Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment. Despite vaccination, pertussis infection continues to cause ϳ195,000 deaths worldwide, primarily of infants (1). Of the estimated 16 million cases of pertussis each year, ϳ95% occur in the developing world. Even in developed countries, the disease incidence has increased dramatically over the last decade, reaching prevaccination levels in some countries (2, 3). This rise has been attributed to shortcomings of the current acellular vaccine (4) as well as pathogen adaptation (5). In both cases, high levels of circulating disease place young infants at risk, as this population is the most susceptible to severe disease. An antibody therapeutic could be used to treat seriously ill infants in the developing world and to prevent disease in high-risk areas.Pertussis toxin (PTx) is one of several virulence factors secreted by the Gram-negative bacterium Bordetella pertussis. PTx is directly responsible for suppression of the innate immune system (6) and for systemic leukocytosis, which is the key clinical indicator of severe disease and appears to be directly responsible for pulmonary hypertension and organ failure (7). In addition, low titers of PTx-neutralizing antibodies correlate with susceptibility to clinical infection (8). We previously developed a binary mixture of two humanized anti-pertussis toxin antibodies which was able to mitigate whooping cough in mouse and baboon models of infection (9). The antibody hu11E6 blocks binding of the toxin to host cells, while the antibody hu1B7 interferes with the catalytic pathway. At a dose selected to demonstrate efficacy but not synergy, the individual antibodies and the mixture were able to completely suppress leukocytosis in a murine ...

An increasing number of web resources are available for aiding in proteomics research. Databases contain repositories of proteins and associated information. A recent article by Chen et al. (Genomics Proteomics Bioinformatics 13(1):36-39, 2015) evaluates a number of MS-based proteomics repositories containing MS and expression data, including repositories devoted to cataloguing high confidence post-translational modifications. Many sites have tools developed by research labs that are shared with the community and online tutorials and videos for learning how to use the tools. This chapter contains a selection of web sites useful for proteomics analyses but is by no means comprehensive. Using a search engine such as Google is the easiest way to find the sites using the name given below.