Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development

Abstract

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.

A New Venue of TNF Targeting

“…In in vitro models of intestinal epithelial barrier dysfunction, the main VNAR was as efficacious as adalimumab. Therefore, these drugs could be considered as a novel alternative class of biological agents[320]. Similar to etanercept, Lenercept is a soluble fusion protein consisting of TNFR1 fused to the hinge region of the IgG1 Fc region.…”

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

“…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.…”

“…Cell-Based Neutralisation Assay. As previously described [22], a TNF-α-sensitive mouse fibrosarcoma cell line (L929 cells) was grown to 90% confluence in Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies Ltd., UK) supplemented with 10% (v/v) Gibco® heat-inactivated fetal bovine serum (Thermo Fisher Scientific, UK) before seeding 5 × 10 3 cells/well into CELLSTAR® sterile 96-well cell culture flat-bottom plates, with a lid (Greiner Bio-One, Germany). Seeded cells were incubated for 48 h at 37°C and 5% (v/v) CO 2 .…”

“…There is a clear need, especially for these refractory patients, for a next-generation potent hTNF-α-neutralising biologic with inherent low immunogenicity. The structurally less complex biologics delivered from the variable new antigen receptor (VNAR) drug platform, and related humanised formats known as soloMERs™ [19][20][21][22], have both previously been reported as having inherently low immunogenicity in a classical dendritic cell-T-cell assay [21]. Their smaller molecular size, simple single-chain format, minimal requirements for posttranslational modification, and excellent stability, may all contribute to this low immunogenicity profile.…”