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Single-domain antibodies for radio nuclear imaging and therapy of esophageal squamous cell carcinoma: a narrative review

Liu, Huifang; Nie, Xu; Tian, Zhenchao; Chen, Dan; Chen, Xueli; Zeng, Qi; Xu, Xinyi

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doi: 10.1097/JBR.0000000000000074
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Abstract

Introduction

Esophageal cancer is one of the most common malignant tumors and the sixth most common cause of death globally.[1] Indeed, an estimated 572,000 new cases and 508,000 deaths caused by esophageal cancer were reported for 2018.[2] Two most common histological types of esophageal cancer are: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC)[3]; the latter represents the histological type of more than 90% of esophageal cancer in China.[4] With regard to mortality in China, esophageal cancer comes in fourth place, just after lung, liver, and stomach cancers.[5] Because the low incidence of ESCC in western countries limits sample availability, research on important molecular pathways and regulatory mechanisms of ESCC is mainly carried out in China and Japan at present.

A number of markers of ESCC tumor cells have been identified, such as epidermal growth factor receptor (EGFR),[6] human epidermal growth factor receptor 2 (HER2),[7] human epidermal growth factor receptor 3 (HER3),[8] hepatocyte growth factor (HGF) receptor (c-Met),[9] vascular endothelial growth factor receptor 2 (VEGFR2),[10] chemokine receptor 4 (CXCR-4),[11] chemokine receptor 7 (CXCR-7),[12] and carcinoembryonic antigen (CEA).[13] ESCC has three molecular subtypes: ESCC1 (n = 50), ESCC2 (n = 36), and ESCC3 (n = 4).[14] Among them, ESCC1 had a higher amplification frequency of SOX2/TP63, in addition, ESCC1 gene expression is similar to the classic subtypes described in the Lung Cancer Genome Atlas (TCGA) description of lung squamous cell carcinoma (LUSC) and head and neck squamous cell carcinoma (HNSCC).[14] ESCC2 showed higher NOTCH1 or ZNF750 mutation rates, more frequent changes in KDM6A and KDM2D inactivation, CDK6 amplification, and PTEN or PIK3R1 inactivation.[14] However, ESCC3 tumors lack sufficient evidence to show cell cycle dysregulation and only one sample of four had a TP53 mutation, TCGA data analysis showed that no tumors had similar characteristics to ESCC3, indicating that this type of squamous tumors may be confined to ESCC.[14] Combination therapy involving surgery, radiotherapy, and chemotherapy is presently the standard treatment strategy for ESCC.[15] However, the prognosis for ESCC remains poor with a 3-year survival rate of only 22%.[16] The primary reason for poor diagnosis and prognosis of ESCC is a lack of typical symptoms and sensitive screening methods, which means that most ESCC patients are already in an advanced stage or have terminal cancer at initial diagnosis. Molecular imaging combines molecular biology with modern medical imaging, yielding a qualitative and quantitative non-invasive research method to investigate molecular-level changes in biological processes. Molecular imaging provides an effective method for early diagnosis, tumor staging, and prognostic assessment of ESCC.[17]

Molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems.[18] Molecular imaging techniques for tumor research include ultrasound, optical imaging, positron emission tomography (PET)/single-photon emission computed tomography (SPECT) imaging, computed tomography (CT), and magnetic resonance imaging (MRI), which use biological probes of specific cells or molecules to monitor specific molecular events in the living body by a non-invasive, quantitative, and visual imaging method. The molecular imaging probe consists of three components: a signal agent that produces the imaging signal, a component with high affinity to the target, and a linker that connects the affinity component and signal agent.[19] An ideal molecular imaging probe with clinical transformation potential should have high affinity, strong specificity, high sensitivity, high imaging contrast, low toxicity, low immunogenicity, and strong stability in vivo.[20] Hence, molecular imaging probes require a suitable affinity component. Although monoclonal antibodies are most widely used as affinity components in molecular imaging probes, their large molecular weights (150 kD) limit the penetration depth of the probe in tumors. With the development of technology, traditional complete antibodies have evolved into monoclonal antibodies, antibody fragments, and single-domain antibodies (sdAbs) (Fig. 1), the molecular weight of affinity components has gradually decreased.[21]

Figure 1
Figure 1:
Schematic illustrations of antibody. (A) Schematic representation of sdAb and antibody domains. Adapted from Oliveira et al.[65] (B) Modification of sdAb. (C) Schematic representation of the route of the sdAb after administration. sdAb = single-domain antibody.

sdAbs have strong targeting affinity and excellent tissue penetration ability, which can effectively overcome the shortcomings of traditional monoclonal antibody probes, namely imaging penetration and sensitivity.[22] This article reviews applications of sdAbs in targeted imaging of ESCC, LUSC, HNSCC, and other cancers for which molecular classifications are similar to ESCC. The sdAbs described in this article correspond to eight specific biomolecules known to be highly expressed in ESCC (Table 1). The results of these studies reveal new prospects for the future development of sdAb-based molecular imaging probes targeting ESCC.

Table 1 - Highly expressed biomolecules in ESCC.
Target Expression site ESCC expression rate (%) Adjacent tissues expression rate Reference
EGFR Cell membrane 36.6–80 Normal Xu et al[9]
HER2 0–55.9 Normal Mimura et al[23]
HER3 46.50 Normal Yamamoto et al[8]
c-Met 34–69.2 Normal Xu et al[9]
VEGFR2 52.13 No Sun et al[24]
CXCR4 37.6–61 No Uchi et al[25], Yang et al[26]
CXCR7 45 No Tachezy et al[27]
CEA 53 Low Ishida et al[28]
CEA = carcinoembryonic antigen, c-Met = hepatocyte growth factor receptor, CXCR = chemokine receptor, EGFR = epidermal growth factor receptor, ESCC = esophageal squamous cell carcinoma, HER = human epidermal growth factor receptor, VEGFR2 = vascular endothelial growth factor receptor 2.

Database search strategy

This article mainly summarizes the application of nanobodies targeting biomarkers of esophageal squamous cell carcinoma in molecular imaging in order to provide new ideas for constructing imaging probes for esophageal squamous cell carcinoma. Non-systematic reviews include English experimental articles and review articles from January 1999 to July 2020. We use the following search strategy to search the PubMed database and other databases to obtain relevant literature. First, through searching for the two keywords of esophageal squamous cell carcinoma and biomarkers in the reference list included in the study, the eight markers overexpressed on the membrane of esophageal squamous cell carcinoma were determined. After searching for the two keywords of nanobody and molecular imaging, the author determined some of the articles by screening the titles and abstracts of the articles, and then screened the keywords of eight markers, namely EGFR, HER2, HER3, c-Met, VEGFR2, CXCR-4, CXCR-7, and CEA. The data extraction process focuses on the application of nanobodies for PET imaging, following by the application of nanobodies for anti-tumor drugs. The author also described the problems need to be solved in the application of nanobodies.

Characteristics of sdAb

Hamers and his team first discovered an sdAb with naturally missing light chains in camel serum in the early 1990s.[29] Also called nanobodies because they contain only one heavy chain variable region and two conventional CH2 and CH3 regions, SdAbs have become widely used in molecular imaging as affinity components for molecular probes because of their unique molecular imaging properties, as described below.

Using the phage display technique, high-affinity sdAbs can be easily screened and obtained from a library.[30] sdAbs have strong binding capacity to recognize and target a unique epitope, depending on their long complementarity determining region 3 (CDR3).[31] An sdAb is the smallest unit of an antibody that retains all antigen-binding competence. sdAbs can quickly penetrate and diffuse into tissues, and are also quickly cleared. These two advantages make sdAbs suitable for non-invasive imaging in vivo.[32] In addition, most sdAbs show high stability in response to high temperatures, high solvent concentrations, and other chemical conditions.[33] Because genes encoding sdAbs share high sequence homology with genes belonging to the human VH families 3 and 4, they exhibit low immunogenicity and good biocompatibility.[34] These beneficial properties make sdAbs an ideal molecular imaging probe with broad application prospects for early tumor diagnosis and disease treatment.

EGFR

EGFR, also known as erbB1, is a member of the HER family of receptors that includes HER2, HER3, and HER4. Combined with its ligands, EGFR can lead to proliferation, invasion, and metastasis of tumor cells.[35] EGFR is overexpressed in many tumors, including HNSCC, breast cancer, lung cancer, colorectal cancer, and ESCC.[6]

In 2011, Vosjan et al[36] reported two radionuclide probes containing an sdAb 7D12 targeting EGFR that displayed clear tumors with a high tumor background ratios (TBRs) in PET imaging. Biodistribution studies showed high radioactive absorption of these probes in kidney and bladder. Increasing probe uptake was observed in tumor tissues from 1 to 3 hours after injection, and probes reached their highest absorption 3 hours after injection (Table 2). An in vitro stability study revealed that both probes were stable in buffer at 4°C and in serum at 37°C. Compared with other radio-labeled monoclonal antibodies used in pre-clinical imaging studies, sdAbs can perform high-contrast imaging at earlier time points and have lower liver uptake.

Table 2 - Overview of sdAb-based probes molecular imaging.
Target SdAb Imaging modality (Label) Maximum uptake in tumor Best imaging time for TBR (h) Excretory organ Disease model Reference
EGFR 7D12 PET (68Ga) 7.2 ± 1.5%ID/g 3 Kidney Human epidermoid cervical carcinoma (A431) Chen and Chen[19]
D10 SPECT/CT (99mTc) 2.3 ± 0.7%ID/g 0.75 Kidney Human epidermoid cervical carcinoma (A431) Xavier et al[38]
HER2 2Rs15d PET/CT (68Ga) 4.34 ± 0.9%IA/g 1 Kidney Human ovarian adenocarcinoma (SK-OV-3) Vaidyanathan et al[39]
5F7 Micro-PET/CT (18F) 36.28 ± 14.1%ID/g 2 Kidney Breast carcinoma (BT474M1) Gonzalez-Sapienza et al[33]
HER3 MSB0010853 PET (89Zr) 6.2 ± 1.1%ID/g 24 Kidney Non-small cell lung carcinoma (H441) Warnders et al[41]
CEA NbCEA5 SPECT/Micro-CT (99mTc) 6.15 ± 2.33%IA/cm3 1 Kidney Human colon adenocarcinoma (LS174T) Vaneycken et al[53]
CEA = carcinoembryonic antigen, EGFR = epidermal growth factor receptor, HER = human epidermal growth factor receptor.

In 2016, Krüwel et al[37] showed that sdAb-based tracers targeting EGFR had higher uptake and lower background in tumors compared with a monoclonal antibody. Moreover, radio-labeling did not affect the binding properties of the sdAb to EGFR. 99mTc-D10 was injected into a subcutaneous human epidermoid carcinoma (A431) mouse model, whereby it accumulated in tumors (Table 2). Imaging revealed small tumors with an average volume of 26.6 mm3 (Fig. 2). Meanwhile, 99mTc-Cetuximab detected an average volume of 40 mm3 for small tumors. Collectively, these results indicated that probes with an sdAb can detect small tumors at the earliest time point, improving the overall prognosis of patients. Therefore, the 99mTc-D10 anti-EGFR probe can be used for clinical non-invasive diagnosis not only to detect small tumors, but also obtain information about EGFR expression levels during diagnosis and disease progression.

Figure 2
Figure 2:
In vivo tumor visualization of small human A431 tumor xenografts with anti-EGFR sdAb 99mTc-D10 in comparison to 99mTc-Cetuximab by SPECT and CT. SPECT imaging was performed 45 minutes after 99mTc-D10 administration and 24 hours after 99mTc-Cetuximab administration. Tumors are indicated by white arrows. Note that a high tumor accumulation with low background signal was achieved with anti-EGFR sdAb 99mTc-D10. Reprinted with permission from Krüwel et al.[37] CT = computed tomography, EGFR = epithelial growth factor, p.i. = post injection, sdAb = single-domain antibody, SPECT = single-photon emission computer tomography.

HER2

HER2 is the most popular target for tumor therapy. It has been confirmed that HER2 is highly expressed in ESCC tumor cell membranes, and this high expression is related to the progression of ESCC.[7] Moreover, HER2 expression is higher in patients with local relapses or distant metastases early after surgery compared with other patients.[7] Therefore, HER2 can be used as a target for the diagnosis and treatment of ESCC.

Xavier et al[38] confirmed that anti-HER2 sdAb 2Rs15d features high tumor uptake and low background signal. The sdAb-based tracer 68Ga-NOTA-2Rs15d could bind to the HER2 target protein with low-nanomolar affinity and showed high metabolic stability in human plasma. Moreover, 68Ga-NOTA-2Rs15d was shown to specifically bind to HER2-positive SK-OV-3 cells. Biodistribution results showed relatively high radioactive absorption in HER2-positive tumors (Table 2), and the tumor-to-muscle ratio of HER2-positive tumors was significantly higher than that of HER2-negative tumors.[38] Finally, PET/CT imaging studies revealed strong tracer uptake in HER2-positive tumors, while mouse toxicity and dosimetry studies demonstrated the safety of this tracer.[38] Therefore, similar sdAb-based tracers can be used for clinical diagnosis of ESCC.

In 2016, Vaidyanathan et al[39] reported that the anti-HER2 sdAb 18F-RL-I-5F7 can specifically target tumors positively expressing HER2, and features high tumor uptake and normal tissue clearance rates. Internalization of the sdAb-based tracer revealed that an sdAb labeled with RL-1 can effectively retain radioactivity. A biodistribution study of 18F-RL-I-5F7 showed that tumor uptake increased from 29.0 ± 3.9% ID/g at 1hour to 36.3 ± 14.1% ID/g at 2 hours.[39] Micro-PET/CT imaging showed high tumor uptake at both time points, whereas significant uptake was not observed in other normal organs with the exception of kidney and bladder (Fig. 3, middle and right).[39] Tumor uptake of 18F-RL-I-5F7 could be reduced by over 90% through pre-injection of trastuzumab (Fig. 3, left).[39] The results of this study indicate that 18F-RL-1-5F7 can be used to image tumors positively expressing HER2. However, the problem of excessive kidney uptake remains to be resolved

Figure 3
Figure 3:
PET/CT images of mice bearing BT474M1 (breast carcinoma cells) xenografts after injection of 18F-RL-I-5F7. Images were obtained at 1 and 2 hours, and at 1hour with blocking of HER2 receptors by pre-administration of trastuzumab. Reprinted with permission from Vaidyanathan et al.[39] CT = computed tomography, HER2 = human epidermal growth factor receptor 2, PET = positron emission tomography.

HER3

HER3 plays important roles in tumor growth, differentiation, and metastasis. Unlike EGFR and HER2, HER3 does not directly promote the proliferation of tumor cells; instead, HER3 binding to heregulin led to the formation of HER2 heterodimers able to activate signaling pathways.[40] In addition to HNSCC, HER3 is also highly expressed in ESCC.[8] Therefore, an anti-HER3 sdAb can be designed in combination with other antibodies for the diagnosis and treatment of ESCC.

Warnders et al[41] developed and demonstrated the sustained anti-tumor activity of a biparatopic sdAb construct, MSB0010853, that could bind to two different HER3 epitopes. Biodistribution of MSB0010853 in the body could be non-invasively monitored through PET imaging.[41] The construct was composed of albumin and two sdAbs that bound to different HER3 epitopes. Biodistribution results revealed that an injection dose of 25 mg yielded the best tumor uptake (5.7% ID/g), and tumor uptake of 89Zr-MSB0010853 peaked (Table 2) within 24 hours and thereafter decreased. PET imaging showed that 89Zr-MSB0010853 in positive tumors remained clearly visible 96 hours after injection (Fig. 4). Moreover, radioactive absorptions of HER3-positive tumors H441 (Table 2) were 2.4 greater than that of the HER3-negative tumor Calu-1 (2.3 ± 0.3% ID/g) and FaDu (5.1 ± 0.4% ID/g) were 2.2 times greater than that of Calu-1 (Fig. 5). The half-life of albumin in humans is longer than in mice, thus 89Zr-MSB0010853 is expected to have a longer circulation time and show sustained anti-tumor activity in humans. This research provides new ideas for the development of drugs for the treatment of ESCC.

Figure 4
Figure 4:
Uptake of single domain antibody 89Zr-MSB0010853 in xenografts mice. (A) Representative coronal PET images of mice bearing H441 xenografts injected with 25 μg of 89Zr-MSB0010853; images were obtained 24, 48, 72, and 96 hours after tracer injection. Arrows indicate xenograft tumors. (B) Small-animal PET data quantification was performed for tumor uptake at 24, 48, 72, and 96 hours. Reprinted with permission from Warnders et al.[41], PET = positron emission tomography.
Figure 5
Figure 5:
Uptake of single domain antibody 89Zr-MSB0010853 in different xenografts mice. (A) Representative coronal positron emission tomography images of mice bearing H441 (non–small cell lung cancer cell line, HER3-positive), FaDu (head and neck squamous cell cancer cell line, HER3-positive), or Calu-1 (non–small cell lung cancer cell line, HER3-negative) xenografts 24 hours after injection of 89Zr-MSB0010853 (25 μg). Arrows indicate xenograft tumors. (B) Corresponding tumor uptake. Data are presented as the mean ± SD. P≤0.05. Reprinted with permission from Warnders et al.[41]

c-Met

The hepatocyte growth factor (HGF) receptor (c-Met) is a receptor tyrosine kinase that is highly expressed in a variety of tumor cells,[42] including ESCC.[9] When combined with HGF, c-Met undergoes autophosphorylation and causes cell growth, invasion, and metastasis.[43] Therefore, this receptor is a promising target for tumor treatment.

Based on previously designed anti-EGFR nanobody-albumin nanoparticles (NANAPs),[44] Heukers et al[45] synthesized a type of albumin nanoparticles decorated with an sdAb targeting c-Met (G2-PEG-NP) in 2013. Their results showed that G2-PEG-NP could inhibit tumor growth, making it potentially suitable for intracellular drug delivery and targeted medicine. Moreover, G2-PEG-NP could specifically bind to cells expressing c-Met. Scratch wound experiments revealed that G2-PEG-NP did not cause cell migration. When HGF and G2-PEG-NP acted together on cells, cell migration induced by HGF was significantly inhibited, suggesting that G2-PEG-NP could potentially inhibit tumor growth. The results of co-staining revealed that internalized G2-PEG-NP was located first in early endosomes and finally in lysosomes. In addition, both nanoparticles and c-Met receptors were degraded during the late stage. Therefore, anti-c-Met NANAPs may be suitable for intracellular delivery and release of therapeutic compounds. Given its characteristics, this type of targeted nanoparticle may be a good candidate for the treatment of tumors overexpressing c-Met.

VEGFR2

VEGFR2, also known as FLK-1, is a subtype of VEGFR that exists in blood vessels and the lymphatic endothelium. Binding of VEGFR2 by VEGF promotes the formation of blood vessels and enhances the invasion and infiltration of tumors. This receptor is highly expressed in a variety of tumor cells, including ESCC, and has thus become a popular target for tumor therapy.[24]

In 2017, Tian et al[10] developed an sdAb-conjugate, V21-DOS47, capable of specifically binding VEGFR2 to deliver drugs to target sites to inhibit angiogenesis. V21-DOS47 kills cells expressing VEGFR2 by inducing cytotoxic activity in the target cell; this method differs from most anti-angiogenic agents. V21-DOS47 has high purity, and binding affinity experiments revealed that V21H4-DOS47 binds to VEGFR2 with approximately five-times higher affinity than V21H1-DOS47. Moreover, V21H4-DOS47 was found to exclusively bind to VEGFR2. Above all, this sdAb-conjugate appears to be a promising anti-cancer drug. With regard to ESCC, this conjugate may be constructed to direct drug delivery to the target site to reduce non-specific side effects.

CXCR4

CXCR4, a chemokine receptor, is a transmembrane G protein-coupled receptor. Similar to CXCR7, CXCR4 is a receptor for the chemokine CXCL12, which can induce chemotaxis and enhance intracellular calcium, proliferation, and gene transcription.[46] Studies have shown that CXCR4 may be highly expressed in ESCC, with a total expression rate of 61%.[11] Therefore, synthesis of an sdAb targeting CXCR4 may be useful to diagnose and treat ESCC.

Jähnichen et al[47] synthesized the anti-CXCR4 sdAbs 238D2 and 238D4 in 2010, and found that the biparatopic sdAb could both inhibit the response induced by CXCL12 and act as an inverse agonist, thereby inhibiting tumor growth. Radioactive 125I-labeled 238D2 and 238D4 were discovered to selectively bind to CXCR4-positive cells. Functional characterization of 238D2 and 238D4 showed that the sdAb could completely inhibit the accumulation of inositol phosphate induced by CXCL12, and these antagonistic effects increased with increasing concentrations of anti-CXCR4 sdAb. Importantly, the anti-CXCR4 biparatopic sdAb exhibited high affinity and antagonistic properties at picomolar concentrations. Moreover, the anti-CXCR4 sdAb was found to act as fast and effectively as stem cell mobilizers.[47] Therefore, anti-CXCR4 sdAbs may provide a new route for the development of therapeutic agents for CXCR4-related diseases.

CXCR7

CXCR7 can bind to chemokines and is a newly identified receptor for CXCL12, which plays a key role in the occurrence and progression of liver, esophagus, head and neck, and other cancers.[12] CXCR7 can act as a co-receptor of CXCR4 or interact with β-arrestin.[48] Therefore, treatment of ESCC may be achieved by constructing an sdAb targeting CXCR7.

Maussang et al[49] demonstrated that CXCR7 is highly expressed in HNSCC, indicating that CXCR7 has potential tumorigenic effects in HNSCC. Thus, they developed an anti-CXCR7 sdAb and showed that an anti-CXCR7 multivalent sdAb could effectively inhibit angiogenic factor secretion and tumor growth. Moreover, they confirmed that the sdAbs NB2 and NB3 (with strong binding) could effectively inhibit the recruitment of β-arrestin2 induced by CXCL12, and exerted effective antagonistic effects, whereas sdAb NB1 (with weak binding) could not. Competitive binding assays revealed that the binding epitopes of NB2 and NB3 differed from that of NB1. Compared with a monovalent sdAb, the affinity of NB4 [NB1 and NB3 combined through serum albumin (Alb8)] and NB5 (NB2 conjugated to Alb8) for CXCR7 was significantly increased, and they could similarly inhibit the recruitment of β-arrestin2 induced by CXCL12. Interestingly, NB4 was approximately 10 times more potent than NB5. In vitro experiments confirmed that NB4 could inhibit secretion of the angiogenic factor CXCL1, but did not inhibit the cell cycle process.[49] An investigation of therapeutic potential in vivo showed that the tumors of mice treated with NB4 grew more slowly and were significantly smaller in size than phosphate buffer saline-injected mice; moreover, NB4 was confirmed to be non-toxic.[49] The anti-CXCR7 sdAb could inhibit CXCL12-induced β-arrestin2 recruitment, and the multivalent sdAb had higher affinity and greater inhibitory effects than the monovalent sdAb, thus establishing a new option for the development of anti-tumor drugs.

CEA

CEA, a member of the immunoglobulin molecule family expressed on the cell surface,[50] is a broad-spectrum tumor marker that is highly expressed in a variety of colon, gastric, pancreatic, and non-small-cell lung cancers,[51] as well as ESCC.[13] CEA is expressed during the fetal period and then becomes quite limited in healthy adults, although it is re-expressed in cancer. Therefore, CEA has become a popular target for cancer therapy.[52]

In 2010, Vaneycken et al[53] constructed a humanized sdAb (NbCEA5) and found that this sdAb-based tracer features high specificity for tumor targeting, rapid renal clearance, and a low background signal. Moreover, enzyme-linked immunosorbent assay results revealed this humanized sdAb can specifically bind to CEA with a high binding capacity. 99mTc-humanized CEA5 grafts could effectively bind purified CEA protein and CEA-expressing CHO cells in vitro. Blood clearance rates and pinhole SPECT/Micro-CT of the 99mTc-sdAb showed high tumor uptake of 99mTc-NbCEA5 (7.09 ± 1.36% IA/cm3) and 99mTc-labeled humanized CEA5 grafts (Table 2) at 1hour after injection. The tumor-to-muscle ratios of NbCEA5 (39.25 ± 27.64) and humanized sdAb (7.74 ± 3.05) were generally high. Collectively, these results indicated excellent clinical application potential of anti-CEA sdAbs for the detection of ESCC.

Developmental prospects of sdAbs

Although sdAbs have been widely used in molecular imaging on account of their high conformational stability, high tolerance to the environment, and easy synthesis, some problems continue to hinder their clinical applications. The following sections briefly introduce these problems and describe possible solutions.

High renal metabolic rate

The kidney is the main metabolic organ for sdAb-based tracers and takes in high radiation doses, so it may cause nephrotoxicity and reduce the sensitivity of detecting specific molecular signals near the esophagus.[54] Research conducted to address this issue found relatively low accumulation of kidney radioactivity in mice lacking megalin.[54] Co-injection of lysine or gelatin serine with the tracer also appeared to reduce kidney uptake.[55] After removal of the histidine tag from an sdAb-based tracer, the tracer retention rate in the kidney was greatly decreased because the pH in the kidney was lower than the pKa of imidazole. Differences in the total charge of an sdAb can be used to explain why histidine tag removal reduces kidney retention.[38]

Short body metabolism time

sdAbs are widely used because of their small size. However, their characteristic rapid elimination is not conducive to treatment effectiveness. Thus, it is necessary to increase the half-life of sdAbs without affecting their characteristics to achieve effective treatments. Binding of an sdAb to albumin to form a multivalent sdAb may be used to extend its half-life.[41] Multivalent sdAbs exhibit faster and deeper tumor penetration compared with monoclonal antibodies. Coupling with a polyethylene glycol (PEG) linker has also been attempted.[45] These methods can effectively extend the half-life of sdAbs to increase the residence time of the drug in the blood, thus permitting lower doses of the drug to be injected and extending the interval between infusions. This provides a new idea for sdAbs to become clinical anti-tumor drugs.[56,57]

Endoscopy is very easy to access the esophagus, so early diagnosis of esophageal cancer is possible, and significant progress has been made.[58] Therefore, fluorescent-labeled sdAbs can be used to detect ESCC. Some studies have used quantum dots to label sdAbs targeting tumor markers. The resulting materials can detect not only primary tumors, but also tumor cells and micrometastatic lesions in different organs with high brightness and sensitivity.[59,60] An sdAb labeled with the near-infrared fluorophore IRDye800CW could clearly identify tumors during operation. Compared with mice injected with a control probe, the TBR of this antibody was clearly higher.[61] Massa et al[62] linked the fluorescent dye Cy5 to an sdAb and visualized HER2-expressing tumors in vivo with high contrast and specificity 1 hour after injection. Fluorescent-labeled sdAbs have been widely used to detect various tumors. Non-invasive or minimally invasive examinations can be performed using endoscopy to detect ESCC. Therefore, fluorescent-labeled sdAbs have great application prospects as ESCC markers and can be developed for clinical diagnosis.

Conclusion

This article focuses on the application of single-domain antibodies in radiological imaging, briefly mentioning the application in fluorescence imaging, and there is little discussion of single-domain antibodies that have been used in clinical practice. Recently review has compiled sdAb currently in clinical trials, among them, only anti-HER2 sdAb have reference significance for esophageal squamous cell carcinoma.[22]

Molecular probes containing an sdAb can specifically bind to the target antigen; be radioactively labeled for PET, SPECT/CT, or PET/CT imaging; and be labeled for fluorescent imaging, yielding high diagnostic accuracy. Molecular imaging studies have shown that probes containing an sdAb have higher tumor-to-background ratios, and most sdAb tracers exhibit the best imaging results within 1 to 3 hours. The signal ratio of tumor-to-normal-tissue was 5–36. The sdAb exhibited rapid blood elimination efficiency and final metabolism by the kidney or liver, and the half-life of the probe was 30 to 143.5 minutes in vivo.[63] The results of single-dose intravenous toxicity studies on sdAb-based tracers indicated good compatibility of this type of probe.

Although sdAb-based probes are widely used, the following problems still need to be solved. First, sdAb-based tracers exhibit high renal uptake and fast clearance rates. At present, co-injection of lysine and gelatin serine with tracers, or removal of histidine tags, has been identified as methods to decrease renal uptake. In addition, short metabolism times in vivo can be prolonged by formation of a multivalent sdAb, or coupling with albumin or a PEG linker. Second, sdAb-based fluorescent probes can help clinicians clearly observe tumor edges during endoscopic diagnosis, but labeling with a fluorescent group may affect the structure of the sdAb.[30] Studies have shown that specific amino acid sequences of an sdAb can be recognized by enzymes, thus allowing the sdAb to be selectively enzyme-catalyzed and modified with a fluorescent group.[64] In addition, sdAbs can be singly and specifically labeled by orthogonal reactions.

As an important technical means for tumor diagnosis, molecular imaging technology has broad prospects and plays a pivotal role in accurate tumor diagnosis. The demand for new molecular imaging probes with clinical advantages is becoming increasingly apparent. sdAbs are used as molecular probes in tumor diagnosis and treatment with the advantages of good penetration, high affinity, and low immunogenicity. With the emergence of highly specific, targeted, sdAb probes, new molecular probes employing an sdAb core are expected to provide novel strategies for precise diagnosis and treatment of ESCC.

Acknowledgments

None.

Author contributions

HL, ZT, and XX participated in manuscript concept. HL, ZT, DC, XC, QZ, and XX designed the manuscript. XN, DC, XC, QZ, and XX participated in definition of intellectual content. HL and ZT searched the literature. HL participated in data collection and integration. HL, XN, and XX participated in manuscript preparation. HL and XN edited the manuscript. HL, XN, DC, XC, QZ, and XX participated in manuscript review. HL and XX served as guarantors. All authors approved the final version of the manuscript.

Financial support

This work was supported, in part, by the National Key Research & Development Program of China (No. 2018YFC0910600); the National Natural Science Foundation of China (Nos. 81871397, 81701853, and 81627807); the Natural Science Basic Research Plan in Shaanxi Province of China (Nos. 2019JQ-519, 2019JQ-201, and 2019JQ-045); and the Fundamental Research Funds for the Central Universities (No. JB191209).

Conflicts of interest

The authors declare that they have no conflicts of interest.

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              Keywords:

              esophageal squamous cell carcinoma; hepatocyte growth factor receptor; human epidermal growth factor receptor; molecular imaging; single-domain antibody

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