Anti-ANGPTL3 / 8 complex antibodies and methods of using the same

Abstract

Anti-angiopoietin-like protein (ANGPTL) 3 / 8 complex antibodies are provided that can be used to treat diseases and disorders related to lipid metabolism and glucose metabolism. [Solution] Antibodies that bind to the human ANGPTL3 / 8 complex are provided, which include light chain determining regions LCDR1, LCDR2, and LCDR3, and heavy chain determining regions HCDR1, HCDR2, and HCDR3, each having a specific amino acid sequence. Pharmaceutical compositions containing one or more anti-ANGPTL3 / 8 complex antibodies of the invention in a pharmaceutically acceptable carrier are also disclosed. Methods of making and using the above, particularly for increasing lipoprotein lipase activity and lowering triglycerides, are also disclosed.

Description

Technical Field

[0001] The present disclosure generally relates to biology and medicine, and more particularly to antibodies (Abs) that bind to the human angiopoietin-like protein (ANGPTL) 3 / 8 complex and thereby neutralize it. Such antibodies can increase lipoprotein lipase (LPL) activity, thereby reducing serum triglyceride (TG), and can be used in the treatment of lipid metabolism-related and glucose metabolism-related diseases and disorders.

Background Art

[0002] ANGPTL is a family of proteins that regulate many physiological and pathophysiological processes. Of particular interest herein is the role of ANGPTL3 and ANGPTL8 in lipid and glucose metabolism.

[0003] Evidence supports the role of ANGPTL3 as a major regulator of lipoprotein metabolism, and ANGPTL3 may regulate TG clearance by inhibiting LPL and endothelial lipase (EL). See Chi et al. (2017) Mol. Metab. 6:1137-1149. Deficiency, inactivation, or loss of ANGPTL3 may result in reduced levels of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and TG. ANGPTL3 may also affect insulin sensitivity and thereby play a role in the regulation of not only lipid metabolism but also glucose metabolism. See Robciuc et al. (2013) Arterioscler. Thromb. Vasc. Biol. 33:1706-1713. The nucleic acid and amino acid sequences of human ANGPTL3 are known. For example, one nucleic acid sequence is found in NCBI reference sequence number NM_014495 (SEQ ID NO: 1), and one amino acid sequence is found in NCBI reference sequence number NP_055310 (SEQ ID NO: 2).

[0004] ANGPTL8 is highly expressed in the liver and adipose tissue and has been reported to inhibit LPL by forming a complex with ANGPTL3 and thereby activating ANGPTL3. See Chi supra. Human ANGPTL8 appears to be induced by feeding. The nucleic acid and amino acid sequences of human ANGPTL8 are known. For example, one nucleic acid sequence is found in NCBI Reference Sequence No. NM_018687 (SEQ ID NO: 3), and one amino acid sequence is found in NCBI Reference Sequence No. NP_061157 (SEQ ID NO: 4).

[0005] There exists an ANGPTL3 / 8 complex having one or more ANGPTL3s bound to one or more ANGPTL8s. Evidence suggests that these complexes more effectively mediate the inhibition of LPL when compared to ANGPTL3 or ANGPTL8 alone. Furthermore, the ANGPTL3 / 8 complex can be made in vitro by co-expressing ANGPTL8 and ANGPTL3 in a mammalian expression system. See Chi supra.

[0006] Antibodies are known that bind to either ANGPTL3 or ANGPTL8 and can be used alone or in combination with each other to treat lipid metabolism-related and glucose metabolism-related diseases and disorders. For example, International Patent Application Publication No. WO2012 / 174178 discloses a fully human monoclonal Ab and antigen-binding fragments thereof that bind to ANGPTL3 and interfere with its activity. Other therapeutic anti-ANGPTL3 antibodies are also known. See, e.g., International Patent Application Publication No. WO2008 / 073300 and U.S. Patent No. 7,935,796. Similarly, International Patent Application Publication No. WO2017 / 027316 discloses a fully human monoclonal Ab or antigen-binding fragments thereof that bind to ANGPTL8 and interfere with its activity. Also, International Patent Application Publication No. WO2017 / 177181 discloses a combination therapy of an anti-ANGPTL3 antibody and an anti-ANGPTL8 antibody.

[0007] Unfortunately, existing antibodies that bind only to either ANGPTL3 or ANGPTL8 do not completely abrogate the effects of these ANGPTLs and / or ANGPTL3 / 8 complexes on lipid and / or glucose metabolism. See, for example, Dewey et al. (2017) N. Engl. J. Med. 377:211-221, and Gusarova et al. (2017) Endocrinology 158:1252-1259. In view thereof, additional Abs, particularly anti-ANGPTL3 / 8 complex Abs, are needed to treat lipid metabolism-related and glucose metabolism-related diseases and disorders, and such Abs have improved pharmacological inhibitory and / or regulatory properties for modulating lipid and / or glucose metabolism. SUMMARY OF THE INVENTION

[0008] To address this need, nucleic acid and amino acid sequences of modified ANGTPL3 / 8 complexes are provided. Accordingly, nucleic acid sequences encoding one or more of modified ANGPTL3 and modified ANGPTL8 (i.e., fusion proteins) are described herein. In some cases, the nucleic acid sequences include a polynucleotide sequence encoding an ANGPTL3 fusion protein having the amino acid sequence of SEQ ID NO: 17. In other examples, the nucleic acid sequences include a polynucleotide sequence encoding an ANGPTL8 fusion protein having the amino acid sequence of SEQ ID NO: 18. In still other examples, the nucleic acid sequences include polynucleotide sequences encoding SEQ ID NOs: 17 and 18.

[0009] Furthermore, nucleic acid constructs are provided that include polynucleotide sequences encoding the ANGPTL3 fusion proteins described herein, the ANGPTL8 fusion proteins described herein, or both, and such constructs can be expression cassettes or vectors.

[0010] In view of the above, there is provided a host cell comprising one or more expression cassettes or vectors described herein. Optionally, the host cell is a eukaryotic cell. Optionally, the polynucleotide sequences of the ANGPTL3 and ANGTPL8 fusion proteins are on separate expression cassettes or vectors, and in other cases, they may be on the same expression cassette or vector.

[0011] Also provided is an ANGPTL3 fusion protein comprising the amino acid sequence of SEQ ID NO: 17 or 19, and active variants or fragments thereof. Similarly, provided is an ANGPTL8 fusion protein comprising the amino acid sequence of SEQ ID NO: 18 or 20, and active variants or fragments thereof.

[0012] Furthermore, there is provided a functional ANGPTL3 / 8 complex, particularly a human ANGPTL3 / 8 complex, wherein the ANGPTL3 part of the complex is native (full-length or truncated) ANGPTL3 or an ANGPTL3 fusion protein described herein, and the ANGPTL8 part of the complex is an ANGPTL8 fusion protein described herein. Optionally, the ANGPTL3 fusion protein comprises the amino acid sequence of SEQ ID NO: 19. Similarly, optionally, the ANGPTL8 fusion protein comprises the amino acid sequence of SEQ ID NO: 20. Furthermore, optionally, the complex can have a 1:1 ratio of ANGPTL3 part to ANGPTL8 part. In other examples, the complex can have a ratio other than 1:1, for example, a ratio of ANGPTL3 part to ANGPTL8 part of 1:2, 1:3, 2:1, or 3:1, respectively.

[0013] Methods for producing a recombinant ANGTPL3 / 8 complex are also provided. The method can include at least one step of expressing one or more polynucleotide sequences of the ANGPTL3 portion and the ANGPTL8 portion described herein in a host cell such as a mammalian expression system, and obtaining the ANGPTL3 / 8 complex therefrom. Optionally, the ANGPTL3 and ANGPTL8 portions are provided on separate expression constructs or vectors. In other examples, ANGPTL3 and ANGPTL8 are provided on one expression construct or vector. The method can also include a step of purifying the resulting ANGPTL3 / 8 complex, which can include not only concentrating the ANGPTL3 / 8 complex, but also removing one or more tags, linkers, and serum albumin from the ANGPTL3 portion and / or the ANGPTL8 portion. The method can also include a step of concentrating the ANGPTL3 / 8 complex before and / or after the purification step.

[0014] Second, Abs against the ANGPTL3 / 8 complex and their use are provided, which include treating lipid metabolism-related and glucose metabolism-related diseases and disorders by binding to the ANGPTL3 / 8 complex and thereby inhibiting ANGPTL3 / 8 complex activity.

[0015] An effective amount of the anti-ANGPTL3 / 8 complex Ab or a pharmaceutically acceptable salt thereof described herein can be used in an individual in need thereof for increasing LPL activity, decreasing TG, and treating lipid metabolism- and / or glucose metabolism-related diseases or disorders.

[0016] The anti-ANGPTL3 / 8 complex Ab described herein binds to the soluble ANGPTL3 / 8 complex, thereby increasing LPL activity and decreasing serum TG levels. Individuals with low TG levels have a lower risk of cardiovascular disease. Advantageously, the anti-ANGPTL3 / 8 complex Ab described herein binds only to the ANGPTL3 / 8 complex at appropriate concentrations and does not bind to ANGPTL3 alone or ANGPTL8 alone. The anti-ANGPTL3 / 8 complex Ab increases the catabolism of TG-rich lipoproteins (TRL), decreases TG and / or non-HDL-C, and thereby is thought to improve the lipid disorder risk factors of atherosclerotic cardiovascular disease (ASCVD) that cannot be addressed by current therapies. Furthermore, since the anti-ANGPTL3 / 8 complex Ab described herein does not bind to ANGPTL3 or ANGPTL8 alone, other actions of these ANGPTLs are not inhibited, and there may be fewer adverse in vivo effects such as a decrease in the relief of EL inhibition.

[0017] In particular, the anti-ANGPTL3 / 8 complex Ab is a human anti-ANGPTL3 / 8 complex Ab. In some cases, the anti-ANGPTL3 / 8 complex Ab can suppress, block, inhibit, interfere with, neutralize, or reduce the in vivo activity of the ANGPTL3 / 8 complex, particularly its LPL inhibitory activity. In some cases, the anti-ANGPTL3 / 8 complex Ab can be full-length or only an antigen-binding fragment (e.g., Fab, F(ab’)2 or scFv fragment). Desirable properties of the anti-ANGPTL3 / 8 complex Ab include TG reduction at low doses of Ab, which persists for at least 21 days.

[0018] In some cases, the anti-ANGPTL3 / 8 complex binds to the human ANGPTL3 / 8 complex and includes light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, where LCDR1 has the amino acid sequence RSSQSLLDSDDGNTYLD (SEQ ID NO: 11), LCDR2 has the amino acid sequence YMLSYRAS (SEQ ID NO: 12), LCDR3 has the amino acid sequence MQRIEFPLT (SEQ ID NO: 13), HCDR1 has the amino acid sequence TFSGFSLSISGVGVG (SEQ ID NO: 14), HCDR2 has the amino acid sequence LIYRNDDKRYSPSLKS (SEQ ID NO: 15), and HCDR3 has the amino acid sequence ARTYSSGWYGNWFDP (SEQ ID NO: 16).

[0019] An Ab comprising a light chain variable region (LCVR) is further provided, where the LCVR has the amino acid sequence of SEQ ID NO: 9. Alternatively, an Ab comprising a heavy chain variable region (HCVR) is further provided, where the HCVR has the amino acid sequence of SEQ ID NO: 10. In some cases, the Ab comprises an LCVR having the amino acid sequence of SEQ ID NO: 9 and an HCVR having the amino acid sequence of SEQ ID NO: 10. In some cases, the Ab comprises a light chain (LC) and a heavy chain (HC), where the LC has the amino acid sequence of SEQ ID NO: 5 and the HC has the amino acid sequence of SEQ ID NO: 6. Alternatively, the Ab comprises a light chain (LC) and a heavy chain (HC), where the LC has the amino acid sequence of SEQ ID NO: 5 and the heavy chain (HC) has the amino acid sequence of SEQ ID NO: 6. In some cases, the Ab is of the IgG4 isotype.

[0020] In some cases, the anti-ANGPTL3 / 8 complex Ab can be a variant of the above Ab, particularly an LC variant having a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22), a D33T mutation (SEQ ID NO: 23), an M56T mutation (SEQ ID NO: 24), an E99Q mutation (SEQ ID NO: 25), or a combination thereof (e.g., D33T and M56T mutations, or D33A and M56T mutations) with respect to the LC having the amino acid sequence of SEQ ID NO: 5.

[0021] Furthermore, provided are antibodies produced by culturing mammalian cells containing a cDNA molecule encoding a polypeptide having the amino acid sequences of SEQ ID NO: 5 and 6 under conditions such that the polypeptide is expressed and recovering the antibody. Further provided are antibodies produced by culturing mammalian cells containing two cDNAs, a first cDNA encoding a polypeptide having the amino acid sequence of SEQ ID NO: 5 and a second cDNA molecule encoding a polypeptide having the amino acid of SEQ ID NO: 6, under conditions such that the polypeptide is expressed and recovering the antibody. In some cases, the anti-ANGPTL3 / 8 complex Ab may be a variant of the above Ab, particularly an LC variant having a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22), a D33T mutation (SEQ ID NO: 23), an M56T mutation (SEQ ID NO: 24), an E99Q mutation (SEQ ID NO: 25), or a combination thereof, with respect to the LC having the amino acid sequence of SEQ ID NO: 5.

[0022] Furthermore, in a standard LPL activity assay, an Ab that binds and neutralizes the human ANGPTL3 / 8 complex at an EC 50 of 0.5 nM or less is provided. Also provided is an Ab that binds to the human ANGPTL3 / 8 complex with a dissociation constant of 1×10 -6 M or less. Further provided are Abs that bind to ANGPTL3 and human ANGPTL8 with a dissociation constant greater than 1×10 -6 M. Further provided are Abs that bind to the human ANGPTL3 / 8 complex with a signal more than 3-fold greater than the non-binding background signal as measured by a single-point ELISA assay, but do not bind to human ANGPTL3 alone or human ANGPTL8 alone with a signal more than 3-fold greater than the non-binding background signal as measured by a single-point ELISA assay. Similarly, at the time point of 14 days after administration, at a dose of 10 mg / kg, an Ab that reduces TG by at least 50% in vivo compared to an IgG control is provided.

[0023] Thirdly, there is provided a pharmaceutical composition comprising the Ab of the present specification or a population of the Abs of the present specification and an acceptable carrier, diluent or excipient. Mammalian cells are also provided that contain a DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, wherein the cells are capable of expressing the Ab of the present specification. Mammalian cells are further provided that contain a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:5, the second DNA molecule comprises a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:6, and the cells are capable of expressing the Ab of the present specification. Optionally, the anti-ANGPTL3 / 8 complex Ab may be a variant of the above Ab, particularly an LC variant having a D31S mutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22), a D33T mutation (SEQ ID NO:23), an M56T mutation (SEQ ID NO:24), an E99Q mutation (SEQ ID NO:25), or a combination thereof, with respect to the LC having the amino acid sequence of SEQ ID NO:5.

[0024] Fourthly, there is provided a method for making an Ab, the method comprising culturing mammalian cells having a DNA molecule having a polynucleotide sequence encoding a polypeptide having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, wherein the cells are capable of expressing the Ab of the present specification under conditions such that the Ab is expressed, and recovering the expressed Ab. Optionally, the polynucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:5 can encode a D31S mutation, a D33A mutation, a D33T mutation, an M56T mutation, an E99Q mutation, or a combination thereof. Methods are also provided herein for treating atherosclerotic cardiovascular disease (ASCVD), chronic kidney disease (CKD), diabetes, hypertriglyceridemia, non-alcoholic steatohepatitis (NASH), obesity, or combinations thereof, the method comprising administering to an individual in need of treatment an effective amount of the Ab of the present specification. There is further provided a method for reducing triglycerides (TG) comprising administering to an individual in need of reducing TG an effective amount of the Ab of the present specification.

[0025] Fifth, an Ab is provided for use in therapy. In particular, the Ab is for use in the treatment of ASCVD, CKD, diabetes, hypertriglyceridemia, NASH, obesity, or combinations thereof. Also provided is a pharmaceutical composition for use in treating ASCVD, CKD, diabetes, hypertriglyceridemia, NASH, obesity, or combinations thereof, which comprises an effective amount of the Ab of the present specification.

[0026] Other advantages, effects, features and purposes will become more readily apparent upon consideration of the following detailed description. Such detailed description refers to the following drawings (s).

Brief Description of the Drawings

[0027]

Figure 1

Mode for Carrying Out the Invention

[0028] References to elements by the indefinite article “a” or “an” do not exclude the possibility of there being more than one element, unless the context clearly requires that there be one and only one element. Thus, the indefinite article “a” or “an” typically means “at least one”.

[0029] Definitions As used herein, “about” means within a statistically meaningful range of values, such as, for example, the stated concentrations, lengths, molecular weights, pH, sequence identities, time frames, temperatures, volumes, etc. (where there may be more than one). Such values or ranges can be within a range of magnitudes typically within 20% of a given value or range, more typically within 10% of a given value or range, and even more typically within 5% of a given value or range. The allowable variations encompassed by “about” depend on the particular system in the study and can be readily understood by one of ordinary skill in the art.

[0030] As used herein, "affinity" means the strength of binding of an Ab to an epitope on the ANGPTL3 / 8 complex.

[0031] As used herein, "angiopoietin-like protein 3" or "ANGPTL3" means a protein having an amino acid sequence comprising SEQ ID NO: 2.

[0032] As used herein, "angiopoietin-like protein 8" or "ANGPTL8" means a protein having an amino acid sequence comprising SEQ ID NO: 4.

[0033] As used herein, "ANGPTL3 / 8 complex" means a multi-protein complex of one or more ANGPTL3 compounds bound to one or more ANGPTL8 compounds.

[0034] As used herein, "anti-ANGPTL3 / 8 complex Ab" or "anti-ANGPTL3 / 8 complex Ab" means an Ab that simultaneously recognizes and binds regions of both ANGPTL3 and ANGPTL8, particularly in the form of ANGPTL3 / 8. Generally, anti-ANGPTL3 / 8 complex Abs do not bind to other ANGPTL family members (e.g., ANGPTL1, ANGPTL2, ANGPTL4, ANGPTL5, ANGPTL6, or ANGPTL7). Further, as described elsewhere, anti-ANGPTL3 / 8 complex Abs do not bind to ANGPTL3 or ANGPTL8 alone at the specified concentrations, as described in the following single-point ELISA assay.

[0035] As used herein, "bind" or "binds" means the ability of a protein to form a chemical bond or attraction of some kind with another protein or molecule, as determined by common methods known in the art. Binding can be characterized by an equilibrium dissociation constant (K D ) of about 1 × 10 -6 M or less (i.e., K D(Indicating that the smaller the value, the stronger the binding). Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. Here, the anti-ANGPTL3 / 8 complex Ab binds only to the ANGPTL3 / 8 complex and does not bind to ANGPTL3 alone or ANGPTL8 alone. Whether the Ab binds only to the ANGPTL3 / 8 complex and not to ANGPTL3 alone or ANGPTL8 alone can be determined by a standard ELISA assay in single-point format as described below, and the binding can be characterized by Biacore as described below. The Abs herein are human, but they may exhibit cross-reactivity with other ANGPTL3 / 8 complexes derived from other species, such as cynomolgus monkey ANGPTL3 / 8 complex, mouse ANGPTL3 / 8 complex, or rat ANGPTL3 / 8 complex.

[0036] As used herein, "effective amount" means the amount or dosage of a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention that, upon administration of a single or multiple doses to an individual, elicits a biological or medical response or a desired therapeutic effect in a tissue, system, animal, mammal, or human being sought by a researcher, veterinarian, physician, or other clinician. In some cases, administration of an effective amount of a compound or composition thereof described herein to an individual in need thereof increases LPL activity. The dosage can include a higher initial loading dose, followed by a lower dose. The dosage can be administered at any therapeutically effective interval, such as multiple times a day, once a day, every other day, three times a week, twice a week, once a week, once every two weeks, once a month, once every two months, etc. The dosage constituting the effective amount can be from 0.01 mg / kg to 100 mg / kg.

[0037] As used herein, "equilibrium dissociation constant" or "K" D" means a quantitative measurement of the Ab affinity for a specific antigen interaction, e.g., the affinity of an Ab for the ANGPTL3 / 8 complex, in particular, a measure of the tendency of an Ab / ANGPTL3 / 8 complex conjugate to reversibly dissociate into its components. Similarly, as used herein, "equilibrium binding constant" or "K a " means K D 's reciprocal.

[0038] As used herein, "functional" means that an ANGPTL3 fusion protein, ANGPTL8 fusion protein or ANGPTL3 / 8 complex has biological activities similar to those of natural ANGPTL3, natural ANGPTL8 or natural ANGPTL3 / 8 complex, including, for example, inhibiting LPL and acting as an antigen against which an Ab is made and directed.

[0039] As used herein, "glucose metabolism-related disease or disorder" means diabetes and the like.

[0040] As used herein, "lipid metabolism-related disease or disorder" means a condition related to abnormal lipid metabolism, e.g., dyslipidemia, hyperlipidemia and hyperlipoproteinemia, including hypertriglyceridemia, hypercholesterolemia, chylomicronemia, mixed lipid disorders (obesity, metabolic syndrome, diabetes, etc.), lipodystrophy, and adipose tissue atrophy. This term also encompasses certain cardiovascular diseases such as atherosclerosis and coronary artery disease, acute pancreatitis, NASH, obesity, etc.

[0041] As used herein, "maximum half-effect concentration" or "EC 50 " means the concentration of an Ab (usually expressed in molar units (M)) that induces a response midway between baseline and maximum after a predetermined period. The EC 50 described herein is ideally 3.0 nM or less.

[0042] As used herein, "nucleic acid construct" or "expression cassette" means a nucleic acid molecule having at least one control sequence operably linked to a coding sequence. In this way, a control sequence such as a promoter operably interacts with a nucleic acid sequence encoding at least one polypeptide of interest, such as the ANGPTL3 fusion protein described herein and / or the ANGPTL8 fusion protein described herein. Such nucleic acid constructs can be in the form of expression cassettes or transfer cassettes. The nucleic acid construct can comprise an oligonucleotide or polynucleotide composed of deoxyribonucleotides, ribonucleotides, or combinations thereof, into which the nucleotide sequences of one or more polypeptides of interest are incorporated.

[0043] As used herein, "operably linked" means that the elements of a nucleic acid construct are configured so as to perform their normal functions. Thus, a control sequence (i.e., a promoter) operably linked to a coding sequence can affect the expression of the coding sequence. The control sequence need not be contiguous with the coding sequence so long as it functions to direct its expression (i.e., maintains the appropriate reading frame). Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0044] As used herein, "control sequence" or "control sequences" means a promoter, polyadenylation signal, transcription and translation termination sequence, upstream regulatory domain, origin of replication, internal ribosome entry site ("IRES"), enhancer, etc., which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient host cell. Not all of these control sequences need always be present so long as the selected coding sequence can be replicated, transcribed, and translated in an appropriate host cell.

[0045] As used herein, the "coding sequence" or "coding sequences" means a nucleic acid sequence encoding one or more polypeptides of interest, which, when placed under the control of appropriate regulatory sequences, is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of RNA) into a polypeptide in vitro or in vivo. The boundaries of the coding sequence(s) are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. The coding sequence includes, but is not limited to, viral nucleic acid sequences, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, or even synthetic DNA sequences.

[0046] With respect to the regulatory and coding sequences, they can be endogenous / similar to the host cell or to each other. Alternatively, the regulatory and coding sequences can be heterologous to the host cell or to each other.

[0047] As used herein, the "promoter" means a nucleotide region composed of nucleic acid regulatory sequences, where the regulatory sequences are derived from a gene, created synthetically, can bind to RNA polymerase, and initiate transcription of the downstream (3' direction) coding sequence. Many promoters, including the endogenous promoter of one or more polypeptides of interest, can be used in nucleic acid constructs. Alternatively, a promoter can be selected based on the desired result. Such promoters include, but are not limited to, inducible promoters, repressible promoters, and constitutive promoters.

[0048] As used herein, "variant" means a polynucleotide or polypeptide having one or more modifications such as addition, deletion, insertion and / or substitution of one or more specific nucleic acid or amino acid residues when compared to a reference nucleic acid or amino acid sequence. Thus, a variant contains one or more changes when compared to a reference nucleic acid or amino acid sequence. Here, the anti-ANGPTL3 / 8 complex Ab can have variations in the LC or HC. In particular, for the LC having the amino acid sequence of SEQ ID NO: 5, the Ab can be an LC variant having a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22), a D33T mutation (SEQ ID NO: 23), an M56T mutation (SEQ ID NO: 24), or an E99Q mutation (SEQ ID NO: 25). Similarly, the LC variant can also be, for the LC having the amino acid sequence of SEQ ID NO: 5, for example, any two combinations of the above, such as D31S and D33A mutations, D31S and D33T mutations, D31S and M56T mutations, D31S and E99Q mutations, D33A and M56T mutations, D33A and E99Q mutations, D33T and M56T mutations, D33T and E99Q mutations, and M56T and E99Q. Furthermore, the LC variant can also be, for the LC having the amino acid sequence of SEQ ID NO: 5, for example, any three combinations of the above, such as D31S, D33A and M56T mutations, D31S, D33A and E99Q mutations, D31S, D33T and M56T mutations, D31S, D33T and E99Q mutations, D33A, M56T and E99Q mutations, and D33T, M56T and E99Q mutations. Furthermore, the LC variant can be, for the LC having the amino acid sequence of SEQ ID NO: 5, for example, any four combinations of the above, such as D31S, D33A, M56T and E99Q mutations, and D31S, D33T, M56T and E99Q mutations.

[0049] As used herein, "vector" means a replicon such as a plasmid, phage or cosmid, to which another nucleic acid sequence such as an expression cassette can be ligated to effect replication of the ligated sequence. A vector can introduce a nucleic acid molecule into a host cell. A vector usually contains one or a few restriction endonuclease recognition sites, enabling insertion of the nucleotide sequence of interest in a determinable manner without loss of the essential biological functions of the vector, and can also be inserted with a selectable marker that can be used to identify and select host cells transformed with the vector. A vector for this purpose can introduce a nucleic acid sequence into a target cell.

[0050] As used herein, "treatment" or "treating" means managing and caring for an individual having a condition as indicated for the purpose that administration of anti-ANGPTL3 / 8 complex Ab is effective in or relieves the symptoms and complications of the individual's condition. Treatment includes administering a compound of the invention or a composition comprising a compound of the invention to an individual in need thereof to prevent the onset of symptoms or complications, to alleviate symptoms or complications, or to eliminate a disease, condition or disorder. Treatment includes administering a compound of the present specification or a composition comprising a compound of the present specification to an individual in need thereof to effect an increase in LPL activity and a decrease in TG. The individual to be treated is an animal, particularly a human.

[0051] As used herein, "patient", "subject" and "individual" are used interchangeably herein and mean an animal, particularly a human. In certain instances, the individual is a human, further characterized by a disease, disorder or condition that would benefit from administration of anti-ANGPTL3 / 8 complex Ab.

[0052] As used herein, "antibody" or "Ab" etc. means a full-length Ab comprising two heavy chains and two light chains having interchain and intrachain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region mainly involved in antigen recognition. Each HC contains an N-terminal HCVR and a heavy chain constant region (HCCR). Each light chain contains a light chain (LC) variable region (LCVR) and an LC constant region (LCCR). Here, the Ab is an immunoglobulin G (IgG)-type Ab, and the IgG isotype can be further classified into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The HCVR and LCVR regions are further classified into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with more conserved framework regions (FRs). Each HCVR and LCVR contains three CDRs and four FRs arranged in the following order, from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, FR4. In this specification, the three CDRs of the HC are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of the LC are referred to as LCDR1, LCDR2, and LCDR3. Most of the residues that form specific interactions with antigens such as the ANGPTL3 / 8 complex are included in the CDRs. Assigning residues to the various CDRs can be done by algorithms such as Chothia, Kabat, or North. The definition of the North CDR is based on affinity propagation clustering by a number of crystal structures (North et al. (2011) J. Mol. Bio. 406:228-256). Here, the CDR is most preferably defined by the sequences listed in the sequence listing based on a combination of multiple definitions including North.

[0053] The isolated DNA encoding the HCVR region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the HCVR to another DNA molecule encoding the heavy chain constant region. The sequences of the heavy chain constant region genes of humans and other mammals are known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification.

[0054] Isolated DNA encoding the LCVR region can be converted into a full-length light chain gene by operably linking the DNA encoding the LCVR to another DNA molecule encoding the light chain constant region. The sequences of the light chain constant region genes of humans and other mammals are known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

[0055] The Abs herein may comprise an IgG4-PAA Fc portion. The IgG4-PAA Fc portion has a mutation from Ser to Pro at position 231 (S231P), a mutation from Phe to Ala at position 237 (F237A), and a mutation from Leu to Ala at position 238 (L238A), as numbered by the absolute positions of SEQ ID NO: 6. The S231P mutation is a hinge mutation that prevents half-antibody formation (the dynamic exchange phenomenon of half-molecules in IgG4 Abs). The F237A and L238A mutations further reduce the already low effector functions of the human IgG4 isotype. However, it is contemplated that the Abs herein may alternatively comprise different Fc portions.

[0056] To reduce the potential induction of an immune response when administered to humans, certain amino acids may require back-mutations to be compatible with the Ab germline sequence.

[0057] The pharmaceutical composition comprising the compound of the invention can be administered parenterally to an individual in need of such treatment. Such individuals have or may be at high risk of having ASCVD. These individuals may have a history of acute coronary syndrome, myocardial infarction (MI), stable or unstable angina, coronary or other arterial revascularization, stroke, transient ischemic attack (TIA), thoracic or abdominal aortic aneurysm, or peripheral arterial disease presumed to be of atherosclerotic origin. Individuals at high risk of ASCVD may further suffer from type 2 diabetes (T2D), CKD, or familial hypercholesterolemia (FH).

[0058] Parenteral administration can be carried out by subcutaneous, intramuscular or intravenous injection using a syringe, optionally a pen-like syringe, or a mechanically-driven syringe. Alternatively, parenteral administration can be performed using an infusion pump. In some cases, a pharmaceutical composition suitable for administration to an individual has a therapeutically effective amount of the compound of the present specification and one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can be prepared by any of a variety of techniques using conventional excipients for pharmaceuticals well known in the art. For example, see Remington, "The Science and Practice of Pharmacy" (D.B. Troy ed., 21 st Ed., Lippincott, Williams & Wilkins, 2006).

[0059] The compounds of the present specification can be used in combination simultaneously, separately, or sequentially with one or more additional therapeutic agents useful for the regulation of LPL activity, the treatment of lipid metabolism-related diseases or disorders including any of the disorders listed above, or the treatment of glucose metabolism-related diseases or disorders. Non-limiting examples of additional therapeutic agents that can be combined with the compounds of the present invention include anti-diabetic drugs such as insulin or insulin analogs, biguanides, sulfonylureas, thiazolidinediones, dipeptidyl peptidase-4 ("DPP-4") inhibitors, or sodium-dependent glucose transporter (SGLT2) inhibitors; incretin compounds such as glucagon-like peptide-1 (GLP-1) or GLP-1 analogs, gastric inhibitory polypeptide (GIP) or GIP analogs, oxyntomodulin (OXM) or OXM analogs; aspirin; antiplatelet agents; H2 receptor blockers; proton pump inhibitors; antihypertensive drugs; lipid-modifying therapeutic agents such as HMG-CoA reductase inhibitors, PCSK9 inhibitors, cholesterol absorption inhibitors, fibrates, niacin, LXR agonists, RXR agonists, ROR agonists, or cholesterol reverse transport modulators; heart failure therapeutic agents such as ACE, angiotensin receptor neprilysin inhibitors, ARBs, or beta-adrenergic antagonists; anti-inflammatory therapeutic agents; hypertension therapeutic agents, atrial fibrillation therapeutic agents; neurodegenerative therapeutic agents; cancer therapeutic agents; therapeutic agents for diabetic cardiomyopathy, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, weight loss, wound healing; nephropathy therapeutic agents; PAD therapeutic agents, or combinations of any of the aforementioned agents, but are not limited thereto. The anti-ANGPTL3 / 8 complex Ab and one or more additional therapeutic agents (s) can be administered together via the same delivery route and device such as a single pill, capsule, tablet, or injectable formulation, or administered simultaneously via separate delivery devices or routes, or administered separately either sequentially.

[0060] Preparation and purification of the ANGPTL3 / 8 complex One aspect of the present disclosure is an ANGPTL3 / 8 complex that can be used to generate an Ab that binds only to the complex and not to ANGPTL3 alone or ANGPTL8 alone. Anti-ANGPTL3 Ab and anti-ANGPTL8 Ab are known, but there are challenges in synthesizing a sufficient amount of functional ANGPTL3 / 8 complex, particularly a human ANGPTL3 / 8 complex, to generate an anti-ANGPTL3 / 8 complex Ab. Additionally or alternatively, such ANGPTL3 / 8 complexes can be used in assays to evaluate the properties of Abs against ANGPTL3, ANGPTL8, and / or the ANGPTL3 / 8 complex.

[0061] Thus, the present disclosure also describes a method of generating ANGPTL8 as an N-terminal or C-terminal serum albumin (e.g., human, mouse, or rabbit) fusion protein. A functional ANGPTL3 / 8 complex can then be produced by co-expressing the ANGPTL8 fusion protein with native ANGPTL3 or an ANGPTL3 fusion protein in a mammalian expression system.

[0062] As noted above, the nucleic acid and amino acid sequences of native human ANGPTL3 and native human ANGPTL8 are known (see, e.g., SEQ ID NOs: 1-2 and 3-4, respectively). However, the ANGPTL3 or ANGPTL8 described herein is modified (i.e., recombinant / synthetic) and thus differs from the native sequences by including additional amino acid sequences to improve the production, secretion, and / or complexation of ANGTPL3 and ANGPTL8.

[0063] For example, as is known in the art, ANGPTL3 can be modified to include one or more linkers and tags. Here, human ANGPTL3 (SEQ ID NO: 2) is modified to include a linker and a FLAG tag such that the ANGPTL3 fusion protein has the amino acid sequence of SEQ ID NO: 17. Optionally, the linker can be about 1 to about 10 amino acids, such as 3 amino acids, particularly 3 Ala residues. The linker and the FLAG tag can be placed at the N-terminus or C-terminus of the ANGPTL3 sequence, particularly the C-terminus such as in SEQ ID NO: 17, where residues 1 to 460 correspond to ANGPTL3 and residues 461 to 471 correspond to the 3-Ala linker and the FLAG tag.

[0064] Similarly, in addition to the sequence of serum albumin, particularly human serum albumin, ANGPTL8 can be modified to include one or more linkers and tags. Here, human ANGPTL8 (SEQ ID NO: 4) is altered to include a linker, an IgG kappa signal peptide, a polyhistidine (His) tag, mature human serum albumin, a linker, and a PreScission® cleavage site, such that the ANGPTL8 fusion protein has the amino acid sequence of SEQ ID NO: 18. Optionally, the linker can be about 1 to about 10 amino acids, such as a rigid polyproline repeat, particularly an Ala-Pro (AP)-10 linker. The signal peptide, His tag, mature HSA, linker, and PreScission® cleavage site can be placed at the N-terminus or C-terminus of the ANGPTL8 sequence, particularly the N-terminus such as in SEQ ID NO: 18, where residues 1 to 20 correspond to the IgG kappa signal peptide, residues 21 to 27 correspond to the His-tag, residues 28 to 612 correspond to HSA, residues 613 to 632 correspond to the AP-10 linker, residues 633 to 643 correspond to the PreScission® cleavage site, and residues 644 to 820 correspond to ANGPTL8.

[0065] Methods for constructing nucleic acid constructs that express the ANGPTL3 fusion protein and / or the ANGPTL8 fusion protein described herein are well known in the art, for example, Balbas & Lorence, “Recombinant Gene Expression: Reviews and Protocols” (2 nd Ed. Humana Press 2004), Davis et al., “Basic Methods in Molecular Biology” (Elsevier Press 1986), Sambrook & Russell, “Molecular Cloning: A Laboratory Manual” (3 rd Ed. Cold Spring Harbor Laboratory Press 2001), Tijssen, “Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes” (Elsevier 1993), and “Current Protocols in Molecular Biology” (Ausubel et al. eds., Greene Publishing and Wiley-Interscience 1995), and can be found in U.S. Patent Nos. 6,664,387, 7,060,491, 7,345,216, and 7,494,805. Since ANGPTL8 does not contain disulfide bonds, mammalian expression systems can be used. Here, the HEK293 expression system or the CHO expression system can be used to produce ANGPTL3 and / or ANGPTL8 fusion proteins, particularly by co-expression.

[0066] In addition to the expression of the ANGPTL3 fusion protein and / or the ANGPTL8 fusion protein, the method can also include purifying the resulting fusion protein based on the specific tags used for each fusion protein, which are well known in the art. With respect to the ANGPTL3 and ANGTPL8 fusion proteins described herein, purification can result in multiple cleavages after tag removal, such that the ANGPTL3 fusion protein has the amino acid sequence of SEQ ID NO: 19 (residues 1-444 correspond to ANGPTL3, and residues 445-455 correspond to the 3-Ala linker and the FLAG-tag), and the ANGPTL8 fusion protein has the amino acid sequence of SEQ ID NO: 20 (residues 1-4 correspond to cleavage residues from the PreScission® cleavage site, and residues 5-182 correspond to a fragment of ANGPTL8), which can readily associate with each other to form a functional ANGPTL3 / 8 complex.

[0067] Preparation and purification of antibodies The anti-ANGPTL3 / 8 complex Ab can be produced in a mammalian cell expression system using the CHO GSKO cell line. Glutamine synthetase (GS) gene knockout allows for stringent selection stringency by eliminating endogenous GS background activity that can permit the survival of cells with low or non-productive production under secretion conditions. The genes encoding the Ab HC and LC herein can be subcloned into individual GS-containing expression plasmids for co-transfection, or both chains can be subcloned into a single GS-containing expression plasmid. The cDNA sequence encoding the HC or LC chain can be fused in-frame with the coding sequence of a signal peptide, which can be the mouse kappa leader sequence, to enhance the secretion of the desired product into the cell culture medium. Expression is driven by the viral cytomegalovirus (CMV) promoter.

[0068] CHO GSKO cells are stably transfected using electroporation and appropriate amounts of recombinant HC and LC expression plasmids, and the transfected cells are maintained in suspension culture at an appropriate cell density. Selection of the transfected cells is achieved by growth in serum-free medium containing 25 μM methionine sulfoximine (MSX) without glutamine, and incubated at 32 - 37 °C and 5% - 7% CO2. The Ab is secreted from the CHO cells into the medium. The Ab can be purified by protein A affinity chromatography, followed by anion exchange, or hydrophobic interaction chromatography (or other appropriate methods), and size exclusion chromatography can be utilized for further purification.

[0069] The Ab from the collected medium is captured on MabSelect SuRe Protein A resin (GE Healthcare). Next, the resin is simply washed with a running buffer such as phosphate buffered saline (PBS, pH 7.4) or a running buffer containing Tris to remove nonspecifically bound substances. Next, the Ab is eluted from the resin with a low pH solution such as 20 mM acetic acid / 5 mM citric acid. Fractions containing ANGPTL3 / 8 Ab can be pooled and held at low pH to inactivate potential viruses. The pH can be neutralized by adding a base such as 0.1 M Tris pH 8.0. The ANGPTL3 / 8 Ab can be further purified by hydrophobic interaction chromatography (HIC) using a resin such as Phenyl HP (GE Healthcare). The anti-ANGPTL3 / 8 Ab can be eluted from the HIC column using a sodium sulfate gradient in 20 mM Tris, pH 8.0. The anti-ANGPTL3 / 8 Ab can be further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare) with isocratic elution in PBS, pH 7.4.

[0070] The compounds described in the present invention are prepared in this manner or by similar methods readily determined by one of ordinary skill in the art.

[0071] Mice possessing human variable light and heavy chain domains are immunized with the above ANGPTL3 / 8 complex, and single antigen-specific B cells are isolated by FACS using ANGPTL3 / 8 (positive) and ANGPTL3 (negative) as markers. Heavy and light chain variable region DNAs are recovered from single B cells by PCR and cloned into an IgG expression vector. The CHO cell supernatant is tested for binding activity after transfection.

Example

[0072] The following non-limiting examples are provided for illustrative purposes only and not for limitation.

[0073] Example 1: Preparation of ANGPTL3 / 8 Complex Expression of ANGPTL3 and ANGPTL8: A nucleotide sequence encoding the amino acid sequence of human ANGPTL3, a linker, and a FLAG tag (SEQ ID NO: 17) are inserted into a mammalian expression vector containing a CMV promoter. Similarly, a nucleotide sequence encoding the amino acid sequence of human ANGTPL8, a mouse IgG kappa signal peptide, a HIS tag, and a mature human serum albumin (HSA)-PreScission® cleavage site (SEQ ID NO: 18) are inserted into a mammalian expression vector containing a CMV promoter. Protein expression is by transient co-transfection of both of the above expression vectors in HEK293F cells cultured in serum-free medium. The culture medium is collected 5 days after transfection and stored at 4 °C for subsequent protein purification.

[0074] Protein purification: Protein purification is carried out at 4°C, and 4 L of culture medium is supplemented with 1 M Tris-HCl (pH 8.0) and 5 M NaCl at final concentrations of 25 mM and 150 mM, respectively. Next, the medium is incubated overnight with 150 ml of Ni-NTA resin (Qiagen). The resin is packed into a column and washed with buffer A (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl). The protein is eluted with a 0 - 300 mM imidazole gradient in buffer A. Fractions containing HIS-HSA-ANGPTL8 / ANGPTL3-Flag are pooled, concentrated, loaded onto a HiLoad® Superdex® 200 column (GE Healthcare Biosciences), and eluted with buffer A. Fractions containing HIS-HSA-ANGPTL8 / ANGPTL3-Flag are pooled again, concentrated, and digested with PreScission® protease (GE Healthcare Biosciences) to remove HSA from the HIS-HSA-ANGPTL8 fusion protein. The protein sample digested with PreScission® is loaded onto a HiLoad® Superdex® 200 column and eluted with storage buffer (20 mM HEPES, pH 8.0, 150 mM NaCl). Fractions containing the ANGPTL3 / 8 complex are pooled and concentrated, and the protein concentration is determined using the Bradford method. The ANGTPL3 / 8 complex is aliquoted and stored at -80°C until further use.

[0075] LPL activity assay: The EnzCheck® LPL assay is performed according to the manufacturer's (ThermoFisher Scientific) instructions. Briefly, ANGPTL3 and the ANGPTL3 / 8 complex are serially diluted in growth medium, incubated for 1 hour to replace the LPL cell medium. Next, EnzCheck® lipase substrate (ELS) is added to LPL-expressing cells and incubated for 30 minutes. Fluorescence is measured at 482 nm / 515 nm (excitation / emission, respectively). The inhibition percentages of ANGPTL3 and 3 / 8 against LPL are calculated.

[0076] Result: Table 1 shows the yields of the ANGTPL3 / 8 complex protein at various stages of purification / concentration.

[0077]

Table 1

[0078] Similarly, Figure 1 shows a 4-20% TG TGX Coomassie-stained gel of the purified and concentrated ANGPTL3 / 8 complex obtained from the column, confirming that the complex forms / assembles. After purification and concentration, ANGTL3 has the amino acid sequence of SEQ ID NO: 19, and ANGPTL8 has the amino acid sequence of SEQ ID NO: 20.

[0079] In the LPL assay, the EC 50 of ANGPTL3 is 21.5 nM, while the EC 50 of the ANGTPL3 / 8 complex is 0.54 nM, confirming that the complex is functional.

[0080] Example 2: Assay Single-point ELISA (SPE) assay: The Ab binding selectivity for either the ANGPTL3 / 8 complex, free ANGPTL3, or free ANGPTL8 is first verified using a standard ELISA assay in single-point format. Briefly, the assay plate is coated with anti-human Fc Ab at a concentration of 2 μg / ml and subsequently blocked with casein. The IgG secreted into the supernatant after expression in CHO cells is then captured on the assay plate. Biotinylated antigen is added at a concentration of 25 nM to enable Ab / antigen binding. The Ab / antigen complex is detected after adding alkaline phosphatase-conjugated NeutrAvidin and an alkaline phosphatase substrate and subsequently measuring the optical density at 560 nm. Positive binding is determined by a signal exceeding three times the non-binding background signal.

[0081] ELISA assay: The Ab binding selectivity for either the ANGPTL3 / 8 complex, free ANGPTL3, or free ANGPTL8 is verified using a standard ELISA assay. Briefly, the assay plate is coated with anti-human Fc Ab at a concentration of 2 μg / ml and subsequently blocked with casein. Next, the Ab from the CHO supernatant after expression in CHO cells is captured on the assay plate, and the Ab concentration becomes 2 μg / ml. Biotinylated antigen (ANGPTL3, ANGPTL8, or ANGPTL3 / 8 complex) is added at various concentrations by serial dilution to allow Ab / antigen binding. The Ab / antigen complex is detected after adding alkaline phosphatase-conjugated neutravidin and an alkaline phosphatase substrate, followed by measuring the optical density at 560 nm.

[0082]

Table 2

[0083] In the SPE assay, the Ab having the LC of SEQ ID NO: 5 and the HC of SEQ ID NO: 6 shows positive binding to the human ANGPTL3 / 8 complex and negative binding to human ANGPTL8 and human ANGPTL3. The Ab having the LC variant with the D31S mutation (SEQ ID NO: 21), D33A mutation (SEQ ID NO: 22), D33T mutation (SEQ ID NO: 24), M56T mutation (SEQ ID NO: 24), or E99Q mutation (SEQ ID NO: 25) and the HC of SEQ ID NO: 6 similarly shows binding to the human ANGPTL3 / 8 complex and negative binding to human ANGPTL8 and human ANGPT.

[0084]

Table 3

[0085] The Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6 showed positive binding to human ANGPTL3 / 8 complex in a concentration-dependent manner up to an antigen concentration of 100 nM in this assay, and showed no detectable binding to human ANGPTL8 and human ANGPTL3 (Table 3).

[0086] In addition to the above anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6, Abs having an LC variant with a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22), or an E99Q mutation (SEQ ID NO: 25) and HC of SEQ ID NO: 6 were assayed. Similarly, in this assay, up to an antigen concentration of 100 nM, they showed positive binding to human ANGPTL3 / 8 complex in a concentration-dependent manner and showed no detectable binding to human ANGPTL8 and human ANGPTL3 (Tables 4 - 6).

[0087]

Table 4

[0088]

Table 5

[0089]

Table 6

[0090] Example 3: In Vitro Receptor Affinity The association kinetics can be determined using a Biacore (registered trademark) T200 instrument (GE Healthcare Bio-Sciences Corp.; Piscataway, NJ). The CM4 sensor chip surface can be prepared by covalent attachment of Human Fab Binder (GE Healthcare Bio-Sciences Corp.). The kinetic experiments can be performed at approximately 25 °C in a running buffer of HBSEP +, 0.01% BSA, pH 7.4. Antibodies can be captured and a series of concentrations of mouse, cynomolgus monkey, or human ANGPTL3 / 8 complex can be injected onto the chip surface at approximately 50 μL / min for approximately 240 seconds with a dissociation time of approximately 800 seconds. To determine the kinetic parameters (e.g., k a k d K D ), the data is double-referenced and fitted to a 1:1 binding model using Biacore T200 evaluation software (GE Healthcare Bio-Sciences Corp.). Table 7 below shows the binding of the anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6 to various ANGPTL3 / 8 complexes from different species at pH 7.4 and temperature 25 °C.

[0091] [Table 7]

[0092] In addition to the above anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6, Abs having an LC variant with a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22), or an E99Q mutation (SEQ ID NO: 25) and HC of SEQ ID NO: 6 are assayed for binding to various ANGPTL3 / 8 complexes from different species at pH 7.4 and temperature 25 °C (Tables 8-10).

[0093] [Table 8]

[0094]

Table 9

[0095]

Table 10

[0096] Example 4: In Vitro Functional Activity LPL Assay Using a cell-based bioassay, the ability of an anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6 to relieve the inhibition of ANGPTL3 / 8 purified protein on LPL activity is evaluated. The ANGPTL3 / 8 inhibitory activity of the Ab is determined using EnzChek™ Lipase Substrate (ThermoFisher). HEK293 cell lines expressing human, cynomolgus monkey, mouse or rat LPL, and purified human, cynomolgus monkey, mouse or rat ANGPTL3 / 8 protein. The generation of HEK293-LPL involves a human embryonic cell line (HEK293-huLPL) that stably expresses human LPL generated using standard methods. Briefly, human LPL is cloned into a lentiviral plasmid with a CMV promoter and blasticidin resistance. The plasmid is used to generate human LPL lentivirus using ViraPower Packaging Mix (Invitrogen). HEK293 cells are incubated with the LPL lentivirus, and clones resistant to blasticidin are selected. After confirming the expression of human LPL mRNA by qPCR and LPL activity using the EnzChek™ substrate, clones are selected. This method is repeated for cynomolgus monkey, mouse, and rat LPL.

[0097] The method modified the method described in Basu et al. (Basu et al. (2011) J. Lipid Res. 52:826-832): (a) HEK293-LPL cells were added at a density of 25,000 cells / well to half of the area of 96-well plates A (human), B (cynomolgus monkey), C (mouse), or D (rat) coated with poly-D-lysine and incubated overnight at 37 °C and 5% CO2. The Ab was serially diluted 9-fold from the starting stock concentration to generate a 10-point CRC, and then added to the purified ANGPTL3 / 8 protein in 96-well plates E (human), F (cynomolgus monkey), G (mouse), or H (rat) (IC 80 concentrations are 0.42 nM for human, 0.38 nM for cynomolgus monkey, 0.13 nM for mouse, or 0.81 nM for rat).

[0098] The medium of the HEK293-LPL cells in plates A, B, C, and D was replaced with the ANGPTL3 / 8 and Ab mixtures from plates E, F, G, and H, respectively, and incubated at 37 °C and 5% CO2 for 1 hour. 10 μl of EnzChek™ lipase substrate (prepared at a concentration of 5 μM in 0.05% Zwittergent (3-(N,N-dimethyloctadecylammonio)propanesulfonate) (Sigma)) was added to the cells, ANGPTL3 / 8, and Ab mixtures in plates A, B, C, and D. Fluorescence at 482 nm excitation and 515 nm emission was measured using a plate reader with a 495 nm cut-off filter. The plates were incubated at 37 °C and 5% CO2 between each time point. Relative fluorescence (directly proportional to LPL activity) was calculated by subtracting the signal at 1 minute from the signal at 31 minutes. The effective concentration (EC 50 ) at which the Ab restores 50% of LPL activity was calculated using Excelfit. The EC 50 concentrations are shown in Table 11.

[0099] The dissociation rate is calculated as follows.

Equation

[0100] Ab is generally EC 50 is low and thus is potent in relieving the inhibition of LPL. Ab also has an advantageous maximum relief % of LPL.

[0101] [Table 11]

[0102] In addition to the above anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6, Abs having an LC variant with a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22), a D33T mutation (SEQ ID NO: 23), or an E99Q mutation (SEQ ID NO: 25) and HC of SEQ ID NO: 6 are assayed for the relief of the inhibition of LPL activity by the ANGPTL3 / 8 purified protein (Table 12).

[0103] [Table 12]

[0104] Example 5: In Vivo Triglyceride Response The effect of the anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6 on serum TG is evaluated in mice transformed with huCETP and hu apolipoprotein A1 (a). Blood is collected from the mice and serum is separated before the start of the experiment. TG in the serum samples is measured using a Cobas® clinical chemistry analyzer (Roche). The animals are assigned to 5 groups of 20 to obtain groups with similar serum TG mean values. Next, each group of 20 is further subdivided into 4 groups of 5 humans with similar serum triglyceride mean values. The Ab is administered to the mice by single subcutaneous injection at 1 mg / kg (n = 20), 3 mg / kg (n = 20), 10 mg / kg (n = 20), or 30 mg / kg (n = 20) to four separate groups of animals. A control antigen-binding inactive isotype-matched mAb is administered to the fifth group of animals by single subcutaneous injection at 30 mg / kg (n = 20).

[0105] At 1 hour (n = 5), 8 hours (n = 5), 1 day (n = 5), 2 days (n = 5), 3 days (n = 5), 7 days (n = 5), 14 days (n = 5), and 21 days (n = 5) after Ab administration, blood is collected from the animals. From the animals in subgroup A, blood is collected at 1 hour and 3 days. From the animals in subgroup B, blood is collected at 8 hours and 7 days. From the animals in subgroup C, blood is collected at 1 day and 14 days. From the animals in subgroup D, blood is collected at 2 days and 21 days. Serum is prepared from the blood and serum TG levels are measured using a Cobas® clinical chemistry analyzer (Roche). The percent change in TG from the time-matched isotype control is calculated for each dose of Ab at each time point. The calculation of percent change is [(R × serum triglyceride - time-matched isotype control serum triglyceride) / (time-matched isotype control serum triglyceride)] × 100. The data is shown in Table 13.

[0106]

Table 13

[0107] The efficacy of reducing TG of the Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6 starts well from the first day, and the good effect persists until the 21st day.

[0108] In addition to the above anti-ANGPTL3 / 8 complex Ab having LC of SEQ ID NO: 5 and HC of SEQ ID NO: 6, assay an Ab having an LC variant with D31S mutation (SEQ ID NO: 21), D33A mutation (SEQ ID NO: 22) or E99Q mutation (SEQ ID NO: 25) and HC of SEQ ID NO: 6, and evaluate the change in TG compared to IgG control in mice (Table 14).

[0109]

Table 14

[0110] Sequence Listing The following nucleic acid sequences and amino acid sequences are referred to as described above in the present disclosure and are provided below for reference.

[0111] SEQ ID NO: 1

[0112] Array number 2 MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE

[0113] Array number 3 ataccttagaccctcagtcatgccagtgcctgctctgtgcctgctctgggccctggcaatggtgacccggcctgcctcagcggcccccatgggcggcccagaactggcacagcatgaggagctgaccctgctcttccatgggaccctgcagctgggccaggccctcaacggtgtgtacaggaccacggagggacggctgacaaaggccaggaacagcctgggtctctatggccgcacaatagaactcctggggcaggaggtcagccggggccgggatgcagcccaggaacttcgggcaagcctgttggagactcagatggaggaggatattctgcagctgcaggcagaggccacagctgaggtgctgggggaggtggcccaggcacagaaggtgctacgggacagcgtgcagcggctagaagtccagctgaggagcgcctggctgggccctgcctaccgagaatttgaggtcttaaaggctcacgctgacaagcagagccacatcctatgggccctcacaggccacgtgcagcggcagaggcgggagatggtggcacagcagcatcggctgcgacagatccaggagagactccacacagcggcgctcccagcctgaatctgcctggatggaactgaggaccaatcatgctgcaaggaacacttccacgccccgtgaggcccctgtgcagggaggagctgcctgttcactgggatcagccagggcgccgggccccacttctgagcacagagcagagacagacgcaggcggggacaaaggcagaggatgtagccccattggggaggggtggaggaaggacatgtaccctttcatgcctacacacccctcattaaagcagagtcgtggcatctcaaaaaaaaaaaaaaaaa

[0114] Sequence number 4 MPVPALCLLWALAMVTRPASAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[0115] Sequence number 5 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0116] Sequence number 6 QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0117] Sequence number 7 gatattgtgatgacccagacccccctgtctctgcctgtcactccgggggaaccggcctcgatctcatgccggtcgagccagtccctgctggactccgatgacgggaacacttatttggattggtacctccaaaagcctggacagagcccgcagctcctgatctacatgctgtcctaccgggcctccggagtgccagaccgcttctcgggaagcggctccggtaccgacttcacactgaagatctcccgcgtggaagctgaggacgtgggcatctactactgtatgcaaagaatcgagttccccctcaccttcggcggcgggactaaggtcgagattaagagaaccgtggccgcaccatccgtgttcatttttcccccgtccgatgaacagctgaagtccggaaccgcctccgtcgtgtgcctgctcaacaacttctacccgagggaagcgaaagtgcagtggaaagtggacaatgcgctgcagtccggaaactcccaagagtccgtgaccgaacaggactccaaggactcaacctactcgctgagctcaacgctgaccctgagcaaggccgactacgagaagcacaaggtctacgcctgcgaagtgacccatcagggtttgagctcgcccgtgaccaagtccttcaaccggggagagtgc

[0118] Sequence number 8

[0119] Array number 9 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIK

[0120] Array number 10 QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNWFDPWGQGTLVTVSS

[0121] Array number 11 RSSQSLLDSDDGNTYLD

[0122] Array number 12 YMLSYRAS

[0123] Array number 13 MQRIEFPLT

[0124] Array number 14 TFSGFSLSISGVGVG

[0125] Array number 15 LIYRNDDKRYSPSLKS

[0126] Array number 16 ARTYSSGWYGNWFDP

[0127] Array number 17 MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK

[0128] Sequence number 18 METDTLLLWVLLLWVPGSTGDHHHHHHDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPLEVLFQGPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[0129] Sequence number 19 SRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK

[0130] Sequence number 20 GPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[0131] Sequence number 21 DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0132] Sequence number 22 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSADGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0133] Sequence number 23 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSTDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0134] Sequence number 24 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0135] Sequence number 25 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIQFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

**Claim 1** An antibody that binds to the human ANGPTL3 / 8 complex comprising a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 21, 22, 23, 24, and 25, and the HC comprises the amino acid sequence of SEQ ID NO:

10. **Claim 2** The antibody binds to and neutralizes the human ANGPTL3 / 8 complex with an EC ５０ of 3.0 nM or less in a standard lipoprotein lipase activity assay, the antibody according to claim 1. **Claim 3** The antibody binds to the human ANGPTL3 / 8 complex with a dissociation constant of 1×10 －６ M or less, the antibody according to claim 1 or 2. **Claim 4** The antibody according to any one of claims 1 to 3, which binds to the human ANGPTL3 / 8 complex up to an antigen concentration of up to 100 nM as measured by an ELISA assay and does not bind to human ANGPTL3 alone or human ANGPTL8 alone. **Claim 5** The antibody according to any one of claims 1 to 4, which reduces triglycerides in vivo by at least 50% at a dose of 10 mg / kg at 14 days after administration compared to an IgG control. **Claim 6** A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5 and an acceptable carrier, diluent, or excipient. **Claim 7** A mammalian cell comprising a cDNA molecule, wherein the cDNA molecule encodes a polypeptide comprising an amino acid sequence of one of SEQ ID NO: 5, 21, 22, 23, 24, and 25, the cDNA molecule encodes a heavy chain (HC) of an antibody comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10, and the cell is capable of expressing the antibody. **Claim 8** A mammalian cell comprising at least two cDNA molecules, wherein the first cDNA molecule encodes a light chain (LC) of an antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 21, 22, 23, 24, and 25, the second cDNA molecule encodes a heavy chain (HC) of the antibody comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10, and the cell is capable of expressing the antibody. **Claim 9** A method for producing an antibody, comprising: (i) culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes a polypeptide containing one amino acid sequence among SEQ ID NOs: 5, 21, 22, 23, 24, and 25, the cDNA molecule encodes a heavy chain (HC) of the antibody containing a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 10, and culturing the mammalian cell under conditions such that the encoded polypeptide is expressed, and then (ii) recovering the antibody.

10. A method for producing an antibody, comprising: (i) culturing a mammalian cell containing at least two cDNA molecules, wherein a first cDNA molecule encodes a light chain (LC) of an antibody containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 21, 22, 23, 24, and 25, a second cDNA molecule encodes a heavy chain (HC) of the antibody containing a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 10, and culturing the mammalian cell under conditions such that the antibody is expressed, and then (ii) recovering the antibody.

11. A medicament for reducing triglyceride, comprising the antibody according to any one of claims 1 to 5, which is used to administer an effective amount of the antibody to a patient in need of reducing triglyceride.

12. Use of the antibody according to any one of claims 1 to 5 in the manufacture of a medicament for use in therapy.

13. Use of the antibody according to any one of claims 1 to 5 in the manufacture of a medicament for use in reducing triglyceride.

14. A pharmaceutical composition for use in reducing triglyceride, comprising the antibody according to any one of claims 1 to 5.

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