Rapid Assembly of Oligo Pools into Medium-Scale T Cell Receptor Lentiviral Libraries 2267508

Abstract Introduction Synthesis and construction of T cell receptor cDNA is one of the main bottlenecks to large-scale T cell receptor (TCR) antigen screening. New technologies allow for massive screening of TCRs against hypothesized antigens[1,2,4], including the multiplexed barcoded pMHC tetramer screening technology[2] developed in our lab. However, validation of discovered TCRs requires synthesis and cloning that costs upwards of $100 per TCR and is unsustainable for antigen screening labs. Methods have been published that seek to overcome this bottleneck[5,6] but have significant start-up costs and take multiple days of benchwork. Herein we describe a workflow that rapidly generates small pools of TCRs at a cost of ∼ $10 per TCR. Methods In this method, we synthesized pools of alpha and beta CDR3s linked in single oligos to ensure pairing fidelity of the two chains. A proprietary set of CDR3-TRV overlap sequences were computationally generated with codon substitution to maximize overlap diversity while maintaining homogenous melting temperature and GC content. Our method only requires 6 steps which can be completed in one day. In our preliminary investigation, we successfully synthesized and assembled a pool of 13 TCRs through a series of assembly PCRs. Results Sequencing of the plasmid pools confirmed that all TCRs were present in the final plasmid pool. Transduced J76 cells were stained with anti-human TCR antibody and tetramer confirming expression of the antigen specific T-cell clones in the pool. Conclusion This novel method overcomes a major bottleneck in antigen screening pipelines by providing rapid synthesis and assembly of TCRs in a pool. Future work will identify the upper limit of TCR assembly for this method and demonstrate its screening utility using tetTCR-seqHD. (1) Dezfulian M, +, Cell 2023; (2) Zhang SQ, +, Nat. Biotech. 2018; (4) Birnbaum M, +, Cell 2014; (5) Moravec Z, +, Nat. Biotech. 2025; (6) Ganglione S, +, BioRxiv 2025 Funding Source NIH R33 IMAT 2021 Topic Categories Technological Innovations in Immunology (TECH)