Abstract 4361: Timing of PD-1 blockade is critical to successful synergy with OX40 costimulation in preclinical mammary tumor models
Authors: Messenheimer David J. , Feng Zipei , Wegmann Keith W. , Jensen Shawn M. , Bifulco Carlo B. , Fox Bernard A. 
Published: July 2016

Abstract
With the recent success of cancer immunotherapies targeting specific inhibitory receptors like PD-1 and CTLA-4, there is great interest in how to combine these drugs with other novel therapies targeting costimulatory receptors that could further augment an anti-tumor response. Recently, we observed that orthotopically-transplanted MMTV-PyMT tumor-bearing mice that received anti-OX40 treatment saw a significant delay in tumor growth (p &lt; 0.001) compared to untreated mice. However when combined concurrently with anti-PD-1 blockade, instead of a synergistic effect, we noted no additive benefit, and in fact saw a significant attenuation (p &lt; 0.05) in survival. These results fit a similar pattern to what we have seen in the 4T1 tumor model, where the anti-tumor effect provided by vaccine plus anti-OX40, was significantly attenuated (p &lt; 0.001) when anti-PD-1 was added concurrently.
We hypothesized that treating with anti-PD-1 antibody would be more effective during the contraction phase of the T cell boost generated by anti-OX40. Thus we delayed anti-PD-1 treatment until after anti-OX40 dosing was complete and saw a significant delay (p &lt; 0.01) in tumor growth and subsequent increase (p &lt; 0.01) in survival in the PyMT transplant model (compared to anti-OX40 alone), with some of the tumors reaching full regression. We had also previously seen a similar significant antitumor effect (p &lt; 0.001) in the 4T1 tumor model. These results were reproduced using delayed treatment with an anti-PD-L1 antibody combined with anti-OX40, demonstrating that blocking either side of the PD-1-PD-L1 interaction is sufficient. Also supporting this hypothesis, we noted a significant increase (p &lt; 0.05 compared to untreated) in PD-1 expression on CD4+ T cells during and after anti-OX40 treatment.
Investigating the effects of the concurrent combination, we noticed a striking increase in IFN-γ in the serum compared to treatment with single agent (p &lt; 0.001 after 3 doses). Serum levels of other cytokines TNF, IL-4, IL-6, and IL-10 were also elevated in the combination treated group compared to anti-OX40 alone. Interestingly, we observed a large increase in PD-L1 expression on both CD4+ and CD8+ T cells and we also noted significant increases (p &lt; 0.05) in inhibitory receptors LAG3, TIM3 and CTLA4 on CD4+ and CD8+ splenic T cells. These may provide additional escape mechanisms for the tumor to evade immune destruction and potentially offer other targets to enhance combination therapy.
Our results demonstrate that the sequence of antibody treatment targeting both costimulatory and inhibitory receptors is critical to success of the combined therapy. These data offer a strong rationale for delaying PD-1 blockade until after costimulation has provided an initial immune boost.
Citation Format: David J. Messenheimer, Zipei Feng, Keith W. Wegmann, Shawn M. Jensen, Carlo B. Bifulco, Bernard A. Fox. Timing of PD-1 blockade is critical to successful synergy with OX40 costimulation in preclinical mammary tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4361.