<p>Supplementary figure 1. Tumor growth kinetics in individual mice treated with trametinib (T) in combination with the immunomodulator-targeting PD1 in the CT26 murine syngeneic model.</p>
<p>ICOS agonist mAbs demonstrate improved antitumor activity in combination with PD-1 blockade. EMT6 (subcutaneous) tumor-bearing BALB/c mice were administered intraperitoneally biweekly with anti-mouse ICOS mAb [7E.17G9 (mIgG1)], anti-PD-1 mAb (RMP1-14), or isotype controls (mIgG1 and rat IgG2a, respectively) alone and in combination for a total of six doses. Mice were evaluated for pharmacodynamic changes (<b>B</b> and <b>C</b>) within tumors, tumor growth (<b>D</b>), and survival (<b>E</b>). As illustrated in <b>A</b>, transcriptional analysis was performed (<i>n</i> = 5–7) on tumor tissue harvested from mice 48 hours after second (<b>B</b>) and third doses (<b>C</b>) of indicated mAbs; raw data in <a href="#SMT4" target="_blank">Supplementary Table S4</a>. Each line in <b>D</b> represents an individual mouse (<i>n</i> = 10/group). Tumor-free mice at study termination are indicated within each subpanel. <b>E,</b> Kaplan–Meier plot illustrating OS in D. <b>F,</b><i>ICOS</i> expression following <i>ex vivo</i> anti–PD-1 (pembrolizumab) or vehicle control treatment of tumor slices from patients with HNSCC for 48 hours. Each symbol represents an individual human tumor sample (<i>n</i> = 50/group). <b>G,</b> Fold change in IFNγ production by TILs from dissociated NSCLC tumor samples (<i>n</i> = 5–6 samples/group) following exposure to anti-CD3 (plate-bound, 0.6 μg/mL) in concert with anti-PD-1 (pembrolizumab, soluble) or feladilimab (plate-bound) alone or in combination for 24 hours. <b>H,</b> A2058 (subcutaneous) tumor-bearing NSG mice were administered intraperitoneally biweekly with feladilimab and anti-PD-1 (pembrolizumab) alone or in combination for a total of six doses and assessed for tumor growth inhibition (<i>n</i> = 10/group). Data in F–H are represented as mean ± s.e.m. Significance in G determined by unpaired Student <i>t</i> test. Despite trends in tumor growth kinetics following combination treatment, the curves in H were not significantly different as determined by one-way ANOVA. ANOVA, analysis of variance; HNSCC, head and neck squamous cell carcinoma; ICOS, inducible T cell costimulator; IFN, interferon; i.p., intraperitoneal injection; mAb, monoclonal antibody; NSCLC, non–small cell lung carcinoma; OS, overall survival; PD-1, programmed cell death protein 1; s.e.m., standard error of the mean; TIL, tumor-infiltrating lymphocyte.</p>
<p>ICOS agonist mAbs demonstrate single-agent antitumor activity. <b>A–C,</b> EMT6 (subcutaneous) tumor-bearing BALB/c mice were administered intraperitoneally biweekly with anti-mouse ICOS mAb [7E.17G9 (mIgG1)] or isotype control (mIgG1) for a total of six doses and evaluated for tumor growth (<b>A</b>), survival (<b>B</b>), and pharmacodynamic changes (<b>C</b>) within tumors. Each line in A represents an individual mouse (<i>n</i> = 10/group). Number of tumor-free mice at study termination are indicated within each subpanel. <b>B,</b> Kaplan–Meier plot illustrating OS in A. <b>C,</b> Percentage of tumor-infiltrating CD8<sup>+</sup> T cells, T<sub>reg</sub> cells, and associated CD8:T<sub>reg</sub> cell ratio 48 hours after the third dose of anti-ICOS mAb (10 μg, ∼study day 7) assessed using flow cytometry (nonspecific binding was blocked with human or mouse Fc block). Each symbol represents an individual mouse. <b>D–F,</b> A549 (subcutaneous) tumor-bearing NSG mice were administered intraperitoneally biweekly with feladilimab or isotype control for a total of six doses. <b>D,</b> A549 tumor growth kinetics for controls (PBS or isotype) and feladilimab-treated groups (0.04 mg/kg, black; 0.4 mg/kg, red). Percentage of tumor-infiltrating ICOS+ CD8<sup>+</sup> T cells (<b>E</b>; assessed using flow cytometry with nonspecific binding blocked using human or mouse Fc block) and associated CD8:T<sub>reg</sub> cell ratio (<b>F</b>) 48 hours after the fourth dose of feladilimab (∼study day 21). Data in C, D, E, and F represent the mean ± s.e.m. Significance was determined by unpaired Student <i>t</i> test. See <a href="#SMF1" target="_blank">Supplementary Fig. S1</a> for flow cytometry gating strategy for C, E, and F. CD8/Treg ratios were calculated on the basis of percent (%) positive CD8s (of live CD45<sup>+</sup>) and FoxP3/CD25<sup>+</sup> Treg cells (of live CD45<sup>+</sup> CD4<sup>+</sup> cells). ICOS, inducible T-cell costimulator; i.p., intraperitoneal injection; mAb, monoclonal antibody; OS, overall survival; s.e.m., standard error of the mean.</p>
<p>Feladilimab-mediated ICOS co-stimulation results in differential phenotypic changes and cytokine production in CD4+ non-Treg and Treg populations.</p>
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