CMKLR1-targeting peptide tracers for PET/MR imaging of breast cancer

Abstract

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...