EP0774000A1 - Anticorps se fixant a une molecule cd4 a conformation modifiee, induite par fixation du virus de l'immunodeficience humaine - Google Patents

Anticorps se fixant a une molecule cd4 a conformation modifiee, induite par fixation du virus de l'immunodeficience humaine

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Publication number
EP0774000A1
EP0774000A1 EP95928088A EP95928088A EP0774000A1 EP 0774000 A1 EP0774000 A1 EP 0774000A1 EP 95928088 A EP95928088 A EP 95928088A EP 95928088 A EP95928088 A EP 95928088A EP 0774000 A1 EP0774000 A1 EP 0774000A1
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EP
European Patent Office
Prior art keywords
antibody
cell
molecule
monoclonal
monoclonal fab
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95928088A
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German (de)
English (en)
Inventor
Robin Bachelder
Norman Letvin
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Beth Israel Deaconess Medical Center Inc
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Beth Israel Hospital Association
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Application filed by Beth Israel Hospital Association filed Critical Beth Israel Hospital Association
Publication of EP0774000A1 publication Critical patent/EP0774000A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • CD4 is a transmembrane glycoprotein, having a molecular weight of 55.000 to 62,000
  • CD4 + T cells a subset of T lymphocytes
  • the CD4 + subset of T cells identifies the T helper or inducer cell population. Upon, antigenic stimulation, this population produces cytokines that promote the proliferation and differentiation of T cells and B cells, thereby inducing effector mechanisms such as antibody production and T cell cytotoxicity.
  • CD4 can also be expressed on other cell types, such as macrophages and certain brain cells.
  • the primary target of certain infectious agents is cells that express CD4 on their surface.
  • This tropism of HIV for CD4 + cells is attributed to CD4 functioning as a membrane-anchored receptor for the virus.
  • HIV binding to CD4, mediated through a surface envelope protein of HIV is believed to target HIV to CD4 + cells.
  • the major envelope protein of HIV is produced as a precursor polypeptide (gpl60), which in mature form is cleaved into an exterior membrane protein (gpl20) and a smaller transmembrane protein (gp41) (Ratner, L. et al. (1985) Nature iL2:277-284).
  • the binding of gpl20 to CD4 is thought to initiate infection of CD4+ cells (Dalgleish, A.G. et al. (1984) Nature 112:763-767; Klatzmann, D. et al. (1984) Nature 312:767-768). and additionally may initiate membrane fusion of infected CD4 + cells with uninfected CD4 + cells (called syncytia formation), which may contribute to cell-cell transmission of the virus and to its cytopathic effects (Habeshaw, J.A. and Dalgleish, A.G. (1989) J. AIDS 2:457-468; Lifson, J.D. and Engleman, E.G. (1989) Immunol. Rev. 109:93- 117).
  • CD4 The nucleotide sequence of human CD4 cDNA, and the deduced amino acid sequence of human CD4 protein, have been reported (see Maddon, P.J. et al. (1985) Cell 42:93-104; and the corrected sequence reported in Liftman, D. et al. (1988) Cell 5_5_:541). Structurally, the CD4 protein can be divided into an extracellular domain (approximately amino acids 1-375), a membrane spanning domain (approximately amino acids 376-395) and a cytoplasmic domain (approximately amino acids 396-433). CD4 is synthesized as a precursor protein with a 25 amino acid signal sequence.
  • the CD4 extracellular domain can further be divided into four tandem regions, termed VI , V2, V3 and V4, having homology to immunoglobulin VJ regions.
  • the VI region spans approximately amino acids 1-1 13
  • the V2 region spans approximately amino acids 114-180
  • the V3 region spans approximately amino acids 181-297
  • the V4 region spans approximately amino acids 298-375 (see Maddon, P.J. et al. (1985) cited supra).
  • the VI region has been identified as the binding site for HIV gpl20 (Arthros, J. et al. (1989) Cell 52:469-481 ; Mizukami, T. et al. (1988) Proc. Natl. Acad. Sci. USA £5:9273-9277; Peterson, A. and Seed, B. (1988) Ce// 54:65-72; Landua. N.R. et al.
  • CD4 VI -specific antibodies see e.g., Sattentau, Q.J. et al. (1986) Science 23.4: 1 120-1 123; Jameson, B.A. et al. (1988) Science 24Q: 1335- 1339; Peterson, A. and Seed, B. (1988) cited supra; Bates, P. A. et al. (1989) Prot. Engng. 3.: 13-21 ; McDougal, J.S. et al. (1986) J. Immunol. 122:2937-2944; and Dalgleish, A.G.
  • antibodies may be limited in their therapeutic utility by an inability to act on CD4 that has already bound HIV gpl20.
  • Certain antibodies which bind other regions of CD4 have been found to have some effect on syncytia formation or viral infection while not substantially inhibiting gpl20 binding to CD4.
  • another potential limitation to the use of anti-CD4 antibodies that bind to native cell-surface CD4 is their immunosuppressive activity.
  • OKT4A and OKT4B have been reported to be immunosuppressive (see e.g., Lamarre, D. et al. (1989) cited supra).
  • CD4 participates not only in the initial binding event with gpl20, but also in virus fusion with the cell membrane (a necessary requirement for viral entry into the cell) and HIV-envelope mediated syncytia formation. It further has been suggested that upon binding of gpl20 to CD4 on the surface of a CD4 + cell, the CD4 molecule may undergo a conformational change that is an intermediate step in the mechanism of viral entry and/or syncytia formation (see e.g., Healey, D. et al. (1990) J. Exp. Med. 122:1233-1242; Celada, F. et al. (1990) J. Exp. Med. 122:1 143-1 150).
  • This invention pertains to a novel conformational form of cell-surface CD4 that is induced upon binding of HIV (or an envelope protein thereof), ligands that bind this novel form of CD4, including antibodies and antibody mimetic agents, isolated molecules expressing one or more epitopes exposed on this novel form of CD4, and uses therefor.
  • One aspect of this invention relates to antibodies, or fragments thereof, that bind a conformationally altered form of a CD4 molecule, preferably human CD4, expressed on the surface of a CD4 + cell upon contact of the cell with HIV, or an envelope protein thereof (e.g., gpl20), thereby providing evidence for an HIV-induced conformational change in cell- surface CD4.
  • the antibodies of the invention are characterized by an ability to bind this conformationally altered form of CD4 on the surface of a cell upon contact of the cell with gpl20 and by an inability to substantially bind native human CD4 on the surface of the cell prior to contact with gpl20.
  • a population of CD4 + cells contacted with gpl20 preferably at least 30 % of the gpl20 + cells are bound by an antibody of the invention. More preferably, at least 50 %, even more preferably at least 70% to 90 %, of the gpl20 + cells are bound by an antibody of the invention.
  • the antibody of the invention is a monoclonal antibody, preferably a human monoclonal antibody.
  • Preferred antibodies of the invention are human monoclonal Fabs designated 3-47 and 3-51 , or antibodies that binds the same epitope recognized by either 3-47 or 3-51. Additionally, antibodies that bind other epitopes exposed, upon gpl20 binding, on the conformationally altered form of human CD4 that also displays either the 3-47 or 3-51 epitope are encompassed by the invention.
  • the invention further provides antibody mimetic agents, such as 3-47 or 3-51 mimetic agents, which are non- antibody compounds having the same epitope binding specificity as an antibody of the invention. Pharmaceutical compositions comprising the antibodies, or fragments thereof, or antibody mimetic agents described herein are also within the scope of the invention.
  • nucleic acid molecules comprising a nucleotide sequence encoding the light chain or heavy chain variable region (VL or VH region) of the antibodies of the invention, preferably encoding the VL or VH region of monoclonal Fab 3-47 or 3-51.
  • the nucleic acid encoding the VL region further encodes a CL region (i.e., a full-length antibody light chain).
  • the nucleic acid encoding the VH region further encodes a CH 1 region (i.e., the first constant domain of an immunoglobulin heavy chain).
  • the nucleic acid encoding the VH region encodes a full-length antibody heavy chain (e.g., includes CHI, CH2 and CH3 regions).
  • the nucleic acid molecules of the invention can be incorporated into expression vectors and introduced into a host cell (e.g., a bacterial or mammalian cell).
  • a host cell e.g., a bacterial or mammalian cell.
  • the monoclonal Fab 3-47 is expressed in host cell.
  • the monoclonal Fab 3-51 is expressed in a host cell.
  • a full-length antibody having the epitope binding specificity (e.g., the VL and VH regions) of monoclonal Fab 3-47 or 3-51 is expressed in a host cell.
  • the isolated nucleic acid molecules, expression vectors and host cells of the invention are useful for producing recombinant antibodies of invention, in particular recombinant antibodies having the epitope binding specificity of monoclonal Fab 3-47 or 3-51.
  • the antibodies, and mimetic agents thereof can be used to detect the presence of a conformationally altered form of CD4 on the surface of a cell, for example to monitor the course of HIV infection or the efficacy of a therapeutic regimen.
  • the cell is contacted with an antibod of the invention and the antibody bound to the surface of the cell is detected.
  • the antibodies and mimetic agents of the invention are further useful for screening agents for their ability to inhibit or induce formation of a conformationally altered form of CD4 on a cell surface.
  • a CD4 + cell can be contacted with gpl20 and an agent to be tested, further contacted with an antibody of the invention, and the amount of antibody bound to the cell surface determined.
  • a reduced amount of binding of the antibody to the gpl20-treated cell in the presence of the agent, as compared to the amount of antibody binding to a gpl20- treated cell in the absence of the agent, can be used as an indicator that the agent inhibits formation of a conformationally altered form of CD4 on the cell surface.
  • a CD4 + cell can be contacted with an agent to be tested, further contacted with an antibody of the invention, and the amount of antibody bound to the cell surface determined.
  • An increase amount of binding of the antibody to the CD4 + cell in the presence of the agent, as compare to the amount of antibody binding to a CD4 + cell in the absence of the agent, can be used as an indicator that the agent induces formation of a conformationally altered form of CD4 on the cell surface.
  • agents which inhibit or induce expression of a conformationally altere form of cell-surface CD4 may be useful therapeutically to treat HIV infection.
  • the antibodies, and antibody mimetic agents, of the invention can also be used to inhibit infection of a cell by human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • the cell is contacted with a therapeutically effective amount of an antibody or mimetic agent of the invention.
  • a cell can be contacted with the antibody or mimetic agent in vitro, or alternatively, the antibody or mimetic agent can be administered to a subject in vivo.
  • Yet another aspect of the invention pertains to isolated molecules that express at least one epitope expressed on a conformationally altered form of human CD4 induced upon contact with HIV, or an envelope protein or peptide thereof.
  • this conformationally altered form of human CD4 is one which displays the epitope bound by either monoclonal Fab 3-47 or monoclonal Fab 3-51.
  • the isolated molecule expresses the same epitope bound by 3-47 or 3-51.
  • the molecule expresses another epitope(s) that is also exposed on this conformationally altered form of CD4 upon gpl 20 binding.
  • the molecule can be a protein or peptide, such as a modified human CD4 protein, or peptide fragment thereof, or a non-human primate CD4 protein, or peptide fragment thereof (e.g., from a rhesus monkey or chimpanzee).
  • the molecule can be an anti-idiotype antibody, or fragment thereof, that binds 3-47 or 3-51.
  • the molecule can be a peptide mimetic that expresses the appropriate altered CD4 epitope.
  • a molecule of the invention expressing an epitope(s) displayed by a conformationally altered form of human CD4 can be incorporated into a pharmaceutical composition, preferably including a pharmaceutically acceptable adjuvant.
  • a mammal can be immunized with such a composition to elicit antibodies which bind a conformationally altered form of human CD4.
  • the molecules of the invention expressing at least one epitope of a conformationally altered form of CD4 can be used to induce an antibody response in a subject that may inhibit infection of cells in the subject by HIV.
  • still another aspect of the invention pertains to methods for inhibiting infection of a cell by HIV involving administering to a subject a therapeutically effective amount of a molecule that expresses at least one epitope exposed on the conformationally altered form of human CD4 such that an antibody response against the epitope(s) expressed by the molecule is induced in the subject.
  • a bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the monoclonal Fab 3-47 has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, MD, on July 19, 1994 and assigned ATCC Designation No. 69658.
  • a bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the monoclonal Fab 3-51 has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, MD, on August 25, 1994 and assigned ATCC Designation No. 69684.
  • Figure 7 is a graphic representation of the titers of human rsCD4-specific antibodies in the serum of four human rsCD4-immunized, HIV-infected humans at the time of, and at various time points after, immunization. The arrows indicate immunization time points.
  • Figure 2 is a schematic representation of the phagemid pComb3 used to construct a combinatorial human immunoglobulin library. Arrows indicate the restriction sites used for cloning.
  • Fab expression is induced through the lacZ promoter with IPTG.
  • the pelB leader sequence directs heavy and light chains to the periplasmic space of induced bacterial cells, where Fab assembly occurs.
  • the heavy chain is expressed as a fusion protein with the Ml 3 coat protein encoded by gene III, which directs the expression of Fab molecules on the virio surface.
  • Figures 3A-J are a series of flow cytometric profiles depicting the binding of various antibodies to human peripheral blood lymphocytes preincubated with PBS (panels A-E) or with recombinant gpl20 (panels F-J).
  • the following antibodies are depicted: control FITC- labelled goat anti-human secondary antibody (panels A and F), 19thy5D7 (specific for the gpl20 binding site of CD4) (panels B and G), L736523 (specific for the V3 loop domain of gpl20) (panels C and H), Fab clone 3-47 (panels D and I) and Fab clone 3-51 (panels E and J).
  • Figure 4 depicts the reactivity of Fab clone 3-47 on a Western blot with both human rsCD4 and CD4 from a human peripheral blood lymphocyte lysate.
  • the lanes show the reactivity of: an irrelevant gpl20-specific antibody (L736523) with human rsCD4 (lane 1), a CD4-specific control antibody (humanized 5A8) with human rsCD4 (lane 2), the monoclona Fab 3-47 with rsCD4 (lane 3), the gpl20-specific antibody (L736523) with a human PBL lysate (lane 4) and the monoclonal Fab 3-47 with a human PBL lysate (lane 5).
  • Molecular weights are indicated in kilodaltons.
  • Figure 5 is a photograph of an immunoprecipitation experiment, depicting the ability of Fab 3-47, but not a negative control monoclonal antibody, to immunoprecipitate a 55 kD cell surface protein (corresponding to the molecular weight of CD4) from H9 cell lysates.
  • Figures 6A-H is a series of flow cytometric profiles depicting the binding of Fab 3-4
  • Figures 7 A-D are a series of flow cytometric profiles depicting the binding of Fab- 347 (panel D), control Fab 2-36 (panel C), a control anti-CD4 antibody ("Hu5A8", panel B) or no antibody ("PBS", panel A) to H9 cells preincubated with live HIV-1, demonstrating th Fab 3-47 binds to H9 cells preincubated with live HIV-1.
  • This invention pertains to a conformationally altered form of a cell-surface CD4 molecule expressed on a CD4 + cell upon binding of HIV, or an envelope protein thereof, to the cell.
  • this conformationally altered form of CD4 can be induced by contacting a CD4 + cell with isolated gpl20 (e.g., soluble recombinant gpl20).
  • isolated gpl20 e.g., soluble recombinant gpl20.
  • ligands including antibodies, antibody fragments and antibody mimetic agents, that bind novel epitopes exposed on CD4 upon contact with HIV or a portion thereof (e.g., gpl20), as well as isolated molecules that express these novel epitopes exposed on CD4, and used therefor.
  • the antibodies of the invention are capable of binding CD4 on the surface of a CD4 + cell following contact of the cell with gpl20, but do not substantially bind CD4 on the surface of the cell prior to contact with gp-120. Accordingly, these antibodies are specific for epitopes exposed on the CD4 molecule upon gpl20 binding.
  • a population of CD4 + cells contacted with gpl20 preferably at least 30 % of the gpl20 + cells are bound by an antibody of the invention. More preferably, at least 50 % of the gpl20 + cells are bound by an antibody of the invention. Even more preferably, at least 70 % to 90 % of the gpl20 + cells are bound by an antibody of the invention.
  • Preferred antibodies of the invention are human monoclonal Fab fragments designated 3-47 and 3-51. These Fab fragments were isolated from a random combinatorial immunoglobulin phage display library prepared from an HIV-infected individual that had been immunized multiple times with soluble recombinant human CD4 in incomplete Freund's adjuvant. The light and heavy chain variable regions of monoclonal Fabs 3-47 and 3-51 differ, demonstrating that they are distinct antibodies.
  • a conformationally altered form of cell-surface CD4 induced upon binding of HIV, or an envelope protein thereof is defined by expression of an epitope recognized by 3-47 or 3-51 (referred to herein as the 3-47 and 3-51 epitopes, respectively) on the CD4 molecule.
  • one aspect of this invention relates to antibodies, or fragments thereof, that bind the 3-47 or 3-51 epitopes or that bind other epitopes that are exposed on the conformationally altered form of CD4 that also expresses the 3-47 and/or 3-51 epitope.
  • the discovery of the 3-47 and 3-51 epitopes provides evidence for an HIV-induced conformational change in the CD4 receptor that can be targeted for diagnostic, screening and therapeutic purposes, as described herein.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as a conformationally altered form of CD4.
  • the invention pertains to polyclonal and, more preferably, monoclonal antibodies.
  • a monoclonal antibody of the invention is a human monoclonal antibody.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, are within the scope of the invention.
  • the simplest naturally occurring antibody e.g., IgG
  • IgG immunoglobulfide-binding protein
  • H heavy chain
  • L light chain inter-connected by disulfide bonds
  • antigen-binding function of an antibody can be performed by fragments of a naturally-occurring antibody.
  • these antigen-binding fragments are also intended to be designated by the term "antibody”.
  • binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) an Fd fragment consisting of the VH and CHI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al., (1989) Nature 241:544-546 ) which consists of a VH domain; (v) an isolated complimentarity determining region (CDR); and (vi) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • Antibody fragments such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibody fragments can be obtained using standard recombinant DNA techniques, as described herein. Furthermore, although the two domains of the Fv fragment are coded for by separate genes, a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bir et al. (1988) Science 242:423-426; and Huston et al. (1988) PNAS £5:5879-5883) by recombinant methods. Such single chain antibodies are also encompassed within the term "antibody”.
  • An antibody of the invention is further intended to include bispecific and chimeric molecules having a binding portion that recognizes a conformationally altered form ofCD4.
  • antibody combining site refers to that structural portion o an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) antigen.
  • immunogen or “reactive with” in its various forms is used herein to refer to binding between an antigenic determinant containing molecule and a molecule containing an antibody combining site, such as a whole antibody molecule or a portion thereof.
  • epipe refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site. The term is also used interchangeably with "antigenic determinant”.
  • epitope When particular epitope is present on a molecule and available for immunological recognition (e.g., binding by an antibody), the epitope is said to be “expressed” or “displayed” by the molecule.
  • the epitopes of particular interest with regard to this invention are not expressed constitutively on the native cell-surface CD4 molecule but rather become expressed (i.e., exposed) on the conformationally altered form of CD4 upon gpl20 binding, as described herein.
  • Such epitopes that are exposed on a molecule upon a conformational change in the molecule are often referred to in the art as "neo-epitopes".
  • epitope binding specificity of an antibody refers to the reactivity of an antibody for a specifi epitope, i.e., antibodies that bind the same epitope are referred to as having the same epitope binding specificity.
  • a specifi epitope i.e., antibodies that bind the same epitope are referred to as having the same epitope binding specificity.
  • the ability of one antibody to competitively inhibit the binding of the other antibody to the antigen can be determined by conventional techniques.
  • An antibody of the invention is typically prepared by immunizing a suitable subject with an appropriate CD4 immunogen and isolating an antibody having the characteristics described herein.
  • monoclonal antibodies are isolated by screening a combinatorial immunoglobulin library prepared from the immunized subject, although monoclonal antibodies may also be isolated by screening conventional antibody- secreting hybridomas.
  • the CD4 immunogen is human recombinant soluble CD4 (rsCD4) and the immunized subject is an HIV-infected human.
  • the immunized subject can be an uninfected subject (e.g., human).
  • a non-human primate such as a rhesus monkey or a chimpanzee
  • a human can be immunized with a non-human primate rsCD4 molecule (e.g., rhesus monkey or chimpanzee CD4).
  • a subject can be immunized with a molecule (e.g., a peptide) that expresses the 3-47 and/or 3-51 epitope(s), described in further detail below.
  • a soluble CD4- gpl20 complex or with CD4 + cells (e.g., peripheral blood lymphocytes) that have been treated (i.e., contacted with) soluble recombinant gpl20. It may also be possible to raise antibodies as described herein by immunizing a mouse transgenic for human CD4 (and therefore tolerant to native human cell-surface CD4) with a CD4 immunogen such as a soluble form of a non-human primate CD4 molecule or an altered form of a soluble human CD4.
  • a CD4 immunogen such as a soluble form of a non-human primate CD4 molecule or an altered form of a soluble human CD4.
  • the unit dose of CD4 immunogen and the immunization regimen will depend upon the species of mammal immunized, its immune status, the body weight of the mammal and the CD4 content of the CD4 immunogen administered.
  • the CD4 immunogen is typically administered with an adjuvant, such as Freund's complete or incomplete adjuvant.
  • an adjuvant such as Freund's complete or incomplete adjuvant.
  • 1 mg of human rsCD4 in incomplete Freund's adjuvant is injected intramuscularly into an HIV-infected individual (with absolute CD4 counts greater than 500) and the individual is boosted several times (e.g., five times) at regular intervals (e.g., every 3-5 weeks).
  • Immunization of a subject with a CD4 immunogen as described above induces a polyclonal anti-CD4 antibody response.
  • the anti-CD4 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized rsCD4.
  • ELISA enzyme linked immunosorbent assay
  • the polyclonal antibod molecules can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • monoclonal antibodies can be prepared and screened.
  • monoclonal antibodies are prepared by constructing a recombinant combinatorial immunoglobulin library, such as a Fab phage display library, using immunoglobulin light chain and heavy chain cDNAs prepared from mRNA derived from lymphocytes of the immunized subject. Methodologies for preparing and screening such a library are described in detail in the Examples. Briefly, mRNA is isolated from a lymphocyte-containing cell population, such as bone marrow lymphocytes. First-strand cDNA is synthesized using primers specific for a constant region of the heavy chain (e.g., CH3) and the constant region of each of the K and ⁇ light chains.
  • a recombinant combinatorial immunoglobulin library such as a Fab phage display library
  • the heavy and light chain cDNAs are amplified by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the amplified DNA is then ligated into appropriate vectors for further manipulation in generating a library of display packages.
  • Oligonucleotide primers useful in amplification protocols may be unique or degenerate and may incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.
  • the immunoglobulin library e.g., a Fab library
  • the display package comprises a system that allows for the sampling of a large, diverse antibody display library, rapid sorting after each affinity separation round, and easy isolation of the antibody genes from the purified display packages.
  • a Fab library e.g., a Fab library
  • the display package comprises a system that allows for the sampling of a large, diverse antibody display library, rapid sorting after each affinity separation round, and easy isolation of the antibody genes from the purified display packages.
  • kits for generating phage display libraries e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene phage display kit, catalog no. 240612
  • examples of methods and reagents particularly amenable for use in generating antibody display library can be found in, for example, Ladner et al.
  • the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFv gene subsequently cloned into the desired expression vector or phage genome.
  • a flexible linker As generally described in McCafferty et al., Nature ( 1990) 24£:552-554, complete VH and VL domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody expressed on the surface of a display package, such as a filamentous phage.
  • the antibody library is screened to identify and isolate packages that express an antibody that binds a conformationally altered form of CD4.
  • the primary screening of the library involves panning with immobilized rsCD4, or, more preferably, an immobilized rsCD4/gpl20 complex (described in further detail in Example 3). Display packages expressing antibodies that bind immobilized CD4, or, more preferably, the rsCD4/gpl20 complex, are selected.
  • Soluble forms of the selected antibodies can then be generated (as described in Example 3) and the soluble antibodies further selected in secondary screenings, e.g., by ELISA, radioimmunoassay and/or flow cytometry (FACS analysis).
  • FACS analysis an antibody that binds a conformationally altered form of CD4 is preferably selected based upon its ability to bind CD4 + cells contacted with recombinant gpl 20, while not substantially binding to CD4 + cells prior to gpl 20 treatment (see Example 4).
  • FACS analysis is used to determine whether an antibody binds cell-surface CD4 in the presence or absence of gpl 20 treatment.
  • FACS analysis can also be used to determine the percentage of gpl20 + , CD4 + cells that bind the antibody.
  • Reagents useful for screening antibodies of the invention have been described in detail in the art and/or are commercially available.
  • full-length and truncated, soluble forms of human CD4 useful in the above-described screening assays are disclosed in PCT patent application PCT/US88/02940 and Fisher, R.A. et al. (1988) Nature 331 :76-78.
  • Recombinant soluble human CD4 is commercially available (e.g., from ABT, Cambridge, MA).
  • HIV gpl 20 for use in the above-described assays can be, for example, gpl 20 isolated from HIV or, more preferably, recombinant gpl 20 expressed by host cells transfected with the gene for HIV gpl 20.
  • a purified, soluble HIV gpl 20 isolated from a unicellular host transfected with a truncated gpl 60 gene encoding HIV gpl 20 is used.
  • Purified recombinant HIV gpl 20 is commercially available (e.g., from Repligen, Cambridge, MA or from Celltech, Berkshire, United Kingdom). Cells producing recombinant gpl20 are described, for example, in Lasky et al.
  • CD4 + cells for use in screening assays include peripheral blood lymphocytes (either unfractionated or a selected CD4 + subpopulation) and tissue culture cells transfected with DNA encoding full-length human CD4 and expressing CD4 on their surface. Suitable transfected CD4 + tissue culture cells are described in Fisher, R.A. et al. (1988) Nature 221:76-78.
  • nucleic acid encoding the selected antibody ca be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.
  • the nucleic acid can be further manipulated (e.g., linked to nucleic acid encoding additional immunoglobulin domains, such as additional constant regions) and/or expressed in a host cell, as described in further detail below.
  • a monoclonal antibody of the invention can be prepared and isolated using a technique which provides for the production o antibody molecules by continuous cell lines in culture.
  • a technique which provides for the production o antibody molecules by continuous cell lines in culture include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495 497) (see also, Brown et al. (1981) J. Immunol 122:539-46; Brown et al. (1980) JBiol Chem 255:4980-83; Yeh et al. (1976) PNAS 26:2927-31; and Yeh et al. (1982) Int. J. Cancer 22:269-75), and the more recent human B cell hybridoma technique (Kozbor et al.
  • an immortal cell line typically myeloma cells
  • lymphocytes typically splenocytes
  • a mammal immunized with a CD4 immunogen as described above
  • the culture supernatants of the resulting hybridoma cells are screened, as described above for screening of recombinant immunoglobulin libraries, to thereby identify an antibody of the invention.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with a CD4 immunogen with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3- x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.
  • ATCC American Type Culture Collection
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • human hybridomas can be made using human lymphocytes (e.g., from an HIV-infected individual immunized with soluble human CD4, as described above) and human B cell- or EBV-hybridoma techniques.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants using the screening assays described above.
  • a primary screen can be performed to select antibodies that bind immobilized human rsCD4 or, more preferably, an immobilized rsCD4/gpl20 complex (e.g., by ELISA).
  • a secondary screen can then be performed to identify antibodies that bind CD4 + cells treated with recombinant gpl 20 but which do not substantially bind CD4 + cells prior to gpl 20 treatment. This secondary screen is preferably performed by FACS analysis.
  • Hybridoma cells that test positive in the above screening assays can be cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium, to thereby produce whole antibodies.
  • Tissue culture techniques and culture media suitable for hybridoma cells are well known (see, e.g., Lerner, Yale J. Biol. Med. and Kenneth, Monoclonal Antibodies, cited supra).
  • Conditioned hybridoma culture supernatant containing the antibody can then be collected.
  • the desired antibody may be produced by injecting the hybridoma cells into the peritoneal cavity of an unimmunized mouse.
  • the hybridoma cells proliferate in the peritoneal cavity, secreting the antibody homolog, which accumulates as ascites fluid (see Lerner, Yale J. Biol. Med. and Kenneth, Monoclonal Antibodies, cited supra).
  • the antibody is harvested by withdrawing the ascites fluid from the peritoneal cavity with a syringe. Accordingly, it will be understood by the ordinary skilled worker that monoclonal antibodies of the invention may be purified with ease from conditioned hybridoma culture supernatant or from ascites.
  • a monoclonal antibody prepared from a murine (or other non-human) hybridoma has the disadvantage that the antibody will be recognized as foreign in a subject of another species (e.g., a human).
  • One approach to circumventing this problem is to engineer a recombinant chimeric or humanized antibody derived from the original non-human monoclonal antibody, as described in further detail below.
  • a human monoclonal directed against a human protein can be generated in transgenic mice carrying human antibody repertoires (see, e.g., Wood et al. PCT publication WO 91/00906, Kucherlapati et al.
  • a human antibody-transgenic mouse can be immunized with a CD4 immunogen as described above and splenocytes from these immunized transgenic mice can then be used to create hybridomas, which are then screened to identify an antibody of the invention as described above. Additionally, double transgenic animals, e.g., transgenic both for human CD4 and human antibody genes, can be immunized with a CD4 immunogen and lymphocytes therefrom used to generate human monoclonal antibodies of the invention.
  • the antibodies of the invention further encompass recombinant forms of antibodies, such as chimeric and humanized antibodies.
  • antibodies produced in non-human subjects are used therapeutically in humans, they are recognized to varying degrees as foreign and an immune response may be generated in the patient.
  • One approach for minimizing or eliminating this problem, which is preferable to general immunosuppression, is to produce chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region.
  • Such antibodies retain the epitope binding specificity of the original monoclonal antibody, but may be less immunogenic when administered to humans, and therefore more likely to be tolerated by the patient.
  • Chimeric monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the constant region of a non-human antibody molecule is substituted with a gene encoding a human constant region, (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al.. European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al. U.S. Patent No.
  • a chimeric antibody can be further "humanized” by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
  • "humanized” chimeric antibodies are provided by Morrison, S. L. (1985) Science 222:1202-1207 and by Oi et al. (1986) BioTechniques 4:214. Such methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain.
  • the cDNA encoding the humanized chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
  • Suitable "humanized” antibodies can be alternatively produced by CDR or CEA substitution (see U.S.
  • this invention provides a derivatized antibody in which an antibody of the invention is functionally linked (by chemical coupling, genetic fusion or otherwise) to one or more other molecular entities, such as another antibody of the invention, a mimetic agent of the invention (described below), a detectable agent, a cytotoxic agent and/or a pharmaceutical agent.
  • an antibody of the invention is functionally linked (by chemical coupling, genetic fusion or otherwise) to one or more other molecular entities, such as another antibody of the invention, a mimetic agent of the invention (described below), a detectable agent, a cytotoxic agent and/or a pharmaceutical agent.
  • An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like.
  • detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like.
  • detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like.
  • detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like.
  • detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like.
  • an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product.
  • the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and di
  • Useful pharmaceutical agents include biologically active peptides, polypeptides and proteins, such as an antibody specific for a human polypeptide other than CD4.
  • Other useful pharmaceutical agents include non-proteinaceous pharmaceutical agents such as HIV reverse transcriptase inhibitors (e.g., 3'-azido-2',3'-dideoxythymidine (“AZT”) and 2',3'-dideoxyinosine (“DDI”) and other antiviral compounds, or immunosuppressive agents (e.g., cyclosporin or FK506).
  • the invention further encompasses non-antibody molecules that mimic the epitope binding specificity of the antibodies described herein. These agents are referred to herein as "antibody mimetic agents".
  • the antibody mimetic agents of the invention are non-antibody compounds that bind an epitope exposed on a conformationally altered form of human CD4 upon binding of HIV, or an envelope protein thereof, to CD4. Accordingly, these compound bind CD4 + cells upon incubation with gpl 20 but do not substantially bind CD4 + cells prior to gpl 20 treatment.
  • Preferred antibody mimetic agents of the invention bind an epitope recognized by the monoclonal Fab 3-47, referred to herein as a "3-47 mimetic agent", or an epitope recognized by the monoclonal Fab 3-51, referred to herein as a "3-51 mimetic agent”.
  • Preferred antibody mimetic agents e.g., 3-47 or 3-51 mimetic agents, inhibit infection of CD4 + cells by HIV.
  • the most preferred antibody mimetic agents of the invention display th properties of one or more antibodies of this invention (e.g., monoclonal Fabs 3-47 or 3-51).
  • An antibody mimetic agent of this invention may be produced by synthesizing a plurality of peptides (e.g., 5-20 amino acids in length), semi-peptidic compounds or non-peptidic, organic compounds, and then screening those compounds for their ability to bind CD4 + cells upon treatment of the cells with gpl 20, using assays described herein.
  • peptide libraries construction and screening see U.S. Patent No. 4,833,092; Scott, J.K. and Smith, G.P. (1990) Science 242:86-90; Devlin, J.J. et al. (1990) Science 249:404-407.
  • the agents can be screened for their ability to competitively inhibit binding of an antibody of the invention to gpl20-treated, CD4 + cells.
  • a 3-47 mimetic agent can be identified based upon its ability to inhibit the binding of the monoclonal Fab 3-47 to gpl20-treated CD4 + cells.
  • FACS analysis is used to determine whether an antibody mimetic agent can competitively inhibit the binding of an antibody of the invention to gpl20-treated CD4 + cells.
  • an antibody of the invention is produced in quantity by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell is transfected with DNA encoding the immunoglobulin light and heavy chains of the antibody in a form suitable for expression of the light and heavy chains in the host cell.
  • Recombinant antibodies may be produced by well known genetic engineering techniques (see, e.g., U.S. Patent No. 4,816,397).
  • DNA encoding the light and heavy chains of a selected antibody of interest can be recovered from the display package (e.g., from the genome of the filamentous phage) and, if desired, further manipulated. Such manipulation may involve conversion of a partial antibody chain to a full-length antibody chain.
  • a Fab expression library is screened, the isolated DNA encoding the heavy chain of the Fab can be converted to a full-length heavy chain gene by operatively linking the DNA to another DNA molecule encoding the additional heavy chain constant regions.
  • the monoclonal Fab fragments 3-47 and 3-51 described herein can be converted to full-length, whole antibodies having the same epitope binding specificity of 3-47 or 3-51.
  • a scFv library is screened, the portions of the isolated DNA encoding the linked VL and VH regions of the scFv can be separated and the separate VL- and VH-encoding DNA molecules can then be operatively linked to other DNA molecules encoding the appropriate light and heavy chain constant regions to produce full-length antibody genes.
  • an antibody of the invention when isolated by screening hybridomas, as described above, cDNA or genomic DNA encoding the immunoglobulin light and heavy chains of a selected antibody, or a portion thereof, can be isolated from the hybridoma cell by standard molecular biology techniques.
  • cDNAs or genomic DNAs encoding partial or full-length light or heavy chains are inserted into expression vectors so that both genes are operatively linked to their own transcriptional and translational control sequences.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Typically, both genes are inserted into the same expression vector.
  • the expression vectors is transfected into a host cell by standard techniques. Prokaryotic or eukaryotic host cells may be used.
  • transfection or "transfected into” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Expression of antibodies in eukaryotic host cells is preferred because such cells are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
  • any antibody produced that is inactive due to improper folding may be renaturable according to well known methods (see e.g., P. S. Kim and R. L. Baldwin (1982) Ann. Rev. Biochem. 51:45989).
  • Host cells can also be used to produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which are encompassed by the term "antibody” as used herein. It will be understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to a conformationally altered form of CD4. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.
  • bifunctiona antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than CD4, or another epitope of CD4.
  • the monoclonal Fab 3-47, or a full-length antibody havin the same epitope binding specificity of 3-47 (e.g., the VL and VH regions of 3-47), is produced recombinantly in a host cell.
  • the partial nucleotide sequence of an isolated nuclei acid molecule (e.g., DNA) encoding the light chain variable region (VL) of 3-47 is shown i SEQ ID NO: 15.
  • the partial nucleotide sequence of an isolated nucleic acid molecule e.g., DNA
  • VL heavy chain variable region
  • SEQ ID NO: 16 The partial nucleotide sequence of an isolated nucleic acid molecule (e.g., DNA) encoding the heavy chain variable region (VL) of 3-47 is shown in SEQ ID NO: 16.
  • bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the monoclonal Fab 3-47 has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, MD, on July 19, 1994 and assigned ATC Designation No. 69658.
  • Oligonucleotide primers described in Example 2 can be used to amplify DNA encoding the light or heavy chain of 3-47 by the polymerase chain reaction using this plasmid as template DNA.
  • the monoclonal Fab 3-51, or a full-length antibod having the same epitope binding specificity of 3-51 i produced recombinantly in a host cell.
  • the partial nucleotide sequence of an isolated nuclei acid molecule (e.g., DNA) encoding the light chain variable region (VL) of 3-51 is shown in SEQ ID NO: 17.
  • the partial nucleotide sequence of an isolated nucleic acid molecule (e.g., DNA) encoding the heavy chain variable region (VL) of 3-51 is shown in SEQ ID NO: 18.
  • bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the monoclonal Fab 3-51 has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, MD, on August 25, 1994 and assigned
  • Oligonucleotide primers described in Example 2 can be us to amplify DNA encoding the light or heavy chain of 3-51 by the polymerase chain reaction using this plasmid as template DNA.
  • a nucleic acid molecule of the invention may encode only the immunoglobulin light and or heavy chain variable region of an antibody of the invention, e.g., the 3-47 or 3-51 Fa More preferably, the nucleic acid molecule also includes nucleotide sequences encoding at least one immunoglobulin constant region operatively linked to the variable region-encodin DNA (i.e., the VL region can be linked to a CL region or the VH region can be linked to on or more CH regions).
  • the nucleic acid molecules of the invention can be inserted into expression vectors and transfected into host cells to express the monoclonal Fab fragments 3 47 or 3-51, or a portion thereof, or a full-length antibody having VL and VH regions derived from 3-47 or 3-51, as described above.
  • the host cell is a bacterial cell (e.g., E. coli).
  • the host cell is a mammalian cell, such as CHO cell or a myeloma cell.
  • an antibody e.g.
  • the antibodies of this invention can be incorporated into pharmaceutical compositions that can be used prophylactically or therapeutically in the prevention or treatment of diseases caused by infectious agents whose primary targets are CD4 + lymphocytes.
  • diseases include AIDS, ARC and HIV infection in humans.
  • the generic term "HIV” is intended to refer to independent isolates from AIDS patients and to laboratory strains derived therefrom.
  • HIV is also intended to include viruses elsewhere identified as human T cell lymphotrophic virus type III (HTLV-III), lymphadenopathy- associated virus (LAV) and AIDS-associated retrovirus (ARV).
  • the compositions may also be useful for treating or preventing AIDS-like diseases caused by other retroviruses, such as simian immunodeficiency virus.
  • Preferred pharmaceutical compositions of this invention include an antibody having the epitope binding specificity of the monoclonal Fab 3-47 or the epitope binding specificity of the monoclonal Fab 3-51.
  • the antibody may be, for example, 3-47 or 3-51 itself, or a whole antibody containing VL and VH regions derived from 3-47 or 3-51 (as described herein).
  • compositions of this invention may further comprise other therapeutics for the prophylaxis or treatment of AIDS, ARC and HIV infection.
  • an antibody of the invention may be used in combination with antiretroviral agents that block reverse transcriptase, such as AZT, DDI, HPA-23, phosphonoformate, suramin, ribavirin and dideoxycytidine, or with agents that inhibit the HIV protease.
  • compositions of this invention may further comprise anti-viral agents such as interferons (including alpha interferon, beta interferon and gamma interferon) or glucosidase inhibitors such as castanospermine, or immunosuppressive agents such as adrenal corticosteroids, azathrioprine, cyclosporin or FK506.
  • anti-viral agents such as interferons (including alpha interferon, beta interferon and gamma interferon) or glucosidase inhibitors such as castanospermine, or immunosuppressive agents such as adrenal corticosteroids, azathrioprine, cyclosporin or FK506.
  • the antibodies of the invention may be administered in combination with anti-CD4 antibodies having differing antigenic specificities than the antibodies described herein, such as anti-CD4 antibodies that bind native cell-surface CD4 in the absence of gpl 20 binding (e.g., that are specific for the VI, V2, V3 or V4 domain of native CD4). They may also be administered in combination with antibodies (anti-idiotypic or otherwise) specific for HIV polypeptides such as g l 20 and gp41.
  • one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents.
  • Such combination therapies may - 20 -
  • the pharmaceutical compositions of the invention comprise an immunotherapeutically effective amount of one or more antibodies according to this invention, or derivatized form(s) thereof and, preferably, a pharmaceutically acceptable carrier.
  • therapeutically effective amount is meant an amount capable of lessening the spread, severity or immunocompromising effects of AIDS, ARC or HIV infection, or of othe diseases caused by infective agents whose primary targets are CD4 + lymphocytes.
  • the therapeutically effective amount is capable of completely preventing infectio by such viruses when used prophylactically.
  • pharmaceutically acceptable carrier refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered.
  • suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • compositions of this invention may be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable and infusible solutions.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the preferred compositions are in the form of injectable or infusible solutions.
  • the preferred pharmaceutical compositions of this invention are similar to those used for passive immunization of humans with other antibodies.
  • the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal).
  • therapeutically effective amount of antibody of this invention will depend, inter alia, upon the administration schedule, the unit dose of antibody administered, whether the antibody is administered in combination wit other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular antibody administered.
  • therapeutically effective amounts per unit dose of an antibody which is an intact antibody typically range from about 0.5 to about 5 mg/kg patient weight, preferably about 2 to about 3 mg/kg patient weight.
  • Unit doses should be administered from once each 3 days to once each 28 days until an antiviral effect is observed, preferably once each week for an indefinite period of treatment.
  • the antiviral effect may be measured by a variety of methods, including assessment of viral load, lymphocyte counts an clinical signs and symptoms. It will be apparent to the skilled artisan, however, that lower or higher dosages and other administration schedules may be employed. Treatment regimens for antibodies that are not intact antibodies (e.g., Fab fragments) may differ, depending on their size and pharmaceutical properties.
  • antibody mimetic agents of the invention can also be incorporated into pharmaceutical compositions in accordance with the foregoing description. Treatment regimens for antibody mimetic agents may differ, depending on their size and pharmaceutical properties. Additionally, an antibody mimetic agent of the invention can be used in combination with an antibody of the invention.
  • the antibodies and antibody mimetic agents of this invention are also useful in diagnostic compositions and methods, such as for the diagnosis of diseases involving changes in helper T cell number. For example, they may be used to monitor the course of, or the efficacy of treatment for, HIV infection. Accordingly, this invention encompasses compositions, kits and methods characterized by a diagnostically effective amount of an antibody or antibody mimetic agent according to this invention.
  • a "diagnostically effective amount" of antibody, or mimetic agent thereof refers to an amount sufficient for monitoring the course of HIV infection or the efficacy of treatment for HIV infection.
  • an amount of antibody or mimetic agent sufficient to allow quantitation of CD4 + cell number in a subject or sample obtained from a subject or to determine the quantity of CD4 + cells expressing a conformationally altered form of CD4 on their surface in a subject or sample obtained from a subject.
  • the antibodies of the invention identify a novel conformational form of cell-surface CD4 that can be targeted for diagnostic, screening or therapeutic purposes. Accordingly, in various embodiments, the antibodies of the invention are useful in methods for detecting cells expressing on their surface a conformationally altered form of a human CD4 molecule, for inhibiting infection of a cell by HIV (e.g., by interfering with viral entry into the cell) and for identifying agents that inhibit or induce the formation of this conformationally altered form of CD4 on the cell surface (which agents themselves may be useful in treating HIV infection).
  • the invention provides a method for detecting a cell expressing on its surface a conformationally altered form of a human CD4 molecule induced upon binding of HIV, or an envelope protein thereof (e.g., gpl20) to the cell.
  • This method involves contacting the cell with an antibody of the invention and detecting the antibody bound to the cell surface to thereby detect a conformationally altered form of a human CD4 molecule expressed on the cell surface.
  • Preferred antibodies for use in the method are the monoclonal Fabs 3-47 and 3-51, or an antibody having the same epitope binding specificity as 3-47 or 3-51.
  • the antibody bound to the cell surface may be detected directly, e.g., the antibody can be directly labelled with a detectable substance (e.g., a derivatized antibody of the invention, as described above, labelled with a fluorescent label or a radioisotope), or alternatively, the antibody bound to the cell surface can be detected indirectly, e.g.. using a labelled secondary antibody that recognizes the antibody of the invention. Since the formation of this conformationally altered form of CD4 on the cell surface is thought to be a intermediate step in the mechanism of infection of CD4 + cells by HIV, this detection metho can be used to monitor the progress of HIV infection, e.g., during a therapeutic regimen.
  • a detectable substance e.g., a derivatized antibody of the invention, as described above, labelled with a fluorescent label or a radioisotope
  • the antibody bound to the cell surface can be detected indirectly, e.g.. using a labelled secondary antibody that recognizes the antibody of the invention. Since the formation
  • the invention provides methods for identifying an agent that inhibits or induces formation of a conformationally altered form of a human CD4 molecule induced on a cell surface.
  • CD4 + cells are contacted with a gpl 20 composition and an agent to be tested, and then further contacted with an antibody of the invention (e.g., the monoclonal Fabs 3-47 or 3-51 , or an antibody having the same epitope binding specificity as 3-47 or 3-51). Subsequently, the amount of antibody bound to the cells is determined (e.g., directly or indirectly, as described above).
  • a reduced amount of binding of the antibody to the gpl20-treated cells in the presence of the agent is used as an indicator that the agen inhibits formation of a conformationally altered form of CD4 on the cell surface.
  • CD4 + cells are contacted with an agent to be tested and then further contacted with an antibody of the invention. Subsequently, the amount of antibody bound to the cells is determined (e.g., directly or indirectly, as described above).
  • CD4 + cells for use in these screening assays can be, for example, CD4 + lymphocytes
  • a gpl 20 composition with which the cell is contacted to induce formation of the conformationally altered form of CD4 on the cell surface is preferably recombinant soluble gpl 20, although HIV itself or cells infected with HIV may also be used to induce the conformational alteration of CD4 on the surface of the CD4 + cell.
  • the amount of antibody binding to the surface of the CD4+ cells can be determined by FACS analysis or other suitable assays known in the art.
  • the invention provides a method for inhibiting infection of a CD4 + cell by HIV (or a related CD4-tropic virus such as SIV), involving contacting the cell with an antibody (or antibody mimetic agent) of the invention.
  • Preferred antibodies for use in the method are the monoclonal Fab 3-47 and 3-51, or antibodies having the same epitope binding specificity as 3-47 or 3-51.
  • the antibody is administered to a subject to inhibit infection of cells by HIV in vivo.
  • these antibodies may inhibit a requisite intermediate step in viral infection subsequent to the induction of a conformational change in cell-surface CD4 that is necessary for viral membrane fusion and viral entry into the cell (i.e., antibody binding to the conformationally altered form of CD4 may inhibit subsequent processes necessary for viral infection). Additionally, since the antibodies of the invention do not bind native CD4 on the surface of CD4 + cells but only bind CD4 + cells upon gpl 20 binding, these antibodies are likely to be less immunosuppressive than anti-CD4 antibodies that bind native cell-surface CD4.
  • a cell is contacted with a therapeutically effective amount of antibody that is sufficient to substantially inhibit infection of cells by HIV.
  • substantially inhibition of infection indicates at least an 80 % reduction in the infection of CD4 + cells by HIV in vitro.
  • any indication of HIV infection and/or replication can be monitored.
  • inhibition of HIV infection can be determined by comparing HIV p24 levels in the presence and absence of the antibody in HIV-infected CD4 + cell cultures. HIV p24 levels may be determined, for example, by ELISA, radioimmunoassay or the like.
  • HIV infection e.g., viral entry into a CD4 + cell
  • CD4 + cells can be infected with a recombinant HIV strain, either in the presence or absence of an antibody of the invention.
  • the recombinant HIV strain used in the assay directs the expression of a detectable gene product (e.g., chloramphenicol acetyl transferase (CAT)) in infected cells and carries a deletion in the viral envelope gene.
  • CAT chloramphenicol acetyl transferase
  • Such virions are capable of infecting target CD4 + cells, but do not direct the synthesis of infectious progeny virions as a result of the deletion in the envelope gene in the viral genome:
  • the number of infected cells can be assessed by measuring the amount of detectable gene product (e.g., CAT) synthesized in cells exposed to the virus.
  • detectable gene product e.g., CAT
  • a reduced amount of detectable gene product e.g., reduced CAT activity
  • the antibody blocks e.g., inhibits
  • syncytia formation in the presence of the antibody can be examined.
  • any known syncytia assay may be used.
  • an HIV laboratory isolate or HIV-infected CD4 + tissue culture cells e.g., H9 are added to cultures of C8166 or CEMX174 cells, and varying amounts of the antibody are added to all but the control cultures.
  • the antibody may be assessed for its ability to inhibit syncytia amon tissue culture cells expressing the HIV env gene product (gpl60). Control cultures (negative controls) are supplemented with nothing, or with an irrelevant antibody of the same isotype as the antibody homolog. After incubation, all the cultures are scored by visual inspection f syncytia. In this way, the ability of the antibody to block syncytia formation is evaluated.
  • agents that inhibit or induce expression of a conformationally altered form of CD4 on the surface of a CD4 + cell may have therapeutic utility in treating HIV infection. Accordingly, such agents can be formulated into pharmaceutical compositions and administered to a subject, e.g., to inhibi infection of cells in the subject by HIV.
  • Another aspect of the invention pertains to isolated molecules that express at least on epitope exposed on a conformationally altered form of human CD4 induced on the surface o CD4 + cells upon contact with gpl 20.
  • the altered conformational form is one tha expresses the 3-47 epitope and/or the 3-51 epitope (i.e., the epitopes bound by the monoclonal Fabs 3-47 and 3-51, respectively).
  • the isolated molecule itself expresses the 3-47 epitope and/or the 3-51 epitope.
  • the molecule may express other epitopes that are exposed on the conformationally altered form CD4 induced upon gpl 20 binding.
  • the term "isolated” refers to a molecule substantially free of cellular material or culture medium when purified from a natural source or produced in a host cell by recombinant DNA techniques, or substantially free of chemical precursors or other chemical when chemically synthesized.
  • the isolated molecule is a protein or peptide.
  • the molecule can be a modified human CD4 protein, or peptide fragment thereof, or a non-human CD4 protein, or peptide fragment thereof, such as a non- human primate CD4 protein or peptide (e.g., from a rhesus monkey or chimpanzee).
  • the molecule can be an anti-idiotype antibody, or fragment thereof, that binds monoclonal Fab 3-47 or 3-51 (i.e., the molecule can be an antibody having specificity for the antigenic binding site of the 3-47 or 3-51 Fab).
  • the molecule can be a peptide mimetic.
  • the peptide mimetic may be a semi-peptidic compound or a non-peptidic, organic compound that mimics the conformation of an epitope exposed on the conformationally altered form of CD4 upon gpl 20 binding, e.g., the 3-47 epitope or the 3-51 epitope.
  • a molecule of the invention expressing an epitope exposed on a conformationally altered form of CD4 can be identified, for example, by screening a library of compounds, with an antibody of the invention and selecting a compound(s) that binds the antibody.
  • the library may be composed of, for example, modified (e.g., mutated) human CD4 proteins, non-human CD4 proteins (e.g., non-human primate CD4 proteins), peptides, semi-peptidic compounds or non-peptidic, organic compounds.
  • a random peptide display library is expressed on the surface of a display package, e.g., filamentous phage, and the peptide library is screened with an antibody of the invention.
  • the library can be screened with the monoclonal Fab 3-47 to identify and isolate peptides that bind 3-47 (i.e., express the 3-47 epitope).
  • the library can be screened with the monoclonal Fab 3-51 to identify and isolate peptides that bind 3-51 (i.e., express the 3-51 epitope).
  • Techniques for preparing random peptide display libraries, and screening thereof with antibodies, are known in the art (see e.g., Parmley, S.F. and Smith, G.P. (1988) Gene 22:305-318; Cwirla, S.E. et al. (1990) Proc. Natl. Acad. Sci. USA £2:6378-6382; Devlin, J.J.
  • Antibody-binding peptides selected from the library can be sequenced by standard techniques and the amino acid sequences of the selected peptides then compared to generate a consensus motif that represents a preferred, or optimal, amino acid sequence recognized by the antibody.
  • the consensus motif of an epitope bound by an antibody of the invention can further be used to design other molecules expressing the epitope.
  • a human or non-human CD4 protein, or peptide fragment thereof can be systematically mutated (e.g., by site-directed mutagenesis) to alter the protein or peptide such that it constitutively expresses an epitope bound by the antibody of the invention (i.e., the CD4 protein is altered to express the epitope in the absence of gpl 20 treatment).
  • the molecules of the invention expressing at least one epitope exposed on a conformationally altered form of cell-surface CD4 are useful for generating antibodies of the invention by immunizing a mammal with the molecule. Accordingly, the invention provides a method for producing an antibody that binds an epitope exposed on a conformationally altered form of a human CD4 molecule involving immunizing a mammal with a molecule of the invention described herein.
  • Polyclonal antibodies can be induced by standard techniques and monoclonal antibodies can be prepared and selected as described previously.
  • the molecules of the invention expressing at least one epitope exposed on the conformationally altered form of CD4 can be incorporated into pharmaceutical compositions.
  • compositions typically include at least the epitope- expressing molecule and a pharmaceutically acceptable carrier.
  • the formulation and administration of such compositions is similar to that described above for antibody compositions of the invention.
  • the molecule is included in the composition in an amount effective for inducing an immune response against the molecule (e.g., an antibody response against the molecule) in a subject.
  • the composition is administered intramuscularly via one or, more preferably, several administrations.
  • a pharmaceutically acceptable adjuvant in the composition.
  • Adjuvant is used in its broadest sense and is intended to include any immune stimulating compound.
  • a typical adjuvant used in the art is Freund's incomplete adjuvant.
  • a suitable adjuvant and carrier composition is a muramyl dipeptide derivative and a carrier which includes a detergent and a combination of free fatty acids.
  • suitable adjuvants and carriers for use in the compositions include, for example, ion exchangers, alumina, aluminun stearate, lecithin, serum proteins (such as human serum albumin), buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine, sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances and polyethylene glycol.
  • Adjuvants for topical or gel base forms may be selected from sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene block polymers, polyethylene glycol
  • the ability of the molecules of the invention to induce an antibody response in a subject directed against one or more epitopes exposed on a conformationally altered form of cell-surface CD4 may be exploited in therapeutic methods for inhibiting infection of cells by HIV in a subject. While not intending to be limited by mechanism, it is thought that these molecules may have therapeutic utility by inducing an antibody response that has anti-viral activity in a subject, e.g., the induced antibodies may inhibit a requisite intermediate step necessary for viral entry into cells and thereby inhibit infection of the cell by HIV.
  • the invention provides a method for inhibiting infectio of a cell by HIV in a subject involving administering to the subject a therapeutically effectiv amount of a molecule that expresses at least one epitope exposed on a conformationally altered form of human CD4, such that an antibody response against the epitope(s) expressed by the molecule is induced in the subject.
  • the subject is immunized with a molecule that expresses the 3-47 epitope and/or the 3-51 epitope.
  • the molecule preferably in a pharmaceutical composition as described above (e.g., including an adjuvant), is administered at a dosage and by a route sufficient to induce an antibody response in the subject.
  • the molecule is typically administered intramuscularly, bu may be administered by another suitable route, e.g., subcutaneously, intradermally, intravenously etc.
  • the molecule may be administered at one time or, more preferably, is administered over a series of treatments. The most effective mode of administration and dosage regimen will depend upon the particular composition and/or adjuvant used for treatment, the severity and course of infection, previous therapy, the patient's health status and response to treatment and the judgement of the treating physician.
  • a more highly immunogenic form of compound may require a lower dosage or treatment time, e.g., when an adjuvant is used.
  • a daily dose of about 0.1 to 1.0 mg of active compound per kilogram of body weight is administered to the subject once a day for about 30 days.
  • the subject may require intermittent boosters (e.g., about 0.1 to 1.0 mg/kg body weight on a weekly to monthly basis).
  • antibodies that bind conformationally altered forms of non-human CD4 molecules induced on the surface of non-human cells upon binding of other CD4-tropic viruses to the cells e.g., altered forms of simian CD4 molecules induced upon binding of simian immunodeficiency virus (SIV)
  • SIV simian immunodeficiency virus
  • an SIV-infected rhesus monkey can be immunized with soluble rhesus monkey or human CD4, and antibodies which bind a conformationally altered form of rhesus monkey CD4 induced on the surface of cells upon SIV binding can be selected as described herein.
  • CD4 conformationally altered form of CD4 described herein (e.g., the form of CD4 that expresses the 3-47 and 3-51 epitopes) is induced upon binding of HIV, or an envelope protein thereof, it is possible that the CD4 neo-epitope(s) expressed upon HIV binding are naturally-occurring cryptic epitopes of the CD4 molecule. Accordingly, it may be possible to induce this altered form of CD4 by other mechanisms in addition to HIV binding (e.g., through other proteins with which CD4 naturally interacts).
  • EXAMPLE 1 Elicitation of rsCD4-specif ⁇ c Antibodies by Immunization of HIV-infected Humans with Human rsCD4
  • HIV-infected humans were immunized with recombinant soluble human CD4 (rsCD4) in an adjuvant and the polyclonal anti-CD4 antibody titer was measured to determine whether an anti-CD4 response was elicited by immunization.
  • rsCD4 recombinant soluble human CD4
  • Human recombinant soluble CD4 immunization of human immunodeficiency virus CHTVV infected individuals Four asymptomatic HIV-infected humans with absolute CD4 counts greater than 500 were immunized and boosted intramuscularly five times with 1 mg human recombinant soluble CD4 (rsCD4) (Biogen, Inc., Cambridge, MA) in incomplete Freund's adjuvant. Determination of rsCD4-specific antibody titers in the serum of rsCD4-immunized. HIV- infected humans
  • the anti-CD4 antibody titer of the four immunized individuals is shown in Figure 1.
  • Antibodies in the serum of all four rsCD4-immunized individuals could be shown to bind to human rsCD4 by standard ELISA. However, these antibodies were present at a relatively low titer. The elicitation of these antibodies by immunization with a self protein suggests that epitopes to which these individuals were not tolerant were presented to the immune system by the human rsCD4 molecule.
  • the bone marrow cells used as a source of mRNA for immunoglobulin gene amplification were obtained from this individual 200 days after the initial immunization, at the time of peak rsCD4-specific antibody titer.
  • a combinatorial immunoglobulin library of 3 x 10" members was expressed in the Ml 3 bacteriophage vector pCOMB3.
  • Fabs are expressed on the surface of recombinant Ml 3 bacteriophage particles through the expression of the heavy chain as a fusion protein with the Ml 3 coat protein, gene III. Fab clones of a given antigen specificity can then be selected by panning these recombinant bacteriophage over the antigen of interest.
  • the following methodology was used to produce the combinatorial immunoglobulin library:
  • heparinized bone marrow cells Twenty ml of heparinized bone marrow cells were harvested from rsCD4 immunized, HIV-infected individuals at the time of peak demonstrable rsCD4-specific antibody titer (200 days after the initial immunization) (see Figure 1). Lymphocytes were isolated from these samples by Ficoll-diatrizoate density gradient centrifugation, rapidly frozen on dry ice, and stored at -70 degrees C prior to RNA extraction.
  • Immunoglobulin heavy chain cDNA was then synthesized using primers specific for the third constant domain of human heavy chains.
  • Immunoglobulin light chain cDNA was similarly synthesized using primers corresponding to the constant domains of human kappa and lambda light chains.
  • the nucleotide sequences of primers used for cDNA synthesis and PCR amplification of immunoglobulin heavy and light chain DNA are shown below (restriction sites incorporated into primers for cloning purposes are underlined):
  • VHA ag gtg cag ctg etc gag tct gg (SEQ ID NO: 1 )
  • VHC ag gtg caa ttg &c_gag tct gg (SEQ ID NO: 3)
  • VHD ag gtg caa ctg etc gag teg gg (SEQ ID NO: 4)
  • VHE ag gtg cag eta &c_gag teg gg (SEQ ID NO: 5)
  • VHF ag gta cag ctg etc gag tea gg (SEQ ID NO: 6)
  • VKl gt gec aga tgt gag_£_£ gtg atg ace cag tct cca (SEQ ID NO: 7)
  • VK2 gt gec aga tgt gag etc gtg ttg acg cag tct cca (SEQ ID NO: 8)
  • VL1 c tgc aca ggg tec tgg gec gag etc gtg ttg acg ca (SEQ ID NO: 9)
  • VL2 c tgc aca ggg tec tgg gec gag etc ata ctg acg ca (SEQ ID NO: 10)
  • CH 1 age ate act agt aca aga ttt ggg etc (SEQ ID NO: 11 )
  • the reverse transcriptase was inactivated by heating the reaction to 65 degrees C for twenty minutes.
  • the resulting cDNA was stored at -20 degrees C prior to PCR amplification. Two rounds of PCR amplification were employed to obtain sufficient quantities of immunoglobulin heavy chain material for cloning.
  • the first round PCR reaction contained 20 ⁇ l heavy chain specific cDNA, 2.5 U Pfu polymerase (Stratagene, La Jolla, CA) Pfu buffer II, 0.2 mM dNTPs (Promega, Madison, Wisconsin), 10 ng heavy chain CH3 constant region primer (SEQ ID NO: 12; Operon, Alameda, CA), 10 ng of one of six variable region primers corresponding to six heavy chain families (SEQ ID NOs: 1-6; Operon. Alameda, CA), and RNase-free water (United States Biochemical, Cleveland, Ohio) to make a final volume of 100 ⁇ l. Twenty-five amplification cycles, utilizing the hot start technique, followed by a 72 degrees C IO minute final extension, were performed, using the following conditions: 94 degrees C for 1.5 minutes, 52 degrees C for 2.5 minutes, 72 degrees C for 3 minutes.
  • One microliter of the first round PCR product was then exposed to twenty-five identical cycles of amplification under the same conditions except substituting the heavy chain CH 1 constant region primer (SEQ ID NO: 11 ) for the CH3 constant region primer (SEQ ID NO: 12).
  • Immunoglobulin light chain DNA was amplified by 35 cycles identical to those described above, except using primers corresponding to the first constant domain of kappa or lambda light chains (SEQ ID NO: 13 or 14), as well as one of four variable region primers specific for different kappa and lambda light chain families (SEQ ID NOs: 7-10).
  • PCR amplified heavy and light chain DNAs were purified from a 1.5 % agarose gel using the Sephaglas BandPrep kit (Pharmacia, Piscataway, NJ). Equal amounts of material from each light chain family were then pooled and digested with the enzymes Xba I and Sac I (Boehringer Mannheim, Indianapolis, IN).
  • the digested material was gel purified as above and ligated with Xba I/Sac I digested pCOMB3 vector (TSI, La Jolla, CA) (illustrated in Figure 2) overnight at 16 degrees C at a 2:1 insert: vector molar ratio in a 150 ⁇ l reaction containing T4 DNA ligase buffer and 10 U T4 DNA ligase (Gibco BRL, Gaithersburg, MD).
  • the ligation products were transformed into electrocompetent XL 1 -blue cells (Stratagene, La Jolla, CA) by electroporation.
  • Recombinant plasmid DNA was isolated from this overnight culture using the Wizard Magic Miniprep Kit (Promega, Madison, WI). Recombinant DNA was then digested with the restriction enzymes Xho I and Spe I (Boehringer Mannheim, Indianapolis, IN), gel purified, and ligated overnight at 16 degrees C with pooled Xho I/Spe I digested, gel purifie light chain PCR products using a 1.6:1 insert:vector ratio.
  • Recombinant Ml 3 bacteriophage were precipitated on ice for one hour in the presenc of four percent polyethylene glycol-8000 (Sigma, St. Louis, MO) and 3 % sodium chloride (Sigma, St. Louis, MO). Precipitated phage were pelleted at 9000 rpm for twenty minutes at 4 degrees C. Finally, the phage pellet was resuspended in 2 ml PBS and stored at -20 degrees C.
  • the human combinatorial immunoglobulin library produced as described in Example 2, was screened for CD4-specific antibodies.
  • Two panning strategies were employed to select CD4-specific Fabs from this immunized individual. In the first strategy, the library was panned against human rsCD4. In the second strategy, the library was panned against preformed CD4/gpl20 complexes captured onto ELISA wells with the anti-CD4 monoclonal antibody 5D4. Clones of interest selected by each of the panning procedures were then converted to soluble Fab expressing clones to facilitate the characterization of thei binding specificity by ELISA. Clones that bound to CD4 but not gpl 20 by ELISA were the selected for further characterization. The following methodology was used: Eanning
  • Unbound rsCD4 was removed by performing three 350 ⁇ l washes with sterile double-distilled water. The wells were again blocked at 37 degrees C for one hour with 350 ⁇ l 3% BSA PBS. After shaking out the block solution, 100 ⁇ l Ml 3 combinatorial phage (10 ⁇ 2 pfu) were added to each well and incubated for two hours at 37 degrees C. Nonadherent phage were removed and each well was washed one time with sterile double-distilled water. Each well was then washed ten times over a period of one hour at room temperature with 350 ⁇ l TBS/0.5 % Tween-20.
  • phage elution buffer 0.1 M HC1, 1 mg/ml BSA-pH 2.2 with glycine. The elution was allowed to proceed for ten minutes at room temperature. The solution was then pipetted up and down several times, transferred to a sterile tube, and neutralized with 6 ⁇ l 2 M Tris base per 100 ⁇ l eluted phage. The panned phage were titered and stored at -20 degrees C in preparation for amplification and further rounds of panning.
  • the panning procedure was repeated until enrichment for antigen-specific clones was achieved, as determined by the percent yield phage (the number of phage eluted divided by the number of phage applied multiplied by 100) after each amplification/panning cycle.
  • Amplification of panned bacteriophage Eluted phage were incubated with 2 ml of an .0 XL 1 -blue culture for fifteen minutes at room temperature. The cells were then grown in a 37 degree C shaking incubator for one hour in ten ml superbroth, 20 ⁇ g/ml carbenicillin, 10 ⁇ g/ml tetracycline. These cells were then grown for an additional hour in a 100 ml volume of superbroth, 50 ⁇ g/ml carbenicillin, 10 ⁇ g/ml tetracycline.
  • the cells were infected with 10 pfu of the Ml 3 helper phage VCSM 13 to direct the assembly of Fab-expressing Ml 3 bacteriophage. Finally, cells which had been infected with the helper phage were selected by growing the culture overnight in the presence of 70 ⁇ g/ml kanamycin.
  • Plasmid DNA was isolated from 3 mL overnight cultures of individual panned clones using the Wizard Magic Miniprep Kit (Promega, Madison, WI). This DNA was digested with Spe I and Nhe I to remove gene III (see Figure 2). The remaining 4.7 Kb vector fragment was then gel purified. Because the Spe I and Nhe I restriction sites are compatible, recircularized plasmid was made by ligating this vector overnight at 16 degrees C. The ligation product was transformed into competent XL 1 -blue cells in preparation for the induction of Fab expression.
  • Combinatorial clones were inoculated into 500 ml superbroth, 50 ⁇ g/ml carbenicillin, 20 mM MgCl2 and grown at 37 degrees C in a shaking incubator to an OO ⁇ QQ/T ⁇ ] 1.0. Soluble Fab expression was then induced by growing these cultures at 30 degrees C overnigh in the presence of 1 mM IPTG (Stratagene, La Jolla, CA) and 4 nM dibutyryl cAMP (Sigma, St. Louis, MO).
  • lysozyme 4 mg/ml lysozyme, immediately followed by 160 ml osmotic shock solution B (50 mM Tris- HC1, pH 8.6, 250 mM sucrose, 0.25 mM EDTA, 2.5 mM MgCl2). After a ten minute incubation on ice, bacterial debris was pelleted by centrifuging the lysate in 35 ml Oakridge tubes at 12,500 rpm for five minutes at 4 degrees C. After transferring the supernatant to a new Oakridge tube, the protease inhibitor AEBSF (Calbiochem, San Diego, CA) was added to a final concentration of 1 mM.
  • AEBSF protease inhibitor
  • the periplasmic extracts of induced cells were applied to goat anti-human F(ab')2 affinity columns.
  • the affinity columns were prepared by incubating 4 mg goat anti-human F(ab')2 (Jackson Immunoresearch, West Grove, PA) with 2 ml Gammabind G Sepharose beads (Pharmacia, Piscataway, N.J.) for one hour at room temperature on a rocking platform. After washing the conjugated beads four times with ten ml 0.2 M sodium borate, pH 9.0, the beads were incubated on a rocker for 1.5 hours with the coupling reagent DMP at a concentration of 20 mM in 0.2 M sodium borate, pH 9.0.
  • the coupled beads were loaded onto Econo-Pac columns (Bio-Rad, Hercules, CA), and sodium azide was removed by washing the column several times with PBS.
  • the column was then equilibrated with 10 column volumes elution buffer (3.5 M sodium thiocyanate) to remove uncoupled IgG.
  • periplasmic extract isolated from a 500 ml induced culture was applied to the column at 4 degrees C. Nonspecifically bound proteins were washed off with 200 ml PBS/1 mM AEBSF (Calbiochem, San Diego, CA).
  • bound Fab was eluted with eight column volumes 3.5 M NaSCN, and the samples were desalted using Centriprep 30 ultrafiltration devices (Amicon, Beverly, MA).
  • Wells of a 96 well Maxisorp plate were coated overnight at 4 degrees C with 1 ⁇ g of the antigen of interest in a 150 ⁇ l volume. After washing eight times with TBS, the wells were blocked for one hour at 37 degrees C with 0.5 % nonfat dry milk/0.05 % Tween- 20/TBS. Blocking solution was then removed, and the Fabs were incubated in the appropriate wells for one hour at 37 degrees C. Unbound Fab was removed by washing eight times with TBS.
  • the library was enriched three-fold for rsCD4- specific clones, as determined by the percent yield of phage (the number of phage eluted divided by the number of phage applied, multiplied by 100). Fifty five clones from this enriched library were converted to soluble Fab expressing clones to facilitate the characterization of their specificity by ELISA. Nine of these clones demonstrated strong reactivity with human rsCD4, but not with recombinant gpl20, by ELISA. The ELISA results for a representative clone, 2-6, are shown below in Table 1.
  • the library was panned against preformed CD4/gpl20 complexes captured onto ELISA wells with the anti-CD4 monoclonal antibody 5D4. After three rounds of panning, the library was enriched two-fold for CD4/ gpl20-specific clones. Thirty-five clones from this enriched library were converted to soluble Fab expressing clones. Nine of these clones were shown to react with human rsCD4, but not with rgpl20, by ELISA. The ELISA results for two representative clones, 3-47 and 3-51 , are shown below in Table 1.
  • the binding characteristics of the fourteen CD4-specific antibodies described in Example 3 were examined.
  • Fab binding to CD4 on the surface of PBLs was assessed by incubating the cells with the antibody, followed by a fluorescein isothiocyanate (F ⁇ TC)-labelled goat anti- human F(ab')2 secondary antibody. Bound antibody was then detected by FACS analysis.
  • F ⁇ TC fluorescein isothiocyanate
  • Human peripheral blood lymphocytes were isolated from 10 ml heparinized blood by Ficoll diatrizoate density gradient centrifugation. The cells were preincubated at 37 degrees C for one hour with either PBS or 35 ⁇ g/ml recombinant gpl 20 (HIV-1 g 2 strain) (kindly provided by Chiron, Emeryville, CA). After washing, the cells were incubated for twenty minutes on ice with the Fab or control antibody at a concentration of 2 ⁇ g/ml. 19thy5D7 is a CD4 domain one-specific antibody, whereas OKT4 recognizes CD4 domain three. L736523 is a gpl 20 specific antibody.
  • the cells were stained at 4 degrees C for twenty minutes with a 1 :50 dilution of either FITC -conjugated F(ab * )2 goat anti-human F(ab')2 (Jackson Immunoresearch, West Grove, PA) or FITC-conjugated F(ab')2 goat anti- mouse F(ab')2(Jackson Immunoresearch, West Grove PA).
  • FITC -conjugated F(ab * )2 goat anti-human F(ab')2 Jackson Immunoresearch, West Grove, PA
  • FITC-conjugated F(ab')2 goat anti- mouse F(ab')2 Jackson Immunoresearch, West Grove PA
  • FIG. 3 Representative results of the surface staining experiments are shown in the series of flow cytometric profiles depicted in Figure 3, panels A-J.
  • panels A-E human peripheral blood lymphocytes preincubated with PBS were stained with various antibodies.
  • panels F- J human peripheral blood lymphocytes preincubated with recombinant gpl 20 were stained with various antibodies.
  • Figure 3 depicts cell staining with the following antibodies: control FITC-labelled goat anti-human secondary antibody (panels A and F), 19thy5D7 (specific for the gpl 20 binding site of CD4) (panels B and G), L736523 (specific for the V3 loop domain of gpl 20) (panels C and H), Fab clone 3-47 (panels D and I) and Fab clone 3-51 (panels E and J).
  • Peripheral blood lymphocytes were isolated from seventy ml heparinized human blood by Ficoll diatrizoate gradient centrifugation. After washing with PBS, the cells were lysed in Triton X-100 lysis buffer (300 mM NaCl, 50 mM Tris-HCL, pH 7.6, 0.5 % Triton XI 00, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 1 mM PMSF, 1.8 mg/ml iodoacetamide) on ice for forty-five minutes with occasional mixing.
  • Triton X-100 lysis buffer 300 mM NaCl, 50 mM Tris-HCL, pH 7.6, 0.5 % Triton XI 00, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 1 mM PMSF, 1.8 mg/ml iodoacetamide
  • the gel was run at 100V for approximately six hours before transferring the proteins overnight at 4 degrees C in transfer buffer (50 mM Tris base, 380 mM glycine, 0.1 % SDS, (20 % methanol) to a nitrocellulose membrane (Stratagene, La Jolla, CA) in a Transphor Transfer eiectrophoresis TE 42 unit (Hoefer Scientific Instruments, San Francisco, CA).
  • transfer buffer 50 mM Tris base, 380 mM glycine, 0.1 % SDS, (20 % methanol
  • the membrane was air dried and blocked in TBST (100 mM Tris-HCl, pH 7.5, 0.9 % NaCl, 0.1 % Tween 20) for two hours with gentle rocking. The membrane was cut into strips and probed for one hour at room temperature with gentle rocking with the appropriate antibody or Fab diluted to a final concentration of 2 ⁇ g/ml in TBST.
  • TSS Tris-buffered saline
  • HRP horseradish peroxidase
  • F(ab')2 goat anti-human F(ab')2
  • Bound Fab was detected using ECL chemiluminescent Western blotting detection reagents (Amersham) according to the manufacturer's instruction.
  • FIG. 4 illustrates the reactivity of an irrelevant gpl 20-specific antibody (L736523) with human rsCD4 (lane 1 ), a CD4- specific control antibody (humanized 5A8) with human rsCD4 (lane 2), the monoclonal Fab 3-47 with rsCD4 (lane 3), the gpl20-specific antibody (L736523) with a human PBL lysate (lane 4) and the monoclonal Fab 3-47 with a human PBL lysate (lane 5).
  • the data demonstrate that clone 3-47 specifically recognizes denatured rsCD4 (lane 3).
  • this Fab clone recognizes a 60 kDa protein, corresponding to the molecular weight of the
  • CD4 molecule from a human PBL lysate (lane 5). These results confirm the CD4 specificity of Fab clone 3-47, and in combination with the cell staining data described above, suggest that Fab clone 3-47 recognizes an epitope exposed on a conformationally altered form of the CD4 molecule exposed upon gpl20 binding to CD4. Thus, Fab clone 3-47 provides direct evidence for an HIV-induced conformational change in the CD4 receptor. The ability of the HIV envelope protein to induce this conformational change in CD4 suggests that HIV entry involves not only envelope binding to the first domain of CD4, followed by fusion with the cell membrane, but a series of events involving multiple regions of the CD4 receptor.
  • the biotinylated H9 cell pellet was lysed in Triton X-100 lysis buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.6, 0.5 % Triton X-100, 100 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 1 mM PMSF, 1.8 mg/ml iodoacetamide) on ice for 45 minutes with occasional mixing. After pelleting cell debris at 12,500 rpm for 15 minutes at 4 °C in a microcentrifuge, the supernatant was transferred to a 1.5 ml eppendorf tube.
  • Triton X-100 lysis buffer 300 mM NaCl, 50 mM Tris-HCl, pH 7.6, 0.5 % Triton X-100, 100 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 1 mM PMSF, 1.8 mg/ml iod
  • the lysate was incubated for 1 hour at 4 °C with 300 ⁇ l of a 50 % suspension of Gammabind G beads (Pharmacia) in PBS with gentle agitation. These samples were centrifuged at 12, 5000 rpm for 1 minute in a microcentrifuge. The precleared lysate was then incubated overnight with Fab 3-47 or a negative control antibody (the gpl20-specific monoclonal antibody L736523) at a concentration of 15 ⁇ g/ml.
  • Fab 3-47 or a negative control antibody the gpl20-specific monoclonal antibody L736523
  • the samples were incubated with 200 ⁇ l of a 75 % suspension of goat anti-human IgG, F(ab')2-conjugated Gammabind G beads in PBS for 1 hour at 4 °C with shaking.
  • the beads were then pelleted as described above, washed three times with high salt wash buffer (0.5 M NaCl, 20 mM Tris-HCl, 1 mM EDTA, 1 % Na-Deoxycholate, 0.5 % NP-4, 30 % Sucrose), followed by two washes with low salt wash buffer (10 mM NaCl, 10 mM Tris-HCl, pH 7.6).
  • Immunoprecipitated proteins were eluted from the beads in nonreducing sample buffer (60 mM Tris-HCl, pH 6.8, 25 % glycerol, 2 % SDS, 0.1 % bromophenol blue) at 95 ° for 10 minutes and loaded onto a 10 % SDS-polyacrylamide gel. After eiectrophoresis, these proteins were transferred overnight to nitrocellulose.
  • sample buffer 60 mM Tris-HCl, pH 6.8, 25 % glycerol, 2 % SDS, 0.1 % bromophenol blue
  • the membrane was blocked with 5 % bovine serum albumin (BSA)/TBST (100 mM Tris-HCl, pH 7.5, 0.9 % NaCl, 0.1 % Tween- 20) and probed with 2 ⁇ g/ml horse radish peroxidase-conjugated avidin (Pierce)/0.3 % BSA/TBST to detect biotinylated proteins. After extensive washing with TBST, HRP- avidin-bound, biotinylated proteins were visualized suing the ECL detection system (Amersham).
  • BSA bovine serum albumin
  • TST 100 mM Tris-HCl, pH 7.5, 0.9 % NaCl, 0.1 % Tween- 20
  • full- length and truncated CD4 molecules were used in enzyme linked immunosorbent assays (ELISAs) with Fab 3-47 and control antibodies.
  • ELISAs enzyme linked immunosorbent assays
  • One ⁇ g of full-length human rsCD4 (comprising amino acids 1-371) or truncated human rsCD4 (comprising amino acids 1-183, corresponding to the VI and V2 domains) was absorbed onto the wells of a Maxisorp plate overnight at 4 °C. After washing eight times with Tris-buffered saline (TBS), the wells were blocked for one hour at 37 °C with 0.5 % nonfat dry milk/0.05 % Tween-20/TBS.
  • TBS Tris-buffered saline
  • Blocking solution was then removed and the Fab fragments were incubated in the appropriate wells for one hour at 37 °C. Unbound Fab was removed by washing eight times with TBS. Bound Fab was then detected by incubating the wells for one hour at 37 °C with a 1 :50,000 dilution of horse radish peroxidase-conjugated goat anti-human IgG F(ab')2 (Jackson Immunoresearch, West Grove, PA). After eight TBS washes, TMP one component substrate solution (KPL, Gaithersburg, MD) was added. The reactions were terminated after twenty minutes by the addition of 2/3 N H2SO4.
  • Fab 3-47 or various control antibodies were incubated with human peripheral blood lymphocytes (PBLs) that had been preincubated with HIV-1 recombinant gpl 20 (rgpl20) at either 4 °C or 37 °C.
  • PBLs peripheral blood lymphocytes
  • the controls included a positive control anti-gpl20 monoclonal antibody (L736523) , a negative control Fab (2-36) and a no antibody control (i.e., PBS alone).
  • Panels A-D represent human PBLs preincubated with rgpl20 at 4 °C.
  • Panels E-H represent human PBLs preincubated with rgpl20 at °37 C.
  • Cells in panels A and D were incubated with PBS alone.
  • Cells in panels B and F were incubated with the control anti-gpl20 antibody.
  • Cells in panels C and G were incubated with the control Fab 2-36.
  • Cells in panels D and H were incubated with Fab 3-47.
  • H9 cells were incubated overnight at 37 C with infectious recombinant (20 ng p24 per 1.5 x 106 cells) in the presence of either Fab 3-47, a positive control anti-CD4 monoclonal antibody (Hu5 A8, a humanized antibody that recognizes domain 2 of CD4) or a negative control Fab (2-36), each at a concentration of 2 ⁇ g/ml.
  • Fab 3-47 a positive control anti-CD4 monoclonal antibody
  • Human5 A8 a humanized antibody that recognizes domain 2 of CD4
  • a negative control Fab (2-36 negative control Fab
  • Panel A represents the PBS control.
  • Panel B represents the cell staining with the positive control anti-CD4 antibody (Hu5A8).
  • Panel C represents cell staining with the negative control Fab 2-36.
  • Panel D represents cell staining with Fab 3-47. The results demonstrate that Fab 3-47 binds to H9 cells preincubated with live HIV-1, indicating the preincubation of H9 cells with live HIV-1 leads to the formation o the conformationally altered form of CD4 that is recognized by Fab 3-47.
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO: 1 : AGGTGCAGCT GCTCGAGTCT GG 22 (2) INFORMATION FOR SEQ ID NO:2:
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO:2 :
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO: 5 :
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 7:
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO:12 : CTCAGTATGG TGGTTGTGC 19 (2) INFORMATION FOR SEQ ID NO:13:
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE cDNA
  • TCCTGTGCAG CCTCTGGATT CACTTTCAGT AGCTACGGCA TGCATTGGGT CCGCCAGNCT 120 CCAGGCAAGG GGCTGGAGTG GGTGGCAGTT ATATCAAATG ACGGAAAAGA TTAGATATAT 180 GCAGACTCGG T

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  • Chemical & Material Sciences (AREA)
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Abstract

Cette invention se rapporte à des anticorps qui se fixent à une forme de CD4 modifée dans sa conformation, induite sur la surface d'une cellule CD4+, par contact de ladite cellule avec le virus de l'immunodéficience humaine (VIH) ou avec une protéine enveloppe de ce virus (par exemple gp120), mais qui ne se lient pas à la forme native de CD4 sur la surface d'une cellule avant d'entrer en contact avec le VIH ou avec une protéine enveloppe de ce virus. Les anticorps préférés de cette invention sont les fragments Fab monoclonaux 3-47 et 3-51. Des compositions pharmaceutiques ainsi que des procédés de diagnostic, de triage et de thérapie utilisant ces anticorps sont également décrits. Les anticorps de cette invention servent à détecter la présence d'une forme de CD4 à conformation modifée sur la surface d'une cellule, à identifier des agents qui inhibent ou qui induisent la production de cette forme de CD4 à conformation modifée après fixation de la protéine gp120, ou à inhiber l'infection de cellules CD4+ par le VIH. Des molécules qui expriment au moins un épitope exprimé sur une forme de CD4 à conformation modifiée, induites sur la surface d'une cellule CD4+, après contact de la cellule avec le VIH, ou avec une protéine enveloppe de ce virus, font également l'objet de cette invention. De telles molécules peuvent être utilisées pour produire des anticorps de cette invention ou pour induire chez un sujet une réponse par anticorps qui inhibe l'infection des cellules de ce sujet par le VIH.
EP95928088A 1994-07-19 1995-07-19 Anticorps se fixant a une molecule cd4 a conformation modifiee, induite par fixation du virus de l'immunodeficience humaine Withdrawn EP0774000A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US27708094A 1994-07-19 1994-07-19
US277080 1994-07-19
US30590394A 1994-09-13 1994-09-13
US305903 1994-09-13
PCT/US1995/009114 WO1996002647A1 (fr) 1994-07-19 1995-07-19 Anticorps se fixant a une molecule cd4 a conformation modifiee, induite par fixation du virus de l'immunodeficience humaine

Publications (1)

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EP0774000A1 true EP0774000A1 (fr) 1997-05-21

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EP95928088A Withdrawn EP0774000A1 (fr) 1994-07-19 1995-07-19 Anticorps se fixant a une molecule cd4 a conformation modifiee, induite par fixation du virus de l'immunodeficience humaine

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EP (1) EP0774000A1 (fr)
JP (1) JPH10505490A (fr)
AU (1) AU3196895A (fr)
CA (1) CA2195238A1 (fr)
WO (1) WO1996002647A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7041871B1 (en) 1995-10-10 2006-05-09 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US6300129B1 (en) * 1990-08-29 2001-10-09 Genpharm International Transgenic non-human animals for producing heterologous antibodies
WO1999015897A1 (fr) * 1997-09-19 1999-04-01 Chiron Corporation Procede de criblage soustractif de proteines servant a identifier des genes
JP5514539B2 (ja) 2006-03-31 2014-06-04 メダレックス・リミテッド・ライアビリティ・カンパニー ヒト抗体の調製に用いるためのキメラ抗体を発現するトランスジェニック動物

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0550516A1 (fr) * 1990-09-28 1993-07-14 Hospital For Joint Diseases Procede inhibant l'action infectieuse du virus de l'immunodeficience humaine
CA2152915A1 (fr) * 1992-12-31 1994-07-21 Jonathan M. Gershoni Anticorps diriges contre des epitopes liants-associes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9602647A1 *

Also Published As

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CA2195238A1 (fr) 1996-02-01
JPH10505490A (ja) 1998-06-02
WO1996002647A1 (fr) 1996-02-01
AU3196895A (en) 1996-02-16

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