This invention provides an antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1. This antibody is then used to identify a molecule which is important for HIV infection. Different uses of the antibody and the molecule are described.

DETAILED DESCRIPTION OF THE INVENTION

Murine hybridomas PA-6 and PA-7, secreting monoclonal antibodies PA-6 and PA-7, respectively, were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on The International Recognition Of The Deposit Of Microorganisms For The Purposes Of Patent Procedure (the "Budapest Treaty") with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, on Mar. 24, 2005 under ATCC Accession Nos. PTA-6637 (PA-6) and PTA-6638 (PA-7), respectively.

This invention provides an antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1. In an embodiment, the antibody is a monoclonal antibody. This invention also provides a hybridoma cell line producing the monoclonal antibody.

In another embodiment, the antibody is a chimeric monoclonal antibody. In a separate embodiment, the antibody is a humanized monoclonal antibody. In a still separate embodiment, the antibody is a human monoclonal antibody.

For the purposes of this invention, a "chimeric" monoclonal antibody is a murine monoclonal antibody comprising constant region fragments (F.sub.c) from a different species. In a preferred embodiment of this invention, the chimeric monoclonal antibody comprises human F.sub.c and murine F.sub.ab. For the purposes of this invention, a "humanized" monoclonal antibody is a murine monoclonal antibody in which human protein sequences have been substituted for all the murine protein sequences except for the murine complementarity-determining regions (CDR) of both the light and heavy chains.

This invention also provides single chain antibodies or an antigen binding antibody fragment capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1. The fragments include but are not limited to Fab and Fab'. The methods to generate the single chain antibodies and antigen binding antibody fragments with particular binding activities are well-known in the art (See for example, Crawley, P (1995) Antibody Patents, in Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc. NY, pages 299 335).

This invention also provides a monoclonal antibody capable of competitively inhibiting the binding of the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1 to its target molecule.

In one embodiment of this invention, the monoclonal antibody is labelled with a detectable marker, for example, a radioactive isotope, enzyme, dye or biotin. This invention provides a pharmaceutical composition comprising the complete or a portion of the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1 and a pharmaceutically acceptable carrier.

For the purposes of this invention "pharmaceutically acceptable carriers" means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, emulsions such as oil/water emulsion, and various type of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

The monoclonal antibodies described and claimed herein are useful for isolating the compound to which the monoclonal antibodies bind.

This invention provides a method of inhibiting HIV-1 infection in a subject comprising administering an effective amount of the above-described pharmaceutical composition to the subject.

This invention provides an isolated nucleic acid molecule encoding the complete or a portion of the light chain of the monoclonal antibody. In one embodiment of this invention, the nucleic acid molecule is a DNA molecule. In another embodiment, the DNA molecule is a cDNA molecule. In a separate embodiment, the DNA molecule is a genomic DNA molecule.

This invention provides an isolated nucleic acid molecule encoding the complete or a portion of the heavy chain of the monoclonal antibody. In one embodiment of this invention, the nucleic acid molecule is a DNA molecule. In another embodiment, the DNA molecule is a cDNA molecule. In a separate embodiment, the DNA molecule is a genomic DNA molecule.

This invention provides an isolated nucleic acid molecule encoding the above-described single chain antibody.

The DNA molecules of the subject invention also include DNA molecules coding for polypeptide analogs, fragments or derivatives of polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all of the properties of the naturally-occurring forms. These include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.

The nucleic acid sequences described and claimed herein are useful for generating new viral and circular plasmid vectors described below.

This invention provides the vector, for example a plasmid or a viral vector, comprising a nucleic acid molecule encoding the complete or a portion of light chain protein of the monoclonal antibody operably linked to a promoter of RNA transcription. This invention also provides a vector, for example a plasmid or a viral vector, comprising a nucleic acid molecule encoding the complete or a portion of heavy chain protein of the monoclonal antibody operably linked to a promoter of RNA transcription.

This invention also provides a vector comprising a nucleic acid molecule encoding the above-described single chain antibody operably linked to a promoter of RNA transcription.

This invention also provide a vector comprising the nucleic acid molecules encoding the complete or a portion of light chain protein and the complete or a portion of heavy chain protein of the monoclonal antibody each operably linked to a promoter of RNA transcription.

This invention also provide a host vector system comprising one or more vectors which comprise either the complete or portion of the light chain or a complete or portion of the heavy chain or a combination thereof in a suitable host cell.

This invention further provides the above host vector system, wherein the suitable host cell is selected from a group consisting of a bacterial cell, an insect cell, a yeast cell or a mammalian cell.

This invention provides the molecule specifically recognized by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1. In an embodiment, the molecule is a glycolipid. In another embodiment, the molecule is a polypeptide.

This invention also provides an isolated nucleic aced molecule encoding the complete or a portion of the polypeptide which is specifically recognize by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1.

This invention provides a multichain polypeptide molecule comprising the polypeptide which is specifically recognized by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1.

This invention also provides an isolated nucleic acid molecule encoding the complete or a portion of a polypeptide of the above-described multichain polypeptide molecule. This invention also provides vectors comprising the nucleic acid molecule encoding the complete or a portion of a polypeptide of the above-described multichain polypeptide molecule operably linked to a promoter of RNA transcription. This invention also provides vectors comprising the nucleic acid molecule encoding the complete or a portion of the above polypeptide molecule operably linked to a promoter of RNA transcription.

This invention also provides a host vector system comprising the above vectors in a suitable host cell.

The suitable host cell includes but is not limited to a bacterial cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.

The above-described molecules specifically recognized by the antibody may be used to vaccinate healthy subjects for prevention or therapy of HIV infection. These molecules are also useful in identifying specific sites to which these molecules bind on the HIV-1 envelope glycoproteins gp120 and gp41. The sites revealed may be used to generate more specific antibodies directed to these sites. Moreover, this information may be used to design drugs which inhibit the binding of the HIV-1 envelope glycoproteins gp120 or gp41 to the molecule.

This invention also provides a soluble protein which comprises a portion of the polypeptide or the multichain polypeptide molecule.

For the purposes of this invention, a "soluble protein" is a protein free of cell membranes and other cellular components. In one embodiment of this invention, the soluble protein is labelled with a detectable marker, for example, a radioactive isotope, enzyme, dye or biotin. The soluble protein is valuable as a product for making a new and useful pharmaceutical composition.

This invention also provides a pharmaceutical composition comprising an effective amount of the above soluble protein to inhibit HIV-1 infection and a pharmaceutically acceptable carrier.

Methods of determining an "effective amount" are well known to those skilled in the art. Simple titration experiments using different amounts of the soluble protein administered to different animal models of HIV-1 infection or to HIV-1 infected human subjects may be performed to determine such an effective amount. The amount administered to the animal model of HIV-1 infection or HIV-1 infected humans which results in a reduction in HIV-1 infection is an effective amount.

This invention also provides a method of inhibiting HIV-1 infection in a subject comprising administering an effective amount of the pharmaceutical composition of the above soluble protein to the subject.

For the purposes of this invention, "administration" means any of the standard methods of administering a pharmaceutical composition known to those skilled in the art. Examples include, but are not limited to, intravenous, intraperitoneal or intramuscular administration.

This invention provides an isolated nucleic acid molecule encoding the soluble protein. In one embodiment of this invention, the nucleic acid molecule is a DNA molecule. Preferably, the DNA molecule is a cDNA molecule.

The nucleic acid sequences which encode the soluble protein are useful for generating new viral and circular plasmid vectors described below. The nucleic acid molecules are valuable in a new and useful method of gene therapy, i.e., by stably transforming cells isolated from a patient with the nucleic acid molecules and then readministering the stably transformed cells to the patient. Methods of isolating cells include any of the standard methods of withdrawing cells from an animal. Suitable isolated cells include, but are not limited to, bone marrow cells. Methods of readministering cells include any of the standard methods of readministering cells to a patient. U.S. Pat. No. 5,366,346, entitled, "Gene Therapy" describes different gene therapy procedures, the contents of which are incorporated herein by reference.

This invention also provides a vector, for example, a plasmid vector or a viral vector, comprising the isolated nucleic acid molecule operably linked to a promoter of RNA transcription.

The vectors described and claimed herein are valuable as products useful for generating stably transformed eukaryotic host cells, and thereby in new and useful methods of growing such host cells under conditions suitable for the production of the soluble protein.

This invention further provides a host vector system comprising the vector having the sequence which encodes the soluble protein in a suitable host cell. In one embodiment of this invention, the suitable host cell is a stably transformed eukaryotic cell, for example, a stably transformed eukaryotic yeast or mammalian cell. Preferably, the stably transformed cell is a mammalian cell.

The host vector system is valuable as a product useful for the large scale synthesis of the soluble protein by growing the host vector system under conditions suitable for the production of protein and recovering the protein so produced. Thus, a method of producing the soluble protein is also provided. This invention further provides the soluble protein produced by this method.

This invention provides a method for identifying inhibitors of HIV-1 infection comprising steps of: (a) contacting an effective amount of a compound with a system which contains HIV-1 gp120, HIV-1 gp41 or a fragment thereof with the molecule specifically recognized by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1 under conditions permitting formation of a complex between the HIV-1 gp120, HIV-1 gp41 or a fragment thereof with the molecule, so as to inhibit such formation; (b) determining the amount of complex formed; and (c) comparing the amount determined in step (b) with the control which is without the addition of the compound, a decrease in the complex formation indicating that the compound is capable of inhibiting HIV-1 infection. In an embodiment, the compound tested is not previously known. This invention also provide the compound identified by the above method.

This invention also provides a pharmaceutical composition comprising the compound identified by the above method and a pharmaceutically acceptable carrier.

This invention provides a method of inhibiting HIV-1 infection in a subject comprising administering an effective amount of the above pharmaceutical composition comprising the compound identified by the above method to the subject.

This invention provides a kit for identifying inhibitors of HIV-1 infection which comprises, in separate compartments: (a) purified HIV-1 gp120, HIV-1 gp41 or a fragment thereof; and (b) the molecule specifically recognized by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1.

This invention provides a transgenic nonhuman mammal which comprises an isolated DNA molecule encoding a molecule specifically recognized by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1.

One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium (Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding the molecule specifically recognized by the monoclonal antibody capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein.sup.+ cell with an appropriate CD4.sup.+ cell without cross reacting with the HIV-1 envelope glycoprotein or CD4 and capable of inhibiting infection by one or more strains of HIV-1 is purified from a vector by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.

The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a pipet puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.

CD4 may be expressed in the above transgenic animal system such that the animal will be susceptible to HIV-1 infection. Accordingly, this invention also provides the above transgenic nonhuman animals further comprising an isolated DNA molecule encoding the full-length or portion of the CD4 molecule sufficient for binding the HIV-1 envelope glycoprotein. These animal model systems are useful for screening compounds which are capable of inhibiting HIV-1 infection. Moreover, they are useful in predicting or evaluating possible therapeutic applications of HIV drugs. Therefore, this invention also provides a method for screening compounds using these transgenic animals.
 

Claim 1 of 8 Claims

1. A method of inhibiting HIV-1 infection of a CD4+ cell which comprises contacting the CD4+ cell with an amount of a monoclonal antibody or portion thereof effective to (a) specifically inhibit 67% or greater of fusion of a CD4+ PM-1 cell to a HeLa cell expressing envelope glycoprotein from HIV-1.sub.JR-FL, and (b) inhibit 18% or less of fusion of a CD4+ SUP-T1 cell to a HeLa cell expressing envelope protein from HIV-1.sub.LAI, wherein the antibody (i) does not crossreact with HIV-1 envelope glycoprotein or CD4, (ii) reacts with an antigen on the surface of a PM-1 cell having an approximate molecular weight of 44 kD, (iii) does not react with an antigen on the surface of a SUP-T1 cell, and (iv) is at least as active as monoclonal antibody PA-7 produced by the hybridoma designated PA-7 (ATCC Accession No. PTA-6638) in inhibiting fusion as recited in (a) above and less active than monoclonal antibody PA-6 produced by the hybridoma designated PA-6 (ATCC Accession No. PTA-6637) in inhibiting fusion as recited in (b) above, so as to thereby inhibit HIV-1 infection of the CD4+ cell.
 

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