Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity

Abstract

The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the K(d) of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

“…Sedimentation equilibrium experiments provide the most direct measure for the equilibrium constants and gave results for the wild type interaction that are consistent with the affinity previously reported (28) (Fig. 4, top panel).…”

“…Each antibody in this class recognizes an antigenic region between amino acids 671 and 721 of PA (17,27). 14B7 and affinity-enhanced derivatives of it have been proposed as potential therapeutic reagents for anthrax (28). The second goal of this work was to identify amino acids in the small loop region of PA domain 4 necessary for interaction with the neutralizing monoclonal antibody 14B7.…”

Immunogenicity of Bacillus anthracis Protective Antigen Domains and Efficacy of Elicited Antibody Responses Depend on Host Genetic Background

“…The higher level of protection afforded by anti-PA1-4 sera relative to that afforded by the truncated forms of PA indicates that all four domains are required for the best level of full protection. The level of protection achieved in our study was comparable to or better than that achieved by other polyclonal antiserum or MAbs (8,17,22,24,45). The observation that host genetics makes a large contribution to the antibody response to PA with regard to the antibody amount, specificity, and isotype suggests that differences in human responses to PA-based vaccines may depend on the genetic background of the individual.…”

“…LF is a zinc metalloprotease that cleaves several mitogen-activated protein kinase kinases. EF is a calmodulin-dependent adenylate cyclase that causes local edema and that impairs neutrophil function (24).…”

Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli -expressed libraries

“…At the heart of the new generation of antibody therapeutics is protein engineering to reduce immunogenicity and increase antigen affinity (2). For example, we have recently reported studies with a series of antibody fragments produced by directed evolution in which toxin neutralization efficacy correlated with antitoxin antibody affinity in an animal model of anthrax intoxication (3).…”