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WO1997049424A9 - Anticorps anti-corecepteur cellulaire pour le virus de l'immunodeficience humaine et procedes d'utilisation - Google Patents

Anticorps anti-corecepteur cellulaire pour le virus de l'immunodeficience humaine et procedes d'utilisation

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Publication number
WO1997049424A9
WO1997049424A9 PCT/US1997/011093 US9711093W WO9749424A9 WO 1997049424 A9 WO1997049424 A9 WO 1997049424A9 US 9711093 W US9711093 W US 9711093W WO 9749424 A9 WO9749424 A9 WO 9749424A9
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antibody
hiv
cell
cells
protein
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  • the field of the invention is infection by and pathogenesis of Human Immunodeficiency Virus.
  • CXCR4 also known as Lestr, LCR-1, and HUMSTR
  • CCR5 which are members of the chemokine receptor family of proteins
  • CCR3 and CCR2b also appear to function as cofactors for some HIV-1 isolates (Doranz et al, 1996, Cell 85:1149- 1158; Berson et al., 1997, J. Virol. 71 :1692-1696; Choe et al, 1996, Cell 85:1135- 1148).
  • CCR5 and CXCR4 as coreceptors for isolates of simian immunodeficiency viruses (SIV) and HIV-2, respectively. This, indicates that the use of chemokine receptors is a general property of all human and nonhuman lentiviruses.
  • CD4-negative cells rapidly and cause extensive cell fusion (Clapham et al., 1992, supra).
  • the highly cytopathic nature of these infections has suggested that these isolates can utilize one or more receptors other than CD4 with high efficiency.
  • HIV-1 strains exhibit distinct tropisms for CD4-positive cells.
  • Macrophage tropic (M-tropic) strains of HIV-1 enter and replicate in macrophages and primary T cells but generally fail to enter T cell lines. These isolates characteristically do not induce muhinucleated giant cells when cultured with certain immortalized T cell lines and are generally non-syncytium inducing (NSI).
  • T cell tropic strains fail to enter macrophages efficiently but readily infect primary T cells and induce syncytia (SI) on some T cell lines (Fauci et al., 1996, Nature 384:529-534).
  • SI syncytia
  • This protein is a member of a family of molecules that bind chemokines which are involved in the trafficking of T cells and phagocytic cells to areas of inflammation (Power and Wells, 1996, Trends Pharmacol. Sci. 17:209-213).
  • the chemokines MlP-alpha and MlP-beta and RANTES all bind to CCR5 while stromal cell derived factor (SDF-1) binds to CXCR4 (Bluel, et al., 1996, Nature 382:829-832; Oberlin et al., 1996, Nature 382:833- 835).
  • CXCR4 fulfills the requirements of an HIV receptor co-factor. It renders a number of murine, feline, simian, quail, and hamster cell lines, as well as human cell lines, which cells are normally resistant to HIV-1 entry, fully permissive for HIV-1 env mediated syncytia formation.
  • the T cell tropic HIV strain fulfills the requirements of an HIV receptor co-factor. It renders a number of murine, feline, simian, quail, and hamster cell lines, as well as human cell lines, which cells are normally resistant to HIV-1 entry, fully permissive for HIV-1 env mediated syncytia formation.
  • the T cell tropic HIV strain fulfills the requirements of an HIV receptor co-factor. It renders a number of murine, feline, simian, quail, and hamster cell lines, as well as human cell lines, which cells are normally resistant to HIV-1 entry, fully permissive for HIV-1 env mediated syncy
  • HIV-1 IIIB is capable of infecting both murine and feline cells which co-express human CD4 and CXCR4.
  • the macrophage tropic strain Ba-L is not capable of infecting cells which co-express both CXCR4 and CD4.
  • Current anti-HIV therapy includes the use of compounds which inhibit various aspects of HIV replication in a cell such as inhibition of replication and/or transcription of viral nucleic acid and inhibition of protein processing. While these therapies, particularly when used in combination with one another, are effective, they are frequently short-lived in that viral strains rapidly develop that are resistant to one or more of the compounds used. There therefore remains an acute need to develop additional therapies and strategies for preventing HIV infection in humans.
  • the invention relates to an antibody capable of binding to a cellular protein, which antibody has antiviral activity by virtue of the fact that the cellular protein to which the antibody binds is a protein which is required for entry of virus into a cell expressing that protein.
  • One aspect of the invention relates to an anti-immunodeficiency virus antibody capable of binding to a cellular protein.
  • the immunodeficiency virus is selected from the group consisting of HIV-1, HIV-2 and
  • the protein to which the antibody of the invention binds is a chemokine receptor protein, preferably, an HIV receptor protein and/or a cellular cofactor for a cellular HIV receptor protein. More preferably, the protein to which the antibody of the invention binds is selected from the group consisting of CXCR4 and CCR5; and most preferably, the protein to which the antibody binds is CXCR4.
  • the antibody is selected from the group consisting of a monoclonal antibody and a synthetic antibody.
  • the antibody is a monoclonal antibody, and more preferably, the antibody is MAb 12G5.
  • the invention also relates to an isolated DNA encoding an anti- immunodeficiency virus antibody capable of binding to a cellular protein.
  • the immunodeficiency virus is selected from the group consisting of HIV-1, HIV-2 and SIV.
  • the protein to which the antibody of the invention binds is a chemokine receptor protein.
  • the protein is an HIV receptor protein and/or a cellular cofactor for a cellular HIV receptor protein. More preferably, the protein is selected from the group consisting of CXCR4 and CCR5; and most preferably, the protein is CXCR4 and the antibody is the monoclonal antibody (MAb) 12G5.
  • the invention also relates to a method of inhibiting infection of a cell by HIV comprising adding to the cell an anti-immunodeficiency virus antibody capable of binding to a cellular protein on the cell, wherein upon binding of the antibody to the cellular protein infection of the cell by HIV is inhibited. Also included in the invention is a method of treating HIV infection in a human comprising administering to the human an anti-immunodeficiency virus antibody capable of binding to a cellular protein on a cell, wherein upon binding of the antibody to the cellular protein, infection of the cell by HIV is inhibited, thereby treating the HIV infection in the human.
  • the invention further includes a method of obtaining an anti- immunodeficiency virus antibody capable of binding to a cellular protein on a cell, the method comprising generating a panel of antibodies directed against HIV infected or SIV infected cell proteins, and screening the antibodies for anti-immunodeficiency virus activity to obtain an antibody capable of binding to a cellular protein, which antibody has anti-immunodeficiency virus activity.
  • Also included in the invention is a method of identifying a target cell for immunodeficiency virus infection, the method comprising adding to a population of cells an anti-immunodeficiency virus antibody capable of binding to a cellular protein on a cell, wherein binding of the antibody to a cell in the population is an indication that the cell is an immunodeficiency virus target cell.
  • a method of identifying a candidate anti- immunodeficiency virus compound comprises isolating a test compound capable of binding to an anti-immunodeficiency virus antibody, which antibody binds to a cellular protein, and assessing the ability of the test compound to inhibit infection of a cell by an immunodeficiency virus in an antiviral assay, wherein inhibition of infection of the cell by the immunodeficiency virus in the presence of the test compound is an indication that the test compound is an anti-immunodeficiency virus compound.
  • the invention also includes a method of measuring the level of expression of CXCR4 on a cell comprising adding to the cell an antibody which binds to the CXCR4 and assessing the amount of antibody bound to the cell, wherein the amount of the antibody bound to the cell is a measure of the level of expression of the CXCR4 on the cell.
  • Figure 1A is a series of graphs graph depicting detection of CXCR4 cell surface glycoprotein expression following staining of MOLT-4, C8166, U87/CD4 and RD/CD4 cells and primary human macrophages (MAC) cultured for 1 and 5 days.
  • Figure IB is an image of Northern blot analysis of the indicated cells using CXCR4 or GAPDH nucleic acid as a probe.
  • Figure 1C is an image of reverse transcriptase (RT) PCR analysis on the indicated cells using CXCR4 or GAPDH nucleic acid as a probe.
  • RT reverse transcriptase
  • Figure 2A is a graph depicting 12G5-mediated inhibition of cell-free infectivity of RD/CD4 cells.
  • Figure 2B is a graph depicting 12G5-mediated inhibition of cell-free infectivity of HeLa/CD4 cells.
  • Figure 3A is a series of graphs depicting infection of CD4 negative cells with HIV-2/vcp.
  • the cell lines indicated in the panels were inoculated with cell- free HIV-2/vcp or HIV-1 /LAI and were monitored for reverse transcriptase (RT) activity in culture supernatant at the indicated time points. Input virus was removed after 24 hours by washing. Except for Sup-Tl , all cell lines were CD4-negative as determined by FACS analysis using a panel of anti-CD4 MAbs and/or by western blot using an anti-CD4 serum.
  • Figure 3B is a graph depicting the failure of an anti-CD4 MAb to inhibit HIV-2/vcp infection of a CD4-negative cell line.
  • BC7 or Sup-Tl cells were inoculated with either HIV-2/vcp or HIV-1 /LAI, respectively, in the presence or absence of anti-CD4 MAb #19.
  • Input virus was removed after 24 hours by washing and cells were maintained in the presence of anti-CD4 MAb for 8 days at which time RT activity was determined. Similar results were seen using OKT4A, another anti-CD4 MAb #19.
  • Figure 3C is a series of images of syncytium induction assays which were performed on the indicated target cells by cocu ivation with HIV-2/vcp-infected BC7 cells or HIV-1/LAI-infected Hut-78 cells in the presence or absence of 10 ⁇ g/ml of anti-CD4 MAb #19. Cultures were photographed after either 24 or 48 hours. As shown, extensive syncytia formation is induced by HIV-2/vcp on CD4-negative BC7 cells which is unaffected by the addition of anti-CD4 MAb.
  • Figure 4A is a series of graphs depicting inhibition of CP-MAC and HIV-2/vcp infection by the 12G5 MAb.
  • Sup-Tl cells or CD4-negative BC7, Nalm6 and Daudi cells were preincubated with the indicated concentrations of 12G5 MAb and were then inoculated with the viruses shown.
  • RT activity in infected cell supernatants was assessed at the indicated times.
  • Dose-dependent inhibition of CP-MAC and HIV- 2/vcp infection by 12G5 MAb is shown.
  • Figure 4B is a series of images depicting inhibition of CP-MAC and
  • HIV-2/vcp syncytia induction by the 12G5 MAb Sup-Tl or BC7 cells were cultured with either CP-MAC-infected Sup-Tl cells, or HIV-2/vcp-infected BC7 cells in the presence or absence of 12G5 (10 ⁇ g/ml). Cells were photographed after 48 hours of culture. Inhibition of syncytium formation by 12G5 is evident in cells infected with either virus.
  • Figure 5A is a series of graphs depicting reactivity of 12G5 MAb with CXCR4.
  • U87 cells stably expressing either CXCR4, CCR1, or the control vector pBABe-puro were evaluated for reactivity with 12G5 (10 ⁇ g/ml) by FACS.
  • the region for positivity, designated Ml was defined using a non-reactive control MAb, and the percent of cells falling within this window for each sample is indicated. As shown, a marked shift in reactivity was observed in the entire population of CXCR4-expressing cells.
  • Figure 5B is a graph depicting reactivity of 12G5 MAb with CXCR4.
  • Control CHO cells or cells stably expressing HA-tagged CXCR4 or the other chemokine receptors indicated were evaluated for reactivity to 125 I-12G5, using protocols described in Pelchen-Matthews et al. (1989, EMBO J. 8:3641-3649).
  • Scatchard type analysis indicated that the K d for 12G5 binding to CHO-CXCR4 cells was 1-5 nM; 125 I-12G5 binding was competed to close to background levels by 100 nM of unlabeled 12G5 but was not influenced by the anti-FIA antibody 12G5.
  • Figure 5C is a series of photographs depicting immunofluorescence confocal microscopy of CHO cells stably expressing CXCR4 or chemokine receptors using 12G5.
  • CHO-K1 cells expressing HA-tagged CXCR4 or the human IL8R-B receptor were stained with 12G5, the anti-human CD4 MAb Q4120, or an antibody against the HA-tag (Pelchen-Matthews et al. (1989, supra). Only cells with HA-tagged
  • CXCR4 is sufficient to render U87 cells susceptible to HIV-2/vcp syncytium induction.
  • U87 cells stably expressing either CXCR4 or CD4, or untransduced cells (control) were cocultured with HIV-2/vcp infected BC7 cells and syncytium formation was assessed by photography. Large syncytia are evident only in U87 cells which express CXCR4. Induction of syncytia was completely inhibited in cells which were preincubated with
  • Figure 6B is a graph depicting the fact that recombinant CXCR4 is sufficient to render U87 cells susceptible to HIV-2/vcp infection.
  • Cells were inoculated with cell-free HIV-2/vcp (1000 TCID 50 units, determined on BC7 cells) and the amount of RT in the culture supernatants was monitored at the indicated time points. Only CXCR4-expressing cells were infected with virus. Extensive syncytia formation and cell killing were also observed in the infected CXCR4-expressing cells which correlated with the time of production of RT.
  • Figure 7 is a graph depicting induction of cell fusion by the HIV-2/vcp envelope glycoprotein in a gene reporter fusion assay.
  • HeLa effector cells were transfected with pCR3.1 expressing either HIV-2/vcp env or BH8 HIV-1 env clones and were then infected with vaccinia virus.
  • QT6 target cells were transfected with constructs expressing CXCR4, IL8R-B or the PCR3.1 expression vector alone, and a plasmid containing the luciferase gene driven by a T7 promoter (Promega Biotech). Where indicated, target cells were also infected with pT4, which constitutively expresses CD4 from the CMV promoter.
  • Luciferase activity as an indication of cell fusion is indicated in terms of relative light units (RLU) as described (Doranz et al., 1996, Cell 85:1149-1158).
  • Figure 8 is a series of graphs depicting downregulation of CXCR4 expression by HIV-2/vcp infection. BC7, Daudi, Nalm6 or Sup-Tl cells that were either uninfected or infected with the indicated viruses were evaluated for surface reactivity by FACS either with an isotope-matched control MAb or MAb 12G5 (10 ⁇ g/ml) and the mean channel fluorescence intensity (MCF) is shown for each cell type.
  • MCF mean channel fluorescence intensity
  • the invention relates to an antiviral antibody which binds to a cellular protein essential for entry of a virus into a cell expressing that protein.
  • the antibody of the invention is an antiviral antibody in that it is an antibody which binds to a specific cellular protein which is essential for virus entry into the cell in which the cellular protein is expressed.
  • the antibody of the invention inhibits entry of the virus into the cell and is therefore termed an antiviral antibody despite the fact that it does not bind to a viral protein, but rather, binds to a cellular protein.
  • the virus against which the antiviral antibody is directed is an immunodeficiency virus, that is, a virus which causes an immunodeficiency disease.
  • an immunodeficiency virus antibody is termed an anti-immunodeficiency virus antibody.
  • immunodeficiency virus should be construed to include any strain of HIV or SIV.
  • HIV as used herein, is meant any strain of a human immunodeficiency virus belonging to the group of either HIV type 1 or HIV type 2.
  • SIV as used herein is meant any of five recognized strains of SIV (SIVmac, SIVsmm, SIVagm, SIVmnd and SIVcpz) which are known to infect non-human primates.
  • the antibody of the invention is an antibody which is capable of binding to a cellular protein required to form a functional cellular receptor for entry of HIV into a cell.
  • the antibody of the invention may be a monoclonal antibody (MAb) or may be antibody which is derived from a phage library or a humanized or a synthetic antibody, or an antibody fragment expressed intracellularly.
  • the antibody of the invention is an antibody which binds to a cellular co-factor required for entry of HIV into a cell.
  • a "cellular co-factor” as used herein is defined as a protein which is required, in association with a cellular receptor for HIV, for entry of HIV into cells. Since the preferred antibody of the invention, MAb 12G5, binds to a protein which is both a cellular cofactor and an HIV-receptor protein, depending upon the cell type to be infected, it will be appreciated that the antibody of the invention should be construed to be one which binds to a cellular protein which may be a cellular cofactor and may also be a receptor protein in its own right.
  • the antibody of the invention is one which binds to a cellular protein which is necessary for vims entry into cells. Since binding of the antibody to the cellular protein serves to block virus entry into cells, the antibody is an antiviral antibody.
  • the antibody of the invention is directed against the cellular protein CXCR4; more preferably, the antibody is a monoclonal antibody and even more preferably, the antibody of the invention is MAb 12G5.
  • the antibody of the invention is useful in a method of inhibiting infection of a cell by HIV as described herein.
  • the antibody is further useful for the generation of antibody derivatives which are useful for inhibiting infection of a cell by HIV also as described herein.
  • the antibody of the invention is useful in a method of screening compounds for anti-HIV activity as described herein. Additional uses for the antibody of the invention include the identification of cells in the body which are potential targets for viral infection. The antibody is thus also useful for the isolation of such cells using flow cytometry technology or other cellular isolation techniques which are common in the art.
  • the invention also relates to methods of use of the antibody of the invention, which methods include diagnostic and therapeutic uses.
  • the antibody or derivatives thereof may be expressed intracellularly, reducing CXCR4 expression on the cell surface and rendering these cells resistant to HIV infection.
  • the antibody of the invention inhibits SDF-1 binding and signalling by CXCR4.
  • the antibody or derivatives thereof may be used to antagonize SDF-1 function in vivo.
  • the antibody of the invention exemplified herein by the MAb 12G5
  • the preferred antibody of the invention was discovered to bind to a cell protein termed CXCR4, which protein is essential for entry of an immunodeficiency virus into cells. Since MAb 12G5 possesses antiviral activity, this antibody is specific for epitopes on CXCR4 which are essential for virus infection.
  • the functional assay by which the antibody is generated is unique in that it facilitates the identification of an antiviral antibody which binds a cellular protein rather than a viral protein.
  • CXCR4 functions as a cellular co-factor for entry of HIV into cells using CD4 as the cellular receptor molecule. In other instances, CXCR4 functions as a cellular receptor for HIV in the absence of CD4. Thus, CXCR4 is both a cellular co-factor, as defined herein, and is a cellular virus receptor protein in its own right for the entry of HIV into certain cells.
  • MAb 12G5 is specific for CXCR4.
  • MAb 12G5 binds to both human and nonhuman cell lines following transient or stable cellular expression of a recombinant CXCR4 protein.
  • this antibody is capable of inhibiting both infection as well as syncytia formation by a number of HIV-1, HIV-2 and SIV isolates. The extent of inhibition of virus replication is dependent upon the test virus and also the target cell.
  • antiviral activity is meant an antibody which when added to an immunodeficiency virus or to a cell to be infected with such a virus, mediates a reduction in the ability of the vims to infect and/or replicate in the cell compared with the ability of vims to infect and/or replicate in the cell in the absence of the antibody.
  • assays for antiviral activity are described in detail in the experimental detail section and include, but are not limited to, reverse transcriptase assays, immunofluorescence assays, assays for formation of syncytia, antigen capture assays and the like.
  • the antibody is a monoclonal antibody
  • cells which are suspected to encode a cellular protein essential for vims entry are first infected with HIV or SIV. Extracts are prepared from the cells and are used to generate a panel of monoclonal antibodies directed against the infected cells.
  • Antibodies are screened for antiviral activity in a functional antiviral assay as described herein, and for the ability to bind cellular rather than viral proteins, also as described herein.
  • Antiviral antibodies which bind cellular proteins are selected and are further characterized with respect to the proteins to which they bind and to their antiviral capabilities.
  • the entire CXCR4 molecule is used to generate anti-CXCR4 antibodies since the entire molecule is in the correct conformation for the generation of anti-CXCR4 antibodies which block vims entry into cells.
  • the invention should not be construed to be limited solely to the use of the entire CXCR4 molecule for the generation of antibodies, it being understood that peptides, which can be made according to well known procedures, may also be used provided they give rise to an antibody having the characteristics described herein.
  • Analogs may differ from naturally occurring proteins by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both.
  • conservative amino acid changes may be made, which although they alter the primary sequence of the peptide, do not normally alter its function.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.
  • Modifications include in vivo, or in vitro chemical derivatization of proteins, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a protein during its synthesis and processing or in further processing steps; e.g., by exposing the protein to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • proteins which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties.
  • Analogs of such proteins include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
  • the proteins of the invention are not limited to products of any of the specific exemplary processes listed herein.
  • the present invention should therefore be constmed to include unmodified or modified CXCR4, which CXCR4 is capable of eliciting the production of an antibody specific for CXCR4 and which antibody has antiviral activity as defined herein.
  • the CXCR4 used to generate the antibody of the invention is CXCR4 located within the context of a cell.
  • the invention should also be constmed to include DNA which encodes the antibody of the invention, or a portion of such antibody.
  • the nucleic acid encoding the antibody may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and the references cited therein. Further, the antibody of the invention may be "humanized” using the technology described in Wright et al., (supra) and in the references cited therein.
  • a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g. , the desired antibody.
  • cDNA copies of the mRNA are produced using reverse transcriptase.
  • cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
  • the procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY).
  • Bacteriophage which encode the desired antibody may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed.
  • the bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell.
  • Bacteriophage which do not express the antibody will not bind to the cell.
  • panning techniques are well known in the art and are described for example, in Wright et al., (supra).
  • a cDNA library is generated from mRNA obtained from a population of antibody-producing cells.
  • the mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same.
  • Amplified cDNA is cloned into Ml 3 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin.
  • this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.
  • synthetic antibody an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be constmed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • the invention thus includes a DNA encoding the antibody of the invention or a portion of the antibody of the invention.
  • DNA is extracted from antibody expressing phage obtained according to the methods of the invention. Such extraction techniques are well known in the art and are described, for example, in Sambrook et al. (supra).
  • the isolated DNA encoding an anti-CXCR4 antibody may be constmcted such that it is useful for intracellular immunization of a cell against immunodeficiency vims infection.
  • single chain fragments from the variable region of the antibody that contain the CXCR4 binding site could be expressed at intracellular sites. This expression is predicted to interfere with the delivery of the native cellular CXCR4 protein to the cell surface, thereby rendering these cells resistant to infection with HIV.
  • This general approach is useful for engineering immune or hematopoietic stem cells resistant to HIV infection and provides a novel gene therapy approach for treatment of HIV infected individuals.
  • isolated DNA refers to a DNA sequence, segment, or fragment which has been purified from the sequences which flank it in a naturally occurring state, e.g. , a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to DNA which has been substantially purified from other components which naturally accompany the DNA, e.g., RNA or DNA or proteins which naturally accompany it in the cell.
  • the invention should also be constmed to include DNAs which are substantially homologous to the DNA isolated according to the method of the invention.
  • DNA which is substantially homologous is about 50% homologous, more preferably about 70% homologous, even more preferably about 80%) homologous and most preferably about 90% homologous to DNA obtained using the method of the invention.
  • Homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50%o homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
  • the DNA sequences 3 ⁇ TTGCC5' and 3 ATGCG5 1 share 50% homology.
  • the protein may be extracted from the surface of the phage on which it is expressed.
  • a substantially pure preparation of a protein comprising, for example, an antibody, may be obtained by cloning an isolated DNA encoding the antibody into an expression vector and expressing the protein therefrom. Protein so expressed may be obtained using ordinary protein purification procedures well known in the art.
  • substantially pure describes a compound, e.g., a protein or polypeptide which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least
  • a compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.
  • cells are treated with the antibody of the invention, or a derivative thereof, either prior to or concurrently with the addition of vims.
  • Inhibition of infection of the cells by the antibody of the invention is assessed by measuring the replication of virus in the cells, by identifying the presence of viral nucleic acids and/or proteins in the cells, for example, by performing PCR. Southern, Northern or Western blotting analyses, reverse transcriptase (RT) assays, or by immunofluorescence or other viral protein detection procedures.
  • the amount of antibody and vims to be added to the cells will be apparent to one skilled in the art from the teaching provided herein.
  • the antibody of the invention, or a derivative thereof is administered to a human subject who is either at risk of acquiring HIV infection, or who is already infected with HIV.
  • a pharmaceutically acceptable formulation such as a saline solution or other physiologically acceptable solution which is suitable for the chosen route of administration and which will be readily apparent to those skilled in the art of antibody preparation and administration.
  • the dose of antibody to be used may vary dependent upon any number of factors including the age of the individual, the route of administration and the extent of HIV infection in the individual.
  • the antibody is prepared for administration by being suspended or dissolved in a pharmaceutically acceptable carrier such as saline, salts solution or other formulations apparent to those skilled in such administration.
  • the antibody is administered in a range of 0.1 ⁇ g to 1 g of protein per dose. Approximately 1-10 doses are administered to the individual at intervals ranging from once per day to once every few years.
  • the antibody may be administered by any number of routes including, but not limited to, subcutaneous, intramuscular, oral, intravenous, intradermal, intranasal or intravaginal routes of administration.
  • the antibody of the invention may be administered to the patient in a sustained release formulation using a biodegradable biocompatible polymer, or by on- site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema).
  • the appropriate pharmaceutically acceptable carrier will be evident to those skilled in the art and will depend in large part upon the route of administration.
  • Derivatives of the antibody of the invention may be prepared using technology common in the art and described, for example in Harlow et al. (1988, In: Antibodies. A Laboratory Manual, Cold Spring Harbor, NY) and in Sambrook et al.
  • the antibody of the invention may also be used in a method of screening compounds for anti-HIV activity.
  • a test compound is first screened for the ability to bind to the antibody of the invention.
  • Compounds which bind to the antibody are likely to share structural and perhaps biological activities with CXCR4 and thus, may serve as competitive inhibitors for inhibition of the interaction of HIV envelope protein with CD4 and or CXCR4 plus CD4.
  • An antibody-binding compound is further tested for antiviral activity by treating cells with the compound either prior to or concurrently with the addition of vims to the cells. Alternatively, the vims and the compound may be mixed together prior to the addition of the mixture to the cells.
  • the ability of the compound to affect vims infection is assessed by measuring vims replication in the cells using any one of the known techniques, such as a RT assay, immunofluorescence assays and other assays known in the art useful for detection of viral proteins or nucleic acids in cells. Generation of newly replicated vims may also be measured using known vims assays such as those which are described herein.
  • the antibody of the invention may also be used in competition assays to screen for compounds that bind to CXCR4 and which therefore prevent binding of the antibody to CXCR4. Such compounds, once identified, may be examined further to determine whether or not they prevent entry of vims into cells. Compounds which prevent entry of virus into cells are useful as anti-viral compounds.
  • Additional uses for the antibody of the invention include the identification of cells in the body which are potential targets for infection by an immunodeficiency vims.
  • target cell for immunodeficiency vims infection a cell which expresses receptor protein(s) for an immunodeficiency virus and which cell is therefore capable of being infected by an immunodeficiency virus.
  • Cells which are potential targets for HIV infection may be identified by virtue of the presence of CXCR4 on their surface.
  • the antibody of the invention facilitates identification of these cells as follows: The antibody of the invention is first combined with an identifiable marker, such as an immunofluorescent or radioactive marker. Cells which are obtained from a human subject are then reacted with the tagged antibody. Binding of the antibody to cells is an indication that such cells are potential targets for HIV infection.
  • CXCR4 is differentially expressed and regulated on human T lymphocytes (Bleul et al., 1997, Proc. Natl. Acad. Sci. USA 94:1925-1930). Further, reactivity of immune cells to MAb 12G5 is high on naive cells and low on memory cells and thus, the pattern of expression of CXCR4 and its utilization by viruses may contribute to immune dysfunction. CXCR4 has also been detected, using the monoclonal antibody of the invention, on some endothelial cells (in atherosclerotic plaques), platelets and some hematopoietic precursor cells. In the case of individuals who are infected with HIV, the identification of target cells provides an immune profile of these individuals which provides useful information regarding the progress of their infection.
  • the antibody is useful for the detection of CXCR4 on a variety of cell types on which CXCR4 may be expressed.
  • CXCR4 is expressed on human neurons (Hesselgesser et al., 1997, Current Biology 7:112-121), including cells in the human brain.
  • the monoclonal antibody of the invention may be useful for monitoring CXCR4 expression levels on a variety of cell types, which expression may be an indication of a disease state in a human, including, but not limited to HIV infection, atherosclerosis, and the like.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited material, and in any case, for a period of at least thirty (30) years after the date of the deposit or for the enforceable life of the patent, whichever period is longer. Applicant's assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • Example 1 Inhibition of HIV by a Monoclonal Antibody to a Coreceptor (CXCR4) is both Cell Type and Virus Strain Dependent.
  • 12G5 is a mouse MAb that specifically recognizes CXCR4 but not other members of the chemokine receptor family, including CCR1-5 and CXCR1 and CXCR2 (interleukin-8 receptors and ⁇ , respectively).
  • FIG. 1A there is shown the levels of cell surface CXCR4 expression determined by flow cytometry following 12G5 staining of the CD4 + T-cell lines MOLT-4 and C8166, the CD4-transfected human rhabdomyosarcoma cell line RD, and the human glioma cell line U87 as well as primary macrophages purified by adherence to plastic and cultured for 1 or 5 days (McKnight et al., 1995, J. Virol. 69:3167-3170; and Simmons et al, 1995, Virology 209:696-700). Flow cytometry was carried out as previously described (McKnight et al., 1996, J. Virol. 70:4598-4606).
  • FIGS. IB and 1C show the results of reverse transcription-PCR (RT-PCR) and Northern blot analyses, respectively, of RNA prepared from the same cell types as shown in Figure 1 A.
  • RNA for RT-PCR was prepared with RNA-zol.
  • cDNA was then prepared from 5 ⁇ g of RNA by using Stratagene RTOPCR kit. One-twentieth of the cDNA prepared was included in the PCR reactions. PCR for CXCR4 used the primers 5'-TAG ATA TCT TAC CAT GGA
  • the positive-strand primer incorporated a 5' tail which encoded an EcoRV site, and the minus strand incorporated a 3' tail encoding a Notl site.
  • Conditions for CXCR4 amplification were 30 cycles of 95 °C for 30 seconds, 50 °C for
  • Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) sequences were amplified by using the positive-strand primers 5'-TGG ATA TTG CCA TCA ATG ACC-3' and the negative-strand primer 5'-GAT GGC ATG GAC TGT GGT CAT G-3'.
  • Conditions for the PCR were 40 cycles of 95 °C for 30 seconds, 65 °C for 1 minute, and 72°C for 30 seconds. Control PCR analysis of the R ⁇ A preparations was consistently negative for both CXCR4 and GAPDH D ⁇ A.
  • mR ⁇ A for Northern blot analysis was prepared from 10 6 to 10 7 cells Pharmacia Quickprep Micro mRNA isolation system), and 1 ⁇ g was fractionated on a 1.2%> agarose- formaldehyde gel. RNA was transferred overnight onto a Genescreen Plus membrane (N ⁇ N) with 10* SSP ⁇ (l ⁇ SSP ⁇ is 0.18 M NaCl, 10 mM NaH 2 PO 4 , and 1 mM ⁇ DTA) and then baked at 80 °C for 2 hours. 32 P-labeled CXCR4 and GAPDH double-stranded probes were prepared by random priming (Amersham) and hybridized to the membrane in Quickhyb solution (Stratagene) for 1 hour at 65 °C.
  • the membrane was washed twice for 15 minutes at room temperature in 2 ⁇ SSC (1 SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate and once for 30 minute at 65 °C in O.l x SSC-01.% sodium dodecyl sulfate. The blot was then exposed at -70°C for 1 week.
  • WI-38t cells are simian vims 40-transformed counterparts of the WI-38 human diploid cell line (Ponten et al, 1962, J. Cell. Comp. Physiol. 61 :145-163).
  • Table 1 summarizes the data shown in Figure 1 and includes a survey of other cell types, including Daudi (a B-cell line).
  • Daudi a B-cell line
  • CXCR4 is expressed widely on human hematopoietic cell types and on some nonlymphoid cell types but is absent from the surfaces of human U87 glioma, SCL skin, and WI-38/t lung cells and is only weakly expression on 5-day-old macrophages derived from blood monocytes.
  • This pattern of CXCR4 expression correlates well with the sensitivity of these CD4 + cell types to infection by T-cell-line- passaged HIV-1 strains such as LAI and RF (Tables 1 and 2) (Chesebro, et al, 1990, supra; and Clapham et al, 1991, supra).
  • HIV-2 strains e.g., ROD
  • some HIV-1 strains e.g., the GUN-1 variant [GUN-lvar]
  • CXCR4 cells types e.g., U87/CD4
  • CXCR4 mRNA was measured by RT-PCT. -, no CXCR4 expressed; +, weak CXCR4 expression; ++, high-level CXCR4 expression.
  • Values are percentages of cells expressing CXCR4 as determined by fluorescence-activated cell sorter analysis of 12G5 staining.
  • CXCR4 expression was estimated after 5 days of culturing PBMCs or macrophages.
  • the percentages of 12G5-positive cells are averages derived from staining 10 batches of PBMCs and 3 batches of macrophages, each from different donors.
  • MAb 12G5 was tested to determine if it could inhibit cell-to-cell fusion induced by a panel of HIV-1 and HIV-2 strains.
  • the HIV-1 and HIV-2 strains tested were chosen because they show distinct tropisms for various CD4 1" cell types (summarized in Table 2), but all infect CD4 + T-cell lines as well as HeLa/CD4 and RD/CD4 cells.
  • the wild-type GUN-1 strain of HIV-1 (GUN-lwt) is dual tropic and, unlike LAI and RF, efficiently infects primary macrophages as well as CD4 + T-cell lines (McKnight et al, 1995, supra; and Simmons et al, 1995, Virology 209:696-700).
  • HIV-2 ROD/B is a variant of the prototype ROD strain (ROD/A) that can infect certain CD4 " human cell types (Clapham et al, 1992, supra) with CXCR4 alone as a receptor (described herein) yet still retains the broad, CD4- dependent tropism characteristic of most T-cell-line-passaged HIV-2 isolates (McKnight et al, 1994, supra).
  • Uninfected target cells e.g., RD/CD4 cells
  • 12G5 dilutions were treated for 30 minutes with 12G5 dilutions before addition of an equal number of H9 cells chronically infected with an appropriate HIV-1 or HIV-2 strain. Cocultivations were incubated at
  • MAb 12G5 was then assessed to determine whether this antibody could inhibit HIV-1- and HIV-2-induced cell-to-cell fusion of CXCR4 + CD4 + T-cell lines as well as other CD4 ' cell types (either CXCR4" of CXCR4 " ).
  • the data presented in Table 3 establish that 12G5 failed to inhibit cell-to-cell induction of fusion by any of the seven viruses on the T-cell lines MOLT-4, Sup Tl, and MT-2, although a slight but consistent reduction in syncytium formation was seen for the HIV-1 GUN-1 strains on MOLT-4 and Sup Tl cells with the highest does of 12G5 (20 ⁇ g/ml).
  • ⁇ Antibody titers were estimated as the highest concentration of 12G5 ( ⁇ g/ml) that inhibited >95% syncytium formation; i.e., >20 means there was no inhibition by 12G5 up to a concentration of 20 ⁇ g ml, whereas 1.25 means that 12G5 blocked >95% of syncytium formation at 1.25 ⁇ g/ml but lower concentrations inhibited less or not at all. Unless otherwise noted, the titers shown refer to >95% syncytium inhibition.
  • MAb 12G5 inhibition of infection of cells by cell-free virus was assessed as follows. RD/CD4 or HeLa CD4 were seeded at 2 x 10 4 cells per well in 24-well trays 2 days before infection. Cells were treated for 30 minutes with 100 ⁇ l of appropriate 12G5 antibody dilutions. Vims supernatant (100 ⁇ l) containing between 50 and 100 focus-forming units was added, and the cultures were incubated for a further 90 minutes. Inocula were then removed, and cells were washed twice before the addition of 1 ml of growth medium and incubation for 4 days. Foci of infection were detected by immunostaining as previously described (Clapham et al, 1992, supra).
  • CD4 + cell types (Table 2) and therefore must use alternative coreceptors for entry into these cells.
  • at least some HIV strains can choose between compatible alternative coreceptors for CD4-dependent entry.
  • several primary dual-tropic HIV-1 isolates (as well as GUN- lwt) infected cat CCC cells expressing human CD4 as long as either CXCR4 or CCR5 was present, while a subset of the primary strains could use CCR3, CCR5, or CXCR4 (Simmons et al, 1996, supra).
  • RANTES binds to a number of CC chemokine receptors (Chaudhuri et al. , 1994, J. Biol. Chem. 269:7835-7838; Neote et al, 1993, Cell 72:415-425; Power et al, 1995, J. Biol. Chem. 270:19495-19500; Samson et al, 1996, Biochemistry 35:3362-3367), including CCR5, a coreceptor for NSI macrophage-tropic HIV-1 strains (Alkhatib et al, 1996, Science 272:1955-1958; Deng et al, 1996, Nature 381 :661-666; Dragic et al,
  • CXCR4 presentation on different cell types may explain the cell-type-dependent inhibition of HIV strains shown here but does not resolve why inhibition on some cell types (e.g., C8166 and HeLa/CD4) was also vims strain dependent. It is thus likely that different strains interact differently with CXCR4 to trigger fusion. Although the natures of the different interactions are currently unknown, this hypothesis is supported by our recent observation that deletions at the N terminus of CXCR4 have different effects depending on the HIV strain tested.
  • Example 2 Cells CD4-positive human T lymphoid cell lines, Sup-Tl, Hut-78, H9, CEM, Molt4- clone ⁇ , and the TxB cell hybrid line, CEMxl74, have been described previously (Hoxie et al, 1986, Science 234: 1 123-1127; Hoxie et al, 1988, J. Virol. 62:2557- 2568; LaBranche et al, 1994, J. Virol. 68:5509-5522). CD4-negative T lymphoid lines HSB and CEMss4 " are described (Weiner et al, 1991, Pathobiol.
  • BC7 was derived from Sup-Tl cells, as described in Table 4.
  • Human B cell lines, Nalm ⁇ , KM3.79, and REH are described (McKnight et al, 1995, J. Virol.69:3167-3170; McKnight et al, 1996, J. Virol. 70:4598-4606) HeLa, RD, and Daudi cells have been described previously (Clapham et al, 1991, supra 5; Clapham et al, 1992, supra).
  • RT-PCR and cloned into pcDNAlneo for expression in mammalian cells were derived by screening for reactivity to the anti-HA MAb, 12CA5.
  • the receptor selectivity of the HA-tagged receptor lines was tested by radioligand binding assays using membranes purified from the respective cells. No significant differences in ligand binding properties were seen between tagged and nontagged receptors.
  • HIV-2/vcp was derived from HIV-2/NIH-z (Zagury et al, 1988, Proc. Natl. Acad. Sci. USA 85:5941-5945) by passaging vims first onto CP-MAC infected Sup-Tl cells that had completely down- regulated their surface expression of CD4 (LaBranche et al, 1994, supra), and then onto BC7 cells. Sequence analysis of the HIV-2/vcp env gene amplified from genomic DNA showed no evidence of recombination in env with CP-MAC.
  • CP-MAC was derived as previously described from the SIVmac molecular clone, BK28 (LaBranche et al, 1994, supra). Additional variants of HIV-2 described for their ability to infect CD4-negative cells included HIV-2/CBL-23, HIV-2/Rod-A, and HIV-2/Rod-B (Clapham et al, 1992, supra). Constructs The HIV-2/vcp env gene was PCR amplified from genomic DNA from HIV-2/vcp-infected HSB cells using the primer pair
  • Antibodies 12G5 was produced by inoculating Balb/c mice with 10 7 living CP-MAC- infected Sup-Tl cells intraperitoneally for 3 weekly injections, and fusion protocols were performed as described (Brass et al, 1994, J. Biol. Chem. 269:2943-2952).
  • Hybridomas were screened in 96-well plates for the ability to inhibit CP-MAC-induced syncytium induction on Sup-Tl cells and were then cloned by limiting dilution.
  • Antibody was purified from ascites using HiTrap Protein G (Pharmacia Biotech).
  • the anti-CD4 MAB #19 was produced using a similar protocol in which the immunizing cell type was uninfected Sup-Tl cells. Specificity of this antibody for CD4 was determined by its ability to detect the 55 kDa CD4 protein by Western blot from lysates of CD4-positive cells.
  • the anti-CD4 MAbs OKT4A and OKT4 were purchased from Ortho Pharmaceuticals, and 12 CA5 was purchased from Boehringer-Mannheim. D47 is an IgG 2a MAB reactive with the HIV-1 /LAI gp 120. Rabbit anti-human CD4 serum was used in these experiments.
  • Viral Infection and Neutralization Assavs Vims infection assays on lymphoid cell lines and U87 cells expressing recombinant Fusin or CD4 were performed by inoculating cultures with 1000 TCID 50 units of either HIV-1 /LAI or HIV-2/vcp. Input virus was washed out after 24 hours and cultures were monitored for infection by serial determinations of reverse transcriptase activity and visual inspection for syncytium formation.
  • target cells were preincubated with varying concentrations of either 12G5 or anti-CD4 MAbs for 30 minutes at 37°C followed by the addition of 100 TCID 50 units of vims, as titered on each target cell. Input vims was removed after 24 hours and cultures were monitored for infection as described herein.
  • H9 cells chronically infected with these vimses were preincubated with 5 ⁇ g/ml of soluble CD4 prior to cocuhivation with target cells as described (Clapham et al, 1992, supra).
  • luciferase gene reporter assay (Doranz et al, supra).
  • HeLa effector cells were transfected with HIV env-containing constmcts and infected with recombinant vaccinia vims encoding the T7 polymerase gene.
  • Target cells were quail QT6 cells that were transfected with a plasmid containing the luciferase gene driven by a T7 promoter (Promega).
  • Selected target cells were also transfected with pT4 which constitutively expresses CD4 from the CMV promoter. Effectors and target cells were mixed and allowed to fuse for 8 hours. Cells were then washed with PBS and lysed in 150 ⁇ l reporter lysis buffer (Promega) and assayed for luciferase activity according to the instructions of the manufacturer.
  • Binding assays with iodinated 12G5 and iodinated 12CA5 were performed essentially as described (Pelchen-Matthews et al, 1989, EMBO J. 8:3641-3649). 12G5 and 12CA5 were labeled with 125 1 using Bolton and
  • HIV-2/vcp a biological variant of HIV-2, termed HIV-2/vcp
  • HIV-2/vcp was shown to infect a number of CD4-negative lymphoid cell lines of T-(BC7, HSB, CEMss4-) and B- (Daudi and Nalm6) cell origin ( Figure 3A) as well as the nonlymphoid rhabdomyosarcoma line RD. As expected, no infection was seen when these cell types were inoculated with the T cell line-tropic isolate HIV-1/LAl ( Figure 3 A).
  • anti-CD4 MAbs were unable to inhibit infection of BC7 (Figure 3B), Daudi or Nalm ⁇ cells, or cell-to-cell fusion between HIV-2/vcp-infected cells and uninfected BC7 cells ( Figure 3C). Similar to other HIV-2 isolates that can infect CD4-negative cells (Clapham et al, 1992, supra), HIV-2/vcp infected the majority of these cell types with extensive cell fusion and killing, indicating the highly efficient use of one or more alternative receptors. 12G5. a Monoclonal Antibody That Inhibits CD4-Independent Infection bv HIV-2
  • the anti-cellular MAB termed 12G5
  • 12G5 the anti-cellular MAB
  • CEMss4- cells are a CD4- negative variant of CEMss generated by serially panning with an anti-CD4 MAB, sorting for CD4-negative cells and cloning by limiting dilution.
  • BC7 cells were cloned by limiting dilution from a culture of Sup-Tl cells chronically infected by HIV-1/NL4-3.
  • BC7 cells exhibit no detectable HIV-1, as determined by soluble p24 production, coculturing with susceptible cell lines, and PCR of genomic DNA with primers specific for the HIV-1 LTR, gag and env. Lysates of BC7 and CEMss4- cells exhibit no detectable CD4 protein by western blot using a rabbit anti-CD4 antiserum.
  • HSB is a CD4-negative and CD8-positive human T-cell line (Weiner et al, 1991 , Pathobiol. 59:361-371).
  • 12G5 In addition to inhibiting cell fusion, 12G5 also neutralized infection by HIV-2/vcp and CP-MAC when preincubated with appropriate target cells.
  • CP-MAC infection of Sup-Tl cells was readily inhibited by antibody concentrations of 1 ⁇ g/ml while somewhat higher concentrations (5-20 ⁇ g/ml) were required to inhibit HIV-2/vcp 5 infection of CD4-negative lines including BC7, Nalm ⁇ , and Daudi ( Figure 4B).
  • HIV-2/Rod-B that have been shown to induce fusion on CD4-negative cell lines including Daudi and RD (Clapham et al, 1992, supra). HIV-2/Rod-A infectivity has been shown to be enhanced by preincubation with soluble CD4 (sCD4) while HIV-2/Rod-B induces syncytia on these CD4-negative cells without sCD4. As shown in Table 5, 12G5 5 inhibited cell fusion at concentrations >5 ⁇ g/ml when H9 cells chronically infected by
  • HIV-2/Rod-A or /Rod-B were cultured in the presence or absence of sCD4, respectively. Inhibition was also observed in the case of another HIV-2 isolate, HIV- 2/CBL-23 that induces syncytia on RD cells following preincubation with sCD4 (Table 5) Clapham et al, 1992). Therefore, in addition to its anti-viral effects on CP-MAC 0 and HIV-2/vcp, 12G5 inhibited infection and/or cell fusion by two other genetically distinct isolates of HIV-2 on CD4-negative target cells.
  • CD4-negative RD or Daudi cells were treated for 30 minutes with varying concentrations of
  • infected cells were preincubated with sCD5 (1 ⁇ g/ml) at 37°C for 60 minutes. 3. Numbers indicate the minimum antibody concentration ( ⁇ g/ml) that inhibited syncytium formation by >95%.
  • a panel of CHO cell lines was used that stably expressed either Fusin, CC- chemokine receptors (CCRl, CCR2b, CCR3, CCR4, or CCR5), or CXC-chemokine receptors (IL8R-A or IL8R-B).
  • the recombinant receptors in these CHO lines were expressed as either untagged or tagged proteins wherein the amino terminus was tagged with the influenza hemagglutinin (HA) epitope.
  • Surface binding assays were performed using 125 I-labeled 12G5 and showed specific binding only to cells that expressed Fusin (Figure 5B).
  • CCR4, and CCR5 were stained with 12G5.
  • Intracellular Fusin was also detectable using 12G5 on cells permeabilized with saponin. Therefore, 12G5 bound specifically to both human and nonhuman cells that expressed recombinant Fusin, strongly suggesting that this antibody reacts specifically with the human Fusin protein.
  • the HIV-2/vcp envelope glycoprotein was PCR amplified from genomic DNA, cloned and expressed in HeLa cells, and cell-to-cell fusion was quantitated as previously described using a luciferase gene reporter assay (Doranz et al, 1996, supra). Quail QT6 target cells were transfected with a luciferase reporter gene and either Fusin alone or Fusin with CD4. As shown ( Figure 7), the HIV-2/vcp envelope glycoprotein fused with Fusin-expressing target cells in the presence and absence of CD4.
  • Cellular receptors for enveloped vimses are characteristically down-regulated from the cell surface following productive infection, rendering infected cells resistant to superinfection by vimses that utilize the same receptor (Weiss, 1993, In the Retroviridae, J.A. Levy, ed. New York, Plenum Press, pp. 1-108). Indeed, the observation that CD4 was selectively down-regulated on HIV-1 infected cells provided the initial evidence that CD4 was a receptor for this virus (McDougal et al, 1986, Science 231 :382-385).
  • Fusin is serving as a primary viral receptor for HIV-2/vcp.
  • CD4 was down-regulated 95% > and 98% on HIV-2/vcp and CP-MAC-infected Sup-Tl cells, respectively.
  • Fusin was also down-regulated 75%> on HIV- 2/vcp-infected Sup-Tl cells, no reduction in 12G5 reactivity was observed on Sup-Tl cells that were infected by CP-MAC ( Figure 8) or HIV-1/LAI. Because CP-MAC

Abstract

L'invention concerne un anticorps anti-virus de l'immunodéficience humaine qui se lie à une protéine cellulaire. On décrit des procédés d'utilisation diagnostique et thérapeutique de cet anticorps.
PCT/US1997/011093 1996-06-25 1997-06-25 Anticorps anti-corecepteur cellulaire pour le virus de l'immunodeficience humaine et procedes d'utilisation Ceased WO1997049424A1 (fr)

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US7345153B2 (en) 1996-01-17 2008-03-18 Progenics Pharmaceuticals, Inc. Compounds capable of inhibiting HIV-1 infection

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US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6025154A (en) 1995-06-06 2000-02-15 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor HDGNR10
WO1996041020A1 (fr) 1995-06-07 1996-12-19 Progenics Pharmaceuticals, Inc. Methode de determination de transfert d'energie resonante par fluorescence servant a identifier la cellule de glycoproteine d'enveloppe de hiv-1
WO1999050461A1 (fr) * 1998-03-30 1999-10-07 Northwest Biotherapeutics, Inc. Applications therapeutiques et diagnostiques basees sur le role du gene cxcr-4 dans l'oncogenese
US6610834B1 (en) * 1998-11-18 2003-08-26 Peter I. Lobo Human IgM antibodies to chemokine receptors
US7138119B2 (en) 2000-09-15 2006-11-21 Progenics Pharmaceuticals, Inc. Compositions and methods for inhibition of HIV-1 infection
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7122185B2 (en) 2002-02-22 2006-10-17 Progenics Pharmaceuticals, Inc. Anti-CCR5 antibody
WO2005105841A2 (fr) 2004-03-12 2005-11-10 Human Genome Sciences, Inc. Récepteur humain (ccr5) hdgnr10 de chimiokine de la protéine g
WO2012178137A1 (fr) 2011-06-24 2012-12-27 Gillies Stephen D Protéines hybrides d'immunoglobuline à chaîne légère et leurs procédés d'utilisation
KR102531889B1 (ko) 2016-06-20 2023-05-17 키맵 리미티드 항-pd-l1 및 il-2 사이토카인

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7345153B2 (en) 1996-01-17 2008-03-18 Progenics Pharmaceuticals, Inc. Compounds capable of inhibiting HIV-1 infection

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