Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice
2012; Elsevier BV; Volume: 53; Issue: 8 Linguagem: Inglês
10.1194/jlr.m028340
ISSN1539-7262
AutoresElena Fattori, Manuela Cappelletti, Paola Lo Surdo, Alessandra Calzetta, Claus Bendtsen, Ni Yan, Shilpa Pandit, Ayesha Sitlani, Giuseppe Mesiti, Andrea Carfı́, Paolo Monaci,
Tópico(s)Viral Infections and Immunology Research
ResumoSuccessful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins. Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins. alkaline phosphatase human cytomegalovirus electrical stimulation knockout immune-histochemistry immunological knockdown low density lipoprotein cholesterol LDL receptor monoclonal antibody 0.05% Tween-20 in PBS room temperature small interfering RNA The development of drugs against novel target proteins requires a significant commitment in terms of time and cost (1DiMasi J.A. Hansen R.W. Grabowski H.G. The price of innovation: new estimates of drug development costs.J. Health Econ. 2003; 22: 151-185Crossref PubMed Scopus (3191) Google Scholar). In many cases, new drugs fail in the clinic due to unacceptable side effects or lack of efficacy (2Schäfer S. Kolkhof P. Failure is an option: learning from unsuccessful proof-of-concept trials.Drug Discov. Today. 2008; 13: 913-916Crossref PubMed Scopus (27) Google Scholar, 3Giacomini K.M. Krauss R.M. Roden D.M. Eichelbaum M. Hayden M.R. 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Pro-protein convertase subtilisin-like/kexin type 9 (PCSK9) has recently emerged as a key determinant of liver low density lipoprotein receptor (LDLR) and LDL-cholesterol (LDL-c) plasma levels, and consequently of cardiovascular health in humans (10Horton J.D. Cohen J.C. Hobbs H.H. PCSK9: a convertase that coordinates LDL catabolism.J. Lipid Res. 2009; 50: S172-S177Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar–13Costet P. Krempf M. Cariou B. PCSK9 and LDL cholesterol: unravelling the target to design the bullet.Trends Biochem. Sci. 2008; 33: 426-434Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). PCSK9 belongs to the mammalian pro-protein convertase family of serine proteases (14Henrich S. Lindberg I. Bode W. Than M.E. Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.J. Mol. Biol. 2005; 345: 211-227Crossref PubMed Scopus (135) Google Scholar, 15Seidah N.G. Prat A. 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The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol.Structure. 2007; 15: 545-552Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar–21Seidah N.G. Benjannet S. Wickham L. Marcinkiewicz J. Jasmin S.B. Stifani S. Basak A. Prat A. Chretien M. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.Proc. Natl. Acad. Sci. USA. 2003; 100: 928-933Crossref PubMed Scopus (918) Google Scholar). The addition of PCSK9 to cultured cell medium has been shown to result in LDLR degradation, both overall and at the cell surface (22Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (524) Google Scholar, 23Cameron J. Holla O.L. Ranheim T. Kulseth M.A. Berge K.E. Leren T.P. 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Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (524) Google Scholar). Together, these data are consistent with a mechanism whereby PCSK9 acts as a chaperon binding LDLR and shuttling the receptor to lysosomes for degradation (11Horton J.D. Cohen J.C. Hobbs H.H. Molecular biology of PCSK9: its role in LDL metabolism.Trends Biochem. Sci. 2007; 32: 71-77Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Genetic knockout (KO) of PCSK9 in mice has been shown to result in reduced circulating cholesterol levels, increase in liver LDLR levels, and accelerated clearance of LDL-c (16Zaid A. Roubtsova A. Essalmani R. Marcinkiewicz J. Chamberland A. Hamelin J. Tremblay M. Jacques H. Jin W. Davignon J. et al.Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.Hepatology. 2008; 48: 646-654Crossref PubMed Scopus (318) Google Scholar, 28Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking PCSK9.Proc. Natl. Acad. Sci. USA. 2005; 102: 5374-5379Crossref PubMed Scopus (561) Google Scholar). Conversely, the introduction into mouse circulation of PCSK9 through parabiosis or by intravenous injection of the recombinant protein caused a strong reduction in hepatic LDLR levels (22Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. 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Higbee J. Xia Z. et al.A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.Proc. Natl. Acad. Sci. USA. 2009; 106: 9820-9825Crossref PubMed Scopus (330) Google Scholar–33Chaparro-Riggers J. Liang H. Devay R.M. Bai L. Sutton J.E. Chen W. Geng T. Lindquist K. Galindo Casas M. Boustany L.M. et al.Increasing serum half-life and extending cholesteroil lowering in vivo by engineering an antibody with pH sensitive binding to PCSK9.J. Biol. Chem. 2012; 287: 11090-11097Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and 65% in a human trial (34Stein E.A. Mellis S. Yancopoulos G.D. Stahl N. Logan D. Smith W.B. Lisbon E. Gutierrez M. Webb C. Wu R. et al.Effect of a monoclonal antibody to PCSK9 on LDL cholesterol.N. Engl. J. Med. 2012; 366: 1108-1118Crossref PubMed Scopus (578) Google Scholar). Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. We show that this polyclonal response does include antibodies, which specifically bind to the target protein and effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target in the treatment of elevated levels of plasma LDL-c. Full-length human and mouse PCSK9 (indicated as hPCKS9 and mPCKS9, respectively) cDNAs were amplified from human or mouse fetal liver, respectively, and cloned under the transcriptional control of human cytomegalovirus (CMV) promoter in pVIJ expression plasmid (35Montgomery D.L. Shiver J.W. Leander K.R. Perry H.C. Friedman A. Martinez D. Ulmer J.B. Donnelly J.J. Liu M.A. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors.DNA Cell Biol. 1993; 12: 777-783Crossref PubMed Scopus (245) Google Scholar). A pVIJ_hPCSK9 and pVIJ_mPCSK9 derivatives with C-terminal V5 and 6-His epitope tags were expressed in stably transfected HEK293 cell lines and purified as previously described (24Fisher T.S. Lo Surdo P. Pandit S. Mattu M. Santoro J.C. Wisniewski D. Cummings R.T. Calzetta A. Cubbon R.M. Fischer P.A. et al.Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation.J. Biol. Chem. 2007; 282: 20502-20512Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Female BALB/c and C57BL6 mice were bred under specific pathogen-free conditions by Charles River Breeding Laboratories (Calco, Como, Italy). In all operations, mice were treated in accordance with European guidelines. Animals were maintained in standard conditions under a 12 h light-dark cycle, provided irradiated food (Mucedola, Settimo Milanese, Italy) and water ad libitum. Six-week-old mice were fully anesthetized with ketamine (Merial Italia, Milano, Italy) at 100 mg/kg of body weight and xylazine (BIO 98, Bologna, Italy) at 5.2 mg/kg. The immunization experiments were all performed at the I.R.B.M. Laboratory Animal Research, which has full AAALAC accreditation. In xeno-DNA protocol mice were electro-injected intramuscularly with pVIJ_hPCSK9 (50 µg/mouse/injection: 3 times at 2 weeks intervals; days 0, 14, and 28) and CpG adjuvant (50 µg/mouse/injection; 3 times at 2 weeks intervals; day 7, 21, and 35: Fig. 1). A 20-mer (5′-TCCATGACGTTCCTGACGTT-3′) with a nuclease-resistant phosphorothioate backbone, which contains two CpG motifs with known immune-stimulatory effects on the murine immune response, was used as CpG in the studies we describe (36Krieg A.M. Yi A.K. Schorr J. Davis H.L. The role of CpG dinucleotides in DNA vaccines.Trends Microbiol. 1998; 6: 23-27Abstract Full Text PDF PubMed Scopus (222) Google Scholar). Control mice were injected only with CpG (50 µg/mouse/injection; 3 times at 2 week intervals; days 7, 21, and 35). For the xeno-protein protocol the hPCSK9 protein formulated with CpG (100 µg protein and 50 µg adjuvant) was injected subcutaneously at the base of the tail on days 0, 3, 6, and 21. Control mice were injected only with CpG with the same schedule (Fig. 1). Both protocols showed a low and comparable reactivity in this as well as in other similar experiments for the control groups. This prompted us to use in the experiments reported in this work a unique “negative-ctrl” group populated by the mice from the two groups. Peripheral blood was collected and direct HDL-c, direct LDL-c, and total cholesterol were measured using the ADVIA 1200 IMS (Bayer Healthcare, Terrytown, NY). Multi-well Maxisorp ELISA plates (Nunc, Roskild, Denmark) were coated overnight with PCSK9 protein at a concentration of 5 µg/ml in 50 mmol/L NaHCO3 (pH 9.6). Plates were then briefly rinsed with washing buffer (PBST, 0.05% Tween-20 in PBS) and incubated for 1 h at 37°C with blocking buffer (3% nonfat dry milk in PBST). Serial dilutions of preimmune or immune sera in PBST (ranging from 1:100 to 1/8,100) were added to the wells and incubated for 2 h at room temperature. Plates were washed and incubated with mAb anti-mouse IgG Fc-specific, alkaline phospatase (AP)-conjugated (Sigma-Aldrich Inc., St. Louis, MO) for 60 min at RT and AP activity detected by incubation with AP substrate solution (Sigma-Aldrich Inc., St. Louis, MO) in 10% diethanolamine/0.5 mmol/L MgCl2. Titers of anti-hPCSK9 antibodies in the serum of immunized mice were computed as follows. Experimental data were acquired as (A405nm-A460nm) for each dilution of each sample. For each animal, prebleeds were included in duplicates at a 1:100-fold dilution together with the serial dilutions. We observed some variability in the prebleeds (A405nm-A460nm) between plates, but not within each plate. The baseline was therefore determined as the mean in addition to 3 standard deviations of the prebleed (A405nm-A460nm) for each plate, individually. Data referring to the same sample were fitted by a monotone Hermite spline. Titers were defined as the dilution at which the spline intersects the base line. When this intersection fell outside the dilution interval, titers were reported as the minimal or maximal dilutions, as appropriate. Plasma mPCSK9 concentration was measured by ELISA as previously described (37Ni Y.G. Di Marco S. Condra J.H. Peterson L.B. Wang W. Wang F. Pandit S. Hammond H.A. Rosa R. Wood D.D. et al.A proprotein convertase subtilisin-like/kexin type 9 (PCSK9)-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces free circulating PCSK9 and LDL-cholesterol in mice and rhesus monkeys.J. Lipid Res. 2011; 52: 78-86Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). High binding 4HBX plates (ThermoLabsystems, Helsinki, Finland) were coated overnight at 4°C with 50 µl of 10 µg/ml of anti-mPCSK9 A1 antibody. Next day, the wells were first blocked for 1 h at room temperature with 250 µl of blocking solution (1% BSA and 0.05% Tween-20 in TBS) and then washed in a plate-washer. Purified mPCSK9 protein diluted in 1% BSA in PBS (as standard) or mouse plasma was added to the wells and incubated at 37°C for 2 h followed by a washing step. Then, 100 μl of 1 µg/ml biotinylated anti-mPCSK9 Fab A2, was added to the plate and after an additional washing step, 75 µl of 1:1,000 Streptavidin/Europium (Perkin Elmer, Waltham, MA) was added. Plates were incubated at room temperature for 20 min, followed by a last washing step and the addition of 100 µl of DELFIA enhancer (Perkin Elmer, Waltham, MA). After 1 h, the plates were read with a Europium reader. Liver expression of mouse LDLR was evaluated by Western blot analysis and immunohistochemical staining. Liver crude protein extracts were prepared using RIPA Lysis buffer 500 μl per mg tissue (Santa Cruz Biotechnology Inc., Santa Cruz, CA) with phenylmethylsulfonyl fluoride, sodium orthovanadate, and proteinase inhibitors. NuPAGE 4-12% Bis-Tris gels (Life Technologies, Carlsband, CA) were loaded with 50 μg protein per lane. Blotted proteins were developed using a FITC-labeled anti mlDLR goat IgG (R and D Systems, Minneapolis, MI) diluted in primary Ab diluent buffer. FITC-labeled anti-Tubulin (Sigma, St Louis, MO) mouse mAb 3 was used to normalize data. Quantification of bands on X-Ray was made with ImageReader LAS 3000 (Fujifilm, Tokio, Japan). Tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections of 10 µl were obtained using a microtome, cleared in xylol, and rehydrated. The unmasking procedure was carried out by incubating the samples in 10 × antigen retrieve solution (DAKO, Glostrup, Denmark) at 99°C for 40 min. After rinsing in PBS, the sections were incubated with blocking solution (15 μl goat serum in 1 ml PBS) for 30 min at RT and then without additional rinse, immediately incubated with rabbit polyclonal anti-LDLR (Abcam Inc., Cambridge, UK) for 1 h at room temperature. Samples were then rinsed in PBS and incubated with goat anti-rabbit IgG Peroxidase-conjugated antibody (Sigma-Aldrich Inc., St. Louis, MO) at the dilution of 1:200 for additional 60 min. After rinsing in PBS, the sections were stained with the diaminobenzidine staining kit (Vector Laboratories Inc., Burlingame, CA) and nuclei were stained with hematoxylin. The sections were dehydrated and mounted with Entellan (Merck KGaA, Darmstadt, Germany). Differences in antibody titers were assessed using Wilcoxon's rank sum test due to lack of normality. Differences in LDL-c, HDL-c, cholesterol, and triglycerides levels were assessed using Welch's t-test. Normality assumptions were confirmed using the Shapiro-Wilk test. Correlations were assessed using Spearman's rank test. All reported p-values are two-sided. We explored different immunization protocols for their ability to raise antibodies against endogenous PCSK9 protein in mice. In a first set of experiments, BALB/c or C57BL6 mice were immunized by electro-injecting into the quadriceps muscles a DNA plasmid expressing mPCSK9 under the control of the human CMV promoter (pVIJ-mPCSK9), in conjunction with a CpG oligonucleotide adjuvant (35Montgomery D.L. Shiver J.W. Leander K.R. Perry H.C. Friedman A. Martinez D. Ulmer J.B. Donnelly J.J. Liu M.A. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors.DNA Cell Biol. 1993; 12: 777-783Crossref PubMed Scopus (245) Google Scholar, 36Krieg A.M. Yi A.K. Schorr J. Davis H.L. The role of CpG dinucleotides in DNA vaccines.Trends Microbiol. 1998; 6: 23-27Abstract Full Text PDF PubMed Scopus (222) Google Scholar). As expected, this syngeneic immunization failed to elicit a detectable antibody response against the target mPCSK9 protein in both mouse strains as assessed by ELISA (data not shown). In a second set of experiments, a xenogeneic approach was evaluated by inducing expression of the hPCSK9 protein, which is 78% identical to mPCSK9. Six-week-old female BALB/c or C57BL6 mice were immunized by electro-injecting into the quadriceps muscles a DNA plasmid expressing hPCSK9 under the control of the human CMV promoter (pVIJ-hPCSK9). The DNA plasmid injections were performed at days 0, 14 and 28 and the CpG adjuvant injections were performed at days 7, 21 and 35 (xeno-DNA protocol; Fig. 1). As a negative control, a second group of mice was injected only with CpG adjuvant at days 7, 21 and 35 (DNA-ctrl protocol; Fig. 1). High titers of anti-hPCSK9 antibodies were detected by ELISA as early as 14 days after immunization (data not shown) and a fraction of these antibodies was also shown to cross-react with the mPCSK9 protein (Fig. 2). Similar effects were observed in BALB/c and C57BL6 mice. However, because anti-mPCSK9 titers were higher in BALB/c than in C57BL6 mice (data not shown), the former mouse strain was used for further immunizations. To compare the efficacy of protein versus DNA immunization protocols, we performed a third set of experiments in which BALB/c mice were immunized by injection of highly purified hPCSK9 (xeno-protein protocol; Fig. 1). Recombinant hPCSK9 protein formulated with CpG adjuvant was subcutaneously injected at the base of the mice tail at days 0, 3, and 6, followed by a fourth injection at day 21. In a control experiment, mice were similarly injected only with CpG at days 0, 3, 6, and 21 (protein-ctrl). Fourteen days after the beginning of the immunization protocol, a very strong anti-hPCSK9 response was detected by ELISA in the serum of mice immunized with the hPCSK9 protein but not in the control animals (data not shown). As previously observed for the xeno-DNA vaccination protocol, a fraction of these antibodies cross-reacted with the endogenous mouse protein (Fig. 2). Of note, anti-mPCSK9 titers in the xeno-protein group were consistently higher than titers in the xeno-DNA group on days 14, 28, and 42 (P < 0.002, P < 0.001, and P = <0.017, respectively). At later times, this difference tended to decrease but it was still statistically significant at day 42. In addition the antibody titers in both groups differed strongly from those in the control animals at all time points (P < 0.001) demonstrating that there was no relevant effect of the CpG adjuvant alone (Fig. 2). Mice immunized by electro-injection of pVIJ-hPCSK9 plasmid-DNA and those immunized by the injection of the recombinant hPCSK9 protein formulated with CpG adjuvant (same protocol described in Fig. 1) were monitored every 14 days for total cholesterol, HDL-c, LDL-c, and triglyceride levels (days 0, 14, 28, 42, and 56). Two weeks after the first injection, mice immunized with hPCSK9 protein showed total cholesterol levels reduced by 40% (Fig. 3A). Subsequently, the total cholesterol concentration gradually increased but after 42 days was still reduced by 28% compared with day 0. The level of HDL-c, the most abundant cholesterol fraction in mice, paralleled that of total cholesterol with a reduction of 42% and 22% of the initial HDL-c value at days 14 and 42, respectively (Fig. 3B). Strikingly, compared with the starting levels, at day 14, circulating LDL-c was reduced by 60% and remained consistently lower than in control mice for at least 8 weeks and approximately 35% lower than the initial value even at day 42 (Fig. 3C). The above changes were highly statistically significant (P < 0.0001 for all). A similar though milder phenotype was obtained in mice immunized with plasmid-DNA expressing the human protein where reduced levels of total cholesterol, HDL-c, and LDL-c (P = 0.005, P = 0.01, and P = 0.0001, respectively) were observed. These levels returned to their basal values by day 28 for LDL-c, HDL-c and total cholesterol (Fig. 3A–C). In contrast to the reduced cholesterol levels, in both protein- or DNA-immunized mice the triglyceride levels, did not significantly vary throughout the same period (Fig. 3D). Together these data demonstrate that immunization of mice with hPCSK9 protein results in an acute decrease of serum cholesterol. Analysis of the anti-mPCSK9 antibody levels reported above reveals that at day 14, the average antibody titers from mice immunized with the protein protocol were higher than the average titers measured on the DNA-immunized group (Fig. 2). Consistently, the average level of circulating LDL-c in mice immunized with the protein protocol was 49% lower than that measured in the DNA-immunized group which, in turn, was 23% lower of the average LDL-c level in the control-DNA group mice (Fig. 3C). Furthermore, in immunized mice, an inverse correlation between LDL-c levels and anti-mPCSK9 antibodies titers was observed with anti-mPCSK9 antibodies and LDL-c levels being respectively at the highest and lowest levels in the protein-immunized mice (Fig. 4). Thus, the immunization procedure using the hPCSK9 protein and, to a lesser extent, the hPCSK9 DNA elicited high anti-mPCSK9 titers that directly correlate with the reduction in plasma LDL-c levels. To monitor the variation on immunization in the levels of circulating mPCSK9, we used a highly sensitive ELISA (37Ni Y.G. Di Marco S. Condra J.H. Peterson L.B. Wang W. Wang F. Pandit S. Hammond H.A. Rosa R. Wood D.D. et al.A proprotein convertase subtilisin-like/kexin type 9 (PCSK9)-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces free circulating PCSK9 and LDL-cholesterol in mice and rhesus monkeys.J. Lipid Res. 2011; 52: 78-86Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This assay uses a biotinylated mPCSK9-specific fragment binding antigen (Fab) and a neutralizing mAb that competes with LDLR/PCSK9 binding and allows an accurate detection of plasma mPCSK9. Two groups of mice each were immunized with hPCSK9 protein formulated with CpG or with CpG only and plasma mPCSK9 levels in the two groups were measured at day 14. In the control mice, the average level of detected circulating mPCSK9 was 238 ng/ml (SD = 50 ng/ml) whereas in the protein-immunized mice, the average level was significantly decreased (84 ng/ml; SD = 32 ng/ml). In line with previous experiments, circulating LDL-c was strongly reduc
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