Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine with beneficial functions in immune regulation and host defense, but when expressed at high levels, is also implicated in a number of human diseases. TNF-α is a critical mediator of the autoimmune process, playing a key role in several inflammatory diseases ranging from rheumatoid arthritis, inflammatory bowel diseases, psoriasis, and uveitis. Rheumatoid arthritis (RA) is a common, chronic inflammatory disorder that causes progressive articular destruction and is associated with comorbidities in vascular, metabolic, bone and even the psychological well-being of sufferers (1, 2). Affecting about 1% of the global population, RA patients are faced with functional disability, pain, joint damage, a reduced quality of life, and even premature mortality, with a corresponding and significant negative economic impact on both the patients and government health systems struggling to treat the disease (3, 4). The management of many autoimmune mediated diseases has significantly improved in the last decade because a better understanding of disease mechanisms has guided the development of new and potent therapies. The mainstay treatment for steroid-refractory RA is via the targeted neutralization/blocking of the TNF-α signaling pathway with anti-TNF-α or anti-TNF receptor biologics. The monoclonal antibody, Adalimumab (Humira®) is the most widely used and commercially successful TNF-α antagonist monoclonal antibody (5–7). However, there still remain some significant limitations with this treatment approach. Approximately 50% of patients fail to see their disease properly controlled with the existing battery of anti-TNF biologics. A failure to respond to treatment or the development of resistance to therapy (through immunogenicity or other mechanisms), the use of progressively higher doses (10–30 mg/kg) with prolonged therapy windows, concomitant, and often significant systemic side-effects (infection, heart problems, malignancy) are just few of the limitations associated with the current anti-TNF biologics in the clinic (8–16). Some of these limitations are in part also related to the molecular mass and structural complexity of mAbs which can increase manufacturing costs and clinically limit routes of administration.

The Variable New Antigen Receptors (VNARs) are the smallest (11 kDa) naturally occurring independent binding domains in the vertebrate kingdom (17–19). They play an integral role in the adaptive immune system in cartilaginous fish and although they are structurally similar to mammalian heavy and light variable chains it has been well-documented that they arose from a distinct evolutionary lineage from Immunoglobulins (20). A lack of CDR2 and the addition of two loops of diversity (HV2 and 4) as well as low percentage sequence homology exemplifies this distinction. Their characteristic protruding paratopes, often referred to as “canyon-binders,” predisposes these domains to access and bind epitopes not normally available to conventional biologics and encourages the selection of highly potent neutralizers specific for enzyme and/or receptor targets (21–23). We have previously demonstrated the ease with which reformatting of VNAR domains can be achieved delivering: multivalent, bi/tri-specific constructs, and serum half-life extension through molecular fusion to an anti-human serum albumin (HSA) VNAR scaffold, NDure™ or via a more traditional route using IgG (mouse or human) Fc domains (24–26). These highly stable constructs can be expressed cost-efficiently and at scale in non-mammalian systems. This menu of format options permits tailored selection of drug modalities optimized for systemic, site-specific, or topical administration of VNAR candidates.

Here, we have demonstrated that an anti-hTNF-α VNAR construct (Quad-X™) previously reported as having potent in vitro activity (24) retains this superiority to the commercially available blockbuster anti-TNF-α, Adalimumab (Humira®) in vivo and is capable of preventing the development of spontaneous polyarthritis in a transgenic mouse model of arthritis at doses below 3 mg/kg. Also, and for the first time, we have shown that sustained in vivo efficacy is possible from a non-Fc fused anti-HSA half-life extended linear VNAR construct following systemic administration in the same RA disease model.

About a decade ago, it was proposed that unlike VHH domains, VNARs might not efficiently form dimeric fusion molecules, and even where dimerization of VNAR was achieved, the resulting dimeric construct was published as having compromised expression levels and limited functional binding activity; postulated to be the result of steric hindrance or occlusion of the epitope by the incorporated linker (35, 37, 38). This speculation was later dismissed following the demonstration that VNARs tolerate both N- and C-terminal molecular fusions to form dimers and trimers without any obvious impact on their inherent biophysical properties or expressibility (39). Since these early reports, VNAR reformatting has become routine, and rational, extensive reformatting can generate tailor-made constructs specifically designed for either systemic, site-specific or topical delivery (24, 25, 40). These fusion constructs have been expressed in E. coli, yeast and mammalian cells at scalable yields and to the best of our knowledge, no evidence of significant aggregation of these domains, even when concentrated up to 150 mg/ml with prolonged storage, has been reported. Extending the half-life of therapeutic VNAR domains via molecular fusion is now achievable with a molecular mass of drug candidates at 25 kDa (dimer) or 40 kDa (trimer) using NDure™ (39, 40) or 80–100 kDa with an Fc fragment (24, 25). The benefits of smaller and simpler biologic drug formats cannot be over-emphasized and include: access to both extracellular and intracellular target antigens, non-systemic alternative routes of administration, reduced immunogenicity and cost/flexibility of production.

Although NDure™ fusion proteins and VNAR-Fc formats had been previously administered systemically in several preclinical proof-of-concept studies without in vivo toxicity [non-human primate (26), rat—manuscript in preparation, and mouse (25)], the Quad-X™ construct has never been in vivo before. This first example of a quadra-valent VNAR has demonstrated exceptional in vitro potency (2–5 picomolar) when compared with conventional anti-TNF-α biologics in the clinic (24, 41–43). In a preliminary MTD study in C57BL/6 mice, the safety of the anti-TNF-α Quad-X™ at 30 mg/kg, administered subcutaneously, was confirmed. There was no clinical evidence of acute or delayed adverse events in these treated mice.

With its unique and protruding binding loops (CDR1 and CDR3), we speculated that these anti-TNF-α domains (VNAR D1 and C4), forming the backbone of the D1-BA11-C4 and D1-Fc-C4 Quad-X™ constructs, may be interacting with unique and previously unseen “recessed” epitopes on the TNF-α molecule, in stark contrast to the epitopes recognized by the planar paratopes of the commercially available biologics including even VHH (35, 44–47). In a comprehensive TNF-α species cross-reactivity characterization study, the lead anti-TNF-α VNARs appeared to bind a unique TNF-α epitope. In addition, and using data collected from the European Medicines Agency Evaluation of Medicines for Human Use scientific report detailing other commercially available TNF-α and TNFR1 antagonist biologics (Tables 1A,B), whilst the binding/neutralization profile of our VNAR domains share some similarity with Humira® and Simponi®, these two biologics also bind/neutralize mouse and rabbit TNF-α, respectively. In a BIAcore™ epitope binning study, a VNAR D1-C4 dimer and VHH TNF30-TNF30 recognized different epitopes on immobilized human TNF-α (data not shown). Although for cost effective drug development, it is beneficial to demonstrate broad species cross-reactivity, facilitating proof-of-concept preclinical studies in inexpensive animal models, restricted cross-reactivity seems to be a feature of improved human efficacy of TNF-α protein antagonist (32–34). In Table 1, there is a striking and restricted cross-reactivity across all listed clinical biologics. This restricted cross-reactivity is no longer considered a limitation to the development of anti-TNF-α biologics as the use of surrogate domains and transgenic animal models of human disease (expressing human TNF-α) is now a well-worn and successful clinical path (32, 48–50). In parallel to the work published here, we have solved the structure of the anti-TNF-α VNAR-human TNF-α complexes and the analysis of this crystallization data has confirmed the binding of VNAR to a novel epitope. Like Humira® (47), one VNAR molecule interacts with two adjacent TNF-α protomers. However, this is where the similarity ends with only 10% of the TNF-α contact residues common to both proteins (manuscript in preparation).

It is sometimes easy to forget that the original regulatory and clinical path of anti-TNF biologics required the use of surrogate binders in poorly predictive anti-inflammatory animal models. This enforced strategy was adopted because molecules such as Humira^Ⓡ (and here the Quad X™) are unable to bind and neutralize, with any therapeutically relevant potency, rodent TNF-α. It is only with the recent development of new transgenic disease models (27) that interrogation of a system that approaches that seen in man is now possible. Although there is strong evidence that models such as Tg197 mice are highly predictive of future efficacy in man, these in vivo pre-clinical data sets are typically less comprehensive than those generated for other indications such as cancer, where the use of multiple, related in vivo models is often possible.

The development of TNF-α transgenic mice (Tg197) carrying a 3'-modified UTR human TNF-α gene construct allows deregulated soluble human TNF-α gene expression in vivo. These mice develop chronic inflammatory polyarthritis resembling human rheumatoid arthritis as early as 4 weeks after birth (27, 51, 52). It is clear that all types of RA and IBD in (human) patients don't begin with dysregulated TNF expression and certainly not with the rapidity of the disease progression seen in Tg197 mice. However, this model has proved to be an invaluable in vivo tool capable of accurately forecasting the therapeutic potency of anti-TNF drugs and accelerating the development of next-generation anti-TNF-α biologic therapies. Where they have been less informative is their ability to predict some of the now well-recognized side effects that are associated with longer term anti-TNF-α therapies such as increased susceptibility to opportunistic infections, malignancies and cardiovascular risks (53–56). While inflammatory disease in the Tg197 mouse model is solely driven by soluble TNF-α, it is worth emphasizing that the soluble TNF-α precursor, transmembrane TNF-α (tmTNF-α), is not only a mediator of immunity but also displays anti-inflammatory and anti-apoptotic reactions (57–59). Whilst establishing the tmTNF-α binding and neutralizing profile of our VNARs may potentially add value to our current knowledge, here it is the clinical importance of targeting soluble TNF-α that has been the focus of these studies, since it is binding and neutralization of the soluble form of TNF-α that has been shown to be the critical and common mechanism of action of all approved anti-TNF-α therapeutic agents (60).

The in vivo efficacy of Quad-X™ and D1-BA11-C4 in a Tg197 transgenic human TNF-α polyarthritis disease model reinforces the validity of our previously reported potent in vitro neutralization data which saw an order of magnitude improvement over Humira® (24). In this present study, clinical efficacy seen with a 1 mg/kg dosing of Quad-X™ is slightly better than, but comparable to, that observed with a 10 mg/kg Humira® dose. In addition, breakthrough of underlying disease was observed for both the 1 and 3 mg/kg Humira® treatment regimens. It has previously been established that a 3 mg/kg Humira® dosing is sub-therapeutic, at least in this disease model (36). Although the Quad-X™ format clearly demonstrated improved efficacy in this transgenic model over Humira®, it would be premature to make assumptions about reducing any longer-term risks such as greater susceptibility to infection in the absence of clinical data. The trimer construct D1-BA11-C4 (40 kDa), is a second rationally designed therapeutic format incorporating on this occasion an anti-HSA binding domain, NDure™ (which cross-reacts with mouse serum albumin) to extend serum half-life close to that of a wild type immunoglobulin Fc region (39). The VNAR BA11 domain located in the middle of the two anti-TNF-α also functions as a spacer alongside the flexible 20 amino acid Gly₄Ser linker flanking both N- and C- terminals of BA11. The presence of a spacer has previously been associated with a significant enhancement in in vitro potency (24), and is an effect that has been reported by other independent researchers (35, 61). This relatively low molecular weight, super-potent trimer may also be well-suited to topical and/or site-specific delivery in autoimmune conditions such as IBD, uveitis, and psoriasis. A combination of topical and site-specific delivery and the absence of an Fc region is typically associated with reduced systemic toxicity (35, 62, 63), further extends the format's utility and compliance.

VNAR domains are evolutionarily distant from human and other lower warm-blooded mammals, but evidence from both in silico and dendritic cell-T-cell assays, capable of identifying the presence or absence of potential T-cell epitopes, support their inherent non-immunogenic features, at least in humans (40), with an established route to humanization confirmed for a number of VNAR domains (39). A humanized VNAR domain, such as NDure™, is often referred to in the literature as a soloMER™ (24, 40, 64).

While dose escalation and in-class switching are approaches utilized by clinicians to manage most anti-TNF-α biologics non-responder patients, dose escalation eventually leads to increased treatment cost, increased systemic toxicity, accelerated immunogenicity, infusion reaction, and eventual termination of therapy (13, 16, 65, 66). The anti-TNF-α Quad-X™, which is about two-thirds of the size of a conventional mAb, and the anti- TNF-α NDure™ fusion product (40 kDa) both clearly have the potential to be new and useful additions to the existing formulary of anti-TNF biologics available to clinicians. Where these drug candidates show real advantage as future disruptive therapies, is through their enhanced in vivo potency and controlled PK/delivery, predicting their use at significantly lower doses than current standard of care biologics. It is hoped that this tailoring of drug use will, in turn, limit the risk of anti-drug antibodies, control/optimize the costs of drug therapy and begin to reduce the systemic toxicity risks [infection and malignancies (53–55, 67, 68)] seen from the practice of extended dose escalation therapies.

All reported animal experiment was conducted at the Biomedcode SA test facility. All procedures conformed to the Presidential Decree No 56/2013 Government Gazette No A' 106 applicable in Greece (EEC Directive 2010/63/ECC) and were approved by the directorate of Agricultural and Veterinary Policy (DAVP) of the Attica Region. Approved license Protocol No. 3798/3-07-2017.

OU, scientific lead on this paper, conducted the majority of the scientific work and wrote the manuscript. JS assisted in the rational in-silico design and preliminary expression of all anti-TNF VNAR constructs described in this publication. CB led the science team at Elasmogen Limited and co-supervised this project alongside AP. Also, CB and AP reviewed the manuscript.