Nanobodies as versatile tools: A focus on targeted tumor therapy,tumor imaging and diagnostics

Abstract

Monoclonal antibodies and vaccines have widely been studied for the immunotherapy of cancer, though their large size appears to limit their functionality in solid tumors, in large part due to unique properties of tumor microenvironment. Smaller formats of antibodies have been developed to throw such restrictions. These small format antibodies include antigen binding fragments, single-chain variable fragments, single variable domain of camelid antibody (so-called nanobody (Nb) or VHH). Since their serendipitous discovery, nanobodies have been studies at length in the fields of research, diagnostics and therapy. These antigen binding fragments, originating from camelid heavy-chain antibodies, possess unusual hallmarks in terms of (small) size, stability, solubility and specificity, hence allowing cost-effective production and sometimes out performing monoclonal antibodies. In addition, these small camelid heavy-chain antibodies are highly adaptable tools for cancer research as they enable specific modulation of targets, enzymatic and non-enzymatic proteins alike. Molecular imaging studies benefit from the rapid, homogeneous tumor accumulation of nanobodies and their fast blood clearance, permitting previously unattainable fast tumor visualization. Moreover, they are endowed with considerable therapeutic potential as inhibitors of receptor-ligand pairs and deliverers of drugs or drug-loaded nanoparticles towards tumors. In this review, we shed light on the current status of nanobodies in diagnosis and imaging of tumor and exploiting nanobodies revert immunosuppressive events, modulation of immune checkpoints, and as deliverers of drugs for targeted tumor therapy.

Keywords

Tumor immunotherapy nanobodies tumor microenvironment single chain antibody tumor diagnostics molecular imaging

1. Introduction

Table 1
Advantages of nanobodies over conventional monoclonal antibodies

Nanobody properties	Advantages	Applications
Small size (15 kd)	Increased blood clearance and high tissue infiltration.	Tumor therapy and in vivo imaging. Toxin neutralization.
Hydrophilic nature	Allows for high expression yields. High solubility. Proteolytic stability.	Easy production. Intrabody production. Oral immunotherapy.
Single-domain nature	Absence of domain mispairing that allows for increased functional size of immune libraries.	Single-cell oligoclonal production.
	Allows for flexible linker design and simultaneous binding of multivalent agents.	Easy production of bispecific or trivalent forms.
	Production in microbial systems, taking advantage of simple fermentation conditions, and offering short development times.	Cost-effective large-scale production.
Sequence stability	Facile genetic manipulation to yield fused moieties with high solubility and low aggregation potential (decreased immunogenicity).	Selective tumor targeting. Selective in vivo imaging.
	Allows for a variety of treatments, storage and formulations.	Harsh engineering.
	Low immunogenicity. Permits repeated or high-dose treatments.	Harsh engineering. Optimized drug manufacturing.
CDR3 loops extended	Effectual refolding after denaturation. High thermodynamic stability.	Harsh engineering. Optimized drug manufacturing. Oral immunotherapy.
	Recessed antigenic sites permits enzyme inhibition and recognition of cryptic epitopes.	Rapid in vivo or in vitro diagnosis of infection. Molecular research.
Variable N-terminal of CDR1	Penetration through the blood-brain barrier.	Management of neurological disorders.

Figure 1.

Representation of a heavy-chain antibody (HcAb) and its antigen binding fragment, called nanobody. A. In comparison to a monoclonal antibody (mAb), which includes two heavy and two light chains, a HcAb only contains heavy chains. Moreover, HcAbs also lack one constant domain, the antigen binding region only consists of a single fragment, called a nanobody. The tail region of the antibodies forms the Fc part and is able to trigger the immune system. B. Schematic representation of the nanobody entity, composed of framework regions (FR1–4) alternated with three complementary determining regions (CDR1–3). Mutations in FR2 region renders the structure more hydrophilic as compared to conventional antibody fragments. Moreover, the CDR3 loop is extended and enables recognition of hidden or buried epitopes.

Table 2

Summary of therapeutic nanobodies in clinical trials

Target	Nanobody	Company/University	Disease	Clinical status	References
VWF	Caplicizumab	Ablynx-Sanofi	aTTP	Approved	NCT02553317
HER2	68-GaNOTA	Universitair Ziekenhuis Brussel	Breast cancer	Phase II	NCT03331601
ADAMTS5	M6495	Ablynx-Merck	Osteoarthritis	Phase I	NCT03583346
TNF	Ozoralizumab	Ablynx-Pfizer	Rheumatoid arthritis	Phase II	NCT01007175
IL-6R	Vobarilizumab	Ablynx-AbbVie	Systemic lupus	Phase II	NCT02437890
BCMA	LCAR-B38M	Nanjing Legend Janssen Biotech	Multiple myeloma	Phase II	NCT03758417
Angiopoietin-2, VEGF	BI 836880	Ablynx-Boehringer Ingelheim	Solid tumors	Phase I	NCT02674152
HER2	[131I]-SGMIB	Camel-IDS	Breast cancer	Phase I	NCT02683083
	Anti-HER2 VHH1

Monoclonal antibodies and vaccines have been extensively studied for the diagnostics and treatment of many diseases including cancer [1, 2]. The large size though limits the function of monoclonal antibodies in solid tumors, in large part due to unique properties of tumor microenvironment such as high pressure of tumor interstitial fluid [3]. In order to tackle such limits, smaller formats of antibodies have been developed, including antigen binding fragments, single-chain variable fragments, single variable domain of camelid antibody (so-called nanobody (Nb) or VHH) Fig. 1 [4, 5]. The existence of efficient heavy-chain-only antibodies in the serum of camelids (dromedaries, camels, llamas, alpacas, guanacos and vicuñas) was revealed serendipitously nearly 30 years ago [6]. These heavy chain-only antibodies (HCAbs) display alike affinity to their cognate antigen, even though these have only one single variable domain (VHH) for antigen recognition [7]. Studies have revealed that these autonomous VHH are the smallest natural intact antigen-binding fragment and retain their full antigen-binding potential [8, 9]. Nanobodies have a very small size (15 kDa) and dimensions in the nanometer range ( $\sim$ 2.5 nm in diameter and $\sim$ 4 nm in height) and have received immense interest for diagnostics and in therapeutics in tumour biology (Table 1). Furthermore, these single domain antibodies are highly stable, and due to the small size show large tissue permeability demonstrating advantageous physicochemical and pharmacological features [10]. Nbs offer multiple applications in therapeutics including tumor immunotherapy, fundamental research, and diagnostics. Less than 30 years after the discovery of functional heavy-chain-only antibodies, the nanobody derivatives are already extensively used by the biotechnology research community. Besides, Nbs can be modified to improve their functionalizations and the modifications include PEGylation and conjugation to the Fc domain, peptide tags, drugs, toxins, aptamers, and radionuclides [7]. The immense efforts in the Nb research field underscoring the remarkable prospects of these molecules formed eventually the basis for the foundation of spin-offs such as Ablynx, Chromotek, Agrosavfe, QvQ, Camel-IDS, Hybrigenics, Confo-Therapeutics, and many other companies offering the technology of generating and selecting Nbs [8, 10]. The focus of all these companies ranges from service providers to developing therapeutic Nbs, currently tested in clinical trials with $\sim$ 9 candidates in advanced stage and more than 15 at the discovery and preclinical stage [11, 12]. Recently, caplacizumab, a bivalent nanobody, received approval from the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for treatment of patients with thrombotic thrombocytopenic purpura [13]. In addition, many nanobodies are in clinical trials (Table 2). In this review, we will shed light on the current status of nanobodies in diagnosis and imaging of tumor and exploiting nanobodies revert immunosuppressive events, modulation of immune checkpoints, and as deliverers of drugs for targeted tumor therapy.

2. Nanobodies in cancer

Cancer is considered a cluster of diseases with different molecular changes, including gene mutations and amplifications, copy number alterations, changes in tumour suppressor and DNA repair genes, and epigenetic modifications. Development of a successful tumour therapy is challenging due to low specificity of the drug and toxic effect on adjacent non-tumour cells. Besides the surrounding tumor microenvironment which include stromal components such as CAFs, TAMs TANs, Endothelial Cells etc. and non-stromal components such as ECM, etc., promote tumorigenesis by limiting drug efficacy in tumors and inducing drug resistance [14, 15, 16]. A dynamic targeted therapy relies on the specific delivery of an active drug to the target using different possible affinity reagents such as those mediated by a lectin-carbohydrate, ligand-receptor or antibody-antigen recognition [17, 18, 19]. Apparently, for maximal effect, the specific receptor targeted by the affinity reagent should be overexpressed at the surface of the diseased cells. Thus, active targeting refers to site-specific ligand-mediated accumulation of drugs into the diseased site due to an increased expression of a specific biomarker for that malignancy [20]. During the past few decades monoclonal antibodies (mAbs) have been widely used as a treatment modality for cancer therapy. The mAbs strongly diminish the growth of tumor cells by binding to their transmembrane receptors, growth factors, or cytokines, through inhibiting the cognate signal transduction or inducing apoptosis [2, 21]. However, the large size ( $\sim$ 150 kDa) and low stability of mAbs, limits their penetration ability into the cellular membrane as well the solid tumors in vivo [22]. Furthermore, mAbs have long half-life (ranging from days to up to 4 weeks) which results in high background levels during molecular imaging. The variable fragments of Camelid heavy-chain only antibodies (HcAbs), called nanobodies, may provide an answer to several of these concerns and to date, various multifunctional nanomedicines have been used to treat cancer [23, 24]. Since their serendipitous discovery 30 years ago camelid heavy-chain antibodies, have received a progressively growing interest [7]. As a result of the beneficial properties of these stable recombinant entities, they are currently highly valued proteins for multiple applications, in tumour biology including fundamental research, diagnostics, and tumor therapeutics. In the following sections we will describe the therapeutic potential of nanobodies in stimulation of T-cell mediated anti-tumor immune response, modulation of immune check points using nanobodies and nanobodies in drug delivery.

Table 3
Bispecific nanobodies in targeted tumor therapy

S. No	Target	Nanobody	Clinical status	References
1.	Muc-1 and CD16	Muc1-Bi-1	Preclinical	[29]
2.	EGFR & CD3	EgA1 VHH	Preclinical	[41]
		LiTE	Preclinical	[28]
3.	HER2	Nanocar	Preclinical	[31]
4.	EGFR	7D12-9G8-Alb	Preclinical	[32]

Figure 2.

Antibody fragments with therapeutic potential. A conventional antibody comprises two heavy and two light chains while a nanobody or HcAb contains heavy chains only. Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. BsAbs combine specificities of two antibodies and simultaneously address different antigens or epitopes. At present, three forms of bi-specifics exist with definite benefits viz., the bi-specific T-cell engager (BiTE), dual-affinity retargeting proteins (DARTs), and tandem diabodies (TandAbs). Abbreviation: bsAb, bispecific antibodies; bsFab, bispecific Fab fragment; HcAb, heavy chain only antibodies.

Figure 3.

Schematic depiction of the bridging of B-cells and CD8 $+$ T-cells by Blinatumomab. Blinatumomab is a BiTE nanobody comprised of two scFv fragments, one specific for CD19 of B cells and the other ScFv fragment specific for CD3 receptor of CD8 $+$ T-cells. Bridging of the two cells viz., B-cells and T-cells by the nanobody blinatumomab induces secretion of cytotoxic granules in T-cells and subsequent activation of pro-apoptotic caspases and induction of apoptosis in B-cells.

2.1 Nanobodies in T-cell activation for cancer immunotherapy

T-lymphocytes and their subsets are crucial component of the adaptive immune system and demonstrate significant antitumor activity [25]. T-cells secrete granzymes and perforins, thereby induce tumor cell death and chemokines promoting recruitment and activation of other immune cells to counter tumor activities [25]. Conversely, tumor cells and the surrounding living and non-living stroma secrete factors such as chemokines etc. promoting suppression of T-lymphocytes to supress the anti-tumor immune response [26]. Nevertheless, many therapeutic approaches involving nanobodies have been employed to offset the tumor induced inactivation and suppression of T-cells. Studies revealed that the checkpoint inhibitors in combination with bi-specific antibodies promotes T-cell linkage with tumour cells which reduces tumour mass and tumor induced immune suppression [27]. Several nanobodies have been thus developed to target tumor tissues (Table 3) [28, 29, 30, 31, 32]. At present, three forms of bi-specifics exist with definite benefits viz., the bi-specific T-cell engager (BiTE), dual-affinity retargeting proteins (DARTs), and tandem diabodies (TandAbs) Fig. 2 [33]. BiTE nanobodies (such as Blinatumomab) are comprised of two scFv fragments and were developed to bridge T-cells with tumour cells. Among the two scFv fragments, one scFv fragment binds T-cell markers (such as CD3) while the other scFv fragment binds antigens on the tumor cells, in this manner bridging tumour cells and T-cells [34]. Blinatumomab is an FDA approved nanobody, developed to treat the aggressive leukemia (B-cell lymphoblastic leukemia (ALL)) [35]. The nanobody was FDA approved and binds T-cells (CD3) and B-cells (CD19) markers connected with a glycine-serine linker and demonstrated significant efficacy Fig. 3. The main problem with blinatumomab is its short half-life, and therefore entails high therapeutic dosage to reach requisite T-cell activation [36].

To overcome the short half-life of BiTEs, Dual affinity retargeting proteins (DARTs) have been developed as an alternative. Unlike BiTEs, DARTs have hetero-dimerised Fv fragments, with the VL of target A and VH of target B on Fv1 fragment and the VL of target B and VH of target A on the Fv2 fragment [37]. These combinations mimic the natural IgG molecule and have increased the half-life/serum retention of these nanobody molecules from 1.25 $\pm$ 0.63 hours (BiTE) to approx. 10 hours (DARTs) [38, 39]. Furthermore, compared to conventional BiTEs, bi-specifics, with considerably augmented stability and solubility would likely demonstrate improved efficacy. Recently, a bi-specific antibody against CD3 of T-cells and Her2 of breast cancer cells was developed. The distinctiveness of this bispecific therapeutic antibody is the combination of CD3 specific scFv-fragment and the single-domain (VHH) specific to Her2 of tumor cells [40]. Mølgaard et al. in 2018 also developed a similar construct and termed it LiTE (Light T-cell Engager). The construct developed had a scFv fragment specific for T-cell CD3 and two anti-EGFR VHH domains [28]. Both the constructs demonstrated excellent tissue penetration, significant specificity to cytolysis of tumour tissue and markedly low cytotoxicity [28]. Furthermore, novel bi-specifics have been developed to specifically target tumour tissues while bridging CD3 ${}^{+}$ T-cells which include asymmetric tandem trimer body for T-cell activation and cancer killing (ATTACK). In the ATTACK construct, CD3 specific scFv was fused with three anti-EGFR nanobodies to create a construct that exhibited improved specificity towards EGFR with a 15-fold increase in efficacy [41]. Moreover, many bi-specific-trivalent constructs and other different antibody constructs are being developed, all which confirm the potential of nanobodies with improved tumour localisation and lower off-target toxicity and side effects [30].

Figure 4.

Schematic depiction of the PD-L1 inhibiting nanobody KNO35 in the treatment of cancer. A. The tumour cells induce immunosuppression by exploiting the immune checkpoints such as PDL1/PD-1 axis. B. Inhibiting the immune checkpoints using nanobodies such as KNO35 reverts the tumor induced immunosuppression and subsequent development of anti-tumor immune response.

2.2 Nanobodies in modulation of immune checkpoints

The effector components of the immune system are regulated by immune checkpoints to prevent autoimmune diseases and induce self-tolerance. However, cancer cells in combination with tumor stroma components exploit this mechanism and evade immune surveillance to suppress the immune response and establish the tumour microenvironment [42]. T-cells and natural killer cells mainly express checkpoint receptors such as CTLA-4, TIM-3, PD-1, LAG-3, and PD-L1 [43, 44]. Several drugs, monoclonal antibodies and small molecules have been developed to inhibit immune checkpoints, promoting antitumor immune response [45]. However, the conventional antibody checkpoint inhibitors exhibit low binding affinity, low penetration, decrease stability and production [46]. KN035 nanobody developed against the PD-L1 receptor demonstrated enhanced affinity (approx. 1000 times) for the PD-L1 receptor compared to the conventional mAb [47]. KN035 binds to the PD-L1 receptor through a loop of 21 amino acids by hydrophobic and ionic interactions with Ile54, Tyr56 and Arg113 residues [48]. Binding of KNO35 to the PD-L1 receptor promotes subsequent activation of T-cells thereby inducing anti-tumor response Fig. 4. Similarly, a nanobody known as sdAbK2 was developed against PD-L1, which has a dual role in diagnosis (nuclear imaging), as well as a therapeutic agent with a binding affinity of 3.75 nM, equivalent to avelumab, an anti-PD-L1 monoclonal antibody [49]. This nanobody also showed excellent performance when labelled with technetium-99m for diagnosis and screening of PD-L1 elevated breast cancer, melanoma and kidney cancer with high signal to noise ratio [50].

Furthermore, a ground breaking approach to synthesise and deliver a checkpoint inhibitor antibody/ nanobody using probiotic bacteria was developed to overcome toxicity issues with conventional checkpoint inhibitors [51]. Besides, advances in synthetic biology facilitated the development of genetically engineered bacteria with genes for desired therapeutic proteins and a lysis circuit operon (Synchronized Lysis Circuit) [52]. These bacteria grow and replicate until a threshold population density is reached and undergo cell lysis releasing the synthesised therapeutics which can further be used as a drug delivery vehicle [53]. Similarly, Gubatri et al. developed a probiotic E. coli Nissle 1917 system expressing genes for dynamic lysis and for nanobodies against PDL-1 and CTLA-4 [54]. This system was further tested in syngeneic mice models and demonstrated significant systemic immune response with enhanced T-cell activation [54]. TIM-3 (T-cell immunoglobulin and mucin domain 3) is also a crucial component of immune checkpoints and several inhibitors have been developed to target TIM-3 during cancer therapy. In vitro studies involving novel nanobody developed to inhibit TIM-3 receptor displayed significant anti-proliferative activity in an acute myeloid leukemia cell line [55]. However, manipulating immune checkpoints can also cause substantial toxicities and off target affects that require expert management.

2.3 Nanobody drug conjugates as tumor therapy

Chemotherapy is the use of drugs to destroy rapidly growing cells in the body, however, the challenge with this approach and to a lesser extent radiotherapy is off-target effects, which leads to acute toxicity in healthy cells [56]. The combinatorial approach involving use of targeted therapy such as an antibody/nanobody with a non-targeted therapy like chemotherapy was an advance on previous approaches [57, 58]. Fang et al. used a VHH7 domain ( $\alpha$ MHC-II nanobody-drug conjugate) as a therapeutic agent, as well as an imaging tool for B-cell lymphoma [59]. The VHH7 domain, when conjugated with the thio-containing maytansine derivative, mertansine (DM1), demonstrated excellent specificity, internalisation and cytotoxicity in a murine model of A20 B-cell lymphoma and showed a decrease in tumour growth and metastasis [59]. Besides, conjugating the same VHH7 domain with near infra-red (NIR) fluorophores, exhibited excellent diagnostic properties in visualising both localised and metastatic tumour cells [59, 60].

Photo-thermal therapy is an emerging approach to induce tumour cell death by combining photosensitisers (nanoparticles) and laser light. These nanoparticles generate heat and reactive oxygen species when absorbed and excited by lasers (NIR), and induce tumour cell death. Branched gold nanoparticles are one such photosensitiser which showed promising results in killing cancer cells [61]. Bieke et al. [20] developed a nanobody conjugated with branched gold nanoparticles against human epidermal growth factor receptor 2 (HER2). These nanobodies showed high binding affinity for HER2 prominent in breast and ovarian cancers and destroyed HER2 positive SKOV3 cells within five minutes of irradiation [20]. These nanobodies may present as an effective targeted photothermal therapy. Photodynamic therapy is a similar approach where tumour cell death is induced by singlet O2 produced upon irradiation [61]. In light of this, a nanobody conjugated with IRDye700DX (photosensitizer) against EGFR was developed and anti-tumour activity was examined in an orthotopic mouse. It was observed that nanobody conjugated with IRDye700DX induced 90% tumour cell necrosis one-hour post injection [62]. Another approach to manipulate nanobodies in therapeutics is the nanobody-enzyme conjugates, which have showed promising activity in tumour elimination. cAb-CEA5: $\beta$ L, a nanobody developed by Virna et al. with an enzyme conjugate Enterobacter cloacae $\beta$ -lactamase, to target tumour-associated carcinoembryonic antigen (CEA) [63]. The enzyme can site-specifically activate the anti-tumour pro-drug 7-(4-carboxybutanamido)-cephalosporin mustard into the active drug phenylenediamine mustard [64]. The flexibility and stability was provided using a llama $\gamma$ 2c hinge for conjugation and it also exhibited prompt clearance from systemic circulation and concentration in tumours, thereby avoiding off-target toxicity [63]. The potency of this strategy warrants further studies to translate this technology from bench to bedside.

3. Nanobodies in diagnostics and imaging

In addition to their use as therapeutic agents nanobodies also make highly effective and non-invasive molecular imaging reagents and diagnostic tools due to their high penetration, quick elimination, low immunogenicity and ease of production and selection [65, 66, 67]. Early diagnosis of tumors is essential to augment odds of survival. Nbs supported by their small size, high stability, and high target specificity and affinity have been engineered into Nb-detective constructs for non-invasive in vivo molecular imaging [68, 69]. The constructs developed reach promptly a maximal contrast between signal in the pathological tissues and that in healthy tissues [66, 69]. For optimal in vivo molecular imaging, promptness in reaching maximal contrast is crucial. Furthermore, Nbs have a very short half-life and the excess of non-targeting Nbs are rapidly cleared from the bloodstream due to rapid clearance via kidney and bladder. This rapid clearance from blood assures a high tumor to background ratio at early time points after administering the Nb probe [70]. Several imaging techniques have been developed to date for clinical application and manifestation, such as single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical, ultrasound, and photo-acoustic imaging positron emission tomography (PET), computed tomography (CT) [71]. In following sections, we will be shedding light on how nanobodies can be manipulated in detecting and defining tumor markers and nanobodies in molecular imaging.

Table 4
Nanobody based probes in tumor imaging

Target	Nanobody	Imaging modality (label)	Disease model	References
HER-2	2Rs15d	SPECT ( ${}^{\text{99m}}$ Tc)	Colon carcinoma (LS174T), Breast cancer (SKBR3),	[93]
			Human ovarian cancer (SKOV3)
	2Rs15d	PET ( ${}^{\text{68}}$ Ga)	Ovarian cancer (SKOV3)	[82]
	11A4	Optical (IRDye800CW)	Breast cancer	[67]
HGF	1E2 and 6E10	PET ( ${}^{\text{89}}$ Zr)	Glioblastoma	[89]
EGFR	8B6	SPECT ( ${}^{\text{99m}}$ Tc)	Epidermoid carcinoma	[83]
		Prostate carcinoma
	7C12 and 7D12	SPECT ( ${}^{\text{99m}}$ Tc), PET ( ${}^{\text{68}}$ Ga),	Epidermoid carcinoma	[84, 85, 86, 91, 92]
		Optical (IRDye800CW)
VCAM-1	cAbVCAM1-5	Ultrasound (microbubble)	Adenocarcinoma (MC38)	[90]
CEA	CEA1	SPECT ( ${}^{\text{99m}}$ Tc)	Colon adenocarcinoma (LS174)	[88]
MMR	$\alpha$ -MMR	SPECT ( ${}^{\text{99m}}$ Tc)	Mammary adenocarcinoma, Lung carcinoma	[87]

3.1 Nanobodies and cancer biomarkers

In addition to tumor therapy, nanobodies can aid in early diagnosis and cancer prevention by identifying or defining tumor biomarkers. Furthermore, study performed by Chen et al. [72] in detection of cancer biomarker alpha-fetoprotein (AFP) guarantees that the application can withstand harsh conditions. The subtlest detection (0.0005 ng/mL) was achieved utilizing anti-AFP nanobodies in immune-PCR [72]. In vitro cell-based ELISA assays involving nanobodies developed for the detection of carbonic anhydrase IX enzyme (CAIX), tumor-associated glycoprotein 72 (TAG-72), HER2, and prostate-specific membrane antigen PMSA, demonstrated momentous outcomes [73, 74, 75, 76]. Besides, use of multiple or a mixture of nanobodies demonstrated an improved performance. In addition, nanobodies used to detect cancer biomarker alpha-fetoprotein (AFP) touched a detection limit of 0.47 ng/mL. Patris et. al. proposed a chip format sandwich ELISA with anti-HER2 nanobodies covalently bound onto a screen-printed electrode (SPE) with a detection limit of 1 $\mu$ g/mL [77]. In order to perform a sandwich ELISA, nanobodies are used for both capturing and detecting different epitopes.

The generation of single domain nanobody is less costly compared to conventional antibodies and therefore a sophisticated and dissolute approach to generate nanobodies against (unknown) cancer biomarkers is by performing immunization with patient samples. Utilising this strategy lead to in the identification of a novel breast cancer-specific biomarker, cytokeratin 19 [78]. Likewise, nanobody based reverse proteomics was exploited in glioblastoma multiforme (GBM) and further mass-spectrometry analysis on nanobody-antigen pairs, the new GBM biomarkers TRIM28 and $\beta$ -actin was revealed [79]. As all these markers are localized intracellularly, they are especially proposed for immunohistochemical-based diagnostic purposes.

3.2 Nanobodies in molecular imaging

Nanobodies have very low molecular weight compared to conventional monoclonal antibodies and have small size that is exceedingly strategic especially in the field of molecular imaging as it enables rapid tumor accumulation and homogenous distribution as well as efficient blood clearance, contributing to high tumor-to-background ratios [80]. Besides, conjugation of several kinds of imaging agents with nanobodies and their high specificity renders their use relatively safe [80, 81]. Many techniques have developed which involve use of nanobodies for imaging tumors including SPECT, PET etc. Single-photon emission computed tomography (SPECT) is a $\gamma$ -ray based technique and nanobodies are conjugated to radionuclides such as ${}^{\text{99m}}$ Tc, ${}^{\text{111}}$ In, ${}^{\text{123}}$ I and ${}^{\text{177}}$ Lu. Conversely, the positron-emitting radioisotopes like ${}^{\text{68}}$ Ga, ${}^{\text{124}}$ I or ${}^{\text{89}}$ Zr are used for positron emission tomography (PET) Table 4 [82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93]. EGFR and HER2 are two interesting targets among the cancer-specific receptors for tumor visualization [85, 94, 82, 95, 96, 97, 98].

8B6 a ${}^{\text{99m}}$ Tc radiolabelled nanobody, developed against the EGFR, is used in the radio-immunodetection of EGFR overexpressing tumours by SPECT [83]. Furthermore, several studies reported that 8B6 is very effective in the detection of human epidermoid carcinoma and human prostate carcinoma. On the other hand, ${}^{\text{89}}$ Zr labelled 1E2 and 6E10, albumin coupled nanobodies have been developed against hepatocyte growth factor (HGF), a prominent growth factor in promoting invasiveness and metastasis in several types of cancers [99]. Xenograft studies of U87 MG glioblastoma demonstrated the effectiveness of these nanobodies in visualizing HGF $+$ tumours using PET. The nanobodies when administered, demonstrate uniform tumour distribution and also suppressed tumour growth [89]. Movahedi et al., on the other hand, developed nanobody constructs against the macrophage mannose receptor MMR, highly expressed by macrophages in the tumor microenvironment, known as tumor associated macrophages (TAMs). This strategy serves as a substitute to image the tumor stroma, especially the hypoxic areas [87]. Though, it was reported that ${}^{\text{99m}}$ Tc-labeled anti-MMR nanobodies used to visualize TAMs accumulated highly in liver and spleen (expressing MMR), compared to tumor accumulation. Nevertheless, the problem was solved by administration for an excess of unlabelled bivalent anti-MMR nanobody, which eliminates extratumoral signals while maintaining the targeting of tumor associated MMR-positive cells Fig. 5 [95, 87, 91, 100].

Figure 5.

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) images obtained using nanobodies that are labelled with distinct radionuclides in preclinical and clinical studies. I. Uptake of ${}^{68}$ Ga-HER2-Nanobody in primary breast carcinoma lesions (arrows) on PET/CT images (top) and PET images (bottom). (A) Patient showed highest tracer uptake. (B) Patient showed modest tracer uptake, which was effortlessly visible from background. (C) Patient showed no uptake, with CT showing marker clip at tumor region. Adapted from [95]. II. Uptake of ${}^{68}$ Ga-HER2-Nanobody in metastatic lesions on PET/CT images (top) and PET images (bottom). (A) Patient, with invaded lymph nodes in mediastinum and left hilar region. (B) Patient with bone metastasis in pelvis. Adapted from [95]. III. SPECT and micro-CT images of transverse, coronal, and sagittal views of fused pinhole of mice bearing A431 and R1M xenografts. A431 (A) and R1M (C) tumors injected with ${}^{\text{99m}}$ Tc-7C12. A431 (B) and R1M (D) tumors injected with ${}^{\text{99m}}$ Tc-7D12. Images were acquired at 1 h after injection. Adapted from [91]. IV. Micro-SPECT/CT images obtained in HER2 $+$ tumor models BT474/M1 (A) and SKOV-3 (B), 1, 4, and 24 hours after injection of ${}^{[\text{131I}]}$ -2Rs15d. Accumulation of radioactivity was observed in kidneys, bladder, and tumor, indicated by white arrows. Adapted from [100]. V. Representative maximum-intensity-projection images at 10, 60, and 90 min after injection of ${}^{68}$ Ga-HER2-Nanobody for different mass subgroups. (A) Patient 4, injected with 0.01 mg of ${}^{68}$ Ga-HER2-Nanobody. (B) Patient 12, injected with 0.1 mg. (C) Patient 17, injected with 1.0 mg. Adapted from [95].

At present, ${}^{\text{68}}$ Ga-NOTA anti-HER2 nanobodies, established as nanobody-based imaging agent for iPET imaging is demonstrating promising outcomes [82]. Evaluation of the ${}^{\text{68}}$ Ga-coupled anti-HER2 nanobody at the preclinical stage demonstrated significant results with a good toxicological profile and a low radiation burden, enabling the construct to enter phase I clinical trials on humans [95]. The nanobody in the phase I clinical trials, recorded well in terms of efficacy, tracer accumulation. Moreover, in terms of safety, no adverse effects or immunogenicity against the administrated nanobody were detected, rendering this construct suitable to enter phase II clinical trials [95].

Recently, a study exploited the extra cellular matrix (ECM) targeting for non-invasive imaging of tumor progression, metastasis, and fibrosis using a nanobody NJB2, specific for an alternatively spliced domain of fibronectin. The study revealed that in in vivo immuno-PET/CT imaging, NJB2 detects primary tumors and metastatic sites with excellent specificity in multiple models of breast cancer, including human and mouse triple-negative breast cancer, and in melanoma. Furthermore, NJB2 was able to detect not only PDAC tumors but also early pancreatic lesions called pancreatic intraepithelial neoplasias, which are challenging to detect by any current imaging modalities, with excellent clarity and signal-to-noise ratios that outperformed conventional 2-fluorodeoxyglucose PET/CT imaging [101]. Also, OA-cb6 nanobody with ${}^{\text{99}}$ mTc (CO) 3 ${}^{+}$ developed against EGFR was evaluated in the A431 human epidermal carcinoma cell line and demonstrated significant radiolabelling and radiochemical purity. Furthermore, in vivo studies revealed favourable bio-distribution of the nanobody in tumor-to-muscle ratio at 4-hr post injection and thus suggests that cb6 nanobody with ${}^{\text{99}}$ mTc (CO) 3 ${}^{+}$ is a promising radiolabelled biomolecule for tumor imaging in cancers with high EGFR overexpression [102].

4. Conclusion and perspective

Nanobodies, nanobody-based heavy chain antibodies, and nanobody-drug conjugates have a huge potential as antitumor therapeutics. Nanobodies may overcome some of the obstacles that hamper therapies with antitumor mAbs. In vivo studies have underscored the favorable biodistribution of nanobodies, including deep penetration into tumors. Numerous nanobody-based biologics have shown antitumor efficacy in preclinical studies in vivo. Moreover, naked nanobodies can antagonize growth factor receptors and block ion channels and ecto-enzymes in the tumor microenvironment. Also, fusion of one or more nanobodies to the hinge and Fc-domains of a human immunoglobulin yields highly soluble and versatile heavy chain antibodies. Importantly, because nanobodies do not bind light chains and because they do not show any tendency to aggregate, nanobody-based bispecific hcAbs do not suffer from the VH–VL pairing problem of bispecifc conventional antibodies. Heavy chain antibodies are roughly half the size of conventional antibodies and, thus, may show better tissue penetration than conventional mAbs, while retaining the capacity to recruit the complement system, NK cells, and macrophages.

Furthermore, molecular imaging is the foundation for accurate diagnosis and pre-treatment staging of various diseases, monitoring the response to therapy and providing surveillance after therapeutic intervention. Recent advances in molecular imaging using nanobody-based probes have enhanced the possibility of high-contrast imaging of different types of tumors within much shorter time period after tracer injection, when compared to the conventional intact antibody based approaches. Nanobodies are smaller than intact antibodies and can penetrate into solid tumor tissue more efficiently, thereby representing a promising class of targeting ligands for non-invasive molecular imaging of specific targets of interest. The maturity in nanobody technology would improve our understanding of the molecular alterations in various types, subtypes and stages of cancers and other diseases, which can help the choice of suitable targeted therapeutics for each individual patient. However, although it has been more than 2 decades since nanobodies were first described, the translation of these promising imaging agents and technologies to the clinic has been very slow. Meanwhile, the development of a new diagnostic probe is an iterative process that requires substantial effort and sometimes luck. The reasons for slow clinical translation is complex and may include considerable regulatory hurdles, limited potential market, lobbying by the manufacturers of other molecular imaging probes, lack of reimbursement strategies for novel imaging agents, etc. Despite these hurdles, the exciting results obtained to date with nanobodies for molecular imaging clearly indicated that these tracers will have multifaceted applications in future clinical practices. Assuming that progress will continue at the present pace, it is likely that the future repertoire of clinicians will include an increasing battery of nanobody-based antitumor therapeutics.

Footnotes

Acknowledgments

The author is thankful to the support and assistance by the Stroke Research Chair (SATCAS) Majmaah University Almajmaah KSA.

Conflict of interest

The author declares no conflict of interest.

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Nanobodies as versatile tools: A focus on targeted tumor therapy,tumor imaging and diagnostics

Abstract

Keywords

1. Introduction

Table 1 Advantages of nanobodies over conventional monoclonal antibodies

Table 3 Bispecific nanobodies in targeted tumor therapy

2.3 Nanobody drug conjugates as tumor therapy

3. Nanobodies in diagnostics and imaging

Table 4 Nanobody based probes in tumor imaging

3.2 Nanobodies in molecular imaging

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Advantages of nanobodies over conventional monoclonal antibodies

Table 3
Bispecific nanobodies in targeted tumor therapy

Table 4
Nanobody based probes in tumor imaging