| Anthrax toxin is composed of three components: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds to the integrin-like I domains of either the tumor endothelial marker 8 (TEM8) or the capillary morphogenesis protein 2 (CMG2) receptor. It was reported that the soluble tumor endothelial marker 8 and capillary morphogenesis protein 2 (sTEM8 and sCMG2, respectively) receptor decoys bind to anthrax toxin protective antigen (PA) and compete with cellular receptors for binding. The model structure of PA-TEM8 complex was generated in our laboratory. We superpose TEM8 variant L56A may serve as a potent antitoxin receptor decoy. TEM8 was reported to be selectively over expressed during tumor angiogenesis. Therefore, TEM8 maybe a specific tumor cell marker. It was reported that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.The aim of our work was to desin the anti anthrax toxin receptor decoy (TEM8 variants) and tumor targeting anthrax toxin (PA variants) base on the crystal structures of PA and its receptors complex. The variants were screened in vitro and in vivo.Section One: Pharmaceutical Study of Anti-anthrax toxin TEM8 Mutant Receptor Decoy based on the Interaction between Anthrax Toxin and Its Receptor In this study, the soluble CMG2 vWA domain (sCMG2), sTEM8 and mutant L56A were obtained by expression and purification on the basis of previous studies. sTEM8, sCMG2 and L56A proteins were tested over a range of concentrations for their capacities to inhibit intoxication of J774A.1 cells by LeTx (PA and LF). The IC50 were respectively 274.6±8.7 nM for sTEM8, 69.5±5.8 nM for L56A, and 20.8±1.5 nM for sCMG2. sCMG2 showed the strongest inhibition on LeTx. In vivo lethal toxin (LeTx) challenge experiments demonstrated sTEM8 could fully protect the rats when the molar ratio of sTEM8 and PA was 5:1. sCMG2 and L56A could fully protect the rats when the molar ratio of sCMG2 and PA was 3:1 and 1:1. The survival time of rat was 115.3±9.7 min, when the molar ratio of L56A and PA was 0.6:1; the survival time of the rat was 91.7±4.1 min, when the molar ratio of sCMG2 and PA was 0.6:1. There was statistical differences between them (P=0.0350), indicating the activity of anti-anthrax toxin of L56A in rats was stronger than that of sCMG2, the current most effective anthrax receptor decoy.In order to explore the real reason for differences of receptor decoy between in vivo and in vitro and to reveal the in vivo action rule of soluble anthrax toxin receptor decoy, we introduced pharmacokinetics to study the absorption, metabolism, distribution and excretion processes in vivo of the three soluble receptor decoy. The method of anti-anthrax toxin receptor decoy in rat serum and tissue by isotope labeling was developed and validated. The concentration and specific activity were respectively 248.51μg·ml-1 and 141.23 kBq·μg-1 for 125I-L56A, 135.91μg·ml-1 and 198.19 kBq·μg-1 for 125I-sTEM8, and 370.89μg·ml-1 and 56.67 kBq·μg-1 for 125I- sCMG2. For body fluids and tissues sample, 125I-L56A, 125I-sTEM8 and 125I- sCMG2 mainly exist in TCA precipitation. Therefore, TCA precipitation radioactivity was used for pharmacokinetic study. The sensitivity, linearity, precision and recovery of the method were investigated and meet the need of pharmacokinetic study. Pharmacokinetics of 125I-L56A, 125I-sTEM8 and 125I-sCMG2 was studied. Three labeled proteins were administered i.v. at dose of 13.5μg/rat. The blood and urine samples at 5min, 30min and 4h after administration were collected for molecular size exclusion radioactive chromatography. The results showed that the three labeled proteins metabolized rapidly and the bind of 125I-L56A and 125I-sTEM8 to serum albumin prolonged their in vivo acting time, and that is, their elimination half-life were extended. However, 125I-sCMG2 does not bind with serum albumin, remaining unchanged. This may be one of the reasons for stronger anti-anthrax toxin activity of L56A in rats compared to sCMG2, although in vitro intoxication of L56A was lower than sCMG2. This has been proved by pharmacokinetic parameters.Non-compartment model was used to calculate pharmacokinetic parameters. The elimination half-life of sCMG2, sTEM8 and L56A were 0.4±0.1h, 0.8±0.1h and 0.8±0.0h, respectively.Tissue distribution was studied at 5min, 30min and 4h after i.v. administration of 125I-L56A, 125I-sTEM8 and 125I-sCMG2 at dose of 13.5μg/rat. The results showed that the target organ of 125I-L56A and 125I-sTEM8 was lung, while the target organ of 125I-sCMG2 was kidney. Now, the studies were proved that lung was the major site during anthrax infections. Therefore, the difference of tissue targeting between L56A and CMG2 also may be a reason for stronger anti-anthrax toxin activity of L56A in rats compared to sCMG2 although in vitro intoxication of L56A was lower than sCMG2.Section Two:Design of anti-tumor PA variants of TEM8 Receptor Selectivity Based on the Interaction between Anthrax Toxin and its ReceptorBased on the structural basis of the difference between PA-TEM8 and PA-CMG2, we compared the ligand binding sites of PA Domain II with TEM8 and CMG2. We presume that 340LAGE343 loop in PA Domain II were key resides resulting in the difference of binding affinity between TEM8 and CMG2, especially resides L340 and A341. We designed a number of PA single point mutants for TEM8 receptor selectivity (L340R, A341E, A341D) and multiple site mutants (L340R+A341D, L340R+A341E, L340R+R659D, L340R+R659D+M662R (BD7)). By cloning, expression and purification, proteins other than A341D were obtained.Mouse macrophage J774A.1 cells high expression of CMG2 were used as a model to screen PA variants for safety evaluation. Human cervical cancer Hella cells were selected as having high TEM8 gene expression for effectiveness evalution. PA BD7 was selectively toxic to Hella cells. BD7 may be useful as a therapeutic agent for cancer treatment. BD7 was more effective and safer than PA R659D /M662R (D7). To further exploit the receptor selectivity of BD7 for specific human tumor cells, several tumor cells were selected, including NCI-H460, DU-145, SMMC-7721 and breast cancer MDA-MB-231 cells. NCI-H460 was the most selective to BD7+FP among these tumor cells.Athymic nude mice were inoculated s.c. with NCI-H460 cells. Four groups of mice were then treated i.p. with high, mid, low doses of BD7+FP, high, mid, low doses of D7+FP59, PA+FP or vehicle alone (PBS), every 3 days for three total injections. The body condition, body weight, tumor growth inhibitiotion and histologic analysis demonstrated that D7+FP was more toxic than BD7+FP to normal tissue of mice, which was BD7+FP was safer than D7+FP. Preliminary experiment showed that BD7 with 0.04 mg/kg~0.2mg/kg might has the anti-tumor effects. BD7+FP 0.15mg+ 0.02mg/kg was the maximum tolerated dose in mice base on anmimal physical state and histological analysis. The growth inhibitor of tumor in vivo was conducting based on this dose.In conclusion, anti anthrax toxin receptor decoy (TEM8 variants) and tumor targeting anthrax toxin (PA variants) was designed base on the crystal structures of PA and its receptors complex. TEM8 variant L56A was potent anti anthrax toxin receptor decoy in vivo. L56A could bind to serum albumin with obvious slower degradation rate compared to sCMG2. Tissue distribution indicated that L56A targeted at lung which was the major site during anthrax infections, while sCMG2 at kidney. These may be the main reasons why L56A had stronger anti-anthrax toxin ability in vivo compared to sCMG2. PA variant BD7 was potent tumor targeting anthrax toxin. L56A and BD7 were both hopeful. This work proved design of anti anthrax toxin receptor decoy (TEM8 variants) and tumor targeting anthrax toxin (PA variants) base on the crystal structures of PA and its receptors complex was feasible. In future, the crystal structures of PA and its receptors complex was target site of anthrax toxin receptor decoy and tumor targeting anthrax toxin. |
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