The development of radiolabeled nanobodies against the next-generation immune checkpoint TIGIT for non-invasive immune phenotyping of tumors

IntroductionImmune checkpoint therapy (ICT) has recently changed the treatment landscape of multiple cancers. However, the highly dynamic tumor environment has resulted in intrinsic and acquired resistance against current ICTs in many patients. Therefore, ICTs against next-generation immune checkpoints, including TIGIT, are being developed. To better predict the treatment outcome of these ICTs, patient stratification using reliable non-invasive diagnostic methods are required. Small antigen binding moieties such as nanobodies (Nbs), are promising candidates for such tracer development.
MethodsLlamas were immunized with recombinant TIGIT proteins, phage-display libraries were constructed and screened for TIGIT specific Nbs. Binding to TIGIT was confirmed by flow cytometry (FC) and ELISA. The affinity of the produced and purified Nbs was determined using surface plasmon resonance (SPR) and FC. Thermostability was assessed by thermofluor assays. Radiochemical purity (RCP) after 99mTc labeling was assessed by iTLC. The 99mTc-Nbs were injected intravenously (i.v.) in naive C57BL/6 mice, in TIGIT overexpressing (TIGIT+) - or wild type (WT) - TC-1 tumor xenografted nude mice and in mice lacking mouse TIGIT. SPECT/CT imaging was performed 1h post-injection (p.i.), followed by ex vivo biodistribution analysis by gamma counting.
Results/DiscussionWe obtained 154 different clones after ELISA screening. Three Nbs with sub-nanomolar binding affinity to mouse but not human TIGIT were produced recombinantly and demonstrated binding to TIGIT expressing CD3+ splenocytes (Fig. 1a,b). The Nbs were evaluated for their potential as a non-invasive imaging tracer upon 99mTc labeling. All 99mTc-Nbs exhibited >98% RCP. Biodistribution data of naive C57BL/6 mice showed significant higher uptake of 99mTc-Nbs in different organs (e.g., spleen, lymph nodes, intestines) compared to mice lacking mouse TIGIT (Fig. 2a). Expected high uptake is seen in the kidneys and the bladder due to renal clearance. In tumor bearing mice, uptake (%IA/g ± SD) of Nb16988 was significantly higher in the TIGIT+ tumor (4.317 ± 1.021) compared to the WT tumor (0.709 ± 0.237) and to the uptake of a control Nb R3B23 in both tumors (0.282 ± 0.065) (Fig. 2b,c). All Nbs showed significant higher TIGIT+-to-WT-tumor ratio compared to the control Nb (Fig. 2d).
ConclusionsThis is the first report to non-invasively detect mouse TIGIT in vivo upon same-day nuclear imaging with radiolabeled Nbs, with Nb16988 as lead supported by the high signal-to-noise ratios. Ongoing experiments with Nb 68Ga or 18F labeling and the development of a human TIGIT specific PET tracer hold promise toward phenotyping tumors in patients who would be eligible for TIGIT-targeted therapies.
AcknowledgementThe authors would like to thank Petra Roman and Elsy Vaeremans for the production of the plasmids and Jan De Jonge for his support with the purification of the Nbs.