| BackgroundColorectal carcinoma is one of the most common malignant tumors which have high incidence, occupying the fourth place in our country and the death rate increasing continuously in recent years. Therefore, efficient diagnostic and therapeutic approaches are important for colorectal carcinoma research. Although in recent years some progress has been made in respect to application of monoclonal antibodies (IgG) for the diagnosis and therapy of colorectal carcinoma, most IgG are of murine origin, so that repeated administration can induce human anti - mice antibodies ( KAMA) , which severely limits the efficacy of antibody in clinical utilization. Moreover, intact IgG are generally too large (Mr 150 000) to penetrate the capillaries of tumor masses. To overcome such deficiencies, antibody gene engineering processes are developed, including humanized antibodies, single chain Fv(scFv), human - murine chimeric antibodies, etc. ScFv, which is composed of immunoglobulin heavy - and light - chain variable antibody, is employed most widely at present. The main advantages of scFv over intact IgG and Fab fragment are their small sizes ( Mr 30 000, amounting to one sixth of intact IgG) , making them penetrate a solid mass rapidly and evenly. In addition, the lack of Fc domains in scFv makes them much less immunogenic responsive and less capable of binding to Fc receptors distributing on normal cells. These characteristics make scFv potentially useful in tumor diagnosis and therapy as a carrier.ND - 1 is a murine monoclonal antibody against tumor - associated antigen LEA, mainly expressed in human colorectal carcinoma,developed by Song Jindan in 1986, which was obtained by immunizing Balb/c mice with CCL - 187 human colorectal carcinoma cell line. The histological determination of nearly one thousand pathologic samples showed that ND -1 can bind specially to well differentiated and moderately differentiated colorectal carcinoma tissues and its specificity is superior to IgG against CEA which has been commercialized in the US. In 2001, ND - 1 scFv against human colorectal carcinoma was constructed by fusing gene of variable region of heavy chain with gene of variable region of light chain by a short peptide ( Gly4Ser)3, developed by Fang Jin and the ND - 1 scFv protein was functionally expressed in E. Coli.In this study, we used 99mTc - labeled ND - 1 scFv in in vivo ra-dioimmuno- imaging, biodistribution and pharmacokinetic studies, trying to determine its in invo biological activity so as to provide evidences for clinical diagnosis and therapy of colorectal carcinoma.Methods6-8 week Balb/c female mice produced ascite after being injected with IC - 2 hybridoma cells into abdominal chamber. Purify ND -1 IgG from the ascite by affinity chromatography using HiTrap Protein G column ( AmPharmacia) , collect the proteins at active peak, and then we can obtain ND -1 IgG. The positive bacteria line with pET -28 a ( + ) - ND - 1 scFv was incubated onto LB liquid cultured matrix with Kanamycin, induced by IPTG to express a fusion protein scFv with His - tag in E. coli. BL21, and purified by metal affinity chromatography using Ni - NTA resin. SDS - PAGE analysis and gel brightness scanning of the purified scFv showed the purity of protein. Indi-rect immunofluorescent methods were used to determine the immune activity. Proteins were stored at -20 ℃ for later use.4-6 week female Balb/c nude mice were xenografted under back skin with human colorectal carcinoma CCL - 187 cells. When the tumors have sizes of 0. 5 - 1. 5cm, the nude mice were injected with 99mTc -ND - 1 scFv by tail vein. At 1h, 3h, 8h, 12h, 24h after injection, radioimmuno - imaging of the nude mice was carried on and after imaging, the nude mice were killed. Eyeball blood, tumor, heart, spleen, colon, skeleton, muscle, kidney, lung were weighed, amounts of radiology were counted, and T/NT ratios in different tissues were calculated.After nude mice bearing CCL - 187 tumors were injected with 99mTc - ND - 1 scFv and 99mTc - ND... |
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