抗CD45人—鼠嵌合抗体的构建、表达与鉴定

发布时间：2018-07-25 06:59

【摘要】： 研究背景急性髓系白血病(AML)是一种常见的恶性血液系统疾病,虽然化放疗和造血干细胞移植技术的应用提高了患者的生存率,但仍有60%-70%遭受该疾病痛苦的病人死于AML。放化疗后的造血干细胞移植只是为许多耐药白血病类型病人提供了一种治愈的机会,尽管有许多研究团队已证明了这种治疗方法的有效、安全和可行性。研究证明针对白血病前体细胞和骨髓造血干细胞进行靶向放疗最安全的方法就是用放射物标记的抗体直接靶向白血病细胞或正常骨髓造血干细胞。用单克隆抗体治疗急性髓性白血病早已有所报道,常用的抗体靶向标志有CD33、CD66和CD45,而现行研究认为抗CD45的抗体是最具研发前景的。 CD45抗原是一种稳定、高表达于所有白细胞和他们的前体细胞以及超过70%的骨髓有核细胞表面上的单链细胞膜糖蛋白,CD45又被称为GP180、T200或白细胞共同抗原(LCA),是一种分子量约为200 kDa的酪氨酸磷酸酶,它稳定表达于除成熟红细胞和血小板外的所有造血细胞,且不存在于非造血组织中,使之成为治疗白血病的一个诱人的靶点。 CD45能广泛的表达于几乎所有的白细胞,包括骨髓的髓系前体细胞和淋巴结中的成熟淋巴细胞以及90%的AML细胞,这些细胞和组织为病人治疗的缓解或复发提供了大量抗体结合位点。由于CD45作为白细胞的标记物能出现在正常和恶性细胞,所以在病人体内应用抗CD45抗体可以通过代谢分布到骨髓、脾脏和淋巴结中与靶抗原结合,用于治疗急性白血病。在放射免疫治疗中,通常的做法是有选择地针对一个特定的抗原将放射性物质运输到肿瘤细胞。由于造血组织来源的细胞对放射线敏感和其上的靶抗原的高度可接近性,对血液系统肿瘤的放免治疗一直被认为是最成功的。在AML和MDS的放免治疗中,CD45抗原由于具有高表达和特异结合性而成为极具吸引力的靶向目标。放射性标记的抗CD45抗体可将辐射物质运输到抗原阳性细胞和周边环绕的抗原阴性细胞上。因此,放射免疫偶联物不需要以高浓度绑定到每一个白血病细胞上或渗入骨髓或肿瘤细胞中引发致死性的DNA损伤。在复发的白血病病人的放免治疗中,即使肿瘤细胞不表达CD45抗原,也会由于围绕其周围的高表达CD45抗原的非恶性造血细胞所介导的旁观者效应而被杀灭。在造血干细胞移植领域,CD45抗原的这些特性,能促进研究者针对它的放射性标记的系列抗体进行更加深入的研究,以便用于临床的治疗。随着CD45分子异构体的研究、流式细胞仪免疫分型的应用及抗CD45单抗的靶向治疗研究等深入发展,CD45分子在免疫学和血液学方面的应用研究受到进一步的关注。可以预期,CD45必将会在临床相关疾病的诊断、治疗和预后等方面发挥更大的作用。然而抗CD45单克隆抗体来源于小鼠,用于人体可诱导抗鼠抗体的产生。因此进行基因工程改造抗体具有重要实践意义。我们实验组已报道过了抗CD45单抗在AML治疗中良好前景,由于治疗中药物仍存在毒性使我们一直致力于不断的改进。在这篇文章中,我们首次报告关于抗CD45人-鼠嵌合抗体的构建,并描述了该基因工程抗体对血液系统疾病治疗的独特优势。为后续将该嵌合抗体和放射性物质如碘-131偶联,用于预定位放射免疫治疗自体和异体造血干细胞移植的进一步研究奠定了坚实的基础。人源化或嵌合单克隆抗体(MoAbs)能与造血细胞表达的抗原产生反应,他们可以直接(“亲本”或“裸”抗体)或通过与毒素、放射性同位素或抗肿瘤药物偶联而用于临床研发中。抗体治疗由于抗体小分子的特异性和亲和力在循环系统中容易与肿瘤细胞结合是急性的骨髓性白血病治疗的理想方案。根据其是否携带其他连接物分为未结合型单抗、抗癌药物结合型单抗及同位素结合型单抗。目前已有五个药物获美国FDA的批准用于临床,其中未结合型均为人-鼠嵌合抗体或人源化抗体。但单独使用未结合型CD45单抗的在人体内应用效果甚微和受到鼠源性的限制,目前国外关于CD45单抗的研究主要是与放射性核素131I及213Bi连接作为造血干细胞移植预处理方案的组成部分,以期减少TBI的剂量,从而减少TBI所导致的全身毒副作用。动物试验及Ⅰ/Ⅱ期临床试验均证实,CD45单抗能选择性的将放射性核素131I及213Bi导入骨髓、肝脾和淋巴结,可部分代替TBI参与预处理方案。Vallera等用90Y标记CD45单抗进行了淋巴瘤动物模型的治疗试验,结果显示,给予适当90Y-Anti-CD45剂量的所有小鼠,第6天肿瘤消失,观察到第135天无1例复发,肝、肾、小肠病理切片检查均未发现明显损害。我们已采用用90Y标记的CD45单抗进行急性白血病的治疗研究,前期实验中选择CDTPA螯合90Y与CD45单抗。CDTPA是一种经典的金属离子鳌合剂,早在1985年Hnatowich就用CDTPA螯合IgG与90Y,所得的产物比活度为1.7μCi/μg,放化纯度达90%以上,24小时的解离率为13%。我们的标记产物90Y-CDTPA-CD45单抗在CDTPA/IgG为20：1条件下,标记率为95%,放化纯度达99%以上；稳定性良好,24小时解离率为8.32%,与国内张锦明等报道的每天11.9%的解离率也相近。通过间接免疫荧光试验也证明,该螯合物的免疫活性没有受到核素的明显影响,与AML细胞的结合率较高,是一个较理想的靶向治疗制剂,为进行下一步的研究奠定了基础。鼠标源性全分子抗体由于具有高度免疫原性和巨大分子量在临床治疗应用局限很多,而且长期使用鼠单抗能诱发人抗鼠抗体(HAMA)的反应导致过敏性反应和损伤对人体健康造成危害。为了减少亲本鼠单抗的免疫原性,提高抗体在体内增强免疫机制的能力,我们设想改造其成为人-鼠嵌合的基因工程抗体。我们用人源抗体的恒定区去取代亲本鼠单抗的C区,由于嵌合抗体中的C区为人源性的,所以该人-鼠嵌合抗体在保留了鼠单抗特异性的同时,又降低了鼠抗体对人体的免疫原性；而且还具有比小鼠抗体更强的介导补体和细胞对靶抗原的杀伤和吞噬作用。此外,在构建嵌合抗体时,我们有目的地选择了抗体的类型,使之能更有效的发挥抗体的效用。我们将该单抗的可变区基因插入到含人恒定区的嵌合抗体专用表达载体pFUSE-CHIg和pFUSE2-CLIg,转染CHO细胞表达、纯化嵌合抗体。现在我们首次研究并报导这种新型抗CD45的鼠／人嵌合抗体(命名Chi-CD45),目前国内外尚无将CD45单抗进行人源化改造的相关报道。体外试验研究证明,抗体的竞争抑制试验结果随着单克隆抗体浓度的改变发生梯度变化。而补体介导的细胞杀伤作用发挥了嵌合抗体恒定区的功能,它能通过增加嵌合抗体的浓度而抑制.Jurkat/人PBMC增殖。本课题利用PCR技术扩增抗CD45单抗的轻链与重链基因,并插入pGEM-T载体中,将阳性克隆进行核苷酸序列分析,目的是获得正确地基因,为进一步研究奠定基础。目的构建抗CD45嵌合抗体的真核表达载体并实现在真核细胞中的表达。方法通过PCR技术分别扩增抗CD45单克隆抗体的轻链与重链可变区基因片段；利用DNAtools,IMGT/QUEST及EBI TOOLS:ClustalW2分析软件对轻链和重链基因分别进行同源性比较。将经过测序确认的基因序列分别构建入嵌合抗体的表达载体中转染CHO细胞,通过Western blotting检测嵌合抗体的表达。对该嵌合抗体进行分子建模,模拟其蛋白质二级、三级结构,并通过相关效应检测进一步验证该基因工程抗体四级结构的完整和功能的有效性。收集细胞培养上清后,通过Protein A亲和层析对表达产物进行纯化；通过FACS检测纯化后的嵌合抗体与靶细胞特异性结合的能力；与亲本单抗的竞争抑制活性；在补体存在条件下的嵌合抗体Fc段介导的CDC效应能力,以及嵌合抗体抑制靶细胞增殖的活力。结果PCR扩增出的VL、VH基因片段经1%琼脂糖凝胶电泳鉴定,其片段大小与理论值相符,DNA测序鉴定显示其序列正确。经分析软件鉴定功能性轻链结构域：从第16位碱基(ATG)开始,其后为57个bp的信号肽序列,编码19个氨基酸；可变区全长为333个碱基,抗CD45单抗有功能的轻链均属于IGκV1-117'01家族,V区匹配率为99.32%,J区为100%。重链结构域分析：从第16位碱基(ATG)开始,其后为57个bp的信号肽序列,编码19个氨基酸；可变区全长为348个碱基,功能性重链属于IGHV2-9-1'01家族。V区匹配率为96.84%,J区为87.23%。重组嵌合抗体的表达载体分别通过特异性引物经PCR扩增鉴定显示其产物片段大小与理论值相符,DNA测序鉴定显示其序列和阅读框正确。共转染CHO细胞经筛选后显示嵌合抗体表达量低,后续实验结果不理想。重组抗体产量低是制备基因工程抗体最大的障碍,高表达细胞株相对于整个转染细胞群是非常稀少的,在稳定培养时产量可形成梯度下降,过度生长的非表达细胞亦可使整个稳转细胞产量下降更明显,甚至形成非表达细胞群。大多数细胞系产生的IgG可合成更多的轻链基因,在对人鼠嵌合抗体杂交细胞系研究中,发现轻链分泌的比率和细胞内的轻链含量存在相关性,它合成的速度比重链要快几倍,而且需要的量更多。有文献报道分步转染的方法,可以使表达有功能的细胞增加5-30%,先用高浓度抗生素稳转轻链基因待该细胞稳定后再进一步转染重链基因。在后续培养过程适量减少培养基用量,延长转染细胞培养时间达7-10天以上均能相对提高抗体浓度,或选用DMSO,丁酸钠等在能抑制细胞增长同时,促进抗体分泌尤其对CHO细胞重组蛋白质有促进效果(产量提高约2倍)。参考相关文献后我们采用了分步转染的方法并用丁酸钠间隔刺激转染细胞,ELISA结果显示嵌合抗体产量有极大提高；分子模拟的各项检测指标显示了构建的抗CD45人-鼠嵌合抗体符合完整有功能的基因工程抗体,Insight II软件模拟了抗CD45抗体的Fab段结构域,该建模模型表明该抗体具有完整的抗原结合凹槽能稳定的发挥抗体的相关功能。转染瘤细胞经检测证实表达成功以后,部分转染瘤细胞放入液氮中冻存,5个月后取出冻存的细胞复苏培养,先后经过一次常规有限稀释克隆和多次ELISA方法鉴定,证明该转染瘤细胞经过液氮冻存及多次传代,仍能够保持良好的体外生长状态。 Western blotting结果显示培养3天的转染瘤细胞分泌的上清中有嵌合抗体表达。收集培养的上清经Protein A亲和层析纯化后,SDS-PAGE鉴定显示在纯化产物中仅见嵌合抗体重链和轻链条带,其分子量大小与理论值相符。FCM检测显示纯化后的嵌合抗体分子的V区能够与多株肿瘤细胞及人PBMC细胞表面CD45抗原特异性的结合,且其结合能力与相同浓度的鼠源性亲本抗体相似。与亲本单抗的竞争抑制作用随着嵌合抗体的浓度增加而梯度增加；嵌合抗体所介导的CDC效应及抑制肿瘤细胞增殖的能力均随该抗体浓度的增加而增强。结论成功地构建了表达抗CD45人-鼠嵌合抗体真核表达载体,并获得了具有生物学活性的嵌合抗体分子。为后续研究该嵌合抗体对血液系统疾病的治疗提供了坚实的基础。
[Abstract]:Background acute myeloid leukemia (AML) is a common malignant blood system disease. Although the application of chemoradiotherapy and hematopoietic stem cell transplantation improves the survival rate of patients, there is still a hematopoietic stem cell transplant after AML. radiotherapy and chemotherapy for patients with 60%-70% suffering from the disease only for a number of patients with drug-resistant leukemia. It provides a chance to cure, although many research teams have demonstrated the effectiveness, safety and feasibility of this treatment. The safest way to target radiotherapy for leukemic precursor cells and bone marrow hematopoietic stem cells is to target leukemia cells or normal bone marrow hematopoiesis with a radiated antibody. Stem cells. The treatment of acute myeloid leukemia with monoclonal antibodies has long been reported. The commonly used antibody targeting markers are CD33, CD66 and CD45, and the current research suggests that anti CD45 antibodies are the most promising.
CD45 antigen is a stable, highly expressed single strand cell membrane glycoprotein on the surface of all leukocytes and their precursors and more than 70% of the bone marrow nucleated cells. CD45 is also known as GP180, T200, or leukocyte common antigen (LCA), a tyrosine phosphatase with a molecular weight of about 200 kDa, which is stably expressed in the removal of mature red cells. All hematopoietic cells outside the platelets do not exist in non hematopoietic tissues, making them an attractive target for the treatment of leukemia.
CD45 can be widely expressed in almost all leukocytes, including myeloid precursor cells in bone marrow and mature lymphocytes in lymph nodes, and 90% AML cells. These cells and tissues provide a large number of antibody binding sites for the relief or recurrence of treatment. As CD45 as a marker for leukocyte, it can appear in normal and malignant cells. Therefore, the use of anti CD45 antibodies in patients can be combined with target antigens through metabolic distribution to bone marrow, spleen and lymph nodes for the treatment of acute leukemia.
In radioimmunotherapy, the usual practice is to selectively transport radioactive substances to a tumor cell for a specific antigen. Because the cells of the hematopoietic tissue are sensitive to the radiating line and the target antigen is highly approachable, the treatment of hematological cancer is always considered the most successful. In AML and MDS In radioimmunoassay, CD45 antigen is a highly attractive target target because of its high expression and specificity. Radioactivity labeled anti CD45 antibodies can transport radiant substances to antigen positive cells and surrounding surrounding antigen negative cells. Therefore, radioimmunoconjugate does not need to bind to every leukemia at high concentration. Lethal DNA damage in or infiltrating into bone marrow or tumor cells. In the radioimmunoassay of recurrent leukemia patients, even if the tumor cells do not express CD45 antigen, they will be killed by the bystander response mediated by non malignant hematopoietic cells surrounding the high expression of CD45 antigen. In the field of hematopoietic stem cell transplantation, C These characteristics of D45 antigens can facilitate researchers to further study their radiolabeled antibodies in order to be used for clinical treatment.
With the research of the CD45 isomer, the application of the immunophenotyping of flow cytometer and the research on the targeting therapy of anti CD45 monoclonal antibody, the application of CD45 in immunology and hematology has been paid more attention. It is expected that CD45 will play a greater role in the diagnosis, treatment and prognosis of clinical related diseases. However, anti CD45 monoclonal antibodies are derived from mice and are used to induce the production of anti mouse antibodies in the human body. Therefore, it is of great practical significance to carry out genetically engineered antibodies. Our experimental group has reported that the anti CD45 monoclonal antibody has a good prospect in the treatment of AML, and we have been committed to the constant toxicity of drugs in the treatment. Improvement.
In this article, we first report on the construction of anti CD45 human mouse chimeric antibody and describe the unique advantage of this gene engineering antibody in the treatment of blood system diseases. The research laid a solid foundation.
Human or chimeric monoclonal antibodies (MoAbs) can react with antigens expressed in hematopoietic cells. They can be used directly ("parent" or "naked" antibody) or in clinical research and development by coupling with toxins, radioisotopes or antitumor drugs. Antibody therapy is in the circulatory system due to the specificity and affinity of anti body small molecules. Combining with tumor cells is an ideal solution for the treatment of acute myeloid leukemia. According to whether or not it carries other connections, it is divided into uncombined monoclonal antibody, anticancer drug binding monoclonal antibody and isotope binding monoclonal antibody. Currently, five drugs have been approved by the FDA in the United States for clinical use, of which unconjugated type is human mouse chimeric antibody or Human derived antibody. But the use of unbound CD45 monoclonal antibody in human body is very small and is limited by mouse origin. The current research on CD45 mAb is mainly composed of the connection of radionuclide 131I and 213Bi as a preconditioning scheme for hematopoietic stem cell transplantation, in order to reduce the dose of TBI and thus reduce TBI Both animal tests and phase I / II clinical trials have confirmed that CD45 mAb can selectively transfer radionuclide 131I and 213Bi into the bone marrow, liver and spleen and lymph nodes, and can partially replace TBI in the pre treatment.Vallera with 90Y labeled CD45 McAbs for the treatment of lymphoid tumor animal models, and the results show that it is given. All mice with appropriate 90Y-Anti-CD45 dose disappeared in sixth days. No 1 cases of recurrence were observed on the 135th day. No obvious damage was found in the liver, kidney, and small intestine pathological sections. We have used the 90Y labeled CD45 monoclonal antibody for the treatment of acute leukemia. In the earlier experiment, the selection of CDTPA chelating 90Y and CD45 McAb.CDTPA is a classic. Metal chelating agent, as early as in 1985, Hnatowich was chelated with CDTPA and IgG and 90Y, the specific activity of the product was 1.7 mu Ci/ mu g, the radiochemical purity was above 90%, and the 24 hour dissociation rate was 13%.. The labeling product 90Y-CDTPA-CD45 McAb was 95% and the radiochemical purity was more than 99% under the CDTPA/IgG 20:1 condition, and the stability was good and 24 was small. The dissociation rate was 8.32%, which was similar to the 11.9% dissociation rate per day reported by Zhang Jinming in China. Indirect immunofluorescence test also proved that the immune activity of the chelate was not obviously affected by the nuclide, and the binding rate with AML cells was higher. It was an ideal target treatment preparation, which laid the foundation for the next step of research.
The mouse derived total molecular antibody is limited in clinical treatment because of its high immunogenicity and large molecular weight, and the response to human anti mouse antibody (HAMA) induced by rat mAb induces hypersensitivity and damage to human health. In order to reduce the immunogenicity of the monoclonal antibody to the parent, improve the antibody in the body. In order to enhance the ability of the immune mechanism, we conceive to transform it into a human mouse chimeric gene engineering antibody. We use the constant region of the human antibody to replace the C region of the parent mouse mAb. Because the C region of the chimeric antibody is humanized, the human mouse chimeric antibody retains the specificity of the mouse monoclonal antibody while reducing the mouse antibody against the human body. The immunogenicity and the killing and phagocytosis of the mediated complement and cell against the target antigen are also stronger than that of the mice. In addition, in the construction of chimeric antibodies, we have selected the type of antibody to make it more effective to use the antibody. We insert the variable region gene of the monopulse into the constant area. The antibody specific expression vector pFUSE-CHIg and pFUSE2-CLIg were transfected into CHO cells to express and purify chimeric antibody.
For the first time, we have studied and reported this new anti CD45 mouse / human chimeric antibody (named Chi-CD45). At present, there is no related report on the human transformation of CD45 McAbs at home and abroad. In vitro studies have shown that the results of the competitive inhibition test of antibodies change with the change of the concentration of monoclonal antibodies. Cytotoxicity plays a role in the constant region of chimeric antibody. It can inhibit the proliferation of.Jurkat/ human PBMC by increasing the concentration of chimeric antibody.
In this study, the PCR technique was used to amplify the light chain and heavy chain genes of anti CD45 monoclonal antibodies and inserted into the pGEM-T vector to analyze the positive clones for nucleotide sequences. The aim is to obtain the correct genes and lay the foundation for further research.
Objective to construct a eukaryotic expression vector against CD45 chimeric antibody and to express it in eukaryotic cells.
Methods the light chain and heavy chain variable region gene fragment of anti CD45 monoclonal antibody was amplified by PCR technique, and the homology of light chain and heavy chain gene was compared with DNAtools, IMGT/QUEST and EBI TOOLS:ClustalW2 analysis software. The gene sequence confirmed by sequencing was constructed into the expression vector of chimeric antibody and transfected to CHO respectively. Cell, the expression of chimeric antibody was detected by Western blotting. Molecular modeling of the chimeric antibody was used to simulate the protein two and three structure, and the integrity and function of the four stage structure of the gene engineering antibody were verified by correlation effect detection. After collecting cell culture supernatant, Protein A affinity chromatography was used to table the table. The product was purified; the ability to detect the specific binding of the purified chimeric antibody to the target cells by FACS, the competitive inhibition activity with the parent monoclonal antibody, the CDC effect mediated by the chimeric antibody Fc segment under the presence of complement, and the activity of the chimeric antibody to inhibit the proliferation of the target cells.
Results the VL, VH gene fragment amplified by PCR was identified by 1% agarose gel electrophoresis, and the size of the fragment was in accordance with the theoretical value. The sequence of DNA sequencing showed that the sequence was correct. The functional light chain domain was identified by the analysis software: from the sixteenth base base (ATG), the sequence of the 57 BP peptide and the 19 amino acids were encoded, and the variable region length was 333. The light chain of anti CD45 monoclonal antibody belongs to the IG kappa V1-117'01 family. The matching rate of V region is 99.32%, and the J area is 100%..
Heavy chain domain analysis: starting from sixteenth base (ATG), followed by 57 BP signal peptide sequences, encoding 19 amino acids, variable region full length of 348 bases, functional heavy chains belonging to the IGHV2-9-1'01 family.V region matching rate of 96.84%, J region 87.23%.
The expression vector of recombinant chimeric antibody was identified by PCR amplification by specific primers. The fragment size of the product was consistent with the theoretical value. DNA sequencing showed that the sequence and reading frame were correct. After screening, the co transfected CHO cells showed that the expression of chimeric antibody was low and the subsequent experimental results were not ideal. The low yield of recombinant antibody was a preparation gene. The highest expression of engineering antibody is that the high expression cell line is very rare compared with the whole transfected cell group. The yield can form a gradient in stable culture, and the overgrowth of non expressed cells can also decrease the output of the whole cell, even form a non expressed cell group.
The IgG produced by most cell lines can synthesize more light chain genes. In the study of human chimeric antibody hybrid cell lines, it is found that the ratio of light chain secretion is correlated with the light chain content in cells. It synthesizes a few times faster and needs more. The functional cells increase 5-30%, and then use high concentration antibiotics to stabilize the light chain gene and then further transfect the heavy chain gene after the cell is stable. In the subsequent culture, a proper amount of the medium is reduced and the cell culture time is more than 7-10 days, or DMSO, or sodium butyrate can be used to inhibit the cell growth. At the same time, it promotes the secretion of antibodies, especially for CHO cell recombinant protein (the yield is increased by about 2 times).
Reference correlation
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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