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Adrianna Latuszek, Ying Hu, Jingtai Cao, Stanley J Wiegand, George D Yancopoulos; Evaluation of complement activity in wild type, C5 knockout and humanized mice for drug discovery. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1324.
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© ARVO (1962-2015); The Authors (2016-present)
Complement has been implicated in ocular inflammatory and retinal degenerative diseases. Blocking C5 cleavage by monoclonal antibody (mAb) is a possible therapeutic strategy for these disorders. The present study was undertaken to better understand the complement activity in mouse vs. human, and setup a system that could further evaluate the function of candidate therapeutic mAbs in vitro and in vivo.
Serum was collected from wild type (WT) mice; complement component knockout mice; C5-/-, C3-/-, and humanized C5 mice (C5hu/hu), all generated with Velocigene technology (Regeneron). Hemolytic assay (CH50) was used to assess the functional activity of the complement system. Normal, C3 or C5-depleted human serum (Quidel Corp.) was used for comparison. Human C3 (400 μg/ml), C5 (50 μg/ml) or murine C5 protein (50 μg/ml) was added to test if it could restore the hemolysis function. Anti-human C5 mAb (10 μg/ml) and anti-mouse C5 mAb (10 μg/ml) were used to confirm the specificity and utility of this system for future drug discovery.
WT mouse sera have approximately 10 times lower complement activity compared to human. Sera from C5-/- and C3-/- mice show lack of complement activity. C5hu/hu mouse serum displays no hemolytic activity and is equivalent to serum from complement component deficient mice. The addition of mouse C5 to C5hu/hu mouse serum rescued hemolysis function. Anti-mouse C5 mAb blocked hemolysis in normal mouse serum. Anti-human C5 mAb blocked hemolysis in normal human serum but not in mouse serum. Hemolysis could not be recovered by adding human C5 to C5-/- mouse serum, whereas the addition of human C3 and C5 together rescued hemolysis in C5-/- mouse serum. Addition of human C3 to C5hu/hu mouse serum rescued hemolysis function.
Our findings demonstrate that wild type mice have dramatically lower complement activity compared to human. Transgenic mice expressing human C5 are functional murine C5 knockouts, possibly because mouse convertase cannot cleave human C5. Our results indicate that C3 and C5 double humanized mouse would have complement activity similar to WT and be able to test human Abs.
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