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. 2018 Aug 3;9:3049. doi: 10.1038/s41467-018-05395-y

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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. 2 — Evaluation of multiple anti-CD3 scFv domains for recruitment of TAC to the TCR-CD3 complex. a, e Schematic representation of evaluated TAC constructs. TAC receptors utilizing the (a) anti-HER2 DARPin are paired with either the UCHT1 or OKT3 anti-CD3 scFv. TAC receptors using the (e) anti-CD19 scFv are paired with either the huUCHT1, F6A, or L2K anti-CD3 scFv. b, f Relative TAC surface expression is measured by flow cytometry. Cells are stained for CD4, CD8, tNGFR and TAC, and gated on either CD4⁺NGFR⁺ or CD8⁺NGFR⁺. Representative data of three independent experiments are presented as histogram analysis of (b) HER2-TAC or (f) CD19-TAC. Surface expression of OKT3 relative to UCHT1 was significantly higher in CD4 cells (p = 0.0007) but not in CD8 cells. huUCHT1 expression is significantly higher compared to either L2K (p = 0.005 (CD4)/0.0002 (CD8)) or F6A (p < 0.0001 (CD4) p < 0.0001 (CD8)). For the gating strategy see Supplementary Fig. 13A. c, g HER2- and CD19-specific TAC-T cells are stimulated with antigen-positive (c) SK-OV-3 and (g) Raji tumor cells, respectively. Data are presented as percent of CD4 or CD8 T cells producing cytokine. Cytokine producing cells are compared from (c) TAC-T cells bearing UCHT1 (square) or OKT3 (inverted triangle), or (g) TAC-T cells bearing huUCHT1 (square), F6A (triangle), or L2K (diamond). Lines represent the mean. Multiple t-test is used to determine significance in all cases. For the gating strategy see Supplementary Fig. 13B. d, h HER2- and CD19-TAC and vector control (vector only carrying tNGFR) T cells are co-cultured with (d) SK-OV-3 and (h) NALM-6 tumor cells, respectively, to measure TAC-T cell-mediated cytotoxicity. Vector control T cells (circles) are compared against d HER2-specific TAC-T cells bearing UCHT1 (square) or OKT3 (triangle), or (h) CD19-specific TAC-T cells bearing huUCHT1 (square), F6A (triangle), or L2K (diamond). Data are from three independent experiments with three different donors, error bars are standard deviation