Cell-Based Genetic Immunopeptidomic Screen for Targeted Epitope Identification
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Solution Overview
Problem
Current methods for identifying MHC-I binding peptides are limited by their inability to target specific pathogen or tumor-derived epitopes, as they sample the entire cellular proteome, making it difficult to evaluate T cell epitopes generated from particular pathogens or tumors.
Innovation Solution
A cell-based genetic immunopeptidomic screen using DNA oligonucleotide synthesis to generate pre-defined libraries for targeted immunopeptidomics, where cells are engineered to express specific HLA alleles and candidate epitope peptides, allowing for the rapid empirical determination of MHC-I binding peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If mass spectrometry is used to identify MHC-I ligands, then large-scale experiments can identify roughly a thousand peptides eluted from any given HLA allele, but the method must sample peptides derived from the entire cellular proteome and cannot be readily adapted to permit targeted evaluation of T cell epitopes generated from a particular pathogen or neo-antigens
Solution Approach 1:
The patent segments the peptide evaluation process by dividing the cellular proteome into specific pathways. It uses TAP-deficient cells to separate classical pathway peptides (from cytosolic proteins) from alternative pathway peptides (from extracellular proteins). This segmentation enables targeted evaluation of specific pathogen or tumor-derived epitopes while maintaining the ability to identify multiple peptides through the segmented evaluation of different protein sources.
2Adaptability or versatility
If cells express multiple HLA alleles, then comprehensive peptide presentation occurs, but the diverse array of MIHC-I molecules and their highly polymorphic nature across the human population complicates understanding of peptide binding rules
Solution Approach 1:
The patent extracts and isolates specific HLA alleles from the complex mixture of multiple HLA alleles expressed in normal cells. By using cells that are deficient in endogenous HLA-I molecules and introducing specific HLA alleles via viral vectors, the method extracts and studies individual HLA alleles in isolation. This allows comprehensive understanding of peptide binding rules for each allele without the complicating factor of polymorphic diversity across the human population.
3Adaptability or versatility
If the entire cellular proteome is sampled for peptide identification, then all possible epitopes are evaluated, but it becomes difficult to evaluate T cell epitopes generated from a particular pathogen or neo-antigens presented by a particular tumour
Solution Approach 1:
The patent applies local quality by focusing the peptide evaluation on specific local sources rather than the entire proteome. It uses TAP-deficient cells to locally evaluate peptides from specific protein sources (pathogen or tumor proteins) while excluding peptides from other sources. This localized evaluation approach maintains comprehensive coverage for the targeted proteins while improving measurement precision for pathogen or tumor-derived epitopes by eliminating interference from other proteomic sources.
Data Source
AI summary
Described herein are methods for identification of peptides that bind MHC-I molecules from within a starting pool of candidate epitope peptides, using a cell-based genetic immunopeptidomic screen.