Background: Molecular targeting remains to be a promising approach in oncology. Overexpression of G protein-coupled receptors (GPCRs) in human cancer is offering a powerful opportunity for tumor-selective imaging and treatment employing nuclear medicine. We utilized novel chemerin-based peptide conjugates for chemokine-like receptor 1 (CMKLR1) targeting in a breast cancer xenograft model.Methods: By conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of five highly specific, high-affinity tracers for hybrid positron emission tomography/magnetic resonance (PET/MR) imaging. A xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. We acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments.Results: Five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be validated by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers [68Ga]Ga-DOTA-AHX-CG34, [68Ga]Ga-DOTA-KCap-CG34 and [68Ga]Ga-DOTA-ADX-CG34 with best tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 3.3% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. In addition, tumor uptake was blocked by excess of unlabeled peptide (6.4-fold, 5.5-fold and 3.4-fold 1 h post-injection), further confirming CMKLR1 specificity. Out of five tracers, we identified these three tracers with moderate, balanced hydrophilicity to be the most potent in receptor-mediated tumor targeting.Conclusion: We demonstrated the applicability of 68Ga-labeled peptide tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging, paving the way for developing them into theranostics for tumor treatment.

This study investigated the role of individual U-II amino acid positions and side chain characteristics important for U-IIR activation. A complete permutation library of 209 U-II variants was studied in an activity screen that contained single substitution variants of each position with one of the other 19 proteinogenic amino acids. Receptor activation was measured using a cell-based high-throughput fluorescence calcium mobilization assay. We generated the first complete U-II substitution map for U-II receptor activation, resulting in a detailed view into the structural features required for receptor activation, accompanied by complementary information from receptor modeling and ligand docking studies. On the basis of the systematic SAR study of U-II, we created 33 further short and linear U-II variants from eight to three amino acids in length, including d- and other non-natural amino acids. We identified the first high-potency linear U-II analogues. Urolinin, a linear U-II agonist (nWWK-Tyr(3-NO)-Abu), shows low nanomolar potency as well as improved metabolic stability.

Purpose Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68 Gallium ( 68 Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68 Ga-DOTA peptide purified from the excess of unlabeled peptide. Procedures The MC1R ligand DOTA-NAPamide was labeled with 68 Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68 Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. Results In biodistribution studies, non-purified 68 Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68 Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. Conclusions We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68 Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

Background: FAP is a membrane-bound protease under investigation as a pan-cancer target given its limited expression in normal adult tissues but high expression on cancer-associated fibroblasts. FAP-2286 and FAPI-46 are radiotracers in clinical development that target FAP through different modalities: FAP-2286 employs a peptide macrocycle, whereas FAPI-46 is a quinoline-based small molecule. Here, the biochemical and cellular properties of FAP-2286 and FAPI-46 were evaluated, as well as their in vivo biodistribution and efficacy. Methods: FAP-2286 and FAPI-46 were assessed in biochemical and cellular assays for affinity to FAP and cellular uptake. Complexes of the compounds with gallium-68 (68Ga) or lutetium-177 (177Lu) were investigated in imaging and efficacy studies using the HEK-FAP xenograft mouse model. Results: FAP-2286 and FAPI-46 displayed potent affinity to human FAP by surface plasmon resonance with equilibrium dissociation constants of 1.1 and 0.04 nM, respectively. In addition, FAP-2286 and FAPI-46 inhibited human FAP protease activity with IC50 of 3.2 and 1.2 nM, respectively, and competitor binding to FAP-expressing cells with IC50 of 2.7 and 1.3 nM, respectively. In a PET imaging study, 68Ga-FAP-2286 or 68Ga-FAPI-46 resulted in comparable tumor uptake at 1 hour after injection (10 MBq; 10.6 vs 10.1 percent injected dose per gram [%ID/g]). In contrast, 177Lu-FAP-2286 and 177Lu-FAPI-46 had significant differences in tumor uptake at 24 hours (30 MBq; 15.8 vs 3.8 %ID/g, respectively; P=0.001) and 72 hours (16.4 vs 1.6 %ID/g respectively; P=0.002) after dosing as observed by SPECT imaging. Consistent with the SPECT imaging data, Alexa Fluor 488-derivatized FAP-2286 was retained in cells and secluded in endosomes out to 72 hours in vitro, whereas Alexa Fluor 488-derivatized FAPI-46 levels decreased over time starting at 8 hours, despite both showing a similar level of binding and initial internalization to HEK-FAP cells. FAP-2286 and FAPI-46 labeled with 177Lu demonstrated antitumor efficacy with mean tumor volumes (MTV) of 107 and 245 mm3, respectively, on day 9 after dosing compared with 952 mm3 for vehicle control (30 MBq; P<0.001). The suppressed tumor growth was maintained on day 23 for FAP-2286- but not for FAPI-46-treated animals (MTV 12 vs 1210 mm3; P<0.0001). At the end of the study (day 41), all mice treated with 177Lu-FAPI-46 had reached the endpoint MTV of >1500 mm³ with a median survival time (MST) of 27.5 days, whereas the MTV of mice treated with 177Lu-FAP-2286 was 106 mm3 with MST not being reached. Conclusions: In preclinical studies, the radiotherapeutic 177Lu-FAP-2286 showed longer tumor retention, resulting in greater tumor inhibition than 177Lu-FAPI-46. The phase 1/2 LuMIERE clinical trial (NCT04939610) will evaluate FAP-2286 as a therapeutic (177Lu-FAP-2286) and imaging (68Ga-FAP-2286) agent in multiple indications. Citation Format: Dirk Zboralski, Aileen Hoehne, Anne Bredenbeck, Matthias Paschke, Jan Lennart von Hacht, Jim Xiao, Andrew D. Simmons, Frank Osterkamp, Thomas Harding, Minh Nguyen. Comparative biodistribution and radiotherapeutic efficacy of the fibroblast activation protein (FAP)-targeting agents FAP-2286 and FAPI-46 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3317.

42Molecular targeting remains to be a promising approach in cancer medicine. Knowledge 43 about molecular properties such as overexpression of G protein-coupled receptors 44 (GPCRs) is thereby offering a powerful tool for tumor-selective imaging and treatment of 45 cancer cells. We utilized chemerin-based peptides for CMKLR1 receptor targeting in a 46 breast cancer xenograft model. By conjugation with radiolabeled chelator 1,4,7,10-47 tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of highly 48 specific and affine tracers for hybrid in vivo imaging with positron emission tomography 49 (PET)/ magnetic resonance (MR) and concomitant biodistribution studies. 50 Methods 51We developed five highly specific and affine peptide tracers targeting CMKLR1 by linker-52 based conjugation of chemerin peptide analogs (CG34 and CG36) with radiolabeled 53 ( 68 Ga) chelator DOTA. Our established xenograft model with target-positive DU4475 and 54 negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging 55 in vivo. Therefore, we acquired small animal PET/MR images, assessed biodistribution by 56 ex vivo measurements and investigated the tracer specificity by blocking experiments. 57 Results 58The family of five CMKLR1-targeting peptide tracers demonstrated high biological activity 59 and affinity in vitro with EC50 and IC50 values being below 2 nM. Our target-positive 60 (DU4475) and target-negative (A549) xenograft model could be confirmed by ex vivo 61 analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three 62 most promising tracers 68 Ga-DOTA-AHX-CG34, 68 Ga-DOTA-KCap-CG34 and 68 Ga-63 DOTA-ADX-CG34 with apparent DU4475 tumor uptake were further analyzed. Hybrid 64 PET/MR imaging along with concomitant biodistribution studies revealed distinct 65 3 CMKLR1-specific uptake (5.1% IA/g, 4.5% IA/g and 6.2% IA/g 1 h post-injection) of our 66 targeted tracers in DU4475 tumor tissue. More strikingly, the tumor uptake could be 67 blocked by excess of unlabeled peptide (6.4-fold, 7.2-fold and 3.4-fold 1 h post-injection) 68 and further confirmed the CMKLR1 specificity. As our five tracers, each with particular 69 degree of hydrophobicity, showed different results regarding tumor uptake and organ 70 distribution, we identified these three tracers with moderate, balanced properties to be the 71 most potent in receptor-mediated tumor targeting. 72 Conclusion 73 With the breast cancer cell line DU4475, we established a model endogenously 74 expressing our target CMKLR1 to evaluate our chemerin-based peptide tracers as highly 75 affine and specific targeting agents. Eventually, we demonstrated the applicability of our 76 68 Ga-labeled tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR 77 imaging and thus developed promising theranostics for tumor treatment.78 79 Keywords 80 Tumor targeting, PET tracer, Chemokine-like receptor 1, peptide ligand, breast cancer 81 82 83 84 Molecular targeting remains to be one of the most promising approa...

2 1 2 2 2 3 ¶ These authors contributed equally to this work.2 4 2 5 2 Abstract 2 6Purpose: Melanocortin receptor 1 is overexpressed in melanoma and may be a 2 7 molecular target for imaging and peptide receptor radionuclide therapy. 68 Gallium 2 8 labeling of DOTA-conjugated peptides is an established procedure in the clinic for 2 9 use in positron emission tomography imaging. Aim of this study was to compare a 3 0 standard labeling protocol against the 68 Ga-DOTA peptide purified from the excess of 3 1 unlabeled peptide.3 2 Procedures: The MC1R ligand DOTA-NAPamide was labeled with 68 Ga using a 3 3 standard clinical protocol. Radioactive peptide was separated from the excess of 3 4 unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide 3 5 and 68 Ga (95 °C, 15 min), the reaction was loaded on a C18 column and separated 3 6by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. 7Radiolabeled products were compared in biodistribution studies and PET imaging 3 8 using nude mice bearing MC1R-expressing B16/F1 xenograft tumors.

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