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. 2022 Feb 2;10:3. doi: 10.1186/s40170-022-00279-8

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — A, B Graphical representation of ADCC data. CA9hu-1 and CA9hu-2 antibody variant-dependent cell-mediated cytotoxicity. Comparison of CA IX-negative versus CA IX-positive target cell line samples, n = 2. C Fit curves determine EC₅₀ of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software. D Humanized antibody variants showed high ADCC activity in Reporter ADCC assay (Promega) tested on cells BT-20, JIMT-1, and 42MGBA naturally expressing CA IX in hypoxia. The signal of the control was set as 100%. E Humanized antibody variants showed high ADCC activity in comparison with irrelevant therapeutic antibody bevacizumab. This figure clearly shows that humanized antibody variants CA9hu-1 and CA9hu-2 retain the ability to activate the ADCC pathway and to mediate the cytotoxic effect on target cells expressing CA IX. Data in the graph represent the mean ± standard deviation values. Statistical significance of differences was assessed using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001), n = 3