Characterization of in Vitro Activity of PCSK9 Monoclonal Antibodies
Objective To investigate the affinity of tafolecimab,evolocumab and alirocumab to proprotein convertase subtilisin/kexin type 9(PCSK9).Methods The affinity of three PCSK9 monoclonal antibodies(tafolecimab,evolocumab and alirocumab)for PCSK9 was examined by the surface plasmon resonance and biolayer interferometry methods.The expression level of low-density lipoprotein receptor(LDLR)in HepG2 cells was detected by the Western blot,and the ability of different mass concentrations(0,6,60,180 μg/mL)of tafolecimab to block recombinant human PCSK9(D374Y)binding to LDLR was investigated.Results The equilibrium dissociation constants of tafolecimab with PCSK9 determined by the surface plasmon resonance and biolayer interferometry methods were 2.90 × 10-10 mmol/L and 5.05 × 10-10 mmol/L,respectively.The affinity of tafolecimab to recombinant human PCSK9(D374Y)was 1.85 times and 2.57 times higher than that of evolocumab,and 5.38 times and 4.71 times higher than that of alirocumab.The half maximal inhibitory concentration(IC50)of tafolecimab for blocking the binding of recombinant human PCSK9(D374Y)to LDLR was 64.42 nmol/L.The concentration for 50％of maximal effect(EC50)of tafolecimab for restoring low-density lipoprotein cholesterin(LDL-C)endocytosis by binding human PCSK9 and recombinant human PCSK9(D374Y)was(15.03±0.63)μg/mL and(7.22±0.63)μg/mL,respectively.Western blot test results showed that the grayscale values of HepG2 cells in different concentrations(0,6,60,180 µg/mL)of tafolecimab were 0.76,0.83,3.21,and 3.28,respectively.Conclusion Tafolecimab,which has a stronger affinity for PCSK9 than alirocumab and evolocumab,can prevent the degradation of LDLR and increase the expression of LDLR on the surface of hepatocytes in a concentration-dependent manner.
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