Molecular Design And Efficacy Evaluation Of Receptor Decoys As Drug Candidates Against Anthrax

Anthrax is a deadly zoonotic infectious disease and its pathogen is Bacillus anthracis. Spores of B. anthracis has high resistance to the environment and is easily preparated, which is a potential biological warfare agent and biological terrorist agent. Virulence factors of B. anthracis mainly include bacterial capsular and exotoxin. Capsules can help anthrax bacteria to evade the host immune system attack so as to promote massive breeding of the bacteria in the body. Exotoxin, concluding three protein components: protective antigen(PA) and lethal factor(LF) and edema factor(EF) of, is the main reason resulting in death of the host. After the combination to the target cell surface receptor(CMG2 or TEM8), PA mediated LF and EF into cells and induce the toxicity.Antibiotics is mainly used for the treatment of anthrax in the clinic, but antibiotics can only kill B. anthracis and at the same time is unable to suppress anthrax toxin.Thus, the fact that the poor treating effect for patients with advanced and severe stage of infection calls for specific drugs targeting anthrax toxin. The present study drugs against anthrax toxin include monoclonal antibodies, specific immunoglobulin, small molecule inhibitors etc. Our previous studies and other laboratory reports showed expression of recombinant soluble anthrax toxin receptor fragment(called “receptor decoy") could competitive binds to anthrax toxin against cell surface receptor, thereby blocking the toxin into cells and is a new type of anthrax toxin inhibitor. In this study, we optimize the design and evaluate the efficacy of receptor molecules gained in our previous study.Our main content includes: extending in vivo half-life of receptor molecules through fusion with Ig G Fc fragment of antibody or human serum albumin(HSA) etc.; improving the affinity of receptor molecules with PA by effectting combinatorial mutagenesis sites of PA affinity critical amino acid; determining receptor drug candidate molecules structure by comparing and analyzing the efficacy of the candidate molecules using cell and animal models, laying the foundation for the researching a new type anti anthrax drugs.CMG2 is the main cell receptor in the process of anthrax infection. The study showed that the affinity of s CMG2(soluble CMG2 fragment) to PA was high, with good resistance to anthrax toxin activity in vitro and in vivo. However, s CMG2 has a short half-life in the plasma which influence it become a formal drug. Fusion of antibody Ig G Fc fragment is a commonly used method to extend half-life of the protein drugs in vivo. Our study firstly constructs the s CMG2 and human Ig G1 Fc fusion expression plasmid, then transient transfected 293 F cells and finally obtained purified fusion protein(CMG2-Fc) after purified by protein G column. In the anthrax toxin sensitive J774 A.1 cell model, the biological activity of CMG2-Fc and s CMG2 were comparative analyzed. The toxin neutralization activity IC50 is respectively 1.45 n M(CMG2-Fc) and 2.03 n M(s CMG2), showing that s CMG2 activity was not affected after the integration of Fc. CMG2-Fc was injected into SD rats by caudal vein and the plasma CMG2-Fc levels were detected by ELISA method. The in vivo half-life of CMG2-Fc was estimated to be 29.98 h, which was about 60 times longer than that of s CMG2. We compare the CMG2-Fc and s CMG2 toxin inhibitory activity In Fisher344 rats models by evaluating in vivo protective effect after anthrax lethal toxin(Le Tx,20 μg r PA+10 μg r LF) attack. The results showed that CMG2-Fc can significantly prolong the survival time of rats, but not protect rats survived. Howerver, s CMG2 can completely protect rats. It is postulated that the reason may be that the Fc’s own some features such as ADCC or CDC effect which influence CMG2-Fc in vivo activity, though it needs further experimental verification. The results showed that the Fc fusion significantly improved s CMG2 half life, but inhibitory activity of the toxin in vivo decreased, preventing it serving as an ideal molecular design for the class of receptors.Fusion with human serum albumin(HSA) is another common strategy to improve the half-life of protein drugs. This research constructed the s CMG2 and human serum albumin(HSA) fusion protein(HSA-CMG2), connected HSA and s CMG2 by 3 × G4 S linker, inserted the gene fragment into the yeast expression vector p MEX9 K, electric transfected Pichia pastoris GS115, undergo methanol induced secretory expression, then obtain the purified protein through blue affinity chromatography and anion exchange chromatography. The half-life of HSA-CMG2 in rats was 5.01 h, which was 10 times longer than that of sCMG2 in vivo. The results of toxin neutralizing activity in J774 A.1 cell model showed that the IC50 of HSA-CMG2 was 1.83 n M, which was similar to that of s CMG2 and CMG2-Fc. We evaluate the protective effect in Fisher344 rat model in vivo.HSA-CMG2 and Le Tx was premixed and was injected into the rat again through the tail vein. The results showed that the toxin molar ratio of 2:1 can protect rat survival. Before the rats injected with Le Tx, HSA-CMG2 and s CMG2 group were all survival in prevention administration in 5 minutes group. However, in 24 hours in advance to medicine, HSA-CMG2 can significantly prolong the survival time of rats, but survival time of s CMG2 group and the control group(challenged) had no significant difference. The above results indicate that the fusion of s CMG2 and HSA not only prolongs the half-life, but also maintains a higher toxin inhibitory activity. It is an ideal candidate for the class of receptor drugs.TEM8 is another anthrax toxin receptor. The inhibitory activity of s TEM8(soluble TEM8 fragment) to anthrax toxin is quite low due to its affinity to PA was 10 times lower than that of CMG2. According to the previous structure prediction, we design s TEM8 mutant L56A(in this study it is called MT2) and the affinity to PA was increased significantly. On this basis, we obtained a new MT4 receptor molecule by combined multiple mutations of the key amino acids which affects s TEM8 and PA affinity. Recombinant MT4 was prepared by the E.coli expression system. In vitro affinity assay revealed that the affinity of MT4 with PA was 0.374 n M, higher than that of s CMG2(1.01 n M) and MT2(0.52 n M). Cell model test showed that IC50 of MT4 was close to s CMG2(2.29 n M and 1.46, respectively), better than MT2(4.45 n M), and was nearly 12 times more than s TEM8(27.2 n M). The rat experiment showed that s CMG2 and MT4 were able to protect the rats 100% when the molar ratio to Le Tx was 1:1, while the protection rate of MT2 was 40%. The results showed that MT4 based on the s TEM8 mutant significantly increased the affinity with PA, and had a similar inhibitory activity with s CMG2.MT4 also has a short half-life in vivo, this study constructed MT4 and Ig G1 Fc fusion protein(MT4-Fc). But the test results showed that the toxin inhibitory activity in cells decreased about 10 times after MT4 fusion with Fc. Due to MT4-Fc and MT4 was respectively prepared in E.coli expression system of 293 F cells, the decreased activity of MT4-Fc may be related to glycosyl modification in the cell expression system. We modified two potential glycosylation mutation site of MT4, the activity of mutants MT4 D and MT4 expressed in E.coli has no significant difference. Neutralization activity to toxin of MT4D-Fc expressed in 293 F cell partial recovered compared to MT4-Fc. Therefore, we need to consider the effect of glycosylation of receptor on toxin neutralizing activity when using class of receptor molecules s TEM8 from mammalian cell.In this study, MT4 was further fused with HSA refered to HSA-CMG2 and HSAMT4 was obtained in yeast expression system. Half-life of HSA-MT4 in rats is about 5 h. Cell model test showed MT4 and HSA-MT4(IC50 2.95 n M) has similar toxin neutralizing activity. Test in rat showed injection of premix of HSA-MT4 with Le Tx can completely protect the animals’ survival. The above results indicate that HSA-MT4 can also be used as a candidate molecule for the class of receptor drugs.In summary, this study constructed and purified a new anti-anthrax receptor molecules HSA-CMG2, prolonging the s CMG2 half-life in vivo, observing ideal toxin inhibitory activity in vitro and in vivo. We also designed receptor like molecule MT4 based on s TEM8, with high affinity to PA and similar toxin inhibitory activity compared with s CMG2. HSA-MT4 resulted from HSA fusion prolonged the MT4 half-life in vivo. HSA-CMG2 and HSA-MT4 can be used as candidate molecules of anti-anthrax receptor drugs, and lay the foundation for research and development of new type of specific anthrax therapeutic drugs with independent intellectual property rights.