title: Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size.
abstract: Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.
authors.affiliation: Universität Zürich, Biochemisches Institut, Winterthurerstrasse, 8057 Zürich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Zahnd
authors.forename: Martin
authors.initials: M
authors.lastname: Kawe
authors.forename: Michael T
authors.initials: MT
authors.lastname: Stumpp
authors.forename: Christine
authors.initials: C
authors.lastname: de Pasquale
authors.forename: Rastislav
authors.initials: R
authors.lastname: Tamaskovic
authors.forename: Gabriela
authors.initials: G
authors.lastname: Nagy-Davidescu
authors.forename: Birgit
authors.initials: B
authors.lastname: Dreier
authors.forename: Roger
authors.initials: R
authors.lastname: Schibli
authors.forename: H Kaspar
authors.initials: HK
authors.lastname: Binz
authors.forename: Robert
authors.initials: R
authors.lastname: Waibel
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat / Q000502:physiology; D000915:Antibody Affinity; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry / Q000627:therapeutic use; D016503:Drug Delivery Systems / Q000379:methods*; D015195:Drug Design; D005260:Female; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000008:administration & dosage / Q000737:chemistry / Q000627:therapeutic use; D051379:Mice; D008819:Mice, Nude; D008970:Molecular Weight; D009369:Neoplasms / Q000188:drug therapy*; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000008:administration & dosage* / Q000138:chemical synthesis* / Q000737:chemistry / Q000627:therapeutic use; D013379:Substrate Specificity / Q000502:physiology; D016896:Treatment Outcome; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D007141:Immunoglobulin Fc Fragments; D011506:Proteins