Nanobody-based targeting of the Macrophage Mannose Receptor for effective in vivo imaging of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are an important component of the tumor stroma and exert several tumor-promoting activities. Strongly pro-angiogenic TAMs that reside in hypoxic tumor areas highly express Macrophage Mannose Receptor (MMR, CD206). In this study, we targeted MMR(+) TAMs using nanobodies, which are single-domain antigen-binding fragments derived from Camelidae heavy-chain antibodies. MMR-specific nanobodies stained TAMs in lung and breast tumor single-cell suspensions in vitro, and intravenous injection of (99m)Tc-labeled anti-MMR nanobodies successfully targeted tumor in vivo. Retention of the nanobody was receptor-specific and absent in MMR-deficient mice. Importantly, co-injection of excess unlabeled, bivalent anti-MMR nanobodies reduced nanobody accumulation in extra-tumoral organs to background levels, without compromising tumor uptake. Within tumors, the (99m)Tc-labeled nanobodies specifically labeled MMR(+) TAMs, as CCR2-deficient mice that contain fewer TAMs showed significantly reduced tumor uptake. Anti-MMR nanobodies also accumulated in hypoxic regions, therefore targeting pro-angiogenic MMR(+) TAMs. Together, our findings provide preclinical proof of concept that anti-MMR nanobodies can be used to selectively target and image TAM subpopulations in vivo.