The use of a CD163-specific nanobody-based immunotracer as a clinical imaging agent to study immunosuppressive tumor-associated macrophages

Research output: Unpublished contribution to conferencePoster


Non-invasive imaging of different immune cells inside the tumor microenvironment could predict the patients’ response to immunotherapy [1]. Unfortunately, only a limited number of good tracers to image immune cells are available in the clinic. In this study, we generated and characterized a cross-reactive nanobody-based immunotracer against CD163, a receptor that is specifically expressed on a subset of immunosuppressive tumor-associated macrophages (TAMs) [2]. This CD163-targeting immunotracer could be used for imaging of TAMs to predict immunotherapy response in cancer patients.
The CD163-targeting nanobody showed binding affinities in the low nanomolar range for both human and mouse CD163 proteins and did not compete with mouse hemoglobin, a ligand of the CD163 receptor. Further, no indirect activation of T cells after macrophage binding was detected for the CD163-targeting nanobody in both spleen and bone marrow of wild-type (WT) and CD163-/- mice (n=3) after overnight incubation. Multiplex analysis of the supernatant showed that incubation with the anti-CD3 antibody (positive control) resulted in the secretion of pro-inflammatory cytokines (IL-2, IL-6, TNF-α and IFN-γ) after T cell activation, while no increase of these cytokines was detected after incubation with the CD163-targeting nanobody. The nanobody was also considered low immunogenic after performing a dendritic-T cell killing assay as no increase in the pro-inflammatory cytokines IFN- γ and IL-5 was measured.
Uptake of the lead immunotracer in macrophage-rich organs was demonstrated via μSPECT/CT imaging in naïve WT mice, while no uptake was shown in CD163-/- mice, supporting the specificity for CD163+ cells. Macrophage-specificity was proven by comparing untreated with macrophage-depleted mice. The signal of the immunotracers in liver and lymph nodes was significantly reduced in macrophage-depleted mice validating the specific expression of CD163 on macrophages. μSPECT/CT imaging of LLC-OVA tumor-bearing mice, with subsequent flow cytometry analysis of the tumor, showed the ability of the i mmunotracer to target TAMs. The translation towards a PET tracer has been optimized and the binding of the 68Ga-labeled PET tracer to CD163 has been confirmed both in vitro via SPR and cell binding studies and in vivo via μPET/CT imaging with ex vivo analysis. In addition, stability studies (n=3) with the [68Ga]Ga-NOTA-CD163-targeting nanobody showed that after 60 min, the tracer was still stable in injection buffer (RCP; 95,5 ± 1,2%) and human serum (RCP; 92,2 ± 2,9%) at 37°C.
At the moment, the predictive value of the CD163-targeting immunotracer during a macrophage-depleting therapy is being investigated in LLC-OVA tumor bearing mice. Mice are treated, 5 days post inoculation, with the CSF1R inhibitor PLX3397 (600mg/kg food) for 3 weeks and scanned at 4 different timepoints. The dynamics of CD163+ TAMs in the tumor will be analyzed via μPET/CT imaging, ex vivo radioactive uptake analysis, flow cytometry and immunohistochemistry.
Altogether, we have developed a cross-reactive immunotracer that is specific for CD163+ macrophages and allows imaging of these immune cells in the tumor microenvironment. The radiolabeled nanobody-based immunotracer could be a promising clinical imaging agent to further study the role of CD163-expressing TAMs, predict immunotherapy responses and contribute to personalized medicine.
This work was supported by the EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (Immune Image GA831514) under grant agreement N° 831514.
Original languageEnglish
Publication statusIn preparation - 5 Oct 2023
EventFourth Annual Immune-Image meeting 2023 - Hotel Alimara, Barcelona, Spain
Duration: 4 Oct 20235 Oct 2023


OtherFourth Annual Immune-Image meeting 2023


  • macrophages
  • CD163
  • imaging


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