Preparation Of McAb Against EGF-PE40, Purification Of EGF-PE40 And Determination Of Antigenicity Of EGF-PE40

Dong BingEGF-PE4O, recombinant toxin consisting of human epidermal growth factor (EGF) fuse to N?terminate of truncated Pseudomonas exotoxin A (PEA), is a novel immunotoxin expressed in E.coli. There are three domains of PEA. Studies have shown that domain La has an important role in binding to target cells, domain Ii is required for efficient translocation of the toxin into the cytosol, and domain III contains the portion of the molecule that ADP ribosylates elongation factor 2 rendering it inactive in protein synthesis and resulting in cell death. Removal of domain Ia results in a truncated Pseudomonas exotoxin molecule whose molecular weight is 40,000 (PE4O). EGF-PE4O can selectively bind to cells overexpressing the EGF receptor (EGFR, characteristic of many human tumors of epithelial origin, such as in cancer cells of the breast, lung and ovary) by ligand EGF and trans-membrane and intoxicates cancer cells by PE4O.Purifying EGF-PE4O from crude extract by series of column is the traditional method by which causes two defects: producing rate decreasing and lose of biological activity. Here we report to purify EGF-PE4O by immunoaffinity chromatography. In this research, the McAb to EGF-PE4O, D7, was prepared and purified, its affinity constant to EGF-PE4O was 2.24 X iO~ (mol/Ly1, and its class and subclass was determined also (IgGl). Purified McAb was coupled to CNBr activated Sepherose 4B and subsequently used to purify EGF-PE4O from the crude extract. EGF-PE4O purified by this method was 80% higher rather than the purity of crude extract (20%). And the purified products activated to cancer cells (I-Iep-2).But continuous injections of EGF-PE4O will cause significant titres of antibody in patients?serum so as to reduce the effect of EGF-PE4O. Previous studies showed that the method to determine responsive antibody level was ELISA which could not measure the antigenicity of heteropolymer thoroughly. In this study, agar double diffusion was used to determine 搉eutralizing antibody?which indicated the thorough antigenicity of heteropolymer. EGF49PE4O was injected i.v. to rabbits every day in a continuous 28 days (one course). The sera of saline injection and EGF-PE4O with Freund抯 adjuvant were used as negative and positive controls, respectively. The results showed that binding antibody level in determined sera was rising as injection times went on. The highest titre got 1:252 at 28 days. At the same time, we used rabbits sera as contrast, which were injected EGF-PE4O and cyclophosphamide (doubled human dose), irnniunology inhibitor, simultaneously, to determine the neutralizing antibody level under low immune body condition. The results indicated that the binding antibody level in sera of this group was lower than that of the group which weren抰 injected cyclophosphaniide. But there was no significant difference (p>0.05) with the experimental dose. The serum, which owned the highest titre of binding antibody level at 28 days, acted with different concentrations of EGF-PE4O (80.. 40.. 20.. 10~. 5.. 2.5 mgfL) and the results showed no precipitation. It meant that there were no neutralizing antibodies could be determined after continuously injection of EGF-PE4O in 28 days. EGF-PE4O of different concentration was mixed with the determined serum, the negative control serum (health rabbit serum) and the positive control serum. After 24 hours acted at the room temperature, the mixtures were acted with the positive control serum by agar double diffusion, respectively. All of them showed precipitation except the mixture of the positive control serum and EGF-PE4O whose concentration was 35 mg/L. It meant that, by contrast with the positive control serum, neutralizing antibodies in determined sera were not sufficient to reduce the concentration of EGF-PE4O.