Validation of Neoantigen In Silico Prediction using QuickSwitch™ Kits

Neoantigens originate from tumours and are not present in normal tissues. Research findings indicate that neoantigens have the potential to activate tumour-specific T-cell-mediated anti-tumour immune responses, making them promising targets for immunotherapy.

Chuwdhury, Gulam Sarwar, et al., 2023 developed ImmuneMirror as a stand-alone open-source pipeline (https://github.com/weidai2/ImmuneMirror/) and a web server (http://immunemirror.hku.hk/App/) incorporating a balanced random forest model for neoantigen prediction and prioritisation; the model was trained and tested using known immunogenic neopeptides collected from 19 published studies. This study is the most extensive examination of neoantigen prediction models, assessing them comprehensively using experimentally validated neopeptides (1). This pipeline was utilised to identify neoantigens originating from somatic mutations in cancer-related genes with common MHC class I subtypes in Pan-Cancer studies and from real-world WES and RNASeq data from GIT cancer patients (1). 

The QuickSwitch^TM Quant HLA-A02:01 Tetramer Kit-PE was used to validate the binding affinity between the chosen neoepitope and MHC HLA-A02:01. The binding affinity between HLA-A02 and neoepitopes derived from various mutations at G245 in TP53 were assessed using the QuickSwitch Quant HLA-A*02:01 Tetramer Kit-PE. Results indicated the neoepitope TP53G245V (YMCNSSCMGV) had a higher reference peptide exchange rate of 97.03% than the wild-type peptide YMCNSSCMGG (80.8%). The binding affinity of neoepitope-TP53G245V (YMCNSSCMGV) was the highest among TP53G245R, TP53G245D, TP53G245C, and TP53G245S. These results provide evidence of the effectiveness and reliability of ImmuneMirror as a cutting-edge tool for predicting neoantigens (1).

A different study by Xiao, Jingyu, et al., 2023 used whole exome sequencing (WES) data of 364 HCC patients in The Cancer Genome Atlas database and predicted 25 highly potential immunogenic neoantigens to human leukocyte antigen (HLA)‐A*02:01 in silico (2). To confirm the binding strength of the six neoantigens, the study utilised QuickSwitch™ Quant HLA-A*02:01 Custom Tetramer-PE kit to identify the frequencies of antigen-specific CD8+ T cells in CTLs. The generated tetramers detected proportions of neoantigen-specific CTLs. The positive frequencies of antigen-specific CD8+ T cells suggested that all six functional HCC neoantigens exhibited a high binding affinity to HLA-A*02:01 (2).

MBL International offers QuickSwitch™ Custom Tetramers kit for you to create your own custom tetramer. This kit is supplied with a tetramer bound with an irrelevant peptide. After adding a peptide exchange factor and your peptide of interest, peptide exchange will occur if there is binding affinity of the peptide with the MHC molecule. By using the QUANT kit, you can quantify the amount of exchange that occurred, thereby gathering binding affinity information for the MHC/peptide complex. This kit can be used to create custom tetramers, or for screening peptides, all in one day. QuickSwitch™ is an assay system that allows discrimination of MHC binding from non-binding peptides.

REFERENCES:

1. Chuwdhury, Gulam Sarwar, et al. “ImmuneMirror: a Machine Learning-based Integrative Pipeline and Web Server for Neoantigen Prediction.” bioRxiv (2023): 2023-02.
2. Xiao, Jingyu, et al. “Engineering In Vitro Organ‐Structured Tumor Model for Evaluating Neoantigen‐Specific T Cell Responses in Hepatocellular Carcinoma.” Advanced Materials Interfaces (2023): 2300155.

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