In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development

Abstract

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

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]. 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)).…”

“…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.…”

“…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.…”

Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody

“…The lead clones were assessed for any tendency to dimerize, tolerance to N-and C-terminal fusions, stability, affinity and relative antigenicity in human dendritic cell assays. In addition, the functionality of the clones was verified in vivo through the extension of serum half-life in a typical drug format [66]. From these analyses, a clone known as BA11 showed insignificant antigenicity, high affinity and high stability for mouse, rat and HSA.…”

“…From these analyses, a clone known as BA11 showed insignificant antigenicity, high affinity and high stability for mouse, rat and HSA. When these attributes were combined with demonstrable functionality in a rat model of pharmacokinetics, the BA11 was established as their clinical candidate [66].…”