Mechanism of TT30 activity, structure, and functional assays. (A) Complement alternative pathway (detailed description is provided in the Introduction). (B-C) TT30 structure and selective inhibition of human CAP and CCP in vitro. (B) TT30 is a fusion protein that combines the first 4 short consensus repeats (SCRs) of Complement Receptor type 2 (CR2) with the first 5 SCRs of factor H. The CR2 domain binds iC3b and C3dg/C3d, while the factor H domain inactivates the CAP. (C) ELISA-based complement pharmacodynamic (PD) assays for assessment of TT30 activity ex vivo. For CAP testing (top panel), serum samples were loaded onto LPS-coated wells under conditions promoting CAP activation, which leads to MAC deposition on surface with expression of activated C9 neo-epitope. Addition of mouse anti-human C9 neo-epitope IgG mAb-AP and an alkaline peroxidase substrate resulted in colorimetric reaction where the amount of complement activation correlated with the color intensity and was measured in terms of absorbance at 405 nm using ELISA Plate Reader. Similar process was followed for the CCP activation (bottom panel), with the exception that the wells were coated with IgM and the buffer diluent contained 0.5mM MgCl2, and 2mM CaCl2.
