Chemotherapy, radiotherapy, and surgery are routine methods to eradicate tumor tissues; however, nowadays more efficient and less harmful methods are in sight. Carefully selected monoclonal antibodies (mAbs) have been shown to exert strong suppression of tumor growth. These effects are provoked by different strategies, such as a direct targeting of malignant cells, delivering cytotoxic moieties, modifying the host immune response, and retargeting the cellular immunity toward malignant cells (1, 2). Since proliferating cancer cells induce and form new blood vessels to meet their needs for nutrients, inhibition of blood vessel formation seems to be an attractive option for cancer therapy (3).

During the past decades, administration of mAbs as cancer therapeutics has increased steadily, and currently, approximately 350 mAbs have entered clinical trial programs and over 70 intact antibodies or fragments thereof received approval from FDA or EMEA for clinical applications (4). Although, the mouse hybridoma technique, developed in 1975, pioneered the identification of mAbs of defined specificity, this first generation of mAbs failed to fulfill its promises to produce therapeutics, mainly because the rodent origin of mAbs provoked severe immune responses in humans. Chimeric, humanized, and fully human mAbs were developed to remedy these immunogenicity problems and nearly all currently marketed antibodies belong to one of these types (5). Despite these improvements, the full potential of mAbs remains curtailed due to their large size, necessity to be produced in a multimeric format, high production and purification costs and their poor diffusion within tissues and the solid tumor (6). Intact functional mAbs are complex, glycosylated proteins with a molecular mass of about 150 kDa. Moreover to be used as therapeutic proteins, they have to be produced and purified in large quantities under GMP conditions. In addition, the vast majority of the administered mAbs usually remains in the bloodstream, and despite having a high specificity for a particular tumor associated antigen, they fail to reach and to associate with their target outside the blood compartment (7).

The miniaturization of mAbs to smaller antigen-binding fragments (Figure 1) avoids many of the above shortcomings as these products overcome

the necessity of using complex expression systems,

In contrast to whole antibodies, microbial expression systems manage to produce functional antigen-binding fragments in high yields. These smaller antigen-binding fragments are produced successfully at an industrial scale in microbial systems (bacteria, yeasts, and fungi), providing access to a faster and larger production of a safer drug at a lower cost. So, antibody fragments including the antigen-binding fragment (Fab ~55 kDa), the single chain variable Fragment (scFv: 26–30 kDa), and single-domain antibody fragments (sdAb ~12–14 kDa) have been expressed from bacteria and yeasts for therapeutic purposes and are indeed being evaluated in clinical trials (8, 9).

The search for smaller antibody fragments eventually led to the development of engineered sdAbs consisting of the variable fragment (about 110 amino acids) of heavy or light chain immunoglobulin polypeptides. These sdAbs from human origin as engineered originally at Domantis (Cambridge, UK, now part of GlaxoSmithKline) may overcome immunogenicity issues and could be administered orally, by inhalation or topically in a gel or cream (10).

Remarkably, an alternative to human immunoglobulin to generate sdAbs was proposed from the serendipitous discovery of heavy chain-only antibodies (HCAb) in sera of camelids. These are naturally occurring, functional antibodies, devoid of light chains, and without the first constant domain, the CH1 (11). They recognize the antigen by virtue of the variable domain of the heavy chain of heavy chain-only antibodies (known as VHH). The recombinant VHH was later on also referred to as nanobody (Nb), because of its dimensions in the low nanometer scale (MW: 12–14 kDa) (12). The amino acid sequence of VHH (as occurring in camelids) is close to sequences of family 3 of human VH and up until now Nbs are not generally immunogenic in patients participating in clinical trial projects (13).

The differences between VH from classical antibodies and VHH from HCAbs in the framework region 2 (FR2) and in the length of the CDRs (complementary determining region) are well established (14). Large hydrophobic amino acids in FR2 of the VH domain that associate with the VL (variable light chain), are substituted in VHHs by smaller and/or more hydrophilic amino acids. These mutations increase the solubility and stability of autonomous VHHs in absence of partner VL domains (14). The hypervariable region that overlaps with CDR1, is extended by four more amino acids toward the N terminal end and this region in a VHH is probably involved directly or indirectly in antigen recognition. Likewise, the CDR3 is on average longer in a VHH than in a VH and part of it can form a protruding loop contacting grooves or concave epitope architectures on the surface of the antigen (15–17). These longer CDR1 and CDR3 loops are regularly connected through an interloop disulfide bond (in camels or dromedaries), which might further contribute favorably to the stability of the domain under stringent conditions such as elevated temperature or pH extremes (18).

Among the main properties of Nbs, we note their

small size (1/10 of intact conventional antibody),

Each of these characteristics leads to a number of beneficial outcomes (14) and their robustness even allows gut passage of orally taken Nbs to reduce the morbidity of rotavirus infected animals (19).

The small size and monomeric behavior of Nbs facilitates their gene manipulation (Table 1) and assists their penetration into tumor tissues where a prevalent high pressure of the interstitial space prevents the transport of larger molecules (whole antibodies) (16). Although the monomeric Nbs are rapidly cleared from the blood via the kidneys (half-life of around 30 min), nonetheless, a high tumor accumulation can be reached (20). The conjugation of Nbs with nuclides or dyes generates tracers for usage in non-invasive, in vivo imaging of tumors for diagnosis or to monitor the therapeutic treatment (21). Furthermore, the monovalent Nb can be easily manipulated to form bivalent, multivalent, bispecific, or bi-paratopic constructs. The fusion of a Nb to another Nb with specificity to albumin increases its half-life blood retention from less than 30 min to 2–3 days (22).

The natural source of Nbs, in addition to their unique properties have attracted a lot of attention and many research groups are currently developing new Nbs as candidates for therapeutic and diagnostic applications (Table 1). Nb ALX0061 and Nb ALX00171 against interleukin 6 receptor and respiratory syncytial virus (RSV) for treatment of rheumatoid arthritis and RSV infection might be on the market soon (8). In addition, several Nb-based therapeutic agents against TNF-α, IL17A (26), VEGF/angiopoietin-2 (Ang-2) (27), CXCR1, CXCR2 (28), vWF (29), RANKL (30) applicable in autoimmune disease, malignant disease, inflammation, hematopoitic disorders, and bone disorder, respectively, are at various stages of clinical trials.

The aforementioned beneficial properties support Nbs as potent agents in targeting a wide variety of disease-related antigens, especially those related to cancer. At present there are many more active projects on the identification and development of new Nbs against cancer specific antigens.

Angiogenesis is the physiologic pathway whereby new blood vessels are formed from existing vessels. These new vessels are induced by various stimulators such as hypoxia, vessel damage, or angiogenesis growth factors that act as environmental triggers (37). This process needs to be controlled under strict conditions and each disturbance in its balance might cause pathologic distress such as tumorigenesis. Tumor angiogenesis is one of the main properties of cancer cells whereby new blood vessels are formed in the vicinity of the tumor so that tumor cells are supplied with the required oxygen and nutrients. Therefore, upregulation of angiogenesis factors stimulates tumor growth and metastasis. Indeed, some of the angiogenic modulators like members of the vascular endothelial growth factor (VEGF) family and the VEGF receptor (VEGFR) family have a direct role in both the pysiological and the pathological conditions (38) (Figure 2).

In the VEGF/VEGFR signaling pathway, the ligands, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PLGF), interact with membrane bound tyrosine kinase receptors VEGFR-1 (FLT-1), VEGFR-2 (FLK-1/KDR), and VEGFR-3 (FLT4) (Figure 2). VEGFs also bind to particular co-receptors, including neurophilin NRP-1 and NRP-2. The association of VEGF-A (known as VEGF) to VEGFR-2 has been discovered to be a key mediator of angiogenesis. VEGF-A, which is expressed in many human tumors, triggers a number of intracellular signaling cascades in endothelial cells leading to formation and enhancement of tumor microvasculature (39). A variety of factors like AKT, Raf, P13K, MEK, and ERK may be involved in the molecular mechanism of the intracellular signaling pathways of angiogenesis (40).

Furthermore, several studies revealed that tumor tissues express additional factors, such as cancer-associated antigens, that have indirect effects on angiogenesis. Tyrosine kinase inhibitors or mAbs, targeting these angiogenic factors are currently used in the clinic. Despite their strong inhibitory potential of angiogenesis, they offer only limited success in treating cancer patients due to the defense mechanisms of the tumor to escape and to resist the anti-angiogenesis therapy, for example by overexpressing other angiogenesis factors (41).

In the following sections, we will discuss in detail the Nbs that target the angiogenesis factors.

Tumor angiogenesis involves a complex network of interactions. It has been demonstrated that some of the transmembrane proteins, such as tyrosine kinase receptors, are one of the best options for Nb targeting. Here, the prevention of ligand association on the tyrosine kinase receptor by Nbs and avoiding the intracellular cascade signaling is the main objective. Some receptors have several extracellular domains [e.g., epidermal growth factor receptor (EGFR), HER2, c-Met] and thus expose multiple potential epitopes for Nb recognition. Possibly, targeting these epitopes might lead to subsequent internalization of the associated molecule inside the cell, which might be an effective route to transport a drug, toxin, nuclide, or any other harmful substance inside cancer cells. Alternatively, Nbs against the ligand of the transmembrane receptor is also a feasible strategy, especially when the signaling pathway is activated by only one ligand. The effectiveness of this approach was exemplified with Nbs against hepatocyte growth factor (HGF) that activate c-Met, but fail to interfere with the EGFR signaling as this receptor is activated by multiple ligands (12).

The high potential of Nbs as magic bullets that can interfere with angiogenesis are expected to bridge easily the gap from bench to clinic. The targeting of angiogenesis with Nbs has been explored to reach various outcomes. For therapeutic objectives, a clear inhibition of tumor growth and metastasis is required, so that Nbs against the receptor or against the ligand can be employed. For diagnosis and monitoring the treatment follow up, the primary target will be the tumor receptor. For these purposes, the Nb itself should likely be manipulated to improve its potential. These modifications include (i) increasing the valency of Nbs via designing bifunctional, bispecific, or bi-paratopic constructs and (ii) increasing the half-life of Nbs either by increasing their hydrodynamic volume (e.g., PEGylation) or by hooking on an abundant compound with long half-life (e.g., decorating the therapeutic Nb directly with albumin or tethering a Nb against serum albumin to the therapeutic Nb). Currently, there are several Nbs that reached different clinical stages and most of these are multivalent Nbs, such as Caplacizumab (28), Vobarilizumab (42), ALX0171 (43), Ozoralizumab (44), and ALX0761 (45).

Vascular endothelial growth factor, because of its strong effect on angiogenesis stimulation, is the main target for development of strategies in anti-angiogenesis therapy. Bevacizumab (Avastin, Genentech, San Francisco, CA, USA), a humanized anti-VEGF-A mAb and potent anti-angiogenesis agent has been approved since 2004, along with other chemotherapeutic drugs (46). Apart from tumor therapy, the VEGF antibody and its antibody fragments have been used to combat other angiogenic disorders, such as age-related macular degeneration (AMD). Aflibercept ((Eleya), Regeneron and Sanofi Aventis, Bridgewater, NJ, USA) is a fusion protein consisting of the VEGF-binding regions of the extracellular domains of human VEGFR-1 or VEGFR-2 fused to the Fc regions of human immunoglobulin G1. This hybrid molecule could block VEGF-A, B, and PLGF in wet AMD and diabetic macular edema and is also being investigated for retinal vein occlusion (46).

Vascular endothelial growth factor is also a major target for development of domain antibodies. Recently, a VEGF dual domain antibody has been reported that seems to be more effective than Avastin or Aflibercept (47). Several studies indicated favorable targeting properties for molecules that combine high affinity and a small molecular size (48).

Following another strategy, Ablynx developed a tri-specific humanized Nb for targeting angiogenesis. In their complex, a Nb against VEGF, a second Nb against Ang-2 and a third Nb against serum albumin for half-life extension are combined. The VEGF and Ang-2 are cross talking and the VEGF upregulates expression of Ang-2. The dual targeting Nb (BI1836880) developed by Ablynx and Boehringer Ingelheim inhibits the VEGF and Tie-2 (the Ang-2 receptor) signaling and prevents the proliferation of endothelial cells. In different in vivo models, this Nb construct seems to be superior in efficacy in comparison to Avastin (49).

In one of our studies, we could select VEGF-binding Nbs with specificity for VEGF-121, an isoform of VEGF-A. This Nb inhibits proliferation and tube formation of human umbilical vein endothelial cells (HUVEC) (50). Farajpour et al. developed a Nb against VEGF-165, this binder not only blocked interaction of VEGF with its receptor in cell ELISA but could also prevent significantly the proliferation of HUVEC in a dose-dependent manner (51). Ebrahimzadeh et al. also developed a Nb (VA12) against VEGF, which exhibited high affinity (3 nM) and stability along with significant anti-angiogenesis potential in a chorioallantoic membrane (CAM) assay (52). The CAM assay is an in vivo angiogenesis model of fertilized chicken eggs, used for studying the neovascularation (38).

The VEGFR-2, type II of transmembrane tyrosine kinase receptor, expressed on endothelial cells and on circulating bone marrow-derived endothelial progenitor cells, is a key receptor in tumorigenesis. The importance of VEGFR-2 signaling in tumor angiogenesis suggests that targeting of this receptor would be a useful therapeutic strategy to inhibit angiogenesis and tumor growth. Ghavamipour et al. developed a set of high-affinity Nbs (K_D down to 0.6 nM) against a conformational epitope corresponding to the VEGF-binding domain of VEGFR-2. To increase the chances to retrieve Nbs with this specificity, a combinatorial screening strategy was applied employing a competition phage ELISA panning (53).

In 2012, Behdani et al. reported the identification of an Nb against VEGFR-2 after phage display and panning on immobilized extra cellular domain of VEGFR-2 (54). VEGFR-2, in contrary to VEGFR-1 is switched on in pathologic conditions such as tumorigenesis. In this study, the Nb could inhibit tube formation of HUVEC cells. The Nb was used to develop pseudo-lentiviral vectors for potential transductional targeting of tumor vasculature (55). In another study, Ma et al. selected a high quality Nb against VEGFR-2 domain 3 (VEGFR-2 D3) after panning with antigen in solution. The affinity, as measured by surface plasmon resonance (SPR) indicated a moderate K_D of 49 ± 1.8 nM for VEGFR-2 domain 3. Nevertheless, this Nb could inhibit the growth and tube formation of HUVEC cells (56).

Placental growth factor is a member of the VEGFs family. This factor is responsible for physiologic angiogenesis but it is also overexpressed in many cancers. Multiple studies have been reporting the anti-angiogenesis effect of PLGF targeting by applying mAbs (57, 58), gene inactivation methods, and antagonist peptides (59). Studies have shown how this factor and its receptor (VEGFR-1) are upregulated in tumor tissues. The mAb against PLGF had inhibitory effects on growth and metastasis in different tumor tissues. This mAb could enhance the effect of chemotherapy agents (57). Currently, a fully human mAb against PLGF called TB-403 entered clinical trials. The results of a phase I dose-escalation study of this humanized anti-PLGF mAb (TB-403, ThromboGenics/BioInvent) plus Bevacizumab in patients with advanced solid tumors were reported. The toxicity profile of this mAb plus Bevacizumab showed promising results (60). Bevacizumab is the first approved angiogenesis inhibitor which has been used for the treatment of metastatic colorectal cancer by inhibiting VEGF-A, one of the other members of VEGF family (61). Recently, our group reported the successful development of a Nb against PLGF. This Nb inhibited the proliferation, migration, and invasion of endothelial and breast cancer. The results of the CAM assay demonstrated the inhibitory role of the Nb against vascular formation in the chicken CAM (38).

Apparently, Nbs can efficiently block their target antigen in cases where cell killing by Fc mediated effector function is not required. This is probably similar to the activity of Lucentis (Ranibizumab) that also acts in absence of any Fc region (62).

Table 2 gives an overview of studies investigating the effects of Nbs on VEGF, VEGFR, PLGF, HER2/HGF, and EGFR families.

Although there are a large number of angiogenesis inhibitors, remaining clinical problems, including resistance from the tumor microenvironment, enhanced tumor hypoxia, and reduced delivery of chemotherapeutic agents, have curtailed their full therapeutic potential. Given that mAbs have shown considerable success in tumor-targeted therapies during the past couple of decades, an increasing focus is now going toward remediation of the therapeutic limitations of antibodies, such as those related to their large and complex structure. The introduction of Nbs, a single-domain antigen-binding fragment has demonstrated that they can overcome some drawbacks of intact antibody-based cancer therapeutics and diagnostics. The Nb seems to become a promising therapeutic agent for cancers. Their angiogenesis inhibiting potential can be employed in various ways—as receptor antagonist, by targeting effective epitopes, and by decorating nano-carrier surfaces (17, 109). The small size of Nbs is highly suitable for generating tracers for non-invasive in vivo diagnosis of malignant cells. In targeting angiogenesis, Nbs should be equipped with a half-life extension moiety to increase the tumor loading capacity. The recognition of distinct epitopes that is often inaccessible for classical antigen-binding fragments might also broaden their therapeutic applications. Despite the challenges related to the efficacy of Nbs resulting from the lack of natural effector functions as present in the Fc region, the Nb holds promises, both for diagnosis and therapy of cancer.

All authors listed have made a substantial intellectual contribution to write the text and have approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JT and handling editor declared their shared affiliation.