Study On Modification Of Monoclonal Antibody Anti-CD3 With MPEG

At present, anti-CD3 monoclonal antibody has been widely used in Graft rejection reaction after organ transplant in clinic treatment, which has significant therapeutic effects. However, body tends to produce specific humoral immune response because of murine anti-human CD3 monoclonal antibody, resulting that the antibody was rapidly removed, and its half-life was significantly shorter, thereby limiting its therapeutic effects. The modification of proteins with PEG is one of the hotspots in the second development of protein drugs. If connected with drugs, PEG can extend half-life and lessen renal burden. In this paper, murine anti-human CD3 monoclonal antibody was modified with low-molecular-weight mPEG in order to reduce its immunogenicity, lessen renal burden, extend its half-life, and increase water solubility.The experiment was started with pepsin degrading murine anti-human CD3 monoclonal antibody, and the antibody fragment Fab' was then separated by gel column and purified by ultrafiltration. The purity was detected by SDS-PAGE. The single-factor tests showed that the optimization of enzymolysis was as following: the mol ratio of pepsin to antibody reached 2:100, digesting 24 h, pH 3.0, temperature 37℃; and the optimization of deoxygenization was as following: mercapto-ethanol concentration was 0.1 mol/L, digesting 30 min. The antibody fragment Fab' of higher purity was obtained, and the purity of the separated Fab' was greater than 95 %.Then the antibody and its fragment Fab' were modified with PEG-SPA and mPEG was coupled to them. The reaction conditions of PEG-modified mouse anti-human CD3 monoclonal antibody and its fragment were optimized by orthogonal factors experiments. The results showed that the technical process was optimized as following: in the borate buffer (pH 9.0, 25℃), the concentration of the antibody was 0.2 mg/mL, the molar ratio of antibody to mPEG was 1:20, reacted by 3h.The experiment was followed by in vivo and in vitro study on T cell proliferation with PEG-modified IgG and its fragment Fab', which aimed to determine the immune activity of them. The results showed that the inhibitory activity of modified anti-CD3 monoclonal antibody dropped 32.28 %, and PEG-modified Fab' dropped 19.92 % while original Fab' dropped 7.96 %. Though the T-cell inhibition rate decreased, the cytotoxicity was not obviously enhanced.Finally the half-life of the antibody and its fragment after modified in the blood were measured by ELISA.The result manifest: The half-life time in vivo has obviously extend. The T_1/2s of antibody, PEG-antibody, Fab', PEG-Fab'were 2.658347h, 8.722217h, 2.209713h, 4.1723h. respectively, T_1/2s of PEG-antibody are 3.28 times thanT_1/2s of antibody, T_1/2s of PEG- Fab' are 1.895 times than T_1/2s of Fab'. In conclusion, we found it feasible to apply low-molecular-weight mPEG to modify antibody, thus reducing its immunogenicity, extending its half-life, and greatly improving the clinic application of monoclonal antibody, especially non-human type to a great extend.