An Anti-hTNF-α Variable New Antigen Receptor Format Demonstrates Superior in vivo Preclinical Efficacy to Humira® in a Transgenic Mouse Autoimmune Polyarthritis Disease Model

Abstract

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.

In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development

“…Here, the serum levels of Humira® in treated mice were lower compared with the Quad-X™-treated samples, with the 1 mg/kg Humira® group showing a severe depletion in drug levels in the pooled serum samples (Figure 1(a) and 2(a)). In addition, the functional anti-hTNF-α drug is absent in the 1 mg/kg Humira®-treated samples with this data further supporting the reported outcome of a preclinical in vivo efficacy study where 1 mg/kg Humira® treatment was ineffective at blocking disease progression in Tg197 mice, but complete control was achieved by Quad-X™ at 0.5 mg/kg [28]. Assessing drug levels in unmasked individual serum samples further reinforced the evidence of anti-Humira® mouse ADA-mediated rapid clearance of Humira® from the systemic circulation of the treated mice (Figures 3(a)-3(f)).…”

“…Journal of Immunology Research (Figure 3(f)) was only 10% of the corresponding Quad-X™ levels (Figure 3(e)) and would support the partial disease control seen in vivo with the 3 mg/kg Humira® dose [28]. The levels of mouse ADA in complex with drugs seen in 1 mg/kg Quad-X™-treated samples were comparable to that seen in 1 mg/kg Humira® samples (Figures 3(c) and 3(d), respectively); however, that level was significantly lower in 3 mg/kg Quad-X™ compared to the corresponding Humira®-treated samples (Figures 3(g) and 3(h), respectively).…”

“…We have previously reported superior in vitro and in vivo efficacy of the anti-TNF-α Quad-X™ over Humira® and have shown that much of this 10x improvement in potency is due to the empirical design of the Quad-X™ format [22,28]. The low inherent immunogenicity of the VNAR domains and its humanised soloMER™ derivatives has been previously confirmed experimentally [21].…”

“…The novel anti-hTNF-α VNAR Quad-X™ is a 103 kDa engineered domain with two biparatopic anti-hTNF-α VNAR domains (VNAR D1 and C4) fused to the hinge region of a wildtype human IgG1 Fc via a short glycine-rich linker, and two additional binding domains (VNAR D1 and C4) Cterminally fused to the CH-3 region of the Fc fragment via a longer flexible glycine-rich linker creating a quadrivalent/biparatopic construct harbouring a wild-type human IgG1 Fc fragment. A second anti-hTNF-α VNAR drug D1-NDure™-C4 is a 38 kDa linear bispecific construct of the two anti-hTNF-α VNAR D1 and C4 domains, respectively, fused via flexible glycine-rich linkers to the N-and C-terminal regions of a humanised anti-human serum albumin VNAR, NDure™ [21,22,28,29]. This linear construct incorporates a c-terminal poly-histidine tail and a protein-L binding site in the framework region of the HSA binding partner (NDure™) allowing flexibility for downstream immunodetection and purification.…”

“…Analysis compared the level of ADA production in trough mouse serum samples for two novel anti-hTNF-α modalities (Quad-X™ and D1-NDure™-C4) with a clinically available anti-hTNF-α monoclonal antibody, Humira®. Furthermore, trough serum drug levels were determined using both direct and indirect ELISA methods, allowing us to better understand and correlate the impact of ADA on the preclinical in vivo efficacy study outcomes of these anti-hTNF-α drugs [28]. The conventional approach for ADA investigation is to assess serum samples collected at the end of a therapeutic cycle (trough level), where the drug level is low enough to minimise any drug interference in a drugsensitive assay but not so low as to limit the clinical usefulness of the assay in detecting ADA [1].…”