Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

Abstract

These authors contributed equally to this work.Keywords: monoclonal antibody, EGFR, panitumumab, ADCC, target-selective activation, Probody TM Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; mAb, monoclonal antibody; EGFR, epidermal growth factor receptor; uPA, urokinase-type plasminogen activator; TKI, tyrosine kinase inhibitor; IgG, Immunoglobulin G; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; CI, confidence interval; LC, light chain; HC, heavy chain; EGFR VIII, EGFR Type III Variant; CRC, colorectal cancer; ECD, extracellular domain; SEC, size exclusion chromatography; CCK-8, Cell Counting Kit 8YunPanitumumab, as a commercially available antibody, is an effective anticancer therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Here, we firstly engineered panitumumab by grafting its variable region into an IgG1 backbone. The engineered panitumumab (denoted as Pan) retained binding activity identical to the parental antibody while exhibiting stronger ADCC activity in vitro and more potent antitumor effect in vivo. To further enhance the target selectivity of Pan, we generated Pan-P by tethering an epitope-blocking peptide to Pan via a tumor-specific protease selective linker. Pan-P showed almost 40-fold weaker affinity compared with Pan, but functional activity was restored to a similar extent as Pan when Pan-P was selectively activated by urokinase-type plasminogen activator (uPA). More importantly, targeted localization of Pan-P was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice, strongly indicating that specific activation also existed ex vivo and in vivo. Furthermore, Pan-P also exhibited effective in vivo antitumor potency similar to Pan. Taken together, our data evidence the enhanced antitumor potency and excellent target selectivity of Pan-P, suggesting its potential use for minimizing on-target toxicity in anti-EGFR therapy.

Preclinical studies of a Pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy

“…Activated PanP-DM1 was obtained by incubating PanP-DM1 with recombinant uPA in vitro as described previously and characterized using SDS-PAGE ( Fig S2). 13,25 Activated PanP-DM1 and activated PanP exhibited mostly equivalent ability to bind to EGFR-overexpressing A431 cells at saturating (10 mg/ml) and subsaturating (1 mg/ml) concentrations, suggesting that attachment of DM1 did not affect the uPA-mediated activation (Fig. 2B).…”

“…25 Briefly, 96-well plates (Nunc) were coated with EGFR-Fc and blocked with phosphate-buffered saline (PBS) containing 10% nonfat dried milk. Indicated concentrations of antibodies were added to the plates and incubated for 1 hour at room temperature.…”

“…It was engineered by fusing the peptide comprising uPA substrate sequence, blocking peptide and Gly-Ser-rich peptide linkers to the light chain N terminus of Pan that derived from panitumumab. 25 In the present study, the maytansine (DM1) was conjugated to PanP through the stable non-reducible thioether linkage. The enhanced anti-tumor effects were assessed using in vitro and in vivo models.…”

Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VLframework using a structure-guided approach

“…42 Significant effort has been devoted to understanding the effect of affinity on biological activity and developing methods to tailor affinity for desired purposes. [43][44][45][46][47] This is exemplified by the ability of the C10 series of anti-EGFR scFv antibodies to block EGFR signaling and inhibit cell growth in a manner that correlates with increased binding affinity. 20,39 These types of data, developed across a large array of antibody/antigen pairs, have led to incorporation of affinity maturation as a critical step in therapeutic antibody development.…”

Optical Control of Antibody Activity by Using Photocleavable Bivalent Peptide–DNA Locks

“…Despite their intrinsic affinities and specificities, however, antibody‐based therapeutics and molecular imaging agents still suffer from background binding and toxicity through binding to receptors in non‐diseased tissue . New molecular strategies are needed to increase the specificity of antibody‐mediated targeting, either by rendering their activity conditional on the presence of other biomarkers or by allowing their local activation by external triggers . Protease‐activatable therapeutic antibodies have been developed by fusing blocking peptides or protein domains to the antigen‐binding domains through protease‐cleavable linkers .…”