Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD‐1 antibody for BRAF ‐mutated melanoma

Kenta Nakamura; Tomonori Yaguchi; Masashi Murata; Yosuke Ota; Asuka Mikoshiba; Yukiko Kiniwa; Ryuhei Okuyama; Yutaka Kawakami

doi:10.1111/cas.16251

. 2024 Jun 18;115(9):2879–2892. doi: 10.1111/cas.16251

Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD‐1 antibody for BRAF ‐mutated melanoma

Kenta Nakamura ^1,^2,^✉, Tomonori Yaguchi ^1,³, Masashi Murata ⁴, Yosuke Ota ⁴, Asuka Mikoshiba ², Yukiko Kiniwa ², Ryuhei Okuyama ², Yutaka Kawakami ^1,^5,^✉

PMCID: PMC11462939 PMID: 38894534

Abstract

Programmed death 1 (PD‐1)/programmed death‐ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti‐PD‐1 antibody that blocks the major adaptive immune‐resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune‐resistant mechanisms, such as cancer cell‐derived immunosuppressive cytokines, and a Toll‐like receptor 7 agonist that enhances innate immune responses that promote antitumor T‐cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF‐mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T‐cell‐stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll‐like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon‐α/natural killer cell pathways and augmenting the T‐cell‐stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF‐mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100‐specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti‐PD‐1 antibody resulted in complete regression of SIY antigen‐transduced BRAF‐mutated melanoma in a CD8 T‐cell‐dependent manner. These findings indicate that a triple‐combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor‐specific T cells may be crucial for effective tumor eradication.

Keywords: BRAF, immune checkpoint inhibitors, melanoma, programmed death 1, Toll‐like receptor 7

In this study, our aim was to develop an effective combination immunotherapy for patients with BRAF‐mutated melanoma by targeting three essential pathways involved in the induction and function of antitumor T cells. In a syngeneic mouse BRAF‐mutated melanoma model, triplet therapy with DSR6424, vemurafenib, and an anti‐PD‐1 antibody was found to effectively eradicate the BRAF‐mutated melanoma among various doublet combination therapies.

graphic file with name CAS-115-2879-g001.jpg

Abbreviations

ELISA: enzyme‐linked immunosorbent assay
IFN: interferon
MAPK: mitogen‐activated protein kinase
MHC: major histocompatibility complex
NK: natural killer
PD‐1: programmed death 1
TLR7: Toll‐like receptor 7
VEGF: vascular endothelial growth factor

1. INTRODUCTION

Immune checkpoint inhibitors (ICIs), such as anti‐programmed death 1 (PD‐1) antibodies, are widely used for patients with various cancers, including melanoma. However, the response rates to anti‐PD‐1 antibodies are still limited, approximately 20%–40% in melanoma. ¹ , ² , ³ Therefore, PD‐1/PD‐ligand 1 (PD‐L1)‐based combination immunotherapies have been exploited for various types of cancers, and some combinations, including chemotherapy, anti‐vascular endothelial growth factor (VEGF) therapy, or other ICIs, have already been approved. ⁴ , ⁵ , ⁶ For melanoma, the combination of ICIs, such as antibodies for PD‐1 plus cytotoxic T‐lymphocyte‐associated protein 4 and for PD‐1 plus lymphocyte‐activation gene 3 (LAG3), has been approved, but further development is required.

In designing combination immunotherapy, primary and adaptive immune resistance mechanisms need to be considered. PD‐1/PD‐L1 interaction is a major adaptive resistance mechanism, and its blockade forms the basis of combination immunotherapy. In addition, primary resistance triggered by cancer cells includes multiple mechanisms, such as insufficient immunogenic antigens (DNA mutation‐derived neoantigens), insufficient T‐cell induction pathways (defects in antigen presentation and T‐cell‐stimulating innate immunity), and the production of immunosuppressive molecules by cancer cells. ⁷ , ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹²

In melanoma, activated oncogene signals, including the NRAS/BRAF/mitogen‐activated protein kinase (MAPK), phosphatidylinositol‐3‐kinase/protein kinase B/phosphatase and tensin homolog (PTEN), and Wnt/β‐catenin pathways, have been reported to cause immunosuppression. We have reported that gain‐of‐function mutations of BRAF, such as the V600E mutation frequently detected in melanoma, not only promote melanoma cell proliferation but also induce the production of various immunosuppressive cytokines. ¹³ Knockdown of the BRAF gene or treatment with MAPK kinase (MEK) inhibitors in human BRAF‐mutated melanoma cells reduced their production of immunosuppressive cytokines, such as interleukin (IL)‐10, IL‐6, and VEGF, inhibiting the T‐cell‐stimulatory activity of dendritic cells (DCs). ¹³ Administration of BRAF inhibitors is effective for patients with BRAF‐mutated melanoma, and an increase in CD8⁺ T cells in tumors has been observed in responders, suggesting that BRAF inhibitors might also have immunomodulating activities. However, drug resistance frequently occurs within 1 year. ¹⁴ , ¹⁵ , ¹⁶ Combination therapy with BRAF and MEK inhibitors has prolonged the time to acquisition of drug resistance ¹⁷ , ¹⁸ but still shows low clinical responses in patients with high lactate dehydrogenase levels and metastases to remote organs. ¹⁹ Clinical trials of combination therapy with BRAF inhibitor vemurafenib, MEK inhibitor cobimetinib, and anti‐PD‐L1 antibody atezorizumab have been reported to prolong progression‐free survival but not overall survival. ²⁰ Therefore, additional treatments are required for the effective treatment of BRAF‐mutated melanoma.

In addition to eliminating the immunosuppression induced by cancer cells, promoting innate immunity to support adaptive immune responses may be important for ICI therapies. Recent reports have highlighted the importance of DC activation in the tumor microenvironment for the induction and function of antitumor T cells. ²¹ , ²² , ²³ , ²⁴ , ²⁵

The gut microbiota has been shown to be involved in favorable responses to ICIs in patients with melanoma. We and others have reported that certain gut microbiota enhance antitumor T‐cell responses in ICI therapies through systemic activation of DCs, including those in tumor tissues. ²⁶

High numbers of natural killer (NK) cells and M1‐like monocytes and macrophages have also been reported to correlate with response to ICI therapy in patients with melanoma. ²⁷ , ²⁸ , ²⁹ These observations suggest the importance of promoting innate immune responses for the induction of tumor antigen‐specific T cells in ICI treatment. Toll‐like receptors (TLRs) are attractive targets to augment various innate immune responses. Among TLR agonists, TLR7 agonists stimulate plasmacytoid DCs (pDCs) to produce high amounts of interferon‐alpha (IFN‐α), which subsequently activate NK cells and conventional DCs (cDCs). ³⁰ , ³¹ Topical application of a TLR7 agonist, imiquimod, on melanoma has been reported to increase the number of lymphocytes infiltrating the tumors and enhance antitumor immune responses through activating NK cells, ³² , ³³ although it is not effective on remote metastases.

In this study, we applied systemic administration of a TLR7 agonist along with the combination of an anti‐PD‐1 antibody and a BRAF inhibitor. Interestingly in BRAF‐mutated murine melanoma models, we found that only triplet therapy effectively regressed BRAF‐mutated melanoma among various mono and doublet combination therapies. Therefore, in addition to the blockade of the adaptive resistance by an anti‐PD‐1 antibody, removal of multiple primary resistance mechanisms by a BRAF inhibitor and additional enhancement of various innate immune responses by a TLR7 agonist may be essential for effective regression of BRAF‐mutated melanoma.

2. MATERIALS AND METHODS

2.1. Animals and cell lines

C57BL/6, Balb/c, and Balb/c nu/nu mice, aged 6–8 weeks, were bred at the animal facilities of Keio University following the guidelines for animal experimentation. Human melanoma cell lines, A375 and C32, were purchased from the American Type Culture Collection and maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin. Additional information on the cell lines can be found in Data S1. All surgical interventions and animal care procedures were approved by the ethics committee of Keio University School of Medicine. The protocol for this study was approved by the Ethics Committee on Animal Research of Keio University School of Medicine (protocol number 250142).

2.2. Cell culture (WST‐1, enzyme‐linked immunosorbent assay [ELISA])

A375 and C32 were cultured at a density of 2 × 10³/well/200 μL in 96‐well plates. Vemurafenib (Sumitomo Dainippon Pharma Co., Ltd.), dissolved in dimethylsulfoxide, was added to the cultures at concentrations ranging from 0.01 to 10 μM. A cell proliferation assay was performed after 24 and 72 h using the WST‐1 cell proliferation reagent (Roche) following the manufacturer's instructions. For more detailed information, please refer to Data S1.

2.3. Conditioned media collection

The supernatant was collected from tumor‐infiltrating CD11c⁺ cells (5 × 10⁵ cells/mL) cultured in a 0.1% bovine serum albumin‐containing medium after 24 h of incubation. All samples were centrifuged to remove cells.

2.4. Cytokine and prostaglandin E2 (PGE2) measurement

Cytokines were quantified in cell culture supernatants and sera using commercially available ELISA kits. For more detailed information, please refer to Data S1.

2.5. In vivo vemurafenib administration

Female Balb/c nu/nu mice, aged 6 weeks, were subcutaneously inoculated with 5 × 10⁶ A375 cells in the flank region. Three days after tumor transplantation, vemurafenib was orally administered at a dose of 30 mg/kg/day once a day on consecutive days. For more detailed information, please refer to Data S1.

2.6. In vivo administration of vemurafenib and a TLR7 agonist

Vemurafenib and a TLR7 agonist were administered in vivo, and tumor‐specific T‐cell responses were then detected. In brief, female Balb/c nu/nu mice, aged 6 weeks, were subcutaneously inoculated with 5 × 10⁶ A375 cells or 5 × 10⁶ C32 cells in the flank region. Vemurafenib was orally administered at a dose of 30 mg/kg/day once a day on consecutive days, starting on the day of tumor transplantation. DSR6434 (Sumitomo Dainippon Pharma Co., Ltd.) was intravenously administered to the mice at a dose of 1 mg/kg once a week, starting on the day of tumor transplantation. For more detailed information, please refer to Data S1.

2.7. Isolation of CD11c ⁺ cells from draining lymph nodes and tumors

Between the 15th and 20th days after tumor transplantation, draining lymph nodes and tumors from each A375 cell‐bearing mouse were resected. For more detailed information, please refer to Data S1.

2.8. Dendritic cell stimulation ability

T cells were positively selected from the splenocytes of normal Balb/c mice using CD90.2 (Thy1.2) MicroBeads (Miltenyi Biotec). DCs were positively selected from A375 cell‐bearing Balb/c nu/nu mice using CD11c MicroBeads (Miltenyi Biotec) and irradiated with 32 Gy. For more detailed information, please refer to Data S1.

2.9. Plasmacytoid DC collection and culture

Plasmacytoid DCs were collected from the spleens of Balb/c mice using the mouse pDC isolation kit (Miltenyi Biotec). For more detailed information, please refer to Data S1.

2.10. Flow cytometry

Tumor tissues and lymph nodes were digested in Roswell Park Memorial Institute 1640 medium containing 1 mg/mL collagenase (Wako) and 0.1 mg/mL DNase (Merck) for 1 h at 37°C. After digestion, cells from tumor tissues were stained with fluorescein‐conjugated monoclonal antibodies for 1 h. The cells were then washed and analyzed using a Gallios flow cytometer (Beckman Coulter, Inc.). For more detailed information, please refer to Data S1.

2.11. ⁵¹Chromium ( ⁵¹Cr)‐release assay for cytotoxicity of natural killer cells

Natural killer cells were collected from the spleens of Balb/c nu/nu mice using an NK cell isolation kit (Miltenyi Biotec) following the manufacturer's instructions. Cytotoxicity assays were then performed using a 4‐h ⁵¹Cr‐release method. For more detailed information, please refer to Data S1.

2.12. DNA extraction from transgenic (Tg) mice and cell lines

Transgenic mice (B6.Cg‐Braf ^tm1Mmcm Pten ^tm1Hwu Tg[Tyr‐cre/ERT2]13Bos/BosJ) were obtained from Jackson Laboratory. Tamoxifen was applied externally to induce spontaneous melanoma oncogenesis in the Tg mice. For more detailed information, please refer to Data S1.

2.13. Direct sequencing

Primers were designed to amplify exon 15 of BRAF, which includes mutational hot spots, following our previous report. ³⁴ For more detailed information, please refer to Data S1.

2.14. Quantitative real‐time polymerase chain reaction

A TaqMan real‐time polymerase chain reaction was used for mRNA expression analysis. Primers specific to the target genes were generated. For more detailed information, please refer to Data S1.

2.15. Three‐agent combination with anti‐PD‐1 antibody assay

Female C57BL/6 mice, aged 6 weeks, were subcutaneously inoculated with 1 × 10⁶ Tg SIY mel cells in the flank region. Tumor size was measured using the formula: volume = 0.5 × (width)² × (length). The SIY‐expressing melanoma cells (1 × 10⁶) were subcutaneously grafted into C57BL/6 mice. Either vemurafenib or the vehicle was administered orally to the mice from post‐graft days 5–15. The TLR7 agonist was administered intravenously on post‐graft days 6 and 13. Either anti‐PD‐1 or isotope antibodies (200 μg/body; Bio X Cell) were administered intraperitoneally on post‐graft days 7, 10, and 13. Tumor diameter was then measured every 2–3 days. Each experimental group consisted of eight mice, and the experiment was conducted in duplicate. The SIY‐expressing melanoma cells (1 × 10⁶) were reimplanted.

2.16. CD8 depletion and pDC depletion

Rat antimouse CD8 or isotype antibodies (Bio X Cell) were administered intraperitoneally at a dose of 200 μg, 1 day before tumor implantation and on days 3, 6, and 9 after tumor implantation. Each experimental group consisted of five mice, and the experiment was conducted in duplicate. Rat antimouse PDCA1 or isotype antibodies (Bio X Cell) were administered intraperitoneally at a dose of 200 μg 1 day before tumor implantation and on days 3 and 6 after tumor implantation.

2.17. Statistical analysis

All results were expressed as means and standard deviations. Statistical analyses were performed using BellCurve for Excel (Social Survey Research Information Co., Ltd.). The unpaired Student's t‐test was used to compare the data among the experimental, treatment, and control groups. Survival curves were generated using the Kaplan–Meier method for time‐to‐event analysis. Statistical significance was considered at a p‐value less than 0.05.

3. RESULTS

3.1. Immunostimulating activity of BRAF inhibitor vemurafenib on human melanoma

In our previous study, the inhibition of IL‐6, IL‐10, and VEGF production, which have immunosuppressive effects on DCs derived from human melanoma cell lines, by BRAF knockdown has been reported. In this study, the immunomodulating activities of vemurafenib were evaluated by examining the in vitro effects of vemurafenib on cell proliferation and the production of multiple immunosuppressive soluble factors, including IL‐6, IL‐8, and PGE2, using BRAF‐mutated (V600E) human melanoma cell lines A375 and C32. Inhibitory effects on cell proliferation were observed in a dose‐dependent manner in both A375 and C32 cell lines, with concentrations higher than 0.5 μM of vemurafenib in a 72‐h WST1 assay (Figure 1A,B). There was no significant reduction in the cell number observed in the 24‐h WST1 assay. However, a significant reduction in the production of immunosuppressive soluble factors such as IL‐6, IL‐8, and PGE2 was observed in a dose‐dependent manner (Figure 1C–H). These findings indicate that vemurafenib has significant inhibitory activities on the production of immunosuppressive factors by BRAF‐mutated human melanoma cells.

The effect of the BRAF inhibitor vemurafenib on the production of immunosuppressive molecules by human melanoma cell lines. (A, B) The BRAF inhibitor inhibited the proliferation of the *BRAF*‐mutated human melanoma cell line A375 in vitro in a dose‐dependent manner, with significant inhibition observed at 0.5 μM or higher. (C) The supernatant was collected when no inhibition of A375 cell proliferation was observed and used for an enzyme‐linked immunosorbent assay (ELISA). The BRAF inhibitor inhibited the production of immunosuppressive cytokines, including interleukin (IL)‐6, IL‐8, and prostaglandin E2 (PGE2), in a dose‐dependent manner. (D–F) The supernatant was collected when no inhibition of C32 proliferation was observed and used for ELISA. (G) The BRAF inhibitor inhibited the production of IL‐8 in a dose‐dependent manner. (H) All data were derived from three independent experiments. Error bars indicate the SD. *p < 0.05 using Student's t‐test.

The immunomodulating effects of vemurafenib were further evaluated in vivo using a nude mouse model implanted with human melanoma cells. The nude mice were subcutaneously implanted with 5 × 10⁶ BRAF‐mutated A375 melanoma cells. Once the tumor volume reached 200 mm³, vemurafenib (30 mg/kg) was administered orally daily, 3 days after tumor inoculation. On day 15, a decrease in human IL‐6 produced by the implanted A375 melanoma was observed in the serum of the vemurafenib‐treated mice compared with the control mice, even when a difference in tumor sizes was not detected (Figure 2A,B). Since IL‐6 has inhibitory activity on DCs through STAT3 activation, ³⁵ , ³⁶ , ³⁷ , ³⁸ and human IL‐6 can affect murine DCs via the murine IL‐6 receptor, ³⁹ the T‐cell‐stimulatory activity of murine DCs in draining lymph nodes and tumors was evaluated in the xenogeneic tumor model. The T‐cell‐stimulatory activity of lymph node DCs was found to be impaired in tumor‐bearing mice compared with non‐tumor‐bearing mice, but it was significantly restored in the vemurafenib‐treated mice (Figure 2C). Similarly, the T‐cell‐stimulatory ability of DCs in tumors was also significantly restored in the vemurafenib‐treated mice (Figure 2D). These findings indicate that vemurafenib has the ability to restore DC functions in vivo, which were impaired by immunosuppressive cytokines from human BRAF‐mutated melanoma cells.

Restoration of impaired dendritic cell (DC) function in human melanoma‐bearing nude mice by the BRAF inhibitor, possibly through reduced interleukin (IL)‐6 production from melanoma cells. (A, B) The serum IL‐6 levels decreased in the BRAF inhibitor‐treated group compared with the control group, even when no noticeable difference in tumor size was observed. (C, D) Implantation of human melanoma cell lines in nude mice impaired the in vivo function of murine DCs. However, systemic administration of the BRAF inhibitor restored DC function to activate T cells at a dose that did not affect tumor growth. All data were derived from three independent experiments. Error bars indicate the SD. *p < 0.05 using Student's t‐test.

3.2. Immunostimulating activity of a combination of a BRAF inhibitor and a TLR7 agonist

Since the combination of vemurafenib, which inhibits cell proliferation and blocks the immunosuppressive activity of BRAF‐mutated melanoma cells, and ICIs such as an anti‐PD‐1 antibody, which block the major mechanism of adaptive immune resistance, does not show significant antitumor effects, ²⁰ additional immune interventions targeting innate immunity were evaluated to promote antigen‐specific T‐cell induction. In this study, the antitumor effects of the combination of vemurafenib and the TLR7 agonist DSR6434 were evaluated. DSR6434, a systemically injectable TLR7 agonist, stimulates innate immune responses via stimulating pDCs to produce high amounts of type 1 interferon (IFN‐α), which subsequently activate IFN‐γ‐producing NK cells. ³¹ Previous studies have shown that topical application of a TLR7 agonist, imiquimod, may help overcome the problem of drug resistance in a murine BRAF‐mutated melanoma model by enhancing T‐cell and NK cell responses after vemurafenib treatment. ³⁰

The antitumor effects of the combination of vemurafenib and TLR7 agonist DSR6434 were evaluated in a mouse model implanted with A375 cells. DSR6434 (1 mg/kg) was administered intravenously once a week in combination with the daily oral administration of vemurafenib to nude mice. Tumor growth was found to be inhibited in mice treated with vemurafenib alone or DSR6434 alone compared with control mice. However, the combination group had a significantly higher progression‐free survival rate than the other groups. A significant additive antitumor effect was observed in the mice treated with vemurafenib and the DSR6434 combination (Figure 3A,B). Similar inhibitory effects on tumor growth were also observed in nude mice implanted with human BRAF‐mutated melanoma C32 cells (Figure S1). DSR6434 did not have direct in vitro effects on cell proliferation or cytokine production of human melanoma cell lines, although TLR7 was expressed on A375 cells (Figure S2A,B). Additionally, it did not affect in vitro T‐cell proliferation upon stimulation with an anti‐CD3 antibody (Figure S2C). These results suggest that the significant inhibition of human melanoma growth observed in vivo in nude mice with the combination of vemurafenib and DSR6434 in an additive manner is likely due to the strong activation of innate immunity and direct antitumor effects.

Synergistically enhanced antitumor effects of the combination of the BRAF inhibitor and the Toll‐like receptor 7 (TLR7) agonist in human melanoma‐bearing nude mice. (A) Progression‐free survival was calculated using a cutoff value of more than 200 mm³. (B) Graph showing the individual tumor growth progression in each group. (C) The combination of the BRAF inhibitor and the TLR7 agonist synergistically enhanced the serum interferon (IFN)‐α level in A375 cell‐bearing nude mice. (D) The A375 cell‐killing activity of natural killer (NK) cells collected from the spleens of the mice was evaluated using the ⁵¹Chromium‐releasing assay. (E) The T‐cell‐stimulatory activity of DCs in regional lymph nodes was evaluated using the mixed‐leukocyte reaction. (F–H) The proportion of DCs and the expression of major histocompatibility complex (MHC) class II on DCs in the tumor increased in the BRAF inhibitor‐treated group, the TLR7 agonist‐treated group, and the coadministration group compared with the control group. All the data in (C–H) were derived from three independent experiments. Error bars indicate the SD. *p < 0.05 using Student's t‐test.

To further clarify the mechanism of antihuman melanoma activities in the combination therapy, the levels of serum mouse IFN‐α and splenic NK cell cytolytic activity against A375 cells were measured. It was found that both parameters were increased in the A375 cell‐implanted nude mice treated with DSR6434 alone or vemurafenib alone, but the combination therapy resulted in a significantly higher increase (Figure 3C,D). It is possible that vemurafenib supports the activation of pDCs by the TLR7 agonist, as pretreatment of A375 cells with vemurafenib partially decreased the in vitro inhibitory activity of A375 cell culture supernatants on IFN‐α production by murine pDCs (Figure S3).

Since vemurafenib administration reduced the immunosuppressive IL‐6 levels that suppress DC function, and the TLR7 agonist increased IFN‐α levels that stimulate DCs, the T‐cell‐stimulatory activity of cDCs in draining lymph nodes of A375 melanoma cell‐bearing nude mice treated with vemurafenib and the DSR6434 combination was evaluated. The combination treatment resulted in an enhancement of T‐cell‐stimulatory activity in DCs in an additive manner (Figure 3E). Furthermore, the number of DCs and expression of major histocompatibility complex (MHC) class II on DCs in tumors were increased in all mice treated with the combination (Figure 3F–H). There were no observed changes in the numbers of tumor‐associated macrophages and myeloid‐derived suppressor cells in the treated mice (data not shown). These findings indicate that the combination of vemurafenib and the systemically administered TLR7 agonist enhances antitumor innate immune responses through the pDC‐IFN‐α‐NK cell and cDC pathways, which may promote the development of tumor antigen‐specific T cells.

3.3. A combination of the BRAF inhibitor, TLR7 agonist, and anti‐PD‐1 antibodies showed synergistic antitumor effects

Based on our findings of significant enhancement of innate immunity, including cDCs, which can enhance the induction and effector function of tumor antigen‐specific T cells, by combination therapy with vemurafenib and TLR7 agonist, a triple combination of vemurafenib, TLR7 agonist, and T‐cell‐stimulating anti‐PD‐1 antibody was then evaluated. To assess tumor antigen‐specific T cells, a syngeneic mice model was developed using a newly established BRAF‐mutated murine melanoma cell line BPmel‐1 derived from spontaneously developed melanoma in gene‐engineered mice (B6.Cg‐Braf ^tm1Mmcm Pten ^tm1Hwu Tg(Tyr‐cre/ERT2)13Bos/BosJ) with BRAF‐mutated/PTEN‐loss genotype. This mouse melanoma cell line had a homozygous BRAF V600E mutation and expressed an immunogenic melanoma‐specific antigen, gp100 (Figure 4A,B). Vemurafenib inhibited cell proliferation of the BRAF‐mutated murine melanoma cells in a 72‐h WST assay and also inhibited IL‐6 production without affecting cell proliferation in a 24‐h WST assay in a dose‐dependent manner, similar to human melanoma cells (Figure 4C–E).

Synergistically enhanced antitumor effects of the combination of the BRAF inhibitor and the Toll‐like receptor 7 (TLR7) agonist accompanied by augmented gp100‐specific CD8⁺ T‐cell induction in *BRAF*‐mutated murine melanoma‐bearing mice. (A, B) A *BRAF*‐mutated murine melanoma cell line was established from gene‐engineered mice with a *BRAF‐*mutated/*PTEN*‐loss genotype. This cell line has a homozygous *BRAF* V600E mutation and expresses gp100, a melanoma‐specific antigen. (C) The BRAF inhibitor inhibited the proliferation of *BRAF*‐mutated murine melanoma cells in vitro in a dose‐dependent manner, with significant inhibition observed at 0.05 μM or higher. (D, E) The supernatant was collected when no inhibition of *BRAF*‐mutated murine melanoma proliferation was observed and used for an enzyme‐linked immunosorbent assay. The BRAF inhibitor inhibited the production of interleukin (IL)‐6 in a dose‐dependent manner. (F) C57BL/6 mice were inoculated with a *BRAF*‐mutated murine melanoma cell line. (G) Gp100‐specific cytotoxic T lymphocyte induction was evaluated by interferon (IFN)‐γ‐releasing assays. All the data in (C–G) were derived from three independent experiments. Error bars indicate the SD. *p < 0.05 using Student's t‐test.

Syngeneic C57/B6 mice subcutaneously implanted with BRAF‐mutated murine melanoma cells were systemically treated with vemurafenib alone (30 mg/kg orally daily), DSR6434 alone (1 mg/kg intravenously weekly), or a combination of vemurafenib and DSR6434. The combination significantly inhibited tumor growth and was accompanied by a significant increase in melanoma antigen gp100‐specific T cells in draining lymph nodes (Figure 4F,G), indicating synergistic augmentation of antitumor T cells by the combination treatment with vemurafenib and a TLR7 agonist.

Lastly, the antitumor effects of triplet therapy with vemurafenib, DSR6434, and an anti‐PD‐1 antibody were evaluated. Three‐drug combination therapy did not induce complete regression of BRAF‐mutated murine melanoma cells (data not shown). This may be due to the nonimmunogenic nature of the melanoma cells with a lower tumor mutation burden that were established from mutated BRAF oncogene Tg mice. To prepare immunogenic mouse melanoma cells similar to human melanoma, we generated SIY antigen‐transfected BRAF‐mutated mouse melanoma cells and tested the drug combination. Syngeneic C57/B6 mice subcutaneously implanted with SIY‐transduced BRAF‐mutated murine melanoma cells were systemically treated with vemurafenib, DSR6434, and an anti‐PD‐1 antibody, alone, in doublet combinations, or in the triplet combination.

Although weak additional antitumor effects were observed with the doublet combinations, such as tumor regression in 4 out of 16 mice in the TLR7 agonist and anti‐PD‐1 antibody combination group, the triplet combination therapy was the only one that demonstrated very strong synergistic antitumor effects, resulting in complete regression of all the tumors (Figure 5A,B). The three‐drug group had a significantly higher progression‐free survival rate than the other groups (Figure 5C).

Synergistically enhanced antitumor effects of the combination of the BRAF inhibitor, Toll‐like receptor 7 (TLR7) agonist, and anti‐PD‐1 antibody. (A) A melanoma cell line expressing the artificial antigen SIY was created and subcutaneously grafted into C57/B6 mice (1 × 10⁶), followed by treatment with combination therapy (n = 8). (B) Graph showing the individual tumor growth progression in each group. (C) Progression‐free survival was calculated using a cutoff of an increase in tumor size >200 mm³. (D) Intraperitoneal administration of 200 μg of rat antimouse CD8 or isotype antibody was performed 1 day before tumor implantation and on days 3, 6, and 9 after tumor implantation (mean tumor size ± SD [n = 5]). (E) A SIY‐expressing murine melanoma cell line was subcutaneously implanted in C57/B6 mice (1 × 10⁶; n = 5) that were then treated with combination therapy. Rat antimouse PDCA1 or isotype antibodies (Bio X Cell) were administered intraperitoneally at a dose of 200 μg 1 day before tumor implantation and on days 3 and 6 after tumor implantation. Tumor sizes were subsequently measured. All the data in (A) and (D) were derived from two independent experiments. Error bars indicate the SD. *p < 0.05 using Student's t‐test.

The antitumor effects of the triplet therapy disappeared upon CD8⁺ T‐cell depletion using a specific antibody, indicating that the antitumor effects are dependent on CD8⁺ T cells (Figure 5D). The number of IFN‐γ producing gp100‐specific CD8⁺ T cells was higher in the three‐drug combination group than in the two‐drug combination group with the BRAF inhibitor and a TLR7 agonist (Figure S5). In addition, depletion of pDCs in mice by the antibody suppressed antitumor effects of the triple‐combination therapy (Figure 5E), indicating that pDCs were involved in the antitumor effects of the triple‐combination therapy. SIY melanoma cells (1 × 10⁶) were reimplanted in eight mice whose tumors had been rejected by the triple therapy. None of these tumors grew (Figure S4), indicating the presence of significant memory T‐cell responses in mice treated with triplet therapy.

These findings indicate that the triple combination of inhibiting oncogene (BRAF)‐dependent cancer cell‐derived immunosuppressive factors, enhancing innate immune responses to promote T‐cell induction and function via the pDC‐IFN‐α‐NK cell/cDC pathways, and blocking the major adaptive immunoresistant mechanism PD‐1/PD‐L1 interaction is essential for eradicating relatively immunoresistant BRAF‐mutated melanoma. However, further clinical trials are warranted to evaluate the effectiveness of these combinations. Furthermore, this study suggests that such triplet immune interventions may be applicable to a variety of cancers by appropriately targeting the immunoresistant mechanisms that are specific to particular cancer subtypes.

4. DISCUSSION

In this study, our aim was to develop an effective combination immunotherapy for patients with BRAF‐mutated melanoma by targeting three essential pathways involved in the induction and function of antitumor T cells. These pathways included the blocking of the major adaptive immune‐resistant mechanism, PD‐1/PD‐L1 interaction, using an anti‐PD‐1 blocking antibody; inhibiting the primary immune‐resistant mechanism triggered by cancer cells by using a BRAF inhibitor to inhibit activated BRAF/MAPK oncogene signaling, which leads to the production of multiple immunosuppressive molecules; and augmenting innate immunity to promote antigen‐specific T‐cell induction, including pDCs, type I IFN, NK cells, and cDCs, by using a TLR7 agonist, DSR6434. First, the in vivo immune‐augmenting effects of the BRAF inhibitor vemurafenib were confirmed. Through the administration of vemurafenib, human immunosuppressive cytokines were reduced, and the T‐cell‐stimulatory activity of DCs was enhanced in vivo in an immunodeficient mouse model implanted with human BRAF‐mutated melanoma cells.

Next, systemic administration of DSR6434 was found to enhance murine IFN‐α and activate murine NK cells in vivo. Furthermore, when combined with vemurafenib, DSR6434 synergistically enhanced the antitumor effects. Lastly, in an immunocompetent syngeneic mouse model with a newly developed murine BRAF‐mutated melanoma, it was observed that the triplet combination of an anti‐PD‐1 antibody, vemurafenib, and DSR6434 exhibited significantly high tumor eradication activity compared with other mono‐ or doublet therapies in syngeneic mouse models implanted with BRAF‐mutated mouse melanoma cells. These findings underscore the importance of triplet combinations for efficiently eradicating relatively resistant cancer cells, including BRAF‐mutated melanoma.

We previously reported that the immunosuppressive activity of human BRAF‐mutated melanoma cells on DCs was reduced by inhibiting the BRAF/MAPK signals, resulting in the activation of T cells in vitro. ¹³ In this study, we confirmed that the BRAF inhibitor vemurafenib effectively inhibited the production of multiple immunosuppressive factors, including IL‐6, IL‐8, and PGE2, by human BRAF‐mutated melanoma cell lines in vitro. In addition, when vemurafenib was administered to nude mice implanted with BRAF‐mutated human melanoma cell lines, the T‐cell‐stimulatory activity of murine DCs, which had been impaired in tumor‐bearing mice in vivo, was found to be restored along with a decrease of human IL‐6 in nude mice, which has suppressive activity on murine DCs.

The BRAF inhibitor vemurafenib had an inhibitory effect on the proliferation of BRAF‐mutated A375 human melanoma cells in vitro and had in vivo antitumor effects in nude mice implanted with A375 (Figure 3A). However, the BRAF inhibitor alone did not have stimulatory activity on NK cells in nude mice (Figure 3D), indicating that the BRAF inhibitor has direct antitumor activity on melanoma cells. We observed further enhancement of in vivo antitumor activity in the nude mouse model treated with a TLR7 agonist via the activation of antitumor immune responses. The BRAF inhibitor thus appears to have dual antitumor mechanisms: (i) a direct antiproliferation effect and (ii) an indirect immunological effect through enhancement of immune response with reduction of immunosuppressive factors. The relative importance of these mechanisms may depend on the tumor models.

Other research groups have also reported that BRAF inhibitors can enhance antitumor T cells via the paradoxical activation of the MAPK signaling pathway ⁴⁰ , ⁴¹ and upregulation of MHC class I expression in melanoma cells. ⁴² , ⁴³ In patients with BRAF‐mutated melanoma, vemurafenib therapy has been shown to increase the number of CD8⁺ T cells in tumors, along with increased expression of melanoma antigens and reduced levels of IL‐6 and IL‐8. ⁴⁴ , ⁴⁵ Therefore, BRAF inhibitors are considered immunoenhancing modulators and could potentially be useful in combination immunotherapy. However, the clinical efficacy of combined treatment with BRAF/MAPK inhibitors and anti‐PD‐1/PD‐L1 antibodies remains controversial. Clinical trials investigating the combination therapy of the BRAF inhibitor vemurafenib, the MEK inhibitor cobimetinib, and the anti‐PD‐L1 antibody atezorizumab have reported that the treatment extended progression‐free survival but not overall survival. ²⁰ Therefore, in this study, our aim was to explore additional interventions to improve the combination of PD blockade and BRAF inhibitors.

One of the additional strategies to improve the inhibitory effects of PD‐1 and BRAF doublet therapy may involve enhancing innate immunity, supporting the induction and function of tumor antigen‐specific T cells. High numbers of NK cells and M1‐like monocytes and macrophages have also been shown to correlate with a positive response to ICI therapy in patients with melanoma. ²⁷ , ²⁸ , ²⁹ During antitumor T‐cell responses, relatively weak T‐cell stimulation with tumor antigens with less costimulatory activity in lymph nodes and repetitive antigen stimulation resulted in exhaustion of the antitumor T‐cell differentiation pathway. In this condition, secondary activation of stem‐like T cells or transitory effector‐like T cells at perivascular niches, APC/DC niches, or tertiary lymphoid structures in the tumor microenvironment appear to be important for generating final effector T cells. ²¹ , ²² , ²³ , ²⁴ , ²⁵ There are prior reports that the activation of cDCs in tumors by specific gut microbiota enhances the antitumor effects of ICIs via the induction of antitumor T cells. ²⁶

TLR7, a receptor for single‐stranded RNAs derived from viruses, can activate innate immunity. It can be activated by small ligands such as guanosine and imidazoquinoline derivatives. ⁴⁶ , ⁴⁷ , ⁴⁸ TLR7 is an attractive target for enhancing innate immune responses via multiple mechanisms. TLR7 agonists have been reported to stimulate pDCs to produce high amounts of IFN‐α, which in turn activates NK cells to produce IFN‐γ. The produced IFNs further stimulate cDCs, which in turn activate tumor antigen‐specific T cells. ³⁰ , ³¹ Peritumoral topical application of imiquimod has been shown to prevent the development of BRAF inhibitor‐resistant melanoma and prolong tumor growth control. This may be attributed to an increase in T cells, NK cells, and IFN‐γ in mice treated with the combination, while BRAF inhibitor monotherapy resulted in relatively rapid regrowth of resistant melanoma. ³⁰ Topical application of imiquimod, a TLR7 agonist, on high‐risk invasive melanoma has also been reported to increase the infiltration of both CD4⁺ and CD8⁺ T cells in melanoma lesions, as well as CD4⁺ T‐cell infiltration in sentinel lymph nodes. ⁴⁹ A phase I/II clinical study investigating combination therapy with imiquimod and IL‐2 showed a favorable clinical outcome in 50.5% of lesion sites, ⁵⁰ although it was not effective on remote metastases.

TLR7 agonists that can be administered systemically have been developed. The systemic administration of TLR7 agonist 852A has shown a delay in tumor growth and lung metastasis in mouse B16 melanoma, along with the production of IFN‐α from pDCs. ⁵¹ Intravenous administration of 852A in patients with refractory solid cancers has shown disease stabilization, along with increased IFN‐α levels and NK cell activation. ⁵² Oral administration of TLR7 agonist SM‐276001 has been found to reduce the growth of mouse colon cancer cell line CT26 and renal cancer cell line Renca while also increasing multiple cytokines, including IFN‐γ, and activating NK and T cells. ⁵³ Intravenous administration of TLR7 agonist SC1 has been shown to inhibit CT26 and B16 melanoma cells while increasing CD8⁺ T‐cell and cDC infiltration in tumors, strongly activating pDCs, and skewing toward M1‐like macrophages. In the same vein, intravenous administration of TLR7 agonist DSP‐0509 has been reported to inhibit CT26 tumor growth in a T‐cell‐dependent manner, increase multiple cytokines, including IFN‐α and tumor necrosis factor‐alpha, and enhance the antitumor effects of the anti‐PD‐1 antibody in CT26 and mouse breast cancer 4 T1 models. ⁵⁴ Therefore, systemic therapy with the TLR7 agonist may be useful for improving tumor immunity.

In this study, the TLR7 agonist DSR6434, which is 300 times more potent than 852A, ⁵⁵ was systemically administered. Intravenous administration of DSR6434 has been reported to enhance the antitumor effects of local ionizing radiation and improve the survival of mice implanted with the CT26 cell line or the mouse fibrosarcoma KHT cell line, with an increase in tumor antigen‐specific T cells. ⁵⁵ We aimed to evaluate various combination immunotherapies using systemic administration of the TLR7 agonist DSR6434, the BRAF inhibitor vemurafenib, and the anti‐PD‐1 antibody for BRAF‐mutated melanomas. In a xenogeneic nude mouse model implanted with human BRAF‐mutated melanoma cells, vemurafenib administration effectively restored the T‐cell‐stimulatory activity of cDCs, possibly by decreasing cross‐reactive immunosuppressive cytokines such as IL‐6 produced by the implanted human melanoma cells. Additional intravenous administration of DSR6434 enhanced the tumor growth inhibition by the BRAF inhibitor, accompanied by an increase in pDC‐derived IFN‐α, enhanced murine NK activity, and enhanced T‐cell‐stimulatory activity of cDCs in draining lymph nodes and tumors. In a syngeneic mouse BRAF‐mutated melanoma model, triplet therapy with DSR6434, vemurafenib, and an anti‐PD‐1 antibody was found to effectively eradicate the BRAF‐mutated melanoma among various doublet combination therapies, although some of the other combinations inhibited tumor growth. These findings indicate that the combination of blocking adaptive resistance with the anti‐PD‐1 antibody and overcoming primary resistance with the BRAF inhibitor alone was not sufficient to eradicate the tumor in this model. However, the additional immune intervention with a TLR7 agonist for enhancing innate immunity was able to induce sufficient antitumor activity for tumor eradication.

In summary, these findings indicate that comprehensive immune interventions, including blocking adaptive and primary immune resistance and enhancing innate immunity for tumor antigen‐specific T cells, may be essential for effective combination cancer immunotherapy. Clinical trials of the triplet combination immunotherapy for patients with BRAF‐mutated melanoma and other cancers beyond BRAF‐mutated cancers are warranted.

AUTHOR CONTRIBUTIONS

Kenta Nakamura: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; validation; visualization; writing – original draft; writing – review and editing. Tomonori Yaguchi: Conceptualization; investigation; methodology; project administration; supervision; validation; writing – original draft; writing – review and editing. Masashi Murata: Conceptualization; resources; writing – original draft. Yosuke Ota: Conceptualization; resources; writing – original draft. Asuka Mikoshiba: Conceptualization; writing – original draft. Yukiko Kiniwa: Conceptualization; writing – original draft. Ryuhei Okuyama: Conceptualization; supervision; writing – original draft; writing – review and editing. Yutaka Kawakami: Conceptualization; funding acquisition; methodology; project administration; supervision; writing – original draft; writing – review and editing.

FUNDING INFORMATION

This work was supported by Grants‐in‐aid for Scientific Research (26221005) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and 15sk0210034h0001, the Project for Development of Innovative Research on Cancer Therapeutics (P‐DIRECT) (15cm0106084h0005), and the Project for Cancer Research and Therapeutic Evolution (P‐CREATE) (16cm0106305h0001) from the Japan Agency for Medical Research and Development (AMED).

CONFLICT OF INTEREST STATEMENT

This study was funded by Sumitomo Pharma Co., Ltd. Masashi Murata and Yosuke Ota are full‐time employees of Sumitomo Pharma Co., Ltd. Yutaka Kawakami and Ryuhei Okuyama are editorial board members of Cancer Science.

ETHICS STATEMENTS

Approval of the research protocol by an Institutional Reviewer Board: N/A.

Informed Consent: N/A.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: The protocol for this study was approved by the Ethics Committee on Animal Research of Keio University School of Medicine (protocol number 250142).

Supporting information

Figure S1.

CAS-115-2879-s003.docx^{(160.8KB, docx)}

Figure S2.

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Figure S3.

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Figure S4.

CAS-115-2879-s001.docx^{(143.1KB, docx)}

Figure S5.

CAS-115-2879-s006.docx^{(125.6KB, docx)}

Data S1.

CAS-115-2879-s002.docx^{(34.7KB, docx)}

ACKNOWLEDGMENTS

We would like to thank Kenji Morii for technical assistance.

Nakamura K, Yaguchi T, Murata M, et al. Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD‐1 antibody for BRAF ‐mutated melanoma. Cancer Sci. 2024;115:2879‐2892. doi: 10.1111/cas.16251

Kenta Nakamura and Tomonori Yaguchi contributed equally to this work.

Contributor Information

Kenta Nakamura, Email: kenta1983@shinshu-u.ac.jp.

Yutaka Kawakami, Email: yutakawa@iuhw.ac.jp, Email: yutakawa@keio.jp.

DATA AVAILABILITY STATEMENT

The datasets used or analyzed in the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

CAS-115-2879-s003.docx^{(160.8KB, docx)}

Figure S2.

CAS-115-2879-s005.docx^{(131.9KB, docx)}

Figure S3.

CAS-115-2879-s004.docx^{(120.3KB, docx)}

Figure S4.

CAS-115-2879-s001.docx^{(143.1KB, docx)}

Figure S5.

CAS-115-2879-s006.docx^{(125.6KB, docx)}

Data S1.

CAS-115-2879-s002.docx^{(34.7KB, docx)}

Data Availability Statement

The datasets used or analyzed in the current study are available from the corresponding author upon reasonable request.

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PERMALINK

Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD‐1 antibody for BRAF ‐mutated melanoma

Kenta Nakamura

Tomonori Yaguchi

Masashi Murata

Yosuke Ota

Asuka Mikoshiba

Yukiko Kiniwa

Ryuhei Okuyama

Yutaka Kawakami

Abstract

Abbreviations

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Animals and cell lines

2.2. Cell culture (WST‐1, enzyme‐linked immunosorbent assay [ELISA])

2.3. Conditioned media collection

2.4. Cytokine and prostaglandin E2 (PGE2) measurement

2.5. In vivo vemurafenib administration

2.6. In vivo administration of vemurafenib and a TLR7 agonist

2.7. Isolation of CD11c + cells from draining lymph nodes and tumors

2.8. Dendritic cell stimulation ability

2.9. Plasmacytoid DC collection and culture

2.10. Flow cytometry

2.11. 51Chromium ( 51Cr)‐release assay for cytotoxicity of natural killer cells

2.12. DNA extraction from transgenic (Tg) mice and cell lines

2.13. Direct sequencing

2.14. Quantitative real‐time polymerase chain reaction

2.15. Three‐agent combination with anti‐PD‐1 antibody assay

2.16. CD8 depletion and pDC depletion

2.17. Statistical analysis

3. RESULTS

3.1. Immunostimulating activity of BRAF inhibitor vemurafenib on human melanoma

FIGURE 1.

FIGURE 2.

3.2. Immunostimulating activity of a combination of a BRAF inhibitor and a TLR7 agonist

FIGURE 3.

3.3. A combination of the BRAF inhibitor, TLR7 agonist, and anti‐PD‐1 antibodies showed synergistic antitumor effects

FIGURE 4.

FIGURE 5.

4. DISCUSSION

AUTHOR CONTRIBUTIONS

FUNDING INFORMATION

CONFLICT OF INTEREST STATEMENT

ETHICS STATEMENTS

Supporting information

ACKNOWLEDGMENTS

Contributor Information

DATA AVAILABILITY STATEMENT

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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2.7. Isolation of CD11c ⁺ cells from draining lymph nodes and tumors

2.11. ⁵¹Chromium ( ⁵¹Cr)‐release assay for cytotoxicity of natural killer cells