Bivalent EGFR-Targeting DARPin-MMAE Conjugates

Abstract

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR designed ankyrin repeat protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Reforming solid tumor treatment: the emerging potential of smaller format antibody-drug conjugate

“…They found that medium-sized conjugates exhibited superior antitumor effects. Furthermore, an additional study on DARPin-drug conjugation specifically investigated on bivalent EGFR-targeting DARPin-MMAE conjugates (DAR 2) and monomeric DARPin-Fc-MMAE conjugates (DAR 4), which effectively killed EGFR-overexpressing A431 cells with sub-nanomolar potency [ 70 ]. Additionally, tumor targeted-DARPin has also been conjugated to anticancer proteins like fragmented human lactoferrin (rtHLF4), Cecropin A (CRPA), Cecropin CMV (CRPC).…”

Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts: Efficacy and Selection of Companion Imaging Counterpart

“…Due to more favorable structural characteristics, ESPs have higher tolerance to pH and temperature changes than mAbs, resulting in better in vitro and in vivo stability of the drug conjugates. Several conjugates of affibody molecules [18,25,26], DARPins [20,[31][32][33] and adnectins [35] with drugs and toxins have been developed and have demonstrated anti-tumor activity in preclinical studies.…”

“…The feasibility of using ESPs for tumor-targeted delivery of drugs and toxins has been studied for affibody molecules [18,[25][26][27], DARPins [20,[28][29][30][31][32][33], adnectins [34,35] and anticalins [36]. The HER2-targeting ESPs described in the literature interacts with different epitopes on the receptor; e.g., the HER2-targeting affibody molecules bind to subdomain III, the DARPin 9_29 binds to subdomain I and the DARPin G3 binds to subdomain IV [37].…”

Recent Advances on Affibody- and DARPin-Conjugated Nanomaterials in Cancer Therapy

“…Moreover, affibodies encompass only 13 amino acid positions that differ between binding members and therefore, much of the knowledge to manipulate and functionalize these proteins is known [8,9]. A few examples can be recalled that highlight the importance of affibodies and DARPins in several biomedical applications that include both therapeutics [10][11][12] and imaging agents for tumors [13,14].…”