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. 2012 Aug 15;72(16):3928-37.
doi: 10.1158/0008-5472.CAN-11-2837. Epub 2012 Jun 12.

BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy

Richard C Koya  1 , Stephen Mok, Nicholas Otte, Kevin J Blacketor, Begonya Comin-Anduix, Paul C Tumeh, Aspram Minasyan, Nicholas A Graham, Thomas G Graeber, Thinle Chodon, Antoni Ribas
Affiliations

BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy

Richard C Koya et al. Cancer Res. .

Abstract

Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma.

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Conflict of interest statement

Conflict of Interest: Antoni Ribas has received honoraria from consulting with Roche-Genentech, which is the maker of vemurafenib.

Figures

Figure 1
Figure 1
Effects of vemurafenib in the murine SM1 BRAFV600E mutant melanoma cell line. A) Detection of BRAFV600E mutation (T1799A) in SM1 by DNA sequencing. B) Human melanoma cell-lines and murine SM1 melanoma cells were exposed to increasing concentrations of vemurafenib for IC50 determination using an MTS assay. C) Cell cycle arrest in SM1 cells induced by vemurafenib (15 µM) after a 72 hour exposure analyzed by flow cytometry. D) Apoptosis marker analysis of SM1 cells exposed to vemurafenib for 72 hours at 15 µM analyzed by flow cytometry. E) Immunoblotting for analysis of signaling molecules after vemurafenib exposure of SM1 cells at increasing concentrations for 1 or 24 hours. F) SM1 tumor implanted mice were injected i.p. daily with 10 mg/kg of vemurafenib or vehicle control and followed for tumor size changes over time.
Figure 2
Figure 2
A) Effects of vemurafenib on murine splenocyte viability. Cell viability assay (MTS) at different time points and doses of vemurafenib in ex vivo activated pmel-1 splenocytes. B) Immunoblotting for analysis of phosphorylated ERK (pERK) and total ERK after vemurafenib exposure of ex vivo gp-100 peptide activated pmel-1 splenocytes at increasing concentrations for 24 hours.
Figure 3
Figure 3
Combined antitumor activity of TCR engineered adoptive cell adoptive cell transfer (ACT) immunotherapy and vemurafenib in the ovalbumin (OVA) model. A) Schematic of the OT-1 TCR engineered ACT model based on adoptively transferring C57BL/6 splenocytes, stably transduced to express a TCR specific for OVA using retroviral transduction, into lymphodepleted mice with previously established flank SM1 cells stably expressing OVA (SM1-OVA). Vemurafenib, or DMSO vehicle control, was started on day +2 from the tumor, and on day +7 mice received the ACT of OVA-specific TCR transduced splenocytes with lymphodepleting radiation therapy the day before, followed by three days of systemic IL-2 therapy. B). Western blot analysis for OVA expression in parental SM1 cells (line 1), SM1-OVA cells (line 2) and SM1-OVA tumor graft (line 3). C) Schematic of the MSCV-based retroviral vector co-expressing the alpha and beta chains of the OT-1 TCR linked by a F2A picornavirus sequence. D) Tetramer analysis for OVA-specific surface TCR expression on splenocytes from C57BL/6 untransduced (left panel) or transduced with the OT-1 TCR-expressing retrovirus. E) Tumor growth curves in C57BL/6 mice with established SM1-OVA tumors. F) Kaplan-Meier actuarial plot of time to mouse sacrifice due to large tumor burden, or to study termination when tumor size was less than 14 mm in maximum diameter, combining results from two replicate experiments in the OVA TCR engineered ACT model.
Figure 4
Figure 4
Combined antitumor activity of TCR engineered adoptive cell adoptive cell transfer (ACT) immunotherapy and vemurafenib in the pmel-1 model. A) Schematic of the pmel-1 model, where C57BL/6 mice with established SM1 tumors received vemurafenib, or DMSO vehicle control, from day +2 after tumor implantation, lymphodepleting radiation therapy on day +7 and the adoptive transfer of pmel-1 splenocytes activated in vitro with gp100 peptide on day +7. This was followed with three days of systemic IL-2 therapy and gp100 peptide pulsed dendritic cell (DC) vaccines on day +7. B). Western blot analysis for gp100 expression in parental SM1 cells exposed to DMSO vehicle control or vemurafenib (PLX4032) at three different concentrations for 1 or 24 hours. Protein loading was normalized to tubulin. C) Tumor growth curves in C57BL/6 mice with established SM1 tumors. D) Kaplan-Meier actuarial plot of time to mouse sacrifice due to large tumor burden, or to study termination when tumor size was less than 14 mm in maximum diameter, combining results from two replicate experiments in the pmel-1 ACT model.
Figure 5
Figure 5
Effects of vemurafenib on the number or distribution of adoptively transferred lymphocytes. A) pmel-1 transgenic T cells were used for ACT in the pmel-1 combined therapy model. Tumors were harvested on day +5 after ACT and representative H&E (left panel) and immunofluorescence for pmel-1 cells stained with anti-Thy1.1-FITC (green, right panels), and nuclei stained with DAPI (blue, right panels). B) Splenocytes and tumor infiltrating lymphocytes harvested at day 5 were counted and analyzed by flow cytometry for gp100 tetramer/Thy1.1/CD3/CD8 staining. C) In vivo bioluminescence imaging of TCR transgenic T cell distribution. Pmel-1 transgenic T cells were transduced with a retrovirus-firefly luciferase and used for ACT. Representative figure at day 5 depicting three replicate mice per group. D) Quantitation of bioluminescence imaging of serial images obtained through day 14 post-ACT of pmel-1 transgenic cells expressing firefly luciferase with 3 mice per group.
Figure 6
Figure 6
Effects of vemurafenib on the cytotoxic and cytokine producing functions of adoptively transferred lymphocytes. A) Effects on cytotoxicity with the in vivo cytotoxic T cell assay. C57BL/6 mice received ACT of 5 × 104 pmel-1 splenocytes and daily vemurafenib or vehicle administered intraperitoneally. On day 16 mice received an intravenous challenge with CFSE-labeled target cells (splenocytes pulsed with gp100 peptide or control OVA peptide). Ten hours later splenocytes were harvested and analyzed by flow cytometry. B) Effects on cytokine production upon antigen re-stimulation. SM1 tumor-bearing C57BL/6 mice received pmel-1 ACT with or without vemurafenib. At day 5 post-ACT, tumors were harvested and TILs isolated for intracellular IFN-γ staining analyzed by intracellular staining by flow cytometry upon 5 hour ex vivo exposure to the gp10025–33 peptide.
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