Novel ImmunoPET Tracers for Noninvasive Imaging of CD4+ Cell Distribution in Cancer Mouse Models

Abstract

Increasing evidence highlights the crucial role of CD4⁺ T cells in orchestrating anti-tumor immunity, underscoring their potential as biomarkers for disease classification and monitoring responses to immunotherapy. However, no established noninvasive method currently exists to quantify CD4⁺ cell infiltration in diseased tissue. Molecular imaging of CD4⁺ cells could provide critical insights into their whole-body distribution and migration dynamics during treatment, thereby supporting personalized therapeutic strategies. The aim of this thesis was to validate novel scFv-CH3 minibody (Mb)-based and VHH single-domain antibody nanobody (Nb)-based PET tracers for noninvasive in vivo imaging of human CD4⁺ cells. With a focus on clinical translation, we evaluated their capacity to visualize, spatially localize, and sensitively detect clinically relevant changes in endogenous CD4⁺ immune cell infiltrates in preclinical cancer models. The parallel development of radiolabeled murine and human CD4-specific minibodies (⁸⁹Zr-mCD4-Mb and ⁸⁹Zr-hCD4-Mb, respectively) enabled noninvasive whole-body imaging of endogenous CD4⁺ cell distributions in models of cancer immunotherapy, including human CD4 receptor knock-in and wild-type mice. These tracers allowed monitoring of treatment-induced immune responses and prediction of therapeutic outcome. In addition, a newly developed ⁶⁴Cu-labeled CD4-specific nanobody (⁶⁴Cu-CD4-Nb1) demonstrated highly sensitive detection and precise spatial localization of subtle alterations in CD4⁺ cell densities across multiple tumor models. The distinct molecular weights of the Mb- and Nb-based probes resulted in differing pharmacokinetic profiles, rendering them suitable for complementary imaging applications. Importantly, neither probe format affected T-cell proliferation or function. Together, these findings identify CD4-targeted Mb and Nb PET tracers as promising candidates for clinical translation across a broad range of tumors and cancer immunotherapy settings.