抗HER2单链抗体与力达霉素强化融合蛋白的构建及其抗肿瘤活性研究
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摘要
乳腺癌是女性最常见的恶性肿瘤之一，占女性肿瘤死亡第二位，其发病率逐年增高，人们迫切需要采用新的治疗方法来提高患者的生存率及生存质量。分子靶向治疗在肿瘤治疗中的地位逐渐上升，成为肿瘤治疗的一个热点。目前已有多种分子靶向治疗的药物应用于乳腺癌的治疗或正在临床试验阶段，如赫赛汀(Herceptin)、细胞周期调节蛋白抑制剂、环氧化酶-2(COX-2)抑制剂、蛋白激酶C(PKC)抑制剂等。
HER2受体是人表皮生长因子受体家族四成员之一，定位于人类17q21，编码分子量为185kD的氨基酸，命名为P185，具有酪氨酸激酶活性，蛋白从结构上分为胞外区、跨膜区和胞内区，后者具有酪氨酸激酶活性，与表皮生长因子受体具有同源性。HER2的功能是作为生长因子受体在细胞分化、粘附和运动中起作用。Her2基因扩增导致HER2高表达，导致细胞恶性转化见于30％转移性乳腺癌患者和部分卵巢癌，肺癌，胃癌和口腔癌患者。HER2过表达增加肿瘤细胞侵袭、转移的能力，通过启动多种转移相关机制而增加转移能力，包括细胞迁移率，体外侵袭力，Ⅳ型胶原酶活性，也影响某些粘附分子如上皮细胞钙粘蛋白等的合成，从而促进转移。妇女患HER-2阳性肿瘤预后差，复发快。因此，HER2高表达可成为判断上述肿瘤恶性程度的独立预后指标。研究表明HER2表达的高低与乳腺癌的临床诊断、药物的疗效及预后判断关系密切。HER2是治疗乳腺癌及卵巢癌的明确靶点。
分子靶向治疗在临床肿瘤治疗方面起重要的影响。赫赛汀(Herceptin)是人源化的HER2胞外段单克隆抗体，代表着治疗女性HER2阳性转移性乳腺癌的重大突破。临床病例单用赫赛汀或与紫杉醇联合用药取得良好治疗效果，并且成为治疗HER2阳性肿瘤的一线用药。赫赛汀在转移性肿瘤中的应用将HER2阳性的地位从不良预后的标志变成良好的治疗成果，并且正在进行的研究将为HER2阳性的转移性乳腺癌提供更多治疗的选择机会。
抗体导向治疗可以利用抗体为载体将药物导向肿瘤病灶，提高药物疗效，降低药物的副作用。研究表明，单链抗体片段组织穿透能力强，较易穿透细胞外间隙到达深部的肿瘤细胞。与完整抗体相比，单链抗体的免疫原性较弱，可降低人体抗鼠抗体反应。但是，另一方面，由于单链抗体片段缺乏Fc段，丧失完整抗体的效应功能，难以杀伤靶细胞。因此，研究小型抗体需要使用“弹头”分子，包括高效的药物、毒素、细胞因子等。
高活性的“弹头”药物力达霉素(lidamycin，LDM，又称C1027)，是从我国湖北省潜江县土壤中分离得到的由一株球孢链霉菌(Streptiomyces globisporus)产生的烯二炔类抗生素，是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素。LDM的分子由两部分组成：一部分为烯二炔结构的发色团(AE)，具有强烈的细胞毒作用，但不稳定；另一部分为110个氨基酸残基组成的辅基蛋白(LDP)，对发色团的稳定性起保护作用。二者可以拆分与重建，重建的力达霉素与天然状态力达霉素具有相同的活性。
Smith在1985年最先将外源基因插入丝状噬菌体f1的基因Ⅲ，使目的基因编码的多肽能以融合蛋白的形式展示在噬菌体表面，从而创建了噬菌体展示技术，从此，这一领域得到了发展。噬菌体技术是一种基因表达产物和亲和选择相结合的技术。噬菌体展示通过与特定的靶抗原结合，可以从大的单链抗体库中快速筛选出特异抗体。经过3轮或更多轮的筛选，与抗原有高度亲和力的单链抗体scFv可以被富集。针对数百种抗原的抗体已经从噬菌体展示文库中得到，包括细胞表面标志，肽类激素，其他人类蛋白及碳水化合物等。噬菌体展示技术的优点在于，一旦抗体库建立，就可以用来筛选针对靶抗原的抗体。一个抗体库可以被数千研究者用作无限抗原的克隆抗体的来源。并且筛选的过程非常迅速，可以在2～3周内完成。运用ELISA、Western-blot方法通过免疫检测鉴定筛选抗体的亲和力。通常，这个过程包括亚克隆与表达scFv片段得到大量抗体，是耗时与繁琐的。为了解决这个问题，最近研究者着重研究利用免疫检测和功能研究筛选噬菌体克隆。本研究利用将单链抗体连接在M13噬菌体衣壳蛋白PⅢ的N端构建噬菌体抗体库。
本研究主要目的是利用噬菌体展示技术筛选HER2高亲和力的单链抗体，与力达霉素构建具有靶向杀伤HER2高表达肿瘤的强化融合蛋白，有助于乳腺癌、卵巢癌等HER2高表达肿瘤的导向治疗。
一、抗HER2噬菌体抗体库构建与筛选
本研究利用纯化的HER2胞外段蛋白免疫小鼠，从脾细胞提取mRNA，利用RT-PCR扩增抗体重链可变区(VH)和轻链可变区(VL)。利用重叠延伸反应(SOE-PCR)将VH、VL用连接肽(Gly_4Ser)_3进行连接，克隆至噬菌粒pCANTAB5E，转染E.coli TG1，得到噬菌体抗体库。以高表达HER2的人乳腺癌细胞SKBR3为抗原进行4轮筛选，得到库容为7.5×10~7的次级抗体库5F-10。从初级抗体库中随机挑取20个克隆进行菌体PCR，结果显示其中15个克隆有scFv片段插入，克隆效率为75％。BstNⅠ酶切随机克隆提取的质粒，酶切图谱显示多样性，表明抗体库具有多样性。用ELISA法鉴定各单克隆与HER2蛋白结合能力，获得与HER2高亲和力克隆scFv6，经序列分析，此基因全长732bp，编码243个氨基酸序列，通过Blast序列比对，此抗体为鼠源性，与以往报道的任何抗体序列都不相同。
二、抗HER2单链抗体与力达霉素强化融合蛋白的构建、表达、纯化及强化融合蛋白活性分析
利用基因重组技术将筛选出来的HER2高亲和力抗体scFv6片段克隆至含有力达霉素辅基蛋白基因的重组质粒pET-LDP，构建含有融合蛋白的质粒pET-Fv-LDP，转化到大肠杆菌BL21(DE3)starTM中，IPTG诱导表达带有组氨酸标签肽(His-tag)的融合蛋白HER2(Fv-LDP)。经SDS-PAGE分析，表达产物分子量40kD，主要以可溶形式表达在周质腔中，表达占全菌蛋白的7％，用Ni离子亲和柱进行纯化，1L菌液诱导后可产生约2mg可溶性融合蛋白。然后与力达霉素发色团AE重组，得到强化融合蛋白HER2(Fv-LDM)。研究其免疫学活性及其对HER2高、低表达肿瘤细胞的细胞毒作用。Western-blot分析显示，融合蛋白与纯化的HER2蛋白胞外段有结合活性。细胞ELISA和免疫荧光结果表明，融合蛋白HER2(Fv-LDP)与SKBR3、SKOV3都有较高的亲和活性，略低于商品化HER2单抗L87，而与MCF7无结合力。0.01M，0.1M，1M的强化融合蛋白可降低SKBR3细胞HER2的mRNA与蛋白表达水平。流式细胞周期测定结果融合蛋白将SKBR3细胞周期阻滞于G1期，而强化融合蛋白将细胞周期阻滞于G2／M期。HE染色和Hoechest染色显示，强化融合蛋白可以诱导细胞凋亡。MTT结果显示，强化融合蛋白对HER2高、低表达的细胞都有强烈的杀伤活性，较好地保留了力达霉素的细胞毒作用。在裸鼠皮下移植人乳腺癌SKBR3肿瘤模型中检测强化融合蛋白HER2(Fv-LDM)的体内抗肿瘤活性，接种肿瘤的第10天和17天分别尾静脉给药2次，观察肿瘤体积和裸鼠体重变化。实验结果表明0.2mg／kg，0.3mg／kg，0.4mg／kg三个剂量的强化融合蛋白HER2(Fv-LDM)对人乳腺癌SKBR3裸鼠移植瘤生长都有抑制作用，抑瘤率分别为60％，70％，89.7％。强化融合蛋白HER2(Fv-LDM)0.3mg／kg，0.4mg／kg的抑瘤率与LDM(0.05mg／kg)抑瘤率56.8％相比有显著差异(P＜0.05)。体内实验结果说明，强化融合蛋白可以提高动物对力达霉素的耐受剂量，同时显著改善治疗效果，是具有很好前景的抗体靶向药物。
Breast cancer is the most common malignancy in women, it now represents thesecond leading cause of death from cancer in women, and the number of new breastcancer cases has been increasing year by year. To increase the patients' survival ratesand their life quality, we need new measures of therapy. Molecular targeted cancertherapy has been developing rapidly. In present, many molecular agents for targetedtherapy have been used for breast cancer therapy in of clinical practice, such asHerceptin, Iressa and inhibitors of cell cycle regulator, COX-2 or PKC.
The Her-2/neu (c-erbB2) proto-oncogene is a component of the four-memberfamily of closely related growth factor receptors that includes the epidermal growthfactor receptor Her-1 (EGFR), Her-3 (c-erbB3) and Her-4 (c-erbB4). The human geneis located on chomosome 17q21 and encodes a 185 kD protein with tyrosine kinaseactivity, also known by the designation P185. Structurelly, the protein hasextracellular, transmembrane, and cytoplasmic domains, the latter of which containsthe tyrosine kinase domain and shares significant homology, although is distinct,from EGFR. Her-2/neu is thought to function as a growth factor receptor and plays arole in cell differentiation, adhesion, and motility. Overexpression of HER-2, usuallyas a result of her-2 gene amplification, can result in malignant transformation of cellsand is seen in tumor tissue in up to 30% of patients with metastatic breast cancer(MBC) and part of patients with ovarian, lung, gastric, and oral cancers. HER-2overexpression is usually associated with a more aggressive tumor phenotype, andwomen with HER-2 positive tumor have a poor overall prognosis and faster relapse.So the overexpression of HER-2 has been an independent prognostic index forevaluating the malignancy of above cancers. Evidently, HER2 become an explicittarget for therapy of breast cancer and ovarian cancer.
Antibody can carry agent to tumor region, so enhances the therapeutic effect anddecreases the toxicity. It has been shown that scFv (single chain variable Fragment)can penetrate into the solid tumor deeply. Compared to the whole antibody, scFvshows low immunogenicity and so induce less human anti-mouse antibody (HAMA)response. On the other hand, because of the absence of Fc, scFv can not execute theeffector function to kill target tumor cells. So a "warhead" molecule is neededincluding drugs, toxins, and cytokines.
Molecular targeted therapy is currently having a tremendous impact on the daily practice of clinical oncology. The advent of Herceptin (trastuzumab), a humanizedmonoclonal antibody against extracellular domain of HER-2, represented a majorbreakthough in the treatment of women with HER-2-positive MBC. Pivotal trials ofHerceptin alone or in combination with taxanes have result in significant clinicalbenefit, and Herceptin plus taxanes as first-line therapy is now the standard of care ofHER-2-positive disease. Already, the use of Herceptin in the metastatic setting haschanged HER-2-positive status from a marker of poor prognosis to one of betteroverall outcome, and ongoing studies should expand further treatment options forpatients with HER-2-positive MBC. This agent was shown to significantly prolongthe progression-free survival as a single agent or in combination with standardchemotherapeutic agents.
Lidamycin (LDM), an antibiotic produced by Streptomyces globisporus strainwhich was isolated in our institute, displayed extremely potent cytotoxicity againsttumor cells. LDM consists of a chomophore and an apoprotein, and the former has theability to attack DNA, whereas the latter plays the role as a protecting protein. Thetwo parts can be separated and reconstructed without affecting its activity.
The first surface expression system was developed by Smith in 1985, whodisplayed the filamentous phage on the surface of bacteriophge. In recent years, thefilamentous bacteriophages have been used extensively for the display of largerepertoires of antibodies on their surface. Phage display enables rapid selection ofantibodies from a large single chain fragment variable (scFv) library by virtue of thebinding to certain target antigen. After three or more rounds of selection, the resultingphage population is markedly enriched for the scFvs that bind to the antigen.Antibodies against hundreds of target antigens have so far been obtained from phagedisplay antibody libraries, including cell-surface markers, peptide hormones, humanproteins, and carbohydrates. One of the prominent advantages of the phage-displaytechnology is that, once a library has been created, it can be used to select antibodiesthat bind to any target antigens of interest. A single phage-antibody library can bedistributed to thousands of users and serve as the source of cloned antibodies againstan unlimited array of antigens. This technology is also efficient, and the process ofselection and primary screening is very rapid and can be completed with 2-3 weeks.The selected phage clones are then subjected to immunodetection to confirm theirbinding activities. ELISA and Western blot are extensively used in immunodetection.Conventionally, the procedure usually involves subcloning and expression of the target fragments encoding the scFv to obtain significant quantities of antibodies,which proves to be time-consuming and laborious. To resolve this problem,researchers have recently foucused on the use of selected phage clones inimmunodetection or function studies. In this study, a phage-display scFv antibodylibrary constructed on the N terminus of pⅢprotein of M13 filamentous phage.
In this study, phage display has been used to select high affinity scFv to HER2,and molecule reconstitution was performed to construct energized fusion proteinwith extremely potent cytotoxicity to HER2 over-expressing tumor cells.
1. Construction and selection of HER2 phage scFv library
In this study, mRNA was isolated from splenocytes of mice immunized withpurified ECD protein of HER2. Immunoglobulin variable fragments VH and VL wereamplified by RT-PCR and were connected by linker (Gly_4Ser)_3 to form the singlechain Fv (scFv) through a peptide by SOE-PCR. Then the scFv gene was cloned inphagemid pCANTAB5E, and transformed into E.coli TG1. The transformed cellswere infected by M13KO7 helper phage to get the primary phage display scFv library.Panning against breast cancer SKBR3 cells line was performed four times. At last, arecombinant phage display scFv library with a titre of 7.5×10~7 was established.Random 20 clones were selected from primary and selected phage antibody library,through PCR identified, scFv fragment was inserted into 15 clones of primary phagelibrary and 20 clones in selected phage library. Random 18 clones' plasmids fromprimary library digested with BstNⅠshowed different pattern, and clones fromselected library showed specially pattern enriched. The affinity of clones to HER2was identified by ELISA.Through 4 rounds of panning and screening, a phage-scFvnamed scFv6 binding to HER2 specifically was selected from the library. Bysequence analysis, the length of this gene is 732 bp, and 243 amino acid is encoded.This murine antibody is different from any other antibodies discovered before bysequence blast analysis.
2. Construction, expression, purification and characterization analysis of energizedfusion protein of HER2(Fv-LDM)
ScFv6 which has high affinity with HER2 antigen was cloned intoreconstruction plasmid pET-LDP, and plasmid pET-Fv-LDP was transformed intoE.coli BL21 (DE3) star~(TM), fusion protein HER2(Fv-LDP) with His-tag wasexpressed when induced by IPTG. Through SDS-PAGE analysis, 7% of soluble fusion protein existed in periplasm of E.coli. Subsequently purified by IMAC(immobilized metal affinity chromatography). About 2 mg of protein was obtainedfrom 1 liter fermentation broth. The purity of this protein exceeded 90%. For thepreparation of energized fusion protein HER2 (Fv-LDM), we added AE to the PBSsolution of the HER2 (Fv-LDP) fusion protein with the molecular ratio of 5:1 for 12h at room temperature. After the reconstitution was completed, separation andpurification were preformed using PD-10 colomn to remove the free AE (SephadexG-25 column). Western-blot assay showed that fusion protein have affinity withpurified protein ECD of HER2. Cell ELISA and cell fluorescence showed thatfusion protein HER2 (Fv-LDP) had high affinity with SKBR3 and SKOV3 cells,but it was lower than that of commercial anti-HER2 mAb L87. By contrast, HER2(Fv-LDP) had little affinity with MCF7 cells. The level of HER2 mRNA andprotein in SKBR3 cells decreaced by the treatment with HER2 (Fv-LDM). Flowcytometry (FCM) assay of cell cycle showed that cells were arrested at G1 phasewith fusion protein HER2 (Fv-LDP) treatment, instead of G2/M phase withenergized fusion protein HER2 (Fv-LDM) treatment. Through Hoechest and HEdying, the appearance of cells and nuclei showed that energized fusion proteinHER2 (Fv-LDM) can induce cell apoptosis of SKBR3、SKOV3 and MCF7. ByMTT assay, HER2 (Fv-LDM) showed potent cytotoxicity to SKBR3, SKOV3,Moreover, the in vivo therapeutic efficacy of the energized fusion protein HER2(Fv-LDM) was investigated in human breast carcinoma SKBR3 xenograft in nudemice. In experiment, the SKBR3 tumor bearing mice were treated by single i.v doseon day 10 and day17 after subcutaneous tumor transplantation, inhibitory effect ofHER (Fv-LDM), LDM, HER2 (Fv-LDP) was detected through measuring thevolume of tumor. At dose 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, HER2 (Fv-LDP)exhibited highly effected potency in retarding growth of SKBR3 with inhibitionrate of 60%, 70%, 89.7%, while the inhibition rate of LDM (0.05mg/kg) is 56.8%.So the energized fusion protein can increase the tolerance dose of LDM, improvethe therapeutic effects, and become an antibody targeted medicine of goodperspective.
HER2受体是人表皮生长因子受体家族四成员之一，定位于人类17q21，编码分子量为185kD的氨基酸，命名为P185，具有酪氨酸激酶活性，蛋白从结构上分为胞外区、跨膜区和胞内区，后者具有酪氨酸激酶活性，与表皮生长因子受体具有同源性。HER2的功能是作为生长因子受体在细胞分化、粘附和运动中起作用。Her2基因扩增导致HER2高表达，导致细胞恶性转化见于30％转移性乳腺癌患者和部分卵巢癌，肺癌，胃癌和口腔癌患者。HER2过表达增加肿瘤细胞侵袭、转移的能力，通过启动多种转移相关机制而增加转移能力，包括细胞迁移率，体外侵袭力，Ⅳ型胶原酶活性，也影响某些粘附分子如上皮细胞钙粘蛋白等的合成，从而促进转移。妇女患HER-2阳性肿瘤预后差，复发快。因此，HER2高表达可成为判断上述肿瘤恶性程度的独立预后指标。研究表明HER2表达的高低与乳腺癌的临床诊断、药物的疗效及预后判断关系密切。HER2是治疗乳腺癌及卵巢癌的明确靶点。
分子靶向治疗在临床肿瘤治疗方面起重要的影响。赫赛汀(Herceptin)是人源化的HER2胞外段单克隆抗体，代表着治疗女性HER2阳性转移性乳腺癌的重大突破。临床病例单用赫赛汀或与紫杉醇联合用药取得良好治疗效果，并且成为治疗HER2阳性肿瘤的一线用药。赫赛汀在转移性肿瘤中的应用将HER2阳性的地位从不良预后的标志变成良好的治疗成果，并且正在进行的研究将为HER2阳性的转移性乳腺癌提供更多治疗的选择机会。
抗体导向治疗可以利用抗体为载体将药物导向肿瘤病灶，提高药物疗效，降低药物的副作用。研究表明，单链抗体片段组织穿透能力强，较易穿透细胞外间隙到达深部的肿瘤细胞。与完整抗体相比，单链抗体的免疫原性较弱，可降低人体抗鼠抗体反应。但是，另一方面，由于单链抗体片段缺乏Fc段，丧失完整抗体的效应功能，难以杀伤靶细胞。因此，研究小型抗体需要使用“弹头”分子，包括高效的药物、毒素、细胞因子等。
高活性的“弹头”药物力达霉素(lidamycin，LDM，又称C1027)，是从我国湖北省潜江县土壤中分离得到的由一株球孢链霉菌(Streptiomyces globisporus)产生的烯二炔类抗生素，是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素。LDM的分子由两部分组成：一部分为烯二炔结构的发色团(AE)，具有强烈的细胞毒作用，但不稳定；另一部分为110个氨基酸残基组成的辅基蛋白(LDP)，对发色团的稳定性起保护作用。二者可以拆分与重建，重建的力达霉素与天然状态力达霉素具有相同的活性。
Smith在1985年最先将外源基因插入丝状噬菌体f1的基因Ⅲ，使目的基因编码的多肽能以融合蛋白的形式展示在噬菌体表面，从而创建了噬菌体展示技术，从此，这一领域得到了发展。噬菌体技术是一种基因表达产物和亲和选择相结合的技术。噬菌体展示通过与特定的靶抗原结合，可以从大的单链抗体库中快速筛选出特异抗体。经过3轮或更多轮的筛选，与抗原有高度亲和力的单链抗体scFv可以被富集。针对数百种抗原的抗体已经从噬菌体展示文库中得到，包括细胞表面标志，肽类激素，其他人类蛋白及碳水化合物等。噬菌体展示技术的优点在于，一旦抗体库建立，就可以用来筛选针对靶抗原的抗体。一个抗体库可以被数千研究者用作无限抗原的克隆抗体的来源。并且筛选的过程非常迅速，可以在2～3周内完成。运用ELISA、Western-blot方法通过免疫检测鉴定筛选抗体的亲和力。通常，这个过程包括亚克隆与表达scFv片段得到大量抗体，是耗时与繁琐的。为了解决这个问题，最近研究者着重研究利用免疫检测和功能研究筛选噬菌体克隆。本研究利用将单链抗体连接在M13噬菌体衣壳蛋白PⅢ的N端构建噬菌体抗体库。
本研究主要目的是利用噬菌体展示技术筛选HER2高亲和力的单链抗体，与力达霉素构建具有靶向杀伤HER2高表达肿瘤的强化融合蛋白，有助于乳腺癌、卵巢癌等HER2高表达肿瘤的导向治疗。
一、抗HER2噬菌体抗体库构建与筛选
本研究利用纯化的HER2胞外段蛋白免疫小鼠，从脾细胞提取mRNA，利用RT-PCR扩增抗体重链可变区(VH)和轻链可变区(VL)。利用重叠延伸反应(SOE-PCR)将VH、VL用连接肽(Gly_4Ser)_3进行连接，克隆至噬菌粒pCANTAB5E，转染E.coli TG1，得到噬菌体抗体库。以高表达HER2的人乳腺癌细胞SKBR3为抗原进行4轮筛选，得到库容为7.5×10~7的次级抗体库5F-10。从初级抗体库中随机挑取20个克隆进行菌体PCR，结果显示其中15个克隆有scFv片段插入，克隆效率为75％。BstNⅠ酶切随机克隆提取的质粒，酶切图谱显示多样性，表明抗体库具有多样性。用ELISA法鉴定各单克隆与HER2蛋白结合能力，获得与HER2高亲和力克隆scFv6，经序列分析，此基因全长732bp，编码243个氨基酸序列，通过Blast序列比对，此抗体为鼠源性，与以往报道的任何抗体序列都不相同。
二、抗HER2单链抗体与力达霉素强化融合蛋白的构建、表达、纯化及强化融合蛋白活性分析
利用基因重组技术将筛选出来的HER2高亲和力抗体scFv6片段克隆至含有力达霉素辅基蛋白基因的重组质粒pET-LDP，构建含有融合蛋白的质粒pET-Fv-LDP，转化到大肠杆菌BL21(DE3)starTM中，IPTG诱导表达带有组氨酸标签肽(His-tag)的融合蛋白HER2(Fv-LDP)。经SDS-PAGE分析，表达产物分子量40kD，主要以可溶形式表达在周质腔中，表达占全菌蛋白的7％，用Ni离子亲和柱进行纯化，1L菌液诱导后可产生约2mg可溶性融合蛋白。然后与力达霉素发色团AE重组，得到强化融合蛋白HER2(Fv-LDM)。研究其免疫学活性及其对HER2高、低表达肿瘤细胞的细胞毒作用。Western-blot分析显示，融合蛋白与纯化的HER2蛋白胞外段有结合活性。细胞ELISA和免疫荧光结果表明，融合蛋白HER2(Fv-LDP)与SKBR3、SKOV3都有较高的亲和活性，略低于商品化HER2单抗L87，而与MCF7无结合力。0.01M，0.1M，1M的强化融合蛋白可降低SKBR3细胞HER2的mRNA与蛋白表达水平。流式细胞周期测定结果融合蛋白将SKBR3细胞周期阻滞于G1期，而强化融合蛋白将细胞周期阻滞于G2／M期。HE染色和Hoechest染色显示，强化融合蛋白可以诱导细胞凋亡。MTT结果显示，强化融合蛋白对HER2高、低表达的细胞都有强烈的杀伤活性，较好地保留了力达霉素的细胞毒作用。在裸鼠皮下移植人乳腺癌SKBR3肿瘤模型中检测强化融合蛋白HER2(Fv-LDM)的体内抗肿瘤活性，接种肿瘤的第10天和17天分别尾静脉给药2次，观察肿瘤体积和裸鼠体重变化。实验结果表明0.2mg／kg，0.3mg／kg，0.4mg／kg三个剂量的强化融合蛋白HER2(Fv-LDM)对人乳腺癌SKBR3裸鼠移植瘤生长都有抑制作用，抑瘤率分别为60％，70％，89.7％。强化融合蛋白HER2(Fv-LDM)0.3mg／kg，0.4mg／kg的抑瘤率与LDM(0.05mg／kg)抑瘤率56.8％相比有显著差异(P＜0.05)。体内实验结果说明，强化融合蛋白可以提高动物对力达霉素的耐受剂量，同时显著改善治疗效果，是具有很好前景的抗体靶向药物。
Breast cancer is the most common malignancy in women, it now represents thesecond leading cause of death from cancer in women, and the number of new breastcancer cases has been increasing year by year. To increase the patients' survival ratesand their life quality, we need new measures of therapy. Molecular targeted cancertherapy has been developing rapidly. In present, many molecular agents for targetedtherapy have been used for breast cancer therapy in of clinical practice, such asHerceptin, Iressa and inhibitors of cell cycle regulator, COX-2 or PKC.
The Her-2/neu (c-erbB2) proto-oncogene is a component of the four-memberfamily of closely related growth factor receptors that includes the epidermal growthfactor receptor Her-1 (EGFR), Her-3 (c-erbB3) and Her-4 (c-erbB4). The human geneis located on chomosome 17q21 and encodes a 185 kD protein with tyrosine kinaseactivity, also known by the designation P185. Structurelly, the protein hasextracellular, transmembrane, and cytoplasmic domains, the latter of which containsthe tyrosine kinase domain and shares significant homology, although is distinct,from EGFR. Her-2/neu is thought to function as a growth factor receptor and plays arole in cell differentiation, adhesion, and motility. Overexpression of HER-2, usuallyas a result of her-2 gene amplification, can result in malignant transformation of cellsand is seen in tumor tissue in up to 30% of patients with metastatic breast cancer(MBC) and part of patients with ovarian, lung, gastric, and oral cancers. HER-2overexpression is usually associated with a more aggressive tumor phenotype, andwomen with HER-2 positive tumor have a poor overall prognosis and faster relapse.So the overexpression of HER-2 has been an independent prognostic index forevaluating the malignancy of above cancers. Evidently, HER2 become an explicittarget for therapy of breast cancer and ovarian cancer.
Antibody can carry agent to tumor region, so enhances the therapeutic effect anddecreases the toxicity. It has been shown that scFv (single chain variable Fragment)can penetrate into the solid tumor deeply. Compared to the whole antibody, scFvshows low immunogenicity and so induce less human anti-mouse antibody (HAMA)response. On the other hand, because of the absence of Fc, scFv can not execute theeffector function to kill target tumor cells. So a "warhead" molecule is neededincluding drugs, toxins, and cytokines.
Molecular targeted therapy is currently having a tremendous impact on the daily practice of clinical oncology. The advent of Herceptin (trastuzumab), a humanizedmonoclonal antibody against extracellular domain of HER-2, represented a majorbreakthough in the treatment of women with HER-2-positive MBC. Pivotal trials ofHerceptin alone or in combination with taxanes have result in significant clinicalbenefit, and Herceptin plus taxanes as first-line therapy is now the standard of care ofHER-2-positive disease. Already, the use of Herceptin in the metastatic setting haschanged HER-2-positive status from a marker of poor prognosis to one of betteroverall outcome, and ongoing studies should expand further treatment options forpatients with HER-2-positive MBC. This agent was shown to significantly prolongthe progression-free survival as a single agent or in combination with standardchemotherapeutic agents.
Lidamycin (LDM), an antibiotic produced by Streptomyces globisporus strainwhich was isolated in our institute, displayed extremely potent cytotoxicity againsttumor cells. LDM consists of a chomophore and an apoprotein, and the former has theability to attack DNA, whereas the latter plays the role as a protecting protein. Thetwo parts can be separated and reconstructed without affecting its activity.
The first surface expression system was developed by Smith in 1985, whodisplayed the filamentous phage on the surface of bacteriophge. In recent years, thefilamentous bacteriophages have been used extensively for the display of largerepertoires of antibodies on their surface. Phage display enables rapid selection ofantibodies from a large single chain fragment variable (scFv) library by virtue of thebinding to certain target antigen. After three or more rounds of selection, the resultingphage population is markedly enriched for the scFvs that bind to the antigen.Antibodies against hundreds of target antigens have so far been obtained from phagedisplay antibody libraries, including cell-surface markers, peptide hormones, humanproteins, and carbohydrates. One of the prominent advantages of the phage-displaytechnology is that, once a library has been created, it can be used to select antibodiesthat bind to any target antigens of interest. A single phage-antibody library can bedistributed to thousands of users and serve as the source of cloned antibodies againstan unlimited array of antigens. This technology is also efficient, and the process ofselection and primary screening is very rapid and can be completed with 2-3 weeks.The selected phage clones are then subjected to immunodetection to confirm theirbinding activities. ELISA and Western blot are extensively used in immunodetection.Conventionally, the procedure usually involves subcloning and expression of the target fragments encoding the scFv to obtain significant quantities of antibodies,which proves to be time-consuming and laborious. To resolve this problem,researchers have recently foucused on the use of selected phage clones inimmunodetection or function studies. In this study, a phage-display scFv antibodylibrary constructed on the N terminus of pⅢprotein of M13 filamentous phage.
In this study, phage display has been used to select high affinity scFv to HER2,and molecule reconstitution was performed to construct energized fusion proteinwith extremely potent cytotoxicity to HER2 over-expressing tumor cells.
1. Construction and selection of HER2 phage scFv library
In this study, mRNA was isolated from splenocytes of mice immunized withpurified ECD protein of HER2. Immunoglobulin variable fragments VH and VL wereamplified by RT-PCR and were connected by linker (Gly_4Ser)_3 to form the singlechain Fv (scFv) through a peptide by SOE-PCR. Then the scFv gene was cloned inphagemid pCANTAB5E, and transformed into E.coli TG1. The transformed cellswere infected by M13KO7 helper phage to get the primary phage display scFv library.Panning against breast cancer SKBR3 cells line was performed four times. At last, arecombinant phage display scFv library with a titre of 7.5×10~7 was established.Random 20 clones were selected from primary and selected phage antibody library,through PCR identified, scFv fragment was inserted into 15 clones of primary phagelibrary and 20 clones in selected phage library. Random 18 clones' plasmids fromprimary library digested with BstNⅠshowed different pattern, and clones fromselected library showed specially pattern enriched. The affinity of clones to HER2was identified by ELISA.Through 4 rounds of panning and screening, a phage-scFvnamed scFv6 binding to HER2 specifically was selected from the library. Bysequence analysis, the length of this gene is 732 bp, and 243 amino acid is encoded.This murine antibody is different from any other antibodies discovered before bysequence blast analysis.
2. Construction, expression, purification and characterization analysis of energizedfusion protein of HER2(Fv-LDM)
ScFv6 which has high affinity with HER2 antigen was cloned intoreconstruction plasmid pET-LDP, and plasmid pET-Fv-LDP was transformed intoE.coli BL21 (DE3) star~(TM), fusion protein HER2(Fv-LDP) with His-tag wasexpressed when induced by IPTG. Through SDS-PAGE analysis, 7% of soluble fusion protein existed in periplasm of E.coli. Subsequently purified by IMAC(immobilized metal affinity chromatography). About 2 mg of protein was obtainedfrom 1 liter fermentation broth. The purity of this protein exceeded 90%. For thepreparation of energized fusion protein HER2 (Fv-LDM), we added AE to the PBSsolution of the HER2 (Fv-LDP) fusion protein with the molecular ratio of 5:1 for 12h at room temperature. After the reconstitution was completed, separation andpurification were preformed using PD-10 colomn to remove the free AE (SephadexG-25 column). Western-blot assay showed that fusion protein have affinity withpurified protein ECD of HER2. Cell ELISA and cell fluorescence showed thatfusion protein HER2 (Fv-LDP) had high affinity with SKBR3 and SKOV3 cells,but it was lower than that of commercial anti-HER2 mAb L87. By contrast, HER2(Fv-LDP) had little affinity with MCF7 cells. The level of HER2 mRNA andprotein in SKBR3 cells decreaced by the treatment with HER2 (Fv-LDM). Flowcytometry (FCM) assay of cell cycle showed that cells were arrested at G1 phasewith fusion protein HER2 (Fv-LDP) treatment, instead of G2/M phase withenergized fusion protein HER2 (Fv-LDM) treatment. Through Hoechest and HEdying, the appearance of cells and nuclei showed that energized fusion proteinHER2 (Fv-LDM) can induce cell apoptosis of SKBR3、SKOV3 and MCF7. ByMTT assay, HER2 (Fv-LDM) showed potent cytotoxicity to SKBR3, SKOV3,Moreover, the in vivo therapeutic efficacy of the energized fusion protein HER2(Fv-LDM) was investigated in human breast carcinoma SKBR3 xenograft in nudemice. In experiment, the SKBR3 tumor bearing mice were treated by single i.v doseon day 10 and day17 after subcutaneous tumor transplantation, inhibitory effect ofHER (Fv-LDM), LDM, HER2 (Fv-LDP) was detected through measuring thevolume of tumor. At dose 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, HER2 (Fv-LDP)exhibited highly effected potency in retarding growth of SKBR3 with inhibitionrate of 60%, 70%, 89.7%, while the inhibition rate of LDM (0.05mg/kg) is 56.8%.So the energized fusion protein can increase the tolerance dose of LDM, improvethe therapeutic effects, and become an antibody targeted medicine of goodperspective.
引文
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27. Better M, Chang CP, Robinson RR, et al. Escherichia coli secretion of an active chemeric antibody fragment. Scicence, 1998; 240:1941-1043
28. Skerr A, Pluckthun A. Assembly of functional immunoglobulin Fv fragment in Escherichia coli. Science, 1988; 1038-1041
29. Huston JS, Levinson D, Mudgett-Hunter M, et al. Protein engineering of antibody binding sites:recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA, 1988; 85(16): 5879-5883
30. Powers DB, Amersdorfer P, Poul MA, et al. Expression of single chain Fv-Fc fusion in Pichia pastoris.J Immuno Meghods, 2001; 251:123-135
31. Tsuda H, Prognostic and predictive value of c-erbB-2(HER-2/neu) gene amplification in human breast cancer. Breast Cancer, 1001; 8:38-44
32. ColomerR, Shamon L A, Tsai MS, et al. Herceptin: from the bench to the clinic.Cancer Invest, 2001; 19(1): 49-54
33. Eisenhauer EA. From the molecular to the clinic inhibiting HER-2 to treat breast cancer. N Engl J Med, 2001; 344:841-842
34. Slamon DJ, Leyland-Jones B,Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastastic breast cancer that overexpresses HER2. N Engl J Med, 2001; 344: 783-792
1. Kaufman DL, Evans GA, et al. Restriction endonuclease cleavage at the termini of PCR products. Biotechniques. 1990 9(3): 304-306.
2. Sblatter OD, Florian F, Bradburya, et al. Analyzing the peripheral blood antibody repertoire of a celiac disease patientus using gphage antibody libraries. Human antibodies. 2000; 9(4): 199-205.
3. Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985; 228(4705): 1315-1317.
4. Winter G, Griffiths AD, Hawkins RE, et al. Monoclonal antibodies can be used to inhibit allo-gr aft rejection. Annu Rev Immunol. 1994, (12): 433-455.
5. Clackson T, Hoogenboom HR, Grifiths AD. Making antibody fragments using phage display libraries. Nature. 1991; 352 (6336), 624-628.
6. Sidhu SS, Weiss GA, Wells JA, et al. High copy displayof large proteins on M13 phage for functional selections. J Mol Biol. 2000; 296: 487-495.
7. Hufton SE, Moerkerk PT, Meulemans EU, et al. Phage display of cDNA repertoires: the pⅥ display system and its applications for the selection of immunogenic ligands. J Immunol Methods 1999; 10: 231(12): 39-51.
8. Waterhouse EP, Griffith AD, Johsonks, et al. Combinatiorial infection and in vivo recombination: A strategy for making large phage antibody repertoires. Nucleic Acids Research. 1993; 21:2265-2273.
9. Geoffroy F, Sodoyer R, Augamel, et al. A new phage display system to construct multicombinatorial libraries of very large antibody repertoires. Gene. 1994; 151:109-118.
10. Barbas CF, Kang AS, Lerner RA, et al. Assembly of combinatorial antibody libraries on phage surface: the gene III site. Proc Natl Acad Sci USA. 1991; 88: 7978-7982.
11. Alting-Mees MA, Short JM. Polycosvectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts. Gene. 1993; 137 (1): 93-100.
12. Chester KA, Begent RH, Robson L. Phage libraries for generation of clinically useful antibodies. Lancet. 1994; 343 (8895):455-456.
13. Barbas CF, Burton DR. Selection and evolution of high-affinity human anti-viral antibodies. Trends Biotechnol. 1996; 14 (7):230-234.
14. Clark MA, Hawkins NJ, Papaioannou A. Isolation of human anti-c-erbB Fabs from a lymph node derived phage display library. Clin Exp immunol. 1997; 109 (1): 166-174.
15. Hoogenboom HR. Designing and optimizing library selection strategies for generating high-affinity antibodies.Trends Biotechnol. 1997; 15 (2):62-70.
16. Haard HJ, van Neer N, Reurs A. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem. 1999; 274 (26): 18218-18230.
17. Roovers RC, Van der Linden E, Zijlema H. Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display. Int J Cancer. 2001; 93(6):832-840.
18. Grifiths AD, Williams SC, Hartley O. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 1994; 13(14):3245-3260.
19. Knappik A, Ge L, Honegger A. Fully synthetic human combinatoryial antibody libraryes (HuCAL)based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 2000; 296 (1):57-86.
20. Silacci M, Brack S, Schirru G, Marlind J, et al. Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics. 2005; 5 (9):2340-2350.
21. Marks JD, Griffiths AD, Malmgvist M. By-passing immunization: building high affinity hum- an antibodies by chain shuffling. Biotechnology. 1992; 10 (7): 779-783.
22. Low NM, Holiger PH, Winter G. Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J Mot Biol. 1996; 260(3): 359-368.
23. Soderlind E, Lagekvist AC, DuenasM, et al. Chaperon in assisted phagedisplay ofantibody fragments on filamentous bacteriophages. Biotechnol. 1993, 11(4): 503-507.
24．王琰，化冰，刘群英，等．半合成抗体库的构建和鉴定．中华微生物学和免疫学杂志1999，19(3)：238-241．
25. McIntosh RS, Asghar S, Watson PE et al. Cloning and analysis of IgGκ and IgGλ, anti-thyroglobulin autoantibodies from a patient with Hashimuto's thyroid- itis. J Immunol. 1996; 167: 927-934.
26. IhaY, HayashiN, SawadaJ, et al. Change in the specificity of antibodies against steroid antigens by introduction of mutations into complemetarity determinng regions of the VH domain.Protein Eng. 1998; 11(5): 361-370.
27. Gram H, Marconi LA, Basbas CF, et al. Inviroselection and affinity maturation of antib- odies from a naitive combinatorial immunoglobulinlibrary. Proc Natl Acad Sci USA. 1992; 89: 3576-3580.
28. Kang AS, Jones TM, Burton DR. Antibody redesign by chain shuffling from random combinational immunoglobulin. Proc Natl Acad Sci USA. 1991; 88: 11120-11123.
29. Jirholt P, Ohlin M, Borrebaech CAK, et al. Exploiting sequence space: shuffling in vivo formed complementary determing regions into a master framework. Gene. 1998; 215(2): 471-476.
30. Azriel-Rosenfeld, R, Valensi, M. Benhar I. A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions. J Mol Biol. 2004; 335: 177-192.
31．侯云霞，董文其，徐伟文，等．抗恶性疟原虫富含组氨酸蛋白Ⅱ单链抗体库的构建及筛选．第一军医大学学报，2001；21(4)：241-244。
32．王卓智，王琰，李竞，等．抗原表位定向选择的人源抗TNF-αFab的鉴定．中华微生物和免疫学杂志，1999：19(3)：242-245．
33. Xia XB, Wan HY, Wang B, et al. The expression of single chain Fv fragment of mon- oclonal antibody against activated platelets in E. coli. Clin J Microbid Immunol. 2000; 20(3): 358-362.
34. Freyre FM, Vazquez JE, Ayala M, et al. Very high expression of anti- carcino- embryonic antigen single chain Fv antibody fragment in the yeast Pichia Pastoris. J Biotechnol. 2000; 76(2-3): 157-263.
35. Calarese DA, Scanlan CN, Zwick MB, et al. Antibody domain exchange is an immuhological solution to carbohydrate cluster recognition. Science. 2003; 300:2065-2071
36. Balass M, Morag E, Bayer E A, et al. Recovery of high affinity phage from an itro-streptavid in matrix in phage display technology.Anal Biochem. 1996; 243 (2):264-269.
37. Rapoport B, Portolano S, Mclachlan SM. Combinatorial libraries: new insights into human organ specific autoantibodies. Immunol Today. 1996; 16: 43-49.
38. Williamson P, Yatchews R. Development of neutralizing human recombinant antibodies to pertuesis toxin. FEMS Immunol Med Microbiol. 1999; 123 (4):313-322.
39. Persson H, Lantto J, Ohlin M. A Focused Antibody Library for Improved Hapten Recognition. J Mol Biol. 2006; 19:57-68.
40. Bender E, Pilkington GR, Burton DR. Human monoclonal Fab fragments from a com- binatorial library prepared from an individual with a low serum titer to a virus. Humam Antibody Hybridomas. 1994; 6: 3-8.
41. Burton DR, Barbas CFHI, Pereeon YAA, et al. A large array of human monoclonal anti -bodies to type I human immunodeficiency virus from combinatorial libraries of asymptomatic individual. Proc Natl Acad Sci USA. 1991; 88: 10134-10143.
42. Mullen LM, Nair SP, Ward JM, et al. Henderson B. Phage display in the study of infectious diseases. Trends Microbiol. 2006; Feb 3.
43. Mutuberria R, Satijn S, Huijbers A, et al. Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries.J Immunol Methods 2004; 287:31-47
44. Cattani P, Rossolini, Creati S, et al. Detection and typing of herpessimplex viruses by using recombinant immunoglobulin fragments produced in bacteria. J Clin Microbioi. 1997; 35 (6): 1504-1509.
45. Williamson RA, Lazzarotto R, SannaPP, et al. Use of recombinant human antibody fragments for detection cytomegalovirus antigenemie. J Clin Microbiol. 1997; 36 (8):2047-2053.
46. Zebedee SL, Barbas CF3rd, Hon YL, et al. Human combinatorial antibody libraries to hepatitis B surface antigen. Proc Natl Acad Sci USA. 1992; 89: 3176-3181.
47. Barbas CF3rd, Bjorling E, Chiodi F, et al. Recombinant Fab fragmentsneutralize human type I immunodeficiency virus in vitro. Proc Natl Acad Sci USA. 1992; 89: 9334-9339.
48. Walter G, Konthur Z, Lehrach H, et al. Automation of phage display for high-throughput antibody development. Target. 2002; 1(1):30-36.
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2. Sjogren S, Inganas M, Lindgren A, et al. Prognostic and predictive value of c-erbB-2 (HER-2/neu) gene amplification in human breast cancer. Breast Cancer, 2001; 8: 38-44
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4. Wright C, Angus B, Nicholson S, et al. Expression of c-erbB-2 oncoprotein: A prognostic indicator in human breast cancer. Caner Res,1989; 49:2087-2090
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6. Holbro T, Civenni G, Hynes NE. The ErbB-2 receptor and their role in cancer progression. Exp Cell Res, 2003; 284(1): 99-110
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15. Chaudhary VK, Batra JK, Gallo MG.. A rapid method of cloning functional variable-region antibody genes in Escherchia coli as single-chain immunotoxins.Proc Natl Acad Sci USA, 1990; 87: 1066-1070
16. Masanori Onda, Qing-cheng Wang, Hong-fen Guo, et al. In vitro and in vivo cytotoxic activeeties of recombinant immunotoxin 8H9(Fv)-PE38 against Breast Cancer, Osteosarcoma, and Neuroblastoma.Cancer Research, 2004; 64: 1419-1424
17. Smith GE Filamentous fusion phage: novel expression vectors that display cloned antigens on the vires surface.Science, 1985; 228: 1315-1317
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27. Ward ES, Gussow D, Grifiths AD. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 1989;341 (6242):544-546
28. Marks JD, Hogenboom H, Bonnert TP, et al. By-passing immunization human antibody from v-gene libraries displayed on phage. J Mol Biol, 1991; 222:581-597
29. Hughes Jones NS, Bye JM, Gorick BD, et al. Synthesis of Rh Fv phage-antibody using VH and VL germline genes. Br J Haematol. 1999; 105: 811-816
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1. Colcher D, Pavlinkova G, Beresford G, et al. Single-chain antibodies in pancreatic cancer. Ann N Y Acad Sci, 1999; 880:263-280
2. Yokota T, Milenic DE, Whitlow M, et al. Rapid tumor penetration of a single-chaiin Fv and comparision with other immunoglobulin forms. Cancer research, 1992; 52:3402-3408
3. Reiter Y. Recombinant immunotoxins in targeted cancer therapy. Adv Cancer Res, 2001; 81: 93-124
4. Bosslet K, Czech J, Hoffmann J. Tumor-selective prodrug activation by fusion protein-mediated catalysis. Cancer research, 1994; 54(8): 2151-2159
5. Penicher ML, Morrison SL. Antibody-cytokine fusion protein for the therapy of cancer. J Immunol Methods, 2001; 248(1-2): 91-101
6. Buchegger F, Roth A, Allal A, et al. Radioimmunotherapy of colorectal cancer liver metastasis: combination with radiotherapy. Ann N Y Acad Sci, 2000; 910: 263-269
7．甄永苏，薛玉川，邵荣光．烯二炔类新抗生素C1027的抗肿瘤作用研究．中国抗生素杂志1994，19(2)：164-168
8．邵荣光，甄永苏．新烯二炔类大分子抗肿瘤抗生素C1027的分子构成与活性关系．药学学报1995；30：336—339．
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14. Bhatia J, Sharma S K, Chester K A, et al. Catalytic activity of an in vivo tumor targeted anti-CEA scFv: carboxypeptidase G2 fusion protein.Int J Cancer, 2000; 85(4):571-577
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16. Dickman S. Antibodies stage a comeback in cancer treatment. Science, 1998; 280: 1196-1197
17. Sblatero D, Bradbury A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol, 2000; 18 (1): 75-80
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19. Fuchs P, Breitling F, Little M, et al. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.Hybridoma,1997;16: 227-233
20. Masanori Onda, Qing-cheng Wang, Hong-fen Guo, et al. In vitro and in vivo cytotoxic activities of recombinant immunotoxin 8H9(Fv)-PE38 against breast cancer, osteosarcoma and neuroblastoma.Cancer research, 2004; 64:1419-1424
21. Chaudhary VK, Janendrya K, Batra, et al. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins (Pseudomonas exotoxin/recombinant immunotoxin). Proc Natl Acad Sci USA, 1990; 287:1066-1070
22. Wels W,Moritz D,Schnidt M, et al. Biotechnological and gene therapeutic strategies in cancer treatment.Gene, 1995; 159(1):73-80
23. Bosslet K, Lorenz P, Sedlacek HH, et al. Molecular and functional characterization of a fusion protein suited for tumor specific prodrug activation. Br J Cancer, 1992;65:234
24. Kerr DE, Vrudhula VM, Svensson HP, et al. Comparison of recombinant and synthetically formed monoclonal antibody-beta-lactamase conjugate for anticancer prodrug activation. Br J Cancer, 1992; 65:234-241
25. Cregg JM, Madden KR. Development of the methylotrophic yeast, Pichia pastoris, as a host system for the production of foreign proteins. Dev Ind Microbiol. 1988; 29:33-41
26. Ward ES, Gussow D, Griffith AD, et al. Binding activities of a repertoire of single immunoglobulin variable domains serected from Escherichia coli. Nature,1989; 341(6242): 544-546.
27. Better M, Chang CP, Robinson RR, et al. Escherichia coli secretion of an active chemeric antibody fragment. Scicence, 1998; 240:1941-1043
28. Skerr A, Pluckthun A. Assembly of functional immunoglobulin Fv fragment in Escherichia coli. Science, 1988; 1038-1041
29. Huston JS, Levinson D, Mudgett-Hunter M, et al. Protein engineering of antibody binding sites:recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA, 1988; 85(16): 5879-5883
30. Powers DB, Amersdorfer P, Poul MA, et al. Expression of single chain Fv-Fc fusion in Pichia pastoris.J Immuno Meghods, 2001; 251:123-135
31. Tsuda H, Prognostic and predictive value of c-erbB-2(HER-2/neu) gene amplification in human breast cancer. Breast Cancer, 1001; 8:38-44
32. ColomerR, Shamon L A, Tsai MS, et al. Herceptin: from the bench to the clinic.Cancer Invest, 2001; 19(1): 49-54
33. Eisenhauer EA. From the molecular to the clinic inhibiting HER-2 to treat breast cancer. N Engl J Med, 2001; 344:841-842
34. Slamon DJ, Leyland-Jones B,Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastastic breast cancer that overexpresses HER2. N Engl J Med, 2001; 344: 783-792
1. Kaufman DL, Evans GA, et al. Restriction endonuclease cleavage at the termini of PCR products. Biotechniques. 1990 9(3): 304-306.
2. Sblatter OD, Florian F, Bradburya, et al. Analyzing the peripheral blood antibody repertoire of a celiac disease patientus using gphage antibody libraries. Human antibodies. 2000; 9(4): 199-205.
3. Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985; 228(4705): 1315-1317.
4. Winter G, Griffiths AD, Hawkins RE, et al. Monoclonal antibodies can be used to inhibit allo-gr aft rejection. Annu Rev Immunol. 1994, (12): 433-455.
5. Clackson T, Hoogenboom HR, Grifiths AD. Making antibody fragments using phage display libraries. Nature. 1991; 352 (6336), 624-628.
6. Sidhu SS, Weiss GA, Wells JA, et al. High copy displayof large proteins on M13 phage for functional selections. J Mol Biol. 2000; 296: 487-495.
7. Hufton SE, Moerkerk PT, Meulemans EU, et al. Phage display of cDNA repertoires: the pⅥ display system and its applications for the selection of immunogenic ligands. J Immunol Methods 1999; 10: 231(12): 39-51.
8. Waterhouse EP, Griffith AD, Johsonks, et al. Combinatiorial infection and in vivo recombination: A strategy for making large phage antibody repertoires. Nucleic Acids Research. 1993; 21:2265-2273.
9. Geoffroy F, Sodoyer R, Augamel, et al. A new phage display system to construct multicombinatorial libraries of very large antibody repertoires. Gene. 1994; 151:109-118.
10. Barbas CF, Kang AS, Lerner RA, et al. Assembly of combinatorial antibody libraries on phage surface: the gene III site. Proc Natl Acad Sci USA. 1991; 88: 7978-7982.
11. Alting-Mees MA, Short JM. Polycosvectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts. Gene. 1993; 137 (1): 93-100.
12. Chester KA, Begent RH, Robson L. Phage libraries for generation of clinically useful antibodies. Lancet. 1994; 343 (8895):455-456.
13. Barbas CF, Burton DR. Selection and evolution of high-affinity human anti-viral antibodies. Trends Biotechnol. 1996; 14 (7):230-234.
14. Clark MA, Hawkins NJ, Papaioannou A. Isolation of human anti-c-erbB Fabs from a lymph node derived phage display library. Clin Exp immunol. 1997; 109 (1): 166-174.
15. Hoogenboom HR. Designing and optimizing library selection strategies for generating high-affinity antibodies.Trends Biotechnol. 1997; 15 (2):62-70.
16. Haard HJ, van Neer N, Reurs A. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem. 1999; 274 (26): 18218-18230.
17. Roovers RC, Van der Linden E, Zijlema H. Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display. Int J Cancer. 2001; 93(6):832-840.
18. Grifiths AD, Williams SC, Hartley O. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 1994; 13(14):3245-3260.
19. Knappik A, Ge L, Honegger A. Fully synthetic human combinatoryial antibody libraryes (HuCAL)based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 2000; 296 (1):57-86.
20. Silacci M, Brack S, Schirru G, Marlind J, et al. Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics. 2005; 5 (9):2340-2350.
21. Marks JD, Griffiths AD, Malmgvist M. By-passing immunization: building high affinity hum- an antibodies by chain shuffling. Biotechnology. 1992; 10 (7): 779-783.
22. Low NM, Holiger PH, Winter G. Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J Mot Biol. 1996; 260(3): 359-368.
23. Soderlind E, Lagekvist AC, DuenasM, et al. Chaperon in assisted phagedisplay ofantibody fragments on filamentous bacteriophages. Biotechnol. 1993, 11(4): 503-507.
24．王琰，化冰，刘群英，等．半合成抗体库的构建和鉴定．中华微生物学和免疫学杂志1999，19(3)：238-241．
25. McIntosh RS, Asghar S, Watson PE et al. Cloning and analysis of IgGκ and IgGλ, anti-thyroglobulin autoantibodies from a patient with Hashimuto's thyroid- itis. J Immunol. 1996; 167: 927-934.
26. IhaY, HayashiN, SawadaJ, et al. Change in the specificity of antibodies against steroid antigens by introduction of mutations into complemetarity determinng regions of the VH domain.Protein Eng. 1998; 11(5): 361-370.
27. Gram H, Marconi LA, Basbas CF, et al. Inviroselection and affinity maturation of antib- odies from a naitive combinatorial immunoglobulinlibrary. Proc Natl Acad Sci USA. 1992; 89: 3576-3580.
28. Kang AS, Jones TM, Burton DR. Antibody redesign by chain shuffling from random combinational immunoglobulin. Proc Natl Acad Sci USA. 1991; 88: 11120-11123.
29. Jirholt P, Ohlin M, Borrebaech CAK, et al. Exploiting sequence space: shuffling in vivo formed complementary determing regions into a master framework. Gene. 1998; 215(2): 471-476.
30. Azriel-Rosenfeld, R, Valensi, M. Benhar I. A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions. J Mol Biol. 2004; 335: 177-192.
31．侯云霞，董文其，徐伟文，等．抗恶性疟原虫富含组氨酸蛋白Ⅱ单链抗体库的构建及筛选．第一军医大学学报，2001；21(4)：241-244。
32．王卓智，王琰，李竞，等．抗原表位定向选择的人源抗TNF-αFab的鉴定．中华微生物和免疫学杂志，1999：19(3)：242-245．
33. Xia XB, Wan HY, Wang B, et al. The expression of single chain Fv fragment of mon- oclonal antibody against activated platelets in E. coli. Clin J Microbid Immunol. 2000; 20(3): 358-362.
34. Freyre FM, Vazquez JE, Ayala M, et al. Very high expression of anti- carcino- embryonic antigen single chain Fv antibody fragment in the yeast Pichia Pastoris. J Biotechnol. 2000; 76(2-3): 157-263.
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