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. Author manuscript; available in PMC: 2022 Sep 3.
Published in final edited form as: Nat Biomed Eng. 2022 Mar 3;6(3):310–324. doi: 10.1038/s41551-022-00852-y

Fig. 2 |. Sensing T cell killing of tumour cells by antibody-GzmB sensor conjugates.

Fig. 2 |

a, Schematic showing αPD1 antibody conjugated to fluorescently quenched peptide substrates for GzmB. Upon incubating these conjugates with transgenic Pmel T cells and B16 tumour cells, secreted GzmB cleaves peptide substrates, separating the fluorescent reporter from the internal quencher and resulting in an increase in sample fluorescence. b, In vitro protease cleavage assays showing normalized fluorescence (relative fluorescence units, RFU) of αPD1-GS after incubation with recombinant GzmB (blue), mouse serum (red), and other bystander proteases (n = 3 technically independent wells). c, Michaelis-Menten curve of initial cleavage velocity (V0) at varying substrate concentrations for GzmB substrate (GS) when unconjugated (in presence of unmodified αPD1) and when conjugated to αPD1. kcat and Km were determined by fit to Michaelis-Menten equation (n = 3 technically independent wells, error bars depict s.e.m.). d, ELISA quantification of GzmB from T cell killing assays in which Pmel T cells were incubated with B16 target cells at different T cell to target cell ratios (one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons, ****P < 0.0001, n = 4 biologically independent wells, error bars depict s.e.m.). e, Bar plot quantifying percent of cell cytotoxicity as measured by lactate dehydrogenase (LDH) assay from cocultures of Pmel T cells with B16 target cells (one-way ANOVA with Dunnett’s post-test and correction for multiple comparisons, **P = 0.004, ***P < 0.001, n = 3 biologically independent wells, error bars depict s.e.m.). f, Activity assays showing sample fluorescence after incubating αPD1-GS, αPD1, and an αPD1 conjugate with control substrates (αPD1-CtrlSub) with cocultures of Pmel T cells with B16 target cells (two-way ANOVA with Tukey’s post-test and correction for multiple comparisons, **P = 0.0075, ****P < 0.0001, n = 3 biologically independent wells, error bars depict s.e.m.). g, Half-life measurements of intact αPD1-GS and unconjugated αPD1 antibody (one phase decay fitting function, n = 3 biological replicates, error bars depict s.e.m.).