A Novel Anti-B7-H3 × Anti-CD3 Bispecific Antibody with Potent Antitumor Activity

Abstract

B7-H3 plays an important role in tumor apoptosis, proliferation, adhesion, angiogenesis, invasion, migration, and evasion of immune surveillance. It is overexpressed in various human solid tumor tissues. In patients, B7-H3 overexpression correlates with advanced stages, poor clinical outcomes, and resistance to therapy. The roles of B7-H3 in tumor progression make it a potential candidate for targeted therapy. Here, we generated a mouse anti-human B7-H3 antibody and demonstrated its binding activity via Tongji University Suzhou Instituteprotein-based and cell-based assays. We then developed a novel format anti-B7-H3 × anti-CD3 bispecific antibody based on the antibody-binding fragment of the anti-B7-H3 antibody and single-chain variable fragment structure of anti-CD3 antibody (OKT3) and demonstrated that this bispecific antibody mediated potent cytotoxic activities against various B7-H3-positive tumor cell lines in vitro by improving T cell activation and proliferation. This bispecific antibody also demonstrated potent antitumor activity in humanized mice xenograft models. These results revealed that the novel anti-B7-H3 × anti-CD3 bispecific antibody has the potential to be employed in treatment of B7-H3-positive solid tumors.

Keywords: B7-H3; bispecific T cell engager; immunotherapy; solid tumor.

Figure 1

B7-H3 is highly expressed on human solid tumor cell lines. (a–l) Expression of B7-H3 on cell lines (A498, U-87 MG, SKOV3, MDA-MB-468, PANC-1, A549, BCPAP, Raji, HepG2, MDA-MB-231, AsPC-1, and K562) was detected by flow cytometry. Gray histogram, isotype control; red histogram, APC-conjugated anti-human CD276 (10 μg/mL). MFI values (control/anti-human CD276) of these cell lines were, respectively, 160/25,090, 62/10,733, 62/4317, 57/1240, 134/2472, 67/2918, 60/3315, 4.5/15, 71/1262, 63/2966, 60/2911, and 26/84. MFI, median fluorescence intensity.

Figure 2

Anti-B7-H3 antibody 10-2#c shows high binding activity to B7-H3 and cell lines with high B7-H3 expression. (a) The binding of 10-2#c to human B7-H3 reconstituted protein was evaluated by ELISA (n = 3), EC₅₀ = 2.216 ng/ml; (b–i) The binding of 10-2# to cell lines (U-87 MG, MDA-MB-231, HepG2, A549, MDA-MB-468, SKOV3, Raji, and K562) was detected by flow cytometry. MFI, median fluorescence intensity. MFI values (control/maximum concentration of 10-2#c) of these cell lines were, respectively, 834/93,630, 366/19,477, 353/16,980, 144/9385, 245/8215, 250/12,400, 125/136, and 140/646.

Figure 3

Generation and characterization of αB7-H3/CD3 bispecific antibody. (a) An illustrative representation of the αB7-H3/CD3 format. The format comprised an anti-CD3 scFv fused to light chain of a monovalent anti B7-H3 via a (G₄S)₃ linker; (b) Coomassie blue-stained SDS-PAGE analysis of purified αB7-H3/CD3 containing three chains with the following molecular weights: 57, 55, and 28 kDa; (c–k) αB7-H3/CD3 bispecific antibody dose-dependently binds to B7-H3+ tumor cells and CD3+ T cells, evaluated using flow cytometry. MFI values (control/maximum concentration of 10-2#c) of these cell lines were, respectively, 205/31,800, 7500/226,444, 480/5700, 350/27,950, 150/8065, 480/1550, 125/155, 50/10,300, and 195/3100.

Figure 4

αB7-H3/CD3 mediates T cell cytotoxicity against B7-H3 expressing tumor cells in vitro. (a) Cytotoxic activity of PBMCs in U-87 MG cells mediated by αB7-H3/CD3 or other antibodies was measured by flow cytometry. n = 3, p-value for mAb mix vs. bsAb: *** p < 0.001; (b–h) αB7-H3/CD3 demonstrated cytotoxic activity against multiple tumor cell lines (U-87 MG, A498, SKOV3, MDA-MB-231, HepG2, K562, and Raji) with various levels of B7-H3 expression, E:T = 10:1. n = 3, mAb mix, anti-CD3 and anti-B7-H3 mAb mixture; bsAb, αB7-H3/CD3.

Figure 5

αB7-H3/CD3 induces T cell activation and proliferation in vitro. U-87 MG and PBMCs were co-cultured with indicated antibodies as described in methods. T cell activation markers (CD69 and GrB) and T cell proliferation were analyzed in CD8+ and CD4+ subsets by flow cytometry. The profile of cytokines released by PBMCs was quantified using ELISA. (a,b) Percentage of CD69 and GrB-positive cells in CD4+ or CD8+ T cell subsets. n = 3; (c) Secretion of cytokines by PBMCs induced by the indicated antibodies. n = 3; (d,e) T cell proliferation was measured using CFSE dilutions. Groups in which U-87 MG was replaced with no tumor or Raji (e). Representative histograms of CFSE-labeled T cells in CD8+ (left) and CD4+ (right) T cell subsets are shown. CD3, anti-CD3 mAb; B7-H3, anti-B7-H3 mAb; mAb mix, anti-CD3 and anti-B7-H3 mAb mixture; bsAb, αB7-H3/CD3. **** p < 0.0001; *** p < 0.001; * p < 0.05.

Figure 6

αB7-H3/CD3 inhibits tumor growth in xenograft models using human PBMC-engrafted NCG mouse models. (a,c) Schematic map of the development of U-87 MG (a) and SKOV3 (c) xenograft model. U-87 MG or SKOV3 were subcutaneously implanted into NCG mice engrafted with human PBMCs. Mice were treated with PBS and doses of αB7-H3/CD3 twice a week post tumor development; (b,d) average tumor growth curve of U-87 MG (b) and SKOV3 (d) xenografts for every group. The tumor volume was plotted against the time in days following antibody administration. The number of mice is shown in figures; (e) images of individual xenograft tumors at the end of experiments; (f) immunofluorescence staining analysis of CD3+ T cells (red) in U-87 MG tumors. Scale bar, 100 μm. bsAb, αB7-H3/CD3.