Mass cytometry immune profiling to detect signatures of clinical activity of bintrafusp alfa (BA) in patients with advanced non-small cell lung cancer (NSCLC).
Authors: McAdams Meredith , Medina Carlos , Healy Connor , Donahue Renee N. , Sigal Natalia , Enrico Elizabeth M. , Marte Jenn , Sweere Jolien , Srinivasan Ramji , Nguyen Ngan , Schlom Jeffrey , Rajan Arun , Gulley James L. 
Published: June 2023

e21016 Background: BA, a bifunctional fusion protein targeting programed death-ligand 1 and transforming growth factor-β, is clinically active in a subset of patients (pts) with advanced NSCLC. In this study, mass cytometry was used to identify immune features associated with response and survival in pts with NSCLC treated with BA. Methods: Peripheral blood mononuclear cells were collected at baseline and after 1 and 3 doses of BA from 12 NSCLC pts treated every 2 weeks in a dose expansion biomarker cohort of a phase I clinical trial (NCT02517398). Samples were analyzed using a 44-marker mass cytometry panel designed to detect immune cell subsets and functional states of the innate and adaptive immune system. Manually gated immune cell subpopulations and unsupervised clusters were analyzed with Kruskal Wallis and Wilcoxon Rank Sum testing for multigroup comparison; and with linear correlation, univariate Cox regression and Kaplan Meier methods for progression free survival (PFS) analysis. Results: Among 12 pts, 3 had an objective response (OR), 6 had stable disease (SD) and 3 developed progressive disease (PD). Immune cell subsets associated with clinical activity by multigroup analyses included memory B cells, CD16+ natural killer (NK) cells, and double negative T cells (DNT). Pts with OR had a 2-fold higher frequency of memory B cells at baseline compared to pts who developed SD or PD. After 1 dose of BA, pts who subsequently developed an OR or SD had a 5-fold increase of CD16+ NK cells compared to pts with PD. After 3 doses of BA, pts who subsequently developed an OR had 4-fold more memory B cells and 3-fold more T-bet+ DNTs compared to pts with PD or SD. Immune cell subpopulations associated with survival included 3 unsupervised clusters annotated by the clustering algorithm as CD16+ NK cells, DNTs and classical monocytes. Fewer CD16+ NK cells at baseline were associated with longer PFS (p = 0.0053). Longer PFS was also associated with higher CD161+ CD127+ DNT cells after 1 dose of BA relative to baseline (p = 0.029), while an expansion of IgG4+ PD-1+ classical monocytes after 3 doses of BA was associated with shorter PFS (p = 0.015). Linear correlation analysis did not find strong associations between any one immune state and survival. Conclusions: Our study demonstrates the ability of mass cytometry immune profiling to detect potential immunological markers of response and survival in NSCLC pts treated with BA. The association between CD16+ NK cell frequencies and clinical benefit could reflect the ability of BA to facilitate antibody-dependent cellular cytotoxicity by NK cells in NSCLC. A high fraction of memory B cells at baseline and after 3 doses identifies pts likely to respond to BA. If validated in larger studies, these novel immune signatures can be used to improve clinical outcomes in pts with NSCLC considering immunotherapy.