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Nanobodies as in vivo, non-invasive, imaging agents

In vivo imaging has become in recent years an incredible tool to study biological events and has found critical applications in diagnostic medicine. Although a lot of efforts and applications have been achieved using monoclonal antibodies, other types of delivery agents are being developed. Among them, VHHs, antigen binding fragments derived from camelid heavy chain–only antibodies, also known as nanobodies, have particularly attracted attention. Indeed, their stability, fast clearance, good tissue penetration, high solubility, simple cloning and recombinant production make them attractive targeting agents for imaging modalities such as PET, SPECT or Infra-Red. In this review, we discuss the pioneering work that has been carried out using VHHs and summarize the recent developments that have been made using nanobodies for in vivo, non-invasive, imaging.


Introduction
In vivo imaging allows the investigation of molecular and cellular events in intact living subjects. Studying the dynamics of biological processes non-invasively and in their native context may offer new insights and provide opportunities for the discovery of novel biology. In vivo imaging has therefore become an indispensable tool in both biomedical research and medical practice. [1][2][3][4][5] A diverse set of in vivo imaging approaches has been developed. These include Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Infra-Red and Near Infra-Red Imaging (IR/NIR), Ultrasound (US), and photoacoustic imaging. 1,[3][4][5][6][7][8] Regardless of the method used, in vivo imaging requires differential accumulation of the relevant signal at the target site to distinguish it from background. 9 The major challenge of non-invasive in vivo imaging of a target of interest is to achieve adequate selectivity and discrimination from the background signal in the targeted area. To achieve specificity, combining the imaging agent (radio)isotope or fluorophore with a carrier such as antibodies, 10 peptides, 11,12 small molecules, 13 and aptamers 14,15 has been the method of choice. In particular, immunoglobulins and immunoglobulin-derived fragments have long been considered attractive candidates, since they can be generated to target a wide range of biomolecules [16][17][18] and recognize them with exquisite specificity.
Nevertheless, monoclonal antibodies (mAbs) are large (150 kDa), which explains their comparatively poor tissue penetration when administered systemically, with a long circulatory half-life. 9,19 Efforts have been devoted to improving the pharmacokinetics of antibodies without compromising their unique affinity and specificity. Antibody fragments such as Fab, F(ab 0 )2, single chain Fv (scFv), or variants like diabodies and minibodies (molecular weight ranging from 25-100 kDa) were bioengineered specifically for the purpose of creating in vivo imaging agents. [20][21][22] More recently, other types of proteins have been investigated as alternatives to antibodies for in vivo imaging. These include nanobodies ( Fig. 1), Affibodies, and anticalins. [20][21][22] Of these, nanobodies have attracted growing interest for use in molecular imaging across various imaging platforms, including SPECT, PET, Infra-red (IR) and ultrasound. 9,23 Nanobodies are antigen binding fragments derived from camelid heavy chain-only antibodies, which fold and function in the absence of light chains. These camelid immunoglobulin (Ig) heavy chains can be shrunk to just their variable domains to yield VHHs or nanobodies. The resulting VHHs retain antigen binding and can be produced in an active form in E. coli, often without the need of an intrachain disulfide bond. Typically, nanobodies have a molecular weight of B12-16 kDa and are considered the smallest immunoglobulin-derived antigen binding fragments. 24 Because of their much smaller size when compared to intact Ig (B150 kDa), Fab (B50 kDa) and scFv (B25 kDa) fragments, nanobodies can penetrate tissues more efficiently and bind to antigens less accessible to conventional antibodies or their smaller derivatives. 19,[25][26][27] Better tissue penetration is not the only advantage of nanobodies as compared to intact conventional Ig. Nanobodies are poorly immunogenic in humans, 19 and some nanobodies have been ''humanized'' to display even less immunogenicity. 25,28,29 Furthermore, their small size and the preponderance of beta sheet secondary structure make them highly stable, even at high temperatures and extremes of pH. 30 Finally, owing to their small size, nanobodies that fail to bind their target are rapidly cleared from the blood, mostly by renal elimination. 19,25,[31][32][33][34] This, in tandem with their usually high affinity, can produce a high tumor-tobackground ratio as early as 1 h after tracer injection. 32,35,36 Although nanobodies possess the many advantages listed above, their relatively small size leads to rapid elimination from the circulation by renal clearance. Their rapid renal clearance from the body might prevent optimal binding at the desired site, 35 as the concentrations of the imaging agent in the circulation drop rapidly. This is often accompanied also by uptake of the imaging agent in the kidneys, making imaging experiments in neighboring tissues difficult because of the high background. 37 Finally, the binding properties of nanobodies may be altered when they are conjugated to produce imaging agents. 38 In this review, we describe how nanobodies are equipped with the corresponding probes for each imaging modality and how rapid clearance and non-targeted organ uptake, problems inherent in the use of nanobodies, can be overcome.

Labeling of nanobodies for in vivo imaging
In order to be observable by any imaging modality (SPECT, PET, or IR), nanobodies must be equipped with a suitable radioisotope or fluorescent dye. Although some aspects of the requisite methods for modification are similar, the imaging modality chosen might require specific considerations with respect to the labeling method used.

Radio-labeling for SPECT and PET imaging
Radiohalogens, like 123/125/131 I for SPECT or 124 I and 18 F for PET, must be covalently attached to the nanobody. Radiometals such as 67 Ga, 99m Tc, and 111 In for SPECT and 64 Cu, 68 Ga, and 89 Zr for PET are complexed via a chelator and thus require a two-step process where the nanobody is first equipped with a chelator and then incubated with the radiometal in solution. The (His)6 tag often present on recombinantly expressed proteins to facilitate affinity purification readily coordinates sitespecifically with 99m Tc-tricarbonyl. 39,40 In either case, a chelator or a prosthetic group must be attached to the nanobody of choice. Various labeling approaches can be divided into two major classes: uncontrolled or site-specific labeling. Uncontrolled labeling relies mainly on lysine and cysteine conjugation via carbodiimide/N-hydroxysuccinimide (DIC/NHS) chemistry or maleimide chemistry, respectively. Although this method is common, straightforward, and easily accessible, the heterogenous mixture of labeled products might lead to reduced binding of the nanobody, especially if the binding site itself is modified. 41 Site-specific strategies aim to obtain homogeneous and more consistent products, with the desired moiety attached to a specific site of one's choosing. Different site-specific methods to modify nanobodies have been described, relying on enzymatic methods, click chemistry, or meticulous and selective incorporation of an unpaired cysteine residue. 24,42 There are a wide range of commercially available chelators with functionalized handles, such as maleimide, NHS ester, azido derivatives, etc. (Fig. 2A). However, this is not the case for the radiohalogens (obtained as salts) for both 18 F and 123/124/125/ 131 I. The Iodogen method allows rapid but uncontrolled iodination of the desired protein via an oxidation reaction of tyrosine sidechains, but a more sophisticated synthetic chemistry is required to prepare radiohalogen reagents prior to their use for the labeling of nanobodies (Fig. 2B). 43,44

Fluorescent dye labeling for IR and NIR
In vivo imaging using fluorescent dyes relies on fluorophores emitting in the near infrared (NIR) or infrared region (IR). An ideal dye should have a maximum emission in the 650 nm to 900 nm range (Fig. 2C). This particular range of wavelengths reduces scattering and nonspecific autofluorescence, produces a good signal to background ratio, and thus improves resolution. 23 The cyanine dye derivatives: Cy5, AF680, IRDye 680RD IRDye800CW, etc. are commonly used for this application. 45,46 However, their high hydrophobicity has pushed the development of more water-soluble dyes, such as the pyrrolopyrrole cyanine family (PPCy) (Fig. 2C). 47 Conjugation of fluorophores can significantly affect pharmacokinetics, biodistribution, quality/stability and aggregation properties of the engineered construct. Uncontrolled and multiple-labeling of fluorophores to nanobodies yields products with abnormal distribution, can contribute adversely to the background signal, produces accumulation in non-targeted organs such as the liver and results in poor contrast for the target of interest. 36,[48][49][50][51] Site-specific labeling of IRDye800CW

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and IRDye680RD dyes by conjugation to a selectively introduced C-terminal cysteine yields imaging agents with improved biodistribution: acceptable kidney uptake, rapid binding to the target of interest and minimal nonspecific uptake (Fig. 3). 49 Nanobodies for NIR/IR in vivo imaging can also be prepared as fusions to a far-red fluorescent protein. This eliminates the need of further handling of the nanobody but comes with a drawback: the considerable size of the fluorescent protein (20-30 kDa) compared to the nanobody (B15 kDa) might disturb binding and the biodistribution of the resulting fusion protein. 23,51 3. Overcoming nanobody challenges: renal accumulation and rapid clearance Long-term renal retention of nanobodies, especially in a radiolabeled form, can cause nephrotoxicity. The bright kidney signals caused by such accumulation make the imaging of molecular targets in the adjoining areas difficult. Different strategies have been developed to reduce renal absorption. For example, co-injection with gelofusin or positively charged

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RSC Chemical Biology amino acids like lysine can reduce renal uptake for radiolabeled biomolecules. 52,53 This strategy has also been applied to nanobodies. 31,54 Another approach consists of removing the affinity tags often used for purification purposes: these tags, typically myc or (His)6 tags, consist of negatively-and positivelycharged amino acids respectively, and can directly impact the degree of renal accumulation of the nanobody. 54,55 Attachment of PEG moieties can decrease renal uptake. The availability of differently sized PEGs allows simple screening and tuning of the desired construct to optimize its imaging characteristics. Rashidian et al. used nanobody constructs bearing differently sized PEG moieties and showed a direct correlation between the size of the PEG moiety and the amount of tracer accumulating in the kidney (Fig. 4). 56 Unmodified VHH X118, a nanobody that targets CD8+T cells, fails to cleanly show the spleen (located near the kidneys), although the spleen is a main reservoir of CD8+T cells. In contrast, the 20 kDa PEG-derivative of VHHX118 readily allows visualization of secondary lymphoid organs.
PEGylation tackles yet another variable associated with the use of nanobodies, which is their rapid clearance from the circulation. While quick elimination from the body allows nanobodies to be used for same-day imaging, it also can lead to poor accumulation in the area of interest. Attachment of PEG of different sizes, e.g. 5, 10 or 20 kDa, increases their molecular weight/Stokes' radius and thus their circulatory dwell time, with an improvement in the signal to noise ratio. 56 Increasing the half-life of nanobodies by increasing their size has been accomplished by methods other than PEGylation. Wardners et al. used a tri-paratopic nanobody construct, consisting of two nanobodies that bind to two different HER3 epitopes and a third that binds to albumin in order to extend the circulatory half-life of the construct. This construct showed less renal accumulation than a 'regular' HER3-targeting nanobody and circulated longer, as measured by radioactivity remaining in the blood. 57 Size and targeting properties must be properly balanced, parameters that cannot be predicted and therefore require systematic experimentation.
Interestingly, the degree of kidney retention for radiohalogenated (fluorinated and iodinated) nanobodies is significantly lower than their radiometal-labeled counterparts. Catabolites of radiohalogenated compounds formed in the kidneys are thought to be non-residualizing and hydrophobic. They are rapidly excreted via the urine. 23 With the exception of this kidney accumulation -which can be overcome at least in part by the methods described hereaccumulation in other non-targeted organs is usually very low. The unique characteristics of nanobodies (efficient tissue penetration, fast tissue diffusion and fast clearance) thus allow same-day imaging with high target-to-background ratios. 9,23 This is impossible to achieve with conventional antibodies, where several days of equilibration between circulation and target tissues are required after administration before it is possible to acquire high quality images.

Radio imaging: SPECT and PET
Radionuclide-based techniques such as PET and SPECT are commonly used because they are sensitive, quantitative, and clinically relevant. 58,59 SPECT and PET have the sensitivity needed to visualize most interactions between physiological targets and ligands, enabling non-invasive detection of tracers down to the picomolar level.
4.1.1. SPECT imaging using nanobodies. Single-photon imaging exploits the g rays emitted by a radioactive atom. A y-emitting element with an energy of 100-250 keV is ideal. 1 The spatial resolution of single-photon imaging is on the order of 8-10 mm, although somewhat better resolution can be obtained with recently developed special-purpose imaging systems. [60][61][62][63] Although 99m Tc, 123/131 I, 111 In and 155 Tb are the four radionuclides suitable and commonly used for SPECT imaging, 64 only two of them have been used with nanobodies: 99m Tc and 111 In. A few examples describe nanobody-based SPECT imaging using 111 In as a radionuclide: Chatalic et al. developed an anti-PSMA nanobody conjugated to the metal chelator diethylenetriaminepentaacetic acid (DTPA), which showed good tumor targeting and minimal uptake by the non-targeted organs. 54 Bala et al. used 111 In in combination with a VCAM-1 targeting nanobody in a study to compare the pharmacokinetics of the imaging agent with the use of the different tracers: 68 Ga, 18 F, 111 In and 99m Tc. 65 The reason for the success of 99m Tc as a radionuclide for SPECT imaging derives not only from its favorable nuclear decay characteristics, but also from its simple and straightforward radiolabeling chemistry. This has allowed the labeling of a wide range of biomolecules. For example, 99m Tc(CO) 3 can be used to directly label nanobodies equipped with a (His)6 tag without further modifications. 32,66 Additionally, the long halflife of 99 Mo and the low cost associated with the 99 Mo/ 99m Tc generator make the production of 99m Tc widely available. 67 Nanobodies against many different markers have been used in combination with 99m Tc for SPECT imaging of cancers. Examples include epidermal growth factor receptor 1 (EGFR) 31,32,66,68,69 and 2 (HER-2), 70 prostate-specific membrane antigen (PSMA), 71 the Macrophage Mannose Receptor (MMR), 37 carcinoembryonic antigen (CEA), 72,73 mesothelin 74 and the M-protein. 75 Nanobodies that target non-cancer markers have also found application for SPECT imaging. De Vos et al. immunized a dromedary with the Lectin-like oxidized low density lipoprotein receptor (LOX-1). 76 This protein is a biomarker for detection and phenotyping of atherosclerotic plaques. They obtained a nanobody, LOX-sdAb, that binds LOX-1 with picomolar affinity. Recombinant expression in E. coli of this nanobody equipped with a C-terminal (His)6-tag, followed by direct radiolabeling using 99m Tc(CO) allowed SPECT imaging in apoliprotein E-deficient mice (ApoEÀ/À). It showed a clear signal of atherosclerotic plaques in the aortic arch. This nanobody is an example of a potentially powerful tool for cardiovascular prognostic and diagnostic use.
Other examples of diseases/conditions imaged using nanobodies for SPECT include diabetes, 77,78 liver inflammation, 79,80 rheumatoid arthritis, 81,82 Gelsolin amyloidosis, 83 imaging of immune cells, 84 or more generally immune checkpoints in cancer. 85 Nanobodies used for SPECT imaging, labeled with 99m Tc, are summarized in Table 1 and show the breadth of nanobodies deployed for such studies.
While these preclinical examples show the utility of nanobodies for SPECT imaging in mouse models, their application for in vivo imaging in humans is not as straightforward. Nanobodies, because of their non-human origin, might induce an immune response when applied in clinical settings. ''Humanization'' of nanobodies is usually considered to advance their application to the clinic. Vaneycken et al. described an interesting approach in which they genetically grafted the CDRs of an anti-CEA nanobody, NbCEA5, onto a humanized nanobody scaffold (Fig. 5). 73 The ''transplant'' was successful, and the grafted nanobody displayed imaging properties similar to the original. This promising work opens the door to wider application of nanobodies in a human setting.
4.1.2. PET imaging using nanobodies. PET is considered the more attractive non-invasive imaging modality over its single-photon cousin, owing to its higher spatial resolution (usually 2-3 mm or lower for PET), higher sensitivity (typically 10 À8 to 10 À10 M tracer concentrations for PET, compared to B10 À6 M for SPECT), and the wide availability of positronemitting radioisotopes. 90 In the context of immuno-oncology, nanobodies and PET have been partnered to investigate the expression and tracking of immunologically relevant molecular targets, such as effector molecules, immune cell populations, and checkpoint molecules within the tumor microenvironment (TME). The application of nanobodies to cancer-relevant models, both for imaging and therapeutic purposes has been reviewed recently, 9,23,91,92 but some highlights are presented in the following.
Nanobody-based PET tracers that target cancer biomarkers, such as human epidermal growth factor receptor 2 (HER2), 33,93,94 HER3, 57 CD20, 95 epidermal growth factor receptor (EGFR) 34 and hepatocyte growth factor (HGF), 96 have been used as imaging agents to evaluate the presence or absence of these biomarkers in preclinical models. The results from these studies showed high tumor-specific uptake of the nanobodybased probes into the TME. A major step towards clinical translation of nanobody-based PET tracers was the phase I clinical trial of 68 Ga-NOTA-labeled anti-HER2 nanobody in breast carcinoma patients. 97 PET/CT images taken at 90 min post-injection were chosen as the optimal imaging time point, showing less background than at earlier time points. Irrespective of non-specific accumulation of tracer in the liver, kidneys

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RSC Chemical Biology and intestines, tumor-specific uptake was above background (Fig. 6). In this clinical trial, Keyaerts et al. achieved safe, noninvasive visualization of HER2 status of both primary lesions and/or metastases. A phase II study of this tracer is underway (NCT03331601). In addition to imaging the tumor's antigen profile, monitoring the immunological landscape in the TME could provide insight into the prognosis of a therapeutic response. Composition of the myeloid compartment, other immune cell types, cellular distribution and activation status could all be monitored A nanobody against macrophage mannose receptor (MMR, CD206), a marker for M2-polarized macrophages the presence of which correlates with an immunosuppressive TME and poor prognosis, showed the presence of tumor-associated macrophages (TAMs) in preclinical models. 98,99 A phase I clinical trial with 68 Ga-NOTA-labeled anti-MMR nanobody is ongoing (NCT04168528). Nanobody-based PET tracers that target CD11b and major histocompatibility complex (MHC) class II positive cells, can track immune infiltrates in both xenogeneic and syngeneic tumor models. 100 In line with these findings, an anti-human MHC class II nanobody detects inflammation in a humanized graftversus-host disease mouse model. 101 The distribution of cytotoxic CD8+T cells could play a pivotal role in predicting the outcome of immune checkpoint blockade (ICB) therapy. Rashidian et al. demonstrated promising results in tracking CD8+T cell responses to ICB in preclinical models, using a 89 Zr-labeled anti-CD8 nanobody. 56,102 Indeed, PET imaging showed a positive correlation between homogeneous  distribution of CD8+T cells and responders to ICB therapy (Fig. 7). Similar to tracking of immune cells, non-invasive imaging of the targets of ICB may lead to better understanding of the intratumoral immunological landscape. Nanobodybased PET tracers against PD-L1 and CTLA-4, developed by Ingram et al., showed distribution of checkpoint molecules in melanoma. 103,104 In yet another example, the extracellular matrix (ECM) of tumors is a critical determinant for understanding the response to therapy. An anti-EIIIB nanobody that recognizes an alternatively spliced variant of fibronectin in the ECM was developed and used for PET imaging of primary, metastatic and fibrotic lesions in preclinical cancer models. 105 An overview of some of the nanobody-based PET tracers is provided in Table 2.

Infra-red imaging in vivo
Infra-red imaging is less sensitive than SPECT and PET. The amount of probe that must accumulate in the targeted area in order for it to be visible is much larger than that for SPECT and PET. Yet, recent development of fluorescent dyes in the infra-red and near infra-red region has led to the emergence of optical imaging as a viable technology for in vivo imaging. 106-108 IR/NIR imaging is flexible, sensitive, fast and relatively inexpensive compared to the radiolabeling approach for SPECT and PET, not to mention the advantage of avoiding radioactivity altogether. 9 Nanobodies conjugated to such dyes are now being used for in vivo imaging. 23,48,[109][110][111][112] For example, Bannas et al. used a nanobody (s+16a) against the toxin-related ADPribosyltransferase ART2, an enzyme present on the surface of mouse T cells, to image lymph nodes in ART2-TG and ART2À/À mice using AlexaFluor 680. 50 For comparison, they also compared images of the cervical and axillary lymph nodes obtained with a nanobody-Fc fusion (s+16mFc) and the monoclonal antibody Nika102 (Fig. 8). Although all three constructs show in vivo fluorescence of the lymph nodes, s+16a shows no

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background signal, while strong background fluorescence was observed for Nika 102 and s+16mFc, particularly visible in the ART2À/À mice (Fig. 8).
Light scattering and light absorption by tissues limits the depth of penetration for NIR/IR imaging, ranging from microns to a few centimeters only. 113,114 Bleaching and blinking of fluorescent dyes also diminish the imaging signal strength over time. For these reasons, NIR/IR in vivo imaging is not suitable for whole-body imaging. Other systems that overcome these limitations are being developed and are being used for in vivo imaging. One such example is single-walled carbon nanotubes (SWCNTs). These nanomaterials present ideal optoelectronic characteristics for imaging in the IR region by emitting in the 850 to 1700 nm region. 115 This spectral window is highly beneficial for biological and especially deep-tissue imaging, due to its reduced absorbance, phototoxicity, reduced background fluorescence and scattering as compared to traditional fluorescent dyes. SWCNTs do not bleach or blink, allowing longer exposure and imaging times. Mann et al. successfully used this type of imaging agent by conjugating it to a GFPspecific nanobody. To link SWCNTs to the nanobody a two-step process was deployed, using a maleimide-bearing oligonucleotide that wrapped itself around the SWCNT, followed by conjugation to the nanobody bearing a free cysteine residue. This construct was then used to monitor and track GFP-modified Kinesin-5 motor proteins in a Drosophila embryo (Fig. 9). 116 One promising area where NIR/IR imaging may find application is in the context of intraoperative imaging to discriminate Fig. 8 Fast clearance of nanobodies allows rapid in vivo imaging after injection. ART2-TG mice and ART2À/À mice were injected with: buffer (control), 50 mg s+16a680, 5mg s+16mFc680 or 10 mg Nika102680. Control and antibody-injected mice (2 and 24 h after injection) were subjected to simultaneous imaging to obtain visually comparable images. Arrowheads indicate cervical and axillary lymph nodes. Adapted from ref. 50.  23 This has dramatically improved surgery for removal of liver metastases, ovarian, cervical and breast cancer and melanoma. 111 Incomplete tissue penetration, which limits nanobody applications for whole body imaging, is less of a problem in this set-up. Indeed, nanobodies have been successfully used for such applications. 23,48,[110][111][112] Van Brussel et al. used a CAIX-specific nanobody, B9, modified via maleimide chemistry with a IRDye800CW dye. CAIX, carbonic anhydrase IX, is a tumor-specific membrane-bound protein expressed in hypoxic tumors. 117,118 Using a xenograft breast cancer mouse model of a ductal carcinoma in situ and mimicking a surgery set-up, strong accumulation of B9 was seen in the tumor, well differentiated from surrounding healthy tissue (Fig. 10A). 110

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Another example reported by van Driel et al. used a nanobody that targets EGFR, 7D12, conjugated to IRDye800CW for the imaging of a tongue tumor (Fig. 10B). Not only were they able to clearly identify the primary tumor, the imaging agent also detected cervical lymph node metastases. 48 Although NIR/IR in vivo imaging might not be as sensitive as the radiolabeling methods, it represents an appealing alternative, especially in the case of intraoperative imaging for cancer surgery. A summary of the different nanobodies used for NIR/IR in vivo imaging and for intraoperative imaging is shown in Table 3.

Conclusions
Here we reviewed recent advances in the field of molecular imaging using nanobody-based probes. The unique properties of nanobodies make them ideal targeting agents, and not just for in vivo imaging. Their rapid clearance from the circulation, their tissue penetration properties, and their low background retention enable highly specific imaging at early time points after administration, even on the same day, with a reduced risk of nonspecific toxicity. Unfortunately, this advantage over the regular antibodies leads generally to an important kidney uptake. However, methods, such as PEGylation, are emerging to overcome this challenge. Although most of the applications reported to date using nanobodies have employed SPECT or PET as the imaging modality, other techniques such as infrared imaging, especially in the case of intraoperative surgery, ultrasound, 121-123 photoacoustic imaging, 124,125 and Magnetic Resonance Imaging (MRI) 126-128 deserve attention.
The results obtained to date with nanobodies for molecular imaging show that these tracers will find many applications in the laboratory as well as in the clinic. The preclinical success of nanobodies justifies further exploration and development to expand molecular imaging, ultimately with a view to clinical success.

Conflicts of interest
There are no conflicts to declare.