MEK Inhibitor Augments Antitumor Activity of B7-H3-Redirected Bispecific Antibody

Abstract

Targeting cancer antigens by T cell-engaging bispecific antibody (BiAb) or chimeric antigen receptor T cell therapy has achieved successes in hematological cancers, but attempts to use it to fight solid cancers have been disappointing, in part due to antigen escape. MEK inhibitor had limited activity as a single agent, but enhanced antitumor activity when combined with other therapies, such as targeted drugs or immunotherapy agents. This study aimed to analyze the expression of B7-H3 in non-small-cell lung cancer (NSCLC) and bladder cancer (BC) and to evaluate the combinatorial antitumor effect of B7-H3 × CD3 BiAb with MEK inhibitor trametinib. We found B7-H3 was highly expressed in NSCLC and BC compared with normal samples and its increased expression was associated with poor prognosis. Treatment with trametinib alone could induce apoptosis in tumor cell, while has no effect on T cell proliferation, and a noticeable elevation of B7-H3 expression in tumor cells was also observed following treatment. B7-H3 × CD3 BiAb specifically and efficiently redirected their cytotoxicity against B7-H3 overexpressing tumor cells both in vitro and in xenograft mouse models. While trametinib treatment alone affected tumor growth, the combined therapy increased T cell infiltration and significantly suppressed tumor growth. Together, these data suggest that combination therapy with B7-H3 × CD3 BiAb and MEK inhibitor may serve as a new therapeutic strategy in the future clinical practice for the treatment of NSCLC and BC.

Keywords: B7-H3; bispecific antibody; bladder cancer; immunotherapy; non-small-cell lung cancer; trametinib.

Figure 1

Expression of B7-H3 on human NSCLC and BC. (A–C) B7-H3 IHC staining patterns. Representative cases of normal lung and bladder samples (A), NSCLC (B), and BC (C) samples including para cancer tissues at different grades (high, medium or low). Scale bar, 50 μm. (D) Immunofluorescence staining of A549, H460, T24 tumor cells for B7-H3. Scale bar, 20 μm.

Figure 2

Effects of MEK inhibitor trametinib in A549, H460, T24, and human T cells. (A) Apoptosis detection with annexin V-FITC/7AAD double staining by flow cytometry. A549 and H460 cells were cultured with 10 μM trametinib for 48 h. (B) Inhibition rate of A549, H460, and T24 cells after exposure to trametinib with various concentrations. After 48 h of treatment, inhibition rate was measured using Cell Counting Kit-8 assays. (C) B7-H3 expression after trametinib treatment with indicated concentrations by flow cytometry. Histograms represent the measured fluorescence of cells incubated with the B7-H3 antibody. (D) Histogram of the mean fluorescence intensity. (E) Western blot analysis of B7-H3, MEK, and P-MEK expression in A549 cells after trametinib treatment with indicated concentrations. Expression of β-actin was used as an internal control. (F) Proliferation (CFSE dilution assay) of human T cells after 48 h of treatment with 1 μM trametinib by flow cytometry. Histograms represent the measured fluorescence of cells incubated with CFSE. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Construction, characterization and cytotoxicity of B7-H3 × CD3 BiAb. (A) The schematic representation of B7-H3 × CD3 BiAb. (B) Schematic diagram of B7-H3 × CD3 BiAb expression vector. (C) SDS-PAGE analysis of B7-H3 × CD3 BiAb. The BiAb was run on reducing and non-reducing SDS-PAGE gels. (D) Dot plot diagram of flow cytometry showing CD4⁺ and CD8⁺ percentage of human T cells after 5 or 10 μg/mL B7-H3 × CD3 BiAb treatment for 48 h. (E–G) Morphology of tumor cells after co-culture with human T cells. A549 (E), H460 (F), or T24 (G) cell lines were co-cultured with T cells for 12 or 24 h at a ratio of E:T = 4:1. Group “BiAb + T cell” was treated with B7-H3 × CD3 BiAb at a concentration of 5 μg/mL. Scale bar, 50 μm. (H) 51Cr-release assays of T cells against A549, H460 and T24 cell lines with 5 μg/mL B7-H3 × CD3 BiAb in different E:T ratios. (I) Quantification of IFN-γ by ELISA in the supernatant 24 h after co-culture of T cells with A549, H460, or T24 cell lines at different E:T ratio. Group “BiAb” was treated with B7-H3 × CD3 BiAb at a concentration of 5 μg/mL. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Antitumor activity by B7-H3 × CD3 BiAb in combination with trametinib in vitro. (A) Morphology of tumor cells after various treatments for 12 h. Scare bar, 50 μm. (B) Real-time cytotoxicity assay of H460 cells after various treatments for 75 h. (C) Activation signal of human T cells after co-culture with tumor cells by adding 1 μM trametinib alone or in combination with 5 μg/mL B7-H3 × CD3 BiAb. Cells were collected 24 h after co-culture with H460 and T24 cell lines and stained with antibodies against CD4, CD8, CD25, CD69 for flow cytometry. (D,E) Killing activity of B7-H3 × CD3 BiAb or in combination with trametinib was detected using the 3D tumorsphere model. A549 (D) and H460 (E) tumorspheres were co-cultured with CFSE labeled T cells including 5 μg/mL B7-H3 × CD3 BiAb alone or in combination with 1 μM trametinib for 12 h. Scare bar, 100 μm.

Figure 5

Antitumor activity by B7-H3 × CD3 BiAb in combination with trametinib in vivo. (A) Experiment design scheme. (B,C) Tumor growth curves from H460 (B) and T24 (C) mice models treated with T cell, T cell with trametinib, the BiAb or the BiAb combined with trametinib. Tumor volume measurements were recorded every 3 days. (D,E) Tumors from H460 (D) and T24 (E) mice models on day 21 are shown. (F) H&E staining images of liver, spleen, kidney, heart, and lung in H460 mice models. (G,H) Bodyweight of H460 (G) and T24 (H) mice treated with T cell, T cell with trametinib, the BiAb or the BiAb combined with trametinib. Bodyweight measurements were recorded every 3 days.

Figure 6

IHC analysis of tumors in H460 mice models. (A) Representative images of CD31 and caspase-3 staining in different groups. Scare bar, 50 μm. (B) Representative images of tumor-infiltrating T cells in different groups. T cells were detected by CD3 staining. Scare bar, 50 μm. (C) Quantification of cleaved caspase 3 in different groups. (D) Quantification of T-cell infiltration in different groups. *P < 0.05, **P < 0.01.

Figure 7

Schematic summary for synergistic effect between MEK inhibitor trametinib and B7-H3 × CD3 BiAb in killing NSCLC and BC cells.