Abstract
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.
Highlights
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), among other inhibitory T-cell surface molecules, attenuate a variety of activated T-cell functions, including cellular proliferation, cytokine secretion, and cytolysis [1]
In the CT26 colon carcinoma model, anti-PD-1 and anti-CTLA-4 combination therapy elicited tumor rejections that were superior to the individual therapies (Fig 1C and 1D)
Antitumor efficacy varied by the source of the mice as tumor-bearing mice from Harlan Laboratories (HAR) were more responsive to therapy than those from Charles River Laboratories (CRL), possibly as a result of intestinal microbiome differences [26, 27] (Fig 1C and 1D)
Summary
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), among other inhibitory T-cell surface molecules, attenuate a variety of activated T-cell functions, including cellular proliferation, cytokine secretion, and cytolysis [1]. CTLA-4 has been shown to inhibit T-cell responses by both intrinsic and extrinsic mechanisms [6,7,8,9,10]. With respect to the intrinsic mechanism, engagement of CTLA-4 on T cells by B7 ligands leads to their functional attenuation. There are multiple extrinsic mechanisms that include the ability of CTLA-4-expressing cells to effectively compete with CD28 for B7 ligands or trans-endocytic removal of costimulatory ligands from antigen-presenting cells (APC) [11]. Treatment of tumor-bearing mice with anti-CTLA-4 antibodies capable of depletion have been shown to substantially reduce Tregs in tumors but not in the periphery, resulting in potentiated antitumor activity as compared to antibodies that lack effector function [13,14,15]
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