Therapeutic mAbs have delivered several blockbuster drugs in oncology and autoimmune inflammatory disease. Revenue for mAbs continues to rise, even in the face of competition from a growing portfolio of biosimilars. Despite this success, there are still limitations associated with the use of mAbs as therapeutic molecules. With a molecular mass of 150 kDa, a two-chain structure and complex glycosylation these challenges include a high cost of goods, limited delivery options, and poor solid tumour penetration. There remains an urgency to create alternatives to antibody scaffolds in a bid to circumvent these limitations, while maintaining or improving the therapeutic success of conventional mAb formats. Smaller, less complex binders, with increased domain valency, multi-specific/paratopic targeting, tuneable serum half-life and low inherent immunogenicity are a few of the characteristics being explored by the next generation of biologic molecules. One novel ‘antibody-like’ binder that has naturally evolved over 450 million years is the variable new antigen receptor (VNAR) identified as a key component of the adaptive immune system of sharks. At only 11 kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery. VNAR attributes include high affinity for target, ease of expression, stability, solubility, multi-specificity, and increased potential for solid tissue penetration. This review article documents the recent drug developmental milestones achieved for therapeutic VNARs and highlights the first reported evidence of the efficacy of these domains in clinically relevant models of disease.

Introduction

It is predicted that in 2018, biologic sales will account for almost half of the world's 100 top-selling drugs. IMS Health, the US-based healthcare information provider, estimates that the biologics' share of the USD 1.2 trillion global spend on medicines in 2017 was USD 221 billion, and predicts that this will increase to USD 239.95 billion by 2025 [13]. In addition, biologics continue to be strongly represented in company pipelines, accounting for 26% of the total new molecular entities approved in 2017 [4]. This growth is primarily being driven by monoclonal antibodies [1]. Monoclonal antibodies (mAbs) have revolutionised the pharmaceutical industry-driving life-changing advancements in treatment outcomes and the discovery of novel targets, and have transformed drug revenues from a growing portfolio of approved products. Despite these well-documented successes, various fixed characteristics of the conventional monoclonal antibody molecule, such as its molecular mass, shape, specificity, affinity, and valency, exert significant limitations on their clinical benefit. These include reduced penetration of solid tumours, drug administration typically limited to systemic injections (not site-specific/topical delivery), and an inability to efficiently bind and/or modulate the activity of intracellular targets. However, antibody engineering is continuously seeking to improve biologics formats to modify properties such as size, ease of production, valency, mechanism of action, potency, off-site/by-stander toxicity, target accessibility, bioavailability, half-life, dosing frequencies, and/or route of administration [58].

Next-generation antibody fragments have shown potential as biologic drugs and include single-chain variable, diabody, Bi-specific T-cell engagers (BITE), and Fab fragments [8]. Moreover, Camelidae heavy chain ‘domain’ antibodies (VHH) that are evolutionarily derived from conventional antibodies that have lost their light-chain partners [9] have progressed successfully into the clinic for several indications. Nature, however, first recognised the potential of smaller binding sites 100s of millions of years earlier than camels, utilising a novel antibody-like structure (IgNAR), as an important component of the Elasmobranch (shark) adaptive immune system and containing two single domain-binding sites (variable new antigen receptors, VNARs) [10,11]. At ∼11 kDa, VNARs are the smallest naturally occurring independent binding domains in the vertebrate kingdom [1012]. Their simple protein architecture allows for flexible reformatting without any loss of their inherent domain properties [1215]. VNAR formats with reduced size but increased domain valency (multivalent mono-specific or multi-specific), improved efficacy, deeper tissue penetration, increased in vivo half-life, and biophysical stability have been demonstrated by several independent research groups [1318].

In this review, we will highlight the different strategies utilised to develop VNARs as therapeutic drugs and show examples where these domain formats retain and/or incorporate desirable attributes of next-generation biologics.

Reformatting VNAR domains for clinical development

The simplicity of the VNAR architecture, which facilitates in vitro molecular engineering, cannot be over-emphasised. VNAR drug libraries can be readily adapted for multiple display technologies, and this flexibility makes the building and screening of immunised, naive, and synthetic libraries straightforward, particularly when compared with antibody fragment libraries for example [1921]. A growing portfolio of high-affinity (nM range), antigen-specific VNARs with favourable characteristics such as stability and solubility have been successfully generated from these libraries [12,14,15,2123]. Rational and step-wise multimerization creating multivalent, bi-paratopic and/or multi-specific VNAR constructs have led to significant improvements in affinity (pM range) and subsequent potency without compromising inherent VNAR properties. Recently, anti-TNF VNAR domains, reformatted into quadrivalent constructs, showed in vitro potency an order of magnitude greater than the world's best-selling drug Adalimumab (Humira®) [15] (Figure 1). At a molecular mass of 40 and 100 kDa, respectively, the Trivalent, Quad-X™ and Quad-Y™ VNAR constructs are the most potent (anti-hTNF-α) VNAR constructs published to date, capable of neutralisation of cytokine-mediated cytotoxicity at concentrations of 3–5 pM. This staggering potency was associated with domain valency, bi-paratopic binding, and the presence of a spacer (linker length and Fc region). While this strategy worked well for TNF, it is worth noting that the choice/optimisation of construct and reformatting strategy will largely depend on the antigen, because the mechanisms of blockade/neutralisation of a soluble target may be different from those required for a membrane-bound or intracellular protein.

Schematic of multivalent VNAR reformatting capacity and their corresponding protein production yields.

VNARs have successfully targeted a growing list of structurally and functionally diverse targets (see Table 1). The first VNAR to demonstrate efficacy in a clinically relevant disease model was raised against a cell surface antigen, induced costimulatory ligand (ICOSL) [18]. ICOSL, also known as B7-related protein (B7RP-1) and B7 homologue (B7h), is constitutively expressed on antigen-presenting cells such as B cells, activated monocytes, and dendritic cells, and is implicated in several autoimmune inflammatory diseases through its interactions with ICOS, CD28, and CTLA4 [18,24,25]. In a murine model of non-infectious uveitis, Fc-fused anti-ICOSL VNARs injected systemically resulted in a marked reduction of inflammation in treated mice [18] with a significant improvement in both clinical score and histology, when compared with systemically administered steroid as a positive control. In the same publication, a monomer of the anti-ICOSL VNAR domain (11 kDa), applied topically to a scratched corneal mouse model (mimicking anterior eye inflammation), was able to penetrate the cornea in 20 min. This was in stark contrast with an anti-ICOSL VNAR-Fc (80 kDa) and a commercial anti-ICOSL monoclonal antibody (150 kDa) which were undetectable in the anterior fluid. In a very recent development, a VNAR isolated against vascular endothelial growth factor (VEGF)165 also demonstrated intraocular penetration after topical administration. This anti-VEGF VNAR was detected in the aqueous humour of healthy eyes of New Zealand rabbits, 3 h post-administration [16]. Affinity maturation of this anti-VEGF165 VNAR variant (a single residue substitution within the CDR3, Pro98Tyr) produced an enhancement in its neutralisation potency seen through an increased inhibitory effect on vascular angiogenesis and tumour growth in an in vivo mouse tumour model [26].

Table 1
List of potential therapeutic VNAR domains
Target Potential application Reference 
AMA1 Diagnosis and therapy [37,38
BAFF Autoimmunity [17
EpCAM Diagnosis and therapy [22
EphA2 Diagnosis and therapy [22
HBeAg of HBV Anti-viral [39
HSA Half-life extension [14
HSA Humanised VNAR (soloMER[34
HTRA1 Arthritis therapy [22
ICOSL Autoimmunity and inflammation [18
TNF-α Anti-inflammatory [15
Tom70 Diagnosis and therapy [40
VEGF165 Anti-angiogenic [16
VHSV Anti-viral [41
Target Potential application Reference 
AMA1 Diagnosis and therapy [37,38
BAFF Autoimmunity [17
EpCAM Diagnosis and therapy [22
EphA2 Diagnosis and therapy [22
HBeAg of HBV Anti-viral [39
HSA Half-life extension [14
HSA Humanised VNAR (soloMER[34
HTRA1 Arthritis therapy [22
ICOSL Autoimmunity and inflammation [18
TNF-α Anti-inflammatory [15
Tom70 Diagnosis and therapy [40
VEGF165 Anti-angiogenic [16
VHSV Anti-viral [41

Abbreviations: AMA1, apical membrane antigen-1 from malarial parasites; BAFF, B-cell-activating factor; EpCAM, epithelial cell adhesion molecule; EphA2, Ephrin type-A receptor 2; HBeAg, Hepatitis B e antigen; HBV, Hepatitis B virus; HSA, human serum albumin; HTRA1, human serine protease; ICOSL, induced costimulatory ligand; TNF, tumour necrosis factor; Tom70, 70 kDa translocase of the outer mitochondrial membrane; VEGF165, vascular endothelial growth factor type-A isoform 165; VHSV, viral haemorrhagic septicaemia virus.

Delivery of biologics to certain tissues like the eye remains a major obstacle. Similarly, for the central nervous system, and the brain, in particular, addressing neuro-oncology and neurodegenerative disorders with biologic therapies is a considerable challenge, due to the impermeable barrier posed by the brain capillary endothelium. The inherent attributes of VNAR domains may hold promise in crossing this blood–brain barrier (BBB) by exploiting or piggy-backing on cerebral endothelial cell receptor-mediated transcytosis, carrying other biologic fusions across the BBB. This approach is currently being explored by a US company, Ossianix Inc. [27,28].

While VNARs may provide alternative approaches to the tissue-specific delivery of biologic therapies, their small size and stable nature may also be ideal characteristics for intracellular delivery. Burgess et al. [29] illustrated the usefulness of VNARs as molecular probes when characterising the regulatory mechanisms of serine/threonine protein kinases and used this information to provide a rational for the structure-guided design of allosteric (small molecule) inhibitors for the cytoplasmic, oncology target, Aurora A kinase. Their data, however, could also be interpreted as early evidence of the potential of intracellularly delivered VNARs to modulate disease outcomes.

Small therapeutic domain size clearly aids site-specific delivery, but it may also increase solid tumour penetration and present opportunities for intracellular binding and modulation of drug targets considered only available to small molecule therapies. For some systemic indications that require sustained neutralisation of antigen, pharmacokinetic parameters, such as serum half-life (typically only a few hours) and consequently dosing frequency, may make these domains unattractive for clinical development. Binding (antibody) fragments have utilised several strategies to extend serum half-life [13,15,3034]. For therapeutic VNARs, an anti-human serum albumin (HSA) binder has provided a half-life extension platform which has been validated in several animal models including non-human primate studies [14,34,35].

Multivalent non-Fc-fused constructs are linked via (Gly4Ser)4 flexible linkers. Molecular fusion of a VNAR to a human IgG1 Fc (VNAR-Fc) is achieved via a VNAR C-terminal (Gly4Ser)2 linker fused to the hinge region of the human Fc. C-terminal VNAR in a Quad-X™ construct is fused to the Fc region via a (Gly4Ser)4 flexible linker. Texts in yellow shows that representative protein expression yields in different systems. VNAR monomers, dimers, and trimers have all shown good periplasmic or cytoplasmic yield in Escherichia coli strains, including Lactococcus lactis. All Fc-based and trimer constructs have also been expressed in HEK293 cells demonstrating scalable yields. CHO expressions were conducted by Evitria AG, Switzerland [15].

Strategies for humanisation of lead VNAR drug candidates

Immunogenicity can be a major developmental obstacle on the path to a successful biologics therapy. All administered protein therapeutics engender cellular and humoral immune responses. Anti-drug antibodies may neutralise the therapeutic effect of the drug or cross-react with autologous proteins. Immunogenicity screening and de-immunisation (humanisation) should be considered at an early stage of any protein drug development programme. Shark VNARs could potentially have offered a challenge for humanisation because of their low overall sequence homology (≈30%) to human VH/VL sequences [11,36]. However, using available VNAR crystallisation data, it was observed that VNAR domains organise their framework regions in a similar manner to human immunoglobulin variable domains, providing a route to de-immunised or humanised versions of VNAR binders [34,36]. For humanised VNARs, predictive in silico immunogenicity and, more importantly, the proliferative T-cell response measured using the ProImmune's REVEAL® Immunogenicity System DC–T-cell assay was comparable to those obtained for well-known human and humanised antibodies used regularly in man [34]. The first VNAR domain to be humanised and developed for clinical use was an HSA-specific domain isolated from an immunised spiny dogfish. This domain was originally referred to as E06 and is a critical ‘enabler’ required to improve the efficacy of other fusion protein products, by increasing their serum half-life and subsequent in vivo potency. Several functional versions of E06, based on the human germ line VL scaffold DPK9, were cloned and fully characterised. From these, a candidate and clinic-ready humanised VNAR (now called a soloMER™) was identified and known by the product name NDure™. NDure™ shows negligible antigenicity in ex vivo models, retained binding as a fusion product (C- and N-terminal or both), extension of serum half-life in vivo, and high levels of stability, expressibility, and solubility [14,34] and is readily amenable to efficient, site-specific, chemical conjugation (Barelle et al., unpublished).

Expression and purification of soloMER™ formats

The production/manufacture of mAbs at scale can only be achieved in expression systems that are capable of complex and diverse post-translational protein modifications (e.g. glycosylation). These requirements certainly play a part in the high production cost of these large proteins and also influence their in vivo potency, biology, and potential immunogenicity if administered clinically [4244]. When VNAR/soloMER domains are expressed as fusion proteins with Fc regions, they also typically require production in a mammalian expression system. However, the complexity of the final product is significantly less than for a mAb which must achieve functional union between heavy and light-chain antibody pairs with a final molecular mass of 150 kDa. In contrast, VNAR/soloMER-Fc fusions (40 kDa) and Quad X or Y formats (50 kDa) only require the spontaneous dimerisation of a single protein product (Figure 1) reducing the complexity of vector design and protein assembly inside the cell with knock-on benefits for simpler protein purification protocols in particular [15]. When VNAR/soloMERs are produced without an Fc region, there is a further reduction in the post-translational modifications required for full binding activity, excepting intra-domain disulfide bond formation. Monovalent, multivalent dimers, and trimer constructs (‘string-of-pearls’) have been successfully expressed at intermediate scale (Figure 1) in both prokaryotic and eukaryotic systems [15,18,21,34]. Purification of these domains was efficiently achieved through the engineered addition of purification/detection tags (histidine, HA, c-myc) [12,14,15,22]. Through soloMERisation, we were have also successfully incorporated a protein L-binding site into the framework 1 region of our humanised domains creating an alternative means of detection and purification that is now internal to, and part of, our final product and parallels the more traditional protein-A affinity purification strategy used successfully for Fc fusions and conventional mAbs [36].

Developing VNARs as biomolecular tools

The same favourable properties of small size and protruding binding-site topology, which make the VNAR scaffold an exciting platform for biologics drug development, may also offer up the possibility of extending their utility to include diagnostic and research reagents [29,45,46].

One example that demonstrates a competitive alternative to more typical antibody analytics and purification is the use of anti-idiotypic (binding site specific) VNAR variants and their application as highly effective ‘capturing tools’ that recognise the epidermal growth factor receptor-specific therapeutic mAbs, cetuximab and matuzumab, respectively [45]. Here, the lead VNARs were isolated from a naive semi-synthetic VNAR yeast surface display library and exhibited preferential binding to the ‘intact’ mAb target with little if any cross-reactivity with mispaired variants. This remarkable binding specificity was attributed to the elongated CDR3 binding loop's accessibility and recognition of the discontinuous epitopes within the antigen-binding site of the mAb heavy and light chains, allowing for strict differentiation of correctly assembled paratopes. Furthermore, retained target recognition in the presence of human and mouse serum and a development timeline of only 3 months emphasised the applicability of VNARs as affinity purification reagents for therapeutic antibodies in complex serum samples [45].

A more recent development of VNAR-binding site engineering, delivering pH-sensitive target recognition, opens-up new avenues for tailor-made affinity-based reagents and enables the use of milder pH elution protocols, relevant to many chromatographic applications. From a histidine-doped semi-synthetic VNAR master library, a pH-switchable VNAR specific for an epithelial cell adhesion molecule was successfully isolated and characterised [47]. The incorporation of ionisable histidine residues within the binding-site interface encouraged pH-dependent release by altering electrostatic interactions and forced a reduction in target binding [48,49]. CDR3-based histidine substitutions modulated a 30-fold increase in the dissociation rate of the VNAR-target complex at pH 5.5 compared with pH 7.4, with no pH-dependent effect observed for the non-engineered control [47].

The versatility of VNARs as biomolecular tools has also been elegantly demonstrated through their ability for molecular mimicry published now for both an antigenic epitope [46] and a receptor motif [17]. VNARs specific for human B-cell-activating factor (BAFF), derived from a VNAR semi-synthetic phage display library, had an inhibitory effect on BAFF activity that was attributed to molecular mimicry, since the most potent VNAR inhibitors contained within their CDR3 region a structural motif (DXL) common to all three BAFF receptors. This receptor antagonism had a measurable inhibitory effect on B-cell development and a corresponding impact on immune-regulation [17].

Conclusion

There seems little doubt that the bullish trends associated with the biologics’ market will continue into the foreseeable future. The length of this drug-class runway will, however, be dependent upon a continued refinement in the performance of biologics and modifications that circumvent known limitations to their utility and in vivo efficacy. Next-generation biologics platforms are already responding to these demands offering new potency through novel targeting and delivery and interestingly are also enhancing our knowledge and understanding of diseases and their pathobiology.

Antibody-like VNARs and their humanised cousins soloMERs™ may be considered by some as the ‘new kids on the block’, particularly when compared with their single-domain antibody counterpart, VHH nanobodies [50]. However, these domains have already shown their potential as the building blocks of protein therapies capable of binding a diverse class of targets with unrelated biology, function, and sites of expression (Table 1). Because the VNARs are amenable to extensive reformatting without compromising their inherent biophysical properties and activity, they are ideally placed to overcome many of the limitations now recognised for mAb therapy. Furthermore, in the intensely competitive world of antibody therapeutics, their unique evolutionary origin (antibody-like molecules that have never had a light-chain partner or CDR 2 region) places them outside of the complex intellectual property landscape that surrounds antibody drug discovery and development, permitting a greater freedom to operate and a correspondingly faster, less encumbered development path.

Abbreviations

     
  • BAFF

    B-cell-activating factor

  •  
  • BBB

    blood–brain barrier

  •  
  • HSA

    human serum albumin

  •  
  • ICOSL

    induced costimulatory ligand

  •  
  • mAbs

    monoclonal antibodies

  •  
  • VEGF

    vascular endothelial growth factor

  •  
  • VNAR

    variable new antigen receptor

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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