Thil Batuwangala

A dual-targeting antibody against EGFR-VEGF for lung and head and neck cancer treatment†

An antibody simultaneously targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), two major tumor growth‐driving machineries, may provide a novel effective strategy for optimizing tumor targeting and maximizing potential clinical benefits. Human domain antibodies selected against VEGF and EGFR were formatted into a fully human dual‐targeting IgG (DT‐IgG) to directly target both antigens in a single molecule. We evaluated the efficacy of DT‐IgG in comparison with bevacizumab and cetuximab alone and in combination in the lung cancer cell line A549 (low EGFR expression and KRAS mutant) and the head and neck squamous cell carcinoma (HNSCC) cell line Tu212 (high EGFR expression and KRAS wild type) in vitro and in vivo. DT‐IgG suppressed Tu212 and A549 cell growth, inhibited EGFR activation and induced apoptosis as effectively as cetuximab, and neutralized VEGF as effectively as bevacizumab. DT‐IgG induced EGFR‐dependent VEGF internalization, constituting a novel antiangiogenesis mechanism. In xenograft models with lung and head and neck cancer cell lines, DT‐IgG displayed efficacy equivalent to bevacizumab in diminishing tumor growth despite its short serum half‐life (36 hr in rats) and both agents may constitute preferable alternatives to cetuximab in KRAS‐mutant tumors. Immunofluorescence staining revealed that localization of DT‐IgG was similar to that of cetuximab, largely associated with EGFR+tumor cells. Our proof of principle study suggests a DT‐IgG against EGFR and VEGF as an alternative therapeutic strategy with potentially enhanced clinical benefit.

Novel Interaction Mechanism of a Domain Antibody Based Inhibitor of Human Vascular Endothelial Growth Factor with Greater Potency Than Ranibizumab and Bevacizumab and Improved Capacity Over Aflibercept.

A potent VEGF inhibitor with novel antibody architecture and antigen binding mode has been developed. The molecule, hereafter referred to as VEGF dual dAb (domain antibody), was evaluated in vitro for binding to VEGF and for potency in VEGF-driven models and compared with other anti-VEGF biologics that have been used in ocular anti-angiogenic therapeutic regimes. VEGF dual dAb is more potent than bevacizumab and ranibizumab for VEGF binding, inhibition of VEGF receptor binding assays (RBAs), and VEGF-driven in vitro models of angiogenesis and displays comparable inhibition to aflibercept (Eylea). VEGF dual dAb is dimeric, and each monomer contains two distinct anti-VEGF domain antibodies attached via linkers to a human IgG1 Fc domain. Mechanistically, the enhanced in vitro potency of VEGF dual dAb, in comparison to other anti-VEGF biologics, can be explained by increased binding stoichiometry. A consistent model of the target engagement has been built based on the x-ray complexes of each of the two isolated domain antibodies with the VEGF antigen.

'In-Format' screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules.

ABSTRACT Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an ‘in-format’ screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC50) when assessed in-format were identified. In contrast, the corresponding dAb monomers had ∼1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening naïve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time.