抗EGFR/VEGF双特异性单链抗体的构建及其抗卵巢癌SKOV-3的研究

目的 将表皮生长因子受体(EGFR)和血管内皮生长因子(VEGF)同时进行靶向治疗,可协同抑制作用,增强抗肿瘤效果.方法 使用重叠聚合酶链式反应(PCR)手段,西妥昔单抗的可变区和贝伐珠单抗的可变区通过G4S柔性肽连接在一起,获得同时靶向EGFR和VEGF的双特异性单链抗体(scDb);通过酶联免疫吸附测定(ELISA)检测scDb与EGFR和VEGF的结合活性,并计算亲和力常数;流式细胞术检测scDb对受体过表达的卵巢癌细胞系SKOV-3的结合能力;MTT法检测scDb对卵巢癌细胞系SKOV-3的增殖抑制;Western Blot研究与单独给药相比 scDb对 SKOV-3相关信号通路的影响;建立卵巢癌SKOV-3细胞荷瘤小鼠动物模型,检测scDb的体内抗肿瘤活性.结果 通过重叠PCR手段和毕赤酵母X-33表达,成功获得scDb,经Western blot鉴定验证了scDb的分子量为55kD,证明表达及装配正确.ELISA检测scDb与EGFR和VEGF均有结合活性,通过EC50计算,得到scDb与EGFR的亲和力常数为1.27nM,与VEGF的亲和力常数为0.75nM.流式细胞术检测scDb与SKOV-3的结合率为72.00%.MTT实验,亲本抗体组与双特异性单链抗体组对SKOV-3均有抑制作用.通过Western blot实验表明,scDb能同时阻断EGFR和激酶结构域受体(KDR)的磷酸化,最终导致丝氨酸/苏氨酸激酶等信号通路被强烈抑制.通过SKOV-3细胞裸鼠移植瘤模型的体内抗肿瘤实验,scDb组在体内仍能发挥较强的抗肿瘤效果,高剂量组肿瘤抑制率可以达到(74.53±9.63)%.结论 该文成功构建并表达了scDb.scDb具有良好的体内外抗肿瘤活性,为抗肿瘤治疗提供了新的思路,有潜在的临床应用前景.     

4-1BBL胞膜外区蛋白增强抗CD3/抗PgP的抗肿瘤作用

目的研究4-1BBL胞膜外区蛋白对抗CD3/抗Pgp微型双功能抗体抗肿瘤作用的影响。方法表达纯化人4-1BBL胞膜外区融合蛋白及抗CD3/抗Pgp微型双功能抗体,体外CytoTox96检测联合应用人4-1BBL胞膜外区融合蛋白、抗CD3/抗Pgp微型双功能抗体及PBL对靶细胞K562/A02细胞的杀伤作用,体内建立裸鼠移植瘤模型检测4-1BBL胞膜外区蛋白作为一种免疫调节蛋白,增强抗CD3/抗Pgp基于PBL的抗肿瘤效果。结果4-1BBL胞膜外区融合蛋白在体外能够增强抗CD3/抗Pgp及PBL对靶细胞K562/A02的杀伤作用,在体内能够增强抗CD3/抗Pgp基于PBL的抗肿瘤作用。结论可溶型4-1BBL可能成为一种有前景的生物治疗佐剂,有助于PBL更高效地靶向杀伤肿瘤细胞。     

Bispecific targeting of thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor‐1 by a heterodimer diabody

Summary. Background and objectives: Thrombin activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor‐1 (PAI‐1) play important roles in fibrinolysis. Both reduce plasmin generation, but they exert their antifibrinolytic effects via different mechanisms. This study reports the cloning and characterization of a heterodimer diabody that inhibits TAFI and PAI‐1 simultaneously. Methods and results: The diabody was derived from two inhibiting monoclonal antibodies, i.e. MA‐33H1F7, an anti‐PAI‐1 antibody that induces non‐inhibitory substrate behavior of PAI‐1, and MA‐T12D11, an anti‐TAFI antibody that inhibits activation of TAFI by the thrombin–thrombomodulin complex. A single‐chain variable fragment (scFv) was derived from MA‐T12D11 that displayed slightly reduced binding and inhibitory properties as compared to MA‐T12D11. Characterization of the diabody revealed a similar affinity for TAFI and PAI‐1 as that of the parental antibodies. Furthermore, the inhibitory properties of MA‐33H1F7 and MA‐T12D11 were fully preserved in the diabody format. In platelet‐free plasma (PFP) clots, addition of the diabody had a stronger effect in shortening lysis times than either MA‐T12D11 or MA‐33H1F7. A similar reduction in clot lysis time was observed in platelet‐rich plasma (PRP) clots. The same effect on clot lysis times in PFP and PRP was also achieved by the combined addition of MA‐T12D11 and MA‐33H1F7. The lysis rate of human model thrombi, made from whole blood, was approximately doubled after addition of the diabody. Moreover, this effect was significantly better than after the combined addition of the individual antibodies. Conclusions: These observations demonstrate that simultaneous inhibition of TAFI and PAI‐1 results in faster lysis of the formed thrombus.     

Engineering Antibodies for the Clinic

In the last ten years recombinant protein drugs such as erythropoeitin or tissue plasminogen activator have become widely used in the clinic. After some early setbacks antibodies look well placed to join them. A decade of antibody engineering is finally beginning to pay off with a string of chimeric and humanized antibodies gaining the Food and Drug Administration approval in the last two years. Here we will report on recent developments in the clinical application of antibodies, in particular, in the treatment of malignant lymphoma. We will also discuss some of the current strategies for the engineering of both whole antibodies (IgG) and recombinant antibody fragments for the next generation of antibody therapeutics.     

抗HBsAg和抗红细胞双特异Diabody的构建及表达

目的构建及表达抗乙肝病毒表面抗原（ＨＢｓＡｇ）和抗红细胞（ＲＢＣ）双特异Ｄｉａｂｏｄｙ，用于血清中ＨＢｓＡｇ的快速检测。方法将抗ＨＢｓＡｇ的人抗体轻重链可变区基因与抗红细胞的鼠抗体重轻链可变区基因交叉配对构建成两个杂合Ｄｉａｂｏｄｙ基因，并将两个配对基因组建在一个表达载体中，成为双特异Ｄｉａｂｏｄｙ表达载体，并在大肠杆菌中分泌表达。结果经ＥＬＩＳＡ检测和红细胞凝集实验测定，证明所表达的双特异性Ｄｉａｂｏｄｙ具有抗ＨＢｓＡｇ和抗ＲＢＣ双重功能，并可使含有ＨＢｓＡｇ的ＲＢＣ悬液产生血球凝集。结论所表达的双特异性Ｄｉａｂｏｄｙ具有双重抗体活性，可用于血清中ＨＢｓＡｇ的快速检测     

人源性抗地高辛单链抗体(scFv)及diabody的获取

目的:从大容量噬菌体抗体库中筛选人源性抗地高辛单链抗体(scFv),并构建双价抗体(diabody)。方法:以固相化的Dig对构建的大容量噬菌体抗体库进行筛选,4轮后挑取集落,用ELISA法鉴定其特异性,并对抗Dig阳性抗体基因进行DNA指纹分析及测序分析。选取活性好的抗体基因进行改造,构建diabody。结果:在抗体库的筛选过程中可见到明显的富集现象,获得4株可与Dig特异性结合的人源单链抗体,经DNA指纹分析及测序分析证明为不同抗体基因,分泌抗Dig抗体的阳性克隆的可变区基因轻链均属于λ第1亚群,重链基因分别属于第3和第4亚群。选取4号克隆进行基因改造,构建diabody的表达载体,获得活性较好的diabody。结论:利用噬菌体抗体技术获得了人源性抗Dig的抗体,并改造成应用前景较好的diabody,为临床诊断及治疗洋地黄类药物的中毒提供了具有实用价值的人源抗体。     

Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.     

抗人CD80双价抗体的构建、表达及生物学功能初步研究

构建及表达抗人CD80双价抗体(dimeric antibody fragment,diabody),并初步研究其生物学功能.PCR法获得轻重链可变区基因,构建表达载体pIRES2-EGFP/diabody.将pIRES2-EGFP/diabody转染中华仓鼠卵巢细胞(CHO),G418加压,筛选稳定高表达细胞株,扩大...     

Development of a recombinant camelid specific diabody against the heminecrolysin fraction of Hemiscorpius lepturus scorpion

Scorpion sting is one of the most important problems in public health practice in many countries. Khuzestan yellow scorpion, scientifically known as Hemiscorpius lepturus, belongs to the family Hemiscorpidae, and is one of the most common and dangerous scorpions in Iran. Hemiscorpius lepturus scorpion has potentially cytotoxic venom. The first treatment for scorpion envenoming is horse antisera. Due to their unique characteristics, camels have been considered as an important source for antiserum and monoclonal antibody (Nanobody) production. In the previous study, we reported a specific nanobody against the hemolytic fraction (heminecrolysin) of Hemiscorpius lepturus venom. In the present study, a recombinant diabody was constructed and produced by fusion of two nanobodies. Results from in vitro and in vivo assays showed that the diabody has a promising ability to neutralize venom activity.