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. 2017 Aug 21;7:8360. doi: 10.1038/s41598-017-08641-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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In vitro bioactivity of BsAb (CD3×HER2). (a) Target-dependent T cell mediated cytotoxicity of BsAb (CD3×HER2) was detected using LDH release assay. Effecters human PBMCs, E:T ratio 10:1, time point of 20 hours. The EC50 values were calculated by fitting the dose-response curve with Graphpad Prism software. R² represents the square of correlation coefficient. (b) T cell activation was detected by staining cells for CD8 and CD69 followed by FACS analysis. Effectors CD3⁺ T cells, target NCI-N87 cell line, E:T ratio 10:1. The upper panel showed the stimulation of human T cells by 100 ng/mL BsAb (CD3×HER2) or anti-CD3 mAb in the presence of target cells, while the bottom panel was lack of target cells. (c) Photographs of the redirection of T cells to cancer cells by 10 ng/mL BsAb (CD3×HER2). Effecters human PBMCs, target SK-BR-3 cell line, E:T ratio 10:1, time point of 20 hours. (d) FACS analysis of the redirection of CD3⁺ cells to cancer cells by BsAb (CD3×HER2). Jurkat (CD3⁺) cells were labeled by PKH26 (PE-A) and NCI-N87 cells were labeled by CFSE (FITC-A) separately. Then the two cells were mixed at equal ratio and treated with 10 ng/mL BsAb (CD3×HER2) or trastuzumab for 30 minutes. Data points in the figure represent the mean of three samples; error bar, SEM.