Method for identifying and validating dominant T helper cell epitopes using an HLA-DM-assisted class II binding assay
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.
1. A method for producing an isolated or purified complex of an 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 polypeptide or 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 polypeptide or optionally 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 polypeptide or optionally denatured polypeptide so that a peptide of about 10 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 of said MHC class II restricted peptide from said polypeptide of interest.
2. A method for producing an isolated 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 eluting or otherwise removing said peptide from said complex and isolating said peptide, thereby producing the isolated MHC class II restricted peptide.
3. The method of claim 1 , wherein the soluble human HLA-DM protein is used in step (b)(ii).
4. The method of claim 1 , wherein the human MHC class II protein is the a DR, DP or DQ protein.
5. The method of claim 4 , wherein the human class II MHC protein is a DR protein.
6. The method of claim 5 , wherein the human class II MHC protein is DR1.
7. The method of claim 1 , wherein, in step (c), the proteolytic digestion is accomplished by a mixture of at least two proteinases.
8. The method of claim 7 , wherein the at least two proteinases are cathepsins that occur naturally in mammalian endo/lysosomes.
9. The method of claim 8 wherein the at least two cathepsins comprise cathepsin B, cathepsin H, cathepsin S or cathepsin L.
10. The method of claim 9 , wherein the at least two cathepsins are cathepsin B and cathepsin H.
11. The method of any one of claims 1 , 2 , and 3 - 10 , further comprising sequencing said MHC class II restricted peptide.