Michael Weidner

Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. Mol Cancer Ther; 12(10); 2031–42. ©2013 AACR.

Abstract 1370: RO5323441, a humanized monoclonal antibody against the placenta growth factor, blocks PlGF-induced VEGFR-1 phosphorylation in vitro and tumor growth in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Solid tumor growth requires new blood vessel formation or angiogenesis. Inhibition of angiogenesis as a therapeutic strategy in oncology has been validated by treatments that block vascular endothelial growth factor (VEGF) or its receptors, such as bevacizumab (Avastin®), which is an anti-VEGF antibody that prolongs survival in colorectal, lung and other cancer patients in combination with chemotherapy. However, since not all tumors are sensitive to VEGF blockade and resistance mechanisms to VEGF therapies can develop, inhibition of additional targets may be necessary in order to control tumor growth and achieve better clinical effects. PlGF is a member of the VEGF family that is found only in very low levels under normal physiological conditions, but is up-regulated in almost all major malignant diseases. PlGF expression has shown to correlate with tumor stages and patient survival in breast cancer, CRC and gastric cancer [1-3]. Pre-clinical data support a role for PlGF in tumor angiogenesis, and demonstrate that blocking PlGF can inhibit tumor growth [4]. RO5323441, a humanized IgG1 monoclonal antibody directed against PlGF, has demonstrated anti-tumor activity in human tumor xenograft models in mice, has a benign preclinical toxicology profile and is being developed for the treatment of multiple advanced cancer indications. RO5323441 binds to both PlGF-1 and PlGF-2 in a dose dependent manner, and is not cross-reactive with murine PlGF or human VEGF. RO5323441 inhibits the binding of human PlGF-1 or PlGF-2 to VEGFR −1 with IC50 values of 0.1 and 0.2 nM, respectively. RO5323441 blocks PlGF-induced VEGFR-1 phosphorylation in Flt-1-transfected HEK293 cells. Antitumor activity has been demonstrated in mutliple tumor models including ACHN and Caki-1 renal cell carcinoma and Huh-7, hepatocellular carcinoma xenografts. Inhibition of established tumors ranged from 43-97% with twice weekly dosing. A refractory model of non-small cell lung cancer has also been identified. Studies to understand the mechanism of action and to identify pharmacodynamic markers and have been carried out in ACHN-tumor bearing mice. References 1. Wei SC, Tsao PN, Yu SC, et al. Placenta growth factor expression is correlated with survival of patients with colorectal cancer. Gut. 2005 May; 54(5):666-72. 2. Parr C, Watkins G, Boulton M et al. Placenta growth factor is over-expressed and has prognostic value in human breast cancer. Eur J Cancer. 2005 Dec;41(18):2819-27. 3. Chen CN, Hsieh FJ, Cheng YM, et al. The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer. Cancer Lett. 2004 Sep 15;213(1):73-82. 4. Fischer C, Jonckx B, Mazzone M, et al. Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell. 2007. 131 : 463-75. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1370.

Abstract 3285: Ang-2-VEGF CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function mediates potent anti-tumoral and anti-angiogenic efficacy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL VEGF-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. In tumors Ang-2 is up-regulated and a bad prognostic factor. Recent data demonstrated that Ang-2 inhibitors, both as a single agent or in combination with chemo- or anti-VEGF therapy mediate anti-tumoral effects. We have recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies (CrossMabs) based on the crossover of the CH1 and CL domains within the Fab region of one half of a bispecific antibody combined with the knobs-into-holes technology to enforce heterodimerization of the Fc portion. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab and recognizing Ang-2 with the other arm based on LC06, a highly Ang-2 selective human IgG1 antibody. The Ang-2-VEGF CrossMAb could be produced with good yields and purity by eukaryotic. Surface Plasmon resonance studies showed that the two different arms of the Ang-2-VEGF CrossMab retained their antigen binding affinity for VEGF-A and Ang-2 and interfered with VEGF-induced HUVEC proliferation and Ang-2 induced Tie2-phosphorylation in a similar manner than the parental antibodies. Crossmab showed very potent tumor growth inhibition in orthotopic (KPL-4) and in subcutaneous xenograft tumors (Colo205). In the orthotopic KPL-4 and the s.c. Colo205 xenograft we observed a strong inhibition of angiogenesis by ex vivo analysis. In the VEGF-induced cornea pocket assay Crossmab resulted in a complete shutdown of angiogenesis. We have generated a bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function simultaneously. Our data indicate that the Ang-2-VEGF CrossMab mediates potent anti-tumoral, anti-metastatic and anti-angiogenic efficacy and represents a promising therapeutic agent for the therapy of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2011-3285