Rational truncation of streptokinase reduces immunogenicity while preserving plasminogen activation: in vitro and in vivo analysis

The clinical utility of streptokinase, a widely used and cost-effective thrombolytic agent, has gained importance post COVID-19 due to increase in cardiovascular diseases globally. But the strong humoral immunogenicity exhibited by it, restricts its usage. Building upon our prior work of developing truncated streptokinase variants and their plasminogen activation study, we report here the results of immunogenicity studies for identification of variants with reduced immunogenicity. A panel of ten rationally truncated and recombinantly expressed streptokinase fragments were screened for antibody recognition and immunogenicity potential. Out of the ten fragments, two N-terminal constructs (amino acid residues 1–300 and 1–173) exhibited approximately 40–45% lower ELISA immunoreactivity compared with full-length streptokinase during initial in vitro screening. These fragments were also already reported to retain >85% of native plasminogen activation activity. In vivo evaluation of the fragments in rabbits demonstrated significantly attenuated humoral responses over a 28-day immunization period, confirmed by longitudinal antibody titration and cumulative area-under-the-curve analysis when compared to the full-length streptokinase. Fragment 1-173 emerged as a promising lower-immunogenic thrombolytic candidate, and this work demonstrates that rational domain minimization can decouple functional competence from immunodominant regions in streptokinase establishing a broadly applicable framework for engineering improved therapeutic proteins.