VNARs: An Ancient and Unique Repertoire of Molecules  That Deliver Small, Soluble, Stable and High Affinity  Binders of Proteins

Barelle, Caroline; Porter, Andy

doi:10.3390/antib4030240

Cited by 34 publications

(37 citation statements)

References 73 publications

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“…The notion that IgNAR are part of the adaptive immune system of sharks has been reported previously ( 16 , 26 ), and although time consuming, immunization still remains our preferred route for the isolation of high-affinity binders or neutralizers. Shark immunization route can be particularly useful for cross-species conserved proteins as sharks are evolutionarily distant from humans, diverging from a common ancestor approximately 450 million years ago and, therefore, the likelihood of immune tolerance to such a protein antigen is much less likely ( 15 , 20 , 46 ).…”

Section: Discussionmentioning

confidence: 99%

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

Ubah¹,

Steven²,

Kovaleva³

et al. 2017

Front. Immunol.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Ubah¹,

Steven²,

Kovaleva³

et al. 2017

Front. Immunol.

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“…The thermodynamic stability of human V H s is comparable to that of V H Hs, but most human V H s do not refold reversibly without aggregating. In contrast, many V H Hs and V-NARs refold reversibly, resist aggregation even when unfolded and retain activity after being heated above their melting temperatures [75, 76]. Both colloidal stability (solubility) and conformational (folding) stability contribute to antibody solution properties [77].…”

Section: Introductionmentioning

confidence: 99%

Engineered Autonomous Human Variable Domains

Nilvebrant

Tessier

Sidhu

2017

CPD

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The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.

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“…Shark derived sdAb offer an important alternative to both conventional antibodies and camelid sdAb for therapeutic, detection, and biotechnology applications [ 6 , 34 , 39 ]. Through CDR grafting, coupled with site directed mutagenesis, we were successful in finding a variant of shark096 that has an increased melting temperature while maintaining high affinity.…”

Section: Resultsmentioning

confidence: 99%

“…Both camelids and sharks produce unique heavy chain antibodies that are able to recognize their cognate antigen with excellent affinity and specificity in the absence of a light chain [ 1 , 2 ]. Binding takes place through an unpaired variable heavy domain which can be expressed recombinantly as a single-domain antibody (sdAb) [ 3 , 4 , 5 , 6 ]. The single domain architecture of sdAb provides recognition reagents with properties such as good solubility, facile production in Escherichia coli , and the ability to refold after heat denaturation.…”

Section: Introductionmentioning

confidence: 99%

Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

Anderson¹,

Teichler²,

Zabetakis³

et al. 2016

PLoS ONE

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Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

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VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

Cited by 34 publications

References 73 publications

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

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

Engineered Autonomous Human Variable Domains

Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

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