1484 T cell receptor sequence clustering correlates with response to combination immunotherapy in fibrolamellar carcinoma

<h3>Background</h3> Fibrolamellar carcinoma (FLC) is a liver tumor that primarily affects otherwise healthy adolescents and young adults for which there is no approved or standard systemic treatment option. Virtually all cases of FLC share an oncogenic fusion between <i>DNAJB1</i> and <i>PRKACA</i>, which creates a shared neoantigen that is potentially targetable. We conducted a clinical trial testing a peptide vaccine against this driver fusion in combination with immune checkpoint blockade (ICB) in FLC patients. Here we analyze the T cell receptor (TCR) repertoire and T cell functional profile in these patients to identify vaccine-induced T cells and novel correlates of response to therapy. <h3>Methods</h3> 12 patients were treated with a 24mer <i>DNAJB1-PRKACA</i> synthetic long peptide vaccine with poly-ICLC in combination with ipilimumab and nivolumab. 3 patients received one cycle of vaccine-only treatment to evaluate safety, followed by combination therapy thereafter. Peripheral blood samples were collected at baseline and throughout treatment. TCR repertoire sequencing at each timepoint was performed using a novel high-throughput paired TCR repertoire sequencing technology, TIRTL-seq. TCR sequence similarity among longitudinally expanded clones was quantified using TCRdist¹ and used to generate clustering networks identifying closely related TCR sequences within and across patients. Select patient samples were also subjected to antigen-specific expansion followed by single-cell gene expression and paired TCR sequencing. <h3>Results</h3> Among patients evaluable for a 12-week immune assessment, 3/12 patients had a PR as a best response to therapy, and 9/12 patients had disease control (SD+PR). We observed significant T cell expansion in peripheral blood post-treatment in all 12 patients, although expansion was more pronounced after ICB than with vaccine alone. The number of longitudinally expanded TCR clones belonging to clusters of related TCR sequences significantly increased over time in patients who responded to therapy compared to patients with PD. We validated multiple vaccine-specific T cell clones, most of which were characterized by longitudinal expansion and activation in response to stimulation with the vaccinating peptide. <h3>Conclusions</h3> Our results point to increased clustering of longitudinally expanded TCR clones as a novel correlate of response to combination immunotherapy. Vaccine-specific clones were most readily identified by combining a longitudinal repertoire sequencing approach with parallel functional studies. Together, our results highlight the utility of deep, paired TCR repertoire sequencing in both identifying tumor-reactive TCRs for potential therapeutic use and discovering correlates of response to immunotherapy. <h3>Trial Registration</h3> ClinicalTrials.gov Identifier NCT04248569. <h3>Reference</h3> Dash P, <i>et al.</i> Quantifiable predictive features define epitope-specific T cell receptor repertoires. <i>Nature</i>. 2017;<b>547</b>:89–93. <h3>Ethics Approval</h3> This study was approved by the Johns Hopkins University School of Medicine Institutional Review Board (IRB), IRB approval number 00222681; and the St. Jude Children’s Research Hospital Institutional Review Board, IRB approval number 21-0790.