Validation of [(125)I]CPCR4.3 as an investigative tool for the sensitive and specific detection of hCXCR4 and mCXCR4 expression in vitro and in vivo

BACKGROUND: The development and clinical translation of [(68)Ga] Pentixafor has substantially promoted the relevance of non-invasive PET imaging of CXCR4 expression in a broad spectrum of diseases, including cancer and inflammation. Its pronounced selectivity for the human receptor (hCXCR4), however, precludes the use of [(68)Ga] Pentixafor for imaging receptor expression and dynamics in CXCR4-related diseases in endogenous mouse models. To overcome this restriction, [(125)I]CPCR4.3, a structurally related pentapeptide ligand, has been evaluated as a preclinical tool for efficient in vitro and in vivo targeting of hCXCR4 and mCXCR4. RESULTS: Compared to the reference [(68)Ga] Pentixafor, [(125)I]CPCR4.3 showed 2.4- to 11-fold increased specific binding to human cancer cell lines with different hCXCR4 expression levels (Jurkat, Daudi, HT-29, SH-5YSY, MCF-7, LNCaP) as well as strong and highly specific binding to mCXCR4 expressing cells (mCXCR4-transfected CHO cells, Eμ-myc 1080, 4 T1), which was not detectable for [(68)Ga]Pentixafor. This is the consequence of the equally high affinity of iodo-CPCR4 to hCXCR4 and mCXCR4 (IC(50) = 5.4 ± 1.5 and 4.9 ± 1.7 nM, respectively) as opposed to [(nat)Ga] Pentixafor (hCXCR4: 42.4 ± 11.6 nM, mCXCR4: > 1000 nM). Additionally, [(125)I]CPCR4.3 showed enhanced tracer internalization (factor of 1.5–2 compared to the reference). In vivo biodistribution studies in immunocompetent Black Six and immunocompromised CD-1 nude mice showed predominant hepatobiliary excretion of [(125)I]CPCR4.3 (logP = 0.51), leading to high activity levels in liver and intestines. However, [(125)I]CPCR4.3 also showed high and specific accumulation in organs with endogenous mCXCR4 expression (spleen, lung, adrenals), even at low receptor expression levels. CONCLUSIONS: Due to its excellent hCXCR4 and mCXCR4 targeting efficiency, both in vitro and in vivo, [(125)I]CPCR4.3 represents a sensitive and reliable tool for the species-independent quantification of CXCR4 expression. Its suboptimal clearance properties will certainly restrict its use for in vivo imaging applications using (123)I (for SPECT) or (124)I (for PET), but due to its high and specific accumulation in mCXCR4 expressing tissues, [(125)I]CPCR4.3 holds promise as a powerful preclinical tool for the investigation and quantification of CXCR4 involvement and kinetics in various murine disease models via, e.g., biodistribution and autoradiography studies.