Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin

Abstract

Targeted delivery of toxins is a promising way to treat disseminated cancer. The use of monoclonal antibodies as targeting moiety has provided proof-of-principle for this approach. However, extravasation and tissue penetration rates of antibody-based immunotoxins are limited due to antibody bulkiness. The use of a novel class of targeting probes, Affibody molecules, provides smaller toxin-conjugated constructs, which may improve targeting. Earlier, we have demonstrated that affitoxins containing a HER2-targeting Affibody moiety and a deimmunized and truncated exotoxin A from Pseudomonas aeruginosa, PE38X8, provide highly selective toxicity to HER2-expressing cancer cells. To evaluate the influence of molecular design on targeting and biodistribution properties, a series of novel affitoxins were labelled with the residualizing radionuclide 111In. In this study, we have shown that the novel conjugates are more rapidly internalized compared with the parental affitoxin. The use of a (HE)3 purification tag instead of a hexahistidine tag enabled significant (p<0.05) reduction of the hepatic uptake of the affitoxin in a murine model. Fusion of the affitoxin with an albumin-binding domain (ABD) caused appreciable extension of the residence time in circulation and several-fold reduction of the renal uptake. The best variant, 111In-(HE)3-ZHER2-ABD-PE38X8, demonstrated receptor-specific accumulation in HER2-expressing SKOV-3 xenografts. In conclusion, a careful molecular design of scaffold protein based anticancer targeted toxins can appreciably improve their biodistribution and targeting properties.

Affibody molecules as engineered protein drugs

“…By randomizing amino acids on two of the three helices, large libraries can be constructed, from which potent binders can be isolated by a variety of display methods, 5, 6, 7 and the scaffold has been engineered for improved synthesis and solubility. 8 Affibody molecules can be selected to a large variety of different proteins, and can be functionalized with genetic fusions to protein modules 9, 10 or by chemical conjugation to toxic molecules, 11 reviewed in Löfblom et al 12 …”

Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs

“…Earlier studies have demonstrated a strong influence of molecular design (sequence of binding domains, valency, composition of linkers and termini) on in vivo targeting and biodistribution properties of targeting agents [ 29 , 30 , 31 , 32 ]. Dependent on design, constructs might interact differently with targeted as well as non-targeted tissues.…”

Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

“…Even if the efficacy of H5E as an endosomolytic agent is not dramatically higher than that H6, the discovery of a humanized Hn version that performs slightly better than H6 in cytosolic delivery exemplifies the potential of optimized histidine tails to promote endosomal escape. In addition, the peptide HEHEHE does not promote hepatic accumulation of associated polypeptides [56,60], probably because of its hydrophilic nature when compared with similar Hn constructs [61]. As a side observation, we demonstrate that the presence of CQ enhances the intracellular permanence of uptaken proteins.…”

“…-based affinity chromatography [46,54]. Upon purification, the presence of H6 or related peptides in the final protein product might have unexpected biological impacts, such as aberrant biodistribution of the protein or protein complex upon systemic injection [55,56]. In this context, and also envisaging potential immunoreactions in protein materials intended for clinical use, the removal of H6 from purified proteins would be a desirable biotechnological step [44].…”