Combination of the Glycoengineered Type II CD20 Antibody Obinutuzumab (GA101) and the MDM2 Selective Antagonist RG7388 Results in Superior Anti-Tumor Activity

Introduction:

Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody that strongly induces direct cell death. As a result of glycoengineering, obinutuzumab has increased affinity for FcgRIII on innate immune effector cells resulting in enhanced induction of ADCC and ADCP. Obinutuzumab has been approved for first line treatment of CLL patients in combination with chlorambucil in the US and Europe and is currently in pivotal clinical trials in indolent NHL and DLCBL. RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound are novel, orally bioavailable, selective MDM2 antagonists which reactivate p53 and thereby mediate cell cycle arrest and subsequent apoptotic cell death in solid and hematologic tumors. RG7388 is currently in clinical trials for the treatment of AML and prostate cancer. Based on the fact that the majority of B lymphoid malignancies including NHL and CLL bear wildtype p53, and the complementary mechanisms of action involving increased apoptosis (MDM2 antagonist) or direct cell death (obinutuzumab), the combination of both compounds has the potential for superior efficacy in treating B lymphoid malignancies.

Experimental methods: The combination of obinutuzumab and RG7388 (or obinutuzumab and rituximab with RG7112, the frontrunner compound with identical mode of action) was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (Annexin V/PI positivity) on p53 wildtype Z138 Mantle cell lymphoma (MCL) and DoHH-2 Diffuse large B-Cell lymphoma (DLBCL) cells by FACS and the impact of MDM2 inhibition on ADCC induction and whole blood B cell depletion. In vivo efficacy of the combination of obinutuzumab or rituximab with RG7122 and RG7388 was evaluated in the s.c. Z138 MCL xenograft model in immunodeficient SCID beige mice.

Results: RG7338 induced concentration-dependent cell death of Z-138 and DOHH-2 cell lines. At concentrations > 10-100 nM RG7388 resulted in enhanced cell death induction of DOHH-2 and Z-138 cells in combination with obinutuzumab. Notably, RG7388 did not influence obinutuzumab mediated ADCC during 4 h up to concentrations of 1000 nM and did not affect obinutuzumab mediated NK cell activation (CD16 downregulation, CD107a upregulation). Similarly, addition of RG7388 did not interfere with obinutuzumab mediated B cell depletion in healthy human whole blood at concentrations up to 1000 nM. In the Z-138 xenograft model, the combination of suboptimal doses of 0.5 mg/kg obinutuzumab or 1 mg/kg rituximab with 150 mg/kg RG7112 (three times a week p.o. for 3 weeks) resulted in superior tumor growth inhibition by obinutuzumab as compared to rituximab including the induction of complete tumor remission. In a second Z-138 study, the combination of the suboptimal dose of 0.5 mg/kg obinutuzumab with 80 mg/kg RG7388 yielded similar anti-tumor activity. In summary, the combination of either obinutuzumab or rituximab with RG7112 or the combination of obinutuzumab with RG7388 showed superior in vivo efficacy with no clinical signs of toxicity.

Conclusions: The combination of obinutuzumab with MDM2 antagonists results in enhanced cell death of p53 wildtype NHL tumor cells while not affecting obinutuzumab mediated ADCC of tumor cells or B cell depletion in whole blood from healthy donors. In vivo the combination of obinutuzumab with MDM2 inhibitors RG7112 and RG7388 results in robust combined anti-tumor efficacy in xenograft models. Taken together, these preclinical data strongly support the investigation of obinutuzumab and RG7388 combination therapy in clinical trials.