Gregory Coia

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.

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.

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.

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.

Construction of recombinant extended single-chain antibody peptide conjugates for use in the diagnosis of HIV-1 and HIV-2

The construction, expression and evaluation of recombinant scFv based HIV diagnostic reagents are described. In a whole-blood, erythrocyte agglutination assay format, recombinant scFv antibodies (expressed in Escherichia coli), linked to a spacer domain and HIV-gp36 or -gp41 peptides, were shown to be able to detect efficiently natural antibodies against HIV in human serum. Performance in trials suggests that these single chain reagents have potential as alternatives to existing Fab-peptide chemical conjugates. We also report the construction of an inducible expression vector, pGC, which can be used both in laboratory experiments and in large-scale fed-batch fermentations. It was found that while the base scFv reagent (lacking a spacer) functioned as well as the Fab peptide conjugate in assays where whole (negative) blood was spiked with mouse monoclonal anti-HIV antibodies (IgG or IgM), clinical assays using human sera showed lower sensitivities and increased false negatives. This deficiency was overcome by inclusion of the natural 1C3 kappa (light) chain domain as a spacer arm between the scFv and HIV peptide tags. This spacer was thought to overcome steric constraints which would otherwise prevent efficient interaction between the reagent (once bound to the surface of red blood cells) and the various serum antibodies against the respective C-terminal peptide epitopes. As a result of this important modification, performance of the extended scFv reagent (for both HIV-1 and HIV-2) equalled that of the current commercial technology in limited trials.

Escherichia coli expression of a bifunctional Fab-peptide epitope reagent for the rapid diagnosis of HIV-1 and HIV-2

BACKGROUND The current format of a rapid whole-blood agglutination assay for HIV relies on a bifunctional molecule which comprises a 1C3 Fab fragment, with specificity for the human red blood cell surface marker (glycophorin A), chemically conjugated to a synthetic peptide that corresponds to a single immunodominant region of HIV envelope glycoprotein. In this assay erythrocyte agglutination occurs if the blood sample contains anti-HIV antibodies. OBJECTIVES To establish whether a bacterially synthesised Fab fragment encoding several C-terminal immunodominant peptide tails can be produced in sufficient purity and yield to function in whole-blood agglutination assays. STUDY DESIGN An E. coli dicistronic Fab expression cassette was constructed comprising of light and heavy chain gene fragments derived from a glycophorin specific monoclonal antibody (1C3), genetically linked with C-terminal immunoreactive peptide epitopes. Expression and purification procedures were established to enable the rapid production of 1C3 Fab-peptide epitope conjugates. RESULTS A recombinant 1C3 Fab fragment was expressed with two different immunological epitope markers, Glu-Glu-Phe (EEF) and FLAG, at the C-terminus of the Fd heavy and kappa light chain, respectively. This model Fab-EEF/FLAG conjugate was detected in culture supernatant by SDS-PAGE gels and Western blots, and could be successfully used in erythrocyte agglutination assays. Furthermore, an HIV specific 1C3 Fab reagent, containing immunoreactive peptide epitopes from the surface glycoproteins of HIV-1 and HIV-2, was also expressed but at lower levels and with increased sensitivity to proteolytic degradation. Nevertheless, this recombinant Fab reagent with dual diagnostic specificity performed very effectively in whole-blood diagnosis of patients infected with either HIV-1 or HIV-2. CONCLUSION A recombinant 1C3 Fab fragment terminated by immunoreactive peptide epitopes can be expressed in E. coli in a soluble, antigen-binding form, and it can successfully mimic the commercial Fab-HIV reagents in whole-blood agglutination assays.

Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling

The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimers disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal.

Cancer-targeting Antibody–Drug Conjugates: Site-specific Conjugation of Doxorubicin to Anti-EGFR 528 Fab' through a Polyethylene Glycol Linker

Antibody–drug conjugates have been prepared to examine the effect that attaching small-molecule drugs to an antibody fragment has on antibody activity. The anticancer drug doxorubicin was covalently attached through a polyethylene glycol linker to a cancer-targeting, anti-epidermal growth factor receptor antibody fragment (Fab′). The reactivity of maleimide was compared with a substituted maleimide derivative (citraconimide) in conjugation reactions with cysteine residues on a Fab′. Introduction of polyethylene glycol increased aqueous solubility of the cytotoxic drug, which led to an improvement in overall yield of the conjugation reaction with the antibody fragment. Antibody–drug conjugates prepared retained activity of the parent antibody, as determined by antigen binding experiments measured by surface plasmon resonance.