Targeting the Active Domain of C1s for Discovery of Antibodies That Inhibit Classical Complement Pathway Activity

Targeting the active domain of C1s for discovery of antibodies that inhibit classical complement pathway activity

 

The classical complement pathway is a biochemical cascade of the innate immune system that provides defense against pathogens. The pathway is typically activated when the C1 protein complex recognizes antibodies bound to pathogenic surfaces, which initiates downstream effector immune functions. In the case of autoimmune disease and transplant rejection, this process can be aberrantly activated and result in complement attack on host cells or transplanted tissue. Thus in certain inflammatory disorders, therapeutic modulatation of complement activation would be desirable. C1s is a central protease of this pathway and was thus chosen as a target for inhibition. Utilizing the serine protease domain of C1s (recombinantly expressed in insect cells), a synthetic bivalent antibody fragment (FAB) library expressed on the surface of phage and the plasma B cell repertoire of hyperimmunized BALB/c mice were mined by high-throughput DNA sequencing to discover antibodies that bind the nearest the active site of the C1s.  Of the antibodies discovered, biochemical characterization studies demonstrated at least one clone (antibody D) that was able to inhibit C1s protease activity towards its natural substrates, C4 and C2, at 20-fold molar excess. Future studies will utilize antibody D to determine classical complement pathway inhibition in molecular and cellular assays with human serum and mammalian cells.