Synthetic antibody fragment lights up human macrophages in PET scans

For decades, scientists have relied on biopsies and histopathology to count macrophages inside tumors, an approach that is invasive, slow, and limited to the small slivers of tissue that a needle happens to capture. Now a team at the University of Alabama at Birmingham, working with collaborators at Yale University and elsewhere, has reported the discovery and preclinical validation of the first immunoPET tracer designed to target human CD68, the gold-standard biomarker that pathologists use to identify macrophages of all types. The work, published in the European Journal of Nuclear Medicine and Molecular Imaging, describes a synthetic antibody fragment, or Fab, that was engineered from scratch, radiolabeled with zirconium-89, and shown to bind specifically to human macrophages in living mice. If the approach translates to patients, it could allow clinicians to visualize the total macrophage burden of a tumor with a single whole-body scan, opening a window onto a cell type that strongly influences prognosis, immunotherapy resistance, and metastatic risk.

The rationale for targeting macrophages directly is rooted in their complicated role in cancer biology. Macrophages are versatile immune cells that maintain tissue homeostasis and defend against pathogens, but within the tumor microenvironment they are notoriously plastic and heterogeneous, adopting functions that range from pro-inflammatory to strongly immunosuppressive. Dense infiltration of tumors by CD68-positive macrophages has been linked in breast cancer to larger tumor size, higher histologic grade, hormone-receptor negativity, lymphovascular invasion, and reduced overall and progression-free survival. Existing imaging probes have struggled to capture this picture accurately. Some tracers, such as those targeting CD163 or CD206, preferentially label the immunosuppressive M2-like subset, while probes aimed at the translocator protein TSPO are not exclusive to macrophages and are affected by genetic polymorphisms that vary between individuals. A copper-64-labeled CD68-Fc fusion protein has been explored, but it targets scavenger receptors indirectly rather than macrophages themselves.

To fill this gap, the researchers turned to phage display, a technique that screens enormous libraries of antibody fragments for those that bind a chosen target. They used a synthetic Fab library, designated Library F, that was deliberately designed without lysine residues in its complementarity-determining regions, the loops that make contact with the antigen. Lysines were replaced with arginines, which preserve the basic character of the loops but lack the reactive nucleophilic amine that would otherwise be chemically modified during radiolabeling. Framework lysines elsewhere in the molecule were retained to allow controlled conjugation to a chelator without disturbing the antigen-binding surface. Screening this library against immobilized recombinant human CD68 yielded 14 unique sequences, of which five, named Fab3, Fab4, Fab7, Fab8, and Fab14, could be expressed successfully in bacterial cultures and purified on Protein A columns.

Characterization of the five candidates revealed important differences. Enzyme-linked immunosorbent assays showed that all five bound human CD68 with high significance compared with a bovine serum albumin control, but Fab3 also bound strongly to BSA itself and cross-reacted with mouse CD68, so it was eliminated. The remaining four Fabs bound human CD68 in a concentration-dependent manner with half-maximal effective concentrations ranging from 1.7 to 15 nanomolar, an affinity range well suited to immunoPET probes, and showed no detectable binding to BSA or mouse CD68. Production metrics then became the deciding factor. Fab4 and Fab7, despite their favorable affinities, expressed inconsistently and lost purity during storage, retaining only 71 and 75 percent monomer after 60 days at 4 degrees Celsius. Fab14 expressed at high yields but with purities of only 60 to 70 percent. Fab8, by contrast, delivered consistent yields of about 1.7 milligrams per liter, the highest purity of 80 to 90 percent straight from Protein A purification, and 96 percent monomer after two months of storage, making it the clear lead candidate.

Converting Fab8 into a PET tracer required attaching a chelator that could hold a positron-emitting radionuclide. The team selected p-SCN-Bn-deferoxamine, or DFO, and zirconium-89, a pairing with a well-established chemistry and a track record of clinical translation. After optimizing the conjugation stoichiometry to a 4:1 ratio of DFO to Fab, which minimized free chelator without inducing protein aggregation, the researchers radiolabeled the conjugate at a specific activity of 7.4 kilobecquerels per nanomole, achieving a radiochemical yield of 99 percent. Stability testing showed that the resulting tracer, [89Zr]Zr-DFO-Fab8, remained more than 95 percent intact monomer in phosphate-buffered saline over 48 hours, and about 80 percent intact in human serum at 37 degrees Celsius over the same period, with the remainder of the radioactivity associated with serum proteins.

In vitro experiments confirmed that the tracer behaved as intended on real cells. The human monocytic leukemia line THP-1 was differentiated into macrophages, which expressed human CD68 on roughly 25 percent of cells compared with about 4 percent of undifferentiated THP-1 cells, as measured by flow cytometry. When fixed macrophages were incubated with the tracer, binding reached about 10 percent of the added activity, five times higher than an irrelevant radiolabeled Fab control and roughly three times higher than in undifferentiated THP-1 cells. Importantly, neither the unlabeled conjugate nor the radiolabeled tracer compromised macrophage viability at the concentrations tested, with only a modest 19 percent decrease at the highest radiotracer concentration of 125 nanomolar, far above the estimated nanomolar circulating levels in the animal studies.

The first in vivo test used a cleverly simple model. Because Fab8 does not cross-react with mouse CD68, the team implanted subcutaneous plugs of Matrigel mixed with either recombinant human CD68 protein or BSA into opposite shoulders of immunocompetent BALB/c mice. PET/CT imaging at 1 and 4 hours after tracer injection showed significantly higher uptake in the human CD68 plugs than in the BSA control plugs, with plug-to-muscle ratios of 11 and 13 versus roughly 5 and 6, respectively. Voxel-level histogram analysis confirmed that the spatial distribution of tracer within the antigen plugs differed significantly from the controls over time. Biodistribution measurements correlated strongly with the imaging data, with a Pearson correlation coefficient of 0.97, and showed the expected renal dominance typical of antibody fragments of this size.

The more biologically demanding test came next. The researchers grew bilateral MDA-MB-231 triple-negative breast cancer xenografts, a tumor line that forms highly vascularized tumors and does not express CD68, in immunodeficient NSG mice. Once the tumors had developed vasculature, they injected differentiated THP-1 macrophages directly into one tumor and undifferentiated THP-1 cells into the other, then administered the tracer systemically. PET imaging at 1 and 6 hours revealed significantly greater uptake in the macrophage-containing tumors, with tumor-to-muscle ratios of about 5.2 and 5.9 compared with 2.7 and 3.6 in the control tumors. Immunofluorescence staining of the excised tumors provided the decisive validation: macrophage-injected tumors contained roughly 660 CD68-positive cells per area versus only 16 in the controls, and the CD68 density correlated with the PET standardized uptake values with a Pearson coefficient of 0.9663. This is the study’s central achievement, a direct, quantitative bridge between a noninvasive PET signal and the histopathological gold standard for pan-macrophage burden.

The authors are candid about the limitations that must be addressed before clinical translation. Kidney uptake was high, as expected for a 50-kilodalton Fab, and about 10 percent aggregation in serum may have contributed to elevated liver and spleen signal through sequestration by the reticuloendothelial system. Because the target uptake in the xenograft model was inherently low, background signal was comparatively prominent, and tumor-to-heart ratios below one suggested incomplete blood clearance at the early imaging timepoints used. Future work will explore polyethylene glycol modification to reduce renal retention, an approach already validated clinically by the approved drug certolizumab pegol, along with better animal models of human macrophage infiltration. Even so, the study establishes a complete pipeline, from phage display discovery through radiopharmaceutical engineering to correlated PET and pathology, for a first-generation human CD68 immunoPET tracer. Such a probe could eventually help select patients for macrophage-targeting therapies, monitor treatment response, and predict resistance to immunotherapy, all from a single noninvasive scan.

Subject of Research: Development of a zirconium-89-labeled anti-CD68 Fab immunoPET tracer for imaging human macrophages

Article Title: A FABulous approach to image human macrophages

Article References: Roohani, B., Vaughn Embs, A., Kumar, R., Lee, S., Katz, S. R., La Prairie, C., Nash, D., Dangarwala, M., Kluger, H. M., Klein, D. E., Zhang, P., Larimer, B. M., Nelson, B., & Marquez-Nostra, B. (2026). A FABulous approach to image human macrophages. European Journal of Nuclear Medicine and Molecular Imaging. https://doi.org/10.1007/s00259-026-08180-y

Image Credits: AI Generated

DOI: 10.1007/s00259-026-08180-y

Keywords: CD68, macrophages, immunoPET, phage display, Fab, zirconium-89, tumor-associated macrophages, PET imaging, triple-negative breast cancer, molecular imaging, radiopharmaceutical, tumor microenvironment