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. 2023 May 19;11(5):e006436. doi: 10.1136/jitc-2022-006436

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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Modular, selective and reversible activation of SAR T cells, irrespective of soluble forms of MCSP tumor antigen. (A) A375 melanoma cells were plated and co-cultured with human SAR T cells (E:T 2:1) and αMCSP/αE3 BiAb (αMCSP, 1 µg/mL). Different concentrations of soluble, recombinant MCSP (rMCSP) were added. The tumor cell lysis over time was assessed using xCELLigence (n=3). The cell index was normalized to the respective time point of T cell addition as indicated by an arrow. (B) Human SAR or E3del control T cells and αMCSP/αE3 BiAb (1 µg/mL) were plated in wells either coated with different concentrations of rMCSP or where different concentrations of soluble rMCSP were added to the medium. After 48 hours the supernatant was taken and analyzed for IFN-γ using ELISA (n=3). (C) Schematic overview of SAR-transduced T cells targeting TYRP1⁺ MCSP⁺ melanoma cells via an αTYRP1/αE3 or αMCSP/αE3 BiAb. (D and E) A modularity stress test was carried out using αMCSP/αE3 or αTYRP1/αE3 BiAb (αMCSP or αTYRP1, 1 µg/mL). SAR or E3del control T cells were co-cultured with A375 tumor cells (E:T 2:1). HER2 CAR T cells were used as a control and co-cultured with HER2⁺ A375 tumor cells (no BiAb was added). At assay start, co-cultures received either αMCSP/αE3 or αTYRP1/αE3 BiAb (first dosage). At 24 hours, the T cells were washed to remove residual BiAb and transferred to freshly plated A375 tumor cells. Co-cultures were then either redosed with the same BiAb, redosed with the BiAb against the other target, or not redosed after initial dosing (second dosage) and incubated for another 24 hours. At 24 (D) or 48 hours (E), supernatants were taken and ELISA for human IFN-γ were performed (n=3). Analyses of differences between groups for (A) were performed using two-way analysis of variance with correction for multiple testing by the Bonferroni method. For statistical analysis of (B), (D) and (E), the unpaired two-tailed Student’s t-test was used. Experiments show mean values±SD calculated from at least three biological replicates and are representative of three independent experiments. BiAb, bispecific antibodies; E:T, effector to target ratio; IFN, interferon; EGFRvIII, epidermal growth factor receptor variant III; MCSP, melanoma-associated chondroitin sulfate proteoglycan; SAR, synthetic agonistic receptor; TYRP1, tyrosinase-related protein 1; HER2, human epidermal growth factor receptor.