【作者】 陈琳；

【导师】 郭亚军；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2011， 硕士

【摘要】 非霍奇金淋巴瘤（Non Hodgkin’s Lymphoma NHL）是临床最常见的淋巴系统恶性肿瘤,其中B细胞来源约占85%。人CD20分子又称为B1或Bp35,仅表达于所有的前B细胞、成熟B细胞和超过95%的恶性B细胞表面,而不表达于造血干细胞、浆细胞和其他正常组织中。其与抗体结合后无显著内化和脱落,也不因与抗体结合而发生抗原调变,从而成为治疗B细胞非霍奇金淋巴瘤的理想靶点。Rituximab（C2B8）是美国基因科技公司研发的以人CD20分子为靶点的人鼠嵌合型IgG1免疫球蛋白,可特异性地与B淋巴细胞表面CD20抗原结合。主要通过以下两种机制发挥抗肿瘤作用:诱导补体介导的细胞毒作用（CDC）和诱导抗体依赖细胞介导的细胞毒作用（ADCC）。尽管Rituximab已显示良好的治疗效果,但其临床反应率仅为50%左右,完全缓解率只有10%,因此进一步研制出具有更强抗肿瘤活性的CD20抗体对临床治疗具有重要的意义。在前期研究中,本实验室获得了Rituximab/CD20抗原肽的复合物共晶,在此基础上通过计算机模拟辅助设计得到三个亲和力提高的突变体:Rituximab_D57R（Rituximab重链57位D突变成R）、RituximabY102R（Rituximab重链102位Y突变成R）、Rituximab_Y102S（Rituximab重链102位Y突变成S）。经体外实验表明:与Rituximab相比,这些亲和力提高的突变体CDC活性没有改变,而ADCC活性显著增强,显示出较亲本抗体Rituximab更强的抗肿瘤活性。在前期实验中,我们还成功获得一株CDC活性显著高于Rituximab的抗CD20人源化抗体hu8E4,经体内实验证实:hu8E4的抗肿瘤作用比Rituximab明显增强。计算机模拟辅助设计提高抗体的亲和力,需要在已获得抗原抗体复合物共晶的基础上,这往往难于获得。而过去研究表明采用传统杂交瘤技术制备的CD20单克隆抗体均与Rituximab具有相似的结合表位,因此我们设想能否通过在具有相似抗原结合表位的单克隆抗体间对应移植亲和力提高的突变位点,从而达到提高抗体亲和力的目的。在本课题研究中,我们利用噬菌体展示技术对hu8E4的抗原表位进行筛选和鉴定,发现其同样与Rituximab具有相似的结合表位。于是我们尝试将能够提高Rituximab亲和力的三个突变位点:D57R、Y102R、Y102S,对应移植到hu8E4上;与此同时,为进一步验证该方法的有效性,我们也将这三个突变位点对应移植到与Rituximab具有相似结合表位的嵌合抗体2H7上。实验结果表明,在得到的hu8E4三个突变体中,hu8E4_D57R和hu8E4_Y102S的亲和力获得了提高,但hu8E4Y102S的亲和力下降;而在C2H7的三个突变体中,C2H7_D57R和C2H7_Y102R的亲和力提高,C2H7_Y102S的亲和力下降。重要的是,我们发现在hu8E4和C2H7的突变体上,亲和力提高的突变位点是一致的。经体外实验表明:hu8E4和C2H7突变体分别具有与亲本抗体相似的CDC活性,但它们的ADCC活性明显增强。综上所述,我们获得了人源化CD20抗体hu8E4的高亲和力突变体,经体外生物学功能鉴定证实具有更强的抗肿瘤作用。而这种在具有相似抗原结合表位的单克隆抗体间对应移植亲和力提高的突变位点,从而提高抗体亲和力及其活性的方法,具有潜在的应用价值。更多还原

【Abstract】 CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target for monoclonal antibodies （mAb）, such as rituximab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows the antibody to persist on the cell surface for extended periods to deliver sustained immunological attack from complement and FcR expressing cells.The chimeric anti-CD20 mAb rituximab was approved for use in relapsed or refractory low-grade or follicular B-cell non-Hodgkin’s lymphoma （NHL） in 1997. Previous studies have suggested that several main mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity （CDC） and antibody-dependent cellular cytotoxicity （ADCC）. Despite the effectiveness of rituximab, only 48% of patients respond to the treatment and complete responses are less than 10%. In addition, a significant number of patients have progressive disease during antibody therapy. There is an urgent need to develop more effective anti-CD20 antibodies to further improve the efficacy of antibody therapy for B-cell lymphomas. In our previous study, we developed the crystal structure of rituximab-Fab in complex with the CD20 epitope peptide. Based on this crystal structure, we used computer-aided design to generate three single-point mutants with higher affinity (Rituximab_D57R、Rituximab_Y102R、Rituximab_Y102S). Our data showed that these high-affinity mutants displayed similar CDC activity to that of rituximab, but their ADCC activity was greater than that of rituximab.In our previous study, a humanized anti-CD20 antibody, hu8E4, was generated by complementarity-determing region grafting method. Hu8E4 was as effective as rituximab in mediating antibody-dependent cellular cytotoxicity in B-lymphoma cells, but it exhibited much more potent complement-dependent cytotoxicity than rituximab. In our present study, we attempted to enhance the affinity of hu8E4 in order to further improve its anti-tumor activity. To employ the computer-aided design to enhance antibody binding affinity, we need to acquire the crystal structure. However, the crystal structure of antigen-antibody complexes was diffcult to obtain. Previous studies have suggested that most of anti-CD20 antibodies which employed by the traditional hybridoma technology compared with rituximab with the same epitope peptide. So we speculated whether the affinity-improving mutations of an antibody could be transplanted into the corresponding places of another antibody to improve its affinity when these two antibodies had similar eiptopes. In our previous study, we employed the phage display peptide libraries technology to select peptides binding to hu8E4. We found that hu8E4 and rituximab recognize the same epitope peptide. So we try to transplant the three affinity-improving mutations （D57R、Y102R、Y102S） to hu8E4 in order to enhance antibody binding affinity. In addition to verify its effectiveness, we also transplant the three affinity-improving mutations （D57R、Y102R、Y102S） to chimeric antibody 2H7 which also compared with rituximab with the same epitope peptide.The results showed that the avidity of hu8E4D57R and hu8E4_Y102S was higher than that of hu8E4, while the avidity of hu8E4_Y102S was lower than that of hu8E4. The three mutants (hu8E4_D57R、hu8E4_Y102R、hu8E4_Y102S) were as effective as hu8E4 in CDC, while they were more effective in mediating ADCC in an affinity-dependent manner. Our data indicated that the avidity of C2H7_D57R and C2H7_Y102R was higher than that of C2H7, while the avidity of C2H7_Y102S was lower than that of C2H7. The three mutants (C2H7D57R、C2H7_Y102R、C2H7_Y102S) were as effective as C2H7 in CDC, while they were more effective in mediating ADCC in an affinity-dependent manner.Here we have obtained the high-affinity mutants of the humanized anti-CD20 monoclonal antibody, hu8E4. The high-affinity mutants showed more potent anti-tumor activity than that of the parental antibody. Our study suggested that the affinity-improving mutations of an antibody could be transplanted into the corresponding places of another antibody to improve its affinity when these two antibodies had similar eiptopes. These high-affinity mutants of hu8E4 might be the promising therapeutic agents for the treatment of B-cell lymphomas.更多还原