Site-directed Chemical Modification of Recombinant Human aFGF Mutant with Polyethylene Glycol

Chinese Chemical Letters Vol. 16, No.8, pp 1013-1016, 20051013http://www.imm.ac .cn/jourmal/ccl.htmlSite-directed Chemical Modification of Recombinant Human aFGFMutant with Polyethylene GlycolQing ZHENGl2, Zhi Feng HUANG', Xiao Ping WU*, Zhi Jian SU, Hua XU',Wen ZHAO, Ya Dong HUANG', Xiao Kun LI2'College of Pharmacy, Jinan University, Guangzhou 510632'Biopharmaceutical R&D Center of Jinan University, Guangzhou 510630Absract: A new product PEGylated rhaFGF was obtained by site-directed chemical modification.When compared with unmodified thaFGF, PEGylated rthaFGF showed comparable bioactivity andsuperior stability at 37°C in mouse serum and the stronger resistant potency to trypsin. This wasaccompanied by a substantial decreasing immunogenicity. Site-specific PEGylation of rhaFGFmay increase its therapeutic potency in humans.Keywords: rhaFGF, chemical modification, PEGylated rthaFGF.Human acid fibroblast growth factor (haFGF) is a cytokine which can stimulate all thecells of mesodermal and neuroectodermal origin to divide.As an efficient tissuerepaired stimulator and a cardio- and neuroprotector, haFGF has a unique potential forwound healing, bone damage curing and cardio- and neural disease treatment-. Busome defection of haFGF, such as low stability, short half life in vivo and strongimmunogenic activity, limited its clinical application.It has been proven thamodification of proteins with polyethylene glycol (PEG) was an efficient method toenhance the stability as well as to lower the immunogenic activity of proteins'. In thispaper, a new product PEGylated rhaFGF was obtained by site-directed chemicalmodification. The results indicated that the mitogenic activity of PEGylated rhaFGFwas comparable to that of native rhaFGF in vitro, while the resistant potency ofPEGylated rthaFGF to temperature and trypsin was much stronger than that of nativerhaFGF and the immunogenicity of PEGylated rhaFGF was lower than that of the latter.It seems the PEGylated rhaFGF generating by site- directed PEGylation of recombinantrhaFGF is more suitable for therapeutic application.ExperimentalBefore site-directed PEGylation of rthaFGF, a rhaFGF mutant was constructed byreplacing the 98 th and the 132 nd cysteine residue with serine residue. The mutant,which retained the bioactivity of rhaFGF, was then copinnated with 5 9 nlvrathylene中国煤化工"E-mail: xp_ _wu@ hotmail.comYHCNMHG1014Qing ZHENG et al.Figure 1 Reaction of rthaFGFg. 132 and mPEG5KCys31CH2OrhaFGF9s, 132+ottomPEGylated rhaFGF9s, 132mPEG5Kglycol-maleimide at the 31st cysteine position (Figure 1)A typical procedure for preparation of PEGylated rhaFGF mutant is as follows:rhaFGF mutant with a free thiol group in PBS (pH=7.2) was allowed to react with30-fold molar excess of 5kD mPEG-maleimide (mPEG5K) at 25°C for 4 h. Thereaction mixture was directly loaded on a Sephadex G-25 (Amersham Phamacia) columnto eliminate salt and loaded on a Heparin SepharoseTM CL-6B (Amersham Phamacia)column previously equilibrated with 20 mmol/LPB (pH=7.2). Washing the columnwith buffer A (20 mmo/LPB(pH=7.2)、After the UV absorbance trace return to thebasal, the column was eluted with buffer B (0.6 mol/L NaCl in 20 mmol/L PB (pH=7.2)).Collecting the peak fraction to get the PEGylated rhaFGF mutant. The purity of theproduct was up to 99%, according to the result of SDS-PAGE analysis.The mitogenic activity of native and PEGylated rhaFGF was assessed by MTTmethod. The stability of native and PEGylated rhaFGF was determined by incubatingthem at a final concentration of 1 mg/mL at 37°C in mouse serum.The remainingmitogenic activity of the treated sample was checked by MTT method. The resistantpotency of native rhaFGF and PEGylated rhaFGF to trypsin was determined bySDS-PAGE of the equal molar samples, which were incubated with trypsin at 37°C for 0,5, 10 and 30 min, followed by densitometry scanning. Immugenicity of native andPEGylated rhaFGF was analyzed as follows: Inject the male BAL B/c mice with nativend PEGylated rhaFGF at doses of 1.875μumol/mouse in PBS (pH 7.4, 50 mmol/L)containing FCA.The mice were immunized again two weeks after the firstimmunization. The specific IgG levels in serum were assessed by ELISA.Result and DiscussionThe mitogenic activity of PEGylated rhaFGF is 55.53% of native rhaFGF. Comparedwith other reported proteins modified by site- directed PEGylation 4, PEGylated rhaFGFshowed comparatively high bioactivity. That the chemical modified position at the 31stCys residue is far from the active site of rhaFGF, wh中国煤化工inal', itcould be the reason for high bioactivity remaining of IfHCNMHGChemical Modification of Recombinant Human aFGF Mutant1015The heat stability of native and PEGylated rhaFGF was assessed by incubating themin mouse serum at 37°C for various periods of time (Figure 2). The mitogenic activityof native rhaFGF diminished in a time-dependent manner. In contrast, PEGylatedrhaFGF was quite stable. After 48 h, PEGylated rhaFGF retained about 70.51% of theirinitial activity.The result indicated that the resistant potency of PEGylated rhaFGF to trypsin washigher than that of the native rhaFGF. Incubated with trypsin for 30 min PEGylatedrhaFGF retained about 32% of its amount, however, the native rhaFGF was almosthydrolyzed.The reason of these results is possible that the PEG sterically hinders rhaFGF fromproteolytic attack6.Figure 2 Stability of PEGylated rhaFGF in mouse serum.8 120100|I80▲5040200244872incubating time (h)a-native rthaFGF; b PEGylated rthaFGFFigure 3 IgG responses in mice to PEGylated rhaFGF.0.10.08.20.0620. 040.021-native rthaFGF; 2-PEGylated rhaFGF; 3-中国煤化工YHCNMHG1016Qing ZHENG et al.PEGylated rhaFGF was also found to have decreased immunogenicity (Figure 3),which could be attributed to reduced degradation by antigen presenting cells, shieldingof some epitopes of peptides after degradation，and prevention of binding to receptorson B cells. Additionally, the transportation of PEGylated rthaFGF from blood toimmune tissues such as spleen, bone marrow, and lymphoid tissue may be limited*.The nonspecific binding and uptake of PEGylated proteins by antigen-presenting cellsmay also be reduced.Detailed studies on tissue distribution and cell-binding arenecessary to clarify these issues.With higher stability and lower immunogenicity, the new product PEGylated is ofgreat interest for developing therapeutic application. The approach used to modifyrhaFGF could be applicable to other recombinant growth factors modification aimed atimproving the stability and immunogenicity of them.AcknowledgmentsThe Hi-tech Research and Development Program of China (2002AA2Z3318), Guangdong NaturalScience Foundation (010424) supported this study.ReferencesT. N. Mellin, R. J. Mennie, D. E. Cashen, et al, Growth Factors, 1992, 7 (1),12141.2. T. D. Bjornsson, D. M. Ryjski, T. J. luczek, et al, Proc. Natl. A cad. Sci. USA, 1991, 88 (19),3. P. Rameshraja, W. Huashan, G. Anil, Adv. Drug Deliv. Review, 2000, 40, 185.Y. Jiang, S. W. Xu, R. Gao, Polymer Bulletin, 2002, 2, 34.P. 1. Wong, B. Hampton, E. Szylobryt et al., J. Biol. Chem, 1995, 270 (43) , 25805.C. TKuan, Q. C.Wang, I. Pastan, J. Biol. Chem., 1994, 269, 7610.7. Q. C. Wang, P L. Hai, W.Debinski et al, Cancer Res., 1993, 53, 4588.8. Y. S. Tsutsumi, O. D. Masanori, S. S. Nagata, Medical science, 2000, 97(15), 8548.Received 27 Augest, 2004中国煤化工MHCNMHG