Novel, anti-hTNF-α VNAR formats with enhanced neutralising potency and multi-functionality, generated for therapeutic development

Obinna C. Ubah, John Steven, Marina Kovaleva, Laura Ferguson, Charlotte Barelle, Andrew J. R. Porter, Caroline J. Barelle

Research output: Contribution to journalArticle

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Abstract

The management of chronic inflammatory diseases such as inflammatory bowel disease (IBD), psoriasis, and rheumatoid arthritis (RA) 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 characterisation of a new class of super-neutralising anti-TNF-α biologics formats, the building blocks of which were originally derived as variable new antigen receptor (VNAR) domains from an immunised nurse shark. These parental small, stable VNAR monomers recognise and neutralise 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-neutralising fusion-proteins able to block TNF-α induced cytotoxicity in the 2-5 picomolar range whilst 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 neutralisers may provide improvements to current therapy options.
Original languageEnglish
Article number1780
Pages (from-to)1-13
Number of pages13
JournalFrontiers in Immunology
Volume8
DOIs
Publication statusPublished - 22 Dec 2017

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Antigen Receptors
Biological Products
Half-Life
Therapeutics
Sharks
Biological Factors
Life Expectancy
Electric Impedance
Inflammatory Bowel Diseases
Psoriasis
Rheumatoid Arthritis
Proteins
Chronic Disease
Nurses
Serum

Keywords

  • vNAR
  • TNF-α
  • phage display
  • cytokine neutralisation
  • chronic inflammation
  • shark IgNAR
  • biparatopic/bispecific binding domain
  • anti-TNF biologics
  • shark immunisation
  • single binding domain

Cite this

Novel, anti-hTNF-α VNAR formats with enhanced neutralising potency and multi-functionality, generated for therapeutic development. / Ubah, Obinna C.; Steven, John; Kovaleva, Marina; Ferguson, Laura; Barelle, Charlotte; Porter, Andrew J. R.; Barelle, Caroline J.

In: Frontiers in Immunology, Vol. 8, 1780, 22.12.2017, p. 1-13.

Research output: Contribution to journalArticle

Ubah, Obinna C. ; Steven, John ; Kovaleva, Marina ; Ferguson, Laura ; Barelle, Charlotte ; Porter, Andrew J. R. ; Barelle, Caroline J. / Novel, anti-hTNF-α VNAR formats with enhanced neutralising potency and multi-functionality, generated for therapeutic development. In: Frontiers in Immunology. 2017 ; Vol. 8. pp. 1-13.
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AU - Barelle, Charlotte

AU - Porter, Andrew J. R.

AU - Barelle, Caroline J.

N1 - ACKNOWLEDGMENTS The authors wish to acknowledge the funding support for this work from MSD/Scottish Universities Life Sciences Alliance (SULSA), Scottish Enterprise, the Biotechnology and Biological Sciences Research Council (BBSRC), and the University of Aberdeen. FUNDING Grateful for support from Biotechnology and Biological Sciences Research Council (BB/K010905/1), Scottish Enterprise [VNAR_001 (2012)], Scottish Universities Life Sciences Alliance/ MSD (MSD01_A_Porter-Teismann), and the College of Life Sciences and Medicine, University of Aberdeen (Fee bursary to OU).

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N2 - The management of chronic inflammatory diseases such as inflammatory bowel disease (IBD), psoriasis, and rheumatoid arthritis (RA) 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 characterisation of a new class of super-neutralising anti-TNF-α biologics formats, the building blocks of which were originally derived as variable new antigen receptor (VNAR) domains from an immunised nurse shark. These parental small, stable VNAR monomers recognise and neutralise 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-neutralising fusion-proteins able to block TNF-α induced cytotoxicity in the 2-5 picomolar range whilst 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 neutralisers may provide improvements to current therapy options.

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KW - phage display

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KW - chronic inflammation

KW - shark IgNAR

KW - biparatopic/bispecific binding domain

KW - anti-TNF biologics

KW - shark immunisation

KW - single binding domain

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