PEP08

Anti‐Ro52 autoantibodies (Ro52‐autoAbs) appear in the sera of connective tissue disease (CTD) patients with interstitial lung disease (ILD). Studies using patient sera have shown a correlation between the generation of Ro52‐autoAbs and the clinical morbidity and severity of CTD with ILD. In this study, we used a single B‐cell manipulating technology and obtained 12 different monoclonal Ro52‐autoAbs (mRo52‐autoAbs) from the selected four patients suffering from severe ILD with a high titer of Ro52‐autoAbs in their sera. Western blot analysis revealed that 11 of 12 mRo52‐autoAbs bound to the coiled‐coil domain of Ro52. Competitive ELISA demonstrated that mRo52‐autoAbs competed with each other to bind to Ro52. Epitope mapping showed that two of them specifically bound to a peptide (PEP08) in the coiled‐coil domain. We then examined the titer of Ro52‐autoAbs in the sera of 192 CTD patients and assessed the relationship between the serum levels of Ro52‐autoAbs that were reactive to PEP08 peptide and the clinical morbidity and severity of ILD. Statistical analysis revealed that the production of PEP08‐reactive Ro52‐autoAbs correlated with the morbidity and severity of ILD in CTD. Assessment of the production of PEP08‐reactive Ro52‐autoAbs in autoimmune diseases is useful for predicting the clinical morbidity of ILD.

Gastrin-releasing peptide receptor (GRPR) is a G-protein-coupled receptor that is overexpressed in several malignancies, rendering it an ideal target for cancer imaging and therapy. In this report, we conducted structure-activity relationship studies by optimizing the substitution of Pro¹⁴ based on an agonist sequence, [d-Phe⁶,Pro¹⁴]Bombesin(6-14), and optimizing the substitution of the C-terminal Leu¹³-NH² based on an antagonist sequence, [d-Phe⁶,des-Met¹⁴]Bombesin(6-14). PEP-1-PEP-5 derived from the agonist sequence and PEP-6-PEP-14 derived from the antagonist sequence were synthesized by a solid-phase approach and were obtained in 10-60% yield. In vitro competition binding assays showed that the K_i values of PEP-1-PEP-14 were in the range of 1.16 nM to 266 nM. PEP-3, PEP-4, PEP-8, PEP-9, and PEP-10 with K_i < 6 nM were selected for elongation with Ga-DOTA complex and Pip linker at their N-terminus. Calcium release assays confirmed the agonist-antagonist nature of Ga-DOTA-Pip-conjugated peptides. Ga-DOTA-Pip-PEP-4, Ga-DOTA-Pip-PEP-9, and Ga-DOTA-Pip-PEP-10 retaining high GRPR binding affinity (K_i < 10 nM) were selected for ⁶⁸Ga labeling for further evaluation. [⁶⁸Ga]Ga-DOTA-Pip-PEP-4, [⁶⁸Ga]Ga-DOTA-Pip-PEP-9, and [⁶⁸Ga]Ga-DOTA-Pip-PEP-10 were obtained in 16-44% decay-corrected radiochemical yields with ≥64 GBq/μmol molar activity and ≥ 97% radiochemical purity. PET imaging and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice at 1 h postinjection. All [⁶⁸Ga]Ga-DOTA-Pip-PEP-4, [⁶⁸Ga]Ga-DOTA-Pip-PEP-9, and [⁶⁸Ga]Ga-DOTA-Pip-PEP-10 were excreted mainly via the renal pathway and enabled clear visualization of tumor xenografts in PET images with good tumor-to-background contrast. The pancreas uptake of the agonist tracer, [⁶⁸Ga]Ga-DOTA-Pip-PEP-4, was much lower than that of the clinically evaluated agonist tracer, [⁶⁸Ga]Ga-AMBA. Similarly, the pancreas uptake of the antagonist tracers, [⁶⁸Ga]Ga-DOTA-Pip-PEP-9, and [⁶⁸Ga]Ga-DOTA-Pip-PEP-10, was also much lower than that of the clinically evaluated antagonist tracers, [⁶⁸Ga]Ga-RM2, [⁶⁸Ga]Ga-SB3, and [⁶⁸Ga]Ga-NeoB. Our data demonstrate that the sequences of Ga-DOTA-Pip-PEP-4, Ga-DOTA-Pip-PEP-9, and Ga-DOTA-Pip-PEP-10 are promising templates for the development of radiopharmaceuticals targeting GRPR-expressing cancer.