Patent Description:
Elevated cholesterol level in serum is an important risk factor resulting in cardiovascular events. At present, cholesterol-lowering drugs with multiple mechanisms of action have been launched or are under development. Among them, anti-PCSK9 monoclonal antibodies have drawn extensive attention because of their good safety and efficacy (<NPL>).

With high specificity in action, mild adverse reaction and other advantages, monoclonal antibody drugs have become the mainstream of drug development in many disease fields, and anti-PCSK9 monoclonal antibody drugs that reduce LDL-C by reducing LDLR endocytosis are a current research hotspot. With a remarkable effect in reducing LDL-C, good safety and convenience in administration, anti-PCSK9 monoclonal antibodies are drugs marking a major breakthrough in the field of lipid-lowering treatment.

There is still a demand for alternative anti-PCSK9 antibodies. Specifically, there is a demand for alternative anti-PCSK9 antibodies that have a high affinity for PCSK9, a reliable cell strain source, high stability, and high efficiency in reducing LDL-C. More specifically, there is a demand for alternative anti-PCSK9 antibodies that efficiently reduce LDL-C and provide a long duration of effect (such as long-lasting inhibition for LDL-C level). Such antibodies will also preferably have physical and chemical properties that are favorable for development, manufacturing or preparation. At present, most of the monoclonal antibody drugs on the market are administrated by intravenous injection. However, subcutaneous injection has become a preferred mode of injection because patients with some certain chronic diseases hope to be self-medicated at home. Since the volume for subcutaneous injection is generally limited to <NUM> to <NUM>, the antibody preparation needs to have a high concentration (≥ <NUM>/mL) to meet the requirements for clinical dosage.

Formulations comprising anti-PCSK9 antibodies are already disclosed (<CIT> (<CIT>)). High-concentration antibody preparations, however, also face a great challenge of high viscosity, because high-viscosity preparations will bring great difficulty to the downstream process and subcutaneous administration of drugs with a syringe. Under high concentrations, proteins are often less stable and are likely to aggregate and granulate. Moreover, the instability of protein conformations will also lead to a significant change in chemical stability such as charge heterogeneity.

In addition, as biomacromolecules, monoclonal antibodies have a very complex structure. In the process of production, expressed antibody molecules will undergo various post-translational modification and degradation, such as N-terminus cyclization, glycosylation, deamidization, isomerization, oxidation, fragmentation and disulfide bond mispairing. These quality attributes may affect the safety and effectiveness of final products, so it is also very important to control the correctness and consistency of product quality.

There is a need in the art to provide a liquid preparation comprising an anti-PCSK9 antibody with low viscosity, stability and high concentration.

PCSK9 is an important and beneficial therapeutic target. The present invention provides a liquid antibody preparation comprising an anti-PCSK9 antibody or a fragment thereof (such as the anti-PCSK9 antibody defined herein). The following embodiments are presented for illustrative purposes; the invention for which protection is sought is defined by the claims.

In one aspect, the present invention provides a liquid antibody preparation, which comprises:.

In one embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL to about <NUM>/mL. In another embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL to about <NUM>/mL. In another embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL. In another embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL. In another embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL. In another embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL. In another embodiment, the concentration of the anti-PCSK9 antibody or the fragment thereof in the liquid antibody preparation disclosed herein is about <NUM>/mL.

In one embodiment, the anti-PCSK9 antibody is any antibody binding to PCSK9 proteins (such as human PCSK9), such as a polyclonal antibody, a monoclonal antibody or a combination of the two. Preferably, in one embodiment, the anti-PCSK9 antibody is a monoclonal antibody. In one embodiment, the anti-PCSK9 antibody or the fragment thereof is the anti-PCSK9 antibody or the fragment thereof defined herein.

In one embodiment, the concentration of the buffer in the liquid antibody preparation disclosed herein is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the buffer in the liquid antibody preparation disclosed herein is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the buffer in the liquid antibody preparation disclosed herein is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the buffer in the liquid antibody preparation disclosed herein is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the buffer in the liquid antibody preparation disclosed herein is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the buffer in the liquid antibody preparation disclosed herein is about <NUM>/mL.

In one embodiment, the buffer is selected from histidine, glutamate, phosphate, acetate, citrate, and tris(hydroxymethyl)aminomethane. In one embodiment, the buffer is histidine.

In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody preparation disclosed herein is about <NUM> mmol/L to <NUM> mmol/L. In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody preparation disclosed herein is about <NUM> mmol/L to <NUM> mmol/L. In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody preparation disclosed herein is about <NUM> mmol/L to <NUM> mmol/L. In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody preparation disclosed herein is about <NUM> mmol/L to <NUM> mmol/L.

In one embodiment, the viscosity inhibitor is selected from sugar alcohols (such as sorbitol), arginine, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate, and combinations thereof. In one embodiment, the viscosity inhibitor is sorbitol. In one embodiment, the viscosity inhibitor is sorbitol with a concentration of about <NUM>% to <NUM>% (w/v). In one embodiment, the viscosity inhibitor is sorbitol with a concentration of about <NUM>% to <NUM>% (w/v), and the liquid preparation does not comprise other viscosity inhibitors. In one embodiment, the viscosity inhibitor is a combination of sorbitol and arginine or arginine salt (preferably arginine hydrochloride). In one embodiment, the viscosity inhibitor is a combination of sorbitol and arginine or arginine salt (preferably arginine hydrochloride), wherein the concentration of sorbitol is about <NUM>% to <NUM>% (w/v), preferably about <NUM>% to <NUM>% (w/v), and more preferably about <NUM>% (w/v), and the concentration of arginine or arginine salt (preferably arginine hydrochloride) is about <NUM> mmol/L to <NUM> mmol/L, preferably about <NUM> mmol/L to <NUM> mmol/L, and more preferably about <NUM> mmol/L. In one embodiment, the viscosity inhibitor is a combination of sorbitol and arginine or arginine salt (preferably arginine hydrochloride), wherein the concentration of sorbitol is about <NUM>% to <NUM>% (w/v), preferably about <NUM>% to <NUM>% (w/v), and more preferably about <NUM>% (w/v), and the concentration of arginine or arginine salt is about <NUM> mmol/L to <NUM> mmol/L, preferably about <NUM> mmol/L to <NUM> mmol/L, and more preferably about <NUM> mmol/L, and the liquid preparation does not comprise other viscosity inhibitors. In one embodiment, the concentration of the surfactant is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the surfactant is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the surfactant is about <NUM>/mL to <NUM>/mL. In one embodiment, the concentration of the surfactant is about <NUM>/mL.

In one embodiment, the surfactant is a nonionic surfactant. In one embodiment, the surfactant is, for example, pluronics, polysorbate-<NUM>, polysorbate-<NUM>, polysorbate-<NUM>, or polysorbate-<NUM>.

In one embodiment, the liquid preparation disclosed herein comprises a solvent, and preferably, the solvent is selected from water for injection, organic solvents for injection (including but not limited to oil for injection, ethanol, and propylene glycol), and combinations thereof.

In one embodiment, the pH of the liquid preparation is about <NUM> to <NUM>. In one embodiment, the pH of the liquid preparation is about <NUM> to <NUM>. In one embodiment, the pH of the liquid preparation is about <NUM> to <NUM>. In one embodiment, the pH of the liquid preparation is about <NUM>.

In one embodiment, the viscosity of the liquid preparation at <NUM> is about <NUM> to <NUM> centipoises. In one embodiment, the viscosity of the liquid preparation at <NUM> is about <NUM> to <NUM> centipoises. In one embodiment, the viscosity of the liquid preparation at <NUM> is about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> centipoises. In one embodiment, the viscosity of the liquid preparation at <NUM> is about <NUM> to <NUM> centipoises. In one embodiment, the viscosity of the liquid preparation at <NUM> is about <NUM> centipoises. In one embodiment, the viscosity of the liquid preparation at <NUM> is about <NUM> centipoises.

In one embodiment, the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection.

In one preferred embodiment, the liquid antibody preparation disclosed herein comprises:.

wherein the pH of the liquid preparation is about <NUM> to <NUM>.

In one more preferred embodiment, the liquid antibody preparation disclosed herein also comprises water for injection.

In one embodiment, after the liquid preparation comprising an anti-PCSK9 antibody disclosed herein is stored at about -<NUM> to about <NUM>, such as -<NUM>, about -<NUM>, about -<NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM> or about <NUM>, for at least <NUM> days, at least <NUM> days, at least <NUM> month, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, or longer, the purity of the anti-PCSK9 antibody or a fragment thereof decreases by no more than <NUM>%, for example, no more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%, as detected by SEC-HPLC.

In one embodiment, after the liquid preparation comprising an anti-PCSK9 antibody disclosed herein is stored at about -<NUM> to about <NUM>, such as -<NUM>, about -<NUM>, about -<NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM> or about <NUM>, for at least <NUM> days, at least <NUM> days, at least <NUM> month, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, or longer, the purity of the anti-PCSK9 antibody or a fragment thereof decreases by no more than <NUM>%, for example, no more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%, as detected by non-reduced CE-SDS.

In one embodiment, after the liquid preparation comprising an anti-PCSK9 antibody disclosed herein is stored at about -<NUM> to about <NUM>, such as -<NUM>, about -<NUM>, about -<NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>, for at least <NUM> days, at least <NUM> days, at least <NUM> month, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, at least <NUM> months, or longer, the charge heterogeneity of the anti-PCSK9 antibody or a fragment thereof changes by no more than <NUM>%, for example, no more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%, as detected by CEX-HPLC.

The preparation disclosed herein has a high antibody concentration, a low viscosity suitable for administration (particularly subcutaneous administration), and a high physical and chemical stability, and is less prone to aggregate and granulate and exhibit heterogeneity change when stored. These advantages are very beneficial to the production of complete, effective, and highly consistent clinical drugs.

The inventors surprisingly found that sorbitol can be used as a viscosity inhibitor for the liquid preparation comprising an anti-PCSK9 antibody, in which case a liquid preparation comprising an anti-PCSK9 antibody or a fragment thereof having low viscosity and high concentration can be obtained without adding other viscosity inhibitors, and the liquid preparation has high stability. In addition, the inventors surprisingly found that a combination of sorbitol and arginine as a viscosity inhibitor can better reduce the viscosity of the liquid preparation comprising an anti-PCSK9 antibody and enable a high-concentration liquid preparation comprising an anti-PCSK9 antibody or a fragment thereof to have lower viscosity suitable for subcutaneous injection lower viscosity suitable for subcutaneous injection, and the liquid preparation comprising an anti-PCSK9 antibody has higher stability (see examples <NUM>-<NUM>).

Therefore, in another aspect, the present invention provides use of sugar alcohol as a viscosity inhibitor for the liquid preparation comprising an anti-PCSK9 antibody.

In one embodiment, the concentration of the sugar alcohol in the liquid preparation comprising an anti-PCSK9 antibody is about <NUM>% to <NUM>% (w/v), preferably about <NUM>% to <NUM>% (w/v), and more preferably about <NUM>% (w/v). In one embodiment, the sugar alcohol is preferably sorbitol.

In another aspect, the present invention provides use of sugar alcohol as a viscosity inhibitor in preparing the liquid preparation comprising an anti-PCSK9 antibody.

In one embodiment, the present invention provides use of sugar alcohol as the only viscosity inhibitor in preparing the liquid preparation comprising an anti-PCSK9 antibody.

In another aspect, the present invention provides use of a combination of sugar alcohol and arginine (or arginine salt, preferably arginine hydrochloride) as a viscosity inhibitor for the liquid preparation comprising an anti-PCSK9 antibody.

In one embodiment, the concentration of the sugar alcohol in the liquid preparation comprising an anti-PCSK9 antibody is about <NUM>% to <NUM>% (w/v), preferably about <NUM>% to <NUM>% (w/v), and more preferably about <NUM>% (w/v); and the concentration of arginine or arginine salt (preferably arginine hydrochloride) is about <NUM> mmol/L to <NUM> mmol/L, preferably about <NUM> mmol/L to <NUM> mmol/L, and more preferably about <NUM> mmol/L. In one embodiment, the sugar alcohol is preferably sorbitol. In another aspect, the present invention provides use of a combination of sugar alcohol and arginine (or arginine salt, preferably arginine hydrochloride) as a viscosity inhibitor in preparing the liquid preparation comprising an anti-PCSK9 antibody.

In one embodiment, the present invention provides use of a combination of sugar alcohol and arginine (or arginine salt, preferably arginine hydrochloride) as the only viscosity inhibitor in preparing the liquid preparation comprising an anti-PCSK9 antibody.

In one embodiment, the concentration of the sugar alcohol in the liquid preparation comprising an anti-PCSK9 antibody is about <NUM>% to <NUM>% (w/v), preferably about <NUM>% to <NUM>% (w/v), and more preferably about <NUM>% (w/v); and the concentration of arginine or arginine salt (preferably arginine hydrochloride) is about <NUM> mmol/L to <NUM> mmol/L, preferably about <NUM> mmol/L to <NUM> mmol/L, and more preferably about <NUM> mmol/L. In one embodiment, the sugar alcohol is preferably sorbitol. In another aspect, the present invention provides a solid preparation obtained by lyophilizing the aforementioned liquid preparation. Before use, the solid preparation can be reconstituted in a suitable solvent to give the liquid preparation disclosed herein.

In another aspect, the present invention provides a method for preparing the liquid preparation disclosed herein, which comprises the following steps:.

In one embodiment, the anti-PCSK9 antibody or the fragment thereof in step a is a separated and purified anti-PCSK9 antibody.

In one embodiment, the anti-PCSK9 antibody or the fragment thereof in step a is provided in the form of a solution, preferably an aqueous solution.

In one embodiment, before step b, the anti-PCSK9 antibody or the fragment thereof is concentrated by being centrifuged using an ultrafiltration centrifuge tube having a molecular weight cutoff of, for example, about <NUM> KD.

In one embodiment, the buffer and the viscosity inhibitor in step b are provided in the form of a solution, preferably an aqueous solution.

In one embodiment, after step b, the anti-PCSK9 antibody or the fragment thereof is concentrated by ultrafiltration and centrifugation and then diluted with the solutions, preferably aqueous solutions, of the buffer and the viscosity inhibitor, and this is repeated until the replacement is completed.

In one embodiment, before step c, the concentration of the anti-PCSK9 antibody or the fragment thereof is adjusted.

The anti-PCSK9 antibody or the fragment thereof, the buffer, the viscosity inhibitor, and the surfactant are as described herein, and doses and/or concentrations thereof are as described for, or can be appropriately determined by, the liquid preparation disclosed herein.

In one embodiment, the method for preparing the liquid preparation disclosed herein comprises the following steps:.

In the embodiment, the anti-PCSK9 antibody is the anti-PCSK9 antibody defined herein.

In some embodiments, an anti-PCSK9 antibody or an antibody fragment (preferably an antigen-binding fragment) binding to PCSK9 or a fragment thereof (preferably human PCSK9 protein) is provided.

In one aspect of the present invention, an anti-PCSK9 antibody and a fragment thereof (such as an antigen-binding fragment) are provided herein. In some embodiments, the anti-PCSK9 antibody inhibits or blocks the activity of PCSK9. In some embodiments, the anti-PCSK9 antibody disclosed herein has one or more of the following properties:.

In some embodiments, the anti-PCSK9 antibody disclosed herein or the antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises:.

In some embodiments, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein comprises a light chain variable region (VL), wherein the VL comprises:.

In some embodiments, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region VH and a light chain variable region VL, wherein,.

In a preferred embodiment, the VH comprises or consists of an amino acid sequence selected from SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>.

In a preferred embodiment, the VL comprises or consists of an amino acid sequence set forth in SEQ ID NO: <NUM>.

In a preferred embodiment, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein comprises:.

In some embodiments, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein.

In a preferred embodiment, the present invention provides an anti-PCSK9 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein.

In a preferred embodiment, the present invention provides an anti-PCSK9 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises (CDRs) LCDR1, LCDR2, and LCDR3; and combinations of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contained in the antibody or the antigen-binding fragment thereof are shown in the following table (Table I):.

In a preferred embodiment, the present invention provides an anti-PCSK9 antibody or an antigen-binding fragment thereof comprising a combination of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 shown in Table I.

In some embodiments, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein,.

In a preferred embodiment, the present invention provides an anti-PCSK9 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein combinations of the heavy chain variable region (VH) and the light chain variable region (VL) contained in the antibody or the antigen-binding fragment thereof are shown in the following table (Table II):.

In some embodiments, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain and/or a light chain, wherein.

In a preferred embodiment, the present invention provides an anti-PCSK9 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein combinations of the heavy chain and the light chain contained in the antibody or the antigen-binding fragment thereof are shown in the following table (Table III):.

In some embodiments, the antibody disclosed herein also encompasses a variant of the amino acid sequence of an anti-PCSK9 antibody, as well as an antibody that binds to the same epitope as any of the antibodies described above.

In certain embodiments, an antibody or an antibody fragment (preferably an antigen-binding fragment) that binds to PCSK9 or a fragment thereof is provided, wherein the antibody binds to an epitope within the fragment of PCSK9. In certain embodiments, an antibody or an antibody fragment that binds to PCSK9 or a fragment thereof is provided, wherein the antibody binds to an epitope within an amino acid fragment comprising human PCSK9 amino acid sequence set forth in SEQ ID NO: <NUM>.

In some embodiments, the heavy chain and/or light chain of the anti-PCSK9 antibody or the fragment thereof disclosed herein further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG.

In one embodiment of the present invention, the amino acid alteration described herein includes amino acid replacement, insertion or deletion. Preferably, the amino acid alteration described herein is an amino acid replacement, preferably a conservative replacement.

In a preferred embodiment, the amino acid alteration described herein occurs in a region outside the CDR (e.g., in FR). More preferably, the amino acid alteration described herein occurs in a region outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the replacement is a conservative replacement. A conservative replacement refers to the replacement of an amino acid by another amino acid of the same class, e.g., the replacement of an acidic amino acid by another acidic amino acid, the replacement of a basic amino acid by another basic amino acid, or the replacement of a neutral amino acid by another neutral amino acid. Exemplary replacements are shown in Table IV below:.

In certain embodiments, the replacement occurs in the CDRs of the antibody. Generally, an obtained variant has modifications (e.g., improvements) in certain biological properties (e.g., increased affinity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody. Exemplary replacement variants are affinity mature antibodies.

In certain embodiments, the antibody disclosed herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed. When the antibody comprises an Fc region, carbohydrate attached thereto can be altered. In some applications, modifications that remove undesired glycosylation sites may be useful, for example, removing fucose modules to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see <NPL>). In other applications, galactosidylation modification can be carried out to modify complement-dependent cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications can be introduced into an Fc region of the antibody disclosed herein, thereby producing an Fc region variant, such that, for example, the efficacy of the antibody in treating diseases is enhanced. The Fc region variant may comprise a human Fc region sequence (such as human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (such as replacement) at one or more amino acid positions. For examples of the Fc variant, see <CIT>, <CIT>, <CIT>; <CIT> and <NPL>), <CIT>, <CIT> and <NPL>), <CIT>, <NPL>); <CIT>; <CIT>; and <CIT>.

In certain embodiments, antibodies modified by cysteine engineering may need to be produced, such as "sulfo-MAb", wherein one or more residues of the antibodies are replaced by cysteine residues. A cysteine-modified antibody can be produced as described, for example, in <CIT>.

In certain embodiments, the antibody disclosed herein can be further modified to comprise other non-protein portions known in the art and readily available. Suitable portions for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly-<NUM>,<NUM>-dioxane, poly-<NUM>,<NUM>,<NUM>-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and glucan or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the anti-PCSK9 antibody or the antigen-binding fragment thereof disclosed herein has one or more of the following properties:.

In some embodiments, the anti-PCSK9 antibody disclosed herein is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.

In some embodiments, the anti-PCSK9 antibody disclosed herein is a monoclonal antibody. In some embodiments, the anti-PCSK9 antibody disclosed herein is humanized. In some embodiments, the anti-PCSK9 antibody disclosed herein is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-PCSK9 antibody disclosed herein is a human consensus framework sequence. In one embodiment, the anti-PCSK9 antibody disclosed herein also encompasses an antibody fragment thereof. Examples of the antibody fragment include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')<NUM>, a diabody, a linear antibody, a single-chain antibody molecule (e.g., scFv), and a multispecific antibody formed from the antibody fragment.

An antibody is digested by papain to produce two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize easily. An F(ab')<NUM> fragment having two antigen-binding sites and still being capable of being cross-linked with an antigen is produced by treatment of pepsin.

In some embodiments, the anti-PCSK9 antibody disclosed herein is a humanized antibody. Different methods for humanizing antibodies are known to those skilled, as summarized by Almagro & Fransson, (<NPL>). Almagro & Fransson distinguish between rational approach and empirical approach. The rational approach is characterized by generating a few engineered antibody variants and assessing their binding or any other property of interest. If the designed variants do not produce expected results, a new round of design and evaluation will be launched. The rational approach includes CDR grafting, resurfacing, superhumanization, and human string content optimization. In contrast, the empirical approach is based on generating large humanized variant libraries, and selects the best clones using enrichment techniques or high-throughput screening. Thus, the empirical approach depends on a reliable selection and/or screening system capable of searching for a large number of antibody variants. In vitro display technologies such as phage display and ribosome display meet these requirements and are well known to those skilled. The empirical approach includes FR library construction, guided selection, framework-shuffling, and humaneering.

In some embodiments, the anti-PCSK9 antibody disclosed herein is a human antibody. The human antibody can be prepared using a variety of techniques known in the art. The human antibody is generally described in <NPL>) and <NPL>).

In some embodiments, the anti-PCSK9 antibody disclosed herein is a chimeric antibody.

The antibody disclosed herein can be isolated by screening antibodies having desired activity in a combinatorial library. For example, various methods for generating phage display libraries and screening antibodies with desired binding characteristics in the libraries are known in the art. The methods are reviewed in, for example,<NPL>), and further described in, for example, <NPL>; <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); and <NPL>).

In some embodiments, the present invention also encompasses an anti-PCSK9 monoclonal antibody ("conjugate or immunoconjugate") conjugated to other substances, such as a detectable label (such as fluorescent dye, enzyme, substrate, bioluminescent substance, radioactive substance, and chemiluminescent portion), a cytotoxic agent or an immunosuppressant. The cytotoxic agent includes any agent that is harmful to cells. Examples of the cytotoxic agent (such as a chemotherapeutic agent) suitable for forming the immunoconjugate are known in the art, see, for example, <CIT>.

In some embodiments, the antibody disclosed herein may be monospecific, bispecific, or multispecific. The multispecific monoclonal antibody may be specific to different epitopes of a target polypeptide or may comprise antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al. <NUM>:<NUM>-<NUM>. The anti-PCSK9 monoclonal antibody may be linked to or co-expressed with another functional molecule (such as another peptide or protein). For example, the antibody or a fragment thereof may be functionally linked to one or more other molecules, such as another antibody or antibody fragment (for example, by chemical coupling, genetic fusion, non-covalent association, or other methods), to produce a bispecific or multispecific antibody having a second or more binding specificities.

In some embodiments, the antibody disclosed herein binds to human PCSK9 protein, such as human PCSK9 protein set forth in SEQ ID NO: <NUM>.

Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human body by therapy.

In another aspect, the present invention relates to a method for inhibiting the binding of PCSK9 to LDL-receptor (LDLR) in a subject, wherein the method comprises administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. The present invention also relates to use of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein in preparing a drug for inhibiting the binding of PCSK9 to LDL-receptor (LDLR) in a subject.

In another aspect, the present invention relates to a method for lowering the cholesterol level of a subject, wherein the method comprises administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. In one embodiment, cholesterol is LDL-cholesterol, preferably serum cholesterol. In another aspect, the present invention relates to a method for lowering the LDL-cholesterol level of a subject, comprising administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. In some embodiments, the present invention relates to a method for lowering the LDL-cholesterol level in the serum of a subject, wherein the method comprises administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. In another aspect, the present invention also relates to use of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein in preparing a drug for lowering the cholesterol level (LDL-cholesterol level or serum LDL-cholesterol level in one embodiment) in a subject.

In another aspect, the present invention relates to a method for preventing or treating a disorder associated with increased LDL-cholesterol level in a subject, wherein the method comprises administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. The present invention also relates to use of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein in preparing a drug for treating a disorder associated with increased LDL-cholesterol level in a subject.

In one aspect, the present invention relates to a method for preventing or treating a cholesterol-related disease, wherein the method comprises administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. The present invention also relates to use of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein in preparing a drug for treating a cholesterol-related disease. Exemplary and non-limiting examples of the cholesterol-related disease will be provided hereinafter. In some embodiments, the cholesterol-related disease is hypercholesterolemia or hyperlipidaemia. In some embodiments, the present invention relates to a method for treating hypercholesterolemia and/or hyperlipidaemia, wherein the method comprises administering to the subject an effective amount of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. In some embodiments, the present invention also relates to use of any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein in preparing a drug for treating hypercholesterolemia and/or hyperlipidaemia.

In one aspect, the present invention relates to a method for preventing or treating any disease or disorder capable of being improved, alleviated, inhibited or prevented by eliminating, inhibiting, or reducing the activity of PCSK9. In some embodiments, diseases or disorders that can be treated or prevented by using statins can also be treated or prevented by using any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein. In some embodiments, diseases or disorders that can benefit from the prevention of cholesterol synthesis or the increase of LDLR expression can also be treated by using any of the anti-PCSK9 antibody preparations described herein or the anti-PCSK9 antibody disclosed herein.

In some embodiments, the method described herein also comprises administering to the subject an effective amount of an additional therapeutic agent in a combination therapy. In one embodiment, the present invention also relates to a combination product that comprises the antibody preparation or the antibody described herein and the additional therapeutic agent. In one embodiment, the additional therapeutic agent can increase the level of LDLR protein. In another embodiment, the additional therapeutic agent can decrease the level of LDL-cholesterol. In another embodiment, the additional therapeutic agent comprises statins. In another embodiment, the additional therapeutic agent is a statin. In some embodiments, the statin is selected from atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and any combination thereof. In certain embodiments, the additional therapeutic agent is used to prevent and/or treat atherosclerosis and/or cardiovascular diseases. In certain embodiments, the additional therapeutic agent is used to lower the risk of cardiovascular event recurrence. In certain embodiments, the additional therapeutic agent is used to increase the level of HDL-cholesterol in a subject. In some embodiments, the subject or individual is a mammal, preferably a human.

The aforementioned combination therapy includes combined administration (wherein two or more therapeutic agents are contained in one preparation or separate preparations) and separate administration, wherein any of the anti-PCSK9 antibody preparations or the anti-PCSK9 antibody disclosed herein can be administered before, concurrently with, and/or after the administration of an additional therapeutic agent and/or an adjuvant.

In order to prevent or treat diseases, the appropriate dosage of the preparation or antibody disclosed herein (used alone or in combination with one or more other additional therapeutic agents) will depend on the type of the disease to be treated, the type of the antibody, the severity and progression of the disease, the purpose for which the antibody is administered (prophylactic or therapeutic), previous treatments, the clinical history of the patient, responses to the preparation or antibody, and the discretion of the attending physician. The antibody is suitably administered to a patient through a single treatment or through a series of treatments. The exemplary dosage range of the antibody includes <NUM>/kg to <NUM>/kg.

In other aspects, the present invention provides use of any of the anti-PCSK9 antibody preparations or the anti-PCSK9 antibody disclosed herein in the production or preparation of a drug used to treat the aforementioned related diseases or disorders.

In certain embodiments, any anti-PCSK9 antibody preparation disclosed herein or the anti-PCSK9 antibody disclosed herein can be prophylactically administered to prevent or alleviate the onset of hypercholesterolemia, hyperlipidemia, cardiovascular diseases, and/or any cholesterol-related disease. In certain embodiments, any anti-PCSK9 antibody preparation disclosed herein or the anti-PCSK9 antibody disclosed herein can be administered to treat existing hypercholesterolemia and/or hyperlipidemia and/or cardiovascular diseases and/or any cholesterol-related disease. In some embodiments, any anti-PCSK9 antibody preparation disclosed herein or the anti-PCSK9 antibody disclosed herein will delay the onset of a disorder and/or symptoms associated with the disorder.

In one aspect, the present invention provides a nucleic acid encoding any aforementioned anti-PCSK9 antibody or a fragment thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cells suitable for preparing an antibody or an antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

In one embodiment, the present invention provides a method for preparing an anti-PCSK9 antibody or a fragment thereof (preferably an antigen binding fragment), wherein the method comprises culturing the host cell under conditions suitable for the expression of the nucleic acid encoding the antibody or the fragment thereof (preferably the antigen-binding fragment), and optionally isolating the antibody or the fragment thereof (preferably the antigen-binding fragment). In a certain embodiment, the method further comprises isolating the anti-PCSK9 antibody or the fragment thereof (preferably the antigen-binding fragment) from the host cell.

In one aspect, the present invention relates to a method for detecting PCSK9 protein in a sample, wherein the method comprises (a) contacting the sample with any of the anti-PCSK9 antibody preparations described herein or the antibody disclosed herein; and (b) detecting the formation of a complex between the anti-PCSK9 antibody or fragment thereof and the PCSK9 protein. In one embodiment, the anti-PCSK9 antibody is detectably labeled.

The present invention also encompasses any combination of the embodiments described herein. Any of the embodiments described herein or any combination thereof is applicable to any and all of the preparations or anti-PCSK9 antibodies or fragments thereof, the methods and the use described herein.

The terms used in the present invention have the following definitions. If no definitions are given herein, the terms used in the present invention have the meanings commonly understood in the art.

For the purpose of explaining this specification, the following definitions will be used, and wherever appropriate, terms used in the singular form may also include the plural form, and vice versa. It should be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

As used herein, the term "and/or" refers to any one of the options or two or more of the options.

As used herein, the term "comprise" or "include" is intended to mean that the described elements, integers or steps are included, but not to the exclusion of any other elements, integers or steps. The term "comprise" or "include" used herein, unless otherwise specified, also encompasses the situation where the entirety consists of the described elements, integers or steps. For example, when referring to "comprise" an antibody variable region of a particular sequence, it is also intended to encompass an antibody variable region consisting of the specific sequence.

Unless otherwise stated, the term "proprotein convertase subtilisin/kexin type <NUM> (PCSK9)", "PCSK9", or "NARC-<NUM>" used herein refers to any natural PCSK9, preferably any natural PCSK9 from any vertebrate (including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats)). This term encompasses "full-length" unprocessed PCSK9 and PCSK9 in any form resulting from intracellular processing or any fragment thereof. This term also includes variants of naturally existing PCSK9, such as splice variants or allelic variants.

The term "activity of PCSK9" or "bioactivity of PCSK9" used herein includes any biological effect of PCSK9. In some embodiments, the activity of PCSK9 includes the ability of PCSK9 to interact with or bind to a substrate or a receptor. In some embodiments, the bioactivity of PCSK9 is the ability of PCSK9 to bind to LDL-receptor (LDLR). In some embodiments, PCSK9 binds to LDLR and catalyzes a reaction involving LDLR. In some embodiments, the activity of PCSK9 includes the ability of PCSK9 to decrease or reduce the availability of LDLR. In some embodiments, the bioactivity of PCSK9 includes the ability of PCSK9 to increase the amount of LDL in a subject. In some embodiments, the bioactivity of PCSK9 includes the ability of PCSK9 to reduce the amount of LDLR capable of binding to LDL in a subject. In some embodiments, the bioactivity of PCSK9 includes the ability of PCSK9 to reduce the amount of LDLR capable of binding to LDL. In some embodiments, the bioactivity of PCSK9 includes any bioactivity caused by PCSK9 signaling.

The term "anti-PCSK9 antibody", "anti-PCSK9", "PCSK9 antibody" or "antibody binding to PCSK9" refers to an antibody capable of binding to a PCSK9 protein or a fragment thereof with sufficient affinity so as to serve as a diagnostic agent and/or a therapeutic agent in targeting PCSK9. In one embodiment, an anti-PCSK9 antibody binds to an unrelated, non-PCSK9 protein to an extent less than about <NUM>% of the extent to which the antibody binds to PCSK9, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (KD) of an anti-PCSK9 antibody is less than or equal to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> (e.g., <NUM>-<NUM> M or less, e.g., <NUM>-<NUM> M to <NUM>-<NUM> M, e.g., <NUM>-<NUM> M to <NUM>-<NUM> M).

"Antibody fragment" refers to a molecule different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. Examples of the antibody fragment include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')<NUM>; a diabody; a linear antibody; a single-chain variable fragment (e.g., scFv); a single-domain antibody; a bivalent or bispecific antibody or a fragment thereof; a Camelidae antibody; and a bispecific antibody or multispecific antibody formed from antibody fragments.

As used herein, the term "epitope" refers to a moiety of an antigen (e.g., PCSK9) that specifically interacts with an antibody molecule.

An "antibody that binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks <NUM>% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks <NUM>% or more of the binding of the antibody to its antigen in a competition assay.

An antibody that competes with a reference antibody to bind to its antigen refers to an antibody that blocks more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another, such as direct or indirect solid-phase radioimmunoassay (RIA), direct or indirect solid-phase enzyme immunoassay (EIA), and sandwich competition assay (see, e.g., <NPL>).

An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the binding of the reference antibody to its antigen. Conversely, the reference antibody inhibits more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the binding of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

An antibody that shows the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having at least more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.

"Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and/or comprises antigen-contacting residues ("antigen contact site"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, which are numbered sequentially from N-terminus. The CDRs located in a heavy chain variable domain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, whereas the CDRs located in a light chain variable domain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of a light chain variable region or a heavy chain variable region, the exact amino acid sequence boundary of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (<NPL>; <NPL>)), Kabat based on antibody sequence variability (<NPL>)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt. fr/ on the World Wide Web), and North CDR definition based on the affinity propagation clustering using a large number of crystal structures.

However, it should be noted that boundaries of CDRs of variable regions of an antibody based on different assignment systems may differ. That is, CDR sequences of variable regions of an antibody defined by different assignment systems differ. Therefore, when it comes to defining an antibody with specific CDR sequences defined in the present invention, the scope of antibody also encompasses such antibodies whose variable region sequences comprise the specific CDR sequences, but having claimed CDR boundaries different from the specific CDR boundaries defined by the present invention due to a different protocol (e.g., different assignment system rules or their combinations) applied.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The smallest overlapping region can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North methods, thereby providing a "minimal binding unit" for antigen binding. The minimal binding unit may be a sub-portion of the CDR. As will be clear to those skilled in the art, residues of the rest CDR sequences can be determined via antibody structure and protein folding. Therefore, any variants of the CDRs given herein are also considered in the present invention. For example, in a CDR variant, the amino acid residues in the minimal binding unit may remain unchanged, while the other CDR residues defined by Kabat or Chothia may be substituted by conservative amino acid residues.

"Functional Fc region" possesses the "effector functions" of Fc regions of native sequences. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, cell surface receptor (e.g., B cell receptor, or BCR) down-regulation, and the like. Such effector functions generally require that the Fc region is associated with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays, such as those disclosed herein.

The term "Fc region" is used herein to define a C-terminus region of an immunoglobulin heavy chain, which comprises at least one portion of a constant region. The term includes Fc regions of native sequences and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is based on an EU numbering system, which is also called EU index as described in<NPL>.

"Antibodies and antigen-binding fragments thereof" applicable to the present invention include but are not limited to polyclonal, monoclonal, monovalent, bispecific, isoconjugate, multispecific, recombinant, heterogenous, heterogenous hybrid, chimeric, humanized (particularly CDR-grafted), deimmunized or human antibodies, Fab fragments, Fab' fragments, F(ab')<NUM> fragments, fragments produced by an Fab expression library, Fd, Fv, disulphide-linked Fv (dsFv), single-chain antibodies (such as scFv), diabodies or tetrabodies (<NPL>), nanobodies (also referred to as single-domain antibodies), anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibody disclosed herein), and an epitope-binding fragment of any of the above.

"Human consensus framework" refers to a framework which represents the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is a selection from subtypes of variable domain sequences.

"Antibody in IgG form" refers to the heavy chain constant region of the antibody belonging to the IgG form. Heavy chain constant regions of all antibodies of the same type are identical, and heavy chain constant regions of antibodies of different types are different. For example, an antibody in the form of IgG1 refers to the Ig domain of its heavy chain constant region being an Ig domain of an IgG1.

The term "therapeutic agent" described herein encompasses any substance that is effective in preventing or treating cholesterol-related diseases, including cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents, etc..

"Human antibody" refers to an antibody having an amino acid sequence which corresponds to the amino acid sequence of an antibody generated by a human or human cell or derived from a non-human source that utilizes human antibody libraries or other human antibody encoding sequences. This definition of a human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues.

"Humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all CDRs correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (such as a non-human antibody) refers to an antibody that has been humanized.

"Individual" or "subject" includes mammals. The mammals include, but are not limited to, domestic animals (e.g., cattle, goats, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is an antibody which has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than <NUM>% or <NUM>% as determined by, e.g., electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF) and capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, <NPL>).

The "percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences (with gaps introduced if necessary) to achieve maximum percent sequence identity without considering any conservative replacement as part of sequence identity. Various methods in the art can be employed to perform sequence alignment so as to determine the percent amino acid sequence identity, for example, using computer software available to the public, such as BLAST, BLAST-<NUM>, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to obtain maximum alignment for the full length of the aligned sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases to, e.g., identify other family member sequences or related sequences.

The term "combination product" refers to a kit with a fixed combination, a non-fixed combination, or a part for combined administration in the form of a dose unit, wherein two or more therapeutic agents can be independently administered simultaneously or separately administered at intervals of time, especially when these intervals allow combination partners to exhibit collaboration, such as synergistic effect. The term "fixed combination" means that the antibody disclosed herein and a combination partner (e.g., other therapeutic agents, such as statin drugs) are simultaneously administered to a patient in the form of a single entity or dose. The term "non-fixed combination" means that the antibody disclosed herein and a combination partner (e.g., other therapeutic agents, such as statins) are simultaneously, concurrently, or sequentially administered to a patient as separate entities, without specific time limitation, wherein such administration provides therapeutically effective levels of the two compounds in the patient. The latter is also applicable to a cocktail therapy, e.g., administration of three or more therapeutic agents. In one preferred embodiment, the drug combination is a non-fixed combination.

The term "combination therapy" means that two or more therapeutic agents are administered to treat cholesterol-related diseases as described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule with a fixed proportion of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a variety of or separate containers (such as tablets, capsules, powder and liquid). The powder and/or liquid can be reconstituted or diluted to a desired dosage before administration. In addition, such administration also includes using each type of therapeutic agents in a sequential manner at approximately the same time or at different times. In any case, the therapeutic regimen will provide the beneficial effect of the drug combination in the treatment of disorders or symptoms described herein.

As used herein, "treatment" (or "treat" or "treating") refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "prevention" (or "prevent" or "preventing") includes the inhibition of the onset or progression of a disease or disorder or a symptom of a specific disease or disorder. In some embodiments, subjects with family histories are candidates for prophylactic regimens. Generally, the term "prevention" (or "prevent" or "preventing") refers to the administration of a drug prior to the onset of disorders or symptoms, particularly in subjects at risk.

The term "cholesterol-related disease" includes any one or more of the following: hypercholesterolemia, hyperlipidaemia, heart disease, metabolic syndrome, diabetes mellitus, coronary heart disease, stroke, cardiovascular diseases, Alzheimers disease, and general dyslipidemia (shown as, for example, increased total serum cholesterol, increased LDL, increased triglyceride, increased VLDL, and/or low HDL). Some non-limiting examples of primary and secondary dyslipidemias that can be treated with an anti-PCSK9 antibody (alone or in combination with one or more other drugs) include metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemias, including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, and familial defective apolipoprotein B-<NUM>; polygenic hypercholesterolemia; remnant removal disease; hepatic lipase deficiency; dyslipidemia secondary to any of the following: dietary indiscretion, hypothyroidism, drugs (including estrogen and progesterone therapies, β blockers and thiazide diuretics); nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia.

The term "hypercholesterolemia" used herein refers to a disorder in which the cholesterol level rises to above a certain level. In some embodiments, the LDL-cholesterol level rises to above a certain level. In some embodiments, the serum LDL-cholesterol level rises to above a certain level.

The term "vector" used herein refers to a nucleic acid molecule capable of proliferating another nucleic acid to which it is linked. The term includes vectors that serve as self-replicating nucleic acid structures as well as vectors binding to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are called "expression vectors" herein.

"Subject/patient sample" refers to a collection of cells or fluids obtained from a patient or a subject.

The source of tissue or cell samples can be solid tissues, e.g., from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluids, amniotic fluids, peritoneal fluids, or interstitial fluids; and cells from a subject at any time during pregnancy or development. Tissue samples may comprise compounds which are naturally not mixed with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

The term "effective amount" refers to an amount or a dosage of the preparation, antibody, or fragment described herein which generates an expected effect in a treated patient after the administration of a single or multiple doses. The effective amount can be easily determined by an attending physician as a person skilled in the art by considering a variety of factors as follows: species such as mammals; its size, age, and general health condition; the specific disease involved; the extent or severity of the disease; response in an individual patient; specific antibody administered; route of administration; bioavailability characteristics of the administered preparation; selected dosage regimen; and use of any concomitant therapy.

The "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result at a necessary dosage for a necessary period of time. The therapeutically effective amount of the preparation, antibody or antibody fragment described herein, or conjugate or composition thereof can vary depending on a variety of factors such as disease state, age, sex and weight of an individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. The therapeutically effective amount is also such an amount in which any toxic or undesired effect of the preparation, antibody or antibody fragment or conjugate or composition thereof is inferior to the therapeutically beneficial effect.

The "prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Generally, since a prophylactic dose is administered in a subject before or at an earlier stage of a disease, a prophylactically effective amount will be less than a therapeutically effective amount.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids are introduced, including progenies of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progenies derived therefrom, regardless of the number of passages. A generation may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant generations having the same function or bioactivity that are screened or selected from the initially transformed cells are included herein.

As used herein, the term "preparation" refers to a composition which is suitably administered to animals, preferably mammals (including humans) and comprises at least one active ingredient and at least one non-active ingredient. "Liquid preparation" refers to a preparation in the form of liquid. The liquid preparation disclosed herein comprises (i) an anti-PCSK9 antibody or a fragment thereof; (ii) a buffer; (iii) a viscosity inhibitor; (iv) a surfactant; and (v) a solvent. The composition of the preparation disclosed herein may be as shown in the aforementioned liquid preparation embodiments. The liquid preparation disclosed herein is preferably an injection, more preferably a subcutaneous injection.

As used herein, the "buffer" refers to a pH buffer. Preferably, the buffer can keep pH of the liquid preparation disclosed herein at about <NUM> to <NUM>, preferably about <NUM> to <NUM>, more preferably about <NUM> to <NUM>, and most preferably about <NUM>. The concentration of the buffer in the liquid preparation is, for example, about <NUM>/mL to <NUM>/mL, about <NUM>/mL to <NUM>/mL, about <NUM>/mL to <NUM>/mL, about <NUM>/mL to <NUM>/mL, about <NUM>/mL to <NUM>/mL, or about <NUM>/mL. Preferably, the buffer is selected from histidine, glutamate, phosphate, acetate, citrate and tris(hydroxymethyl)aminomethane.

As used herein, the "viscosity inhibitor" refers to a substance that can reduce the viscosity of the liquid preparation comprising an anti-PCSK9 antibody. The concentration of the viscosity inhibitor in the liquid preparation is, for example, about <NUM> mmol/L to <NUM> mmol/L, about <NUM> mmol/L to <NUM> mmol/L, about <NUM> mmol/L to <NUM> mmol/L, or about <NUM> mmol/L to <NUM> mmol/L. Preferably, the viscosity inhibitor is selected from sugar alcohol, arginine, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate, and combinations thereof. More preferably, the viscosity inhibitor is sorbitol or a combination of sorbitol and arginine or arginine salt (preferably arginine hydrochloride). Preferably, the concentration of sorbitol in the liquid preparation is about <NUM>% to <NUM>% (w/v); and the concentration of arginine or arginine salt (preferably arginine hydrochloride) in the liquid preparation is about <NUM> mmol/L to <NUM> mmol/L, preferably about <NUM> mmol/L to <NUM> mmol/L, and more preferably about <NUM> mmol/L.

As described herein, the "surfactant" refers to a substance which can significantly change the interface state of a solution system after being added in a small amount. The concentration of the surfactant in the liquid preparation is, for example, about <NUM>/mL to <NUM>/mL, about <NUM>/mL to <NUM>/mL, or about <NUM>/mL to <NUM>/mL, and more preferably about <NUM>/mL. Preferably, the surfactant is a nonionic surfactant, such as pluronics, polysorbate-<NUM>, polysorbate-<NUM>, polysorbate-<NUM>, or polysorbate-<NUM>.

As used herein, the term "solvent" refers to a liquid that is used to dissolve or suspend active ingredients and non-active ingredients to form a liquid preparation. Solvents that can be used in the present invention include, but are not limited to, water for injection, organic solvents for injection (including but not limited to oil for injection, ethanol, and propylene glycol), and combinations thereof.

As used herein, the term "sugar alcohol" refers to a corresponding polyol which is obtained by reduction reaction, such as catalytic hydrogenation, of monosaccharide. Sugar alcohols include, but are not limited to, sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, etc..

The term "about" used in combination with a numerical value is intended to encompass the numerical values in a range from a lower limit less than the specified numerical value by <NUM>% to an upper limit greater than the specified numerical value by <NUM>%.

As used herein, "w/v" refers to "weight/volume", for example, "<NUM>% w/v" means <NUM>/<NUM> = <NUM>/mL = <NUM>/mL.

The present invention will be further illustrated below with reference to examples, and the following examples should not be construed as limiting the present invention. The scope of the present invention is defined by the claims.

According to the instructions of the manufacturer, PCSK9 antigen (SEQ ID NO: <NUM>) was labeled with biotin using the sulfosuccinimidylbiotin labeling kit from Pierce. The FITC-labeled goat anti-human immunoglobulin F(ab') kappa chain antibody (LC-FITC) was purchased from Southern Biotech, polyethylene avidin (SA-PE) was purchased from Sigma, and streptavidin-<NUM> (SA-<NUM>) was purchased from Molecular Probes. Streptomycin microbeads and a cellular immunomagnetic bead separation column were purchased from Miltenyi LS.

Eight synthetic yeast-based antibody presentation libraries (from Adimab) were amplified according to an existing method (<CIT>; <CIT>; <CIT>), with the diversity of each library reaching <NUM> × <NUM><NUM>. Briefly, the first two rounds of screening employed magnetic-activated cell sorting using the MACS system available from Miltenyi. First, yeast cells (approximately <NUM>×<NUM><NUM> cells/library) from each library were incubated in FACS buffer (phosphate buffer, containing <NUM>% bovine serum albumin) for <NUM> at room temperature, and the buffer contained <NUM> biotin-labeled PCSK9 antigen as prepared above. The cells were washed once with <NUM> of pre-cooled FACS buffer and resuspended in <NUM> of the same buffer, followed by addition of <NUM>µL of streptomycin microbeads and incubation at <NUM> for <NUM>. The mixture was centrifuged at <NUM> rpm for <NUM>. After discarding the supernatant, the cells were resuspended in <NUM> of FACS buffer. The resulting cell suspension was loaded on a Miltenyi LS column. After loading, the column was washed three times with FACS buffer, <NUM> each time. The Miltenyi LS column was removed from the magnetic field and eluted with <NUM> of growth medium. The eluted yeast cells were collected and incubated overnight at <NUM>.

The next round of sorting was performed using a flow cytometer, wherein approximately <NUM> × <NUM><NUM> yeast cells screened by the MACS system were washed three times with FACS buffer and cultured in PCSK9 antigens or PCSK9-Fc fusion antigens labeled by a low concentration of biotin (<NUM>-<NUM>) at room temperature. The culture media were discarded. After the cells were washed twice with FACS buffer, the cells were mixed with LC-FITC (<NUM>:<NUM> dilution) and SA-<NUM> (<NUM>:<NUM> dilution) or EA-PE (<NUM>:<NUM> dilution) and cultured at <NUM> for <NUM>. The cells were washed twice with pre-cooled FACS buffer, resuspended in <NUM> of buffer and transferred into a separator tube with a filter. The cells were sorted using FACS ARIA (BD Biosciences).

Several rounds of screening were then performed to give competitive ligands and remove nonspecific conjugates (such as the membrane protein of CHO cells). After the last several rounds of sorting, the collected yeast cells were plated and cultured at <NUM> overnight, and target monoclonal antibodies were picked out. Sanger sequencing was employed to sequence variable regions of the obtained antibodies. About <NUM> antibodies with unique variable region sequences were obtained and identified one by one.

These anti-PCSK9 antibody proteins were obtained by yeast expression and protein A affinity chromatography purification.

The yeast cells expressing the anti-PCSK9 antibodies obtained by screening were induced by shaking at <NUM> for <NUM> to express the anti-PCSK9 antibodies. After the induction, the yeast cells were centrifuged at <NUM> rpm for <NUM>, and the supernatant was collected. The anti-PCSK9 antibodies in the supernatant were purified with Protein A and eluted with acetic acid buffer at pH <NUM>, and the anti-PCSK9 antibodies were harvested. The antibodies were digested with papain and purified with KappaSelect (GE Healthcare) to produce corresponding Fab fragments.

According to a conventional method in the art, a gene DNA encoding the anti-PCSK9 antibodies was obtained from the aforementioned yeast cells expressing the anti-PCSK9 antibodies and, based on the conventional method, the gene DNA was cloned to a new expression vector (pCDNA3. The aforementioned expression vector containing a target antibody gene and a transfection reagent Lipofectamine TM2000 (purchased from Invitrogen) was transiently transfected into cultured human embryonic kidney cells <NUM> according to a scheme provided by the manufacturer. The medium was discarded, and the cells were diluted to <NUM> × <NUM><NUM>/mL with fresh medium. The cells were cultured at <NUM>, <NUM>% CO<NUM> for <NUM> days, with fresh medium fed every <NUM>. After <NUM> days, the cells were centrifuged at <NUM> rpm for <NUM>. The supernatant was purified with Protein A to achieve an antibody purity of greater than <NUM>%.

The ForteBio affinity assay was performed according to the prior art (<NPL>). Briefly, a sensor was equilibrated offline in assay buffer for <NUM>, and was equilibrated online for <NUM> to establish a baseline. The purified antibody obtained as described above was loaded on line onto an AHQ sensor. The sensor was then exposed to <NUM> PCSK9 antigens for <NUM>, before transferring the sensor to the assay buffer for dissociation for <NUM>. The kinetic analysis was performed using a <NUM>:<NUM> binding model.

The assay of equilibrium affinity is as described above (Estep, et al. The biotin-labeled PCSK9 antigen (b-PCSK9) with a final concentration of <NUM>-<NUM> pM obtained above was added to phosphate-buffered saline (PBSF) containing <NUM>% IgG-free BSA, and the anti-PCSK9 Fab or mAb obtained above was serially diluted <NUM> to <NUM> folds to give an Fab or mAb solution with a concentration of <NUM>-<NUM>. The antibodies (diluted in <NUM> phosphate-buffered saline) were coated on a MSD-ECL plate at <NUM> overnight or at room temperature for <NUM>. <NUM>% BSA was added, and the antibodies were immobilized at <NUM> rpm for <NUM> at room temperature, and were then washed <NUM> times with a washing buffer (PBSF + <NUM>% Tween <NUM>). The samples were added to the plate, incubated at <NUM> rpm in a shaker at room temperature for <NUM> and then washed once. The plate was added with <NUM> ng/mL sulfotag-labeled streptomycin (diluted in PBSF) and incubated at room temperature for <NUM>. After washing the plate <NUM> times with the buffer, antigens binding on the plate were identified using MSD Sector Imager <NUM>. The percentage of unbinding antigens was obtained by the antibody titration method, and it was discovered that the binding of the anti-PCSK9 Fab or mAb to the antigens follows a quadratic equation of pharmacokinetics.

The identification of epitope binding adopted a standard sandwich interactive blocking analysis method. A target-specific control IgG was fixed on an AHQ sensor, and an irrelevant human IgG1 antibody was used to occupy an empty Fc-binding site on the sensor. The sensor was immersed in <NUM> target antigen PCSK9 solution for <NUM>, and was then put into a second <NUM> anti-PCSK9 antibody or ligand solution prepared as above. Data were read and processed by ForteBio data analysis software <NUM> (ForteBio). If the antigen could be bound by a second antibody or ligand after being bound by an antibody, it was suggested that there was an unbound epitope (non-competitive), otherwise epitope blocking (competitive or ligand blocking) was indicated. Through the aforementioned screening and identification, some antibodies that can block the binding of PCSK9 to LDLR and bind to both human and murine PCSK9 were obtained. To obtain an anti-PCSK9 antibody with higher affinity, the antibody ADI-<NUM> was optimized by the following method.

This method was to introduce mutations into antibody heavy chain regions by the conventional mismatched PCR method. In the PCR process, the probability of base pair mismatch was increased to about <NUM> bp by adding <NUM> highly mutated base analogs dPTP and <NUM>-oxo-dGTP.

The resulting mismatched PCR product was constructed into a vector containing a heavy chain constant region by homologous recombination. By this method, a secondary pool with the capacity of <NUM> × <NUM><NUM> was obtained under screening pressure including PCSK9 antigen titer, unlabeled antigen competition, and competition with the parent antibody. Three rounds of screening were successfully performed by FACS.

CDRH3 genes of progeny antibodies obtained by VHmut were constructed into a CDRH1/CDRH2 gene pool with the diversity of <NUM> × <NUM><NUM>, and <NUM> rounds of screening were carried out for the genes. The first round of screening adopted MACS, while the second and third rounds adopted FACS. Antibody-antigen conjugates were subjected to pressurized screening for screening out antibodies with the highest affinity.

The first round of optimization: The first step was to increase the affinity of the anti-PCSK9 antibody ADI-<NUM> (named "parent" antibody) with human-murine cross-activity and ligand competition. In short, mutations were introduced into the parent antibody (by employing the "mismatched PCR" method) to establish a secondary yeast-based antibody presentation library. Finally, a secondary library with a size of about <NUM> × <NUM><NUM> was generated for the subsequent enriching of antibodies with higher affinity. Screening pressure included PCSK9 antigen titer, unlabeled antigen competition, and competition with the parent antibody. The FACS technique was also used to screen a target population (see <NPL>). After <NUM> to <NUM> rounds of enriching, the obtained yeasts were plated to give monoclonal antibodies. After this, <NUM> progenies with improved affinity, ADI-<NUM>, ADI-<NUM> and ADI-<NUM>, were obtained. Assayed by ForteBio Octet, the KD ranges of the three antibodies were <NUM> to <NUM>. The two progeny antibodies ADI-<NUM> and ADI-<NUM> were used in the second round of affinity optimization.

The second round of affinity optimization: The second step was to improve the affinity of the two anti-PCSK9 monoclonal antibodies ADI-<NUM> and ADI-<NUM> (named "parent" antibody) with human-murine cross-activity and ligand competition. In short, a secondary yeast-based antibody presentation library was established again for each parent antibody. The CDR-H3 and light chains (LC) of the parent antibodies were combined with CDR-H1 and CDR-H2 of genes in an existing yeast library (named "H1/H2" optimization). Finally, <NUM> libraries with a size of about <NUM> × <NUM><NUM> were generated for the subsequent enriching of antibodies with higher affinity. The screening method was the same as that in the first round of screening. After <NUM> to <NUM> rounds of enriching, the yeasts were plated to give monoclonal antibodies. After this, progeny antibodies with improved affinity were obtained, among which ADI-<NUM>, ADI-<NUM> and ADI-<NUM> were variants of CDR-H1 and CDR-H2 regions of ADI-<NUM>, and ADI-<NUM>, ADI-<NUM> and ADI-<NUM> were variants of VH regions of ADI-<NUM>. The related sequence information of the antibodies is shown in Tables A-C. The affinity of these antibodies for human PCSK9 was increased by <NUM> times, and the KD ranges were between <NUM>-<NUM> pM and <NUM> pM (Table <NUM> and Table <NUM>), as detected by the ForteBio method and MSD-SET assay. The affinity of part of the antibodies was about ten times higher than that of control antibodies. The identification of other antibody functions can further reduce the number of antibodies so as to facilitate preclinical development.

The sequence information and number of each anti-PCSK9 antibody involved herein are shown in Tables A-C below:.

The PCSK9 protein (biotin-labeled PCSK9 protein) described in example <NUM> was diluted to <NUM> nmol/L with PBS solution (phosphate-buffered saline solution) to serve as a working solution. The anti-PCSK9 antibodies (ADI-<NUM>, ADI-<NUM>, ADI-<NUM>, ADI-<NUM>, ADI-<NUM> and ADI-<NUM>) obtained in example <NUM> were diluted with PBS solution to concentrations of <NUM> nmol/L, <NUM> nmol/L, <NUM> nmol/L, <NUM> nmol/L and <NUM>. <NUM> nmol/L, respectively, and according to the same method, solutions of control antibodies (Alirocumab, Evolocumab, Bococizumab and Lodelcizumab) at each concentration were prepared. The PCSK9 working solution was mixed with an equal volume of each gradiently diluted anti-PCSK9 antibody sample or control sample. CHO cells (CHO-LDLR) overexpressing human LDLR were resuspended in PBS solution and counted. The cell concentration was adjusted to <NUM> × <NUM><NUM> cells/mL with PBS solution. The CHO cells were inoculated into a U-bottom <NUM>-well plate, with <NUM>µL of cell medium added in each well. <NUM>µL of a mixed solution of PCSK9 and each anti-PCSK9 antibody was added. Each well contains <NUM> × <NUM><NUM> cells. The mixture was centrifuged at <NUM> at room temperature for <NUM>, and the supernatant was discarded. Anti-His-FITC (R&D Systems) was diluted with PBS solution at a ratio of <NUM>:<NUM> to a final concentration of <NUM>µg/mL, added to a <NUM>-well plate, at <NUM>µL per well, and put in an ice bath for <NUM>. Centrifugation was performed at <NUM> at room temperature for <NUM>, the supernatant was gently discarded, and <NUM>µL of PBS solution was added into each well; then centrifugation was performed at <NUM> at room temperature for <NUM>, and the supernatant was gently discarded. This operation was repeated <NUM> times. PBS solution was added to each well at <NUM>µL/well, and the cells were pipetted for several times with a pipette for resuspension. The fluorescence signal value of cells was detected by a flow cytometer.

For the fluorescence signals detected in the experiment, see Table <NUM> below.

The raw data in Table <NUM> were analyzed and plotted using GraphPad Prism6, obtaining <FIG>. According to the results, the anti-PCSK9 antibodies have a capability of blocking the binding of PCSK9 to LDLR equivalent to that of the control antibodies.

A HepG2 cell cryopreservation tube was taken out of a liquid nitrogen storage tank and rapidly thawed at <NUM> under a water bath. The cell suspension was transferred into a <NUM> centrifuge tube, and <NUM> of room-temperature growth medium (<NUM>% DMEM + <NUM> % FBS, both purchased from Gibco) was slowly added. After <NUM> of centrifugation at <NUM> r/min at room temperature, the deposited cells were resuspended in fresh growth medium, transferred into a culture flask, and cultured at <NUM>, <NUM>% CO<NUM>. HepG2 cells in the log phase were washed twice with PBS solution and then digested for <NUM> with <NUM> of <NUM>% trypsin (purchased from Gibco), and the cells were resuspended in <NUM> of growth medium to quench the reaction. After the cell concentration was adjusted to <NUM> × <NUM><NUM> cells /mL with the growth medium, the HepG2 cells were inoculated into a black clear-bottom <NUM>-well plate coated with poly-D-lysine (purchased from Nunc) at <NUM>µL/well, and incubated in an incubator at <NUM>, <NUM>% CO<NUM> for <NUM> to <NUM>. The growth medium was discarded and replaced by assay medium (DMEM + <NUM>% FBS) at <NUM>µL/well, and the HepG2 cells were cultured in an incubator at <NUM>, <NUM>% CO<NUM> overnight. The antibody samples (ADI-<NUM>, ADI-<NUM>, ADI-<NUM> and ADI-<NUM>) were diluted to <NUM> nmol/L with the assay medium. With <NUM> nmol/L as the initial concentration, <NUM>-fold gradient dilution was performed in the same manner for both the positive control antibodies (Alirocumab, Evolocumab and Lodelcizumab) and the negative control antibodies (LDL, PCSK9 + LDL and IgG). <NUM>µL of each obtained concentration-gradient sample was mixed with an equal volume (<NUM>µL) of <NUM> nmol/L PCSK9, thus obtaining each mixture. The blank control was <NUM>µL of assay medium. The liquid in the <NUM>-well plate was pipetted out, and <NUM>µL of the mixture and blank control was added into each well and incubated in an incubator at <NUM>, <NUM>% CO<NUM> for <NUM>. The culture plate was taken out, added with <NUM>µg/mL BODIPY-labeled LDL solution (purchased from life technologies) diluted with the assay medium at <NUM>µL/well, and cultured at <NUM>, <NUM>% CO<NUM> for <NUM>. The medium was discarded, and <NUM>µL of PBS solution was added to each well to wash the plate and then discarded. After washing twice, each well was added with <NUM>µL of PBS solution. The fluorescence values were read using a Spectra Max I3 microplate reader.

The obtained raw fluorescence data are listed in Table <NUM> below, which were analyzed and plotted using GraphPad Prism6, thus obtaining <FIG>. It can be seen from the results that the fluorescence values obtained in the case where HepG2 cells were treated with anti-PCSK9 antibodies (ADI-<NUM>, ADI-<NUM>, ADI-<NUM>, and ADI-<NUM>) were enhanced by about two times compared with the fluorescence values obtained in the absence of the anti-PCSK9 antibodies. These data prove that each anti-PCSK9 antibody disclosed herein can enhance the capability of restoring LDLR in HepG2 cells and inhibit the decrease of LDL-C uptake induced by PCSK9, resulting in increased LDL-C uptake in HepG2 cells, which is superior to the positive control antibodies at gradients of <NUM> and <NUM>.

PCSK9 can directly bind to LDLR to promote the internalization of LDLR which is transported to lysosomes for degradation after entering hepatocytes, thus reducing LDLR expressed on the surface of cells and increasing the level of LDL-C in serum. Anti-PCSK9 antibodies can block the binding of PCSK9 to LDLR, thus reducing the ability of PCSK9 to consume LDLR. In this experiment, the bioactivity of anti-PCSK9 antibodies against the internalization of LDLR into cells was determined by co-incubating CHO-LDLR cells with the anti-PCSK9 antibodies and a PCSK9 protein solution, detecting the fluorescence values of LDLR with the aforementioned flow cytometer, and comparing the fluorescence values of the anti-PCSK9 antibodies and the positive control antibody (Evolocumab).

RPMI <NUM> cell medium (Gibco) was used to prepare the PCSK9 protein described in example <NUM> into a concentration of <NUM>µg/mL. <NUM>µL of <NUM> anti-PCSK9 antibodies (ADI-<NUM> and ADI-<NUM>) were uniformly mixed with a solution of PCSK9 (<NUM>µg/mL) and then the mixture was incubated for <NUM>. The positive control antibody was treated in the same way. CHO cells and CHO-LDLR cells were separately centrifuged at <NUM> at room temperature for <NUM>. The cells were resuspended in PBS solution with the cell density adjusted to <NUM> × <NUM><NUM> cells/mL. The cell suspensions were added to U-bottom <NUM>-well plates at <NUM>µL/well. The aforementioned mixed sample was added to the plate at <NUM>µL/well in quadruplicate, pipetted uniformly, and incubated at <NUM> for <NUM>. The mixture was washed <NUM> times with <NUM>µL of PBS solution, and centrifuged at <NUM> at room temperature for <NUM>. <NUM>µL of anti-LDLR-PE (Sino Biological, Item No. <NUM>-R301-P) was diluted with <NUM>µL of PBS solution, and then added to a U-bottom <NUM>-well plate at <NUM>µL/well and incubated away from light for <NUM>. The mixture was washed <NUM> times with <NUM>µL of PBS solution, and centrifuged at <NUM> for <NUM>. The obtained cells were resuspended in cell medium, and the fluorescence signal of the PE fluorescence-labeled LDLR protein on the surface of the CHO-LDLR cells was detected using a flow cytometer. The results are shown in Table <NUM>. The raw data in Table <NUM> were analyzed and plotted using GraphPad Prism6, obtaining <FIG>.

It can be seen from the results in Table <NUM> that the antibodies obtained herein effectively block the internalization of LDLR in cells.

The tested antibody (anti-PCSK9 antibody ADI-<NUM>) was administered to SPF SD rats (Vital River) according to a conventional method in the art, wherein the female rats were about <NUM> to <NUM> in weight and about <NUM> to <NUM> weeks old, and the male rats were about <NUM> to <NUM> in weight and about <NUM> to <NUM> weeks old. The tested antibody was administered to each group once. The dosage regimen is shown in Table <NUM>.

The blood of each group of animals was collected via the jugular vein according to a conventional method at the following time points: <NUM> before administration (D1) and <NUM> (D4), <NUM> (D8), <NUM> (D15), <NUM> (D22), <NUM> (D29), and <NUM> (D36) after administration. The blood samples were collected into anticoagulant-free tubes, coagulated on ice and then centrifuged at <NUM>-<NUM> at <NUM> rpm/min for <NUM>, and serum was collected and assayed for LDL-C and HDL-C using a Hitachi-<NUM> automatic biochemical analyzer. According to the data of blood lipid analysis, the change rate of LDL-C and HDL-C (% LDL-C and % HDL-C) relative to those before administration (baselines) at each time point were calculated. According to the results, it was found that after <NUM>-<NUM>/kg of the anti-PCSK9 antibody disclosed herein was subcutaneously administered to the rats once, the LDL-C and HDL-C levels in serum decreased dose-dependently (<FIG>). For example, <NUM> days, <NUM> days, <NUM> days and <NUM> days after administration, the LDL-C and HDL-C levels significantly decreased in comparison with the baseline levels. In addition, it was also found that after <NUM>/kg of Evolocumab was subcutaneously administered to the rats once, the LDL-C and HDL-C levels in serum did not significantly decrease.

For other antibodies disclosed herein, the same method can also be applied for the above assay.

The tested antibody (anti-PCSK9 antibody ADI-<NUM>) was administered to cynomolgus monkeys (Guangdong Blooming-Spring Biological Technology Development Co. ) according to a conventional method in the art, wherein the female animals were about <NUM> to <NUM> in weight and about <NUM> to <NUM> years old, and the male animals were about <NUM> to <NUM> in weight and about <NUM> to <NUM> years old. The dosage regimen is shown in Table <NUM>, wherein the tested antibody was administered to groups <NUM>-<NUM> once and group <NUM> once a week, <NUM> times in total.

For groups <NUM>-<NUM>, blood was collected from the subcutaneous vein or the inguinal femoral artery/inguinal vein in the contralateral forelimb or hind limb of the limb on which the drug was administered according to a conventional method at the following time points: <NUM> before administration and <NUM> (D2), <NUM> (D4), <NUM> (D6), <NUM> (D8), <NUM> (D15), <NUM> (D22), <NUM> (D29), <NUM> (D36), <NUM> (D43), <NUM> (D50) and <NUM> (D57) after administration. With regard to group <NUM>, blood was collected at the following time points according to the aforementioned method: <NUM> before the first administration and <NUM> (D2), <NUM> (D4), <NUM> (D6), <NUM> (D8, before the second administration) and <NUM> (D15, before the third administration) after administration. For the last administration, blood was collected <NUM> before administration and <NUM> (D2), <NUM> (D4), <NUM> (D6), <NUM> (D8), <NUM> (D15), <NUM> (D22), <NUM> (D29), <NUM> (D36), <NUM> (D43), <NUM> (D50) and <NUM> (D57) after administration.

Whole blood was collected into tubes containing coagulant and separation gel, placed on ice for coagulation, and then centrifuged at <NUM>-<NUM> at <NUM> rpm/min for <NUM>, and serum was collected. After all blood samples were collected, total cholesterol (TC), LDL-C and HDL-C were assayed. According to the data of blood lipid analysis, the change rate of LDL-C and HDL-C (% LDL-C and % HDL-C) relative to those before administration (baselines) at each time point were calculated. According to the results, it was found that after a single subcutaneous administration of the anti-PCSK9 antibody (ADI-<NUM>) disclosed herein to the cynomolgus monkeys at <NUM>/kg, <NUM>/kg and <NUM>/kg, both the LDL-C (<FIG>) and TC (<FIG>) levels in serum significantly decreased in an obvious dose-effect manner, and were significantly lower than the baseline levels <NUM> to <NUM> days after the administration. This indicates that the antibody disclosed herein can effectively alleviate conditions and/or disorders related to LDL-C and TC, such as lowering blood lipid level. The administration of the anti-PCSK9 antibody had no significant influence on the HDL-C level in the serum of the cynomolgus monkeys on the whole (<FIG>).

However, it was surprisingly found that after separate single subcutaneous administration of the anti-PCSK9 antibody disclosed herein and Evolocumab to the cynomolgus monkeys at <NUM>/kg, Evolocumab only significantly lowered LDL-C in comparison with the baseline level in <NUM> days after the administration, shorter than <NUM> days after the administration achieved by the same dose of the antibody disclosed herein. That is, the anti-PCSK9 antibody disclosed herein significantly lowered LDL-C for a longer period than Evolocumab.

A liquid preparation comprising an anti-PCSK9 antibody with a concentration of <NUM>/mL prepared according to formula A.

The anti-PCSK9 antibody (ADI-<NUM>) obtained in example <NUM> was added to an ultrafiltration centrifuge tube (molecular weight cutoff: <NUM> KD), after concentration by centrifugation, the anti-PCSK9 antibody was diluted with an aqueous solution of formula A without polysorbate <NUM> and the anti-PCSK9 antibody (<NUM>/L histidine, <NUM> mmol/L arginine, <NUM>% sorbitol, pH <NUM>) and then concentrated; and this operation was repeated until the replacement was completed. The concentration of the replaced protein was adjusted to <NUM>/mL. Then an aqueous solution of polysorbate <NUM> (<NUM>/L, <NUM>/<NUM> by volume) was added to adjust the final concentration of polysorbate <NUM> to <NUM>/L. After being aseptically filtered, the semi-finished product was aliquoted into vials and the vials were then capped with rubber stoppers and aluminum-plastic caps, and the product was obtained.

Comparative example: An preparation comprising anti-PCSK9 antibody liquid with a concentration of <NUM>/mL prepared according to formula B.

The anti-PCSK9 antibody (ADI-<NUM>) obtained in example <NUM> was added to an ultrafiltration centrifuge tube (molecular weight cutoff: <NUM> KD), after concentration by centrifugation, the anti-PCSK9 antibody was diluted with an aqueous solution of formula B without polysorbate <NUM> and the anti-PCSK9 antibody (<NUM>/L histidine, <NUM> mmol/L arginine, pH <NUM>) and then concentrated; and this operation was repeated until the replacement was completed. The concentration of the replaced protein was adjusted to <NUM>/mL. Then an aqueous solution of polysorbate <NUM> (<NUM>/L, <NUM>/<NUM> by volume) was added to adjust the final concentration of polysorbate <NUM> to <NUM>/L. After being aseptically filtered, the semi-finished product was aliquoted into vials and the vials were then capped with rubber stoppers and aluminum-plastic caps, and the product was obtained.

The viscosity was measured with a cone-and-plate viscometer (Brookfield, CAP1000+/<NUM>+. For details, visit http://www. sinoinstrument. com/product_details-<NUM>-<NUM>-<NUM>-<NUM>. html) at room temperature of <NUM>-<NUM>. A rotor and a rotational speed were selected (rotor: cp-<NUM>; rotational speed: <NUM> rpm). The rotor was connected to a connecting nut, with the position adjusted, and samples were added. The Run button was pushed to start the measurement of the viscosity of the samples. According to the above experiment, the viscosity of preparation A was <NUM> centipoises, and the viscosity of preparation B was <NUM> centipoises. Surprisingly, the viscosity of preparation A was significantly lower than that of preparation B, indicating that the combination of arginine and sorbitol is more beneficial to reduce the viscosity of a high-concentration antibody preparation.

In order to inspect the stability of preparation A and preparation B, an accelerated stability test was carried out. The product of preparation A and preparation B aliquoted in the plugged and capped vials were examined for accelerated stability at <NUM> ± <NUM>/<NUM>% RH ± <NUM>% RH for <NUM> months. Meanwhile, the change in protein purity was assayed by SEC-HPLC and non-reduced CE-SDS as follows.

The sample was diluted to a concentration of <NUM>/mL; a hydrophilic silica gel size exclusion chromatography column TSKG <NUM> SWxl was used; the amount of injected protein was <NUM>µg; the mobile phases were <NUM> mmol/L Na<NUM>HPO<NUM>•<NUM><NUM>O, <NUM> mmol/L NaCl, and <NUM> mmol/L arginine; the pH was <NUM>; the flow velocity was <NUM>/min; the detection wavelength was <NUM>; the column temperature was <NUM>; and the area normalization method was used for calculation.

About <NUM>µg of sample was taken and added with a sample buffer (pH <NUM>) to adjust the total volume to <NUM>µL. The reduced sample was added with <NUM>µL of β-mercaptoethanol, and the non-reduced sample was added with <NUM>µL of <NUM> mmol/L NEM. After mixing, the mixture was heated at <NUM> for <NUM> and detected.

By SEC-HPLC, based on the percentage decrease of an SEC main peak, the changes in protein purity were assayed to be a decrease by <NUM>% after <NUM> month and a decrease by <NUM>% after <NUM> months for preparation A; and a decrease by <NUM>% after <NUM> month and a decrease by <NUM>% after <NUM> months for preparation B. Based on the percentage decrease of non-reduced CE-SDS, the changes in protein purity were assayed to be a decrease by <NUM>% after <NUM> weeks and a decrease by <NUM>% after <NUM> month for preparation A; and a decrease by <NUM>% after <NUM> weeks and a decrease by <NUM>% after <NUM> month for preparation B. It can be seen that the purity change rate of preparation A is significantly slower than that of preparation B. Relative to preparation B, preparation A has higher stability.

At different time points, preparation A and preparation B which had undergone the accelerated stability test in example <NUM> were sampled for charge heterogeneity assay. Beckman uncoated capillaries and a Beckman PA800 plus capillary electrophoresis apparatus were adopted; the sampling voltage was <NUM> psi; the sampling time was <NUM>; the separation voltage was <NUM> kV; and the analysis time was <NUM>. The area normalization method was used to calculate the peak area of acidic components, basic components, and the main peak as a percentage of the total peak area. The CZE main peak of preparation A dropped by <NUM>% after <NUM> weeks and by <NUM>% after <NUM> month. The CZE main peak of preparation B dropped by <NUM>% after <NUM> weeks and by <NUM>% after <NUM> month.

Claim 1:
A liquid antibody preparation, comprising:
(i) an anti-PCSK9 antibody or an antigen-binding fragment thereof;
(ii) a buffer;
(iii) a viscosity inhibitor selected from sugar alcohols, arginine, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate and combinations thereof; and
(iv) a surfactant,
wherein the anti-PCSK9 antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein
(a) the heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: <NUM>;
and
(b) the light chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: <NUM>.