title: Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems.
abstract: BACKGROUND
  Antibodies 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.
  
  
  RESULTS
  An 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Department of Pathology and Diagnostics, University of Verona, Verona, Italy. pietro.decri@gmail.com.
authors.forename: Pietro
authors.initials: P
authors.lastname: Della Cristina
authors.affiliation: Department of Pathology and Diagnostics, University of Verona, Verona, Italy. monica.castagna@univr.it.
authors.forename: Monica
authors.initials: M
authors.lastname: Castagna
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy. alessiolombardi64@yahoo.it.
authors.forename: Alessio
authors.initials: A
authors.lastname: Lombardi
authors.affiliation: Department of Pathology and Diagnostics, University of Verona, Verona, Italy. erika.barison@gmail.com.
authors.forename: Erika
authors.initials: E
authors.lastname: Barison
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy. giovannitagliabue@virgilio.it.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Tagliabue
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy. ceriotti@ibba.cnr.it.
authors.forename: Aldo
authors.initials: A
authors.lastname: Ceriotti
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy. ikoutris@yahoo.com.
authors.forename: Ilias
authors.initials: I
authors.lastname: Koutris
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy. luana.dileandro@univaq.it.
authors.forename: Luana
authors.initials: L
authors.lastname: Di Leandro
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy. francesco.giansanti@cc.univaq.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Giansanti
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy. vago.riccardo@hsr.it.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Vago
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy. rodolfo.ippoliti@univaq.it.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ippoliti
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, (Leukaemia Busters), Southampton General Hospital, Southampton, UK. beef@leukaemiabusters.org.uk.
authors.forename: Sopsamorn U
authors.initials: SU
authors.lastname: Flavell
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, (Leukaemia Busters), Southampton General Hospital, Southampton, UK. DavidF@leukaemiabusters.org.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Flavell
authors.affiliation: Department of Pathology and Diagnostics, University of Verona, Verona, Italy. marco.colombatti@univr.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Colombatti
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy. msfabbrini@gmail.com.|Istituto Nazionale di Genetica Molecolare-INGM, Milan, Italy. msfabbrini@gmail.com.
authors.forename: Maria Serena
authors.initials: MS
authors.lastname: Fabbrini
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002051:Burkitt Lymphoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics / Q000378:metabolism*; D000076985:Saporins; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D051918:Sialic Acid Binding Ig-like Lectin 2; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A