Gene expression based outcome of CT-26 tumor in C57/BL6 mice after treatment with anti-PD-1 antibody, GITR-L, combination or Bispecific molecules

Co-stimulatory receptors such as GITR play key roles in regulating the effector functions of T cells. Agonistic anti-GITR antibodies have been investigated as a cancer immunotherapy with demonstrated efficacy in pre-clinical models. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect which may be due to a suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a novel bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (anti-PD-1-GITR-L) that induces a PD-1-dependent and FcγR-independent GITR clustering, resulting in an enhanced activation, proliferation, and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1 directed GITR-L construct that has a different mechanism of action (MoA) than the co-administration of anti-PD-1 plus GITR-L and demonstrated a dose-dependent immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models. Combination of anti-PD-1-GITR-L with TGFβ inhibition or chemotherapy (gemcitabine), respectively, resulted in tumor remission in immunogenically cold and checkpoint blockade resistant mouse models. Dose-dependent correlations between target saturation and Ki67 and TIGIT up-regulation on memory T cells have been observed following a single dose of anti-PD-1-GITR-L bispecific in mice and non-human primates. Anti-PD-1-GITR-L thus represents a novel bispecific approach for directing GITR agonism for cancer immunotherapy. 5 mice per treatment arm were treated using 3 doses of test agents on days 7, 9 and 11 after tumor inoculation (tumors approx 100mm3 day 7). Test articles are isotype control 0.3mg/kg, anti-muPD-1 0.3mg/kg, isotype-muGITR-L 0.43mg/kg, 1:1 combination (anti-muPD-1 0.3mg/kg plus isotype-muGITR-L 0.43mg/kg), and anti-muPD-1-muGITR-L bispecific 0.43mg/kg. FFPE samples collected days 8 and 10 for FFPE production of tumors and draining lymph nodes. RNA run on NanoString mouse PanCancer immune profiling panel C3400.