TY - JOUR
T1 - Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting
AU - Westerlund, Kristina
AU - Honarvar, Hadis
AU - Tolmachev, Vladimir
AU - Eriksson Karlström, Amelie
N1 - Publisher Copyright:
© 2015 American Chemical Society.
Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 2015/8/19
Y1 - 2015/8/19
N2 - 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, ZHER2:342-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 (Tm), both in HP1:HP2 and in ZHER2:342-SR-HP1:HP2 (Tm = 86-88 °C), and the high binding affinity between ZHER2:342-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 (KD) for ZHER2:342-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.
AB - 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, ZHER2:342-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 (Tm), both in HP1:HP2 and in ZHER2:342-SR-HP1:HP2 (Tm = 86-88 °C), and the high binding affinity between ZHER2:342-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 (KD) for ZHER2:342-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.
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U2 - 10.1021/acs.bioconjchem.5b00292
DO - 10.1021/acs.bioconjchem.5b00292
M3 - Article
C2 - 26086597
AN - SCOPUS:84939628085
VL - 26
SP - 1724
EP - 1736
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
SN - 1043-1802
IS - 8
ER -