Abstract 5329: Blocking multiple coinhibitory pathways for immunotherapy of established melanoma
Authors: Cai Guifang , Xia Bu , Fu Buyin , Freeman Gordon J. 
Published: February 2011

Abstract
Previous studies have shown that coinhibitory molecules like PD-L1 and B7-H3 are upregulated on a variety of tumor cells and high expression correlates with poor prognosis. Recent studies also showed that coinhibitory receptors like CTLA4, PD-1, LAG3 and CD160 were up-regulated in exhausted CD8 T cells during chronic infection, and blocking of coinhibitory pathways could reverse the exhaustion phenotype and restore CD8 T cell effector functions. Our previous studies have shown that HVEM binding to CD160 delivered a coinhibitory signal to T cell activation. Deletion of the CRD1 domain of HVEM (HDD1) converted HVEM from a dominant coinhibitory molecule into an exclusively costimulatory molecule, making HDD1 a good candidate to boost anti-tumor immune responses.
Our preliminary studies showed that HVEM was up-regulated in human melanoma cell lines, and mice challenged with B16 melanoma had increased expression of HVEM on a subset of B cells. To determine if targeting the HVEM pathway could enhance anti-melanoma immune responses, we transduced B16F10 tumor cells with the mutant costimulatory HVEM (HDD1). Our data showed that engagement of HDD1 enhanced NF-kB signaling, which correlated with enhanced susceptibility of B16 cells to irradiation-induced apoptosis. Our in vivo data showed that tumor vaccination with irradiated B16-HDD1 combined with dual blockade of CTLA4 and PD1 using UC10 and 29F.1A12 mAbs (triple treatment, on day 0, 3, 6 and 9) significantly enhanced survival from 0 to 75% when the treatment started the same day as tumor challenge (50,000 live B16F10 cells subcutaneously). In contrast, treatment with any component alone or in dual combination resulted in suboptimal efficacies ranging from 30-40% survival. The enhanced survival with triple treatment was associated with enhanced infiltration of neutrophils, CD4 and CD8 T cells into tumor sites. Trp2 tumor antigen-specific CD8 T cell production of IFN-gamma was also increased in triple treated mice compared to control groups. In addition, in an established tumor model, when the triple treatment started on day 10 after the tumor was established, triple treatment gave 30% survival rate. When another anti-CTLA4 mAb, 9H10, was used instead of UC10, survival with triple treatment was increased to 80%. In addition, depletion of CD4 T cells using GK1.5 mAb further enhanced survival to tumor challenge; however, this correlated with enlarged draining lymph nodes associated with severe skin irritation, indicating possible autoimmune responses due to depletion of regulatory CD4 T cells. Depletion of CD8 T cells during triple treatment only slightly reduced the efficacy of triple treatment, indicating that other effector cells such as CD4 and NK cells also contribute to the anti-tumor immune response.
In summary, our data demonstrated that triple treatment could be used as an efficient immunotherapy for melanoma through enhancing tumor specific immune responses.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5329.