Balancing Selectivity and Efficacy of Bispecific Epidermal Growth Factor Receptor (EGFR) × c-MET Antibodies and Antibody-Drug Conjugates

Abstract

Bispecific antibodies (bsAbs) and antibody-drug conjugates (ADCs) have already demonstrated benefits for the treatment of cancer in several clinical studies, showing improved drug selectivity and efficacy. In particular, simultaneous targeting of prominent cancer antigens, such as EGF receptor (EGFR) and c-MET, by bsAbs has raised increasing interest for potentially circumventing receptor cross-talk and c-MET-mediated acquired resistance during anti-EGFR monotherapy. In this study, we combined the selectivity of EGFR × c-MET bsAbs with the potency of cytotoxic agents via bispecific antibody-toxin conjugation. Affinity-attenuated bispecific EGFR × c-MET antibody-drug conjugates demonstrated high in vitro selectivity toward tumor cells overexpressing both antigens and potent anti-tumor efficacy. Due to basal EGFR expression in the skin, ADCs targeting EGFR in general warrant early safety assessments. Reduction in EGFR affinity led to decreased toxicity in keratinocytes. Thus, the combination of bsAb affinity engineering with the concept of toxin conjugation may be a viable route to improve the safety profile of ADCs targeting ubiquitously expressed antigens.

Keywords: antibody engineering; anticancer drug; epidermal growth factor receptor (EGFR); sortase A; toxicity.

FIGURE 1.

A, BLI of monovalent oa Fab SEED (AG) antibodies to soluble c-MET ECD, including a schematic antibody representation. B10 and F06 are parental and B10v5 and CS06 are the affinity-matured variants thereof. Affinities for c-MET are given as the dissociation equilibrium constant (K_D) (nm). B, BLI of monovalent oa scFv SEED (GA) antibodies to soluble EGFR ECD, including a schematic antibody representation. Variants of humanized cetuximab with low (225-L), medium (225-M), and high (225-H) affinity as well as humanized matuzumab (mAb 425) were analyzed. Affinities for EGFR are given in nm. C, simultaneous binding of soluble recombinant c-MET and EGFR by bsAb B10v5 × 225-H analyzed by BLI. Biotinylated c-MET ECD is captured to streptavidin octet biosensors. bsAb and EGFR-ECD are associated in two steps employing buffer and non-related isotype control (anti-HEL). Shown is a schematic representation of bispecific EGFR × c-MET antibody using the SEED technology.

FIGURE 2.

Inhibition of c-MET and EGFR phosphorylation by EGFR × c-MET bsAbs during ligand stimulation. Phosphorylated c-MET (A) and phosphorylated EGFR (B) were quantified in A549, A431, and primary keratinocytes (NHEK) using ECL. Cells were treated with varying concentrations of bsAbs and a non-related isotype SEED control (anti-HEL) with subsequent stimulation with 100 ng/ml HGF (A) or 100 ng/ml EGF (B). Triangles and dotted lines indicate respective receptor phosphorylation levels for stimulated (upright triangle) and non-stimulated cells (inverted triangle). Dose-response curves were fitted using a 3PL model in GraphPad Prism version 5 (GraphPad Software). Error bars, S.D.

FIGURE 3.

In vitro selectivity of EGFR × c-MET bsAbs in comparison with cetuximab. A, EBC-1 as a tumor model cell line with high to moderate c-MET and EGFR expression and T47D as an epithelial model cell line with low EGFR expression and no c-MET expression were mixed in a ratio of 1:30. To distinguish the two cell lines, EBC-1 cells were stained with the green membrane dye PKH2. The cell mixture was incubated with 30 nm bsAb or cetuximab and subjected to flow cytometric analysis. Antibody binding was detected by PE-labeled anti-human Fc secondary antibody. Representative dot plots for green versus yellow fluorescence are shown. B, in vitro selectivity was defined as the ratio of mean fluorescence intensity of the EBC-1 and the T47D cell population. Displayed are means with S.D. of two independent experiments. Asterisks, significant difference of groups in comparison with the selectivity of cetuximab (***, p < 0.001, with one-way analysis of variance with Dunnett's test). Error bars, S.D.

FIGURE 4.

Cytotoxicity of EGFR × c-MET bispecific SEED antibody-drug conjugates generated by covalent, site-directed conjugation of the tubulin inhibitor MMAE C-terminally to both heavy chains in comparison with cetuximab as ADC and anti-HEL ADC as corresponding reference constructs. After incubation of bispecific ADCs on EGFR-overexpressing tumor cells A431 (A) and MDA-MB-468 (B), on primary keratinocytes (NHEK.f-c.; C) as normal epithelial cell line, and on c-MET-overexpressing cells MKN45 (D) and EBC-1 (E) as well as HepG2 (F) as liver cell line, cell viability was assessed using the ATP-based luminescence CellTiter-Glo® assay. The assay was run in duplicates in three independent experiments, and curves were fitted by sigmoidal curve fitting using GraphPad Prism version 5 (GraphPad Software). Error bars, S.D.