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. 2015 Jan 26;7(2):377–389. doi: 10.1080/19420862.2015.1007816

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© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC

© Yariv Mazor, Vaheh Oganesyan, Chunning Yang, Anna Hansen, Jihong Wang, Hongji Liu, Kris Sachsenmeier, Marcia Carlson, Dhanesh V Gadre, Martin Jack Borrok, Xiang-Qing Yu, William Dall'Acqua, Herren Wu, and Partha Sarathi Chowdhury

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 6. — Concurrent binding of DuetMab to 2 antigens on the same cell as determined by FACS analysis. (A) Preferential binding by concurrent engagement to 2 antigens (CD4 and CD70) on a single cell as determined by flow-cytometry. Cell-bound DuetMab was detected with PE-labeled anti-human Fc_γ. Binding of CD4-CD70 DuetMab to 1:1:1 premix of CD4⁺CD70⁺ (red triangle), CD4⁻CD70⁺ (green square) and CD4⁺CD70⁻ (blue circle) lymphocytes labeled with different tracer dyes. (B) Detection of total DuetMab binding (y-axis) and of free CD4 binding sites on cell bound anti CD4-anti CD70 DuetMab using biotin labeled sCD4 (x-axis). (C) Detection of total DuetMab binding (y-axis) and of free CD70 binding sites on cell bound anti CD4-anti CD70 DuetMab using biotin labeled sCD70 (x-axis). Each point in (B) and (C) refers to a different DuetMab concentration. These were 0, 0.001, 0.005, 0.02, 0.08, 0.3 and 1.25 nM. (D) Preferential binding of EGFR-HER2 DuetMab to SK-OV3 in a 1:1:1 premix of SK-OV3 (EGFR^MedHER2^Med in red triangle, SKBr3 (EGFR^LowHER2^Hi in green square) and A431(EGFR^HiHER2^Low in blue circle) cells, each labeled with different tracer dyes. The number of EGFR and HER2 sites, respectively, on the 3 cells as determined by QIFIKIT are as follows. A431- 83.3 × 10⁴ ± 5.2 × 10⁴ and 3.3 × 10⁴ ± 0.15 × 10⁴; SK-OV3 – 14.4 × 10⁴ ± 0.7 × 10⁴ and 34.5 × 10⁴ ± 1.4 × 10⁴; and SKBr3- 4.2 × 10⁴ ± 0.3 × 10⁴ and 176 × 10⁴ ± 7.2 × 10⁴. The maximum number of bivalent binding possible on each cell line is indicated in bold. (E) Detection of total DuetMab binding (y-axis) and of free EGFR binding sites on cell bound EGFR-HER2 DuetMab using biotin labeled sEGFR (x-axis). (C) Detection of total DuetMab binding (y-axis) and of free HER2 binding sites on cell bound EGFR-HER2 DuetMab (x-axis). Each point in (E) and (F) refers to a different DuetMab concentration. These were 0, 0.02, 0.08, 0.3, 1.25 and 5 nM. Binding of DuetMab was monitored by PE labeled anti-human Fc and binding of biotinylated soluble ligands were monitored by BV421 labeled streptavidin.