抗CD45人—鼠嵌合抗体的构建、表达与鉴定
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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。
Background
AML kills 60% to 70% of affected adults despite improvements in induction chemotherapy and HCT. High-dose chemoradiotherapy followed by stem cell transplantation offers the only chance of cure for many patients with treatment-resistant leukemia. Several groups have documented the feasibility, safety, and efficacy of treating AML. Therefore, targeted radiation to leukemic progenitor and stem cells residing in hematopoietic bone marrow was the safest resolution to use radiolabeled antibodies directed to epitopes expressed on leukemic cells or normal hematopoietic bone marrow cells.For treatment of acute myeloid leukemia, monoclonal antibodies have been directed to CD33,CD66, and CD45, and studies using anti-CD45 have shown the most promising results.
CD45 antigen is stably expressed at a high density on the surface of all leukocytes, their precursors and more than 70% of nucleated cells in normal bone marrow, with an average copy number of approximately 200,000 molecules per cell. CD45 (also called GP180, T200, or leukocyte common antigen) is a～200-kDa tyrosine phosphatase that is stably expressed at a high density on the surface of virtually all hematopoietic cells except mature erythrocytes and platelets. At least 90% of myeloid leukemias express CD45 and the antigen is not found on tissues of nonhematopoietic origin, making CD45 an attractive target for treatment of leukemia.
The abundant expression of CD45 on virtually all leukocytes, including myeloid precursors in bone marrow and mature lymphocytes in lymph nodes as well as more than 90% of AML samples, provides a high number of Ab binding sites in these tissues for patients in remission or relapse. It is expressed by both normal and malignant cells, and therefore anti-CD45 antibodies can deliver to marrow, spleen, and lymph nodes in patients with acute leukemia.
In radioimmunotherapy, a specific antigen is targeted to selectively deliver radioactivity to cancer cells. Radioimmunotherapy of blood-borne cancers has been the most successful because of high accessibility of the target antigen expressed on inherently radiosensitive cells. The CD45 antigen acts as an attractive alternative target for radioimmunotherapy of AML and MDS.Radiolabeled anti-CD45 Ab can delivery radiation to antigen-positive or surrounding antigen-negative cell in myeloid tissues. Therefore, the radioimmunoconjugate does not need to bind to every leukemia cell at a high density or penetrate homogeneously into bone marrow or tumor cell masses to induce lethal DNA damage. For patients in remission, even blasts that do not express CD45 may be killed if they are surrounded predominantly by nonmalignant hematopoietic cells, because of the bystander effect. The characteristics of the CD45 antigen have prompted investigations of radiolabeled antibodies against CD45 with transplantation therapy. With the CD45 molecular isomer research, the development of flow cytometry immunophenotyping and anti-CD45 monoclonal antibody application,CD45 molecules in the application of immunology and hematology research would get further attention. We can expecte, CD45 could play greater role in clinical diagnosis, treatment, prognosis. Our group has documented the promise of anti-CD45 monoclonal antibodies (Ab) administered in the setting of allogeneic HCT for AML, but toxicity remains high. So we have been committed to continuous improvement.
In this article, we report in the first time the construction of anti-CD45 mouse/human chimeric antibody, and describe its merits on the blood system diseases. To lay solid foundation for further study on this chimera label with 131I on the pretargeted radioimmunotherapy (PRIT) to use autologous and allogeneic haematopoietic stem cell transplantation.
Humanized or chimeric monoclonal antibodies (MoAbs) have been developed that react with antigens expressed by hematopoietic cells. They have been used either unlabeled('native' or 'naked'antibodies) or conjugated with toxins, radioisotopes, or antitumor drugs. According to whether they carry the material into other connections not bound monoclonal antibody, anti-cancer drug conjugated monoclonal antibody and isotope conjugated monoclonal antibody. So far, five drugs by the U.S.FDA approval for clinical, which are no-binding human-mouse chimeric antibody or humanized antibody. But use no-binding CD45 monoclonal antibody had little effect in the human body and with the limitation of murine application, the current research on the CD45 monoclonal antibodies mainly on antibodies connect with radionuclides 131I and 213Bi as part of hematopoietic stem cell transplantation conditioning to reduce the dose of TBI and its side effects. Animal testing andⅠ/Ⅱclinical trials have confirmed, CD45 monoclonal antibody can be selectively deliver radionuclides 213Bi and 131I into bone marrow, liver, spleen and lymph nodes. Vallera et al. used 90Y labeled anti-CD45 monoclonal antibodies treat for experimental animal models of lymphoma, the results showed that give appropriate doses of 90Y-Anti-CD45 to mice, tumors disappeared after 6 days, and without one case happen to recurrence until 135 days. Liver, kidney, small intestine biopsy found no apparent damage. We have used anti-CD45 monoclonal antibody labeled with 90Y for the treatment of acute leukemia, preliminary experiments to select CDTPA to chelate 90Y and anti-CD45 monoclonal antibody. CDTPA is a classic metal ion chelating agent, as early as 1985,Hnatowich took CDTPA chelae IgG and 90Y, we obtained specific activity was 1.7μCi/μg, the radiochemical purity more than 90%,24 hours dissociation was 13%.The products 90Y-CDTPA-CD45 monoclonal antibody under the conditions of 20:1 CDTPA/IgG, the labeling efficiency was 95%, the radiochemical purity was more than 99%; good stability,24-hour dissociation rate was 8.32%. These results in accordance with Zhang Jinming et al.reported that the dissociation rate was 11.9%. Indirect immunofluorescence assay also proved that the immunological activity of the chelates have not been obviously affected by the combination with the high rate of nuclides. The binging rate with AML cells was good. It is a better targeted therapeutic agents and laying the basis for the further study. Antibody therapy is ideally suited to the treatment of acute myeloid leukemia because of the ready accessibility of neoplastic cells in the circulation.
But mouse-derived whole-molecule antibodies have been demonstrated to have many limitations for therapeutic applications due to high immunogenicity and large molecular weight, and human anti-mouse antibody (HAMA) responses that can cause allergic reaction and damage in the human body.In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effect or mechanism in vivo, we herein developed its chimera. Genetically engineered antibody could have more important practical significance. To remove these difficulties, humanized antibody with a human antibody constant region was genetically engineered. Humanized human-mouse chimeric antibody has employed the constant region of human antibody to replac C region of murine monoclonal antibody. Human-mouse chimeric antibody not only retains the specificity of mouse monoclonal antibody but also decreases the immunogenicity. This genetically engineered antibody has more advantage than the mouse antibody in the function of complement mediated cell killing of target antigens and phagocytic effect. In addition, with construction of chimeric antibodies, we purposely chose the type of antibody, making it more effective to play the effectiveness of antibodies. Therefore, the variable region genes of this MoAb were cloned and ligated into chimeric antibody expression vector pFUSE-CHIg-hGl and pFUSE2-CLIg-hk, generating chimeric anti-human CD45 MAb (chi-mAb CD45)expression vectors,transfected CHO cells,expressed and purified chimeric antibody.
Now we first generated and report a novel anti-CD45 mouse/human chimeric antibody (Chi-CD45).Untill now, it is no reported about reconstruction of humanized anti-CD45 monoclonal antibody at home and abroad. In vitro assay, it revealed that pro-competitive inhibition with the monoclonal antibody showed the change by gradient. The effects of CDC mediated by chimeric antibody and ability to inhibit Jurkat/human PBMC proliferation were enhanced with the increase of the concentrations of chimeric antibody.
Aim To construct the eukaryotic expression vector of chimeric anti-CD45 mouse/human chimeric antibody and realize its expression.
Methods VH gene and VL gene of anti-CD45 monoclonal antibody were amplified by PCR. Use DNAtools, IMGT/QUEST and EBI TOOLS:ClustalW2 analysis software to compare the homology of the light chain and heavy chain gene respectively.After identification by DNA sequencing and PCR, the recombinant expression vectors transfected into CHO cells, and then Western blotting was used to identify the expression of chimeric antibody. Molecule modelling simulated protein secondary and tertiary structure of chimeric antibody, and fatherly verified its structural integrity and functional effectivity.
The supernatants of transfected CHO cells were harvested and purified by a HiTrap Protein A HP.The purified antibody samples were identified by SDS-PAGE. Furthermore, the assays of the ability of V region of chimeric antibody binding to surface CD45 on CD45-expressing tumor cells and human PBMC cells, pro-competitive inhibition with murine-derived monoclonal antibody, C region of chimeric antibody mediating CDC to CD45-expressing tumor cells and inhibit the activity of target cells were valuated by FACS.
Results The PCR amplified gene fragments of VL and VH were identified by 1% agarose gel electrophoresis and DNA sequencing, and the length and sequence of amplified fragments were identical to the theoretical values. Competitive V-gene PCR is a convenient and rapid method to determine the sequence of functional antibody V-genes in hybridoma that express the endogenous aberrant myeloma mRNA. The primer VL5'5 hybridized the variable VL genes belong to IGKV1-117*01 family by IMGT/QUEST system analyze, the identity was 99.32%.VJ genes showed characteristic according to IGKJ1*01 family, the homology was 100.00%.Sequence analysis revealed these functional VL gene products anti-CD45 variable region 333bp long,before it has a 57 bp signal peptide.
The recombinant expression vectors were respectively amplified by the specific primers, and both the lengths of amplified fragments and the reading frames were identical to the theoretical values. The expression of chimeric antibody screening revealed that took the way of co-transfected CHO cells the yield was very low, follow-up results were not satisfactory. The low yield of recombinant antibody genetically engineered antibody preparation biggest obstacle, high expression cell line transfected cells relative to the whole group is very rare,cultured in a stable output can form gradient descent, excessive growth of non-expressing cells stable transfected cells can yield a whole was more obvious, and even the formation of non-expression of cell groups. During cell lines produced IgG most of them can produce more light chain genes. In the research of human-mouse hybrid cell line study, we found the correlation of the ratio of light chain secretion and intracellular content of light-chain. The speed of produce is faster than the synthesis proportion of heavy chain, and the concentration has the same result. Some literature reports step by step transfection can increase the functional cells expression to 5～30%. At first we used high concentration antibiotics to transfect light chain gene to CHO cells stably, and then transfected heavy chain gene. Reduction the amount of medium volume in the follow-up culture process, extend the cultured time of the transfected cells for 7 to 10 days or use etc can increase the relative concentration of antibodies. DMSO/sodium can inhibit cell growth,promote antibody secretion on the CHO cells (promote the yield of recombinant proteins approximately 2-fold).
We studied on the relevant literature and used the step by step transfection method. We designed an alternate inducing methods, which used medium with or withour NaBu, ELISA results showed that production of chimeric antibodies have greatly improved. The detection index of molecular modeling shows the anti-CD45 antibody was according to the functional chimeric antibody. InsightⅡsoftware simulated Fab segment domain of anti-CD45 antibody, the model showed that the antibody has antigen-binding groove completely can exert the antibody-related functions.
The detection of transfected cells expression confirmed successfully. We freezing the part of the transfected cells into the liquid nitrogen, after 5 months recovered them. After conventional limiting dilution cloning and ELISA, we identified that after frozen in liquid nitrogen and passaged several times,the cells still able to maintain good growth conditions in vitro.Western blotting demonstrated that the expression products were mainly in the supernatants of culture for 3d. The supernatants of tranfected cells were purified by a HiTrap Protein A HP. SDS-PAGE showed that there were only two bands of heavy chain and light chain in the purified products, and the molecular weight of purified antibody was identical to the theoretical values. FACS indicated that the purified antibodies were able to specially bind to surface target antigen on CD45-expressing tumor cells and human PBMC cells, and the capability of binding was similar to that of murine-derived monoclonal antibody at the same concentrations. The assay of pro-competitive inhibition with the monoclonal antibody showed the change of gradient. The effects of CDC mediated by chimeric antibody and ability to inhibit tumor cell proliferation were enhanced with the increase of the concentrations of chimeric antibody.
Conclusion:We successfully constructed the recombinant eukaryotic expression vector of anti-CD45 mouse/human chimeric antibody, and the complete chimeric antibodies having biologic activity were obtained.To lay solid foundation for further study on on the blood system diseases.
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。
Background
AML kills 60% to 70% of affected adults despite improvements in induction chemotherapy and HCT. High-dose chemoradiotherapy followed by stem cell transplantation offers the only chance of cure for many patients with treatment-resistant leukemia. Several groups have documented the feasibility, safety, and efficacy of treating AML. Therefore, targeted radiation to leukemic progenitor and stem cells residing in hematopoietic bone marrow was the safest resolution to use radiolabeled antibodies directed to epitopes expressed on leukemic cells or normal hematopoietic bone marrow cells.For treatment of acute myeloid leukemia, monoclonal antibodies have been directed to CD33,CD66, and CD45, and studies using anti-CD45 have shown the most promising results.
CD45 antigen is stably expressed at a high density on the surface of all leukocytes, their precursors and more than 70% of nucleated cells in normal bone marrow, with an average copy number of approximately 200,000 molecules per cell. CD45 (also called GP180, T200, or leukocyte common antigen) is a～200-kDa tyrosine phosphatase that is stably expressed at a high density on the surface of virtually all hematopoietic cells except mature erythrocytes and platelets. At least 90% of myeloid leukemias express CD45 and the antigen is not found on tissues of nonhematopoietic origin, making CD45 an attractive target for treatment of leukemia.
The abundant expression of CD45 on virtually all leukocytes, including myeloid precursors in bone marrow and mature lymphocytes in lymph nodes as well as more than 90% of AML samples, provides a high number of Ab binding sites in these tissues for patients in remission or relapse. It is expressed by both normal and malignant cells, and therefore anti-CD45 antibodies can deliver to marrow, spleen, and lymph nodes in patients with acute leukemia.
In radioimmunotherapy, a specific antigen is targeted to selectively deliver radioactivity to cancer cells. Radioimmunotherapy of blood-borne cancers has been the most successful because of high accessibility of the target antigen expressed on inherently radiosensitive cells. The CD45 antigen acts as an attractive alternative target for radioimmunotherapy of AML and MDS.Radiolabeled anti-CD45 Ab can delivery radiation to antigen-positive or surrounding antigen-negative cell in myeloid tissues. Therefore, the radioimmunoconjugate does not need to bind to every leukemia cell at a high density or penetrate homogeneously into bone marrow or tumor cell masses to induce lethal DNA damage. For patients in remission, even blasts that do not express CD45 may be killed if they are surrounded predominantly by nonmalignant hematopoietic cells, because of the bystander effect. The characteristics of the CD45 antigen have prompted investigations of radiolabeled antibodies against CD45 with transplantation therapy. With the CD45 molecular isomer research, the development of flow cytometry immunophenotyping and anti-CD45 monoclonal antibody application,CD45 molecules in the application of immunology and hematology research would get further attention. We can expecte, CD45 could play greater role in clinical diagnosis, treatment, prognosis. Our group has documented the promise of anti-CD45 monoclonal antibodies (Ab) administered in the setting of allogeneic HCT for AML, but toxicity remains high. So we have been committed to continuous improvement.
In this article, we report in the first time the construction of anti-CD45 mouse/human chimeric antibody, and describe its merits on the blood system diseases. To lay solid foundation for further study on this chimera label with 131I on the pretargeted radioimmunotherapy (PRIT) to use autologous and allogeneic haematopoietic stem cell transplantation.
Humanized or chimeric monoclonal antibodies (MoAbs) have been developed that react with antigens expressed by hematopoietic cells. They have been used either unlabeled('native' or 'naked'antibodies) or conjugated with toxins, radioisotopes, or antitumor drugs. According to whether they carry the material into other connections not bound monoclonal antibody, anti-cancer drug conjugated monoclonal antibody and isotope conjugated monoclonal antibody. So far, five drugs by the U.S.FDA approval for clinical, which are no-binding human-mouse chimeric antibody or humanized antibody. But use no-binding CD45 monoclonal antibody had little effect in the human body and with the limitation of murine application, the current research on the CD45 monoclonal antibodies mainly on antibodies connect with radionuclides 131I and 213Bi as part of hematopoietic stem cell transplantation conditioning to reduce the dose of TBI and its side effects. Animal testing andⅠ/Ⅱclinical trials have confirmed, CD45 monoclonal antibody can be selectively deliver radionuclides 213Bi and 131I into bone marrow, liver, spleen and lymph nodes. Vallera et al. used 90Y labeled anti-CD45 monoclonal antibodies treat for experimental animal models of lymphoma, the results showed that give appropriate doses of 90Y-Anti-CD45 to mice, tumors disappeared after 6 days, and without one case happen to recurrence until 135 days. Liver, kidney, small intestine biopsy found no apparent damage. We have used anti-CD45 monoclonal antibody labeled with 90Y for the treatment of acute leukemia, preliminary experiments to select CDTPA to chelate 90Y and anti-CD45 monoclonal antibody. CDTPA is a classic metal ion chelating agent, as early as 1985,Hnatowich took CDTPA chelae IgG and 90Y, we obtained specific activity was 1.7μCi/μg, the radiochemical purity more than 90%,24 hours dissociation was 13%.The products 90Y-CDTPA-CD45 monoclonal antibody under the conditions of 20:1 CDTPA/IgG, the labeling efficiency was 95%, the radiochemical purity was more than 99%; good stability,24-hour dissociation rate was 8.32%. These results in accordance with Zhang Jinming et al.reported that the dissociation rate was 11.9%. Indirect immunofluorescence assay also proved that the immunological activity of the chelates have not been obviously affected by the combination with the high rate of nuclides. The binging rate with AML cells was good. It is a better targeted therapeutic agents and laying the basis for the further study. Antibody therapy is ideally suited to the treatment of acute myeloid leukemia because of the ready accessibility of neoplastic cells in the circulation.
But mouse-derived whole-molecule antibodies have been demonstrated to have many limitations for therapeutic applications due to high immunogenicity and large molecular weight, and human anti-mouse antibody (HAMA) responses that can cause allergic reaction and damage in the human body.In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effect or mechanism in vivo, we herein developed its chimera. Genetically engineered antibody could have more important practical significance. To remove these difficulties, humanized antibody with a human antibody constant region was genetically engineered. Humanized human-mouse chimeric antibody has employed the constant region of human antibody to replac C region of murine monoclonal antibody. Human-mouse chimeric antibody not only retains the specificity of mouse monoclonal antibody but also decreases the immunogenicity. This genetically engineered antibody has more advantage than the mouse antibody in the function of complement mediated cell killing of target antigens and phagocytic effect. In addition, with construction of chimeric antibodies, we purposely chose the type of antibody, making it more effective to play the effectiveness of antibodies. Therefore, the variable region genes of this MoAb were cloned and ligated into chimeric antibody expression vector pFUSE-CHIg-hGl and pFUSE2-CLIg-hk, generating chimeric anti-human CD45 MAb (chi-mAb CD45)expression vectors,transfected CHO cells,expressed and purified chimeric antibody.
Now we first generated and report a novel anti-CD45 mouse/human chimeric antibody (Chi-CD45).Untill now, it is no reported about reconstruction of humanized anti-CD45 monoclonal antibody at home and abroad. In vitro assay, it revealed that pro-competitive inhibition with the monoclonal antibody showed the change by gradient. The effects of CDC mediated by chimeric antibody and ability to inhibit Jurkat/human PBMC proliferation were enhanced with the increase of the concentrations of chimeric antibody.
Aim To construct the eukaryotic expression vector of chimeric anti-CD45 mouse/human chimeric antibody and realize its expression.
Methods VH gene and VL gene of anti-CD45 monoclonal antibody were amplified by PCR. Use DNAtools, IMGT/QUEST and EBI TOOLS:ClustalW2 analysis software to compare the homology of the light chain and heavy chain gene respectively.After identification by DNA sequencing and PCR, the recombinant expression vectors transfected into CHO cells, and then Western blotting was used to identify the expression of chimeric antibody. Molecule modelling simulated protein secondary and tertiary structure of chimeric antibody, and fatherly verified its structural integrity and functional effectivity.
The supernatants of transfected CHO cells were harvested and purified by a HiTrap Protein A HP.The purified antibody samples were identified by SDS-PAGE. Furthermore, the assays of the ability of V region of chimeric antibody binding to surface CD45 on CD45-expressing tumor cells and human PBMC cells, pro-competitive inhibition with murine-derived monoclonal antibody, C region of chimeric antibody mediating CDC to CD45-expressing tumor cells and inhibit the activity of target cells were valuated by FACS.
Results The PCR amplified gene fragments of VL and VH were identified by 1% agarose gel electrophoresis and DNA sequencing, and the length and sequence of amplified fragments were identical to the theoretical values. Competitive V-gene PCR is a convenient and rapid method to determine the sequence of functional antibody V-genes in hybridoma that express the endogenous aberrant myeloma mRNA. The primer VL5'5 hybridized the variable VL genes belong to IGKV1-117*01 family by IMGT/QUEST system analyze, the identity was 99.32%.VJ genes showed characteristic according to IGKJ1*01 family, the homology was 100.00%.Sequence analysis revealed these functional VL gene products anti-CD45 variable region 333bp long,before it has a 57 bp signal peptide.
The recombinant expression vectors were respectively amplified by the specific primers, and both the lengths of amplified fragments and the reading frames were identical to the theoretical values. The expression of chimeric antibody screening revealed that took the way of co-transfected CHO cells the yield was very low, follow-up results were not satisfactory. The low yield of recombinant antibody genetically engineered antibody preparation biggest obstacle, high expression cell line transfected cells relative to the whole group is very rare,cultured in a stable output can form gradient descent, excessive growth of non-expressing cells stable transfected cells can yield a whole was more obvious, and even the formation of non-expression of cell groups. During cell lines produced IgG most of them can produce more light chain genes. In the research of human-mouse hybrid cell line study, we found the correlation of the ratio of light chain secretion and intracellular content of light-chain. The speed of produce is faster than the synthesis proportion of heavy chain, and the concentration has the same result. Some literature reports step by step transfection can increase the functional cells expression to 5～30%. At first we used high concentration antibiotics to transfect light chain gene to CHO cells stably, and then transfected heavy chain gene. Reduction the amount of medium volume in the follow-up culture process, extend the cultured time of the transfected cells for 7 to 10 days or use etc can increase the relative concentration of antibodies. DMSO/sodium can inhibit cell growth,promote antibody secretion on the CHO cells (promote the yield of recombinant proteins approximately 2-fold).
We studied on the relevant literature and used the step by step transfection method. We designed an alternate inducing methods, which used medium with or withour NaBu, ELISA results showed that production of chimeric antibodies have greatly improved. The detection index of molecular modeling shows the anti-CD45 antibody was according to the functional chimeric antibody. InsightⅡsoftware simulated Fab segment domain of anti-CD45 antibody, the model showed that the antibody has antigen-binding groove completely can exert the antibody-related functions.
The detection of transfected cells expression confirmed successfully. We freezing the part of the transfected cells into the liquid nitrogen, after 5 months recovered them. After conventional limiting dilution cloning and ELISA, we identified that after frozen in liquid nitrogen and passaged several times,the cells still able to maintain good growth conditions in vitro.Western blotting demonstrated that the expression products were mainly in the supernatants of culture for 3d. The supernatants of tranfected cells were purified by a HiTrap Protein A HP. SDS-PAGE showed that there were only two bands of heavy chain and light chain in the purified products, and the molecular weight of purified antibody was identical to the theoretical values. FACS indicated that the purified antibodies were able to specially bind to surface target antigen on CD45-expressing tumor cells and human PBMC cells, and the capability of binding was similar to that of murine-derived monoclonal antibody at the same concentrations. The assay of pro-competitive inhibition with the monoclonal antibody showed the change of gradient. The effects of CDC mediated by chimeric antibody and ability to inhibit tumor cell proliferation were enhanced with the increase of the concentrations of chimeric antibody.
Conclusion:We successfully constructed the recombinant eukaryotic expression vector of anti-CD45 mouse/human chimeric antibody, and the complete chimeric antibodies having biologic activity were obtained.To lay solid foundation for further study on on the blood system diseases.
引文
[1]Vanhove M, Usherwood YK, Hendershot LM. Unassembled Ig Heavy Chains Do Not Cycle from BiP In Vivo but Require Light Chains to Trigger Their Release. Immunity.,2001; 15(1):105-14.
[2]Lee YK, Brewer JW, Hellman R, et al.BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly. Molecular Biology of the Cell,Vol.10,2209-2219, July 1999
[3]Goto Y, Hamaguchi K.The Role of the Intrachain Disulfide Bond in the Conformation and Stability of the Constant Fragment of the Immunoglobulin Light Chain1.J. Biochem,1979; 86(5),1433-41
[4]Bergman LW, Harris E, Kuehl WM. Glycosylation Causes an Apparent Block in Translation of Immunoglobulin Heavy Chain. J Biol Chem.,1981; 256(2): 701-6.
[5]Lenny N, Green M. Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA. J Biol Chem.,1991; 266(30):20532-7.
[6]Jiang Z, Huang Y, Sharfstein ST. Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells:A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression. Biotechnol Prog.,2006; 22(1):313-8.
[7]Zuberbuhler K, Palumbo A, Bacci C, et al. A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells. Protein Eng Des Sel.2009; 22(3):169-74.
[8]Kennard ML, Goosney DL, Monteith D, et al. The Generation of Stable, High MAb Expressing CHO Cell Lines Based on the Artificial Chromosome Expression(ACE)Technology. Biotechnol Bioeng.,2009; 104(3):540-53.
[9]Majors BS, Betenbaugh MJ, Pederson NE, et al. Enhancement of Transient Gene Expression and Culture Viability Using Chinese Hamster Ovary Cells Overexpressing Bcl-xL. Biotechnol Bioeng.,2008; 101(3):567-78.
[10]Wulhfard S, Tissot S, Bouchet S, et al. Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells. Biotechnol Prog.,2008; 24(2):458-65.
[11]Schlatter S, Stansfield SH, Dinnis DM, et al. On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells. Biotechnol Prog.,2005; 21(1):122-33.
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[2]Fukuhara K, Okumura M, Shiono H,et al. A study on CD45 isoform expression during T-cell development and selection events in the human thymus. Hum Immunol,2002,63(5):394-404
[3]肖溶,CD45分子及在急性白血病中应用的研究进展,国外医学内科学分册,2004；31(3)：93-96
[4]朱志刚,沈关心,朱慧芬等。抗CD4人-鼠嵌合抗体的表达和抗增殖效应,中华微生物学和免疫学杂志,2002；22(3)：134-138
[5]朱志刚,沈关心,朱慧芬等。抗CD4单克隆抗体可变区基因克隆及序列分析,免疫学杂志；1999；15(2)：142
[6]朱志刚,沈关心,毛平等。抗CD4单克隆抗体可变区基因克隆及测序,广东医学；1999；21(8)636-638
[7]扶云碧,孟凡义。单克隆抗体治疗恶性血液病的临床应用,中华血液学杂志,2005；26(8)：209-210
[8]朱志刚,孟凡义。单克隆抗体在造血干细胞移植预处理的临床应用,国际输血及血液学杂志,2008；31(4)：351-355
[9]苏娜,沈关心主编.抗体工程[M].北京：科学出版社,1996
[10]王硕,蒋琳,沈关心等.抗转铁蛋白受体抗体单抗可变区基因克隆和序列分析.[J].细胞与分子免疫学杂志,1998；14(1)：13-16
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[12]瞿秋霞,陈永井,葛彦,等.抗人CD40-人-鼠嵌合抗体的构建及其表达[J].中国免疫学杂志,2006；22(2)：189-192
[13]葛彦,陈永井,瞿秋霞,张学光.抗人CD154人-鼠嵌合抗体Fab段的构建和表达.细胞与分子免疫学杂志.2007；23(6)：569-571
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[15]王玉刚,黄英,沈倍奋等.用RLM-RAEC法克隆抗体CD20单克隆抗体可变区基因及其信号肽基因[J].细胞与分子免疫学杂志.2005；21(4)：466-469
[16]扶云碧,李贵平,孟凡义.90Y标记单克隆抗体CD45的研究,南方医科大学学报,2006；26(8)：1230-1232
[17]李贵平,扶云碧,孟凡义等.90Y间接法标记单克隆抗体的实验研究,放射免疫学杂志,2008；21(6)：481-483
[18]龚非力,主编.医学免疫学(第二版)[M]北京：科学出版社,2004
[19]王秀红,周素芳.基因工程抗体研究进展.生物技术通讯,2007,VOL.18NO.2 MAR.
[20]萨姆布鲁克J,拉塞尔DW(黄培堂,等译).分子克隆实验指南[M].第三版.北京：科学出版社,2002：2-137
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[1]Mulford DA, Jurcic JG. Antibody-based treatment of acute myeloid leukaemia. Expert Opin Biol Ther. 2004,4(1):95-105.
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[18]Pagel JM, Appelbaum FR, Eary JF,etal.131I-anti-CD45 antibody plus busulfan and cyclophosphamide before allogeneic hematopoietic cell transplantation for treatment of acute myeloid leukemia in first remission. Blood.2006,107(5):2184-2191.
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[2]Lee YK, Brewer JW, Hellman R, et al.BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly. Molecular Biology of the Cell,Vol.10,2209-2219, July 1999
[3]Goto Y, Hamaguchi K.The Role of the Intrachain Disulfide Bond in the Conformation and Stability of the Constant Fragment of the Immunoglobulin Light Chain1.J. Biochem,1979; 86(5),1433-41
[4]Bergman LW, Harris E, Kuehl WM. Glycosylation Causes an Apparent Block in Translation of Immunoglobulin Heavy Chain. J Biol Chem.,1981; 256(2): 701-6.
[5]Lenny N, Green M. Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA. J Biol Chem.,1991; 266(30):20532-7.
[6]Jiang Z, Huang Y, Sharfstein ST. Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells:A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression. Biotechnol Prog.,2006; 22(1):313-8.
[7]Zuberbuhler K, Palumbo A, Bacci C, et al. A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells. Protein Eng Des Sel.2009; 22(3):169-74.
[8]Kennard ML, Goosney DL, Monteith D, et al. The Generation of Stable, High MAb Expressing CHO Cell Lines Based on the Artificial Chromosome Expression(ACE)Technology. Biotechnol Bioeng.,2009; 104(3):540-53.
[9]Majors BS, Betenbaugh MJ, Pederson NE, et al. Enhancement of Transient Gene Expression and Culture Viability Using Chinese Hamster Ovary Cells Overexpressing Bcl-xL. Biotechnol Bioeng.,2008; 101(3):567-78.
[10]Wulhfard S, Tissot S, Bouchet S, et al. Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells. Biotechnol Prog.,2008; 24(2):458-65.
[11]Schlatter S, Stansfield SH, Dinnis DM, et al. On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells. Biotechnol Prog.,2005; 21(1):122-33.
[1]Schraven B, H. Kirchgessner, B. Gaber, Y. Samstag, and S.Meuer.1991. A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45,the protein tyrosine kinase p56 Ick and pp32, a possible common substrate. Eur. J. Immunol.21:2469.
[2]Fukuhara K, Okumura M, Shiono H,et al. A study on CD45 isoform expression during T-cell development and selection events in the human thymus. Hum Immunol,2002,63(5):394-404
[3]肖溶,CD45分子及在急性白血病中应用的研究进展,国外医学内科学分册,2004；31(3)：93-96
[4]朱志刚,沈关心,朱慧芬等。抗CD4人-鼠嵌合抗体的表达和抗增殖效应,中华微生物学和免疫学杂志,2002；22(3)：134-138
[5]朱志刚,沈关心,朱慧芬等。抗CD4单克隆抗体可变区基因克隆及序列分析,免疫学杂志；1999；15(2)：142
[6]朱志刚,沈关心,毛平等。抗CD4单克隆抗体可变区基因克隆及测序,广东医学；1999；21(8)636-638
[7]扶云碧,孟凡义。单克隆抗体治疗恶性血液病的临床应用,中华血液学杂志,2005；26(8)：209-210
[8]朱志刚,孟凡义。单克隆抗体在造血干细胞移植预处理的临床应用,国际输血及血液学杂志,2008；31(4)：351-355
[9]苏娜,沈关心主编.抗体工程[M].北京：科学出版社,1996
[10]王硕,蒋琳,沈关心等.抗转铁蛋白受体抗体单抗可变区基因克隆和序列分析.[J].细胞与分子免疫学杂志,1998；14(1)：13-16
[11]王硕,蒋琳,叶庆,朱慧芬,杨敬,邵静芳,沈关心.抗CD71人-鼠嵌合抗体(D2C)的构建和表达,分子与细胞免疫学,392(4)：665-668
[12]瞿秋霞,陈永井,葛彦,等.抗人CD40-人-鼠嵌合抗体的构建及其表达[J].中国免疫学杂志,2006；22(2)：189-192
[13]葛彦,陈永井,瞿秋霞,张学光.抗人CD154人-鼠嵌合抗体Fab段的构建和表达.细胞与分子免疫学杂志.2007；23(6)：569-571
[14]王玉刚,黄英,沈倍奋等.抗CD20嵌合抗体的构建与表达.细胞与分子免疫学杂志.2006；22(3)363-367
[15]王玉刚,黄英,沈倍奋等.用RLM-RAEC法克隆抗体CD20单克隆抗体可变区基因及其信号肽基因[J].细胞与分子免疫学杂志.2005；21(4)：466-469
[16]扶云碧,李贵平,孟凡义.90Y标记单克隆抗体CD45的研究,南方医科大学学报,2006；26(8)：1230-1232
[17]李贵平,扶云碧,孟凡义等.90Y间接法标记单克隆抗体的实验研究,放射免疫学杂志,2008；21(6)：481-483
[18]龚非力,主编.医学免疫学(第二版)[M]北京：科学出版社,2004
[19]王秀红,周素芳.基因工程抗体研究进展.生物技术通讯,2007,VOL.18NO.2 MAR.
[20]萨姆布鲁克J,拉塞尔DW(黄培堂,等译).分子克隆实验指南[M].第三版.北京：科学出版社,2002：2-137
[1]Clarke E, Potter MN, Oakhill A, et al. A laboratory comparision of T cell depletion by CD34+ cell immunoaffinity selection and in vitro Campath-1M treatment:clinical implications for bone marrow transplantation and donor leukocyte therapy. BMT,1997,20(7):599-605
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