Anti-tumoral, anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2

Abstract

Vascular 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.

Tumor suppressor microRNA-613 inhibits glioma cell proliferation, invasion and angiogenesis by targeting vascular endothelial growth factor A

“…Ang-2 is another important protein to regulate tumor angiogenesis ( 19 ). The present study demonstrated that Ang-2 and VEGFA serve synergistic roles in tumor angiogenesis ( 33 ). Therefore, it was concluded that VEGFA may be a direct target for miR-613, implicating miR-613 as a novel target for glioma therapy.…”

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

“…Similar findings were recent published. 29 Bs4Ab platform allows modifications of the Fc similar to IgG1 to extend half-life and to modulate FcgRs functions Under some therapeutic conditions, it may be desired to either reduce or increase antibody Fc-mediated effector functions. For example, for antibodies that target cell-surface molecules, especially those on immune cells, removing Fc effector function is preferred.…”

“…14 Dual blockage of VEGF and Ang2 has already been demonstrated to result in potent tumor growth inhibition in xenograft mouse models by limiting angiogenesis and increasing apoptosis. [14][15][16][17] In addition, the effects of neutralization of VEGF and Ang2 with a bispecific monovalent CrossMab antibody (RG7221, vanucizumab) 15 in combination with chemotherapy are currently being investigated in a Phase 2 clinical trial (ClinicalTrials.gov NCT02141295) of patients with metastatic colorectal cancer. Therefore, the work described herein does not focus on confirming that dual targeting of VEGF and Ang2 can potentiate or differentiate the activity over the individual parental antibodies or their combination, but instead describes the molecular strategies that have been used to design the Bs4Ab bispecific antibody platform and to provide additional evidences of its applicability.…”

Variable heavy–variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly

“…For proof-of-concept, we generated the three possible domain crossover antibodies (as described ( Schaefer et al , 2011 ; Klein et al , 2016 ) with target specificities directed against Angiopoietin 2 (Ang-2) and Vascular Endothelial Growth Factor (VEGF-A) based on the parental antibodies Ang-2i-LC06 ( Thomas et al , 2013 ; Scheuer et al , 2016 ) and bevacizumab ( Presta et al , 1997 ; Ferrara et al , 2004 ) on a human IgG1 framework with knobs-into-holes including an additional stabilizing disulfide bond between the CH3 (constant heavy 3) domains ( Merchant et al , 1998 ; Carter, 2001 ; Kuglstatter et al , 2017 ). The domain crossover was introduced into the VEGF binding arm using the described elbow sequence ( Klein et al , 2016 ) together with the knob (T366W) and an additional cysteine (S345C), whereas the hole (T366S, L368A, Y407V) was introduced into the CH3 domain of the Ang-2 binder together with an additional cysteine (Y349C).…”