Extended Data Fig. 6. Expression/in vitro/in vivo characterization of GITR and PD-1 in heterozygous and homozygous GEM, and anti-muPD-1-muGITR-L enhanced survival of CT26, EMT6 and JC tumor bearing mice in comparison to monotherapies and combination.

(A and B) Mouse and human target expression on anti-CD3 activated CD4⁺CD44⁺CD62L⁺ T cells isolated from spleens of indicated mice (A. n = 2 & B. n = 2). (C and D) IL-2 secretion following treatment with mouse surrogate and chimeric bispecifics (C: mGITR-L-mPD-1 and hGITR-L-mPD-1, and D: mGITR-L-mPD-1 and mGITR-L-hPD-1) using anti-CD3 activated splenocytes isolated from indicated mice. Color of bispecific domains indicated: purple = variable domain of anti-muPD-1, green = muGITR-L, yellow = huGITR-L and orange = variable domain of anti-huPD-1. A single experiment was conducted with 2 replicates and mean values were plotted. (E) Representative human and Mouse PD-1 and GITR expression by IHC (representative staining of n = 4 tissues) on spleens isolated from indicated mice. Scale bars indicate 100 µm (original magnification 20x). (F and G) Tumor baseline study of MC-38 cell line in huGITR (F) and huPD-1 (G) homozygous vs wild-type C57BL/6 mice following inoculation of 1 and 3 × 10⁶ cells/mouse. Each point on the curve represents the mean tumor volume for each group (n = 5 mice per group for huGITR & n = 7 mice per group for hu PD-1). Data are presented as mean values +/- SEM. (H to J) Mice survival following dosing of anti-muPD-1-muGITR-L bispecific. Results depict cumulative survival curves with indicated treatments: (H) CT26, (I) EMT6 and (J) JC models (n = 10 mice per group, ~ 100 mm³ tumor at time of dosing). Statistical significance was calculated with log-rank test with post-hoc analysis for multiple comparisons (statistics refer to anti-PD-1-GITR-L vs combo).

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