Next-generation flexible formats of VNAR domains expand the drug platform's utility and developability.

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 150 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 11 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.

A novel anti-B7-H3 chimeric antigen receptor from a single-chain antibody library for immunotherapy of solid cancers.

B7-H3 (CD276) has emerged as a target for cancer immunotherapy by virtue of consistent expression in many malignancies, relative absence from healthy tissues, and an emerging role as a driver of tumor immune inhibition. Recent studies have reported B7-H3 to be a suitable target for chimeric antigen receptor-modified T cell (CAR-T) therapy using CARs constructed from established anti-B7-H3 antibodies converted into single-chain Fv format (scFv). We constructed and screened binders in an scFv library to generate a new anti-B7-H3 CAR-T with favorable properties. This allowed access to numerous specificities ready formatted for CAR evaluation. Selected anti-human B7-H3 scFvs were readily cloned into CAR-T and evaluated for anti-tumor reactivity in cytotoxicity, cytokine, and proliferation assays. Two binders with divergent complementarity determining regions were found to show optimal antigen-specific cytotoxicity and cytokine secretion. One binder in second-generation CD28-CD3ζ CAR format induced sustained in vitro proliferation on repeat antigen challenge. The lead candidate CAR-T also demonstrated in vivo activity in a resistant neuroblastoma model. An empirical approach to B7-H3 CAR-T discovery through screening of novel scFv sequences in CAR-T format has led to the identification of a new construct with sustained proliferative capacity warranting further evaluation.

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties.

VNAR domains are the binding regions of new antigen receptor proteins (IgNAR) which are unique to sharks, skates, and rays (Elasmobranchii). Individual VNAR domains can bind antigens independently and are the smallest reported adaptive immune recognition entities in the vertebrate kingdom. Sharing limited sequence homology with human immunoglobulin domains, their development and use as biotherapeutic agents require that they be humanized to minimize their potential immunogenicity. Efforts to humanize a human serum albumin (HSA)-specific VNAR, E06, resulted in protein molecules that initially had undesirable biophysical properties or reduced affinity for cognate antigen. Two lead humanized anti-HSA clones, v1.10 and v2.4, were subjected to a process of random mutagenesis using error-prone PCR. The mutated sequences for each humanized VNAR variant were screened for improvements in affinity for HSA and biophysical properties, achieved without a predicted increase in overall immunogenicity.

Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents.

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.

Phage Display Derived IgNAR V Region Binding Domains for Therapeutic Development.

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.

(13)CO2 breath tests in non-invasive hepatological diagnosis.

In liver diagnostics, a simple, non-invasive test with high sensitivity and specificity is permanently being sought in order to assess the degree of liver damage. In addition to liver biopsy, algorithms using blood parameters or elastometry are used in clinical practice. However, these methods do not provide information about the true liver reserve, so the liver breath test seem to be a promising diagnostic tool. The basis of this test depends on the ability of particular hepatocyte enzyme systems to metabolise a tested substance labelled with a stable carbon isotope. The kinetics of (13)CO2 elimination with expiratory air then permits quantitative assessment of the functional liver reserve and the degree of organ damage. In this paper the most commonly used tests, grouped according to the main metabolic pathways, are described. The usefulness of liver breath tests in specific clinical situations, both as a diagnostic and prognostic tool, is presented.

The effect of drugs and stimulants on gastric myoelectrical activity.

Electrogastrography (EGG) is a non-invasive diagnostic method useful for the registration and analysis of gastric myoelectrical activity. Abnormalities within an electrogastrogram were found to correlate with a number of disorders and symptoms, like functional dyspepsia, diabetic gastroparesis and terminal hepatic or renal failure. The EGG is also a valuable diagnostic method enabling the evaluation of the effect of drugs on gastric myoelectrical activity, which can be intentional, as in the case of prokinetics, or can have an adverse character. Our review focuses on drugs with a proven impact on gastric myoelectrical activity and hence on the electrogastrogram. The paper assembles and discusses the results of investigations dealing with changes in the electrogastrograms evoked by various drugs. Moreover, the mechanisms of the influence on the gastric myoelectrical activity of drugs, curative substances and stimulants are presented.

Pragmatically on the sense of taste - a short treatise based on culinary art.

The sense of taste is essential for proper functioning of the organism. The authors describe, in an accessible way, the complex mechanisms of taste perception. The structure of particular taste receptors, variants of their activation, as well as physical and chemical factors modifying the sensation of taste, are presented. Exquisite culinary examples are given in order to facilitate the reader with the understanding of why, at the level of the cerebral cortex, a virtually infinite number of combinations of taste sensations can be perceived. The discourse is spiced up by reflections of the eminent philosopher of taste, J.A. Brillat-Savarin, who convinces us that food intake should be not only a physiological act, but also a refined pleasure.