Design of CG001 and its inhibitory effect on complement pathways. (A) Schematic of CG001. It is composed of the extracellular domain of CRIg (green CRIg_EX; PDB, 2ICC¹³), the short consensus repeat (SCR) 1-5 of FH (red FH_SCR1-5; image showing SCR1-4 from PDB, 2WII¹⁴ in panel B), and the Fc fragment of human IgG4 (purple IgG4 Fc; PDB, 4C54¹⁵). (B) Composite image of the binding of CRIg_EX (green; PDB, 2ICF¹³) and FH_SCR1-4 (red; PDB, 2WII¹⁴) to C3b (sky blue). Diagrams were generated with PyMOL molecular visualization software. (C) Comparison of CG001 with other CRIg- or FH-derived fusion proteins to inhibit AP activation using the Wieslab complement system AP kit. The IC₅₀ was 0.83 ± 0.12 nM for CG001; 1.35 ± 0.49 nM for FH-CRIg-Fc; 239.3 ± 123.5 nM for CRIg-Fc; 121.6 ± 36.2 nM for FH-Fc; and 24.55 ± 0.31 for CRIg-Fc + FH-Fc. (D-E) Effect of CG001 on protecting erythrocytes from complement-mediated hemolysis. (D) CP was activated by 0.2% sheep erythrocyte hemolysin and 1% NHS to induce the hemolysis of sheep erythrocytes (sRBCs), whereas (E) AP was directly activated by 12% NHS to induce the hemolysis of rabbit erythrocytes (rRBCs). The IC₅₀ was 446.2 ± 89.06 nM for CG001; 1083 ± 168.18 nM for FH-CRIg-Fc; 5458 ± 1036.81 nM for FH-Fc; and 9269 ± 307.45 nM for CRIg-Fc + FH-Fc for CP. The IC₉₅ was 34.19 ± 3.77 μM for CG001; 68.66 ± 45.85 μM for FH-CRIg-Fc; 111.07 ± 24.13 μM for FH-Fc; and 176.11 ± 7.15 μM for CRIg-Fc + FH-Fc for CP. CRIg showed little inhibitory activity against CP. In addition, the IC₅₀ was 88.88 ± 8.31 nM for CG001; 167 ± 8.66 nM for FH-CRIg-Fc; 416.1 ± 52.83 nM for CRIg-Fc; 1231 ± 146.83 nM for FH-Fc; and 195.4 ± 16.34 nM for CRIg-Fc + FH-Fc for AP. The IC₉₅ was 206.83 ± 7.83 nM for CG001; 419.23 ± 139.03 nM for FH-CRIg-Fc; 2160 ± 345.98 nM for CRIg-Fc; 5040 ± 1846.65 nM for FH-Fc; and 551.05 ± 158.17 nM for CRIg-Fc + FH-Fc for AP (E). Values represent the mean ± standard deviation (SD), and the experiments were performed at least 3 times.