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: Crossreactive 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 MMRexpressing 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. Conclusion: 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. Dur ing tumor development, myeloid cells are attracted to the tumor stroma. These infiltrating immune cells are versatile, adopting different activation states in response to a changing microenvironment, leading to subsets of tumor-associated macrophages (TAMs) with specialized functions (1,2). Two main morphologically distinct TAM subsets can be distinguished on the basis of major histocompatibility complex (MHC) class II expression levels in multiple mouse tumor models. Tumor promotion has been linked with an accumulation of M2-oriented MHC II low TAMs in lung and breast carcinoma (3,4). Accordingly, MHC II low TAMs were found to reside primarily in less oxygenated zones, express hypoxia-regulated genes, and facilitate the angiogenic switch (5). Interestingly, the macrophage mannose receptor (MMR, CD206), a typical M2...

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology

“…3A (16)). A 18 F-labeled variant was successfully developed as a PET tracer for MMR expression in mice (17), confirming its potential for clinical translation.…”

An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy

“…A fully human imaging agent specific for human CD8 can be arrived at through humanization of existing anti-CD8 monoclonal antibodies or de novo isolation of fully human antibodies by phage display (42). Alternative scaffolds, such as single domain camelid antibodies labeled with 18 F or 64 Cu for PET, have demonstrated utility in the detection of the macrophage mannose receptors, MHC Class II, and CD11b expressed on myeloid cells in preclinical models (43, 44). However, such agents might require humanization to reduce potential immunogenicity.…”

Targeting mannose receptor expression on macrophages in atherosclerotic plaques of apolipoprotein E-knockout mice using 111In-tilmanocept

“…The radiolabelled single-domain antigen-binding fragments derived from Camelidae heavy-chain antibodies (known as nanobodies) specifically targeting the macrophage MR have shown high potential for clinical implementation for imaging of the tumour-promoting macrophages [25, 26] and rheumatoid arthritis [27]. Effective targeting of imaging agents to MR-expressing cells is a potential approach for atherosclerotic plaque imaging.…”