人鼠嵌合型抗人CD19抗體Hm2E8b的研制及功能研究
發(fā)布時(shí)間：2019-06-04 19:04
【摘要】：單克隆抗體(單抗)特異性高,因此已經(jīng)廣泛用于疾病的診斷和治療,但是目前研制的單抗多為鼠源性的,在治療中反復(fù)使用,作為異源蛋白在人體內(nèi)可誘發(fā)產(chǎn)生人抗鼠免疫球蛋白抗體(HAMA),大大限制了它的臨床應(yīng)用。只有將其進(jìn)行人源化的改造,才能有效應(yīng)用于臨床治療。人鼠嵌合型抗體是指通過(guò)基因工程的技術(shù)和方法,將鼠源性單抗的可變區(qū)與人抗體恒定區(qū)拼接形成的抗體。目前較為成熟的做法就是將鼠單抗的可變區(qū)構(gòu)建成單鏈抗體與人Ig的Fc段結(jié)合,相對(duì)于其它人源化抗體具有以下優(yōu)點(diǎn)：技術(shù)路線簡(jiǎn)單,易于操作；鼠源抗體的親和力和特異性得到了很好的保留；抗體的完整性好,在體內(nèi)的潴留時(shí)間長(zhǎng)。 CD19分子是B淋巴細(xì)胞表面發(fā)揮特異性信號(hào)轉(zhuǎn)導(dǎo)的受體,存在于B細(xì)胞成熟的各個(gè)階段,在大多數(shù)的非霍奇金淋巴瘤(NHL)和許多白血病包括急性淋巴細(xì)胞白血病(ALL)和慢性淋巴細(xì)胞白血病(CLL)細(xì)胞上高表達(dá),目前已成為免疫治療的一個(gè)重要靶點(diǎn)。目前針對(duì)B系惡性腫瘤的治療,已經(jīng)有一些CD19特異性抗體治療,包括非結(jié)合抗體,藥物共軛抗體以及雙特異性的抗體如CD19-CD3等,在體外實(shí)驗(yàn)、動(dòng)物模型、以及早期的臨床實(shí)驗(yàn)中取得了較為理想的結(jié)果。但是還遠(yuǎn)沒能滿足臨床應(yīng)用的需要,技術(shù)上也還存在不少問(wèn)題,因此有必要研究更多的CD19抗體。 ZCH-4-2E8(簡(jiǎn)稱2E8),是本院自行研制的鼠源性抗CD19單抗,本課題在前期研究的基礎(chǔ)上,對(duì)此單抗進(jìn)行進(jìn)一步的基因工程改造,構(gòu)建可以表達(dá)人鼠嵌合型2E8抗體(Hm2E8b)的真核表達(dá)載體,轉(zhuǎn)染至CHO細(xì)胞中表達(dá),檢測(cè)Hm2E8b的生物學(xué)活性,為該抗體免疫毒素的研制和臨床應(yīng)用以及進(jìn)一步人源化改造打下基礎(chǔ)。 方法： 1.ZCH-4-2E8輕、重鏈信號(hào)肽和可變區(qū)基因克隆和序列分析。 1)抽提2E8細(xì)胞RNA,并以此為模板,通過(guò)5'-RACE方法擴(kuò)增出2E8單抗輕鏈和重鏈的可變區(qū)片段,建立TA克隆,送測(cè)序。 2)將測(cè)序結(jié)果與2E8輕鏈和重鏈的已知序列進(jìn)行比對(duì),并通過(guò)SignalP3.0信號(hào)肽預(yù)測(cè)服務(wù)軟件對(duì)2E8輕鏈和重鏈的信號(hào)肽進(jìn)行預(yù)測(cè)。 2.真核表達(dá)載體pHMCH3-Hm2E8b的構(gòu)建。 1)以pAc-κ-CH3載體為模板,設(shè)計(jì)引物PCR擴(kuò)增出Fc片段,建立TA克隆,測(cè)序正確后,連入pcDNA3.1+載體,構(gòu)建pHMCH3載體。 2)通過(guò)重疊延伸PCR的方法擴(kuò)增出scFv2E8片段,建立TA克隆,送測(cè)序,連入pHMCH3載體,構(gòu)建真核表達(dá)載體pHMCH3-Hm2E8b。 3. Hm2E8b融合蛋白的表達(dá)和活性檢測(cè) 1)通過(guò)脂質(zhì)體轉(zhuǎn)染的方法,以pHMCH3-Hm2E8b轉(zhuǎn)染CHO細(xì)胞,轉(zhuǎn)染后24小時(shí)G418加壓篩選。 2)加壓篩選14天左右,RT-PCR,細(xì)胞免疫熒光,流式細(xì)胞儀分析法檢測(cè)陽(yáng)性克隆。 3)對(duì)陽(yáng)性克隆進(jìn)行單克隆化,流式細(xì)胞儀分析法篩選出高效穩(wěn)定表達(dá)細(xì)胞株,命名為CHO-Hm2E8b。 4)收集上清SPA Sepharose親和層析法純化嵌合抗體Hm2E8b, BCA法測(cè)定抗體濃度。 5) SDS-PAGE和Western-Blot檢測(cè)細(xì)胞培養(yǎng)上清中的重組蛋白Hm2E8b的表達(dá),并確定分子量大小。 6)滴定實(shí)驗(yàn)和競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)了解嵌合抗體Hm2E8b的親和力。 7)CDC實(shí)驗(yàn)檢測(cè)嵌合抗體Hm2E8b能否激活補(bǔ)體殺傷靶細(xì)胞。 結(jié)果： 1.ZCH-4-2E8輕、重鏈信號(hào)肽和可變區(qū)基因克隆和序列分析：經(jīng)SignalP3.0信號(hào)肽預(yù)測(cè)服務(wù)軟件分析,2E8輕鏈信號(hào)肽全長(zhǎng)60bp,編碼20個(gè)氨基酸,2E8重鏈信號(hào)肽全長(zhǎng)57bp,編碼19個(gè)氨基酸。可變區(qū)序列與通用引物測(cè)序序列比較發(fā)現(xiàn)VL2E8序列有3處有差異,分別是3bp(T-C、18bp(G-A)、19bp(A-T), VH2E8有5處存在差異,分別是lbp (G-C)、4bp(G-A)、6bp(G-C)、7bp(A-C)、13bp(G-C)。 2.真核表達(dá)載體pHMCH3-Hm2E8b的構(gòu)建：通過(guò)PCR擴(kuò)增人IgGl的Fc片段,將其插入真核表達(dá)載體pCDNA3.1+,構(gòu)建pHMCH3載體。通過(guò)SOE-PCR擴(kuò)增scFv2E8片段,將其插入pHMCH3載體,構(gòu)建真核表達(dá)載體pHMCH3-Hm2E8b。 3. Hm2E8b嵌合抗體的表達(dá)和功能的初步研究：以重組載體pHMCH3-Hm2E8b轉(zhuǎn)染CHO細(xì)胞,G418加壓培養(yǎng)2周獲得能表達(dá)Hm2E8b抗體蛋白的CHO細(xì)胞。RT-PCR檢測(cè)轉(zhuǎn)染的CHO細(xì)胞cDNA可擴(kuò)增出目的條帶,細(xì)胞免疫熒光法檢查可見轉(zhuǎn)染的CHO細(xì)胞內(nèi)含有目的蛋白。流式細(xì)胞儀分析法在轉(zhuǎn)染CHO細(xì)胞培養(yǎng)上清中檢測(cè)到有活性的人鼠嵌合型CD19抗體Hm2E8b,陽(yáng)性細(xì)胞率為93.32%,且嵌合型抗體Hm2E8b對(duì)其親本抗體2E8-FITC有明顯的阻滯作用。對(duì)陽(yáng)性細(xì)胞進(jìn)行單克隆化,流式分析法篩選出高效穩(wěn)定表達(dá)株CHO-Hm2E8b,該細(xì)胞株G418加壓培養(yǎng)3-4周后陽(yáng)性細(xì)胞數(shù)可達(dá)96%以上。SPA Sepharose親和層析法純化Hm2E8b抗體,BCA法測(cè)濃度濃縮純化抗體為2mg/ml, CHO-Hm2E8b細(xì)胞培養(yǎng)上清濃度約為15-PAGE和Western-Blot鑒定Hm2E8b抗體單體分子量約50KDa,上清中還存在130KDa和200KDa大小的多聚體。CDC實(shí)驗(yàn)發(fā)現(xiàn)Hm2E8b能夠靶向殺傷CD19陽(yáng)性的細(xì)胞Nalm6,該作用存在劑量依賴性。 結(jié)論： 成功研制了有活性的抗人CD19人鼠嵌合型抗體Hm2E8b,該抗體能夠通過(guò)CDC作用殺傷靶細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392
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