Development and evaluation of nanobody tracers for noninvasive nuclear imaging of the immune-checkpoint TIGIT

Introduction T cell Ig and ITIM domain receptor (TIGIT) is a next-generation immune checkpoint predominantly expressed on activated cells NK cells, exhibiting an unfavorable prognostic association with various malignancies. Despite the emergence of multiple TIGIT-blocking agents entering clinical trials, only fraction patients responded positively to anti-TIGIT therapy. Consequently, urgent demand arises for noninvasive techniques quantify monitor TIGIT expression, facilitating patient stratification enhancing therapeutic outcomes. Small antigen binding moieties such as nanobodies, are promising candidates tracer development. Methods We generated panel anti-human or anti-mouse nanobodies from immunized llamas. In addition, we designed single-chain variable fragment derived clinically tested monoclonal antibody Vibostolimab targeting TIGIT, assessed its performance alongside nanobodies. vitro characterization studies were performed, including ability affinity recombinant TIGIT. After Technetium-99m labeling, evaluated in vivo their detect expression using SPECT/CT imaging, followed by ex biodistribution analysis. Results Nine selected low sub-nanomolar affinities thermostable. A six-fold higher uptake TIGIT-overexpressing tumor was demonstrated one hour post- injection labeled compared irrelevant control nanobody. Though exhibited superior TIGIT-expressing peripheral blood mononuclear , behavior yielded lower tumor-to-background ratios at injection, indicating that better suited imaging than fragment. good affinity, high specificity on-target mice this setting, tumor- infiltrating lymphocytes within MC38 tumors remained elusive. This likely due levels model. Discussion The excellent rapid bearing TIGIT- overexpressing showed diagnostic potential noninvasively image tumor. These findings hold promise translation aid selection improve therapy response.