Method for Identifying and Validating Dominant T Helper Cell Epitopes Using an HLA-DM-Assisted Class II Binding Assay

Abstract

Rational design of immunotherapeutics relies on clear knowledge of the immunodominant epitopes of antigens. Current methods for identifying kinetically stable peptide-MHC complexes are in many cases inadequate for a number of reasons. Disclosed herein is a reductionistic system incorporating known participants of MHC class II antigen processing in solution to generate peptide pools from antigens, including those for which no immunodominant epitope has yet been identified, that are highly enriched for proteolytic fragments containing their immunodominant epitopes. HLA-DM-mediated editing contributes significantly to immunodominance and is exploited in discovering immunodominant epitopes from novel or previously uncharacterized antigens, particularly antigens associated with pathogens, tumors or autoimmune diseases.

What is claimed is: 
     
         1 . A method for producing an isolated or purified complex of an immunogenic or immunodominant MHC class II restricted peptide from a polypeptide of interest, comprising:
 (a) optionally, denaturing the polypeptide of interest to produce a denatured polypeptide;   (b) incubating the optionally denatured polypeptide in the presence of
 (i) a soluble human MHC class II protein or an active homologue thereof from another mammalian species; and 
 (ii) soluble human HLA-DM protein or an active homologue thereof from another mammalian species; 
    such that the denatured polypeptide binds to the peptide binding groove of the MHC class II protein, forming a complex with said class II protein;   (c) proteolytically digesting exposed regions of said optionally denatured polypeptide so that a peptide of about 10 to about 26, or about 12 to about 26, amino acid residues remains bound to the peptide binding groove of said MHC class II protein, thereby producing said complex; and   (d) further isolating or purifying said complex,   
       thereby producing said isolated or purified complex. 
     
     
         2 . A method for producing an isolated immunogenic or immunodominant MHC class II restricted peptide from a polypeptide of interest, comprising producing the complex in accordance with the method of  claim 1 , and further comprising the step of:
 (e) eluting or otherwise removing said peptide from said complex and isolating said peptide,   
       thereby producing the isolated peptide. 
     
     
         3 . A method for identifying or characterizing an immunogenic MHC class II restricted polypeptides of a protein, comprising isolating said peptide in accordance with the method of  claim 2 : and further comprising:
 (f) identifying or characterizing isolated peptide.   
     
     
         4 . The method of any of  claims 1 - 3 , wherein the soluble human HLA-DM protein is used in step (b)(ii) 
     
     
         5 . The method of any of  claims 1 - 4  wherein the human MHC class II protein is the a DR, DP or DQ protein. 
     
     
         6 . The method of  claim 5  wherein the human class II MHC protein is a DR protein. 
     
     
         7 . The method of  claim 6  wherein the human class II MHC protein is DR1. 
     
     
         8 . The method of any of  claim 1 - 7  wherein, in step (c), the proteolytic digestion is accomplished by a mixture of at least two proteinases. 
     
     
         9 . The method of  claim 8  wherein the at least two proteinases are cathepsins that occur naturally in mammalian endo/lysosomes. 
     
     
         10 . The method of  claim 9  wherein the at least two cathepsins comprise cathepsin B, cathepsin H, cathepsin S or cathepsin L. 
     
     
         11 . The method of  claim 10  wherein the at least two cathepsins are cathepsin B and cathepsin H. 
     
     
         12 . The method of any of  claims 3 - 11  wherein said identifying or characterizing step comprises sequencing said peptide. 
     
     
         13 . The method of any of  claims 1 - 12  wherein said immunodominant epitope is one that can stimulate and/or is recognized by T helper cells. 
     
     
         14 . The method of any of  claims 1 - 12  wherein said immunodominant epitope is an epitope of (i) a self antigen that is associated with or responsible for an autoimmune disease or (ii) an antigen of a pathogen or pathogenic cell. 
     
     
         15 . The method of  claim 14  wherein said immunodominant epitope is of a pathogen or pathogenic cell selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a protozoal antigen, a plasmodial antigen, a helminthic antigen, and a tumor-associated antigen. 
     
     
         16 . A method of producing an immunogenic composition comprising an immunogenic or immunodominant MHC class II restricted peptide from a polypeptide of interest, comprising the steps of:
 (i) producing the isolated peptide in accordance with the method of any of  claims 2  and  4 - 15 ,   (ii) rendering said peptide into an immunogenic form; and   (iii) optionally, adding an adjuvant or other immunostimulatory agent, thereby producing said immunogenic composition.   
     
     
         17 . The method of  claim 16 , wherein said rendering step (ii) comprises coupling said peptide to an immunogenic carrier, producing a peptide multimer of at least two repeating units of said peptide. 
     
     
         18 . A method of inducing an immune response in a host against an antigen, comprising administering to said host an effective amount of an immunogenic composition produced in accordance with  claim 16  or  17 , wherein the peptide is an immunogenic or immunodominant epitope of said antigen. 
     
     
         19 . The method of any of  claims 16 - 18  wherein said immunodominant epitope is an epitope of (i) a self antigen that is associated with or responsible for an autoimmune disease or (ii) an antigen of a pathogen or pathogenic cell. 
     
     
         20 . The method of  claim 19  wherein said immunodominant epitope is of a pathogen or pathogenic cell selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a protozoal antigen, a plasmodial antigen, a helminthic antigen, and a tumor-associated antigen. 
     
     
         21 . A kit comprising:
 (a) a soluble human MHC class II protein or an active homologue thereof from another mammalian species; and   (b) a soluble human HLA-DM protein or an active homologue thereof from another mammalian species; and   (c) at least two proteinases capable of proteolytically digesting exposed regions of a denatured polypeptide of interest.