A novel chimeric protein for prevention and treatment of hiv infection

Abstract

This invention relates to bispecific fusion proteins effective in viral neutralization. More specifically, such proteins have two different binding domains, an inducing-binding domain and an induced-binding domain, functionally linked by a peptide linker. Such proteins, nucleic acid molecules encoding them, and their production and use in preventing or treating viral infections are provided. One prototypical bispecific fusion protein is sCD4-SCFv(17b), in which a soluble CD4 fragment (containing domains D1 and D2) is fused to a single chain Fv portion of antibody 17b via a linker.

We claim: 
     
         1 . A neutralizing bispecific fusion protein capable of binding to two sites on a target protein, comprising a first binding domain capable of binding to an inducing site on the target protein, thereby exposing an induced epitope; a second binding domain capable of forming a neutralizing complex with an induced epitope of the target protein; and a linker connecting the first domain to the second domain. 
     
     
         2 . The protein according to  claim 1 , wherein the first binding domain comprises a binding domain of a variable region of an antibody heavy or light chain. 
     
     
         3 . The protein according to  claim 2 , wherein the antibody binding domain mimics a biological activity of a CD4 molecule in binding to the inducing site and exposing the inducing epitope. 
     
     
         4 . The protein according to  claim 1 , wherein the first binding domain is derived from a CD4 molecule. 
     
     
         5 . The protein according to  claim 1 , wherein the second binding domain comprises a binding domain of a variable region of an antibody heavy or light chain. 
     
     
         6 . The protein according to  claim 1 , wherein the target protein is a viral envelope protein of a virus. 
     
     
         7 . The protein according to  claim 6 , wherein the viral envelope protein is gp120. 
     
     
         8 . The protein according to  claim 7 , wherein the second binding domain mimics a biological activity of an HIV coreceptor molecule in binding to gp120. 
     
     
         9 . The protein according to  claim 8 , wherein the second binding domain is a peptide fragment of a chemokine receptor selected from the group consisting of CXCR4, CCR2B, CCR3, and CCR5, CCR8, CCR9, CX 3 CR1, US28, or the chemokine receptor related proteins including STRL33, GPR15, APJ, ChemR23, and BLTR. 
     
     
         10 . The protein according to  claim 5 , wherein the binding domain of the antibody is capable of binding to at least one coreceptor binding determinant of gp120. 
     
     
         11 . The protein according to  claim 1 , wherein the linker maintains the second binding domain in binding proximity to the induced epitope when the first binding domain is bound to the inducing site. 
     
     
         12 . The protein according to  claim 11 , wherein the linker is:
 (a) substantially flexible;   (b) 15-100 angstroms (Å) long;   (c) 10-100 amino acid residues in length; or   (d) any two or more of (a), (b), and (c).   
     
     
         13 . An isolated nucleic acid molecule encoding a protein according to  claim 1 . 
     
     
         14 . A transgenic eukaryotic cell comprising the isolated nucleic acid molecule according to  claim 13 . 
     
     
         15 . A method for producing in a eukaryotic cell a functional recombinant bispecific fusion protein capable of binding two sites on a target protein, comprising the steps of:
 a) transfecting the eukaryotic cell with a recombinant nucleic acid molecule according to  claim 13 ;   b) culturing the transfected eukaryotic cell under conditions that cause production of the protein; and   c) recovering the protein produced by the cultured eukaryotic cell.   
     
     
         16 . The method of  claim 15 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         17 . The method of  claim 15 , wherein recovering the protein comprises:
 a) identifying the protein by the presence of a molecular tag; and   b) separating the protein having the molecular tag so identified from molecules without the tag, so as to recover the protein produced by the cultured eukaryotic cell.   
     
     
         18 . A method for inactivating a gp120 protein and/or for blocking and preventing the binding of a viral or recombinant gp120 protein to soluble CD4 or lymphocyte CD4, comprising the step of:
 contacting the gp120 protein with a protein according to  claim 7 , or a variant protein, analog or mimetic thereof.   
     
     
         19 . A method for neutralizing a human immunodeficiency virus, comprising the step of:
 contacting the human immunodeficiency virus with a protein according to  claim 7 , or a variant protein, analog or mimetic thereof.   
     
     
         20 . A method for inhibiting HIV virus replication or infectivity in a subject, comprising administering to the subject an amount of the protein according to  claim 7 , or a variant protein, analog or mimetic thereof, sufficient to inhibit HIV virus replication or infectivity. 
     
     
         21 . The method according to  claim 20 , wherein the protein is administered in the form of a pharmaceutical composition. 
     
     
         22 . A composition comprising the protein of  claim 1 , or a variant protein, analog or mimetic thereof. 
     
     
         23 . A pharmaceutical composition comprising the protein according to  claim 1 , or a variant protein, analog or mimetic thereof, and a pharmaceutically acceptable carrier. 
     
     
         24 . A kit for treatment and/or prevention of HIV infection, comprising a clinically effective dose of the neutralizing bispecific fusion protein of  claim 1 .