High-concentration composition and novel anti-ANGPTL3 antibody suitable for subcutaneous administration

Abstract

The present invention relates to a novel monovalent anti-ANGPTL3 antibody for use in medicine, specifically for reducing plasma triglyceride levels in patients in need thereof, such as patients suffering from or at risk of cardiovascular diseases such as hypertriglyceridemia and / or atherosclerotic cardiovascular disease (ASCVD), as well as a pharmaceutical composition suitable for subcutaneous administration, and a kit comprising such a compound and composition.

Description

Technical Field

[0001] Anti-ANGPTL3 antibodies for use in medicine and compositions comprising such compounds.

[0002] Incorporation by reference of a sequence listing This application is filed together with an electronic form of the sequence listing. The entire content of the sequence listing is incorporated herein by reference.

Background Art

[0003] Human angiopoietin-like protein 3 (hANGPTL3) is a 460-amino acid-long protein first identified by Conklin et al. ([Non-Patent Document 1]). hANGPTL3 circulates in plasma as a trimer formed by two coiled-coil domains present in the N-terminal portion of the protein. hANGPTL3 is part of a family of angiopoietin-like proteins that affect lipid metabolism by inhibiting lipoprotein lipase (LPL) and endothelial lipase (EL).

[0004] hANGPTL3 is the most abundantly expressed member of this family and is a well-described inhibitor of LPL. It mainly exerts its action in the non-fasting state. LPL is an important enzyme fixed to endothelial cells and hydrolyzes triglycerides from triglyceride-rich lipoparticles (VLDL and chylomicrons). The higher the LPL activity, the more triglycerides are removed from the circulation.

[0005] When LPL is inhibited by hANGPTL3, the plasma triglyceride and LDL concentrations increase.

[0006] Inhibition of LPL by hANGPTL3 is thought to function through interaction with human angiopoietin-like protein 8 (hANGPTL8) and possibly human angiopoietin-like protein 4 (hANGPTL4). hANGPTL8 does not have an in vitro inhibitory function in the absence of hANGPTL3, while hANGPTL3 can inhibit LPL alone. hANGPTL4 is also known to inhibit LPL independently of hANGPTL3 and 8. hANGPTL3 is generally expressed by the liver, while hANGPTL4 and 8 show different expression patterns in different organisms. hANGPTL4 and 8 are mainly expressed by external stimuli, while hANGPTL3 shows high constitutive expression from the liver.

[0007] There are different cleavage forms in circulation ([Non-Patent Document 2]). Full-length hANGPTL3 containing the N-terminal domain and cleaved hANGPTL3 all inhibit LPL and EL and increase triglyceride levels.

[0008] Loss-of-function variants in the gene encoding hANGPTL3 are associated with hypolipidemia and protection against atherosclerotic cardiovascular disease. The monoclonal antibody against hANGPTL3, evinacumab (Evkeeza®) ([Patent Document 1]), has shown benefits in patients with homozygous familial hypercholesterolemia at a dose of 15 mg per kilogram of body weight every 4 weeks ([Non-Patent Document 3]).

[0009] Due to the high dose required for treatment, the currently available antibody product Evkeeza® (the recommended dose of EVKEEZA is a 15 mg / kg dose administered by intravenous infusion over 60 minutes every 4 weeks using an inline or add-on filter) cannot be used for self-administration, and a more optimal treatment scenario may be beneficial for the patient population.

[0010] In the art, improved anti-hANGPTL3 antibodies are needed.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0013] The present invention relates to a monovalent antibody that binds to human ANGPTL3 (hANGPTL3) and can reduce hANGPTL3-mediated inhibition of LPL, an antigen-binding fragment thereof, a medicament, and its use in a pharmaceutical composition containing such a compound.

[0014] In one aspect, the monovalent antibody and its antigen-binding fragment can bind to hANGPTL3 and can reduce the concentration of triglycerides in human plasma.

[0015] In one aspect, the monovalent antibody and its antigen-binding fragment can bind to hANGPTL3 and can reduce the inhibition of LPL by reducing the concentration of hANGPTL3 in human plasma.

[0016] In one aspect, the monovalent antibody and its antigen-binding fragment can bind to hANGPTL3 and can inhibit hANGPTL3-mediated inhibition of LPL.

[0017] In one aspect, the monovalent antibody and antigen-binding fragment thereof can bind to hANGPTL3, inhibit the hANGPTL3-mediated inhibition of LPL, and by reducing the concentration of hANGPTL3 in human plasma, reduce the hANGPTL3-mediated inhibition of LPL.

[0018] In one particular aspect, the invention relates to a monovalent antibody or antigen-binding fragment thereof that can bind to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), a) the heavy chain of the antibody or antigen-binding fragment thereof is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, the CDR1 sequence, and ● a CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, the CDR2 sequence, and ● a CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, the CDR3 sequence, or b) the heavy chain of the antibody or antigen-binding fragment thereof is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 24), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, the CDR1 sequence, and ● a CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 25), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, the CDR2 sequence, and ● a CDR3 sequence of amino acid residues EGWYDNWNDP (SEQ ID NO: 26), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, the CDR3 sequence, and The light chain of the antibody or its antigen-binding fragment is ● A CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted with different amino acid residues, and the CDR1 sequence, ● A CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), wherein 0, 1, or 2 of these amino acid residues may be substituted with different amino acid residues, and the CDR2 sequence, A CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein 0, 1, or 2 of these amino acid residues may be substituted with different amino acid residues, and the CDR3 sequence, and includes.

[0019] In one aspect, the monovalent antibody and its antigen-binding fragment may be formulated into a high-concentration composition.

[0020] In one aspect, the monovalent antibody and its antigen-binding fragment are suitable for subcutaneous administration.

Brief Description of the Drawings

[0021]

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[0022] Brief Description of Sequences SEQ ID NO:1 represents the amino acid sequence of human ANGPTL3.

[0023] SEQ ID NO: 2, 3, and 4 represent the amino acid sequences of complementarity-determining regions (CDRs) 1-3 of the heavy chain of tmAb1, respectively.

[0024] SEQ ID NO: 5 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb1.

[0025] SEQ ID NO: 6 represents the amino acid sequence of the heavy chain of tmAb1 containing a C-terminal Gly-Lys deletion (des-(Gly-Lys)).

[0026] SEQ ID NO: 7 represents the amino acid sequence of the truncated heavy chain (tHC) of tmAb1, tmAb2, tmAb3, and tmAb4, also containing a C-terminal Gly-Lys deletion (des-(Gly-Lys)).

[0027] SEQ ID NO: 8 represents the amino acid sequence of an alternative truncated heavy chain (tHC) that differs from SEQ ID NO: 7 in that it does not have a C-terminal Gly-Lys deletion.

[0028] SEQ ID NO: 9, 10, and 11 represent the amino acid sequences of complementarity-determining regions (CDRs) 1-3 of the light chain of tmAb1, tmAb2, tmAb3, and tmAb4, respectively.

[0029] SEQ ID NO: 12 represents the amino acid sequence of the light chain variable domain (VL) of tmAb1, tmAb2, tmAb3, and tmAb4.

[0030] SEQ ID NO: 13 represents the amino acid sequence of the light chain of tmAb1, tmAb2, tmAb3, and tmAb4.

[0031] SEQ ID NO: 14, 15, and 16 represent complementarity-determining regions (CDRs) 1-3 of the heavy chain of tmAb2, respectively.

[0032] SEQ ID NO: 17 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb2.

[0033] SEQ ID NO: 18 represents the amino acid sequence of the heavy chain of tmAb2.

[0034] SEQ ID NO: 19, 20, and 21 represent the amino acid sequences of complementarity-determining regions (CDRs) 1-3 of the heavy chain of tmAb3, respectively.

[0035] SEQ ID NO: 22 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb3.

[0036] SEQ ID NO: 23 represents the amino acid sequence of the heavy chain of tmAb3.

[0037] SEQ ID NO: 24, 25, and 26 represent the amino acid sequences of complementarity-determining regions (CDRs) 1-3 of the heavy chain of tmAb4, respectively.

[0038] SEQ ID NO: 27 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb4.

[0039] SEQ ID NO: 28 represents the amino acid sequence of the heavy chain of tmAb4.

Mode for Carrying Out the Invention

[0040] When an anti-ANGPTL3 antibody such as Evkeeza (registered trademark), which is used for inhibiting the inhibition of LPL by ANGPTL3 to reduce triglycerides in human plasma, is administered intravenously (i.v.), dose-dependent accumulation of ANGPTL3 was observed (Ahmad et al. (2019) Circulation Aug 6;140(6):470-486 Supplementary Information Figure 2).

[0041] This phenomenon is known as antibody-mediated antigen accumulation and can significantly increase the antibody dosage required to achieve therapeutic efficacy. The need for a high therapeutic effective dose can pose challenges to the administration route. For example, to administer a high dose of an antibody subcutaneously (s.c.), the antibody needs to have sufficiently good properties regarding solubility in solution, self-association, and viscosity.

[0042] So-called sweep antibodies have been developed to generally increase the removal of soluble antigens to which such antibodies bind (Igawa et al. (2016) Immun. Rev. 270:132-151), but oligomeric target proteins such as ANGPTL3 present specific challenges to this concept when several antibodies bind to the same target, thereby presenting multiple binding opportunities to Fc receptors. This adds avidity to the antibody-Fc receptor affinity in the antibody recycling mechanism, thereby facilitating the uptake of immune complexes (ICs) into endosomes. However, at the same time this challenges pH-dependent antigen release and sweep in endosomes / lysosomes and also challenges antibody recycling by inducing clearance of multi-component immune complexes (ICs) generated when several antibodies bind to a single multimeric antigen. The presence of two or more Fab moieties on each antibody molecule exposes several binding opportunities to the same antibody for oligomeric targets, potentially generating complexes and heterogeneous IC networks, the size of which correlates with the immune response and IC clearance (Opolka-Hoffmann et al. (2021) mAbs, 13:1, 1995929, St. Clair et al. (2017) PLOS one, 12(1):e0170556).

[0043] The inventors have found that by utilizing a monovalent antibody format that further has pH-dependent binding characteristics, an improved viscosity profile of the antibody can be achieved, thereby providing the possibility of administering the antibody by subcutaneous administration. Surprisingly, by switching to a monovalent format having only one ANGPTL3 binding site per molecule, a triglyceride-lowering ability comparable to that of Evkeeza® having two ANGPTL3 binding sites per molecule was obtained. The smaller the size of the antibody disclosed herein, the more enhanced the possibility of subcutaneous administration. The antibodies disclosed herein have biophysical properties that enable a concentration in a solution of at least 70 mg / ml, in contrast to Evkeeza® prepared in an intravenous infusion bag at a concentration of at most 20 mg / ml.

[0044] Accordingly, the present invention relates to a monovalent antibody and an antigen-binding fragment thereof that bind to hANGPTL3 and can reduce hANGPTL3-mediated inhibition of LPL, as well as a medicament and a pharmaceutical composition suitable for subcutaneous administration, and to the use of such a compound in a pharmaceutical composition comprising such a compound.

[0045] In one aspect, the monovalent antibody or an antigen-binding fragment thereof can bind to hANGPTL3 and reduce the concentration of triglycerides in human plasma.

[0046] In one such aspect, the monovalent antibody and an antigen-binding fragment thereof can bind to hANGPTL3 and reduce the inhibition of LPL by reducing the concentration of hANGPTL3 in human plasma.

[0047] In another such aspect, the monovalent antibody and an antigen-binding fragment thereof are antagonists that can bind to hANGPTL3 and inhibit hANGPTL3-mediated inhibition of LPL.

[0048] In one aspect, the monovalent antibody and antigen-binding fragment thereof can bind to hANGPTL3, inhibit hANGPTL3-mediated inhibition of LPL, and reduce hANGPTL3-mediated inhibition of LPL by reducing the concentration of hANGPTL3 in human plasma.

[0049] In one aspect, the monovalent antibody or antigen-binding fragment thereof does not increase the concentration of hANGPTL3 in human plasma after administration.

[0050] In one aspect, the monovalent antibody or antigen-binding fragment thereof decreases the concentration of hANGPTL3 in human plasma after administration.

[0051] In one aspect, the monovalent antibody or antigen-binding fragment thereof provides a reduction in viscosity, a reduction in gel formation, and a low tendency for self-association over a wide pH range in solution compared to the corresponding bivalent antibody.

[0052] In one particular aspect, the invention relates to a monovalent antibody or antigen-binding fragment thereof that can bind to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), a) the heavy chain of the antibody or antigen-binding fragment thereof is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), wherein 0 or 1 of these amino acid residues may be substituted by a different amino acid residue, the CDR1 sequence, and ● a CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by a different amino acid residue, the CDR2 sequence, and ● a CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by a different amino acid residue, the CDR3 sequence, or b) the heavy chain of the antibody or antigen-binding fragment thereof is ● The CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 24), wherein 0 or 1 of these amino acid residues may be substituted by a different amino acid residue, and the CDR1 sequence, ● The CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 25), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by a different amino acid residue, and the CDR2 sequence, ● The CDR3 sequence of amino acid residues EGWYDNWNDP (SEQ ID NO: 26), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by a different amino acid residue, and the CDR3 sequence, and The light chain of the antibody or its antigen-binding fragment is ● The CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by a different amino acid residue, and the CDR1 sequence, ● The CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), wherein 0, 1, or 2 of these amino acid residues may be substituted by a different amino acid residue, and the CDR2 sequence, The CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein 0, 1, or 2 of these amino acid residues may be substituted by a different amino acid residue, and the CDR3 sequence, and

[0053] In one aspect, the present monovalent antibody and its antigen-binding fragment are suitable for subcutaneous administration.

[0054] In one aspect, the present monovalent antibody and its antigen-binding fragment may be formulated into a high-concentration composition.

[0055] In one aspect, the present monovalent antibody or its antigen-binding fragment may be prepared into a composition, and the concentration of the present antibody or its antigen-binding fragment is 25 mg / ml or more, preferably 50 mg / ml or more, for example 70 mg / ml or more.

[0056] To facilitate a better understanding of the present invention, certain terms are first defined.

[0057] The terms "a" or "an" are intended to mean "one or more." When preceding a listing of steps or elements, the term "comprise," and its variants such as "comprises" and "comprising," are intended to mean that the addition of further steps or elements is optional and not excluded.

[0058] The term "about" is used herein to mean approximately, roughly, or around. When the term "about" is used in conjunction with a numerical range, the range is modified by extending the boundaries above and below the recited numerical values. Generally, the term "about" can modify numerical values that are 10 percent above or below (higher or lower), above and below the recited value.

[0059] The term "antagonistic antibody," as used herein, refers to an antibody that inhibits or reduces the biological activity of an antigen to which the antibody binds, such as hANGPTL3. In some embodiments, the antibody or its antigen-binding fragment specifically binds to hANGPTL3, and as a result, by partially or completely blocking its inhibitory effect on LPL, for example, reduces the triglyceride level in the plasma of a human patient.

[0060] The term "antibody" includes, but is not limited to, full-length antibodies that include at least four polypeptide chains, two heavy chains (HC) and two light chains (LC) connected by disulfide bonds, and antibodies that include at least three polypeptide chains, two heavy chains (HC) and one light chain (LC) connected by disulfide bonds. One of the heavy chains can be a truncated heavy chain.

[0061] One class of immunoglobulins with specific pharmaceutical benefits is IgG. In humans, the IgG class may be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequences of their heavy chain constant regions. Light chains can be divided into two types, kappa chains and lambda chains, based on differences in their sequence compositions. An IgG molecule is composed of two heavy chains linked by two or more disulfide bonds, and two light chains each attached to a heavy chain by a disulfide bond.

[0062] The IgG heavy chain contains a heavy chain variable domain (V H ) and up to three heavy chain constant (C H ) domains: C H 1, C H 2, and C H 3. The light chain may contain a light chain variable domain (V L ) and a light chain constant domain (C L ). The V H and V L regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) or hypervariable regions (HvRs), interspersed with more conserved regions called framework regions (FRs). The V H and V L domains typically consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable domain and the light chain variable domain containing the hypervariable regions (CDRs) form a structure that can interact with an antigen, while the constant region of the antibody can mediate binding to host tissues or factors of the immunoglobulin. Such host tissues or factors include, but are not limited to, various cells of the immune system (effector cells), Fc receptors, and C1q, which is the first component of the C1 complex of the classical complement system.

[0063] The antibody of the present invention may be a monoclonal antibody (mAb) such as a monovalent monoclonal antibody. The antibody of the present invention may be produced and purified using various methods known to those skilled in the art. For example, the antibody may be produced from hybridoma cells. The antibody may be produced by B cell proliferation. The antibody or its antigen-binding fragment may be recombinantly expressed in a mammalian or microbial expression system, or by in vitro translation. Also, the antibody or its antigen-binding fragment may be recombinantly expressed as a cell surface binding molecule, for example, by phage display, bacterial display, yeast display, mammalian cell display, or ribosome or mRNA display.

[0064] The antibody or its antigen-binding fragment may be defined in terms of its complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" as used herein refers to the region of the antibody in which amino acid residues involved in antigen binding are located. CDRs can be identified as the regions of highest variability in amino acid alignments of antibody variable domains. Databases such as the Kabat database can be used for CDR identification, and this CDR is defined, for example, as including amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain, and amino acid residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Typically, the numbering of amino acid residues in this region is carried out by the method described in Kabat et al. above. In this specification, phrases such as "Kabat position", "Kabat residue", and "according to Kabat" refer to this numbering system for the heavy chain variable domain or the light chain variable domain, and the Kabat numbering is used herein unless the context is inconsistent.

[0065] The numbering of amino acid residues in the constant region of the antibody is carried out by referring to the numbering used in the crystal structure of human IgG1 (Edelman et al. (1969) Proc. Natl. Acad. Sci. USA 63:78-85), and terms such as "EU position", "EU residue", and "according to the EU index" herein refer to the numbering of the heavy and light chain constant domains according to Edelman et al. (1969) Proc. Natl. Acad. Sci. USA 63:78-85.

[0066] References to positions in the heavy (or light) chain, such as "valine (V) at position 250", mean that valine is found at position 250 of both heavy chains, unless otherwise specified or inconsistent in the context (as long as a given residue position exists in both heavy chains).

[0067] The term "antigen-binding fragment" of an antibody, as described herein, refers to one or more fragments of an antibody that specifically bind to an antigen, such as ANGPTL3, specifically hANGPTL3, or retain the ability to recognize these antigens. Examples of antigen-binding fragments include a single V H , a single V L domain, and monovalent molecules that include both the Fc receptor interaction region (but are not limited thereto).

[0068] The antibody fragments of the present invention may be made, for example, by deletion of one or more amino acids from the N-terminus and / or C-terminus of the heavy and / or light chains. Also, the fragments may be generated by one or more internal deletions.

[0069] The present invention encompasses variants of the antibodies disclosed herein or antigen-binding fragments thereof that may include one or more amino acid substitutions and / or deletions and / or insertions in the individual sequences disclosed herein.

[0070] The "substitution" variant preferably involves the replacement of one or more amino acids having the same number of amino acids. The substitution may be, but is not limited to, a conservative substitution. For example, for conservative substitutions, an amino acid may be substituted with an amino acid having similar biochemical properties. For example, a basic amino acid may be substituted with another basic amino acid (e.g., from lysine to arginine), an acidic amino acid may be substituted with another acidic amino acid (e.g., from glutamic acid to aspartic acid), a neutral amino acid may be substituted with another neutral amino acid (e.g., from threonine to serine), a charged amino acid may be substituted with another charged amino acid (e.g., from glutamic acid to lysine), a hydrophilic amino acid may be substituted with another hydrophilic amino acid (e.g., from asparagine to glutamine), a hydrophobic amino acid may be substituted with another hydrophobic amino acid (e.g., from alanine to valine), a polar amino acid may be substituted with another polar amino acid (e.g., from serine to threonine), an aromatic amino acid may be substituted with another aromatic amino acid (e.g., from phenylalanine to tryptophan), and an aliphatic amino acid may be substituted with another aliphatic amino acid (e.g., from leucine to isoleucine).

[0071] Preferred variants include those containing a structural analog of an amino acid residue in place of the amino acid residue found in the sequence.

[0072] The antibodies and antigen-binding fragments thereof disclosed herein include, but are not limited to, regions that can interact with cell surface receptors called Fc receptors, such as the crystallizable region (the "Fc region," also known as the "Fc domain"). The Fc region is the C-terminal region of the antibody, which is the hinge and constant C H 2 and C HIt comprises three domains. The Fc domain may interact with cell surface receptors called Fc receptors, as well as several proteins of the complement system. The Fc region enables the antibody to interact with the immune system. In one aspect of the invention, the antibody is engineered to include modifications within the Fc region, typically to modify one or more of its functional properties such as plasma half-life, complement fixation, Fc-receptor binding, protein stability, and / or antibody-dependent cell cytotoxicity, or the lack thereof (e.g., T252L / T254S / T256F Ghetie et al., (1997) Nature Biotechnol. 15:637-640, M428L / N434S, Zalevsky et al., (2010) Nat. Biotechnol. 28:157-159, H433K / N434F, Ober (2014) (US8834871), T307A / E380A / N434A, Petkova et al. (2006) Int. Immunol 18:1759-1769, T250A / M428L, Hinton et al. (2006) J. Immunol. 176:346-356, M252Y / S254T / T256E, US2003 / 0190311, Dall’Acqua et al. (2006) J. Biol. Chem. 281:23514-23524, N434H, Zheng et al., (2011) Clin. Pharm. & Ther. 89:283-290, M252Y / T256D, T256D / T307Q, T256D / T307W, Mackness et al. (2019) mAbs 11:1276-1288, residue numbering follows the EU index). Further, the present antibody and its antigen-binding fragments may also be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to modify one or more functional properties of the antibody.

[0073] Antibodies such as IgG1 antibodies may carry a modified Fc domain comprising one or more, preferably all, of the substitutions that increase binding to certain Fc-gamma receptors, such as Q311R and P343R, in combination with L234Y, P238D, T250V, V264I, T307P, and A330K (residue numbering follows the EU index). Alternatively, other amino acid substitutions known in the art to increase FcRn and / or Fc-gamma receptor binding may optionally be used in combination with the substitutions described above. See, for example, International Publication No. WO 2016 / 125495, as well as International Publication Nos. WO 2013 / 002362, WO 2014 / 104165, WO 2012 / 115241, WO 2014 / 030728, WO 2014 / 163101, and WO 2017 / 104783, all of which are incorporated herein by reference.

[0074] IgG1 antibodies may carry a modified Fc domain comprising substitutions that increase the plasma half-life. In one embodiment, such substitutions include the combination of M252Y, S254T, and T256E (residue numbering according to the EU index). See also US 2003 / 0190311, which is incorporated herein by reference.

[0075] The term "binding affinity" is a measure of the strength of non-covalent interactions between two molecules, e.g., between an antibody or a fragment thereof and an antigen. This term is used to describe monovalent interactions. The binding affinity between two molecules via monovalent interactions, e.g., between an antibody or a fragment thereof and an antigen, can be quantified by determining the equilibrium dissociation constant (K D ). K D can be determined by measuring the kinetics of complex formation and dissociation, e.g., by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). The rate constants corresponding to the association and dissociation of a monovalent complex are the association rate constant k a (or k on ), and the dissociation rate constant k d(or k off ) is referred to as K D is given by the formula K D = k d / k a and is related to k a and k d via this relationship.

[0076] According to the above definition, binding affinities associated with different molecular interactions, such as the binding affinities of different antibodies to a given antigen, may be compared by comparing the K D values of the individual antibody / antigen complexes.

[0077] The value of the dissociation constant can be determined directly by well-known methods. Standard assays for evaluating the binding ability of a ligand, such as an antibody towards a target, are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as SPR.

[0078] A competitive binding assay can be performed to compare the binding of an antibody to a target with the binding of the target in the presence of another ligand of the target, such as another antibody.

[0079] Unless there is a contradiction in the context, K D is preferably determined by surface plasmon resonance as described herein (see Example 7).

[0080] A "cross-reactive" antibody binds to ANGPTL3 from all of the indicated species (e.g., human, mouse, and cynomolgus monkey) with comparable affinity, specifically within a factor of 100, such as within a factor of 50, within a factor of 20, or within a factor of 10, etc., with a K D defined by the factor X. K DWithin the range, the highest affinity for a particular enumerated species means that it does not exceed X times the lowest affinity measured for binding to a different enumerated species. One of ordinary skill in the art would use any method for measuring affinity to verify that the cross-reactive antibody binds to target antigens from all enumerated species within the given K D coefficient range described herein, provided that the same conditions apply to the measurement of K D for all enumerated species. Preferably, the K D value is measured using SPR, particularly at 25°C. Preferably, the affinity is measured using a cross-reactive antibody such as a Fab fragment.

[0081] The term "elevated plasma triglyceride concentration" as used herein refers to plasma levels of 150 - 500 mg / dL.

[0082] The term "substantially elevated plasma triglyceride concentration" as used herein refers to plasma levels greater than 500 mg / dL.

[0083] The term "normal plasma triglyceride concentration" as used herein refers to plasma levels less than 150 mg / dL.

[0084] The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides, which is determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of exact matches between the smaller of two or more sequences with gap adjustments (if any) handled by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be readily calculated by known methods. In the present invention, Needleman from EMBOSS-6.6.0 (Needleman et al. J. Mol. Biol. 1970; 48: 443-453) was used, with parameters 10 and 0.5 for gap start and gap extension respectively (gapopen = 10, gapextend = 0.5), to determine similarity and identity.

[0085] The term "monovalent antibody", as used herein, refers to an antibody that can bind to one antigen molecule and cannot perform antigen cross-linking.

[0086] The term "one-armed", as used herein, refers to a specific type of monovalent antibody composed of an antibody single light chain, heavy chain, and a truncated heavy chain lacking at least the heavy chain Fv region.

[0087] The term "monospecific" antibody, as used herein, refers to an antibody that can bind to a single epitope.

[0088] As used herein, the term "human antibody" is intended to include antibodies having variable domains in which at least a portion of the framework region and / or at least a portion of the CDR region is derived from human germline immunoglobulin sequences. For example, a human antibody may have a variable domain in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Further, when an antibody includes a constant region, the constant region or a portion thereof is also derived from human germline immunoglobulin sequences. Preferably, the human antibody is a monoclonal antibody.

[0089] The term "heavy chain" includes full-length heavy chains. A full-length heavy chain includes a variable domain, V H , as well as three constant domains, C H 1, C H 2, and C H 3. The V H domain is at the amino terminus of the polypeptide, the CH domains are at the carboxyl terminus, and C H 3 is closest to the -COOH terminus. The C-terminus of the heavy chain may include a C-terminal Gly-Lys deletion (des-(Gly-Lys)).

[0090] The term "light chain" as used herein includes full-length light chains. A full-length light chain includes a variable domain, V L , and a constant domain, C L . The variable domain of the light chain is at the amino terminus of the polypeptide. The light chains described herein include kappa chains and lambda chains.

[0091] The term "immune complex" as used herein refers to a complex formed by one or more antibodies when bound to one or more target antigens. An example of this is one or more anti-ANGPTL3 antibodies bound to two or more ANGPTL3 molecules. This term is used interchangeably with immune complex.

[0092] The term "isoelectric point" or "pI", as used herein, refers to the pH value at which the overall net charge of a protein, such as an antibody, is zero. A protein has many charged groups, and at the pI, the sum of all these charges is zero. At a pH above the pI, the overall net charge of the protein is negative, while at a pH value below the pI, the overall net charge of the protein is positive.

[0093] The pI may be either a theoretically or experimentally determined pI.

[0094] Those skilled in the art are aware of methods for determining the pI of a protein. Most commonly, the pI of a protein is calculated based on its amino acid sequence. A number of (online) tools are available that enable the determination of the isoelectric point of a protein, such as "ExPASy Compute pI / Mw". See Protein Identification and Analysis Tools on the ExPASy Server; Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.; John M.Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607.

[0095] The pI may also be determined experimentally, and charge variants can be separated using charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis or capillary isoelectric focusing (cIEF) gel electrophoresis.

[0096] The term "multimerization domain", as used herein, refers to each region of the heavy chain of a monovalent antibody, for example. A "multimerization domain" enhances stable interactions between entities of a chimeric monovalent antibody or antigen-binding fragment thereof. Preferably, the multimerization domain promotes the formation of a desired heteromultimeric monovalent antibody by enhancing the interaction between a first heavy chain and a second heavy chain, and substantially reduces the likelihood of formation of an undesired homomultimeric antibody. The multimerization domain may interact via an immunoglobulin sequence, leucine zipper, hydrophobic region, hydrophilic region, or free thiol that forms an intermolecular disulfide bond between chimeric molecules of a chimeric heteromultimer. The free thiol can be introduced at the interface of one or more interacting polypeptides by substituting a naturally occurring residue of the polypeptide with cysteine, for example, at a position that allows the formation of a disulfide bond between a first heavy chain and a second heavy chain. The multimerization domain may include an immunoglobulin constant region. Potential multimerization domains are disclosed in PCT / US90 / 06849, in which hybrid immunoglobulins are described. Additionally, the multimerization region may be engineered such that the steric interactions not only enhance stable interactions, but also further enhance the formation of heterodimers via homodimers from a mixture of monomers. For example, for the interface between a first polypeptide and a second polypeptide for hetero-oligomerization, see PCT / US96 / 01598, which discloses a "protuberance-into-cavity" strategy, and particularly WO 98 / 50431. A "protuberance" is constructed by replacing a small amino acid side chain from the interface of a first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory "cavity" of the same or similar size as the protuberance is optionally created on the interface of the second polypeptide by replacing a large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine). The immunoglobulin sequence is an immunoglobulin constant domain.The immunoglobulin portion in the antibodies disclosed in this specification may be obtained from the IgG1, IgG2, IgG3, or IgG4 subtype.

[0097] There are many techniques for generating chimeric, hybrid, or asymmetric immunoglobulins, and the cleavage technique used in this application (Merchant et al., (2013) PNAS USA 110:E2987-2996) may be used in combination with any of the chimeric formats (e.g., DEEK (De Nardis et al., (2017) J. Biol. Chem. 292:14706-14717), ART-Ig and FAST-Ig (Shiraiwa et al., (2019) Methods 154:10-20, Yamaguchi et al., (2020) Blood 136:19), or DuoBody (Labrijn et al., (2013) PNAS USA 110:5145-5150).

[0098] The term "plasma half-life," as used herein, refers to the time required for half of the amount of a substance administered to a patient to be metabolized or removed from the patient's serum or plasma by normal biological processes.

[0099] The term "surface-exposed amino acid residue", as used herein, refers to an amino acid residue whose side chain can contact solvent molecules (generally, mostly water molecules). However, the side chain does not necessarily have to be in complete contact with the solvent molecules. When even a part of the side chain is in contact with the solvent molecules, the amino acid residue is defined as an "amino acid located on the surface". Amino acid residues located on the surface of a polypeptide may also include amino acid residues located near the surface of an antibody, and thereby may be affected by the mutual charges from other amino acid residues whose side chains are in contact with the solvent molecules even partially. A person skilled in the art can prepare a homology model or a three-dimensional molecular model based on machine learning of a polypeptide or an antibody by performing homology modeling or machine learning using, for example, commercially available or publicly available software. Alternatively, it is possible to use methods such as X-ray crystallographic analysis for three-dimensional molecular model generation. Amino acid residues that can be exposed on the surface can be determined using the coordinates from a three-dimensional molecular model of an antibody using a computer program such as MOE (Chemical Computing Group) or Bioluminate (Schrodinger). The surface-exposed sites may be determined using algorithms known in the art (see, for example, Lee and Richards (1971) J. Mol. Biol. 55: 379-400, Connolly, J. Appl. Cryst. (1983) 16: 548-558). The surface-exposable sites can be determined using software suitable for protein modeling and analysis of three-dimensional structural information obtained from antibodies. Software available for such purposes includes, for example, MOE (Chemical Computing Group) or Bioluminate (Schrodinger). Solvent-accessible surface (Å 2The area is calculated using a water probe with a probe radius of 1.4 Å. Further, the method for determining the surface-exposed region and area using software for a personal computer is described by Pacios (Pacios, Comput. Chem. 18(4):377-386(1994), J. Mol. Model. 1:46-53(1995)). Based on the information as described above, appropriate amino acid residues located on the surface of the antibody in contact with the solvent can be selected.

[0100] As used herein, the term "treatment" refers to medical therapy for any human subject in need thereof. The subject is expected to have undergone a physical examination by a physician who has given a provisional or definitive diagnosis indicating that the use of the particular treatment is beneficial to the health of the human subject. The timing and purpose of the treatment may vary from individual to individual according to the current state of the subject's health. For this reason, the treatment may be prophylactic, palliative, symptomatic, and / or curative. With respect to the present invention, prophylactic, palliative, symptomatic, and / or curative treatments may represent separate aspects of the present invention.

[0101] As used herein, the terms "treating", "treat", or "treatment", and variations thereof, include administering a therapeutically effective amount of an antibody disclosed herein sufficient to reduce or eliminate at least one symptom of a disorder. However, "treatment" need not be a cure.

[0102] As used herein, the terms "preventing", "prevent", or "prevention", or variations thereof, refer to protecting a subject from the onset of at least one symptom of a disease or reducing the severity of the symptoms of a disorder.

[0103] As used herein, the term "truncated heavy chain" lacks at least a portion of the variable domain such that it cannot bind to hANGPTL3 and, optionally, lacks cysteine residues suitable for heavy chain / light chain pairing (such as the IgG1 Cys220 EU residue) in the first constant IgG domain (C H 1) or a heavy chain lacking a portion thereof. In one embodiment, the "truncated heavy chain" refers to a heavy chain lacking the entire variable domain, C H 1, and a portion of the hinge region such that the IgG1 Asp221 (EU residue) is the N-terminal amino acid residue. For clarity, a heavy chain in which only the C-terminal glycine-lysine residues at positions 446 and 447 are removed is not considered a "truncated heavy chain".

[0104] In another embodiment, the "truncated heavy chain" differs from the full-length heavy chain in that the Fab region is absent and the C-terminal glycine-lysine residues (EU residues) at positions 446 and 447 of SEQ ID NO: 8 are either present (SEQ ID NO: 8) or absent (SEQ ID NO: 7).

[0105] In one such embodiment, the monovalent antibody disclosed herein comprises a heavy chain and a truncated heavy chain, wherein both chains are of the IgG1 isotype or based on the IgG1 isotype, the Fab region and the Gly-Lys C-terminal residues (EU residues) at positions 446 and 447 are not present in the truncated heavy chain, and the Gly-Lys C-terminal residues (EU residues) at positions 446 and 447 of the heavy chain are not present.

[0106] In a preferred embodiment, the truncated heavy chain comprises SEQ ID NO: 7.

[0107] As used herein in the context of a pharmaceutical composition, the term "high concentration" means a concentration of 50 mg / ml or greater.

[0108] Production of Monovalent Antibodies The monovalent antibodies or antigen-binding fragments thereof disclosed herein can be produced in one host cell line expressing three different polypeptide chains, specifically, a heavy chain, a truncated heavy chain, and a light chain. For non-limiting examples of such antibodies, see Figure 2. No further arm exchange or in vitro refolding of the antibody is required to obtain a fully functional molecule.

[0109] In one embodiment, the monovalent antibody or antigen-binding fragment thereof includes a so-called knob-in-hole substitution that facilitates the formation of heterodimers of two different heavy chains, such as a full-length heavy chain and a truncated heavy chain. There are various sets of knob-in-hole substitutions. In a preferred embodiment, the heavy chain includes T366W (EU residue), and the truncated heavy chain includes T366S, L368A, and Y407V (EU residue). See also International Publication No. WO 98 / 50431, which is incorporated herein by reference.

[0110] In a preferred embodiment, the monovalent antibody includes a light chain, a full-length heavy chain, and a truncated heavy chain (221-445, EU residues), all of which are co-expressed and assembled in a host cell suitable for antibody production.

[0111] In another preferred embodiment, the monovalent antibody includes a light chain, a C-terminal des-Gly-Lys heavy chain (1-445, EU residues), and a C-terminal des-Gly-Lys truncated heavy chain (221-445, EU residues), all of which are co-expressed and assembled in a host cell suitable for antibody production.

[0112] The complex comprising hANGPTL3 and the antibody disclosed herein is observed at neutral pH (pH 7.4). Thus, the affinity of the monovalent antibody is sufficient for efficient binding to hANGPTL3. Binding of two to three monovalent antibodies to the trimeric hANGPTL3 antigen is shown, and a more homogeneous and less heterogeneous hANGPTL3 / antibody immune complex is obtained compared to the Evinacumab SIA antibody / hANGPTL3 immune complex (see Example 6).

[0113] In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein provides an antibody / hANGPTL3 immune complex that is smaller and less heterogeneous compared to the evinacumab SIA antibody / hANGPTL3 immune complex. The smaller and / or less heterogeneous immune complex is expected to reduce immunogenicity compared to the case where the antibody / hANGPTL3 complex is larger and / or more heterogeneous.

[0114] Increased cellular uptake of antibody-antigen complexes The monovalent antibodies disclosed herein are capable of pH-dependent binding to hANGPTL3 and include substitutions that increase cellular uptake of the antibody-antigen complex.

[0115] pH-dependent antibody The monovalent antibody or antigen-binding fragment thereof disclosed herein has lower antigen-binding activity in the acidic pH range than in the neutral pH range. As a result, due to the decreased affinity for hANGPTL3 at low pH (see Example 7), the hANGPTL3 / antibody complex dissociates completely or almost completely at low pH (pH 6.0).

[0116] In one embodiment, the antibody or antigen-binding fragment thereof has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D and is defined as the ratio of K D (pH6) / K D (pH7.4) value of 20 or more.

[0117] In one embodiment, the antibody or antigen-binding fragment thereof has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D and is defined as the ratio of K D (pH6) / K D (pH7.4) value of 30 or more.

[0118] In one embodiment, the antibody or antigen-binding fragment thereof has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 DK defined as the ratio to D (pH6) / K D (The value of pH7.4) is 40 or more.

[0119] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is 50 or more.

[0120] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is 60 or more.

[0121] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is 70 or more.

[0122] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is 80 or more.

[0123] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is 90 or more.

[0124] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value is 95 or more.

[0125] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value is 100 or more.

[0126] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value is 110 or more.

[0127] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value is 120 or more.

[0128] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value is 130 or more.

[0129] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D(pH 7.4) value is 140 or more.

[0130] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D The K defined as the ratio of D (pH 6) / K D (pH 7.4) value is 150 or more.

[0131] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D The K defined as the ratio of D (pH 6) / K D (pH 7.4) value is 160 or more, for example, 166 or more.

[0132] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D The K defined as the ratio of D (pH 6) / K D (pH 7.4) value is 170 or more.

[0133] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D The K defined as the ratio of D (pH 6) / K D (pH 7.4) value is 180 or more.

[0134] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D The K defined as the ratio of D (pH 6) / K D (pH 7.4) value is 190 or more.

[0135] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 Dand the K of the antigen at pH 7.4 D K defined as the ratio to D (pH6) / K D (pH7.4) value is 200 or more.

[0136] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D K defined as the ratio to D (pH6) / K D (pH7.4) value is in the range of 40 to 500.

[0137] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D K defined as the ratio to D (pH6) / K D (pH7.4) value is in the range of 40 to 400.

[0138] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D K defined as the ratio to D (pH6) / K D (pH7.4) value is in the range of 40 to 300.

[0139] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D K defined as the ratio to D (pH6) / K D (pH7.4) value is in the range of 40 to 200.

[0140] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D K defined as the ratio to D (pH6) / K D (pH7.4) value is in the range of 90 to 500.

[0141] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D The K D (pH6) / K D (pH7.4) value is in the range of 90 to 400.

[0142] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D The K D (pH6) / K D (pH7.4) value is in the range of 90 to 300.

[0143] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D The K D (pH6) / K D (pH7.4) value is in the range of 90 to 200.

[0144] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D The K D (pH6) / K D (pH7.4) value is in the range of 80 to 250.

[0145] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D The K D (pH6) / K D (pH7.4) value is in the range of 80 to 200.

[0146] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 DK defined as the ratio to D (pH6) / K D (The value of pH7.4) is in the range of 80 to 170.

[0147] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is in the range of 90 to 250.

[0148] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is in the range of 90 to 200.

[0149] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is in the range of 90 to 170.

[0150] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is in the range of 90 to 166.

[0151] In one embodiment, the present antibody or its antigen-binding fragment is the K of the antigen at pH6 D and the K of the antigen at pH7.4 D K defined as the ratio to D (pH6) / K D (The value of pH7.4) is in the range of 95 to 250.

[0152] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value ranges from 95 to 200.

[0153] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value ranges from 95 to 170.

[0154] In one embodiment, the present antibody or its antigen-binding fragment has a K of the antigen at pH 6 D and a K of the antigen at pH 7.4 D such that the K D (pH6) / K D (pH7.4) value ranges from 95 to 166.

[0155] Improved Fc receptor binding In the context of the present invention, it is desirable to increase the cellular uptake of the antibody-antigen complex in order to reduce the level of circulating hANGPTL3.

[0156] In some embodiments, the increase in cellular uptake of the antibody-antigen complex is enhanced by increasing the affinity of the monovalent antibody towards the cell surface. By introducing additional positive charges in the Fc portion of the antibody, the uptake increases due to the increased interaction with the negatively charged surface of the cell, and thus the cellular uptake of the antibody and the antibody-antigen complex is increased overall.

[0157] In some embodiments, the increased cellular uptake of the antibody-antigen complex is enhanced by increasing the affinity of the monovalent antibody for human FcγRIIb (CD32). Amino acid substitutions in the Fc portion of the antibody increase the affinity for human FcγRIIb (CD32) at pH 7.4, while this substitution does not affect the affinity of the antibody for other Fc receptors on the cell surface.

[0158] In some embodiments, the increased cellular uptake of the antibody-antigen complex is enhanced by increasing the avidity of the antibody-antigen immune complex for human FcγRIIb (CD32). Amino acid substitutions in the Fc portion of the antibody increase the avidity of the antibody-antigen immune complex for human FcγRIIb (CD32) at pH 7.4, while this substitution does not affect the avidity for other Fc receptors on the cell surface.

[0159] In one embodiment, the monovalent antibody or its antigen-binding fragment promotes the clearance of hANGPTL3 from the circulation by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to the case where the monovalent antibody or its antigen-binding fragment is not administered. The circulating levels of hANGPTL3 are well known in the art and can be measured by the in vitro assays described herein (see Example 5).

[0160] Inhibitory effect of the monovalent antibody The inhibitory activity of the monovalent antibody or its antigen-binding fragment described herein against hANGPTL3-mediated LPL inhibition can be determined by methods such as those described in Example 4 herein.

[0161] In one embodiment, the monovalent antibody or its antigen-binding fragment described herein can reduce the triglyceride concentration in human plasma.

[0162] In one embodiment, the monovalent antibody or its antigen-binding fragment described herein can suppress the increase in triglyceride concentration in human plasma.

[0163] Indications Diseases or disorders treatable by administering a monovalent antibody or antigen-binding fragment thereof disclosed herein are any disease or condition that is improved, ameliorated, inhibited, or prevented, or the incidence of which is reduced, by removing, inhibiting, reducing, or otherwise interfering with hANGPTL3 activity, compared to a disease or condition without anti-hANGPTL3 antibody treatment (e.g., an hANGPTL3-mediated disease or disorder).

[0164] Examples of treatable diseases or disorders include, but are not limited to, hyperlipidemias, hyperlipoproteinemias, and dyslipidemias involved in lipid metabolism, such as atherosclerosis, diabetic dyslipidemia, hypertriglyceridemia, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), caused by, for example, decreased LDL receptor (LDLR) activity and / or LDL receptor deficiency (e.g., homozygous familial hypercholesterolemia with LDLR- / -), modified ApoC2, ApoE deficiency, increased ApoB, increased production of very low density lipoprotein (VLDL) and / or decreased removal, etc.

[0165] The monovalent antibody or antigen-binding fragment thereof disclosed herein is also useful for the prevention or treatment of dyslipidemia, hyperlipoproteinemias, and / or diseases or disorders associated with or resulting from dyslipidemia, including but not limited to cardiovascular diseases or disorders such as atherosclerosis, atherosclerotic cardiovascular disease, angina pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, etc.

[0166] Pharmaceutical Composition In another aspect, the present invention provides a composition comprising a compound of the present invention, such as an antibody or an antigen-binding fragment thereof described herein. For example, the present invention provides a pharmaceutical composition comprising one or more antibodies of the present invention formulated with a pharmaceutically acceptable carrier.

[0167] Accordingly, one aspect of the present invention is to provide a pharmaceutical composition comprising such a monovalent antibody or antigen-binding fragment present at a concentration of 0.25 mg / ml to 250 mg / ml, such as 1 mg / ml to 250 mg / ml, such as 25 mg / ml to 250 mg / ml, and the composition has a pH of 4.0 to 9.0. The pharmaceutical composition may further comprise one or more of a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer, or a surfactant, and various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0168] In one embodiment, the pharmaceutical composition is an aqueous composition. Such compositions are typically solutions or suspensions, but may also include colloids, dispersants, emulsions, and multiphase materials. The term "aqueous composition" is defined as a composition containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0169] In another embodiment, the pharmaceutical composition is a lyophilized composition to which a solvent and / or diluent is added prior to use.

[0170] In a further aspect, the pharmaceutical composition comprises an aqueous solution of a monovalent antibody or an antigen-binding fragment thereof and a buffer, the antibody is present at a concentration of 1 mg / ml or more, and the composition has a pH of about 5.0 to about 8.0.

[0171] In a further aspect, the pharmaceutical composition comprises an aqueous solution of a monovalent antibody or an antigen-binding fragment thereof and a buffer solution, the antibody is present at a concentration of 1 mg / ml to 150 mg / ml, and the composition has a pH of about 5.0 to about 8.0.

[0172] In one such embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 50 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 60 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 70 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 80 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 90 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 100 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 110 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 120 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 130 mg / ml or more. In one embodiment, the monovalent antibody or an antigen-binding fragment thereof may be prepared in the composition, wherein the concentration of the antibody or an antigen-binding fragment thereof is 140 mg / ml or more.

[0173] In one embodiment, the monovalent antibody or antigen-binding fragment thereof may be prepared in a composition, wherein the concentration of the antibody or antigen-binding fragment thereof is 150 mg / ml or more.

[0174] In one embodiment, the present invention relates to an injection device having the contents of the composition. In some embodiments, the pharmaceutical composition of the present invention is intended for use in an injection device and / or for containment within an injection device. In some embodiments, the injection device is a disposable, pre-filled, multi-dose pen of the FlexTouch® type (supplier: Novo Nordisk A / S, Denmark). In some embodiments, the injection device is a single-shot device.

[0175] In some embodiments, the injection device is a fixed-dose device, such as one configured to deliver a plurality of predetermined doses of a drug, and is sometimes referred to as a multi-dose fixed-dose device or a fixed-dose, multi-shot device.

[0176] Administration and Dosage The monovalent antibody or antigen-binding fragment thereof disclosed herein may be administered parenterally, for example, intravenously, for example, intramuscularly, for example, subcutaneously. In a preferred embodiment, the antibody or antigen-binding fragment thereof is administered subcutaneously. The monovalent antibody or antigen-binding fragment thereof may be administered prophylactically. The monovalent antibody or antigen-binding fragment thereof may be administered therapeutically (as required).

[0177] The dose of the compound to be delivered may be about 0.01 mg to 500 mg of the compound per day.

[0178] Also, a suitable dose may be adjusted based on the properties of the antibody, including its in vivo plasma half-life or mean residence time and its biological activity, for a particular compound.

[0179] Compositions comprising the monovalent antibodies or antigen-binding fragments thereof disclosed herein can be administered for prophylactic treatment and / or, in some embodiments, for therapeutic treatment. In therapeutic use, pharmaceutical compositions such as those disclosed herein are administered to a subject already suffering from a disease such as any of the diseases described herein in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective amount." As will be understood by those skilled in the art, the effective amount for this purpose will depend on the severity of the disease or injury and the weight and general condition of the subject.

[0180] Embodiment In one embodiment, the monovalent antibody or antigen-binding fragment thereof disclosed herein results in a lower viscosity in solution than the corresponding bivalent antibody (containing the same heavy chain).

[0181] In one embodiment, the monovalent antibody or antigen-binding fragment thereof disclosed herein results in a higher physical stability in solution than the corresponding bivalent antibody (containing the same heavy chain).

[0182] In one embodiment, the monovalent antibody or antigen-binding fragment thereof results in a lower viscosity and self-association tendency over a wide pH range than the viscosity and self-association tendency of the corresponding bivalent antibody (containing the same heavy chain).

[0183] In one embodiment, the monovalent antibody is designated as tmAb1 or an antigen-binding fragment thereof, tmAb2 or an antigen-binding fragment thereof, tmAb3 or an antigen-binding fragment thereof, and tmAb4 or an antigen-binding fragment thereof.

[0184] The present invention is further illustrated by the following embodiments. 1. A monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), wherein the antibody or antigen-binding fragment thereof has a K of the antigen at pH 6D and the K of the antigen at pH 7.4 D defined as the ratio to D (pH6) / K D (pH7.4) value is 40 or more, for example, 50, 60, 70, 80, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 165, 166, 170, 180, 190, 200 or more, a monovalent antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof can reduce the concentration of hANGPTL3 in human plasma. 2. The antibody or the antigen-binding fragment thereof has a K of the antigen at pH 6 D and the K of the antigen at pH 7.4 D defined as the ratio to D (pH6) / K D (pH7.4) value is in the range of 90 to 105, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, the antibody or the antigen-binding fragment thereof according to Embodiment 1. 3. The antibody or the antigen-binding fragment thereof according to any one of the foregoing embodiments, which comprises a region capable of interacting with a cell surface Fc receptor. 4. The antibody or the antigen-binding fragment thereof according to any one of the foregoing embodiments, which comprises an Fc region, for example, one or two Fc regions. 5. The antibody isotype is IgG, and C H 2 or C H at least one negatively charged or neutrally surface-exposed amino acid residue in the 3 domain is substituted with a positively charged amino acid residue, whereby the isoelectric point of the antibody or the antigen-binding fragment thereof is increased as determined using fractionation electrophoresis, the antibody or the antigen-binding fragment thereof according to any one of the foregoing embodiments. 6. The antibody isotype is IgG and comprises arginine (R) (EU residue) at positions 311 and / or 343 of the heavy chain, the antibody or the antigen-binding fragment thereof according to any one of the foregoing embodiments. 7. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody isotype is IgG and comprises arginine (R) (EU residue) at positions 311 and 343 of the heavy chain. 8. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody isotype is IgG and the Fc region is modified to promote binding to FcγRIIb. 9. The antibody or an antigen-binding fragment thereof, in the heavy chain, tyrosine (Y) at position 234, aspartic acid (D) at position 238, valine (V) at position 250, isoleucine (I) at position 264, proline (P) at position 307, and / or lysine (K) at position 330 (EU residues), and comprises an antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments. 10. The antibody or an antigen-binding fragment thereof, in the heavy chain, tyrosine (Y) at position 234, aspartic acid (D) at position 238, valine (V) at position 250, isoleucine (I) at position 264, proline (P) at position 307, and lysine (K) at position 330 (EU residues), and comprises an antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments. 11. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody isotype is IgG and the Fc region is modified to promote binding to FcRn. 12. The antibody or an antigen-binding fragment thereof, in the heavy chain, tyrosine (Y) at position 252, threonine (T) at position 254, and glutamic acid (E) at position 256 (EU residues), and comprises an antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments. 13. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof can reduce the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL). 14. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is an antagonist that can inhibit the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL). 15. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof can reduce the concentration of hANGPTL3 in human plasma. 16. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is an antagonist that can inhibit the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL) and can reduce the concentration of hANGPTL3 in human plasma. 17. a) The heavy chain of the antibody or antigen-binding fragment thereof is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, and the CDR1 sequence, ● a CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, and the CDR2 sequence, ● a CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, and the CDR3 sequence, or b) The heavy chain of the antibody or antigen-binding fragment thereof is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 24), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, and the CDR1 sequence, ● A CDR2 sequence of the amino acid residue SISSHSTYIYYADSVKG (SEQ ID NO: 25), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted with different amino acid residues, and the CDR2 sequence, ● A CDR3 sequence of the amino acid residue EGWYDNWNDP (SEQ ID NO: 26), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted with different amino acid residues, and the CDR3 sequence, The light chain of the antibody or its antigen-binding fragment is, ● A CDR1 sequence of the amino acid residue RASQNIRSPYLA (SEQ ID NO: 9), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted with different amino acid residues, and the CDR1 sequence, ● A CDR2 sequence of the amino acid residue GVSSRAA (SEQ ID NO: 10), wherein 0, 1, or 2 of these amino acid residues may be substituted with different amino acid residues, and the CDR2 sequence, ● A CDR3 sequence of the amino acid residue QQYDDHPYT (SEQ ID NO: 11), wherein 0, 1, or 2 of these amino acid residues may be substituted with different amino acid residues, and the CDR3 sequence, and the antibody or its antigen-binding fragment according to any one of the foregoing embodiments. 18. The antibody or its antigen-binding fragment according to embodiment 17, wherein the substitution is a conservative substitution. 19. The heavy chain of the antibody or its antigen-binding fragment is, ● A CDR1 sequence of the amino acid residue SYWMT (SEQ ID NO: 2), ● A CDR2 sequence of the amino acid residue SISSHSTYIYYADSVKG (SEQ ID NO: 3), ● A CDR3 sequence of the amino acid residue EGWYDNWFDP (SEQ ID NO: 4), and The light chain of the antibody or its antigen-binding fragment is, ● A CDR1 sequence of the amino acid residue RASQNIRSPYLA (SEQ ID NO: 9), ● A CDR2 sequence of the amino acid residue GVSSRAA (SEQ ID NO: 10), ● An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, comprising the CDR3 sequence of the amino acid residue QQYDDHPYT (SEQ ID NO: 11). 20. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the heavy-chain variable domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5, and the light-chain variable domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12. 21. a) The heavy-chain variable domain comprises SEQ ID NO: 5, and the light-chain variable domain comprises SEQ ID NO: 12, or b) The heavy-chain variable domain comprises SEQ ID NO: 17, and the light-chain variable domain comprises SEQ ID NO: 12, or c) The heavy-chain variable domain comprises SEQ ID NO: 22, and the light-chain variable domain comprises SEQ ID NO: 12, or d) The heavy-chain variable domain comprises SEQ ID NO: 27, and the light-chain variable domain comprises SEQ ID NO: 12. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments. 22. A monovalent antibody or an antigen-binding fragment thereof that can bind to hANGPTL3 (SEQ ID NO: 1), wherein a) The heavy chain of the antibody or its antigen-binding fragment is ● The CDR1 sequence of the amino acid residue SYWMT (SEQ ID NO: 2), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, the CDR1 sequence, and ● The CDR2 sequence of the amino acid residue SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, the CDR2 sequence, and ● The CDR3 sequence of the amino acid residue EGWYDNWFDP (SEQ ID NO: 4), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, the CDR3 sequence, or b) The heavy chain of the antibody or its antigen-binding fragment is ● The CDR1 sequence of amino acid residues 31 - 35 (SYWMT) of SEQ ID NO: 24, wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, and the CDR1 sequence, ● The CDR2 sequence of amino acid residues 50 - 66 (SISSHSTYIYYADSVKG) of SEQ ID NO: 25, wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, and the CDR2 sequence, ● The CDR3 sequence of amino acid residues 99 - 110 (EGWYDNWNDP) of SEQ ID NO: 26, wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, and the CDR3 sequence, and the light chain of the antibody or its antigen - binding fragment is ● The CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, and the CDR1 sequence, ● The CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), wherein 0, 1, or 2 of these amino acid residues may be substituted by different amino acid residues, and the CDR2 sequence, ● The CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein 0, 1, or 2 of these amino acid residues may be substituted by different amino acid residues, and the CDR3 sequence, and a monovalent antibody or its antigen - binding fragment. 23. The antibody or its antigen - binding fragment according to embodiment 22, wherein the substitution is a conservative substitution. 24. The antibody or its antigen - binding fragment according to any one of embodiments 17 - 23, wherein the antibody or its antigen - binding fragment comprises a region capable of interacting with cell - surface Fc receptors. 25. The antibody or its antigen - binding fragment according to any one of embodiments 17 - 24, wherein the antibody or its antigen - binding fragment comprises an Fc region. 26. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 24, wherein the antibody or antigen-binding fragment thereof is capable of reducing the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL). 27. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 24, wherein the antibody or antigen-binding fragment thereof is an antagonist capable of inhibiting the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL). 28. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 24, wherein the antibody or antigen-binding fragment thereof is capable of reducing the concentration of hANGPTL3 in human plasma. 29. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 24, wherein the antibody or antigen-binding fragment thereof is an antagonist capable of inhibiting the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL) and capable of reducing the concentration of hANGPTL3 in human plasma. 30. The heavy chain of the antibody or antigen-binding fragment thereof ● comprises the CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), ● comprises the CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), ● and comprises the CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4), and the light chain of the antibody or antigen-binding fragment thereof ● comprises the CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), ● comprises the CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), ● and comprises the CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11). The monovalent antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments. 31. An antibody or an antigen-binding fragment thereof according to any of embodiments 22 to 30, wherein the heavy chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 5, 17, 22, 27, or 32, and the light chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 12. 32. An antibody or an antigen-binding fragment thereof according to any of embodiments 22 to 31, wherein the heavy chain variable domain comprises SEQ ID NO: 5 and the light chain variable domain comprises SEQ ID NO: 12. 33. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the isotype of the antibody is IgG1, IgG2, IgG3, or IgG4. 34. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the isotype of the antibody is based on IgG1. 35. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the isotype of the antibody is based on IgG1 and glycine and / or lysine (EU residues) at positions 446 and / or 447 in the heavy chain are each deleted. 36. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the isotype of the cleaved heavy chain of the antibody is based on IgG1 and glycine and lysine (EU residues) at positions 446 and 447 of the heavy chain are each deleted. 37. The antibody isotype is IgG and C H 2 or C H 3 At least one negatively charged or neutrally charged amino acid residue exposed on the surface in the domain is substituted with a positively charged amino acid residue, whereby, as determined using fractionation electrophoresis, the isoelectric point of the present antibody or its antigen-binding fragment is increased. An antibody or an antigen-binding fragment thereof according to any of embodiments 17 to 36. 38. An antibody or an antigen-binding fragment thereof according to any of embodiments 17 to 37, wherein the antibody or its antigen-binding fragment comprises arginine (R) at positions 311 and / or 343 in the heavy chain (EU residues). 39. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 38, wherein the antibody or antigen-binding fragment thereof comprises arginine (R) (EU residue) at positions 311 and 343 in the heavy chain. 40. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 39, wherein the antibody isotype is IgG and the Fc region is modified to promote binding to FcγRIIb. 41. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 40, wherein the antibody or antigen-binding fragment thereof comprises one or more of the following in the heavy chain: tyrosine (Y) at position 234, aspartic acid (D) at position 238, valine (V) at position 250, isoleucine (I) at position 264, proline (P) at position 307, and lysine (K) at position 330 (EU residues). 42. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 41, wherein the antibody or antigen-binding fragment thereof comprises the following in the heavy chain: tyrosine (Y) at position 234, aspartic acid (D) at position 238, valine (V) at position 250, isoleucine (I) at position 264, proline (P) at position 307, and lysine (K) at position 330 (EU residues). 43. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 42, wherein the antibody isotype is IgG and the Fc region is modified to promote binding to FcRn. 44. The antibody or antigen-binding fragment thereof according to any one of embodiments 17 to 43, wherein the antibody or antigen-binding fragment thereof comprises the following in the heavy chain: tyrosine (Y) at position 252, threonine (T) at position 254, and glutamic acid (E) at position 256 (EU residues). 45. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, comprising a first and a second heavy chain, each comprising a multimerization domain, wherein the sequences of the heavy chains are different and the heavy chains form a multimer by interaction of the multimerization domains. 46. The antibody comprises a first and a second heavy chain, wherein the first heavy chain comprises a tryptophan (W) at position 366, the second heavy chain comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407 (EU residue), and is an antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments. 47. An antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody comprises a first and a second heavy chain, and the first and second heavy chains comprise a lysine (K) at position 214 (EU residue). 48. The antibody comprises a first and a second heavy chain, and the first and second heavy chains comprise a lysine (K) at position 214, arginines (R) at positions 311 and 343, a tyrosine (Y) at position 234, an aspartic acid (D) at position 238, a valine (V) at position 250, an isoleucine (I) at position 264, a proline (P) at position 307, a lysine (K) at position 330, a tyrosine (Y) at position 252, a threonine (T) at position 254, and a glutamic acid (E) at position 256, and the first heavy chain further comprises a tryptophan at position 366, and the second heavy chain further comprises a serine at position 366, an alanine at position 368, and a valine at position 407. and is an antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments. Optionally, a glycine and a lysine at positions 446 and / or 447 of the first and / or second heavy chain are each deleted (EU residues), and the antibody or an antigen-binding fragment thereof described in any of the foregoing embodiments. 49. The antibody according to any of the foregoing embodiments, comprising a first heavy chain according to SEQ ID NO: 6, a second heavy chain according to SEQ ID NO: 7 or 8, and a single light chain according to SEQ ID NO: 13. 50. A monovalent antibody or an antigen-binding fragment thereof that can bind to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), comprising a first heavy chain, a second heavy chain, and a light chain, wherein the first heavy chain comprises SEQ ID NO: 6, the second heavy chain comprises SEQ ID NO: 7, and the light chain comprises SEQ ID NO: 13, and the monovalent antibody or an antigen-binding fragment thereof. 51. The antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or an antigen-binding fragment thereof can bind to hANGPTL3 in a pH-dependent manner. 52. The antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or an antigen-binding fragment thereof can reduce the size of an immune complex formed between the antibody or an antigen-binding fragment thereof and hANGPTL3. 53. The antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or an antigen-binding fragment thereof can increase the homogeneity of an immune complex formed between the antibody or an antigen-binding fragment thereof and hANGPTL3. 54. The antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or an antigen-binding fragment thereof can reduce the hANGPTL3 plasma concentration. 55. The antibody or an antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or an antigen-binding fragment thereof can reduce the triglyceride concentration in plasma without increasing the hANGPTL3 plasma concentration. 56. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is humanized or human. 57. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof is a one-armed antibody. 58. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding fragment thereof has cross-species reactivity. 59. The antibody or antigen-binding fragment thereof according to embodiment 58, wherein the antibody or antigen-binding fragment thereof is capable of binding to mouse ANGPTL3, rat ANGPTL3, porcine ANGPTL3, canine ANGPTL3, and / or cynomolgus monkey ANGPTL3. 60. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any of the foregoing embodiments and one or more pharmaceutically acceptable carriers. 61. The antibody or antigen-binding fragment thereof according to any of embodiments 1 to 59 or the composition according to embodiment 60 for use in medicine. 62. The antibody or antigen-binding fragment thereof according to any of embodiments 1 to 59 or the composition according to embodiment 60 for use in the treatment or prevention of an increase in triglyceride concentration in human plasma. 63. The antibody or antigen-binding fragment thereof according to any of embodiments 1 to 59 or the composition according to embodiment 60 for use in suppressing an increase in triglyceride concentration in human plasma. 64. A method of treating a patient with an elevated triglyceride concentration in human plasma, the method comprising administering to the patient the antibody or antigen-binding fragment thereof according to any of embodiments 1 to 59 or the composition according to embodiment 60. 65. A method of treating a patient with a highly elevated triglyceride concentration in human plasma, the method comprising administering to the patient the antibody or antigen-binding fragment thereof according to any of embodiments 1 to 59 or the composition according to embodiment 60. 66. A method for reducing the triglyceride concentration in human plasma, comprising administering to a patient in need thereof an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60. 67. A method for suppressing an increase in the triglyceride concentration in plasma, comprising administering to a patient in need thereof an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60. 68. Use of an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 for manufacturing a medicament for treating a patient with elevated triglycerides. 69. Use of an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 for manufacturing a medicament for treating a patient with highly elevated triglycerides. 70. The use or method according to any one of Embodiments 61 to 69, or the composition according to Embodiment 60, wherein the patient is diagnosed with atherosclerotic cardiovascular disease (ASCVD) and / or type 2 diabetes. 71. An antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 for use in the treatment or prevention of cardiovascular disease. 72. An antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 for use in the treatment or prevention of atherosclerotic cardiovascular disease (ASCVD). 73. A method for treating or preventing cardiovascular disease in a patient, comprising administering to the patient an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60. 74. A method for treating or preventing atherosclerotic cardiovascular (ASCVD) disease in a patient, comprising administering to the patient an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60. Use of an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 for manufacturing a medicament for treating or preventing cardiovascular diseases. 76. Use of an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 for manufacturing a medicament for treating or preventing atherosclerotic cardiovascular disease (ASCVD). 77. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 59 or the composition according to Embodiment 60 and an instruction manual. 78. An antibody or an antigen-binding fragment thereof according to any one of Embodiments 45 to 48, wherein the first heavy chain is a cleaved heavy chain. 79. An antibody or an antigen-binding fragment thereof according to any one of Embodiments 45 to 50, wherein the second heavy chain is a cleaved heavy chain.

Examples

[0185]

Table 1

[0186] Initial screening of phage panning : Biotinylated hANGPTL3 was coated on M280-streptavidin Dynabeads (Thermo Fisher Scientific #11205D). A phage display library (10 13After depleting the pfu / mL (super) for non-specific binders with uncoated streptavidin Dynabeads, beads coated with hANGPTL3 were added for binding at pH 7.4. After washing 10 times with PBS and 0.05% Tween 20 and 3 times with PBS, the beads with phage bound to ANGPLT3 were suspended in 1 mL of PBS and mixed with 9 mL of actively growing E. coli XL-Blue culture (OD 600 = 0.6) in 2×YT medium (2% glucose, 25 μg / ml chloramphenicol, 10 μg / ml tetracycline) and incubated at 37 °C for 30 minutes. All cells were centrifuged and resuspended overnight at 37 °C in a 24 cm × 24 cm plate with 2×YT medium. The E. coli cells were infected with M13K07 helper phage to amplify and produce phage. After 24 hours, the E. coli cells were removed and the phage was harvested by precipitation with PEG / NaCl solution and then resuspended in PBS buffer for the next round of panning.

[0187] Antibody identification : After 4 rounds of panning, single colonies were picked for Sanger sequencing and ELISA. The top hits were selected for gene synthesis in a full antibody format. After expression and purification (see Example 2), the full antibodies were tested for ANGPTL3 binding at pH 7.4 and pH 6 by SPR analysis (see Example 7). Antibody mAb0 was identified as having the most ideal binding to hANGPTL3.

[0188] Construction of a library for incorporating pH-dependent binding To incorporate a pH switch into the variable domains of mAb0 that binds to ANGPTL3, computational analysis was performed on the homology model. Briefly, all mAb0 residues within 7.5 angstroms of positively charged residues in the interface were identified. The identified positions were sequentially changed to histidine, another charged residue (positive or negative), or a stabilizing substitution, such as proline. The stereochemistry and intramolecular interactions of the amino acid substitutions were computationally evaluated from the homology model. Next, a library was constructed using degenerate codons with approximately 10 7 of diversity and having 16 mutant sites.

[0189] Phage panning for identification of pH-dependent variants of mAb0 Biotinylated hANGPTL3 was coated on M280-streptavidin Dynabeads. The resulting phage library (10 12 pfu / mL) was depleted with uncoated streptavidin Dynabeads. The depleted phage library was added to beads coated with hANGPTL3 (17 - 460) for binding at pH 7.4. After washing three times with PBT and three times with PB, the bound phage was eluted twice with elution buffer at pH 5.5 and then incubated for 15 minutes. The eluted phage particles were then amplified and subjected to additional screening rounds.

[0190] The eluate was transferred to a 1.5 mL microcentrifuge tube and neutralized with 1 M Tris-HCl (pH 8.0). Half of the neutralized phage solution was mixed with 1 mL of actively growing E. coli NEB 5-alpha F’ (OD 600 = 0.8) in 2×YT medium containing 10 μg / mL of tetracycline and incubated at 37 °C for 1 hour. 1×10 10The pfu M13K07 helper phage was added and incubated for 1 hour. The infected E. coli was amplified in 50 mL of 2×YT medium containing 50 μg / mL carbenicillin and 25 μg / mL kanamycin by shaking at 200 rpm and growing overnight at 37°C. The next day, the phage was harvested for the subsequent panning round by precipitation with PEG / NaCl solution and resuspension in PBS buffer.

[0191] Identification of antibodies showing pH-dependent binding to hANGPTL3 The phage pool from 3 rounds of panning was collected for next-generation sequencing. The most enriched Abs sequences were selected for gene synthesis and then subjected to expression and purification (see Example 2). The prepared antibodies were analyzed by SPR essentially as in Example 7. The full-length His6-tag hANGPTL3 was captured by the anti-His antibody immobilized on the SPR sensor chip, and the anti-hANGPTL3 antibodies were flowed at pH 6.0 and pH 7.4, respectively. Antibodies with relatively weaker affinity at pH 6.0 than at pH 7.4 were selected. Antibodies containing the variable domains of the heavy and light chains of tmAb0.1 and tmAb1 were identified using this approach. These antibodies retained the inhibitory activity against hANGPTL3.

[0192] Example 2: Recombinant expression of antibodies and their fragments Antibodies and their fragments were expressed using transient transfection of HEK293 suspension cells (293Expi, Invitrogen) essentially according to the manufacturer's instructions. 293Expi cells were typically subcultured every 3 - 4 days in Expi293F expression medium (Invitrogen, catalog number A1435104) supplemented with 1% P / S (GIBCO catalog number 15140-122). Expi293F cells were transfected at a cell density of 2.5 - 3 mill / mL using Expifectamine. For each liter of Expi293F cells, a total of 1 mg of plasmid DNA (V H -C H1 (for Fab) or (for mAb HC) and the LC plasmid in a 1:1 ratio) were diluted into 50 mL of Optimem (GIBCO, catalog number 51985 - 026, dilution A), and 2.7 mL of Expifectamine was diluted into 50 mL of Optimem (dilution B) to perform the transfection. For Fab and mAb co - transfection, V H -C H 1 and the LC plasmid (Fab) and the HC and LC plasmids (mAb) were each used in a 1:1 ratio. Dilutions A and B were mixed and incubated at room temperature for 10 - 20 minutes. Thereafter, the transfection mixture was added to Expi293F cells, and the cells were incubated at 37 °C in a humidified incubator using orbital rotation (85 - 125 rpm). One day after transfection, the transfected cells were supplemented with 5 ml of ExpiFectamine 293 transfection enhancer 1 and 50 ml of ExpiFectamine 293 transfection enhancer 2. The cell culture supernatant was typically harvested 4 - 5 days after transfection by centrifugation and then filtered.

[0193] The expression of monovalent antibodies was essentially performed as described above, but using the co - transfection of three different plasmids. A one - armed antibody consists of one heavy chain (HC), one truncated heavy chain (tHC), and one LC. Thus, the transfection was set up using a DNA mixture of the HC, tHC, and LC plasmids in a 1:1:1 ratio.

[0194] The monovalent antibodies designated tmAb1, tmAb2, tmAb3, and tmAb4 comprise HC, tHC, and LC based on the human IgG1 kappa isotype. The constant regions of tmAb1-4 HC and tHC were engineered to contain the following substitutions compared to wild-type IgG1: L234Y, P238D, T250V, M252Y, S254T, T256E, V264I, T307P, Q311R, A330K, and P343R (EU residues). To ensure proper HC and tHC heterodimerization for efficient tmAb1-4 production, the knob-in-hole technology (U.S. Patent Application No. 20030078385) was used. That is, the T366W mutation (knob) was inserted into the HC and the T366S, L368A, Y407V mutations (hole) were inserted into the tHC, or vice versa.

[0195] Example 3: Purification and characterization of antibodies or their fragments All purification steps were carried out at 4°C. For laboratory scale, Milli-Q water was used for buffer preparation. The HPLC system used for SE-HPLC analysis was an Agilent 1100. Aggregation and LC / MS were evaluated for QC.

[0196] Fab capture was performed by HiTrap KappaSelect (Cytiva) affinity chromatography using a binding buffer in 1×PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. One-step elution was performed using 0.1 M glycine, pH 2.8. The eluted Fab was further purified, buffer-exchanged into a formulation buffer at pH 7.4 (20 mM HEPES, 150 mM NaCl) by size-exclusion chromatography on a 320 mL Superdex 200 26 / 60 column (Cytiva), concentrated by a centrifugal ultrafiltration device (30KD C.O.), and stored at -80°C.

[0197] To evaluate the quality of the purified Fab, SDS-PAGE and high-performance size-exclusion chromatography (SE-HPLC) analyses were performed. LC / MS was carried out to verify the identity of the Fab protein. The molecular weight (MW) of all Fabs was shown to be consistent with the theoretical MW of the heavy and light chains, respectively.

[0198] Purification and characterization of antibodies Antibody purification was performed by affinity chromatography using Protein A MabSelect PrismA resin (Cytiva). For small-scale antibody production, protein A-based purification was performed in 96-well plates, while for larger production, the AktaXpress chromatography system (Cytiva) was used with an equilibration buffer composed of 20 mM sodium phosphate pH 7.2, 150 mM NaCl, and an elution buffer composed of 10 mM formic acid pH 3.5. The cell supernatant was directly applied to a pre-equilibrated MabSelect SuRe column without any adjustment. The column was washed with approximately 5 column volumes of the equilibration buffer, and the antibody was eluted at a uniform concentration with approximately 2 - 5 column volumes of the elution buffer. The eluted antibody was directly applied to a 320 mL Superdex 200 26 / 60 column (Cytiva) for removal of aggregates and buffer exchange to 20 mM HEPES, 150 mM NaCl pH 7.4

[0199] The purified antibodies were characterized using different methods such as analysis by SDS-PAGE / Coomassie, size exclusion high performance liquid chromatography (SE-HPLC) and liquid chromatography mass spectrometry (LC-MS). SDS-PAGE / Coomassie analysis was performed using NuPage 4-12% bis-tris gel (Invitrogen, catalog number NP0321BOX). Here, all antibodies showed the expected light and heavy chain components. Intact molecular mass determination was performed using a setup for liquid chromatography electrospray ionization time-of-flight mass spectrometry on an Agilent 6210 instrument with a desalting column MassPREP (Waters, catalog number USRM10008656). The buffer systems used were an equilibration buffer composed of 0.1% formic acid in LC-MS grade H 2 2O and an elution buffer composed of 0.1% formic acid in LC-MS grade acetonitrile (ACN). Analysis was performed with and without N-glycosidase F (Roche Diagnostics, catalog number 11365177001) and a reducing agent (i.e., mercaptoethanol or DTT). All antibodies showed the expected intact molecular mass according to the sequence and one heavy chain N-glycan. Purity was determined based on SE-HPLC. The final protein purity was based on an SE-HPLC method setup on an Agilent LC 1100 / 1200 system, using a BIOSep-SEC-S3000 300×7.8 mm column (Phenomenex, catalog number 00H-2146-K0), and a running buffer composed of 200 mM sodium phosphate pH 6.9, 300 mM NaCl, and 10% isopropanol. UV280 and fluorescence (Ex 280 nm / Em 354 nm) detectors were used for detection. The present antibody eluted as a single symmetric peak with a retention time reflecting the size of the antibody. Purity estimates were all between 95-99% for the different antibodies. To measure the final protein concentration, a NanoDrop spectrophotometer (Thermo Scientific) was used together with the specific extinction coefficient for each of the antibodies.

[0200] Example 4: Assay for determining inhibition of rhANGPTL3-mediated LPL inhibition The inhibition of hANGPTL3-mediated LPL inhibition was measured in a recombinant hANGPTL3-LPL assay. In this assay, the in vitro enzymatic activity of lipoprotein lipase (LPL) was measured using EnzChek™ Lipase as the substrate. The activity of bovine LPL was inhibited by adding recombinant hANGPTL3 (rhANGPTL3). This inhibition can then be blocked by an rhANGPTL3 inhibitory compound such as an anti-hANGPTL3 antibody disclosed herein. The apparent affinity of the anti-hANGPTL3 antibody was measured in comparison to evinacumab SIA.

[0201] rhANGPTL3-LPL enzyme activity procedure All solutions were prepared using assay buffer (DPBS (1×) (Gibco 14190-144) + 1% fatty acid-free BSA (MP Biomedicals 05033)), except for the EnzChek™ / Zwittergent reagent prepared with ultrapure water.

[0202] A dilution series of the anti-rhANGPTL3 antibody was prepared as follows. The starting concentration was 3 μM and it was diluted 3-fold in assay buffer. This was repeated 7 times to prepare the dilution series. The mixing of compounds for analysis is described below.

[0203] Assay buffer and 10 μL of each dilution were mixed with 10 μL of 1.2 μM rhANGPTL3 (SEQ ID NO:1) and 10 μL of 60 nM heparin (Sigma H3149) in an assay plate (96-well half-area black plate, Corning 3993) and pre-incubated for 1 hour at room temperature. After incubation, 10 μL of 1.8 μM human ApoCII (chemically synthesized residues 59 - 79) and 10 μL of 60 nM bovine LPL (Sigma L2254) were added to each well and incubation was continued for 15 minutes at room temperature. Finally, 10 μL of EnzChek™ / Zwittergent substrate (4.8 μM EnzChek™ Lipase (Thermo Fisher Scientific E33955), 0.03% Zwittergent 3-18 (Merck 41570)) was added to each well and fluorescence was measured kinetically (every 20 minutes for 20 minutes) using an excitation wavelength of 485 nm and an emission wavelength of 535 nm in an Envision Multimode Plate Reader (PerkinElmer).

[0204] In addition to the diluted compounds, the plate also included six wells in which the compound was replaced with 10 μL of assay buffer, and six wells in which both the compound and rhANGPTL3 were each replaced with 10 μL of assay buffer. These wells were used to establish the levels of LPL activity that were completely inhibited and not inhibited, respectively. Additionally, to evaluate the effect that the anti-ANGPTL3 antibody has directly on LPL activity in the absence of rhANGPTL3, the plate then included one well per compound using the highest compound concentration but without rhANGPTL3.

[0205] Each plate included a duplicate dilution series of evinacumab SIA.

[0206] Statistical analysis The LPL activity in each well of the assay plate was calculated by performing a set of linear regressions over a window of five consecutive time points from the kinetic data, selecting the fit with the highest gradient (Vmax) and a determination correlation exceeding 0.95, and calculating the gradient of the selected line. The Vmax values were then normalized using the average of the Vmax from six fully inhibited LPL activity control wells as the value 0 and the average of the Vmax from six uninhibited LPL activity control wells as the value 1. The apparent affinity of rhANGPTL3 for the tested compounds was calculated from the non-linear four-parameter logistic (4PL) curve fitting of the normalized Vmax~concentration data from the dilution series of the compounds. The fitting was performed using the LL.4 function from the drc R package. The value 0 (i.e., the normalized average of the Vmax of the control wells at six fully inhibited LPL activity levels) was used as the common lower asymptote in the fitting. The apparent affinity of the compounds was extracted from the 4PL fitting as the "e" parameter named 50 , and the relative apparent affinity was calculated using the average of the eptacizumab SIA IC 50 on the plate as the denominator. Furthermore, for each compound, it was determined that the normalized Vmax of the control wells evaluating the direct effect of the compound on LPL activity in the absence of rhANGPTL3 was not significantly different from 1 (i.e., the average of the normalized Vmax of the uninhibited LPL activity control wells). 50 The average relative IC

[0207] Results Table 1 shows the mean relative IC 50 and standard deviation of rhANGPTL3-mediated LPL inhibition for five compounds. Include the number of replicates forming the basis of the mean for completeness. For eptacizumab SIA, the mean relative IC 50 is 1.0 by definition, but the standard deviation of 58 replicates is calculated from the individual results. tmAb4 was only analyzed when the standard deviation could not be calculated. Compared to eptacizumab SIA, this monovalent antibody showed similar or slightly reduced activity towards the inhibition of rhANGPTL3, which is the relative IC measured for eptacizumab SIA50 It is shown as a ratio exceeding 1 when compared with the value.

Table 2

[0208] Example 5: In vivo evaluation of anti-ANGPTL3 antibodies in normal mice Using evinacumab SIA and the monovalent anti-ANGPTL3 antibodies designated as tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4, a test was conducted to measure the effect of ANGPTL3 inhibition on the plasma triglyceride levels and endogenous ANGPLT3 (250 - 300 μg / ml at T = 0 hour) levels of healthy male C57bl6 / 6J mice from Janvier Labs, France as described below.

[0209] The monovalent antibodies tested were not modified at positions 234, 238, 250, 252, 254, 256, 264, 307, 311, 330, 343 within the Fc region, that is, they contained wild-type human IgG1 residues at these positions, which differed from tmAb1 - 4.

[0210] Due to the lack of functionality of Fc substitution in mice, wild-type human IgG1 residues at these positions (specifically, 234L, 238P, 250T, 252M, 254S, 256T, 264V, 307T, 311Q, 330A, 343P) were used. The effect of the Fc substitution introduced into tmAb1 - 4 is specific to human receptors (see Example 2).

[0211] Blood sampling Blood samples were collected from 12 mice in each group at 0 h, 48 h, 168 h, 240 h, and 336 h. Blood samples (120 μL) were collected by puncturing the sublingual vein (lingual blood) and transferred to tubes coated with EDTA (Microvette® VetMed 200 K3E, Sarstedt nr 09.1293.100). The blood was centrifuged at 6000 G for 5 minutes within 20 minutes at 4 °C. Plasma samples were transferred to 25 μl for triglyceride measurement, 25 μl for mANGPTL3 and hIgG measurement (antibody plasma exposure measurement) into microtubes.

[0212] Triglycerides were measured on a COBAS 6000 multi-analyzer according to the manufacturer's recommendations (Roche Diagnostics).

[0213] Mouse plasma samples were analyzed for mouse ANGPTL3 using an ELISA from R&D Systems (Mouse Angiopoietin-like 3 ELISA Kit - Quantikine ELISA, catalog number MANL30) according to the kit protocol. To avoid matrix effects, the samples were diluted at least 250-fold in the calibrator diluent from the kit. The assay range extends from 62.5 to 4000 pg / ml. Samples measured above the ULOQ were further diluted with calibrator diluent. The LLOQ was 15.625 pg / ml (250 × 62.5 pg / ml).

[0214] Analysis of mouse ANGPTL3 in mouse plasma Mouse plasma samples are analyzed for mouse ANGPTL3 using an ELISA (Mouse Angiopoietin-like 3 ELISA Kit - Quantikine ELISA, Catalog number MANL30) from R&D Systems according to the kit protocol. To avoid matrix effects, the samples are diluted at least 250-fold in the calibrator diluent from the kit. The assay range extends from 62.5 to 4000 pg / ml. Samples measured above the ULOQ are further diluted with the calibrator diluent. The LLOQ is 15.625 pg / ml (250 × 62.5 pg / ml).

[0215] Principle of LOCI assay for determining anti-ANGPTL3 antibody concentration Plasma samples are analyzed for anti-ANGPTL3 antibody (hIgG) using a luminescent oxygen channeling immunoassay (LOCI). Donor beads are coated with streptavidin, while acceptor beads are conjugated with a first monoclonal mouse anti-human IgG Fc antibody (Southern Biotech, Catalog number 9040-01) specific for the Fc region of the anti-ANGPTL3 antibody. The other antibody used is polyclonal from goat and specific for the human IgG Fc region (Jackson ImmunoResearch, code: 109-005-098). This polyclonal antibody is biotinylated in-house. Three reactants (donor beads with streptavidin, acceptor beads conjugated with the target-specific Ab, and another biotinylated target-specific Ab) are combined with the anti-ANGPTL3 antibody (hIgG) to form a two-site immune complex. Upon irradiation of the complex, single oxygen atoms are released from the donor beads. These are channeled to the acceptor beads, inducing chemiluminescence that is measured by an EnVision plate reader. The amount of light is proportional to the concentration of the anti-ANGPTL3 antibody (tmAb0.1).

[0216] Analysis of hIgG in mouse plasma After applying 1 μL of plasma sample / calibrator / control to the wells of a 384-well LOCI plate, apply a 15 μL mixture of acceptor beads (0.5 μg / well) coated with mAb anti-hIgG and biotinylated pAb anti-hIgG. Incubate the plate at room temperature (RT) for 1 hour. Then, add 30 μL of donor beads (2 μg / well) coated with streptavidin to each well and incubate at RT for 30 minutes. Read the plate on an Envision plate reader at 21 - 22°C using a filter with a bandwidth of 520 - 645 nm after excitation with a 680 nm laser. The total measurement time per well is 210 milliseconds including a 70 millisecond excitation time. The calibrator material is an anti-ANGPTL3 antibody diluted in mouse plasma (untreated K2EDTA plasma, filtered through 0.22 μm from Bioreclamation). Since the calibrator varies for each antibody, estimate the lower limit of quantification for each run (generally 100 - 500 pM).

[0217] The assay range extends from 41 - 30,000 pM. Test mouse samples undiluted and dilute 20-fold in mouse plasma. Further dilute samples measured above the ULOQ in mouse plasma.

[0218] In vivo study 1 Test groups: Gr.1: 11 mice were dosed with vehicle (i.v.). Gr.2: 11 mice were dosed with evinacumab SIA 40 nmol / kg (i.v.). Gr.3: 11 mice were dosed with tmAb0.1 40 nmol / kg (i.v.). Gr.4: 11 mice were dosed with tmAb0.2 40 nmol / kg (i.v.). Gr.5: 11 mice were dosed with tmAb0.3 40 nmol / kg (i.v.). Gr.6: 11 mice were dosed with tmAb0.4 40 nmol / kg (i.v.).

[0219] The vehicle was 20 mM HEPES, 150 mM sodium chloride, pH = 7.4, the injection volume was 5 ml / kg, and the concentration of the dosing solution was 8 nmol / ml.

[0220] Results TG level With evinacumab SIA and tmAb0.1, all antibodies showed a 50 - 60% decrease in triglyceride (TG) levels after 48 hours, indicating the highest ability to reduce TG. Evinacumab SIA, tmAb0.1, and tmAb0.3 were still able to reduce TG by 58 - 67% after 168 hours, while the effectiveness of tmAb0.2 and tmAb0.4 was not as significant (31 - 38%). Only after 336 hours were evinacumab SIA and tmAb0.1 observed to have significantly lower TG levels compared to the vehicle group, both falling below the baseline by 40 - 45%. See Figure 3.

[0221] ANGPTL3 level Evinacumab SIA increased the total plasma ANGPTL3 level approximately 10 - fold from the baseline after 48 hours and then slowly declined towards the baseline. In contrast, tmAb0.1, tmAb0.2, and tmAb0.3 all decreased the total plasma ANGPTL3 level below the baseline throughout the test period. See Figures 4A and 4B.

[0222] Antibody level All antibodies were present in plasma in the nanomolar range, and evinacumab SIA and tmAb0.1 showed the highest exposure levels remaining above 200 nM throughout the test period. See Figure 5.

[0223] Conclusion From this study, tmAb0.1 is considered comparable to evinacumab SIA in terms of TG - lowering efficacy.

[0224] These results are remarkable considering that tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4 are monovalent antibodies (i.e., each has only one antagonistic ANGPTL3 binding site). Furthermore, the low exposure of tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4 compared to evinacumab SIA (Figure 5) indicates that despite the low plasma concentration of the monovalent antibodies, they can reduce plasma triglyceride levels to levels similar to those obtained upon use of evinacumab SIA.

[0225] Also, tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4 show the ability to remove the target ANGPTL3 in plasma. In contrast, administration of evinacumab SIA resulted in the accumulation of total ANGPTL3 in plasma. The inventors expect that combining the Fc substitutions described herein with the Fab of tmAb0.1 - 0.4 (resulting in tmAb1 - 4) will significantly reduce the levels of circulating hANGPTL3 in the human setting.

[0226] Example 6: Determination of ANGPTL3 antibody complexes by SE-UPLC chromatography Immune complexes were analyzed using size - exclusion ultra - performance liquid chromatography (SE - UPLC) on a Waters ACQUITY UPLC System. Complexes were formed by mixing antibodies at concentrations in the range of 0.2 - 2.0 mg / ml with rhANGTPL3 at a molar ratio of 1:3. After equilibration of the antibody - antigen mixture for 1 hour, it was analyzed on an ACQUITY UPLC Protein BEH SEC Column, 450 Å, 2.5 μm. Each sample was injected twice using an injection volume of 20 μL and a flow rate of 0.3 mL / min in a running buffer composed of either A) 300 mM NaCl, 10 mM sodium citrate, pH 6.0, or B) 300 mM NaCl, 10 mM Tris / HCl, pH 7.4. The sample chamber was cooled at 4 °C and the column temperature was kept constant at 22 °C ± 2 °C. The elution profile was monitored by UV - VIS absorbance at 276 nm.

[0227] Quantification of the size of immune complexes and pH-dependent target binding The sizes of the complexes were evaluated by their retention times and mean retention times (MRT), i.e., the mass midpoints of the chromatographic traces. On the other hand, the pH responsiveness of the target binding was evaluated by subtracting the mean retention time at pH 6.0 from the mean retention time at pH 7.4.

[0228] Results The results of the SE-UPLC analysis are shown in FIGS. 6A-6E and Table 2 below. It is clear that Evinacumab SIA, as well as tmAb1, tmAb2, tmAb3, and tmAb4, form immune complexes with the target antigen rhANGTPL3. However, Evinacumab SIA forms larger and more polydisperse immune complexes compared to tmAb1-4 (FIGS. 6A-6E).

[0229] Furthermore, as can be seen by comparing the MRT shifts in Table 2, the formation of tmAb1 immune complexes is quite sensitive to changes in pH. The use of tmAb1 showed that the complexes are less homogeneous (more uniform) compared to those formed by Evinacumab SIA. Well-defined antibody / antigen complexes are important for target sweep by complex uptake into cells and increased antibody recycling. By having two or more binding sites to Fc receptors on the cell surface, the avidity increases, leading to increased uptake of the complex. Furthermore, the complex size and complexity correlate with immune responses that are thought to be less prominent with smaller antibody / antigen complexes. Almost complete dissociation is observed at pH 6.0. The higher the dissociation at pH 6.0, the better the sweep ability that the antibody is thought to have. The ability to completely release the antigen when the antibody-antigen complex is taken up by cells is important for the sweep behavior of the antibody. The antibody can release the antigen and be recycled into the circulation for continued function / binding of the antigen (Igawa et al. (2016) Immun. Rev. 270:132-151).

Table 3

[0230] Example 7: Characterization of the interaction kinetics of monovalent antibodies against ANGPTL3 protein The binding assay was performed using a Biacore 8K (GE Healthcare) that measures molecular interactions in real time through surface plasmon resonance. The experiment was conducted at 37 °C, and the samples were stored at 15 °C in the sample compartments. The signal (RU, response units) reported by the Biacore is directly correlated with the mass on the surface of the individual sensor chip in four consecutive flow cells. The anti-HPC4 monoclonal antibody was immobilized on both flow cells in the active channel of a C1 sensor chip according to the manufacturer's instructions. The capture of the purified trimeric target protein, human ANGPTL3 (SEQ ID NO: 1 residues 17-220 with a C-terminal HPC4 tag), was carried out by diluting the protein in a running buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2 , 0.05% v / v P20, 0.1% BSA pH 7.4 or 20 mM histidine, 150 mM NaCl, 1 mM CaCl 2 , 0.05% v / v P20, 0.1% BSA, pH 6.0), injecting it onto flow cell 2, and creating a reference surface on flow cell 1 with only the anti-HPC4 antibody immobilized. The binding of the monovalent antibody to the captured target was carried out by injecting the analyte (monovalent antibody) onto both flow cells, enabling a comparative analysis of the binding of different monovalent antibodies to the captured target compared to the binding to the reference surface. The monovalent antibody was serially diluted in the running buffer, injected at 30 μl / min for 240 seconds, and dissociated for 300 seconds. The C1 surface was regenerated after each injection cycle of the analyte via injection of 20 mM Tris, 150 mM NaCl, 50 mM EDTA, 0.05% P20, pH 7.4. This regeneration step removed the captured target and any bound monovalent antibody from the immobilized capture antibody surface, enabling subsequent binding of the next interaction sample pair. In this regeneration procedure, the directly immobilized anti-HPC4 capture antibody was not removed from the chip surface.

[0231] The binding affinity between the monovalent antibody and the target was quantified by determination of the equilibrium dissociation constant (K D ) by measuring the kinetics of complex formation and dissociation. ka (Association rate) and k d Rate constants corresponding to the association and dissociation of monovalent complexes such as (dissociation rate), were obtained by global fitting data to a 1:1 Langmuir model using local Rmax, using Biacore evaluation software for data analysis. K D is given by the equation K D = k d / k a and is related to k a and k d through this relationship.

[0232] Binding curves were processed by double referencing (subtraction of the reference surface signal and blank buffer injection on the captured bispecific protein before data analysis). This enabled correction for instrument noise, bulk shift, and drift during sample injection.

[0233] Table 3 shows the results of the measurement of the binding constant K D (equilibrium dissociation constant) for the interaction between human ANGPTL3 and tmAb1 - 4. K D The calculated ratio is expressed as the value at pH 6.0 / the value at pH 7.4.

[0234] When the HC G26D mutation was introduced into tmAb1 (resulting in tmAb2), K D increased 2 - fold due to a slow association rate. Weak binding at pH 7.4 was associated with weak binding at pH 6.0. All the variants listed were characterized by being too low to measure or having low (μM range) affinity at pH 6.0.

Table 4

[0235] Example 8: Monovalent format reduces self-association Antibodies need to have a low self - association tendency over a wide pH range to ensure the success of bioprocessing steps and formulation development. Here, to investigate the effect of changing the antibody format from bivalent to monovalent, the self - association of tmAb1 and its bivalent form designated as "mAb1" was evaluated.

[0236] Materials and methods tmAb1 and mAb1 were prepared as initial protein stock solutions at a concentration of 40 mg / ml in 10 mM L - histidine, 10 mM NaCl, pH 6.0. The protein stock solution was mixed with the pH screen stock in a 1:1 ratio (10 μl of protein stock: 10 μl of pH screen) to reach a final pH value of 3.3, 3.7, 4.2, 5.2, 5.6, 6.0, 6.4, 6.9, 7.2, 7.5, 8.1, 8.4, and a final protein concentration of 20 mg / ml. The pH screen stock solution consisted of 70 mM succinic acid and 70 mM L - histidine and was adjusted to the desired pH. The protein stock solution and the pH screen stock solution were mixed in a 384 - well plate (corning 3540), sealed with a transparent plastic film, and incubated overnight at room temperature in the dark. Then, the plate was centrifuged at 1400 rpm (corresponding to 169×g) for 5 minutes and then analyzed using a dynamic light scattering (DLS) plate reader (Wyatt DynaPro) with a 5 - second acquisition time and 40 acquisitions per sample. The hydrodynamic radius (R h ) was derived using the instrument software and plotted as a function of pH (see also Dingfelder et al. (2022) In: Houen G. (eds) Therapeutic Antibodies. Methods in Molecular Biology, vol 2313. Humana, New York, NY).

[0237] Results and discussion The hydrodynamic radius (R h) The increase suggests that this has unusually high self-association, which is likely to impair further expression of this molecule. In particular, in the pH range of 6.4 to 8.4, a significant increase in R h is observed, which may also cause problems in vivo. By changing the molecular format from the divalent form to the monovalent form, a dramatic decrease in the self-association tendency with only a slight increase at pH 6.4 to 8.4 was observed. The slight increase in the self-association of tmAb1 is low and considered acceptable. See Table 4 and Figure 7. This data supports the preparation of a highly concentrated composition (formulation) for subcutaneous administration.

Table 5

[0238] Example 9: Monovalent format reduces gel formation High physical stability at high protein concentrations (50 - 175 mg / ml) is typically important for the successful development of antibodies as pharmaceuticals. This is required during many steps in development, including the final steps of the purification process, the formulation process, and subcutaneous administration. Here, the physical stability at high protein concentrations, physiological pH, low ionic strength, and high ionic strength is evaluated.

[0239] Materials and methods tmAb1 and its divalent form designated as "mAb1" were buffer-exchanged into high ionic strength formulations (20 mM Hepes, 150 mM NaCl, pH 7.4) and low ionic strength formulations (20 mM Hepes, pH 7.4). Subsequently, the samples were concentrated in an Amicon 30 kDa centrifugal spin filter at room temperature to reach 70 mg / ml. The protein concentration was determined by absorbance at 280 nm.

[0240] Results and discussion tmAb1 was easily concentrated to 70 mg / ml, but for mAb1 (bivalent), it could not be concentrated beyond 30 mg / ml in a high ionic strength formulation. As visually observed for mAb1, phase separation occurred due to gel formation in the high ionic strength formulation, preventing concentration to formulation-related concentrations. See Figure 8.

[0241] This data supports that a high-concentration formulation suitable for subcutaneous administration can be prepared, in which the concentration of the monovalent antibody tmAb1 or its antigen-binding fragment can be 70 mg / ml or higher.

[0242] Example 10: Monovalent format reduces viscosity A low solution viscosity of the antibody solution is essential for the final production process, formulation, and subcutaneous administration. Here, the effect of the change from the bivalent format (mAb1) to the monovalent format (tmAb1) on the solution viscosity is evaluated.

[0243] Materials and methods tmAb1 and mAb1 were buffer-exchanged into a high ionic strength formulation (20 mM Hepes, 150 mM NaCl, pH 7.4) and a low ionic strength formulation (20 mM Hepes, pH 7.4). Subsequently, the samples were concentrated in a room-temperature Amicon 30 kDa centrifugal spin filter at a target of 70 mg / ml.

[0244] Viscosity was measured using a Rheosense Initium instrument using 50 μl of the sample and a temperature of 5°C. A single shear rate of 5,000 1 / s was used for comparison because no shear effect was observed during the test from shear rates of 1,000 to 10,000 1 / s, which suggest a Newtonian solution.

[0245] Results and discussion In the low ionic strength formulation, mAb1 has a high viscosity of 10.2 mPa*s, while tmAb1 has a significantly lower viscosity of 6.6 mPa*s (see Table 5). In the high ionic strength formulation, tmAb1 has a similar viscosity of 5.8 mPa*s, suggesting that the effect of the ionic strength of the formulation is limited. However, for mAb1, phase separation visually observed due to gel formation occurred, so it could not be concentrated above 30 mg / ml in the high ionic strength formulation.

[0246] These results show how the poor properties of the high-concentration mAb1 solution can be rescued by changing from a bivalent to a monovalent antibody format. mAb1 is considered non-druggable, while tmAb1 has good and acceptable biophysical properties, supporting the preparation of a high-concentration formulation suitable for subcutaneous administration.

Table 6

[0247] Example 11: In vivo evaluation of tmAb1 in healthy cynomolgus monkeys The purpose of this study was to determine the pharmacokinetics of the anti-ANGPTL3 monoclonal antibody tmAb1 and its effect on triglycerides.

[0248] tmAb1 was administered as a single intravenous bolus injection to cynomolgus monkeys with normal blood lipids at three different dose levels (0.3, 3, and 30 mg / kg). The injection volume was 0.3 mL / kg. All animals had not received any therapeutic antibodies or other protein-based therapeutic agents.

[0249] Blood sampling and sample analysis: Blood samples (0.5 mL) were collected from all animals at pre-dose, 10 minutes, 30 minutes, 1, 2, 4, 8, 14, 24, 48, 72, 96, 120, 144, 168, 192, and 240 hours (h) post-dose. Blood was collected from the femoral vein and placed in Teklab K containing 1.75 mg of EDTA / mL of blood 3Collected in an EDTA tube (part number 3K200PP). The sample was thoroughly mixed by inverting and immediately placed on wet ice for a maximum of 30 minutes before centrifugation. The sample was centrifuged at approximately 2000 g for 10 minutes at 2 - 8 °C, and the plasma aliquot was transferred to a Micronic tube (catalog number MP32022). The samples were analyzed to quantify tmAb1 and triglyceride concentrations.

[0250] Triglyceride Triglycerides were measured on a COBAS 6000 multi-analyzer according to the manufacturer's recommendations (Roche Diagnostics).

[0251] Human IgG measurement (tmAb1), principle of the assay : Plasma samples were analyzed for anti-ANGPTL3 hIgG (tmAb1) using a Luminescent Oxygen Channeling Immunoassay (LOCI). Donor beads were coated with streptavidin, while acceptor beads were conjugated with a custom monoclonal mouse anti-human IgG that does not cross-react with cynomolgus IgG. The biotinylated recombinant antibody fragment (nanobody / V H HH) that cross-reacts with human IgG-Fc (not cynomolgus) is from ThermoFisher (CaptureSelect, catalog: 7103322500). The three reactants (donor beads with streptavidin, acceptor beads conjugated with the target-specific Ab, and biotinylated target-specific antibody fragment) were combined with the anti-ANGPTL3 antibody (hIgG) to form a two-site immune complex. Upon irradiation of the complex, single oxygen atoms are released from the donor beads. These are channeled to the acceptor beads, inducing chemiluminescence that is measured by an EnVision plate reader (PerkinElmer) according to the manufacturer's instructions. The amount of light is proportional to the concentration of the anti-ANGPTL3 antibody (tmAb1).

[0252] Human IgG measurement (tmAb1), assay procedure : After applying 2 μL of plasma sample / calibrator / control to the wells of a 384-well LOCI plate, a 15 μL mixture of acceptor beads (0.5 μg / well) coated with MAb anti-hIgG and biotinylated antibody fragments was applied. The plate was incubated for 1 hour at room temperature (RT). Then, 30 μL of donor beads (2 μg / well) coated with streptavidin were added to each well and incubated for 30 minutes at RT. The plate was read on an Envision plate reader at 21 - 22 °C using a filter with a bandwidth of 520 - 645 nm after excitation with a 680 nm laser. The total measurement time per well was 210 milliseconds including an excitation time of 70 milliseconds. The calibrator was tmAb1 diluted with PBS + 1% cynomolgus plasma. The assay range extended from 11 - 20,000 pM. Cynomolgus samples were tested after dilution 100-fold with PBS plasma. Samples measured above the upper limit of quantification (ULOQ) were further diluted with PBS + 1% cynomolgus plasma.

[0253] Results PK The pharmacokinetics (PK) of tmAb1 are shown in Figure 9. The PK was linear over 240 hours at all dose levels and had signs of extensive non-linearity across all dose levels. Half-lives calculated using non-compartmental analysis and the "linear-up log-down" method, based on 240-hour observations, were 119, 107, and 107 hours geometric mean for the 0.3, 3, and 30 mg / kg groups, respectively, and were within the range of 90.9 - 171 for all animals.

[0254] TG The effect on TG was evaluated over a period of 0 - 240 hours (Figure 10). 3 mg / kg and 30 mg / kg provided a rapid decrease in TG levels, resulting in a decrease of approximately 40 - 50% after 4 hours. The maximum decrease seen during the test varied between 56 - 78%. Generally, the effect on TG lasted for at least 240 hours. At a dose level of 0.3 mg / kg, a transient effect of 28 - 44% on TG levels was seen, and after 4 hours and 12 hours, the TG levels returned to the baseline level.

[0255] Example 12: Generation of tmAb2, tmAb3, tmAb4 A CDR single-site mutagenesis library of the variable domain sequence of tmAb1 was generated to explore the possibility of further modulation of pH-dependent target binding (see Examples 6 and 7) and LPL activity (Example 4). The most performant molecules were evaluated as tmAbs with respect to the effects on formulation (Examples 8, 9, and 10), activity and target binding parameters (Examples 4 and 7), IC formation (Example 6), and in vivo effector function (Example 5), and as a result, tmAb2, tmAb3, and tmAb4 were selected.

Claims

**Claim 1** A monovalent antibody or an antigen-binding fragment thereof that can bind to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), wherein a) the heavy chain of the antibody or its antigen-binding fragment is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, a CDR1 sequence, ● a CDR2 sequence of amino acid residues SISSHSTYIYYADSVRKG (SEQ ID NO: 3), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, a CDR2 sequence, ● a CDR3 sequence of amino acid residues EGWYDWNWFD P (SEQ ID NO: 4), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, a CDR3 sequence, and includes, or b) the heavy chain of the antibody or its antigen-binding fragment is ● a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 24), wherein 0 or 1 of these amino acid residues may be substituted by different amino acid residues, a CDR1 sequence, ● a CDR2 sequence of amino acid residues SISSHSTYIYYADSVRKG (SEQ ID NO: 25), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, a CDR2 sequence, ● a CDR3 sequence of amino acid residues EGWYDWNWND P (SEQ ID NO: 26), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, a CDR3 sequence, and the light chain of the antibody or its antigen-binding fragment is ● a CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein 0, 1, 2, or 3 of these amino acid residues may be substituted by different amino acid residues, a CDR1 sequence, ● a CDR2 sequence of amino acid residues GVSSRA A (SEQ ID NO: 10), wherein 0, 1, or 2 of these amino acid residues may be substituted by different amino acid residues, a CDR2 sequence, ● A CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein 0, 1, or 2 of these amino acid residues may be substituted with different amino acid residues, and a monovalent antibody or an antigen-binding fragment thereof containing the CDR3 sequence.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the substitution is a conservative substitution.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable domain has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5, and the light chain variable domain has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

12.

4. a) The heavy chain variable domain contains SEQ ID NO: 5, and the light chain variable domain contains SEQ ID NO: 12, or b) The heavy chain variable domain contains SEQ ID NO: 17, and the light chain variable domain contains SEQ ID NO: 12, or c) The heavy chain variable domain contains SEQ ID NO: 22, and the light chain variable domain contains SEQ ID NO: 12, or d) The heavy chain variable domain contains SEQ ID NO: 27, and the light chain variable domain contains SEQ ID NO:

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The antibody or antigen-binding fragment thereof contains, in the heavy chain, tyrosine (Y) at position 252, threonine (T) at position 254, and glutamic acid (E) at position 256 (EU residues). The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The antibody or antigen-binding fragment thereof contains, in the heavy chain, tyrosine (Y) at position 234, aspartic acid (D) at position 238, valine (V) at position 250, isoleucine (I) at position 264, proline (P) at position 307, and / or lysine (K) at position 330 (EU residues). The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The antibody or antigen-binding fragment thereof contains arginine (R) at positions 311 and 343 (EU residues) in the heavy chain. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is an antagonist capable of inhibiting the hANGPTL3-mediated inhibition of lipoprotein lipase (LPL) and reducing the concentration of hANGPTL3 in human plasma.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises an Fc region.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the isotype of the antibody is based on IgG1, IgG2, IgG3, or IgG4.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, a cleaved heavy chain, and a light chain, the heavy chain comprises SEQ ID NO: 6, the cleaved heavy chain comprises SEQ ID NO: 7, and the light chain comprises SEQ ID NO:

13.

12. A monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), comprising a heavy chain, a cleaved heavy chain, and a light chain, wherein the heavy chain comprises SEQ ID NO: 6, the cleaved heavy chain comprises SEQ ID NO: 7, and the light chain comprises SEQ ID NO:

13.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and one or more pharmaceutically acceptable carriers.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the composition according to claim 13 for use in medicine.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the composition according to claim 13 for use in the treatment or prevention of an increase in triglyceride concentration in human plasma.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the composition according to claim 13 for use in the treatment or prevention of cardiovascular diseases such as atherosclerotic cardiovascular disease (ASCVD).

17. The method or use according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof or the composition according to claim 13 is administered subcutaneously.

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the composition according to claim 13 and an instruction manual.

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