Indium-111-Labeled Single-Domain Antibody forCXCR4 Imaging Using Single-Photon Emission Computed Tomography.

in Bioconjugate chemistry by Muriel Aline Spahn, Stephanie Mareike Anbuhl, Kaat Luyten, Tom Van Loy, Matti F Pronker, Christopher Cawthorne, Christophe M Deroose, Dominique Schols, Raimond Heukers, Guy Bormans, Frederik Cleeren

TLDR

  • The study aimed to identify a promising single-domain antibody (sdAb) construct for CXCR4 PET imaging, and found that VUN400-C showed high CXCR4-specific tumor uptake and favorable pharmacokinetic properties.
  • The study's findings suggest that VUN400-C has potential as a vector for CXCR4 PET imaging applications.

Abstract

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 [Ga]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 forIn-labeling. Their binding affinity against human CXCR4 (hCXCR4) was assessed by using a previously described BRET-based displacement assay. Theprofile 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 ([In]In-DOTAGA-VUN400-C-Direct and [In]In-DOTAGA-VUN400-C) were tested in mice bearing xenografts derived from U87.CD4, U87.CXCR4, and U87.CD4.CXCR4 cells throughbiodistribution 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, [In]In-DOTAGA-VUN400-C-Direct and [In]In-DOTAGA-VUN401-C-Direct displayed the highest cellular uptake, achieving 10.4 ± 1.6% and 11.5 ± 1.1%, respectively. In naive mice, [In]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,= 3, 60 min p.i.), but average plasma stability (40.6 ± 9.4%,= 3, 60 min p.i.). In a xenografted tumor model, [In]In-DOTAGA-VUN400-C-Direct showed only minor uptake (SUV0.71 ± 0.002,= 3, 60 min p.i.). [In]In-DOTAGA-VUN400-C demonstrated nearly identical plasma stability (41.08 ± 5.45%,= 4) but showed high and specific uptake in the CXCR4-expressing xenografted tumor (SUV3.75 ± 1.08 vs SUV= 0.64 ± 0.19,= 5, 60 min p.i.), which could be blocked by coinjection of AMD3100 (5 mg/kg) (SUV0.55 ± 0.32 vs SUV= 0.39 ± 0.07,= 2, 60 min p.i.). In conclusion, all six sdAbs exhibited highaffinity against hCXCR4. Among these, [In]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.

Overview

  • The study aims to screen multiple CXCR4-targeting single-domain antibody (sdAb) constructs to identify a promising molecule for the development of a CXCR4 fluorine-18-labeled tracer.
  • The study generated five CXCR4-specific sdAb constructs with a cysteine-containing C-terminal tag and one probe without, and coupled them to maleimide-DOTAGA for In-labeling.
  • The study focused on evaluating the binding affinity, plasma stability, and biodistribution of these constructs in mice, and identifying the most promising sdAb construct for future CXCR4 PET imaging applications.

Comparative Analysis & Findings

  • All six sdAb constructs exhibited high affinity against human CXCR4 (hCXCR4), with [In]In-DOTAGA-VUN400-C-Direct and [In]In-DOTAGA-VUN401-C-Direct displaying the highest cellular uptake in radioactive binding assays.
  • [In]In-DOTAGA-VUN400-C-Direct showed a favorable biodistribution profile in naive mice, with low uptake across all organs except the kidneys, but average plasma stability.
  • [In]In-DOTAGA-VUN400-C demonstrated high and specific uptake in the CXCR4-expressing xenografted tumor, which could be blocked by coinjection of AMD3100, indicating its potential as a promising vector for CXCR4 PET imaging applications.

Implications and Future Directions

  • The study's findings suggest that VUN400-C has potential as a vector for CXCR4 PET imaging applications, and its derivative [In]In-DOTAGA-VUN400-C could be further developed as a CXCR4-targeted probe.
  • Future studies could evaluate the efficacy of [In]In-DOTAGA-VUN400-C in different tumor models and investigate its potential as a diagnostic tool for cancer patients.
  • The study highlights the importance of developing specific and stable CXCR4-targeted probes for PET imaging, and future research could focus on optimizing the design and labeling of sdAb constructs for optimal imaging performance.
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