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.