Combined targeted therapy and immunotherapy in the treatment of advanced melanoma.

Recently, a CTLA-4 antibody and BRAF inhibitors showed survival benefits in advance melanoma treatment. The documentation of immune infiltrates in melanomas early during BRAF inhibitor therapy provides a scientific rationale for combination therapy with both treatments with possible synergistic benefits. This commentary reviews immune responses induced by BRAF inhibitors.

After decades of negative clinical trials in patients with advanced melanoma, two agents have recently shown a significant survival benefit in separate phase 3 clinical trials. One agent is the immune-stimulating human anti-CTLA-4 antibody ipilimumab. Ipilimumab binds to CTLA-4, thereby blocking negative immunogenic signaling stimulated by B7 molecules on antigen-presenting cells (APCs). This enables CD28 on T-cells to bind to CD80/86 on APCs, leading to activation and proliferation of T-cells. Clinical trials evaluating ipilimumab in metastatic melanoma patients have shown response rates of 10–15%, with significant improvements in median overall survival when compared with a GP-100 vaccine (27.8 vs. 17.2 mo) or in combination with dacarbazine chemotherapy compared with dacarbazine alone (19.3 vs. 8.1 mo). The second agent to show a significant improvement in overall survival when compared with dacarbazine was BRAF inhibitor (BRAFi) targeted therapy. Selective BRAFi target V600-mutated-BRAF, which constitutively activates the mitogen-activated-protein-kinase (MAPK) pathway and occurs in approximately 50% of metastatic melanoma patients. BRAFi have response rates of approximately 50%, although by 6 mo 50% of patients develop resistance to the BRAFi principally through oncogenic-rerouting. These two new clinically efficacious agents have different mechanisms of action in treatment of metastatic melanoma; one is an immune-modulator enhancing the body’s immune-response against the tumor and the other inhibits a signal transduction pathway and directly inhibits tumor proliferation. Ipilimumab has low but durable response rates, while BRAFi have high response rates and durability in few patients. Currently interest has turned to combining both agents to achieve more durable responses in BRAF mutant melanoma patients. To investigate the effects of the selective BRAFi on immune-responses, we recently analyzed biopsies of metastatic melanoma patients taken before and early during treatment with a BRAFi. These studies showed, a dramatic increase in the number of tumor-infiltrating lymphocytes (TILS) in biopsies following commencement of BRAFi treatment. Using immunohistochemistry, we showed that intratumoral density of CD4/CD8+ T-cells significantly increased with BRAFi treatment. The increase in intratumoral T-cell density following BRAFi treatment correlated with a reduction in tumor-size and an increase in necrosis. Furthermore, in biopsies of progressing lesions, the density of the CD4/CD8+ T-cells reduced to levels observed in the pre-BRAF inhibitor samples. Our findings lead us to conclude that BRAFi may increase the ability of cytotoxic T-cells to infiltrate metastatic melanoma and hence the combination with immunotherapies may lead to more favorable outcomes in BRAF-mutant metastatic melanoma patients.

The mechanisms responsible for this increase in T-cell infiltration following commencement of BRAFi therapy remain unknown. The melanoma may become more “visible” to the immune system following BRAF inhibition. Boni et al., reported that in vitro treatment of BRAF mutant cell lines with a BRAFi induced the expression of melanoma cell surface antigens GP-100/MART-1, which increased the recognition of the melanoma cells by antigen-specific T-cells. Additionally, our study and those of other authors showed increased levels of necrosis and apoptosis in response to BRAFi and both forms of cell death are known to induce an immune-response. Other mechanisms of increased immune-reaction may result from the disruption of the immunosuppressive tumor-stroma microenvironment that MAPK overexpression has helped maintain. This hypothesis is supported by data from Suminoto et al. who demonstrated that BRAFV600E shRNA reduced the secretion of immunosuppressive chemokines IL-6/10 by BRAF-mutant cell lines. Furthermore, the supernatant from these untreated cell lines inhibited the production of inflammatory chemokines IL-12 and TNF-? by dendritic cells. Also disruption to the tumor-stroma maintained immunosuppressive and anti-inflammatory microenvironment by BRAFi therapy may have allowed T-cells to infiltrate the tumor more readily.

A potentially more clinically important issue is whether the CD8+ T-cells seen in BRAFi-treated melanoma biopsies are functionally active. Lymphocytes may be rendered anergic by a variety of mechanisms including contact with ligands of negative signaling receptors PD-1, CTLA-4, TIM-3 as well as activation of FoxP3+ T regulatory cells. Granzyme B expression identifies activated T-cells in the tumor-microenvironment and importantly we observed an increase in Granzyme B expressing T-cells in melanomas with BRAFi therapy. However, the magnitude of the increase was less than that of the total CD8+ T-cells possibly indicating at least a portion of the T-cells may be non-functional. Understanding the functional status of these T-cells may provide important insights into the selection of the most effective immunotherapy combination.

The optimal criteria for the most appropriate drug to be combined with a BRAFi would include low/absent toxicity, induction of strong responses and a short latency period. Our study showed that the increase in TILS was lost once the tumor progresses on BRAFi therapy, suggesting that an immunotherapy with a shorter latency period may be needed to exploit the BRAFi response. The obvious choice would appear to be the CTLA-4 antibody Ipilimumab as it has proven efficacy and responses can be rapid. Initial clinical trials are underway which combine BRAFi and ipilimumab (ClinicalTrials.gov Identifier:NCT01400451). Another possible combination is the antibody against PD1, an immune antagonist, which has shown similar responses but lower toxicity than Ipilimumab in early phase clinical trials. In addition, in vitro studies have shown that the combination of CTLA-4 and PD1 antibodies drastically improved the response rates above those of either agent alone. Both PD-1 and CTLA-4 antibodies are being trialled together with the hope of synergistic effects (ClinicalTrials.gov Identifier:NCT01024231), although not with BRAFi as yet.

The use of BRAFi to debulk and sensitize a patient’s tumor to immunotherapies is an exciting prospect and we wait to see the results of these combination therapy clinical trials. However, irrespective of these results, much remains unknown about the functional phenotype of the TILs and the mechanisms controlling their proliferation and trafficking. A greater understanding of these factors may provide new insights into rationale therapies and biomarkers to select patients for the appropriate inhibitor and immunotherapy combination.

Figure 1.Disruption to the tumor microenvironment through BRAF inhibition may improve immunotherapy efficacy. A) The immunosuppressive tumor microenvironment is maintained by the tumor and stroma which release immunosuppressive chemokines and through the negative regulation of T-cells. B) BRAF inhibitors may be used to sensitize melanomas to immunotherapies such as anti-CTLA-4 and anti-PD-1 antibodies. BRAF inhibition may reduce tumor suppressive chemokines produced by the tumor and recruited monocytes. Additionally, BRAF inhibitors induce necrosis and increase cell surface antigens which may increase the visibility of the tumor to the immune system. BRAF inhibitors can be combined with immunotherapies which may increase tumor specific T-cell activation and proliferation. C) Pre-treatment biopsy showing a low density of CD8+ lymphocytes. D) Pre-treatment biopsy showing a low density of Granzyme B+ lymphocytes. E) Post BRAF inhibitor biopsy showing a high density of CD8+ lymphocytes. F) Post BRAF inhibitor biopsy showing a high density of Granzyme B+ lymphocytes.