【作者】 沈笛颖；

【导师】 汤永民；

【作者基本信息】 浙江大学 ， 儿科学， 2011， 博士

【摘要】 单克隆抗体(单抗)特异性高,因此已经广泛用于疾病的诊断和治疗,但是目前研制的单抗多为鼠源性的,在治疗中反复使用,作为异源蛋白在人体内可诱发产生人抗鼠免疫球蛋白抗体(HAMA),大大限制了它的临床应用。只有将其进行人源化的改造,才能有效应用于临床治疗。人鼠嵌合型抗体是指通过基因工程的技术和方法,将鼠源性单抗的可变区与人抗体恒定区拼接形成的抗体。目前较为成熟的做法就是将鼠单抗的可变区构建成单链抗体与人Ig的Fc段结合,相对于其它人源化抗体具有以下优点：技术路线简单,易于操作；鼠源抗体的亲和力和特异性得到了很好的保留；抗体的完整性好,在体内的潴留时间长。CD19分子是B淋巴细胞表面发挥特异性信号转导的受体,存在于B细胞成熟的各个阶段,在大多数的非霍奇金淋巴瘤(NHL)和许多白血病包括急性淋巴细胞白血病(ALL)和慢性淋巴细胞白血病(CLL)细胞上高表达,目前已成为免疫治疗的一个重要靶点。目前针对B系恶性肿瘤的治疗,已经有一些CD19特异性抗体治疗,包括非结合抗体,药物共轭抗体以及双特异性的抗体如CD19-CD3等,在体外实验、动物模型、以及早期的临床实验中取得了较为理想的结果。但是还远没能满足临床应用的需要,技术上也还存在不少问题,因此有必要研究更多的CD19抗体。ZCH-4-2E8(简称2E8),是本院自行研制的鼠源性抗CD19单抗,本课题在前期研究的基础上,对此单抗进行进一步的基因工程改造,构建可以表达人鼠嵌合型2E8抗体(Hm2E8b)的真核表达载体,转染至CHO细胞中表达,检测Hm2E8b的生物学活性,为该抗体免疫毒素的研制和临床应用以及进一步人源化改造打下基础。方法：1.ZCH-4-2E8轻、重链信号肽和可变区基因克隆和序列分析。1)抽提2E8细胞RNA,并以此为模板,通过5’-RACE方法扩增出2E8单抗轻链和重链的可变区片段,建立TA克隆,送测序。2)将测序结果与2E8轻链和重链的已知序列进行比对,并通过SignalP3.0信号肽预测服务软件对2E8轻链和重链的信号肽进行预测。2.真核表达载体pHMCH3-Hm2E8b的构建。1)以pAc-κ-CH3载体为模板,设计引物PCR扩增出Fc片段,建立TA克隆,测序正确后,连入pcDNA3.1+载体,构建pHMCH3载体。2)通过重叠延伸PCR的方法扩增出scFv2E8片段,建立TA克隆,送测序,连入pHMCH3载体,构建真核表达载体pHMCH3-Hm2E8b。3. Hm2E8b融合蛋白的表达和活性检测1)通过脂质体转染的方法,以pHMCH3-Hm2E8b转染CHO细胞,转染后24小时G418加压筛选。2)加压筛选14天左右,RT-PCR,细胞免疫荧光,流式细胞仪分析法检测阳性克隆。3)对阳性克隆进行单克隆化,流式细胞仪分析法筛选出高效稳定表达细胞株,命名为CHO-Hm2E8b。4)收集上清SPA Sepharose亲和层析法纯化嵌合抗体Hm2E8b, BCA法测定抗体浓度。5) SDS-PAGE和Western-Blot检测细胞培养上清中的重组蛋白Hm2E8b的表达,并确定分子量大小。6)滴定实验和竞争结合实验了解嵌合抗体Hm2E8b的亲和力。7)CDC实验检测嵌合抗体Hm2E8b能否激活补体杀伤靶细胞。结果：1.ZCH-4-2E8轻、重链信号肽和可变区基因克隆和序列分析：经SignalP3.0信号肽预测服务软件分析,2E8轻链信号肽全长60bp,编码20个氨基酸,2E8重链信号肽全长57bp,编码19个氨基酸。可变区序列与通用引物测序序列比较发现VL2E8序列有3处有差异,分别是3bp(T-C、18bp(G-A)、19bp(A-T), VH2E8有5处存在差异,分别是lbp (G-C)、4bp(G-A)、6bp(G-C)、7bp(A-C)、13bp(G-C)。2.真核表达载体pHMCH3-Hm2E8b的构建：通过PCR扩增人IgGl的Fc片段,将其插入真核表达载体pCDNA3.1+,构建pHMCH3载体。通过SOE-PCR扩增scFv2E8片段,将其插入pHMCH3载体,构建真核表达载体pHMCH3-Hm2E8b。3. Hm2E8b嵌合抗体的表达和功能的初步研究：以重组载体pHMCH3-Hm2E8b转染CHO细胞,G418加压培养2周获得能表达Hm2E8b抗体蛋白的CHO细胞。RT-PCR检测转染的CHO细胞cDNA可扩增出目的条带,细胞免疫荧光法检查可见转染的CHO细胞内含有目的蛋白。流式细胞仪分析法在转染CHO细胞培养上清中检测到有活性的人鼠嵌合型CD19抗体Hm2E8b,阳性细胞率为93.32%,且嵌合型抗体Hm2E8b对其亲本抗体2E8-FITC有明显的阻滞作用。对阳性细胞进行单克隆化,流式分析法筛选出高效稳定表达株CHO-Hm2E8b,该细胞株G418加压培养3-4周后阳性细胞数可达96%以上。SPA Sepharose亲和层析法纯化Hm2E8b抗体,BCA法测浓度浓缩纯化抗体为2mg/ml, CHO-Hm2E8b细胞培养上清浓度约为15-PAGE和Western-Blot鉴定Hm2E8b抗体单体分子量约50KDa,上清中还存在130KDa和200KDa大小的多聚体。CDC实验发现Hm2E8b能够靶向杀伤CD19阳性的细胞Nalm6,该作用存在剂量依赖性。结论：成功研制了有活性的抗人CD19人鼠嵌合型抗体Hm2E8b,该抗体能够通过CDC作用杀伤靶细胞。更多还原

【Abstract】 Immunotherapy using monoclonal antibodies is an effective and safe method for the treatment of human lymphoid maglignancies. CD19 is a 95kDa transmembrane glycoprotein, expressed on most of B-lineage acute lymphoblastic leukemia, chronic lymphocytic leukemia, and lymphomas. Several CD19-recoginizing antibodies have been evaluated for the treatment of B-lineage malignancies in vitro, in mouse models, and in clinical trials, including unconjugated antibodies, antibody-drug conjugated, and bispecific antibody targeting CD19 and CD3.2E8, a murine IgM-type anti-CD19 antibdoy, was generated in our laboratory previously. We have demonstrated that the 2E8 and antibody-norcantharidin conjugated immunotoxin (2E8-NCTD) could specifically target the CD19 expressing B-lineage leukemia cells. However, as 2E8 is a murine MAb, it is immunogenic and does not mediate effector function in human due to the murine origin of its constant region. In the present study, we have connected the VH and VL domain by a short peptide linker to form a sigle-chain Fv (scFv) antibody fragment, then fused to the Fc (hinge,CH2,CH3) domains of human IgG1. The results revealed that the scFv-Fc fusion protein retained the specific antigen-binding affinity of the parental antibody.Methods:1. Confirm the sequence of the ZCH-4-2E8 signal peptides and variable domains: The sequence of the ZCH-4-2E8 signal peptides and variable domains was amplified by 5’RACE; the gene was cloned to pGEM?-T-Easy vector to establish the 2E8-cDNA clone and sequenced. The sequences of the signal peptides were predicted by Signal 1P3.0 software.2. Construction of recombinant expression vector pHMCH3-Hm2E8:1) The Fc fragment of human IgGl was amplified from a baculovirus expression vector pAc-κ-CH3 by PCR and subcloned into pGEM?-T-Easy vector to generate TA-Fc. After sequencing, the Fc domain was subcloned into backbone vector pcDNA3.1(+)to generate the vector pHMCH3.2) The genes encoding the VH and VL of 2E8 were constructed in to scFv through splicing overlap extension and subcloned into pGEM?-T-Easy vector to generate TA-scFv2E8 After sequencing, the scFv2E8 domain was subcloned into pHMCH3 to generate the expression vector pHMCH3-Hm2E8b.3. Stable transfection of CHO cells by recombinant expression vector and expression of the chimeric antibody Hm2E8b:1) The recombinant vector was transfected to CHO cells by using lipofectamineTM 2000. Transfected cells were selected in the presence of G418 (600μg/ml).2) Two weeks after selection, the total RNA from the Hm2E8b gene transfected CHO cells (Hm2E8b-CHO) was extracted and analysed by RT PCR.3) Hm2E8b-CHO was incubated with fluorescein isothiocyanante-conjugated goat anti-human IgG Fc antibody (GAH-Fc-FITC) and observed under a muticolor fluorescence microscope.4) The supernatant of Hm2E8b-CHO was detected by flow cytometric analysis.5) The positive clone was isolated by limiting dilution, the highest-producing clone was chosen.6) The supernatant was harvested, and then purified by SPA Sepharose columns. The purified chimeric antibody Hm2E8b was identified by SDS-PAGE and Western-Blot.7) Study on the function of Hm2E8b antibody:The ability of Hm2E8b killing target cells was analyzed by complement dependent cytotoxicity (CDC). Results:1. Confirm the sequence of the ZCH-4-2E8 signal peptides and variable domains:The sequences of the signal peptides were predicted by Signa11P3.0 software. The signal peptides of light and heavy chains were 60bp and 57bp, encoding 20 and 19 amino acid residues, respectively. Compared with the sequences of the variable region genes of the 2E8 antibody obtained before, there were 3 (at 3bp(T-C),18bp(G-A) and 19bp(A-T)) and 5 (at lbp(G-C),4bp(G-A),6bp(G-C),7bp(A-C) and 13bp(G-C)) differences in positions exsisted in VL2E8 and VH2E8, respectively.2. Construction of recombinant expression vector pCDNA3.1 (+)-L2E8H-scFv2E8-Fc:The Fc domain and scFv2E8 were amplified successfully and subcloned into pCDNA3.1 (+)to generate eukaryogenic expression vector pHMCH3, then confirmed by DNA sequencing.3. Stable transfection of CHO cells by reeombinant expression vector and expression of the chimeric antibody Hm2E8b:The recombinant expression vector was transinfected into CHO cell and selected by G418 for two weeks. The band of the Hm2E8b gene was amplified from the CHO-Hm2E8b cells by RT-PCR. Under multicolor fluorescence microscope, the fluorescence could be clearly observed in the cytoplasm of CHO-Hm2E8b cells. The supernatant of Hm2E8b-CHO was detected by flow cytometric analysis. The positive rate of Hm2E8b on CD19+Nalm6 cells was 93.32%with mean fluorescence intensity (MFI) of 20.14%. Hm2E8b could block its parental murine antibody 2E8-FITC on Nalm-6 cells indicating that the binding activity and specificity of the engineered antibody Hm2E8b were not significantly changed. The supernatant was purified, and then identified by SDS-PAGE and Western-Blot analysis. The molecular weights of Hm2E8b monomer and dimmer were approximately 50KDa and 130KDa, and a 200KDa multiner antibody was identified simultaneously. Hm2E8b could kill the target cells through the effects of CDC Conclusions:The human-mouse chimeric antibody Hm2E8b was successfully expressed in CHO cells with excellent binding activities and the ability of killing the target cells, comparable with its parental antibody 2E8 of murine origin.更多还原