On-Resin DIAMSAR-Conjugated CD38-Targeted Peptides and Their Inverso and Dimeric-Inverso Analogs for PET Imaging of...

CD38 is an established biomarker of multiple myeloma (MM), and peptide-based radiopharmaceuticals targeted to this receptor offer a route to molecularly specific imaging. In this work, we identified a novel CD38-targeted peptide sequence (HAPWFRGGGGS) through phage display and synthesized it using automated solid-phase peptide synthesis. The peptide was modified by introducing a PEG₄ spacer and on-resin conjugation of the DIAMSAR chelator, which forms a stable complex with Copper-64 (Cu-64), yielding DIAMSAR-PEG₄-HAPWFRGGGGS (Monomer_L). [⁶⁴Cu]Cu-Monomer_L was radiolabeled with high molar activity (>98% yield, ∼65 MBq/nmol) but showed suboptimal serum stability (∼45% intact at 2 h). To improve in vivo stability, the l-amino acid sequence was replaced with d-amino acid (Monomer_D), resulting in >90% serum stability and enhanced binding affinity toward CD38, as demonstrated by molecular docking and cell-binding assays in CD38-expressing MOLP2 human MM cells. To further increase avidity, a dimeric analog (Dimer_D) was designed by linking two Monomer_D units via a PEG₄ linker. In viable MOLP2 MM cells, tracer uptake ranked as [⁶⁴Cu]Cu-Dimer_D > [⁶⁴Cu]Cu-Monomer_D > [⁶⁴Cu]Cu-Monomer_L, and was markedly reduced by excess unlabeled peptide, confirming CD38-specific binding. Binding specificity and functional engagement of CD38 were further supported by antibody-blocking, enzymatic activity inhibition, and cellular internalization studies. Replacement of L-with d-amino acids improved binding affinity, lowering the K_d from 1043 nM ([⁶⁴Cu]Cu-Monomer_L) to ∼740 nM ([⁶⁴Cu]Cu-Monomer_D). The dimerization further lowered the K_d (∼730 nM) with markedly higher B_max (6993 fmol/mg vs 3024 fmol/mg), consistent with avidity-driven enhancement in receptor engagement. In vivo small animal dynamic PET/CT and ex vivo biodistribution were performed in disseminated and subcutaneous MOLP2-CBR-GFP MM models with naïve controls. Uptake increased with peptide valency, showing maximum femoral uptake of 1.52 ± 0.35 and 2.93 ± 0.68% ID/mL for [⁶⁴Cu]Cu-Monomer_D and [⁶⁴Cu]Cu-Dimer_D, respectively, whereas naïve mice exhibited <1% ID/mL over 0-2 h post injection (3-4 MBq; 45-50 pmol). In the subcutaneous model, [⁶⁴Cu]Cu-Dimer_D enabled clear tumor visualization at 2 h post injection with 4.66 ± 0.20% ID/mL uptake and a T/M ratio of 10.6 ± 3.1. Ex vivo tissue biodistribution confirmed higher femoral uptake (2.26 ± 0.42% ID/g) and femur-to-muscle ratio (18.17 ± 3.26). Autoradiography of excised tissues corroborated tracer localization to tumor-rich regions. Overall, [⁶⁴Cu]Cu-Dimer_D demonstrates high stability, avidity, and translational promise as a CD38-targeted PET tracer for MM.