Linker Optimization in Lu-177 Labeled αvβ6-Integrin Binding Peptide Trimers for Targeted Radionuclide Therapy of Cancer

Theranostic radiopharmaceuticals exploit the same molecular target for diagnosis and therapy. Among emerging pan-cancer targets, αvβ6-integrin is highly expressed on various malignant cell types and can be imaged clinically with Ga-68-Trivehexin. However, therapeutic αvβ6-integrin-directed radioligands remain scarce. Here, Lu-177-labeled multimerics of the cyclic nonapeptides Tyr2, sequence c(YRGDLAYp(NMe)K), were constructed using the tetrafunctional chelator DOTPI by means of CuAAC-based and evaluated as αvβ6-integrin-targeted radiotherapeutics. PEG linkers of increasing length (PEG0, PEG3, PEG7, PEG11) were introduced between the chelator and peptides to modulate pharmacokinetics and cellular processing. The Lu-177-labeled conjugates displayed comparable polarity (log D7.4 ≈ −2) and αvβ6-integrin affinities determined by ELISA (IC50 ∼ 0.2–0.5 nM), indicating minimal effects of PEG length on in vitro parameters. In αvβ6-positive H2009 cells, PEG linkers markedly enhanced and prolonged receptor-mediated uptake and internalization, consistent with improved multivalent engagement. In H2009 xenograft mice, PEG linkers reduced early blood-pool activity and increased tumor uptake at 24 h, while gelofusine (4% succinylated gelatin in Ringer’s acetate) efficiently mitigated the elevated renal retention (up to 92% reduction). Performance gains plateaued beyond PEG7, identifying the PEG7 trimer (P7) as the preferred lead due to favorable tumor uptake/retention. P7 exhibited high selectivity (65- to 671-fold) over other integrin subtypes, αvβ6-integrin-dependent radiotoxicity to tumor cells, and sustained tumor retention for up to 6 days according to μSPECT.