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. 2022 Sep 22;13:5552. doi: 10.1038/s41467-022-33284-y

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© The Author(s) 2022

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. 1 — a Schematic diagram of engineering bispecific antibodies. A tetravalent bsAb (14-H-06) and a bivalent bsAb (14-crs-06) were engineered from two parental IgGs that bind to two distinct epitopes on the RBD. The modules for antibody engineering are illustrated in the box. Fv: variable fragment; scFv: single-chain Fv, constructed as VH-(G₄S)₃-VL; CH1-CL: constant region 1 for heavy chain (CH1) and constant region for light chain (CL); CL-CH1: the crossover format of CH1-CL; KiH: the Knob-into-Hole design with the T366W (Knob) + S354C mutations in the heavy chain CH3 region on one arm and the T366S + L368A + Y407V (Hole)+ Y349C mutations in the CH3 region on the other arm. b Purities of indicated antibodies were analyzed by SEC. c The in-tandem BLI-based assay to test antibody blocking of RBD epitopes. The His-tagged RBD was captured onto the Ni-NTAbiosensors. The first antibodies, either bsAbs or parental antibodies, were used to bind the RBD. The second antibodies were tested for their abilities to bind pre-blocked RBD. d, e The epitope pre-blocking effects using bispecific antibodies 14-H-06 (left) and 14-crs-06 (right) as 1^st antibodies and parental antibodies as 2^nd antibodies (d), or using parental antibodies as 1^st antibodies and the bispecific antibodies 14-H-06 (left) and 14-crs-06 (right) as 2^nd antibodies (e). The segments of each sensorgram were color coded to show individual binding events.