A novel high-throughput screening approach for the detection of cytotoxic T-cell receptor epitopes
Sharma, G1,2, Holt, RA1,2
1University of British Columbia, Vancouver BC
2BC Cancer Agency, Vancouver BC
One of the key challenges in the effort to develop more potent adoptive tumor-infiltrating lymphocyte (TIL) therapies is the lack of knowledge regarding the tumor antigens that elicit anti-cancer cytotoxic T-cell CTL responses. Existing methods for T-cell receptor (TCR) antigen discovery are limited in scalability and therefore require biased selection of candidate epitopes as a starting point. Typically, only a small number of these pre-selected candidate epitopes verify as bona fide tumor antigens. In order to delve deeper and map the entire landscape of CTL/tumor interactions in an unbiased fashion, new tools with greater high-throughput capacity are needed.
We have developed such a method by employing a granzyme-B (GzmB)-cleavable fluorescent reporter gene linked to large libraries of short peptide-encoding "minigene" sequences. Minigene-reporter cassettes are delivered into a pool of surrogate target cells by lentiviral gene transfer. When co-cultured with CTL populations-of-interest, transduced cells carrying putatively antigenic minigenes receive a dose of GzmB and are isolated by fluorescence-activated cell sorting. These minigenes are then identified by deep sequencing.
Proof-of-principle experiments have shown that peak GzmB signal precedes cellular degradation in targeted cells by several hours, that the delivery of GzmB to target cells is highly specific, and that spiked-in model antigens can be detected out of libraries of decoy sequence when they are expressed in as few as 1 per 10,000 target cells. We expect to able to use this approach to screen minigene libraries derived from tumor transcriptomes against patient TIL in order to reveal all targets available to adoptive cell therapy. Moreover, using random library screening, we aim to characterize the cross-reactivity of known TCRs in order to estimate the breadth of protection and the risk of off-target effects afforded by potential therapeutic TCRs.