Abstract A002: cloneTCR: A high-throughput TCR assembly platform for highly multiplexed screening

Abstract Manufacturing T cell receptors (TCRs) de novo has previously been considered costly and time-consuming, limiting the widespread application of TCR transgenic T cell immunotherapies. Here, we introduce a fully automated, cost-effective, and high-throughput TCR cloning platform capable of assembling and quality controlling thousands of TCRs per week. Our pipeline leverages the widely adopted AIRR (VDJ sequence) format as input, generating scripts automatically for streamlined operation of our lab automation. The platform is highly modular and adaptable, accommodating both human and murine V(D)J sequences alongside a broad spectrum of modular constant regions. These constant regions are available in germline-encoded or engineered versions, all designed for orthogonal compatibility. Additionally, the system supports multiple vector formats, enabling diverse delivery solutions such as lentiviral vectors, transposons, direct in vitro transcription, and recombination-based vectors targeting loci like AAVS1 and TRAC. A key innovation of our platform is the integration of Oxford Nanopore-based bulk long-read sequencing for quality control, enabling precise assessment of library uniformity and cloning fidelity. Compared to existing approaches, our method achieves superior library uniformity (frequency 2.5x around the median) and high sequence accuracy of 95%. With its unparalleled capacity to clone and quality-check 2,000 TCRs a week, our platform sets a new benchmark for high-throughput TCR production and screening. It enables large-scale, multiplexed exploration of entire tumor-infiltrating lymphocyte (TIL) repertoires, paving the way for transformative advances in T cell immunotherapy. Citation Format: Moritz Hamberger, Marie-Therese Neuhoff, Michael Platten, Edward Wilhelm Green. cloneTCR: A high-throughput TCR assembly platform for highly multiplexed screening [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A002.