Della Cristina Pietro, Castagna Monica, Lombardi Alessio, Barison Erika, Tagliabue Giovanni, Ceriotti Aldo, Koutris Ilias, Di Leandro Luana, Giansanti Francesco, Vago Riccardo, Ippoliti Rodolfo, Flavell Sopsamorn U, Flavell David J, Colombatti Marco, Fabbrini Maria Serena
Department of Pathology and Diagnostics, University of Verona, Verona, Italy.
Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy.
Microb Cell Fact. 2015 Feb 13;14:19. doi: 10.1186/s12934-015-0202-z.
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.
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.
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.
针对在恶性细胞表面过表达的特定抗原产生的抗体已与蛋白质毒素结构域进行化学偶联，以获得能够选择性杀死癌细胞的免疫毒素（ITs）。由于最新一代免疫毒素由与抗体片段基因融合的毒性结构域组成，这些抗体片段赋予ITs靶向选择性特异性，我们从杂交瘤4KB128中拯救出一种靶向CD22的重组单链可变片段（scFv），CD22是B系白血病和淋巴瘤表达的一种标志物抗原。我们使用两种均能阻断蛋白质翻译的酶毒素构建了几种ITs，一种是细菌来源的（铜绿假单胞菌外毒素A的截短版本，PE40），具有EF - 2 ADP - 核糖基化活性，另一种是植物核糖体失活蛋白皂草素，能够特异性地使23/26/28S核糖体RNA脱嘌呤。选择PE40是因为它已被广泛用于构建重组ITs，这些ITs已在临床试验中进行了评估。皂草素也已进行临床评估，并且最近已在毕赤酵母表达系统中成功高水平表达。本研究的目的是评估各种IT形式的最佳微生物表达。
获得了一种名为4KB的抗CD22 scFv，它表现出预期的结合活性，也被CD22 + 靶细胞内化，并且也受到亲本单克隆CD22抗体的竞争。设计了几种融合构建体并在大肠杆菌或毕赤酵母中表达，然后对得到的融合蛋白进行亲和纯化。在CD22 + 人Daudi细胞上进行蛋白质合成抑制试验，结果表明所选的ITs具有活性，其IC50值（相对于对照抑制蛋白质合成50％的浓度）在纳摩尔范围内。
我们对不同融合构建体在大肠杆菌或毕赤酵母表达系统中的产量以及每种构建体的特异性杀伤效力进行了系统比较。我们的结果证实，大肠杆菌是表达细菌来源重组融合毒素的首选系统，而我们进一步证明，基于皂草素的ITs在酵母密码子使用优化后，在毕赤酵母培养物中表达和回收效果最佳。
