The monoclonal antibodies ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) 
have shown remarkable antitumor activity in an increasing number of cancers. 
When combined, ipilimumab and nivolumab have demonstrated superior activity in 
patients with metastatic melanoma (CHECKMATE-067). Here we describe the 
preclinical development strategy that predicted these clinical results. 
Synergistic antitumor activity in mouse MC38 and CT26 colorectal tumor models 
was observed with concurrent, but not sequential CTLA-4 and PD-1 blockade. 
Significant antitumor activity was maintained using a fixed dose of anti-CTLA-4 
antibody with decreasing doses of anti-PD-1 antibody in the MC38 model. 
Immunohistochemical and flow cytometric analyses confirmed that CD3+ T cells 
accumulated at the tumor margin and infiltrated the tumor mass in response to 
the combination therapy, resulting in favorable effector and regulatory T-cell 
ratios, increased pro-inflammatory cytokine secretion, and activation of 
tumor-specific T cells. Similarly, in vitro studies with combined ipilimumab and 
nivolumab showed enhanced cytokine secretion in superantigen stimulation of 
human peripheral blood lymphocytes and in mixed lymphocyte response assays. In a 
cynomolgus macaque toxicology study, dose-dependent immune-related 
gastrointestinal inflammation was observed with the combination therapy; this 
response had not been observed in previous single agent cynomolgus studies. 
Together, these in vitro assays and in vivo models comprise a preclinical 
strategy for the identification and development of highly effective antitumor 
combination immunotherapies.