Abstract A65: Improved immunological responses against melanoma in vivo with T lymphocyte adoptive cell therapy coupled with targeted inhibition of mutated and activated BRAF
Authors: Koya Richard C. , Mok Stephen , Otte Nicholas , Chodon Thinle , Comin-Anduix Begonya , Ribas Antoni 
Published: June 2012

Abstract
Vemurafenib blocks activated BRAF with V600E driver mutation and induces unprecedented high response rates in patients with metastatic melanoma. However, most patients have response durations limited to only several months. Conversely, immunotherapy based on adoptive transfer of T cells has induced low frequency, but highly durable tumor responses. We previously reported (Clin Cancer Res. 16(24), 2010) that T lymphocytes exposed to high concentrations of vemurafenib had preserved viability and function, providing a compelling rationale for a combined targeted drug/immunotherapy approach.
We first established an implantable murine melanoma cell line driven by the V600E BRAF oncogene (SM1) from a spontaneously arising melanoma in transgenic mice harboring the V600E BRAF mutation under the control of tyrosinase promoter. SM1 cells exposed to vemurafenib had partial in vitro sensitivity (IC50 of 14 uM) resulting from inhibition of MAPK pathway signaling (as demonstrated by Immunoblotting), while murine lymphocytes were spared. In vitro assays indicated that SM1 cells undergo apoptosis and cell cycle arrest at G1 phase in the presence of vemurafenib. Mice implanted with SM1 tumors responded significantly with daily i.p. injection of vemurafenib, confirming its efficacy in vivo. We then tested the combination of vemurafenib and immunotherapy in vivo using two models of adoptive cell transfer (ACT) therapy. OT-1 T cell receptor-expressing lymphocytes targeting ovalbumin (OVA) present in SM1-OVA tumors or pmel-1 lymphocytes targeting the melanoma-associated-antigen gp100 (endogenously expressed by SM1) combined with daily i.p. injections of vemurafenib resulted in superior antitumor responses compared to either therapy alone. We then quantified the adoptively transferred T cells in spleen and tumor biopsies by tissue immunostaining. There were no significant differences in numbers of T cells infiltrating the tumors with or without vemurafenib. Further analysis with two different molecular imaging-based in vivo T cell tracking (1-Luciferin bioluminescence and 2-Positron Emission Tomography with dFAC tracer) confirmed that vemurafenib did not significantly alter the expansion, distribution or tumor accumulation of the adoptively transferred T cells. Also, vemurafenib did not alter SM1 antigen presentation as demonstrated by lack of significant differences in gp100 expression and MHC-I levels in SM1, as well as, in vivo cytotoxic activity of adoptively transferred cells against their cognate antigen. We then performed immunophenotypic analysis of transferred T cells. Vemurafenib skewed these cells towards a phenotype resembling central memory T cells, a favorable characteristic for superior and prolonged tumor control. Further functional analysis of tumor infiltrating lymphocytes indicated increased interferon-gamma secretion as assayed by intracellular staining and FACS.
In conclusion, our data derived from two independent models combining BRAF targeted therapy and immunotherapy indicated favorable changes in T cell function/immunophenotype and support the testing of such combination in patients with BRAFV600 mutant metastatic melanoma.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A65.