Identifying peptides having T-cell-exposed motifs with known frequency of occurrence in a reference database
The present invention provides methods and systems for identifying and classifying epitopes and use of that information to analyze proteins and peptides within proteins, especially potential epitopes, and to use the information to design synthetic peptides and proteins, analyze biopharmaceutical proteins, and diagnose autoimmune conditions. Peptides which are bound in MHC grooves comprise two sets of amino acids: those that face inwards into the groove and determine the binding affinity to the MHC molecule (the groove exposed motifs or GEM) and those which do not interact with the groove but rather are on the obverse side exposed outwardly to the T-cells (the T-cell exposed Motifs or TCEM). The present invention utilizes information related to the identity and physiochemical characteristics of the GEM and TCEM.
1. A method to identify peptides in a target protein having one or more T-cell-exposed motifs with a known frequency of occurrence in a reference database of T-cell-exposed motifs and synthesize a biomolecule encoding the identified peptides comprising:
establishing a reference database of T-cell-exposed motifs by:
assembling a database of reference proteins comprising at least 40,000 proteins,
extracting all sequential 9-mer and 15-mer peptide sequences with a single amino acid displacement from said reference proteins, wherein said T-cell-exposed motifs are those subsets of amino acids within a peptide which, if bound in a MHC molecule, are directed outwards and exposed to T-cell binding, comprising for a possible MHC I binding 9-mer peptide, amino acid positions 4, 5, 6, 7, 8 of a 9-mer and comprising, for a possible MHC II binding 15-mer peptide, amino acid positions 2, 3, 5, 7, 8 or -1, 3, 5, 7, 8 based on a 15-mer peptide with a central core of 9 amino acids numbered 1-9 and positions outside that core numbered as negative (N terminal) or positive (C terminal),
identifying said T-cell-exposed motifs in said peptide sequences, and
categorizing said T-cell: exposed motifs based on the frequency of occurrence of said T-cell-exposed motifs in said reference database; and
determining the relative frequency of each of said T-cell-exposed motifs, thereby providing said reference database of T-cell-exposed motifs;
extracting peptide sequences from said target protein and identifying T-cell exposed motifs in said target protein;
comparing said T-cell-exposed motifs from said target protein to the frequency of occurrence of the same T-cell-exposed motifs in said reference database of T-cell-exposed motifs to identify one or more peptides in said target peptide containing one or more T-cell-exposed motifs having a known frequency of occurrence in said reference database; and
cloning said one or more peptides from said target peptide into an expression vector and recombinantly synthesizing a biomolecule selected from the group consisting of a protein and nucleic acid encoding the one or more peptides.
2. The method of claim 1 wherein said reference proteins are selected from the group consisting of immunoglobulin proteins, proteins of the human proteome other than immunoglobulins, protein allergens and microorganism proteins.
3. The method of claim 1 , wherein said categorizing further comprises determining whether said T-cell-exposed motifs in said target protein occur with a frequency of greater than 1 in 64 in said reference database of T-cell-exposed motifs.
4. The method of claim 1 , wherein said categorizing further comprises determining whether said T-cell-exposed motifs in said target protein occur with a frequency of greater or less than 1 in 1024 in said reference database of T-cell-exposed motifs.
5. The method of claim 1 , further comprising the step of characterizing the overall immunogenicity of said target protein by ranking of the frequency of occurrence of a plurality of said T-cell-exposed motifs in said target protein.
6. The method of claim 1 , further comprising determining the predicted WIC binding affinity of said peptide sequences comprising said T-cell-exposed motifs derived from said target protein by determining the WIC allele-specific binding of the groove exposed motifs corresponding to each T-cell-exposed motif and modifying the one or more peptide sequences to have an increased or decreased WIC binding affinity.
7. The method of claim 1 , further comprising
designing modifications in the one or more peptides selected from said target protein wherein said modifications are selected to provide a lower or higher frequency of representation of T-cell-exposed motifs as compared to said reference database of T-cell-exposed motifs.