In-depth cross-validation of human and mouse CD4-specific minibodies for noninvasive PET imaging of CD4+ cells and response prediction to cancer immunotherapy

ABSTRACT

Increasing evidence emphasizes the pivotal role of CD4+ T cells in orchestrating cancer immunity. Noninvasive
in vivo imaging of the temporal dynamics of CD4+ T cells and their distribution patterns might provide novel
insights into their effector and regulator cell functions during cancer immunotherapy (CIT).
Methods: We conducted a comparative analysis of 89Zr-labeled anti-mouse (m) and anti-human (h)
CD4-targeting minibodies (Mbs) for in vivo positron emission tomography (PET)/magnetic resonance imaging
(MRI) of CD4+ T cells in human xenografts, syngeneic tumor-bearing wild-type (WT), and human CD4+
knock-in (hCD4-KI) mouse models.
Results: Both 89Zr-CD4-Mbs yielded high radiolabeling efficiencies of >90%, immunoreactivities of >70%, and
specific in vitro binding to their target antigens. The specificity of in vivo targeting of 89Zr-hCD4-Mb was
confirmed by PET/MRI, revealing ~4-fold greater 89Zr-hCD4-Mb uptake in subcutaneous hCD4+ hematopoietic
peripheral blood acute lymphoblastic leukemia tumors (HPB-ALL) than in solid hCD4- diffuse histiocytic
lymphomas (DHL) and 89Zr-mCD4-Mb uptake in hCD4+ HPB-ALL tumors. In a comparative cross-validation
study in anti-programmed death ligand (αPD-L1)/anti-4-1BB-treated orthotopic PyMT mammary
carcinoma-bearing hCD4-KI and WT mice, we detected 2- to 3-fold enhanced species-specific 89Zr-hCD4-Mb
or 89Zr-mCD4-Mb uptake within CD4+ cell-enriched secondary lymphatic organs (lymph nodes and spleens).
The 89Zr-hCD4-Mb uptake in the PyMT tumors was more pronounced in hCD4-KI mice compared to the WT
control littermates. Most importantly, MC38 adenocarcinoma-bearing mice treated with a combination of
αPD-L1 and anti-lymphocyte-activation gene 3 (αLag-3) antibodies exhibited ~1.4-fold higher 89Zr-mCD4-Mb
uptake than mice that were not responsive to therapy or sham-treated mice.
Conclusion: CD4 PET/MRI enabled monitoring of the CD4+ cell distribution in secondary lymphatic organs
and the tumor microenvironment, capable of predicting sensitivity to CIT. Our imaging approach will provide
deeper insights into the underlying molecular mechanisms of CD4-directed cancer immunotherapies in
preclinical mouse models and is applicable for clinical translation.