title: PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments.
abstract: Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing (18)F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population.
  
  
  METHODS
  Cross-reactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer (18)F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models.
  
  
  RESULTS
  Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. (18)F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a (99m)Tc-labeled counterpart. Compared with MMR- and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the (18)F tracer for MMR and macrophages, respectively.
  
  
  CONCLUSIONS
  Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. (18)F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma.
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium anneleen.blykers@gmail.com.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Blykers
authors.affiliation: Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Myeloid Cell Immunology (MCI), VIB, Brussels, Belgium.
authors.forename: Steve
authors.initials: S
authors.lastname: Schoonooghe
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Catarina
authors.initials: C
authors.lastname: Xavier
authors.affiliation: Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Myeloid Cell Immunology (MCI), VIB, Brussels, Belgium.
authors.forename: Kevin
authors.initials: K
authors.lastname: D'hoe
authors.affiliation: Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Myeloid Cell Immunology (MCI), VIB, Brussels, Belgium.
authors.forename: Damya
authors.initials: D
authors.lastname: Laoui
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Matthias
authors.initials: M
authors.lastname: D'Huyvetter
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium Department of Nuclear Medicine, UZ Brussel, Brussels, Belgium; and.
authors.forename: Ilse
authors.initials: I
authors.lastname: Vaneycken
authors.affiliation: Laboratory for Radiopharmacy, KU Leuven, Leuven, Belgium.
authors.forename: Frederik
authors.initials: F
authors.lastname: Cleeren
authors.affiliation: Laboratory for Radiopharmacy, KU Leuven, Leuven, Belgium.
authors.forename: Guy
authors.initials: G
authors.lastname: Bormans
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium Department of Nuclear Medicine, UZ Brussel, Brussels, Belgium; and.
authors.forename: Johannes
authors.initials: J
authors.lastname: Heemskerk
authors.affiliation: Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Myeloid Cell Immunology (MCI), VIB, Brussels, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Raes
authors.affiliation: Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Myeloid Cell Immunology (MCI), VIB, Brussels, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: De Baetselier
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium Department of Nuclear Medicine, UZ Brussel, Brussels, Belgium; and.
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.affiliation: Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium Laboratory of Myeloid Cell Immunology (MCI), VIB, Brussels, Belgium.
authors.forename: Jo A
authors.initials: JA
authors.lastname: Van Ginderachter
authors.affiliation: In Vivo Cellular and Molecular Imaging laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium Department of Nuclear Medicine, UZ Brussel, Brussels, Belgium; and.
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
mesh_terms: D000818:Animals; D001345:Autoradiography; D002161:Camelids, New World; D005462:Fluorine Radioisotopes / Q000737:chemistry*; D005464:Fluorobenzenes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037181:Lectins, C-Type / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009369:Neoplasms / Q000378:metabolism*; D049268:Positron-Emission Tomography / Q000379:methods*; D019275:Radiopharmaceuticals / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000737:chemistry*; D014018:Tissue Distribution; D013721:Triethylenephosphoramide / Q000031:analogs & derivatives
chemical_list: D005462:Fluorine Radioisotopes; D005464:Fluorobenzenes; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D019275:Radiopharmaceuticals; D011956:Receptors, Cell Surface; D061905:Single-Domain Antibodies; C015890:fluorobenzotepa; D013721:Triethylenephosphoramide
keywords: 18F; PET; camelid single-domain antibody fragment (sdAb); macrophage mannose receptor (MMR); tumor microenvironment