Method of selecting hla-dp4 ligands and the applications thereof

Abstract

A method of selecting a test molecule that binds to HLA-DP4 by (i) incubating HLA-DP4 with the test molecule and a labeled peptide of formula (I) Z 1 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 Z 2 thereby forming respective complexes, wherein Z 1 and Z 2 , which may be identical or different, are each either zero or from 1 to 100 amino acids; X 1 is an aromatic or hydrophobic amino acid, or S; X 6 is an aromatic or hydrophobic amino acid, or C; X 9 is an aromatic or hydrophobic amino acid, or C, D, Q, S, T, or E; and X 2 , X 3 , X 4 , X 5 , X 7 and X 8 are each an amino acid; (ii) separating the respective complexes formed; (iii) detecting the HLA-DP4/labeled peptide complexes; and (iv) selecting the test molecule that exhibits a binding activity of IC 50 <1000 nM, which corresponds to the concentration of the test molecule that inhibits 50% of the competitive HLA-DP4 binding of the labeled peptide.

1 - 26 . (canceled) 
     
     
         27 . A method of identifying an HLA-DP4-ligand peptide or a modified HLA-DP40-ligand peptide, which comprises at least one of the following steps:
 (a) establishing an HLA-DP4-binding matrix by calculating for all the mutants of a tracer peptide, representing all the substitutions of the residues at positions 1, 4, 6 or 9 of said tracer peptide with the other 19 natural amino acids, the ratio of the IC 50  values for said mutant peptides and for said tracer peptide, wherein said ratios correspond to the concentration of these molecules which inhibit 50% of the binding of the tracer peptide;   (b) evaluating the HLA-DP4-binding of peptides of at least 9 amino acids included in said amino acid sequence, by calculating, for each fragment of 9 amino acids of said peptide, the sum of the scores for HLA-DP4-binding of the residues at positions 1, 4, 6 and 9 of said fragment, using the binding matrix established in (a), and/or   (c) identifying the HLA-DP4-ligand peptides corresponding to those to which the loss of HLA-DP4-binding, relative to said tracer peptide, is smallest, i.e. those for which the sum of the binding scores has the lowest value expressed as a logarithm (log);   wherein said tracer peptide is at least one peptide selected from the group of consisting of the peptides exhibiting a signal/background noise ratio greater than 5, at a concentration of 10 nM, in a direct HLA-DP4-binding assay, and corresponding to a formula (I) Z 1 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 Z 2 ; wherein   Z 1  and Z 2 , which may be identical or different, are zero or each represent a peptide of 1 to 100 natural or synthetic amino acids;   X 6  represents an aromatic or hydrophobic amino acid, or a cysteine;   X 1  represents an aromatic or hydrophobic amino acid and/or X 9  represents an aromatic or hydrophobic amino acid, or a cysteine (C), an aspartic acid (D), a glutamine (Q), a serine (S), a threonine (T) or a glutamic acid (E); and   X 2 , X 3 , X 4 , X 5 , X 7  and X 8  each represent a natural or synthetic amino acid.   
     
     
         28 . The method of  claim 27 , which comprises steps (a), (b), and (c). 
     
     
         29 . The method of  claim 27 , comprising selecting an HLA-DP4 peptide or modified peptide that exhibits an HLA-DP4-binding activity of less than 1000 nM. 
     
     
         30 . The method of  claim 27 , which identifies an HLA-DP4 ligand peptide. 
     
     
         31 . The method of  claim 27 , which identities a modified HLA-DP4 ligand peptide. 
     
     
         32 . The method of  claim 27 , wherein the HLA-DP4 peptide or modified peptide sequence corresponds to formula (I) Z 1 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 Z 2 , wherein
 Z 1  and Z 2 , which may be identical or different, are zero or each represent a peptide of 1 to 100 natural or synthetic amino acids;   X 6  represents an aromatic or hydrophobic amino acid, or a cysteine;   X 1  represents an aromatic or hydrophobic amino acid; and/or   X 9  represents an aromatic or hydrophobic amino acid, or else a cysteine (C), an aspartic acid (D), a glutamine (Q), a serine (S), a threonine (T) or a glutamic acid (E); and   X 2 , X 3 , X 4 , X 5 , X 7  and X 8  each represent a natural or synthetic amino acid,   on condition that said HLA-DP4-ligand peptide of general formula (I) does not correspond to any of the sequences SEQ ID NOS: 1 to 17.   
     
     
         33 . The method of  claim 32 , wherein
 X 6  is selected from the group consisting of L, I, W, F, M, Y and C, and   X 1  is selected from the group consisting of A, V, L, I, W, F, M and Y; and/or   X 9  is selected from the group consisting of A, V, L, I, P, W, F, M, Y, C, D, Q, S, T and E.   
     
     
         34 . The method of  claim 33 , wherein the peptide or modified peptide hinds specifically to the HLA-DPB1*0401 molecule and
 X 6  is different from C, and/or   X 1  is different from A and from V, and/or   X 9  represents W or Y or   X 9  is different from E and from C, and/or   X 4  is different from K and from R.   
     
     
         35 . The method of  claim 33 , wherein the peptide or modified peptide hinds specifically to the DPB1*0402 molecule and X 6  represents C, and/or X 1  represents A or V, and/or X 9  represents E or C or X 9  is different from Y and W, and/or X 4  represents K or R.