a method for neutralizing HIV comprising contacting the HIV with an effective amount of a human bs-mAb as described above.
The present invention is further directed to a cell line producing a human bs-mAb as above. In one embodiment, the cell line is a hybrid hybridoma. The cell line may also be a mammalian or bacterial cell transfected with DNA encoding heavy and light chains of a bs- mAb as above.
The present invention also provides a composition useful for neutralizing HIV, preventing infection with HIV or treating a subject infected with HIV, comprising an effective amount of one or more bs- human monoclonal antibodies as above. The composition may further comprise a drug useful in the treatment of HIV infection or AIDS. Also provided is a pharmaceutical composition comprising the bs- mAb as above and a pharmaceutically acceptable excipient.
The present invention is also directed to a method for preventing infection of a subject by HIV-1, or for treating a subject infected with HIV-1, comprising administering to the subject an effective amount of a human bs-mAb as above, or administering an effective amount of a composition or pharmaceutical composition, as above.
The present invention also provides a method for producing a hybrid hybridoma cell which makes a human bs- mAb specific for two neutralizing epitopes of the gpl20 protein of HIV, comprising:
(a) selecting two hybridoma cell lines each of which produces a human mAb specific one of the neutralizing epitopes;
(b) fusing the hybridoma cell lines, thereby producing the heterohybridoma cell .
Preferably, one of the neutralizing epitopes recognized by the bs-mAb is from the V3 loop of the gpl20 glycoprotein, both of the neutralizing epitopes are from the V3 loop of the gpl20 glycoprotein, or one of the neutralizing epitopes is from the V3 loop of the gpl20 glycoprotein and the other of the neutralizing epitopes is from the CD4-binding domain of the gpl20 glycoprotein. Also provide is a method for producing a human bs-mAb specific for two neutralizing epitopes of the gpl20 protein of human immunodeficiency virus, comprising: (a) producing a cell line using a method as described above ;
(b) culturing the cell line; and
(c) recovering the human bs-mAb from the culture.
4. DESCRIPTION OF THE FIGURES
Figure 1 shows elution profiles of two HIV- specific bs-mAbs purified by HPLC. Panel A shows the HPLC purification of supernatant of the hybrid hybridoma designated 729/447D. Panel B shows the HPLC purification of supernatant of the hybrid hybridoma designated 71- 41/447D. Supernatants were first purified on Protein A columns and applied to a Bakerbond Abx HPLC column. The elution gradient was 10 mM MES, pH 5.6 -60% of 500 mM sodium acetate, pH 7.0, run at 1 ml/min over 1 hour. The elution of protein was monitored by measuring absorbance at a wavelength of 280 nm. Proteins not binding to the column eluted in the early fraction (peak 1) . Parental mAbs 729-D and 71-31 eluted in the second fraction (peak 2) , whereas parental mAb 447-52-D eluted in the fourth fraction (peak 4) . Peak 3 contains the bs-mAbs. Figure 2 is a SDS-polyacrylamide gel electrophoresis pattern (under reducing conditions) of samples obtained from an HPLC column. Single heavy (H) chain bands in Fractions 2 and 4 indicate the presence of parental mAbs, 729-D and 447-D, respectively. Double H chain bands in fraction 3 represent the bs-mAbs 729/447D. L indicates the presence of light chains.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a bispecific human monoclonal antibody (bs-mAb) specific to two different neutralizing epitopes of HIV and pharmaceutical compositions comprising such an antibody, methods of producing such bs-mAb and methods of using such bs-mAb in - li ¬ the prophylaxis or treatment of HIV infection and AIDS
5.1. DEVELOPMENT OF NEUTRALIZING HUMAN bs-mAbS SPECIFIC FOR DIFFERENT EPITOPES OF THE V3 REGION AND CD4-BD OF gpl2Q
The present invention provides a human bs-mAb specific for different epitopes of the V3 region and the CD4-bd of the viral gpl20 protein which antibodies neutralize HIV-1. The bs-mAbs are typically generated by the fusion of two parent lines each producing a human mAb. Preferred parent cells lines are human hybridomas or mouse- human heterohybridomas, or human lymphoblastoid cell lines which produce a human mAb specific for HIV. Preferred cell lines are those shown in Table I, below, produced by the present inventors, each of which produces a human mAb which neutralizes HIV-1. Sixteen of these mAbs mediate the
TABLE I
NEUTRALIZING HUMAN MONOCLONAL ANTIBODIES SPECIFIC FOR HIV-1 gp!20 EPITOPES
Human mAb Isotvpe Epitope
A. GPGR GP GPGR
Figure imgf000014_0001
418-D igGi,κ HIGPGRA
419-D igGi,λ KRIHIGP
447-52-D igG3,λ GPGR
453-D IgGl,λ IHIGPGR
504-10-D igGi,κ IHIGPGR
537-D IgGl,λ IGPGR
694/98-D igGi,λ GRAF
782-D lgGi,λ not tested
838-D IgGl,λ not tested
908-D IgGl,λ not tested
B. Anti CD4-Binding Domain
448-D IgGl,λ Discontinuous
559/64-D IgGl, Discontinuous
588-D IgGl, K Discontinuous
654-D IgGl,λ Discontinuous
728/29-D IgGl,λ Discontinuous
729-D igGi,κ Discontinuous
855-D igGi,λ Discontinuous
860-D IgGl, K Discontinuous
* Reactive with 15- -mer from V3; MN (RKRIHIGPGRAF7.
According to the present invention, any one of three different classes of bs-mAbs is preferred. The first class is a bs-mAb directed against two different epitopes of the gpl20 V3 region. Such a bs-mAb may be specific for two V3 epitopes of the most common HIV strain, HIVMN. In another embodiment, the bs-mAb is made by fusing two cell lines, the first of which produces a mAb which reacts strongly with the V3 region of HTLVUIB or HIVpj- and the second of which makes a mAb highly specific for the V3 region of HIVMN. This second type of doubly V3-specific bs- mAb has a broader range of reactivity against different HIV strains than does the first type described above.
Bs-mAbs of the second class are directed against two different epitopes of the CD4-bd of gpl20. The parent lines producing human mAbs are selected on the basis of the specificity of their anti-CD4bd mAbs such that the parent mAbs react with different sites within the CD4bd.
A third class of bs-mAbs is specific for two different neutralizing domains of gpl20: the V3 loop and the CD4-bd. In a preferred embodiment, heterohybridomas making mAbs which act synergistically in the neutralization of HTLVIIIB and/or HIVMN are fused to make bs-mAbs specific for both a V3 epitope and a CD4-bd epitope.
A bs-mAb of the present invention is tested for its ability to bind to HIV-infected cells and to mediate neutralization of viral infection. The neutralization is preferably tested using laboratory strains of HIV as well as various primary clinical HIV isolates. The neutralizing activity of the bs-mAb is generally compared with that of the two parental mAbs.
5.1.1. ANTIBODIES SPECIFIC FOR TWO
EPITOPES OF THE V3 REGION
Parent anti-V3 mAbs are selected for producing a bs-mAb on the basis of their HIV neutralizing activity, as well as their cross-reactivity between different HIV strains. The mAb 447-D is a preferred choice as a parent supplying one of the antigen binding portions of a bs-mAb due to its unique specificity for the most conserved region of the V3 loop and due to its broad HIV strain reactivity. Another unusual feature of the 447-D mAb is its IgG3 isotype. By combining an IgG3 heavy chain (or a portion thereof) with an IgGl heavy chain (or portion thereof) in making a bs-mAb increases the ease of purifying an IgG3/IgGl bs-mAb relative to the ease of purifying a bs-mAb having a single heavy chain isotype. In general, the bs- mAb of the present invention is purified from parental type mAbs produced by hybrid hybridoma cells using standard purification techniques well-known in the art, including high performance liquid chromatography (see below) .
Another criteria for selecting parental mAbs for production of a bs-mAb is their binding activity to primary HIV isolates. The present inventors' group recently showed that all 14 anti-V3 human mAbs which bound to gpl20 V3 peptides neutralized HIVMN (Gorny, M.K. et al., 1993, J". Immunol . 150 : 635 ) . Furthermore, there was a significant correlation between antibody affinity to the peptide and the 50% neutralizing dose for HIVMN. Thus, by testing the binding activity of a mAb to limiting concentrations of gpl20 of one or more HIV primary isolates, it is possible to predict to a degree of certainty which mAbs may be more efficient in neutralizing the virus. The V3-specific mAb used to produce a bs-mAb of the present invention is preferably one which is specific for an epitope of at least 4 contiguous amino acids from the amino acid sequence YNKRKRIHIGPGRAFYTTKNIIG (SEQ ID NO:l) . Other preferred mAbs are those specific for a peptide from within SEQ ID NO:l selected from the following group: KRIHI, KRIHIGP, IHIGPGR, HIGPGR, IGPGR, HIGPGRA, GPGR, GRAF, and RKRIHIGPGRAFYTR. A most preferred V3- specific mAb is specific for the GPGR epitope and is characterized by a broad cross-reactivity among HIV strains, such as the human mAb designated 447-D (or 447- 52D) . Also preferred is a mAb specific for GPXR (SEQ ID NO:7) , wherein X is any amino acid residue
The preferred binding assay for testing and selecting a mAb for bs-mAb production is based on a previously used antigen-capture ELISA. The stock virus, which is derived from supernatants of primary isolate cultures, is treated with Empigen® and then used as a source of gpl20. The gpl20 antigen is immobilized onto a solid phase carrier, preferably the surface of the wells of a 96-well microplate, with an antibody specific for a C- terminal portion of gpl20. The binding of an anti-V3 mAb to the immobilized gpl20 is scored as the ratio of "positive" to "negative" wells (the P/N ratio) . The level of gpl20 in a given virus stock is measured by another antigen-capture ELISA in order to normalize the concentration of gpl20 from different stocks.
These assays were recently used in the present inventors' laboratory to test mAb binding to gpl20 of five different primary isolates (see Table V, below) . For example, all 16 mAbs listed in Table I are tested against at least 20 primary HIV isolates to select the mAbs which bind to the broadest range of viral isolates and have the highest P/N ratio.
The reactive mAbs are preferably also tested by flow cytometry for binding to cells infected with the HIV of the primary isolates. Known staining procedures are used (Tyler, D. S. et al., 1990, J". Immunol . 145 : 3276) . For example, cells from the primary cocultures are incubated with mAbs and then stained with enzyme-labeled anti-human immunoglobulins. Flow cytometric analysis is performed using, for example, a FACScan® flow cytometer. In this way, it is possible to select an anti-V3 mAb with the desired characteristics to serve as a "partner" for 447-D in the production of a bs-mAb.
5.1.2. ANTIBODIES SPECIFIC FOR TWO
DIFFERENT EPITOPES PRESENT IN THE CD4-BINDING DOMAIN
The human mAbs which bind to gpl20 and block attachment of virus to the CD4 molecule on the surface of target cells recognize a conformational epitope. The CD4- bd is does not act as a linear epitope but rather involves several constant domains including the C2, C3 and C4 domains of CD4. Target epitopes for an anti-CD4-bd mAb which is a preferred parent for producing a bs-mAb can be determined with some approximation using the "Sodroski mutants" of HIV which have single amino acid changes in the conserved residues of gpl20 (Olshevsky, U. et al . , 1990, J". Virol . 64:5701) . Recently, the present inventors' group mapped the target epitopes of three anti-CD4-bd mAbs, termed 448-D, 559-D and 588-D. Recognition of the envelope glycoproteins by 448-D was affected by different changes in the gpl20 amino acid residues to a greater extent than was recognition by mAbs 559-D and 588-D (McKeating, J. A. et al . , 1992, Virology 150:134) . Using the same mutants, three more mAbs have been mapped more recently. The mAb 654-D appeared to be unique among the anti-CD4-bd mAbs in terms of its binding to mutants. A detailed description of anti-CD4-bd human mAbs and methods of their production and use, as well as synergistic interactions between such anti- CD4-bd antibodies and between anti-CD4-bd antibodies and anti-V3 loop antibodies, is provided in co-pending, commonly assigned U.S. patent application Serial Number 07/776,772, filed October 17, 1991, which reference is hereby incorporated by reference in its entirety. By first performing epitope mapping studies, it is possible to avoid selecting parental mAbs which recognize the same binding site. Thus by using epitope mapping, it was determined that mAbs 559-D, 588-D, 728/29-D and 729-D may interfere with each other if combined in a bs-mAb. Of these four mAbs, the ones which bind strongly to gpl20 of primary isolates and/or to infected cells are used in combination with either 654-D or 448-D for producing a bs-mAb. Since all the anti-CD4-bd mAbs described above are of the IgGl isotype, the bs-mAbs are distinguishable from the parental mAbs on the basis of their different light chains: K versus λ. Thus, for example, the mAbs 654-D (IgGl,λ) or 448-D (IgGl,λ) can be used for making bs-mAbs with a selected mAb from the group having K light chains (559-D, 588-D or 729-D. The molecular genetic approaches described herein utilizing leucine zipper peptides for the production of bs- mAbs (see Section 5.3, below) should allow the use of different combinations of parental mAbs which do not have distinct H chain or L chain isotypes. 5.1.3. BS-mAbs SPECIFIC FOR A V3 LOOP
EPITOPE AND A CD4-BINDING DOMAIN EPITOPE
Preferred mAbs for constructing a bs-mAb which recognizes a V3 loop epitope and a CD4-bd epitope are those which (a) bind strongly to primary HIV isolates, (b) are broadly reactive with various V3 peptides and (c) have potent virus neutralization activity against laboratory strains of HIV. Recently, the present inventors' group and Tilley's laboratory provided the first descriptions of synergistic activity between two neutralizing anti-HIV mAbs, one specific for V3 and one for the CD4-bd, in the neutralization of HIVIIIB (Buchbinder, A. et al . , 1992, AIDS Res . Hum . Retrovir. 8:425; Tilley, S.A. et al . , 1992, AIDS Res . Hum. Retrovir. 8:461) .
The synergistic interactions of two mAbs in neutralization are determined using the microtiter neutralization assay. Two combinations, 654-D + 447-D and 729-D + 447-D, have already been tested against HIVI1IB and have demonstrated synergy (see Examples, below) .
Synergistic activity against two other strains of HIV-1, HIVMN and HIVAla, may also be tested to determine if the effective mixtures synergize in the neutralization of both virus strains or against only one. Results are analyzed for synergistic, additive or antagonistic activity using the computer program of Chou and Chou (DOSE-EFFECT ANALYSIS WITH MICROCOMPUTERS, Biosoft, Cambridge, 1987) .
Synergistic neutralizing activity between two mAbs is attributable to the ability of one neutralizing mAb to enhance the binding, and therefore the activity, of the other. A mAb to the CD4-bd may cause a stereochemical change in the gpl20 molecule so that the V3 loop is better exposed for binding of an anti-V3 mAb (Mckeating, J. A. et al., 1992, Virology 191 : 732 ) . Alternatively, the antibodies may act in the opposite sequence. (Such synergistic activities are described in co-pending commonly assigned U.S. patent application Serial Number 07/776,772, filed October 17, 1991 and incorporated by reference herein . )
5.1.4. PRODUCTION OF HYBRID-HYBRIDOMAS
One way of producing bs-mAbs is by creating hybrids between two hybridomas, for example two mouse-human heterohybridomas, each of which produces a human mAb specific for an HIV neutralizing epitope. Hybrid- hybridomas are preferably produced by a fusion technique developed in the present inventors' laboratory (Gorny, M.K. et al . , 1989, Proc . Natl . Acad. Sci . USA 86:1624; Gorny, M.K. et al . , 1991, Proc . Na tl . Acad. Sci . USA 88 : 3238 ) . For selecting hybridomas which produce bs-mAbs, one "parent" hybridoma is back-selected for hypoxanthine, aminopterin and thymidine (HAT) -sensitivity prior to fusion. This procedure is carried out by growing cells in the presence of 8-azaguanine (30 μg/ml) which induces an enzyme deficiency for hypoxanthine guanine phosphoribosyl transferase (HGPRT) . The hybridoma cells lacking HGPRT fail to grow in medium containing HAT, since both the DNA and RNA biosynthetic pathways are blocked. The other
"parent" hybridoma (or lymphoblastoid cell line) remains HAT-resistant, but is pretreated with a lethal dose (5 mM) of the irreversible biochemical inhibitor, iodoacetamide (Suresh, M. R. et al . , 1986, Meth . Enzymol . 121 : 210 ) . Cells treated in this way are rendered non-viable unless "rescued" by fusion. In the case where HAT-sensitive hybridoma cells are fused with lymphoblastoid cells, the selecting medium contains HAT and ouabain. Lymphoblastoid cells that result from Epstein Barr virus transformation of peripheral blood lymphocytes are ouabain-sensitive and are killed when unfused.
Both parental cell lines are preferably mixed at a ratio of about 1:1, and cell fusion is induced using polyethylene glycol with dimethyl sulfoxide (DMSO) . The fused cells are plated at a density of about 8 x 104 cells/well on a feeder layer of about 1 x 104 murine peritoneal cells. Two to three weeks post-fusion, all culture wells are screened for production of bs-mAbs in the supernatant using one or more techniques (described below) and the positive cultures are expanded in 24-well plates. Hybrid-hybridomas that produce the highest level of antibodies as measured in ELISA, are cloned at 100 to 1 cell/well. After cloning, the positive wells are expanded into flasks.
To obtain high levels of bs-mAbs, hybrid hybridomas are grown in the peritoneum of SCID mice. Mice are primed by intraperitoneal (i.p.) injection of 0.5 ml pristane (2,6,10,14 tetramethyldecanoic acid; Sigma Chemicals) . After 7-14 days, about 1-5 x 106 hybridoma cells are injected i.p. and ascites fluid is collected a few weeks later. A single mouse may yield 2-10 ml of ascites fluid. Antibody concentrations are generally between 1 and 10 mg/ml.
5.1.5. SCREENING ASSAYS FOR HIV-SPECIFIC ANTIBODIES
5.1.5.1. Direct ELISA
The presence of antibodies specific for peptides or recombinant proteins of HIV is determined using a direct ELISA. For example, mAbs specific for the V3 loop are detected by ELISA using 20-23-mer peptides that represent the V3 regions of HIV-strains MN, RF, and HXB2 (American Biotechnologies, Cambridge, MA) . Antibodies to the CD4-bd, which are specific for a discontinuous epitope, are identified with recombinant gpl20 from HIVIIIB (rgpl20IIIB) prepared in baculovirus-infected insect cells (Repligen, Cambridge, MA) . These antigens are coated onto plastic microplates at a concentration ranging from about 0.2 to 1.0 μg/ml. The culture supernatants or purified mAbs are tested for reactivity to these antigens by a standard direct ELISA, such as that previously described by the present inventors' group (Gorny, M.K. et al . , 1989, Proc . Natl . Acad. Sci . USA 86:1624; Gorny, M.K. et al . , 1991, Proc. Natl . Acad. Sci . USA 88:3238) .
The hybrid hybridoma cells producing bs-mAbs are screened preferably by either a single antigen ELISA, a double isotype ELISA, or by an antigen-isotype ELISA. In a single antigen ELISA, the culture supernatant is tested separately for its binding to plates coated with one of two different antigens. The double isotype ELISA is used when bs-mAbs consist of two different isotypes, such as IgGl and IgG3. In such an assay, an antibody to one of the isotypes e . g. , anti-IgGl, is coated onto the plate, whereas the antibody for the second isotype, e . g. , anti-IgG3, is enzyme-labeled. A positive well indicates the presence of a bs-mAb that binds to both the anti-IgGl and anti-IgG3 reagents . The antigen-isotype ELISA is employed when the bs-mAbs consist of two different isotypes with two specificities that recognize two different peptides. For example, a bs-mAb may consist of a portion having the IgGl isotype and specificity for an epitope of the CD4-bd and a portion having the IgG3 isotype and specificity for an epitope of the V3 loop. To detect such an antibody, the V3 peptide is coated onto the plate and the binding of the bs- mAb is detected using an enzyme-labeled anti-IgGl reagent. In the reverse format, rgpl20IIIB peptide is coated onto the plastic and the reactivity of the bs-mAb is detected using an enzyme-labeled anti-IgG3 reagent.
5.1.5.2. Antigen Capture ELISA
(a) Binding of human mAbs to primary isolates of HIV-1 Preferred bs-mAbs are made using parental mAbs selected for their binding to the majority of HIV-1 primary isolates. The present inventors have found that antibodies specific for antigens present in viral lysates at low concentrations are more readily detected using antigen - capture ELISAs than in direct ELISAs . Therefore, to detect binding activity of a human mAb to the gpl20 of a primary isolate of HIV-1, wells of microtiter plates (preferably Immulon-2) are coated with a commercially available sheep antibody specific for the C-terminus of gpl20. Subsequently, the supernatants from primary cultures (see below) treated with 1% Empigen® detergent and containing known levels of gpl20, are added to coated plates and incubated. After washing, human mAbs are added, followed by rabbit anti-human IgG coupled to alkaline phosphatase (or any of a large number of enzymes useful in ELISAs) and then a chromogenic substrate for the alkaline phosphatase (or for the other enzyme) . The reaction can be further enhanced using the ELAST® Amplification system.
(b) p24 assay
This assay has been developed by the present inventors and is used for screening supernatants from primary cultures or as a read-out for a microtiter neutralization assay. The assay, as currently practiced, has a limit of sensitivity of 150 pg/ml . One human mAb specific for p24 (Gorny et al . , 1989, supra) is used to coat a microtiter plate, which is then blocked and washed. Supernatants from primary cultures or from a neutralization assay are then added to the wells and the plate is incubated for about 3 hours. The color reaction is developed using an enzyme-conjugated rabbit anti-p24 antibody. Diluted recombinant p24 in the range of about 0.2 to 20 ng/ml is used to determine the standard curve.
(c) gpl20 assay
This assay was established in the present inventors' laboratory to determine the concentration of gpl20 in stocks of primary HIV isolates treated with the Empigen® detergent . The wells of microtiter plates are coated with a sheep antiserum specific for the C-terminus of gpl20. After washing and blocking, the plates are incubated with various dilutions of lysed virus. An aliquot of an affinity purified pool of human mAbs directed to the CD4-bd is added, followed by an enzyme- (e.g., alkaline phosphatase) labeled anti-human IgG. A standard curve is constructed using rgpl20IIIB (Repligen) as a reference reagent in a range of about 0.1-20 ng/ml. The standard curve is used to estimate the concentration of gpl20 in a sample.
5.1.6. PURIFICATION OF bs-mAbs
Production of bs-mAbs by the hybrid hybridoma method described above results from the recombination of H and L chains, which process can also lead to the formation of multiple isoforms (Suresh et al . , supra) . which can functionally interfere with true bs-mAbs and should therefore be removed.
Antibodies are purified from culture supernatants using well-known methods. Thus, the antibodies are preferably first by precipitated with 50% saturated ammonium sulfate and the resulting precipitate dissolved in phosphate buffered saline (PBS) and dialyzed against several changes of PBS. Antibodies are further purified, first by Protein A affinity chromatography (Pharmacia) and subsequently by ion-exchange HPLC, for example, using a 7.75 x 100 mm Bakerbond Abx column (J.T.Baker, Phillipsburg, NJ) . Samples are then preferably dialyzed against 10 mM 2- (N-morpholine) ethanol sulfonic acid (MES) and chromatographed in a linear gradient of 10 mM MES, pH 5.6, 500 mM sodium acetate (NaAc), pH 7.0, over a 60 minute period. Immunoglobulins are collected in separate fractions every minute and then tested for antigen binding or binding to an isotype-specific antibody in the double isotype or antigen-isotype ELISA described above. Fractions with bispecific and parental mAb activity are pooled, rechromatographed by ion-exchange HPLC and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . 5.1.7. POLYACRYLAMIDE GEL ELECTROPHORESIS
Samples of purified mAbs and bs-mAbs are diluted into a buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.002% bromphenol blue, 10 mM Tris HCl, pH 6.8, '• and are boiled for 3 minutes. A 20 μl volume is electrophoresed on a 10% polyacrylamide gel according to standard methods (Laemmli, E.K., 1970, Nature 227:680) . Proteins are stained preferably using Coomassie blue.
5.1.8. DETERMINATION OF DISSOCIATION • CONSTANT
To determine the dissociation constants (Kd) for the binding of antibodies, the method of Friguet et al . is preferably employed (Friguet, B. et al . , 1985, J. Immun . Meth . 77:305) . Briefly, purified bs-mAbs and parental mAbs are diluted to concentrations ranging from 0.5 to 2.5 μg/ml . To establish the equilibrium phase in solution, the antibodies are incubated 16 hr at room temperature with antigen (V3 loop peptides or rgpl20mB) at various concentrations ranging between about 10 nM and 10 μM. Each mixture is added to microwells coated directly with the homologous antigen at a concentration of about 1 μg/ml . The excess of antibodies not complexed with antigen in solution is measured by ELISA. Absorbance and antigen concentration values are plotted according to the Friguet modification of Klotz (Friguet et al. , supra) to determine the Kd. The Kd is generally taken as the average value of three or more determinations.
5.1.9. PRIMARY ISOLATES OF HIV-1
Three different methods for the production of primary HIV isolates may be used to generate viral proteins which are optimal for testing the binding activity of human mAbs to gpl20. The binding capacity of the antibody serves as an indicator of the suitability of a mAb for bs-mAb production.
One method (Gartner, S. et al., In: TECHNIQUES IN HIV RESEARCH, A. Aldovini et al . , eds. , Stockton Press, New York, 1990, p. 53) is based on the coculture of infected and uninfected lymphocytes. Peripheral blood lymphocytes (PBLs) derived from seropositive patients and from uninfected subjects are first stimulated independently for three days with phytohemagglutinin (PHA) and are then cocultured at a 1:3 ratio in the presence of medium supplemented with interleukin-2 (IL-2) . The cells are fed about twice a week with fresh medium. The culture supernatants are collected prior to feeding and used either as stock virus for neutralization assays (after titration) or treated with 1% Empigen® detergent. Supernatants with the highest p24 levels (as measured by the antigen-capture ELISA) are be pooled and used as a source of viral proteins.
The second method is designed to avoid possible selection of variants which may occur during coculture. Donors' PBLs and CD4+ T cells from infected patients are stimulated directly to enhance HIV-1 production (Hausner, M.A. et al . , 1993. J. Immunol Meth . 157:181) . The patient PBLs are incubated for 1 hour at room temperature in an activated AIS MicroCELLector® CD4 capture flask (Applied Immune Sciences, Menlo Park, CA) in which anti-CD4 mAbs are covalently attached to the plastic surface. After incubation, non-adherent cells are removed by rinsing the flask and the adherent CD4+ cells are cultured in the flask in medium supplemented with human IL-2 and an anti-CD3 antibody, such as the mAb OKT3, for 5 days. The supernatant is collected and treated as previously described.
Another source of primary HIV isolates is patient plasma. Cell-free virus in the plasma is expected to reasonably reflect the virus actually replicating in the host (although passage through PBLs derived from healthy donors may again cause some unwanted selection) . Therefore, about 1 ml of plasma from a patient with high circulating p24 levels is incubated with about 2.5 x 106 PHA-stimulated normal donor PBLs overnight. After washing, the cultures are maintained in medium supplemented with IL- 2 and fed bi-weekly. Supernatants are collected before feeding and tested for p24 antigen. These supernatants can be used either as a source of primary isolates for neutralization or as viral proteins after treatment with Empigen® detergent.
5.1.10. NEUTRALIZATION ASSAYS
5.1.10.1. Neutralization of laboratory
HIV strains
The present inventors have established the neutralizing activity of over 20 human mAbs specific for HIV-1
(Gorny, M.K. et al . , 1993, J. Immunol . 150 : 635 ; Gorny, M. K. et al . , 1992, J. Virol . 66:7538) using a well-known neutralization assay (Nara, P.L., In: TECHNIQUES IN HIV RESEARCH, A. Aldovini et al . , eds., Stockton Press, New York, 1990, p. 77) . The present inventors have further simplified the Nara assay by developing a p24 ELISA to replace the standard read-out which required the microscopic counting of syncytia. The p24 antigen levels and number of syncytia were found to give comparable results (r=0.95) .
Briefly, various serial two-fold dilutions of a bs- mAb and, separately, its parental mAbs are incubated for 1 hr at room temperature with culture supernatants of HIVMN or HIVmB which have a titer of about 100-200 syncytium-forming units/ml. The virus/antibody mixture is added to cells of the CEM-SS line previously adhered to the wells of a poly-L- lysine-coated 96-well plate, and the plate is incubated for 1 hr at 37°C. The washed cells are then cultivated for 3 days and the amount of p24 produced in culture supernatants is assayed by antigen-capture ELISA. At no time during this assay is human plasma used, and the antibodies do not remain in the culture medium. The concentration at which 50% and 90% of the input virus is neutralized is calculated using the IBM- PC program developed by Chou et al . ( supra) . 5.1.10.2. Neutralization of primary HIV isolates
Since primary isolates of HIV-1 differ from laboratory strains, a different assay system is used to determine the neutralization of primary virus isolated from
PBLs of HIV-seropositive subjects. Stocks of primary isolates from the supernatants of PBL cocultures are first titered using a p24-based assay system. Antibodies (bs-mAbs, parental mAbs and control mAbs specific for unrelated antigens) at different concentrations are added to 100 TCID50 of primary HIV isolates in a volume of 100 μl and incubated for 1 h at room temperature. PBL (106) derived from seronegative donors, which cells have been stimulated with PHA for 3 days, are then exposed to the virus/mAb mixture or to medium alone after which the cells are incubated for 1 h at 37°C. The resultant mixtures are transferred to 24-well plates, washed once and then cultured for 7 days. The culture supernatants are tested for p24 levels by antigen-capture ELISA. Neutralization is defined by the method used for neutralization of laboratory strains.
Finally, another neutralization assay is preferably utilized to study the effect of bs-mAbs on viruses which are derived from primary isolates which have been maintained in culture for prolonged periods of time (versus a short period, as above) . Antibodies (bs-mAbs, parental mAbs or control mAbs) at 10-fold dilutions are added to cocultures of 106 PHA-stimulated PBLs derived from infected and uninfected individuals. Antibody-containing cocultures of uninfected and HIVMN-infected PHA-blasts serve as positive virus controls. The cultures are maintained for 21 days, and fed every 3 days. The collected supernatants are assayed for p24.antigen. The results are expressed as the antibody concentration needed for 50% neutralization.
5.1.10.3. Synergy analysis To select the mAbs which will result in the most potent bs-mAbs, combinations of different mAbs are tested to determine antagonistic, additive or synergistic effects in the neutralization of laboratory HIV strains. "Cooperation" of mAbs is determined in terms of the concentration of mAbs which achieve a given effect when used alone as compared with the concentration needed to obtain the same effect when the mAbs are used in combination. Human mAbs to the V3 loop and to the CD4-bd, alone and in combinations, are tested for 50 and 90% neutralization. Results are analyzed using the computer program of Chou et al . ( supra) .
5.2. BIOLOGIC FUNCTION OF bs-mAbs
Each bs-mAb of the present invention, produced as described herein, is preferably also tested for its ability to bind to virus-infected cells using flow cytometric methods described herein (see, Tyler et al . , supra) . To minimize non¬ specific binding and high backgrounds, a biotinylated form of each bs-mAb is preferably used together with fluoresceinated - avidin. Controls consist of staining uninfected cells or staining infected cells with biotinylated human mAbs specific for an unrelated antigen, such as a cytomegalovirus (CMV) antigen. Functional studies include neutralization assays with (1) various laboratory isolates ( e . g. , IIIB and Ala) and with (2) primary HIV isolates from infected adults.
The mAbs described in Table I, above were selected for their specificities to various laboratory strains of HIV (Gorny, M.K. et al . , 1993, J. Immunol . 150 : 635 ; Gorny, M.K. et al . , 1992, J. Virol . 66: 7538 ; Karwowska, S. et al . , 1992, AIDS Res . Hum. Retrovir. 8:1099) .
Bs-mAbs are preferably tested against the same laboratory virus strains to prove that neutralizing activity is a property of the bs-mAb molecule. This activity is then compared to the parental mAb activity, to test whether incorporation of two different specificities into one antibody molecule results in greater virus neutralization capacity. Use of laboratory isolates allows better standardization of the neutralization assay, and easier repetition and adaptation to different combinations of control Abs. However, this assay alone is not sufficiently informative about the clinical utility of a given bs-mAb of this invention, because a laboratory strain of HIV represents one unique genome and phenotype of virus and does not reflect the complexity of the HIV-1 variants present in a single infected individual.
For the above reason, testing of the bs-mAb on primary HIV isolates is important for evaluation of natural immunity against HIV-1 infection, especially when mAbs or bs- mAbs are considered. as candidates for passive administration in the immunoprophylaxis or therapy of HIV infection. Primary isolates comprise a mixture of closely related viral genomes which result from a high misincorporation rate caused by reverse transcriptase errors (Nara, P. L. et al . , 1991, FASEB J. 5:2437; Wain-Hobson, S., 1989, AIDS 3:sl3) . Variation among HIV isolates occurs within a given individual during the course of infection, as well as between individuals (Epstein, L.G. et al . , 1991, Virology 180 : 583 ; Meyerhans, A. et al. ,
1989, Cell 58:901) . Similarly, virus variation occurs in the course of its amplification during in vi tro culture. Selection of virus variants adapted to tissue culture conditions induces and stabilizes alterations that are not necessarily representative of those which exist in an infected subject in vivo (Wain-Hobson, supra; Cheynier, R. et al . ,
1990, Dev. Biol . Standard 72:349) . For instance, the primary culture of HIV with IL-2 treated PBLs may preferentially select for lymphocytotropic viruses, at the expense of macrophage-tropic viruses. Despite the foregoing, primary isolates remain the most representative virus population available for testing the neutralizing activity of different anti-HIV antibodies in vi tro.
Because it appears that primary isolates from PHA- stimulated lymphocyte cultures are difficult to neutralize (Posner, M.R. et al . , 1993, J. AIDS 6: 7) , two types of assays are preferably performed. In the first assay, the virus is exposed to the bs-mAb for a brief period of time while in the second assay the bs-mAbs remain in the culture throughout the test.
In the first assay, a stock of primary HIV isolates which has been titered using a p24-based assay, is incubated with the test bs-mAbs for about 1 hour. Cultures without bs- mAbs or with non-neutralizing mAbs can serve as negative antibody controls, while cultures containing dilutions of known polyclonal serum pools can be used as positive antibody controls. The mixture of virus and antibody is added to PHA- stimulated normal PBLs, the cells are then washed to remove antibody, and the cells then maintained in culture for 7 days . Effective virus neutralization is indicated by lack of p24 antigen in cultures treated with antibodies versus the presence of virus in cultures not treated or treated with a non-neutralizing mAb. Results may be expressed as the amount of IgG necessary for 50 and 90% neutralization.
The second assay is designed to mimic the situation in vivo under the most optimal conditions. A bs-mAb and control antibody, at 10-fold dilutions, are added to the co-cultures of PHA-blasts from infected and uninfected donors. The cultures are maintained for 21 days and the supernatants collected every 3 days and assayed for p24 antigen. The cultures are fed with media supplemented with the appropriate antibody to maintain a constant antibody level . Decreased levels of p24 antigen in culture supernatants (p24 antigen levels reflect the amount of replicating virus) may be explained as follows: (1) Cell-free virus is bound and neutralized by antibodies; (2) virus binding to CD4+ T cells is blocked; (3) the fusion or penetration of the virus into susceptible cells is inhibited; and/or (4) cell-to-cell transmission of the virus is prevented by the bs-mAb.
5.3. PRODUCTION OF HUMAN bs-mAbs USING MOLECULAR GENETIC METHODS
In a preferred embodiment, bispecific F(ab')2 heterodimeric antibody fragments, rather than intact bs-mAbs, are produced in transfected cells using leucine zipper peptides from the Fos and Jun proteins. The biological function of the new constructs, including binding affinity to HIV antigens and the ability to neutralize laboratory strains and primary isolates of HIV, are compared with intact bs-mAbs.
The production of bs-F(ab')2 in large quantity is preferably carried out in mammalian cells. An E. coli expression system is useful for further enhancement of production, and for producing bs-mAbs which can be utilized in animal studies and in the clinic.
5.3.1. cDNA CLONING OF IMMUNOGLOBULIN GENES
The general scheme is as follows. mRNA is purified from cells producing human mAb, such as heterohybridoma cells, for example, 729-D, by lysis of the cells in a buffer containing SDS and proteinase K, followed by oligo-dT chromatography of the lysate. DNA and mRNA are removed by high and low salt washes, respectively, and polyA+ RNA is eluted in TE buffer (no salt) . The polyA+ RNA is then precipitated with ethanol and used directly in a reverse transcription reaction to convert it to first strand cDNA. First strand cDNA is used to amplify the specific light chain and Fd regions using the polymerase chain reaction (PCR) . Primers specific for the signal sequences of the immunoglobulin H or L chain is used in conjunction with primers in the hinge region or the region 3' to the L chain coding sequence. cDNA fragments encoding the L chain and or the Fd fragment are cloned into pUC18 and several clones of each are sequenced using dideoxy technology and modified T7 DNA polymerase (Sequenase, US Biochemical) . This technology is well established and has been used by the present inventors to clone and sequence seven different human mAb H and L chain genes, including 447-D.
5.3.2. ASSEMBLY AND GENETIC FUSION OF Fd FRAGMENTS TO fos OR iun
The leucine zipper regions of the Fos and Jun proteins are assembled from oligonucleotides. Since each of these segments is approximately 140 bp in length, four oligonucleotides from 70-75 bases long are required. Each 75mer is phosphorylated using T4 polynucleotide kinase and annealed to the appropriate 70-mer. The annealed pairs of oligonucleotides are then mixed and ligated together using T4 DNA ligase. The 140bp fragment is then purified using agarose gel electrophoresis. The assembled and ligated oligonucleotides are then joined to the Fd antibody fragment by recombinant PCR and cloned into mammalian expression vectors using the Ncol and Notl restriction sites introduced during PCR.
5.3.3. EXPRESSION OF Fab' -ZIPPER
The respective Fd-zipper and L chain genes are inserted, as an Ncol-Notl fragment, into an expression vector, preferably pSJ301. The pSJ301 vector contains the cytomegalovirus immediate early gene ( CMVie) promoter/enhancer for high level transcription of the immunoglobulin genes, a linker region containing a consensus translation initiation signal ("Kozak" sequence) as well as cloning sites for inserting genes, and the bovine growth hormone (bGH) polyadenylation signal for efficient RNA polyadenylation. The expression cassettes for the L chain genes from two heterohybridomas, for example from 729-D and 447-D, and the Fd zipper gene are combined into a single plasmid using a strategy based on the compatible cohesive ends produced by the restriction enzymes BamHl and Bglll which flank the CMV promoter - polyA-i- region. This is accomplished by combining a Pvul-BamHl fragment from the L chain expression vector with a Bglll-Pvul fragment from the Fd expression vector.
5.3.4. STABLE EXPRESSION OF BIVALENT MOLECULES
Stable expression of the bivalent molecules is accomplished in a mammalian cell line, for example, the hfr-Chinese hamster ovary (CHO) cell line DG44. The combined expression vectors, for example the 447-D F(ab'- zipper) and 729-D F (ab' -zipper) expression vectors, are transfected separately into CHO cells by calcium phosphate coprecipitation and selected in nucleoside-free medium. Expression levels are measured after each subcloning step in methotrexate and only the highest producing colonies are carried forward. In this manner, the present inventors have routinely created cell lines stably producing, in under six months, antibody in quantities greater than 100 mg/liter/4 days.
The F(ab) '2 homodimers are purified by HPLC and/or affinity chromatography using anti-L chain antibody (anti-λ for 447-D; anti-λ for 729-D) immobilized on agarose. Bivalent F(ab')2 fragments are generated by mild reduction, mixing and reoxidation of the purified preparation.
5.3.5. PRODUCTION OF F(ab' -ZIPPER) IN E. COLI
The ability of transformed E. coli cells to secrete F(ab' -zipper) either to the periplasm or to the medium depends on several factors, most importantly the strength of the promoter used, the temperature, and the ability to tightly control expression. In this case the pelB leader is preferably used to secrete the individual chains expressed from the lac promoter. Combined vectors are constructed using a similar strategy to that described above for the mammalian expression vectors. Expression of the F (ab' -zipper) is induced by an appropriate inducer for the lac promoter, for example isopropylthio-jS-D-galactoside (IPTG) , at 25°C.
Bacterially produced F (ab' -zipper) can be reoxidized directly after purification by dialyzing against redox buffer. The resulting product should contain mostly heterodimers due to the preferential dimerization of proteins to which Fos and Jun are genetically fused. The heterodimer 729D x 447D, for example, is purified using an HPLC system. A combination of ELISA and SDS-PAGE are used to identify the peaks. 5.4. IN VIVO SCREENING OF bs-mAbs
The mAbs of the present invention may be screened for anti-HIV activity using a recently developed mouse model (CITE) . SCID mice, lacking T lymphocytes are infused with about 2 x 107 human peripheral blood lymphocytes i.p. After about 2-4 weeks, the mice are injected i.p. with the bs-mAbs being tested. Other animals may receive control antibodies, the parental mAbs or a mixture of the two parent mAbs. Shortly after this step, the animals are challenged with a preparation of HIV. At various time points thereafter, in particular about 6 weeks, the animals are examined for virus growth. This can be performed in any of a number of ways, including immunoassay of serum or tissues for p24 antigen, PCR of lymphocytes to detect HIV sequences, co-culture of the animals' lymphocytes with PHA- stimulated human lymphocytes, as described herein, for growth of HIV, and the like. Neutralizing mAbs that are useful for therapy of humans are expected to neutralize the virus and limit infection in this SCID mouse model.
5.5. CLINICAL USES OF bs-mAbsd
Bs-mAbs which incorporate two anti-viral specificities into a single molecule and neutralize HIV-1 with augmented potency over the parental mAbs are particularly useful in prophylaxis of HIV-1 infection. Passive immunotherapy with the bs-mAbs of the present invention is useful in the prevention of transmission of the virus from an infected mother to a newborn, by treating the mother during pregnancy, and in particular, prior to delivery. Another application for bs-mAbs of the present invention is in the prophylaxis of health care workers exposed to the virus through needle sticks. Needle injury is a common problem for medical staff in hospitals. While the risk of HIV-1 transmission by definite inoculum from a known HIV-positive subject is relatively low (below 1 %) , the lack of effective treatment is a source of concern and a cause of much anxiety (Gerberding, J.L., 1989, Infec . Dis . Clin . N. Amer. 3 : 735 ) . The immediate post-exposure administration of one or more bs-mAbs to a person injured via a contaminated needle or otherwise exposed to HIV may protect against successful HIV-1 infection. By the term "treating" is intended the administering to subjects of a bs-mAb, or an HIV antigen-binding fragment or derivative thereof, such as the bs-F(ab')2 fragment described above, for purposes which may include prevention, amelioration, or cure of HIV infection or AIDS.
Bs-mAb compositions within the scope of this invention include all compositions wherein the bs-mAb or derivative is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.01 to 100 mg/kg/body wt. The preferred dosages comprise 1 to 100 mg/kg/body wt. The most preferred dosages comprise 10 to 100 mg/kg/body wt. Administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
In addition to the bs-mAb or derivative thereof, a pharmaceutical composition according to the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations comprise suitable solutions for administration by injection, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active bs-mAb together with the excipient. Suitable formulations for parenteral administration include aqueous solutions of the bs-mAb in water-soluble form, for example, as water-soluble salts. In addition, suspensions of the bs-mAb as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions that may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
Suitable injectable solutions include intravenous subcutaneous and intramuscular injectable solutions. The active compound may also be administered in the form of an infusion solution or as a nasal inhalation or spray.
For intravenous administration, the bs-mAb is preferably administered by drip infusion in a buffered aqueous solution. The bs-mAb may be administered in single or divided doses.
The pharmaceutical composition of the present invention may be comprised of a bs-mAb as described herein in combination with another agent or medicament useful in treating HIV infection or symptoms associated with HIV infection or AIDS. Medicaments are considered to be provided "in combination" with one another if they are provided to the patient concurrently or if the time between the administration of each medicament is such as to permit an overlap of biological activity. Thus, the bs-mAbs of the present invention may be administered in combination with known anti-retroviral agents, for example, AZT, ddA, ddT, ddl, and ddC. Many additional anti-retroviral drugs known in the art which inhibit the activity of various HIV genes are included within the scope of this invention. Also included in such combination therapeutic methods are anti-retroviral peptides, soluble CD4 peptides, or immunotoxins (Vitetta, E. et al., Science 215:644-650 (1983) ; Pastan, I. et al . , Cell 47:641-648 (1986) ; Olsnes, S. et al., Immunol . Today 10 : 291-295 (1989) ; Oeltmann, T.N. et al . , FASEB J. 5:2334-2337 (1991)) comprising such antibodies or CD4 peptides. Anti-bacterial or anti- parasitic drugs, such as drugs directed to Pneumocystis carinii disease, and drugs useful for alleviating any symptoms associated with AIDS, are included within the scope of the combination treatment methods and bs-mAb compositions of the present invention.
In addition to treatment, the bs-mAbs of the present invention may be used diagnostically to detect HIV in cells or body fluids, or to characterize the viral strain infecting a subject. The bs-mAbs are used in the same manner in which mAbs or polyclonal anti-HIV sera are used in immunodiagnosis.
5.6. DETERMINATION OF THE FREQUENCY AND SEQUENCE
CHANGES OF ESCAPE MUTANTS SELECTED BY bsmAbs
Because of genetic variation, an HIV-1 population is not a homogeneous virus population but is rather a mixture of closely related variants (Wain-Hobson, S., 1992, Curr. Top. Micro . Immunol . 176:181) . These variants are subjected to selection by the host immune system resulting in the emergence of variants which can evade immune defenses. Studies performed in chimpanzees, and in vi tro using neutralizing antibodies or patient sera have demonstrated the immune selection of neutralization- resistant mutants (Reitz et al . , supra; Cotropia, J. et al . , 1992, In: VACCINES 92 : MODERN APPROACHES TO NEW VACCINES INCLUDING PREVENTION OF AIDS, F. Brown et al . . eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 157) . Escape mutants selected by neutralizing antibodies exhibited sequence changes in proteins which affected the antibody binding sites. The bs-mAbs of the present invention with specificity against the V3 loop and/or the CD4-bd can be used for the selection of HIV escape mutants in vi tro . Use of these antibodies allows (1) analysis of the genetic variation in HIV-1 that simultaneously alters the phenotypes of two gpl20 domains; (2) definition of the susceptibility for mutations of two neutralizing epitopes, and (3) determination of the threshold level of bs-mAbs that block the selection of neutralization-resistant variants.
A single neutralizing mAb may select a resistant HIV variant through a change or changes which can affect the epitope and shroud it from the mAbs. In the case of a bs-mAb specific for two different epitopes in one molecule, more than one set of changes may be necessary to select a neutralization-resistant mutant. The rate of mutations in the HIV-1 genome is relatively constant, approximately one mutation per genome per replication cycle (Dougherty, J.P. et al . , 1988, J. Virol . 62 : 2817) . While the frequency of appearance of escape mutants in the presence of one mAb will be as high as 10"5, in the presence of bs-mAb, the frequency will be in the order of 10"10. Thus, bs-mAbs are expected to be advantageous over single mAbs and induce the emergence of fewer variants during the same time frame.
The present invention allows one to establish the frequency of appearance and the selection of HIV-1 escape mutants propagated in the presence of bs-mAbs as compared to the two parental mAbs. Bs-mAbs may select escape mutants differently than parental mAbs. This comparative analysis is based on: (1) the quantitative difference in the appearance of escape mutants as measured by the number of wells in a 96-well plate where p24 antigen is present, (2) the time period during which the escape mutants appear, and (3) the pattern of sequence changes in gpl20.
A quantitative assay was developed by the present inventors in which CEM-SS cells are infected with the molecularly characterized viral inoculum of HXB2D. A virus stock is prepared for standardizing the virus input for selecting escape mutants. The viral clone of HXB2D virus is obtained by transfecting H9 cells with the complete molecular proviral clone, pHXB2D, using a lipofection transfection procedure according to the manufacturer's instructions (GIBCO BRL, Gaithersburg, MD) . The transfected H9 cells are monitored for productive viral infection by the detection of p24 using an antigen-capture assay. About seven days post-transfection, when the cells are producing virus, the supernatant is harvested, filtered and used as a stock of cell-free virus. The infectious titer of the stock is determined by end-point titration using 10-fold serial dilutions of virus (50 μl) incubated with 100 μl of CEM-SS cells at 2 x 105 cells per well in microtiter plates at 37°C for 7 days. The plates are scored for the presence of p24 antigen and the 50% TCID50 is calculated.
To determine the frequency of escape mutants, a quantitative analysis is performed using 96-well microtiter plates. CEM-SS cells at a density of 105 cells/well are exposed to 100 and 1000 TCID50 of HXB2D virus stock for 1 hr at 37°C. The unbound virus is removed by washing, and the cells are then cultured in the presence of various dilutions of either neutralizing bs-mAbs, parental mAbs or non-neutralizing irrelevant (control) human mAb at concentrations of 10 and 100 μg/ml. The cultures are fed periodically (preferably biweekly) with medium containing the appropriate antibody and the supernatant is monitored once a week for the presence of p24 antigen. While the first escape mutants may appear after only 2 weeks, the cultures are preferably monitored for six weeks.
The percentage of wells containing p24 antigen indicates the frequency of appearance of escape mutants selected by either the bs-mAbs or the parental mAbs at their respective concentrations. The time period during which the escape mutants appear may also indicate a difference in the selection effect between neutralizing bs- Abs and parental mAbs. Virus is recovered from those wells with a high p24 level as cell-free supernatant and the progeny viruses are propagated in CEM-SS cells in the absence of antibodies. The virus stock is then tested for resistance to neutralization by the selecting mAbs. Recently, the present inventors' group characterized a human serum-selected HIV-1 escape mutant which was resistant to neutralization by CD4-bd mAbs. Virus was recovered after two weeks from the cultures containing the lowest serum dilutions (1/160 and 1/320) . Over a 7 week period no evidence of viral replication was observed at the highest concentration of selecting serum (1/40) .
Determination of a bs-mAb concentration which blocks the selection of escape mutants provides useful information for selecting bs-mAbs and their doses for use in a passive immunotherapy approach.
In another embodiment, the escape mutants are subjected to nucleic acid sequencing for comparative analysis of mutations which occurred during culture in the presence of either the bs-mAb or the parental mAbs. DNA is prepared by standard methods from cultures containing neutralization escape mutants. The polymerase chain reaction (PCR) assay is performed according to the supplier's specifications. All oligonucleotide primers are chosen from HIV-HXB2CG GenBank sequence files. Gpl20 gene amplifications are carried out in two steps by using a model 480 thermal cycler (Perkin Elmer/Cetus) . For gpl20 cloning, the first amplification step consists of 30 cycles of PCR using oligonucleotide primers REC-07 and REC-09 (Smith, M.S. et al . , 1993, J. Infec . Dis . 167:445.) . The second amplification step of 30 cycles uses 5% of the product from the first amplification, together with primers REC-27 and REC-12. The 1550-base-pair gpl20 PCR products are purified by agarose gel electrophoresis and are subcloned in an expression vector, preferably M13mpl8. Pure single stranded DNA is prepared from this vector multiplied in infected E. coli cells and is sequenced directly using the Sequenase kit version 2.0 (U.S. Biochemical) .
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
6. EXAMPLE: SPECIFICITY OF HUMAN mAbs AND SYNERGY IN HIV NEUTRALIZATION
The list of some HIV-neutralizing human mAbs developed in the present inventors' laboratory is presented in Table
I, above. Sixteen mAbs recognize the V3 loop and eight bind to the CD4-bd of gpl20. All of these mAbs neutralize HIVMN (50% neutralization) at concentrations ranging from 10 to 4700 ng/ml (Gorny, 1993, supra) .
The mAb 447-52-D, which is the most broadly reactive with diverse HIV-1 strains (Gorny et al . , 1992, supra) , was tested for its ability to interact with other mAbs specific for the CD4-bd or with other V3-specific mAbs in neutralizing HIV. The combination of 447-52-D with either of two anti-CD4-bd mAbs, 654-D and 729-D, showed synergistic activity in the neutralization of HIVIIIB (Table
II, below) . For instance, while it required >5 and 2.5 μg/ml of 447-52-D (anti-V3) or 654-D (anti-CD4-bd) , respectively, to neutralize HIVIIIB, a 1:1 mixture of these two mAbs gave 50% neutralization at 0.9 μg/ml of each mAb. The combination index (CI) , a measure of synergy (Chou, T. et al . , 1984, Adv. Enzyme Regul . 22 : 27 ) , was significantly below 1.0 for 50% neutralization indicating strong synergistic activity. Similar CI values were observed for the combination of 447-52-D and 729-D. The interaction of mAbs 729-D and 694/98-D, however, was additive for 50% neutralization, yielding a CI value of close to 1.0. TABLE II
NEUTRALIZING DOSES OF ANTI-gpl20 HUMAN mAbs AND THEIR COMBINATIONS AGAINST HIVmB mAb 50% Neutralizing CIa
Concentration (μcr/ml)
447-D alone >5.0
654-D alone 2.5
654D + 447D (1:1) 0.90 0.23
447-D alone 5.0
729-D alone 1.25
729D + 447D (1:1) 0.65 0.25
694/98- -D alone 0.7
729-D alone 5.0
729D - 694D (1:1) 3.7 1.05
Combination Index (CI) was calculated according to Chou et al . ( supra) . A CI value which is less than 1.0 indicates synergy; a CI value of around 1.0 demonstrates additivity.
7. EXAMPLE: PRODUCTION OF HYBRID HYBRIDOMAS
In view of the above synergy results, the present inventor initiated a program of producing hybrid hybridomas which would secrete bs-mAbs. The hybridoma cell line 447- 52-D was back selected for HAT-sensitivity and was fused separately with two hybridomas producing mAbs to the CD4-bd (654-D and 729-D) . The 447-52-D cell line was also fused with hybridomas 694/98-D and 782-D making mAbs to the V3 loop. 694/98-D and 782-D were chosen for the production of bs-mAbs on the basis of their strong reactivity and high affinity to HIVIUB and HIVRF, respectively, whereas 447-52-D had the most prominent activity against HIVMN.
Thus, it was expected that bs-mAbs made with 447- 52-D and 694/98D or 782-D would have broadened specificity and an ability to neutralize a broader array of HIV-1 variants.
A "control" cell line producing a non- neutralizing human mAb to p24, designated 71-31, was fused to the cell line producing 447-52-D to construct a "control bs-mAb" .
Table III describes the production of (a) hybrid hybridomas ("quadromas") derived from the fusion of two hybridomas, and (b) one trioma (71-31/447D) obtained by fusion of an EBV transformed cell line with a hybridoma. The frequency of wells containing hybrid hybridomas ranged from 8% to 65%. The percentage of wells containing hybridomas which produced bs-mAbs ranged between 3% and 57%.
The hybrid hybridomas and trioma were cloned at 100, 10 and 1 cell/well to stabilize the production of bs- mAbs. After cloning, the positive wells were expanded into flasks.
TABLE III
GENERATION OF BISPECIFIC mAbs SPECIFIC FOR HIV-1
Wells with
Hybrid- Wells Hybrid- Wells with specific Abs Hybridoma Plated Hybridomas IgGl IgG3 bs-mAbsa
654D/447D 192 105 51 44 32 (30%)
729D/447D 144 64 28 56 28 (44%)
694D/447D 192 126 107 82 72 (57%)
782D/447D 144 92 4 57 73 (3%)
71-31/447D 132 11 2 5 1 (9%)
The production of bs-mAbs was screened by a double isotype ELISA in which an Ab to one of the subclass of the bs-mAb was coated on the plate, whereas the Ab to the second subclass was enzyme labeled. EXAMPLE: PURIFICATION AND ANALYSIS OF bs- mAbs
Two hybrid hybridoma cell lines, 729/447D and 71-31/447D (see Table III) , were expanded in order to generate large volumes of culture supernatant . The IgG concentration in each of the two supernatants was 49.0 and 37.0 μg/ml, respectively. The antibodies in these supernatants were concentrated and purified on Protein A- Sepharose affinity columns. The total amount of IgG obtained from the two supernatant pools were 14.0 mg for 729/447D and 11.0 mg for 71-31/447D. Such affinity- purified material (2.5 mg) was applied to a Bakerbond Abx HPLC column. The fractions, collected each minute, were tested by an antigen-isotype and single isotype ELISA. Figure 1 (Panels A and B) shows results of representative HPLC separations of the antibodies derived from 729/447-D (panel A) and from 71-31/447D (Panel B) .
The early fractions (peak 1) lacked antibodies specific for the V3MN peptide or the rgpl20IIIB antigen, and contained proteins which did not bind to the column.
Fractions of the second peak bound to the rgpl20 or p24 antigen only, and contained the parental mAbs, 729-D and 71-31, respectively.
Peak 4 contained the second parental mAb, 447-52-D based on binding to the V3MN peptide. In the case of . the 71-31/447D bs-mAb (Figure 1, panel B) fractions from peak 4 contained antibody which bound to the V3MN peptide and reacted with both anti-IgG3 (the isotype of 447-52D) and anti-IgGl (the isotype of 71-31) . This indicated the presence of some bs-mAb in this peak.
Peak 3 contained bs-mAbs only. This conclusion was based on the strong reactivity of fractions included in this peak in an antigen-isotype ELISA in which the antibodies bound V3MN peptide while the of the IgGl isotype was associated with antibody to p24 but not V3MN.
The fractions comprising peaks 3 and 4 of bs-mAb 729/447-D were re-chromatographed by HPLC and, along with fractions from peak 2, were reduced and examined by SDS- PAGE (Figure 2) . The peak 3 fractions had showed bands representing the two H chains (an IgGl H chain and an IgG3 H chain) of the bs-mAb. In contrast, fractions from peak 2 and peak 4 each showed a single band, each of different mobility, indicative of H chains of the parental mAbs.
Purified fractions of peak 2, 3 and 4 of 729/447D were tested in a neutralization assay against HIVmB. The results appear in Table IV, below. Protein A-purified parental mAbs 729-D and 447-52-D were mixed together at a 1:1 ratio for comparison with the HPLC-purified bs-mAb. The results indicated that the mixture of the two parental mAbs showed synergism for 50% neutralization and resulted in increased potency of each parental mAb. Synergy was demonstrated when the combination index (CI) , calculated according to the method of Chou et al . ( supra) was significantly below 1.0. Here, the CI for this mixture was 0.1, though the CI for the bs-mAb purified by HPLC was 10- fold lower. Furthermore, the final concentration of bs-mAb needed to achieve the same activity (50% neutralization) was 9-fold lower than the mixture of two parental mAbs.
TABLE IV
NEUTRALIZATION OF HIVUIB BY INDIVIDUAL gpl20-SPECIFIC HUMAN mAbS, MIXTURES OF mAbs AND BISPECIFIC mAbS
mAb 50% Neutralizing CIa
Concentration (μq/ml)
447-D 30.1
729-D 4.7 729D + 447D (1:1) 0.9 0.1
729/447D bs-mAbb 0 . 1 0 . 01
a See Table III b The 729/447D bs-mAb was HPLC purified, whereas parental mAbs were Protein A-purified These results revealed the advantage of bs-mAbs over the mixture of parental mAbs in neutralizing HIV. Purification of bs-mAbs is feasible using HPLC technology, and may require a re-chromatography step. Larger quantities of antibodies for use in in vi tro studies, animal experiments and clinical applications may be obtained by using larger HPLC columns. Alternatively, molecular genetic approaches, as disclosed herein, are available for large scale production of pure intact bs-mAbs or antigen-binding fragments thereof.
EXAMPLE: SELECTION OF PARENT mAbs FOR PRODUCING bs-mAbs SPECIFIC FOR V3 LOOP AND CD4-BINDING DOMAIN
For optimal selection of mAbs to produce bs-mAbs recognizing both the V3 loop and the CD4-bd, a group of mAbs was tested in a binding ELISA using primary HIV isolates as antigen. Five primary viral isolates derived from different HIV-positive individuals were used in an antigen capture ELISA. The solid phase was created using a capture antibody specific for the C-terminus of gpl20, and envelope glycoproteins were exposed for binding with the test mAbs.
The p24 level in primary isolate stocks was between 40 and 75 ng/ l, while the level of gpl20 ranged between 2.4 and 4.8 ng/ml. Table V presents the results expressed as a ratio of optical density of bound mAb to the average background.
Several anti-V3 mAbs bound to antigens of the primary isolates. The most cross-reactive mAb against various V3 peptides, 447-52-D, recognized and bound to all the tested primary isolates . This result strengthens the notion that 447-52-D is a preferred partner for production of bs -mAbs .
TABLE V
BINDING OF HUMAN MONOCLONAL ANTIBODIES TO PRIMARY HIV ISOLATES
PRIMARY ISOLATES3 mAbb WM8 BW9 CBIO ON6 CN12
Anti-V3 3 Loop:
257-2D 0.75° 0.98 1.21 0.89 2.24d
268-10D 0.79 0.83 1.15 0.82 1.15
311-11D 0.82 0.91 1.14 0.99 1.18
386-10D 0.99 1.15 2.26 1.53 1.89
391/95-D 1.59 1.54 3.94 1.98 2.46
412-D >8.2 1.76 2.32 1.79 1.01
419-D 2.87 1.90 2.38 2.03 2.14
447-52D 2.73 1.89 2.65 1.87 2.59
453-D 1.76 1.46 1.42 1.78 1.44
537-D 0.90 0.96 1.63 0.97 1.04
Anti-CD4- -Binding Domain:
558/64D 2.12 1.72 2.54 1.93 2.27
654-D 2.46 2.74 3.43 2.27 2.16
728/29-D 2.28 1.89 2.69 2.09 2.52
729-D 2.29 1.99 3.07 2.14 2.90
a gpl20 of primary HIV isolates was captured on an ELISA microplate using an anti-gpl20 antibody specific for the C- terminus and incubated with the mAbs as shown. b All mAbs were used at an IgG concentration of 10 μg/ml except in the case of 558/64D which was used at 4.2 μg/μl. c Results are expressed as a ratio of the absorbance of the bound antibody to the absorbance of the average background. Average Background: 0.245 ± 0.058 d underlined values indicates positive binding i.e., ≥ x + 3 S.D. = 0.419 (ratio ≥ 1.71)
On the basis of this binding assay, a second partner is preferably 419-D, 412-D or 391/95-D. Additional studies with other primary isolates may be used to determine which of these three mAbs is a more preferred parent for a bs-mAb. Previous analysis of these three mAbs indicated that while they were restricted in their cross- reactivity to V3 peptides, they were quite cross-reactive in binding to various primary isolates. This result demonstrates the importance of evaluating reactivity of antibodies with primary HIV isolates for selecting partners for production of bs-mAbs of preferred specificity. All four of the anti-CD4-bd mAbs examined recognized each of the five primary isolates. Thus, other criteria, including epitope fine specificity and isotype are utilized for selecting parent mAbs for the production of bs-mAbs which recognize CD4-bd.
10. EXAMPLE: PRODUCTION OF SPECIFIC F(ab')2
HETERODIMERS OF TWO HUMAN mAbs USING MOLECULAR TECHNIQUES
In collaboration with Dr. S. Johnson from Medlmmune, Gaithersburg, MD, the present inventors cloned genes coding variable regions of several human mAbs from hybridoma cells by PCR. VH and VL genes for anti-V3 loop mAbs (257-D, 268-D, 447-D and 694/98-D) and for anti-CD4-bd mAbs (559-D, 588-D, and 654-D) have also been cloned and expressed. Bispecific F(ab')2 heterodimers of two human mAbs will be produced: 447-D and 729-D, using the methods described in Section 5.3, above. These two parental mAbs synergistically neutralize HIVIIIB. Bispecific mAbs obtained by hybrid hybridoma cell lines (see Section 7, above) have improved neutralizing activity relative to the parental mAbs.
The bispecific F(ab')2 heterodimer of 729D x 447D will be compared with the 729/447D bs-mAb, described herein, in neutralization assays against laboratory and primary isolates of HIV. If such bispecific heterodimers produced recombinantly show similar functional activity, the molecular genetic approach will be utilized for large- scale production.
One potential disadvantage is that the F(ab'- zipper)2 bispecific heterodimer may have a shorter half-life in vivo than an intact antibody. This may be overcome by further modifications, for example, by alteration of charge properties or the addition of polyethylene glycol to form a complex, or by the additional through recombinant techniques of an Fc portion.
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 invention 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.

Claims

WHAT IS CLAIMED IS:
1. A bispecific human monoclonal antibody capable of neutralizing HIV, which antibody has two different human variable regions, wherein one of said variable regions is specific for an epitope of the HIV glycoprotein gpl20.
2. A bispecific human monoclonal antibody according to claim 1, each variable region of which is specific for a different epitope of the V3 loop of said gpl20.
3. A bispecific human monoclonal antibody according to claim 1, in which one variable region is specific for an epitope of the V3 loop and the other variable region is specific for an epitope of the CD4- binding domain of said gpl20.
4. A bispecific human monoclonal antibody according to claim 1, wherein said epitope is an epitope of the V3 loop and is a peptide of at least four contiguous amino acids from the amino acid sequence YNKRKRIHIGPGRAFYTTKNIIG (SEQ ID NO:l) .
5. A bispecific human monoclonal antibody according to claim 1, wherein the epitope is a V3 loop epitope, and which epitope is:
(a) a fragment of SEQ ID NO:l selected from the group consisting of HIGPGR, HIGPGRA,
IGPGRAF, GPGRAFY, PGRAFYT, GRAFYTT, GPGRAF, RKRIHIG, KRIHI, KRIHIGP, IHIGPGR, IHIGP, IGPGR, GPGR, GRAF, and RKRIHIGPGRAFYTR;
(b) RKRIHIGPGRAFYTR (SEQ ID NO:2) ; (c) HIGPIHIGP (SEQ ID NO:3);
(d) GPGRVI (SEQ ID NO:4) ;
(e) GPGRTL (SEQ ID NO:5) ;
(f) GPGRVW (SEQ ID NO:6) ; or (g) GPXR (SEQ ID NO:7) , wherein X is any amino acid residue.
6. A bispecific human monoclonal antibody according to claim 1 wherein said one variable region is specific for a V3 loop epitope and is derived from a human monoclonal antibody selected from the group of human monoclonal antibodies consisting of 257-2D, 268-10D, 311- 11D, 386-10D, 391/95-D, 412-D, 418-D, 419-D, 447-52D, 453- D, 504-10-D, 537-D, 694/98-D, 782-D, 838-D and 908-D.
7. A bispecific human monoclonal antibody according to claim 3 wherein
(a) said one variable region is derived from a human monoclonal antibody selected from the group of human monoclonal antibodies consisting of 257-2D, 268-10D, 311-11D, 386-
10D, 391/95-D, 412-D, 418-D, 419-D, 447-52D, 453-D, 504-10-D, 537-D, 694/98-D, 782-D, 838-D and 908-D; and
(b) the other variable region derived from a human monoclonal antibody selected from the group of human monoclonal antibodies consisting of 448-D, 559/64-D, 588-D, 654-D, 728/29-D, 729-D, 855-D and 860-D.
8. A bispecific human monoclonal antibody according to claim 1, wherein one of said variable regions is specific for an epitope of the CD4-binding domain of HIV glycoprotein gpl20.
9. A bispecific human monoclonal antibody according to claim 8 wherein said one variable region is derived from a human monoclonal antibody selected from the group of human monoclonal antibodies consisting of 448-D, 559/64-D, 588-D, 654-D, 728/29-D, 729-D, 855-D and 860-D.
10. A bispecific human monoclonal antibody according to claim 1, selected from the group of bispecific human monoclonal antibodies 729/447D, 654D/447D, 694D/447D or 782D/447D.
11. A cell line producing a bispecific human monoclonal antibody according to claim 1.
12. A cell line producing a bispecific human monoclonal antibody according to claim 2.
13. A cell line producing a bispecific human monoclonal antibody according to claim 3.
14. A cell line producing a bispecific human monoclonal antibody according to claim 8.
15. A cell line according to claim 11 which is a hybrid hybridoma.
16. A method for neutralizing HIV comprising contacting said HIV with an effective amount of a bispecific human monoclonal antibody according to claim 1.
17. A method for preventing infection of a subject by HIV-1, or for treating a subject infected with HIV-1, comprising administering to said subject an effective amount of a bispecific human monoclonal antibody according to claim 1.
18. A composition useful for neutralizing HIV, preventing infection with HIV or treating a subject infected with HIV, comprising an effective amount of one or more bispecific human monoclonal antibodies according to claim 1.
19. A composition according to claim 18 further comprising one or more medicaments useful in the treatment of HIV infection or AIDS.
20. A method for preventing infection of a subject by HIV, or for treating a subject infected with HIV, comprising administering to said subject an effective amount of a composition according to claim 18, thereby preventing said infection or treating said subject.
21. A method for preventing infection of a subject by HIV, or for treating a subject infected with HIV, comprising administering to said subject an effective amount of a composition according to claim 19, thereby preventing said infection or treating said subject.
22. A pharmaceutical composition comprising the bispecific monoclonal antibody of claim 1 and a pharmaceutically acceptable excipient.
23. A method for producing a hybrid hybridoma cell which produces a bispecific human monoclonal antibody specific for two neutralizing epitopes of the gpl20 protein of HIV, comprising:
(a) selecting two hybridoma cell lines each of which produces a human monoclonal antibody specific for one of said neutralizing epitopes;
(b) fusing said hybridoma cell lines, thereby producing said hybrid hybridoma cell .
24. A method according to claim 23, wherein one of said neutralizing epitopes is from the V3 loop of said gpl20 glycoprotein.
25. A method according to claim 23, wherein both of said neutralizing epitopes are from the V3 loop of said gpl20 glycoprotein.
26. A method according to claim 23, wherein one of said neutralizing epitopes is from the V3 loop of said gpl20 glycoprotein and the other of said neutralizing epitopes is from the CD4-binding domain of said gpl20 glycoprotein.
27. A method for producing a bispecific human monoclonal antibody specific for two neutralizing epitopes of the gpl20 protein of HIV, comprising:
(a) producing a cell line using a method according to claim 23;
(b) culturing said cell line; and
(c) recovering said bispecific human monoclonal antibody from said culture.
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US9982052B2 (en) 2014-08-05 2018-05-29 MabQuest, SA Immunological reagents
US9982053B2 (en) 2014-08-05 2018-05-29 MabQuest, SA Immunological reagents
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US11214617B2 (en) 2016-01-22 2022-01-04 MabQuest SA Immunological reagents

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