Characterization of CD163-specific nanobody-based immunotracers for non-invasive imaging of immunosuppressive macrophages.

Introduction
Non-invasive imaging of different immune cells inside the tumor microenvironment could predict the patients’ response to immunotherapy. Unfortunately, only a limited amount of good immunotracers to image immune cells are available in the clinic. In this study, we generated and characterized a cross-reactive nanobody (Nb) against the marker CD163, a receptor that is specifically expressed on a subset of immunosuppressive tumor-associated macrophages (TAMs). This CD163-targeting immunotracer could be used for imaging of TAMs to predict immunotherapy response in cancer patients.

Methods
A Nb cross-reactive for mouse and human CD163 was tested for affinity towards recombinant protein via surface plasmon resonance and on cells via flow cytometry. An in vivo biodistribution experiment with pinhole μSPECT/CT was conducted in naïve C57BL/6 wild-type (WT) and CD163 knock-out (-/-) mice to select a lead Nb showing specificity for CD163+ cells. To test macrophage-specific binding of the radiolabeled Nb, a biodistribution experiment in macrophage-depleted mice (using the CSF1R inhibitor PLX3397, 600 mg/kg food) was performed. Next, TAM-targeting was assessed in a lung carcinoma model (LLC-OVA) with verification via flow cytometry. Finally, the anti-CD163 Nb was conjugated to p-SCN-Bn-NOTA and test-labeled with gallium-68 (68Ga).

Results/Discussion
The anti-CD163 Nb shows affinities in the low nanomolar range for both human and mouse CD163 proteins (Figure 1A). Uptake of the lead Nb-based immunotracer in macrophage-rich organs is demonstrated in naïve WT mice, with no uptake in CD163-/- mice, supporting specificity for CD163+ cells (Figure 1B). Macrophage-specificity is proven by comparing untreated and macrophage-depleted mice. Untreated mice show high uptake of the immunotracer in macrophage-rich organs. The signal of the immunotracer in liver and lymph nodes significantly declines in macrophage-depleted mice validating the specific expression of CD163 on macrophages (Figure 1C). LLC-OVA tumor-bearing mice show Nb uptake in the center of the tumor on μSPECT/CT, correlating with high CD163 expression levels on TAMs (Figure 1D-E). Translation towards a PET tracer has been optimized and validated with 68Ga-labeling.

Conclusions
Altogether, we have developed a cross-reactive immunotracer that is specific for CD163+ macrophages to image these immune cells in the tumor microenvironment. The PET tracer is available and ready to be used for μPET/CT imaging with 68Ga. When the predictive value of CD163-expressing TAMs is established, the radiolabeled Nb could be a promising clinical imaging agent to predict immunotherapy response and contribute to personalized medicine.

Acknowledgement
This work was supported by the EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (Immune Image GA831514) under grant agreement N° 831514.
Y. Lauwers and T.W.M De Groof contributed equally. J.A. Van Ginderachter and N.Devoogdt share senior authorship.