Drug Conjugates Based on a Monovalent Affibody Targeting Vector Can Efficiently Eradicate HER2 Positive Human Tumors in an Experimental Mouse Model

Abstract

The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, ZHER2:2891, fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, ZHER2:2891-ABD-E3-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. ZHER2:2891-ABD-E3-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC50 values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcDM1, was counteracted by incorporation of hydrophilic and negatively charged glutamate residues near the site of mcDM1 conjugation. A dose-escalation experiment showed that increasing doses up to 15.1 mg/kg gave a proportional increase in uptake in xenografted HER2-overexpressing SKOV3 tumors, after which the tumors became saturated. Experimental therapy with four once-weekly injection of 10.3 or 15.1 mg/kg led to efficient regression of tumors in all animals and complete regression in some. Weight loss was detected for some animals in the group receiving the highest dose, suggesting that it was close to the maximum tolerated dose. In conclusion, the monovalent HER2-targeting affibody drug conjugate presented herein have potent anti-tumor activity in vivo.

The Influence of Domain Permutations of an Albumin-Binding Domain-Fused HER2-Targeting Affibody-Based Drug Conjugate on Tumor Cell Proliferation and Therapy Efficacy

“…Since the rates of internalization for both protein carriers were similar (Supplementary Figure S3), the results suggest that the architecture, including the molecular size, influences the in vitro potency in the absence of an inhibitory or stimulating effect on proliferation. In particular, it appears that a smaller size results in a more potent compound, something we have also observed in an earlier study [38]. In the earlier study, it was also found that addition of hydrophilic glutamic acids next to the cysteine where DM1 was attached resulted in a lower uptake in, e.g., the liver, probably due to a shielding of the hydrophobicity of DM1.…”

Imaging-Guided Therapy Simultaneously Targeting HER2 and EpCAM with Trastuzumab and EpCAM-Directed Toxin Provides Additive Effect in Ovarian Cancer Model

“…The higher IC 50 value in SKOV3 cells compared to OV-CAR3 cells might be due to several factors, such as differences in EpCAM expression level [84] and differences in sensitivity to toxin treatment. It was previously observed for HER2-targeting affibody-drug and affibody-toxin conjugates that SKOV3 cells are more resistant to cytotoxic action despite high expression of HER2 receptors [82,85,86]. The results of the in vivo specificity experiment demonstrated EpCAM-mediated accumulation of Ec1-LoPE in human xenografts in mice (Figure 6).…”

Comparative Preclinical Evaluation of HER2-Targeting ABD-Fused Affibody® Molecules 177Lu-ABY-271 and 177Lu-ABY-027: Impact of DOTA Position on ABD Domain

“…One cannot exclude the fact that shielding of the C-terminal part of ABD by conjugation to a hydrophilic DOTA-molecule plays an important role in reducing kidney uptake, potentially by reduction of scavenger receptor uptake in the proximal tubuli. A strong influence of the valency of the targeting part and hydrophilicity of a linker to cytotoxic payload affects the biodistribution pattern and renal uptake of Affibody-ABD conjugates with maytansine [33].…”