| The risk of prostate cancer increases steeply with the growing rate of the elderly and change of life style. It has been much harm to the man. Currently available therapeutic modalities for prostate cancer, such as surgery, radiation, hormone therapy, and chemotherapy, have failed to cure patients because these therapies are not sufficiently tumor-specific, resulting in dose-limiting toxicity. The use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed antibody-directed enzyme prodrug therapy (ADEPT) has attracted considerable interest since the concept was first described in 1987. ADEPE may improve both the efficiency and the specificity of cytotoxic drugs to achieve selective targeting with minimized host organ toxicity. Y -seminoprotein is known to be expressed in a tissue-specific manner. It is a protein expressed exclusively by benign, hyperplastic, and malignant prostatic epithelium. We have been doing experimental research on ADEPT of prostate cancer in recent years. Anti- y seminoprotein monoclonal antibody wasThe Fourth Military Medical University 6produced using conventional hybridoma technology. This hybridoma secretes murine IgGl antibody at relatively high specificity and affinity. We have constructed antibody-enzyme conjugate, therefore ADEPE may offer great promise in this regard. However, when rodent MAbs were used therapeutically, a human antimurine antibody response (HAMA) developed in treated patients. Effector functions of mouse antibodies also proved to be less efficient in the human context. These factors greatly limited their usefulness and dampened interest in Mabs as therapeutic drugs.The resurgence of interest in antibody-based therapeutics is a direct consequence of the introduction of genetically engineered antibodies. Antibody engineering includes chimeric and humanized antibodies, antibody fragments, antibody libraries, antibody fusion proteins and transgenic organisms as bioreactors. As a consequence of refinements in antibody technology, the field of genetically engineered immunoglobulins has matured into an elegant and important drug and reagent development platform. We describe here our research of antibody fragment using antibody engineering to reduce the immunogenicity of murine MAb.The PCR primer sets were designed on the basis of conserved flanking base sequences that exist at the beginning of V gene ( corresponding to the FR1 in the V region ) and at the proximal constant regions. K and Fd gene were amplified by RT-PCR from a murine hybridoma cell line ?87 secreting anti- v seminoprotein monoclonal antibody. Respectively, K and Fd gene were cloned into the eukaryotic expression vector pcDNAS. The expressions of K and Fd activities were clearly observed by immunofluorescent staining analysis when plasmids were tranfected into HeLa cells. K and Fd were cloned into phagemid pCombS to construct the Fab expression vector pComb3-Fab. Phagemids were transformed into XL 1-Blue. Phage displaying Fab was produced from bacteria. For soluble Fab preparation, phagemid DNA was digested with Nhel and Spel. The resulting fragment lacking the gene IIIThe Fourth Military Medical University 7protein was self-Iigated and formed soluble Fab explession vector pComb3-Fabs. Following transformation of XL 1-Blue, the soluble Fab was secreted from bacteria. Soluble Fab exhibited two bands of approximately less than 30KD in reducing SDS-PAGE. The antibody fragment activity was identified. ELISA revealed the antibody fragment had good binding activity to Y seminoprotein antigen . Western blot clearly demonstrated the specificity of the Fab antibody to Y seminoprotein. The binding activity of antibody fragment was obviously detected by immunocytochemistry. Affinity of produced Fab preliminarily measured by non-competitive enzyme immunoassay was about 0. 2 X 109\f'. |
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