A new class of PentixaFor- and PentixaTher-based theranostic agents with enhanced CXCR4-targeting efficiency

Non-invasive PET imaging of CXCR4 expression in cancer and inflammation as well as CXCR4-targeted radioligand therapy (RLT) have recently found their way into clinical research by the development of the theranostic agents [(68)Ga]PentixaFor (cyclo(D-Tyr(1)-D-[NMe]Orn(2)(AMBS-[(68)Ga]DOTA)-Arg(3)-Nal(4)-Gly(5)) = [(68)Ga]DOTA-AMBS-CPCR4) and [(177)Lu/(90)Y]PentixaTher (cyclo(D-3-iodo-Tyr(1)-D-[NMe]Orn(2)(AMBS-[(177)Lu/(90)Y]DOTA)-Arg(3)-Nal(4)-Gly(5)) = [(177)Lu/(90)Y]DOTA-AMBS-iodoCPCR4). Although convincing clinical results have already been obtained with both agents, this study was designed to further investigate the required structural elements for improved ligand-receptor interaction for both peptide cores (CPCR4 and iodoCPCR4). To this aim, a series of DOTA-conjugated CPCR4- and iodoCPCR4-based ligands with new linker structures, replacing the AMBA-linker in PentixaFor and PentixaTher, were synthesized and evaluated. Methods: The in vitro investigation of the novel compounds alongside with the reference peptides PentixaFor and PentixaTher encompassed the determination of hCXCR4 and mCXCR4 affinity (IC(50)) of the respective (nat)Ga-, (nat)Lu-, (nat)Y- and (nat)Bi-complexes in Jurkat and Eμ-myc 1080 cells using [(125)I]FC-131 and [(125)I]CPCR4.3 as radioligands, respectively, as well as the evaluation of the internalization and externalization kinetics of selected (68)Ga- and (177)Lu-labeled compounds in hCXCR4-transfected Chem-1 cells. Comparative small animal PET imaging studies (1h p.i.) as well as in vivo biodistribution studies (1, 6 and 48h p.i.) were performed in Daudi (human B cell lymphoma) xenograft bearing CB17 SCID mice. Results: Based on the affinity data and cellular uptake studies, [(68)Ga/(177)Lu]DOTA-r-a-ABA-CPCR4 and [(68)Ga/(177)Lu]DOTA-r-a-ABA-iodoCPCR4 (with r-a-ABA = D-Arg-D-Ala-4-aminobenzoyl-) were selected for further evaluation. Both analogs show app. 10-fold enhanced hCXCR4 affinity compared to the respective references [(68)Ga]PentixaFor and [(177)Lu]PentixaTher, four times higher cellular uptake in hCXCR4 expressing cells and improved cellular retention. Unfortunately, the improved in vitro binding and uptake characteristics of [(68)Ga]DOTA-r-a-ABA-CPCR4 and -iodoCPCR4 could not be recapitulated in initial PET imaging studies; both compounds showed similar uptake in the Daudi xenografts as [(68)Ga]PentixaFor, alongside with higher background accumulation, especially in the kidneys. However, the subsequent biodistribution studies performed for the corresponding (177)Lu-labeled analogs revealed a clear superiority of [(177)Lu]DOTA-r-a-ABA-CPCR4 and [(177)Lu]DOTA-r-a-ABA-iodoCPCR4 over [(177)Lu]PentixaTher with respect to tumor uptake (18.3±3.7 and 17.2±2.0 %iD/g, respectively, at 1h p.i. vs 12.4±3.7%iD/g for [(177)Lu]PentixaTher) as well as activity retention in tumor up to 48h. Especially for [(177)Lu]DOTA-r-a-ABA-CPCR4 with its low background accumulation, tumor/organ ratios at 48h were 2- to 4-fold higher than those obtained for [(177)Lu]PentixaTher (except for kidney). Conclusions: The in-depth evaluation of a series of novel CPCR4- and iodoCPCR4 analogs with modified linker structure has yielded reliable structure-activity relationships. It was generally observed that a) AMBA-by-ABA-substitution leads to enhanced ligand internalization, b) the extension of the ABA-linker by two additional amino acids (DOTA-Xaa(2)-Xaa(1)-ABA-) provides sufficient linker length to minimize the interaction of the [M(3+)]DOTA-chelate with the receptor, and that c) introduction of a cationic side chain (Xaa(2)) greatly enhances receptor affinity of the constructs, obliterating the necessity for Tyr(1)-iodination of the pentapeptide core to maintain high receptor affinity (such as in [(177)Lu]PentixaTher). As a result, [(177)Lu]DOTA-r-a-ABA-CPCR4 has emerged from this study as a powerful second-generation therapeutic CXCR4 ligand with greatly improved targeting efficiency and tumor retention and will be further evaluated in preclinical and clinical CXCR4-targeted dosimetry and RLT studies.