Abstract 4041: Coupling neoantigen specific T cell clonotypes and their molecular phenotypes at the single cell level in resectable anti-PD-1 treated NSCLC
Authors: Caushi Justina X. , Ji Zhicheng , Zhang Jiajia , Asmar Margueritta El , Anagnostou Valsamo , Cottrell Tricia R. , Chan Hok Yee , Guo Haidan , Merghoub Taha , Wolchok Jedd D. , Naidoo Jarushka , Marrone Kristen A. , Chaft Jamie E. , Hellmann Matthew D. , Gabrielson Edward , Taube Janis M. , Brahmer Julie R. , Velculescu Victor E. , Zhao Ni , Danilova Ludmila , Cope Leslie , Forde Patrick M. , Pardoll Drew M. , Ji Hongkai , Yegnasubramanian Srinivasan , Smith Kellie N. 
Published: August 2019

Abstract
We recently completed a phase II study evaluating the safety and efficacy of neoadjuvant anti-PD-1 in non-small cell lung cancer (NSCLC). To evaluate anti-tumor T cell responses, we used the recently described MANAFEST (Mutation Associated Neoantigen Functional Expansion of Specific T cells) assay, which links antigen specificity with unique CD8+ TCR Vβ CDR3 identities, in 7 patients from the trial. We then carried out single cell sequencing of tumor infiltrating T lymphocytes (TIL) to enumerate the genome wide digital gene expression of each single cell (VDJ+DGE analysis), and more particularly those with Vβ CDR3 regions identical to those identified by MANAFEST. Neoantigen-specific TCRs were detected in all patients with major pathological response (MPR) and in 3 of 4 patients without MPR. VDJ+5’DGE single cell sequencing demonstrated differential phenotypes of TIL obtained from surgical resection, and these T cells had varying degrees of peripheral perturbations during treatment. In PBMC from patient MD043-011, the MANAFEST assay detected a T cell response specific for a CARM1 R208W mutation, despite no evidence of pathological response in this patient. These neoantigen-specific T cells possessed a single TCR Vβ CDR3 at the aa level and represented 3.4% of TIL. Upon analysis of TIL with the VDJ+DGE platform, the same CDR3 was detected in 32 TIL, all of which possessed the identical TCR Vα at the aa level. However, analysis of CDR3 sequences at the nucleotide level revealed 3 distinct clones encoding this antigen-specific TCR. Differential gene expression profiling demonstrated a unique and discriminating expression profile for each clonotype, with one clone expressing CTL activation markers such as granzyme k, 4-1BB, and PD-1, the second clone expressing IL10 family member IL26 and genes that inhibit effector and memory CTL function, such as CISH, TOX, and SLAMF1, and the third clone expressing no known activation markers. These findings demonstrate the existence of different intratumoral T cell clones with distinct functions yet identical specificity for a tumor neoantigen. We propose that these differential expression programs are induced because these clones were differentially activated under different conditions in the TME, primed in distinct anatomical locations, or at different times during disease progression despite recognizing the same antigen. Analyses to evaluate the prevalence of this phenomenon in the larger cohort are ongoing. In conclusion, the coupling of MANAFEST with single cell VDJ+ DGE analysis enabled the first comprehensive phenotypic profiling of neoantigen-specific T cells using the TCR as a molecular barcode. Ultimately, this integrative approach may provide key insights in predicting and understanding not only clinical response to neoadjuvant PD-1 blockade in NSCLC, but also other early stage cancers agnostic of tissue of origin.
Citation Format: Justina X. Caushi, Zhicheng Ji, Jiajia Zhang, Margueritta El Asmar, Valsamo Anagnostou, Tricia R. Cottrell, Hok Yee Chan, Haidan Guo, Taha Merghoub, Jedd D. Wolchok, Jarushka Naidoo, Kristen A. Marrone, Jamie E. Chaft, Matthew D. Hellmann, Edward Gabrielson, Janis M. Taube, Julie R. Brahmer, Victor E. Velculescu, Ni Zhao, Ludmila Danilova, Leslie Cope, Patrick M. Forde, Drew M. Pardoll, Hongkai Ji, Srinivasan Yegnasubramanian, Kellie N. Smith. Coupling neoantigen specific T cell clonotypes and their molecular phenotypes at the single cell level in resectable anti-PD-1 treated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4041.