FIGURE 2 from Activating Inducible T-cell Costimulator Yields Antitumor Activity Alone and in Combination with Anti-PD-1 Checkpoint Blockade
Authors: Yadavilli Sapna , Waight Jeremy D. , Brett Sara , Bi Meixia , Zhang Tianqian , Liu Yao-Bin , Ellis Catherine , Turner David C. , Hahn Ashleigh , Shi Hong , Seestaller-Wehr Laura , Jing Junping , Xie Qing , Shaik Jafar Sadik , Ji Xiao , Gagnon Robert , Fieles William , Hook Laura , Grant Steven , Hopley Stephanie , DeYoung M. Phillip , Blackwell Christina , Chisamore Michael , Biddlecombe Robert , Figueroa David J. , Hopson Christopher B. , Srinivasan Roopa , Smothers James , Maio Michele , Rischin Danny , Olive Daniel , Paul Elaine , Mayes Patrick A. , Hoos Axel , Ballas Marc 
Published: August 2023

<p><i>In vitro</i> functional characteristics of antihuman ICOS agonist mAb feladilimab. <b>A,</b> Detection of ICOS on naïve or preactivated (48 hours anti-CD3/CD28) peripheral blood T cells from healthy human donors using feladilimab (3 μg/mL, soluble) and subsequent FITC-conjugated anti-human IgG secondary antibody. Each symbol represents an individual donor. See <a href="#SMF1" target="_blank">Supplementary Fig. S1</a> for flow cytometry gating strategy. Confocal microscopy illustrating kinetics of ICOS cellular localization using feladilimab (3 μg/mL, soluble) as the antibody for staining (<b>B</b>) and T cell–DC interactions following coincubation with feladilimab and CD3/CD28 (<b>C</b>). <b>C,</b> Stimulated T cells exhibit ICOS polarization and mobilization toward neighboring DCs (dark), localizing with related costimulatory receptor CD28; (I–III) denote image overlays, with (IV) combining all markers. <b>D,</b> Representative Western blot analyses of AKT pathway phosphorylation in activated CD4<sup>+</sup> T cells following treatment with soluble feladilimab or isotype control (1 and 10 μg/mL, soluble) for 0–24 hours; uncropped images available in <a href="#SMF12" target="_blank">Supplementary Fig. S12A</a>. As illustrated in <b>E</b>, CD4<sup>+</sup> non-T<sub>reg</sub> (CD4<sup>+</sup> CD25<sup>−</sup>) and T<sub>reg</sub> cells (CD4<sup>+</sup> CD25<sup>+</sup> CD127<sub>low</sub>) were isolated from healthy donor peripheral blood and stimulated using plate-bound anti-CD3 (1 μg/mL) ± feladilimab or isotype control (each at 5 μg/mL) for 72 hours. <b>F,</b> RNA-based analysis (Nanostring) of T<sub>reg</sub>-associated marker (<i>FOXP3</i>, <i>ICOS</i>, <i>TIGIT</i>, and <i>CTLA4</i>) expression by the stimulated cell subsets (each symbol represents an individual donor). <b>G,</b> Cytokine-based analysis of T-cell subsets following stimulation with plate-bound anti-CD3 and a dose range of feladilimab or isotype control; see <a href="#SMF1" target="_blank">Supplementary Fig. S1</a> for gating strategy. <b>H,</b> IFNγ production in the supernatant of PBMC cultures from patients with NSCLC following plate-bound feladilimab and anti-CD3 (0.6 μg/mL) stimulation (24 and 48 hours for healthy donors; 72 hours for patients with NSCLC). Data in A and H represent the mean ± s.e.m; significance determined by unpaired Student <i>t</i> test. Where shown, significance was determined by one-way ANOVA. AKT, protein kinase B; Fc, fragment crystallizable; ANOVA, analysis of variance; DC, dendritic cell; ICOS, inducible T cell costimulator; IFN, interferon; Ig, immunoglobulin; mAB, monoclonal antibody; NSCLC, non–small cell lung carcinoma; PBMC, peripheral blood mononuclear cells; s.e.m., standard error of the mean.</p>