Noninvasive nuclear imaging of the immune checkpoint TIGIT using different nanobody-scaffolds and radionuclides

Introduction
Immune checkpoint therapy (ICT) has revolutionized cancer treatment, yet therapy resistance persists due to the dynamic tumor environment. ICTs targeting next-generation immune checkpoints, such as TIGIT, are being explored. However, reliable noninvasive diagnostic tracers for whole-body PET imaging of these checkpoints are needed for patient stratification and therapy follow-up. Radiolabeled small antibody fragments such as nanobodies (Nbs) and their engineered variants, emerge as promising candidates for such tracer development.
Methods
An anti-human TIGIT Nb was evaluated previously (1). A ‘Minabody (Mnb)’ derived from this Nb, was produced recombinantly by fusing it genetically to the CH3 domain of mIgG2a (Figure 1A). Affinities of the Nb and Mnb were assessed via surface plasmon resonance. Radiolabeling was performed upon conjugation with [18F]SFB as a prosthetic group, or with 64Cu after NOTA-conjugation. The radiotracers were injected intravenously in nude mice bearing wild type (WT) and human TIGIT-overexpressing TC-1 tumors in flanks. PET/CT imaging and ex vivo biodistribution analysis was conducted: at 1h post-injection with [18F]FB- hTIGIT-Nb (9.8±0.7 GBq/μmol, n=4) or [64Cu]Cu-NOTA-hTIGIT-Nb (20.4±1.2 GBq/μmol, n=4); at 1h, 4h, 8h, 24h and 48h post-injection with [64Cu]Cu-NOTA-hTIGIT-Mnb (28.1±1.1 GBq/μmol, n=3).
Results/Discussion
Mnb displays higher affinity to human TIGIT than Nb due to avidity effects (0.235 nM vs. 1.947 nM) (Figure 1B). PET/CT imaging showed specific tumor targeting and low background signal (except kidneys) with both [18F]FB-hTIGIT-Nb and [64Cu]Cu-NOTA-hTIGIT-Nb (Figure 2A, 2B). Uptake (%IA/g±SD) was significantly higher in hTIGIT overexpressing tumors (1.090±0.093 and 3.256±0.149) compared to WT tumors (0.361±0.154, 0.329±0.130). Notably, [64Cu]Cu-NOTA-hTIGIT-Nb demonstrated nearly threefold higher specific uptake than with [18F]FB-hTIGIT-Nb, albeit with significant higher kidney retention (166.996±14.886 vs. 13.621±1.743).
[64Cu]Cu-NOTA-hTIGIT-Mnb exhibit high blood accumulation with no significant early-time differences between uptake in hTIGIT overexpressing and WT tumors (1h, 4h) (Figure 2C). Although uptake is significant in hTIGIT overexpressing tumors at later time points (8h, 24h, 48h), higher background (uptake
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Abstract #794 EMIM 2024 Late LB-09a - Preclinical Studies in Cancer Therapy & Diagnosis
in WT tumor and liver) persisted compared to [64Cu]Cu-NOTA-hTIGIT-Nb.
Conclusion
While both 18F- and 64Cu-labeled anti-hTIGIT Nb could to detect hTIGIT in tumor-bearing mice, 64Cu-labeled Nb displayed superior specific uptake. Despite Mnb’s higher affinity for hTIGIT than Nb, 64Cu-labeled Nb outperformed Mnb for same-day nuclear imaging, allowing noninvasive detection of human TIGIT as early as 1h post-injection. Mnb is currently being evaluated for its potential as a therapeutic compound.
Novelty
This is the first study reporting anti-hTIGIT Nb and Nb-based Mnb for PET/CT imaging with a side-by-side comparison of different PET radionuclides.
Impact
The translation of this Nb into a PET tracer hold promises toward phenotyping tumors in patients who would be eligible for TIGIT-targeted therapies.
Disclosure
I or one of my co-authors have the following financial interest or relationship(s) to disclose regarding the subject matter of this presentation: Some of the co-authors hold patent applications relating to the use of nanobodies for in vivo imaging and therapy. Some of the co-authors are shareholders of Abscint NV and Precirix NV.
Acknowledgment
This research is funded by FWO-SB 1S61021N and 1S61023N.
The Molecubes β-CUBE PET/CT system was funded via FWO-Hercules grant I005622N.