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ABSTRACTVascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.

Angiogenesis, the process by which new vessels form from existing ones, plays an important role in many developmental processes and pathological conditions. We study angiogenesis in the context of a highly controllable experimental environment: the cornea micropocket assay. Using a multidisciplinary approach that combines experiments, image processing and analysis, and mathematical modelling, we aim to provide mechanistic insight into the action of two angiogenic factors, vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF). We use image analysis techniques to extract quantitative data, which are both spatially and temporally resolved, from experimental images, and we develop a mathematical model, in which the corneal vasculature evolves in response to both VEGF-A and bFGF. The experimental data are used for model parametrization, while the mathematical model is used to assess the utility of the cornea micropocket assay and to characterize proposed synergies between VEGF-A and bFGF.

VEGF 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. Several publications described that VEGF and Ang-2 are up-regulated in several hematological tumors like B-CLL, AML or multiple myeloma. In particular, Ang-2 may qualify as a novel prognostic marker in AML and CLL. We have generated TAvi6, a novel bispecific antibody targeting VEGF-A and Ang-2 and tested its anti-tumor and anti-angiogenic activity. TAvi6 is a tetravalent IgG-like bispecific antibody based on bevacizumab and targets Ang-2 with 2 disulfide-stabilized scFvs (LC06) fused to the C-terminus of the heavy chain.TAvi 6 was profiled in biochemical and cellular (angiogenesis) assays. Antitumoral and anti-angiogenic efficacy was assessed in established s.c. Colo205, s.c. Calu-3 and orthotopic i.m.f.p. KPL-4 xenografts in SCID beige mice. Mice were treated with bevacizumab or antibody LC06 (10 mg/kg), the respective combination (each 10 mg/kg) and TAvi6 (13.3 mg/kg). In addition, TAvi6 was evaluated for inhibition of metastasis to the lung quantified by Alu-PCR. Tumors were explanted for histological analysis of angiogenesis and inhibition of angiogenesis was assessed in the cornea angiogenesis assay.In biochemical assays (affinity, Tie2-Ang-2 interaction) and cellular assays (Tie2 phosphorylation, HUVEC proliferation, tube formation) TAvi6 shows properties identical to the parental antibodies bevacizumab and LC06. In the orthotopic KPL-4-003 xenograft tumor growth inhibition was 79% for bevacizumab; 39% for LC06; 90% for the combination and 91% for TAvi6. In the s.c. Colo205-009 xenograft TGI was 66% for bevacizumab; 50% for LC06; 78% for the combination and 87% for TAvi6. TAvi6 was able to suppress growth of tumors refractory to 1st-line treatment with bevacizumab (Colo205 xenograft) and suppressed metastasis to the lung significantly (KPL-4 xenograft). In the s.c. Calu-3 xenograft we observed a strong inhibition of angiogenesis by in vivo and ex vivo imaging and an advantage of TAvi6 compared to the antibody combination. Furthermore, TAvi6 potently inhibited VEGF-induced angiogenesis in the mouse corneal angiogenesis assay.We have generated a novel tetravalent IgG-like bispecific antibody targeting VEGF-A and Ang-2 simultaneously. TAvi6 shows identical properties compared to the respective parental antibodies. In particular, TAvi6 blocks angiogenesis in vivo efficacously and mediates strong tumor growth inhibition in various xenograft models with a slight advantage of TAvi6 over the combination of the respective single agents bevacizumab and LC06 in several models. Further studies are foreseen to assess the application of bispecific antibodies targeting Ang-2 and VEGF as novel therapeutic approach for the treatment of hematological cancers.Weidner: Roche: Employment, Equity Ownership. Scheuer:Roche: Employment, Patents & Royalties. Thomas:Roche: Employment, Patents & Royalties. Baehner:Roche: Employment, Patents & Royalties. Seeber:Roche: Employment, Patents & Royalties. Kettenberger:Roche: Employment, Patents & Royalties. Schanzer:Roche: Employment, Patents & Royalties. Brinkmann:Roche: Employment, Patents & Royalties. Regula:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Abstract 4304