| There is a need for therapeutic reagents to biothreat agents such as Bacillus anthracis. A major virulence component of B. anthracis is a secreted tri-partite toxin including protective antigen (PA), lethal factor (LF) and edema factor (EF). The individual proteins are non-toxic, however combinations of PA and LF (lethal toxin, LeTx) or PA and EF (edema toxin) produce toxins capable of inducing toxic shock or edema, respectively. Neutralization of PA toxin has largely been attributed to antibody responses engaged to domain 4 of PA, a region key to binding to the host cell receptor. However, a LeTx neutralizing murine mAb, F20G75, has been generated in our lab and has been shown to bind via a defined epitope within the 2beta2-2beta3 loop region in domain 2 of PA (Gubbins et al., 2006). Herein, a novel intronless two-plasmid human IgG1/k mammalian cell antibody expression system was developed for the generation of murine-human chimeric mAbs. Within the system, PCR-amplified heavy and light antigen-specific variable (binding) domains of murine origin were genetically fused to human constant (effector) domains. Full-length, functional chimeric antibody, huG1-F20G75, was generated by co-transfection of these heavy and light chain plasmids into HEK 293 cells for expression. A series of immunochemical tests including affinity fine specificity and biological tests were used to show the chimeric mAb retained the properties of the murine mAb. Characterization via ELISA, Western immunobloting and pepscan analysis shows binding to the identical defined epitope on domain 2 of PA. Affinity analysis via surface plasmon resonance indicates the chimeric mAb retained the original binding affinity. Finally, in vitro testing indicates the chimeric antibody maintains the LeTx neutralizing activity of the parental mAb. This antibody expression system has broad use in the development of recombinant mAbs with high specificity to emerging pathogens and reduced immunogenicity as therapeutics. |
