摘要
肿瘤细胞对不同的化学相关或不相关的抗肿瘤药物产生不敏感性,即多药耐药(Multidrug resistance,MDR)。MDR是造成肿瘤化疗失败的主要原因,产生MDR的原因通常是因为药物失活或被外排泵排出肿瘤细胞。MDR产生的主要分子机制是细胞表面存在一种叫做P-糖蛋白(P-glycoprotein,Pgp)的跨膜糖蛋白,由MDR1基因编码,Mr为170000。Pgp蛋白属于ATP结合盒(ATP-binding cassette,ABC)转运蛋白超家族,是ATP依赖性的药物外排泵,能将各种不同的药物排出细胞,从而产生MDR。已经证实Pgp蛋白与肿瘤复发和预后差高度相关,是评价肿瘤预后的重要指标之一。以Pgp为治疗靶点已经成为克服肿瘤耐药的新策略。
Diabody是双特异性抗体(Bispecific antibody)的一种构建形式之一。通常是两个具有不同抗原结合位点的抗体可变区序列,在同一个启动子控制下组成两个顺反子,同一顺反子中VH和VL用一个短Linker连接。表达产物中同一顺反子上的V_H和V_L因Linker太短而存在空间位阻,不能配对,只能与另一顺反子上的V_H和V_L配对,共价结合形成二聚体。Diabody分子量在55-60 kDa,肿瘤穿透性强;缺乏Fc段,副作用小;Diabody能够将体内细胞毒性T细胞靶向到肿瘤细胞表面,直接发挥细胞毒杀伤效应,避免了使用外源性非特异性细胞毒性物质,并且不受MHC限制。抗CD3/抗Pgp Diabdoy具有良好的临床应用前景。但是,由于Diabody自身结构特点,两条肽链是通过非共价键结合在一起,容易解离而失去活性。向Diabody引入二硫键,可以大大增强其稳定性,并且不影响抗体亲和力,甚至亲和力增加。
T细胞活化需要协同刺激信号,否则,T细胞不但不能有效活化,还会处于无能状态。B7分子是协同刺激分子最主要的家庭成员,它包括两种分子:CD80和CD86。文献报道,化疗药物阿糖胞苷(ARA-C)能够诱导肿瘤细胞高表达B7分子。因此,我们推测抗CD3/抗Pgp Diabdoy和ARA-C联合应用可以增强细胞毒性T细胞的活性,达到更好的治疗效果。
本实验室构建了抗CD3/抗Pgp微型双功能抗体(Diabody),并进行原核可溶性表达,体外实验证实具有良好的生物学活性,体内实验证实能有效地介导细胞毒性T淋巴细胞杀伤耐药移植瘤细胞。抗CD3/抗Pgp Diabody带有15肽的Etag纯化标记,可能引起免疫原性,影响其临床应用价值。并且抗CD3/抗Pgp Diabody在体内实验中,停药一周后肿瘤复发,不能彻底根除肿瘤。可能与体外活化的T细胞在体内不能维持其持续活化的状态,以及Diabody不稳定有关。本实验主要对抗CD3/抗Pgp Diabody进行了两项改进,分别为:①通过基因工程技术将抗CD3/抗Pgp Diabody的纯化标签Etag去除,降低免疫原性。与ARA-C联合应用治疗耐药裸鼠移植瘤。②向抗CD3/抗Pgp Diabody的抗CD3或抗Pgp可变区引入一对二硫键,增强其稳定性,提高疗效。
实验结果显示,去除Etag的抗CD3/抗Pgp Diabody生物学活性没有明显改变,与ARA-C联合应用,疗效大大增强,在观察窗内肿瘤没有复发。向抗CD3/抗Pgp Diabody的抗Pgp可变区引入一对二硫键后,二硫键不能正确配对。而向抗CD3/抗Pgp Diabody的抗CD3可变区引入一对二硫键后(dsCD3-Diabody),二硫键能够正确配对,在原核系统可溶性表达。dsCD3-Diabody抗原结合活性没有明显改变,介导细胞毒性T细胞发挥杀伤效应的能力亦没有明显改变,而体内外稳定性大大增强,介导细胞毒性T细胞杀伤耐药移植瘤的作用增强一倍。
近二十年来,已经证实存在肿瘤干细胞,肿瘤干细胞的存在可能是肿瘤复发难治的根源。最近研究显示,急性髓系白血病(AML)细胞上PgP蛋白的表达明显高于其他亚群的细胞,这样白血病干细胞(LSC)就容易逃脱化疗药物的杀伤作用,重新生长并增殖。研究同时发现,LSC上的Pgp蛋白虽然数量增多,但是其功能是降低的,这可能是ABCB1逆转剂临床治疗效果并没有预期那么好的原因之一。我们研制的抗CD3/抗Pgp Diabody是通过识别Pgp蛋白的三维结构,与之特异性结合,从而介导细胞毒性T细胞发挥细胞杀伤效应。因此,抗CD3/抗Pgp Diabody较ABCB1逆转剂具有更好的临床应用价值。
A major issue in the treatment of cancer in terms of a poor response or relapse is the development of multidrug resistance(MDR) by the tumor cells. Pgp,encoded by the MDR1 gene,is a 170 kDa transporter consisting of 1280 amino acids that is located in the plasma membrane and is responsible for cancer resistance to multiple chemotherapeutic agents.The level of Pgp expression is an adverse prognostic factor for complete remission and survival in malignant diseases.Pgp may therefore act as a potential therapeutic target for cancer intervention.
Immunotherapy with bispecific antibodies(BiAbs) is a very promising approach for targeting to tumors.T cells play a pivotal role acting against tumors by directly eliminating the tumor cells through the formation of cytotoxic T cell-tumor cell synapses.However,the complexity of T cell recognition offers a variety of strategies for tumor cells to evade specific T cell recognition.Anti-CD3/anti-TAA(tumor associated antigen) BiAbs can possess two specificities,one directed at the T cell and the other at the cancer cell.This enables them to serve as mediators between T cells and cancer cells,bypassing the conventional T cell recognition process. Bispecific diabodies are the smallest BiAbs with about one-third the size of IgG.Bispecific diabodies are dimers,one chain comprising a V_H domain from antibody A and a V_L domain from antibody B,connected by a short peptide linker,and vice versa.The linker is too short to allow pairing between domains of the same chain,thus driving the pairing between complementary domains on different chains and forming two antigen binding sites that point away from each other.In addition the relatively small size of diabodies(55 kDa) facilitates penetration into solid tumors as compared to larger whole antibodies.Diabodies lack Fc domains thus eliminating the undesirable side-effects they have in immunotherapy.Further,diabodies can be readily produced by secretion from bacteria at a yield of up to 1g/L,thus possessing a considerable potential for application in a clinical setting.
A critical and important factor contributing to the therapeutic effect of recombinant antibodies is stability.The two chains of diabodies are associated non-covalently and are therefore capable of dissociation.By introducing a disulphide bond into the recombinant Fv fragment,between two conserved framework residues,a significant improvement in the antibody stability is achieved.The disulphide bond locks the two peptide chains covalently while retaining full or even improved antigen binding activity.The disulphide bond stabilized Fv fragment or BsAbs may thus have the same,or even higher antitumor activity,compared to its non-stabilized counterpart.
In our previous study it was suggested that an anti-Pgp/anti-CD3 diabody might be an effective agent in the treatment of MDR tumors. However,the diabody contains an Etag peptide,which can lead to immunogenicity,the poor stability of the diabody limits the antitumor response and reduces its effectiveness in cancer therapy.The required dose of anti-Pgp/anti-CD3 diabody was relatively high and a rapid tumor relapse occurred only one week after therapy.
In this study,we generated an anti-Pgp/anti-CD3 diabody without Etag by the technology of gene egineering,which retain the full binding activity. It was reported that ARA-C can induce CD80 or CD86 expression in tumor cells.We used diabody in combination with ARA-C to inhibit MDR xenografts, and achieve the better therapeutic effect.During the observation window, no tumor was detected.To enhance the stability,We introduced cysteine residues into the CD3(dsCD3-Diabody) or Pgp(dsPgp-Diabody) V-domain to covalently lock the two chains together.The dsPpg-diabody failed to form disulphide bond properly.The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly.Compared with the parent diabody,the dsCD3-Diabody obtained was more stable in human serum at 37℃,without loss of affinity or cytotoxicity activity in vitro. Furthermore,the dsCD3-Diabody showed improved tumor localization and a two-fold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts.
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