Systematic generation of antigen specific human monoclonal antibodies with therapeutical activities using active immunization.

Presenting a panel of human hybridomas secreting serospecific antibodies which confer a high degree of protection against fatal infection with Pseudomonas aeruginosa, we report an efficient approach for a systematic generation of antigen specific human monoclonal antibodies with biological activity. This approach is based on active immunization and antigen specific panning. Individuals were immunized with polysaccharides isolated from LPS of Pseudomonas aeruginosa conjugated to toxin A. Specific B cells were isolated and enriched by panning of blood samples taken at the time point with the highest frequency of fuseable cells. These cells were then transformed with Epstein-Barr virus. Arising lymphoblastoid cell lines were screened for the secretion of anti-LPS antibodies by enzyme-linked immunosorbent assay and fused to a murine-human heteromyeloma cell line. Hybridomas were selected for high levels of antibody secretion and binding to intact bacteria as determined by an immunofluorescence microscopy assay. The observation that protective capacity of an antibody was associated with its ability to bind to LPS determinants accessible on the bacterial cell surface allowed for an effective screening for therapeutically interesting human monoclonal antibodies. Out of four immunized individuals, 15 lymphoblastoid cell lines with anti-LPS activity could be isolated, and 8 hybridomas, which cover the majority of the common Pseudomonas aeruginosa serotypes, were characterized further. The generation of monoclonal anti-Pseudomonas aeruginosa toxin A, anti-Klebsiella capsular polysaccharides, and anti-Escherichia coli LPS antibodies suggests that the success of this approach is not limited to the generation of human monoclonal antibodies of a particular specificity or to the use of antigens of a particular character.