Therapeutic vaccines have been in development for decades with the goal of priming and boosting an immune response against specific antigens in cancer patients. More recently, vaccination with mutation-derived neoantigens has emerged as is an attractive approach to treat cancer patients. However, appropriately selecting immunogenic antigens and neoepitopes that successfully prime the immune system of cancer patents remains a challenge. In fact, despite extensive efforts throughout the years, many vaccines have proven to be ineffective in mediating a clinically relevant anti-tumor immune response in humans. With the advent of emerging new technologies, including the combination of next-generation sequencing and improved bioinformatics tools, a new era of vaccine development is underway. Hereto, we present a novel, highly sensitive, multiplex approach, known as Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA), that combines high-throughput TCR repertoire sequencing with conventional immune monitoring techniques to assess T-cell specificity to large numbers of query antigens. MIRA maps antigen specificity to TCR sequence at an unprecedented sensitivity (1 in ~10 million T-cells) and scale (more than 400 antigens at a time). To validate this approach, we assessed the response of more than 10 million naïve human T-cells from each of 50 healthy donors against a panel of 366 neoepitopes. These include post-translationally modified peptides, derived from over 130 prevalent mutations in greater than 50 cancer indications. From three billion total input T-cells, we identified tens of thousands of TCRB sequences from responsive T-cell clones, of which at least one TCR was yielded for the majority of queried neoepitopes. The TCRs identified were skewed toward a small number of query antigens, with 12 neoepitopes accounting for over 50% of the TCRs identified. This suggests a higher precursor frequency of specific T-cells in the naïve repertoire or a higher immunogenicity of these epitopes or both. By performing MIRA experiments in parallel using transgenes versus peptides, we can further validate which of the neoepitopes were also presented via cells’ natural antigen presentation machinery. Of note, we also paired 52% of the TCRB sequences with their cognate TCRA subunit sequence thus permitting reconstitution of individual TCRs. Generating immunogenicity data to antigens of interest at scale facilitates an opportunity to better inform antigen selection in vaccine programs and improve vaccine design. Once a vaccine has been designed and administered to a patient, MIRA offers a unique solution to assess peptides based on their ability to elicit robust T responses. By creating a post-vaccination, patient-specific MIRA, a permanent record or signature of that patient’s response to the vaccine is created. This information can then be leveraged to identify early biomarkers of response to the vaccine and monitor this response longitudinally by simply using single chain TCRB deep sequencing from a small amount of a patient’s peripheral blood.

Citation Format: Peter Ebert, Mark Klinger, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins. Optimization of cancer vaccine development by using Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA) [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B115.