Radiolabeled sdAbs for Molecular Imaging

Single domain antibodies (sdAbs) fulfill the requisites of an ideal probe for successful molecular imaging. Radiolabeled sdAbs are already used in several contexts, such as to evaluate breast cancer diagnosis in a preclinical setting, to discriminate between moderate and high epidermal growth factor receptor expression for the improved prognosis of cancer therapy, and to monitor the status of inflammatory responses by visualizing dendritic cells. Creative Biolabs provides customized radiolabeled sdAb development services for molecular imaging. We will provide the solution according to your detailed analysis method and desired label.

Molecular Imaging with Radiolabeled sdAbs

Single-photon emission computed tomography (SPECT) is based on γ-rays and sdAbs are linked to radionuclides such as ^99mTc, ¹⁷⁷Lu, ¹²³I and ¹¹¹In. These γ-rays are recorded by the detectors of a dedicated γ-camera or SPECT instrument, which can be converted into an image upon signal processing to pinpoint the localization of the radiolabeled sdAb. When using β-emitting radioisotopes such as ¹³¹I and ¹⁷⁷Lu, a therapeutic effect can also be pursued by their ionization and DNA damaging activity. Such a radionuclide-based construct enabling both imaging and therapeutic use is termed theranostic.

Table.1 Currently approved targeted radionuclide therapies in oncology. (D' Huyvetter, 2014)

On the other hand, the positron-emitting radioisotopes ¹⁸F, ⁶⁸Ga, ¹²⁴I or ⁸⁹Zr have normally used for positron emission tomography (PET) purposes. Among them, the PET isotopes ¹⁸F and ⁶⁸Ga are particularly suited to imaging with sdAbs due to their short half-lives (68 and 110 min, respectively), which match up well with the biological half-life of sdAbs. PET has greater advantages over SPECT concerning sensitivity and resolution. At present, most of the literature reports on radionuclide-based imaging with sdAbs have used ^99mTc for SPECT imaging. The widespread interest in the use of ^99mTc is primarily due to its excellent nuclear decay characteristics, viable coordination chemistry for radiolabeling different biomolecules and convenient availability from cost-effective ⁹⁹Mo/^99mTc generators. Moreover, due to the presence of the hexahistidine tag on the sdAb, it can easily be radiolabeled with ^99mTc(CO)3 without any chemical modification of the protein.

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Radiolabeled sdAbs hold great promise to play an important role in the development and implementation of personalized targeted therapy. There are apparent advantages of the use of radiolabeled sdAbs as companion diagnostics:

• Radiolabeled counterpart reflects not only the level of expression of a target in tumors but also its accessibility.
• The safety of lead sdAb is normally well-known, which reduces the number of necessary tests for their imaging tracer derivatives.
• Labeling with different radioisotopes is well-established and straightforward.

Creative Biolabs provides customized radiolabeled sdAbs for worldwide clients. Our custom products meet the following requirements:

• Tailored products according to requirements and testing platform
• Remain stable in vivo
• Accumulate specifically and in sufficient amounts into the tissue of interest
• An appropriately fast imaging time point and sufficiently extended imaging window should be attained

Radio-immunodetection is a powerful method for evaluation of pharmacokinetics and target engagement in development of new biotherapeutics. We trust that radiolabeled sdAbs will have future potential as theranostic tools. If you are interested in our customized labeled nano-tracer development services, please directly contact us for more information.

Published Data

1. PET/CT Imaging of Protumorigenic Macrophages with ⁶⁸Ga-Labeled anti-MMR-sdAb

Fig.1 The in vivo biodistribution of [⁶⁸Ga]Ga-NOTA-anti-MMR-sdAb.^2,4

This study developed a cross-reactive anti-macrophage mannose receptor (MMR) sdAb, labeled with ⁶⁸Ga, to form [⁶⁸Ga]Ga-NOTA-anti-MMR-sdAb. The tracer was produced with a radiochemical purity of 99 ± 1%, radiochemical yield of 76 ± 2%, and an apparent molar activity of 57 ± 11 GBq/μmol. In vivo biodistribution demonstrated rapid renal clearance and significant accumulation in MMR-expressing organs and tumors, yielding tumor-to-blood and tumor-to-muscle ratios of 6.80 ± 0.62 and 5.47 ± 1.82, respectively. The estimated effective dose was 0.027 mSv/MBq for males and 0.034 mSv/MBq for females, suggesting a radiation dose of 5.0 and 6.3 mSv for a 185 MBq dose. Toxicity studies revealed no adverse effects. This tracer shows great promise for PET/CT imaging of protumorigenic macrophages in the tumor microenvironment.

2. PET Imaging of Fibroblast Activation Protein-α or Folate Receptor-α Using [¹⁸F]FB-Labeled Single Domain Antibodies

Fig.2 Synthesis of [¹⁸F]FB-sdAb.^3,4

This study developed a semi-automated radiofluorination strategy for sdAbs targeting fibroblast activation protein (FAP)-α and folate receptor (FR)-α, using N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). [¹⁸F]SFB was synthesized via a fully automated three-step, one-pot reaction, achieving a radiochemical purity (RCP) >90% and a decay-corrected radiochemical yield (RCY) of 44 ± 4% in 54 minutes. The subsequent manual conjugation with anti-FAP-α and anti-FR-α sdAbs resulted in [¹⁸F]FB-sdAbs with RCP >95%, end-of-synthesis activity >600 MBq, and molar activity >10 GBq/μmol. The RCY d.c. were 9% for [¹⁸F]FB-anti-FR-α-sdAb and 5 ± 2% for [¹⁸F]FB-anti-FAP-α-sdAb. This semi-automated approach provides a generic method for radiofluorination of sdAbs. The radiofluorinated sdAbs exhibited favorable biodistribution, showing potential as PET tracers for FAP-α and FR-α imaging.

References

1. D’Huyvetter, Matthias, et al. "Radiolabeled nanobodies as theranostic tools in targeted radionuclide therapy of cancer." Expert opinion on drug delivery 11.12 (2014): 1939-1954. Distributed under Open Access license CC BY 3.0, without modification.
2. Xavier, Catarina, et al. "Clinical translation of [⁶⁸ Ga] Ga-NOTA-anti-MMR-sdAb for PET/CT imaging of protumorigenic macrophages." Molecular imaging and biology 21 (2019): 898-906.
3. Dierick, Herlinde, et al. "Generic semi-automated radiofluorination strategy for single domain antibodies:[¹⁸F] FB-labelled single domain antibodies for PET imaging of fibroblast activation protein-α or folate receptor-α overexpression in cancer." EJNMMI Radiopharmacy and Chemistry 9.1 (2024): 54.
4. Distributed under Open Access license CC BY 4.0, without modification.

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