D-Peptide-Based Probe for CXCR4-Targeted Molecular Imaging and Radionuclide Therapy

Positron emission tomography (PET) imaging of the C-X-C chemokine receptor 4 (CXCR4) with [(68)Ga]PentixaFor has intrinsic diagnostic value and is used to select patients for personalized CXCR4-targeted radionuclide therapy with its therapeutic radiopharmaceutical companion [(177)Lu]PentixaTher. However, a CXCR4-targeting radiopharmaceutical labeled with fluorine-18 is still of high value due to its favorable characteristics over gallium-68. Furthermore, clinical results with [(177)Lu]PentixaTher are promising, but there is still room for improvement regarding pharmacokinetics and dosimetry profile. Therefore, this study aimed to develop innovative CXCR4-targeting radiopharmaceuticals, both for diagnostic and therapeutic purposes, starting from a D-amino acid-based peptide probe (DV1-k-(DV3)) that conserves high CXCR4 binding affinity after radiolabeling. AlF-NOTA-DV1-k-(DV3) showed similar in vitro binding affinity to human CXCR4 (hCXCR4) compared to [(nat)Ga]PentixaFor (half-maximal inhibitory concentration (IC(50)): 5.3 ± 0.9 nM and 8.6 ± 1.1 nM, respectively) and also binds to murine CXCR4 (mCXCR4) (IC(50): 33.4 ± 13.5 nM) while [(nat)Ga]PentixaFor is selective for hCXCR4 (IC(50) > 1000 nM for mCXCR4). Both the diagnostic radiotracers based on the DV1-k-(DV3) vector platform, [(18)F]AlF-NOTA-DV1-k-(DV3) and [(68)Ga]Ga-DOTA-DV1-k-(DV3), and their therapeutic companion [(177)Lu]Lu-DOTA-DV1-k-(DV3) were successfully produced in high yield, demonstrated high in vitro and in vivo stability, and have the same favorable pharmacokinetic profile. Furthermore, in wild-type mice and a hCXCR4-expressing tumor model, [(18)F]AlF-NOTA-DV1-k-(DV3) shows CXCR4-specific targeting in mCXCR4-expressing organs such as liver (mean standardized uptake value (SUV(mean)) 8.2 ± 1.0 at 75 min post-injection (p.i.)), spleen (SUV(mean) 2.5 ± 1.0 at 75 min p.i.), and bone (SUV(mean) 0.4 ± 0.1 at 75 min p.i., femur harboring bone marrow) that can be blocked with the CXCR4 antagonist AMD3100. However, in a hCXCR4-expressing tumor model, tumor uptake of [(18)F]AlF-NOTA-DV1-k-(DV3) was significantly lower (SUV(mean) 0.6 ± 0.2) compared to [(68)Ga]PentixaFor (SUV(mean) 2.9). This might be explained by the high affinity of [(18)F]AlF-NOTA-DV1-k-(DV3) toward both mCXCR4 and hCXCR4. High mCXCR4 expression in mouse liver results in a large fraction of [(18)F]AlF-NOTA-DV1-k-(DV3) that is sequestered to the liver, resulting despite its similar in vitro affinity for hCXCR4, in lower tumor accumulation compared to [(68)Ga]PentixaFor. As CXCR4 is not expressed in healthy human liver, the findings in mice are not predictive for the potential clinical performance of this novel class of CXCR4-targeting radiotracers. In conclusion, the DV1-k-(DV3) scaffold is a promising vector platform for translational CXCR4-directed research.