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

Abstract

Anti-drug antibodies (ADAs), specific for biotherapeutic drugs, are associated with reduced serum drug levels and compromised therapeutic response. The impact of ADA on the bioavailability and clinical efficacy of blockbuster anti-hTNF-α monoclonal antibodies is well recognised, especially for adalimumab and infliximab treatments, with the large and complex molecular architecture of classical immunoglobulin antibody drugs, in part, responsible for the immunogenicity seen in patients. The initial aim of this study was to develop solid-phase enzyme-linked immunosorbent assays (ELISA) and an in vitro cell-based method to accurately detect ADA and estimate its impact on the preclinical in vivo efficacy outcomes of two novel, nonimmunoglobulin VNAR fusion anti-hTNF-α biologics (Quad-X™ and D1-NDure™-C4) and Humira®, a brand of adalimumab. Serum drug levels and the presence of ADA were determined in a transgenic mouse model of polyarthritis (Tg197) when Quad-X™ and Humira® were dosed at 1 mg/kg and D1-NDure™-C4 was dosed at 30 mg/kg. The serum levels of the Quad-X™ and D1-NDure™-C4 modalities were consistently high and comparable across all mice within the same treatment groups. In 1 mg/kg and 3 mg/kg Quad-X™- and 30 mg/kg D1-NDure™-C4-treated mice, an average trough drug serum concentration of 8 μg/mL, 50 μg/mL, and 350 μg/mL, respectively, were estimated. In stark contrast, Humira® trough serum concentrations in the 1 mg/kg treatment group ranged from <0.008 μg/mL to 4 μg/mL with trace levels detected in 7 of the 8 animals treated. Trough serum Humira® and Quad-X™ concentrations in 3 mg/kg treatment samples were comparable; however, the functionality of the detected Humira® serum was significantly compromised due to neutralising ADA. The impact of ADA went beyond the simple and rapid clearance of Humira®, as 7/8 serum samples also showed no detectable capacity to neutralise hTNF-α-mediated cytotoxicity in a murine fibrosarcoma (L929) cell assay. The neutralisation capacity of all the VNAR constructs remained unchanged at the end of the experimental period (10 weeks). The data presented in this manuscript goes some way to explain the exciting outcomes of the previously published preclinical in vivo efficacy data, which showed complete control of disease at Quad-X™ concentrations of 0.5 mg/kg, equivalent to 10x the in vivo potency of Humira®. This independent corroboration also validates the robustness and reliability of the assay techniques reported in this current manuscript, and while it comes with the caveat of a mouse study, it does appear to suggest that these particular VNAR constructs, at least, are of low inherent immunogenicity.

Shark New Antigen Receptor (IgNAR): Structure, Characteristics and Potential Biomedical Applications

“…Results of their experiments demonstrated the efficiency and potentiality of the VNAR binding domain for the treatment of auto-inflammatory conditions [ 29 ]. Obinna C. Ubah and colleagues designed VNAR fusion anti-hTNF-α Quad-X™ that showed improvement in potency over Humira ® [ 30 ]. These findings from previous studies identified the usefulness of the VNAR complex to the protein that is a good target for those diseases.…”

“…Tumor necrosis factor-alpha (TNF-α) is a cytokine involved in systemic inflammation [ 12 , 42 ] and is identified to be a good target to treat arthritis [ 13 ]. The structure of anti-TNF-α VNAR-human TNF-α complexes has been solved and novel epitopes have been discovered by crystallization analysis where VNAR interacts with two adjacent TNF-α protomers [ 13 , 30 ]. Another VNAR isolated from sharks has shown promising features to alleviate the progress of arthritis in animal models, and average in vivo arthritis inhibition and histopathology scores are 88% and 86% using Quad-XTM at doses of 0.5 and 30 mg/kg, respectively [ 13 ], demonstrating the valuable application of IgNAR in drug discovery again.…”

“…The report from the previous study revealed the low inherent immunogenicity of the VNAR. Impact of anti-drug antibodies (ADAs) detected on preclinical in vivo efficacy using non-immunoglobulin VNAR fusion anti-hTNF-α biologics (Quad-X™ and D1-NDure™-C4) and Humira ® , a brand of adalimumab [ 30 ], demonstrating the promising application of VNAR in biomedical industries.…”

Accurate and rapid microfluidic ELISA to monitor Infliximab titers in patients with inflammatory bowel diseases

“…7,[16][17][18] The standard measurement of IFX and ADAs levels in patient's plasma is usually performed by ELISA, which provides adequate sensitivity and reproducibility. 8,15,19 However, a cELISA does not provide the physician with a result for IFX quantification within the time frame of the medical appointment, making accurate therapeutic adjustments impossible. This in turn leads to a possible increase of undesired immune responses for the patient and consequent costs for the healthcare system.…”

Bamboo Shark as a Small Animal Model for Single Domain Antibody Production

“…Single domain antibodies (sdAbs) are preferred over traditional antibodies because of their simple architecture, high thermal and chemical stability, good solubility, high tissue penetration, easy modularity, and straightforward expression in Escherichia coli ( Holliger and Hudson, 2005 ; Simmons et al, 2006 ; Koenning et al, 2017 ; Steven et al, 2017 ; Ubah et al, 2017 ; Ubah et al, 2020 ; Stocki et al, 2021 ). SdAbs are derived from the antigen-binding variable domain (VHH) of camel heavy chain–only antibody or the variable domain (vNAR) of cartilaginous fish immunoglobulin new antigen receptor (IgNAR) ( Koenning et al, 2017 ).…”