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. Author manuscript; available in PMC: 2021 Apr 8.
Published in final edited form as: J Am Chem Soc. 2020 Mar 30;142(14):6554–6568. doi: 10.1021/jacs.9b11622

Figure 3.

Figure 3.

SpyCatcher T cells are capable of in vitro lytic function against a host of different antigen-expressing tumor lines. (A) Schematic representation of armed SpyCatcher immune receptor lysis (left) and on-demand lysis (right). (B) SpyCatcher or untransduced (Unt) T cells were armed with various concentrations of Herceptin-ST and cocultured in the presence of SKOV3 (Her2+) tumor cells. (C) SpyCatcher T cells were armed with antigen-specific and nonspecific IgGs and cocultured with either MDA-MB-468 (EGFR+/Her2-) or Ramos (CD20+/EGFR-) tumor cells. (D) SpyCatcher T cells armed with antigen-specific DARPins were coculture with SKOV3, MDA-MB-468, or A1847 (EpCAM+) tumor cells expressing luciferase. (E) DARPin9.26-ST armed SpyCatcher T cells were incubated with or without SKOV3 tumor cells (E/T = 3:1) for 24 h, removed from culture, and incubated in media ± 2000 nM DARPin9.26-ST. Receptor arming was analyzed via anti-myc staining and flow cytometric analysis. (F) Unarmed SpyCatcher T cells were incubated with SKOV3 tumor cells for 4 h followed by the addition of Herceptin-ST or Herceptin-STDA (time = 0). (G) SpyCatcher T cells were armed with either DARPin9.26-ST or DARPin9.26-STDA at various concentrations and cocultured with SKOV3 tumor cells expressing luciferase. (B, C: MDA-MB-468, F) Lysis was measured using real-time cell analysis. (C: Ramos, D, G) Residual luciferase expression was calculated after 20 h. All cocultures were carried out using a 7:1 E/T ratio. Error bars represent the mean ± standard deviation. Data points are averages of three replicates. A representative T-cell donor is shown. *P < 0.05, **P < 0.01, and ***P < 0.001.