RESUMEN
The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding topographies, making it an ideal candidate for accessing cryptic epitopes otherwise intractable to conventional antibodies. These attributes, coupled with high physicochemical stability and amenability to phage display, facilitate the selection of VNAR binders to challenging targets. Here, we explored the unique attributes of these single domains for potential application as bioprocessing reagents in the development of the SEED-Fc platform, designed to generate therapeutic bispecific antibodies. A panel of unique VNARs specific to the SEED homodimeric (monospecific) 'by-products' were isolated from a shark semi-synthetic VNAR library via phage display. The lead VNAR candidate exhibited low nanomolar affinity and superior selectivity to SEED homodimer, with functionality being retained upon exposure to extreme physicochemical conditions that mimic their applicability as purification agents. Ultimately, this work exemplifies the robustness of the semi-synthetic VNAR platform, the predisposition of the VNAR paratope to recognise novel epitopes and the potential for routine generation of tailor-made VNAR-based bioprocessing reagents.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Anticuerpos Biespecíficos/química , Sitios de Unión , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Indicadores y Reactivos/metabolismo , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Anticuerpos de Dominio Único/químicaRESUMEN
Therapeutic mAbs have delivered several blockbuster drugs in oncology and autoimmune inflammatory disease. Revenue for mAbs continues to rise, even in the face of competition from a growing portfolio of biosimilars. Despite this success, there are still limitations associated with the use of mAbs as therapeutic molecules. With a molecular mass of 150â kDa, a two-chain structure and complex glycosylation these challenges include a high cost of goods, limited delivery options, and poor solid tumour penetration. There remains an urgency to create alternatives to antibody scaffolds in a bid to circumvent these limitations, while maintaining or improving the therapeutic success of conventional mAb formats. Smaller, less complex binders, with increased domain valency, multi-specific/paratopic targeting, tuneable serum half-life and low inherent immunogenicity are a few of the characteristics being explored by the next generation of biologic molecules. One novel 'antibody-like' binder that has naturally evolved over 450 million years is the variable new antigen receptor (VNAR) identified as a key component of the adaptive immune system of sharks. At only 11â kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery. VNAR attributes include high affinity for target, ease of expression, stability, solubility, multi-specificity, and increased potential for solid tissue penetration. This review article documents the recent drug developmental milestones achieved for therapeutic VNARs and highlights the first reported evidence of the efficacy of these domains in clinically relevant models of disease.
Asunto(s)
Receptores de Antígenos/química , Receptores de Antígenos/metabolismo , Animales , Biosimilares Farmacéuticos , Glicosilación , Humanos , SolubilidadRESUMEN
Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains.