Background CD4+ T cells play essential roles in the development and regulation of autoimmune diseases and cancer. Recently, we have developed single-domain antibodies that bind with high affinity to human CD4 receptor without affecting T cell function. The lead candidate was radiolabeled with NODAGA and Cu-64 (64Cu-αCD4-sdAb) for PET imaging. The aim of this study was to evaluate the applicability of our 64Cu-αCD4-sdAb to visualize and differentiate minimal changes in the CD4+ immune infiltrate during cancer immunotherapy and in inflamed tissue.
Methods 64Cu-αCD4-sdAb was administered intravenously in two orthotopic cancer models and a cutaneous Delayed-Type-Hypersensitivity-Reaction (cDTHR) model. In the first approach, PyMT breast cancer and B16 melanoma cells were implanted in C57BL/6J human-CD4 knock-in (hCD4KI) or wildtype (WT) mice. A subgroup of animals was additionally treated with therapeutic αPD1/α41BB antibodies. To induce cDTHR, hCD4KI mice were sensitized at the abdomen and repetitively challenged at one ear with trinitrochlorobenzene (TNCB). 64Cu-αCD4-sdAb PET/MRI was conducted 24 h after the 5th TNCB-ear challenge and organs of interest were harvested for ex vivo biodistribution, immunohistochemistry or mass cytometry (CyTOF) analyses immediately after the last imaging time-point.
Results Dynamic 64Cu-αCD4-sdAb PET/MRI of PyMT tumors over 6 h revealed the best differentiation between hCD4KI and WT mice at 1.5 h and 3 h imaging time-point post tracer injection. In line with ex vivo analyses of intratumoral CD4+ cell densities, αPD1/α41BB-treated PyMT tumors of hCD4KI mice showed significantly higher 64Cu-αCD4-sdAb uptake (0.46±0.01%ID/ml) compared to untreated littermates (0.30±0.03%ID/ml) and PyMT tumors of WT mice (0.21±0.02%ID/ml). In contrast, B16 tumors with histologically almost absent CD4+ cell infiltrates yielded an uptake of 0.23±0.02%ID/ml similar to WT mice. In the cDTHR model, the 64Cu-αCD4-sdAb uptake in the inflamed ears was 3-fold higher compared to control ears. Importantly, a control sdAb tracer did not reveal such differences in the inflamed and non-inflamed ears, excluding relevant non-specific perfusion effects. Immunohistochemistry revealed a rare number of CD4+ cells in the inflamed tissues being detectable by out extremely sensitive PET/MRI approach.
Conclusions Due to the important role of CD4+ cells in health and disease, precise non-invasive imaging approaches to visualize and monitor CD4+ cells in vivo are urgently needed. Our newly developed 64Cu-αCD4-sdAb PET tracer is able to detect small amounts of CD4+-cell infiltrates in both cancer and cDTHR models, pointing out its potential to serve as a versatile probe for a broad range of T cell-associated diseases.
Ethics Approval All animal experiments were carried out in accordance with the German Animal Welfare Act and with consent of regulatory authorities (Regierungspräsidium Tübingen).
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