Suppression of MUC1-Overexpressing Tumors by a Novel MUC1/CD3 Bispecific Antibody

Abstract

Mucin1 (MUC1) is abnormally glycosylated and overexpressed in a variety of epithelial cancers and plays a critical role in tumor progression. MUC1 has received remark attention as an oncogenic molecule and is considered a valuable tumor target for immunotherapy, while many monoclonal antibodies (mAbs) targeting MUC1-positive cancers in clinical studies lack satisfactory results. It would be highly desirable to develop an effective therapy against MUC1-expressing cancers. In this study, we constructed a novel T cell-engaging bispecific antibody (BsAb) targeting MUC1 and CD3 with the Fab-ScFv-IgG format. A high quality of MUC1-CD3 BsAb can be acquired through a standard method. Our study suggested that this BsAb could specifically bind to MUC1- and CD3-positive cells and efficiently enhance T cell activation, cytokine release, and cytotoxicity. Furthermore, our study demonstrated that this BsAb could potently redirect T cells to eliminate MUC1-expressing tumor cells in vitro and significantly suppress MUC1-positive tumor growth in a xenograft mouse model. Thus, T cell-engaging MUC1/CD3 BsAb could be an effective therapeutic approach to combat MUC1-positive tumors and our MUC1/CD3 BsAb could be a promising candidate in clinical applications for the treatment of MUC1-positive cancer patients.

Keywords: BsAb; Mucin1; epithelial cancers; immunotherapy; oncogenic molecule.

Figure 1

Generation of MUC1/CD3 BsAb. (A) Schematic design of MUC1/CD3 BsAb. (B) SEC-HPLC analysis of the purified MUC1/CD3 BsAb. The percentage of the area of peak 1 and peak 2 is 0.7% and 99.3% respectively, which represents the high purity of the purified BsAb. (C) SDS/PAGE and Coomassie blue staining results of the purified MUC1/CD3 BsAb under non-reducing and reducing conditions. (D) Cell binding analysis of MUC1/CD3 BsAb to MUC1 and CD3-positive cells by flow cytometry, HeLa (MUC1-positive cells), Jurkat (CD3-positive cells), NOZ (CD3 and MUC1-negative cells).

Figure 2

MUC1/CD3 BsAb can induce potent T cell-mediated MUC1-expressing cell lysis. (A) Tumor cell lines: HeLa, MCF7, SKOV3, and NOZ were first stained by MUC1/CD3 BsAb and followed by a secondary FITC anti-human IgG Fc antibody. MUC1 expression was detected by flow cytometry. (B) Detection of tumor cell lysis after 48 h of incubation with human PBMCs (E/T 10:1) or T cells (E/T 5:1) with serially diluted antibodies. (C) Detection of T cell-mediated cytotoxicity in the presence of serially diluted antibodies with different E/T ratios (2:1 or 8:1) in human T cell and tumor cell co-culture cytotoxic assay.

Figure 3

MUC1/CD3 BsAb can induce robust T cell activation. (A) Detection of BsAb-induced T cell and NK cell activation (CD69 and CD25). HeLa cells were co-cultured with human PBMCs with an effector-to-target ratio of 10:1 for 24 h with serially diluted antibodies. After incubation, PBMCs were collected, stained, and analyzed for CD69 and CD25 expression via flow cytometry. (B) Detection of BsAb-induced CD107a expression on CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and NK cells. HeLa cells were co-cultured with human T cells or PBMCs with an effector-to-target ratio of 10:1 with various concentrations of BsAb as indicated, then a CD107a antibody was added to the culture medium. After another 4 h, cells were collected and analyzed for CD107a expression via flow cytometry. For detailed gating strategy of CD25, CD69, and CD107a expression, please refer to the supplementary materials. (C) Measurement of cytokine produced in the co-culture supernatant via ELISA. HeLa cells were co-cultured with human T cells with an effector-to-target ratio of 8:1 in the presence of serially diluted BsAb. After 48 h of incubation, cell-free supernatant was obtained and the secretion of IFN-γ, TNF-α, and IL-2 was detected via ELISA. The data shown here are representative of three individual experiments.

Figure 3

MUC1/CD3 BsAb can induce robust T cell activation. (A) Detection of BsAb-induced T cell and NK cell activation (CD69 and CD25). HeLa cells were co-cultured with human PBMCs with an effector-to-target ratio of 10:1 for 24 h with serially diluted antibodies. After incubation, PBMCs were collected, stained, and analyzed for CD69 and CD25 expression via flow cytometry. (B) Detection of BsAb-induced CD107a expression on CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and NK cells. HeLa cells were co-cultured with human T cells or PBMCs with an effector-to-target ratio of 10:1 with various concentrations of BsAb as indicated, then a CD107a antibody was added to the culture medium. After another 4 h, cells were collected and analyzed for CD107a expression via flow cytometry. For detailed gating strategy of CD25, CD69, and CD107a expression, please refer to the supplementary materials. (C) Measurement of cytokine produced in the co-culture supernatant via ELISA. HeLa cells were co-cultured with human T cells with an effector-to-target ratio of 8:1 in the presence of serially diluted BsAb. After 48 h of incubation, cell-free supernatant was obtained and the secretion of IFN-γ, TNF-α, and IL-2 was detected via ELISA. The data shown here are representative of three individual experiments.

Figure 4

MUC1/CD3 BsAb can efficiently suppress MUC1-expressing tumors in B-NDG mice. (A) Schematic schedule of the in vivo study. Each B-NDG mouse was engrafted with 2.5 × 10⁶ HeLa cells in the right flank via subcutaneous injection on day −9. On day 0, the mice were randomized into three groups as indicated. For BsAb- and PBS (vehicle)-treated groups, each mouse was intraperitoneally injected with 1 × 10⁷ T cells. One hour later, the mouse was given 200 µL of a BsAb (250 µg/mL) solution or PBS via intraperitoneal injection, respectively. The PBS or antibody treatment was given twice per week for a total of 7 doses. Mice body weights and tumor sizes were measured twice per week. (B) Time course of tumor growth of different groups. (C) Tumor weight comparison between different groups. (D) Time course of body weight of different groups. Experimental data are presented as mean ± SEM (p < 0.05 (*)).