Anthrax Prevention Fc Fusion Protein

Anthrax is a serious infection caused by Bacillus anthracis, a lage Gram-positive, rod-shaped, aerobic and spore-forming bacterium. Anthrax is so terrible, not due to bacteria, but rather anthrax toxins released by Bacillus anthracis. In fact,it is sensitive to most antibiotics, such as Penicillin, ciprofloxacin and doxycycline, which can kill Bacillus anthracis. There is a 5-7 days incubation period for anthrax, and which shows no any symptom, however, Bacillus anthracis often wantonly breeding in body during this perid and release tremendous toxins. Althought antibiotics can kill many Bacillus anthracis later, tremendous toxins released by bacterium has already be above the level which human body can endure.The gene ecoding anthrax toxin located in Bacillus anthracis pX01 plasmid, those toxin proteins consist of three proportions, namely, protective antigen (PA), lethal factor (LF) and edema factor (EF). PA mediated LF/EF into the cells, when PA combined with PA receptors(ATR/CMG2) on the target cell membrane, seven ligand-receptor units self-assemble into a heptameric ring-like complex channel, and LF or EF binding PA enter target cell by this channel. Therefore, designing molecule which interfer PA binding PA receptors is a good means to cure anthrax.According to the mechanism of anthrax toxin invading cell, several fusion genes were constructed, namely, combining PA domain 4(596-735aa) with human IgG1 Fc gene, CMG2(1-225aa) with Fc, and TEM8(1-227aa) with Fc. Compared with other PA antibody, those molecules have following characteristics. (1) CMG2 and TEM8 are nature physiological molecules in human body, the immunogenicity problem does not exist. (2) those fusion molecules have long half-life due to Fc fragment. (3) APC cell could easily intake Bacillus anthracis protective antigen by Fc. (4) CMG2Fc/TEM8Fc molecules could avoid PA mutations which interfere with PA monoclonal antibody neutralization without significantly compromising the function of this toxin subunit. (5) those fusion proteins could be easily purified by protein A affinity chromatography.This study successfully obtained PA4-Fc, CMG2-Fc and TEM8-Fc gene expression vector by molecular cloning technique. By transfecting CHO-K1 cells and G418 pressure selection, three monoclonal cell lines expressed those proteins were got. ELISA results showed that those protein in CHO cells have higher expression(≈20-30mg/L). A total of about 400mg protein were harvested by serum-free bottle culture (PA4-Fc≈120mg, TEM8-Fc≈180mg, CMG2-Fc≈100mg). Reduction and non-reducing protein electrophores results show that it is a single band for CMG2-Fc(95.4kDa by Ms assay) and TEM8-Fc(111kDa by Ms assay).However, there are three bands for PA4-Fc. Deglycosylation result shows that it isn`t caused by heterogeneous glycosylation of protein. By adding protease inhibitors and selecting host cells, the study showed that transfection of BHK-21 host cell could reduce degradation of PA4-Fc, however, which cause glycosylation heterogeneity. In this study, association of CMG2-Fc with PA, TEM8-Fc with PA, and PA4-Fc with TEM8 were messured by ELISA and GST pull down method. Those results show that they all have strong association. The affinity of CMG2-Fc with PA(KD=1.31×10-11 mol/L, messured by fortéBio QK) is higher than TEM8-Fc(KD=3.26×10-8 mol/L), and the KD of PA4-Fc with TEM8 is 2.89×10-8 mol/L (messured by BIA2000).Murine RAW264.7 and J774A.1 macrophages are sensitive to anthrax lethal toxin. The ability of three Fc-fusion proteins against anthrax toxin were assayed by murine J774A.1 macrophages. Results show that CMG2-Fc and TEM8-Fc could inhibit anthrax toxin from attacking murins macrophage, in which the protective effect of CMG2-Fc was better than TEM8-Fc (IC50 were 0.49 nM and 500 nM). PA4-Fc can not antagonize the damage caused by anthrax toxin, which was probably because there are too many PA receptor on cell surface, and PA4-Fc has poor binding capacity with PA receptor than wild-type PA, so PA4-Fc can not completely inhibit PA from binding PA receptor, And when a small amount of anthrax toxins entered into cells, the cells could be killed.Fisher 344 rats are commonly used for evaluating drug`s prevention and treatment of anthrax toxin. It could be killed by a single injection of small amount of anthrax toxin (40μg PA and 8μg LF), so Fisher 344 rats were selected to evaluate CMG2-Fc inhibiting anthrax toxin in vivo. When the mole ratio of CMG2-Fc with PA is 2:1, CMG2-Fc could protect F344 rats against anthrax toxin attacking. The result shows that CMG2-Fc as anti-anthrax toxin drugs may be further researched and evaluated.In addition, the study also compared the immunogenicity of PA4-Fc with PA by immune rabbits, and detected the role of Fc fragment on PA4 immunogenicity by immune BALB/c mice. Cell protection tests showed that PA4-Fc immuneserum, PA immuneserum and PA4 immuneserum were able to antagonize anthrax toxin to injure mouse macrophage cell. However, protective effect of PA4-Fc immuneserum was weaker than PA (IC50 were 1/22 and 1/53) and PA4 (IC50 were 1/57 and 1/170). The phenomenon may be due to PA immuneserum not only blocks the binding domain of PA with the receptor, but also inhibits the sites which PA self-assembly and binding LF / EF.To further enhance CMG2-Fc binding ability with PA, according to crystallography structure of CMG2 and PA, this study designs eight CMG2-Fc mutatioin, namely, N57L, E117Q, T118K, Y119Q, H121E, K125E, D148K and Y158Q. In addition, to observe the disulfide bonds(C38-C218) effect of CMG2 on the impact of PA with CMG2, this study also built CMG2 (1-217aa)-Fc cut molecules, and obtained those protein by mammalian cell expression. Cell protection assays show that E117Q mutation enhanceed the affinity of CMG2-Fc and PA , and was more effective against anthrax toxin attack (IC50 = 0.15 nM) than wtCMG2-Fc, and T118K, H121E, and D148K mutations severely reduced the CMG2-Fc binding PA, and losted the ability of inhibiting anthrax toxin, suggested that these loci have an important role in the function of CMG2. In addition, compared with CMG2-Fc protein molecule, the truncated CMG2(1-217aa)-Fc shows weak binding capacity, and it′s ability to inhibit anthrax toxin(IC50 = 20nM) was smaller than CMG2-Fc (IC50 =0.49 nM), prompted that the extracellular disulfide bond of CMG2 is critical for maintenance CMG2 structures and binding PA.