Objectives

This study presents the first head-to-head preclinical comparison of a Nanobody (Nb) and a minibody-like format ('Minabody', Mnb) derived from the same anti-human TIGIT Nb for Positron Emission Tomography (PET) imaging.

• It demonstrates that the 64Cu-labeled Nb PET tracer outperforms the 64Cu-labeled Mnb format, showing faster, more specific tumor targeting and lower background signals in a mouse model.
• It compares three PET radionuclides (Copper-64 (64Cu), Gallium-68 (68Ga), Fluorine-18 (18F)) for Nb imaging, concluding that 64Cu provides superior tumor uptake and image contrast/resolution for preclinical Nb imaging.

Methodology

The study employed protein engineering and radiochemistry methods:

• An anti-hTIGIT Nb was reformatted into a bivalent Mnb (Nb-CH3 fusion, ~55.7 kDa).
• Both Nb and Mnb were conjugated with the chelator p-SCN-Bn-NOTA (NOTA) via lysine residues.
• In vitro binding affinities (KD) were determined using Surface Plasmon Resonance (SPR) against recombinant hTIGIT protein and flow cytometry on hTIGIT-expressing TC-1 cells.
• Radiolabeling: Nb and Mnb were labeled with 64Cu via NOTA chelation. Nb was also labeled with 68Ga (via NOTA) and 18F (using the prosthetic group N-succinimidyl-4-[18F]fluorobenzoate, [18F]SFB).
• Radiochemical purity (RCP) was assessed by radio-SEC and radio-iTLC. Stability was tested in human serum and via EDTA challenge.
• Radioligand binding assays confirmed binding post-labeling.
• In vivo evaluation: PET/CT imaging and ex vivo biodistribution were performed in immunodeficient mice bearing hTIGIT-positive and TIGIT-negative TC-1 tumors at various time points post-injection.
• Statistical analysis: Unpaired t-tests and One-way ANOVA were used to determine significance.

Results

• Binding Affinity: Mnb showed higher affinity (KD=0.33 nM protein, 0.31 nM cells) than Nb (KD=0.95 nM protein, 2.70 nM cells), likely due to avidity. NOTA conjugation and radiolabeling did not significantly impair binding (e.g., [64Cu]Cu-NOTA-Nb KD=1.32 nM, [64Cu]Cu-NOTA-Mnb KD=0.65 nM on protein).
• In Vivo Nb vs. Mnb (64Cu): [64Cu]Cu-NOTA-Nb showed specific uptake in hTIGIT+ tumors from 1h post-injection (p.i.), lasting up to 48h. [64Cu]Cu-NOTA-Mnb showed specific uptake only from 8h p.i., with high initial blood pool signal and significant liver uptake. Nb achieved higher target-to-background ratios; peak hTIGIT+ tumor-to-negative tumor ratio was 11.45±0.99 at 8h for Nb vs. 1.75±0.16 at 48h for Mnb. Peak tumor-to-blood ratio was 23.05±4.22 at 8h for Nb vs. 13.89±1.04 at 48h for Mnb.
• Radionuclide Comparison for Nb (1h p.i.): All tracers showed specific hTIGIT+ tumor uptake. [64Cu]Cu-NOTA-Nb showed highest uptake (3.26±0.15 %IA/g) and best image resolution. [68Ga]Ga-NOTA-Nb had comparable uptake (3.13±1.29 %IA/g) but lower resolution. [18F]FB-hTIGIT-Nb had lower uptake (1.68±0.13 %IA/g) but better resolution than 68Ga. 18F-Nb showed lower renal retention (80.60±17.51 %IA/g) compared to 64Cu-Nb (118.07±19.84 %IA/g) and 68Ga-Nb (166.99±14.89 %IA/g).

Discussions

This study provides valuable insights by directly comparing Nb and Mnb formats for PET imaging, a comparison previously lacking.

• Limitation: The use of an overexpression tumor model (TC-1 hTIGIT) might not fully represent the challenges of imaging endogenous, potentially low-level TIGIT expression in clinical scenarios. The superior performance of the Nb might be less pronounced, or the Mnb's avidity advantage might become more relevant, in low-antigen density settings.
• Unclear Mnb Off-Target Uptake: The significant uptake of [64Cu]Cu-NOTA-Mnb in TIGIT-negative tumors and the liver warrants further investigation. While the authors suggest radionuclide-dependent effects observed in other minibody studies and discount FcRn binding by the CH3 dimer, the exact mechanism remains unclear and limits the Mnb format's utility. Exploring different chelators or radionuclides (like 124I, as mentioned) for the Mnb could clarify this.
• Alternative Formats: Investigating a bivalent Nb format (~30 kDa) could be informative, potentially combining the avidity benefits of the Mnb with the more favorable pharmacokinetics and lower off-target binding of the Nb.
• Radionuclide Choice: The conclusion favoring 64Cu for Nb imaging is well-supported by the preclinical data (resolution, uptake, imaging window). However, the practical limitations of 64Cu (availability, cost) compared to 68Ga (generator) and 18F (widespread cyclotron production) should be acknowledged for clinical translation. The lower uptake with the [18F]SFB-labeled Nb might warrant exploring alternative 18F-labeling strategies like Al18F-RESCA, despite potential yield/purity trade-offs, to confirm if the observation is method-dependent.

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Reference: Optimizing antibody PET imaging a comparative preclinical analysis of nanobody and minibody like PET tracers