The efficacy of NP11-4-derived immunotoxin scFv-artesunate in reducing hepatic fibrosis inducedby Schistosoma japonicum in mice

Schistosomiasis is one of the most prevalent parasitic diseases in China, and hepatic fibrosis caused by schistosome infection is the principal cause of death. The aim of this study was to evaluate the efficacy of NPll-4- derived immunotoxin scFv-artesunate on Schistosoma japonicum-induced hepatic fibrosis. A single-chain variable fragment （scFv） was generated from the murine anti-Schistosoma japonicum （S. japanicum） monoclonal antibody NP11-4. The scFv was expressed as a soluble protein and purified by Ni-affinity chromatography. After conjuga- tion with artesunate, the binding ability with soluble egg antigens （SEA） was determined by an enzyme-linked immunosorbent assay （ELISA）. The biological activity of purified scFv, scFv-artesunate （immunotoxin）, and artesunate was detected in vivo. Image-Pro Plus software was used to analyze the size of egg granuloma and the extent of liver fibrosis. The recombinant scFv expession vector was constructed and expressed successfully. After purification by a His-trap Ni-affinity column, the scFv yield was approximately 0.8 mg/L of culture medium. ELISA results showed that chemical conjugation did not affect the binding activity of the immunotoxin. Our animal experiments indicated that the immunotoxin could significantly reduce the size of egg granuloma in the liver and inhibit hepatic fibrosis. The immunotoxin could be used as a promising candidate in the targeted therapy of S. .japonicum-induced hepatic fibrosis.

Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC_(50) of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M_(21) with a IC_(50) of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

STUDY OF ANTI-CD9 AND CD 10 IMMUNOTOXINS KILLING LEUKEMIC CELLS AND THE EFFECT ON HEMATOPOIESIS

Immuntoxins were synthesized by conjugating a plant toxic ricin with to three different monclonal antibodiesMoAb) directed against markers of human pre- B lymphocyte leukemic cells. It is useful to eliminate residual leukemic cells from bone marrow for preventing leukemia relapse after autologous bone marrow transplantation(ABMT). In the present work, the elimination of human leukemic cell line (Nalm-6) by three immunotoxins (anti-CD9 and anti-CD10) were observed. In addition, the proliferation of hematopoietic progenitors (CFU-GM. BFU E and CFU- mix) were not apparently inhibited by the immunotoxins in the range of effective concentrations. The possibility for utilizating immunotoxins in ABMT was discussed.

Immunotoxin depletion of T cells and its effect on hematopoietic progenitor cells in human cord blood

Objective To study the selective toxicity of immunotoxin (IT) on T cells in cord blood and simultaneously determine its effect on hematopoietic progenitor cells Methods The percentage of CD 5 and CD 8 T cell subsets in cord blood (CB) and bone marrow (BM) as well as peripheral blood (PB) was measured by immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti alkaline phosphatase (APAAP complexes) One way mixed lymphocyte cultures (MLC) were performed to compare the proliferative response of CB with that of PB The proliferative capability of cord blood T cells and T lymphocyte transformation capacity were evaluated in the presence of anti CD 8 or anti CD 5 immunotoxin by one way MLC and colorimetric MTT (tetrazolium) assay, respectively The effect of IT on the growth of hematopoietic progenitor cell of colony forming unit granulocyte and macrophage (CFU GM), burst forming unit erythroid(BFU E), multipotential hemotapoietic progenitors (CFU Mix) from CB were estimated by colony forming assays Results A certain proportion of CD 5 and CD 8 T cells existed in CB The alloproliferative capacity of CB was similar to that of PB CD 5: Ricin at a dosage of 1×10 10 -1×10 8 mmol/L and CD 8: Ricin concentration in the range of 1×10 9 -1×10 8 mmol/L effectively decreased both the proliferative capability of T cells in MLC during CB and T cell transformation Over the dosage of 1×10 10 -1×10 9 mmol/L, both kinds of IT didn't obviously affect the growth of hematopoietic progenitor cells Conclusion CD 5: Ricin and CD 8: Ricin may effectively deplete T cells and may not significantly inhibit the function of hemaptopoietic cells at a specific dosage

Application of anti-CD103 immunotoxin for saving islet allograft in context of transplantation

Background Previous studies using knockout mice document a key role for the integrin CD103 in promoting organ allograft rejection and graft-versus-host disease. However, a determination of whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in these processes has proven problematic due to the lack of reagents that efficiently deplete CD103＋ cells from wild type hosts. To circumvent this problem, in the present study, we invented an anti-CD103 immunotoxin （M290-SAP）. We investigated whether M290-SAP has capacity to eliminate CD103-expressing cells in vivo and protect transplanted islets from destroying by host immune cells.Methods Flow cytometry was used to analyze the efficacy of M290-SAP in depleting CD103-expressing cells in vivo.Then using allogenic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, the therapeutic efficacy of CD103-expressing cell depletion was addressed.Results M290-SAP dramatically reduces the frequency and absolute numbers of CD103-expressing leukocytes in peripheral lymphatic tissues of treated mice. Balb/c islets transplanted into streptozotocin-induced diabetic C57BL/6 mice under single M290-SAP treatment showed an indefinite survival time compared with untreated mice, M290-treated mice and IgG-SAP treated mice （mean survival time, ＞100 days vs. ＜20 days）. C57BL/6 islets transplanted into hyperglycemic NOD mice under single M290-SAP treatment showed a pronounced delay in allograft rejection compared with untreated mice （mean survival time 12-13 days vs. ＜7 days）. Immunological analysis of mice with long-term islet allograft survival revealed an obvious atrophy thymus and severe downregulation of alloimmunity of CD8 subpopulation response to allogenic stimulation.Conclusion Regardless of the underlying mechanisms, these data document that depletion of CD103-expressing cells represents a viable strategy for therapeutic intervention in islet allograft rejection.

Immunotoxins and Cancer Therapy

In the past decade,an increased amount of clinicallyloriented research involving immunotoxins has been published. Immunotoxins are a group of artificially-made cytotoxic molecules targeting cancer cells.These molecules composed of a targeting moiety,such as a ligand or an antibody,linked to toxin moiety,which is a toxin with either truncated or deleted cell-binding domain that prevents it from binding to normal cells.Immunotoxins can be divided into two categories:chemically conjugated immunotoxins and recombinant ones.The immunotoxins of the first category have shown limited efficacy in clinical trials in patients with hematologic malignancies and solid tumors.Within the last few years,single-chain immunotoxins provide enhanced therapeutic efficacy over conjugated forms and result in improved antitumor activity.In this review,we briefly illustrate the design of the immunotoxins and their applications in clinical trials.Cellular & Molecular Immunology.2005;2(2):106-112.

Construction of Luffin A Immunotoxin and Its in vitro Inhibition Against Human Melanoma Cell M_(21)

1 Introduction In recent years, significant progress has been made in applying immunotoxin (IT)in the therapy of leukemia and marrow transplantation. By 1990, several ITs havebeen put into clinical trials under the permission of FDA (Foodand Drug Administration, USA).

Induction of islet transplantation tolerance with anti-CD_4, anti-CD_8 immunotoxins and donor soluble antigen

Objective To induce islet grafting tolerance by intravenous injection of anti CD 4, anti CD 8 immunotoxins and donor soluble antigen Methods Fourteen days or 7 days prior to transplantation, the immunotoxin of anti CD 4, anti CD 8 200?μg respectively, and donor soluble antigen 500?μg were intravenously injected and then 500 donor islets were transplanted under the left renal subcapsular space of diabetes recipients (Sprague Dawley rats) Results The islet grafting survival time for those recipients pretreated with immunotoxin and donor soluble antigen was >60 days ( P <0 01) The immunotoxins, donor soluble antigen treatment alone might only slightly prolong the grafting survival time Conclusion The anti CD 4, anti CD 8 immunotoxins jointly used with donor soluble antigen can induce donor specific immunotolerance

Construction, expression, purification and functional analysis of recombinant 6C6 immunotoxin to human breast-tumor cells

The 28 ku membrane protein is usually over-expressl m human bnasl bmast cancer and other tumor cells. licould be a larget for tumor therapy . By using genetie engineermg teehmgues.a 606 immunotoxin (sefv606-PE40) was construeted by joining the 606 single-chain antibdy (SeFv606) with the truncll Pseudonwnas exotoxin A (PE40), SeFv606 contains both the heavy and light-chnia variable domams of 606 monoelonal antibody. Which speeifieally ree-ognizes the 28 ku protein. The bacterial expression level og 606 imnmmotoxin is 3.3%. about 5.5 mg ml baeterial lysate.lsing singlc-step llisTrap (Nr2 chelating) column chronnetogaphy, the reeombinant peptide was obtained with a purit of 33.2%.This baeterial espressed 606 immunotosin binuls to MDA-231 human breast-tumer ccll surfaee and kill these cells with a median lethal dosage of 92 ngnd.

Immunotoxin Therapy for Lung Cancer

INTRODUCTIONLung cancer is the leading cause for cancer-related deaths in both genders throughout the world. In the United States alone, there were 224,390 estimated new lung cancer cases and 158,080 estimated deaths in 2016.

融合PE38的抗CD70纳米抗体免疫毒素的制备及其对肾透明细胞癌786-O细胞的杀伤作用

目的:构建靶向CD70分子的重组免疫毒素,通过表达、纯化制备PE38与抗CD70纳米抗体重组蛋白,体外抗肿瘤实验探究重组蛋白是否对高表达CD70分子的阳性肿瘤细胞具有杀伤活性。方法:通过基因工程手段,将CD70纳米抗体Nb 2B3基因片段通过一个连接子与pET21a-PE38基因片段相连,获得重组表达载体pET21a-Nb 2B3-PE38并转入BL21(DE3)感受态细胞中进行表达、纯化与鉴定。用间接ELISA及FACS法检测Nb 2B3-PE38与CD70分子的结合活性,MTT法检测Nb 2B3-PE38对高表达CD70分子的肾透明细胞癌786-O细胞的体外杀伤活性,AnnexinⅤ-FITC/PI双染法检测Nb 2B3-PE38对786-O细胞凋亡的影响。结果:成功构建抗CD70纳米抗体重组免疫毒素Nb 2B3-PE38,纯化获得纯度>90%的重组蛋白,SDS-PAGE及WB检测结果表明目的蛋白正确表达,分子量为56000。纯化后的Nb 2B3-PE38能与重组CD70抗原及786-O细胞表面的CD70分子特异性结合;25μg/mL Nb 2B3-PE38即对786-O细胞产生极显著的杀伤作用(P<0.001),并且促进786-O细胞的细胞凋亡(P<0.01),其杀伤效应强于阳性对照顺铂(P<0.01)。结论:成功制备了特异性靶向CD70分子的免疫毒素Nb 2B3-PE38,其能够有效杀伤786-O细胞并诱导细胞凋亡且效果强于顺铂。

抗T细胞免疫毒素的T细胞清除效率及其对造血细胞的影响

抗T细胞免疫毒素主要用于同种异体骨髓移植中清除T细胞,减轻移植物抗宿主反应(GVHD),提高移植成功率,也可用于白血病自体骨髓移植中清除残留白血病细胞,减少移植后白血病复发的可能性。本实验结果表明抗T细胞免疫毒素在10^(-8)M浓度对靶细胞的杀伤达3.0log以上,即可清除99.99%的靶细胞,并可有效地抑制周血T细胞转化功能和骨髓T细胞分泌IL—2的功能,同时对骨髓粒单系和红系造血细胞的增殖无明显的抑制作用。

相思子毒素的分子特点及其在临床中的应用前景

相思子毒素是从豆科植物 (AbrusPrecatoriusL .)种子中分离的一种细胞毒性蛋白。它由A ,B两条链组成 ,由一个二硫键相连 ,B链具有半乳糖凝集活性 ,可与细胞膜上受体结合 ,帮助A链进入细胞内 ,A链进入细胞催化 6 0S大亚基的 2 8SrRNA的第 4 32 4位脱去腺嘌呤而使 6 0S核糖体亚基失活 ,从而使细胞蛋白合成被抑制。本文综述了有关相思子毒素的分子结构特点。

核糖核酸酶特殊生物学功能及药理作用研究新进展

核糖核酸酶作为一种模型蛋白被普遍用于分子生物学研究。近年来的一系列研究表明 ,核糖核酸酶及其结构类似物具有许多重要的生物学功能 ,如控制肿瘤血管生成 ,杀灭肿瘤细胞及抑制病毒 (包括 HIV- 1病毒 )的复制等 ,引起人们的广泛关注。核糖核酸酶家族大多具有裂解 RNA的活性 ,因此可与抗体连接构成具有显著的靶向性及人源化的免疫毒素 ,被用来杀伤癌细胞及其它异常增殖细胞。

重组免疫毒素hIL-2-LuffinP1的构建及表达

目的 构建人分泌型IL 2与免疫毒素LuffinP1的重组基因原核表达载体,并对其产物进行鉴定及纯化。方法 采用基因工程原理,将IL 2 LuffinP1的重组基因片段克隆入表达载体pET2 0b(+ )中,经酶切及DNA序列测定鉴定正确,转化表达宿主菌Origami(DE3 ) pLysS中,经IPTG诱导表达含His标签的融合蛋白 重组免疫毒素hIL 2 LuffinP1。结果 成功构建了免疫毒素表达载体pET2 0b(+ ) hIL 2 LuffinP1,获得纯化的重组免疫毒素,并经Westernblot鉴定正确。结论 hIL 2 LuffinP1的成功构建,为进一步研究它在抗移植排斥中的作用奠定了基础。

二硫键稳定的抗肝癌单链抗体-PE38融合基因的构建、表达与功能检测

目的:制备高特异性有杀伤活性的新型抗肝癌免疫毒素。方法:应用适当的酶切位点,将二硫键稳定的人源化抗肝癌单链抗体(dsFv)基因及PE38基因克隆入原核表达载体pTIG中。诱导表达上清经Ni-NTA亲和层析柱纯化,用MTT比色法检测纯化表达产物的细胞毒活性。结果:目的融合蛋白在大肠杆菌中得到可溶性表达,表达产物的M,为66 000,表达 量占菌体总可溶性蛋白量的21％。细胞毒实验表明,抗肝癌免疫毒素对肝癌细胞具有杀伤作用,而对正常肝细胞无损伤。结论:抗肝癌dsFv与PE38融合基因的成功构建及表达,为其作为抗肝癌药物的进一步研究打下了基础。

Luffin P1-KDEL的构建、表达、纯化及鉴定

目的构建植物毒素Luffin P1的原核表达质粒PET32a(+)-Luffin P1,并对其进行诱导表达,以及表达蛋白的纯化及鉴定。方法采用基因工程技术将重组基因片段Luffin P1克隆到表达载体pET32a(+)中,经酶切及DNA序列分析正确后,转化至表达宿主菌BL21(DE3)中,经IPTG诱导表达Luffin P1融合蛋白,并用His标签抗体对该融合蛋白进行Western blot鉴定,对鉴定正确的融合蛋白进行纯化,肠激酶酶切及再纯化。结果成功的构建了原核表达载体pET32a(+)-Luffin P1,获得了纯度较高的Luffin P1蛋白。结论通过基因工程合成Luffin P1蛋白是成功的,并为进一步对Luffin P1功能研究及制备免疫毒素的弹头鉴定了实验基础。

中华眼镜蛇膜毒素与McAb偶联物对白血病细胞的杀伤作用

用中华眼镜蛇膜毒素(CT)与抗人T细胞McAb Wu71偶联制备免疫毒素Wu71:CT。经SDS-聚丙烯酰胺凝胶电泳、免疫双扩散和间接免疫荧光检测,证实结合物中McAb和CT均保持活性。在无Ca^(2+)、Mg^(2+)的培养基中将人白血病T淋巴细胞系CEM细胞与Wu71:CT共同孵育,先加入Ca^(2+)以抑制CT的溶细胞作用,而不影响McAb与靶细胞的结合;一定时间后用Mg^(2+)和金属螯合剂EGTA恢复CT的溶胞作用,结果在Wu71:CT 0.5×10^(-6)mol/L浓度下靶细胞CEM 82.4％被杀伤,而抗原阴性的非靶细胞Raji仅死亡23.3％,表明Wu71:CT有较好的特异性杀伤作用。扫描电镜显示,免疫毒素作用下的CEM细胞膜破裂、萎缩、崩溃,而非靶细胞大部分保持完整。提示McAb与CT偶合物在白血病和肿瘤的导向治疗中可能具有潜在价值。

抗隐孢子虫子孢子ScFv-PE40免疫毒素表达质粒的构建及其在大肠杆菌中的表达

用PCR方法扩增抗隐孢子虫子孢子ScFv片段,插入表达载体pET-28a中,构建重组质粒pET-28-ScFv,然后,将绿脓杆菌外毒素(PE40)片段定向克隆到ScFv基因的下游,构建免疫毒素表达质粒pET28ScFv-PE40。经酶切分析、PCR检测和测序进行鉴定。成功地构建了免疫毒素表达质粒pET28ScFv-PE40。将上述质粒转化受体菌BL21(DE3)后,经IPTG诱导,成功地表达了目的蛋白。免疫毒素ScFv-PE40大小约为66kDa,表达量约占菌体蛋白总量的14%。