Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation.
Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Elodie
E
Weider
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Delia
D
Susan-Resiga
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Rachid
R
Essalmani
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Josée
J
Hamelin
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Marie-Claude
MC
Asselin
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Surendra
S
Nimesh
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Yahya
Y
Ashraf
Planet Biotechnology Inc., Hayward, California 94545-2740, and.
Keith L
KL
Wycoff
the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
Jianbing
J
Zhang
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
Annik
A
Prat
From the Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, University of Montreal, Montreal, Quebec H2W 1R7, Canada, seidahn@ircm.qc.ca.
Nabil G
NG
Seidah
D000818:Animals; D008078:Cholesterol, LDL / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D000091362:PCSK9 Inhibitors; D000071449:Proprotein Convertase 9 / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects*; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000494:pharmacology*
D008078:Cholesterol, LDL; C573412:LDLR protein, human; D000091362:PCSK9 Inhibitors; D011973:Receptors, LDL; D061905:Single-Domain Antibodies; C472125:PCSK9 protein, human; C472126:Pcsk9 protein, mouse; D000071449:Proprotein Convertase 9
Familial Hypercholesterolemia; LDL Receptor; cholesterol regulation; cysteine-histidine-rich-domain (CHRD); inhibitor; low-density lipoprotein (LDL); proprotein convertase subtilisin/kexin type 9 (PCSK9); single-domain antibody (sdAb,nanobody)