RU2799796C2 - Antibodies against angptl3/8 complex and methods of their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is presented: a complex of angiopoietin-like peptides (ANGPTL)3/8 to obtain antibodies that bind to the complex (ANGPTL)3/8, a nucleic acid encoding a polypeptide with the sequence of SEQ ID NO:19, an expression cassette and an expression vector containing nucleic acid, as well as the host cell to obtain the complex.

EFFECT: invention makes it possible to obtain a complex of angiopoietin-like peptides (ANGPTL)3/8, for obtaining antibodies that bind to the (ANGPTL)3/8 complex.

6 cl, 1 dwg, 14 tbl, 5 ex

Description

[1] This invention relates generally to biology and medicine, and more particularly to antibodies (Ab, Ab) that bind and thereby neutralize human angiopoietin-like protein (ANGPTL) 3/8 complexes. Such Abs can increase the activity of lipoprotein lipase (LPL, LPL) and thereby reduce the level of triglycerides (TG, TG) in serum, as a result of which they can be used to treat diseases and disorders associated with lipid and glucose metabolism.

[2] ANGPTL are a family of proteins that regulate a number of physiological and pathophysiological processes. Of particular interest in this document is the role of ANGPTL3 and ANGPTL8 in lipid and glucose metabolism.

[3] Evidence supports the role of ANGPTL3 as a major regulator of lipoprotein metabolism, and that it can regulate TG clearance by inhibiting LPL and inhibiting endothelial lipase (EL, EL). See Chi et al. (2017) Mol. Metab. 6:1137-1149. Deficiency, inactivation, or loss of ANGPTL3 can lead to low levels of low-density lipoprotein cholesterol (LDL-C, LDL-C), high-density lipoprotein cholesterol (HDL-C, HDL-C), and TG. ANGPTL3 can also influence insulin sensitivity, thus playing a role in modulating not only lipid metabolism but also glucose metabolism. See Robciuc et al. (2013) Arterioscler. Thromb. Vasc. Biol. 33:1706-1713. The nucleotide and amino acid sequences of human ANGPTL3 are known. For example, one nucleotide sequence can be found in NCBI Reference Sequence No. NM_014495 (SEQ ID NO:1), and one amino acid sequence can be found in NCBI Reference Sequence No. NP_055310 (SEQ ID NO:2).

[4] ANGPTL8 is highly expressed in liver and adipose tissue and is reported to inhibit LPL by complexing with ANGPTL3 and thereby activating it. See Chi, cited higher. Human ANGPTL8 appears to be induced by food intake. The nucleotide and amino acid sequences of human ANGPTL8 are known. For example, one nucleotide sequence can be found in NCBI Reference Sequence No. NM_018687 (SEQ ID NO:3), and one amino acid sequence can be found in NCBI Reference Sequence No. NP_061157 (SEQ ID NO:4).

[5] There are ANGPTL3/8 complexes that have one or more ANGPTL3 associated with one or more ANGPTL8. The data suggest that these complexes mediate LPL inhibition more efficiently than ANGPTL3 or ANGPTL8 alone. Moreover, ANGPTL3/8 complexes can be generated in vitro by co-expression of ANGPTL8 and ANGPTL3 in a mammalian expression system. See Chi, cited above.

[6] Antibodies that bind to either ANGPTL3 or ANGPTL8 are known and can be used alone or in combination with each other to treat diseases and disorders associated with lipid and glucose metabolism. For example, International Patent Application Publication No. WO 2012/174178 discloses a fully human monoclonal Ab and antigen-binding fragments thereof that bind to and interfere with ANGPTL3 activity. Other therapeutic antibodies against ANGPTL3 are also known. See, for example, International Patent Application Publication No. WO 2008/073300 and US Patent No. 7,935,796. Similarly, International Patent Application Publication No. WO 2017/027316 discloses a fully human monoclonal Ab or antigen-binding fragments thereof that bind to and interfere with ANGPTL8 activity. In addition, International Patent Application Publication No. WO 2017/177181 discloses combination therapy using anti-ANGPTL3 Abs and anti-ANGPTL8 Abs.

[7] Unfortunately, existing Abs that only bind to ANGPTL3 or ANGPTL8 do not completely suppress the effect of these ANGPTL 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 this regard, there is a need for additional Abs, especially Abs against the ANGPTL3/8 complex, for the treatment of diseases and disorders associated with lipid and glucose metabolism, while such Abs have improved pharmacological inhibitory and/or regulatory properties for modulating lipid and/or glucose metabolism.

[8] To meet this need, nucleotide and amino acid sequences have been proposed for the modified ANGTPL3/8 complex. Accordingly, this document describes nucleic acid sequences encoding one or more modified ANGPTL3 and modified ANGPTL8 (ie, fusion proteins). In some instances, the nucleic acid sequences comprise a polynucleotide sequence encoding an ANGPTL3 fusion protein having the amino acid sequence of SEQ ID NO:17. In other instances, the nucleic acid sequences comprise a polynucleotide sequence encoding an ANGPTL8 fusion protein having the amino acid sequence of SEQ ID NO:18. In other instances, the nucleic acid sequences comprise a polynucleotide sequence encoding SEQ ID NOS: 17 and 18.

[9] In addition, nucleic acid constructs are provided that contain a polynucleotide sequence encoding an ANGPTL3 fusion protein as described herein, an ANGPTL8 fusion protein as described herein, or both, and such constructs may be a cassette or an expression vector.

[10] In view of the foregoing, host cells are provided that include one or more expression cassettes or vectors as described herein. In some cases, the host cells are eukaryotic cells. In some cases, the polynucleotide sequences of the ANGPTL3 and ANGTPL8 fusion proteins are in separate cassettes or expression vectors, while in other cases they may be in the same cassette or expression vector.

[11] ANGPTL3 fusion proteins are also provided, which contain the amino acid sequence of SEQ ID NO:17 or 19, as well as active variants or fragments thereof. Similarly, ANGPTL8 fusion proteins are provided that contain the amino acid sequence of SEQ ID NO:18 or 20, as well as active variants or fragments thereof.

[12] In addition, functional ANGPTL3/8 complexes, especially human ANGPTL3/8 complexes, are provided, wherein the ANGPTL3 fragment of the complex is a native (full-length or truncated) ANGPTL3 or ANGPTL3 fusion protein as described herein, and wherein the ANGPTL8 fragment of the complex is an ANGPTL8 fusion protein as described herein. In some cases, the ANGPTL3 fusion protein contains the amino acid sequence of SEQ ID NO:19. Similarly, and in some cases, the ANGPTL8 fusion protein contains the amino acid sequence of SEQ ID NO:20. Moreover, and in some cases, the complexes may have a 1:1 ratio of ANGPTL3 fragment to ANGPTL8 fragment. In other cases, the complexes may have ratios other than 1:1, such as a 1:2, 1:3, 2:1, or 3:1 ratio of ANGPTL3 fragment to ANGPTL8 fragment, respectively.

[13] Methods for obtaining recombinant ANGTPL3/8 complexes have also been proposed. The methods may include at least the step of expressing one or more polynucleotide sequences for an ANGPTL3 fragment and an ANGPTL8 fragment, as described herein, in a host cell, such as in a mammalian expression system, to obtain ANGPTL3/8 complexes therefrom. In some cases, fragments of ANGPTL3 and ANGPTL8 are proposed in separate constructs or expression vectors. In other instances, ANGPTL3 and ANGPTL8 are provided in the same construct or expression vector. The methods may also include the step of purifying the resulting ANGPTL3/8 complexes, which may include not only concentrating the ANGPTL3/8 complexes, but also removing one or more tags, linkers, and serum albumin from the ANGPTL3 fragment and/or the ANGPTL8 fragment. The methods may also include the step of concentrating the ANGPTL3/8 complexes before and/or after the purification step.

[14] Secondly, an Ab to the ANGPTL3/8 complex is proposed, as well as its use, which includes the treatment of diseases and disorders associated with lipid and glucose metabolism by binding and inhibiting the activity of the ANGPTL3/8 complex.

[15] An effective amount of Ab against the ANGPTL3/8 complex described herein, or a pharmaceutically acceptable salt thereof, can be used to increase LPL activity, lower TG levels, and treat diseases or disorders associated with lipid and/or glucose metabolism in an individual in need thereof.

[16] Anti-ANGPTL3/8 complex antibody described herein binds the soluble ANGPTL3/8 complex, thereby increasing LPL activity and decreasing serum TG levels. Individuals with lower triglyceride levels have a lower risk of developing cardiovascular disease. Advantageously, the Ab against the ANGPTL3/8 complex described herein binds only to the ANGPTL3/8 complex and not only to ANGPTL3 or only to ANGPTL8 at appropriate concentrations. Ab against the ANGPTL3/8 complex is believed to increase the catabolism of TH-rich lipoproteins (THBLP, TRL), which reduces TG and/or non-HDL-C (non-HDL-C), thereby improving risk factors for dyslipidemia in atherosclerotic cardiovascular disease (ASCVD), which is not eliminated by modern methods of treatment. Moreover, since the Ab against the ANGPTL3/8 complex described herein does not bind only to ANGPTL3 or ANGPTL8, other effects of these ANGPTL are not inhibited, which may lead to fewer undesirable effects in vivo , such as reduced EL derepression.

[17] Specifically, the Ab against the ANGPTL3/8 complex is an Ab against the human ANGPTL3/8 complex. In some cases, Abs against the ANGPTL3/8 complex can abolish, block, inhibit, hinder, neutralize, or reduce the in vivo activity of the ANGPTL3/8 complex, especially its LPL inhibitory activity. In some cases, the Ab against the ANGPTL3/8 complex may be full-length or may be only an antigen-binding fragment ( eg Fab fragment, F(ab') ₂ or scFv). Desirable properties of Abs against the ANGPTL3/8 complex include a reduction in TG levels at low doses of Abs that persists for at least 21 days.

[18] In some cases, Ab against the ANGPTL3/8 complex binds to the human ANGPTL3/8 complex and contains complementarity-determining light chain regions LCDR1, LCDR2 and LCDR3, and complementarity-determining heavy chain regions HCDR1, HCDR2 and HCDR3, while LCDR1 has the amino acid sequence RSSQSLLDSDDGNTYLD (SEQ ID NO:11), LCDR2 has the amino acid sequence YMLSYRAS (SEQ ID NO:12), and LCDR3 has the amino acid sequence MQRIEFPLT (SEQ ID NO:13), while 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).

[19] Additionally proposed Ab containing the variable region of the light chain (LCVR), while LCVR has the amino acid sequence of SEQ ID NO:9; or an Ab containing a heavy chain variable region (HCVR), wherein the HCVR has the amino acid sequence of SEQ ID NO:10. In some cases, the Ab contains LCVR with the amino acid sequence of SEQ ID NO:9 and HCVR with the amino acid sequence of SEQ ID NO:10. In some cases, Ab contains a light chain (LC) and a heavy chain (HC), while LC has the amino acid sequence of SEQ ID NO:5 or HC has the amino acid sequence of SEQ ID NO:6. Alternatively, Ab contains a light chain (LC) and a heavy chain (HC), with LC having the amino acid sequence of SEQ ID NO:5 and HC having the amino acid sequence of SEQ ID NO:6. In some cases, At is an IgG4 isotype.

[20] In some cases, the Ab against the ANGPTL3/8 complex may be a variant of the Ab described above, especially 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) to an LC having the amino acid sequence of SEQ ID NO: 5.

[21] In addition, an Ab is provided, which is obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes polypeptides having the amino acid sequences of SEQ ID NO:5 and 6, under conditions under which the polypeptides are expressed, and extracting the Ab. Alternatively, an Ab is provided that is obtained by culturing a mammalian cell containing two cDNA molecules, wherein the first cDNA molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:5, and the second cDNA molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:6, under conditions under which the polypeptides are expressed, and extracting the At. In some cases, the Ab against the ANGPTL3/8 complex may be a variant of the Ab described above, in particular 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 in relation to an LC having the amino acid sequence of SEQ ID NO:5.

[22] In addition, an Ab has been proposed that binds to and neutralizes the human ANGPTL3/8 complex in a standard LPL activity assay with an EC ₅₀ of 0.5 nM or less. Also proposed is an Ab that binds to the human ANGPTL3/8 complex with a dissociation constant less than or equal to 1×10 ^-6 M. At the same time, an Ab is proposed that binds to human ANGPTL3 and human ANGPTL8 with a dissociation constant of more than 1×10 ^-6 M. In addition, an Ab is proposed that binds to the human ANGPTL3/8 complex with a signal greater than 3 times the background signal, character non-binding as measured by a single-point calibrated ELISA, but does not bind to human ANGPTL3 alone or to ANGPTL8 alone with a signal greater than 3-fold greater than the background signal of no binding as measured by a single-point calibrated ELISA. Similarly, an Ab is proposed that reduces TG in vivo by at least 50% compared to an IgG control at a dose of 10 mg/kg at 14 days post-dose.

[23] Third, a pharmaceutical composition is provided that contains an Ab as described herein, or a population of Abs as described herein, and an acceptable carrier, diluent, or excipient. Also provided is a mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, wherein the cell is capable of expressing the Ab described herein. Additionally, a mammalian cell is provided that contains a first DNA molecule and a second DNA molecule, wherein the first DNA molecule contains a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:5, and the second DNA molecule contains a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:6, and the cell is capable of expressing At described in this document. In some cases, the Ab against the ANGPTL3/8 complex may be a variant of the Ab described above, in particular 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 in relation to an LC having the amino acid sequence of SEQ ID NO:5.

[24] Fourth, a method for producing Ab is provided, which includes cultivating a mammalian cell with a DNA molecule having a polynucleotide sequence encoding polypeptides having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, wherein the cell is capable of expressing the Ab described herein under conditions in which Ab is expressed, and isolating the expressed Ab. In some instances, a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:5 may encode a D31S mutation, a D33A mutation, a D33T mutation, an M56T mutation, an E99Q mutation, or a combination thereof. This document also provides a method for the treatment of ASCVD, chronic renal failure (CKD), diabetes, hypertriglyceridemia, non-alcoholic steatohepatitis (NASH, NASH), obesity, or a combination thereof, the method comprising administering to an individual in need thereof an effective amount of Ab described herein. Additionally, a method for reducing the level of TG is provided, which includes administering to an individual in need thereof an effective amount of Abs described herein.

[25] Fifth, Ab has been proposed for use in therapy. In particular, Ab is intended for the treatment of ASCVD, CKD, diabetes, hypertriglyceridemia, NASH, obesity, or a combination thereof. Also provided is a pharmaceutical composition for use in the treatment of ASCVD, CKD, diabetes, hypertriglyceridemia, NASH, obesity, or a combination thereof, which contains an effective amount of the Ab described herein.

[26] Benefits, effects, features, and objects other than those set forth above will become more apparent upon consideration of the detailed description below. In such a detailed description, reference is made to the following drawing(s), while:

[27] FIG. 1 is an image of an SDS-PAGE gel showing analysis of the ANGPTL3/8 complex under reducing and non-reducing conditions.

[28] Mentioning an element in the singular does not preclude the presence of more than one of the elements, unless the context clearly suggests the presence of one and only one of the elements. Accordingly, terms in the singular usually mean "at least one".

[29] Definitions

[30] As used herein, the term "about" means within a statistically significant range of a value or values, such as, for example, ascertained concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such a value or range may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The tolerance covered by the term "about" will depend on the particular system under study and can be easily estimated by one of skill in the art.

[31] Used in the context of this document, the term "affinity" refers to the strength of binding of Ab to an epitope on the ANGPTL3/8 complex.

[32] Used in the context of this document, the term "angiopoietin-like protein 3" or "ANGPTL3" means a protein having an amino acid sequence containing SEQ ID NO:2.

[33] Used in the context of this document, the term "angiopoietin-like protein 8" or "ANGPTL8" means a protein having an amino acid sequence containing SEQ ID NO:4.

[34] Used in the context of this document, the term "ANGPTL3/8 complex" means a multi-protein complex of one or more ANGPTL3 compounds that are associated with one or more ANGPTL8 compounds.

[35] As used herein, the term "Ab against ANGPTL3/8 complex" or "complex Ab against ANGPTL3/8" means an Ab that simultaneously recognizes and binds to a region on both ANGPTL3 and ANGPTL8, especially when they are in the form of an ANGPTL3/8 complex. In general, Abs against the ANGPTL3/8 complex do not normally bind to other members of the ANGPTL family ( eg , ANGPTL1, ANGPTL2, ANGPTL4, ANGPTL5, ANGPTL6, or ANGPTL7). Moreover, as noted elsewhere in this document, Abs against the ANGPTL3/8 complex will also not bind to ANGPTL3 alone or to ANGPTL8 alone at certain concentrations, as described below in the single point calibration ELISA assay.

[36] As used herein, the term "bind" or "links" means the ability of a protein to form a type of chemical bond or force of attraction with another protein or molecule, as determined by conventional methods known in the art. Binding can be characterized by an equilibrium dissociation constant (K _D ) of about 1×10 ^-6 M or less (ie, lower K _D means tighter binding). Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As stated in this document, Ab against the ANGPTL3/8 complex binds only to the ANGPTL3/8 complex and does not bind separately to ANGPTL3 alone or ANGPTL8 alone. Whether Ab binds only to the ANGPTL3/8 complex, and not only to ANGPTL3 or only to ANGPTL8 alone, can be determined using standard single-point ELISA assays, as described below, and binding can be characterized using Biacore technology, as described below. Although the Abs described herein are human, they may, however, be cross-reactive with other ANGPTL3/8 complexes from other species, such as the cynomolgus macaque ANGPTL3/8 complex, the mouse ANGPTL3/8 complex, or the rat ANGPTL3/8 complex.

[37] As used herein, the term “effective amount” means an amount or dose of a compound or a pharmaceutical composition containing it that, when administered in single or multiple doses to an individual, will produce a biological or therapeutic response, or a desired therapeutic effect in a tissue, system, animal, mammalian, or human body under the supervision of a researcher, veterinarian, physician, or other healthcare professional. In some cases, an effective amount of a compound described herein, or compositions comprising it, for an individual in need thereof, may result in an increase in LPL activity. The dose may include a higher initial loading dose followed by a lower dose. The dose can be administered at any therapeutically effective interval, for example, several 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 dose constituting an effective amount may be from 0.01 mg/kg to 100 mg/kg.

[38] As used herein, the term “equilibrium dissociation constant” or “K _D ” refers to a quantitative measurement of the affinity of Ab for a particular antigen interaction, such as the affinity of Ab for the ANGPTL3/8 complex, specifically the measure of the propensity of an At/ANGPTL3/8 complex to reversibly dissociate into its constituent parts. Similarly, as used here, "equilibrium association constant" or "K _a "means the reciprocal of K _D.

[39] As used herein, the term "functional" means that an ANGPTL3 fusion protein, an ANGPTL8 fusion protein, or an ANGPTL3/8 complex have biological activity similar to that of native ANGPTL3, native ANGPTL8, or native ANGPTL3/8 complex, including, for example, inhibition of LPL or acting as an antigen to which an Ab can be generated and targeted.

[40] Used in the context of this document, the term "disease or disorder associated with glucose metabolism" means diabetes and the like.

[41] used in the context of this document, the term “illness or violation associated with lipid metabolism” means a state associated with anomalous metabolism of lipids, such as dyslipidemia, hyperlipidemia and hyperlipoproteinemia, including hyperthyglyceridemia, hypercholesterolemia, chilomicronemia, mixed dylipidemia (survival, metabolism, metabolism, metabolism, metabolism. Syndrome, diabetes, etc.), lipodistrophy and lipoatrophy. The term also covers certain cardiovascular diseases such as atherosclerosis and coronary heart disease, acute pancreatitis, NASH, obesity, and the like.

[42] Used in the context of this document, the term "half-maximal effective concentration" or "EC ₅₀ "means the concentration of Ab (usually expressed in molar units (M)), which causes a response halfway between baseline and maximum after a predetermined period of time. The EC ₅₀ described herein is ideally 3.0 nM or less.

[43] As used herein, the term "nucleic acid construct" or "expression cassette" means a nucleic acid molecule having at least a regulatory sequence operably linked to a coding sequence. Thus, a regulatory sequence, such as a promoter, is in operative interaction with nucleic acid sequences encoding at least one polypeptide of interest, such as the ANGPTL3 fusion proteins described herein and/or the ANGPTL8 fusion proteins described herein. Such nucleic acid constructs may be in the form of an expression or transfer cassette. The nucleic acid construct may include an oligonucleotide or polynucleotide consisting of deoxyribonucleotides, ribonucleotides, or combinations thereof, containing the nucleotide sequences for one or more polypeptides of interest.

[44] Used in the context of this document, the term "operably linked" means that the elements of the nucleic acid construct are configured to perform their normal functions. Thus, regulatory sequences (ie, promoters) operably linked to a coding sequence are capable of influencing the expression of the coding sequence. Regulatory sequences do not need to be in contact with a coding sequence if they function to direct its expression (ie, maintain the correct reading frame). Thus, for example, intermediate untranslated but still transcribed sequences may be present between the promoter and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[45] As used herein, the term "regulatory sequence" or "regulatory sequences" means promoters, polyadenylation signals, transcription and translation termination sequences, regulatory domains upstream of a coding sequence, replication origins, internal ribosome entry regions ("IRES"), enhancers, and the like, which together provide for replication, transcription, and translation of a coding sequence in a recipient host cell. Not all of these regulatory sequences need to be present if the selected coding sequence is capable of being replicated, transcribed and translated in the appropriate host cell.

[46] As used herein, the term "coding sequence" or "coding sequences" means a nucleic acid sequence that encodes one or more polypeptides of interest, and 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 when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence(s) are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxy) end. The coding sequence may include, 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.

[47] With regard to regulatory and coding sequences, they may be native/similar to the host cell or to each other. Alternatively, the regulatory and coding sequences may be heterologous to the host cell or to each other.

[48] As used herein, the term "promoter" refers to a nucleotide region consisting of a nucleic acid regulatory sequence, wherein the regulatory sequence is derived from a gene or synthetically generated and is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. A number of promoters can be used in nucleic acid constructs, including a native promoter for one or more polypeptides of interest. Alternatively, promoters may be selected based on the desired outcome. Such promoters may include, but are not limited to, inducible promoters, repressible promoters, and constitutive promoters.

[49] Used in the context of this document, the term "variant" means a polynucleotide or polypeptide having one or more modifications, such as the addition, deletion, insertion and/or substitution of one or more specific nucleic acids or amino acid residues compared to the reference nucleotide or amino acid sequence. Thus, a variant includes one or more changes from the reference nucleotide or amino acid sequence. As stated in this document, the Ab against the ANGPTL3/8 complex may have an LC or HC variation. In particular, Ab may 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) to an LC having the amino acid sequence of SEQ ID NO:5. Similarly, an LC variant may be a combination of any two of the above mutations, such as, for example, 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, cations D33T and E99Q, as well as M56T and E99Q, again in relation to the LC having the amino acid sequence of SEQ ID NO:5. Moreover, the LC variant may be a combination of any three of the above mutations, such as, for example, D31S, D33A and M56T mutations, D31S, D33A and E99Q mutations, D31S, D33T and M56T mutations, D31S, D33T and E99Q, D33A mutations, M56T and E99Q mutations, and mutations D33T, M56T and E99Q, again with respect to the LC having the amino acid sequence of SEQ ID NO: 5. In addition, the LC variant may be a combination of any four of the above mutations, such as, for example, mutations D31S, D33A, M56T and E99Q; and mutations D31S, D33T, M56T and E99Q, again in relation to the LC having the amino acid sequence of SEQ ID NO:5.

[50] As used herein, the term "vector" means a replicon, such as a plasmid, phage, or cosmid, to which another nucleic acid sequence, such as an expression cassette, can be attached to cause replication of the attached sequence. The vector is capable of transferring nucleic acid molecules into host cells. Vectors typically contain one or a small number of restriction endonuclease recognition sites, where the nucleotide sequence of interest can be inserted in a defined manner without losing the basic biological function of the vector, and a selectable marker that can be used to identify and select host cells transformed by the vector. Therefore, the vector may be capable of transferring nucleic acid sequences to target cells.

[51] As used herein, the term “treatment” or “treat” means the management and care of an individual having a pathology for which the administration of Abs against the ANGPTL3/8 complex is recommended in order to control the symptoms, or alleviate the symptoms and complications of such pathologies. Treatment includes administering to an individual in need thereof a compound or composition containing a compound described herein to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate a disease, pathology or disorder. Treatment includes administering to an individual in need thereof a compound or compositions containing those described herein resulting in an increase in LPL activity and a decrease in TG. The subject to be treated is an animal, especially a human.

[52] Used in the context of this document, the terms "patient", "subject" and "individual" are used in this document interchangeably and mean an animal, in particular a human. In some instances, the subject is a human and is further characterized by a disease, disorder, or condition that may be ameliorated by administration of Ab against the ANGPTL3/8 complex.

[53] Used in the context of this document, the term "antibody" or "Ab" and the like means a full-length Ab containing 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 primarily responsible for antigen recognition. Each HC contains an N-terminal HCVR and a HC constant region (HCCR). Each light chain contains a light chain variable region (LC)-(LCVR) and a constant region LC-(LCCR). As stated herein, an Ab is an immunoglobulin G (IgG) type Ab, and the IgG isotype can be further divided into subclasses ( eg , IgG1, IgG2, IgG3, and IgG4). The HCVR and LCVR regions can be further subdivided into regions of hypervariability called complementarity determining regions ("CDRs") interspersed with regions that are more conserved and called framework regions ("FRs"). Each LCVR and HCVR contains three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this document, the three heavy chain CDRs are referred to as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. The CDRs contain most of the residues that form specific interactions with an antigen, such as the ANGPTL3/8 complex. The assignment of residues to different CDRs can be done using algorithms such as, for example, Chothia, Kabat or North. North's CDR determination is based on affinity spread clustering with a large number of crystal structures (North et al. (2011) J. Mol. Bio. 406:228-256). In this document, CDRs are best referred to by the sequences listed in the Sequence Listing, which are based on a combination of several definitions, including North's.

[54] An isolated DNA encoding an HCVR region can be converted into a full-length heavy chain gene by operably linking the DNA encoding HCVR with another DNA molecule encoding heavy chain constant regions. The human, as well as other mammalian, heavy chain constant region gene sequences are known in the art. DNA fragments covering these regions can be obtained, for example, using standard PCR amplification.

[55] An isolated DNA encoding an LCVR region can be converted into a full length light chain gene by operably linking the DNA encoding an LCVR with another DNA molecule encoding a light chain constant region. The human light chain constant region gene sequences, as well as those of other mammals, are known in the art. DNA fragments covering these regions can be obtained, for example, using standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

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

[57] In order to reduce the potential induction of an immune response when dosed to humans, some amino acids may require backmutations to match the Ab germline sequences.

[58] Pharmaceutical compositions containing the compounds described herein can be parenterally administered to an individual in need of such treatment. Such an individual may have ASCVD or be at high risk of developing ASCVD. These individuals may have an acute coronary syndrome, a history of myocardial infarction (MI, MI), stable or unstable angina, coronary or other arterial revascularization, stroke, transient ischemic attack (TIA, TIA), thoracic or abdominal aortic aneurysm, or peripheral arterial disease presumably of atherosclerotic origin. Individuals at high risk of ASCVD may additionally have type 2 diabetes mellitus (DM2, T2D), CKD, or familial hypercholesterolemia (FH).

[59] Parenteral administration can be by subcutaneous, intramuscular, or intravenous injection using a syringe, optionally a pen, or a mechanical injector. Alternatively, parenteral administration may be via an infusion pump. In some cases, pharmaceutical compositions suitable for administration to an individual contain a therapeutically effective amount of a compound described herein and one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions may be prepared by any of a variety of methods using conventional pharmaceutical excipients that are well known in the art. See, for example, Remington, “The Science and Practice of Pharmacy” (DB Troy ed., 21 ^st Ed., Lippincott, Williams & Wilkins, 2006).

[60] The compounds described herein may be used in simultaneous, separate or sequential combination with one or more additional therapeutic agents useful in modulating LPL activity, treating diseases or disorders associated with lipid metabolism, or treating diseases or disorders associated with glucose metabolism, including any of the disorders listed above. Non-limiting examples of additional therapeutic agents that can be combined with the claimed compounds include, but are not limited to, anti-diabetic agents such as insulin or insulin analogs, biguanides, sulfonylureas, thiazolidinediones, dipeptidyl peptidase-4 ("DPP-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 drugs such as HMG-CoA reductase inhibitors, PCSK9 inhibitors, cholesterol absorption inhibitors, fibrates, niacin, LXR agonists, RXR agonists, ROR agonists, or cholesterol reverse transport modulators; drugs for the treatment of heart failure, such as ACE inhibitors, angiotensin/neprilysin receptor inhibitors, ARBs, or B-adrenergic antagonists; drugs for anti-inflammatory therapy; drugs for the treatment of hypertension, drugs for the treatment of atrial fibrillation; drugs for the treatment of neurodegeneration; anticancer drugs; drugs for the treatment of diabetic cardiomyopathy, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, weight loss, wound healing; drugs for the treatment of nephropathy; drugs for the treatment of PAD; or combinations of any of the above agents. Ab against the ANGPTL3/8 complex and one or more additional therapeutic agents can be administered either together using the same delivery route and means, such as a single pill, capsule, tablet or injectable dosage form; either administered separately or at the same time by different means of delivery or routes; or entered sequentially.

[61] Preparation and Purification of ANGPTL3/8 Complexes

[62] One aspect of the disclosure is that ANGPTL3/8 complexes can be used to create an Ab that binds only to the complex and does not bind separately to ANGPTL3 only or ANGPTL8 only. Although Abs against ANGPTL3 and Abs against ANGPTL8 are already known, there is a problem in synthesizing sufficient amounts of functional ANGPTL3/8 complexes, especially human ANGPTL3/8 complexes, to generate Abs against the ANGPTL3/8 complex. Additionally or alternatively, such ANGPTL3/8 complexes can be used in assays to evaluate the properties of Abs directed to ANGPTL3, ANGPTL8 and/or ANGPTL3/8 complexes.

[63] Thus, the disclosure also describes methods for generating ANGPTL8 by converting it to N- or C-terminal ( eg , human, mouse, or rabbit) serum albumin. Functional ANGPTL3/8 complexes can then be generated by co-expression of the ANGPTL8 fusion protein with native ANGPTL3 or ANGPTL3 fusion protein in a mammalian expression system.

[64] As noted above, the nucleotide and amino acid sequences of native human ANGPTL3 and native human ANGPTL8 are known (see, for example , SEQ ID NOS: 1-2 and 3-4, respectively). However, ANGPTL3 or ANGPTL 8, as described herein, are modified ( i.e., recombinant/synthetic) and therefore differ from native sequences by containing additional amino acid sequences to improve production, secretion, and/or complexation of ANGTPL3 and ANGTPL8.

[65] For example, ANGPTL3 can be modified to include one or more linkers and labels known in the art. As stated herein, 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. In some cases, the linker may contain from about 1 to about 10 amino acids, for example 3 amino acids, especially 3 Ala residues. The linker and FLAG tags can be placed at the N- or C-terminus of the ANGPTL3 sequence, especially the C-terminus, as in SEQ ID NO:17, with residues 1-460 corresponding to ANGPTL3 and residues 461-471 corresponding to the 3-Ala linker and FLAG tag.

[66] Similarly, ANGPTL8 can be modified to include one or more linkers and tags in addition to the sequence for serum albumin, especially human serum albumin. As stated herein, human ANGPTL8 (SEQ ID NO:4) is modified by including a linker, an IgG kappa signal peptide, a polyhistidine (His) tag, mature human 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. In some cases, the linker may contain from about 1 to about 10 amino acids, for example, a rigid polyproline repeat, especially the Ala-Pro (AP)-10 linker. The signal peptide, His tag, mature HSA, linker and PreScission® cleavage site can be placed at the N- or C-terminus of the ANGPTL8 sequence, especially at the N-terminus as in SEQ ID NO:18, where residues 1-20 correspond to the IgG kappa chain signal peptide, residues 21-27 correspond to the His-tag, residues 28-612 correspond to HSA, residues 6 13-632 correspond to the AP-10 linker, residues 633-643 correspond to the PreScission® cleavage site, and residues 644-820 correspond to ANGPTL8.

[67] Methods for constructing nucleic acid constructs for expressing an ANGPTL3 fusion protein and/or an ANGPTL8 fusion protein as described herein are well known in the art and can be found, for example, in Balbás & Lorence, “Recombinant Gene Expression: Reviews and Protocols” ( ^2nd Ed. Humana Press 2004); Davis et al. , “Basic Methods in Molecular Biology ” (Elsevier Press 1986); Sambrook & Russell, “Molecular Cloning: A Laboratory Manual” ( ^3rd 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 US Pat. Nos. 6,664,387; 7,060,491; 7,345,216 and 7,494,805. Because ANGPTL8 does not contain disulfide bonds, mammalian expression systems can be used. As described herein, HEK293 expression systems or CHO expression systems can be used to generate ANGPTL3 and/or ANGPTL8 fusion proteins, especially by co-expression.

[68] In addition to expressing the ANGPTL3 fusion protein and/or the ANGPTL8 fusion protein, the methods may also include purifying the resulting fusion proteins based on the specific label used for each fusion protein, which are well known in the art. With respect to the ANGPTL3 and ANGTPL8 fusion proteins described in these documents, purification may result in a series of post-tag removal truncations such that the ANGPTL3 fusion protein has the amino acid sequence of SEQ ID NO:19 (with residues 1-444 corresponding to ANGPTL3 and residues 445-455 corresponding to the 3-Ala linker and FLAG tag) and the ANGPTL fusion protein 8 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 the ANGPTL8 fragment) that readily bind to each other to form functional ANGPTL3/8 complexes.

[69] Obtaining and Purifying Antibodies

[70] Abs against the ANGPTL3/8 complex can be generated in a mammalian cell expression system using the CHO-GSKO cell line. Knockout of the glutamine synthetase (GS, GS) gene provides strong selection stringency by eliminating endogenous background GS activity that may allow the survival of low- or unproductive cells under selection conditions. The genes encoding the HC and LC Ab described herein can be subcloned into separate HS-containing expression plasmids for co-transfection, or both strands can be subcloned into a single HS-containing expression plasmid. The cDNA sequence encoding HC or LC is fused in frame with the coding sequence for a signal peptide, which may be a murine kappa chain leader, to enhance secretion of the desired product into the cell culture medium. Expression is driven by the cytomegalovirus (CMV) viral promoter.

[71] CHO - GSKO - cells are stably transfected using electroporation and an appropriate amount of recombinant HC and LC expression plasmid, and the transfected cells are maintained in suspension culture at an appropriate cell density. The selection of transfected cells is carried out by growing without glutamine in serum-free medium containing 25 μm methionine sulfoximine (MSK, MSX), and incubated at 32°C-37°C and 5-7% CO ₂ . Abs are secreted into the medium from CHO cells. Abs can be purified by protein A affinity chromatography followed by anion exchange or hydrophobic interaction chromatography (or other suitable methods), and size exclusion chromatography can be used for further purification.

[72] Abs from harvested media are captured on MabSelect SuRe Protein A resin (GE Healthcare). The resin is then washed rapidly with a running buffer such as phosphate buffered saline (PBS) pH 7.4 or running buffer containing Tris to remove non-specifically bound material. The Ab is then eluted from the resin with a low pH solution such as 20 mM acetic acid/5 mM citric acid. Fractions containing Ab against ANGPTL3/8 are pooled and can be kept at low pH to inactivate potential viruses. The pH can be increased as needed by adding a base such as 0.1 M Tris pH 8.0. Abs against ANGPTL3/8 can be further purified by hydrophobic interaction chromatography (HIC) using resins such as Phenyl HP (GE Healthcare). Anti-ANGPTL3/8 Abs can be eluted from the HIC column using a sodium sulfate gradient in 20 mM Tris, pH 8.0. Abs against ANGPTL3/8 can be further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare) eluting isocratically in PBS, pH 7.4.

[73] The compounds described herein are obtained by this or a similar method, which can be easily determined by a person skilled in the art.

[74] Mice bearing human light and heavy chain variable domains are immunized with the ANGPTL3/8 complex described above and single antigen-specific B cells are isolated by FACS using ANGPTL3/8 (positive) and ANGPTL3 (negative) as markers. Heavy chain and light chain variable region DNA is isolated from individual B cells by PCR and cloned into IgG expression vectors. After transfection, CHO cell supernatants are assayed for binding activity.

EXAMPLES

[75] The following non-limiting examples are offered for purposes of illustration and not limitation.

[76] Example 1: Creation of ANGPTL3/8 Complexes

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

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

[79] Analysis of LPL activity. The EnzCheck® LPL assay is performed according to the manufacturer's instructions (ThermoFisher Scientific). Briefly, ANGPTL3 and ANGPLT3/8 complexes are serially cultured and replaced with LPL cells during 1 hour of incubation. EnzCheck® Lipase Substrate (ELS) is then added to LPL-expressing cells and incubated for 30 minutes. Fluorescence is measured at 482 nm/515 nm (excitation/emission, respectively). Calculate the % inhibitory effect of ANGPTL3 and 3/8 on LPL.

[80] Results:

[81] Table 1 shows the yield of the ANGTPL3/8 complex protein at various purification/concentration steps.

[82] Table 1: Yield of the ANGPTL3/8 Complex Protein.

Start (L) Yield after Ni-NTA Resin (mg) Yield after ^1st Superdex® 200 Column (mg) Yield after ^2nd Superdex® 200 column (mg) 4 105 47 12

[83] Similarly, in FIG. 1 shows a 4-20% TG TGX gel, stained with Coomassie, with a sample of the purified and concentrated ANGPTL3/8 complex obtained from the columns, which confirms that the complexes are forming/assembling. 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.

[84] 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 the functionality of the complexes.

[85] Example 2: Analyzes

[86] Single Point Calibration ELISA Assay (SPE ELISA): The binding selectivity of Ab to ANGPTL3/8 complex, free ANGPTL3, or free ANGPTL8 is initially tested using standard single point ELISA assays. Briefly, assay plates are coated with anti-human Fc Ab at a concentration of 2 μg/ml and then 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 allow Ab/antigen binding. The Ab/antigen complexes are detected after addition of alkaline phosphatase-conjugated neutravidin and alkaline phosphatase substrate and subsequent measurement of absorbance at 560 nm. Positive binding is defined by a signal greater than 3 times the background signal characteristic of no binding.

[87] ELISA assay: The selectivity of Ab binding to ANGPTL3/8 complex, free ANGPTL3, or free ANGPTL8 is tested using standard ELISA assays. Briefly, assay plates are coated with anti-human Fc Ab at a concentration of 2 μg/ml and then blocked with casein. The Ab from the supernatant of CHO cells, after being expressed in CHO cells, is captured on the assay plate, resulting in an Ab concentration of 2 µg/mL. The biotinylated antigen (ANGPTL3, ANGPTL8 or ANGPTL3/8 complex) is added at various concentrations by serial dilutions to allow Ab/antigen binding. The Ab/antigen complexes are detected after addition of alkaline phosphatase-conjugated neutravidin and alkaline phosphatase substrate and subsequent measurement of absorbance at 560 nm.

[88] Table 2: Selectivity of the Binding of Ab to the ANGPTL3/8, ANGPTL8 and ANGPTL3 Complex in the APAATC Assay.

Binding of the ANGPTL3/8 Complex ANGPTL8 binding ANGPTL3 binding At 2.302 (positive) 0.087 (negative) 0.074 (negative) D31S 0.928 0.072 0.058 D33A 0.898 0.118 0.076 D33T 0.661 0.070 0.060 M56T 0.775 0.085 0.060 E99Q 1.538 0.100 0.083 Average Background Signal with No Binding 0.09 0.07 0.08 3 x Background 0.27 0.21 0.24

Values are optical density (OD) at 560 nm, and the background signal, representative of the absence of binding, across all test plates is reflected on average in the "Background signal" line. This negative control shows background signal in the absence of At.

[89] In an APAATK assay, an Ab having an LC with SEQ ID NO:5 and a HC with SEQ ID NO:6 shows positive binding to the human ANGPTL3/8 complex and negative binding to human ANGPTL8 and human ANGPTL3. Abs with LC variants having a D31S mutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22), a D33T mutation (SEQ ID NO:24), an M56T mutation (SEQ ID NO:24), or an E99Q mutation (SEQ ID NO:25) and HC with the sequence of SEQ ID NO:6 similarly show binding to the human ANGPTL3/8 complex and negatively e binding to human ANGPTL8 and human ANGPTL3.

[90] Table 3: ELISA Analysis of Ab Binding at Different Concentrations of ANGPTL3, ANGPTL8 and ANGPTL3/8 Complex.

hANGPTL8 hANGPTL3 hANGPTL3/8 complex Conc. antigen [nM] At Control At Control At Control 100 0.072 0.076 0.081 0.091 2.705 0.063 33.3 0.050 0.060 0.052 0.063 2.411 0.056 11.1 0.045 0.051 0.046 0.052 1.708 0.047 3.70 0.051 0.048 0.046 0.052 1.042 0.046 1.23 0.043 0.048 0.043 0.052 0.510 0.046 0.412 0.046 0.050 0.043 0.067 0.231 0.046 0.137 0.047 0.053 0.048 0.055 0.110 0.044 0.046 0.051 0.054 0.060 0.069 0.078 0.053

The control is a buffer without At.

[91] An Ab having an LC with the sequence of SEQ ID NO:5 and an HC with the sequence of SEQ ID NO:6 shows positive binding to the human ANGPTL3/8 complex depending on its concentration and no detectable binding to human ANGPTL8 and human ANGPTL3 up to an antigen concentration of 100 nM in this assay (Table 3).

[92] In addition to the aforementioned Ab against the ANGPTL3/8 complex having LC with SEQ ID NO:5 and HC with SEQ ID NO:6, Ab with LC variants having D31S mutation (SEQ ID NO:21), D33A mutation (SEQ ID NO:22) or E99Q mutation (SEQ ID NO:25) and HC with SEQ ID NO:6 are being investigated. and similarly show positive binding to the human ANGPTL3/8 complex in a concentration dependent manner and show no binding to human ANGPTL8 and human ANGPTL3 up to an antigen concentration of 100 nM in this assay (Tables 4-6).

[93] Table 4: ELISA Analysis of D31S Ab variant Binding at Different Concentrations of ANGPTL3, ANGPTL8 and ANGPTL3/8 Complex.

hANGPTL8 hANGPTL3 hANGPTL3/8 complex Conc. antigen [nM] At Control At Control At Control 100 0.098 0.076 0.084 0.081 2.471 0.092 33.3 0.063 0.065 0.069 0.064 2.226 0.068 11.1 0.062 0.059 0.062 0.062 1.735 0.053 3.70 0.056 0.055 0.060 0.057 1.134 0.049 1.23 0.058 0.049 0.059 0.058 0.575 0.048 0.412 0.062 0.049 0.058 0.057 0.234 0.047 0.137 0.080 0.077 0.057 0.076 0.137 0.068 0.046 0.096 0.085 0.060 0.064 0.149 0.075

[94] Table 5: ELISA Analysis of D33A Ab variant Binding at Different Concentrations of ANGPTL3, ANGPTL8 and ANGPTL3/8 Complex.

hANGPTL8 hANGPTL3 hANGPTL3/8 complex Conc. antigen [nM] At Control At Control At Control 100 0.078 0.076 0.082 0.081 2.388 0.092 33.3 0.049 0.065 0.067 0.064 2.179 0.068 11.1 0.048 0.059 0.060 0.062 1.635 0.053 3.70 0.048 0.055 0.056 0.057 1.040 0.049 1.23 0.049 0.049 0.057 0.058 0.491 0.048 0.412 0.047 0.049 0.056 0.057 0.196 0.047 0.137 0.053 0.077 0.062 0.076 0.101 0.068 0.046 0.074 0.085 0.062 0.064 0.111 0.075

[95] Table 6: E99Q Ab Variant Binding ELISA Analysis at Different Concentrations of ANGPTL3, ANGPTL8 and ANGPTL3/8 Complex.

hANGPTL8 hANGPTL3 hANGPTL3/8 complex Conc. antigen [nM] At Control At Control At Control 100 0.098 0.076 0.084 0.081 2.471 0.092 33.3 0.063 0.065 0.069 0.064 2.226 0.068 11.1 0.062 0.059 0.062 0.062 1.735 0.053 3.70 0.056 0.055 0.060 0.057 1.134 0.049 1.23 0.058 0.049 0.059 0.058 0.575 0.048 0.412 0.062 0.049 0.058 0.057 0.234 0.047 0.137 0.080 0.077 0.057 0.076 0.137 0.068 0.046 0.096 0.085 0.060 0.064 0.149 0.075

[96] Example 3: In Vitro Receptor Affinity

[97] Binding kinetics can be determined using the Biacore® T200 instrument (GE Healthcare Bio-Sciences Corp.; Piscataway, NJ). The surface of the CM4 sensor chip can be prepared by covalently binding a human Fab Binder (GE Healthcare Bio-Sciences Corp.). Kinetic experiments can be carried out at about 25° C. in HBSEP+ working buffer, 0.01% BSA at pH 7.4. Abs can be captured on the chip surface and series of concentrations of mouse, cynomolgus macaque or human ANGPTL3/8 complex can be injected onto the chip surface at a rate of about 50 µl/min for about 240 seconds with a dissociation time of about 800 seconds. To determine kinetic parameters ( eg , _ka , k _d , K _D ), data were double normalized and matched to a 1:1 binding model using Biacore T200 evaluation software (GE Healthcare Bio-Sciences Corp.). Table 7 below shows the binding of Ab against the ANGPTL3/8 complex, having LC with the sequence of SEQ ID NO:5 and HC with the sequence of SEQ ID NO:6, with various ANGPTL3/8 complexes from different species at pH 7.4 and a temperature of 25°C.

[98] Table 7: Binding of At to Cross Species Complexes ANGPTL3/8 at pH 7.4 and Temperature 25°C.

Binding of the ANGPTL3/8 Complex to Ab k _a (1/Msec) k _d (1/sec) K _D (M) SD N Mouse 4.75 x 10 ⁵ 1.72 x 10 ^-4 3.66 x 10 ^-10 6.19 x 10 ^-11 3 Javanese macaque 4.91 x 10 ⁵ 9.91 x 10 ^-5 2.13 x 10 ^-10 6.30 x 10 ^-11 3 Human 4.77 x 10 ⁵ 6.31 x 10 ^-5 1.35 x 10 ^-10 2.69 x 10 ^-11 2

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

[100] Table 8: Binding of Variant At D31S to Cross Species Complexes ANGPTL3/8 at pH 7.4 and Temperature 25°C.

Binding of the ANGPTL3/8 Complex to Ab D31S k _a (1/Msec) k _d (1/sec) K _D (M) SD N Mouse 4.96 x 10 ⁵ 1.08 x 10 ^-4 2.18 x 10 ^-10 1.88 x 10 ^-11 3 Javanese macaque 4.62 x 10 ⁵ 1.68 x 10 ^-4 3.65 x 10 ^-10 1.50 x 10 ^-11 3 Human 4.97 x 10 ⁵ 5.75 x 10 ^-5 1.16 x 10 ^-10 1.91 x 10 ^-11 3

[101] Table 9: Binding of Variant At D33A to Cross Species Complexes ANGPTL3/8 at pH 7.4 and Temperature 25°C.

Binding of the ANGPTL3/8 Complex to Ab D33A k _a (1/Msec) k _d (1/sec) K _D (M) SD N Mouse 5.15 x 10 ⁵ 3.47 x 10 ^-4 6.80 x 10 ^-10 7.07 x 10 ^-11 3 Javanese macaque 4.35 x 10 ⁵ 2.65 x 10 ^-4 6.09 x 10 ^-10 1.21 x 10 ^-11 3 Human 4.57 x 10 ⁵ 9.19 x 10 ^-5 2.01 x 10 ^-10 1.75 x 10 ^-11 3

[102] Table 10: Binding of E99Q Variant Ab to ANGPTL3/8 Cross Species Complexes at pH 7.4 and Temperature 25°C.

Binding of the ANGPTL3/8 Complex to Ab E99Q k _a (1/Msec) k _d (1/sec) K _D (M) SD N Mouse 5.96 x 10 ⁵ 1.51 x 10 ^-4 2.54 x 10 ^-10 1.79 x 10 ^-11 3 Javanese macaque 6.20 x 10 ⁵ 7.23 x 10 ^-5 1.16 x 10 ^-10 8.58 x 10 ^-12 3 Human 6.74 x 10 ⁵ 3.26×10 ^-5 4.84×10 ^-11 6.50×10 ^-12 3

[103] Example 4: In Vitro LPL Functional Activity Assay

[104] A cell-based bioassay is used to evaluate the ability of an anti-ANGPTL3/8 complex Ab having an LC of SEQ ID NO:5 and an HC of SEQ ID NO:6 to suppress LPL inhibition of activity by the purified ANGPTL3/8 protein. The inhibitory activity of Ab against ANGPTL3/8 was determined using EnzChek™ lipase substrate (ThermoFisher). HEK293 cell lines expressing human, cynomolgus, mouse, or rat LPL and purified human, cynomolgus, mouse, or rat ANGPTL3/8 protein were used. The generation of HEK293-LPL includes a human embryonic cell line stably expressing human LPL, which is produced (HEK293-huLPL) using standard methods. Briefly, human LPL is cloned into a lentivirus 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 LPL lentivirus and clones resistant to blasticidin are selected. The clone is selected after confirmation of human LPL mRNA expression by quantitative PCR and LPL activity using the EnzChek™ substrate. This process is repeated for LPL cynomolgus macaque, mouse and rat.

[105] The methods are modified from those described in Basu et al. (Basu et al. (2011) J. Lipid Res. 52:826-832): (a) HEK293-LPL cells are added to half the area of a 96-well plate coated with Poly-D-Lysine A (human), B (cynomolgus macaque), C (mouse), or D (rat) at a density of 25,000 cells/well and incubated overnight at 37°C , 5% CO ₂ . Ab is serially diluted nine times from the original stock concentration to give a ten-point CRC and then added to purified ANGPTL3/8 proteins (at an IC ₈₀ concentration of 0.42 nM human, 0.38 nM cynomolgus, 0.13 nM mouse, or 0.81 nM rat) in 96-well plates E (human), F (cynomolgus), G (mouse) or H (rat).

[106] Media for HEK293-LPL cells in plates A, B, C and D are replaced with mixtures of ANGPTL3/8 and Ab from plate E, F, G and H, respectively, and incubated for 1 hour at 37°C, 5% CO ₂ . 10 µl of EnzChek™ lipase substrate (prepared at a concentration of 5 µM in 0.05% Zwittergent (3-(N, N-dimethyloctadecylammonio)propanesulfonate) (Sigma)) is added to the cells, ANGPTL3/8 and Ab mixture in plates A, B, C, and D. The analyzer plate is used to measure fluorescence at 482 nm Excitation wavelength and Emission 515 nm with a 495 nm cutoff filter. Between time points, the plates are incubated at 37°C, 5% CO ₂ . Relative fluorescence (directly proportional to LPL activity) is calculated by subtracting the signal at 1 min from the signal at 31 min. The effective concentration at which Ab restores LPL activity by 50% (EC ₅₀ ) is calculated using Excel Fit. EC ₅₀ concentrations are shown in Table 11.

[107] The percentage of derepression is calculated as follows:

= (RFU - RFU(MIN))/(RFU(MAX) - RFU(MIN)) x 100,

where MAX=cells only (LPL) and MIN=cells (LPL) + ANGPTL3/8 SC (conditioned medium).

[108] At usually has a low level of EC ₅₀ and, thus, is able to suppress derepressirovanie LPL. At also has a suitable % maximum LPL derepression.

[109] Table 11: EC ₅₀ At and % Maximum LPL Derepression for LPL Human, Cynomolgus Macaque, Mouse and Rat, and the ANGPTL3/8 Complex Human, Javan Macaque, Mouse and Rat.

LPL and ANGPTL3/8 Different Types EC ₅₀ At (nM) % Max. Derepression LPL human LPL and human ANGPTL3/8 0.28 102% LPL cynomolgus macaque and ANGPTL3/8 cynomolgus macaque 1.31 96% LPL mice and ANGPTL3/8 mice 1.38 103% LPL rat and ANGPTL3/8 rat 2.77 105%

[110] In addition to the above Ab against the ANGPTL3/8 complex having an LC of SEQ ID NO:5 and an HC of SEQ ID NO:6, Abs with LC variants having 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:23) 5) and HC with the sequence of SEQ ID NO:6, were examined for the ability to suppress the inhibition of LPL activity by the purified ANGPTL3/8 protein (Table 12).

[111] Table 12: EC ₅₀ Variant At and % Max. LPL derepressions for Human and Mouse LPL and the Human and Mouse ANGPTL3/8 Complex.

LPL and ANGPTL3/8 Different Types D31S
EC ₅₀ At
(nM),
% Max. Derepression LPL D33A
EC ₅₀ At
(nM),
% Max. Derepression LPL D33T
EC ₅₀ At
(nM),
% Max. Derepression LPL E99Q
EC ₅₀ At
(nM),
% Max. Derepression LPL human LPL and human ANGPTL3/8 0.86, 106% 1.14, 103% 1.56, 105% 0.49, 106% LPL mice and ANGPTL3/8 mice 1.79, 110% 4.07, 106% 4.55, 114% 1.65, 110%

[112] Example 5: In Vivo Triglyceride Response

[113] The effect of Ab against the ANGPTL3/8 complex, having LC with the sequence of SEQ ID NO:5 and HC with the sequence of SEQ ID NO:6, on the level of TG in serum is assessed in mice transgenic for huCETP and huApolipoprotein A1 (a). Mice are bled and serum is isolated before the start of the experiment. TG is measured in serum samples using a Cobas® chemistry analyzer (Roche). Animals were divided into 5 groups of 20 animals each to obtain groups with similar mean serum TG values. Each group of 20 animals is then subdivided into 4 groups of 5 animals with similar mean serum triglyceride values. Ab is administered to mice by a single subcutaneous injection at a dose of 1 mg/kg (n=20), 3 mg/kg (n=20), 10 mg/kg (n=20) or 30 mg/kg (n=20) to 4 separate groups of animals. The control antigen-binding mAb corresponding to the null isotype is administered by a single subcutaneous injection at a dose of 30 mg/kg (n=20) to the fifth group of animals.

[114] Animals are bled 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 administration of At. Blood is taken from animals of subgroup A after 1 hour and 3 days. Blood is taken from animals of subgroup B after 8 hours and 7 days. Blood is taken from animals of subgroup C after 1 day and 14 days. Blood is taken from animals of subgroup D after 2 days and 21 days. Serum was prepared from blood and serum TG levels were measured using a Cobas® clinical chemistry analyzer (Roche). The percentage change in TG from the time-matched isotype control is calculated for each Ab dose at each time point. The percentage change calculation is [(Rx serum triglyceride - timed isotype control serum triglyceride)/(timed isotype control serum triglyceride)] x 100. Data are presented in Table 13.

[115] Table 13: Percentage Ab Reduction in TG Levels Compared to IgG Control at Various Ab Doses at Different Time Points.

Time Post Dose 1 hour 8 ocloc'k 1 day 2 days 3 days 7 days 14 days 21 day 1 mg/kg -22 -33 -76* -66* -61* 41 6.4 -20 3 mg/kg -26 -71* -84* -77* -86* -25 19.7 -19 10 mg/kg -27 -62* -87* -89* -88* -88* -64.4* -28 30 mg/kg -23 -74* -92* -87* -93* -93* -89.1* -75*

Dunnett's test was used for each data set to compare each experimental group with time-matched controls, and a p value < 0.05 was considered statistically significant. Groups that are statistically significant compared to time-matched controls are indicated in the table with an asterisk (*).

[116] The efficacy of Ab having LC of SEQ ID NO:5 and HC of SEQ ID NO:6 in reducing TG is good from Day 1 and the beneficial effect is maintained until Day 21.

[117] In addition to the aforementioned Ab against the ANGPTL3/8 complex having an LC with the sequence of SEQ ID NO: 5 and an HC with the sequence of SEQ ID NO: 6, Abs with LC variants having a D31S mutation (SEQ ID NO: 21), a D33A mutation (SEQ ID NO: 22) or an E99Q mutation (SEQ ID NO: 25) and an HC with the sequence of SEQ ID NO: 6, also were studied to assess the change in TG levels compared with control IgG in mice (Table 14).

[118] Table 14: Percent Reduction in TG Levels Compared to Control IgG at Different Doses of Ab Variants at Different Time Points.

Time Post Dose 1 day 7 days 15 days 21 day D31S 3 mg/kg -83* -38* -25 -12 D31S 10 mg/kg -90* -91* -37* -21 D33A 3 mg/kg -74* -77* -34* -10 D33A 10 mg/kg -78* -86* -76* -60* E99Q 3 mg/kg -82* -49* -5 15 E99Q 10 mg/kg -86* -91* -61* 6

Dunnett's test was used for each data set to compare each experimental group with time-matched controls, and a p value < 0.05 was considered statistically significant. Groups that are statistically significant compared to time-matched controls are indicated in the table with an (*).

SEQUENCE LIST

[119] The following nucleotide and amino acid sequences are mentioned in the disclosure above and are provided below for reference.

[120] SEQ ID NO:1

atatatagagttaagaagtctaggtctgcttccagaagaaaacagttccacgttgcttgaaattgaaaatcaagataaaaatgttcacaattaagctccttcttttttattgttcctctagttatttcctccagaattgatcaagacaattcatcatttgattctctatctccagagccaaaatcaagatttgctatgttagacgatgtaaaaattttagccaat ggcctccttcagttgggacatggtcttaaagactttgtccataagacgaagggccaaattaatgacatatttcaaaaactcaacatatttgatcagtcttttttgatctatcgctgcaaaccagtgaaatcaaagaagaagaaaaggaactgagaagaactacatataaactacaagtcaaaaatgaagaggtaaagaatgtcacttgaactcaactcaaaacttga aagcctcctagaagaaaaaattctacttcaacaaaaagtgaaatatttagaagagcaactaactaacttaattcaaaatcaacctgaaactccagaacacccagaagtaacttcacttaaaacttttgtagaaaaaacaagataatagcatcaaagaccttctccagaccgtggaagaccaatataaacaattaaaccaacagcatagtcaaataaaagaaatagaaa atcagctcagaaggactagtattcaagaacccacagaaatttctctatcttccaagccaagagcaccaagaactccctttcttcagttgaatgaaataagaaatgtaaaacatgatggcattcctgctgaatgtaccaccatttataacagaggtgaacatacaagtggcatgtatgccatcagacccagcaactctcaagttttcatgtctactgtgatgttatatcaggtag tccatggacattaattcaacatcgaatagatggatcacaaaacttcaatgaaacgtgggagaactacaaatatggttttgggaggcttgatggagaattttggttgggcctagagaagatatactccatagtgaagcaatctaattatgttttacgaattgagttggaagactggaaagacaacaaacattatattgaatattctttttacttgggaaatcacgaaaccaactat acgctacatctagttgcgattactggcaatgtccccaatgcaatcccggaaaacaaagatttggtgttttctacttgggatcacaaagcaaaaggacacttcaactgtccagagggttattcaggaggctggtggtggcatgatgagtgtggagaaaacaacctaaatggtaaatataacaaccaagagcaaaatctaagccagagaggaga agaggattatcttggaagtctcaaaatggaaggttatactactctataaaatcaaccaaaatgttgatccatccaacagattcagaaagctttgaatgaactgaggcaaatttaaaaggcaataatttaaacattaacctcattccaagttaatgtggtctaataatctggtattaaatccttaagagaaagcttgagaaatagattttttttcttaaagtcactgt ctatttaagattaaacatacaatcacataaccttaaagaataccgtttacatttctcaatcaaaattcttataatactatttgttttaaattttgtgatgtgggaatcaattttagatggtcacaatctagattataatcaataggtgaacttattaaataacttttctaaataaaaatttagagacttttattttaaaaggcatcatatgagctaatatcacaactttccca gtttaaaaaactagtactctcttgttaaaactctaaacttgactaaatacagaggactggtaattgtacagttcttaaatgttgtagtattaatttcaaaactaaaaatcgtcagcacagagtatgtgtaaaaatctgtaatacaaatttttaaactgatgcttcattttgctacaaaataatttggagtaaatgtttgatatga tttatttatgaaacctaatgaagcagaattaaatactgtattaaaataagttcgctgtctttaaacaaatggagatgactactaagtcacattgactttaacatgaggtatcactataccttatttgttaaaatatatactgtatacattttatatattttaacacttaatactatgaaaacaaataattgtaaaggaatcttgtcagattacagtaagaatga acatatttgtggcatcgagttaaagtttatttcccctaaatatgctgtgattctaatacattcgtgtaggttttcaagtagaaataaacctcgtaacaagttactgaacgtttaaacagcctgacaagcatgtatatatgtttaaaattcaataaacaaagacccagtccctaaattatagaaatttaaattattcttgcatgtttatcga catcacaacagatccctaaatccctaaatccctaaagattagatacaaattttttaccacagtatcacttgtcagaatttatttttaaatatgattttttaaaactgccagtaagaaattttaaattaaacccatttgttaaaggatatagtgcccaagttatggtgacctaccttgtcaatacttagcattatgtatttcaaattatccaatatacatgtcat atatatttttatatgtcacatatatataaaagatatgtatgatctatgtgaatcctaagtaaatattttgttccagaaaagtacaaaataataaaggtaaaaataatctataattttcaggaccacagactaagctgtcgaaattaacgctgatttttttagggccagaataccaaaatggctcctctcttcccccaaaattggacaatttcaaatgcaaaata attcattatttaatatatgagttgcttcctctatt

[121] SEQ ID NO:2

MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQ QHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG YSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE

[122] SEQ ID NO:3

ataccttagaccctcagtcatgccagtgcctgctctgtgcctgctctgggccctggcaatggtgacccggcctgcctcagcggcccccatgggcggcccagaactggcacagcatgaggagctgaccctgctcttccatgggaccctgcagctgggccaggccctcaacggtgtgtacaggaccaccggggggacggctgacaaaaggccaggaac agcctgggtctctctatggccgcacaatagaactcctggggcaggaggtcagccggggccgggatgcagcccaggaacttcgggcaagcctgttggagactcagatggaggaggatattctgcagctgcaggcagaggccacagctgaggtgctgggggaggtggcccaggcacagaaggtgctacgggcacagcgtgcagcggctagaagtccagctga ggagcgcctggctgggccctgcctaccgagaatttgaggtcttaaaggctcacgctgacaagcagagccacatcctatgggccctcacaggccacgtgcagcggcagaggcggggagatggtggcacagcagcatcggctgcgacagatccaggagagactccacacagcggcgctcccagcctgaatctgcctggatggaactgaggaccaatcatg ctgcaaggaacacttccacgccccgtgaggcccctgtgcagggaggagctgcctgttcactgggatcagccagggcgccgggccccacttctgagcacagagcagagacagacgcaggcggggacaaaggcagaggatgtagccccattgggggaggggtggaggaaggacatgtaccctttcatgcctacacacccctcattaaagcagagtcgt ggcatctcaaaaaaaaaaaaaaaaa

[123] SEQ ID NO:4

MPVPALCLLWALAMVTRPASAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[124] SEQ ID NO:5

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

[125] SEQ ID NO:6

QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[126] SEQ ID NO:7

gatattgtgatgacccagaccccctgtctctgcctgtcactccgggggaaccggcctcgatctcatgccggtcgagccagtccctgctggactccgatgacgggaacacttatttggattggtacctccaaaagcctggacagagcccgcagctcctgatctacatgctgtcctaccgggcctccggagtgccagaccgcttct cgggaagcggctccggtaccgacttcacactgaagatctcccgcgtggaagctgaggacgtgggcatctactactgtatgcaaagaatcgagttccccctcaccttcggcggcgggactaaggtcgagattaagagaaccgtggccgcaccatccgtgttcatttttcccccgtccgatgaacagctgaagtccggaaccgcctccgtc gtgtgcctgctcaacaacttctacccgagggaagcgaaagtgcagtggaaagtggacaatgcgctgcagtccggaaactcccaagagtccgtgaccgaacaggactccaaggactcaacctactcgctgagctcaacgctgaccctgagcaaggccgactacgagaagcacaaggtctacgcctgcgaagtgacccatcagggttt gagctcgccgtgaccaagtccttcaaccggggagagtgc

[127] SEQ ID NO:8

caagtcacattgaaggagagcggtccgaccctggtcaagccgactcagaccctgaccctgacgtgcactttctcgggcttctcattgtccatttctggagtgggcgtgggatggatcagacagcccccggggaaggccctcgagtggctcgcgctgatctaccgcaacgacgacaaacgctactccccctcactgaaatcccgg ctgaccatcactaaggatacgtccaagaaccaggtcgtgttgaccctcaccaacatggatcccgtggatactgccacctactattgtgcacggacctatagcagcggttggtacggaaactggttcgacccgtggggccagggaactcttgtgacggtgtcctccgcaagcaccaagggtccttctgtgttccccctggcgccgtgctc gcggagcacctcagagtccaccgccgccctcggctgccttgtgaaggactacttcccggagccagtcaccgtgtcctggaacagcggggccctgacttccggcgtgcacaccttccctgcggtgctgcagagctcaggcctctattcgctgtcatccgtcgtgaccgtgccttcctcgtccctgggcactaagacct acacttgcaacgtggaccataagcccagcaacaccaaagtggacaagagagtggaatccaaatacggaccgccatgtccgccctgccccgccccggaagctgccggggggacccagcgtgttcctgttcccacctaagccgaaggacactctgatgatctcaaggactcccgaagtcacttgcgtggtcgtggacgtgtcccagg aggacccgaagtccagtttaattggtacgtggatggtgtcgaggtccacaacgccaagaccaagcctcgcgaggaacagttcaattccacctaccgggtcgtgtccgtcctgaccgtgctgcatcaggactggctgaacggaaaggagtacaagtgcaaagtgtccaacaagggactcccttcctccatcgaaaagaccatcagcaa ggccaagggccagcctcgcgaaccacaagtctacaccctgcccccatcgcaagaggaaatgaccaagaaccaagtgtcgctgacatgcctcgtcaagggattctacccgtcggatattgcggtggaatgggagtccaacggacagcccgagaacaactacaagaccaccccgccggtgttggactccgacggctcctttttcctgtactcccggct cactgtggacaagtcgcggtggcaggaggggaacgtgttctcctgttccgtgatgcacgaagctctgcacaaccactacacccagaagtcgctgagcctctcactggga

[128] SEQ ID NO:9

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIK

[129] SEQ ID NO:10

QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNWFDPWGQGTLVTVSS

[130] SEQ ID NO:11

RSSQSLLDSDDDGNTYLD

[131] SEQ ID NO:12

YMLSYRAS

[132] SEQ ID NO:13

MQRIEPLT

[133] SEQ ID NO:14

TFSGFSLSISGVGVG

[134] SEQ ID NO:15

LIYRNDDKRYSPSLKS

[135] SEQ ID NO:16

ARTYSSGWYGNWFDP

[136] SEQ ID NO:17

MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQ QHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG YSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK

[137] SEQ ID NO:18

METDTLLLWVLLLWVPGSTGDHHHHHHDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEP QNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETTFFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQ AALGLAPAPAPAPAPAPAPAPAPPLEVLFQGPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQI QERLHTAALPA

[138] SEQ ID NO:19

SRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRT SIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENN LNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK

[139] SEQ ID NO:20

GPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[140] SEQ ID NO:21

DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

[141] SEQ ID NO:22

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSADGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

[142] SEQ ID NO:23

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSTDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

[143] SEQ ID NO:24

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

[144] SEQ ID NO:25

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIQFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC.

Claims (7)

1. Complex of angiopoietin-like peptides (ANGPTL)3/8 for obtaining antibodies that bind to the complex (ANGPTL)3/8, where the complex contains a polypeptide with the amino acid sequence of SEQ ID NO:19 and a polypeptide with the amino acid sequence of SEQ ID NO:20. 2. Nucleic acid encoding a polypeptide with the sequence of SEQ ID NO:19. 3. An expression cassette containing a nucleic acid according to claim 2. 4. An expression vector containing the nucleic acid according to claim 2. 5. The host cell for obtaining the complex according to claim 1, containing the expression cassette according to claim 3 or the vector according to claim 4. 6. The host cell of claim 5, wherein the host cell is a mammalian cell.

Images