Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein

• Published in: Biotechnology and applied biochemistry 2001-12, Vol.34 (3), p.183-187
• Main Authors: Hexham, J. Mark, King, Vicki, Dudas, Debra, Graff, Patrick, Mahnke, Marion, Wang, Y. Karen, Goetschy, Jean-Francois, Plattner, David, Zurini, Mauro, Bitsch, Francis, Lake, Phil, Digan, Mary Ellen
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - toxicity; antibody engineering; Biological and medical sciences; biopharmaceuticals; Biotechnology; CD3 antigen; CD3 Complex - immunology; Cell Survival - drug effects; Cloning, Molecular; diphtheria toxin; Diphtheria Toxin - chemistry; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Humans; Immunoglobulin Fragments - immunology; Immunoglobulin Variable Region - immunology; Immunotoxins - chemistry; Immunotoxins - genetics; Immunotoxins - toxicity; Industrial applications and implications. Economical aspects; Jurkat Cells; Methionine - genetics; Mutagenesis; Other active biomolecules; Production of active biomolecules; Recombinant Proteins - chemistry; Recombinant Proteins - toxicity; Sequence Analysis, Protein - methods; tolerance; transplantation
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1042/BA20010073

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin‐based anti‐(human CD3) immunotoxin, DT389–scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1–389 of diphtheria toxin, scFv is single‐chain variable‐region antibody fragment and UCHT1is an anti‐(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N‐terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N‐terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N‐terminally homogeneous molecule, a panel of seven N‐terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N‐terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389–scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N‐terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N‐terminally homogeneous variants of DT389–scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.