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. 2020 Jun 24;11:3196. doi: 10.1038/s41467-020-16838-w

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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. 5 — Dose–response curves for T-cell killing of FOLR1-positive tumor cells after 48 h mediated by TCB using PBMCs as effector cells with an E:T of 10:1. a, b Comparison of Prot-FOLR1-TCB (noncleavable and precleaved with matA site) for tumor cells with different FOLR1 expression levels (a HeLa cells, b Skov-3 cells). The cytotoxicity induced by the antibodies is shown. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (representative experiment for one PBMC donor, n = 2 different human PBMC donors). c, d The cytotoxicity induced by the Prot-FOLR1-TCB containing MMP-matA site is shown. The Prot-FOLR1-TCB containing an MMP-matA site (gray circles) or a noncleavable linker (blue triangles pointing up) is shown. FOLR1-TCB (red triangles, pointing down), precleaved Prot-FOLR1-TCB (orange squares) and a nontargeted TCB (black circles) are used as controls. The cytotoxicity of the Prot-FOLR1-TCB with a noncleavable linker was only measured for four concentrations using Skov-3 cells as the target cells. The nontargeted TCB was only used at the three highest concentrations for both cell lines. The pretreatment of the Prot-FOLR1-TCB (orange squares) was done by incubation of Prot-FOLR1-TCB with recombinant human matriptase for 24 h at 37 °C. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (representative experiment for one PBMC donor, n = 3 different human PBMC donors). e, f Granzyme B release was quantified by FACS analysis after incubation of PBMCs with 10 nM of Prot-FOLR1-TCB (cleavable linker vs. noncleavable linker) and FOLR1-positive target cells for 48 h. Each point represents the mean value of triplicates. Standard error is indicated by error bars (representative experiment for one PBMC donor, n = 3 different human PBMC donors). g FOLR1-positive tumor cell (CellPlayer™ MDA-MB-231 NucLight Red) growth inhibition mediated by Prot-FOLR1-TCB containing MMP-matA site and human PBMCs. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (representative experiment for one PBMC donor, n = 2 different human PBMC donors).