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Labeling of Anti-HER2 Nanobodies with Astatine-211: Optimization and the Effect of Different Coupling Reagents on Their in Vivo Behavior

Journal article
Authors Y. Dekempeneer
Tom Bäck
Emma Aneheim
H. Jensen
J. Puttemans
C. Xavier
M. Keyaerts
Stig Palm
Per Albertsson
T. Lahoutte
V. Caveliers
Sture Lindegren
M. D'Huyvetter
Published in Molecular Pharmaceutics
Volume 16
Issue 8
Pages 3524-3533
ISSN 1543-8384
Publication year 2019
Published at Institute of Clinical Sciences, Department of Radiation Physics
Pages 3524-3533
Language en
Keywords nanobody, targeted alpha therapy, breast cancer, astatine-211, HER2, targeted radionuclide therapy, free at-211, receptor, radioimmunotherapy, pharmacokinetics, dosimetry, i-125(-), linkers, growth, sdab, Research & Experimental Medicine, Pharmacology & Pharmacy
Subject categories Clinical Medicine


The use of nanobodies (Nbs) as vehicles in targeted alpha therapy (TAT) has gained great interest because of their excellent properties. They combine high in vivo affinity and specificity of binding with fast kinetics. This research investigates a novel targeted therapy that combines the alpha-particle emitter astatine-211 (At-211) and the anti-HER2 Nb 2Rs15d to selectively target HER2+ cancer cells. Two distinctive radiochemical methodologies are investigated using three different coupling reagents. The first method uses the coupling reagents, N-succinimidyl 4-(1,2-bis-tert-butoxycarbonyl)guanidinomethyl-3-(trimethylstannyl)benzo ate (Boc(2)-SGMTB) and N-succinimidyl-3-(trimethylstannyl)-benzoate (m-MeATE), which are both directed to amino groups on the Nb, resulting in random conjugation. The second method aims at obtaining a homogeneous tracer population, via a site-specific conjugation of the N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide (MSB) reagent onto the carboxyl-terminal cysteine of the Nb. The resulting radioconjugates are evaluated in vitro and in vivo. 2Rs15d is labeled with At-211 using Boc(2)-SGMTB, m-MeATE, and MSB. After astatination and purification, the binding specificity of the radioconjugates is validated on HER2+ cells, followed by an in vivo biodistribution assessment in SKOV-3 xenografted mice. alpha-camera imaging is performed to determine uptake and activity distribution in kidneys/tumors. 2Rs15d astatination resulted in a high radiochemical purity >95% for all radioconjugates. The biodistribution studies of all radioconjugates revealed comparable tumor uptake (higher than 8% ID/g at 1 h). [At-211]SAGMB-2Rs15d showed minor uptake in normal tissues. Only in the kidneys, a higher uptake was measured after 1 h, but decreased rapidly after 3 h. Astatinated Nbs consisting of m-MeATE or MSB reagents revealed elevated uptake in lungs and stomach, indicating the presence of released At-211. alpha-Camera imaging of tumors revealed a homogeneous activity distribution. The radioactivity in the kidneys was initially concentrated in the renal cortex, while after 3 h most radioactivity was measured in the medulla, confirming the fast washout into urine. Changing the reagents for Nb astatination resulted in different in vivo biodistribution profiles, while keeping the targeting moiety identical. Boc(2)-SGMTB is the preferred reagent for Nb astatination because of its high tumor uptake, its low background signals, and its fast renal excretion. We envision [At-211]SAGMB-2Rs15d to be a promising therapeutic agent for TAT and aim toward efficacy evaluation.

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