CpG-based immunotherapy impairs antitumor activity of BRAF inhibitors in a B-cell-dependent manner

Abstract

Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.

Figure 1

BRAF inhibitor-induced antitumor responses are impaired when combined with CpG-based peptide vaccines. (a) A previously established murine melanoma cell line bearing BRAF and PTEN mutation (BP) was provided by Dr Wargo (MD Anderson Cancer Center) and maintained in RPMI 1640 with 10% FCS and 100 μg/ml Normocin (Invivogen, San Diego, CA, USA). To generate gp100-expressing murine cell line (BP/gp100), BP cells were transduced with the lentiviral vector encoding full-length human gp100 as previously described. Tumor growth was induced by subcutaneous injection of 5 × 10⁵ BP/gp100 cells in 6- to 8-week-old female C57BL/6 mice (from Charles River NCI, Frederick, MD, USA). Seven days after tumor challenge, tumor-bearing mice were treated with a selective BRAF inhibitor PLX4720 (Plexxikon, Berkeley, CA, USA; 100 mg/kg in 3% DMSO and 1% methylcellulose by oral gavage daily), tumor vaccine with 50 μg CpG (CpG-ODN-2216 synthesized by Invitrogen Life Technologies (Carlsbad, CA, USA) and injected intratumorally once per week for 3 weeks) and 100 μg synthetic, high-affinity H-2D^b-restricted hgp100_25–33 peptide (KVPRNQDWL, purchased from Peptides International (Louisville, KY, USA) at a purity >95% and injected subcutaneously), or both. For groups containing the tumor vaccine, 1 × 10³ Pmel-1T cells (isolated from Pmel-1 TCR/Thy1.1 mice from in-house breeding colonies) were intravenously administered and 100 μg anti-CD40 (Bioxcell, West Lebanon, NH, USA) was intraperitoneally injected on day 7, and 1 × 10⁵ IU rhIL-2 protein (Prometheus Laboratories Inc, San Diego, CA, USA) were intraperitoneally injected on days 7, 8 and 9. (b) Tumor growth was induced by subcutaneous injection of 5 × 10⁵ BP cells in 6- to 8-week-old female C57BL/6 mice. Seven days after tumor challenge, tumor-bearing mice were treated with PLX4720, tumor vaccine with 50 μg CpG and 100 μg H-2K^b-restricted mouse P15E_134–141 peptide (KSPWFTTL, purchased from Peptides International at a purity >95% and injected subcutaneously), or both. For groups containing the tumor vaccine, 100 μg anti-CD40 was intraperitoneally injected on day 7, and 1 × 10⁵ IU rhIL-2 protein were intraperitoneally injected on days 7, 8 and 9. Tumor-bearing mice treated with vehicle (3% DMSO and 1% methylcellulose) were used as control. Tumor growth was monitored every 3 days by measuring the perpendicular diameters of tumors. N=5 mice per group. Data expressed as mean±s.e.m. *P<0.05, **P<0.01, two-way ANOVA plus post hoc Turkey test.

Figure 2

CpG negates the antitumor activity of BRAF inhibitors in a B-cell-dependent manner. To induce spontaneous melanoma in genetically modified mice, Tyr:CreER; PTEN^lox/lox; BRAF^V600E/+ (BP) mice on a C57BL/6 background (6–8 weeks of age) were treated with 50 mg ml⁻¹ 4-hydroxytamoxifen (Sigma, St Louis, MO, USA) to induce the expression of Cre as previously described. Mice with measurable tumors received indicated antitumor treatments. (a, b) Tumor-bearing BP mice were treated with daily oral gavage of 100 mg/kg PLX4720 (PLX), intraperitoneal injection of 50 μg CpG once per week for 3 weeks, or both. Tumor growth was monitored every 3 days by measuring the perpendicular diameters of tumors. N=5 mice per group. (c) Single-cell suspensions were prepared from the spleens of tumor-bearing BP mice treated with PLX, CpG or both, and stained with anti-CD4, CD8 and CD19 antibodies (BD Biosciences, San Jose, CA, USA). The percentages of various immune cell populations (T cells and B cells) in stained samples were analyzed by flow cytometry using a FACSCANTO II cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA). CD19⁺ B cells in spleen in each group were shown. N=5 per group. (d) Tumor-bearing BP mice were simultaneously treated with PLX, CpG or both, as described in (a, b). Also, 50 μg of anti-mouse CD20 Ab IgG2a kindly provided by Biogen Idec (San Diego, CA, USA) were injected intravenously in tumor-bearing BP mice once per week for 2 weeks to deplete B cells either alone or together with PLX and CpG. Isotype control antibody purchased from Sigma were similarly injected as a control. Tumor sizes were monitored every 3 days (Control group: N=7; PLX group: N=6; anti-CD20 group: N=5; CpG group: N=4; PLX+CpG group: N=6; PLX+CpG+anti-CD20 group: N=6). Data expressed as mean±s.e.m. *P<0.05, ***P<0.001, two-way ANOVA (b, d) or one-way ANOVA (c) plus post hoc Turkey test.

Figure 3

CpG treatment dampens BRAF inhibitor-induced tumor regression through B-cell-derived TNFα. (a) Sera samples from tumor-bearing BP mice treated as in Figure 2d were collected and measured by MILLIPLEX mouse cytokine/chemokine panels I (premixed 32-plex) and II (premixed 8-plex) according to the manufacturer's protocol (EMD Millipore, Billerica, MA, USA). The concentration of each cytokine and chemokine in serum was determined using a Luminex 200 system (Luminex Corporation, Austin, TX, USA). Serum expression of TNFα from different treatment groups are shown. N=3 per group. (b) Melanoma tumor cells A375 and 2400 were seeded at 1000 cells per well and stimulated with 1 μm PLX, 20 ng/ml TNFα (Bioxcell) or both. Three days later, cell viability was determined using CellTiter Blue assay according to the manufacturer’s protocol (Promega, Madison, WI, USA). Data expressed as mean±s.e.m. of triplicates from two independent experiments. (c) Tumor-bearing BP mice were treated with vehicle, PLX, CpG, or combination of PLX and CpG as described in Figure 2. To neutralize TNFα, mice were injected with 100 μg of rat anti-mouse TNFα IgG1 (Bioxcell) intraperitoneally once per week for 2 weeks, either alone or together with PLX and CpG. Tumor sizes were monitored every 3 days. (Control group: N=5; PLX group: N=4; anti-TNFα group: N=5; CpG group: N=4; PLX+CpG group: N=6; PLX+CpG+anti-TNFα group: N=6). Data expressed as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA (a, b) or two-way ANOVA (c) plus post hoc Turkey test.