Development and evaluation of an 18F-labeled nanobody to target SARS-CoV-2's spike protein

Abstract

COVID-19, caused by the SARS-CoV-2 virus, has become a global pandemic that is still present after more than two years. COVID-19 is mainly known as a respiratory disease that can cause long-term consequences referred to as long COVID. Molecular imaging of SARS-CoV-2 in COVID-19 patients would be a powerful tool for studying the pathological mechanisms and viral load in different organs, providing insights into the disease and the origin of long-term consequences and assessing the effectiveness of potential COVID-19 treatments. Current diagnostic methods used in the clinic do not allow direct imaging of SARS-CoV-2. In this work, a nanobody (NB) - a small, engineered protein derived from alpacas - and an Fc-fused NB which selectively target the SARS-CoV-2 Spike protein were developed as imaging agents for positron emission tomography (PET). We used the tetrazine ligation to ¹⁸F-label the NB under mild conditions once the NBs were successfully modified with trans-cyclooctenes (TCOs). We confirmed binding to the Spike protein by SDS-PAGE. Dynamic PET scans in rats showed excretion through the liver for both constructs. Future work will evaluate in vivo binding to the Spike protein with our radioligands.

Keywords: COVID-19; PET; SARS-CoV-2; alpaca; biodistribution; nanobodies; tetrazine ligation.

Figure 1

(A) study outline. The protein is first modified with TCO and radiolabeled with an ¹⁸F-Tz. The radiolabeled protein is injected intraveneously (i.v.) into rats and evaluated by PET. (B) Labeling of TCO-W25 with [¹⁸F]Tz1 and [¹⁸F]Tz2 using the tetrazine ligation (C) Labeling of TCO-W25Fc with [¹⁸F]Tz1.

Figure 2

W25 binding to spike in cells and TCO modifications of W25 using TCO-PEG₄-NHS and TCO-NHS ester. (A) H1299 cells were transfected with GFP-Spike (gold) and after 24 h the cells were image in vivo in a Celldiscoverer 7 automatic microscope. (B) H1299 cells transfected with GFP-Spike (Green) of SARS-CoV-2 were incubated 1 h with 1 μg/ml of myc-tagged W25 directly after the cells were washed, fixed and immunostained (myc-W25 in red), nucleus were stained with DAPI to unveil transfected cells (blue) scale bars are 20 um. (C) TCO modifications of W25 using TCO-PEG₄-NHS and TCO-NHS ester. A maximum of 2.59 TCOs/NB was obtained using TCO-PEG₄-NHS ester, and a maximum of 1.53 TCO/NB was obtained with TCO-NHS ester.

Figure 3

(A) FPLC analytical GF traces of hexapro (spike protein) and hexapro incubated with TCO-W25 and TCO-W25Fc. Samples: ∼10 μM Hexapro + ∼30 μM W25 of ∼10 μM W25Fc, in a volume of 100 μl incubation on ice. SEC: Superdex 200 increase 10/300 GL. Buffer: 10 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.5 ml/min, 1 ml fractions (B) SDS-PAGE results of TCO-W25 and TCO-W25Fc incubated with Hexapro. In vivo PET in rats.

Figure 4

Time activity curves (TACs) and examples of PET images of 90 min PET scans in rats with (A) [¹⁸F]1W25, (B) [¹⁸F]2W25 and (C) [¹⁸F]1W25Fc. Excretion through the liver was observed for all three compounds.