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. 2022 Jan 1;18(1):166-179.
doi: 10.7150/ijbs.64094. eCollection 2022.

Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity

Sumei Ren  1   2 , Xiaodong Wang  1 , Guangyi Jin  1
Affiliations

Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity

Sumei Ren et al. Int J Biol Sci. .

Abstract

The use of large molecules for immunotherapy has led to exciting developments in cancer treatment, such as the development of PD-1/PD-L1 antibodies. However, small molecule targeted therapies still lack effective immune-functional classes. Ideal anticancer drugs should simultaneously generate immune memory when killing cancer cells to prevent tumor relapse and metastasis. To this end, we carried out a rationally designed strategy to develop novel classes of small molecule compounds with bifunctional targeting and immunostimulatory abilities by conjugating targeting compounds with TLR7 agonists, generating immune-targeting conjugates (ImmunTacs). GY161, as a representative ImmunTac, was synthesized via chemical conjugation of ibrutinib with a TLR7 agonist. In vitro, GY161 stimulated the production of cytokines by mouse spleen lymphocytes, promoted the maturation of dendritic cells (DCs), and inhibited the growth and induced the apoptosis of B16 melanoma cells by regulating the c-Met/β-catenin pathway. In vivo, GY161 enhanced the frequency of CD8+ T cells in spleens and tumors, suppressed the growth of B16 melanoma cell-derived tumors and prolonged the survival time of mice. In summary, GY161 could prevent melanoma progression through direct tumor killing and by triggering specific immunity. These results strongly suggest that ImmunTacs are a reliable and promising strategy for developing small molecule immunogenic anticancer drugs.

Keywords: TLR7 agonist; ibrutinib; immunotherapy; melanoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Scheme 1
Scheme 1
Route of GY100 and GY161 synthesis.
Figure 1
Figure 1
GY161 stimulated TLR7 signaling. (A) HEK-Blue hTLR7 cells were treated overnight with the compounds at the indicated concentrations, and the final OD values were recorded at 650 nm. (B) Effects of the compounds on HEK reporter cells expressing human TLR8. The data are presented as the mean ± SE (n = 3). Combo: a physical mixture of ibrutinib and GY100 (1:1 molar ratio).
Figure 2
Figure 2
Mouse spleen lymphocytes were treated with the compounds for 24 h, and IL-6, IL-12, TNF-α and IFN-γ secretion was quantified by ELISA. The data are presented as the mean ± SE (n = 3). Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).
Figure 3
Figure 3
GY161 could induce BMDC maturation. Maturation of BMDCs was evaluated by measuring the expression of the surface molecules CD40, CD80 and CD86 by flow cytometry after 24 h of coculture with 0.1% DMSO (control) or compounds (10.0 µM). Significant differences among the groups were analyzed using one-way ANOVA with Bonferroni post hoc test. The data are presented as the mean ± SE, where *P < 0.05, **P< 0.01, ***P<0.001. Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).
Figure 4
Figure 4
(A) Effects of compounds on mouse splenic lymphocyte and BMDC proliferation. Lymphocytes and BMDCs were treated with the compounds for 48 h. (B) Antiproliferative effects of selected compounds on DaudiB, A20, B16, A375 and MCF-10A cells. The cells were incubated with serial dilutions of the compounds at various concentrations (2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 µM) for 48 h. The cells were incubated with DMSO as a control. The data are presented as the mean ± SE. Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).
Figure 5
Figure 5
Effects of compound GY161 on B16 and A375 cells. (A, B) B16 cells were treated with selected compounds for 24 h, and cell apoptosis and cell cycle progression were analyzed by flow cytometry. (C, D) A375 cells were treated with selected compounds for 24 h, and cell apoptosis and cell cycle progression were analyzed by flow cytometry. Cells were treated with DMSO as control. Significant differences among the groups were analyzed using one-way ANOVA with Dunn's post hoc test. The data are presented as the mean ± SE. *P < 0.05, **P< 0.01, ***P<0.001. Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).
Figure 5
Figure 5
Effects of compound GY161 on B16 and A375 cells. (A, B) B16 cells were treated with selected compounds for 24 h, and cell apoptosis and cell cycle progression were analyzed by flow cytometry. (C, D) A375 cells were treated with selected compounds for 24 h, and cell apoptosis and cell cycle progression were analyzed by flow cytometry. Cells were treated with DMSO as control. Significant differences among the groups were analyzed using one-way ANOVA with Dunn's post hoc test. The data are presented as the mean ± SE. *P < 0.05, **P< 0.01, ***P<0.001. Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).
Figure 6
Figure 6
Effects of the selected compounds (20.0 μM) on c-Met and downstream signaling pathways in B16 cells. (A) A heatmap of the expression of ten genes in the control and GY161 groups. (B) The relative expression levels of Met, Ctnnb1 and Mitf were detected by qPCR. (C, D) c-Met protein expression and the inhibition of c-Met/β-catenin signaling in GY161-treated B16 cells were assessed by western blotting. Cells were treated with DMSO as control. Significant differences among the groups were analyzed using a T test (for B) and one-way ANOVA with Dunn's post hoc test (for C and D). The data are presented as the mean ± SE. *P< 0.05, **P< 0.01, ***P<0.001, ****P<0.0001 versus control. Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).
Figure 7
Figure 7
GY161 reduces tumor burden in B16 tumor-bearing mice. (A) Treatment scheme. Six- to 8-week-old C57BL/6J mice were inoculated with 2 × 105 B16 cells. (B) Tumor growth curves were measured with a digital caliper (n = 5 mice per group). (C) The body weights of the group mice over time. (D) Kaplan-Meier survival plots of the treated mice (n = 6 mice per group). GY161 prolonged the survival time of mice. The control group was treated with an equal volume of solvent (5% DMSO+30% PEG300+65% ddH2O). Significant differencesces among the groups were analyzed using two-way ANOVA with Bonferroni post hoc test (for B) and log-rank (Mantel-Cox) test (for D). **P< 0.01, ***P<0.001, ****P<0.0001.
Figure 8
Figure 8
The effect of GY161 (20 mg/kg) on T lymphocyte numbers in spleens and tumors. Mice were treated as shown in Figure 7A. Spleens and tumors were harvested on day 17, and T cells were analyzed by flow cytometry. Numbers of CD8+ and CD8+IFN-γ+ T were calculated. Significant differencesces among the groups were analyzed using one-way ANOVA with Bonferroni post hoc test. The data are presented as mean ± SE; *P<0.05, **P<0.01, ***P<0.001.
Figure 9
Figure 9
Proposed mechanism by which GY161 regulates B16 cells in vitro. We proposed that GY161 inhibited tumor growth by regulating the c-Met/β-catenin pathway, which is associated with B16 cell proliferation and survival.
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