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Emerging Cell Therapies: Realizing the Vision of NextGen Cell Therapeutics | EK15


High-throughput screening for rare antigen-reactive TCRs using natively-paired TCRab expression libraries generated from millions diverse primary T cells.


Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Description

High-throughput screening for rare antigen-reactive TCRs using natively-paired TCRab expression libraries generated from millions diverse primary T cells. Matthew J. Spindler1, Ayla L. Nelson1, James M. Heather2, Ellen K. Wagner1, Adam S. Adler1, David S. Johnson1 1 GigaMune, Inc., 1 Tower Place, Suite 750, South San Francisco, CA 94080, USA 2 Massachusetts General Hospital, 275 Cambridge St., Cambridge, MA 02114, USA ABSTRACT Engineered TCR-T cells can provide potent cancer therapies by targeting HLA presented peptides derived from tumor associated antigens, oncoviral antigens, and neoantigens. Current clinical trials are limited to a handful of antigen-reactive TCRs with the vast majority targeting HLA-A0201 presented peptides. The efficient identification of clinical candidate TCRs is needed to broaden the treatable patient population. Single cell sorting of pHLA-binding T cells using multimers and ex vivo antigen expansion are the current gold standards for identifying antigen-reactive primary T cells. However, functional validation of the TCRs identified by these approaches requires resource intensive cloning of each individual TCRab pair. Additionally, primary T cells, especially tumor infiltrating lymphocytes (TILs), are a limited resource, which restricts the number of antigens that can be screened. To address this, we developed a microfluidic approach to capture and functionally express natively-paired TCRab libraries from millions of single T cells. Unlike DNA barcoding approaches that mark single cells by adding a sequence tag, we physically link the TCRa-TCRb chains to generate sequencing and full-length expression libraries which we introduce into Jurkat cells for functional testing. Using these methods, we are building expression libraries from healthy and cancer patient samples; these libraries include over 2.9 million TCRab clonotypes from six healthy PBMC donors and over 0.5 million from expanded melanoma TIL samples. We applied pMHC binding and cellular activation screens to identify and validate 14 TCRs reactive to common viral and tumor associated antigens with starting frequencies of

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