Angiopoietin-like 4 antibody and method of use

Abstract

To provide: isolated antibodies or fragments thereof that bind to a human angiopoietin-like 4 protein (ANGPTL4); and pharmaceutical compositions containing the isolated antibodies and fragments.SOLUTION: There are provided: an isolated anti-ANGPTL4 antibody or fragment thereof, the isolated antibody or fragment blocking LPL binding to ANGPTL4 protein or binding to a human ANGPTL4 protein with a KD of less than or equal to 45 pM, as measured by ForteBio kinetic binding assays, or less than or equal to 24 pM, as measured by solution equilibrium titration assay (SET); and a pharmaceutical composition containing the isolated antibody and fragment.SELECTED DRAWING: None

Description

[Technical field]

[0001] 2. Background of the Invention Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that acts as an angiopoietin receptor. A member of the poietin-like family. ANGPTL4 forms dimers and tetramers. It is a homo-oligomeric protein capable of binding to macrophages, fat, muscle, and ANGPT is a homo-oligomeric protein expressed by various cell types including liver cells and hepatocytes. L4 also expresses hepatic fibrinogen / angiopoietin-related protein (HFARP) ( Kim et al. (2000) Biochem. J. 346:603-610), PPAR gamma-angiopoietin-related Protein GAR (Yoon, et al. (2000) Mol. Cell Biol., 20:5343-5349), and and fasting-induced adipocyte factor (FIAF) (Kerten et al. (2000) J. Biol. Chem., 275 ANGPTL4 is also known as ANGPTL4-1, which is a ubiquitous agonist that binds to the N-terminal coiled-coil domain. It contains a fibrinogen-like domain at the C-terminus and a fibrinogen (FBN)-like domain at the C-terminus (Kim et al. (2000 ) Biochem. J. 346:603-610).

[0002] Lipoprotein lipase (LPL) is involved in maintaining the levels of lipoproteins in the blood. It plays a central role in lipoprotein metabolism through tissue-specific regulation of the activity of . The coiled-coil region of ANGPTL4 mediates lipoprotein lipase (LPL)-mediated It is known that AN inhibits triglyceride (TG) clearance. Loss-of-function mutations in ANGPTL4 (e.g., as seen in human subjects), gene deletions ( e.g., as seen in transgenic mice), and inhibition by antibodies (e.g., as seen in mice and cynomolgus monkeys) have all been observed to decrease plasma triglycerides. Furthermore, ANGPTL4 antibodies are also known to activate LPL. Conversely, injection of ANGPTL4 into mice results in a rapid increase in circulating triglycerides, at a rate greater than that seen when injecting angiopoietin-like protein 3 (ANGPTL3 ) (Yoshida et al. (2002) J Lipid Res 43:1770-1772).

[0003] The anti-ANGPTL4 antibodies and antigen-binding fragments described in the present invention block, for example, the inhibition of LPL by ANGPTL4, thereby decreasing plasma triglycerides and initiating, promoting, or enhancing the activation of LPL. These antibodies are expected to prevent and improve acute and chronic symptoms of diseases characterized by elevated triglyceride levels, such as primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, type II diabetes, etc. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0004] SUMMARY OF THE INVENTION The present invention relates to monoclonal antibodies that bind to human angiopoietin-like 4 protein (hereinafter, sometimes referred to as "ANGPTL4"), as well as pharmaceutical compositions and treatment methods comprising the same.

[0005] The isolated anti-ANGPTL4 antibodies or antigen-binding fragments described herein bind to ANGPTL4 with an equilibrium dissociation constant (K D ) of 100 pM or less. For example, the isolated antibodies or antigen-binding fragments described herein bind to human ANGPTL4 with a K of 150 nM or less, 50 nM or less, 10 nM or less, 750 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, 100 pM or less, 75 pM or less, 65 pM or less, 60 pM or less, 55 p M or less. More specifically, the isolated antibodies or antigen-binding fragments described herein also bind to human ANGPTL4 with a K D of 45 pM or less as determined by the ForteBio kinetic binding assay, or a K of 24 pM or less as determined by solution equilibrium titration assay (SET), and also bind to cynomolgus ANGPTL4 with a K of 87 pM or less as determined by the ForteBio kinetic binding assay, or a K D of 22 pM or less as determined by SET. D D D

[0006] The present invention relates to isolated antibodies or antigen-binding fragments thereof that bind to human ANGPTL4. The present invention also relates to isolated antibodies or antigen-binding fragments thereof that bind to ANGPTL4 and further compete with the antibodies listed in Table 1 for binding. The present invention also further relates to isolated antibodies or antigen-binding fragments thereof that bind to the same epitope as the antibodies listed in Table 1.

[0007] The binding affinity of the isolated antibodies and antigen-binding fragments described herein is determined by solution equilibrium ​​​​​​​​​​​It can be determined by equilibrium titration (SET). In the art, the SET method is known and will be described in more detail below. Alternatively, the binding affinity of the isolated antibodies or fragments described herein can also be determined by a Biacore assay. In the art, the Biacore reaction rate assay method is known and will be described in more detail below. Using the isolated anti-ANGPTL4 antibodies and antigen-binding fragments described herein, the binding of ANGPTL4 to lipoprotein lipase (LPL) can be inhibited at an EC50 of 100 nM or less, 50 nM or less, 35 nM or less, 25 nM or less, 10 nM or less, or 3 nM or less. Using the isolated anti-ANGPTL4 antibody or its antigen-binding fragment, the circulating triglyceride (TG) level can be reduced. The isolated anti-ANGPTL4 antibodies or antigen-binding fragments described herein can be monoclonal antibodies, human antibodies or humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fv fragments, F(ab’)2 fragments, or scFv fragments, and / or IgG isotypes. The isolated anti-ANGPTL4 antibodies or antigen-binding fragments described herein can also include frameworks in which the amino acids have been replaced with those of an antibody framework derived from each of the human VH germline sequence or the human VL germline sequence.

[0008] It can be determined by equilibrium titration (SET). In the art, the SET method is known and will be described in more detail below. Alternatively, the binding affinity of the isolated antibodies or fragments described herein can also be determined by a Biacore assay. In the art, the Biacore reaction rate assay method is known and will be described in more detail below. 00 nM or less, 50 nM or less, 35 nM or less, 25 nM or less, 10 nM or less, or 3 nM M or less of EC 50 to inhibit the binding of ANGPTL4 to lipoprotein lipase (LPL). It can be inhibited.

[0009] Using the isolated anti-ANGPTL4 antibody or its antigen-binding fragment, the circulating triglyceride (TG) level can be reduced.

[0010] The isolated anti-ANGPTL4 antibodies or antigen-binding fragments described herein can be monoclonal antibodies, human antibodies or humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, F v fragments, F(ab’)2 fragments, or scFv fragments, and / or IgG isotypes and can be. It can be.

[0011] The isolated anti-ANGPTL4 antibodies or antigen-binding fragments described herein can also include frameworks in which the amino acids have been replaced with those of an antibody framework derived from each of the human VH germline sequence or the human VL germline sequence. It can also include.

[0012] Another aspect of the present invention relates to an isolated antibody or an antigen-binding fragment thereof having the complete heavy and light chain sequences of the humanized antibodies set forth in Table 1. More specifically, the isolated antibody or its antigen-binding fragment may have the heavy and light chain sequences of NEG276, NEG276-LALA, NEG278, NEG310 NEG313, NEG315, NEG318, NEG319.

[0013] A further aspect of the present invention relates to an isolated antibody or an antigen-binding fragment thereof having the heavy and light chain variable domain sequences of the humanized antibodies set forth in Table 1. More specifically, the isolated antibody or its antigen-binding fragment may have the heavy and light chain variable domain sequences of NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, NEG319.

[0014] The present invention also relates to an isolated antibody or an antigen-binding fragment thereof that binds to human ANGPTL4 and comprises a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112, and 132, a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133, and a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 9, 34, 54, 74 94, 114, and 134. In another aspect, such an isolated antibody or an antigen-binding fragment thereof may comprise a light chain CDR1 selected from the group consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142, a light chain CDR2 selected from the group consisting of SEQ ID NOs: 18, 43 63, 83, 103, 123, and 143, and a light chain CDR3 selected from the group consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144. further comprising a light chain CDR3 selected therefrom.

[0015] The present invention also provides a light chain CDR1 selected from the group consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142, a light chain CDR2 selected from the group consisting of SEQ ID NOs: 18, 43, 63, 83, 103, 123, and 143, and a light chain CDR3 selected from the group consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144, and relates to an isolated antibody or an antigen-binding fragment thereof that binds to human ANGPTL4.

[0016] The present invention also provides an isolated antibody or an antigen-binding fragment thereof that binds to ANGPTL4, having HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 7, 8, and 9, and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 17, 18, and 19; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 32, 3 3, and 34, and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 42, 43, and 44; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 5 2, 53, and 54, and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 62, 63, and 64; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 5 2, 53, and 54, and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 62, 63, and 64; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 72, 73, and 74, and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 82, 83, and 84; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 92, 93, and 94, and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: ​​comprise column numbers 102, 103, and 104; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 112, 113, and 114, and LCDR1, LCDR2 , LCDR3 comprise SEQ ID NOs: 122, 123, and 124; or HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 132, 133, and 134, and LC DR1, LCDR2, LCDR3 comprise SEQ ID NOs: 142, 143, and 144, a single relates to an isolated antibody or an antigen-binding fragment thereof.

[0017] The present invention also relates to antibodies or antigen-binding fragments having HCDR1, HCDR2, and HCDR3 of the heavy chain variable domains of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138 defined by Chothia, and LCDR1, LCDR2, and LCDR3 of the light chain variable domains of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148. In another aspect of the present invention, the antibody or antigen-binding fragment may have HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain sequences of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138 defined by Kabat, and LCDR1,

[0018] In one aspect of the present invention, an isolated antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NOs: is capable, in which case the heavy chain variable domain and the light chain variable domain combine to form an antigen-binding site for AN GPTL4. In particular, the light chain variable domain sequence can be selected from SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148, in which case the isolated antibody or antigen-binding fragment thereof binds to ANGPTL4.

[0019] The present invention also relates to an isolated antibody or antigen-binding fragment thereof that contains a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148 and binds to ANGPTL4. The isolated antibody or antigen-binding fragment can further contain a heavy chain variable domain sequence, in which case the light chain variable domain and the heavy chain variable domain combine to form an antigen-binding site for ANGPTL4. In particular, the isolated antibody or antigen-binding fragment thereof that binds to ANGPTL4 can have heavy and light chain variable domains that contain the sequences of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148, respectively.

[0020] The present invention further relates to an isolated antibody or antigen-binding fragment thereof that contains a heavy chain variable domain having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138, in which case the antibody binds to ANGPTL4. In one aspect, the isolated antibody or antigen-binding fragment thereof also has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148.

[0021] The present invention further relates to an isolated antibody or antigen-binding fragment thereof that contains a heavy chain variable domain having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138, in which case the antibody binds to ANGPTL4. In one aspect, the isolated antibody or antigen-binding fragment thereof also has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148, in which case the antibody binds to ANGPTL4. In one aspect, the isolated antibody or antigen-binding fragment thereof also has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: ​​also includes a light chain variable domain having at least 90% sequence identity. In a further aspect of the invention , the isolated antibody or antigen-binding fragment has HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined by Kabat and set forth in Table 1 .

[0022] The invention also relates to an isolated antibody or antigen-binding fragment thereof having a light chain variable domain having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148, wherein the antibody binds to ANGPT L4 .

[0023] In another aspect of the invention, an isolated antibody or antigen-binding fragment thereof that binds to ANGPTL4 may have a heavy chain comprising the sequences of SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, and 140. The isolated antibody may also include a light chain that, in combination with the heavy chain, can form an antigen-binding site for human ANGPTL4. In particular, the light chain may have a sequence comprising SEQ ID NOs: 25, 50, 7 0, 90, 110, 130, and 150. In particular, an isolated antibody or antigen-binding fragment thereof that binds to ANGPTL4 may have a heavy chain and a light chain comprising the respective sequences of SEQ ID NOs: 15 and 25; 28 and 25; 40 and 50; 60 and 70; 80 and 90; 100 and 110; 120 and 130; or 140 and 150 .

[0024] The invention still further relates to a heavy chain having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, and 140 . ​​​​​Also related to an isolated antibody or an antigen-binding fragment thereof, wherein the antibody binds to ANGPTL 4. In one aspect, the isolated antibody or an antigen-binding fragment thereof also has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150 and includes a light chain.

[0025] The present invention further relates to an isolated antibody or an antigen-binding fragment thereof that includes a light chain having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 15 0, wherein the antibody binds to ANGPTL4.

[0026] The present invention further relates to an isolated antibody or an antigen-binding fragment thereof that competes for binding with the antibodies or antigen-binding fragments described herein, e.g., the humanized antibodies NEG276, NEG276-LALA, NEG278, N EG310, NEG313, NEG315, NEG318, and NEG319. In one embodiment, when two antibodies or antigen-binding fragments are present at equimolar concentrations, the isolated antibody or antigen-binding fragment of the present invention can inhibit the binding of AN GPTL4 by a humanized antibody selected from NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, and NEG319 by more than 50%. In another embodiment, when two antibodies or antigen-binding fragments are present at equimolar concentrations, the isolated antibody or antigen-binding fragment of the present invention is NEG276, NEG276-LALA, N

[0027] EG278, NEG310, NEG313, NEG315, NEG318, and NEG can inhibit the binding of ANGPTL4 by a humanized antibody selected from 319 by more than 80%. In yet other embodiments, when two antibodies or antigen-binding fragments are present at equimolar concentrations, the isolated antibody or antigen-binding fragment of the invention inhibits the binding of ANGPTL4 by a humanized antibody selected from NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, and NEG319 by more than 85% (or 90%, 95%, 98%, or 99%).

[0028] The invention also relates to a composition comprising the isolated antibody or antigen-binding fragment thereof described herein. The invention similarly relates to an antibody composition combined with a pharmaceutically acceptable carrier. Specifically, the invention further includes, for example, a pharmaceutical composition comprising an antibody of Table 1, such as NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, NEG319, or an antigen-binding fragment thereof. The invention also relates to a pharmaceutical composition comprising a combination of two or more of the isolated antibodies or antigen-binding fragments thereof of Table 1.

[0029] The invention also relates to an isolated nucleic acid sequence encoding a heavy chain variable domain having a sequence selected from SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138. In particular, the nucleic acid has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 14, 27, 39, 59, 79, 99, 119, and 139. % sequence identity) with a sequence selected from the group consisting of SEQ ID NOs: 14, 27, 39, 59, 79, 99, 119, and 139.​​​​​​​​​​​ In a further aspect of the invention, the sequence has SEQ ID NO: 14, 27, 39, 59, 79, 99, 119, or 139.

[0030] The present invention also relates to SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148. The present invention also relates to an isolated nucleic acid sequence encoding a light chain variable domain having a selected sequence. The nucleic acids consist of SEQ ID NOs: 24, 31, 49, 69, 89, 109, 129, and 149. A sequence at least 90% identical to a sequence selected from the group consisting of In a further embodiment of the invention, the sequence has SEQ ID NO: 24. , 31, 49, 69, 89, 109, 129, or 149.

[0031] The present invention also relates to SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148. A light chain variable domain having at least 90% sequence identity to a sequence selected from the group consisting of: The present invention also relates to an isolated nucleic acid comprising a sequence encoding a polypeptide comprising the

[0032] The present invention also relates to vectors comprising one or more of the nucleic acid molecules described herein. Also relates to.

[0033] The present invention also relates to a recombinant DNA sequence encoding the heavy chain of the above-described antibody, and and a second recombinant DNA sequence encoding the light chain of the antibody, In this case, the DNA sequence is operably linked to a promoter and is expressed in the host cell. It is contemplated that the antibody may be a humanized antibody. It is also contemplated that the cell may be a non-human mammalian cell.

[0034] The cells are intended to be human cells. The cells are further intended to be present in a subject. In one embodiment, the cells are intended to be endothelial cells. In other embodiments, the cells can be one or more of adipose, muscle, and liver cells. The subject is still further intended to be human. The present invention also relates to a method of treating, ameliorating, or preventing an ANGPTL4-related disorder in a patient, the method comprising administering to the patient a composition comprising an effective amount of an antibody or an antigen-binding fragment thereof described herein. In one aspect, the ANGPTL4-related disorder is associated with hypertriglyceridemia (e.g., severe hypertriglyceridemia (e.g., plasma triglyceride concentration > 500 mg / dL), hypertriglyceridemia associated with obesity, and type V hypertriglyceridemia). In other aspects, the ANGPTL4-related disorder is associated with primary lipid abnormalities, metabolic syndrome, and type II diabetes. The patient is also intended to be human. Any of the aforementioned isolated antibodies or their antigen-binding fragments can be monoclonal antibodies or their antigen-binding fragments.

[0035]

[0036]

[0037] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention pertains.

[0038] "ANGPTL4 protein" or "ANGPTL4 antigen" or "ANGPTL4" The term "」 is used interchangeably and refers to angiopoietin-like 4 (ANGP TL4) proteins in different species. For example, human ANGPTL4 has the sequence shown in Table 1 (SEQ ID NO: 1) and is described in previous reports and literature (Nature, Vol. 386, p. 73-77, 1997; Genomics , Vol. 54, No. 2, p. 191-199, 1998; Biochem. J., Vol. 339, Part 1, P. 177-184, 1 999; Genbank accession number: NP002534). ANGP TL4 contains an N-terminal coiled-coil domain and a C-terminal fibrinogen (FBN)-like domain (Kim et al. (2000) Biochem. J. 346:603-610). ANGPTL 4 is a homo-oligomeric protein capable of forming dimers and tetramers, expressed by cell types including macrophages, adipose, muscle, and hepatocytes, and is a homo-oligomeric protein known to inhibit triglyceride (TG) clearance mediated by lipoprotein lipase (LPL). In addition, in the context of the present invention, the term "ANGPTL4" includes mutants of the natural angiopoietin-like 4 (ANGPTL4) protein that have an amino acid sequence substantially the same as the amino acid sequence of the natural primary structure (amino acid sequence) described in the above reports. As used herein, the term "mutant of the natural human angiopoietin-like 4 (ANGPTL4) protein having a substantially the same amino acid sequence" refers to such mutant proteins.

[0039]

[0040] ​​​​​​As used herein, the term "antibody" means an intact antibody and any antigen-binding fragment (i.e., an "antigen-binding portion") or single chains thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and each VL comprises three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with an antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to factors including host tissues or various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). That is, an "antigen-binding portion"). An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and each VL comprises three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with an antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to factors including host tissues or various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). can effect. As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., human oxidized LDL receptor (ANGPTL4)). The antigen-binding function of an antibody can be effected by fragments of the intact antibody. The antigen-binding portion or antigen-binding fragment of an antibody host tissues or various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). can effect.

[0041] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., human oxidized LDL receptor (ANGPTL4)). The antigen-binding function of an antibody can be effected by fragments of the intact antibody. The antigen-binding portion or antigen-binding fragment of an antibody host tissues or various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). host tissues or various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). host tissues or various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Examples of binding fragments included within the term "original binding fragment" are Fab fragments, VL domains, monovalent fragments consisting of VH domains, CL domains, and CH1 domains; divalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region, the F(ab )2 fragment; the Fd fragment consisting of the VH domain and the CH1 domain; the Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; single domain antibody (dAb) fragments consisting of a VH domain or a VL domain (Ward et al., 1989 Nature 341:544-546); and isolated complementarity determining regions (CDRs). Furthermore, the two domains of the Fv fragment, the VL domain and the VH domain, are encoded by separate genes, but can be attached by an artificial peptide linker that allows them to be made as a single protein chain using recombinant methods, in which case

[0042] the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv), see, for example, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Pro c. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies contain one or more antigen-binding portions or antigen-binding fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are also screened for utility in the same form as intact antibodies.

[0043] Antigen-binding fragments also include single domain antibodies, maxibodies, minibodies, intrabodies,

[0043] ​I, diabody, triabody, tetrabody, v-NAR, and bis-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of the antibody can also be grafted onto a scaffold based on a polypeptide such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). The antigen-binding fragment can be incorporated into a single-chain molecule that forms a pair of antigen-binding regions together with a complementary light-chain polypeptide, and includes a pair of tandem Fv segments (VH-CH1-VH-CH1) (Zapata et al., 1995 Protein Eng. 8(10):1057-1062; and U.S. Patent No. 5,641,870). The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites via weak non-covalent forces, and the greater the interaction, the stronger the affinity. The term "high affinity" as used herein for an antibody or its antigen-binding fragment (e.g., Fab

[0044] fragment) generally refers to an antibody or antigen-binding fragment with a KD of 10 M or less. (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of the antibody can also be grafted onto a scaffold based on a polypeptide such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0045] The term "amino acid" as used herein includes naturally occurring and synthetic amino acids, as well as naturally occurring amino acid derivatives. (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of the antibody can also be grafted onto a scaffold based on a polypeptide such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). The term "high affinity" as used herein for an antibody or its antigen-binding fragment (e.g., Fab fragment) generally refers to an antibody or antigen-binding fragment with a KD of 10 -9 M or less.

[0046] (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of the antibody can also be grafted onto a scaffold based on a polypeptide such as type III fibronectin Also refers to amino acid analogs and amino acid mimetics that function in a similar manner to the amino acids. Naturally occurring amino acids include, in addition to the amino acids encoded by the genetic code, amino acids that are modified later, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, namely, hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and compounds having an alpha carbon bonded to methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds having a structure different from the general chemical structure of amino acids but that function in a similar manner to naturally occurring amino acids.

[0047] As used herein, the term "binding specificity" refers to the ability of an individual antigen-binding site to react with only one antigenic determinant.

[0048] The phrase "specifically (or selectively) binds to" an antibody (e.g., an ANGPTL4-binding antibody) refers to a binding reaction that determines the presence of a cognate antigen (e.g., human ANGPTL4 or cynomolgus ANGPTL4) within a heterogeneous population of proteins and other biological substances. As used herein, the phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen."

[0049] The term "mediated by ANGPTL4" means that ANGPTL4 is a lipoprotein Inhibits triglyceride (TG) clearance mediated by lipoprotein lipase (LPL), thereby referring to the fact that it is known to increase triglyceride levels.

[0050] As used herein, "ANGPTL4-related disorder", "ANGPTL4-related condition", or similar terms refer to any number of conditions or diseases in which a reduction of ANGPTL4-mediated LPL inhibition and lipoprotein modulation is sought. These conditions are those involved in lipid metabolism such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including atherosclerotic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia (e.g., severe hypertriglyceridemia (e.g., plasma triglyceride concentration > 500 mg / dL), hypertriglyceridemia associated with obesity, and type V hypertriglyceridemia), hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipodystrophy, lipoatrophy, as well as, for example, decreased LPL activity and / or LPL deficiency, decreased LDL receptor activity and / or LDL receptor deficiency, ApoC2 modification, ApoE deficiency, increased ApoB, increased production of very low density lipoprotein (VLDL) and / or decreased catabolism of VLDL, certain drug treatments (e.g., lipid abnormalities induced by glucocorticoid treatment), ) Conditions, including any other conditions caused by any genetic predisposition, diet, lifestyle, etc. including but not limited to.

[0051] Other ANGPTL4-related diseases or disorders associated with or resulting from hyperlipidemia, hyperlipoproteinemia, and / or dyslipidemia include, but are not limited to, atherosclerotic diseases such as atherosclerosis, aneurysms, hypertension, angina pectoris, stroke, cerebrovascular diseases, congestive heart failure, coronary artery diseases, myocardial infarction, peripheral vascular diseases, etc.; acute pancreatitis; non-alcoholic steatohepatitis (NASH); glycemic disorders such as diabetes; obesity, etc. The term "chimeric antibody" refers to an antibody molecule in which (a) the antigen-binding site (variable region) is modified, replaced, or exchanged by a constant region that is different in class, effector function, and / or species, or a constant region in which class, effector function, and / or species are modified, or a completely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., so as to link to it; or (b) the variable region or a part thereof is modified, replaced, or exchanged by a variable region with different antigen specificity or modified antigen specificity.

[0052] For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulin. Due to the replacement with the human constant region, the chimeric antibody can retain its specificity in antigen recognition while reducing its antigenicity in humans compared to the original mouse antibody.

[0053] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a conservatively modified variant is a nucleic acid that encodes the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, refers to an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, any of the codons GCA, GCC, GCG, and GCU encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be modified to any of the corresponding codons described without modifying the encoded polypeptide. Such nucleic acid variations are one type of conservatively modified variation, a "silent variation". Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon within a nucleic acid (except for the AUG codon, which is typically the only codon for methionine, and the TGG codon, which is typically the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence. In the case of polypeptide sequences, a "conservatively modified variant" includes individual substitutions, deletions, or additions to the polypeptide sequence that result in substitution of an amino acid with a chemically similar amino acid. In the art, tables of conserved substitutions presenting functionally similar amino acids are well known. Such conservatively modified variants are the polymorphic variants, interspecies homologs, interspecies analogs, and alleles of the invention. interspecies analogs, and alleles of the invention. For a particular nucleic acid sequence, a conservatively modified variant is a nucleic acid that encodes the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, refers to an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, any of the codons GCA, GCC, GCG, and GCU encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be modified to any of the corresponding codons described without modifying the encoded polypeptide. Such nucleic acid variations are one type of conservatively modified variation, a "silent variation". Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon within a nucleic acid (except for the AUG codon, which is typically the only codon for methionine, and the TGG codon, which is typically the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence. One of ordinary skill in the art will recognize that each codon within a nucleic acid (except for the AUG codon, which is typically the only codon for methionine, and the TGG codon, which is typically the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence. In the case of polypeptide sequences, a "conservatively modified variant" includes individual substitutions, deletions, or additions to the polypeptide sequence that result in substitution of an amino acid with a chemically similar amino acid.

[0054] In the art, tables of conserved substitutions presenting functionally similar amino acids are well known. Such conservatively modified variants are the polymorphic variants, interspecies homologs, interspecies analogs, and alleles of the invention. interspecies analogs, and alleles of the invention. is part of the same body and is added to alleles, and is not something that excludes them. The following eight groups: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V ); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) contain amino acids that are conservative substitutions for each other (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

[0055] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. An epitope usually consists of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually has specific charge characteristics in addition to specific three-dimensional structural features. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, but binding to the latter is not lost.

[0056] As used herein, the term "human antibody" is intended to include antibodies having a variable region in which both the framework region and the CDR region are derived from human-derived sequences. Further, if the antibody contains a constant region, the constant region is also such a human sequence, e.g., then, from the human germline sequence or a mutated version of the human germline sequence derived. The human antibodies of the present invention may contain amino acid residues not encoded by the human sequence (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro, or mutations introduced by somatic mutations in vivo).

[0057] A "humanized" antibody is, for example, a mouse monoclonal antibody that has low immunogenicity when administered as a therapeutic agent in humans, but retains the antigen-specific reactivity of the non-human antibody. See, for example, Robello et al., Transplantation, 68: 1417-1420. This can be achieved, for example, by retaining the non-human antigen-binding region and replacing the remaining part of the antibody with their human counterparts (i.e., the constant region and the parts not involved in the binding of the variable region). See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 8 1:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1989; Verhoeyen et al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of human engineering techniques include, but are not limited to, the Xoma technology disclosed in U.S. Patent No. 5,766,886.

[0058] The terms "identical" or "identity" percent in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences being the same. Using one of the following sequence comparison algorithms, or comparing for maximum correspondence over a comparison window or designated region measured by manual alignment and visual inspection, if the sequences are determined, the specified percentage of amino acid residues or nucleotides of the two sequences are the same (i.e., 60% identity over the designated region, or, if not designated, over the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity), the two sequences are "substantially identical". Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids), or more preferably over a region of 100 - 500, or 1000 nucleotides or more (or 20, 50, or 200 amino acids or more). In sequence comparison, one sequence typically serves as a reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, based on the program parameters, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence compared to the reference sequence.

[0059]

[0060] ​​​​​​​​​​​​As used herein, a "comparison region" refers to a segment of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 consecutive positions, any one of which can be used to compare an array, after optimal alignment of two arrays, with a reference array of the same number of consecutive positions. Array alignment methods for comparison are well known in the art. Optimal array alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)).

[0061] ​​​​​​​​​​​​​​​Algorithms suitable for determining percent sequence identity and percent sequence similarity Two examples of which are, respectively, Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, the BLAST algorithm and the BLAST 2.0 algorithm. Software for performing BLAST analyses is published by the National Center for Biotec hnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or satisfy a positive-valued threshold score T when aligning words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs that contain them. The word hits are extended in either direction along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using, for nucleotide sequences, parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, the scoring matrix is used to calculate the cumulative score. When the cumulative alignment score drops by an amount X from its achieved maximum; when the cumulative score drops below zero due to the cumulative of one or more negative-score residue alignments; or when either end of the sequence is reached score can increase. The cumulative score is calculated using, for nucleotide sequences, parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, the scoring matrix is used to calculate the cumulative score. When the cumulative alignment score drops by an amount X from its achieved maximum; when the cumulative score drops below zero due to the cumulative of one or more negative-score residue alignments; or when either end of the sequence is reached score can increase. The cumulative score is calculated using, for nucleotide sequences, parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, the scoring matrix is used to calculate the cumulative score. When the cumulative alignment score drops by an amount X from its achieved maximum; when the cumulative score drops below zero due to the cumulative of one or more negative-score residue alignments; or when either end of the sequence is reached score can increase. The cumulative score is calculated using, for nucleotide sequences, parameters M (reward score for pairs of matching residues; always >0) and N (penalty In this case, the expansion of word hits in each direction is stopped. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X, which are the BLA In the STN program (for nucleotide sequences), a word length (W) of 11, an expected value (E) of 10, M = 5, and N = -4 are used as defaults, and comparisons of both strands are performed. For the BLASTP program for amino acid sequences, a word length of 3, and an expected value (E) of 10 , and a BLOSUM62 scoring matrix of 50 (see Henikoff and Henikoff, P roc. Natl. Acad. Sci. USA 89:10915, 1989) alignment (B), an expected value (E) of 10 , M = 5, and N = -4 are used as defaults, and comparisons of both strands are performed.

[0062] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., see Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)) that produces an indicator of the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance. For example, if the minimum total probability when comparing a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001, the nucleic acid is considered to be similar to the reference sequence.

[0063] The percent identity between two amino acid sequences is also determined using the PAM120 weighted residue table, 1 ALIGN using a gap length penalty of 2 and a gap penalty of 4. The authors, E. Meyers and W. Miller (Comput. A ppl. Biosci., 4:11-17, 1988) can also be used to determine the In addition, the percent identity between the two amino acid sequences was calculated using the Blossom 62 matrix. PAM250 matrix and 16, 14, 12, 10, 8, 6, or uses a gap weight of 4 and length weights of 1, 2, 3, 4, 5, or 6. Within the GCG software package (available on the internet at gcg.com) The GAP program of Needleman and Wunsch (J. Mol. Biol. 48:444-4 53, 1970).

[0064] Other than the percentage of sequence identity mentioned above, the two nucleic acid sequences or polypeptides may have substantial Another indication of identity to the first nucleic acid is the sequence encoded by the first nucleic acid, as described below. The polypeptide immunologically binds to an antibody against a polypeptide encoded by a second nucleic acid. Thus, for example, if two peptides have only conservative substitutions, Thus, if different, the polypeptide is typically substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is the The ability of two molecules or their complements to hybridize to one another under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primer One advantage of this is that sequences can be amplified using

[0065] The term "isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to ANGPTL4 substantially does not contain antibodies that specifically bind to antigens other than ANGPTL4). However, an isolated antibody that specifically binds to ANGPTL4 may have cross-reactivity with other antigens. Furthermore, an isolated antibody may substantially not contain other intracellular substances and / or chemical substances. For example, an isolated antibody that specifically binds to ANGPTL4 substantially does not contain antibodies that specifically bind to antigens other than ANGPTL4. However, an isolated antibody that specifically binds to ANGPTL4 may have cross-reactivity with other antigens. Furthermore, an isolated antibody may substantially not contain other intracellular substances and / or chemical substances. For example, an isolated antibody that specifically binds to ANGPTL4 substantially does not contain antibodies that specifically bind to antigens other than ANGPTL4.

[0066] The term "isotype" refers to the antibody class (e.g., IgG such as IgM, IgE, IgG1 or IgG4) brought about by the heavy chain constant region gene. Isotype also includes a modified version of one of these classes, and the modification has been made to modify the Fc function, for example, to enhance or reduce effector function or binding to Fc receptors. For example, an isolated antibody that specifically binds to ANGPTL4 substantially does not contain antibodies that specifically bind to antigens other than ANGPTL4. Isotype also includes a modified version of one of these classes, and the modification has been made to modify the Fc function, for example, to enhance or reduce effector function or binding to Fc receptors. For example, an isolated antibody that specifically binds to ANGPTL4 substantially does not contain antibodies that specifically bind to antigens other than ANGPTL4. Isotype also includes a modified version of one of these classes, and the modification has been made to modify the Fc function, for example, to enhance or reduce effector function or binding to Fc receptors.

[0067] As used herein, the term "K assoc " or "K a " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K " or "K di s " or "K d " as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "K " as used herein is intended to refer to the dissociation constant obtained from the ratio of K D to K d (i.e., K a / K ), and is expressed as molar concentration (M). The K d of an antibody / K a The K DValues can be determined using methods well established in the art. The method for determining the K D of an antibody involves measuring surface plasmon resonance using a biosensor system such as the Biacore® system, or measuring the affinity in solution by solution equilibrium titration ( SET). This includes the step of measuring the affinity in solution by solution equilibrium titration (

[0068] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to preparations of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0069] As used herein, the term “nucleic acid” is used interchangeably with the term “polynucleotide” and refers to polymers in the form of deoxyribonucleotides or ribonucleotides and their single-stranded or double-stranded forms. The term “nucleic acid” includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic nucleic acids, naturally occurring nucleic acids, and non-naturally occurring nucleic acids that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly

[0070] phosphorothioates, phosphoramidates, methylphosphonates, chiral methyl

[0070] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly In particular, as detailed below, degenerate codon substitutions include: The third position of one or more selected (or all) codons may contain mixed bases and / or This can be achieved by creating a sequence in which the nucleotides are replaced with deoxyinosine or deoxyinosine residues. (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). .

[0071] The term "operably linked" refers to two or more polynucleotides (e.g., DNA A) refers to the functional relationship between the segments. The term "operably linked" refers to the functional relationship between the segments. Typically, the term refers to the functional relationship of a regulatory sequence to a sequence to be transcribed. The promoter or enhancer sequence may be selected so that it is suitable for use in an appropriate host cell or other expression system. operably linked to a coding sequence when the coding sequence stimulates or modulates transcription of the coding sequence in the Generally, a promoter transcriptional regulatory sequence operably linked to the sequence to be transcribed. The enhancer is physically adjacent to the transcribed sequence, i.e., it is cis-acting. Some transcriptional regulatory sequences, such as transcription factors, are physically attached to the coding sequences whose transcription they enhance. They do not have to be adjacent or closely spaced.

[0072] As used herein, the term "optimized" refers to a nucleotide sequence that is optimized for expression in a production cell. Or the production organism, generally a eukaryotic cell, for example a Pichia cell, a Chinese ha It means that it has been modified to encode the amino acid sequence using preferred codons in murine ovarian cells (CHO), or human cells. The optimized nucleotide sequence is engineered to retain, in whole or to the greatest extent possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence. The optimized sequences herein have been engineered to have preferred codons in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated herein. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as being optimized. As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms "polypeptide" and "protein" apply to both naturally occurring and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0073] As used herein, the term "recombinant human antibody" refers to an antibody isolated from a transgenic or translchromosomal animal (e.g., a mouse) with respect to the human immunoglobulin genes, or from a hybridoma prepared therefrom, an antibody isolated from a host cell transformed to express a human antibody, e.g., a transfectoma, an antibody isolated from a recombinant DNA molecule encoding a human antibody, or a combination thereof. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0074] As used herein, the term "recombinant human antibody" refers to an antibody isolated from a transgenic or translchromosomal animal (e.g., a mouse) with respect to the human immunoglobulin genes, or from a hybridoma prepared therefrom, an antibody isolated from a host cell transformed to express a human antibody, e.g., a transfectoma, an antibody isolated from a recombinant DNA molecule encoding a human antibody, or a combination thereof. Antibodies isolated from alternative combinatorial human antibody libraries, and other antibodies prepared, expressed, created, or isolated by any means involving splicing of all or part of the human immunoglobulin gene sequences to other DNA sequences, such as by recombinant means, including all human antibodies. Such recombinant human antibodies have variable regions in which the framework regions and CDR regions have sequences derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are related to and derived from the VH and VL sequences of the human germline, but may be sequences that do not naturally occur within the germline repertoire of in vivo human antibodies. Splicing of all or part of the human immunoglobulin gene sequences to other DNA sequences, and other antibodies prepared, expressed, created, or isolated by any means, such as by recombinant means Antibodies prepared, expressed, created, or isolated by any means involving splicing of all or part of the human immunoglobulin gene sequences to other DNA sequences, such as by recombinant means Antibodies prepared, expressed, created, or isolated by recombinant means, including all human antibodies These recombinant human antibodies have variable regions in which the framework regions and CDR regions have sequences derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are related to and derived from the VH and VL sequences of the human germline, but may be sequences that do not naturally occur within the germline repertoire of in vivo human antibodies. In vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis) In vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis) In vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis) The amino acid sequences of the VH and VL regions of the recombinant antibody are related to and derived from the VH and VL sequences of the human germline, but may be sequences that do not naturally occur within the germline repertoire of in vivo human antibodies. The amino acid sequences of the VH and VL regions of the recombinant antibody are related to and derived from the VH and VL sequences of the human germline, but may be sequences that do not naturally occur within the germline repertoire of in vivo human antibodies. The amino acid sequences of the VH and VL regions of the recombinant antibody are related to and derived from the VH and VL sequences of the human germline, but may be sequences that do not naturally occur within the germline repertoire of in vivo human antibodies.

[0075] The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It is to be understood that such a term is not intended to refer only to a particular target cell, but also to progeny cells of such a cell. In subsequent generations, certain modifications may occur due to mutations or environmental influences, and thus such progeny cells may not actually be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein. The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It is not intended to refer only to a particular target cell, but also to progeny cells of such a cell. In subsequent generations, certain modifications may occur due to mutations or environmental influences. In subsequent generations, certain modifications may occur due to mutations or environmental influences. Such progeny cells may not actually be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein.

[0076] The term "subject" includes humans and non-human animals. Non-human animals include non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles, etc. , and all vertebrates (e.g., mammals and non-mammals). Unless otherwise specified , in this specification, the terms "patient" or "subject" are used interchangeably. In this specification the terms "cyno" or "cynomolgus" used refer to cynomolgus monkeys (Macaca fascicularis).

[0077] In one embodiment, the terms "treating" or "treatment" of any disease or disorder (e.g., ANGPT L4-related disorder) as used herein refer to ameliorating the disease or disorder (i.e., slowing, stopping, or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, which may not be distinguishable by the patient, including a parameter . In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder physically (e.g., stabilizing distinguishable symptoms), physiologically (e.g., stabilizing physical parameters), or both physically and physiologically . In yet another embodiment, "treating" or "treatment" refers to preventing or delaying the occurrence, onset, or progression of the disease or disorder . . . . . . .

[0078] In the context of the applications described herein, including, for example, ANGPTL4-related disorders, "prevention" means, for example, any action that prevents or slows the worsening of ANGPTL4-related disease parameters in patients at risk of such worsening, as described below. The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it is ligated. One type of vector is a "plasmid" that refers to a circular double-stranded DNA loop capable of ligating additional DNA segments. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), capable of ligating additional DNA segments into the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors) are capable of self-replication in the host cells into which they are introduced. Other vectors (e.g., non-mammalian episomal vectors) can integrate into the genome of the host cell upon introduction into the host cell and thereby replicate with the host genome. Further, certain vectors are capable of directing the expression of a gene to which they are operably linked. In this specification, such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA methods are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, the terms "plasmid" and "vector" can be used interchangeably in this specification. However, the present invention provides equivalent functions.

[0079] ​​​​​​​​​​​​​​​​Other forms of expression vectors are also contemplated, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). BRIEF DESCRIPTION OF THE DRAWINGS

[0080]

Figure 1-1

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Figure 6

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Figure 11

Mode for Carrying Out the Invention

[0081] Detailed Description The present invention is in part based on the discovery of antibody molecules that specifically bind to ANGPTL4 and inhibit its biological activity. The present invention provides antibodies in a full IgG format (e.g., humanized antibodies ​ NEG276, NEG276-LALA, NEG278, NEG310, NEG3 13, NEG315, NEG318, NEG319), as well as Fab fragments and the like, relate to both of these antigen-binding fragments.

[0082] Accordingly, the present invention provides an antibody that specifically binds to ANGPTL4 (e.g., human ANGPTL4), a pharmaceutical composition, methods for producing and using such an antibody and composition. .

[0083] ANGPTL4 protein The present invention provides an antibody that specifically binds to ANGPTL4 and inhibits its biological activity, including the ability to activate lipoprotein lipase (LPL). Conversely,

[0084] Angiopoietin-like 4 protein (ANGPTL4) is a member of the angiopoietin family of secreted proteins. ANGPTL4 is a homo-oligomeric protein capable of forming dimers and tetramers, and is a homo-oligomeric protein expressed by cell types including macrophages, adipose, muscle, and hepatocytes. ANGPTL4 is also known as hepatocyte fibrinogen / angiopoietin-related protein (HFARP) (Kim et al. (2000) Biochem. J. 346:603-610), PPAR gamma angiopoietin-related protein (PGAR) (Yoon, et al. (2000) Mol. Cell Biol., 20:5343-5349), and fasting-induced adipocyte factor (FIAF) (Kerten et al. (2000) J. Biol. Chem., 275:2 4 is also known as hepatocyte fibrinogen / angiopoietin-related protein (HFARP) (Kim et al. (2000) Biochem. J. 346:603-610), PPAR gamma angiopoietin-related protein (PGAR) (Yoon, et al. (2000) Mol. Cell Biol., 20:5343-5349), and fasting-induced adipocyte factor (FIAF) (Kerten et al. (2000) J. Biol. Chem., 275:2 is also known as 8488-28493). ANGPTL4 contains an N-terminal coiled-coil domain and a C-terminal fibrinogen (FBN)-like domain (Kim et al. (2000) Biochem. J. 346:603-610).

[0085] Lipoprotein lipase (LPL) plays a central role in lipoprotein metabolism in maintaining normal lipoprotein levels in the blood and determining when and in which tissues triglycerides (TG) are taken up through tissue-specific regulation of its activity. The coiled-coil region of ANGPTL4 is known to inhibit triglyceride (TG) clearance mediated by lipoprotein lipase (LPL). Thus, loss-of-function mutations of ANGPTL4 (e.g., as seen in human subjects), gene deletions (e.g., as seen in transgenic mice), and inhibition by antibodies (e.g., as seen in mice and cynomolgus monkeys) have all been observed to decrease plasma triglycerides. Furthermore, ANGPTL4 antibodies are also known to activate LPL. Conversely, injection of ANGPTL4 into mice results in a rapid increase in circulating triglycerides, at a rate greater than when angiopoietin-like protein 3 (ANGPTL3) is injected (Yoshida et al. (2002) J Lipid Res 43:1770-1772). The anti-ANGPTL4 antibodies and antigen-binding fragments described in the present invention, for example, block the inhibition of LPL by ANGPTL4, thereby reducing plasma triglycerides.

[0086] ​​​​​​​​​Thereby initiate, promote, or enhance the activation of LPL. These antibodies are useful for preventing and treating diseases characterized by elevated triglyceride levels, such as primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, type II diabetes, etc., both acute and chronic symptoms. It is expected to improve them.

[0087] The anti-ANGPTL4 antibodies and antigen-binding fragments described in the present invention, for example, block the inhibition of LPL by ANGPTL4, thereby reducing plasma triglycerides and thereby initiate, promote, or enhance the activation of LPL. These antibodies are useful for preventing and treating diseases characterized by elevated triglyceride levels, such as primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, type II diabetes, etc., both acute and chronic symptoms. It is expected to improve them.

[0088] ANGPTL4 antibodies and antigen-binding fragments The present invention provides antibodies that specifically bind to ANGPTL4. In some embodiments, the present invention provides antibodies that specifically bind to human and cynomolgus ANGPTL4. The antibodies of the present invention include, but are not limited to, humanized antibodies and Fabs isolated as described in the examples.

[0089] The present invention provides antibodies that specifically bind to an ANGPTL4 protein (e.g., human and cynomolgus ANGPTL4), wherein the antibodies comprise a VH domain having the amino acid sequences of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138. The present invention also provides antibodies that specifically bind to an ANGPTL4 protein, which are listed in Table 1 An antibody comprising a VH CDR having an amino acid sequence of any one of the VH CDRs obtainable is also provided. In particular, the present invention provides an antibody that specifically binds to an ANGPTL4 protein (e.g., human and cynomolgus monkey ANGPTL4), and that comprises one, two, three or more VH CDRs having an amino acid sequence of any one of the VH CDRs listed in Table 1 below or, alternatively, consisting of these).

[0090] The present invention also provides an antibody that specifically binds to an ANGPTL4 protein and that comprises a VL domain having an amino acid sequence of SEQ ID NOs: 23 , 48, 68, 88, 108, 128, and 148. The present invention also provides an antibody that specifically binds to an ANGPTL4 protein (e.g., human and cynomolgus monkey ANGPTL4) and that comprises a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 2 below . In particular, the present invention provides an antibody that specifically binds to an ANGPTL4 protein (e.g., human and cynomolgus monkey ANGPTL4) and that comprises one, two, three or more VL CDRs having an amino acid sequence of any one of the VL CDRs listed in Table 1 below or, alternatively, consisting of these).

[0091] Other antibodies of the invention have been mutated, but still contain amino acids having at least 60, 70, 80, 85, 90, or 95% identity to the CDR regions depicted within the sequences listed in Table 1 within the CDR regions. In some embodiments, other antibodies of the invention have within the CDR regions amino acids having at least 60, 70, 80, 85, 90, or 95% identity to the CDR regions depicted within the sequences listed in Table 1 within the CDR regions ​​​​Compared to this, it includes an amino acid sequence of a mutant in which one, two, three, four, or five or fewer amino acids are mutated. It includes the amino acid sequence of the mutant.

[0092] The present invention also provides nucleic acid sequences encoding VH, VL, full-length heavy chains, and full-length light chains of antibodies that specifically bind to ANGPTL4 proteins (e.g., human and cynomolgus monkey ANGPTL4). Such nucleic acid sequences can be optimized for expression in mammalian cells (e.g., Table 1 shows nucleic acid sequences optimized for the heavy and light chains of the antibodies of the present invention). It encodes the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to ANGPTL4 protein (e.g., human and cynomolgus monkey ANGPTL4). Such nucleic acid sequences can be optimized for expression in mammalian cells (e.g., Table 1 shows nucleic acid sequences optimized for the heavy and light chains of the antibodies of the present invention). For example, Table 1 shows nucleic acid sequences optimized for the heavy and light chains of the antibodies of the present invention. It shows nucleic acid sequences optimized for the heavy and light chains of the antibodies of the present invention.

[0093] Table 1: Examples of ANGPTL4 antibodies, Fab, and ANGPTL4 proteins

Table 1

[0094] Other antibodies of the invention have mutated amino acids or nucleic acids encoding amino acids but still include antibodies having at least 60, 65, 70, 75, 80, 85, 90, or 95 percent identity with the sequences listed in Table 1. Some embodiments have variable regions that retain substantially the same antigen-binding activity and have one, two, three, four, or five or fewer amino acids different compared to the variable regions shown in the sequences listed in Table 1 when It contains the amino acid sequence of a mutant that mutates an acid.

[0095] Since each of these antibodies can bind to ANGPTL4, the VH sequence, VL sequence, full length light chain sequence, and full length heavy chain sequence (amino acid sequence and nucleotide sequence encoding the amino acid sequence) can be "mixed and matched" to create other ANGPTL4-binding antibodies of the present invention. Such "mixed and matched" ANGPTL4-binding antibodies can be examined using binding assays known in the art (e.g., ELISA and other assays described in the Examples section). When mixing and matching these chains, the VH sequence derived from a specific VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, the full length heavy chain sequence derived from a specific full length heavy chain / full length light chain pairing is replaced with a structurally similar full length heavy chain sequence. Similarly, the VL sequence derived from a specific VH / VL pairing is replaced with a structurally similar VL sequence. Similarly, the full length light chain sequence derived from a specific full length heavy

[0096] chain / full length light chain pairing is replaced with a structurally similar full length light chain sequence. Thus, in one aspect, the present invention provides an isolated antibody or an antigen- binding region thereof having a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98, 118 and 138, and a light chain variable domain comprising an amino acid sequence

[0097] More specifically, in certain embodiments, the present invention provides an isolated antibody or an antigen-binding region thereof having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148.

[0098] In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148. In another embodiment, the present invention provides an isolated antibody having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences each selected from the group consisting of SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148.

[0099] As used herein, the terms "complementary determining region" and "CDR" refer to the sequences of amino acids within the variable region of an antibody that confer antigen specificity and antigen-binding affinity. Generally, there are three CDRs within each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs within each light chain variable region (LCDR1, LCDR2, LCDR3). As used herein, the terms "complementary determining region" and "CDR" refer to the sequences of amino acids within the variable region of an antibody that confer antigen specificity and antigen-binding affinity. Generally, there are three CDRs within each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs within each light chain variable region (LCDR1, LCDR2, LCDR3). As used herein, the terms "complementary determining region" and "CDR" refer to the sequences of amino acids within the variable region of an antibody that confer antigen specificity and antigen-binding affinity. Generally, there are three CDRs within each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs within each light chain variable region (LCDR1, LCDR2, LCDR3). As used herein, the terms "complementary determining region" and "CDR" refer to the sequences of amino acids within the variable region of an antibody that confer antigen specificity and antigen-binding affinity. Generally, there are three CDRs within each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs within each light chain variable region (LCDR1, LCDR2, LCDR3). Yes.

[0100] The exact amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al., (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National In stitutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme). Among the many well-known schemes, any one can be used to easily determine.

[0101] For example, according to Kabat, the CDR amino acid residues of the antibody FF1 within the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-66 (HCDR2), and 99 -104 (HCDR3), and the CDR amino acid residues within the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-55 (LCDR2), and 89-9 7 (LCDR3). According to Chothia, the CDR amino acids within VH are numbered 26-32 (HCDR1), 52-57 (HCDR2), and 99-104 ( HCDR3), and the amino acid residues within VL are numbered 26-32 (LCDR1 ), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDR amino acid residues within human VH are 26-35 (HCDR1), 50-66 (HCDR2), 99-104 (HCDR3), and the amino acid residues within VL are 26-32 (LCDR1 ), 50-52 (LCDR2), and 91-96 (LCDR3). . The CDR amino acid residues within human VH are 26-35 (HCDR1), 50-66 (HCDR2), and 90 - 104 (HCDR3), and amino acid residues 24 - 34 in human VL (LCDR1), 50 - 55 (LCDR2), and 89 - 97 (LCDR3). It consists of CDR1), 50 - 55 (LCDR2), and 89 - 97 (LCDR3).

[0102] In another aspect, the present invention provides an ANGPTL4 - binding antibody comprising CDR1, CDR2 , and CDR3 of the heavy and light chains described in Table 1, or combinations thereof. The amino acid sequence of the VH CDR1 of the antibody is shown in SEQ ID NOs: 7, 32, 52, 72, 92, 112 , and 132. The amino acid sequence of the VH CDR2 of the antibody is shown in SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133. The amino acid sequence of the VH CDR3 of the antibody is shown in SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134. The amino acid sequence of the VL CDR1 of the antibody is shown in SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142. The amino acid sequence of the VL CDR2 of the antibody is shown in SEQ ID NOs: 18, 43, 63, 83, 103, 123, and 143. The amino acid sequence of the VL CDR3 of the antibody is shown in SEQ ID NOs: 19, 44, 64, 84, 104, 12 4, and 144. These CDR regions are represented using the Kabat system. represented.

[0103] Alternatively, as defined using the Chothia system (Al - Lazikani et al., (1997), JMB 273 927 - 948 ), the amino acid sequence of the VH CDR1 of the antibody is shown in SEQ ID NOs: 10, 35, 55, 75, 95, 115, and 135. The amino acid sequence of the VH CDR2 of the antibody is shown in SEQ ID NOs: 11, 36, 56, 76, 96, 116, and 136. The amino acid sequences of the VH CDR3 of the antibodies are shown in SEQ ID NOs: 12, 37, 57, 77, , 97, 117, and 137. The amino acid sequences of the VL CDR1 of the antibodies are , as shown in SEQ ID NOs: 20, 45, 65, 85, 105, 125, and 145. The amino acid sequences of the VL CDR2 of this antibody are shown in SEQ ID NOs: 21, 46, 66, 86, 106, 12 6, and 146. The amino acid sequence of the VL CDR3 of the antibody is shown in SEQ ID NO:2. 2, 47, 67, 87, 107, 127, and 147.

[0104] Each of these antibodies is capable of binding to ANGPTL4 and has antigen-binding specificity Considering that the CDR1, CDR2, and CDR3 regions are the major contributors to the Thus, each antibody can be used to generate other ANGPTL4 binding molecules of the invention, including VHC DR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3. Preferably, the VH CDR1 sequence, VH R2, and VL CDR3 are included, but may also contain VH CDR2, VH R3, and VL CDR4. CDR2 sequence, and VH CDR3 sequence, and VL CDR1 sequence, VL CDR The VL CDR3 sequences, as well as the VL CDR3 sequences, can be "mixed and matched" (i.e. (i.e., CDRs from different antibodies can be mixed and matched). "Mixed and matched" ANGPTL4 binding antibodies can be used in a variety of binding assays known in the art. Binding assays described in the Examples (e.g., ELISA, SET, Biacor e) can be used to determine if VH CDR sequences are mixed and matched. CDR1, CDR2, and / or CDR3 sequences derived from a particular VH sequence , replace them with structurally similar CDR sequences. Similarly, when mixing and matching VL CDR sequences, the CDR1 sequence, CDR2 sequence, and / or CDR3 sequence derived from a specific VL sequence shall be replaced with a structurally similar CDR sequence. It will be readily apparent to those skilled in the art that novel VH and VL sequences can be created by replacing one or more VH CDR region sequences and / or VL CDR region sequences with structurally similar sequences derived from the CDR sequences shown herein for the monoclonal antibodies of the present invention. In addition to the foregoing, in one embodiment, the antigen-binding fragment of the antibody described herein can include VH CDR1, VH CDR2, and VH CDR3, or VL CDR1, VL CDR2, and VL CDR3, in which case the fragment binds to ANGPTL4 as a single variable domain. When mixing and matching, the CDR1 sequence, CDR2 sequence, and / or CDR3 sequence derived from a specific VL sequence shall be replaced with a structurally similar CDR sequence. It will be readily apparent to those skilled in the art that novel VH and VL sequences can be created by replacing one or more VH CDR region sequences and / or VL CDR region sequences with structurally similar sequences derived from the CDR sequences shown herein for the monoclonal antibodies of the present invention. In addition to the foregoing, in one embodiment, the antigen-binding fragment of the antibody described herein can include VH CDR1, VH CDR2, and VH CDR3, or VL CDR1, VL CDR2, and VL CDR3, in which case the fragment binds to ANGPTL4 as a single variable domain. In some embodiments of the present invention, the antibody or its antigen-binding fragment can have the heavy and light chain sequences of the humanized antibodies listed in Table 1. More specifically, the antibody or its antigen-binding fragment can have the heavy and light chain sequences of NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, and NEG319. In other embodiments of the present invention, the antibody or antigen-binding fragment that specifically binds to ANGPTL4 has the CDR1 of the heavy chain variable region, the CDR2 of the heavy chain variable region, the CDR3 of the heavy chain variable region, the CDR1 of the light chain variable region, the CDR2 of the light chain variable region, and the CDR3 of the light chain variable region defined by Kabat and listed in Table 1. In some embodiments of the present invention, the antibody or its antigen-binding fragment can have the heavy and light chain sequences of the humanized antibodies listed in Table 1.

[0105] In some embodiments of the present invention, the antibody or its antigen-binding fragment can have the heavy and light chain sequences of the humanized antibodies listed in Table 1. More specifically, the antibody or its antigen-binding fragment can have the heavy and light chain sequences of NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, and NEG319. In other embodiments of the present invention, the antibody or antigen-binding fragment that specifically binds to ANGPTL4 has the CDR1 of the heavy chain variable region, the CDR2 of the heavy chain variable region, the CDR3 of the heavy chain variable region, the CDR1 of the light chain variable region, the CDR2 of the light chain variable region, and the CDR3 of the light chain variable region defined by Kabat and listed in Table 1.

[0106] In other embodiments of the present invention, the antibody or antigen-binding fragment that specifically binds to ANGPTL4 has the CDR1 of the heavy chain variable region, the CDR2 of the heavy chain variable region, the CDR3 of the heavy chain variable region, the CDR1 of the light chain variable region, the CDR2 of the light chain variable region, and the CDR3 of the light chain variable region defined by Kabat and listed in Table 1. It includes the CDR2 of the domain and the CDR3 of the light chain variable region. In yet other embodiments of the present invention an antibody or antigen-binding fragment that specifically binds to ANGPTL4 has CDR1 of the heavy chain variable region, CDR as defined by Chothia and described in Table 1, CDR2 of the heavy chain variable region, CDR 3 of the heavy chain variable region, CDR1 of the light chain variable region, CDR2 of the light chain variable region, and also includes CDR3 of the light chain variable region.

[0107] In a specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4, including CDR1 of the heavy chain variable region of SEQ ID NO: 7, CDR2 of the heavy chain variable region of SEQ ID NO: 8, SEQ ID NO: 9 of CDR3 of the heavy chain variable region, CDR1 of the light chain variable region of SEQ ID NO: 17, SEQ ID NO: 18 of CDR2 of the light chain variable region, and an antibody including CDR3 of the light chain variable region of SEQ ID NO: 19 is included.

[0108] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 including CDR1 of the heavy chain variable region of SEQ ID NO: 32, CDR2 of the heavy chain variable region of SEQ ID NO: 33, CDR3 of the heavy chain variable region of SEQ ID NO: 34, CDR1 of the light chain variable region of SEQ ID NO: 42, SEQ ID NO: 43 of CDR2 of the light chain variable region, and an antibody including CDR3 of the light chain variable region of SEQ ID NO: 44 is included.

[0109] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 including CDR1 of the heavy chain variable region of SEQ ID NO: 52, CDR2 of the heavy chain variable region of SEQ ID NO: 53, CDR3 of the heavy chain variable region of SEQ ID NO: 54, CDR1 of the light chain variable region of SEQ ID NO: 62, SEQ ID NO: 63 of CDR2 of the light chain variable region, and an antibody including CDR3 of the light chain variable region of SEQ ID NO: 64 is included.

[0110] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4, and includes CDR1 of the heavy chain variable region of SEQ ID NO: 72, CDR2 of the heavy chain variable region of SEQ ID NO: 73, CDR3 of the heavy chain variable region of SEQ ID NO: 74, CDR1 of the light chain variable region of SEQ ID NO: 82, SEQ ID NO: 83 of the CDR2 of the light chain variable region, and CDR3 of the light chain variable region of SEQ ID NO: 84. The antibody includes.

[0111] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4, and includes CDR1 of the heavy chain variable region of SEQ ID NO: 92, CDR2 of the heavy chain variable region of SEQ ID NO: 93, CDR3 of the heavy chain variable region of SEQ ID NO: 94, CDR1 of the light chain variable region of SEQ ID NO: 102, SEQ ID NO: 103 of the CDR2 of the light chain variable region, and CDR 3 of the light chain variable region of SEQ ID NO: 104. The antibody includes.

[0112] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4, and includes CDR1 of the heavy chain variable region of SEQ ID NO: 112, CDR 2 of the heavy chain variable region of SEQ ID NO: 113, CDR3 of the heavy chain variable region of SEQ ID NO: 114, CDR 1 of the light chain variable region of SEQ ID NO: 122, CDR2 of the light chain variable region of SEQ ID NO: 123, and CDR of the light chain variable region of SEQ ID NO: 124 3. The antibody includes.

[0113] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4, and includes CDR1 of the heavy chain variable region of SEQ ID NO: 132, CDR 2 of the heavy chain variable region of SEQ ID NO: 133, CDR3 of the heavy chain variable region of SEQ ID NO: 134, CDR 1. An antibody comprising the CDR2 of the light chain variable region of SEQ ID NO: 143 and the CDR3 of the light chain variable region of SEQ ID NO: 144.

[0114] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and comprises the CDR1 of the heavy chain variable region of SEQ ID NO: 10, the CDR2 of the heavy chain variable region of SEQ ID NO: 11, the CDR3 of the heavy chain variable region of SEQ ID NO: 12, the CDR1 of the light chain variable region of SEQ ID NO: 20, the CDR2 of the light chain variable region of SEQ ID NO: 21, and the CDR3 of the light chain variable region of SEQ ID NO: 22. In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and

[0115] comprises the CDR1 of the heavy chain variable region of SEQ ID NO: 35, the CDR2 of the heavy chain variable region of SEQ ID NO: 36, the CDR3 of the heavy chain variable region of SEQ ID NO: 37, the CDR1 of the light chain variable region of SEQ ID NO: 45, the CDR2 of the light chain variable region of SEQ ID NO: 46, and the CDR3 of the light chain variable region of SEQ ID NO: 47. In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and comprises the CDR1 of the heavy chain variable region of SEQ ID NO: 55, the CDR2 of the heavy chain variable region of SEQ ID NO: 56,

[0116] the CDR3 of the heavy chain variable region of SEQ ID NO: 57, the CDR1 of the light chain variable region of SEQ ID NO: 65, the CDR2 of the light chain variable region of SEQ ID NO: 66, and the CDR3 of the light chain variable region of SEQ ID NO: 67. In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and comprises the CDR1 of the heavy chain variable region of SEQ ID NO: 75, the CDR2 of the heavy chain variable region of SEQ ID NO: 76, the CDR3 of the heavy chain variable region of SEQ ID NO: 77, the CDR1 of the light chain variable region of SEQ ID NO: 85, the

[0117] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and comprises the CDR1 of the heavy chain variable region of SEQ ID NO: 75, the CDR2 of the heavy chain variable region of SEQ ID NO: 76, The CDR3 of the heavy chain variable region of SEQ ID NO: 77, the CDR1 of the light chain variable region of SEQ ID NO: 85, the CDR2 of the light chain variable region of SEQ ID NO: 86, and the CDR3 of the light chain variable region of SEQ ID NO: 87. The antibody contains.

[0118] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and contains the CDR1 of the heavy chain variable region of SEQ ID NO: 95, the CDR2 of the heavy chain variable region of SEQ ID NO: 96, the CDR3 of the heavy chain variable region of SEQ ID NO: 97, the CDR1 of the light chain variable region of SEQ ID NO: 105, the CDR2 of the light chain variable region of SEQ ID NO: 106, and the CDR 3 of the light chain variable region of SEQ ID NO: 107.

[0119] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and contains the CDR1 of the heavy chain variable region of SEQ ID NO: 115, the CDR 2 of the heavy chain variable region of SEQ ID NO: 116, the CDR3 of the heavy chain variable region of SEQ ID NO: 117, the CDR 1 of the light chain variable region of SEQ ID NO: 125, the CDR2 of the light chain variable region of SEQ ID NO: 126, and the CDR3 of the light chain variable region of SEQ ID NO: 127.

[0120] In another specific embodiment, the present invention is an antibody that specifically binds to ANGPTL4 and contains the CDR1 of the heavy chain variable region of SEQ ID NO: 135, the CDR 2 of the heavy chain variable region of SEQ ID NO: 136, the CDR3 of the heavy chain variable region of SEQ ID NO: 137, the CDR 1 of the light chain variable region of SEQ ID NO: 145, the CDR2 of the light chain variable region of SEQ ID NO: 146, and the CDR3 of the light chain variable region of SEQ ID NO: 147.

[0121] In certain embodiments, the present invention is specifically described in Table 1 and binds specifically to ANGPTL4 It comprises an antibody or antigen-binding fragment that binds. In a preferred embodiment, the antibody or antigen-binding fragment that binds to ANGPTL4 is NEG276, NEG276-LALA, NEG2 78, NEG310, NEG313, NEG315, NEG318, NEG319 .

[0122] Homologous antibody In yet another embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an amino acid sequence homologous to the sequence described in Table 1, wherein the antibody binds to the ANGPTL4 protein (e.g., human and cynomolgus ANGPTL4) and retains the desired functional properties of the antibodies described in Table 1.

[0123] For example, the present invention provides an isolated antibody or a functional antigen-binding fragment thereof comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 3 8, 58, 78, 98, 118, and 138 and having at least 80%, at least 90%, or at least 95% identity; the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 23, 48, 68, 88, 108, 128, 1 48 and having at least 80%, at least 90%, or at least 95% identity; and the antibody specifically binds to ANGPTL4 (e.g., human and cynomolgus ANGPTL4). In certain aspects of the present invention, the heavy and light chain sequences further comprise HCDR1 sequences, HCDR2 sequences, HCDR3 sequences, LCDR1 sequences, LCDR2 sequences, and LCDR3 sequences as defined by Kabat, e.g., SEQ ID NOs: 7, 8, 9, 17, 18, and 19, respectively. In certain aspects of the present invention, In certain other embodiments, the heavy and light chain sequences are the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined by Chothia, e.g., SEQ ID NOs: 10, 11, 12, 20, 21, and 2 2, respectively.

[0124] In other embodiments, the VH amino acid sequence and / or the VL amino acid sequence is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 %, or 99% identical to the sequences shown in Table 1. In other embodiments, the VH amino acid sequence and / or the VL amino acid sequence can be identical except for amino acid substitutions at one, two, three, four, or five or fewer amino acid positions. Antibodies having VH and VL regions with greater than 80% identity to the VH and VL regions of the antibodies described in Table 1 are obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of the nucleic acid molecules encoding SEQ ID NOs: 13, 38, 58, 78, 98, 118, 118, or 138, and SEQ ID NOs: 23, 48, 68, 88, 108, 128, or 148, respectively, followed by examining the encoded modified antibodies for retention of function using the functional

[0125] assays described herein. In other embodiments, the full-length heavy chain amino acid sequence and / or the full-length light chain amino acid sequence is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97 A full-length heavy chain of any of 00, 120, or 140, and SEQ ID NO: 25, 25, A full-length light chain of any of 50, 70, 90, 110, 130, or 150, and having a high (i.e., 80% or more) identity with the full-length heavy chain and full-length light chain, the antibody having After mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of a nucleic acid molecule encoding such a polypeptide, using the functional assays described herein it can be obtained by examining the encoded modified antibody for retention of function.

[0126] In other embodiments, the full-length heavy chain nucleotide sequence and / or the full-length light chain nucleotide sequence may be 60%, 70%, 80%, 90%, 95%, 96%, 97 %, 98%, or 99% identical to the sequences shown in Table 1.

[0127] In other embodiments, the nucleotide sequence of the heavy chain variable region and / or the nucleotide sequence of the light chain variable region may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences shown in Table 1.

[0128] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using the mathematical algorithms described in the following non-limiting examples.

[0129] In addition, or alternatively, the protein sequences of the invention can be further used, for example, as a "query sequence" to identify related sequences by performing a search against publicly available databases. For example, such a search can be performed using the BLAST program (version 2.0) by Altschul et al., 1990 J. Mol. Biol. 215:403-10. It can be performed.

[0130] Antibodies with conservative modifications In certain embodiments, the antibodies of the invention have a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR 3 sequence, and a light chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein one or more of these CDR sequences have an amino acid sequence designated based on the antibodies described herein or their conservative modifications, and the antibody retains the desired functional properties of the ANGPTL4-binding antibodies of the invention.

[0131] Accordingly, the invention provides an isolated antibody or an antigen-binding fragment thereof consisting of a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, and a light chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein the CDR1 amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112, and 132, and their conservative modifications, the CDR2 amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133, and their conservative modifications, the CDR3 amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 9, 34, and their conservative modifications, and the CDR1 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs: 10, 35, 54, 74, 94, 114, and 134, and their conservative modifications, the CDR2 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs: 11, 36, 55, 75, 95, 115, and 135, and their conservative modifications, Selected from the group consisting of 54, 74, 94, 114, and 134, and their conservative modifications, wherein the CDR1 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142, and their conservative modifications, and the CDR2 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs: 18, 43, 63, 83, 103, 1 23, and 143, and their conservative modifications, and the CDR3 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144, and their conservative modifications, to provide an antibody or antigen-binding fragment thereof that specifically binds to ANGPTL4. In other embodiments, the antibodies of the invention are optimized for expression in mammalian cells and have full-length heavy and light chain sequences, one or more of which have amino acid sequences designated based on the antibodies described herein or their conservative modifications and retain the desired functional properties of the ANGPTL4-binding antibodies of the invention. Thus, the invention provides an isolated antibody consisting of a full-length heavy chain and a full-length light chain, optimized for expression in mammalian cells, wherein the full-length heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 28, 40, 60, 8 0, 100, 120, and 140, and their conservative modifications, and the full-length light chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150, and their conservative modifications, and specifically binds to ANGPTL4 (e.g., human and cynomolgus ANGPTL4).

[0132] In other embodiments, the antibodies of the invention are optimized for expression in mammalian cells and have full-length heavy and light chain sequences, one or more of which have amino acid sequences designated based on the antibodies described herein or their conservative modifications and retain the desired functional properties of the ANGPTL4-binding antibodies of the invention. Thus, the invention provides an isolated antibody consisting of a full-length heavy chain and a full-length light chain, optimized for expression in mammalian cells, wherein the full-length heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 28, 40, 60, 8 0, 100, 120, and 140, and their conservative modifications, and the full-length light chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150, and their conservative modifications, and specifically binds to ANGPTL4 (e.g., human and cynomolgus ANGPTL4). Accordingly, the invention provides an isolated antibody consisting of a full-length heavy chain and a full-length light chain, optimized for expression in mammalian cells, wherein the full-length heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 28, 40, 60, 8 0, 100, 120, and 140, and their conservative modifications, and the full-length light chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150, and their conservative modifications, and specifically binds to ANGPTL4 (e.g., human and cynomolgus ANGPTL4). 0, 100, 120, and 140, and their conservative modifications, and the full-length light chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150, and their conservative modifications, and specifically binds to ANGPTL4 (e.g., human and cynomolgus ANGPTL4). GPTL4 (e.g., human, and cynomolgus ANGPTL4) to provide an anti body that binds specifically.

[0133] Antibodies that bind to the same epitope The present invention provides antibodies that bind to the same epitope as the ANGPTL4-binding antibodies described in Table 1. Thus, in an ANGPTL4 binding assay (such as the ANGPTL4 binding assay described in the Examples), based on their ability to compete with other antibodies of the present invention (e.g., competitively inhibit the binding of other antibodies of the present invention in a statistically significant manner), additional antibodies can be identified. Based on the ability of a test antibody to inhibit the binding of an antibody of the present invention to the ANGPTL4 protein, it is possible for the test antibody to compete with that antibody for binding to ANGPTL4, and such antibodies are, according to non-limiting theory, believed to be able to bind to the same or related (e.g., structurally similar or spatially proximate) epitopes on the ANGPTL4 protein as the antibody with which it competes. In certain embodiments, the antibodies that bind to the same epitope on ANGPTL4 as the antibodies of the present invention are humanized antibodies. Such humanized antibodies can be prepared and isolated as described herein. As used herein, an antibody "competes" for binding when, in the presence of an equimolar concentration of a competing antibody, the competing antibody inhibits the binding of an antibody or antigen-binding fragment of the present invention to ANGPTL4 by more than 50% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%). In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or

[0134] In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more epitopes of ANGPTL4. In some embodiments, the epitope to which the antibody or antigen-binding fragment binds is a linear epitope. In other embodiments, the antibody or The epitope to which the antigen-binding fragment binds is a non-linear conformational epitope is.

[0135] Engineered antibodies and modified antibodies As a starting material for engineering modified antibodies with modified properties from a starting antibody, as shown herein Antibodies having one or more of the VH sequence and / or the VL sequence can be used to further prepare the antibodies of the present invention Antibodies can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), for example, within one or more CDR regions, and / or One or more residues within one or more framework regions. Additionally, or alternatively, antibodies can be engineered by modifying residues within the constant region For example, by modifying the effector function of the antibody can also be engineered .

[0136] One type of variable region engineering that can be performed is CDR grafting. Antibodies Interact with the target antigen mainly through amino acid residues located within six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs The diversity among individual antibodies is greater than that of the sequences outside the CDRs. Since CDR sequences contribute to most antibody-antigen interactions, an expression vector containing CDR sequences derived from a specific naturally occurring antibody grafted into a framework sequence derived from a different antibody with different properties can be constructed to express recombinant antibodies that mimic the properties of the specific naturally occurring antibody (e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 198 is possible 4:569-575). 6 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad. U.S.A. 86:10029-1 0033; U.S. Patent No. 5,225,539 by Winter, and Queen et al., U.S. Patent No. 5,530,101; No. 5,585,089; same No. 5,693,762; and No. 6,180,370; see also ).

[0137] Accordingly, another embodiment of the present invention is a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112 , and 132, a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133, and a CDR3 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 13 4, each comprising a heavy chain variable region, and a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142 , a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 43, 63, 83, 103, 123, and 143, and a CDR3 sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144 , each having a light chain variable region, and an isolated antibody or an antigen-binding fragment thereof. Accordingly, such an antibody contains the VH CDR sequences and VL CDR sequences of monoclonal antibodies, but may contain different framework sequences derived from these antibodies. relates to. Accordingly, such an antibody contains the VH CDR sequences and VL CDR sequences of monoclonal antibodies, but may contain different framework sequences derived from these antibodies.

[0138] ​Such framework sequences are based on published D-RNA sequences, including germline antibody gene sequences. The sequences can be obtained from the NA database or published references. For example, human heavy chains and Germline DNA sequences of the human and light chain variable region genes were obtained from the human germline sequence database. The VBase database (available on the internet at mrc-cpe.cam.ac.uk / vbase) , as well as Kaba, the contents of each of which are expressly incorporated herein by reference. t, EA et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edi tion, US Department of Health and Human Services, NIH Publication No. 91-3242 ;Tomlinson, IM et al., 1992 J. Mol. Biol. 227:776-798; and Cox, JPL e et al., 1994 Eur. J Immunol. 24:827-836.

[0139] Exemplary framework sequences for use in the antibodies of the invention include those of selected antibodies of the invention. The framework sequences used by the antibody of the present invention, for example, The consensus sequence and / or framework sequence used in the present study are structurally similar to the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence. The sequence, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence are The framework sequences are derived from the sequences found in the germline immunoglobulin genes. Alternatively, the CDR sequences can be grafted into framework regions having the same sequence as the CDR sequences. 、It can also be grafted into a framework region containing one or more mutations as compared to the germline sequence. For example, in certain cases, it has been found beneficial to mutate residues within the framework region to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 by Queen et al.). Frameworks that can be utilized as a scaffold for constructing the antibodies and antigen-binding fragments described herein thereon include, but are not limited to, VH1A, VH1B, VH3, Vk1, Vl2, and Vk2. In the art, additional frameworks are known and can be found, for example, in the vBase database at vbase.mrc-cpe.cam.ac.uk / index.php?&MMN_position=1:1 on the Internet.

[0140] Thus, embodiments of the present invention include a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions within the framework region of such a sequence, and a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions within the framework region of such a sequence. Relating to isolated ANGPTL4-binding antibodies or antigen-binding fragments thereof further comprising a domain .

[0141] Another type of variable region modification is within the VH CDR1 region, within the VH CDR2 region, and / or within the VH CDR3 region, known as "affinity maturation", and / or within the VL CDR1 region, within the VL CDR2 region, and / or within the VL CDR3 region, mutate the amino acid residues therein, thereby improving one or more binding specificities (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and in vitro assays or in vivo assays described herein and presented in the examples can be used to assess the effect on antibody binding or other functional properties of interest. Conservative modifications (discussed above) can be introduced. Mutations can be amino acid substitutions, additions, or deletions. Further, typically 1, 2, 3, 4, or 5 or fewer residues within the CDR region are also modified. Thus, in another embodiment, the invention provides an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112, and 132, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 7, 32, 52, 72, 92, 112, 112, and 132, consisting of a VH CDR1 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133, or SEQ ID NOs: 8, 33, 53, 73, 93 can be used to assess the effect on antibody binding or other functional properties of interest. Conservative modifications (discussed above) can be introduced. Mutations can be amino acid substitutions, additions, or deletions. Further, typically 1, 2, 3, 4, or 5 or fewer residues within the CDR region are also modified.

[0142] 112, and 132, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 7, 32, 52, 72, 92, 112, 112, and 132, consisting of a VH CDR1 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133, or SEQ ID NOs: 8, 33, 53, 73, 93 113, and 133 selected from the group consisting of, or SEQ ID NOs: 8, 33, 53, 73, 93​​​​​​​​​​ 、an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions, as compared to 113 and 133, in the VH CDR2 region selected from the group consisting of SEQ ID NOs: 9, 34, 54, 74, 94, 114, 114, and 134, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions, as compared to SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134, in the VH CDR3 region; an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions, as compared to SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142, in the VL CDR1 region; an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 43, 63, 83, 103, 123, and 143, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions, as compared to SEQ ID NOs: 18, 43, 63, 83, 103, 123, and 143, in the VL CDR2 region; and an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions, as compared to SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144, in the VL CDR3 region, and an isolated ANGPTL4-binding antibody comprising a heavy chain variable region having the VL CDR3 region, or an antigen-binding fragment thereof, is provided.

[0143] Thus, in another embodiment, the invention provides an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 35, 55, 75, 95, 115, and 135, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions as compared to SEQ ID NOs: 10, 3 5, 55, 75, 95, 115, and 135, and having a VH CDR1 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 36, 56, 76, 96, 116, and 136 , or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions as compared to SEQ ID NOs: 11, 36, 56, 76, 96 , 116, and 136, and having a VH CDR2 region, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 37, 57, 77, 97, 117, and 137 , or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions as compared to SEQ ID NOs: 12, 37, 57, 77, 97, 117, and having a VH CDR3 region, and a heavy chain variable region , and an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 45, 65, 85, 105, 125, and 145 , or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions , or amino acid additions as compared to SEQ ID NOs: 20, 45, 65, 85, 105, 125, and 145, and having a VL CDR1 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146 , or an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146 as compared to SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146 , or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions as compared to SEQ ID NOs: 20, 45, 65, 85, 105, 125, and 145, and having a VL CDR1 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146 , or an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146 as compared to SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146 Compared with, having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or an amino acid sequence having an amino acid addition in the VL CDR2 region, and SEQ ID NO: 22 , an amino acid selected from the group consisting of 47, 67, 87, 107, 127, and 147 sequence, or compared with SEQ ID NO: 22, 47, 67, 87, 107, 127, and 147 having 1, 2, 3, 4, or 5 amino acid substitutions, amino acid deletions, or amino acid additions in the VL CDR3 region, and a light chain variable region comprising the same isolated ANGPTL4-binding antibody or antigen-binding fragment thereof is provided.

[0144] Grafting of the antigen-binding domain to an alternative framework or scaffold As long as the resulting polypeptide contains at least 1 binding region that specifically binds to ANGPTL4 , a wide variety of antibody / immunoglobulin frameworks or scaffolds can be employed. Such frameworks or scaffolds include the five major idiotypes of human immunoglobulins or fragments thereof, preferably humanized immunoglobulins of other animal species having aspects. In this regard, single-chain antibodies such as those identified in camelids are of particular interest. New frameworks , scaffolds, and fragments are continuously being discovered and developed by those skilled in the art.

[0145] In one aspect, the present invention relates to creating non-immunoglobulin-based antibodies using a non-immunoglobulin scaffold onto which the CDRs of the present invention can be grafted. As long as they contain a binding region specific for the target ANGPTL4 protein , known or future non-immunoglobulin ​The globulin framework and non-immunoglobulin scaffolds can be employed. Known non-immunoglobulin frameworks and non-immunoglobulin scaffolds include fibronectin (C ompound Therapeutics, Inc., Waltham, MA), an ngiogenin (Molecular Partners AG, Zurich, Switzerland ), domain antibodies (Domantis, Ltd., Cambridge, MA; and Ablynx nv, Zwijnaarde, Belgium), lipocalins (P ieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., S eattle, WA), maxibodies (Avidia, Inc., Mountain V iew, CA), protein A (Affibody AG, Sweden), and af yllins (gamma-crystallin or ubiquitin) (Scil Proteins Gm bH, Halle, Germany), but are not limited thereto.

[0146] The fibronectin scaffold is based on the type III fibronectin domain (e.g., the 10th module of type III fi bronectin (10 Fn3 domain)). The type III fib ronectin domains are themselves packed against each other to form the core of the protein and further contain loops that connect the beta strands to each other and are exposed to the solvent (similar to CDR s) and have 7 or 8 beta strands that are distributed between two beta sheets . There are at least 3 such loops at each edge of the beta sheet sandwich , An edge is a protein boundary perpendicular to the direction of the beta strand (see U.S. Patent No. 6,8 18,418). The overall fold is closely related to the fold of the heavy chain variable region, which is the smallest functional antibody fragment containing the entire antigen recognition unit in camel and llama Ig G. However, these fibronectin-based scaffolds are not immunoglobulins . Due to this structure, non-immunoglobulin antibodies mimic antigen-binding properties whose nature and affinity are similar to those of antibodies. These scaffolds can be used in in vitro loop randomization strategies and shuffling strategies similar to the process of antibody affinity maturation in vivo . These fibronectin-based molecules can be used as scaffolds that can replace the loop regions of the molecule with the CDRs of the present invention using standard cloning methods . . .

[0147] Ankyrin technology is based on using proteins with repeat modules derived from ankyrin as scaffolds with variable regions that can be used for binding to different targets. An ankyrin repeat module is a 33-amino acid polypeptide consisting of two anti-parallel alpha-helices and beta-turns . The binding of the variable region is mostly optimized by using ribosome display . . .

[0148] Avimers are derived from natural A domain-containing proteins such as LRP-1. These domains are originally used for protein-protein interactions, and in humans, more than 250 proteins are structurally based on the A domain. Avimers are linked via amino acid linkers . . ​​It consists of a large number (2 to 10) of different "A domain" monomers and can bind to a target antigen. Avimers can be created, for example, using the methods described in U.S. Patent Application Publication No. 2004 / 0175756; No. 2005 / 0053973; No. 2005 / 0048512; and No. 2006 / 0008844.

[0149] An affibody, which is an affinity ligand, is a small, simple protein consisting of a three-helix bundle based on one of the scaffolds of the IgG-binding domain of protein A. Protein A is a surface protein derived from the bacterium Staphylococcus aureus. This scaffold domain is one of 13 that are randomized to create an affibody library with multiple ligand variants (see, for example, U.S. Patent No. 5,831,012). Protein A is a surface protein derived from the bacterium Staphylococcus aureus. This scaffold domain is one of 13 that are randomized to create an affibody library with multiple ligand variants (see, for example, U.S. Patent No. 5,831,012). This scaffold domain is one of 13 that are randomized to create an affibody library with multiple ligand variants (see, for example, U.S. Patent No. 5,831,012). This scaffold domain is one of 13 that are randomized to create an affibody library with multiple ligand variants (see, for example, U.S. Patent No. 5,831,012). The affibody molecule mimics an antibody and has a molecular weight of 6 kDa compared to the 150 kDa molecular weight of an antibody. Despite its small size, the binding site of the affibody molecule is similar to that of an antibody. Despite its small size, the binding site of the affibody molecule is similar to that of an antibody.

[0150] Anticalins are products developed by Pieris ProteoLab AG. Anticalins are generally a broad group of small, robust proteins derived from lipocalins that are involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Anticalins are products developed by Pieris ProteoLab AG. Anticalins are generally a broad group of small, robust proteins derived from lipocalins that are involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Anticalins are products developed by Pieris ProteoLab AG. Anticalins are generally a broad group of small, robust proteins derived from lipocalins that are involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Anticalins are products developed by Pieris ProteoLab AG. Anticalins are generally a broad group of small, robust proteins derived from lipocalins that are involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Some natural lipocalins occur in human tissues or human body fluids. The protein architecture involves hypervariable loops at the top of a rigid framework. Associate with immunoglobulins. However, in contrast to antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain that is only slightly larger than a single immunoglobulin domain, with 160 - 180 amino acid residues. The set of four loops that form the binding pocket shows significant structural plasticity and allows various side chains. Thus, the binding site can be re - formed in a unique process to recognize target molecules of different shapes with high affinity and specificity. To develop anticalins by mutagenizing the set of four loops, a protein of the lipocalin family, the bilin - binding protein (BBP) of the large white butterfly (Pieris Brassicae) is used. One example of a patent application describing anticalins is in PCT International Publication No. WO 1999 / 16873 pamphlet. Affilin molecules are small non - immunoglobulin proteins designed for specific affinity to proteins and small molecules. New affilin molecules can be very rapidly selected from two libraries, each based on a different human - derived scaffold protein. Affilin molecules show no structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma - crystallin, a structural protein of the human lens, and the other is a protein of the "ubiquitin" superfamily. Both human scaffolds are very small, show high thermal stability, and are nearly resistant to pH changes and denaturing agents. This high

[0151] The stability is mainly due to the unfolding of the beta-sheet structure of the protein. For the protein derived from gamma-crystallin, examples are described in Pamphlet of International Publication No. 2001 / 04144, and for the "ubiquitin-like" protein, examples are described in Pamphlet of International Publication No. 2004 / 106368.

[0152] A protein epitope mimetic (PEM) is a medium-sized cyclic peptide-like molecule (molecular weight: 1 - 2 kDa) that mimics the beta-hairpin secondary structure of a protein, which is a major secondary structure involved in protein-protein interactions.

[0153] The present invention provides a fully human antibody that specifically binds to the ANGPTL4 protein. Compared with a chimeric antibody or a humanized antibody, the human ANGPTL4-binding antibody of the present invention has further reduced antigenicity when administered to a human subject.

[0154] Antibodies from camelid animals Antibody proteins obtained from members of the camelid family, including New World members such as the llama species (Lama paccos, Lama glama, and Vicugna vicugna), and the camel (dromedary) (Camelus bactrianus and Camelus dromaderius) have been characterized in terms of size, structural complexity, and antigenicity to human subjects. Certain IgG antibodies derived from mammals of this family found in nature lack a light chain and thus have a typical four-chain quaternary structure with two heavy chains and two light chains, as in the case of antibodies from other animals. Structurally different. See PCT / EP93 / 02214 (published as WO 94 / 04678 on 3 March 1994). See also WO 94 / 04678.

[0155] The region of the camelid antibody, a small single variable domain identified as a VHH, results in a low molecular weight protein with high affinity for the target, thus resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". It can be obtained by genetic engineering. See US Patent No. 5,759,808, issued 2 June 1998. See also Stijlemans, B. et al., 2004 J Biol Chem 279:1256-1261; Dumoulin, M. et al., 2003 Nature 424:783-788; Pleschberger, M. et al., 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al., 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al., 1998 EMBO J 17:3512-3520. Libraries of engineered camelid antibodies and antibody fragments are commercially available, e.g., from Ablynx, Ghent, Belgium. Similar to other non-human-derived antibodies, the amino acid sequence of camelid antibodies can be recombinantly modified to obtain sequences more similar to human sequences, i.e., nanobodies can be "humanized". Thus, the natural low antigenicity of camelid antibodies towards humans can be further reduced.

[0156] ​​​​​​​​​​​​The molecular weight of a camelid nanobody is approximately one tenth of the molecular weight of a human IgG molecule, and the physical diameter of the protein is only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to large antibody proteins, i.e., camelid nanobodies are useful as reagents for detecting antigens that would otherwise remain hidden in forms using classical immunological techniques and are useful as potential therapeutic agents. Thus, yet another consequence of the small size is that camelid nanobodies can inhibit target proteins as a result of binding to specific sites within the grooves or narrow clefts of target proteins, and thus can be used in an ability that more closely resembles the function of classical low molecular weight drugs than the function of classical antibodies. The small size and compact nature also result in camelid nanobodies that are extremely thermostable, stable to extreme pH and proteolytic digestion, and have low antigenicity. Another consequence is that camelid nanobodies can easily move from the circulatory system to tissues, cross the blood-brain barrier, and treat disorders that affect nerve tissue. Nanobodies can also facilitate drug transport across the blood-brain barrier. See U.S. Patent Application Publication No. 20040161738, published on August 19, 2004. Combining these features with the low antigenicity towards humans indicates great therapeutic potential. Furthermore, these molecules can be fully expressed in prokaryotic cells such as Escherichia coli (E. coli) and used as fusion proteins with bacteriophages.

[0157] ​​​​​​​​​​​ is expressed and is functional.

[0158] Therefore, the feature of the present invention is a llama antibody or llama nanobody having a high affinity for ANGPTL4. In certain embodiments of the present specification the llama antibody or llama nanobody is produced natively in llamas, i.e., produced by llamas after immunization with ANGPT TL4 or a peptide fragment thereof using the techniques described herein for other antibodies. Alternatively the ANGPTL4-binding llama nanobody is also engineered, i.e., for example using the panning procedures with ANGPTL4 as the target described in the examples herein to select from a phage library presenting appropriately mutagenized llama nanobody proteins. The engineered nanobody can further be customized by genetic engineering such that the half-life in the recipient subject is between 45 minutes and 2 weeks . In a specific embodiment, the llama antibody or llama nanobody is obtained, for example as described in PCT / EP93 / 02214, by grafting the CDR sequences of the heavy or light chains of the human antibodies of the present invention onto the nanobody or single domain antibody framework sequences.

[0159] Bispecific molecules and multivalent antibodies In another aspect, the present invention features a bispecific molecule or multispecific molecule comprising an ANGPTL4-binding antibody or a fragment thereof of the present invention. The antibody or antigen-binding region thereof of the present invention creates a bispecific molecule that binds to at least two different binding sites or target molecules ​ It can also be derivatized and linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor). The antibodies of the present invention can actually be derivatized to create multispecific molecules that bind to three or more different binding sites and / or target molecules, and can also be linked to two or more other functional molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the present invention, an antibody of the present invention can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, such that a bispecific molecule results. Accordingly, the present invention includes bispecific molecules that include at least a first binding specificity for ANGPTL4 and a second binding specificity for a second target epitope. For example, the second target epitope is a different epitope of ANGPTL4 that is different from the first target epitope. In addition, in the present invention where the bispecific molecule is multispecific, the molecule can further include a third binding specificity in addition to the first and second target epitopes. In one embodiment, the bispecific molecule of the present invention includes, as a binding specificity, at least one antibody or a fragment thereof that includes, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv. It can be derivatized to create multispecific molecules that bind to three or more different binding sites and / or target molecules, and can also be linked to two or more other functional molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the present invention, an antibody of the present invention can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, such that a bispecific molecule results. It can be derivatized to create multispecific molecules that bind to three or more different binding sites and / or target molecules, and can also be linked to two or more other functional molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the present invention, an antibody of the present invention can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, such that a bispecific molecule results. (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise).

[0160] Thus, the present invention includes bispecific molecules that include at least a first binding specificity for ANGPTL4 and a second binding specificity for a second target epitope. For example, the second target epitope is a different epitope of ANGPTL4 that is different from the first target epitope. For example, the second target epitope is a different epitope of ANGPTL4 that is different from the first target epitope.

[0161] In addition, in the present invention where the bispecific molecule is multispecific, the molecule can further include a third binding specificity in addition to the first and second target epitopes. In addition, in the present invention where the bispecific molecule is multispecific, the molecule can further include a third binding specificity in addition to the first and second target epitopes.

[0162] In one embodiment, the bispecific molecule of the present invention includes, as a binding specificity, at least one antibody or a fragment thereof that includes, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv. b', F(ab')2, Fv, or single-chain Fv. It includes antibody fragments. The antibody can also be a dimer of the light chain or the heavy chain, or an Fv or single-chain construct as described in Ladner et al., U.S. Patent No. 4,946,778, any minimal fragment thereof.

[0163] A diabody is a bivalent bispecific molecule in which a VH domain and a VL domain connected by a linker that is too short to allow pairing between two domains on the same chain are expressed on a single polypeptide chain. The VH domain and the VL domain pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, for example, Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 Structure 2:1121-1123). Diabodies can be prepared by expressing two polypeptide chains with VHA-VLB and VHB-VLA structures (VH-VL configuration), or VLA-VHB and VLB-VHA structures (VL-VH configuration) in the same cell. Most diabodies can be expressed in a soluble form in bacteria. A single-chain diabody (scDb) is prepared by connecting two diabody-forming polypeptide chains with a linker of about 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36). ScDb is a soluble and active monomer in bacteria iger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 S tructure 2:1121-1123). The diabody is expressed in the same cell by r, 1997 Cancer Immunol. Immunother., 45(3-4):128-30; Wu et al., 1996 Immunot (Holliger and Winter, 1997 Cancer Immunol. Immuno ther., 45(34):128-30;Wu et al., 1996 Immunotechnology, 2(1):21-36;Pluckthu n and Pack, 1997 Immunotechnology, 3(2):83-105; Ridgway et al., 1996 Protein Eng., 9(7):617-21. (Lu et al., 2004 J. Biol. Chem., 279(4):2856- (See 65).

[0164] Other antibodies that may be incorporated within the bispecific molecules of the invention include murine monoclonal antibodies, chimeric monoclonal antibodies, and humanized monoclonal antibodies.

[0165] Bispecific molecules can be prepared by covalently binding the constituent binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be conjugated to a corresponding The isomers can be generated separately and then conjugated to one another. When the target substance is a protein or peptide, various coupling or cross-linking agents can be used. Examples of cross-linking agents include protein A, protein B, and protein C. Rubodiimide, N-Succinimidyl-S-acetyl-thioacetate (SATA), 5 ,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (S PDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane -1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648 ). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; B rennan et al., 1985 Science 229:81-83; and Glennie et al., 1987 J. Immunol. 13 9:2367-2375. Conjugating agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Roc kford, IL).

[0166] When the binding specificities are antibodies, they can be conjugated by sulfhydryl bonding the C-terminal hinge regions of the two heavy chains. In certain embodiments, prior to conjugation, the hinge region is modified to contain an odd number of sulfhydryl residues, for example, one sulfhydryl residue.

[0167] Alternatively, any binding specificities can be encoded within the same vector and expressed and assembled within the same host cell. This method is particularly useful when the bispecific molecule is an mAb×mAb fusion protein, an mAb×Fab fusion protein, a Fab×F(ab’)2 fusion protein, or a ligand×Fab fusion protein. The bispecifics of the present invention The specific molecule may be a single-chain molecule containing one single-chain antibody and a binding determinant, or may be a single-chain bispecific molecule containing two binding determinants. The bispecific molecule may include at least two single-chain molecules. For methods of preparing bispecific molecules, see, for example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. The binding of the bispecific molecule to its specific target can be confirmed, for example, by enzyme immunoassay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition assay), or Western blot assay. In each of these assays, in general, the presence of a particular protein-antibody complex is detected by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. In another aspect, the present invention provides a multivalent compound comprising at least two identical or different antigen-binding portions of the antibodies of the present invention that bind to ANGPTL4. The antigen-binding portions can be linked together via protein fusion or covalent or non-covalent linkage. Alternatively, methods of linking for bispecific molecules are also described. For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0168] For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. For example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition assay), or Western blot assay. For example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition assay), or Western blot assay. For example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition assay), or Western blot assay. For example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition assay), or Western blot assay. For example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition assay), or Western blot assay.

[0169] In another aspect, the present invention provides a multivalent compound comprising at least two identical or different antigen-binding portions of the antibodies of the present invention that bind to ANGPTL4. The antigen-binding portions can be linked together via protein fusion or covalent or non-covalent linkage. The antigen-binding portions can be linked together via protein fusion or covalent or non-covalent linkage. The antigen-binding portions can be linked together via protein fusion or covalent or non-covalent linkage. The tetravalent compound can be obtained, for example, by crosslinking the antibodies of the invention (antibodies of the antibodies of the invention) with an antibody that binds to the constant region of the antibodies of the invention, such as the Fc region or the hinge region. The trimerization domain is described, for example, in European Patent No. 1012280, a patent by Borean. The pentamerization module is described, for example, in PCT / EP97 / 05897. can be obtained by crosslinking.

[0170] For the trimerization domain, for example, it is described in European Patent No. 10 12280. For the pentamerization module, for example, it is described in PCT / EP97 / 05897.

[0171] Antibodies with extended half-life The present invention provides antibodies that specifically bind to the ANGPTL4 protein and have an extended half-life in vivo. bind specifically.

[0172] Many factors can affect the half-life of proteins in vivo. For example, filtration in the kidney, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenic responses (e.g., neutralization of proteins by antibodies and uptake by macrophages and dendritic cells). Various strategies can be used to extend the half-life of the antibodies of the present invention. For example, by chemical linkage with polyethylene glycol (PEG), reCODE PEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin binding ligands, and carbohydrate shields, genetic fusion with proteins that bind to serum proteins such as albumin, IgG, FcRn, and transferrin, nanobodies, Fab, DARPin, avimers, aff can be extended. For example, polyethylene glycol (PEG), reCODE PEG antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin binding ligands, and carbohydrate shields, genetic fusion with proteins that bind to serum proteins such as albumin, IgG, FcRn, and transferrin (transferring), nanobodies, Fab, DARPin, avimers, aff to bind to serum proteins such as albumin, IgG, FcRn, and transferrin proteins, nanobodies, Fab, DARPin, avimers, aff By conjugating (genetically or chemically) with other binding moieties that bind to serum proteins, such as the ibody and anti - carin, the half - life of the antibodies of the present invention can be extended by rPEG, albumin, albumin domains, albumin - binding proteins, and genetic fusions with Fc, or by incorporation into nanocarriers, sustained - release formulations, or medical devices. To extend the in vivo serum circulation of antibodies, inactive polymer molecules, such as high - molecular - weight PEG, can be attached to antibodies or their fragments via site - specific conjugation of PEG to the N - terminus or C - terminus of the antibody, or via the epsilon - amino groups present on lysine residues, with or without a multifunctional linker. Typically, to PEGylate an antibody, the antibody or its fragment is reacted with PEG, such as a reactive ester or aldehyde derivative of polyethylene glycol (PEG), under conditions where one or more PEG groups attach to the antibody or antibody fragment. PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water - soluble polymer). The term "polyethylene glycol" as used herein is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1 - C10) alkoxy - polyethylene glycol or aryloxy - polyethylene glycol or polyethylene glycol - maleimide.

[0173] In certain embodiments, the PEGylated antibody is a deglycosylated antibody. It can be a linear polymer or a branched polymer. By conjugating (genetically or chemically) with other binding moieties that bind to serum proteins, such as the ibody and anti - carin, the half - life of the antibodies of the present invention can be extended by rPEG, albumin, albumin domains, albumin - binding proteins, and genetic fusions with Fc, or by incorporation into nanocarriers, sustained - release formulations, or medical devices. To extend the in vivo serum circulation of antibodies, inactive polymer molecules, such as high - molecular - weight PEG, can be attached to antibodies or their fragments via site - specific conjugation of PEG to the N - terminus or C - terminus of the antibody, or via the epsilon - amino groups present on lysine residues, with or without a multifunctional linker. Typically, to PEGylate an antibody, the antibody or its fragment is reacted with PEG, such as a reactive ester or aldehyde derivative of polyethylene glycol (PEG), under conditions where one or more PEG groups attach to the antibody or antibody fragment. PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water - soluble polymer). The term "polyethylene glycol" as used herein is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1 - C10) alkoxy - polyethylene glycol or aryloxy - polyethylene glycol or polyethylene glycol - maleimide. In certain embodiments, the PEGylated antibody is a deglycosylated antibody. It can be a linear polymer or a branched polymer. The term "polyethylene glycol" as used herein is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1 - C10) alkoxy - polyethylene glycol or aryloxy - polyethylene glycol or polyethylene glycol - maleimide. In certain embodiments, the PEGylated antibody is a deglycosylated antibody. It can be a linear polymer or a branched polymer. By conjugating (genetically or chemically) with other binding moieties that bind to serum proteins, such as the ibody and anti - carin, the half - life of the antibodies of the present invention can be extended by rPEG, albumin, albumin domains, albumin - binding proteins, and genetic fusions with Fc, or by incorporation into nanocarriers, sustained - release formulations, or medical devices. To extend the in vivo serum circulation of antibodies, inactive polymer molecules, such as high - molecular - weight PEG, can be attached to antibodies or their fragments via site - specific conjugation of PEG to the N - terminus or C - terminus of the antibody, or via the epsilon - amino groups present on lysine residues, with or without a multifunctional linker. Derivatization will be used that results in minimal loss of biological activity. The degree of conjugation will be monitored closely by SDS-PAGE and mass spectrometry to confirm proper conjugation of the PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion chromatography or ion exchange chromatography. PEG-derivatized antibodies can be assayed for binding activity and in vivo efficacy using methods well known to those of skill in the art, such as the immunoassays described herein. In the art, methods for PEGylating proteins are known and can be applied to the antibodies of the present invention. See, for example, European Patent No. 0154316 by Nishimura et al. and European Patent No. 0401384 by Ishikawa et al. Other modified PEGylation techniques include reconstituting chemically orthogonal directed engineering (ReCODE PEG) that incorporates chemically specified side chains into biosynthetic proteins via a reconstitution system containing tRNA synthetase and tRNA. This technique enables the incorporation of more than 30 new amino acids into biosynthetic proteins in E. coli cells, yeast cells, and mammalian cells. The tRNA incorporates unnatural amino acids at any position where the amber codon is placed and converts the amber from the stop codon to a codon that signals the incorporation of a chemically specified amino acid.

[0174]

[0175] The recombinant PEGylation technology (rPEG) can also be used to extend the serum half-life. . This technology involves genetically fusing an unstructured protein tail of 300 - 600 amino acids to an existing pharmaceutical protein. Since the apparent molecular weight of such an unstructured protein chain is about 15 times its actual molecular weight, the serum half-life of the protein is greatly increased. In contrast to conventional PEGylation that requires chemical conjugation and repurification, the manufacturing process is greatly simplified and the product is homogeneous.

[0176] Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to extend the active lifespan and improve the stability of therapeutic peptides and therapeutic proteins. PSA is a polymer of sialic acid (sugar). When used for drug delivery of proteins and therapeutic peptides, during conjugation, the polysialic acid provides a protective microenvironment . This extends the active lifespan of the therapeutic protein in circulation and prevents it from being recognized by the immune system . PSA polymers are found naturally in the human body. PSA has been adopted by certain species of bacteria that have evolved over millions of years to coat their walls with PSA. These naturally polysialylated bacteria can then, through molecular mimicry, disable the body's defense system. PSA, the ultimate natural stealth technology , can be easily produced in large quantities from such bacteria with predetermined physical characteristics. Since bacterial PSA is chemically identical to PSA in the human body , it remains completely non-immunogenic even when coupled to proteins.

[0177] ​​​ Another technique involves the use of hydroxyethyl starch (“HES”) derivatives conjugated to an antibody HES is a modified natural polymer derived from waxy maize starch and can be metabolized by enzymes in the body. HES solutions are typically administered to replenish blood volume and improve the rheological properties of blood HESylation of an antibody not only increases the stability of the molecule but also allows for an extended circulation half-life by reducing renal clearance, resulting in increased biological activity By modifying different parameters such as the molecular weight of HES a wide range of HES-antibody conjugates can be customized

[0178] Antibodies with an extended half-life in vivo can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain or its FcRn-binding fragments (preferably the Fc domain fragment or the hinge Fc domain fragment). See, for example, WO 98 / 23289, WO 97 / 34631; and U.S. Pat. No. 6,277,375

[0179] Furthermore, an antibody or antibody fragment can be conjugated to albumin (e.g., human serum albumin; HSA) to make the antibody more stable in vivo or to extend its half-life in vivo Techniques are well known in the art and are described, for example, in WO 93 / 15199, WO 93 / 152 000, and WO 01 / 77137; and European Patent No 000, and WO 01 / 77137; and European Patent No ​​​​See U.S. Patent No. 413,622. Additionally, in the context of the bispecific antibodies described above the specificity of the antibody is such that one binding domain of the antibody binds to ANGPTL4, while the second binding domain of the antibody can be designed to bind to serum albumin, preferably HSA.

[0180] Strategies for extending the half-life are particularly useful in nanobodies, fibronectin-based binders, and other antibodies or proteins where an extended half-life in vivo is desired.

[0181] Antibody Conjugates The present invention provides antibodies or fragments thereof that specifically bind to the ANGPTL4 protein, which are recombinantly fused or chemically conjugated (including both covalent conjugation and non-covalent conjugation) to a heterologous protein or heterologous polypeptide (or a fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30 at least 40, at least 50, at least 60, at least 70, at least 80 at least 90, or at least 100 amino acids) to create a fusion protein. In particular, the present invention provides fusion proteins comprising an antigen-binding fragment of an antibody described herein (e.g., a Fab fragment, an Fd fragment, an Fv fragment, an F(ab)2 fragment a VH domain, a VH CDR, a VL domain, or a VL CDR) and a heterologous protein, heterologous polypeptide, or heterologous peptide. In the art, it is common to fuse a protein, polypeptide, or peptide to an antibody or antibody fragment ​​​​​​​​or methods of conjugation are known. For example, U.S. Patent No. 5,336,603 Specification, No. 5,622,929 Specification, No. 5,359,046 Specification, No. 5 ,349,053 Specification, No. 5,447,851 Specification, and No. 5,112, 946 Specification; European Patent Nos. 307,434 and 367,166 ; WO 96 / 04388 Pamphlet and WO 91 / 06570 Pamphlet ; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341. See also.

[0182] Additional fusion proteins can be created via techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). By employing DNA shuffling, the activity of the antibodies of the present invention or fragments thereof can be modified (e.g., antibodies or fragments thereof with high affinity and low dissociation rate). Generally, U.S. Patent Nos. 5,605,7 93, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al ., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. ., 1998, Trends Biotechnol. 16:76-82; Hansson, 1999, J. Biotechnol. 71:1-16; and Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91:10747-10751. See also. ., 1998, Trends Biotechnol. 16:76-82; Hansson, 1999, J. Biotechnol. 71:1-16; ., 1998, Trends Biotechnol. 16:76-82; Hansson, 1999, J. Biotechnol. 71:1-16; ., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo a nd Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications is , incorporated herein by reference in its entirety). Antibodies or their fragments, or the encoded antibodies or their fragments, can be modified by, prior to recombination, random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding antibodies or fragments thereof that specifically bind to the ANGPTL4 protein can be recombined with one or more components, motifs, regions, portions, domains, fragments, etc. of one or more heterologous molecules.

[0183] Further, antibodies or their fragments can be fused to marker sequences such as peptides that facilitate purification. In a preferred embodiment, the amino acid sequences of the markers are, among many of which are commercially available, hexahistidine peptides such as the tags provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311). As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine provides for facile purification of the fusion protein. Other peptide tags useful for purification are epitopes derived from the influenza hemagglutinin protein (Wilson et al., 1 A hemagglutinin ("HA") tag corresponding to 984, Cell 37:767), and a "Flag" tag including but not limited to these.

[0184] In other embodiments, the antibodies of the invention or fragments thereof are conjugated to a diagnostic or detectable agent. Such antibodies can be useful for monitoring or prognosticating as part of clinical testing procedures, such as determining the occurrence, onset, progression, and / or severity of a disease or disorder, or the effectiveness of a particular treatment. Such diagnoses and detections are performed by conjugating the antibody to a variety of enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, but not limited to these; bridging molecule families such as streptavidin / biotin and avidin / biotin, but not limited to these; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin, but not limited to these; luminescent materials such as luminol, but not limited to these; bioluminescent materials such as luciferase, luciferin, and aequorin, but not limited to these; iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 etc. useful. to a variety of enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, but not limited to these; bridging molecule families such as streptavidin / biotin and avidin / biotin, but not limited to these; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin, but not limited to these; luminescent materials such as luminol, but not limited to these; bioluminescent materials such as luciferase, luciferin, and aequorin, but not limited to these; iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 ; bridging molecule families such as streptavidin / biotin and avidin / biotin, but not limited to these; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin, but not limited to these; luminescent materials such as luminol, but not limited to these; bioluminescent materials such as luciferase, luciferin, and aequorin, but not limited to these; iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 etc. Iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 ; technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 c), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103 Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 9 0Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68G e, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54 Mn, 75Se, 113Sn, and 117Tin, etc., but not limited to these radioactive materials; and coupling to detectable substances including, but not limited to, positron-emitting metals and non-radioactive paramagnetic metal ions using various positron tomography methods can be achieved. The present invention further encompasses the use of antibodies or fragments thereof conjugated to a therapeutic moiety. The antibody or fragment thereof can be conjugated to a cytotoxic agent, such as a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, such as an alpha emitter, etc. The cytotoxic or cytopathic agent includes any agent harmful to cells. Furthermore, the antibody or fragment thereof can also be conjugated to a therapeutic moiety or a drug moiety that modifies a given biological response. The therapeutic moiety or drug moiety is not considered to be limited to classical chemically therapeutic agents. For example, the drug moiety can be a protein, peptide, or polypeptide having a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis

[0185] The present invention further encompasses the use of antibodies or fragments thereof conjugated to a therapeutic moiety. The antibody or fragment thereof can be conjugated to a cytotoxic agent, such as a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, such as an alpha emitter, etc. The cytotoxic or cytopathic agent includes any agent harmful to cells.

[0186] Furthermore, the antibody or fragment thereof can also be conjugated to a therapeutic moiety or a drug moiety that modifies a given biological response. The therapeutic moiety or drug moiety is not considered to be limited to classical chemically therapeutic agents. For example, the drug moiety can be a protein, peptide, or polypeptide having a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis is a toxin such as diphtheria toxin; tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis is a factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis ​​​Cytotoxic agents, anti-angiogenic agents; or biologic response modifiers such as, for example, lymphokines, and the like may include proteins.

[0187] Furthermore, the antibody may be conjugated to a therapeutic moiety such as a macrocyclic chelating agent useful for conjugating radioactive metal ions such as alpha-emitters such as 213Bi, radioactive metal ions including 131In, 131Lu, 131Y, 131Ho, 131Sm, but not limited to these, to the polypeptide. In certain embodiments, the macrocyclic chelating agent may be 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’- tetraacetic acid (DOTA), which may be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are incorporated herein by reference in their entirety from Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50. .

[0188] Techniques for conjugating a therapeutic moiety to an antibody are well known, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Con trolled Drug Delivery(2nd Ed.), Robinson et al.,(eds.), pp.623-53(Marcel De kker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Ther apy: A Review", Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al.(eds.), pp.475-506(1985); "Analysis, Results, And Future Pros pective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", Mono clonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp.3 03-16(Academic Press 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58 See also.

[0189] Antibodies can also be attached to solid supports that are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0190] Methods for producing the antibodies of the present invention ​​Nucleic acid encoding an antibody The present invention provides a substantially purified nucleic acid molecule encoding a polypeptide comprising a segment or domain of the ANGPTL4-binding antibody chain described above. Some of the nucleic acids of the present invention encode nucleotide sequences encoding heavy chain variable regions shown in SEQ ID NOs: 13, 38, 58, 78, 98, 118, or 138, and / or SEQ ID NOs: 23, 48, 6 8, 88, 108, 128, or 148, and / or nucleotide sequences encoding light chain variable regions shown in SEQ ID NOs: 23, 48, 6 8, 88, 108, 128, or 148. In a specific embodiment, the nucleic acid molecule is the nucleic acid molecule identified in Table 1 Some of the other nucleic acid molecules of the present invention are substantially (e.g., at least 65, 80%, 95%, or 99%) identical to the nucleotide sequence of the nucleic acid molecule identified in Table 1 Containing a nucleotide sequence. When expressed from a suitable expression vector, the polypeptides encoded by these polynucleotides are capable of exhibiting ANGPTL4 antigen-binding ability

[0191] The present invention also provides polynucleotides encoding at least one CDR region derived from the heavy or light chain of the ANGPTL4-binding antibody shown above, and usually all three CDR regions Other polynucleotides encode all or substantially all of the variable region sequences of the heavy and / or light chains of the ANGPTL4-binding antibody shown above Due to the degeneracy of the code, various nucleic acid sequences will encode each of the immunoglobulin amino acid sequences

[0192] The nucleic acid molecules of the present invention can encode both the variable and constant regions of an antibody. The present invention ​​​​​​​​Some of the nucleic acid sequences are SEQ ID NO: 15, 28, 40, 60, 80, 100, 120 , and nucleotides encoding a heavy chain sequence that is substantially (e.g., at least 80%, 90%, or 99%) identical to the heavy chain sequences shown in SEQ ID NO: 140. Some other nucleic acid sequences are SEQ ID NO: 25, 50, 70, 90, 110, 130, and 150, and nucleotides encoding a light chain sequence that is substantially (e.g., at least 80%, 90%, or 99%) identical to the light chain sequences shown in SEQ ID NO: 10.

[0193] The polynucleotide sequences can be made by de novo solid-phase DNA synthesis or by mutagenesis by PCR on existing sequences encoding ANGPTL4-binding antibodies or binding fragments thereof (e.g., the sequences described in the examples below). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method by Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method by Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method by Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method by U.S. Patent No. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be performed, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Protocols, Innis et al. (Eds.), Academic Press, San Diego, CA, 1990; and Current Protocols in Molecular Biology, Ausubel et al. (Eds.), Greene Publishing and Wiley-Interscience, NY, NY, 1992. ) The direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method by Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method by Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method by Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method by U.S. Patent No. 4,458,066. The introduction of mutations into polynucleotide sequences by PCR can be performed, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Protocols, Innis et al. (Eds.), Academic Press, San Diego, CA, 1990; and Current Protocols in Molecular Biology, Ausubel et al. (Eds.), Greene Publishing and Wiley-Interscience, NY, NY, 1992. The direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method by Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method by Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method by Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method by U.S. Patent No. 4,458,066. The introduction of mutations into polynucleotide sequences by PCR can be performed, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Protocols, Innis et al. (Eds.), Academic Press, San Diego, CA, 1990; and Current Protocols in Molecular Biology, Ausubel et al. (Eds.), Greene Publishing and Wiley-Interscience, NY, NY, 1992. Protocols, Innis et al. (Eds.), Academic Press, San Diego, CA, 1990; and Current Protocols in Molecular Biology, Ausubel et al. (Eds.), Greene Publishing and Wiley-Interscience, NY, NY, 1992. ​Applications, Innis et al.(Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Ap plications 1:17, 1991 can be carried out as described therein.

[0194] The present invention also provides an expression vector and a host cell for producing the ANGPTL4-binding antibody described above. A variety of expression vectors can be employed to express a polynucleotide encoding an ANGPTL4-binding antibody chain or a binding fragment. Using either a virus-based expression vector or a non-viral expression vector, an antibody can be produced in mammalian host cells. Non-viral vectors and non-viral systems include plasmids, typically episomal vectors with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, for example, Harrington et al., Nat Genet 15:345, 1997). For example, non-viral vectors useful for the expression of ANGPTL4-binding polynucleotides and ANGPTL4-binding polypeptides in mammalian (e.g., human) cells are pThioHis A, pThioHis B, and pThioHis C, pcDNA3.1 / His, pEBVHis A, pEBVH is B, and pEBVHis C (Invitrogen, San Diego, C A), MPSV vectors, and others known in the art for expressing other proteins. (See, for example, Harrington et al., Nat Genet 15:345, 1997). For example, within mammalian (e.g., human) cells of ANGPTL4-binding polynucleotides and ANGPTL4-binding polypeptides useful non-viral vectors are pThioHis A, pThioHis B, and pThioHis C, pcDNA3.1 / His, pEBVHis A, pEBVH is B, and pEBVHis C (Invitrogen, San Diego, C A), MPSV vectors, and other proteins to be expressed, in the art It includes a number of other known vectors. Useful viral vectors include retroviruses, adeno viruses, adeno-associated viruses, herpesvirus-based vectors, SV40-based ve ctors, papillomaviruses, Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992 for reference.

[0195] The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Expression vectors typically contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an ANGPTL4-binding antibody chain or fragment. In some embodiments, an inducible promoter is employed to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, the arabinose, lacZ, metallothionein promoter, or heat shock promoter. Cultures of the transformed organisms can be grown under non-inducing conditions without biasing the population of coding sequences whose expression products are well tolerated by the host cell. In addition to the promoter, other regulatory elements may also be required or desired for efficient expression of the ANGPTL4-binding antibody chain or fragment. These elements typically include the ATG initiation codon and adjacent ribosome-binding site or other sequences. In addition, the efficiency of expression can be enhanced by incorporating enhancers suitable for the cell line being used. It can also be enhanced by (for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and see Bittner et al., Meth. Enzymol., 153:516, 1987 ). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0196] The expression vector may also provide for the position of a secretion signal sequence to form a fusion protein with the polypeptide encoded by the inserted ANGPTL4-binding antibody sequence. The inserted ANGPTL4-binding antibody sequence is often ligated to a signal sequence prior to incorporation into the vector. Vectors used to receive sequences encoding the light chain variable domain and heavy chain variable domain of an ANGPTL4-binding antibody sometimes also encode a constant region or portions thereof. Such vectors allow for the expression of the variable region as a fusion protein with the constant region, thereby resulting in the production of intact antibodies or fragments thereof. Such constant regions are typically human constant regions. Host cells carrying and expressing an ANGPTL4-binding antibody chain can be prokaryotic cells in some cases or eukaryotic cells in some cases. Escherichia coli (E. coli) is one prokaryotic host useful for cloning and expressing the polynucleotide of the present invention. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and Salmonella

[0197] of the present invention. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and Salmonella ella (Salmone lla) species, Serratia species, and various Pseudomonas species and other Enterobacteriaceae. In these prokaryotic hosts, expression vectors containing expression control sequences (e.g., origins of replication) compatible with the host cells can also typically be prepared. In addition, any number of various well-known promoters exist, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system derived from phage lambda. The promoter is typically, optionally, regulated in expression by an operator sequence, but also has a ribosome-binding site sequence and the like for initiating and terminating transcription and translation. Other microorganisms, such as yeast, can also be employed to express the ANGPTL 4-binding polypeptide of the present invention. Insect cells combined with baculovirus vectors can also be used. In some preferred embodiments, mammalian host cells are used to express and produce the ANGPTL4-binding polypeptide of the present invention. For example, they can be hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the 1D6.C9 myeloma hybridoma clone described in the examples), or mammalian cell lines that carry an exogenous expression vector (e.g., the SP2 / 0 myeloma cells exemplified below). They include any normal non-immortalized animal cells or normal immortalized animal cells or non-normal immortalized animal cells or human cells. For example, they include intact immunoglobulin including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas.

[0198] In some preferred embodiments, mammalian host cells are used to express the ANGPTL4-binding polypeptide of the present invention. For example, they can be hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the 1D6.C9 myeloma hybridoma clone described in the examples), or mammalian cell lines that carry an exogenous expression vector (e.g., the SP2 / 0 myeloma cells exemplified below). They include any normal non-immortalized animal cells or normal immortalized animal cells or non-normal immortalized animal cells or human cells. For example, they include intact immunoglobulin including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. They include any normal non-immortalized animal cells or normal immortalized animal cells or non-normal immortalized animal cells or human cells. For example, they include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. A number of suitable host cell lines capable of secreting phosphorus have been developed. For the use of mammalian tissue cell cultures for expressing polypeptides, see, for example, Winnacker, FRO M GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987, which is generally discussed in. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain a promoter derived from a mammalian gene or a promoter derived from a mammalian virus. Suitable promoters can be constitutive promoters, cell type-specific promoters, stage-specific promoters, and / or modulatable promoters or regulatable promoters. Useful promoters include the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter , the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and combinations of promoters-enhancers known in the art, including but not limited to these. The method of introducing an expression vector containing the target polynucleotide sequence depends on the species of the cell host

[0199] ​​​​​​It varies according to the type. For example, calcium chloride transfection is generally used for prokaryotic cells whereas calcium phosphate treatment or electroporation can be used for other cell hosts (generally, see Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microin jection, gene gun method, virosomes, immunoliposomes, polycation:nucleic acid con jugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and transduction ex vivo. For the long-term high-yield production of recombinant proteins, stable expression is often desired. For example, a cell line that stably expresses an ANGPTL4-binding antibody chain or binding fragment can be prepared using the expression vector of the present invention, which contains a viral origin of replication or an endogenous expression element, and a selectable marker gene. After introducing the vector, the cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The purpose of the selectable marker is to confer resistance to selection and allow cell growth by its presence, thereby successfully expressing the introduced sequence in selective medium. Cells that are stably transfected with resistance can be propagated using tissue culture methods suitable for the cell type . .

[0200] Production of Monoclonal Antibodies of the Present Invention Monoclonal antibodies (mAbs) can be produced by conventional monoclonal antibody methods, for example, Kohler and Standard somatic hybridization techniques according to Milstein, 1975 Nature 256: 495 It can be prepared by various techniques, including those for producing monoclonal antibodies. Many techniques can be employed, such as viral transformation or oncogenic transformation of B lymphocytes. It can be utilized.

[0201] Animal systems for preparing hybridomas include mouse, rat, and rabbit systems. The production of hybridomas in mice is a well-established procedure. In the art, immunization protocols and techniques for isolating immunized spleen cells for fusion are known. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. They are also known.

[0202] The chimeric or humanized antibodies of the present invention can be prepared based on the sequences of mouse monoclonal antibodies prepared as described above. DNA encoding the heavy chain immunoglobulin and the light chain immunoglobulin is obtained from the mouse hybridoma of interest and can be manipulated using standard molecular biological techniques to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to create a chimeric antibody, methods known in the art (e.g., see U.S. Patent No. 4,816,567 by Cabilly et al.) can be used to link the mouse variable region to the human constant region. To create a humanized antibody, methods known in the art can be used to insert the mouse CDR regions into the human framework. For example, U.S. Patent No. 5,225,539 by Winter, as well as U.S. Patent No. 5,530,101 by Queen et al.; For example, U.S. Patent No. 5,225,539 by Winter, as well as U.S. Patent No. 5,530,101 by Queen et al.; For example, U.S. Patent No. 5,225,539 by Winter, as well as U.S. Patent No. 5,530,101 by Queen et al.; For example, U.S. Patent No. 5,225,539 by Winter, as well as U.S. Patent No. 5,530,101 by Queen et al.; See the specifications of Patent No. 5585089, Patent No. 5693762, and Patent No. 6180370. Please refer to them.

[0203] In certain embodiments, the antibodies of the invention are humanized antibodies. Such humanized antibodies that target ANGPTL4 can be produced using transgenic mice or transchromosomal mice that possess parts of the human immune system rather than the murine system. These transgenic mice and transchromosomal mice include mice that are referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice". HuMAb mice (registered trademark) (Medarex, Inc.) contain a mini-locus of human immunoglobulin genes that encodes rearranged non-human heavy (μ and γ) chain immunoglobulin sequences and human κ light chain immunoglobulin sequences, along with a targeted mutation that inactivates the endogenous μ chain locus and the endogenous κ chain locus (see, for example, Lonberg, N., et al., 1994 Nature 368(6474): 856-859). Thus, the mice exhibit reduced expression of mouse IgM or mouse Igκ and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic hypermutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg, N., et al., 1994 supra; Lonberg, N., 1994 Handbook of Experimental Pharmacology 113:

[0204] ​​​​​​​​​​​49-101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Har ding, F. and Lonberg, N., 1995 Ann. N. Y. Acad. Sci. 764: 536-546, which are reviewed in (HuMAb mice preparation and use, and genomic modifications carried by such mice are all specifically incorporated herein by reference in their entirety, Taylor, L. et al., 1992 Nucleic Acids Research 20: 6287-6295; Chen, J. et al., 1993 International Immunology 5: 647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94: 3720-3724; Choi et al., 1993 Nature Genetics 4: 117-123; Chen, J. et al., 1993 EMBO J. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152: 2912-2920; Taylor, L. et al., 1994 International Immunology 5: 79-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14: 845-851. Further, all of the specifications of U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,916,771; 5,939,598; 6,075,181; 6,114,598; 6,118,046; 6,130,364; 6,140,086; 6,180,370; 6,215,058; 6,242,560; 6,281,335; 6,300,129; 6,303,340; 6,319,696; 6,329,158; 6,342,666; 6,368,801; 6,372,937; Specification of Japanese Patent No. 877,397; Specification of Japanese Patent No. 5,661,016; Specification of Japanese Patent No. 5,814,318 Specification of Japanese Patent No. 5,874,299; and Specification of Japanese Patent No. 5,770,429; Specification of U.S. Patent No. 5,545,807 by Surani et al.; all by Lonberg and Kay, PCT International Publication No. 92 / 103918 Pamphlet, International Publication No. 93 / 12227 Pamphlet, International Publication No. 94 / 25585 Pamphlet, International Publication No. 9 7113852 Pamphlet, International Publication No. 98 / 24884 Pamphlet, and International Publication No. 9 99 / 45962 Pamphlet; and also refer to PCT International Publication No. 01 / 14424 Pamphlet by Korman et al.

[0205] In another embodiment, any mouse carrying a human heavy chain transgene and a human light chain transgenic chromosome, or a mouse carrying a human immunoglobulin sequence on the transgene and the transgenic chromosome can be used to provide the human antibodies of the present invention. In this specification, such a mouse is referred to as a "KM mouse", which is described in detail in PCT International Publication No. 02 / 43478 Pamphlet by Ishida et al.

[0206] In the art, even more alternative transgenic animal systems are available for expressing human immunoglobulin genes and can be used to provide the ANGPTL4-binding antibodies of the present invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can also be used. Regarding such mice, for example, refer to U.S. Patent No. 5,939,598 by Kucherlapati et al. ​​​​​​​Specification; the specification of Japanese Patent No. 6,075,181; the specification of Japanese Patent No. 6,114,598; the specification of Japanese Patent No. 6, 150,584; and the specification of Japanese Patent No. 6,162,963. It is described in.

[0207] Furthermore, in the art, alternative transgenic chromosomal animal systems that express the human immunoglobulin gene are available and can be used to provide the ANGPTL4-binding antibodies of the present invention. For example, a mouse that has both human heavy-chain transchromosome and human light-chain transchromosome, a mouse called "TC mouse" can be used. Such mice are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97:722-727. In the art, cows that have human heavy-chain and light-chain transchromosomes are also described (Kuroiwa et al., 2002 Nature Biotechnology 20:8 89-894) and can be used to provide the ANGPTL4-binding antibodies of the present invention. The humanized antibodies of the present invention can also be prepared using the phage display method for screening a library of human immunoglobulin genes. In the art, such phage display methods for isolating human antibodies have been established or are described in the following examples. For example, U.S. Patent No. 5

[0208] by Ladner et al., U.S. Patent No. 5,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571, 698; U.S. Patent No. 5,427,908 by Dower et al.; and such phage display methods for isolating human antibodies have been established or are described in the following examples. For example, U.S. Patent No. 5 by Ladner et al., U.S. Patent No. 5,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571, ,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571, 698; U.S. Patent No. 5,427,908 by Dower et al.; and See U.S. Patent No. 5,580,717 to McCafferty et al.; U.S. Patent No. 5,96 9,108 to the same; and U.S. Patent No. 6,172,197; as well as Griffit hs et al., U.S. Patent No. 5,885,793; U.S. Patent No. 6,521,404 ; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313; U.S. Patent No. 6,58 2,915; and U.S. Patent No. 6,593,081.

[0209] The humanized antibodies of the present invention can also be prepared using SCID mice reconstituted with human immune cells so as to generate a human antibody response upon immunization. Such ma ice are described, for example, in U.S. Patent No. 5,476,996 to Wilson et al. ; and U.S. Patent No. 5,698,767.

[0210] Framework or Fc Manipulation The engineered antibodies of the present invention include, for example, engineered antibodies modified to framework residues within VH and / or VL so as to improve the properties of the antibody. Such framework modifications are typically made so as to reduce the immunogenicity of the antibody. For example, one approach is to "revert to germline mutations" one or more framework residues to the corresponding germline sequences. More specifically, an antibody that has undergone somatic mutations may contain framework residues that differ from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline conformations can be done by For example, by site-directed mutagenesis, somatic mutations can be "reverted" to the germline sequence. Such "reverted" antibodies are also intended to be encompassed by the present invention. This can be done. Such "reverted" antibodies are also intended to be encompassed by the present invention.

[0211] Another type of framework modification involves mutating residues within one or more framework regions, or alternatively residues within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in US Patent Application Publication No. 20030153043 by Carr et al. This is described in more detail in the specification of US Patent Application Publication No. 20030153043 by Carr et al.

[0212] In addition to or alternatively to the modifications made within the framework or CDR regions, the antibodies of the present invention can include modifications within the Fc region, typically to modify one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity. Further, the antibodies of the present invention can be chemically modified (e.g., by attaching one or more chemical moieties to the antibody), and their glycosylation can be modified, also to modify one or more functional properties of the antibody. Each of these embodiments will be described in more detail below. The numbering of residues within the Fc region is according to the EU index of Kabat. This can be done (e.g., by attaching one or more chemical moieties to the antibody), and their glycosylation can be modified, also to modify one or more functional properties of the antibody. Each of these embodiments will be described in more detail below. The numbering of residues within the Fc region is according to the EU index of Kabat. This can be done (e.g., by attaching one or more chemical moieties to the antibody), and their glycosylation can be modified, also to modify one or more functional properties of the antibody. Each of these embodiments will be described in more detail below. The numbering of residues within the Fc region is according to the EU index of Kabat. In one embodiment, the number of cysteine residues in the hinge region is modified, for example, by modifying the hinge region of CH1 to increase or decrease the number. This approach is by Bodmer et al. This is the EU index of Kabat.

[0213] In one embodiment, the number of cysteine residues in the hinge region is modified, for example, by modifying the hinge region of CH1 to increase or decrease the number. This approach is by Bodmer et al. is further described in U.S. Patent No. 5,677,425. The hinge of CH1 The number of cysteine residues in the region, for example, facilitates the assembly of the light and heavy chains or is modified to increase or decrease the stability of the antibody.

[0214] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody to shorten it. More specifically, the binding of the antibody to Staphylococcyl protein A (SpA) is impaired compared to the binding of the native Fc-hinge domain to SpA, and one or more amino acid mutations are introduced into the CH2 domain of the Fc-hinge fragment - into the interface region between the CH3 domains. This approach is further described in detail in U.S. Patent No. 6 ,165,745 to Ward et al.

[0215] In another embodiment, the antibody is modified to extend its biological half-life. Various methods are possible. For example, the following mutations: one or more of T252L, T254S, T256F described in U.S. Patent No. 6,277,37 5 to Ward can be introduced. Alternatively, to extend the biological half-life, the antibody can be modified within the CH1 region or the CL region to contain a salvage receptor-binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 to Presta et al. and 6,12 1,022.

[0216] In still other embodiments, at least one amino acid residue is the effector function of the antibody​​​​​​ The Fc region is modified by replacing the different amino acid residues to be modified. For example, while the antibody modifies its affinity for the effector ligand, one or more amino acids are replaced with different amino acid residues so as to retain the antigen binding ability of the parental antibody. The effector ligand whose affinity is modified therefor can be, for example, an Fc receptor or the C1 component of complement. This method is further described in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al. and is described in more detail therein.

[0217] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with different amino acid residues so that the antibody modifies its binding to C1q and / or reduces or eliminates complement-dependent cytotoxicity (CDC). This method is further described in U.S. Patent No. 6,194,551 by Idugsie et al. and is described in more detail therein.

[0218] In another embodiment, one or more amino acid residues are modified to thereby modify the ability of the antibody to bind to complement. This method is further described in PCT International Publication No. 94 / 29351 by Bodmer et al. and is described in more detail therein.

[0219] In yet another embodiment, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase the affinity of the antibody for the Fcγ receptor. This method is further described in PCT International Publication No. WO 00 / 42072 by Presta Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have also been mapped, and variants with improved binding are also described (see Shields, R.L. et al., 2001 J. Biol. Chem. 276:6591-6604). ).

[0220] In yet another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. Glycosylation can be modified, for example, to increase the affinity of the antibody for an "antigen". Such carbohydrate modifications can be achieved, for example, by modifying one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the loss of a variable region framework glycosylation site, thereby eliminating glycosylation at that site. Such deglycosylation can increase the affinity of the antibody for the antigen . Such techniques are described in more detail in U.S. Patent Nos. 5,714,350 to Co et al.; and 6,350,861 to the same. In addition, or alternatively, antibodies with modified glycosylation types can be produced, such as afucosylated

[0221] antibodies with reduced amounts of fucosyl residues or antibodies with increased amounts of bisected GlcNac structures . Such modifications of the glycosylation pattern can increase the ADCC activity of the antibody . is supported. Such carbohydrate modifications can be achieved, for example, by expressing an antibody in a host cell with a modified glycosylation mechanism. In the art cells with a modified glycosylation mechanism have been described and can be used as host cells for producing an antibody with modified glycosylation by expressing the recombinant antibody of the present invention thereof. For example, European Patent No. 1,176,195 by Hang et al. describes an antibody expressed in such a cell line that exhibits low fucosylation, a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted. PCT International Publication No. 03 / 035835 pamphlet by Presta describes a mutant CHO cell line, Lecl3 cells, which reduces the ability to attach fucose to Asn(297)-linked carbohydrates and also results in low fucosylation of the antibody expressed in the host cell thereof (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT International Publication No. 99 / 54342 pamphlet by Umana et al. describes a cell line engineered to express a glycosyltransferase (e.g., beta(1,4)-N-acetyl glucosaminyltransferase III (GnTIII)) that modifies glycoproteins such that the antibody expressed in the engineered cell line exhibits an increase in the bisected GlcNac structure which results in an increase in the ADCC activity of the antibody (see also Umana et al., 1999 Nat. Biotech. 17:176-18 0). ). ). ). ). ). ). ).

[0222] Methods of engineering modified antibodies As discussed above, ANGPTL4-binding antibodies having the VH and VL sequences or full-length heavy and full-length light chain sequences provided herein can be used to generate novel ANGPTL4-binding antibodies by modifying the full-length heavy chain sequence and / or full-length light chain sequence, VH sequence and / or VL sequence, or the constant regions attached thereto. Accordingly, in another aspect of the invention, structural features of the ANGPTL4-binding antibodies of the invention are used to create structurally related ANGPTL4-binding antibodies that bind to human ANGPTL4 and also inhibit at least one functional property of ANGPTL4 (e.g., inhibiting the binding of ANGPTL4 to the ANGPTL4 receptor, inhibiting ANGPTL4-dependent cell proliferation), while retaining at least one functional property of the antibodies of the invention. For example, one or more CDR regions of the antibodies of the invention, or mutations thereof, can be recombinantly

[0223] combined with known framework regions and / or other CDRs to create additional ANGPTL4-binding antibodies of the invention, as discussed above, that are recombinantly engineered. Other types of modifications include those described in the previous section. The starting materials for the engineering methods are one or more of the VH and / or VL sequences provided herein, or one or more of their CDR regions. It is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and / or VL sequences provided herein, or one or more of their CDR regions, to create the engineered antibody. Instead, the sequences contained within the sequences provided herein, one or more of the VH and / or VL sequences, or one or more of their CDR regions, can be used. of their CDR regions can be used. The information to be used as a starting material for creating a "second-generation" array derived from the original array is used, and then the "second-generation" array is prepared and expressed as a protein.

[0224] Accordingly, in another embodiment, the present invention provides an antibody sequence of a heavy chain variable region having a CDR1 sequence selected from the group consisting of SEQ ID NO: 7, 32, 52, 72, 92, 1 12, and 132, a CDR2 sequence selected from the group consisting of SEQ ID NO: 8, 33, 53, 73, 93, 113, and 133, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 9, 34, 54, 74, 94, 114, and 134, and an antibody sequence of a light chain variable region having a CDR1 sequence selected from the group consisting of SEQ ID NO: 17, 42, 62, 8 2, 102, 122, and 142, a CDR2 sequence selected from the group consisting of SEQ ID NO: 1 18, 43, 63, 83, 103, 123, and 143, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 19, 44, 64, 84, 104, 124, and 1 44, the method comprising creating an ANGPTL4-binding antibody, and modifying at least one amino acid residue in the antibody sequence of the heavy chain variable region and / or also the antibody sequence of the light chain variable region to create at least one modified antibody sequence, and expressing the modified antibody sequence as a protein. Accordingly, in another embodiment, the present invention provides an antibody sequence of a heavy chain variable region having a CDR1 sequence selected from the group consisting of SEQ ID NO: 10, 35, 55, 75, 95, 115, and 135, a CDR2 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5 6, 76, 96, 116, and 136, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5 6, 76, 96, 116, and 136, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5 6, 76, 96, 116, and 136, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5 6, 76, 96, 116, and 136, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5

[0225] Accordingly, in another embodiment, the present invention provides an antibody sequence of a heavy chain variable region having a CDR1 sequence selected from the group consisting of SEQ ID NO: 10, 35, 55, 75, 95, 115, and 135, a CDR2 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5 6, 76, 96, 116, and 136, and / or a CDR3 sequence selected from the group consisting of SEQ ID NO: 11, 36, 5 from the group consisting of SEQ ID NO: 12, 37, 57, 77, 97, 117, and 137 and an antibody sequence of a heavy chain variable region having a CDR3 sequence selected therefrom, and SEQ ID NO: 20, 45, 6 5, 85, 105, 125, and 145, a CDR1 sequence selected from the group consisting of SEQ ID NO: 21, 46, 66, 86, 106, 126, and 146, a CDR2 sequence selected from the group consisting of SEQ ID NO: 22, 47, 67, 87, 107, 127, and 147, and an antibody sequence of a light chain variable region having a CDR3 sequence selected from the group consisting of SEQ ID NO: to prepare an ANGPTL4-binding antibody, and modifying at least one amino acid residue in the antibody sequence of the heavy chain variable region and / or the antibody sequence of the light chain variable region to create at least one modified antibody sequence, and providing a method for expressing the modified antibody sequence as a protein Accordingly, in another embodiment, the present invention provides an ANGPTL4-binding antibody optimized for expression in mammalian cells, comprising a full-length heavy chain antibody sequence having a sequence selected from the group consisting of SEQ ID NO: 15, 28, 40, 60, 80, 1 00, 120, and 140, and a full-length light chain antibody sequence having a sequence selected from the group consisting of SEQ ID NO: 25, 50, 70, 90, 110, 130, and 150, preparing an ANGPTL4-binding antibody, and modifying at least one amino acid residue in the full-length heavy chain antibody sequence and / or the full-length light chain antibody sequence to create at least one modified antibody sequence, and providing a method for expressing the modified antibody sequence as a protein. In one embodiment, the modification of the heavy chain or light chain is within the framework region of the heavy chain or light chain .

[0226] Thus, in another embodiment, the present invention provides a method for preparing an ANGPTL4-binding antibody optimized for expression in mammalian cells, comprising a full-length heavy chain antibody sequence having a sequence selected from the group consisting of SEQ ID NO: 15, 28, 40, 60, 80, 1 00, 120, and 140, and a full-length light chain antibody sequence having a sequence selected from the group consisting of SEQ ID NO: 25, 50, 70, 90, 110, 130, and 150, preparing an ANGPTL4-binding antibody, and modifying at least one amino acid residue in the full-length heavy chain antibody sequence and / or the full-length light chain antibody sequence to create at least one modified antibody sequence, and providing a method for expressing the modified antibody sequence as a protein. In one embodiment, the modification of the heavy chain or light chain is within the framework region of the heavy chain or light chain to provide a method for expressing the modified antibody sequence as a protein. In one embodiment, the modification of the heavy chain or light chain is within the framework region of the heavy chain or light chain to prepare an ANGPTL4-binding antibody, and modifying at least one amino acid residue in the full-length heavy chain antibody sequence and / or the full-length light chain antibody sequence to create at least one modified antibody sequence, and providing a method for expressing the modified antibody sequence as a protein sequence. In one embodiment, the modification of the heavy chain or light chain is within the framework region of the heavy chain or light chain to create at least one modified antibody sequence, and providing a method for expressing the modified antibody sequence as a protein . In one embodiment, the modification of the heavy chain or light chain is within the framework region of the heavy chain or light chain .

[0227] The modified antibody sequences can also be prepared by screening an antibody library that fixes the minimal essential binding determinants described in US Patent Application Publication No. 2005 / 02555 52 and has diversity in the CDR1 sequences and the CDR2 sequences. Screening can be carried out according to any screening technique suitable for screening antibodies from an antibody library, such as phage display technology etc. Screening can be performed according to any screening technique suitable for screening antibodies from an antibody library, such as phage display technology etc. Screening can be carried out according to any screening technique suitable for screening antibodies from an antibody library, such as phage display technology etc. Screening can be performed according to any screening technique suitable for screening antibodies from an antibody library, such as phage display technology

[0228] Using standard molecular biology techniques, modified antibody sequences can be prepared and expressed The antibodies encoded by the modified antibody sequences specifically bind to human, cynomolgus monkey, rat and / or mouse ANGPTL4; and the antibodies inhibit ANGPTL4-dependent cell proliferation in the F36E cell proliferation assay and / or the Ba / F3-ANGPTL4R cell proliferation assay, including but not limited to one, some, or all of the functional characteristics of the ANGPTL4-binding antibodies described herein and retain antibodies that retain one, some, or all of the functional characteristics of the ANGPTL4-binding antibodies described herein and retain antibodies that retain one, some, or all of the functional characteristics of the ANGPTL4-binding antibodies described herein and retain antibodies that retain one, some, or all of the functional characteristics of the ANGPTL4-binding antibodies described herein and retain antibodies that retain one, some, or all of the functional characteristics of the ANGPTL4-binding antibodies described herein

[0229] In certain embodiments of the method of manipulating the antibodies of the present invention, mutations can be introduced randomly or selectively along all or part of the ANGPTL4-binding antibody coding sequence, and the resulting modified ANGPTL4-binding antibodies can be screened for the binding activity and / or other functional characteristics described herein In certain embodiments of the method of manipulating the antibodies of the present invention, mutations can be introduced randomly or selectively along all or part of the ANGPTL4-binding antibody coding sequence, and the resulting modified ANGPTL4-binding antibodies can be screened for the binding activity and / or other functional characteristics described herein In certain embodiments of the method of manipulating the antibodies of the present invention, mutations can be introduced randomly or selectively along all or part of the ANGPTL4-binding antibody coding sequence, and the resulting modified ANGPTL4-binding antibodies can be screened for the binding activity and / or other functional characteristics described herein In certain embodiments of the method of manipulating the antibodies of the present invention, mutations can be introduced randomly or selectively along all or part of the ANGPTL4-binding antibody coding sequence, and the resulting modified ANGPTL4-binding antibodies can be screened for the binding activity and / or other functional characteristics described herein In the art, mutagenesis methods are described. For example, PCT International Publication No. WO 02 / 092780 by Short describes saturation mutagenesis, synthetic ligation assembly 02 / 092780 describes saturation mutagenesis, synthetic ligation assembly Methods for creating and screening for mutations in antibodies using Leu, or combinations thereof, are described. Alternatively, PCT International Publication No. 03 / 074679 by Lazar et al. describes methods for optimizing the physiochemical properties of antibodies using computer screening methods. In certain embodiments of the invention, the antibody is engineered to remove deamidation sites. Deamidation is known to cause structural and functional changes within peptides or proteins. Deamidation can result in a decrease in biological activity, as well as alterations in the pharmacokinetics and antigenicity of protein pharmaceuticals (Anal Chem. 2005 March 1;77(5):1432 - 9). In certain embodiments of the invention, the antibody is engineered to increase its pI and improve its drug - like properties. The pI of a protein is a key determinant of the overall biophysical properties of the molecule. Antibodies with a low pI are known to have low solubility, low stability, and a tendency to aggregate. Furthermore, the purification of antibodies with a low pI can be difficult and problematic, especially at scales up for clinical use. By increasing the pI of the anti - ANGPTL4 antibodies or Fabs of the invention, their solubility is improved, and it becomes possible to formulate the antibodies at high concentrations (>100 mg / ml). Formulating the antibodies at high concentrations (e.g., >100 mg / ml) provides the advantage that high doses of the antibody can be administered via intravitreal injection to the eyes of patients.

[0230]

[0231] ​​​​​​​​​​​​​​​Allows for less frequent dosing, a significant advantage for treating chronic diseases, including cardiovascular disorders. Increasing the pI can also increase the FcRn-mediated linkage of the IgG version of the antibody. It also increases cycling, which allows the drug to remain in the body for longer and be more effective at meeting demand. Finally, due to the increased pI, the antibody The overall stability was significantly improved, resulting in improved shelf life and in vivo The pI is preferably 8.2 or greater.

[0232] The functional properties of the modified antibodies can be evaluated using assays such as those shown in the Examples (e.g., ELISA). Standard applications available in the art and / or described herein, such as The effect can be assessed using an assay.

[0233] Prophylactic and Therapeutic Uses The antibodies that bind ANGPTL4 described herein can be administered to a subject in need thereof. By administering an effective amount of the antibody or antigen-binding fragment of the present invention, ANGPTL4 levels can be increased. The present invention relates to treating a disease or disorder associated with elevated serum albumin and / or increased ANGPTL4 activity. The present invention provides a method for the administration of a medicament to a subject in need thereof, comprising administering to the subject a therapeutically effective concentration of the medicament. and administering an effective amount of an antibody of the present invention to treat ANGPTL4-associated cardiovascular disorders. The present invention provides a method for administering an effective amount of an antibody of the present invention to a subject in need thereof. The present invention provides a method for treating an ANGPTL4-associated cardiovascular disorder by administering

[0234] The antibodies of the present invention can be used, inter alia, to treat patients with abnormally high ANGPTL4 protein levels, and / or a reduction in ANGPTL4 protein level is desired, any number of conditions or disorders related to ANGPTL4, including but not limited to these, can be prevented treated, and ameliorated. These conditions are those involved in lipid metabolism, such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including atherosclerotic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia (e.g., severe hypertriglyceridemia (e.g., with TG>1000 mg / dL), hypertriglyceridemia associated with obesity, and type V hypertriglyceridemia), hypercholesterolemia, chylomicronemia, combined dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipodystrophy, lipoatrophy, and, for example, a decrease in LPL activity and / or deficiency of LPL, a decrease in the activity of LDL receptor and / or deficiency of LDL receptor, modification of ApoC2, deficiency of ApoE, increase in ApoB, increase in production of very low density lipoprotein (VLDL) and / or decrease in disappearance of VLDL, certain drug treatments (e.g., dyslipidemia induced by glucocorticoid treatment), and other conditions caused by any genetic factors, diet, lifestyle, etc.,

[0235] Other ANGPTL4-related diseases or disorders associated with or resulting from hyperlipidemia, hyperlipoproteinemia, and / or dyslipidemia include cardiovascular diseases or disorders such as atherosclerosis, aneurysms, hypertension, angina pectoris, stroke, cerebrovascular diseases, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular diseases, etc.; acute

[0236] The antibodies of the present invention can also be used in combination with other agents for preventing, treating, or ameliorating ANGPTL4-related disorders. For example, statin therapy can be used in combination with the ANGPTL4 antibodies and antigen-binding fragments of the present invention to treat patients with disorders associated with triglyceride levels. levels. levels. levels.

[0237] Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others. The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others. The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others. The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others. The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others. The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others. The present invention provides a pharmaceutical composition comprising an ANGPTL4-binding antibody (intact or binding fragment) formulated together with a pharmaceutically acceptable carrier. The composition may additionally contain, for example, one or more other therapeutic agents suitable for the treatment or prevention of cardiovascular disorders. The pharmaceutically acceptable carrier can be used to enhance or stabilize the composition or to facilitate the preparation of the composition. The pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others.

[0238] The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or mode of administration will vary depending on the desired result. Administration is preferably intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered in proximity to the target site. The pharmaceutically acceptable carrier is to be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody which is a bispecific molecule and a multispecific molecule, may be formulated with acids and other agents that can inactivate the compound. It can be coated with a material that protects from the action of the natural state.

[0239] The composition shall be sterile and fluid. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, and in the case of a dispersion, the required particle size can be maintained and can be maintained by using a surfactant. In many cases, it is preferable to include an isotonic agent in the composition, such as a polyhydric alcohol such as sugar, mannitol or sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be brought about by incorporating an agent that delays absorption, such as aluminum monostearate or gelatin into the composition.

[0240] The pharmaceutical composition of the present invention is well-known in the art and can be prepared according to methods routinely practiced. For example, see Remington: The Science and Practice of Pharm acy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978 See. The pharmaceutical composition is preferably manufactured under GMP conditions. In the pharmaceutical composition of the present invention, it is typical to employ a therapeutically effective dose or a therapeutically effective dose of the ANGPTL4-binding antibody. The ANGPTL4-binding antibody is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. Administration is carried out by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the desired optimal response (e.g., a therapeutic response). For example, a single bolus can be administered, or multiple divided doses can be administered over a period of time, and the dosage can be proportionally reduced or increased as indicated by the requirements of the treatment situation. For ease of administration and uniformity of dosage (dosage), it is particularly advantageous to formulate the parenteral composition in dosage unit form. As used herein, the term "dosage unit form" refers to physically discrete units suitable as unitary dosages for the subjects to be treated. Each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0241] The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors including the activity of the particular composition of the invention being employed, or their esters, salts, or amides, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition being employed, and the age, sex, weight, condition, general health, and medical history of the patient being treated.

[0242] The physician or veterinarian will initiate the administration of the antibodies of the invention employed in the pharmaceutical composition at a level that is less than the level required to achieve the desired therapeutic effect and will titrate the dosage upward until the desired effect is achieved.​​​​​​​​​ The dosage can be gradually increased. Generally, the dosage of the composition of the present invention effective for treating the cardiovascular disorders described herein will vary depending on many different factors including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The treatment dosage needs to be titrated to optimize safety and efficacy. In the systemic administration of an antibody, the dosage ranges from about 0.0001 to 100 mg per kg of the host body weight, more typically in the range of 0.01 to 15 mg per kg of the host body weight. In the intravitreal administration of an antibody, the dosage can range from 0.1 mg per eye to 5 mg per eye. For example, 0. 1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 .0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml, 2.1 mg / ml, 2.2 mg / ml, 2.3 m g / ml, 2.4 mg / ml, 2.5 mg / ml, 2.6 mg / ml, 2.7 mg / ml 、2.8 mg / ml, 2.9 mg / ml, 3.0 mg / ml, 3.1 mg / ml, 3.2 mg / ml, 3.3 mg / ml, 3.4 mg / ml, 3.5 mg / ml, 3.6 mg / m l, 3.7 mg / ml, 3.8 mg / ml, 3.9 mg / ml, 4.0 mg / ml, 4. .1 mg / ml, 4.2 mg / ml, 4.3 mg / ml, 4.4 mg / ml, 4.5 mg / ml, 4.6 mg / ml, 4.7 mg / ml, 4.8 mg / ml, 4.9 mg / ml, 5.0 mg / ml. ml, 4.6 mg / ml, 4.7 mg / ml, 4.8 mg / ml, 4.9 mg / ml, or 5.0 mg / ml. Exemplary treatment regimens involve systemic administration once every two weeks, or once a month, or once every 3 - 6 months. Exemplary treatment regimens involve systemic administration once every two weeks, or once a month, or once every 3 - 6 months, or as-needed (PRN) systemic administration.

[0243] Antibodies are typically administered multiple times. The interval between single administrations can be weekly, monthly, or yearly. The interval can also be irregular, as indicated by measuring the blood level of the ANGPTL4-binding antibody in the patient. Additionally, the physician may determine alternative dosing intervals and administer monthly or at intervals necessary to be effective. In some methods of systemic administration, the dosage is adjusted to achieve a plasma concentration of the antibody of 1 - 1000 μg / ml, and in some methods, to achieve 25 - 500 μg / ml. Alternatively, the antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary according to the half-life of the antibody in the patient. Generally, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies. The dosage and administration frequency can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively low- frequency intervals over a long period. Some patients are treated continuously for the rest of their lives. In therapeutic applications, in some cases, relatively high dosages are required at relatively short intervals until disease progression is reduced or terminated, preferably until the patient A prophylactic regimen can be administered.

Example

[0244] The following examples are presented to further illustrate the present invention and are not presented to limit its scope. Other variations of the present invention will be readily apparent to those skilled in the art and are also encompassed by the appended claims.

[0245] Example 1: Preparation of purified recombinant human ANGPTL4 for use in antibody characterization experiments as an antigen The full-length human ANGPTL4 polypeptide (amino acids 26 - 406, matching NM_139314.2 in the NCBI sequence), the N-terminal signal peptide derived from human IgG-kappa (MKTFILLLWVLLLWVIFLLPGATA) (SEQ ID NO: 152), as well as the C-terminal FLAG epitope (DYKDDDDKH) (SEQ ID NO: 153), the hexahistidine purification tag (HHHHHH) (SEQ ID NO: 154), and the Avi tag (i.e., the BirA biotinylation sequence GGGLNDIFEAQKIEWHE) (SEQ ID NO: 155) were cloned into the mammalian cell expression vector pRS5a to create the plasmid pRS-Ikk-hANGPTL4(26 - 406)-FLAG-6HIS-Avi, which contains amino acids 26 - 406 of human ANGPTL4 following a 20-amino acid IKK signal sequence and together with a carboxyl-terminal Flag tag, 6HIS tag, and Avi tag (Table 2, SEQ ID NO: 156).

[0246] ​​​​​​​​​​​For some preparations, human ANGPTL4 protein was expressed, purified, and biotinylated using the following procedure (Method 1): HEK293T cells adapted to suspension were cultured in serum-free FreeStyle293 expression medium (Life Technologies, catalog number 12338-018) and transfected with the plasmid pRS-Ikk-hANGPTL4(26-406)-FLAG-His6-Avi using polyethyleneimine transfection reagent (Polysciences, catalog number 23966). Five hours after transfection, heparin (Alfa Aesar, catalog number A16198) was added to the culture medium to a final concentration of 0.5 mg / ml. The cells were then cultured for 72-96 hours, and then the cell culture supernatant was harvested by centrifugation at 4°C and sterile filtered using a 0.22 μm filter (Thermo, catalog number 567-0010). The filtered cell culture supernatant was then concentrated to approximately 100 ml by tangential flow filtration. The concentrated supernatant was diluted to a volume of 1 liter with TBS-glycerol buffer (50 mM Tris-HCl, 150 mM NaCl, and 15% glycerol, pH 7.4), and the sample was concentrated to approximately 200 ml by tangential flow filtration. Next, anti-Flag M2 agarose resin (Sigma, catalog number 220102-177) pre-equilibrated with TBS-glycerol buffer was added to the sample, and the resulting solution was gently mixed at 4°C for 1 hour. The agarose resin was then washed 5 times with 25 ml of TBS-glycerol, and the bound ANGPTL4 protein was eluted with 0.2 mg / ml Flag peptide (Sigma, catalog number 220176-317). adapted to suspension were cultured in serum-free FreeStyle293 expression medium (Life Technologies, catalog number 1 2338-018) and transfected with the plasmid pRS-Ikk- hANGPTL4(26~406)-FLAG-His6-Avi using polyethyleneimine transfection reagent (Pol ysciences, catalog number 23966). Five hours after transfection, heparin (Alfa Aesar, catalog number A16198) was added to the culture medium to a final concentration of 0.5 mg / ml. Next, the cells were cultured for 72-96 hours, and then the cell culture supernatant was harvested by centrifugation at 4°C and sterile filtered using a 0.22 μm filter (Thermo, catalog number 567-0010). The filtered cell culture supernatant was then concentrated to approximately 10 0 ml by tangential flow filtration. The concentrated supernatant was diluted to a volume of 1 liter with TBS-glycerol buffer (50 mM Tris- HCl, 150 mM NaCl, and 15% glycerol, pH 7.4), and the sample was concentrated to approximately 200 ml by tangential flow filtration . Next, anti-Flag M2 agarose resin (Sigma, catalog number 220102-177) pre-equilibrated with TBS-glycerol buffer was added to the sample, and the resulting solution was gently mixed at 4°C for 1 hour. Next, the agarose resin was washed 5 times with 25 ml of TBS-glycerol, and the bound ANGPTL4 protein was eluted with 0.2 mg / ml Flag peptide (Sigma, catalog number 220176-317 . pre-equilibrated with TBS-glycerol buffer was added to the sample, and the resulting solution was gently mixed at 4°C for 1 hour. Next, the agarose resin was washed 5 times with 25 ml of TBS-glycerol, and the bound ANGPTL4 protein was eluted with 0.2 mg / ml Flag peptide (Sigma, catalog number 220176-317 washed 5 times with 25 ml of TBS-glycerol, and the bound ANGPTL4 protein was eluted with 0.2 mg / ml Flag peptide (Sigma, catalog number 220176-317 washed 5 times with 25 ml of TBS-glycerol, and the bound ANGPTL4 protein was eluted with 0.2 mg / ml Flag peptide (Sigma, catalog number 220176-317 ) was eluted with 20 ml of TBS - glycerol. Peroxide - free Tw een - 20 (AppliChem, catalog number A1284,0025) was added to the eluted pro tein solution to a final concentration of 0.1%, and the resulting solution was pre - equilibrated in TBS - glycerol containing 0.1% Tween - 20 (Buffer A). It was then loaded onto a 5 ml HiTrap heparin column (GE Lifesciences, catalog number 1 7 - 0407 - 01). The column was washed with 50 ml of Buffer A followed by 50 ml of Buffer A containing 300 m M NaCl. Then, the ANGPTL4 protein was eluted with 20 ml of Buffer A containing 600 mM NaCl. The eluted protein was concentrated using a centrifugal concentrator (Amicon Ultra, catalog number UFC 903024) with a molecular weight cut - off of 30 kD. The purity of the purified ANGPTL4 protein, as evaluated by SDS - PAGE, was >90%.

[0247] For some applications, the ANGPTL4 protein was site - specifically biotinylated on the Avi tag at the C - terminus using 10 μg of purified biotin - protein ligase (BirA) (Avidity) per 1 mg of ANGPTL4. The buffer was supplemented with ATP at a final concentration of 10 mM, magnesium acetate at 10 mM, and d - biotin at 0.5 M. The reaction mixture was incubated at 30 °C for 2 hours and then overnight at 4 °C. Then, it was loaded onto a HiLoad Superdex 200 column ( 26 mm×600 mm) (GE Lifesciences, catalog number 28 - 9893 - 36 ​​​​​​​​​) and loaded. Using SDS-PAGE, the fractions from the Superdex 200 column were analyzed, the fractions containing ANGPTL4 were pooled, and using a centrifugal concentrator they were concentrated.

[0248] For other preparations, human ANGPTL4 protein was expressed, purified, and biotinylated using the following procedure (Method 2). Using the standard polyethyleneimine (PEI) transfection method, the plasmid pRS-Ikk-hANGPTL4(26 ~406)C-Flag6HisAvi was transiently transfected into HEK293T cells. The cells were grown by suspension culture in Freestyle 293 expression medium, and the transfection was carried out at a final cell concentration of 1×10 cells per ml in 4 liters of medium using a 1-liter flask. 6 Five hours after transfection, heparin was added at a final concentration of 500 μg / ml. The cells were grown at 37°C and 5% CO2 for 72 hours. The cells were then pelleted by centrifugation, and the supernatant was passed through a 0.22-μm sterile filter. The clarified supernatant was concentrated and buffer was exchanged to buffer B (50 mM Tris-HCl, 15 0 mM NaCl, 10% glycerol, 10 mM imidazole, pH 7.4) using tangential flow filtration (TFF). The concentrated sample was then passed through a 5-ml Ni-NTA affinity column equilibrated with buffer C (50 mM Tris HCl, 150 mM NaCl, 10% glycerol, 10 mM imidazole, 0.1% n -octyl-β-maltoside, pH 7.4). After loading the sample, the baseline at 280 nm After loading the sample, the baseline at 280 nm The column was washed with the same buffer until the absorbance was reached. Then, by using an imidazole gradient (1 0 mM to 500 mM), the bound ANGPTL4 protein was eluted out. The elution fractions containing human ANGPTL4 were pooled, concentrated using an Amicon concentrator (with a molecular weight cut-off of 10 kD), and the buffer was exchanged to a storage buffer (50 mM Tris-HCl, 150 mM NaCl, 15% glycerol, pH 7.4) using a PD-10 column. The samples were aliquoted, snap-frozen in liquid nitrogen, and stored at -8 0 °C. The purity of the purified human ANGPTL4 protein, as evaluated by SDS-PAGE, was >90%.

[0249] For some applications, the purified ANGPTL4 protein prepared as described above was site-specifically biotinylated as follows. In a 50 mM bicine (pH 8.3) buffer, the purified protein at a final concentration of about 1 mg / mL was incubated at 30 °C for 1 hour and then at 4 °C overnight in the presence of 10 mM ATP, 10 mM magnesium acetate, 0.1 mM biotin, and BirA biotin ligase (Avidit y). Then, the protein was concentrated using an Amicon concentrator (with a molecular weight cut-off of 10 kD), and the buffer was exchanged to a storage buffer (50 mM Tris -HCl, 150 mM NaCl, 15% glycerol, pH 7.4) using a PD-10 column. The samples were aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C.

[0250] Example 2: N of purified recombinant human ANGPTL4 for use in antibody characterization experiments ​​​​​​​Preparation of the C-terminal coiled-coil domain protein Expression of the N-terminal coiled-coil domain of human ANGPTL4 (amino acids 26 - 161) , purification, and biotinylation were carried out using essentially the same method as described for full-length human ANGPTL4 in Method 2 of Example 1 . The sequence of the purified N-terminal domain protein of human ANGPTL4 is shown in Table 2 (SEQ ID NO: 157).

[0251]

Table 2

[0252] Example 3: Preparation and screening of monoclonal antibodies Recombinant human ANGPTL4 protein was prepared in-house as described in Example 1 and used as an immunogen to generate anti-ANGPTL4 hybridoma clones . Bcl-2 transgenic mice were immunized with recombinant human ANGPTL4 according to a standard rapid immunization protocol . Hybridomas were created using a standard electrofusion-based method .

[0253] CHO-K1PD cells that stably express human ANGPTL4 fused to a transmembrane domain were created using standard methods . Due to the presence of the transmembrane domain, these cells present ANGPTL4 on the cell surface . Thus, the binding of antibodies on the surface of these cells to ANGPTL4 can be detected using flow cytometry .

[0254] The hybridoma supernatant was used to detect the binding of the antibodies present in the supernatant to the surface of CHO-K1PD cells ​​Screening was performed by detecting binding to the presented human ANGPTL4. Binding of the antibody to cells was detected using a fluorescently labeled anti-mouse secondary antibody and flow cytometry . Parent CHO-K1PD cells that do not express ANGPTL4 were used as a negative control . For hybridomas that bound ANGPTL4, the antibody was purified from the cell supernatant using standard methods, and the resulting enriched supernatant was examined in a flow cytometry assay using CHO-K1PD / ANGPTL4 cells and CHO-K1PD parental cells.

[0255] The ANGPTL4 antibody titer in the hybridoma supernatant was determined by using a standard direct ELISA assay in which recombinant human ANGPTL4 protein was immobilized on the surface of an ELISA plate . Confirmed positive hybridomas were subcloned, and the sequences of the monoclonal antibodies produced by these hybridomas were determined using standard methods .

[0256] Subsequently, using the method described in Example 7 below, the monoclonal antibodies 1 4P18, 17B1, 19C16, and 37P1 were shown to inhibit the inhibition of human lipoprotein lipase mediated by human ANGPTL4 . The nucleotide and amino acid sequences of the heavy and light chain variable regions of 14P18, 17B1, 19C16, and 37P1 were determined using standard methods .

[0257] Example 4: Humanization of Monoclonal Antibodies In the art, the process of humanization is well described (Jones, et al 1986, Q Queen, et al 1989, Riechmann, et al 1988, Verhoeyen, Milstein and Winter 1988). The term "humanization" is described as the introduction of the antigen-binding site of a non-human antibody, e.g., a mouse-derived antibody, into a human acceptor framework, e.g., a human germline sequence ( Retter, et al 2005). The main rationale for humanizing an antibody is to minimize the risk of generating an immunogenic response against the antibody when administered as a therapeutic agent in humans ( Rebello, et al 1999). The antigen-binding site consists of the complementarity-determining regions (CDRs) (Chothia and Lesk 1987, Kabat, et al 1991) and positions within the framework regions of the variable domains (VL and VH) that directly or indirectly affect binding. Framework residues that can directly affect binding can be found, for example, within the so-called "a

[0258] tter" loop region located between CDR2 and CDR3. Residues that indirectly affect binding can be found, for example in the so-called "Berne band" (Foote and Winter 1992). These are thought to support the conformation of the CDRs. These positions outside the CDRs are considered when selecting an acceptor framework suitable for minimizing the number of deviations in the final humanized antibody, in light of the human germline acceptor sequences within the framework regions. Example 5: Optimization of Antibody Sequences and Affinity Maturation These are thought to support the conformation of the CDRs. These positions outside the CDRs are considered when selecting an acceptor framework suitable for minimizing the number of deviations in the final humanized antibody, in light of the human germline acceptor sequences within the framework regions. antibody, in light of the human germline acceptor sequences within the framework regions. These positions outside the CDRs are considered when selecting an acceptor framework suitable for minimizing the number of deviations in the final humanized

[0259] Example 5: Optimization of Antibody Sequences and Affinity Maturation Certain amino acid sequence motifs are known to undergo post-translational modifications (PTMs) such as glycosylation (e.g., NxS / T [where x is any amino acid other than , P]), oxidation of free cysteine, deamination (e.g., deamination of N in the N-G sequence), or isomerization (e.g., in the DG sequence). When present within the CDR regions, these motifs are ideally removed by site-directed mutagenesis in order to increase the homogeneity of the product. In the art, the process of affinity maturation is well described. Among many display systems, phage display (Smith 1985) and display on eukaryotic cells such as yeast (Boder and Wittrup 1997) are the most commonly applied systems for selecting for antibody-antigen interactions. The advantages of these display systems are that they are suitable for a wide range of antigens and the selection stringency can be easily adjusted. In phage display, scFv fragments or Fab fragments can be presented, and in yeast display, scFv, Fab, or full-length IgG can be presented. These commonly applied methods enable the selection of desired antibody variants from large-scale libraries with a diversity exceeding 1×10 . Small-scale libraries with a diversity of, for example, 1,000 can be screened by microarray and ELISA. Non-targeted antibody variant libraries or random antibody variant libraries can be created, for example, by error-prone PCR (Cadwell and Joyce 1994), which is very simple.

[0260] In the art, the process of affinity maturation is well described. Among many display systems, phage display (Smith 1985) and display on eukaryotic cells such as yeast (Boder and Wittrup 1997) are the most commonly applied systems for selecting for antibody-antigen interactions. The advantages of these display systems are that they are suitable for a wide range of antigens and the selection stringency can be easily adjusted. In phage display, scFv fragments or Fab fragments can be presented, and in yeast display, scFv, Fab, or full-length IgG can be presented. These commonly applied methods enable the selection of desired antibody variants from large-scale libraries with a diversity exceeding 1×10 . Small-scale libraries with a diversity of, for example, 1,000 can be screened by microarray and ELISA. Non-targeted antibody variant libraries or random antibody variant libraries can be created, for example, by error-prone PCR (Cadwell and Joyce 1994), which is very simple. The advantages of these display systems are that they are suitable for a wide range of antigens and the selection stringency can be easily adjusted. In phage display, scFv fragments or Fab fragments can be presented, and in yeast display, scFv, Fab, or full-length IgG can be presented. These commonly applied methods enable the selection of desired antibody variants from large-scale libraries with a diversity exceeding 1×10 . Small-scale libraries with a diversity of, for example, 1,000 can be screened by microarray and ELISA. Non-targeted antibody variant libraries or random antibody variant libraries can be created, for example, by error-prone PCR (Cadwell and Joyce 1994), which is very simple. The advantages of these display systems are that they are suitable for a wide range of antigens and the selection stringency can be easily adjusted. In phage display, scFv fragments or Fab fragments can be presented, and in yeast display, scFv, Fab, or full-length IgG can be presented. These commonly applied methods enable the selection of desired antibody variants from large-scale libraries with a diversity exceeding 1×10 . Small-scale libraries with a diversity of, for example, 1,000 can be screened by microarray and ELISA. 7 Non-targeted antibody variant libraries or random antibody variant libraries can be created, for example, by error-prone PCR (Cadwell and Joyce 1994), which is very simple. The advantages of these display systems are that they are suitable for a wide range of antigens and the selection stringency can be easily adjusted. In phage display, scFv fragments or Fab fragments can be presented, and in yeast display, scFv, Fab, or full-length IgG can be presented. These commonly applied methods enable the selection of desired antibody variants from large-scale libraries with a diversity exceeding 1×10 . Small-scale libraries with a diversity of, for example, 1,000 can be screened by microarray and ELISA.

[0261] Non-targeted antibody variant libraries or random antibody variant libraries can be created, for example, by error-prone PCR (Cadwell and Joyce 1994), which is very simple. The advantages of these display systems are that they are suitable for a wide range of antigens and the selection stringency can be easily adjusted. In phage display, scFv fragments or Fab fragments can be presented, and in yeast display, scFv, Fab, or full-length IgG can be presented. These commonly applied methods enable the selection of desired antibody variants from large-scale libraries with a diversity exceeding 1×10 is convenient but results in a limited approach in some cases. Another strategy is the CDR directed diversification. One or more positions within one or more CDRs can be, for example, synthesized using degenerate oligonucleotides (Thompson, et al 1996), trinucleotide mutagenesis (TRIM) (Kayushin, et al 1996), or any other method known in the art to specifically target.

[0262] Example 6: Expression and Purification of Humanized Antibodies DNA sequences encoding humanized VL and VH domains, including codon optimization for Homo Sapiens, were ordered from GeneArt (Life Technologies, Inc., Regensburg, Germany). The sequences encoding the VL and VH domains were subcloned into expression vectors suitable for expression and isolation by mammalian cells by cut-and-paste from the vectors by GeneArt. The heavy and light chains were cloned into separate expression vectors to allow co-transfection. Elements of the expression vectors include a promoter (the cytomegalovirus (CMV) enhancer-promoter), a signal sequence that facilitates secretion, a polyadenylation signal and a transcription terminator (derived from the bovine growth hormone (BGH) gene), elements that allow replication in episomes and in prokaryotes (e.g., the SV40 origin and ColE1 elements or other elements known in the art), and elements that allow selection (the ampicillin resistance gene and zeocin marker).

[0263] Human embryonic kidney cells (HEK293-T) that constitutively express SV40 large T antigen ATCC 11268) are one of the preferred host cell lines for transient expression of humanized IgG proteins and / or optimized IgG proteins. Transfection is carried out using PEI (polyethyleneimine, MW: 25,000, linear; Polyscien ces, USA, catalog number 23966) as the transfection reagent. The PEI stock solution is prepared by carefully dissolving 1 g of PEI in 900 ml of cell culture grade water at room temperature (RT ). To facilitate the dissolution of PEI, the solution is acidified to pH 3 - 5 by adding HCl, followed by neutralization to a final pH of 7.05 with NaOH . Finally, the volume is adjusted to 1 L, the solution is filtered through a 0. 22 μm filter, aliquoted, and frozen at -80 °C until further use. HEK 293T cells are cultured in a serum-free culture medium, a Novartis patented product for cell transfection and growth, and ExCell VPRO serum-free culture medium (SAFC Biosciences, USA as the production / feed medium. Cells prepared for transient transfection are cultured by suspension culture. For small-scale (<5 L) transfection, cells are grown in Corning shaking flasks (Corni ng, Tewksbury, MA) on an orbital shaker (100 - 120 rpm) in an incubator humidified with 5% CO2 (seeding flask). Cells in the seed culture are in the exponential growth phase ( 5 × 10 cells per ml ). For large-scale transfection, cells are grown in a bioreactor with appropriate agitation and aeration conditions. The transfection procedure involves mixing the plasmid DNA encoding the protein of interest with the PEI solution and adding it to the cell culture. After transfection, the cells are incubated for a certain period of time to allow protein expression. The expressed protein is then harvested from the cell culture supernatant by methods such as filtration, centrifugation, and chromatography. The harvested protein is further purified to obtain a highly pure product suitable for various applications. Quality control measures are implemented throughout the process to ensure the integrity and functionality of the expressed protein. These measures may include protein quantification, SDS-PAGE analysis, Western blotting, and functional assays. The resulting purified protein can be used for research purposes, such as studying protein structure and function, or for biopharmaceutical applications, such as drug development and therapeutic use. The use of HEK 293T cells as a host for transient protein expression offers several advantages, including high transfection efficiency, relatively easy culture conditions, and the ability to produce large amounts of protein in a short period of time. However, it is also important to optimize the transfection conditions and culture parameters to achieve the best results. This may involve adjusting the ratio of plasmid DNA to PEI, the cell density at transfection, the duration of transfection, and the culture medium composition. Additionally, appropriate quality control measures should be in place to ensure the quality and consistency of the expressed protein. Overall, the use of HEK 293T cells for transient protein expression is a widely used and effective method in biotechnology and biopharmaceutical research and production. For large-scale transfection, cells are grown in a bioreactor with appropriate agitation and aeration conditions. The transfection procedure involves mixing the plasmid DNA encoding the protein of interest with the PEI solution and adding it to the cell culture. After transfection, the cells are incubated for a certain period of time to allow protein expression. The expressed protein is then harvested from the cell culture supernatant by methods such as filtration, centrifugation, and chromatography. The harvested protein is further purified to obtain a highly pure product suitable for various applications. Quality control measures are implemented throughout the process to ensure the integrity and functionality of the expressed protein. These measures may include protein quantification, SDS-PAGE analysis, Western blotting, and functional assays. The resulting purified protein can be used for research purposes, such as studying protein structure and function, or for biopharmaceutical applications, such as drug development and therapeutic use. The use of HEK 293T cells as a host for transient protein expression offers several advantages, including high transfection efficiency, relatively easy culture conditions, and the ability to produce large amounts of protein in a short period of time. However, it is also important to optimize the transfection conditions and culture parameters to achieve the best results. This may involve adjusting the ratio of plasmid DNA to PEI, the cell density at transfection, the duration of transfection, and the culture medium composition. Additionally, appropriate quality control measures should be in place to ensure the quality and consistency of the expressed protein. Overall, the use of HEK 293T cells for transient protein expression is a widely used and effective method in biotechnology and biopharmaceutical research and production. orbital shaker (100 - 120 rpm) in an incubator humidified with 5% CO2 (seeding flask). Cells in the seed culture are in the exponential growth phase ( 5 × 10 cells per ml ). 5Cells per milliliter between 3×10 6 cells (cell density) are maintained and shall exhibit a viability of >90% for transfection. For small scale (<5L) transfection, an aliquot of cells is taken from the seed culture and adjusted to 1.4×10 cells per milliliter in 36% of the final volume with Novartis serum-free 6 culture medium. The DNA solution (Solution 1: for 1 L of transfection, 0.5 mg of heavy chain expression plasmid and 0.5 mg of light chain expression plasmid) is prepared by diluting the DNA to 1 mg / l (final volume) in 7% of the final culture volume followed by gentle mixing. To prevent bacterial contamination, this solution is filtered using a 0.22 μm filter (e.g., Millipore Stericup). Next, a 3 mg / L (final volume) PEI solution is also diluted in 7% of the final culture volume and gently mixed (Solution 2). Both solutions are incubated at room temperature (RT) for 5 - 10 minutes. Thereafter, while gently mixing, Solution 2 is added to Solution 1 and incubated at room temperature for an additional 5 - 15 minutes. Then, the transfection mix is added to the cells and cell culture is continued for 4 - 6 hours. Finally, the remaining 50% of the total production volume is

[0264] achieved by addition of ExCell® VPRO serum-free culture medium. Cell culture is continued for 11 days after transfection. The collected cell supernatant was filtered through a Stericup filter. Sterile filter through a 0.22 μm filter and store at 4° C. until further processing. 5ml of freshly poisoned HiTrap Protein A (0.25M NaOH) Using the MabSelect® SuRe column, Conducted on "AKTA 100 Explorer Air" chromatography system The column was filled with 5 column volumes of phosphate buffered saline (PBS, Gibco, Life Sciences). Technologies, Carlsbad, Calif.), then sterile filtered. The filtered supernatant was loaded at 4.0 ml / min. The column was washed with 13 column volumes of PBS. The antibody was then washed with 50 mM citrate, 70 mM NaCl, pH 3.2. The column was eluted with 10 ml of 1 M Tris-HCl, pH 7.0. The eluate was collected in 3 ml fractions and diluted with 1 M Tris-HCl, pH 7.0. The pH was adjusted to 7 with H10. Fractions containing the antibody were pooled and sterile filtered (Millipore). pore Steriflip, 0.22um) and a spectrophotometer (NanoDrop N OD 280nm was measured using a 350 nm OD-1000, and the OD 280 and protein composition were calculated. Protein concentrations were calculated based on the molar extinction coefficient calculated from the column. By size exclusion chromatography with multi-angle light scattering detection (SEC-MALS), Aggregation was examined and purity was confirmed by gel electrophoresis (SDS-PAGE), endotoxin assay (LA The product was analyzed by HPLC, HPLC, and mass spectrometry (MS). If necessary, a second purification step was performed. First, the antibody from the first purification was loaded onto a freshly sanitized (0.5 M NaOH) gel filtration column. (gel filtration column)(Hi Load 16 / 60 Superdex 200 was loaded into a (120 mL, GE-Healthcare). The column was equilibrated with PBS and run with PBS buffer at a flow rate of 1 ml / min. Eluents were collected in 1.2 ml fractions. Proteins containing the antibody were pooled and the resulting purified antibody was analyzed as described for the first purification step.

[0265] Using the method described above, the following humanized antibodies: NEG276, NEG276-L ALA, NEG278, NEG310, NEG318, NEG318-LALA, NEG 319, NEG313, and NEG315 were prepared, expressed, and purified. The frameworks and parental antibodies of these humanized antibodies are shown in Table 3, and the nucleotide and amino acid sequences are shown in Table 1. All humanized antibodies were prepared using a modified Fc region (human IgG1-LAL A) in which the Leu234-Leu235 sequence within the heavy chain was replaced with Ala234-Ala235, except for NEG276-LALA and NEG318-LALA, which were prepared as human IgG1 antibodies. The human IgG1-LALA antibody is known to have reduced effector function compared to wild-type human IgG1 antibody.

[0266]

Table 3

[0267] Example 7: Human lipoprotein lipase assay Using the standard polyethyleneimine (PEI) transfection method, Free HEK 293T cells cultured in Style expression medium (Invitrogen) were transfected with a mammalian expression plasmid encoding the full-length human lipoprotein lipase (LPL) polypeptide (matching NM_0 00237.2 from the NCBI sequence). Twenty-four hours after transfection, heparin was added to the culture medium to a final concentration of 3 U / ml to enhance the release of hLPL secreted from the cell surface. Sixty hours after transfection, the culture medium was collected, filtered using a 0.2 μm filter and glycerol was added to a final concentration of 10% v / v. The resulting solution was loaded onto a 5 ml Heparin Sepharose HiTrap column (GE) pre-equilibrated with buffer A (50 mM Tris-HCl, 200 mM NaCl, 10% v / v glycerol, pH 7.2). The column was washed with buffer A and then human LPL protein was eluted with a step gradient of 500 mM NaCl, 1 M NaCl, and 2 M NaCl in buffer A. The human LPL with the highest purity and greatest catalytic activity eluted with 2 M NaCl. The aliquot of purified human LPL was snap-frozen and stored at -80°C until use. The ability of the antibodies of the present invention to block the inhibition of human lipoprotein lipase by ANGPTL4 was evaluated using the following protocol. A 384-well assay plate (Corning, catalog number 3573) and a sample plate (Greiner Bio-one, catalog number 781201) were coated with 1% bovine serum albumin (BSA) (0.1 per well). v / v glycerol, pH 7.2) was loaded onto a 5 ml Heparin Sepharose HiTrap column (GE) pre-equilibrated with buffer A (50 mM Tris-HCl, 200 mM NaCl, 10% v / v glycerol, pH 7.2). The column was washed with buffer A and then human LPL protein was eluted with a step gradient of 500 mM NaCl, 1 M NaCl, and 2 M NaCl in buffer A. The human LPL with the highest purity and greatest catalytic activity eluted with 2 M NaCl. The aliquot of purified human LPL was snap-frozen and stored at -80°C until use. The column was washed with buffer A and then human LPL protein was eluted with a step gradient of 500 mM NaCl, 1 M NaCl, and 2 M NaCl in buffer A. The human LPL with the highest purity and greatest catalytic activity eluted with 2 M NaCl. The aliquot of purified human LPL was snap-frozen and stored at -80°C until use. The human LPL with the highest purity and greatest catalytic activity eluted with 2 M NaCl. The aliquot of purified human LPL was snap-frozen and stored at -80°C until use. The human LPL with the highest purity and greatest catalytic activity eluted with 2 M NaCl. The aliquot of purified human LPL was snap-frozen and stored at -80°C until use. The aliquot of purified human LPL was snap-frozen and stored at -80°C until use.

[0268] The ability of the antibodies of the present invention to block the inhibition of human lipoprotein lipase by ANGPTL4 was evaluated using the following protocol. A 384-well assay plate (Corning, catalog number 3573) and a sample plate (Greiner Bio-one, catalog number 781201) were coated with 1% bovine serum albumin (BSA) (0.1 per well). type number 781201) were coated with 1% bovine serum albumin (BSA) (0.1 It was washed at room temperature for 30 minutes with ml). Subsequently, the plate was washed twice with a 0.05% T ween-20 solution.

[0269] ANGPTL4 antibody in 100 mM HEPES, pH 7.0 (a serial dilution of 20 μl per well with a final assay concentration ranging from 0 .02 nM to 500 nM) was added to the sample plate. Subsequently, 20 μl of human ANGPTL4 protein (a final assay concentration of 10 nM) was added in assay buffer (100 mM HEPES, 2 mM of MgCl2, pH 7.0), and the plate was incubated at room temperature for 20 minutes while gently shaking. Next, lipoprotein lipase diluted in assay buffer (20 μl) was added, and the plate was incubated at room temperature for 10 minutes while gently shaking.

[0270] Coupling enzyme mix containing acyl-CoA oxidase (Sekisui Diagnos tics, model number T-17), acyl-CoA synthetase (Sekisui Diag nostics, model number T-16), horseradish peroxidase (Sekisui Diagnostics), ATP (Sigma, model number A7699) and coenzyme A ( MP Biomedicals, model number 100493) was prepared. Catalase agarose beads (Sigma, model number C9284) were added to the coupling enzyme mix, and the mixture was incubated at 4°C for 30 minutes while shaking, and then the catalase agarose beads were removed by centrifugation.

[0271] Human VLDL (Millipore, catalog number LP1) was diluted in assay buffer and incubated with catalase agarose beads for 30 minutes, and the beads were removed by centrifugation from the solution. Amplex Red (Invitrogen, catalog number A12222) in assay buffer was added to a concentration of 33 μM.

[0272] Coupling enzyme mix (20 μl) was added to the solution in the sample plate containing LPL, ANGPTL4, and ANGPTL4 antibody, and the resulting 54 μl of solution was transferred to an assay plate. To initiate the lipoprotein lipase reaction, VLDL / Amplex Red solution (18 μl) was added, and resorufin fluorescence was continuously monitored for 30 minutes using an EnVision multimode plate reader (Perkin Elmer). The final assay concentrations were 9.4 nM ANGPTL4, approximately 4 nM human lipoprotein lipase, 2.3 μg / ml human VLDL, 0.75 mM ATP, 90 μM coenzyme A, 0.5 U / ml ACO, 1.25 U / ml ACS, 1.2 U / ml HRP, and 10 μM Amplex Red. .

[0273] Using the resulting resorufin fluorescence vs. time data, the lipoprotein lipase enzyme activity (initial rate) was determined for each sample. Enzyme activity was normalized using a control sample without LPL (blank ground control) or a control sample without ANGPTL4 or ANGPTL4 antibody (LPL activity control), and this was expressed as a percentage relative to the LPL control activity. Using GraphPad Prism software, different Plot the enzyme activity data for the ANGPTL4 antibody concentration and fit the data to generate an EC value for the increase in lipoprotein lipase enzyme activity mediated by the ANGPTL4 antibody. In this assay, human ANGPTL4 at a concentration of 10 nM 50 typically inhibited LPL activity by 70 - 95%. The ANGPTL 4 antibody of the present invention dose-dependently reversed the inhibition of LPL by ANGPTL4. The EC50 results from this assay are shown in Table 4. Representative data for the selected antibodies of the present invention are shown in Figure 1.

[0274] [Table 4]

[0275] Example 8: Preparation of cynomolgus ANGPTL4 protein, mouse ANGPTL4 protein, and rat ANGPTL4 protein, as well as human ANGPTL3 protein for use in antibody characterization The sequence of cynomolgus ANGPTL4 was determined by amplifying the genetic sequence derived from a cynomolgus liver cDNA library (Biochain, catalog number C1534149 - Cy, lot number B409051). The primers, 5-UT-Cyno5’- ATCCCCGCTCCCAGGCTAC-3’ (SEQ ID NO: 158) and 3-UT-c yno5’-CAGCAAGGAGTGAAG-CTCCATGCC-3’ (SEQ ID NO: 1 59), were designed based on the 5’ untranslated region and 3’ untranslated region of human ANGPTL4 cDNA (NCBI sequence NM_139314.2 ). The gel-purified P ) and 3’ untranslated regions. The gel-purified PCR product was cloned into a pCR2.1-TOPO vector (Invitrogen) and sequenced. The mouse ANGPTL4 protein was prepared by cloning the mouse ANGPTL4 cDNA (obtained from Open Biosystems) into a pET-28a(+) vector (Novagen) and expressing it in E. coli BL21(DE3) cells. The rat ANGPTL4 protein was prepared by cloning the rat ANGPTL4 cDNA (obtained from Open Biosystems) into a pET-28a(+) vector (Novagen) and expressing it in E. coli BL21(DE3) cells. The human ANGPTL3 protein was prepared by cloning the human ANGPTL3 cDNA (obtained from Open Biosystems) into a pET-28a(+) vector (Novagen) and expressing it in E. coli BL21(DE3) cells. ​​​​​​The CR product was ligated into pCR4-Blunt-TOPO (Life Technologies, Catalog No. K2875-40) and sequenced. The cloned crab quail ANGPTL4 cDNA encoded a 406-amino acid protein with 95% homology to human ANGPTL4. The nucleic acid sequence encoding amino acids 26-406 of crab quail ANGPTL4 was subcloned into pRS5, a mammalian expression vector, to generate plasmid pRS-Ikk-cynoANGPTL4(26-406)-FLAG-6HIS-Avi, which has a 20-amino acid Ikk signal sequence, amino acids 26-406 of crab quail ANGPTL4, and a carboxyl-terminal FLAG tag, 6HIS tag, and Avi tag (SEQ ID NO: 160 in Table 5). In Example 1, crab quail ANGPTL4(26-406)-FLAG-6HIS-Avi protein was expressed and purified using the same method as described for human ANGPTL4(26-406)-FLAG-6HIS-Avi protein. For some applications, purified crab quail ANGPTL4 protein was site-specifically biotinylated using the same materials as described for human ANGPTL4 protein in Example 1. The purity of the purified crab quail ANGPTL4 protein, as evaluated by SDS-PAGE, was >90%.

[0276] In Example 1, crab quail ANGPTL4(26-161)-FLAG-6HIS-Avi protein was prepared using the same method. The corresponding expression construct encoded the crab quail ANGPTL4(26-161)-FLAG-6HIS-Avi protein. For some applications, the same materials as described for human ANGPTL4 protein in Example 1 were used to site-specifically biotinylate the purified crab quail ANGPTL4 protein. The purity of the purified crab quail ANGPTL4 protein, as evaluated by SDS-PAGE, was >90%. The purity of the purified crab quail ANGPTL4 protein, as evaluated by SDS-PAGE, was >90%. The purity of the purified crab quail ANGPTL4 protein, as evaluated by SDS-PAGE, was >90%.

[0277] Using a similar method, crab quail ANGPTL4(26-161)-FLAG-6HIS-Avi protein was prepared. The corresponding expression construct encoded the The sequence of the difficult monkey ANGPTL4 (26-161) protein is shown in Table 5 (SEQ ID NO 161).

[0278] The expression, purification, and biotinylation of mouse ANGPTL4 (amino acids 26-410) and rat ANGPTL4 (amino acids 24-405) were carried out in Method 2 of Example 1 using a method essentially the same as the method described for human ANGPTL4. The sequences of the mouse ANGPTL4 protein and the rat ANGPTL4 protein encoded by the corresponding expression constructs are shown in Table 5 (SEQ ID NOs 162 and 163, respectively). The purity of the purified mouse ANGPTL4 protein and the purified rat ANGPTL4 protein, as evaluated by SDS-PAGE, was >90%.

[0279] ANGPTL3 (SEQ ID NO: 5, Table 1) is a human protein closely related to ANGPTL4. To enable the assessment of the possible binding of the antibodies of the present invention to ANGPTL3, a method similar to the method described for human ANGPTL4 in Method 2 of Example 1 was used to express, purify, and biotinylate human ANGPTL3 (14-460)-FLAG-His-Avi protein (SEQ ID NO: 164, Table 5).

[0280]

Table 5

[0281] Example 9: Characterization of Antibody Binding Specificity by Direct ELISA Assay A direct ELISA assay was performed to characterize the antibody binding specificity of the selected antibodies ​​Applied. The assay was performed as follows. The plates were incubated with blocking buffer (PBS, pH 7.4, 5% w / v bovine serum albumin) with constant shaking (600 rpm) at 22 °C for 1 hour to block 384-well Meso Scale Discovery (MSD) plates coated with streptavidin. Then, the plates were washed three times with a plate washer (BioTek) using wash buffer (PBS, pH 7.4 and 0.05% v / v Tween-20). Following washing, biotinylated human ANGPTL4 proteins: full-length human ANGPTL4 (hANGPTL4), human ANGPTL4 coiled-coil domain (hANGPTL4-CCD), and full-length human ANGPTL3 (hANGPTL3) diluted in assay buffer (PBS without CaCl2 or MgCl2, pH 、 7.4, 0.5% w / v fatty acid-free bovine serum albumin, and 0.02% v / v Tween-20) were immobilized on the surface by incubation at a concentration of 1 nM (15 μL per well) at 22 °C for 1 hour. Then, the plates were washed three times as described above. Then, antibodies diluted to a concentration of 1 nM in assay buffer were applied to the MSD plates (15 μL per well), and the plates were incubated at 22 °C for 1 hour with constant shaking (60 0 rpm). The bound antibodies were detected with a 1:500 dilution of Sulfo-tagged goat anti-human IgG, 15 μL per well . Then, the plates were washed three times as described above. Then, antibodies diluted to a concentration of 1 nM in assay buffer were applied to the MSD plates (15 μL per well), and the plates were incubated at 22 °C for 1 hour with constant shaking (60 0 rpm). The bound antibodies were detected with a 1:500 dilution of Sulfo-tagged goat anti-human IgG, 15 μL per well It was detected by adding L. Subsequently, the plate was incubated for 1 hour with constant shaking (600 rpm). The plate was washed three times and then 15 μL per well of 1× concentration of MSD read buffer T was added and the plate was developed using a Sector Imager 6000 (Meso Scale Discovery y). For analysis, the data was transferred to Microsoft Excel and plotted using GraphPad Prism v6. These experiments showed that all of the test antibodies of the invention in this assay bind to full-length human ANGPT L4 and the N-terminal domain of human ANGPTL4, but do not bind to full-length human ANGPTL 3. The ANGPTL3-specific reference antibody was used as a positive control for the ANGPTL3 binding assay (Figure 2).

[0282] Example 10: Determination of the dissociation constant of an antibody by solution equilibrium titration (SET) assay The SET assay was performed as follows. In a 96-well polypropylene plate, a constant concentration of ANGPTL4 antibody (10 pM) was mixed with different concentrations of non-biotinylated human, cynomolgus monkey, mouse or rat full-length ANGPTL4 protein, or human ANGPTL4 N-terminal domain protein (five-fold serial dilutions in the range of 0.01 pM to 100 nM) 、 in SET buffer (PBS without CaCl2 or MgCl2, pH 7.4 0.5% w / v bovine serum albumin (fatty acid-free ), and 0.02% v / v Tween-20). The final reaction volume was 80 μL. Adhesive ​​​Using a film, the plates were sealed and incubated at 22 °C for 14 h with a constant shaking (300 rpm). At the same time, the plates were incubated with 50 μL of blocking buffer (PBS, pH 7.4, 5% w / v bovine serum albumin) per well at 4 °C to block 384-well Meso Scale Discovery (MSD ) plates coated with streptavidin. Using a plate washer (BioTek), the blocked MSD plates were washed three times with wash buffer (PBS, pH 7.4 and 0.05% v / v Tween-20). Biotinylated ANGPTL4 (full-length human ANGPTL4, full-length cynomolgus ANGPTL4, full-length mouse ANGPTL4, or also full-length rat ANGPTL4, or human ANGPTL4 N-terminal domain) protein (1 nM, 15 μL per well) was immobilized on the surface of streptavidin-coate...

Claims

**Claim 1**: A polynucleotide encoding an anti-ANGPTL4 antibody or an antigen-binding fragment thereof, comprising: (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 9, and a light chain variable region comprising LCDR1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19; or (b) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 10, HCDR2 of SEQ ID NO: 11, and HCDR3 of SEQ ID NO: 12, and a light chain variable region comprising LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR3 of SEQ ID NO:

22. **Claim 2**: The polynucleotide according to claim 1, wherein the antibody or the antigen-binding fragment thereof is a monoclonal antibody, a humanized antibody, a single-chain antibody, a Fab fragment, an Fv fragment, an F(ab')2 fragment or an scFv fragment. **Claim 3**: The polynucleotide according to claim 1 or 2, wherein the antibody or the antigen-binding fragment thereof is of the IgG1 or IgG4 isotype. **Claim 4**: The polynucleotide according to any one of claims 1 to 3, wherein the antibody or the antigen-binding fragment thereof comprises: (i) a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 23, and (ii) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

13. **Claim 5**: The polynucleotide according to claim 4, wherein the antibody comprises: (i) a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, and (ii) a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

25. **Claim 6**: The polynucleotide according to claim 5, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO:

25. **Claim 7**: The antibody is: (i) a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 28, and (ii) a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 The polynucleotide according to claim 4, comprising the same.

8. The polynucleotide according to claim 7, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28 and a light chain comprising the amino acid sequence of SEQ ID NO:

25.

9. The polynucleotide according to any one of claims 1 to 8, wherein the antibody or an antigen-binding fragment thereof is a bispecific antibody or an antigen-binding fragment thereof.

10. The polynucleotide, wherein (i) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 24, and (ii) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14 The polynucleotide according to any one of claims 1 to 4, comprising the same.

11. The polynucleotide, wherein (i) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 27, and (ii) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 30 The polynucleotide according to any one of claims 1 to 4, comprising the same.

12. The polynucleotide, wherein (i) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 29, and (ii) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 31 The polynucleotide according to any one of claims 1 to 4, comprising the same.

13. The polynucleotide, wherein (i) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 16, and (ii) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 26 The polynucleotide according to any one of claims 1 to 4, comprising

14. A host cell comprising the polynucleotide according to any one of claims 1 to 13.

15. An expression vector comprising the polynucleotide according to any one of claims 1 to 13.

16. The expression vector according to claim 15, wherein the expression vector is a virus-based vector.

17. The expression vector according to claim 15, wherein the expression vector is a non-viral expression vector.

18. The expression vector according to any one of claims 15 to 17, wherein the expression vector further comprises a nucleic acid sequence encoding a secretion signal.

19. A method for producing an anti-ANGPTL4 antibody or an antigen-binding fragment thereof, comprising: culturing a host cell comprising the polynucleotide according to any one of claims 1 to 13 or the expression vector according to any one of claims 15 to 18 under conditions suitable for producing the anti-ANGPTL4 antibody or an antigen-binding fragment thereof.

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