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The management of chronic inflammatory diseases, such as inflammatory bowel disease, psoriasis, and rheumatoid arthritis has significantly improved over the last decade with the clinical availability of anti-TNF-α biologics. Despite this undoubted treatment success, a combination of acquired resistance together with an increased risk of systemic complications, means that a significant number of patients either fail to find a suitable targeted therapy or frustratingly discover that an approach that did work is no longer efficacious. Here, we report the isolation and characterization of a new class of super-neutralizing anti-TNF-α biologics formats, the building blocks of which were originally derived as variable new antigen receptor (VNAR) domains from an immunized nurse shark. These parental small, stable VNAR monomers recognize and neutralize tumor necrosis factor (TNF)-α, in cell-based assays, at nanomolar concentrations. However, the simple, single-chain molecular architecture of VNARs allows for easy and multiple reformatting options. Through reformatting, we achieved a 50,000-fold enhancement in in vitro efficacy with super-neutralizing fusion proteins able to block TNF-α induced cytotoxicity in the 2–5 pM range while retaining other functionality through the addition of fusion proteins known to extend serum half-life in vivo. In an in vitro intestinal epithelial barrier dysfunction efficacy model, the lead VNAR domains, restored barrier function and prevented paracellular flux with comparable efficacy to adalimumab (Humira®). In addition, all multivalent VNAR constructs restored trans-epithelial electrical resistance (TEER) to approximately 94% of the untreated control. Reformatted VNAR domains should be considered as a new class of biologic agents for the treatment of hTNF-α driven diseases; either used systemically with appropriate half-life extension or alternatively where site-specific delivery of small and stable neutralizers may provide improvements to current therapy options.

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae. These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media. Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.

Induced costimulatory ligand (ICOSL) plays an important role in the activation of T cells through its interaction with the inducible costimulator, ICOS. Suppression of full T cell activation can be achieved by blocking this interaction and has been shown to be an effective means of ameliorating disease in models of autoimmunity and inflammation. In this study, we demonstrated the ability of a novel class of anti-ICOSL antigen-binding single domains derived from sharks (VNARs) to effectively reduce inflammation in a murine model of non-infectious uveitis. In initial selections, specific VNARs that recognized human ICOSL were isolated from an immunized nurse shark phage display library and lead domains were identified following their performance in a series of antigen selectivity and in vitro bioassay screens. High potency in cell-based blocking assays suggested their potential as novel binders suitable for further therapeutic development. To test this hypothesis, surrogate anti-mouse ICOSL VNAR domains were isolated from the same phage display library and the lead VNAR clone selected via screening in binding and ICOS/ICOSL blocking experiments. The VNAR domain with the highest potency in cell-based blocking of ICOS/ICOSL interaction was fused to the Fc portion of human IgG1 and was tested in vivo in a mouse model of interphotoreceptor retinoid-binding protein-induced uveitis. The anti-mICOSL VNAR Fc, injected systemically, resulted in a marked reduction of inflammation in treated mice when compared with untreated control animals. This approach inhibited disease progression to an equivalent extent to that seen for the positive corticosteroid control, cyclosporin A, reducing both clinical and histopathological scores. These results represent the first demonstration of efficacy of a VNAR binding domain in a relevant clinical model of disease and highlight the potential of VNARs for the treatment of auto-inflammatory conditions.

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive “hammer-hug” selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and >18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with <2% high molecular weight species present after 7 weeks at 4 °C and viscosity <15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

Tumor necrosis factor-alpha (TNF-α), an established pro-inflammatory cytokine plays a central role in the induction and progression of several chronic inflammatory and autoimmune diseases. Targeting TNF-α as a treatment modality has shown tremendous success, however there are several limitations associated with the current anti-TNF-α biologic drugs including: immunogenicity, life-threatening infections, resistance to treatment, complexity of manufacture and cost of treatment. Here, we report the in vivo efficacy of novel anti-TNF-α formats generated from molecular engineering of variable new antigen receptors (VNARs), originally derived from the immune system of an immunized nurse shark. Two anti-TNF-α VNAR formats, a tandem multivalent trimer, D1-BA11-C4 and an Fc-fused quadrivalent D1-Fc-C4 (Quad-X™) construct were tested in a clinically relevant, preclinical mouse efficacy model of polyarthritis (Tg197) and compared to the commercial anti-TNF-α “best in class” therapy, Adalimumab (Humira®). Both VNAR formats bind and neutralize TNF-α through an epitope that appears to be different from those recognized by other anti-TNF biologics used clinically. All doses of Quad-X™, from 0.5 to 30 mg/kg, significantly blocked the development of polyarthritis. At 0.5 mg/kg Quad-X™, the arthritis score was improved by 76% and the histopathology score by 63%. At 3 mg/kg Quad-X™, control of disease was almost complete at 90% (arthritis) and 88% (histopathology). In marked contrast, 1 mg/kg Humira® saw profound disease breakthrough with scores of 39 and 16% respectively, increasing to a respectable 82 and 86% inhibition at 10 mg/kg Humira®. We have previously reported the superior potency of anti-TNF-α VNARs in vitro and in these studies translate this superiority into an in vivo setting and demonstrate the potential of VNAR formats to meet the requirements of next-generation anti-TNF-α therapies.