Donald I.H. Stewart

Heterologous biopharmaceutical protein expression in Streptomyces

The commercial production of human proteins in recombinant microorganisms for therapeutic use is well established. Systems have been developed to exploit the natural ability of certain bacteria to secrete properly folded, bioactive proteins into the extracellular medium. The streptomycetes are a relatively well-characterized group of nonpathogenic filamentous bacteria that have the capacity to secrete large amounts of protein. In particular, Streptomyces lividans has the ability to secrete human proteins at a commercially viable level, thanks to relatively well-established plasmid-based expression system, a high-biomass fermentation process and a low level of endogenous protease activity.

Preparation and Characterization of Monopegylated Human Granulocyte-Macrophage Colony-Stimulating Factor

ABSTRACT Conjugates of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) attached to polyethylene glycol (PEG) chains were prepared using amine-reactive chemistry. Molecular masses of the PEGs were 20, 30, and 40 kDa. The monopegylated forms were isolated by anion-exchange chromatography and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography, mass spectrometry, reverse-phase high-performance liquid chromatography (HPLC), peptide mapping, in vitro cell proliferation bioassays, and rat pharmacokinetic studies. The pegylation site of the purified monopegylated products was identified as the N-terminus of the protein. All forms of pegylated GM-CSF were able to stimulate TF-1 cell proliferation in a colorimetric bioassay at concentrations equal to or lower than that of GM-CSF. Pharmacokinetic studies in rats demonstrated 32-fold, 27-fold, and 40-fold extensions in elimination half-lives for 20, 30, and 40 kDa PEG-GM-CSF, respectively, as compared with nonmodified GM-CSF.

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding its antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human VH repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the VH3 (41%), VH4 (39%), and VH1 (14%) subgroups used more frequently than the VH5 (3.9%) or VH7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human VH genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.

Development of a Competitive Enzyme Linked Immunosorbent Assay to Identify Epitope Specific Antibodies in Recipients of the U.S. Licensed Anthrax Vaccine

Abstract Vaccination with anthrax vaccine adsorbed (AVA) results in the production of protective antigen (PA) specific antibodies, which play an important protective role against anthrax toxins. Analyzing the specificity of serum antibodies generated in response to AVA vaccination can provide insight into the mechanisms of protective immunity against this important pathogen. The goal of this study was to develop a competitive enzyme linked immunosorbent assay (cELISA) to test human immune serum for antibodies specific for a known lethal toxin neutralizing epitope in PA. PA‐specific antibodies in sera from individuals who received the six‐dose AVA vaccine series competed for binding to immobilized PA with monoclonal antibody F20G75, which binds to a linear epitope in domain 2 of PA and neutralizes lethal toxin activity in vitro. These results suggest that antibodies in human AVA vaccinee serum recognize the same epitope as F20G75, or one in close proximity to it, and may serve a protective role against anthrax lethal toxin. This assay may be used for serological confirmation of successful immunization against anthrax and for the identification of antibodies in human vaccinee serum that recognize protective epitopes on PA.