RU2791034C2 - Antibodies against angptl3/8 complex and their application methods - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to antibodies, which bind to a human ANGPTL3/8 complex and, thereby, neutralize them; to pharmaceutical compositions containing the mentioned antibodies, to mammalian cells expressing antibodies to ANGPTL3/8 complex, to a method for the production of antibodies to ANGPTL3/8 complex, to a method for reduction in a level of triglycerides, as well as treatment of diseases related to lipid metabolism.

EFFECT: invention allows for an increase in activity of lipoproteinlipase and, thereby, efficient reduction in a level of triglycerides, which can be further used for the treatment of diseases and disorders related to lipid metabolism.

36 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 above . 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 either 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 to modulate lipid metabolism and /or glucose.

[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 can be cassette or 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 their active variants or fragments. 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, in particular 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 the fragment The ANGPTL8 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. Furthermore, and in some cases, 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, needing it.

[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 thought 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 heart disease. vascular disease (ASCVD, ASCVD), 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, 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 ( 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 the extraction of 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:22). : 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. However, an Ab is proposed that binds to human ANGPTL3 and human ANGPTL8 with a dissociation constant greater than 1×10 ^-6 M In addition, an Ab has been proposed that binds to the human ANGPTL3/8 complex with a signal greater than 3 times the background signal of no binding as measured by a single point calibration ELISA assay, but does not bind to human ANGPTL3 alone or only with ANGPTL8 with a signal greater than 3 times the background signal of no binding, as measured by a single-point calibrated ELISA assay. 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. Further provided is a mammalian cell comprising 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 an amino acid the sequence of SEQ ID NO:6, and the cell is capable of expressing the 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:22). : 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 culturing 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 Ab, described in this document, under conditions in which Ab expression occurs, and the release of 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 treating 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 in this document. 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] Used in the context of this document, the term "about" means within a statistically significant range of value or values, such as, for example, the established 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 the given value or range. The tolerance covered by the term "about" will depend on the particular system under study and can be readily estimated by one of ordinary 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 in the case when they are in the form of the 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 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, which, when administered single or multiple doses to an individual, will produce a biological or therapeutic response, or a desired therapeutic effect in a tissue, system, organism. animal, mammal, or human under the care 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] Used in the context of this document, the term "equilibrium dissociation constant" or "K _D "means a quantitative measurement of the affinity of Ab for a particular interaction of antigen, such as the affinity of Ab for the ANGPTL3/8 complex, in particular the measure of the propensity of the Ab conjugate/ANGPTL3/8 complex to a reversible division into its component 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 the ANGPTL3 fusion protein, ANGPTL8 fusion protein, or ANGPTL3/8 complex have biological activity similar to that of native ANGPTL3, native ANGPTL8, or native ANGPTL3/8 complex, including, for example, inhibition LPL or acting as an antigen to which Ab can be created and directed.

[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 "disease or disorder associated with lipid metabolism" means a condition associated with abnormal lipid metabolism, such as dyslipidemia, hyperlipidemia and hyperlipoproteinemia, including hypertriglyceridemia, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome , diabetes, etc. ), lipodystrophy 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 operable 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 in this document. 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 able to influence 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, upstream regulatory domains, origins of replication, internal ribosome entry regions ("IRES" ), enhancers, and the like, which together allow for replication, transcription, and translation of the coding sequence in the 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] Used in the context of this document, 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 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] Used in the context of this document, the term "promoter" means a nucleotide region consisting of a regulatory nucleic acid sequence, while the regulatory sequence is derived from a gene or created synthetically and is capable of binding RNA polymerase and initiating transcription of the 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, the 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 , mutations D33T and M56T, mutations D33T and E99Q, and 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, mutations D31S, D33A and M56T, mutations D31S, D33A and E99Q, mutations D31S, D33T and M56T, mutations D31S, D33T and E99Q, D33A , mutations M56T and E99Q, and mutations D33T, M56T and E99Q, again in relation 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, whereby the nucleotide sequence of interest can be inserted in a specific 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] Used in the context of this document, the term "treatment" or "treat" means the management of an individual and the care of an individual having a pathology for which the administration of Ab 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 Fc portion of IgG4-PAA has a Ser mutation to Pro at position 231 (S231P), a Phe mutation to Ala 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, antidiabetic agents such as insulin or insulin analogs, biguanides, sulfonylurea, thiazolidinediones, dipeptidyl peptidase-4 inhibitors ("DPP-4", "DPP- 4") 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 only binds 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 linker 3 -Ala and FLAG label.

[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 described herein, human ANGPTL8 (SEQ ID NO:4) is modified to include 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, in particular at the N-terminus as in SEQ ID NO:18, with 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 613-632 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 to express the ANGPTL3 fusion protein and/or the 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 truncations after deletion of tags 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 correspond to the 3-Ala linker and 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 the ANGPTL8 fragment ), which readily bind to each other to form ANGPTL3/8 functional 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, again combined, concentrated 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 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 of SEQ ID NO:6 similarly show binding to the human ANGPTL3/8 complex and negative binding to human ANGPTL8 and human ANGPTL3.

[90] Table 3: ELISA Analysis of Ab Binding at Different Concentrations of ANGPTL3, ANGPTL8 and the 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 100 nM antigen concentration in this assay (Table 3).

[92] 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, Ab with LC variants having the D31S mutation (SEQ ID NO:21), the mutation D33A (SEQ ID NO:22) or mutation E99Q (SEQ ID NO:25) and HC with the sequence of SEQ ID NO:6, are investigated and similarly show positive binding to the human ANGPTL3/8 complex in a concentration dependent manner and do not show binding to Human ANGPTL8 and human ANGPTL3 up to 100 nM antigen concentration 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° WITH.

[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 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 the D31S mutation (SEQ ID NO:21), the mutation D33A (SEQ ID NO:22) or mutation E99Q (SEQ ID NO: 25) and HC with the sequence of SEQ ID NO:6, were examined for binding to various ANGPTL3/8 complexes from different species at pH 7.4 and temperature 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 (Javanese macaque), C (mice) 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 solution to obtain a ten-point CRC and then added to the purified ANGPTL3/8 proteins (at an IC ₈₀ concentration of 0.42 nM for human, 0.38 nM for cynomolgus macaque, 0.13 nM for mouse or 0.81 nM for 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 5 µM in 0.05% Zwittergent (3-(N, N-dimethyloctadecylammonio)propanesulfonate) (Sigma)) is added to cells, a mixture of ANGPTL3/8 and Ab in plates A, B, C and D. The plate analyzer is used to measure fluorescence at 482 nm Excitation and 515 nm Emission with a 495 nm cut-off 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. The 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 with the sequence of SEQ ID NO:5 and an HC with the sequence of SEQ ID NO:6, Abs with LC variants having the D31S mutation (SEQ ID NO:21), the mutation D33A (SEQ ID NO:22) mutation D33T (SEQ ID NO:23) or mutation E99Q (SEQ ID NO:25) 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) in 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] Blood sampling from animals is done after 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 (*).

[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 above 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 the D31S mutation (SEQ ID NO: 21), the mutation D33A (SEQ ID NO:22) or mutations in E99Q (SEQ ID NO:25) and HC with the sequence of SEQ ID NO:6 were also investigated to assess the change in TG levels compared to IgG control 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 after 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

atatatagagttaagaagtctaggtctgcttccagaagaaaacagttccacgttgcttgaaattgaaaatcaagataaaaatgttcacaattaagctccttctttttattgttcctctagttatttcctccagaattgatcaagacaattcatcatttgattctctatctccagagccaaaatcaagatttgctatgttagacgatgtaaaaattttagccaatggcctccttcagttgggacatggtcttaaagactttgtccataagacgaagggccaaattaatgacatatttcaaaaactcaacatatttgatcagtctttttatgatctatcgctgcaaaccagtgaaatcaaagaagaagaaaaggaactgagaagaactacatataaactacaagtcaaaaatgaagaggtaaagaatatgtcacttgaactcaactcaaaacttgaaagcctcctagaagaaaaaattctacttcaacaaaaagtgaaatatttagaagagcaactaactaacttaattcaaaatcaacctgaaactccagaacacccagaagtaacttcacttaaaacttttgtagaaaaacaagataatagcatcaaagaccttctccagaccgtggaagaccaatataaacaattaaaccaacagcatagtcaaataaaagaaatagaaaatcagctcagaaggactagtattcaagaacccacagaaatttctctatcttccaagccaagagcaccaagaactactccctttcttcagttgaatgaaataagaaatgtaaaacatgatggcattcctgctgaatgtaccaccatttataacagaggtgaacatacaagtggcatgtatgccatcagacccagcaactctcaagtttttcatgtctactgtgatgttatatcaggtagtccatggacattaattcaacatcgaatagatggatcacaaaacttcaatgaaacgtgggagaactacaaatatggttttgg gaggcttgatggagaattttggttgggcctagagaagatatactccatagtgaagcaatctaattatgttttacgaattgagttggaagactggaaagacaacaaacattatattgaatattctttttacttgggaaatcacgaaaccaactatacgctacatctagttgcgattactggcaatgtccccaatgcaatcccggaaaacaaagatttggtgttttctacttgggatcacaaagcaaaaggacacttcaactgtccagagggttattcaggaggctggtggtggcatgatgagtgtggagaaaacaacctaaatggtaaatataacaaaccaagagcaaaatctaagccagagaggagaagaggattatcttggaagtctcaaaatggaaggttatactctataaaatcaaccaaaatgttgatccatccaacagattcagaaagctttgaatgaactgaggcaaatttaaaaggcaataatttaaacattaacctcattccaagttaatgtggtctaataatctggtattaaatccttaagagaaagcttgagaaatagattttttttatcttaaagtcactgtctatttaagattaaacatacaatcacataaccttaaagaataccgtttacatttctcaatcaaaattcttataatactatttgttttaaattttgtgatgtgggaatcaattttagatggtcacaatctagattataatcaataggtgaacttattaaataacttttctaaataaaaaatttagagacttttattttaaaaggcatcatatgagctaatatcacaactttcccagtttaaaaaactagtactcttgttaaaactctaaacttgactaaatacagaggactggtaattgtacagttcttaaatgttgtagtattaatttcaaaactaaaaatcgtcagcacagagtatgtgtaaaaatctgtaatacaaatttttaaactgatgcttcattttgctac aaaataatttggagtaaatgtttgatatgatttatttatgaaacctaatgaagcagaattaaatactgtattaaaataagttcgctgtctttaaacaaatggagatgactactaagtcacattgactttaacatgaggtatcactataccttatttgttaaaatatatactgtatacattttatatattttaacacttaatactatgaaaacaaataattgtaaaggaatcttgtcagattacagtaagaatgaacatatttgtggcatcgagttaaagtttatatttcccctaaatatgctgtgattctaatacattcgtgtaggttttcaagtagaaataaacctcgtaacaagttactgaacgtttaaacagcctgacaagcatgtatatatgtttaaaattcaataaacaaagacccagtccctaaattatagaaatttaaattattcttgcatgtttatcgacatcacaacagatccctaaatccctaaatccctaaagattagatacaaattttttaccacagtatcacttgtcagaatttatttttaaatatgattttttaaaactgccagtaagaaattttaaattaaacccatttgttaaaggatatagtgcccaagttatatggtgacctacctttgtcaatacttagcattatgtatttcaaattatccaatatacatgtcatatatatttttatatgtcacatatataaaagatatgtatgatctatgtgaatcctaagtaaatattttgttccagaaaagtacaaaataataaaggtaaaaataatctataattttcaggaccacagactaagctgtcgaaattaacgctgatttttttagggccagaataccaaaatggctcctctcttcccccaaaattggacaatttcaaatgcaaaataattcattatttaatatatgagttgcttcctctatt

[121] SEQ ID NO:2

MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE

[122] SEQ ID NO:3

ataccttagaccctcagtcatgccagtgcctgctctgtgcctgctctgggccctggcaatggtgacccggcctgcctcagcggcccccatgggcggcccagaactggcacagcatgaggagctgaccctgctcttccatgggaccctgcagctgggccaggccctcaacggtgtgtacaggaccacggagggacggctgacaaaggccaggaacagcctgggtctctatggccgcacaatagaactcctggggcaggaggtcagccggggccgggatgcagcccaggaacttcgggcaagcctgttggagactcagatggaggaggatattctgcagctgcaggcagaggccacagctgaggtgctgggggaggtggcccaggcacagaaggtgctacgggacagcgtgcagcggctagaagtccagctgaggagcgcctggctgggccctgcctaccgagaatttgaggtcttaaaggctcacgctgacaagcagagccacatcctatgggccctcacaggccacgtgcagcggcagaggcgggagatggtggcacagcagcatcggctgcgacagatccaggagagactccacacagcggcgctcccagcctgaatctgcctggatggaactgaggaccaatcatgctgcaaggaacacttccacgccccgtgaggcccctgtgcagggaggagctgcctgttcactgggatcagccagggcgccgggccccacttctgagcacagagcagagacagacgcaggcggggacaaaggcagaggatgtagccccattggggaggggtggaggaaggacatgtaccctttcatgcctacacacccctcattaaagcagagtcgtggcatctcaaaaaaaaaaaaaaaaa

[123] SEQ ID NO:4

MPVPALCLLWALAMVTRPASAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[124] SEQ ID NO:5

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

[125] SEQ ID NO:6

QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[126] SEQ ID NO:7

Gatattgtgatgacccagacccccctgtctctgcctgtcactccgggggaaccggcctcgatctcatgccggtcgagccagtccctgctggactccgatgacgggaacacttatttggattggtacctccaaaagcctggacagagcccgcagctcctgatctacatgctgtcctaccgggcctccggagtgccagaccgcttctcgggaagcggctccggtaccgacttcacactgaagatctcccgcgtggaagctgaggacgtgggcatctactactgtatgcaaagaatcgagttccccctcaccttcggcggcgggactaaggtcgagattaagagaaccgtggccgcaccatccgtgttcatttttcccccgtccgatgaacagctgaagtccggaaccgcctccgtcgtgtgcctgctcaacaacttctacccgagggaagcgaaagtgcagtggaaagtggacaatgcgctgcagtccggaaactcccaagagtccgtgaccgaacaggactccaaggactcaacctactcgctgagctcaacgctgaccctgagcaaggccgactacgagaagcacaaggtctacgcctgcgaagtgacccatcagggtttgagctcgcccgtgaccaagtccttcaaccggggagagtgc

[127] SEQ ID NO:8

caagtcacattgaaggagagcggtccgaccctggtcaagccgactcagaccctgaccctgacgtgcactttctcgggcttctcattgtccatttctggagtgggcgtgggatggatcagacagcccccggggaaggccctcgagtggctcgcgctgatctaccgcaacgacgacaaacgctactccccctcactgaaatcccggctgaccatcactaaggatacgtccaagaaccaggtcgtgttgaccctcaccaacatggatcccgtggatactgccacctactattgtgcacggacctatagcagcggttggtacggaaactggttcgacccgtggggccagggaactcttgtgacggtgtcctccgcaagcaccaagggtccttctgtgttccccctggcgccgtgctcgcggagcacctcagagtccaccgccgccctcggctgccttgtgaaggactacttcccggagccagtcaccgtgtcctggaacagcggggccctgacttccggcgtgcacaccttccctgcggtgctgcagagctcaggcctctattcgctgtcatccgtcgtgaccgtgccttcctcgtccctgggcactaagacctacacttgcaacgtggaccataagcccagcaacaccaaagtggacaagagagtggaatccaaatacggaccgccatgtccgccctgccccgccccggaagctgccgggggacccagcgtgttcctgttcccacctaagccgaaggacactctgatgatctcaaggactcccgaagtcacttgcgtggtcgtggacgtgtcccaggaggaccccgaagtccagtttaattggtacgtggatggtgtcgaggtccacaacgccaagaccaagcctcgcgaggaacagttcaattccacctaccgggtcgtgtccgtcctgaccgtgctgcatcaggactggctgaacggaaaggagtacaagtgcaaagtgtccaacaagggactcccttcct ccatcgaaaagaccatcagcaaggccaagggccagcctcgcgaaccacaagtctacaccctgcccccatcgcaagaggaaatgaccaagaaccaagtgtcgctgacatgcctcgtcaagggattctacccgtcggatattgcggtggaatgggagtccaacggacagcccgagaacaactacaagaccaccccgccggtgttggactccgacggctcctttttcctgtactcccggctcactgtggacaagtcgcggtggcaggaggggaacgtgttctcctgttccgtgatgcacgaagctctgcacaaccactacacccagaagtcgctgagcctctcactggga

[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

MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK

[137] SEQ ID NO:18

METDTLLLWVLLLWVPGSTGDHHHHHHDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPLEVLFQGPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[138] SEQ ID NO:19

SRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK

[139] SEQ ID NO:20

GPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA

[140] SEQ ID NO:21

DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

[141] SEQ ID NO:22

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSADGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

[142] SEQ ID NO:23

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSTDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

[143] SEQ ID NO:24

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

[144] SEQ ID NO:25

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIQFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

--->

SEQUENCE LIST

<110> ELI LILLY AND COMPANY

<120> ANTIBODIES AGAINST ANGPTL3/8 COMPLEX AND METHODS OF THEIR USE

<130> X21977

<150> US 62/783,260

<151> 2018-12-21

<150> US 62/783,265

<151> 2018-12-21

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 2951

<212> DNA

<213> Homo sapiens

<400> 1

atatatagag ttaagaagtc taggtctgct tccagaagaa aacagttcca cgttgcttga 60

aattgaaaat caagataaaa atgttcacaa ttaagctcct tctttttatt gttcctctag 120

ttatttcctc cagaattgat caagacaatt catcatttga ttctctatct ccagagccaa 180

aatcaagatt tgctatgtta gacgatgtaa aaattttagc caatggcctc cttcagttgg 240

gacatggtct taaagacttt gtccataaga cgaagggcca aattaatgac atatttcaaa 300

aactcaacat atttgatcag tctttttatg atctatcgct gcaaaccagt gaaatcaaag 360

aagaagaaaa ggaactgaga agaactacat ataaactaca agtcaaaaat gaagaggtaa 420

agaatatgtc acttgaactc aactcaaaac ttgaaagcct cctagaagaa aaaattctac 480

ttcaacaaaa agtgaaatat ttagaagagc aactaactaa cttaattcaa aatcaacctg 540

aaactccaga acacccagaa gtaacttcac ttaaaacttt tgtagaaaaa caagataata 600

gcatcaaaga ccttctccag accgtggaag accaatataa acaattaaac caacagcata 660

gtcaaataaa agaaatagaa aatcagctca gaaggactag tattcaagaa cccacagaaa 720

tttctctatc ttccaagcca agagcaccaa gaactactcc ctttcttcag ttgaatgaaa 780

taagaaatgt aaaacatgat ggcattcctg ctgaatgtac caccattttat aacagaggtg 840

aacatacaag tggcatgtat gccatcagac ccagcaactc tcaagttttt catgtctact 900

gtgatgttat atcaggtagt ccatggacat taattcaaca tcgaatagat ggatcacaaa 960

acttcaatga aacgtgggag aactacaaat atggttttgg gaggcttgat ggagaatttt 1020

ggttgggcct agagaagata tactccatag tgaagcaatc taattatgtt ttacgaattg 1080

agttggaaga ctggaaagac aacaaacatt atattgaata ttctttttac ttgggaaatc 1140

acgaaaccaa ctatacgcta catctagttg cgattactgg caatgtcccc aatgcaatcc 1200

1260

gtccagaggg ttattcagga ggctggtggt ggcatgatga gtgtggagaa aacaacctaa 1320

1380

ggaagtctca aaatggaagg ttatactcta taaaatcaac caaaatgttg atccatccaa 1440

cagattcaga aagctttgaa tgaactgagg caaatttaaa aggcaataat ttaaacatta 1500

acctcattcc aagttaatgt ggtctaataa tctggtatta aatccttaag agaaagcttg 1560

agaaatagat tttttttc ttaaagtcac tgtctattta agattaaaca tacaatcaca 1620

taaccttaaa gaataccgtt tacatttctc aatcaaaatt cttataatac tatttgtttt 1680

aaattttgtg atgtgggaat caattttaga tggtcacaat ctagattata atcaataggt 1740

gaacttatta aataactttt ctaaataaaa aatttagaga cttttatttt aaaaggcatc 1800

atatgagcta atatcacaac tttcccagtt taaaaaacta gtactcttgt taaaactcta 1860

aacttgacta aatacagagg actggtaatt gtacagttct taaatgttgt agtattaatt 1920

tcaaaactaa aaatcgtcag cacagagtat gtgtaaaaat ctgtaataca aatttttaaa 1980

ctgatgcttc attttgctac 2040

aaacctaatg aagcagaatt aaatactgta ttaaaataag ttcgctgtct ttaaacaaat 2100

ggagatgact actaagtcac attgacttta acatgaggta tcactatacc ttatttgtta 2160

aaatatatac tgtatacatt ttatatattt taacacttaa tactatgaaa acaaataatt 2220

gtaaaggaat cttgtcagat tacagtaaga atgaacatat ttgtggcatc gagttaaagt 2280

ttatatttcc cctaaatatg ctgtgattct aatacattcg tgtaggtttt caagtagaaa 2340

taaacctcgt aacaagttac tgaacgttta aacagcctga caagcatgta tatatgttta 2400

aaattcaata aacaagacc cagtccctaa attatagaaa tttaaattat tcttgcatgt 2460

ttatcgacat cacaacagat ccctaaatcc ctaaatccct aaagattaga tacaaatttt 2520

ttaccacagt atcacttgtc agaatttatt tttaaatatg attttttaaa actgccagta 2580

agaaatttta aattaaaccc atttgttaaa ggatatagtg cccaagttat atggtgacct 2640

acctttgtca atacttagca ttatgtattt caaattatcc aatatacatg tcatatatat 2700

ttttatgt cacatatata aaagatatgt atgatctatg tgaatcctaa gtaaatattt 2760

tgttccagaa aagtacaaaa taataaaggt aaaaataatc tataattttc aggaccacag 2820

actaagctgt cgaaattaac gctgattttt ttagggccag aataccaaaa tggctcctct 2880

cttcccccaa aattggacaa tttcaaatgc aaaataattc attatttaat atatgagttg 2940

cttcctctat t 2951

<210> 2

<211> 460

<212> PROTEIN

<213> Homo sapiens

<400> 2

Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser

5 10 15

Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu

20 25 30

Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn

35 40 45

Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr

50 55 60

Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln

65 70 75 80

Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu

85 90 95

Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu

100 105 110

Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu

115 120 125

Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln

130 135 140

Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu

145 150 155 160

Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys

165 170 175

Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln

180 185 190

His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile

195 200 205

Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg

210 215 220

Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp

225 230 235 240

Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr

245 250 255

Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val

260 265 270

Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg

275 280 285

Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr

290 295 300

Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile

305 310 315 320

Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu

325 330 335

Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly

340 345 350

Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn

355 360 365

Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp

370 375 380

Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly

385 390 395 400

Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys

405 410 415

Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu

420 425 430

Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys

435 440 445

Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu

450 455 460

<210> 3

<211> 888

<212> DNA

<213> Homo sapiens

<400> 3

ataccttaga ccctcagtca tgccagtgcc tgctctgtgc ctgctctggg ccctggcaat 60

ggtgacccgg cctgcctcag cggcccccat gggcggccca gaactggcac agcatgagga 120

gctgaccctg ctcttccatg ggaccctgca gctgggccag gccctcaacg gtgtgtacag 180

gaccacggag ggacggctga caaaggccag gaacagcctg ggtctctatg gccgcacaat 240

agaactcctg gggcaggagg tcagccgggg ccgggatgca gccggaac ttcgggcaag 300

cctgttggag actcagatgg aggaggatat tctgcagctg caggcagagg ccacagctga 360

ggtgctgggg gaggtggccc aggcacagaa ggtgctacgg gacagcgtgc agcggctaga 420

agtccagctg aggagcgcct ggctgggccc tgcctaccga gaatttgagg tcttaaaggc 480

tcacgctgac aagcagagcc acatcctatg ggccctcaca ggccacgtgc agcggcagag 540

600

ggcgctccca gcctgaatct gcctggatgg aactgaggac caatcatgct gcaaggaaca 660

cttccacgcc ccgtgaggcc cctgtgcagg gaggagctgc ctgttcactg ggatcagcca 720

gggcgccggg ccccacttct gagcacagag cagagacaga cgcaggcggg gacaaaggca 780

gaggatgtag ccccattggg gaggggtgga ggaaggacat gtaccctttc atgcctacac 840

acccctcatt aaagcagagt cgtggcatct caaaaaaaaa aaaaaaaa 888

<210> 4

<211> 198

<212> PROTEIN

<213> Homo sapiens

<400> 4

Met Pro Val Pro Ala Leu Cys Leu Leu Trp Ala Leu Ala Met Val Thr

5 10 15

Arg Pro Ala Ser Ala Ala Pro Met Gly Gly Pro Glu Leu Ala Gln His

20 25 30

Glu Glu Leu Thr Leu Leu Phe His Gly Thr Leu Gln Leu Gly Gln Ala

35 40 45

Leu Asn Gly Val Tyr Arg Thr Thr Glu Gly Arg Leu Thr Lys Ala Arg

50 55 60

Asn Ser Leu Gly Leu Tyr Gly Arg Thr Ile Glu Leu Leu Gly Gln Glu

65 70 75 80

Val Ser Arg Gly Arg Asp Ala Ala Gln Glu Leu Arg Ala Ser Leu Leu

85 90 95

Glu Thr Gln Met Glu Glu Asp Ile Leu Gln Leu Gln Ala Glu Ala Thr

100 105 110

Ala Glu Val Leu Gly Glu Val Ala Gln Ala Gln Lys Val Leu Arg Asp

115 120 125

Ser Val Gln Arg Leu Glu Val Gln Leu Arg Ser Ala Trp Leu Gly Pro

130 135 140

Ala Tyr Arg Glu Phe Glu Val Leu Lys Ala His Ala Asp Lys Gln Ser

145 150 155 160

His Ile Leu Trp Ala Leu Thr Gly His Val Gln Arg Gln Arg Arg Glu

165 170 175

Met Val Ala Gln Gln His Arg Leu Arg Gln Ile Gln Glu Arg Leu His

180 185 190

Thr Ala Ala Leu Pro Ala

195

<210> 5

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 5

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Met Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 6

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Ile Ser

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Tyr Arg Asn Asp Asp Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Leu Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Tyr Ser Ser Gly Trp Tyr Gly Asn Trp Phe Asp Pro

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

210 215 220

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440 445

gly

<210> 7

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 7

gatattgtga tgacccagac ccccctgtct ctgcctgtca ctccggggga accggcctcg 60

atctcatgcc ggtcgagcca gtccctgctg gactccgatg acgggaacac ttatttggat 120

tggtacctcc aaaagcctgg acagagcccg cagctcctga tctacatgct gtcctaccgg 180

gcctccggag tgccagaccg cttctcggga agcggctccg gtaccgactt cacactgaag 240

atctcccgcg tggaagctga ggacgtgggc atctactact gtatgcaaag aatcgagttc 300

cccctcacct tcggcggcgg gactaaggtc gagattaaga gaaccgtggc cgcaccatcc 360

gtgttcattt ttcccccgtc cgatgaacag ctgaagtccg gaaccgcctc cgtcgtgtgc 420

ctgctcaaca acttctaccc gagggaagcg aaagtgcagt ggaaagtgga caatgcgctg 480

cagtccggaa actcccaaga gtccgtgacc gaacaggact ccaaggactc aacctactcg 540

ctgagctcaa cgctgaccct gagcaaggcc gactacgaga agcacaaggt ctacgcctgc 600

gaagtgaccc atcagggttt gagctcgccc gtgaccaagt ccttcaaccg gggagagtgc 660

<210> 8

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

caagtcacat tgaaggagag cggtccgacc ctggtcaagc cgactcagac cctgaccctg 60

acgtgcactt tctcgggctt ctcattgtcc atttctggag tgggcgtggg atggatcaga 120

cagcccccgg ggaaggccct cgagtggctc gcgctgatct accgcaacga cgacaaacgc 180

tactccccct cactgaaatc ccggctgacc atcactaagg atacgtccaa gaaccaggtc 240

gtgttgaccc tcaccaacat ggatcccgtg gatactgcca cctactattg tgcacggacc 300

tatagcagcg gttggtacgg aaactggttc gacccgtggg gccagggaac tcttgtgacg 360

gtgtcctccg caagcaccaa gggtccttct gtgttccccc tggcgccgtg ctcgcggagc 420

acctcagagt ccaccgccgc cctcggctgc cttgtgaagg actacttccc ggagccagtc 480

accgtgtcct ggaacagcgg ggccctgact tccggcgtgc acaccttccc tgcggtgctg 540

cagagctcag gcctctattc gctgtcatcc gtcgtgaccg tgccttcctc gtccctgggc 600

actaagacct acacttgcaa cgtggaccat aagcccagca acaccaaagt ggacaagaga 660

gtggaatcca aatacggacc gccatgtccg ccctgccccg ccccggaagc tgccggggga 720

cccagcgtgt tcctgttccc acctaagccg aaggacactc tgatgatctc aaggactccc 780

gaagtcactt gcgtggtcgt ggacgtgtcc caggaggacc ccgaagtcca gtttaattgg 840

tacgtggatg gtgtcgaggt ccacaacgcc aagaccaagc ctcgcgagga acagttcaat 900

tccacctacc gggtcgtgtc cgtcctgacc gtgctgcatc aggactggct gaacggaaag 960

gagtacaagt gcaaagtgtc caacaaggga ctcccttcct ccatcgaaaa gaccatcagc 1020

aaggccaagg gccagcctcg cgaaccacaa gtctacaccc tgcccccatc gcaagaggaa 1080

atgaccaaga accaagtgtc gctgacatgc ctcgtcaagg gattctaccc gtcggatatt 1140

gcggtggaat gggagtccaa cggacagccc gagaacaact acaagaccac cccgccggtg 1200

ttggactccg acggctcctt tttcctgtac tcccggctca ctgtggacaa gtcgcggtgg 1260

caggagggga acgtgttctc ctgttccgtg atgcacgaag ctctgcacaa ccactacacc 1320

cagaagtcgc tgagcctctc actggga 1347

<210> 9

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 9

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Met Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 10

<211> 123

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Ile Ser

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Tyr Arg Asn Asp Asp Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Leu Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Thr Tyr Ser Ser Gly Trp Tyr Gly Asn Trp Phe Asp Pro

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu

5 10 15

asp

<210> 12

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

Tyr Met Leu Ser Tyr Arg Ala Ser

5

<210> 13

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 13

Met Gln Arg Ile Glu Phe Pro Leu Thr

5

<210> 14

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Thr Phe Ser Gly Phe Ser Leu Ser Ile Ser Gly Val Gly Val Gly

5 10 15

<210> 15

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

Leu Ile Tyr Arg Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu Lys Ser

5 10 15

<210> 16

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

Ala Arg Thr Tyr Ser Ser Gly Trp Tyr Gly Asn Trp Phe Asp Pro

5 10 15

<210> 17

<211> 471

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser

5 10 15

Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu

20 25 30

Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn

35 40 45

Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr

50 55 60

Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln

65 70 75 80

Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu

85 90 95

Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu

100 105 110

Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu

115 120 125

Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln

130 135 140

Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu

145 150 155 160

Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys

165 170 175

Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln

180 185 190

His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile

195 200 205

Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg

210 215 220

Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp

225 230 235 240

Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr

245 250 255

Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val

260 265 270

Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg

275 280 285

Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr

290 295 300

Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile

305 310 315 320

Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu

325 330 335

Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly

340 345 350

Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn

355 360 365

Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp

370 375 380

Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly

385 390 395 400

Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys

405 410 415

Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu

420 425 430

Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys

435 440 445

Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu Ala Ala Ala Asp

450 455 460

Tyr Lys Asp Asp Asp Asp Lys

465 470

<210> 18

<211> 820

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

5 10 15

Gly Ser Thr Gly Asp His His His His His His Asp Ala His Lys Ser

20 25 30

Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala

35 40 45

Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu

50 55 60

Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys

65 70 75 80

Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu

85 90 95

Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly

100 105 110

Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys

115 120 125

Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg

130 135 140

Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr

145 150 155 160

Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe

165 170 175

Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe

180 185 190

Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys

195 200 205

Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg

210 215 220

Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala

225 230 235 240

Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala

245 250 255

Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys

260 265 270

Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala

275 280 285

Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu

290 295 300

Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val

305 310 315 320

Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe

325 330 335

Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val

340 345 350

Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr

355 360 365

Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu

370 375 380

Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val

385 390 395 400

Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys

405 410 415

Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn

420 425 430

Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro

435 440 445

Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys

450 455 460

Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu

465 470 475 480

Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val

485 490 495

Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg

500 505 510

Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu

515 520 525

Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser

530 535 540

Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val

545 550 555 560

Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp

565 570 575

Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu

580 585 590

Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala

595 600 605

Ala Leu Gly Leu Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro

610 615 620

Ala Pro Ala Pro Ala Pro Ala Pro Leu Glu Val Leu Phe Gln Gly Pro

625 630 635 640

Gly Arg Ala Ala Pro Met Gly Gly Pro Glu Leu Ala Gln His Glu Glu

645 650 655

Leu Thr Leu Leu Phe His Gly Thr Leu Gln Leu Gly Gln Ala Leu Asn

660 665 670

Gly Val Tyr Arg Thr Thr Glu Gly Arg Leu Thr Lys Ala Arg Asn Ser

675 680 685

Leu Gly Leu Tyr Gly Arg Thr Ile Glu Leu Leu Gly Gln Glu Val Ser

690 695 700

Arg Gly Arg Asp Ala Ala Gln Glu Leu Arg Ala Ser Leu Leu Glu Thr

705 710 715 720

Gln Met Glu Glu Asp Ile Leu Gln Leu Gln Ala Glu Ala Thr Ala Glu

725 730 735

Val Leu Gly Glu Val Ala Gln Ala Gln Lys Val Leu Arg Asp Ser Val

740 745 750

Gln Arg Leu Glu Val Gln Leu Arg Ser Ala Trp Leu Gly Pro Ala Tyr

755 760 765

Arg Glu Phe Glu Val Leu Lys Ala His Ala Asp Lys Gln Ser His Ile

770 775 780

Leu Trp Ala Leu Thr Gly His Val Gln Arg Gln Arg Arg Glu Met Val

785 790 795 800

Ala Gln Gln His Arg Leu Arg Gln Ile Gln Glu Arg Leu His Thr Ala

805 810 815

Ala Leu Pro Ala

820

<210> 19

<211> 455

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 19

Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu

5 10 15

Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn

20 25 30

Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr

35 40 45

Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln

50 55 60

Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu

65 70 75 80

Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu

85 90 95

Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu

100 105 110

Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln

115 120 125

Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu

130 135 140

Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys

145 150 155 160

Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln

165 170 175

His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile

180 185 190

Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg

195 200 205

Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp

210 215 220

Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr

225 230 235 240

Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val

245 250 255

Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg

260 265 270

Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr

275 280 285

Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile

290 295 300

Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu

305 310 315 320

Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly

325 330 335

Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn

340 345 350

Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp

355 360 365

Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly

370 375 380

Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys

385 390 395 400

Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu

405 410 415

Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys

420 425 430

Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu Ala Ala Ala Asp

435 440 445

Tyr Lys Asp Asp Asp Asp Lys

450 455

<210> 20

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

Gly Pro Gly Arg Ala Ala Pro Met Gly Gly Pro Glu Leu Ala Gln His

5 10 15

Glu Glu Leu Thr Leu Leu Phe His Gly Thr Leu Gln Leu Gly Gln Ala

20 25 30

Leu Asn Gly Val Tyr Arg Thr Thr Glu Gly Arg Leu Thr Lys Ala Arg

35 40 45

Asn Ser Leu Gly Leu Tyr Gly Arg Thr Ile Glu Leu Leu Gly Gln Glu

50 55 60

Val Ser Arg Gly Arg Asp Ala Ala Gln Glu Leu Arg Ala Ser Leu Leu

65 70 75 80

Glu Thr Gln Met Glu Glu Asp Ile Leu Gln Leu Gln Ala Glu Ala Thr

85 90 95

Ala Glu Val Leu Gly Glu Val Ala Gln Ala Gln Lys Val Leu Arg Asp

100 105 110

Ser Val Gln Arg Leu Glu Val Gln Leu Arg Ser Ala Trp Leu Gly Pro

115 120 125

Ala Tyr Arg Glu Phe Glu Val Leu Lys Ala His Ala Asp Lys Gln Ser

130 135 140

His Ile Leu Trp Ala Leu Thr Gly His Val Gln Arg Gln Arg Arg Glu

145 150 155 160

Met Val Ala Gln Gln His Arg Leu Arg Gln Ile Gln Glu Arg Leu His

165 170 175

Thr Ala Ala Leu Pro Ala

180

<210> 21

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Ser Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Met Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 22

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Ala Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Met Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 23

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Thr Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Met Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 24

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 25

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Met Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Gln Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<---

Claims (58)

1. An antibody that binds to the human ANGPTL3/8 complex, containing the complementarity-determining light chain regions, LCDR1, LCDR2, and LCDR3, and the complementarity-determining regions of the heavy chain, 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). 2. An antibody according to claim 1, containing a light chain variable region (LCVR), where LCVR has the amino acid sequence of SEQ ID NO: 9. 3. An antibody according to claim 1, containing a heavy chain variable region (HCVR), where HCVR has the amino acid sequence of SEQ ID NO: 10. 4. An antibody according to any one of claims 1-3, where LCVR has the amino acid sequence of SEQ ID NO: 9, and HCVR has the amino acid sequence of SEQ ID NO: 10. 5. An antibody according to any one of claims 1 to 4, wherein the antibody is an IgG4 isotype. 6. An antibody that binds to the human ANGPTL3/8 complex, containing a light chain (LC) and a heavy chain (HC), where LC contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 21, 22, 23, 24 and 25, and HC contains the amino acid sequence of SEQ ID NO: 6. 7. An antibody according to claim 6, where LC has the amino acid sequence of SEQ ID NO: 5, and HC has the amino acid sequence of SEQ ID NO: 6. 8. An antibody according to claim 6, where LC has the amino acid sequence of SEQ ID NO: 21, and HC has the amino acid sequence of SEQ ID NO: 6. 9. An antibody according to claim 6, where LC has the amino acid sequence of SEQ ID NO: 22, and HC has the amino acid sequence of SEQ ID NO: 6. 10. An antibody according to claim 6, where LC has the amino acid sequence of SEQ ID NO: 23, and HC has the amino acid sequence of SEQ ID NO: 6. 11. An antibody according to claim 6, where LC has the amino acid sequence of SEQ ID NO: 24, and HC has the amino acid sequence of SEQ ID NO: 6. 12. An antibody according to claim 6, where LC has the amino acid sequence of SEQ ID NO: 25, and HC has the amino acid sequence of SEQ ID NO: 6. 13. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes polypeptides containing the amino acid sequences of SEQ ID NOs: 5 and 6, under conditions such that the polypeptides are expressed, and isolating antibodies. 14. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes an antibody comprising the amino acid sequences of SEQ ID NOs: 21 and 6, under conditions such that the antibody is expressed, and isolating antibodies. 15. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes an antibody comprising the amino acid sequences of SEQ ID NOs: 22 and 6, under conditions such that the antibody is expressed, and isolating antibodies. 16. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes an antibody comprising the amino acid sequences of SEQ ID NOs: 23 and 6, under conditions such that the antibody is expressed, and isolating antibodies. 17. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes an antibody comprising the amino acid sequences of SEQ ID NOs: 24 and 6, under conditions such that the antibody is expressed, and isolating antibodies. 18. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing a cDNA molecule, wherein the cDNA molecule encodes an antibody comprising the amino acid sequences of SEQ ID NOs: 25 and 6, under conditions such that the antibody is expressed, and isolating antibodies. 19. An antibody that binds to the human ANGPTL3/8 complex, obtained by culturing a mammalian cell containing at least two cDNA molecules, where the first cDNA molecule encodes an antibody light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 21, 22, 23, 24 and 25, and the second cDNA molecule encodes an antibody heavy chain containing the amino acid sequence of SEQ ID NO: 6 under such conditions that the antibody is expressed and the antibody is isolated. 20. An antibody according to any one of claims 1-19, wherein said antibody binds and neutralizes the human ANGPTL3/8 complex in a standard lipoprotein lipase activity assay with an EC ₅₀ of 3.0 nM or less. 21. An antibody according to any one of claims 1-19, wherein the antibody binds to the human ANGPTL3/8 complex with a dissociation constant less than or equal to 1×10 ^-6 M. 22. An antibody according to any one of claims 1 to 19, wherein the antibody binds to human ANGPTL3 and human ANGPTL8 with a dissociation constant greater than 1 x 10 ^-6 M. 23. An antibody according to any one of claims 1 to 19, wherein said antibody exhibits positive binding to the human ANGPTL3/8 complex in a concentration dependent manner and does not exhibit detectable binding to human ANGPTL3 and human ANGPTL8 up to an antigen concentration of 100 nM. 24. An antibody according to any one of claims 1 to 19, wherein the antibody reduces triglyceride levels in vivo by at least 50% compared to an IgG control at a dose of 10 mg/kg 14 days post-dose. 25. A pharmaceutical composition for lowering triglyceride levels, comprising said antibody according to any one of claims 1 to 24 and an acceptable carrier, diluent or excipient. 26. A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides containing the amino acid sequences of SEQ ID NO: 5 and 6, where the cell is capable of expressing the antibody of claim 13. 27. A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides containing the amino acid sequences of SEQ ID NO: 21 and 6, where the cell is capable of expressing the antibody of claim 14. 28. A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides containing the amino acid sequences of SEQ ID NO: 22 and 6, where the cell is capable of expressing the antibody of claim 15. 29. A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides containing the amino acid sequences of SEQ ID NO: 23 and 6, where the cell is capable of expressing the antibody of claim 16. 30. A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides containing the amino acid sequences of SEQ ID NO: 24 and 6, where the cell is capable of expressing the antibody of claim 17. 31. A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding polypeptides containing the amino acid sequences of SEQ ID NO: 25 and 6, where the cell is capable of expressing the antibody of claim 18. 32. A method for producing an antibody, comprising culturing a mammalian cell according to claims 26 to 31 under conditions under which the antibody is expressed, and isolating the expressed antibody. 33. A method of treating a disease associated with lipid metabolism, selected from the group consisting of: atherosclerotic cardiovascular disease (ASCVD), diabetes, obesity, non-alcoholic steatohepatitis (NASH), chronic renal failure (CRF) or hypertriglyceridemia, or a combination thereof, including administering to a patient in need thereof an effective amount of said antibody according to any one of claims 1-24. 34. A method for lowering triglyceride levels, comprising administering to a patient an effective amount of said antibody according to any one of claims 1-24. 35. An antibody according to any one of claims 1 to 24 for use in the treatment of ASCVD, diabetes, obesity, NASH, CKD, or hypertriglyceridemia, or a combination thereof. 36. Pharmaceutical composition for use in the treatment of ASCVD, diabetes, obesity, NASH, CKD or hypertriglyceridemia, or a combination thereof, containing an effective amount of said antibody according to any one of claims 1-24.

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