Abstract Introduction: Macrophages are valuable imaging targets for inflammatory diseases, as they may reflect prognosis and response to therapy. Current noninvasive imaging agents that target different markers of macrophages are either nonspecific or preferentially identify the immunosuppressive macrophage subset. Here, we report the development of the first positron emission tomography (PET) tracer that targets human CD68 (huCD68), the clinically established pan-macrophage marker and its evaluation in the tumor microenvironment (TME). Methods: Fab8, previously identified through phage display, was conjugated with p-SCN-Bn-DFO chelator and radiolabeled with 89Zr to obtain 89ZrZr-DFO-Fab8. Binding specificity of the tracer was evaluated in vitro using THP-1 macrophages, and THP-1 undifferentiated cells was used as a control. NSG mice (n = 3) were inoculated with MDA-MB-231 cells, a triple negative breast cancer cell line that does not express huCD68, subcutaneously to both shoulders. After tumor growth, the mice were then intratumorally injected with THP-1 macrophages on the right tumor to establish a new model of human macrophages in the TME. As a control, undifferentiated THP-1 cells were injected into the left tumors at 1 h before tracer injection. These mice were then injected with 100 µL of 930 kBq (6.3 µg mass dose) of 89ZrZr-DFO-Fab8 and PET imaging was performed at 1 and 6 h post-injection (p.i.). The specific uptake of 89ZrZr-DFO-Fab8 was determined by calculating tumor-to-heart Standardized Uptake Value Ratios (SUVR). The presence of CD68 in the tumors were biologically validated by immunofluorescence (IF) staining. Results: 89ZrZr-DFO-Fab8 was produced with radiochemical yield 95%. 89ZrZr-DFO-Fab8 had significantly higher binding in THP-1 macrophages (10 ± 1.5 %) than in THP-1 undifferentiated cells (3.4 ± 0.11 % ; p = 0.016). The SUVR of 89ZrZr-DFO-Fab8 in THP-1 macrophages was determined to be 0.34 ± 0.05 and 0.48 ± 0.07 and in undifferentiated THP-1 was 0.18 ± 0.03 and 0.29 ± 0.03, at 1 and 6 h p.i. respectively. Tracer uptake in the THP-1 macrophage tumors was significantly higher than that in the undifferentiated THP-1 tumors at 1 and 6 h p.i. (p = 0.0120 and p = 0.0041, respectively). IF results showed more huCD68 staining in the THP-1 macrophage tumors vs the THP-1 undifferentiated tumors, further validating our immunoPET results. Conclusion: We have developed the first immunoPET tracer targeting human CD68. 89ZrZr-DFO-Fab8 binds specifically to huCD68 in both an in vitro and in vivo model, making it a promising tracer for noninvasive imaging of macrophage burden in various diseases for future clinical studies. Citation Format: Ayla Vaughn Embs, Borna Roohani, Robin Kumar, Dajah Nash, Chloe La Prairie, Mann Dangarwala, Harriet M. Kluger, Ping Zhang, Benjamin Larimer, Bernadette V. Marquez-Nostra. ImmunoPET imaging of human tumor associated macrophages abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4998.
Embs et al. (Fri,) studied this question.