The Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST) Assay: A Sensitive Platform for Monitoring Antitumor Immunity

Abstract

Mutation-associated neoantigens (MANA) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing in vitro cytokine production such as ELISpot and intracellular cytokine staining have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates T-cell receptor sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigenspecific clonotypic amplifications. This assay can be adapted for all types of antigens, including MANAs via tumor exomeguided prediction of MANAs. Following in vitro identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices.

Liquid biopsy in the era of immuno-oncology: is it ready for prime-time use for cancer patients?

“…Lastly, some promising tests have been developed in the field of immuno-oncology using T cells for the analysis [135][136][137][138][139]. T cells can easily be isolated from the patients' blood by density centrifugation and selection of CD4 þ or CD8 þ cells by flow cytometry (using fluorescence or magnetic beads) [135].…”

Molecular T-Cell Repertoire Analysis as Source of Prognostic and Predictive Biomarkers for Checkpoint Blockade Immunotherapy

“…The prediction of the TCR antigen specificity based on the TCR amino-acid sequence alone is extremely challenging. This issue has been recently addressed by generating a large database of antigen-specific TCR sequences and elaborating algorithms that accurately identify the patterns of sequence motifs that correlated with antigen specificity [89]. By this approach, it has been possible to identify neoantigen-specific T-cell clones in the neoadjuvant setting and to track neoantigen-specific T-cell clones in blood upon anti-PD-1 therapy [75].…”

“…Then, the authors addressed the hypothesis that a substantial component of antitumor immunity after PD-1 blockade is directed toward mutation-associated neoantigens. To this end, they used a web-based bioinformatics platform that identify mutation-associated neoantigen-specific T-cell clones [89], and demonstrated that in a patient showing a complete pathological response, T-cell clones specific for mutation-associated neoantigens were rapidly expanded in peripheral blood after neoadjuvant PD-1 blockade [73].…”

Progress Toward Identifying Exact Proxies for Predicting Response to Immunotherapies

“…A technology named MANAFEST (Mutation-Associated Neoantigen Functional Expansion of Specific T Cells), incorporates multiple techniques including WES, T cell receptor sequencing and comprehensive bioinformatics, proposes actionable tumor mutations and even includes peptidestimulated cultures, to ultimately achieve an all-encompassing, anti-tumor immunity surveillance. This approach is uniquely poised to gauge a patient's own immune response to their tumor, but whether its clinical utility will be superior in patients with high or low neoantigen burden, remains to be determined (Danilova et al, 2018). Different mutation loads in tumors are also present in virus-associated cancers, characterized by a specific immunological profile (Sivanandam et al, 2019).…”