Abstract 656: Antitumor activity of combined therapy with the class I BRAF inhibitor PLX4032 and immunotherapy
Authors: Koya Richard C. , Mok Stephen , Otte Nicholas , Comin-Anduix Begonya , Chodon Thinle , Ribas Antoni 
Published: June 2012

PLX4032 is a potent inhibitor of V600E BRAF with response rates of up to 80% in patients with metastatic melanoma. However, drug resistance develops in most of patients leading to response durations of only several months. Immunotherapy for melanoma has induced low frequency, but highly durable tumor responses, which may last years. Therefore, the concept of a combined approach of BRAF inhibition and immunotherapy is very attractive. We have established a novel animal model which allows the testing of this concept in fully immunocompetent mice.

An implantable murine melanoma cell line driven by the V600E BRAF oncogene (SM1) was established from a spontaneously arising murine melanoma in transgenic mice backcrossed to C57BL/6 and harboring the V600E BRAF mutation under the control of tyrosinase promoter. The in vitro IC50 of PLX4032 against SM1 was 14μM, which indicates partial resistance to the drug and therefore useful as a model for combined therapy. In vitro assays indicated that SM1 cells undergo apoptosis and cell cycle arrest at G1 phase in the presence of the PLX4032. Mice implanted with tumors responded significantly with daily i.p. injection of PLX4032, confirming its efficacy in vivo, though there was no complete eradication of the tumor. Cultured primary murine T lymphocytes exposed to PLX4032 at different time points (24, 48 and 72h) with a broad range of concentration (0.1 to up to 100 μM) showed no evidence of cytotoxicity. In vivo real-time T cell tracking by luciferase bioluminescence imaging, as well as PET/CT, confirmed that PLX4032 did not adversarially affect the viability, engraftment or tumor infiltration of T cells in mice. We then tested the combination of PLX4032 and immunotherapy in vivo using two models of adoptive cell transfer (ACT) therapy. SM1 cells stably expressing the chicken ovalbumin (OVA) model antigen were implanted in C57BL6 mice. Daily i.p. PLX4032 combined with ACT of OVA-specific TCR transgenic cells (OTI) generated by retroviral transduction demonstrated superior effects of the combined treatment (tumor size at day 14 after ACT plus PLX4032: 114.9±23.6 mm2, PLX4032: 203.2±6.2, OTI ACT: 192.5±6.2, and vehicle control 241.3±3.1, p&lt;0.05). We also attested this combination in the pmel-1 model, which is based on the ACT of TCR transgenic cells against the relevant melanoma antigen gp100, which is endogenously expressed by SM1. The combined treatment PLX4032 plus ACT of pmel-1 transgenic T cells also resulted in a superior tumor shrinkage (124.7±1.7 mm2) compared to PLX4032 alone (157.4±7.4), pmel-1 T cell ACT alone (203.8±2.1) or vehicle control (250.8±3.8), p&lt;0.05.

In conclusion, our data derived from two independent models combining BRAF targeted therapy and immunotherapy support the testing of such combinations in patients with V600E BRAF mutant metastatic melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2011-656