Methods for identifying polypeptide targets and uses thereof for treating immunological diseases

Abstract

The present invention provides methods for identifying viral virulence factors and for identifying cellular polypeptides to which the viral polypeptides bind. The cellular polypeptide is useful as a therapeutic target or as a therapeutic agent for treating diseases and disorders, including immunological diseases or disorders.

1 . A method of identifying a cellular polypeptide to which a viral polypeptide binds comprising:
 (a) contacting a cell, or a fraction or a supernatant of the cell, and a fusion protein comprising a viral polypeptide fused to an affinity tag comprising a polypeptide tag, under conditions and for a time sufficient that permit a viral polypeptide moiety of the fusion protein to interact with a polypeptide associated with the cell, or the fraction or the supernatant of the cell, to provide a fusion protein:cellular polypeptide complex, wherein the viral polypeptide exhibits at least one virulence trait;   (b) isolating the fusion protein:cellular polypeptide complex; and   (c) determining the amino acid sequence of the cellular polypeptide or of at least one cellular polypeptide fragment comprising at least eight amino acids, and thereby identifying a cellular polypeptide to which a viral polypeptide binds.   
     
     
         2 . The method of  claim 1  further comprising prior to step (a), (i) identifying in the genome of a virus, a polynucleotide sequence that encodes a viral polypeptide, which viral polypeptide comprises at least 40 amino acids; and (ii) producing a fusion protein comprising the viral polypeptide fused to an affinity tag sequence. 
     
     
         3 . The method of  claim 2 , wherein the at least one virulence trait is selected from (a) the trait that expression of a mutant viral polypeptide in a cell infected by the virus correlates with a decrease in virulence of the virus; (b) the trait that absence of expression of the viral polypeptide in a cell infected by the virus correlates with a decrease in virulence of the virus; (c) the trait that the viral polypeptide is secreted by a cell infected with the virus or the viral polypeptide is associated with a cellular membrane of a cell infected by the virus; and (d) the trait that the polynucleotide sequence in the virus genome is located in a genomic region that encodes at least one other viral polypeptide that is a viral virulence factor. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1  wherein the viral polypeptide (A) is secreted by a cell infected with the virus, (B) is associated with a cellular membrane, or (C) is intracellular. 
     
     
         6 .- 11 . (canceled) 
     
     
         12 . The method according to  claim 1  wherein prior to step (b) the cell is subjected to at least one stimulus, wherein the at least one stimulus is selected from (a) an antibody that specifically binds to a cognate antigen expressed by the cell; (b) a phorbol ester; (c) concanavalin A; (d) a cytokine; (e) a chemokine; and (f) ionomycin. 
     
     
         13 . (canceled) 
     
     
         14 . The method according to  claim 1  wherein the fraction of the cell is selected from a cell lysate, a cell extract, or at least one isolated cell organelle. 
     
     
         15 . The method according to  claim 1  wherein the affinity tag further comprises a detectable moiety. 
     
     
         16 .- 18 . (canceled) 
     
     
         19 . The method according to  claim 1  wherein the affinity tag further comprises a protease recognition sequence. 
     
     
         20 . (canceled) 
     
     
         21 . The method according  claim 1 , wherein the polypeptide tag is selected from a hemagglutinin peptide; a calmodulin binding polypeptide, a streptavidin binding peptide, an immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least one immunoglobulin binding staphylococcal protein A domain, and Softag™. 
     
     
         22 . (canceled) 
     
     
         23 . The method according to  claim 21  wherein the immunoglobulin Fc polypeptide is a human IgG immunoglobulin Fc polypeptide or an immunoglobulin mutein Fc polypeptide wherein the immunoglobulin mutein Fc polypeptide is a human IgG1 immunoglobulin mutein Fc polypeptide. 
     
     
         24 .- 25 . (canceled) 
     
     
         26 . The method according to  claim 1  wherein the affinity tag comprises a first polypeptide tag and a second polypeptide tag. 
     
     
         27 . The method according to  claim 26  wherein the affinity tag further comprises a protease recognition sequence. 
     
     
         28 . The method according to  claim 27  wherein the protease recognition sequence is located between the first polypeptide tag and the second polypeptide tag. 
     
     
         29 . The method according to  claim 28  wherein the first polypeptide tag and the second polypeptide tag are selected from a hemagglutinin peptide; a calmodulin binding polypeptide, a streptavidin binding peptide, an immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least one immunoglobulin binding staphylococcal protein A domain, and Softag™. 
     
     
         30 . The method according to  claim 26  wherein the affinity tag further comprises a third polypeptide tag. 
     
     
         31 . The method according to  claim 30  wherein the first polypeptide tag, the second polypeptide tag, and the third polypeptide tag are selected from a hemagglutinin peptide; a calmodulin binding polypeptide, a streptavidin binding peptide, an immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least one immunoglobulin binding staphylococcal protein A domain, and Softag™. 
     
     
         32 . (canceled) 
     
     
         33 . The method according to  claim 30  wherein the affinity tag comprises at least one protease recognition sequence. 
     
     
         34 .- 36 . (canceled) 
     
     
         37 . The method according to  claim 30  wherein the affinity tag further comprises a fourth polypeptide tag. 
     
     
         38 . The method according to  claim 37  wherein the first, second, third, and fourth polypeptide are selected from a hemagglutinin peptide; a calmodulin binding polypeptide, a streptavidin binding peptide, an immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least one immunoglobulin binding staphylococcal protein A domain, and Softag™. 
     
     
         39 . (canceled) 
     
     
         40 . The method according to  claim 37  wherein the fourth polypeptide tag is the same as the first, second, or third polypeptide tag. 
     
     
         41 .- 43 . (canceled) 
     
     
         44 . The method according to  claim 33  wherein the affinity tag further comprises a second protease recognition sequence. 
     
     
         45 .- 46 . (canceled) 
     
     
         47 . The method according to  claim 1  wherein step (c) comprises (i) cleaving the isolated cellular polypeptide with a protease to generate a plurality of polypeptide fragments of the cellular polypeptide; (ii) determining the amino acid sequence of at least one polypeptide fragment, wherein the fragment comprises at least eight amino acids; and (iii) comparing the amino acid sequence of the at least one polypeptide fragment with the amino acid sequence of a known cellular polypeptide, thereby identifying the cellular polypeptide to which the viral polypeptide binds. 
     
     
         48 . The method of  claim 47  wherein the amino acid sequence is determined by a method comprising liquid chromatography and mass spectrometry. 
     
     
         49 . The method of  claim 1  wherein step (b) comprises (i) contacting the fusion protein:cellular polypeptide complex and a cognate ligand of the affinity tag under conditions and for a time sufficient to permit formation of a cognate ligand:fusion protein:cellular polypeptide complex; and (ii) isolating the fusion polypeptide:cellular polypeptide from the cognate ligand:fusion protein:cellular polypeptide complex. 
     
     
         50 . The method of  claim 1  wherein the fusion protein is recombinantly expressed. 
     
     
         51 . The method of  claim 1  wherein the fusion protein is recombinantly expressed by the cell of step (a). 
     
     
         52 . The method of  claim 1  further comprising identifying a cell type that comprises a cellular polypeptide to which the viral polypeptide binds comprising:
 (i) contacting the fusion protein and a biological sample comprising at least one cell, or a fraction of the cell or a supernatant of the cell, under conditions and for a time sufficient to permit the viral polypeptide moiety of the fusion protein to interact with the at least one cell, or the cell fraction or the cell supernatant; 
 (ii) determining the presence or absence of binding of the fusion protein to the at least one cell, or the fraction or the supernatant thereof; 
 (iii) isolating the cell to which the fusion protein binds; and 
 (iv) characterizing the cell, and therefrom determining the cell type that comprises a cellular polypeptide to which the viral polypeptide binds. 
 
     
     
         53 . A method for identifying a cellular polypeptide to which a viral polypeptide binds comprising:
 (a) identifying in the genome of a virus, a polynucleotide sequence that encodes a viral polypeptide, which viral polypeptide comprises at least 40 amino acids;   (b) introducing into a cell a recombinant expression construct comprising a promoter operatively linked to a polynucleotide encoding the viral polypeptide fused in frame with an affinity tag;   (c) isolating from the cell, or from a fraction of the cell, or from a supernatant of the cell, a fusion protein:cellular polypeptide complex;   (d) isolating the fusion protein:cellular polypeptide complex; and   (e) determining the amino acid sequence of the cellular polypeptide or of at least one cellular polypeptide fragment comprising at least eight amino acids, and thereby identifying a cellular polypeptide to which a viral polypeptide binds.   
     
     
         54 . A method for identifying a cellular polypeptide to which a viral polypeptide binds comprising:
 (a) contacting a cell, or a fraction or a supernatant of the cell, and a fusion protein comprising a viral polypeptide moiety fused to an affinity tag moiety, under conditions and for a time sufficient that permit the viral polypeptide moiety of the fusion protein to interact with a polypeptide associated with the cell, or the fraction or the supernatant of the cell, to provide a fusion protein:cellular polypeptide complex, wherein the viral polypeptide has at least one virulence trait, and wherein the affinity tag comprises at least a first polypeptide tag, a second polypeptide tag, and at least one protease recognition sequence;   (b) isolating the fusion protein:cellular polypeptide complex, wherein said step of isolating comprises:   (i) contacting the fusion protein:cellular polypeptide complex with a first cognate ligand of the first polypeptide tag under conditions and for a time sufficient to permit the affinity tag moiety of the fusion protein to interact with the first cognate ligand to provide a first cognate ligand:fusion protein:cellular polypeptide complex;   (ii) contacting the first cognate ligand:fusion protein:cellular polypeptide complex with a protease capable of cleaving the fusion protein at or near the protease recognition sequence to provide a cleaved fusion protein:cellular polypeptide complex;   (iii) contacting the cleaved fusion protein:cellular polypeptide complex with a second cognate ligand that specifically binds to the second polypeptide tag, under conditions and for a time sufficient that permit the second cognate ligand and the cleaved fusion protein:cellular polypeptide complex to interact to form a second cognate ligand:cleaved fusion protein:cellular polypeptide complex; and   (iv) isolating the cleaved fusion protein:cellular polypeptide complex from the second cognate ligand:cleaved fusion protein:cellular polypeptide complex; and   (c) determining the amino acid sequence of the cellular polypeptide or of at least one polypeptide fragment of the cellular polypeptide, wherein the at least one polypeptide fragment comprises at least eight amino acids, and thereby identifying a cellular polypeptide to which a viral polypeptide binds.   
     
     
         55 . The method of  claim 54  further comprising prior to the step of contacting the cell, or a fraction or a supernatant of the cell, and a fusion protein the steps of (a) identifying in the genome of a virus, a polynucleotide sequence that encodes a viral polypeptide, which viral polypeptide comprises at least 40 amino acids; and (b) producing a fusion protein comprising the viral polypeptide fused to an affinity tag sequence. 
     
     
         56 . A method of identifying a cellular polypeptide to which a viral polypeptide binds comprising:
 (a) identifying in the genome of a virus, a polynucleotide sequence that encodes a viral polypeptide, wherein the viral polypeptide comprises at least 40 amino acids;   (b) producing a fusion protein comprising the viral polypeptide fused to an affinity tag sequence, wherein the affinity tag sequence comprises a first polypeptide tag sequence, a second polypeptide tag sequence, and a protease recognition sequence located between the first and second polypeptide tag sequences;   (c) contacting the fusion protein and a cell, or a fraction or a supernatant thereof, under conditions and for a time sufficient that permit the viral polypeptide moiety of the fusion protein to interact with a polypeptide associated with the cell, or the fraction or the supernatant thereof, to provide a fusion protein:cellular polypeptide complex;   (d) isolating the fusion protein:cellular polypeptide complex, wherein said step of isolating comprises:   (i) contacting the fusion protein:cellular polypeptide complex with a first cognate ligand of the first polypeptide tag sequence under conditions and for a time sufficient to permit the affinity tag moiety of the fusion protein to interact with the first cognate ligand to provide a first cognate ligand: fusion protein:cellular polypeptide complex;   (ii) contacting the first cognate ligand:fusion protein:cellular polypeptide complex with a protease capable of cleaving the fusion protein at or near the protease recognition sequence to provide a cleaved fusion protein:cellular polypeptide complex;   (iii) contacting the cleaved fusion protein:cellular polypeptide complex with a second cognate ligand that specifically binds to the second polypeptide tag, under conditions and for a time sufficient that permit the second cognate ligand and the cleaved fusion protein:cellular polypeptide complex to interact to form a second cognate ligand:cleaved fusion protein:cellular polypeptide complex; and   (iv) isolating the cleaved fusion protein:cellular polypeptide complex; and   (e) determining the amino acid sequence of the cellular polypeptide or of at least one cellular polypeptide fragment comprising at least eight amino acids, and therefrom identifying a cellular polypeptide to which a viral polypeptide binds.   
     
     
         57 . A method of identifying an agent for treating an immunological disease or disorder comprising:
 (a) identifying a cellular polypeptide to which a viral polypeptide binds according to the method of  claim 1 , wherein interaction between the cellular polypeptide and the viral polypeptide alters immunoresponsiveness of an immune cell;   (b) contacting (i) the cellular polypeptide, or a cell comprising the cellular polypeptide; (ii) the viral polypeptide; (iii) and a candidate agent, under conditions and for a time sufficient that permit the cellular polypeptide and the viral polypeptide to interact;   (c) determining the level of binding of the viral polypeptide to the cellular polypeptide in the presence of the candidate agent to the level of binding of the viral polypeptide to the cellular polypeptide in the absence of the candidate agent, wherein a decrease in the level of binding of the viral polypeptide to the cellular polypeptide in the presence of the candidate agent compared with the level of binding of the viral polypeptide to the cellular polypeptide in the absence of the candidate agent thereby identifies an agent for treating an immunological disease or disorder.   
     
     
         58 . The method according to  claim 57  wherein the agent is selected from (a) an antibody, or antigen-binding fragment thereof, (b) a viral polypeptide/Fc polypeptide fusion protein; (c) a peptide/Fc polypeptide fusion protein; (d) a small molecule; (e) a small interfering RNA (siRNA); (f) an antisense polynucleotide; and (g) an aptamer. 
     
     
         59 .- 61 . (canceled) 
     
     
         62 . A method of treating a disease or disorder comprising administering to a subject in need thereof (a) a pharmaceutically suitable carrier; and (b) an agent identified according to the method of  claim 57 , wherein the disease or disorder is an immunological disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, or a proliferative disease or disorder. 
     
     
         63 . The method according to  claim 62  wherein the agent is an antibody, or antigen-binding fragment thereof. 
     
     
         64 . A method for guiding the selection of a therapeutic agent for treating a disease or medical disorder, comprising:
 (a) identifying a viral polypeptide that increases the virulence of a virus in a host infected with the virus;   (b) identifying a cellular polypeptide to which the viral polypeptide binds, wherein binding of the viral polypeptide to the cellular polypeptide alters at least one biological activity of a cell;   (c) identifying one or more agents that inhibit binding of the viral polypeptide to the cellular polypeptide;   (d) categorizing the capability of the one or more agents identified in step (c) to alter at least one biological effect of a cell, wherein altering the at least one biological effect reduces the risk of developing a disease or medical disorder or reduces at least one symptom of a disease or medical disorder in a host; and   (e) selecting at least one agent from step (d) for testing in preclinical and clinical methods, and therefrom guiding the selection of a therapeutic agent for treating a disease or disorder.   
     
     
         65 . The method according to  claim 64  wherein the at least one biological activity of the cell is immunoresponsiveness and the cell is an immune cell. 
     
     
         66 .- 68 . (canceled)