Efficient production of human recombinant antibodies against hepatitis virus using a novel micro-well array system

虽然随着各种药物的发展，由病原微生物引起的传染病似乎大大减少了，但新出现的传染病，如在全球范围内蔓延的艾滋病或在亚洲爆发的SARS，已经对人类构成了威胁。抗体(Abbs)长期以来一直被认为是治疗病原微生物的理想试剂，但由于难以有效地产生针对各种微生物的人源化单抗，其应用受到了极大的限制。在本项目中，我们试图开发一种新型的微孔阵列系统来高效地寻找抗原(Ag)特异性的人类B细胞，并将该系统应用于制备人抗乙肝病毒表面抗原(HBs)的单抗。我们开发了一种微孔阵列芯片，该芯片由高度集成的微腔(250,000个半平方厘米的微腔)组成，其中微腔被设计成单个B细胞的大小。为了检测抗原特异的B细胞，INTR…在细胞中引入更多的非细胞钙离子指示剂Fluo-4，通过B细胞受体(BCR)作为B细胞活化的标志，并通过定制的DNA阵列检测(Ca2+)i的增加。将乙肝表面抗原免疫的健康志愿者的B细胞用芯片上的乙肝表面抗原刺激，用扫描仪检测反应细胞，用显微操作获取细胞。从采集的B细胞中克隆了Ig-Hand-L链可变区基因，并在293T细胞中制备了重组抗体。分别用ELISA法和小鼠体内模型检测其与乙肝表面抗原的结合能力和中和乙肝病毒的活性，从而获得了几株高亲和力和高中和活性的人重组抗体。该微孔阵列系统有望成为一种高通量的人抗各种感染性微生物单抗的制备系统，并有助于发现针对新出现的传染病的抗体药物。较少

Although infectious diseases caused by pathogenic microbes seemed extraordinarily lessened after the development of various drug discovery, newly emerging infectious diseases, such as worldwide-spreading AIDS or SARS broken out in Asia, have been a threat to humans. Antibodies (Abs) have been long expected as a desirable therapeutic reagent against the pathogenic microbe, their use has been extremely limited because of the difficulties to efficiently generate humanized monoclonal Abs to various micobes. In the present project, we have tried to develop a novel micro-well-array system to efficiently find the antigen (Ag)-specific human B cells and applied this sytem to produce human monoclonal Abs against hepatitis B virus-surface antigen (HbsAg).We have developed a micro-well-array chip consists of highly integrated micro chambers (250,000 wells in half square centimeter) on a silicon chip, of which chambers were designed to a size of a single B cell. To detect Ag-specific B cells, intr … More acellular Ca2^+ indicator, Fluo-4, was introduced to the cells as a marker for B cell activation via B cell receptor (BCR), and the increases in (Ca2^+)i were detected by the customized DNA-array.We applied this system to produce human monoclonal Abs against HbsAg. B cells from healthy volunteers who had been immunized with HBsAg were stimulated with HBsAg on a chip and the responding cells were detected by the scanner and harvested by micromanipulation. The cDNA of variable regions for Ig Hand L chains were cloned from the harvested B cells and the recombinant Abs were generated in 293T cells. The ability to bind to HbsAg or to neutralize the HB virus activity was assessed by ELISA or in vivo mouse model, respectively.As a result, we produced several human recombinant Abs with high binding affinity to HbsAg, as well as high neutralizing activity. This micro-well array system should be a high throughput system for producing human monoclonal Abs against various infectious microbes and contribute to discover antibody-medicine against newly emerging infectious diseases. Less