Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting
2015 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 26, no 8, 1724-1736 p.Article in journal (Refereed) Published
In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z<inf>HER2:342</inf>-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T<inf>m</inf>), both in HP1:HP2 and in Z<inf>HER2:342</inf>-SR-HP1:HP2 (T<inf>m</inf> = 86-88 °C), and the high binding affinity between Z<inf>HER2:342</inf>-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 105 M-1 s-1), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K<inf>D</inf>) for Z<inf>HER2:342</inf>-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of 111In- and 125I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for 125I- and 111In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for 125I-HP2 and 3.83 ± 0.39%ID/g for 111In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.
Place, publisher, year, edition, pages
2015. Vol. 26, no 8, 1724-1736 p.
epidermal growth factor receptor 2; indium 111; iodine 125; peptide nucleic acid; sortase; tetraxetan; tyrosine, amino acid sequence; animal tissue; Article; chimera; circular dichroism; conformational transition; dissociation constant; hybridization; isotope labeling; melting point; mouse; nonhuman; plasma clearance; protein secondary structure; radioactivity; reversed phase high performance liquid chromatography; solid phase synthesis; surface plasmon resonance; transition temperature; ultraviolet radiation, Mus
Organic Chemistry Other Basic Medicine
IdentifiersURN: urn:nbn:se:kth:diva-174607DOI: 10.1021/acs.bioconjchem.5b00292ISI: 000359962900039PubMedID: 26086597ScopusID: 2-s2.0-84939628085OAI: oai:DiVA.org:kth-174607DiVA: diva2:877321
FunderSwedish Cancer Society, 2012/354Swedish Research Council, 521-2012-2228, 621-2013-5135
QC 201512072015-12-072015-10-072015-12-07Bibliographically approved