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Journey in Light & Shadows of Nanobodies - the Application of Nanobodies in Medical Imaging

In recent years, innovations in medical treatments have brought benefits to many patients.
Life expectancy has increased significantly for patients with different forms of locally
advanced or metastatic cancer, such as melanoma, lung cancer, and many hematological

Despite these encouraging results, a large number of patients still
experience severe toxicities during treatment or are unresponsive to many therapies.
A deeper understanding of the cellular and molecular mechanisms behind this phenomenon
will help us develop more effective therapies.

Non-invasive immune imaging technology can provide important and comprehensive
information through precise targeting, helping to analyze the unique molecular patterns
of each patient. It is a key component of precision medicine, playing an important role in 
staging, typing and providing personailized treatment approaches to patients.1
Nanobody-based immunoimaging tracers have strong specificity, fast uptake,
and high tumor/background ratios. This blog will introduce the advantages of nanobodies
in medical imaging and the clinical and preclinical research of nanobody probes
developed for some representative targets (Figure 1).

Figure 1. Schematic diagram of nanobodies used in human PET imaging.

Advantages of Nanobodies in Medical Imaging

Heavy chain antibodies (HCAbs) naturally exist in cartilaginous fish such as camelids and
nurse sharks. Their variable regions (VHH) are nanobodies. VHHs are the smallest
antigen-binding units in nature and can be obtained through immune libraries, natural
libraries or synthetic libraries. Their molecular weight is about 12-15 kDa only and their
diameter is < 4 nm. They are one-tenth the size of traditional antibodies while maintaining
strong antigen recognition ability.

The unique physical and chemical properties of VHHs make them stand out in the field of
immunoimaging. Its main advantages are (Figure 2):

(1) VHHs are highly similar to human type 3 VH sequences, are less likely to cause drug
resistance reactions in the human body, and have low immunogenicity;
(2) VHHs have small molecular weight, deep tissue penetration, and high blood clearance rate;
(3) short half-life, can achieve a higher tumor/background ratio, and high-contrast images
can be obtained 1 hour after injection; commonly using labels such as 18F, 68Ga and 99mTc;
(4) The paratope of VHHs has a convex structure and the CDR3 loop is long, which can
bind to epitopes that are difficult for traditional antibodies to bind;
(5) VHHs have high thermal stability and can withstand harsh environments;
(6) It is easy to modify and transform;
(7) It can be produced by microbial fermentation, with high yield and small batch-to-batch variation.

Figure 2. Advantages of nanobodies used in immunoimaging

Examples of Applications of Nanobodies in Tumor Imaging

1. Tumor marker HER2 imaging

According to the expression of different proteins on the surface of cancer cells, breast
cancer can be divided into four categories: estrogen positive, progesterone positive,
human epidermal growth factor receptor 2 (HER2) positive and triple negative.

HER2-positive breast cancer accounts for 20% to 30% of the total incidence of breast cancer.
The expression level of HER2 can reflect the development and treatment of this type of
breast cancer. However, during the process of disease development and treatment,
the expression level of HER2 is constantly changing, so tracking the expression level
of HER2 requires continuous biopsy. However, breast cancer is highly heterogeneous,
and biopsy results cannot fully reflect the HER2 expression of primary tumors and metastases.
Tissue biopsy may also increase the probability of distant metastasis of breast cancer.
Therefore, non-invasive detection of HER2 is crucial2.

Molecular imaging methods based on nanobodies are a rising star in imaging
HER2-positive breast cancer. They have the advantages of non-invasiveness,
high specificity, good tissue penetration, high tumor/background ratio, and can effectively
avoid false positive and false negative results. They can realize the detection of HER2
Accurate staging and assessment of tumor heterogeneity in positive breast cancer2.

Figure 3. PET/CT image (upper panel) and PET image (lower panel) showing uptake of 68-GaNOTA-Anti-HER2 VHH1 in primary
breast cancer lesions (arrows). (A) Patient 14 had the highest tracer uptake (SUV mean, 11.8). (B) Patient 15 shows moderate tracer
uptake, still with a high tumor-to-background ratio (SUV mean, 4.9). (C) Imaging of patient 6 shows no uptake (SUV mean, 0.9),
and the tumor area on the CT image is indicated by an arrow.

The HER2 nanobody tracer 68-GaNOTA-Anti-HER2 VHH1 has achieved encouraging
results in phase I clinical trials. The accumulation level of 68-GaNOTA-Anti-HER2
VHH1 in tumors significantly exceeded the background value, and in High accumulation
in HER2-positive metastases. Therefore, this tracer successfully entered Phase II
clinical research, with the purpose of uating the uptake of this tracer by brain
metastases in breast cancer patients (Figure 3) (NCT03331601)3.

Based on the excellent performance of 68-GaNOTA-Anti-HER2 VHH1, Precirix has
further developed the [131I]-SGMIB Anti-HER2 VHH1 tracer, which is currently undergoing
clinical phase I trials to uate the safety, tolerability and effective dose (NCT02683083)4.

In addition, another nanobody imaging agent 99mTc-NM-02 is undergoing phase I
clinical research (NCT04040686)5. 10 breast cancer patients were injected with
3-12 MBq/kg 99mTc-NM-02. No adverse reactions were reported, and 99mTc -
The uptake of NM-02 is positively correlated with the expression of HER-2.

Recently, 99mTc-MIRC208, developed by the School of Clinical Oncology at Peking University,
is also undergoing clinical trials with a total of 200 subjects, aiming to study the feasibility
and specificity of the nanobody tracer 99mTc-MIRC208 for imaging HER2-positive
patients (NCT04591652) .

2. Immune detection point PD-1PD-L1 imaging

Programmed death ligand 1 (PD-L1, gene name: CD274) is a cell surface protein typically
expressed in normal tissues to balance and downregulate immune responses by
cytotoxic T lymphocytes (CTLs). This process is mediated by the interaction between
PD-L1 and programmed cell death protein 1 (PD-1), an immunomodulatory protein found
on the surface of CTLs, which when bound to PD-L1 , the cytotoxicity of CTL will be inhibited.
Immune checkpoint blockade therapy triggers T cell (re)activation to kill cancer cells6.

The developers of 68Ga-NOTA-(hPD-L1) explored in their research that when the
complementarity determining region (CDR) of VHH contains lysine residues,
after reacting the nanobody with a non-specific chelating agent, the nanobody molecular
imaging agent Whether the performance will be affected.

The researchers used VHH and NOTA chelators to randomly couple on their lysine residues,
or used Sortase A for site-specific coupling. The results of imaging about 90 minutes after
injection into mice showed that the specific tumor uptake of randomly bound VHH was 1.77±0.29%IA/g,
and the specific tumor uptake of site-specifically bound VHH was 1.89± 0.40%IA/g.

The remarkable stability of both chelates suggests that random lysine chelation may be
an attractive strategy more conducive to the clinical translation of radiolabeled Nanobodies.

Figure 4. Imaging data of 99mTc-NM-01 in (NSCLC) patients. The patient's right upper lobe tumor showed high [18F]FDG uptake (1);
and high 99mTc-NM-01 uptake (2); the mediastinal lymph nodes showed high [18F]FDG uptake (3);
and low 99mTc-NM-01 Uptake (4); demonstrates heterogeneous expression of PD-L1 between primary tumor sites and distant disease sites in the same patient6.

99mTc-NM-01 has previously completed an early phase I clinical trial (NCT02978196)
in China (Figure 4), uating the safety and dosology of 99mTc-labeled anti-PD-L1
nanobodies in non-small cell lung cancer (NSCLC). Compared with the gold standard
tissue biopsy, 99mTc-NM-01 imaging results have good consistency and have the potential
to reliably and non-invasively assess PD-L1 expression.

Currently, this imaging agent is undergoing a new round of clinical trials (NCT04992715
and NCT04436406) in the UK, aiming to uate the diagnostic performance of 99mTc-NM-01.

Another clinical trial (NCT03638804) is currently using an 89Zr–Fc fusion nanobody (Enafolimab)
to analyze targeted uptake and biodistribution in human subjects harboring PD-L1-positive
tumors. This monitoring is performed by PET imaging, while factors such as the safety
and necessary dosage of this Fc-fusion nanobody are also being uated.

This article explains why nanobodies are considered to be the “panacea” in molecular
imaging of tumors, and reviews the latest progress of nanobody imaging agents for
some targets (HER2 and PD-L1). With the current trend of integration of diagnosis and
treatment, nanobodies play a favorable role in this new era: in tumor diagnosis,
in assessment and prediction before customizing and starting treatment regimens,
in dynamic monitoring during treatment, and in detecting tumor recurrence.

Nanobodies may also be used to monitor a variety of other diseases, such as amyloidosis,
viral infections, etc., well beyond those mentioned in this article. In addition to
traditional PET/CT or SPECT imaging, the application of nanobodies can be further
extended to super-resolution imaging to study protein structure, function, and
protein-protein interactions15. In summary, nanobodies are a versatile toolkit that can
play a central role in clinical applications and basic science.



1. Vaneycken, I. et al. Immuno-imaging using nanobodies. Current Opinion in Biotechnology 22, 877-881 (2011).

2. Xavier, C. et al. 18F-nanobody for PET imaging of HER2 overexpressing tumors. Nuclear medicine and biology 43, 247-252 (2016).

3. Keyaerts, M. et al. Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma. Journal of Nuclear Medicine 57, 27-33 (2016).

4. Ge, S. et al. radionuclide molecular imaging targeting HER2 in breast cancer with a focus on molecular probes into clinical trials and small peptides. Molecules 26, 6482 (2021).

5. Altunay, B. et al. 99mTc‑labeled single-domain antibody for SPECT/CT assessment of HER2 expression in diverse cancer types. European Journal of Nuclear Medicine and Molecular Imaging 50, 1005-1013 (2023).

6. Berland, L. et al. Nanobodies for medical imaging: about ready for prime time? Biomolecules 11, 637 (2021).

7. Bao, G., Tang, M., Zhao, J. & Zhu, X. Nanobody: a promising toolkit for molecular imaging and disease therapy. EJNMMI research 11, 1-13 (2021).

8. Gao, H. et al. Nuclear imaging-guided PD-L1 blockade therapy increases effectiveness of cancer immunotherapy. Journal for ImmunoTherapy of Cancer 8, e001156, doi:10.1136/jitc-2020-001156 (2020).

9. Maute, R. L. et al. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proceedings of the National Academy of Sciences 112, E6506-E6514, doi:doi:10.1073/pnas.1519623112 (2015).

10. Qin, S. et al. A preclinical study: correlation between PD-L1 PET imaging and the prediction of therapy efficacy of MC38 tumor with 68Ga-labeled PD-L1 targeted nanobody. Aging (Albany NY) 13, 13006 (2021).

11. Bridoux, J. et al. Anti-human PD-L1 nanobody for immuno-PET imaging: validation of a conjugation strategy for clinical translation. Biomolecules 10, 1388 (2020).

12. Jiang, J. et al. uation of 64Cu radiolabeled anti-hPD-L1 Nb6 for positron emission tomography imaging in lung cancer tumor mice model. Bioorganic & Medicinal Chemistry Letters 30, 126915 (2020).

13. Li, D. et al. Immuno-PET imaging of 89Zr labeled anti-PD-L1 domain antibody. Molecular pharmaceutics 15, 1674-1681 (2018).

14. Xing, Y. et al. Early Phase I study of a 99mTc-labeled anti–programmed death ligand-1 (PD-L1) single-domain antibody in SPECT/CT assessment of PD-L1 expression in non–small cell lung cancer. Journal of Nuclear Medicine 60, 1213-1220 (2019).

15. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nature methods 9, 582-584 (2012).

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