Combined treatment with dabrafenib and trametinib with immune-stimulating antibodies for BRAF mutant melanoma

Abstract

The combination of targeted therapy with BRAF and MEK inhibitors has become the standard of care in patients with BRAF (V600E) mutant melanoma, but responses are not durable. In addition, the impressive clinical benefits with anti-PD-1 and anti-PD-L1 antibodies (Ab) in patients with heavily pretreated metastatic melanoma and the synergistic effect of dabrafenib, trametinib and anti-PD-1 compared with single therapy alone groups support the idea that combining dabrafenib, trametinib and immunotherapy based on PD-1 blockade could be an interesting approach in the treatment of metastatic melanoma. With our mouse model of syngeneic BRAF (V600E) driven melanoma (SM1), we tested whether the addition of an immunostimulatory Ab targeting CD137 (4-1BB) and/or CD134 (OX40) would enhance the antitumor effect of dabrafenib, trametinib and anti-PD-1 or anti-PD-L1 therapy. In vitro studies showed that the combination group of dabrafenib, trametinib and anti-PD-1 increases CD8(+) tumor infiltrating lymphocytes (TILs), as well as CD4(+) T cells and tumor-associated macrophages (TAMs). An upregulation of PD-L1 was observed in the combination of dabrafenib, trametinib and anti-PD-1 therapy. Combination of dabrafenib, trametinib and anti-PD-1, with either anti-CD137 or anti-CD134, showed a superior antitumor effect, but the five-agent combination was not superior to the four-agent combinations. In conclusion, the combination of dabrafenib, trametinib, anti-PD1 or anti-PD-L1 therapy results in robust antitumor activity, which is further improved by adding the immune-stimulating Ab anti-CD137 or anti-CD134. Our findings support the testing of these combinations in patients with BRAF (V600E) mutant metastatic melanoma.

Keywords: PD-1; checkpoint blockade; immunotherapy; melanoma; targeted therapy.

Figure 1.

Enhanced in vivo antitumor activity with dabrafenib (D) + trametinib (T) combined with PD-1 checkpoint blockade against SM1 tumors. In vivo tumor growth curves. SM1 bearing C57BL/6 mice were treated when tumors were 3–5 mm with D 30 mg/kg and T 0.15 mg/kg combination via oral gavage daily, 4 doses of 200 μg of anti-PD-1 (PD-1), D + PD-1, T + PD-1, D + T + PD-1, D + T + anti-CD137 (CD137), PD-1 + CD137 or vehicle + isotype control Ab (4 mice in each group). This is representative graph of a three times repetition of this experiment.

Figure 2.

Increased tumor infiltrating effector and helper T-cells with dabrafenib and trametinib in combination with anti-PD-1 in SM1 tumors. (A) Quantification of tumor infiltrating effector cells (TILs). TILs and splenocytes harvested at day 5 after starting treatment were counted and analyzed by flow cytometry for CD3/CD8⁺ staining (six mice in each group). (B) Representative flow data of percentage of CD3⁺CD8⁺ TILs in tumors is shown. (C) Quantification of tumor infiltrating helper T-cells. TILs and splenocytes harvested at day 5 after starting treatment were counted and analyzed by flow cytometry for CD3/CD4⁺ staining (six mice in each group). (D) Representative flow data of percentage of CD3⁺CD4⁺ TILs in tumors is shown. (E) Quantification of PD-L1 expression. TILs and splenocytes harvested at day 5 after starting treatment were counted and analyzed by flow cytometry for SSC/PD-L1 staining (six mice in each group). (F) Representative flow data of percentage for SSC/PD-L1 in tumors is shown.

Figure 3.

Dabrafenib and trametinib changed the cellular components of the tumor microenvironment. On day 5 post-treatment, tumors and spleens were isolated and stained with fluorescent labeled antibodies, analyzed by FACS. (A) MO-MDSC (CD11b⁺Ly6C^Hi Ly6G^Lo) presented as percentage of CD11b⁺ cells in tumors and spleens. (B) PMN-MDSC (CD11b⁺Ly6C^LowLy6G^Hi) presented as percentage of CD11b⁺ cells in tumors and spleens. (C) Representative flow data of percentage of CD11b⁺Ly6C^Hi Ly6G^Lo and CD11b⁺Ly6C^LowLy6G^Hi in tumors is shown. (D) Analysis of macrophages (F4/80⁺CD11b⁺). *p = 0.03 Vehicle vs. D + T, p = 0.03 Vehicle vs. D + T + PD-1, both in tumors. (E) Representative flow data of percentage of F4/80⁺CD11b⁺ cells in tumors is shown. (F) Analysis of T regulatory cells (Tregs, CD4⁺CD25⁺FOXp3⁺). *p = 0.02 Vehicle vs. D + T, p = 0.03 Vehicle vs. D + PD-1, p = 0.03 Vehicle vs. D + T + PD-1. (G) Representative flow data of percentage of CD4⁺CD25⁺ cells in tumors is shown.

Figure 4.

Addition of immune activating antibodies to CD137 or CD134 to the combination of dabrafenib, trametinib and anti-PD-1. (A) Effects of anti-PD-L1 and anti-CD137 in combination with dabrafenib and trametinib. Tumor growth curves of established SM1 tumors in C57BL/6 mice that received dabrafenib, trametinib, anti-PD-L1 and anti-CD137 antibody. Treatment of anti-PD-L1, anti-CD137 or isotype antibody control was started at the same time with dabrafenib and trametinib when the tumor diameter reached 5 mm (4 mice in each group). This is representative graph of a three times repetition of this experiment. (B) Effects of quintuple combination of dabrafenib, trametinib, anti-PD-L1, anti-CD137 and anti-CD134. Tumor growth curves of established SM1 tumors in C57BL/6 mice that received dabrafenib, trametinib, anti-PD-L1, anti-CD137 and anti-CD134. Treatment of anti-PD-L1, anti-CD137, anti-CD134 or isotype antibody control was started at the same time with dabrafenib and trametinib when the tumor diameter reached 5 mm (4 mice in each group). This is representative graph of a three times repetition of this experiment.