Method and system for neoantigen analysis
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Publication Number
US-11977057-B2
Publication Date
2024-05-07
Expiration Date
Abstract
A method for characterizing a target peptide through a detection approach such as mass spectrometry is provided, including: introducing at least one guard molecule to mix with the target peptide; and applying the detection approach for the characterization of the target peptide. Each guard molecule is configured to have similar characteristics as the target peptide, yet is still distinguishable therefrom by the detection approach, such as having a mass spectrometry-distinguishable different M/z value compared with the target peptide. The method can be used to characterize a neoantigen peptide through mass spectrometry, upstream of which the method can further include steps for tissue sample preparation, HLA molecules enrichment, elution, clean-up, and purification. Some or all of these steps can be configured to be executed in a substantially automatic manner with little or no manual intervention. A system for implementing the neoantigen analysis method is further provided.
Core Innovation
The invention relates to a “Valid-NEO” integrated neoantigen analysis method and system in which one or more guard molecules, including guard peptides, are mixed with a target peptide prior to liquid chromatography coupled to mass spectrometry (LC-MS). Each guard is configured to be similar to the target for sample-preparation co-behavior while remaining distinguishable in mass spectrometry by having an m/z value distinguishable from the target peptide.
The method includes subjecting the guard peptide(s) and the target peptide to liquid chromatography and using the guard peptide(s) to block nonspecific binding of the target peptide to any surface or any substance. After LC, the target peptide is detected by mass spectrometry analysis based on the distinguishable m/z values of the guard peptides, enabling characterization of the target peptide for specified neoantigen peptides.
The disclosed clinical workflow for tissue samples includes frozen tissue grinding and HLA enrichment using anti-HLA antibody immobilized column processing, followed by low-pH elution, trap-column cleanup, and size exclusion chromatography (SEC)-based purification. The system is described as having substantial automation and minimal manual intervention, and it includes components such as the grinding device (UniCeller), HLA enrichment column, trap column, SEC column, and the mass spectrometer.
The document further describes use of MaxRec pre-conditioning peptides to saturate nonspecific binding and improve recovery/sensitivity, and it reports detection/quantification of specified neoantigen peptides with reproducibility assessment. Reported performance includes an average sensitivity increase up to about 27-fold, and downstream analysis is integrated with the Valid-NEO pipeline for characterizing neoantigen peptides.
Claims Coverage
The claim set centers on three main inventive features in an LC-MS workflow for target peptide characterization, with additional dependent refinements for guard composition and upstream tissue preparation.
Guard peptide mixing for target peptide characterization
A method for characterization of a target peptide that mixes at least one guard peptide with the target peptide, wherein the at least one guard peptide has a different amino acid composition and sequence from the target peptide.
Nonspecific binding blocking during liquid chromatography
A method in which the at least one guard peptide and the target peptide are subjected to liquid chromatography, wherein the at least one guard peptide blocks nonspecific binding of the target peptide to any surface or any substance.
Distinguishable guard m/z enabling mass spectrometry detection
A method in which the target peptide is detected by mass spectrometry analysis, wherein each of the at least one guard peptide is configured to have an m/z value that is distinguishable from the target peptide by the mass spectrometry analysis.
Heavy isotope-labeled guard peptide feature
The at least one guard peptide includes at least one amino acid residue that is a heavy isotope-labeled amino acid.
Single-residue difference between guard and target
Only one amino acid residue differs between a guard peptide and a target peptide.
Frozen tissue sample preparation for neoantigen peptide characterization
The method further includes treating a frozen tissue sample by grinding frozen tissue to obtain a frozen single-cell tissue powder and providing a treated tissue sample prior to mixing the at least one guard peptide with the target peptide.
HLA enrichment using an anti-HLA antibody immobilized column
The method further includes enriching human leukocyte antigen (HLA) molecules from a prepared treated tissue sample by passing the sample through an HLA enrichment column containing a matrix with immobilized anti-HLA antibodies before mixing.
Trap-column cleanup to obtain a cleaned eluate
The method further includes, after elution and before subjecting the at least one guard peptide and the target peptide to liquid chromatography, passing the eluate through a trap column that binds neoantigen peptides with lower or no affinity for impurities, then eluting the trap column to produce a cleaned eluate.
The claims collectively cover mixing sequence-different guard peptides with a target peptide, blocking nonspecific binding during liquid chromatography, and detecting the target by mass spectrometry using distinguishable guard m/z values, with dependent refinements for guard composition and tissue/HLA enrichment and cleanup steps.
Stated Advantages
Improved sensitivity and recovery as described using MaxRec pre-conditioning peptides.
Average sensitivity increase reported up to about 27-fold.
Enables detection and quantification of specified neoantigen peptides with reproducibility assessment.
Documented Applications
Clinical tissue sample neoantigen analysis workflow for characterizing target neoantigen peptides, including specified KRAS, TP53, and IDH2 neoantigen peptides, using an LC-MS Valid-NEO approach with guard peptides and HLA enrichment.
Integrated Valid-NEO system including frozen tissue grinding, HLA enrichment, trap-column cleanup, and SEC-based purification as part of upstream clinical tissue processing.
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