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Biotechnology and Applied Biochemistry (2001) 34, (183–187) (Printed in Great Britain)

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

J. Mark Hexham*¹, Vicki King*, Debra Dudas*, Patrick Graff†, Marion Mahnke†, Y. Karen Wang‡, Jean-Francois Goetschy§, David Plattner†, Mauro Zurini†, Francis Bitsch†, Phil Lake* and Mary Ellen Digan*

*Transplantation Research, Novartis Pharmaceuticals, 556 Morris Ave, Summit, NJ 07901, U.S.A., †Core Technologies, Novartis Pharma AG, Basel, 4002, Switzerland, ‡Core Technologies, Novartis Pharmaceuticals, 556 Morris Ave, Summit, NJ 07901, U.S.A. and §Transplantation Research, Novartis Pharma AG, Basel, 4002, Switzerland

Key words: antibody engineering, biopharmaceuticals, diphtheria toxin, tolerance, transplantation.

Abbreviations used: DT, diphtheria toxin; DT389, amino acids 1–389 of DT; Mab, monoclonal antibody; MAP, methionine aminopeptidase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium inner salt); scFv, single-chain variable-region antibody fragment; UCHT1, an anti-(human CD3) monoclonal antibody.

¹To whom correspondence should be sent, at the following address: Functional Genomics, Novartis Pharmaceuticals, 556 Morris Ave, Summit, NJ 07901, U.S.A. (e-mail mark.hexham@pharma.novartis.com).

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.

Received 20 August 2001; accepted 27 September 2001

Portland Press Ltd © 2001