Abstract 2460: Dissecting the in vitro and in vivo mechanism of action of obinutuzumab (GA101) in preclinical models using an immune effector-dead version of obinutuzumab
Authors: Herter Sylvia , Herting Frank , Muth Gunter , van Puijenbroek Erwin , Ferrara Claudia , Lang Sabine , Bacac Marina , Moessner Ekkehard , Umana Pablo , Klein Christian 
Published: August 2015

Abstract
Introduction: Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody mediating superior direct cell death induction, ADCC and ADCP compared to rituximab while CDC is strongly reduced. Obinutuzumab is approved for 1st line treatment of CLL in combination with chlorambucil in the US and Europe. The contribution of these mechanisms of action (MOA) to the efficacy of obinutuzumab in patients is unclear. In order to further dissect the MOA GA101 and rituximab and variants with abolished FcγR and C1q binding were assessed in vitro and in vivo.
Methods: Glycoengineered GA101, non-glycoengineered wild type (wt) GA101, effector-dead GA101 and rituximab with P329G LALA mutations in the Fc were generated and characterized i) for binding to FcγRs and CD20 positive cells by FACS, ii) for cell death induction by AnnexinV/PI staining, iii) in ADCC, ADCP and CDC assays, and iv) for depletion of CD19 positive B cells in whole blood from healthy volunteers. Anti-tumoral efficacy was assessed using established 300 mm2 s.c. SU-DHL4 xenograft tumors in Scid beige mice (30 mg/kg, q7d x 4, ip).
Results: Variants of GA101 and rituximab retain CD20 binding and cell death inducing properties of the respective parental antibody. The introduction of P329G LALA in the Fc part abolishes binding to Fcγ receptors and C1q, so that they do not mediate ADCC, ADCP or CDC any longer, while direct cell death induction is not affected as compared to GA101 and rituximab, respectively. In whole blood assays GA101 showed the best B cell depletion in terms of potency and absolute B cell depletion followed by wtGA101 and rituximab. Notably, GA101 P329G LALA retained significant B cell depletion, while rituximab P329G LALA did not mediate B cell depletion. Thus, in whole blood B cell depletion, ADCC/ADCP plays an important role, but direct cell death induction is equally important for the MOA of GA101, whereas it does not contribute to B cell depletion of rituximab. In the SU-DHL4 xenograft model GA101 (TGI &gt; 100%, non-parametric TCR (npTCR) 0 with 95% CI 0-0) and wtGA101 (TGI &gt; 100%, npTCR 0, 95% CI 0-0.006) showed equivalent efficacy with complete tumor remission in 9/10 mice for GA101 and 8/10 mice for wtGA101. GA101 P329G LALA showed anti-tumoral efficacy (TGI 62%, npTCR 0.43, 95% CI 0.30-0.60) comparable to rituximab (TGI 71%, npTCR 0.37, 95% CI 0.29-0.50), while rituximab P329G LALA showed only residual anti-tumoral activity (TGI 26%, npTCR 0.73, 95% CI 0.44-0.92).
Summary: These data indicate that in whole blood B cell depletion and xenograft models, direct cell death and ADCC/ADCP through the glycoengineered Fc part are required for anti-tumoral efficacy of obinutuzumab. Most notably, the effector dead version of obinutuzumab that completely lacks ADCC, ADCP and CDC activity, but has retained direct cell death induction, is as efficacious as rituximab in vivo supporting the role of direct effects for the efficacy of obinutuzumab.
Citation Format: Sylvia Herter, Frank Herting, Gunter Muth, Erwin van Puijenbroek, Claudia Ferrara, Sabine Lang, Marina Bacac, Ekkehard Moessner, Pablo Umana, Christian Klein. Dissecting the in vitro and in vivo mechanism of action of obinutuzumab (GA101) in preclinical models using an immune effector-dead version of obinutuzumab. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2015-2460