Stewart D. Nuttall

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics.

Protein affinity maturation using molecular evolution techniques to produce high-affinity binding proteins is an important step in the generation of reagents for cancer diagnosis and treatment. Currently, the most commonly used molecular evolution processes involve mutation of a single gene into complex gene repertoires followed by selection from a display library. Fd-bacteriophage are the most popular display vectors, but are limited in their capacity for library presentation, speed of processing and mutation frequency. Recently, the potential of ribosome display for directed molecular evolution was recognised and developed into a rapid and simple affinity selection strategy using ribosome complexes to display antibody fragments (scFv). Ribosome display and selection has the potential to generate and display large libraries more representative of the theoretical optima for naïve repertoires (10(14)). Even more important is the application of ribosome display for the affinity maturation of individual proteins by rapid mutation and selection cycles. These display strategies can apply to other members of the immunoglobulin superfamily; for example single V-domains which have an important application in providing specific targeting to either novel or refractory cancer markers. We discuss the application of ribosome display and selection in conjunction with variable domain (CTLA-4) libraries as the first step towards this objective and review affinity maturation strategies for in vitro ribosome display systems.

Selection and Affinity Maturation of IgNAR Variable Domains Targeting Plasmodium falciparum AMA1

The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide‐bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (VNAR) domains bind antigen independently, and are candidates for the smallest antibody‐based immune recognition units. We have previously produced a library of VNAR domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of VNAR antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen‐1 (AMA1) from Plasmodium falciparum. Two related VNAR clones were selected, characterized by long (16‐ and 18‐residue) CDR3 loops. These recombinant VNARs could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (KD= ∼2 × 10−7 M). One clone, designated 12Y‐2, was affinity‐matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed ∼10‐fold enhanced affinity over 12Y‐2 and was Plasmodium falciparum strain‐specific. Importantly, we demonstrated that this monovalent VNAR co‐localized with rabbit anti‐AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications. Proteins 2004;00:000–000.

Crystal Structure of the Amyloid-β p3 Fragment Provides a Model for Oligomer Formation in Alzheimer's Disease

Alzheimers disease is a progressive neurodegenerative disorder associated with the presence of amyloid-β (Aβ) peptide fibrillar plaques in the brain. However, current evidence suggests that soluble nonfibrillar Aβ oligomers may be the major drivers of Aβ-mediated synaptic dysfunction. Structural information on these Aβ species has been very limited because of their noncrystalline and unstable nature. Here, we describe a crystal structure of amylogenic residues 18–41 of the Aβ peptide (equivalent to the p3 α/γ-secretase fragment of amyloid precursor protein) presented within the CDR3 loop region of a shark Ig new antigen receptor (IgNAR) single variable domain antibody. The predominant oligomeric species is a tightly associated Aβ dimer, with paired dimers forming a tetramer in the crystal caged within four IgNAR domains, preventing uncontrolled amyloid formation. Our structure correlates with independently observed features of small nonfibrillar Aβ oligomers and reveals conserved elements consistent with residues and motifs predicted as critical in Aβ folding and oligomerization, thus potentially providing a model system for nonfibrillar oligomer formation in Alzheimers disease.

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine‐specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K d=1.31±0.26×10−7 M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody‐like molecule.

Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides

We have designed and engineered the human cytotoxic T‐lymphocyte associated protein‐4 (CTLA‐4) variable (V‐like) domain to produce a human‐based protein scaffold for peptide display. First, to test whether the CTLA‐4 CDR‐like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14‐residue intra‐disulfide‐linked neuropeptide. Upon expression as periplasmic‐targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild‐type V‐domain were produced. These mutations in CTLA‐4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation‐dependent anti‐CTLA‐4 monoclonal antibody showed that the V‐domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild‐type and mutated CTLA‐4 proteins on the surface of fd‐bacteriophage as fusions with the geneIII protein. CTLA‐4 displayed on phage bound specifically to immobilized CD80‐Ig and CD86‐Ig and in one‐step panning enriched 5,000 to 2,600‐fold respectively over wild‐type phage. Bacteriophage displaying CTLA‐4 with somatostatin in CDR3 (CTLA‐4R‐Som3) specifically bound somatostatin receptors on transfected CHO‐K1 cells pre‐incubated with 1μg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 μM somatostatin successfully competed with CTLA‐4R‐Som3. CTLA‐4R‐Som3 also activated as well as binding preferentially to non‐glycosylated receptor subtype Sst4. The ability to substitute CDR‐like loops within CTLA‐4 will enable design and construction of more complex libraries of single V‐like domain binding molecules. Proteins 1999;36:217–227.

Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype.

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen‐driven affinity‐matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an “upright” conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3‐specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low‐sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen‐binding surface.

Engineering of an anti‐epidermal growth factor receptor antibody to single chain format and labeling by sortase A‐mediated protein ligation

Sortase‐mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three‐component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo‐glycine acceptor molecule. We describe cloning of the single‐chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG‐biotin onto sc528. EGFR is an important cancer target and is over‐expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA‐biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen‐specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site‐specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody–receptor interaction. Biotechnol. Bioeng. 2012; 109:1461–1470.

Display scaffolds: protein engineering for novel therapeutics.

Protein scaffolds represent a new generation of universal binding frameworks for use as future immunopharmaceuticals to complement the expanding repertoire of therapeutic monoclonal antibodies. Here, we review recent literature describing advances in protein scaffold development, including efforts to engineer the minimal immunoglobulin-based binding-domain and molecular library design. Several diverse protein folds are currently under development on the basis of modular construction, a strategy also observed in families of naturally evolved immune receptors. We describe potential therapeutic and intracellular applications where scaffold-specific features provide distinct advantages for targeting of non-conventional antigens and comment on the scientific progress and validation of several designed scaffolds in the voyage towards first-in-human trials.

Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody.

The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V(NAR)) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V(NAR)s possess advantages in stability, size (~14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V(NAR) domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V(NAR) was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.