title: Multimeric disintegrin protein polymer fusions that target tumor vasculature.
abstract: Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, §Medical Technology Division, Malaysian Nuclear Agency, Bangi, 43000 Kajang Selangor, Malaysia.
authors.forename: Siti M
authors.initials: SM
authors.lastname: Janib
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Gustafson
authors.forename: Radu O
authors.initials: RO
authors.lastname: Minea
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Swenson
authors.forename: Shuanglong
authors.initials: S
authors.lastname: Liu
authors.forename: Martha K
authors.initials: MK
authors.lastname: Pastuszka
authors.forename: Lye Lin
authors.initials: LL
authors.lastname: Lock
authors.forename: Honggang
authors.initials: H
authors.lastname: Cui
authors.forename: Francis S
authors.initials: FS
authors.lastname: Markland
authors.forename: Peter S
authors.initials: PS
authors.lastname: Conti
authors.forename: Zibo
authors.initials: Z
authors.lastname: Li
authors.forename: J Andrew
authors.initials: JA
authors.lastname: MacKay
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy*; D045744:Cell Line, Tumor / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019483:Disintegrins / Q000737:chemistry / Q000494:pharmacology; D004549:Elastin / Q000737:chemistry; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008819:Mice, Nude; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry* / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D001672:Biocompatible Materials; D019483:Disintegrins; D016023:Integrins; D010455:Peptides; D011108:Polymers; D011993:Recombinant Fusion Proteins; C551792:vicrostatin; D004549:Elastin