PE类重组免疫毒素的研究进展

PE类重组免疫毒素是将经过修饰加工后失去与细胞结合能力的PE分子与导向分子(载体)通过DNA融合技术而成的新型导向药物。因其可特异性识别、结合和杀伤靶细胞,因而被用来治疗肿瘤、自身免疫性疾病以及AIDS等疾病。目前有许多种PE类免疫毒素正在进行临床试验,尤其治疗恶性肿瘤方面已取得显著的疗效,预期这些药物在未来几年将取得更长足的发展。本文对目前PE重组免疫毒素的研究进展作一综述。     

免疫毒素的抗肿瘤研究

免疫毒素是由具有导向能力的载体（抗体或细胞因子）和具有细胞毒性的分子（毒素）偶联而成的具有特异性细胞杀伤能力的杂合分子,是一种靶向药物。免疫毒素首先利用肿瘤细胞上导向分子的受体与细胞结合,然后进入细胞,再由毒素发挥蛋白质合成抑制作用,最终导致靶细胞死亡。与其他抗肿瘤药物相比,免疫毒素具有毒性强和特异性高的优点,在肿瘤治疗中显示出巨大的应用前景。简要概述了免疫毒素的作用机理、制备及其抗肿瘤研究进展。     

抗CEA免疫毒素的表达、纯化、复性与生物学活性的研究

以抗癌胚抗原（Carcinoembryonic antigen, CEA）单链抗体与假单胞菌外毒素（Pseudomonas exotoxin A, PEA）的截短和修饰形式PE38/KDEL构建重组免疫毒素CEA/PE38/KDEL,并在大肠杆菌菌株BL21(DE3)-star中表达。采用镍离子螯合层析法纯化变性的包涵体样品,并用连续梯度透析的方法对纯化后的包涵体进行复性。采用流式细胞术鉴定复性产物与靶细胞的结合活性,结果表明免疫毒素CEA/PE38/KDEL具有与靶细胞特异性结合的活性。以MTT法检测免疫毒素对肿瘤细胞的体外杀伤活性,结果表明该免疫毒素对SW1116和CNE_2细胞具有特异性杀伤活性。证明了经包涵体复性的抗CEA免疫毒素CEA/PE38/KDEL对表达CEA抗原的肿瘤细胞具有良好的结合和杀伤活性。     

白细胞介素2免疫毒素研究进展

应用基因工程技术已研制出多种免疫毒素,IL-2免疫毒素是研究最为成功的例子之一,包括IL-2与绿脓杆菌外毒素的融合蛋白,IL-2与白喉毒素的融合蛋白等.这类毒素以IL-2为导向分子,IL-2受体阳性细胞为靶细胞,治疗移植排斥反应、自身免疫性疾病、T细胞淋巴瘤等疾病,在Ⅰ/Ⅱ期临床试验中已取得了较好的疗效.但是这类免疫毒素对人体均有较强的抗原性,使治疗时间和效果均受限制,为此研制了人源化的IL-2免疫毒素,能在体内外杀伤IL-2受体阳性细胞.     

假单胞菌外毒素免疫原性的改善策略

重组免疫毒素(recombinant immunotoxin, RIT)是靶向杀伤肿瘤细胞的药物,由抗体与毒素分子连接而成的融合蛋白。其特异性抗体和靶细胞表面的抗原结合,介导毒素分子进入细胞起到杀伤细胞的作用。多种细菌和植物毒素已被用于制备免疫毒素。假单胞菌外毒素(pseudomonas exotoxin, PE)是构建免疫毒素的优选分子,被证明具有极高的细胞毒性。由于PE分子的免疫原性强、穿透能力差等原因,使其疗效低于预期。近年来利用各种方法降低RIT的免疫原性,去掉B细胞表位和T细胞表位,成为研究重点。现就PE的结构、功能以及免疫原性的降低等作一概述。     

Seragen的融合毒素将成为抗癌药物

有些白血病和淋巴瘤很难治疗.Seragen Inc.'s(Hopkinton,MA)的白细胞介素-2(IL-2)融合毒素对这类顽症显出抗肿瘤效应.此外,其副作用还小于目前常用的治癌药物. 融合蛋白能以极高的精确度识别、穿透并摧毁致病细胞.这种重组蛋白由白喉毒素和生长因子(或称生长激素)配基组成.这些分子能与那些表面带有配基受体的细胞结合.在使用方法和作用方式上,融合毒素与单抗型免疫毒素相同.差异之处在于定靶方式.在     

白喉毒素A片段的表达纯化与单克隆抗体制备

白喉毒素 (Diphtheriatoxin ,DT)A片段 (DTA)是白喉毒素的酶活性区 ,也是DT类免疫毒素的关键结构域。DTA蛋白及其单克隆抗体在免疫毒素的毒性机理、检测与纯化研究等方面具有重要价值。通过在E .coli中表达了DTA ,经Q SepharoseFF和Ni²⁺ Sepharose两步层析纯化 ,得到纯度约为 90 %的融合蛋白。以DTA为抗原免疫BalB c小鼠 ,获得了分泌抗DTA特异单抗的杂交瘤细胞株 3B6和 3B9。单抗为IgG1亚型 ,滴度达 1∶10⁶ 以上 ,与DTA的结合可被抗DT马血清竞争抑制。抗DTA单抗用于免疫印迹试验 ,或制备成免疫亲和柱纯化基于DT的重组免疫毒素 ,均获得较好效果 ,为免疫毒素的研究奠定了良好基础     

免疫毒素研究与一种新型耐热DNA多聚酶

1991年3月26日,苏联分子诊断研究中心(Research Center of Molecular Diagnostics)主任Severin教授和Kiselev教授来病毒学研究所作学术访问。 Severin主任主要介绍了免疫毒素的研究情况,他的研究小组分离出一种免疫毒素,它可以和IgG抗体结合,结合状态的免疫毒素是没有活性的,但当IgG特异性地和靶细胞结合后,毒素释放,从而特异性     

免疫毒素设计的新思路——基因免疫毒素

免疫毒素设计的新思路——基因免疫毒素严明张祖传（中国科学院上海生物化学研究所,上海２０００３１）关键词免疫毒素基因免疫毒素免疫毒素是一种导向性药物,它是由生物来源的毒蛋白和抗肿瘤抗体或某些细胞表面受体的配基偶联而成,以抗体或配基为载体,把毒素定向带到...     

受体特异性蛋白:对Mabs药物生产的一个挑战

尽管受体特异性蛋白还没有广泛地应用,然而利用其在体内通过特异性受体结合到靶药物上(而不必用 Mabs),从而识别特异性细胞的尝试已越来越多。例如 Seragen 股份有限公司(马萨诸塞州霍普金顿)和波士顿、马萨诸塞的几家医院的研究人员已经成功地利用白细胞介素-2(IL-2)和部分白喉毒素的融合蛋白杀死动物中的活性 T 细胞和阻止所谓延缓型过敏性(DTH)反应。上述研究小组用 IL-2基因替代白喉毒素中的受体结合编码区,并在大肠杆菌中生产了     

Comparison of protein synthesis inhibition kinetics and cell killing induced by immunotoxins

Immunotoxins comprised of a monoclonal antibody covalently coupled to recombinant ricin A chain or to a binding-defective form of diphtheria toxin were compared with respect to their rates of protein synthesis inhibition and efficiencies of killing target cells. Protein synthesis inhibition rates were established by measuring the incorporation of L-[14C]leucine in toxin-treated cells relative to untreated cells at several times after exposure of cells to an immunotoxin. Cell killing was assessed by a limiting dilution assay which measures the number of cells surviving toxin treatment relative to untreated cells. At equivalent protein concentrations, the diphtheria toxin immunotoxin inhibited protein synthesis significantly more rapidly than the ricin A immunotoxin but, contrary to previous predictions, achieved a significantly lower cell kill. Thus, the kinetics of protein synthesis inactivation do not necessarily correlate with killing efficiencies. Possible explanations for these results are that the effect of the diphtheria toxin immunotoxin on protein synthesis is partially reversible or that the diphtheria toxin immunotoxin enters the cytosol at a faster rate than the ricin A immunotoxin but also is degraded at a faster rate.     

Development of a diphtheria toxin mutant conjugate directed against antigen-specific B cells expressing high affinity surface Ig

Modification of a mutant diphtheria toxin, possessing reduced binding capacity, with TNP groups resulted in an Ag-toxin conjugate capable of eliminating TNP-specific B cells. Previous experimental approaches to the elimination of Ag-specific B cells have involved the conjugation of Ag to holoricin molecules or ricin A chain. Holoricin conjugates possess efficacy, but display high nonspecific toxicity. A chain conjugates, which appear specific, lack high potency. In developing the diphtheria toxin-based conjugate, we found high potency for target anti-TNP hybridoma cells and for spleen cells isolated from TNP-immunized mice. The similar intoxication of nontarget cells required concentrations approximately three orders of magnitude higher. Additionally, it was found that the TNP-specific agent may have selectively depleted B cells producing high affinity IgG anti-TNP antibodies.     

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.     

Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.     

A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.     

Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins

Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3–10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudo-monas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.     

Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin

Immunotoxins are antibody–toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.     

Antibody-mediated routing of diphtheria toxin in murine cells results in a highly efficacious immunotoxin

The chemical coupling of diphtheria toxin to an antimurine Thy1 antibody resulted in the most efficacious immunotoxin to date. At 1 micrograms/ml the immunotoxin inhibited protein synthesis of a Thy+ AKR murine cell at a rate of 1.4 logs/h, within the order of magnitude of the efficacy of native toxins. This is unusual since murine cells are highly resistant to diphtheria toxin. The conjugate is highly specific; Thy- AKR cells display no intoxication at 1 microgram/ml even after 18 h. The effects of ammonia, acid pulsing of external media, and low temperature reveal some similarities and some differences between intoxication of sensitive cells by toxin and of murine cells by the antibody-toxin conjugate. The differences that result in the high efficacy of the antibody-toxin conjugate appear to result from the antibody-mediated routing. These results imply that murine cells possess an acidic compartment which can mediate toxin cytosolic entry. Unlike the Thy antigen, the toxin receptor on murine cells is unable to route the toxin to this cellular site.     

Construction and preliminary investigation of a plasmid containing a novel immunotoxin DT390-IL-18 gene for the prevention of murine experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a neuropathological animal model for multiple sclerosis. Antigen-presenting cells (APCs) expressing interleukin-18 receptor (IL-18R) were shown to be crucial in the beginning and progress of EAE. In this study we tested the effect of a novel recombinant immunotoxin targeting IL-18R-bearing APC for EAE prevention. The novel eukaryotic plasmid DT390-IL-18-SRalpha, encoding recombinant immunotoxin DT390-IL-18, was constructed. The immunotoxin consisted of IL-18 as the targeting moiety, and a truncated diphtheria toxin (DT) as the toxic moiety. Transfection assay and proliferation inhibition assay proved the immunotoxin could be expressed in vitro and was toxic to the activated mouse T cells. To evaluate the preventive effect of DT390-IL-18-SRalpha on EAE in vivo, cationic liposome-embedded DT390-IL-18-SRalpha was injected into the hind limbs of EAE mice. DT390-IL-18-SRalpha-treated mice showed a delayed manifestation of EAE and decreased symptoms compared to the mice treated with plasmid DT390-SRalpha or phosphate-buffered saline alone. A significant reduction in infiltrating inflammatory cells was detected in the brain tissues from immunotoxin-treated mice as compared with the controls by hematoxylin-eosin staining. This study suggested that the recombinant immunotoxin DT390-IL-18 could be expressed in vitro and in vivo, and prevented murine EAE effectively.