【作者】 陈汐敏；

【导师】 冯振卿；

【作者基本信息】 南京医科大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 原发性肝癌（primary liver cancer，PLC）是世界第5位高发的肿瘤，也是致死率第3位的恶性肿瘤，其中70%-85%为肝细胞癌（hepatocellular carcinoma，HCC）。肝细胞癌由于缺乏有效的治疗手段，严重威胁着人类生命和健康。随着现代分子生物学技术和基因工程技术的迅速发展，为肝癌的生物治疗开辟了全新的领域。生物治疗已成为继手术、放疗、化疗后肿瘤治疗的第四种模式，并显示出了良好的应用前景，主要包括：分子靶向治疗、免疫治疗、基因治疗、内分泌治疗、细胞治疗等。分子靶向治疗是通过靶向肿瘤细胞表面特异性分子的配体与药物、放射性核素或生物毒素等偶联，靶向杀伤肿瘤细胞。由于其能选择性作用于肿瘤局部，具有疗效高、副作用小等优点。其中，基于特异性抗体、偶联化疗药物的肝癌靶向治疗成为新的研究热点。抗体靶向治疗的关键因素之一是高特异性肿瘤抗原的筛选。理想状态下，它应在肿瘤细胞高表达，而在正常组织中不表达，并且其与相应抗体结合后有较好地内化能力，能够使与之结合的抗体很快内化入细胞中，进而使抗体携带的药物能够特异性地进入肿瘤细胞中而发挥作用。已有研究表明HCC的发病机制十分复杂，其发生、发展和转移与多种基因的突变、细胞信号传导通路和新生血管增生异常等机制密切相关，其中多个关键性环节，是进行抗体靶向治疗的理论基础和潜在的重要靶点。研究表明，肝细胞生长因子（hepatocyte growthfactor，HGF)可与其受体c-Met结合，该信号通路在肝癌的形成、侵袭、转移等过程中都发挥着重要作用，c-Met在肝癌组织中高表达，而在其他正常组织中低表达或不表达，因此，c-Met可作为肝癌靶向治疗的重要靶点，将成为肝癌治疗的一个新途径。目前临床化疗药物的品种较多，其中阿霉素是临床最早使用的肿瘤化疗药物之一，使用范围广，是肝癌化疗的常用药物之一，但是其毒副反应较大，最常见的是心脏毒性和骨髓抑制，限制了其在临床肿瘤治疗中的应用。本研究旨在将抗c-Met抗体与阿霉素偶联，利用抗体的靶向性，有效地将药物靶向到肿瘤部位，有希望降低阿霉素剂量，减轻化疗不良反应，提高化疗效果。为此，本课题研究内容如下：1．全人源免疫型Fab噬菌体抗体库的构建2．人源抗c-Met Fab抗体的筛选、表达和鉴定3．人源抗c-Met Fab抗体分子与阿霉素的偶联及偶联物特性分析4．人源抗c-Met Fab-阿霉素对肿瘤细胞的细胞毒作用分析5．人源抗c-Met Fab-阿霉素对荷人肝癌移植瘤裸鼠的体内抑瘤效果分析6．人源抗c-Met Fab-阿霉素在荷人肝癌移植瘤裸鼠体内的定位及分布研究方法：1．分离肝癌患者外周血淋巴细胞，扩增所有的抗体重、轻链可变区基因，构建全人源噬菌体抗体库。基于细胞表面抗原的差减筛选，对构建的噬菌体抗体库进行富集筛选，制备人源抗c-Met Fab质粒，并测定抗体的基因序列。2．重组质粒pComb3X-MetFab，转入大肠杆菌诱导表达Fab，通过诱导条件的优化，筛选最适条件大量表达，选择合适条件采用Protein L亲和柱纯化细菌超声裂解上清，并对纯化后的人源抗c-Met Fab抗体进行鉴定。3．用化学合成的方法，将人源抗c-Met Fab抗体和阿霉素偶联，得到偶联产物MetFab-DOX，并用HPLC进行初步鉴定。通过FACS、细胞ELISA及免疫荧光检测，分析MetFab-DOX对c-Met不同表达水平细胞的结合能力。同时观察MetFab-DOX中阿霉素进入细胞内的过程，并与游离阿霉素进行比较。4．将MetFab-DOX及游离阿霉素分别作用于c-Met阳性表达的肝癌细胞和c-Met阴性表达的细胞，MTT法检测MetFab-DOX、游离阿霉素对细胞的毒性作用，并比较分析二者对不同c-Met表达水平细胞毒性作用的差异。5．建立人肝癌移植瘤裸鼠模型，分别应用MetFab-DOX和高、低剂量阿霉素进行治疗，观察MetFab-DOX与阿霉素的抑瘤效果，并比较二者化疗药物毒副作用的差异。6．裸鼠肝癌移植瘤组织冰冻切片，免疫荧光观察MetFab-DOX在肿瘤组织中的定位。用Cy5.5进行标记MetFab，运用活体成像技术观察MetFab在荷瘤小鼠中的定位。荷瘤裸鼠分别注射游离阿霉素和MetFab-DOX，荧光分光光度法测定并比较给药后不同时间点阿霉素在动物体内各主要脏器和肿瘤组织中的分布。研究结果：1．构建了全人源免疫型Fab抗体库，库容量为2.0×109。筛选出1株人源抗c-Met Fab抗体，并进行了序列测定。选择诱导前加入2%葡萄糖、SB培养基、25℃、1mmol/L IPTG作为最佳诱导表达条件，以最适条件进行Protein L柱的亲和纯化，最终得到纯度较高的人源抗c-Met Fab抗体，产量可达5mg/L。2．采用化学键合的方法将人源抗c-Met Fab抗体分子与阿霉素偶联，分离纯化得到偶联物MetFab-DOX，HPLC分析结果证实二者偶联成功。体外释放实验表明，在pH7.2中性条件下，MetFab-DOX比较稳定，240h累积释放率约为16.9%；在pH4.0酸性条件下，MetFab-DOX能较快释放出游离的阿霉素，96h累积释放率为81.3%，远高于pH7.2时96h的累积释放率12.1%。3．FACS分析表明MetFab-DOX能与不同c-Met表达水平的肝癌细胞特异性结合，抗体的结合效率因细胞表面c-Met表达水平的差异而不同，且与不表达c-Met的NIH3T3细胞基本不结合，显示了较好地靶向性。通过细胞免疫荧光及细胞ELISA检测，证实MetFab-DOX与MetFab具有相同的特性，能与表达c-Met的肝癌细胞特异性结合。跟踪阿霉素的红色荧光观察到MetFab-DOX进入细胞并且释放阿霉素的过程与游离阿霉素不同。游离阿霉素可较快地直接进入细胞核，对细胞无选择性；而MetFab-DOX先与细胞表面的c-Met结合，进入胞浆释放阿霉素定位到核上，具有较好地靶向性。4．对c-Met阳性表达的肝癌细胞，MetFab-DOX及游离阿霉素都具有细胞毒性作用；对c-Met阴性表达的NIH3T3细胞，游离阿霉素有较强的细胞毒性作用，而MetFab-DOX几乎无细胞毒性作用。5．分别用MetFab-DOX和游离阿霉素进行裸鼠人肝癌移植瘤模型的研究，观察到MetFab-DOX与游离阿霉素对肿瘤生长都有明显抑制作用，阿霉素高剂量组的平均抑瘤率为90.38%，阿霉素低剂量组为59.67%，MetFab-DOX组为65.40%。同时MetFab-DOX能明显减轻阿霉素单独给药所引起的体重下降，与阿霉素高、低剂量组相比，差异均有统计学意义（P<0.05）。MetFab-DOX还能明显减轻阿霉素所引起的心、肺、肾的毒副作用。6．肿瘤组织免疫荧光检测发现，MetFab-DOX能够结合在肿瘤局部。活体成像实验结果表明MetFab能够富集于肿瘤局部，并且注射后48h仍能够在肿瘤局部观察到MetFab-Cy5.5的荧光。荧光分光光度法检测结果显示，给药后，MetFab-DOX组裸鼠肿瘤组织中的阿霉素含量均高于游离阿霉素给药组，而其心脏及肾脏中的阿霉素含量均低于游离阿霉素给药组，差异均有统计学意义（P<0.05）。结论：本课题筛选出1株人源抗c-Met Fab抗体，并成功将其与阿霉素进行偶联，制备MetFab-DOX。MetFab-DOX不仅对体外培养的肝癌细胞有细胞毒作用，而且在肝癌的裸鼠模型中，能特异性靶向肿瘤细胞，对肝癌移植瘤生长有明显的抑瘤效果，同时明显减轻了阿霉素的毒副作用。该研究结果为该抗体偶联化疗药物应用到肝癌的临床治疗奠定了基础，为肝癌分子靶向治疗提供了一个候选靶向药物。更多还原

【Abstract】 Among primary liver cancers, hepatocellular carcinoma (HCC) represents themajor histological subtype, accounting for70%to85%of the total liver cancerburden worldwide. HCC is the fifth most common tumor worldwide, but due to itspoor prognosis, it ranks as the third most common cause of death from cancer. Theglobal incidence of HCC is greater than a million cases a year. Despite therapeuticadvances, the treatment to patients with HCC still falls short of affectivity orcredibility. In the last2decades, a new therapeutic modality directed against tumormolecular targets, defined as “targeted therapy,” has been developed rapidly.Targeted cancer therapy is promising to minimize the nonspecific toxicity and toimprove therapeutic efficiency compared to conventional chemotherapy.Antibody-targeted chemotherapy is designed for selective delivery of cytotoxic drugsto tumor cells by linking them to monoclonal antibodies, thereby enhancing antibodytherapeutic activity while minimizing the systemic effects of a cytotoxic drug. Severalmonoclonal antibodies (mAb) have established roles in cancer chemotherapy due totheir specificities for tumor-associated antigens and their manageable off-targettoxicities.Antibody drug conjugates (ADCs) are composed of three key elements that caninfluence the therapeutic index: antibody, linker, and drug. The choice of targetantigen (expression in cancer versus normal tissues) and the ability to internalize theantigen-antibody complex are crucial as they determine tumor cell–specific deliveryof cytotoxic drug. c-Met，the ligand of hepatocyte growth factor (HGF)，is involved inorgan regeneration, as demonstrated for liver and kidney, embryogenesis, hematopoiesis, muscle development, and in the regulation of migration and adhesionof normally activated B cells and monocytes. Furthermore, numerous studiesindicated the involvement of c-Met overexpression in malignant transformation andinvasiveness of malignant cells. c-Met was overexpression in hepatocellularcarcinoma tissues and cells and low or no expression in normal tissues. As a result, itcan be used as a target for antibody drug conjugate in HCC therapy.Doxorubicin (DOX) is an effective and widely used chemotherapeutic agent indifferent cancers. It may produce free radicals, leading to serious side effects,especially cardiomyopathy to congestive heart failure. Therefore, DOX is a preferredcandidate for drug targeting research to evaluate both effectiveness and toxicity.The aim of our study was preparing anti-c-Met Fab and conjugating it withdoxorubicin, bringing the drug target to the tumor tissue by virtue of the specificity ofthe antibody against c-Met on the surface of the cells. Consequently, the conjugatecan decrease the side effect of doxorubicin and increase the therapy effect of HCC.The research plan is as following:1. Constructing immunized human Fab antibody phage library2. Screening, expressing and purifying human anti-c-Met Fab antibody3. Conjugating doxorubicin with human anti-c-Met Fab antibody and identifying thecharacteristics of conjugation in vitro4. Observing the cytotoxicity of the conjugation on HCC cells in vitro5. Evaluating the anti-tumor effect of the conjugation on the mouse xenograft modelof HCC6. Observing the distribution and localization of the conjugation in the mousexenograft modelMethods1. An immunized phage-display Fab library was constructed from peripheral blood lymphocytes from40patients with HCC. The immunized antibody library was usedto select and produce antibodies that bound to c-Met protein of cell surface. Afterscreening, the clone with plasmid of pCOMB3X-MetFab showed highest aviditywith c-Met protein was sequenced and chosen for the next experiment.2. The plasmid pCOMB3X-MetFab was transformed into competent E.coli Top10F’,and the positive insert was identified by PCR amplification of the Fab fragmentfrom bacterial colony. After optimizing the parameters of both expression andpurification, large-scale anti-c-Met Fab was produced by the Protein L affinitychromatography.3. The MetFab and DOX were conjugated via chemical synthesis and confirmed byHPLC. The binding efficacy of the conjugate (MetFab-DOX) was assessed invarious cell lines that are either positive or negative for c-Met by FACS, ELISA andimmunofluorescence observation. The cellular distribution of free DOX andMetFab-DOX was observed by fluorescence microscopy.4. HCC cells with c-Met-positive expression and NIH3T3cells with c-Met-negativeexpression were treated with doxorubicin and MetFab-DOX. Cytotoxicity ofMetFab-DOX was analyzed by the MTT method in vitro and compared with that ofdoxorubicin.5. The mouse xenograft model of HCC was constructed and treated with doxorubicinand MetFab-DOX. The anti-tumor effect in vivo was evaluated and the side effectof each group was observed.6. The localization of conjugation was confirmed by immunofluorescence staining offrozen tumor tissue section from the mouse xenograft model of HCC in vitro.Furthermore, the localization of conjugation was confirmed by optical tumorimaging in vivo with cy5.5labeled MetFab. The tissue distribution of conjugationwas confirmed in a nude mouse xenograft model of HCC by spectrofluorometermehtod. The drug concentrations of doxorubicin in organs at different times werecompared between doxorubicin treated and MetFab-DOX treated mice. Results1. The immunized antibody library, with a diversity of2.0×109, was constructed andused to select and produce anti-Met Fab fragment. One clone, with plasmid ofpCOMB3X-MetFab showed highest avidity with c-Met protein was sequenced andchosen for the next experiment. The optimal Fab antibody production was carriedout by using the SB culture medium adding2%glucose before induction,25℃induction temperature, and1mmol/L IPTG. The expected protein was purified fromthe sonication supernatant with high purity by Protein L affinity chromatographywith adding350mmol/L NaCl in binding buffer and150mmol/L NaCl in elutionbuffer. The final yield was5mg per liter liquid of bacteria.2. The MetFab and DOX were conjugated via chemical synthesis and confirmed byHPLC. The drug release patterns of the conjugate were analyzed under pH7.2andpH4.0, respectively. The conjugation was stable at pH7.2, and the accumulationrelease rate was12.1%after96h and16.9%after240h. While the conjugation canrelease doxorubicin quickly at pH4.0, and the accumulation release rate was81.3%after96h.3. FACS result also demonstrated that the MetFab-DOX could specifically recognizeand bind c-Met protein in live cells with various c-Met expression levels, butcouldn’t bind NIH3T3cells with c-Met negative expression. Immunofluorescencemicroscopy observation and ELISA analysis showed that the affinity ofMetFab-DOX to the c-Met expression HCC cells was smaller compared withMetFab. The entry and partition routes of MetFab-DOX were distinct from that offree DOX. The MetFab-DOX conjugate was clearly distributed in the membrane,cytoplasm and perinuclear zone after incubation, while free doxorubicin quicklylocated into the nulei.4. MetFab-DOX only specifically bound to HCC cell lines expressing c-Met and hadcytotoxic effect on these cells, but not to the NIH3T3cells that does not expressc-Met to a substantial level. 5. Experiments in vivo confirmed that MetFab-DOX exerted anti-tumor effectsimilarly as free DOX. The tumor inhibition rate was65.4%in MetFab-DOXgroup, but significantly reduced the side effects of free DOX(P<0.05), such as theweight loss, the pathological changes in heart, lung and kidney.6. Frozen tumor tissue sectioning showed that MetFab-DOX could bind the tumortissue after24h injection into mice bearing HCC xenograft. Optical tumor imagingin vivo demonstrated that MetFab could target the tumor tissue. After48h ofinjection, the fluorescent signal of MetFab-Cy5.5was still strong in the tumor site.Doxorubicin concentrations detected by spectrofluorometer in the tumor tissue andplasma were higher in mice given MetFab-DOX than in those given free DOX,while the condition was verse in the heart and kidney.ConclusionAs a conclusion, a human anti-Met Fab was screened and conjugated withdoxorubicin successfully. The MetFab-DOX had cytotoxity effect on HCC cells withc-Met expression in vitro. Furthermore, MetFab-DOX effectivly targeted HCC cellswith c-Met expression in mice bearing HCC xenograft, had similar anti-tumor effectas doxorubicin in vivo, changed the distribution of doxorubicin in vivo and reducedthe side effects of doxorubicin effectively. MetFab-DOX may have therapeuticpotential for targeted treatent of HCC.更多还原