Three Is\na Magic Number: Tailored Clickable Chelators\nUsed to Determine Optimal RGD-Peptide Multiplicity in αvβ6-Integrin\nTargeted <sup>177</sup>Lu-Labeled Cancer Theranostics

The cellular adhesion receptor αvβ6-integrin\nis highly\nexpressed in many cancers, e.g., pancreatic, lung, head-and-neck,\ncervical, bladder, and esophageal carcinoma. Multimerization of αvβ6-integrin-specific\nRGD peptides increases the target affinity and retention but affects\nbiodistribution and pharmacokinetics. Amide formation of the terminal\ncarboxylic acid moieties of the square-symmetrical bifunctional chelator\nDOTPI with 3-azidopropylamine yields derivatives with 4, 3, and 2\nterminal azides and zero, 1, and 2 remaining carboxylic acids, respectively,\nwhereby formation of the 2-cis-isomer is preferred according to NMR\ninvestigation of the Eu(III)-complexes. Cu(II)-catalyzed alkyne–azide\ncycloaddition (CuAAC) of the alkyne-functionalized αvβ6-integrin\nbinding peptide cyclo[YRGDLAYp(<i>N</i>Me)K(pent-4-ynoic\namide)] (Tyr2) yields the respective di-, tri-, and tetrameric conjugates\nfor Lu-177-labeling. In mice bearing αvβ6-integrin-expressing\nxenografts of H2009 (human lung adenocarcinoma) cells, the Lu-177-labeled\ntrimer’s tumor-to-blood ratio of 112 exceeds that of the tetramer\n(10.4) and the dimer (54). Co-infusion of gelofusine (succinylated\ngelatin) reduces the renal uptake of the trimer by 89%, resulting\nin a 10-fold better tumor-to-kidney ratio, while no improvement of\nthat ratio is observed with arginine/lysine, <i>para</i>-aminohippuric acid (PAH), and hydroxyethyl starch (HES) coinfusions.\nSince the Lu-177-labeled Tyr2-trimer outperforms the dimer and the\ntetramer, such trimers are considered the best lead structures for\nthe ongoing development of αvβ6-integrin targeted anticancer\ntheranostics.