Method for identifying and validating dominant T helper cell epitopes using an HLA-DM-assisted class II binding assay

摘要

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.

主权项

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.