人鼠嵌合型抗人CD19抗体Hm2E8b的研制及功能研究
发布时间：2019-06-04 19:04
【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。
[Abstract]:The monoclonal antibody (monoclonal antibody) is highly specific and has been widely used for the diagnosis and treatment of the disease, but the currently developed monoclonal antibody is mouse-derived and is repeatedly used in the treatment, and can induce the human anti-mouse immunoglobulin antibody (HAMA) in the human body as a heterologous protein, And the clinical application thereof is greatly limited. The effect can be used for clinical treatment only if it is modified to humanize. The human-mouse chimeric antibody refers to an antibody which is formed by splicing the variable region of the mouse-derived monoclonal antibody and the constant region of the human antibody through the technology and the method of gene engineering. The mature method is to construct the variable region of the mouse monoclonal antibody into a single-chain antibody and the Fc section of the human Ig, and has the advantages that the technical route is simple, the operation is easy, and the affinity and specificity of the mouse source antibody are well preserved; The integrity of the antibody is good, and the retention time in the body is long. The CD19 molecule is a receptor for specific signal transduction on the surface of B-lymphocytes, and is present in the mature of B-cells. The high expression of the majority of non-Hodgkin's lymphoma (NHL) and many of the leukemia, including acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), has now become an important part of immunotherapy For the treatment of B-line malignancies, there have been some CD19-specific antibody therapies, including non-binding antibodies, drug co-antigen antibodies, and bispecific antibodies such as CD19-CD3 and the like, and are ideal in in vitro experiments, animal models, and early clinical trials. As a result, the need for clinical application is still far from being met, and there are many problems in the technology, so it is necessary to study more CD19 The antibody. ZCH-4-2E8 (2E8) is a rat-derived anti-CD19 monoclonal antibody developed by our hospital. Expression in the cell, the detection of the biological activity of Hm2E8b, the development and clinical application of the antibody immunotoxin, and further humanized modification make Basic. Method: 1.ZCH-4-2E8 light, heavy chain signal peptide and can Variable region gene cloning and sequence analysis.1) Extract the 2E8 cell RNA and use this as a template to amplify the variable light chain and heavy chain variable of the 2E8 monoclonal antibody through the 5 '-RACE method. the sequence results are compared with the known sequences of the 2E8 light chain and the heavy chain and the service software is predicted by the SignalP3.0 signal peptide the signal peptide of the 2E8 light chain and the heavy chain is predicted.2. Eukaryotic The expression vector pHCMCH3-Hm2E8b was constructed.1) Using the pAc-1-CH3 vector as the template, the primer PCR was designed to amplify the Fc fragment, the TA clone was established, and the sequence was correct. (2) amplifying the scFv2E8 fragment by the method of overlapping extension PCR, establishing a TA clone, carrying out sequencing, and connecting to the pHMC; H3 vector to construct the eukaryotic expression vector pHCMCH3- Hm2E8b.3. Hm2E8b fusion protein expression and activity detection 1) by liposome transfection, pHCMC H3-Hm2E8b transfected CHO cells, followed by 24-hour G418 pressure screening after transfection. The positive clones were detected by RT-PCR, cell immunofluorescence and flow cytometry (FCM) for 14 days. The high-efficiency and stable expression cell line (CHO-Hm2E8b.4) was collected by flow cytometry. Purification of the chimeric antibody Hm2E8b by the SPA Sepharose affinity chromatography, and the concentration of the antibody was determined by the BCA method. The recombinant protein Hm2E in the culture supernatant of cell culture was detected by Western-Blot 8b expression and determination of molecular weight size.6) titration experiment and competitive binding assay to learn about The affinity of the antibody Hm2E8b.7) The CDC assay detects whether the chimeric antibody Hm2E8b can activate the complement-killing target cell. Results: 1.ZCH-4-2E8 light, heavy chain signal peptide and variable region gene clone and sequence analysis: Predicted service software analysis, the full length of the 2E8 light chain signal peptide is 60 bp, encodes 20 amino acids, the full length of the 2E8 heavy chain signal peptide is 57 bp, and encodes 19 amino acids. The sequence of the variable region and the universal primer sequencing sequence shows that there are three differences in the VL2E8 sequence, which are 3bp (T-C, 18bp (G-A), 19bp (A-T), VH2E8, respectively. There were 5 bp (G-C),4 bp (G-A),6 bp (G-C),7 bp (A-C),13 bp (G-C).2. Eukaryotic expression vector pHMCH3. Construction of Hm2E8b: The Fc fragment of human IgGl was amplified by PCR and inserted into the eukaryotic expression vector pCDNA3.1 + to construct the pHMCH And 3, amplifying the scFv2E8 fragment through SOE-PCR, inserting the scFv2E8 fragment into the pHMCH3 vector, and constructing the expression and function of the eukaryotic expression vector pHCMCH3-Hm2E8b.3. Hm2E8b chimeric antibody. Preliminary study: The recombinant vector pHCMCH3-Hm2E8b was transfected into CHO cells and G418 was cultured for 2 weeks to obtain the Hm2E8b antibody. CHO cells were detected by RT-PCR. The target protein was amplified by RT-PCR. The target protein was found in the transfected CHO cells by means of immunofluorescence. The positive cell rate of Hm2E8b was 93.32%, and the chimeric antibody Hm2E8b had a significant blocking effect on its parent antibody 2E8-FITC. The high-efficiency stable expression strain CHO-Hm2E8b was screened by a flow-flow analysis method. The cell line G418 was cultured for 3-4 weeks and the number of positive cells can reach more than 96%. The Hm2E8b antibody was purified by the SPA Sepharose affinity chromatography, and the concentration of the purified antibody was 2 mg/ ml. The concentration of the culture supernatant of CHO-Hm2E8b was about 15-PA. The molecular weight of the Hm2E8b antibody was determined by GE and Western-Blot, with a molecular weight of about 50 KDa and 13 in the supernatant. 0KD A and 200 KDa size multimers. The CDC experiment found that Hm2E8b was able to target the killer CD19-positive cells, Nalm.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
本文编号：2492936
[Abstract]:The monoclonal antibody (monoclonal antibody) is highly specific and has been widely used for the diagnosis and treatment of the disease, but the currently developed monoclonal antibody is mouse-derived and is repeatedly used in the treatment, and can induce the human anti-mouse immunoglobulin antibody (HAMA) in the human body as a heterologous protein, And the clinical application thereof is greatly limited. The effect can be used for clinical treatment only if it is modified to humanize. The human-mouse chimeric antibody refers to an antibody which is formed by splicing the variable region of the mouse-derived monoclonal antibody and the constant region of the human antibody through the technology and the method of gene engineering. The mature method is to construct the variable region of the mouse monoclonal antibody into a single-chain antibody and the Fc section of the human Ig, and has the advantages that the technical route is simple, the operation is easy, and the affinity and specificity of the mouse source antibody are well preserved; The integrity of the antibody is good, and the retention time in the body is long. The CD19 molecule is a receptor for specific signal transduction on the surface of B-lymphocytes, and is present in the mature of B-cells. The high expression of the majority of non-Hodgkin's lymphoma (NHL) and many of the leukemia, including acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), has now become an important part of immunotherapy For the treatment of B-line malignancies, there have been some CD19-specific antibody therapies, including non-binding antibodies, drug co-antigen antibodies, and bispecific antibodies such as CD19-CD3 and the like, and are ideal in in vitro experiments, animal models, and early clinical trials. As a result, the need for clinical application is still far from being met, and there are many problems in the technology, so it is necessary to study more CD19 The antibody. ZCH-4-2E8 (2E8) is a rat-derived anti-CD19 monoclonal antibody developed by our hospital. Expression in the cell, the detection of the biological activity of Hm2E8b, the development and clinical application of the antibody immunotoxin, and further humanized modification make Basic. Method: 1.ZCH-4-2E8 light, heavy chain signal peptide and can Variable region gene cloning and sequence analysis.1) Extract the 2E8 cell RNA and use this as a template to amplify the variable light chain and heavy chain variable of the 2E8 monoclonal antibody through the 5 '-RACE method. the sequence results are compared with the known sequences of the 2E8 light chain and the heavy chain and the service software is predicted by the SignalP3.0 signal peptide the signal peptide of the 2E8 light chain and the heavy chain is predicted.2. Eukaryotic The expression vector pHCMCH3-Hm2E8b was constructed.1) Using the pAc-1-CH3 vector as the template, the primer PCR was designed to amplify the Fc fragment, the TA clone was established, and the sequence was correct. (2) amplifying the scFv2E8 fragment by the method of overlapping extension PCR, establishing a TA clone, carrying out sequencing, and connecting to the pHMC; H3 vector to construct the eukaryotic expression vector pHCMCH3- Hm2E8b.3. Hm2E8b fusion protein expression and activity detection 1) by liposome transfection, pHCMC H3-Hm2E8b transfected CHO cells, followed by 24-hour G418 pressure screening after transfection. The positive clones were detected by RT-PCR, cell immunofluorescence and flow cytometry (FCM) for 14 days. The high-efficiency and stable expression cell line (CHO-Hm2E8b.4) was collected by flow cytometry. Purification of the chimeric antibody Hm2E8b by the SPA Sepharose affinity chromatography, and the concentration of the antibody was determined by the BCA method. The recombinant protein Hm2E in the culture supernatant of cell culture was detected by Western-Blot 8b expression and determination of molecular weight size.6) titration experiment and competitive binding assay to learn about The affinity of the antibody Hm2E8b.7) The CDC assay detects whether the chimeric antibody Hm2E8b can activate the complement-killing target cell. Results: 1.ZCH-4-2E8 light, heavy chain signal peptide and variable region gene clone and sequence analysis: Predicted service software analysis, the full length of the 2E8 light chain signal peptide is 60 bp, encodes 20 amino acids, the full length of the 2E8 heavy chain signal peptide is 57 bp, and encodes 19 amino acids. The sequence of the variable region and the universal primer sequencing sequence shows that there are three differences in the VL2E8 sequence, which are 3bp (T-C, 18bp (G-A), 19bp (A-T), VH2E8, respectively. There were 5 bp (G-C),4 bp (G-A),6 bp (G-C),7 bp (A-C),13 bp (G-C).2. Eukaryotic expression vector pHMCH3. Construction of Hm2E8b: The Fc fragment of human IgGl was amplified by PCR and inserted into the eukaryotic expression vector pCDNA3.1 + to construct the pHMCH And 3, amplifying the scFv2E8 fragment through SOE-PCR, inserting the scFv2E8 fragment into the pHMCH3 vector, and constructing the expression and function of the eukaryotic expression vector pHCMCH3-Hm2E8b.3. Hm2E8b chimeric antibody. Preliminary study: The recombinant vector pHCMCH3-Hm2E8b was transfected into CHO cells and G418 was cultured for 2 weeks to obtain the Hm2E8b antibody. CHO cells were detected by RT-PCR. The target protein was amplified by RT-PCR. The target protein was found in the transfected CHO cells by means of immunofluorescence. The positive cell rate of Hm2E8b was 93.32%, and the chimeric antibody Hm2E8b had a significant blocking effect on its parent antibody 2E8-FITC. The high-efficiency stable expression strain CHO-Hm2E8b was screened by a flow-flow analysis method. The cell line G418 was cultured for 3-4 weeks and the number of positive cells can reach more than 96%. The Hm2E8b antibody was purified by the SPA Sepharose affinity chromatography, and the concentration of the purified antibody was 2 mg/ ml. The concentration of the culture supernatant of CHO-Hm2E8b was about 15-PA. The molecular weight of the Hm2E8b antibody was determined by GE and Western-Blot, with a molecular weight of about 50 KDa and 13 in the supernatant. 0KD A and 200 KDa size multimers. The CDC experiment found that Hm2E8b was able to target the killer CD19-positive cells, Nalm.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
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