| Summary: | Positron emission tomography (PET) imaging of the C-X-C chemokine receptor 4 (CXCR4) with [<sup>68</sup>Ga]PentixaFor has intrinsic diagnostic value and is used to select patients for personalized CXCR4-targeted radionuclide therapy with its therapeutic radiopharmaceutical companion [<sup>177</sup>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 [<sup>177</sup>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 [<sup>nat</sup>Ga]PentixaFor (half-maximal inhibitory concentration (IC<sub>50</sub>): 5.3 ± 0.9 nM and 8.6 ± 1.1 nM, respectively) and also binds to murine CXCR4 (mCXCR4) (IC<sub>50</sub>: 33.4 ± 13.5 nM) while [<sup>nat</sup>Ga]PentixaFor is selective for hCXCR4 (IC<sub>50</sub> > 1000 nM for mCXCR4). Both the diagnostic radiotracers based on the DV1-k-(DV3) vector platform, [<sup>18</sup>F]AlF-NOTA-DV1-k-(DV3) and [<sup>68</sup>Ga]Ga-DOTA-DV1-k-(DV3), and their therapeutic companion [<sup>177</sup>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, [<sup>18</sup>F]AlF-NOTA-DV1-k-(DV3) shows CXCR4-specific targeting in mCXCR4-expressing organs such as liver (mean standardized uptake value (SUV<sub>mean</sub>) 8.2 ± 1.0 at 75 min post-injection (p.i.)), spleen (SUV<sub>mean</sub> 2.5 ± 1.0 at 75 min p.i.), and bone (SUV<sub>mean</sub> 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 [<sup>18</sup>F]AlF-NOTA-DV1-k-(DV3) was significantly lower (SUV<sub>mean</sub> 0.6 ± 0.2) compared to [<sup>68</sup>Ga]PentixaFor (SUV<sub>mean</sub> 2.9). This might be explained by the high affinity of [<sup>18</sup>F]AlF-NOTA-DV1-k-(DV3) toward both mCXCR4 and hCXCR4. High mCXCR4 expression in mouse liver results in a large fraction of [<sup>18</sup>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 [<sup>68</sup>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.
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