抗CD3/抗Pgp微型双功能抗体的构建和表达

Construction and Expression of Anti-CD3/ Anti-Pgp Diabody

构建和表达抗CD3 抗Pgp微型双功能抗体 ,并测定该微型双功能抗体的生物学活性。采用PCR和overlapPCR方法构建抗CD3 抗Pgp微型双功能抗体 ,并用双脱氧终止法测定DNA序列 ;采用亲和层析法纯化该产物 ,并用Westernblot和分子排阻层析鉴定纯化产物 ;采用免疫荧光法、放射免疫分析法鉴定纯化产物与靶细胞的结合活性。DNA序列测定结果表明 :抗CD3 抗Pgp微型双功能抗体已构建成功 ,表达可溶性产物的产量达 2mg L以上 ,纯化产物中二聚体的比例达 90 % ,具有与Jurkat(CD3 + )和K5 62 A0 2细胞 (Pgp+ )结合的活性 ,与抗CD3ScFv及抗PgpScFv的亲合常数相当。成功地构建了抗CD3 抗Pgp微型双功能抗体 ,并获得高效表达 。

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the 'pump', and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3 +) and K562/A02 cells(Pgp +).The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab′) 2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP×anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.