Indium-111-Labeled Single-Domain Antibody for <i>In Vivo</i> CXCR4 Imaging Using Single-Photon Emission Computed Tomography

C-X-C chemokine receptor type 4 (CXCR4) is highly expressed in a range of pathologies, including cancers like multiple myeloma and non-Hodgkin lymphoma, inflammatory diseases such as rheumatoid arthritis, and viral infections like HIV. Currently, the most advanced radiotracer for CXCR4 imaging in clinics is [⁶⁸Ga]PentixaFor. However, its structure is prone to modifications, complicating the development of a specific CXCR4 fluorine-18-labeled tracer with good pharmacokinetic properties. This study aimed to screen multiple CXCR4-targeting variable domains of heavy-chain-only antibody (VHH or single-domain antibody (sdAb)) constructs to identify the most promising sdAb as a vector molecule for the future development of a CXCR4 fluorine-18 tracer. We have generated five CXCR4-specific sdAb constructs with a cysteine-containing C-terminal tag (C-Direct tag) (VUN400-C-Direct, VUN401-C-Direct, VUN410-C-Direct, VUN411-C-Direct, and VUN415-C-Direct) and one probe (VUN400-C) without. The reduced sdAbs were coupled to maleimide-DOTAGA for ¹¹¹In-labeling. Their binding affinity against human CXCR4 (hCXCR4) was assessed by using a previously described BRET-based displacement assay. The <i>in vivo</i> profile was assessed using naive mice. Based on the plasma stability (60 min post injection (p.i.)), we selected VUN400-C-Direct and its derivative VUN400-C for further evaluation. These compounds ([¹¹¹In]In-DOTAGA-VUN400-C-Direct and [¹¹¹In]In-DOTAGA-VUN400-C) were tested in mice bearing xenografts derived from U87.CD4, U87.CXCR4, and U87.CD4.CXCR4 cells through <i>ex vivo</i> biodistribution studies and SPECT/CT imaging. The six sdAb constructs were labeled with a high radiochemical conversion (75-97%) and purity (>95%). In radioactive binding assays using U87.CD4.CXCR4 cells, [¹¹¹In]In-DOTAGA-VUN400-C-Direct and [¹¹¹In]In-DOTAGA-VUN401-C-Direct displayed the highest cellular uptake, achieving 10.4 ± 1.6% and 11.5 ± 1.1%, respectively. In naive mice, [¹¹¹In]In-DOTAGA-VUN400-C-Direct showed the most favorable biodistribution profile, with low uptake across all organs except the kidneys (Standardized Uptake Value (SUV) > 50, <i>n</i> = 3, 60 min p.i.), but average plasma stability (40.6 ± 9.4%, <i>n</i> = 3, 60 min p.i.). In a xenografted tumor model, [¹¹¹In]In-DOTAGA-VUN400-C-Direct showed only minor uptake (SUV_U87.CXCR4 0.71 ± 0.002, <i>n</i> = 3, 60 min p.i.). [¹¹¹In]In-DOTAGA-VUN400-C demonstrated nearly identical plasma stability (41.08 ± 5.45%, <i>n</i> = 4) but showed high and specific uptake in the CXCR4-expressing xenografted tumor (SUV_{U87.CD4.CXCR4} 3.75 ± 1.08 vs SUV_U87.CD4 = 0.64 ± 0.19, <i>n</i> = 5, 60 min p.i.), which could be blocked by coinjection of AMD3100 (5 mg/kg) (SUV_{U87.CD4.CXCR4} 0.55 ± 0.32 vs SUV_U87.CD4 = 0.39 ± 0.07, <i>n</i> = 2, 60 min p.i.). In conclusion, all six sdAbs exhibited high <i>in vitro</i> affinity against hCXCR4. Among these, [¹¹¹In]In-DOTAGA-VUN400-C showed high CXCR4-specific tumor uptake and favorable pharmacokinetic properties, indicating VUN400-C's potential as a promising vector for future CXCR4 PET imaging applications with fluorine-18.