This technology involves the use of peptide-receptive MHC-I molecules in complex made using the TAPBPR chaperone. The peptide receptive MHC-I can be immobilized on chromatography columns or magnetic beads. They can provide unprecedented levels of highly specific peptide recovery
The MHC-I proteins are extremely polymorphic (more than 13,000 different alleles have been identified in the human population to date), and each allele can display an estimated 1,000-10,000 different peptides. This makes the prediction of actively presented MHC-I peptides challenging. The current state-of-the art for the identification of MHC-I displayed pepties is the use of Liquid Chromatography-Tandem Mass spectroscopy (LC/MS/MS). This technique allows the identification of the masses of thousands of peptides extracted from MHC molecules in relevant biological samples. The derived peptide sequences can be cross-referenced with protein sequence databases.
Although LC/MS/MS is an extremely powerful technique, the recovery of peptides from purified MHC molecules prior to LC/MS/MS using standard acid elution protocols is subject to losses during the peptide purification process. Up to 80% of relevant peptides are not recovered in most applications. This is a major challenge, as the lost peptides can be important targets for both understanding antigen processing and presentation processes and the development of immunotherapies to combat bacterial and viral infections and cancer.
MHC-I peptide libraries for peptidome analysis have historically been generated by acid extraction of the peptides from cells or by MHC-I immunoprecipitation and acid extraction. In addition to the 80% loss mentioned above, an unacceptably high percentage of peptides derived using the acid extraction from cell lysate or cell surface is nonspecific for the MHC-I. The comparison of peptide repertoires extracted from MHC-I-expressing and β2-microglobulin knockout cells revealed that approximately 50% of the peptides identified as MHC-I peptide ligands are false positives not from an MHC class I binding groove.
In addition, cells can express multiple (up to six) different MHC-I alleles, further complicating analysis. And such cells need to express high levels of MHC-I - which tumor and infected cells downregulate.
The use of highly purified, recombinant peptide receptive MHC-I molecules made using TAPBPR efficiently overcomes these bottlenecks. It provides the high concentration of MHC-I molecules necessary for reliable and sensitive peptidome analysis in samples of any origin. The use of affinity tags attached to the recombinant proteins provides the specific binding for MHC-I molecules and diminishes contamination with non-specific peptides.
The method is modular and flexibile enough to support a towards a range of applications, including performing immune repertoire characterization of patient samples. Examples are below:
In this example, patient samples are collected at field sies and applied to columns containing peptide-receptive MHC-I made using TAPBPR. MHC-I molecules enriched with bound ligands can be purified and sent to centralized clinical/research centers for MHC-I epitope identification (e.g. using LC/MS/MS), cross referencing with epitope databases, or using machine learning techniques to predict specificities of enriched peptide ligands for various MHC-I alleles.
These peptides can then be used to produce diagnostic MHC-I tetramers for the immunodominant epitopes that can be sent back to the field for diagnostic and longitudinal surveys as well as testing the eficiency of vaccine or drug trials. The epitope data can also be used in vaccine development.
Identfication of MHC-I antigenic peptides
Generation of MHC-I peptide libraries from infected patients
Generation of MHC-I peptide libraries from cancer patients
Generation of MHC-I peptide libraries from autoimmune patients
Identification of Immunodominant Epitopes
Minimal loss of antigenic peptide
Ease of use
Minimal false positives
|United States Of America||Published Application||20230059548||02/23/2023||2019-975|
|European Patent Office||Published Application||4085069||11/09/2022||2019-975|
MHC-I epitope, T cell receptor specificity, T cell antigen recognition, CD8 T cell epitope, MHC-I peptide library