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. 2018 Feb 21;9:741. doi: 10.1038/s41467-017-02696-6

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© The Author(s) 2018

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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Fig. 6 — Design and bifunctional target binding ability of anti-PDL1-TGFβRII. a Schematic representation of PD-L1 and TGFβ entrained independent and cooperative mechanisms of immune tolerance. Schematic structure and targets of a-PDL1-TGFβRII are shown. a-PDL1-TGFβRII was designed by fusing the C terminus of the heavy chain of a human a-PD-L1 antibody with a ligand-binding sequence of the extracellular domain of TGFβ Receptor II (TGFβRII ECD) via a flexible linker peptide, (GGGGS)_3. b SDS-PAGE under non-reducing (NR) and reducing (R) conditions was used to compare the full-length, heavy chain and light chain of a-PDL1-TGFβRII and a-PD-L1 antibody. Figure shows the results of SDS-PAGE analyses of each of two separate a-PDL1-TGFβRII Y-traps (Ab1 and Ab2) and their respective human a-PD-L1 antibody (atezolizumab and avelumab). c SEC-HPLC analysis of purified a-PDL1-TGFβRII; d ELISA showing the comparative ability of a-PDL1-TGFβRII and a-PD-L1 antibody to bind PD-L1. Biotinylated recombinant human PD-L1 (rh B7-H1-biotin; 0–100 ng ml⁻¹) was added to plates coated with a-PDL1-TGFβRII or a-PD-L1 antibody (1 μg ml⁻¹), followed by detection with HRP-Avidin. Plates coated with non-specific IgG-TGFβRII showed no binding to PD-L1 and served as a negative control to analyze the binding ability of the test samples. e ELISA showing the comparative ability of a-PDL1-TGFβRII and a-PD-L1 antibody to bind TGFβ. Recombinant human TGFβ (rhTGFβ1; 0–2,000 pg ml⁻¹) was added to plates coated with a-PDL1-TGFβRII or a-PD-L1 antibody (1 μg ml⁻¹), which was detected by biotinylated a-TGFβ1 and HRP-Avidin. Plates coated with nonspecific IgG-TGFβRII and rhTGFβRII-Fc served as positive controls to analyze the binding ability of the test samples to TGFβ. f ELISA showing the ability of a-PDL1-TGFβRII to simultaneously bind both PD-L1 and TGFβ. Anti-PDL1-TGFβRII or a-PD-L1 antibody (0–100 ng ml⁻¹) was added to PD-L1-Fc coated plates (1 μg ml⁻¹), followed by rhTGFβ1 (100 ng ml⁻¹) that was detected by a biotinylated anti-human TGFβ1 antibody. For d–f, the data show the optical density (OD) values (mean of three replicate wells for each assay condition) from a representative of two independent experiments