Extended Data Fig. 8. The anti-huPD-1-muGITR-L and anti-muPD-1-huGITR-L bispecifics have different bioactivity in vivo in comparison to anti-PD-1 plus isotype-GITR-L combination and monotherapies in B16F10 tumor syngeneic models in huPD-1 and huGITR HO Tg mice, and bioactivity, anti-tumor efficacy and toxicity of gemcitabine and TGFβ blockade in combination with anti-PD-1-GITR-L in 4T1 mouse syngeneic tumor model.
(A and B) Growth inhibition of B16F10 in huPD-1 HO Tg mice (A) and in huGITR HO Tg mice (B) following indicated treatments and doses (i.p. frequency indicated by arrows). Each point on the curve represents the mean tumor volume for each group (n = 8 mice). (C and D) Flow cytometry analysis of tumor infiltrated T cells collected from both models (120 hours post dose, n = 5 mice). Ki67+, CD226+, KCNA3+ and SLAMF6-TIM3+ are shown as a percent of CD8+ T cells ((C) huPD-1 HO Tg, and (D) huGITR HO Tg). Data are presented as mean values +/- SEM. Statistical significance was calculated with two-way ANOVA with Tukey’s correction for multiple comparisons (statistics refer to anti-PD-1-GITR-L vs combo). (E) In vitro cell killing of 4T1 mouse syngeneic cell line to selected chemotherapeutic agents (fluorouracil, paclitaxel, doxorubicin and gemcitabine) using CellTiter-Glo cell viability assay (n = 3 technical cell culture replicates within a single experiment). (F) Dose range titration of gemcitabine in 4T1 model at indicated doses (i.p. Q3DX4). (G and H) Growth inhibition of 4T1 cells in syngeneic mice by combination of anti-muPD-muGITR-L with anti-TGFβ (G) or gemcitabine (H) following indicated treatments and doses (i.p., frequency indicated by arrows). Data are presented as mean values +/- SEM. (I) Change of mice body weight following indicated treatments. Gemcitabine was dose i.p. Q3DX4 and anti-muPD-1-muGITR-L bispecific was dose i.p. 3qW for 1 week. Each point on the curve represents the mean tumor volume for each group (n = 10 mice). Statistical significance was calculated with two-way ANOVA with Tukey’s correction for multiple comparisons [statistic refer to gemcitabine (100 mg/kg) vs vehicle, anti-PD-1-GITR-L+ TGFβ vs anti-PD-1-GITR-L; and anti-PD-1-GITR-L vs gemcitabine].