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。     

流式细胞术检测人巨细胞病毒活动性感染检测方法的建立及检测效果评价

探讨流式细胞术检测人巨细胞病毒活动性感染检测方法的可行性及效果评价。分离人外周血白细胞,以商品化的小鼠抗人巨细胞病毒pp65抗原单克隆抗体为一抗,FITC标记的羊抗小鼠IgG抗体为二抗,采用流式细胞术对外周血人巨细胞病毒pp65抗原进行检测。同时采用间接免疫荧光法对外周血人巨细胞病毒pp65抗原进行检测。采用配对χ2检验对两种方法的检测效果进行评价。临床送检的65份疑似为人巨细胞病毒感染病人外周血标本中,间接免疫荧光法检出阳性9份,流式细胞术检出阳性11份,两种方法阳性检出率差异无统计学意义(P0.05)。采用流式细胞术可定量检测外周血人巨细胞病毒pp65抗原,与间接免疫荧光法检测结果无统计学差异,可在临床推广使用。     

抗肿瘤免疫毒素稳定性的研究进展

免疫毒素 (immunotoxins)是由某些细菌和植物产生的毒素蛋白与抗体或生长因子等靶向分子连接而成 ,用于杀伤表面带有特定抗原或受体的细胞。在试验中 ,免疫毒素在肿瘤周围环境中的稳定性是影响其抗肿瘤效果的主要因素之一。本文主要对几种由不同形式的抗体Fv段构建的免疫毒素的稳定性进行了比较 ,并对定点突变和PEG化学修饰提高免疫毒素稳定性的方法做一简要介绍。     

抗胃癌免疫毒素表达质粒的构建、表达及对肿瘤细胞的特异杀伤

以含单链抗体 ( Sc Fv) 3H1 1基因全长的质粒 DNA为模板 ,利用 PCR技术扩增 3H1 1 Sc Fv基因片段 ,扩增片段及绿脓杆菌外毒素 PE38表达质粒 p YR39- 1 - PE38经 H ind /N de 酶切、连接 ,转化大肠杆菌 BL2 1,构建免疫毒素的表达质粒 p YR3H1 1 - PE38.转化菌在 IPTG诱导下 ,表达免疫毒素 3H1 1 - PE38,3H1 1 - PE38经纯化、变性、复性处理后 ,通过 MTT法检测其对胃癌细胞MGC80 3的杀伤活性 .结果表明 ,3H1 1 - PE38浓度不变 ,其杀伤率在一定的范围内随作用时间的延长而增加 ,当浓度为 5× 1 0 -8mol/L,作用时间为 60 h时 ,其对胃癌 MGC80 3细胞的杀伤率达74 .2 % ,而同等条件下抗 DNA免疫毒素 p Ig2 0 - PE38的杀伤率仅为 9.2 % ;作用时间一定 ( 60 h) ,免疫毒素浓度与杀伤率呈正相关 ,在 1 0 -10 mol/L以下 ,杀伤率几乎为零 ,而浓度高于 5× 1 0 -8mol/L时 ,杀伤率超过 70 % . 3H1 1 - PE38能够有效杀伤与之特异结合的胃癌细胞 ,具有潜在的应用前景     

抗肿瘤免疫毒素稳定性的研究进展

免疫毒素（immunotoxins)是由某些细菌和植物产生的毒素蛋白与抗体或生长因子等靶向分子连接而成,用于杀伤表面带有特定抗原或受体的细胞,在试验中,免疫毒素在肿瘤周围环境中的稳定性是影响其抗肿瘤效果的主要因素之一,本文主要对几种由不同形式的抗体Fv段构建的免疫毒素的稳定性进行了比较,并对定点突变和PEG化学修饰提高免疫毒素稳定性的方法做一简要介绍。     

高纯度白介素-3与假单胞菌外毒素融合蛋白的表达、纯化及质谱鉴定

旨在利用简单的表达纯化工艺,获得纯度较高、有活性的重组免疫毒素IL3-PE38KDEL并对其物理学及免疫学性质进行鉴定。利用PQE30-IL3-PE38KDEL/SG12009工程菌表达重组免疫毒素IL3-PE38KDEL,在提取包涵体后经一步强阳离子柱层析得到纯度较高的免疫毒素复性纯化产物,用Westenblotting、质谱、氨基测序等先进技术对产物鉴定。结果显示,发酵后目的蛋白表达量占总菌体的17.56%,经强阳离子柱层析纯化复性后的目的蛋白纯度达到90%以上,Western blotting、质谱、氨基测序结果均表明,纯化产物分子量为54.9kD且具有相应的免疫学活性,序列与预期序列完全一致。纯化复性方法对重组免疫毒素IL3-PE38KDEL的生物性质及纯度有较大影响,因此选择一种适当的纯化复性方法至关重要。     

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。     

含Arg9的人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达和内化作用的研究

目的：构建、表达和纯化带有转膜结构域Arg9的ScFv14/EGFP融合蛋白, 并对纯化产物的亲和 活性和内化作用进行研究。 方法： 将Arg9的编码序列分别重组到ScFv14/EGFP基因的5'端或3'端或二者之间,将它们分别克隆入原核表达载体pET32a,转化大肠杆菌BL21(DE3)LysS进行诱导表达和纯化,用间接ELISA方法检测表达产物与HBsAg的亲和活性,并用间接免疫荧光检测纯化蛋白的内化活性。 结果：经测序及酶切鉴定证实四种融合基因序列完全正确.SDS-PAGE和Western blot证实四种融合基因成功表达和纯化.间接ELISA检测证实四种融合蛋白均具有HBsAg结合活性,间接免疫荧光检测显示N端带有Arg9的融合蛋白有较强的内化作用,而且不内化进入HBsAg非表达的细胞。 结论：成功构建、表达和纯化了ScFv14/EGFP融合蛋白和三种带有Arg9的ScFv14/EGFP融合蛋白,纯化产物均具有与抗原HBsAg亲和的活性,N端带有Arg9的融合蛋白与靶细胞作用有较强的内化作用。     

抗A型产气荚膜梭菌α毒素全人源双价单链抗体的构建、表达及其活性的初步研究

目的:为防治A型产气荚膜梭菌α毒素引起的肠毒血症及气性坏疽等相关疾病,构建并高效表达中和α毒素(CPA)的特异性双价单链抗体,并对其生物学活性进行初步研究。方法:以全人源噬菌体抗体库中筛选得到的抗A型产气荚膜梭菌α毒素单链抗体sc Fv基因为模板,PCR的方法扩增两条单链抗体片段并通过引物设计引入中间连接肽G4S或(G4S)3,亚克隆至原核表达载体p ET-28a(+),转化E.coli BL21,IPTG诱导表达、鉴定及表达产物的层析纯化;Western blot和间接ELISA方法检测与抗原的免疫结合活性;通过体外检测抗体抑制CPA水解卵磷脂的活性和溶血活性以及体内小鼠攻毒保护试验初步研究双价单链抗体的生物学活性。结果:双酶切鉴定及基因测序结果表明构建的双价单链抗体sc(Fv)2-5和sc(Fv)2-15均正确,诱导表达后经12%SDS-PAGE分析,两者均以包涵体形式表达且蛋白分子量符合理论值大小,Western blot和间接ELISA分析结果显示,构建的双价单链抗体与抗原CPA具有特异结合活性,且sc(Fv)2-15与抗原的结合活性明显高于sc(Fv)2-5和sc Fv,体外与体内生物活性试验结果进一步证明,sc(Fv)2-15中和毒素的能力较sc(Fv)2-5和sc Fv具有明显优势。结论:成功制备了抗A型产气荚膜梭菌α毒素的全人源双价单链抗体,为进一步研究该毒素引发的各类疾病的诊断和治疗奠定了基础。     

一株针对人EGFR的单链抗体克隆与哺乳细胞表达

目的:制备特异性抗人表皮生长因子受体(EGFR)的单链抗体(sc Fv),鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法:从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5'RACE技术扩增轻链和重链可变区(VL、VH)基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果:获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论:成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。     

Arg9-peptide facilitates the internalization of an anti-CEA immunotoxin and potentiates its specific cytotoxicity to target cells

Arginines-containing membrane translocational signals (MTS), such as Tat(47-57) and VP22(267-300), and synthetic arginine-rich peptides have been reported to be efficient carriers for transporting various types of biomolecules into cytoplasmic and nuclear compartments of living cells. Among those arginines-containing MTS, a 9-mer arginine peptide (Arg9) was proved the most economical and efficient. We fused Arg9-peptide to an anti-CEA (Carcinoembryonic antigen) immunotoxin, PE35/CEA(Fv)/KDEL, at the position between the toxin moiety and the binding moiety. Strong binding and internalization of this fusion protein was observed in all detected cell lines, but little cytotoxicity to the cells that lack the CEA molecules on the cell surface was detected. However, the cytotoxicity besides the binding activity of the fusion protein to specific tumor cells expressing large amount of CEA molecules on the cell surface was improved markedly, indicating that the Arg9-peptide is capable of facilitating the receptor-mediated endocytosis of this immunotoxin, which leads to the increase of the specific cytotoxicity of this immunotoxin.     

Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni(2+) metal affinity chromatography and gel filtration. The biological activity of PE38KDEL was evaluated by the inhibition assay of protein synthesis in rabbit reticulocyte lysate system, and the cytotoxicity was tested in Hut 102 and hepatocellular cell lines by the MTS assay. PE38KDEL can significantly inhibit luciferase synthesis in cell-free protein synthesis assay and was slightly cytotoxic in the Hut 102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.     

Cytotoxic and apoptotic properties of a novel nano-toxin formulation based on biologically synthesized silver nanoparticle loaded with recombinant truncated pseudomonas exotoxin A

Bacterial toxins have received a great deal of attention in the development of antitumor agents. Currently, these protein toxins were used in the immunotoxins as a cancer therapy strategy. Despite the successful use of immunotoxins, immunotherapy strategies are still expensive and limited to hematologic malignancies. In the current study, for the first time, a nano-toxin comprised of truncated pseudomonas exotoxin (PE38) loaded silver nanoparticles (AgNPs) were prepared and their cytotoxicity effect was investigated on human breast cancer cells. The PE38 protein was cloned into pET28a and expressed in Escherichia coli, BL21 (DE3), and purified using metal affinity chromatography and was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AgNPs were biologically prepared using cell-free supernatant of E. Coli K12 strain. Nanoparticle formation was characterized by energy dispersive spectroscopy, transmission electron microscopy, and dynamic light scattering. The PE38 protein was loaded on AgNPs and prepared the PE38-AgNPs nano-toxin. Additionally, in vitro release indicated a partial slow release of toxin in about 100 hr. The nano-toxin exhibited dose-dependent cytotoxicity on MCF-7 cells. Also, real-time polymerase chain reaction results demonstrated the ability of nano-toxin to upregulate Bax/Bcl-2 ratio and caspase-3, -8, -9, and P53 apoptotic genes in the MCF-7 tumor cells. Apoptosis induction was determined by Annexin-V/propidium flow cytometry and caspases activity assay after treatment of cancer cells with the nano-toxin. In general, in the current study, the nano-toxin exhibit an inhibitory effect on the viability of breast cancer cells through apoptosis, which suggests that AgNPs could be used as a delivery system for targeting of toxins to cancer cells.     

Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples

Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified “HV4” and “LV4” in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.     

Construction and expression of novel immunotoxin cpIL-4(13D)-PE38KDEL with increased activity

Interleukin-4 receptors (IL-4Rs) are expressed on a wide variety of human cancer cells, and therefore it may be a good option to treat IL-4R-bearing tumors with IL-4-fusing immunotoxins. In this study, the gene encoding human interleukin-4 mutein cpIL-4(13D) was obtained through overlapping polymerase chain reaction. A chimeric immunotoxin was constructed by genetically fusing the mutein cpIL-4(13D) to a modified version of Pseudomonas exotoxin A (PE38KDEL) and was expressed in Escherichia coli AD494 (DE3). The expression level of the fusion protein was about 30% of the total bacterial protein assessed by SDS-PAGE analysis. After purification by affinity chromatography and anion exchange chromatography, the chimeric protein was tested for its cytotoxicity. Our data show that cpIL-4(13D)-PE38KDEL has improved cytotoxicity on IL-4R-bearing tumor cells in comparison with other IL-4-fusing immunotoxins and might be useful in treating tumors with a large number of IL-4Rs.     

Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

Convergent potency of internalized gelonin immunotoxins across varied cell lines, antigens, and targeting moieties

Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ～ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ～ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.     

Construction,expression and characterization of a dual cancer-specific fusion protein targeting carcinoembryonic antigen in intestinal carcinomas

The abundantly expressed carcinoembryonic antigen (CEA) on several cancer types is an attractive target for antibody-directed therapy. However, CEA is also present in some normal tissues. Here, we produced a dual functioning protein, designated as CAtin that exhibits both specific binding and killing functions, by fusing a tumor-specific apoptosis-inducing molecular Apoptin to C-terminus of an anti-CEA single-chain disulfide-stabilized Fv antibody (scdsFv). The CAtin proteins were expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS–PAGE and Western blotting revealed that the recombinant protein was well-expressed and the yield was approximately 250 mg/L. We demonstrated by flow cytometry and immunofluorescence assays that CAtin could bind specifically to human colon carcinoma cells (LoVo), but almost not to human uterine cervix (Hela). The results suggest that CAtin is active and specific toward CEA-positive cells and may potentially be used in CEA-targeted cancer therapy.     

Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL

Pseudomonas exotoxin (PE) is a 66,000 molecular weight protein secreted by Pseudomonas aeruginosa. PE is made up of three domains, and PE40 is a form of PE which lacks domain Ia (amino acids 1-252) and has very low cytotoxicity because it cannot bind to target cells. The sequence Arg-Glu-Asp-Leu-Lys (REDLK) at the carboxyl terminus of Pseudomonas exotoxin has been shown to be important for its cytotoxic activity (Chaudhary, V. K., Jinno, Y., FitzGerald, D. J., and Pastan, I. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312). In this study, we tested the effect of altering the carboxyl sequence of PE from REDLK to the characteristic endoplasmic reticulum retention sequence, KDEL, or to KDEL repeated three times (KDEL)3. We also made similar changes at the carboxyl terminus of two chimeric toxins in which domain I of PE (amino acids 1-252) was either replaced with transforming growth factor alpha (TGF alpha) to make TGF alpha-PE40 or with a single chain antibody (anti-Tac) reacting with the human interleukin 2 receptor to make anti-Tac(Fv)-PE40. Statistical analyses of our results demonstrate that PE and its derivatives ending in KDEL or (KDEL)3 are significantly more active than PE or derivatives ending in REDLK. We have also found that brefeldin A, which is known to perturb the endoplasmic reticulum, inhibits the cytotoxic action of PE. Our results suggest that the altered carboxyl terminus may enable the toxin to interact more efficiently with a cellular component involved in translocation of the toxin to the cytosol.