Characterization of CD163-specific nanobodies as immunotracers to non-invasively visualize immunosuppressive macrophages in cancer models

: Characterization of CD163-specific nanobodies as immunotracers to non-invasively visualize immunosuppressive macrophages in cancer models

Abstract

Introduction: In the last few decades immunotherapy has opened a new era in cancer treatment. However, challenges such as variations in the patients’ responsiveness and developing resistance limit the widespread use of immunotherapy. To this end, non-invasive whole-body imaging technologies have become an extremely useful technique to monitor the patient’s response to immunotherapy. This study will focus on the imaging of immunosuppressive tumor-associated macrophages (TAMs) using radiolabeled nanobodies (Nbs) against the CD163 receptor and macrophage mannose receptor (MMR or CD206) which are expressed on TAMs. Since MMR receptor is also expressed on other cell types such as liver endothelial cells, the CD163 receptor can be more beneficial in detecting immunosuppressive macrophages as it is only expressed on cells of the monocyte/macrophage lineage. The aims of this project are in vivo characterization of radiolabeled anti-CD163 and anti-MMR Nbs, adaptation of the Nb sequence and translation to a PET tracer. Methods: The functionality and stability of the CD163 Nb were assessed in vitro. The anti-CD163 and anti-MMR Nbs were radiolabeled with ⁹⁹ᵐTc. The functionality of the resulting radiolabeled Nbs was assessed for their in vivo and ex vivo biodistribution in naïve, CD163 knock-out, macrophage depleted and tumor-bearing (MC38 and B16-F10) mice. For translation of the anti-CD163 Nb to a PET tracer, NOTA-coupling and radiolabeling with 68Ga and 67Ga were performed for the Nb. Results: The anti-CD163 Nb showed high affinity towards recombinant hCD163 and mCD163 proteins. Anti-CD163 Nb was successfully radiolabeled with ⁹⁹ᵐTc with radiochemical purity of >99 %. The resulting radiolabeled Nb was functional and in vivo biodistribution studies revealed its selectivity for CD163 receptor and macrophages with significant contrast. The biodistribution studies in tumor-bearing mice revealed low uptake of the radiolabeled anti-CD163 Nb in both MC38 and B16-F10 Nb. NOTA-coupling of the Nb and radiolabeling were more successful for 67Ga compared to 68Ga. The NOTA-coupling and 68Ga-labeling were not successful for anti-CD163 Nb. Conclusion: We have shown the specificity of the [⁹⁹ᵐTc]-anti-CD163-Nb as a targeting vehicle for the CD163 receptor and the macrophages. Both radiolabeled anti-CD163 and anti-MMR Nbs show low accumulation in chosen tumor models. The in vivo biodistribution studies in tumor-bearing mice, and optimization of the anti-CD163 Nb NOTA-coupling require further investigations. Keywords: Immunotherapy; Nanobody; Tumor-associated macrophages; CD163; Positron emission tomography

Date of Award	2023
Original language	English
Supervisor	Yoline Lauwers (Advisor)