Robert Alexander Irving

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.

Affinity maturation of recombinant antibodies using E. coli mutator cells.

BACKGROUND Phage libraries can display repertoires of antibodies which are greater in number than the mammalian immune response. However, the selected antibodies often have low binding affinity to their target antigen or hapten (KD below 10(-6) M), which is characteristic of the primary immune repertoire. There is a need for procedures to mimic somatic hypermutation through antigen driven affinity maturation, thereby increasing the affinity of selected immunoglobulins. OBJECTIVE To investigate the effectiveness of mutation and affinity selection of recombinant antibody genes with mutator E. coli cells, incorporating phage-display strategies. STUDY DESIGN Unique human scFvs were selected from a naive Fd-phage library. These genes were mutated by propagation in mutD5 mutator E. coli cells (mutD5-FIT) which were competent for Fd (M13) based phagemid transfections and generated point mutations (transversions and transitions) in the scFv genes. Individual phage-displayed scFvs were affinity selected from the mutation library and were assayed as soluble scFvs by ELISA and BIAcore for binding to antigen. RESULTS The in vivo mutation of phage-displayed scFvs in E. coli mutD5-FIT, combined with affinity selection against antigen, produced scFv molecules with improved binding activity. The point mutations which resulted in single amino acid substitutions frequently produced ten fold increases in apparent binding affinity. Structural comparisons revealed that these point mutations were in framework regions (adjacent to the CDRs) and within the CDRs. In one case the apparent affinity of an anti-glycophorin scFv after mutation in the VL framework region close to CDR3 increased by 10(3). However, this increase in apparent affinity was accompanied by an increased propensity to dimerise and form aggregates. CONCLUSIONS A strategy for the rapid affinity maturation of scFv and Fab antibody fragments has been developed which utilises mutator strains of E. coli and incorporates phage display of antibody repertoires (libraries).

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.

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.

Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B

A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following production of the scFv protein using an E. coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA). This gene construct was introduced into E. coli mutator cells and the transformed cells were used as an inoculum for liquid cultures. After five cycles of growth at 37 degrees C, each followed by dilution and re-inoculation of fresh media, recombinant phage were recovered. Nucleotide sequence analysis of the scFv gene in phage selected on HBsAg-coated magnetic beads identified amino acid substitutions which produced an increase of greater than 10-fold in apparent production levels. Competitive ELISA studies showed that the selected scFv mutants appeared to have similar affinity to HBsAg as the parent scFv. The apparent increase in production was not the result of improved surface characteristics of regions uniquely exposed in scFvs, as the sites did not correlate with the variable/constant interface of the scFv variable region normally masked in Fabs or IgGs.

High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal

We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins. The vector contains the lambda pRpL promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production. The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane. A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l. This represents the highest yields reported to date for Ab fragments with a native N terminus. An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process. The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product. The purified Ab VH fragment showed binding affinity (Ka less than 10(4)/M) to its target antigen (neuraminidase).

Protein affinity maturation in vivo using E. coli mutator cells

This protocol provides a simple in vivo strategy for introducing random mutations to a target DNA sequences using E. coli mutator cells. The method has been used in our laboratory for affinity maturation of proteins encoded by target DNA sequences. Selection conditions can be modified for antibody fragments with increased production levels. Growth conditions in E. coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, approximately equivalent to one codon change per scFv fragment.

Panning and selection of proteins using ribosome display

Eukaryotic ribosome complexes can be used as a means to display a library of proteins, and isolate specific binding reagents by screening against target molecules. Here we present, as an example, a method for the display of a library of immunoglobulin variable-like domains (VLDs) for the production of stable mRNA/ribosome/protein complexes. These complexes are produced by the addition of specific in vitro transcriptional promoter elements and translation control sequences to the template DNA. Furthermore, an appropriate spacer (anchor) domain is included for efficient folding of the nascent translated protein, which remains attached to the ribosome complex. Ribosome complexes are panned against hen egg lysozyme-conjugated magnetic beads and genes encoding specific, binding, V-like domains are recovered by RT-PCR and cloned into an Escherichia coli expression vector.