Recombinant soluble adenovirus receptor

Abstract

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

1 . An isolated polypeptide comprising an amino acid sequence which corresponds to adenovirus binding domain D1 of human CAR (coxsackievirus and adenovirus receptor) protein.  
     
     
         2 . The isolated polypeptide of  claim 1  wherein the amino acid sequence corresponds to residues 20-144 of human pre-CAR protein with the exception of two amino acid substitutions, L20M and S21G.  
     
     
         3 . An isolated polypeptide comprising an amino acid sequence which corresponds to extracellular domains D1 and D2 of human CAR (coxsackievirus and adenovirus receptor) protein.  
     
     
         4 . The isolated polypeptide of  claim 3  wherein the amino acid sequence corresponds to residues 20-237 of human pre-CAR protein with the exception of two amino acid substitutions, L20M and S21G.  
     
     
         5 . A recombinant DNA molecule comprising a nucleotide sequence which encodes adenovirus binding domain D1 of human CAR (coxsackievirus and adenovirus receptor) protein.  
     
     
         6 . The recombinant DNA molecule of  claim 5  wherein the nucleotide sequence corresponds to nucleotide 121-493 of human CAR cDNA.  
     
     
         7 . A bacterial cell transformed with a recombinant DNA molecule comprising a nucleotide sequence which encodes adenovirus binding domain D1 of human CAR (coxsackievirus and adenovirus receptor).  
     
     
         8 . The bacterial cell of  claim 7  wherein the nucleotide sequence corresponds to nucleotide 121-493 of human CAR cDNA.  
     
     
         9 . A recombinant DNA molecule comprising a nucleotide sequence which encodes adenovirus binding domains D1 and D2 of human CAR (coxsackievirus and adenovirus receptor) protein.  
     
     
         10 . The recombinant DNA molecule of  claim 9  wherein the nucleotide sequence corresponds to nucleotide 121-770 of human CAR cDNA.  
     
     
         11 . A bacterial cell transformed with a recombinant DNA molecule comprising a nucleotide sequence which encodes adenovirus binding domains D1 and D2 of human CAR (coxsackievirus and adenovirus receptor).  
     
     
         12 . The bacterial cell of  claim 11  wherein the nucleotide sequences corresponds to nucleotide 121-770 of human CAR cDNA.  
     
     
         13 . A recombinant DNA molecule comprising a first nucleotide sequence which corresponds to nucleotide 121-491 of human CAR (coxsackievirus and adenovirus receptor) cDNA, fused in frame to a second nucleotide sequence which encodes a polypeptide which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria.  
     
     
         14 . The recombinant DNA molecule of  claim 13  wherein the second nucleotide sequence is located 3′ to the first nucleotide sequence.  
     
     
         15 . The recombinant DNA molecule of  claim 14  wherein the second nucleotide sequence encodes the polypeptide sequence LEDPAANKARKEAELAAATAEQ.  
     
     
         16 . A bacterial cell transformed with a recombinant DNA molecule comprising a first nucleotide sequence which corresponds to nucleotide 121-491 of human CAR (coxsackievirus and adenovirus receptor) cDNA, fused in frame to a second nucleotide sequence which encodes a polypeptide which facilitates folding of D1 into a functional, soluble domain.  
     
     
         17 . The bacterial cell of  claim 16  wherein the second nucleotide sequence is located 3′ to the first nucleotide sequence.  
     
     
         18 . The bacterial cell of  claim 17  wherein the second nucleotide sequence encodes the polypeptide sequence LEDPAANKARKEAELAAATAEQ.  
     
     
         19 . An isolated fusion protein comprising a first amino acid sequence which corresponds to amino acids 20-144 of human pre-CAR protein (D1) fused in frame to a second amino acid sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria.  
     
     
         20 . The isolated fusion protein of  claim 19  wherein the second amino acid sequence is located C-terminal to the first amino acid sequence.  
     
     
         21 . The isolated fusion protein of  claim 19  wherein the second amino acid sequence comprises LEDPAANKARKEAELAAATAEQ.  
     
     
         22 . A therapeutic method for treating a patient infected with a virus which binds to human CAR protein, the method comprising: 
 a) providing a therapeutic composition comprising CAR D1; and    b) administering the therapeutic composition to the patient.    
     
     
         23 . The therapeutic method of  claim 22  wherein administration is topical.  
     
     
         24 . The therapeutic method of  claim 23  wherein administration is to a localized region.  
     
     
         25 . The therapeutic method of  claim 24  wherein the localized region is ocular.  
     
     
         26 . The therapeutic method of  claim 25  wherein administration is with an eye dropper.  
     
     
         27 . The method of  claim 23  wherein the localized region is an upper respiratory tract.  
     
     
         28 . The method of  claim 27  wherein the administration is with an inhaler.  
     
     
         29 . A method of  claim 23  wherein the localized region is gastro-intestinal.  
     
     
         30 . The method of  claim 29  wherein administration is via ingestion.  
     
     
         31 . The method of  claim 22  wherein the virus is selected from the group consisting of adenovirus subgroup A, adenovirus subgroup C, or coxsackievirus subgroup B.  
     
     
         32 . A method for identifying a molecule, or a portion thereof, which binds specifically to the D1 domain of human CAR, comprising: 
 a) providing a binding assay for detecting the binding of a molecule to the D1 domain of human CAR;    b) screening candidate binding molecules for the ability to bind specifically to the D1 domain of human CAR in the binding assay of step a); and    c) identifying molecules which specifically bind to the D1 domain of human CAR.    
     
     
         33 . A method for identifying the binding domain of a molecule, the molecule being characterized by the ability to bind specifically to the D1 domain of human CAR, comprising: 
 a) providing a binding assay for detecting the binding of a molecule to the D1 domain of human CAR;    b) providing a molecule which is predetermined to bind specifically to the D1 domain of human CAR;    c) providing an altered form of the molecule of step b);    d) conducting the binding assay of step a) incorporating, independently, the molecule of step b) and the molecule of step c); and    e) comparing the binding activities determined in step d) for the molecule of step b) and the molecule of step c), a difference in binding activities being indicative of the participation of an altered portion of the molecule of step c) in D1 domain-specific binding.    
     
     
         34 . The method of  claim 33  wherein the molecule of step b) is an adenovirus knob protein.  
     
     
         35 . The method of  claim 33  wherein the molecule of step c) is a protein generated by a mutation in the molecule of step b), the mutation being selected from the group consisting of a substitution, a truncation and a deletion.  
     
     
         36 . The method of  claim 33  wherein the molecule of step c) is a chimeric protein, one component of which is the molecule of step b), or a portion thereof.  
     
     
         37 . A method for identifying antiviral compounds which interfere specifically with the interaction between the D1 domain of human CAR and a viral attachment protein which binds specifically to the D1 domain, comprising: 
 a) providing a binding assay system for the quantitative determination of binding between the viral attachment protein and an isolated D1 polypeptide;    b) introducing candidate antiviral compounds into the binding assay system of step a); and    c) identifying antiviral compounds which substantially inhibit binding of the viral attachment protein to the isolated D1 polypeptide.    
     
     
         38 . The method of  claim 37  wherein the viral attachment protein is selected from the group consisting of the adenovirus knob proteins.  
     
     
         39 . A method for specifically targeting a cell for infection by a virus which binds to the D1 domain of human CAR, comprising the steps of: 
 a) selecting a target cell bearing a predetermined surface protein;    b) providing a molecule which binds specifically to the predetermined surface protein;    c) covalently attaching a polypeptide comprising the D1 domain of human CAR to the molecule of step b) to form an adapter bridge;    d) contacting the adapter bridge of step c) with the virus under conditions appropriate for the binding of D1 to the virus to produce a virus-adaptor bridge complex; and    e) contacting the virus-adaptor bridge complex with the target cell under conditions appropriate for binding of the virus-adaptor bridge complex to the target cell.    
     
     
         40 . The method of  claim 39  wherein the virus is an adenovirus.  
     
     
         41 . The method of  claim 39  wherein the virus has been modified for therapeutic purposes.  
     
     
         42 . The method of  claim 39  wherein the D1 domain has been modified for therapeutic purposes.  
     
     
         43 . A method for treating an infection in a patient caused by a virus that binds to human CAR, the method comprising: 
 a) providing a therapeutic composition comprising an Ad12 fiber knob protein, or a portion thereof; and    b) administering the therapeutic composition to the patient.