Making the Immune Status Visible – Generation and Characterization of Nanobodies Targeting Immune Markers for in vivo Diagnostics

Abstract

Immunotherapies offer promising treatment options for cancer patients, yet responsiveness is highly dependent on the individual immune status. Thus, profiling of immune activity in the tumor microenvironment (TME) is of increasing importance to guide therapeutic decisions through response prediction and efficacy monitoring. In this context, Nanobodies (Nbs), with their small size, high stability and solubility have proven to be promising building blocks for the development of molecular imaging probes for non-invasive immune profiling. In this work, novel Nbs were developed against immune regulatory molecules with emerging relevance as predictive biomarkers, namely the T cell activation markers hCD69 and hOX40, the inhibitory checkpoints hVTCN1 and hVISTA and the myeloid-specific receptor hSIRPα. For this purpose, Nbs were selected from immune or synthetic libraries and characterized with respect to their specificity, affinity, stability and functionality. Based on these results, we chose the lead candidates hCD69-Sb4, hOX40-Nb O18, hVTCN1-Nbs VT1 and VT57, hVISTA-Nbs BV18 and BV36 as well as hSIRPα-Nb S36 for further development of imaging probes. These Nbs were functionalized using different strategies: i) cysteine-maleimide conjugation, ii) SortaseA (SrtA)-mediated transpeptidation and iii) lysine-to-arginine substitution. All three approaches enabled site-specific and flexible fluorescence- or radioisotope-conjugation without impairing Nb performance. Furthermore, optical imaging (OI) of fluorescently labeled hOX40-Nb O18AF647 and positron emission tomography (PET) of radiolabeled 64Cu-hSIRPα-S36K>R Nb demonstrated successful specificity and functionality as in vivo imaging probes. Considering the importance of T cell activation dynamics as well as the presence of checkpoint molecules and pro-tumorigenic myeloid cells in the TME for immunotherapy outcome, we propose that imaging probes based on the herein developed Nbs will contribute to monitor immune dynamics and hold translational potential for patient stratification in the context of personalized immuno-oncology.

Die Dissertation ist gesperrt bis zum 22. Oktober 2027 !