Method for the characterization of peptide:MHC binding polypeptides
Inventors
Schuster, Heiko • Hutt, Meike • Weinschenk, Toni • Bunk, Sebastian • Schoor, Oliver • BACKERT, Linus • Hofmann, Martin • FRITSCHE, Jens • Unverdorben, Felix • SCHIMMACK, Gisela • SALOPIATA, Florian
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12480958-B2
Publication Date
2025-11-25
Expiration Date
Abstract
The present invention relates to a method for the characterization of peptide:MHC binding polypeptides, e.g. by mass spectrometry and an analysis of the recognized peptide space, i.e. in order to identify peptides that can be bound in the context of their presentation by MHC, and those who cannot be bound.
Core Innovation
The invention describes a method for identifying the binding of a peptide: MHC-binding polypeptide that binds to a target peptide in complex with a major histocompatibility complex (MHC) molecule as a peptide: MHC complex. The peptide: MHC-binding polypeptide comprises a T-cell receptor (TCR) and/or an antibody or a pMHC binding fragment thereof. The method is applied to identify binding to off-target peptide: MHC complexes, in particular from a cellular lysate sample.
In the method, a first fraction of a cellular lysate comprising a first off-target peptide: MHC complex is contacted with the pMHC binding polypeptide so that the at least one peptide binding domain binds to the first off-target peptide: MHC complex. A first off-target peptide is isolated from the first off-target peptide: MHC complex bound to the peptide binding domain, and the first off-target peptide is identified by peptide sequencing.
The method further includes a second fraction containing a second off-target peptide: MHC complex, where the second fraction is contacted with an MHC pan-specific antibody or an antigen binding fragment capable of binding to an MHC molecule irrespective of the nature of the bound peptide. After contacting with the MHC pan-specific antibody, a second off-target peptide is isolated from the second off-target peptide: MHC complex bound to the MHC pan-specific antibody or fragment and identified by peptide sequencing.
The identifying steps comprise peptide sequencing, and the first off-target peptide and the second off-target peptide comprise an identical amino acid sequence, thereby enabling comparison of peptide capture associated with the pMHC binding polypeptide and peptide: MHC isolation mediated by an MHC pan-specific antibody.
Claims Coverage
The partial content provides one independent claim (clm-00001). It covers a two-part peptide: MHC off-target identification workflow using a pMHC-binding polypeptide and a separate MHC pan-specific antibody isolation, followed by peptide sequencing with an identical peptide criterion.
Off-target peptide identification from cellular lysate using a pMHC binding polypeptide
A method for identifying binding of a pMHC binding polypeptide comprising a T-cell receptor (TCR) and/or an antibody or a pMHC binding fragment thereof, to off-target peptide: MHC complexes in a sample comprising a cellular lysate, comprising contacting and isolating a first off-target peptide: MHC complex bound to the peptide binding domain and identifying the first off-target peptide by peptide sequencing.
MHC pan-specific antibody peptide: MHC isolation as a parallel identification step
A method further comprising contacting a second fraction of the cellular lysate comprising a second off-target peptide: MHC complex with an MHC pan-specific antibody or an antigen binding fragment thereof capable of binding to an MHC molecule irrespective of the nature of the bound peptide, isolating a second off-target peptide from the second off-target peptide: MHC complex bound to the MHC pan-specific antibody or fragment, and identifying the second off-target peptide by peptide sequencing.
Identical amino acid sequence criterion linking the two peptide identifications
The identifying steps in the method are performed by peptide sequencing such that the first off-target peptide and the second off-target peptide comprise an identical amino acid sequence.
Across the independent claim, inventive coverage centers on using a pMHC-binding polypeptide to capture off-target peptide: MHC complexes from a cellular lysate, using peptide sequencing to identify the captured off-target peptides, and incorporating a parallel peptide: MHC isolation using an MHC pan-specific antibody where the two identified off-target peptides match by identical amino acid sequence.
Stated Advantages
Documented Applications
No documented applications found
Interested in licensing this patent?