Fig 1: 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.
Fig 2: 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.
Fig 3: 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.
Fig 4: 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.
Fig 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.
Supplier Page from Sino Biological, Inc. for Human B7-H3 / CD276 Protein (ECD,His Tag)