Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels Ϸ2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR ؊/؊ mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.antibody ͉ LDL-C ͉ LDLR ͉ PCSK9 ͉ hypercholesterolemia P roprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated as an important regulator of LDL metabolism (1, 2). Human genetic studies provide strong validation for the role of PCSK9 in modulating LDL cholesterol (LDL-C) levels and the incidence of coronary heart disease (CHD) in man. Gain-of-function (GOF) mutations in the PCSK9 gene are associated with elevated serum LDL-C levels (Ͼ300 mg/dL) and premature CHD (3), whereas loss-of-function (LOF) mutations are associated with low serum LDL-C (Յ100 mg/dL) (4). Strikingly, subjects harboring the heterozygous LOF mutations exhibited an 88% reduction in the incidence of CHD over a 15-year period relative to noncarriers of the mutations (5). Moreover, despite a complete loss of PCSK9 and serum LDL-C of Ͻ20 mg/dL, the 2 subjects carrying compound heterozygote LOF mutations appear healthy (6, 7).PCSK9 belongs to the subtilisin family of serine proteases and consists of a prodomain, catalytic domain, and C-terminal V domain (8). Expressed highly in the liver, PCSK9 is secreted after autocatalytic cleavage of its zymogen form (1). The prodomain remains noncovalently associated with the catalytic domain and seems to inhibit further proteolytic enzyme activity (8, 9). Secreted PCSK9 modulates LDL-C levels by posttranslational downregulation of hepatic LDL receptor (LDLR) protein (1). The precise mechanism is unknown, but a direct interaction between repeat A of the LDLR EGF homology domain and the PCSK9 catalytic domain is required (10, 11). Proteolytic cleavage of the LDLR by PCSK9 does not occur (12, 13); rather, the PCSK9:LDLR complex is endocytosed and directed to the endosome/lysosome compartment for degradation (14, 15). Current understanding of the LDLR pathway asserts that apolipoprotein B (apoB) and E (apoE) containing lipoprotein particles endocytosed with the LDLR are transported to the acidic environment of the endosome, where they dissociate from the receptor and are subsequently catabolized in lysosomes, while t...

Random peptide libraries and antigen-fragment libraries (also known as gene-fragment libraries) have been used to identify epitopes on protein antigens. These technologies promise to make significant contributions to diagnostic and vaccine development. Researchers in a number of labs have shown that phage selected from libraries with protective antibodies, raised against whole antigen, can be used as immunogens to stimulate antibody responses that bind native antigen and provide protection in vivo. Others have used the sera of patients with idiopathic diseases to screen libraries, and by this approach have identified candidate antigens involved in immune disease. These may prove useful for diagnosis and, possibly, in determining disease etiology.

surface of the HDL particle, LCAT maintains a chemical gradient that facilitates a unidirectional net fl ow of cholesterol from the cell surface of peripheral tissues to HDL particles in the blood ( 2 ). Mature HDL particles ultimately transport cholesterol (in the form of CE) back to the liver in pathways that involve cholesteryl ester transfer protein, LDL/VLDL, LDL receptor, and scavenger receptor class B type I. Hence, LCAT has been hypothesized to play a central role in driving HDL remodeling and reverse cholesterol transport (RCT) from the peripheral tissues to the liver.Loss-of-function mutations in the LCAT protein, along with their corresponding phenotypes, have been described ( 3 ). Familial LCAT defi ciency (FLD) is caused by a complete loss of LCAT activity, either through an absence of the LCAT protein itself or the presence of a mutant LCAT protein lacking any corresponding LCAT activity. In patients, FLD is characterized by HDL defi ciency along with corneal opacity, anemia, and loss of renal function. Fisheye disease (FED) is a partial LCAT defi ciency and is characterized by the presence of LCAT protein with decreased LCAT activity. Patients with FED manifest with low HDL and corneal opacity later in life. Interestingly, a defi nitive correlation between loss-of-function LCAT mutations and coronary heart disease remains elusive ( 4 ).Glomset ( 5, 6 ) fi rst described LCAT enzymatic activity in 1962 and subsequently purifi ed the protein from plasma. LCAT purifi ed from plasma has a mass of ‫ف‬ 65 kDa as assessed by SDS-PAGE. Later analysis showed that LCAT is a 416-amino-acid protein with a calculated molecular mass of ‫ف‬ 47 kDa, and that the extra mass of plasma LCAT is a result of N-linked carbohydrates at Asn20, Asn84, Asn272, and Asn384. Glycosylation of LCAT has been shown to be important for its activity and secretion ( 7,8 ). LCAT is predicted to have an ␣ / ␤ hydrolase fold with Ser181, LCAT (EC 2.3.1.43) is a plasma enzyme that catalyzes the conversion of cholesterol into long-chain cholesteryl esters (CEs). This reaction occurs on the surface of the HDL particle where, after activation by ApoA-I (the structural protein of HDL particles), LCAT hydrolyzes phosphatidylcholine (lecithin) at the sn -2 position and subsequently transfers the fatty acyl to cholesterol ( 1 ). The increased hydrophobicity of the resulting CE forces it into the interior core of the HDL particle and in the process transforms HDL from nascent discoidal particles into larger CEenriched spherical particles. By reducing cholesterol on the 11 June 2015. Published, JLR Papers in Press, July 20, 2015 DOI 10.1194 The high-resolution crystal structure of human LCAT Abbreviations: CE, cholesteryl ester; Endo H, endoglycosidase H; FED, fi sh-eye disease; FLD, familial LCAT defi ciency; mAb, monoclonal antibody; RCT, reverse cholesterol transport; RMSD, root-mean-square deviation . Abstract LCAT is intimately involved in HDL maturation All authors are or have been employees and shareholders of Amgen. X-ray diffraction da...

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