Abstract 3110: An enhanced and cost-efficient method for neoantigen immunogenicity screening method: Double Barcode Neoepitope ScanTM

Abstract

Abstract Neoantigens, HLA bound peptides generated by tumor-specific mutations, have emerged as promising targets for cancer immunotherapeutic. However, the significant challenge in neoantigen-based therapies is that the neoantigens are rare and difficult to predict. Therefore, in addition to prediction algorithms, the optimization of validation methods is essential for the reliable selection of highly immunogenic neoepitopes. In this study, we have developed a cost-effective screening method to validate large scale of neoantigen candidates. The DNA library-based methods have been employed for antigen selection and T cell receptor identification. We improved the DNA library-based neoantigen screening method by adding two barcodes to each minigene transcribing multiple neoantigen candidates and pooled synthesis of minigenes. We initially designed the barcodes to allow analysis in a 10 by 10 format for screening of hundreds of neoantigens so that PCR with each barcode result in amplification of ten candidates. The second barcode allowed the deconvolution of immunogenicity results without the need for synthesis of individual peptides. In this method B cell was used as antigen-presenting cell, which were co-transfected with mRNA for a minigene pool, 41BBL, and OX40L after expansion with CD40L/IL4/IL21. The resulting B cells were co-cultured with T cells and T cell responses were evaluated using ELISpot. To validate the method the TESLA consortium reported tetramer positive neoantigens were included in minigene. In results, the neoantigens including each barcode were amplified to form a pool of ten neoantigens as a minigene and confirmed that the neoantigens were equally amplified by nested PCR. The neoantigen pools synthesized as mRNA were transfected for presentation in B cells. The result of the targeting a neoantigen pool containing tetramer-positive neoantigens of the TESLA consortium showed a positive for ELISpot. In conclusion, our optimized screening method, employing the double barcode system, offers a time- and cost-efficient approach for the selection of immunogenic neoantigens without the need for individual peptides. This method will facilitate large-scale screening to identify actual neoantigens with immunogenic potential, contributing to the development of neoantigen-targeted vaccines. Citation Format: Seong Gwang Kim, Min-Ho Jung, Hyun-Ji Hwang, Sejin Oh, Seong-Eui Hong, Soonmyung Paik, Hae-Suk Kim. An enhanced and cost-efficient method for neoantigen immunogenicity screening method: Double Barcode Neoepitope ScanTM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3110.