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. 2020 Apr 28;10(3):30. doi: 10.1038/s41408-020-0292-7

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© The Author(s) 2020

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. 4 — a, b FcγRIIIa (a) and FcγRIIa (b) crosslinking by DuoHexaBody-CD37 was analyzed in a bioluminescent Reporter Bioassay using Daudi target cells and engineered FcγRIIIa- or FcγRIIa-expressing Jurkat effector T cells that express luciferase upon FcγR crosslinking. Luciferase production is presented as relative luminescence units (RLU). Error bars represent the mean ± SD of duplicate measurements. c ADCC by 2 µg/ml DuoHexaBody-CD37 was evaluated in a classical ⁵¹Cr release assay using Daudi target cells and PBMCs from 12 healthy human donors as a source of effector cells (E:T of 100:1). The percentage lysis was calculated relative to a Triton X-100 control (100% lysis) and no antibody control (0% lysis). ****P < 0.00001, paired T-test with two-tailed 95% confidence intervals. d Dose-response ADCC (mean percentage lysis ± SD of three replicate samples) induced by DuoHexaBody-CD37, WT IgG1-CD37-010 and WT IgG1-CD37-016 shown for one representative responsive donor as described in c. Error bars represent the mean ± SD of triplicate measurements. e ADCP induced by DuoHexaBody-CD37 using Daudi target cells and monocyte-derived h-MDM from healthy human donors as a source of effector cells. Calcein AM-labeled Daudi cells opsonized with DuoHexaBody-CD37 were incubated with CD11b + h-MDM at an E:T ratio of 2:1 and ADCP was analyzed by flow cytometry after a 4 h co-culture. The amount of h-MDMs that phagocytosed Daudi cells is presented as percent CD11b+/calcein AM+/CD19^- double positive cells. CD19 was used to exclude macrophages with bound instead of phagocytosed tumor cells. The percentage CD11b⁻/calcein AM+ cells was determined as an indicator of the amount of non-phagocytosed Daudi cells; presented here as a depleted cell fraction relative to a no antibody control sample. Data from one representative donor out of three is shown. f ADCP of pHRodo-labeled Daudi target cells by h-MDM induced by DuoHexaBody-CD37 over time at an E:T ratio of 1:1, shown for one out of three representative donors. Red fluoresence indicates phagocytosed Daudi target cells by h-MDM. ADCP was quantified by the total sum of the red fluorescent intensity in the image (RCUxµm²/image) and presented as the mean ± SD of duplicate measurements. g Phase contrast- and red fluorescent images of DuoHexaBody-CD37-opsonized (1 µg/ml) pHRodo-labeled Daudi target cells co-cultured with h-MDM effector cells at 0 and 4 h incubation as described in f.