Overcoming resistance to alpha PD-1 of MMR-deficient tumors with high tumor-induced neutrophils levels by combination of alpha CTLA-4 and alpha PD-1 blockers

Background Clinical studies have highlighted the efficacy of anti-programmed death 1 (alpha PD-1) monoclonal antibodies in patients with DNA mismatch repair-deficient (MMRD) tumors. However, the responsiveness of MMRD cancers to alpha PD-1 therapy is highly heterogeneous, and the origins of this variability remain not fully understood. Methods 4T1 and CT26 mouse tumor cell lines were inactivated for the MMRD gene Msh2, leading to a massive accumulation of mutations after serial passages of cells. Insertions/deletion events and mutation load were evaluated by whole exome sequencing. Mice bearing highly mutated MMRD tumor or parental tumors were treated with alpha PD-1 and tumor volume was monitored. Immune cell type abundance was dynamically assessed in the tumor microenvironment and the blood by flow cytometry. Neutrophils were depleted in mice using alpha LY6G antibody, and regulatory T (Treg) cell population was reduced with alpha CD25 or anti-cytotoxic T-lymphocytes-associated protein 4 (alpha CTLA-4) antibodies. Patients with MMRD tumors treated with immune checkpoint blockade-based therapy were retrospectively identified and neutrophil-to-lymphocyte ratio (NLR) was evaluated and examined for correlation with clinical benefit. Results By recapitulating mismatch repair deficiency in different mouse tumor models, we revealed that elevated circulating tumor-induced neutrophils (TIN) in hypermutated MMRD tumors hampered response to alpha PD-1 monotherapy. Importantly, depletion of TIN using alpha Ly-6G antibody reduced Treg cells and restored alpha PD-1 response. Conversely, targeting Treg cells by alpha CD25 or alpha CTLA-4 antibodies limited peripheral TIN accumulation and elicited response in alpha PD-1-resistant MMRD tumors, highlighting a crosstalk between TIN and Treg cells. Thus, alpha PD-1+alpha CTLA-4 combination overcomes TIN-induced resistance to alpha PD-1 in mice bearing MMRD tumors. Finally, in a cohort of human (high microsatellite instability)/MMRD tumors we revealed that early on-treatment change in the NLR ratio may predict resistance to alpha PD-1 therapy. Conclusions TIN countered alpha PD-1 efficacy in MMRD tumors. Since alpha CTLA-4 could restrict TIN accumulation, alpha PD-1+alpha CTLA-4 combination overcomes alpha PD-1 resistance in hosts with hypermutated MMRD tumors displaying abnormal neutrophil accumulation.