Mapping and reconstitution of a conformational discontinuous binding surface

Abstract

The structure of conformational, discontinuous binding surfaces that associate with a binding molecule, preferably the epitopes of monoclonal antibodies (mAbs) may be discovered. The binding molecule is used to select specific peptides from a peptide library that, in turn, are used as a binding surface (epitope) defining database that is applied via a novel computer algorithm to analyze the crystalline-structure of the original binding surface (antigen). An antigenic epitope-mimetic that is recognized by its original mAb may be reconstituted based on the segments of the epitope identified in the prediction. The basic elements of the binding domain on gp120 that is recognized by broadly neutralizing antibody b12 are disclosed, as in their use in making vaccines for preventing or treating HIV.

What is claimed is: 
     
       1. A method for improved prediction of the region on the surface of a proteinaceous material representing a binding surface that associates with a predetermined binding molecule, comprising:
 (a) screening a peptide library with said predetermined binding molecule to identify a plurality of peptides that bind to said binding molecule; 
 (b) determining the amino acid sequence of each identified peptide; 
 (c) assigning a symbol to each class of amino acid residue represented in the library and presenting each said sequence as a string of said symbols; 
 (d) calculating the frequency of occurrences of each tandem pair of symbols that exist in the strings of symbols presented in step (c); 
 (e) identifying those tandem pairs of symbols, the number of occurrences of which is statistically significant; 
 (f) mapping on a three-dimensional model of the proteinaceous material those pairs of amino acids represented by the tandem pairs of symbols identified in step (e), wherein a pair of amino acids is two amino acids, at least one of which is accessible to the surface of the proteinaceous material and whose alpha carbons are separated by no more than a predetermined distance; and 
 (g) determining clusters of amino acid pairs mapped in (f), each amino acid pair in the cluster being topographically related to at least one other pair in the cluster, 
 whereby each said cluster is a predicted region on the surface of the proteinaceous material representing said binding surface. 
 
     
     
       2. A method of identifying a basic element of a binding surface on a proteinaceous material, which binding surface associates with a predetermined binding molecule, comprising:
 (a) identifying a cluster of amino acids predicted to represent a binding surface by means of the process of  claim 1 ; 
 (b) identifying the outermost amino acids of binding pairs in the cluster so as to define the perimeter of the predicted binding surface; 
 (c) identifying all other amino acids on the surface of the proteinaceous material situated within the perimeter of the predicted binding surface or within a predetermined distance therefrom; and 
 (d) identifying basic elements of the binding surface, each said element being a linear segment of the proteinaceous material whose first and last residues are amino acids identified in (b) or (c) and none of the intermediate amino acids thereof are amino acids on the surface of the proteinaceous material not identified in (b) or (c), any amino acids identified in (b) or (c) that are not part of a said linear segment being a basic element of a single amino acid. 
 
     
     
       3. A method of producing a binding surface mimetic, comprising:
 (a) identifying the basic elements of the binding surface by means of the process of  claim 2 ; 
 (b) displaying one or more of the basic elements in such a manner as to substantially maintain the relative spatial orientation of the amino acids of (a), thereby identifying a molecule that is mimetic of said binding surface; and 
 (c) producing the mimetic molecule. 
 
     
     
       4. A method in accordance with  claim 3 , wherein said step (b) comprises connecting two or more of the basic elements either directly or by means of linkers or by substitution onto an appropriate scaffold in such a manner as to substantially maintain the relative spatial orientation of the amino acids of (a), thereby identifying a molecule that is mimetic of said binding surface. 
     
     
       5. A method in accordance with  claim 1 , wherein said proteinaceous material is gp120 and said predetermined binding molecule is a broadly neutralizing antibody against gp120. 
     
     
       6. A method of producing a binding surface mimetic, comprising:
 (a) identifying the basic elements of the binding surface by means of the process of  claim 5 ; 
 (b) displaying one or more of the basic elements in such a manner as to substantially maintain the relative spatial orientation of the amino acids of (a), thereby identifying a molecule that is mimetic of said binding surface; and 
 (c) producing the mimetic molecule. 
 
     
     
       7. A method in accordance with  claim 6 , wherein said step (b) comprises connecting two or more of the basic elements either directly or by means of linkers or by substitution onto an appropriate scaffold in such a manner as to substantially maintain the relative spatial orientation of the amino acids of (a), thereby identifying a molecule that is mimetic of said binding surface. 
     
     
       8. A method in accordance with  claim 2 , wherein said proteinaceous material is gp120 and said predetermined binding molecule is a broadly neutralizing antibody against gp120. 
     
     
       9. A method of producing a binding surface mimetic, comprising:
 (a) identifying the basic elements of the binding surface by means of the process of  claim 8 ; 
 (b) displaying one or more of the basic elements in such a manner as to substantially maintain the relative spatial orientation of the amino acids of (a), thereby identifying a molecule that is mimetic of said binding surface; and 
 (c) producing the mimetic molecule. 
 
     
     
       10. A method in accordance with  claim 9 , wherein said step (b) comprises connecting two or more of the basic elements either directly or by means of linkers or by substitution onto an appropriate scaffold in such a manner as to substantially maintain the relative spatial orientation of the amino acids of (a), thereby identifying a molecule that is mimetic of said binding surface. 
     
     
       11. A method of producing a molecule that is mimetic of a binding surface of gp120 that is a discontinuous epitope recognized by the b12 antibody, which molecule includes one or more of the basic elements of the binding surface, comprising:
 (a) displaying one or more of the peptides comprising sequences selected from the group consisting of:
 (i) amino acids 254-257 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (ii) amino acids 368-376 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (iii) amino acids 382-386 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; and 
 (iv) amino acids 418-424 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade, 
   
       in such a manner as to substantially maintain the relative spatial orientation that the amino acids thereof have when they are positioned at their native positions in gp120, thereby obtaining a molecule that has a structure that is mimetic of said binding surface, wherein extra residues from gp120 that linearly extend said peptide sequences may be used to maintain said relative spatial orientation, with the proviso that said extra residues do not consist of the entire native gp120 sequence that is between said peptide sequences in native gp120; and
 (b) producing the binding surface mimetic obtained in step (a). 
 
     
     
       12. A method in accordance with claim 11, wherein said step (a) comprises connecting two or more of said peptides, each in forward or reverse sequence, either directly or by means of linkers or by substitution onto an appropriate scaffold, in such a manner as to substantially maintain the relative spatial orientation that the amino acids thereof have when they are positioned at their native positions in gp120, thereby obtaining a binding surface mimetic that has a structure that is mimetic of said binding surface, wherein extra residues from gp120 that linearly extend said peptide sequences may be used to maintain said relative spatial orientation, with the proviso that said extra residues do not consist of the entire native gp120 sequence that is between said peptide sequences in native gp120. 
     
     
       13. A method in accordance with claim 12, wherein said two or more of said peptides is three or more of said peptides. 
     
     
       14. A method in accordance with claim 12, wherein said two or more of said peptides is all four of said peptides. 
     
     
       15. A method of producing a molecule that is mimetic of a binding surface of gp120 that is a discontinuous epitope recognized by the 17b antibody, which molecule includes one or more of the basic elements of the binding surface, comprising:
 (a) displaying one or more of the peptides comprising sequences selected from the group consisting of:
 (i) amino acids 116-124 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (ii) amino acids 200-208 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (iii) amino acids 419-423 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; and 
 (iv) amino acids 432-438 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade, 
   
       in such a manner as to substantially maintain the relative spatial orientation that the amino acids thereof have when they are positioned at their native positions in gp120, thereby obtaining a molecule that has a structure that is mimetic of said binding surface, wherein extra residues from gp120 that linearly extend said peptide sequences may be used to maintain said relative spatial orientation, with the proviso that said extra residues do not consist of the entire native gp120 sequence that is between said peptide sequences in native gp120; and
 (b) producing the binding surface mimetic obtained in step (a). 
 
     
     
       16. A method in accordance with claim 15, wherein said step (a) comprises connecting two or more of said peptides, each in forward or reverse sequence, either directly or by means of linkers or by substitution onto an appropriate scaffold, in such a manner as to substantially maintain the relative spatial orientation that the amino acids thereof have when they are positioned at their native positions in gp120, thereby obtaining a binding surface mimetic that has a structure that is mimetic of said binding surface, wherein extra residues from gp120 that linearly extend said peptide sequences may be used to maintain said relative spatial orientation, with the proviso that said extra residues do not consist of the entire native gp120 sequence that is between said peptide sequences in native gp120. 
     
     
       17. A method in accordance with claim 16, wherein said two or more of said peptides is three or more of said peptides. 
     
     
       18. A method in accordance with claim 16, wherein said two or more of said peptides is all four of said peptides. 
     
     
       19. A method of producing a molecule that is mimetic of a binding surface of gp120 that is a discontinuous epitope recognized by the CG10 antibody, which molecule includes one or more of the basic elements of the binding surface, comprising:
 (a) displaying one or more of the peptides comprising sequences selected from the group consisting of:
 (i) amino acids 119-123 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (ii) amino acids 205-207 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (iii) amino acids 381-382 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; 
 (iv) amino acids 421-423 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade; and 
 (v) amino acids 430-435 of gp120 of isolate HXB2 of clade B (SEQ ID NO:1) or the corresponding amino acids of the sequence or consensus sequence of gp120 of another HIV isolate or clade, 
   
       in such a manner as to substantially maintain the relative spatial orientation that the amino acids thereof have when they are positioned at their native positions in gp120, thereby obtaining a molecule that has a structure that is mimetic of said binding surface, wherein extra residues from gp120 that linearly extend said peptide sequences may be used to maintain said relative spatial orientation, with the proviso that said extra residues do not consist of the entire native gp120 sequence that is between said peptide sequences in native gp120; and
 (b) producing the binding surface mimetic obtained in step (a). 
 
     
     
       20. A method in accordance with claim 19, wherein said step (a) comprises connecting two or more of said peptides, each in forward or reverse sequence, either directly or by means of linkers or by substitution onto an appropriate scaffold, in such a manner as to substantially maintain the relative spatial orientation that the amino acids thereof have when they are positioned at their native positions in gp120, thereby obtaining a binding surface mimetic that has a structure that is mimetic of said binding surface, wherein extra residues from gp120 that linearly extend said peptide sequences may be used to maintain said relative spatial orientation, with the proviso that said extra residues do not consist of the entire native gp120 sequence that is between said peptide sequences in native gp120. 
     
     
       21. A method in accordance with claim 20, wherein said two or more of said peptides is three or more of said peptides. 
     
     
       22. A method in accordance with claim 20, wherein said two or more of said peptides is four or more of said peptides. 
     
     
       23. A method in accordance with claim 20, wherein said two or more of said peptides is all five of said peptides.