Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development

Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. potential as a combination anthrax prophylactic and treatment. Here we statement the high resolution X-ray structures of three high affinity single chain antibodies in the 14B7 family; 14B7 and two high affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA Chlorantraniliprole structure, M18 in complex with PAD4 at 3.8 ? resolution. These structures provide insights into the mechanism of neutralization and on the effect of various mutations on antibody affinity and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4. Introduction Anthrax remains a significant threat as a biological weapon due in large part to its ease of both large-scale manufacture and weaponization in the spore state. Following spore inhalation, anthrax is usually lethal in humans due to the combined actions of secreted toxins.1, 2 An effective countermeasure strategy requires an effective anti-toxin therapy 3C19 to be used in combination with antibiotics, or as a stand alone treatment of an antibiotic resistant strain of anthrax.20 We, as well as others, have been developing a combination prophylactic-post exposure therapeutic for anthrax based on an engineered antibody against the anthrax protective antigen (PA) toxin.7C25 Briefly, the PA toxin facilitates host cellular targeting and transport of the lethal factor (LF) and edema factor (EF) into the cytoplasm. LF is usually a protease that targets mitogen-activated protein kinase kinases (MAPKKs) and EF functions as an adenylate cyclase. The action of LF and EF in the cytoplasm of target cells triggers a series of biochemical events that lead to cell death.1, 2 The intoxication process is initiated when monomeric full-length protective antigen (PA83) is processed by host proteases to form the PA63 fragment, which binds as a heptamer with high affinity to the TEM8 and CMG2 cellular receptors on host cells such as macrophages. Post-exposure administration of high affinity antibodies that block the PA-receptor conversation has been shown to be effective in reducing mortality in animal models.21C25 Anti-PA antibodies can also serve as prophylactics to prevent infection from spore inhalation, even though mechanism of prophylaxis is not well understood.20, 26C29 The 14B7 Chlorantraniliprole murine monoclonal antibody (KD = 4.3 nM),11 originally developed at USAMRIID,12 was shown to delay time-to-death following exposure to anthrax spores in a guinea pig model.24 14B7 is known to recognize the receptor-binding region of PA and thereby block PA-host cell interactions.30 Originally, we used phage display to isolate an affinity enhanced version of the 14B7 variant called 1H, exhibiting a KD of 250 pM.13 A humanized version of this antibody is currently in advanced clinical development.20 The approximately 20-fold affinity enhancement of 1H compared to 14B7 is usually achieved with two mutations, Q55L and S56P, in CDR L2. In subsequent studies, an even higher affinity variant of 14B7 called M18 was isolated from a library of random mutants screened by bacterial display and circulation cytometry.11 M18 has 10 mutations (light chain I21V, L46F, S56P, S76N, Q78L, and L94P; heavy chain S30N, T57S, K64E, and T68I) and exhibits a KD of 35 pM. Chlorantraniliprole Crystallographic studies of antibody fragments in complex with a protein antigen Chlorantraniliprole have been ongoing for more than 25 years.31C40 Generally, antibodies to protein antigens target a discontinuous epitope around the antigen.32 It is also common for all those 6 complementarity determining regions (CDRs) of the antibody to interact with the antigen32,40C42 and, on occasion, for framework residues to make contact as well.32 Shape complementarity along the conversation surface appears to be important,35,40,43 and a nonpolar hotspot is generally found to contribute the majority of the binding energy. A study of the affinity maturation of antibodies to lysozyme revealed that improved shape complementarity and burial of nonpolar surface at the expense of polar surface were generally correlated with increased affinity.35 In addition, Rabbit polyclonal to Catenin alpha2 structural studies with small molecule haptens have indicated that affinity.