Recognition of human gastrointestinal cancer neoantigens by circulating PD-[1.sup.+] lymphocytes

Tumor-resident lymphocytes can mount a response against neoantigens expressed in microsatellite-stable gastrointestinal (GI) cancers, and adoptive transfer of neoantigen-specific lymphocytes has demonstrated antitumor activity in selected patients. However, whether peripheral blood could be used as an alternative minimally invasive source to identify lymphocytes targeting neoantigens in patients with GI cancer with relatively low mutation burden is unclear. We used a personalized high-throughput screening strategy to investigate whether PD-1 expression in peripheral blood could be used to identify [CD8.sup.+] or [CD4.sup.+] lymphocytes recognizing neoantigens identified by whole-exome sequencing in 7 patients with GI cancer. We found that neoantigen-specific lymphocytes were preferentially enriched in the [CD8.sup.+]PD-[1.sup.+/hi] or [CD4.sup.+]PD-[1.sup.+/hi] subsets, but not in the corresponding bulk or PD-[1.sup.-] fractions. In 6 of 7 individuals analyzed we identified circulating [CD8.sup.+] and [CD4.sup.+] lymphocytes targeting 6 and 4 neoantigens, respectively. Moreover, neoantigen-reactive T cells and a T cell receptor (TCR) isolated from the [CD8.sup.+]PD-[1.sup.+] subsets recognized autologous tumor, albeit at reduced levels, in 2 patients with available cell lines. These data demonstrate the existence of circulating T cells targeting neoantigens in GI cancer patients and provide an approach to generate enriched populations of personalized neoantigen-specific lymphocytes and isolate TCRs that could be exploited therapeutically to treat cancer.