Document resource
Background Neoantigens (NeoAg) are ideal targets for effective anti-tumor immune responses and immunotherapies; however, for their clinical application, more accurate and efficient methods for their identification are required. We have developed the ‘IPV’ (identify-prioritize-validate) approach, which includes a computational algorithm to prioritize candidate NeoAg followed by an in vitro stimulation assay with peripheral blood mononuclear cells (PBMCs) to functionally validate NeoAg-specific T cell responses. We now report the use of a novel T cell receptor (TCR) sequencing method (TIRTL-seq) that allow us to identify, clone and validate NeoAg-specific TCRs from PBMC of cancer patients.Methods A patient with uterine Leiomyosarcoma (Hu_436) with low mutational burden (0.8 mutations per megabase) was selected for this study. We used the ‘IPV’ approach to identify NeoAg with some modifications. 1 TCR sequencing was performed by TIRTL-seq,2 and candidate TCRs were synthesized and expressed in a reporter cell line to validate their capacity to recognize NeoAg.3 Results 34 prioritized candidate mutations were tested by ELISPOT assay following in vitro stimulation with measurable T cell responses detected to 5 as NeoAg. In parallel, TCR sequencing from pre- and post-in vitro stimulation PBMC samples was performed by TIRTL-seq.2 Synthetic reconstruction of TCR genes from expanded clonotypes and subsequent functional testing identified 14 NeoAg-specific TCRs recognizing 4 distinct NeoAg in the context of MHC-II. The majority of TCRs tested were able to discriminate between the NeoAg peptide and corresponding wildtype peptide.Conclusions The IPV platform enable to identify and verify multiple NeoAg in cancer patient with low mutational burden cancer. Neoantigen-specific CD4 T cells response can be found from in vitro expansions from peripheral blood of patients. IPV + TIRTL facilitates the identification and subsequent validation of diverse NeoAg-specific TCR clones rapidly and in a cost-effective manner.References Miller AM, Koşaloğlu-Yalçın Z, Westernberg L, Montero L, Bahmanof M, Frentzen A, Lanka M, Logandha Ramamoorthy Premlal A, Seumois G, Greenbaum J, Brightman SE, Soria Zavala K, Thota RR, Naradikian MS, Makani SS, Lippman SM, Sette A, Cohen EEW, Peters B, Schoenberger SP. A functional identification platform reveals frequent, spontaneous neoantigen-specific T cell responses in patients with cancer. Sci Transl Med. 2024 Feb 28;16(736):eabj9905. doi: 10.1126/scitranslmed.abj9905. Epub 2024 Feb 28. Erratum in: Sci Transl Med. 2024 May 8;16(746):eadp8153. doi: 10.1126/scitranslmed.adp8153. PMID: 38416845.Pogorelyy MV, Kirk AM, Adhikari S, Minervina AA, Sundararaman B, Vegesana K, Brice DC, Scott ZB; SJTRC Study Team; Thomas PG. TIRTL-seq: deep, quantitative, and affordable paired TCR repertoire sequencing. bioRxiv [Preprint]. 2024 Oct 31:2024.09.16.613345. doi: 10.1101/2024.09.16.613345. PMID: 39345544; PMCID: PMC11430070.Cetin M, Pinamonti V, Schmid T, Boschert T, Mellado Fuentes A, Kromer K, Lerner T, Zhang J, Herzig Y, Ehlert C, Hernandez-Hernandez M, Samaras G, Torres CM, Fisch L, Dragan V, Kouwenhoven A, Van Schoubroeck B, Wils H, Van Hove C, Platten M, Green EW, Stevenaert F, Felix NJ, Lindner JM. T-FINDER: A highly sensitive, pan-HLA platform for functional T cell receptor and ligand discovery. Sci Adv. 2024 Feb 2;10(5):eadk3060. doi: 10.1126/sciadv.adk3060. Epub 2024 Feb 2. PMID: 38306432; PMCID: PMC10836725.Ethics Approval The project was review and approved by IRB (0009940) in accordance with the requirements of the code of federal regulation of human Subjects (45 CRF and 21 CRF 50 and 56).