Flow cytometry analyses of complement inhibition by the selective alternative pathway inhibitor SH-01

SH-01, a novel recombinant protein derived from Trypanosoma brucei gambiense ISG65 (PMID: 37105991), selectively inhibits the alternative pathway (AP). Here we further characterise SH-01 for its suitability as therapeutic for complement pathway disorders such as paroxysmal nocturnal hemoglobinuria (PNH). We deposit the following flow cytometry data (A) C3b deposition analysis by flow cytometry Flow cytometry analysis with sensitized sheep erythrocytes incubated in C6-depleted human serum shows that SH-01 does not prevent C3b surface deposition. (B) Identification of SH-01 immunogenic epitopes. Spleens from mice immunized with SH-01 were harvested and B-cells isolated. These antibody-presenting cells were analyzed in a flow cytometry-based competitive binding assay, utilizing AlexaFluor-594 labeled SH-01 (SH-01AF594) alone, SH-01AF594 in complex with C3d (the proteolytically liberated thioester domain of C3b), and SH-01AF594 in complex with a mAb-derived Fab (3E12) from a hybridoma cell line. (C)Flow cytometry analysis of PNH erythrocytes Flow cytometry analysis of hemolysis and C3b deposition on PNH patient erythrocytes
Conclusion:
(A)SH-01 does not inhibit C3b deposition during classical and lectin pathway activation but prevents degradation to iC3b. (B) Most B-cell presented antibodies were non-neutralizing for the interaction with C3 (C)SH-01 effectively prevents lysis of PNH erythrocytes Unstained control samples