• The classical complement pathway begins with the activation of the C1 complex, which is a heterodimer consisting of a recognition molecule C1q, C1r and C1s. After the activated C1 complex binds to C4b, C2 is further cleaved to form C4b2a, a key C3 convertase in the classical complement pathway, and finally activates the complement system. C1s has been marketed with antibody drugs, which can inhibit the classical pathway of complement by inhibiting C1s, and inhibit excessive immune activity to treat cold agglutinin disease. C1-INH (a protein present in its natural state) regulates the activation of the C1 complex and regulates the enzyme activity of C1r and C1s, thereby preventing or activating their cleavage of the downstream protein C2-C3, etc., and then regulating the activation of the complement system.
• Protein expression analysis: Human C1S was only detectable in serum of homozygous B-hC1S mice.
• mRNA expression analysis: Mouse C1s mRNA was only detectable in liver of wild-type C57BL/6JNifdc mice. Human C1S mRNA was only detectable in liver of homozygous B-hC1S mice.

Strain specific analysis of C1S mRNA expression in wild-type C57BL/6JNifdc mice and B-hC1S mice by RT-PCR. Liver mRNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hC1S mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse C1s mRNA and human C1S primers. Mouse C1s mRNA was detectable only in wild-type mice. Human C1S mRNA was detectable only in homozygous B-hC1S mice but not in wild-type mice.

Strain specific C1S expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hC1S mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hC1S mice (H/H) (female, 6-week-old, n=3). Protein expression level of C1S was analyzed by ELISA (Thermo, EH57RB). Human C1S was exclusively detectable in homozygous B-hC1S mice

Functional activity of the classical pathway of homozygous B-hC1S mice. Serum was collected from homozygous B-hC1S mice (H/H) (male, n=3, 7 weeks old) and the functional activity was evaluated with the complement kit (Classical Complement Pathway Mouse Assay, Hycult Biotech, HIT420). Sutimlimab-analog (BIVV009) and Claseprubart-analog (DNTH103) are inhibitors targeting C1s. The activity was normalized to the PBS group. Significance was determined by one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001.