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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I3 - Pooled, low-cost synthesis of large-scale T cell receptor libraries

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Despite the critical role that T cell receptors (TCRs) play in immune responses, much is still unknown regarding the precise molecular rules that govern interactions between TCRs and their cognate antigens. A central factor limiting the scale of TCR-antigen pairing studies is the prohibitive cost of TCR library construction – at present, each TCR sequence being studied is often ordered individually as an expensive, full length gene block. To address this challenge, we have developed a novel platform which leverages the inherent modularity of TCR sequences and advances in Golden Gate assembly to enable one-pot production of large paired-chain TCR libraries. Our modular approach achieves a >50-fold reduction in TCR sequence assembly cost relative to conventional methods while maintaining high throughput, fidelity, and ease of usage, enabling researchers to accessibly study libraries containing thousands of custom-synthesized TCRs.

Materials and

Methods:  The alpha and beta chains of the TCR are each composed of a conserved constant domain, one of multiple possible variable domains (TRAV/TRBV), and a short hypervariable loop (CDR3a/b) unique to each TCR. Our protocol hierarchically combines TRAV, TRBV, CDR3a, and CDR3b domains through two nested Golden Gate reactions to form a library of full-length, paired-chain TCR sequences (Fig. 1) In the first step, we react a mixture of 48 TRBV-containing vectors with a pool of >5,000 CDR3a/b-containing fragments. The vector products from this first reaction are then reacted with a mixture of 45 TRAV-containing vectors to form the full paired-chain TCR sequence. Extensive computational and experimental optimization enables both assembly steps to be performed in a pooled format, in which thousands of custom TCR sequences are conveniently assembled in parallel within a single reaction mixture.

Results, Conclusions, and Discussions: We employed Markov Chain Monte Carlo (MCMC) optimization combined with publicly available ligation fidelity datasets to computationally design four highly orthogonal Golden Gate overhang sets for the TRBV and TRAV vectors. We then created a pool of mock vectors and a complementary pool of mock inserts, each containing one overhang pair, to experimentally validate the fidelity of the designed overhang sets. After reacting the vector and insert pools together in a pooled Golden Gate reaction, we sequenced the final assembly products to determine on- and off-target pairing frequencies.

We observed that each overhang almost exclusively paired with its on-target Watson-Crick complement and that off-target pairings were rare. (Fig. 2B) Hence, after filtering sequencing reads to only include circular, transformable products, we observed near-perfect assembly fidelity across all four overhang pools. (Fig. 2B, 2C) Additionally, due to the high efficiency of Golden Gate cloning and the absence of PCR steps in our approach, we avoided library amplification bias and observed very minimal, tolerable depletion levels across all vectors in the pool. (Fig. 2C) Building on these promising results, we began conducting realistic TCR assembly tests using proper TRAV/TRBV vectors. We recently demonstrated successful assembly of full-length TCRs at a small scale and are preparing to soon perform a large-scale 5,000 TCR assembly test.

Overall, we have developed a novel platform which enables users to easily and accurately translate a custom list containing thousands of TCR sequences into a synthetic TCR library at a cost of <$1 per TCR and only using readily available Golden Gate reagents. These TCR libraries can then be transduced into immortalized TCR-expressing cell lines, enabling researchers to conduct TCR-antigen pairing experiments and/or therapeutic TCR screens at an unprecedentedly large scale.

Acknowledgements (Optional):  Pryor, J. M. et al. Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PLOS One 15, e0238592 (2020).