P2.04-24 Transcriptional Profiling of Neoantigen Specific T Cells in Resectable NSCLC Treated with Neoadjuvant Anti-PD-1

Abstract

Neoadjuvant nivolumab has a manageable safety profile and can be effective in patients with resectable non-small cell lung cancer (NSCLC). To characterize the immune response in these patients, we sought to evaluate the existence and dynamics of neoantigen specific tumor-infiltrating T cells and identify their molecular phenotype including co-inhibitory checkpoint expression. We evaluated peripheral blood and tumor infiltrating lymphocytes from seven patients treated with nivolumab. To identify neoantigen-specific T cell responses, we used MANAFEST (Mutation Associated Neoantigen Functional Expansion of Specific T cells), an assay we developed that links antigen specificity with unique CD8+ TCR Vβ CDR3 identities. We then carried out single cell TCRseq/RNAseq of tumor infiltrating T lymphocytes (TIL) to enumerate the genome wide digital gene expression and T cell clonotypic identity of each single cell (VDJ+DGE analysis), and particularly those with Vβ CDR3 regions identical to those identified as neoantigen-specific by MANAFEST. Neoantigen-specific TCRs were detected in peripheral blood in all 3 patients with major pathologic response (MPR) and in 3 of 4 patients without MPR. Several of these clonotypes were found in the resected tumor and underwent peripheral expansions upon PD-1 blockade. In one notable patient, MD043-011, MANAFEST detected a T cell clonotype specific for a CARM1 R208W mutation, despite this patient having no evidence of pathologic response. This neoantigen-specific clonotype represented 3.4% of TIL. Two years later, this patient recurred with a solitary brain metastasis. Single cell analyses of TIL in the primary lung lesion and brain metastasis revealed the same neoantigen-specific T cell clonotype was detected in the metastatic lesion. Strikingly, this clonotype exhibited a differential expression profile in the primary and recurrent lesion, with the clonotype in the primary tumor having an enrichment and upregulation of heat shock proteins indicating molecular stress and the clone in the metastatic lesion having an upregulation of checkpoint molecules, including CTLA4, TIM3, and LAG3. T cell cloning and validation experiments, as well as identification of transcriptional programs associated with MPR, are ongoing. The coupling of MANAFEST with single cell VDJ+ DGE analysis enabled us to characterize antigen specific clonotypes after differential expansion using the TCR as a molecular barcode. The presence of alternate co-inhibitory immune checkpoints on neoantigen-specific TIL from non-responding tumors suggests a potential driver of resistance to anti-PD-1 in early stage NSCLC. Ultimately, this integrative approach may provide key insights in predicting and understanding clinical response to neoadjuvant PD-1 blockade in NSCLC.