This proposal capitalizes on recent methodological advances in immunology and protein engineering in order to analyze and characterize the antibody responses of humans to Streptokinase (SK). SK is a plasminogen activator protein isolated from pathogenic streptococci (Lancefield group C) and has been used for many years as a thrombolytic agent to treat diseases involving clot formation, such as heart attack. However, SK has potential side effects, since it is immunogenic. Patients treated with SK develop high titers of antibodies which neutralize SK activity. Further, some develop anaphylactic shock, immune complex diseases and delayed-type hypersensitivity. It is therefore crucial to elucidate the structural basis of SK antigenicity in humans in order to ascertain which regions of the molecule are more immunogenic than others and to study the molecular mechanisms responsible for antibody:.mediated neutralization of SK activity. These goals will be attained by producing monoclonal antibodies (mAbs) to SK in order to generate probes to map the antigenic epitopes of SK and to investigate the relationship between the structures composing the antigenic epitopes and the plasminogen activation function. The peptide sequences recognized by these mAbs will be studied using genetically engineered truncated SK fragments. These mAbs and recombinant SK fragments will be used as molecular probes to characterize the antigenic epitopes of SK in humans by analyzing the crossreactivity of human anti-SK antibodies with mAbs and recombinant SK fragments. Detailed analysis of antigenic epitopes of SK in humans and the elucidation of the structural basis for its plasminogen activation function should eventually enable us to uncouple its antigenic and functional structure(s) and to design a non-antigenic, effective plasminogen activator for therapeutic use.
