Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems

Microbial Cell Factories

Research

Riccardo Vago¹  Ilias Koutris¹  Luana Di Leandro¹  Rodolfo Ippoliti¹  Francesco Giansanti¹  Erika Barison²  Pietro Della Cristina²  Monica Castagna²  Marco Colombatti²  Alessio Lombardi³  Giovanni Tagliabue³  Aldo Ceriotti³  Maria Serena Fabbrini⁴  Sopsamorn U Flavell⁵  David J Flavell⁵ 

[1] Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy;Department of Pathology and Diagnostics, University of Verona, Verona, Italy;Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy;Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy;Istituto Nazionale di Genetica Molecolare-INGM, Milan, Italy;The Simon Flavell Leukaemia Research Laboratory, (Leukaemia Busters), Southampton General Hospital, Southampton, UK;

关键词: Recombinant immunotoxins;    Anti-CD22;    Pseudomonas exotoxin A;    Saporin;    Bacterial/eukaryotic expression systems;   

DOI  :  10.1186/s12934-015-0202-z

 received in 2014-10-21, accepted in 2015-01-27,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundAntibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats.ResultsAn anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range.ConclusionsWe undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in E. coli or P. pastoris expression systems and also with regard to each constructs specific killing efficacy. Our results confirm that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin whereas we further demonstrate that saporin-based ITs are best expressed and recovered from P. pastoris cultures after yeast codon-usage optimization.

【 授权许可】

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© Della Cristina et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.