Novel anti-ANGPTL3 antibodies suitable for high concentration compositions and subcutaneous administration

Abstract

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

Description

Technical Field

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

[0002] Incorporation by Reference of Sequence Listing

[0003] This application is submitted with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference. Background Art

[0004] Human angiopoietin-like protein 3 (hANGPTL3) is a 460 amino acid long protein first identified by Conklin et al. (Conklin, Gilbertson et al. (1999) Genomics 62(3):477-482). hANGPTL3 circulates in plasma as a trimer formed by two helix-helix domains present in the N-terminal portion of the protein. hANGPTL3 is part of the angiopoietin-like protein family and affects lipid metabolism by inhibiting lipoprotein lipase (LPL) and endothelial lipase (EL).

[0005] hANGPTL3 is the most abundantly expressed member of the family and is a well-described inhibitor of LPL. It exerts its effects primarily in the non-fasting state. LPL is anchored in endothelial cells and is the key enzyme responsible for the hydrolysis of triglycerides from triglyceride-rich lipid particles (VLDL and chylomicrons). The higher the LPL activity, the more triglycerides are removed from the circulation.

[0006] Inhibition of LPL by hANGPTL3 leads to elevated triglyceride and LDL concentrations in plasma.

[0007] Inhibition of LPL by hANGPTL3 is thought to act through interaction with human angiopoietin-like protein 8 (hANGPTL8) and possibly with human angiopoietin-like protein 4 (hANGPTL4). In the absence of hANGPTL3, hANGPTL8 has no inhibitory function in vitro, whereas hANGPTL3 can inhibit LPL by itself. hANGPTL4 is also known to inhibit LPL independently of hANGPTL3 and 8. hANGPTL3 is normally expressed by the liver, whereas hANGPTL4 and 8 show different expression patterns in different organisms; hANGPTL4 and 8 are primarily expressed upon external stimulation, whereas hANGPTL3 shows higher constitutive expression from the liver.

[0008] Different cleavage forms exist in circulation (Ono, Shimizugawa et al. (2003) J Biol Chem 278(43):41804-41809). Both full-length hANGPTL3 and the cleavage form of hANGPTL3 containing the N-terminal domain inhibit LPL and EL, leading to elevated triglyceride levels.

[0009] Loss-of-function variants in the gene encoding hANGPTL3 are associated with hypolipidemia and protection against atherosclerotic cardiovascular disease. is a monoclonal antibody against hANGPTL3 (WO2012 / 174178) that showed benefit in patients with homozygous familial hypercholesterolemia at a dose of 15 mg / kg body weight every 4 weeks (Rall et al., (2020) NEJM, 383(8), 711-720).

[0010] The high doses required for treatment make currently available antibody products (The recommended dose of EVKEEZA is 15 mg / kg administered by intravenous infusion over 60 minutes every 4 weeks with an inline or attached filter) is not available for self-administration, and a more optimized treatment scenario may be beneficial for this patient population.

[0011] There is a need in the art for improved anti-hANGPTL3 antibodies. Summary of the invention

[0012] The present invention relates to monovalent antibodies and antigen-binding fragments thereof capable of binding to human ANGPTL3 (hANGPTL3) and reducing hANGPTL3-mediated LPL inhibition, and their use in medicine and in pharmaceutical compositions comprising such compounds.

[0013] In one aspect, the monovalent antibody and antigen-binding fragment thereof is capable of binding hANGPTL3 and is capable of reducing triglyceride concentration in human plasma.

[0014] In one aspect, the monovalent antibody and antigen-binding fragment thereof are capable of binding to hANGPTL3 and are capable of reducing the concentration of hANGPTL3 in human plasma, thereby reducing the inhibition of LPL.

[0015] In one aspect, the monovalent antibody and antigen-binding fragment thereof is capable of binding hANGPTL3 and is capable of inhibiting hANGPTL3-mediated LPL inhibition.

[0016] In one aspect, the monovalent antibody and antigen-binding fragment thereof are capable of binding to hANGPTL3 and are capable of inhibiting hANGPTL3-mediated LPL inhibition and reducing hANGPTL3 concentration in human plasma, thereby reducing hANGPTL3-mediated LPL inhibition.

[0017] In a specific aspect, the invention relates to a monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), wherein

[0018] a) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0019] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 2), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0020] CDR2 of amino acid residue SISSHSTYIYYADSVKG (SEQ ID NO: 3)

[0021] Sequences in which zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0022] a CDR3 sequence of the amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues; or

[0023] b) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0024] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 24), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0025] CDR2 of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 25)

[0026] Sequences in which zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0027] A CDR3 sequence of the amino acid residues EGWYDNWNDP (SEQ ID NO: 26), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues;

[0028] and

[0029] The light chain of the antibody or antigen-binding fragment thereof comprises:

[0030] a CDR1 sequence of the amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0031] a CDR2 sequence of the amino acid residues GVSSRAA (SEQ ID NO: 10), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues, and

[0032] - A CDR3 sequence of the amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues.

[0033] In one aspect, the monovalent antibodies and antigen-binding fragments thereof can be formulated as high concentration compositions.

[0034] In one aspect, the monovalent antibodies and antigen-binding fragments thereof are suitable for subcutaneous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A-Figure 1D SEQ ID NOs: 2-28 are shown in tabular format.

[0036] Figure 2 A (non-limiting) schematic diagram of a monovalent antibody is shown.

[0037] Figure 3 Shown are the effects of administration of tmAb0.1, tmAb0.2, tmAb0.3, tmAb0.4 and ivizumab sequence identical analog (SIA) on triglyceride concentrations in mouse plasma at different time points.

[0038] Figure 4A and Figure 4B Shown are the effects of administration of tmAb0.1, tmAb0.2, tmAb0.3, tmAb0.4, ivizumab sequence identical analog (SIA), and vehicle on total ANGPTL3 concentrations in mouse plasma. Figure 4B Focusing on the 100,000-400,000 pg / mL ANGPTL3 plasma concentration range, this range is lower than the ANGPTL3 plasma levels in mice treated with ivizumab SIA, so ivizumab SIA data are not shown in the figure.

[0039] Figure 5 Shown are the mouse plasma concentrations of tmAb0.1, tmAb0.2, tmAb0.3 and tmAb0.4 and ivizumab sequence identical analog (SIA) after administration thereof.

[0040] Figure 6A-6E SE-UPLC profiles of antibody-ANGPTL3 complexes are shown, including the ivizumab SIA at different pH values ​​( Fig. 6A )、tmAb1( Figure 6B )、tmAb2( Figure 6C )、tmAb3( Fig.6D )、tmAb4( Fig. 6E ). The dotted line represents the complex analyzed at pH 6. The solid line represents the complex analyzed at pH 7.4.

[0041] Figure 7 Self-association of tmAbl and its divalent form, termed "mAbl", at different pH values ​​is shown.

[0042] Figure 8 Pictures of mAbl and tmAbl in high and low ionic strength formulations are shown. Gel formation was observed for mAbl (divalent) after concentration at high ionic strength (see white arrows).

[0043] Fig. 9 Shown are the levels of tmAbl in plasma samples from cynomolgus monkeys.

[0044] Fig.10 Shown are the levels of triglycerides in plasma samples from cynomolgus monkeys administered tmAbl at three different doses.

[0045] Sequence Description

[0046] SEQ ID NO: 1 represents the amino acid sequence of human ANGPTL3.

[0047] SEQ ID NOs: 2, 3 and 4 represent the amino acid sequences of complementarity determining regions (CDRs) 1-3 of the heavy chain of tmAb1, respectively.

[0048] SEQ ID NO: 5 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAbl.

[0049] SEQ ID NO: 6 represents the amino acid sequence of the heavy chain of tmAbl comprising a C-terminal Gly-Lys deletion (des-(Gly-Lys)).

[0050] SEQ ID NO: 7 represents the amino acid sequence of the truncated heavy chain (tHC) of tmAbl, tmAb2, tmAb3 and tmAb4 also comprising a C-terminal Gly-Lys deletion (des-(Gly-Lys)).

[0051] SEQ ID NO:8 represents the amino acid sequence of an alternative truncated heavy chain (tHC) that differs from SEQ ID NO:7 by not having a C-terminal Gly-Lys deletion.

[0052] SEQ ID NOs: 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.

[0053] SEQ ID NO: 12 represents the amino acid sequence of the light chain variable domain (VL) of tmAb1, tmAb2, tmAb3, and tmAb4.

[0054] SEQ ID NO: 13 represents the amino acid sequence of the light chain of tmAb1, tmAb2, tmAb3 and tmAb4.

[0055] SEQ ID NOs: 14, 15 and 16 represent complementarity determining regions (CDRs) 1-3 of the heavy chain of tmAb2, respectively.

[0056] SEQ ID NO: 17 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb2.

[0057] SEQ ID NO: 18 represents the amino acid sequence of the heavy chain of tmAb2.

[0058] SEQ ID NOs: 19, 20 and 21 represent the amino acid sequences of complementarity determining regions (CDRs) 1-3 of the heavy chain of tmAb3, respectively.

[0059] SEQ ID NO: 22 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb3.

[0060] SEQ ID NO: 23 represents the amino acid sequence of the heavy chain of tmAb3.

[0061] SEQ ID NOs: 24, 25 and 26 represent the amino acid sequences of complementarity determining regions (CDRs) 1-3 of the heavy chain of tmAb4, respectively.

[0062] SEQ ID NO: 27 represents the amino acid sequence of the heavy chain variable domain (VH) of tmAb4.

[0063] SEQ ID NO: 28 represents the amino acid sequence of the heavy chain of tmAb4. DETAILED DESCRIPTION

[0064] Intravenous (iv) administration of an anti-ANGPTL3 antibody such as for inhibiting ANGPTL3 inhibition of LPL Dose-dependent accumulation of ANGPTL3 has been observed after lowering triglycerides in human plasma (Ahmad et al. (2019) Circulation Aug 6; 140(6):470-486, Supplementary Information Figure 2 ).

[0065] This phenomenon is known as antibody-mediated antigen accumulation and can lead to a substantial increase in the required antibody dose to achieve therapeutic efficacy. The need for high therapeutically effective doses can pose challenges to the route of administration. For example, subcutaneous (sc) administration of high doses of antibodies requires that the antibodies have sufficiently good properties in terms of solubility, self-association, and solution viscosity.

[0066] So-called clearing antibodies have been developed to generally increase the elimination of soluble antigens bound by such antibodies (Igawa et al. (2016) Immun. Rev. 270: 132-151), but oligomeric target proteins such as ANGPTL3 pose a particular challenge to this concept when several antibodies bind to the same target, thereby providing multiple binding opportunities for the Fc receptor. This increases the avidity of antibody-Fc receptor affinity in the antibody recycling mechanism, thereby facilitating the uptake of immune complexes (ICs) into endosomes. However, this also poses challenges for pH-dependent antigen release and clearance in endosomes / lysosomes, and also for antibody recycling by triggering the clearance of multicomponent immune complexes (ICs) generated when several antibodies bind to a single multimeric antigen. The presence of two or more Fab portions on each antibody molecule provides oligomeric targets with several opportunities to bind to the same antibody, potentially generating complex and heterogeneous IC networks, the size of which correlates with immune response and IC clearance (Opolka-Hoffmann et al. (2021) mAbs, 13:1, 1995929; St. Clair et al. (2017) PLOS one, 12(1):e0170556).

[0067] The inventors have discovered that improvements in the viscosity profile of the antibody can be achieved by utilizing a monovalent antibody format that further has pH-dependent binding properties, thereby allowing the antibody to be administered subcutaneously. Surprisingly, switching to a monovalent format with only one ANGPTL3 binding site per molecule resulted in a triglyceride-lowering potency comparable to that of a monovalent format with two ANGPTL3 binding sites per molecule. The smaller size of the antibodies disclosed herein further enhances their potential for subcutaneous administration. The antibodies disclosed herein have biophysical properties that allow concentrations in solution to be at least 70 mg / ml, whereas To be prepared in an IV bag at a concentration of up to 20 mg / ml.

[0068] Thus, the present invention relates to monovalent antibodies and antigen-binding fragments thereof capable of binding hANGPTL3 and reducing hANGPTL3-mediated LPL inhibition, and their use in medicine and in pharmaceutical compositions comprising such compounds, including pharmaceutical compositions suitable for subcutaneous administration.

[0069] In one aspect, the monovalent antibody or antigen-binding fragment thereof is capable of binding hANGPTL3 and reducing triglyceride concentration in human plasma.

[0070] In one such aspect, the monovalent antibody and antigen-binding fragment thereof is capable of binding to hANGPTL3 and reducing the concentration of hANGPTL3 in human plasma, thereby reducing the inhibition of LPL.

[0071] In another such aspect, the monovalent antibody and antigen-binding fragment thereof is an antagonist capable of binding hANGPTL3 and inhibiting hANGPTL3-mediated LPL inhibition.

[0072] In one aspect, the monovalent antibody and antigen-binding fragment thereof are capable of binding to hANGPTL3 and inhibiting hANGPTL3-mediated LPL inhibition and reducing hANGPTL3 concentration in human plasma, thereby reducing hANGPTL3-mediated LPL inhibition.

[0073] In one aspect, the monovalent antibody or antigen-binding fragment thereof does not increase the concentration of hANGPTL3 in human plasma following administration.

[0074] In one aspect, the monovalent antibody or antigen-binding fragment thereof reduces the concentration of hANGPTL3 in human plasma following administration.

[0075] In one aspect, the monovalent antibody or antigen-binding fragment thereof provides reduced viscosity, reduced gel formation, and low self-association tendency in solution over a wide pH range compared to the corresponding bivalent antibody.

[0076] In a specific aspect, the invention relates to a monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), wherein

[0077] a) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0078] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 2), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0079] CDR2 of amino acid residue SISSHSTYIYYADSVKG (SEQ ID NO: 3)

[0080] Sequences in which zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0081] a CDR3 sequence of the amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues; or

[0082] b) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0083] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 24), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0084] CDR2 of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 25)

[0085] Sequences in which zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0086] A CDR3 sequence of the amino acid residues EGWYDNWNDP (SEQ ID NO: 26), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues;

[0087] and

[0088] The light chain of the antibody or antigen-binding fragment thereof comprises:

[0089] a CDR1 sequence of the amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0090] a CDR2 sequence of the amino acid residues GVSSRAA (SEQ ID NO: 10), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues, and

[0091] - A CDR3 sequence of the amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues.

[0092] In one aspect, the monovalent antibodies and antigen-binding fragments thereof are suitable for subcutaneous administration.

[0093] In one aspect, the monovalent antibodies and antigen-binding fragments thereof can be formulated as high concentration compositions.

[0094] In one aspect, the monovalent antibody or antigen-binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen-binding fragment thereof is 25 mg / ml or higher, preferably 50 mg / ml or higher, such as 70 mg / ml or higher.

[0095] In order that the present invention may be more easily understood, some terms are first defined.

[0096] The terms “a” or “an” are intended to mean “one or more.” When the terms “comprise” and variations such as “comprising” precede recitation steps or elements, it is intended to mean that adding further steps or elements is optional and not excluded.

[0097] The term "about" is used herein to mean approximately, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" can modify a numerical value up or down (higher or lower) to 10% above or below the stated value.

[0098] As used herein, the term "antagonist antibody" 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 antigen-binding fragment thereof specifically binds to hANGPTL3, thereby partially or completely blocking its inhibitory effect on LPL, thereby, for example, reducing triglyceride levels in the plasma of human patients.

[0099] The term "antibody" includes, but is not limited to, a full-length antibody comprising at least four polypeptide chains: two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, and an antibody comprising at least three polypeptide chains: two heavy chains (HC) and one light chain (LC) linked by disulfide bonds. One of the heavy chains may be a truncated heavy chain.

[0100] One class of immunoglobulins of particular pharmaceutical interest is IgG. In humans, the IgG class can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4, based on the sequence of the constant region of its heavy chain. The light chain can be divided into two types: kappa chain and lambda chain, based on differences in its sequence composition. The IgG molecule consists of two heavy chains interconnected by two or more disulfide bonds and two light chains each connected to a heavy chain by a disulfide bond.

[0101] The IgG heavy chain may include a heavy chain variable domain (V H ) and up to three heavy chain constant (C H ) Domain: C H 1. C H 2 and C H 3. The light chain may include a light chain variable domain (VL ) and the light chain constant domain (C L ). H and V L The V regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) or hypervariable regions (HvR), interspersed with regions that are more conserved, termed framework regions (FR). H and V L The domain is usually composed of three CDRs and four FRs, which are arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable domains containing the hypervariable region (CDR) constitute a structure capable of interacting with the antigen, while the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including but not limited to various cells of the immune system (effector cells), Fc receptors, and the first component of the C1 complex of the classical complement system, C1q.

[0102] The antibody of the present invention can be a monoclonal antibody (mAb), such as a monovalent monoclonal antibody. The antibody of the present invention can be produced and purified using various methods known to those skilled in the art. For example, the antibody can be produced from a hybridoma cell. The antibody can be produced by B cell amplification. The antibody or its antigen-binding fragment can be recombinantly expressed in a mammalian or microbial expression system or by in vitro translation. The antibody or its antigen-binding fragment can also be recombinantly expressed as a cell surface-bound molecule by, for example, phage display, bacterial display, yeast display, mammalian cell display, or ribosome or mRNA display.

[0103] Antibodies or antigen-binding fragments thereof can be defined according to their complementary determining regions (CDRs). The term "complementarity determining region" or "CDR" as used herein refers to the region of an antibody in which the amino acid residues involved in antigen binding are located. CDRs can be identified as regions with the highest variability in the amino acid alignment of the antibody variable domains. Databases can be used for CDR identification, such as the Kabat database, for example, CDRs are defined as 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, US Department of Health and Human Services, NIH Publication No. 91-3242). Typically, the numbering of amino acid residues in this region is performed by the method described by Kabat et al. (supra). Phrases such as "Kabat position," "Kabat residue," and "according to Kabat" herein refer to this numbering system for a heavy chain variable domain or a light chain variable domain, and unless contradicted by context, numbering according to Kabat is used herein.

[0104] The numbering of amino acid residues in the antibody constant region is carried out by reference to the numbering used for the crystal structure of human IgG1 (Edelman et al. (1969) Proc. Natl. Acad. Sci. USA 63:78-85), and phrases such as "EU position", "EU residue" and "according to the EU index" in this article 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.

[0105] Reference to a position in a heavy chain (or light chain), such as "Valine (V) at position 250," means that Valine is present at position 250 in both heavy chains (as long as the given residue position is present in both heavy chains), unless otherwise specified or contradicted by context.

[0106] The term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to or recognize an antigen, such as ANGPTL3 described herein, particularly hANGPTL3. Examples of antigen-binding fragments include, but are not limited to, fragments comprising a single V H , Single V L domain and a monovalent molecule that interacts with an Fc receptor.

[0107] Antibody fragments of the invention can be prepared, for example, by deleting one or more amino acids from the N-terminus and / or C-terminus of the heavy and / or light chain. Fragments can also be generated by one or more internal deletions.

[0108] The present invention encompasses variants of the antibodies or antigen-binding fragments thereof disclosed herein, which may comprise one or more amino acid substitutions and / or deletions and / or insertions in the respective sequences disclosed herein.

[0109] "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids. The substitution can be, but is not limited to, conservative substitutions. For example, for conservative substitutions, an amino acid can be substituted with an amino acid with similar biochemical properties, for example, a basic amino acid can be substituted with another basic amino acid (e.g., lysine is substituted with arginine), an acidic amino acid can be substituted with another acidic amino acid (e.g., glutamic acid is substituted with aspartic acid), a neutral amino acid can be substituted with another neutral amino acid (e.g., threonine is substituted with serine), a charged amino acid can be substituted with another charged amino acid (e.g., glutamic acid is substituted with lysine), a hydrophilic amino acid can be substituted with another hydrophilic amino acid (e.g., asparagine is substituted with glutamine), a hydrophobic amino acid can be substituted with another hydrophobic amino acid (e.g., alanine is substituted with valine), a polar amino acid can be substituted with another polar amino acid (e.g., serine is substituted with threonine), an aromatic amino acid can be substituted with another aromatic amino acid (e.g., phenylalanine is substituted with tryptophan), and an aliphatic amino acid can be substituted with another aliphatic amino acid (e.g., leucine is substituted with isoleucine).

[0110] Preferred variants include those in which, instead of an amino acid residue appearing in the sequence, a structural analog of that amino acid residue is included.

[0111] The antibodies and antigen-binding fragments thereof disclosed herein contain regions that can interact with cell surface receptors called Fc receptors, such as, but not limited to, the crystallizable region ("Fc region", also referred to as "Fc domain"). The Fc region is a region that contains the hinge and constant C H 2 and C HThe Fc domain is the C-terminal region of an antibody that contains three domains. The Fc domain can interact with cell surface receptors called Fc receptors and some proteins of the complement system. The Fc region enables antibodies to interact with the immune system. In one aspect of the invention, antibodies may be engineered to include modifications within the Fc region, generally to alter one or more functional properties thereof, such as plasma half-life, complement fixation, Fc receptor binding, protein stability and / or antigen-dependent cellular cytotoxicity, or the lack of these properties, among others (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, residues are numbered according to the EU index). Furthermore, antibodies and antigen-binding fragments thereof may be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or modified to alter their glycosylation, again to alter one or more functional properties of the antibody.

[0112] Antibodies such as IgG1 antibodies may carry a modified Fc domain comprising one or more, preferably all of the following substitutions that will result in increased binding to certain Fc-γ receptors, such as a combination of Q311R and P343R with L234Y, P238D, T250V, V264I, T307P and A330K (residues are numbered according to the EU index). Alternatively, other amino acid substitutions known in the art to result in increased binding to FcRn and / or Fc-γ receptors may be used, optionally in combination with the above-mentioned substitutions. See, for example, WO2016 / 125495 and WO2013 / 002362, WO2014 / 104165, WO2012 / 115241, WO2014 / 030728, WO2014 / 163101 and WO2017 / 104783, all of which are incorporated herein by reference.

[0113] IgG1 antibodies may carry a modified Fc domain comprising a substitution that will result in an extended plasma half-life. In one embodiment, such substitutions include a combination of M252Y, S254T, and T256E (residues are numbered according to the EU index). See also US2003 / 0190311, which is incorporated herein by reference.

[0114] The term "binding affinity" is a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or fragment thereof and an antigen). The term is used to describe monovalent interactions. The equilibrium dissociation constant (K D ), the binding affinity between two molecules (e.g., an antibody or fragment thereof and an antigen) through a monovalent interaction can be quantified. K can be determined by measuring the kinetics of complex formation and dissociation, for example, by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constants k and k, respectively. a (or k 缔合 ) and the dissociation rate constant k d (or k 解离 ). Through equation K D =k d / k a , K D With k a and k d Associated.

[0115] According to the above definition, by comparing the K of each antibody / antigen complex D Values ​​can be used to compare the binding affinities associated with different molecular interactions, such as the binding affinities of different antibodies to a given antigen.

[0116] The value of the dissociation constant can be determined directly by well-known methods. Standard tests for evaluating the binding ability of ligands such as antibodies to targets are known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of antibodies can also be assessed by standard tests known in the art, such as SPR.

[0117] Competitive binding assays can be performed in which binding of an antibody to a target is compared to binding of that target in the presence of another ligand for that target, such as another antibody.

[0118] Unless contradicted by the context, K D Preferably it is determined by surface plasmon resonance as described herein (see Example 7).

[0119] "Cross-species reactive" antibodies bind with comparable affinity to ANGPTL3 from all specified species (e.g., human, mouse, and cynomolgus monkey), particularly K D In the range of a factor of 100, for example, in the range of a factor of 50, in the range of a factor of 20, or in the range of a factor of 10. In the defined factor X, K D In the range, it is meant that the highest affinity for a particular listed species is no more than X times the lowest affinity measured for binding to a different listed species. Those skilled in the art will appreciate that any method of measuring affinity can be used to verify that a cross-species reactive antibody is within a given K range as described herein. D factors to target antigens from all listed species, as long as the K D The same conditions are applied to all measurements. Preferably, SPR is used to measure K D Values, in particular at 25° C. Preferably, cross-species reactive antibodies are used as Fab fragments to measure affinity.

[0120] As used herein, the term "elevated triglyceride concentration in plasma" refers to plasma levels of 150-500 mg / dL.

[0121] As used herein, the term "highly elevated triglyceride concentrations in plasma" refers to plasma levels above 500 mg / dL.

[0122] As used herein, the term "normal triglyceride concentration in plasma" refers to plasma levels below 150 mg / dL.

[0123] The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides determined by comparing sequences. In the art, "identity" also represents the degree of sequence correlation between polypeptides, as determined by the number of matches between residue strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between two or more sequences and the smaller sequence therein, wherein the gap comparison (if any) is solved by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be easily calculated by known methods. In the present invention, Needleman (Needleman et al., J.Mol.Biol.1970; 48:443-453) from EMBOSS-6.6.0 is used to determine similarity and identity, wherein parameters 10 and 0.5 (gapopen=10, gapextend=0.5) are used for opening and extending the gap, respectively.

[0124] As used herein, the term "monovalent antibody" refers to an antibody that is capable of binding to one antigen molecule without being able to undergo antigen cross-linking.

[0125] The term "one-arm" as used herein refers to a specific type of monovalent antibody composed of a single light chain, a heavy chain, and a truncated heavy chain lacking at least the Fv region of the heavy chain.

[0126] The term "monospecific" antibody as used herein refers to an antibody capable of binding to a single epitope.

[0127] As used herein, the term "human antibody" is intended to include antibodies having variable domains in which at least a portion of the framework regions and / or at least a portion of the CDR regions are derived from human germline immunoglobulin sequences. For example, a human antibody may have variable domains in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region or a portion thereof is also derived from a human germline immunoglobulin sequence. Preferably, the human antibody is a monoclonal antibody.

[0128] The term "heavy chain" includes a full-length heavy chain. A full-length heavy chain includes the variable domain V H and three constant domains C H 1. C H 2 and C H 3. V H The domain is located at the amino terminus of the polypeptide, while the C H The domain is located at the carboxyl terminus, where C H 3 is closest to the -COOH terminus. The C-terminus of the heavy chain may comprise a C-terminal Gly-Lys deletion (des-(Gly-Lys)).

[0129] As used herein, the term "light chain" includes a full-length light chain. A full-length light chain includes the variable domain VL and constant domain C L The variable domain of the light chain is located at the amino terminus of the polypeptide. The light chains described herein include kappa chains and lambda chains.

[0130] As used herein, the term "immune complex" refers to a complex formed by one or more antibodies when bound to one or more target antigens. An example of one of these is one or more anti-ANGPTL3 antibodies bound to two or more ANGPTL3 molecules. The term is used interchangeably with "immune complex".

[0131] As used herein, the term "isoelectric point" or "pi" refers to the pH at which the total net charge of a protein, such as an antibody, is zero. There may be many charged groups in a protein, and at the pI, the sum of all these charges is zero. At pH values ​​above the pI, the total net charge of the protein is negative, while at pH values ​​below the pI, the total net charge of the protein is positive.

[0132] The pi may be a theoretical or an experimentally determined pi.

[0133] 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. Many (online) tools are available that allow 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 MR, Appel R.D., Bairoch A.; (In) John M. Walker (ed.): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607.

[0134] The pi may also be determined experimentally, and the charge variants may be separated, for example, using a charge-based separation technique such as isoelectric focusing (IEF) gel electrophoresis or capillary isoelectric focusing (cIEF) gel electrophoresis.

[0135] As used herein, the term "multimerization domain" refers to the region of each heavy chain of, for example, a monovalent antibody. A "multimerization domain" promotes stable interactions between entities of a chimeric monovalent antibody or its antigen-binding fragment. Preferably, the multimerization domain promotes the interaction between the first heavy chain and the second heavy chain, thereby enhancing the formation of the desired heterologous multimeric monovalent antibody and significantly reducing the probability of formation of unwanted homologous multimeric antibodies. The multimerization domain can interact through immunoglobulin sequences, leucine zippers, hydrophobic regions, hydrophilic regions, or free sulfhydryl groups, which form intermolecular disulfide bonds between the chimeric molecules of the chimeric heteromultimer. Free sulfhydryl groups can be introduced into the interface of one or more interacting polypeptides by replacing naturally occurring residues in the polypeptide with, for example, cysteine ​​at a position that allows the formation of a disulfide bond between the first and second heavy chains. The multimerization domain may comprise an immunoglobulin constant region. Possible multimerization domains are disclosed in PCT / US90 / 06849, in which hybrid immunoglobulins are described. In addition, the polymerized region can be engineered so that the spatial interaction not only promotes stable interaction, but also further promotes the formation of heterodimers from a monomer mixture to exceed homodimers. See, for example, PCT / US96 / 01598, which discloses a "projection into the cavity" strategy for the interface between the first and second polypeptides for hetero-oligomerization, and particularly see WO98 / 50431. "Projection" is constructed by replacing the small amino acid side chains on the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). Optionally, by replacing the large amino acid side chains with smaller amino acid side chains (such as alanine or threonine), a compensatory "cavity" with the same or similar size as the projection is created on the interface of the second polypeptide. The immunoglobulin sequence is an immunoglobulin constant domain. The immunoglobulin part in the antibody disclosed herein can be obtained from IgG1, IgG2, IgG3 or IgG4 subtypes.

[0136] There are many techniques for generating chimeric, hybrid or asymmetric immunoglobulins, and the truncation technology used in this application (Merchant et al., (2013) PNAS USA 110:E2987-2996) can potentially be used in combination with any chimeric form (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).

[0137] As used herein, the term "plasma half-life" refers to the time required for half the amount of a substance administered to a patient to be metabolized or eliminated from the patient's serum or plasma by normal biological processes.

[0138] As used herein, the term "surface exposed amino acid residue" refers to an amino acid residue whose side chain can be in contact with solvent molecules (usually, it may be mainly water molecules). However, the side chain does not necessarily have to be in contact with the solvent molecules in its entirety, and even when a portion 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". The amino acid residues located on the surface of the polypeptide may also include amino acid residues located close to the antibody surface, thereby having mutual charge influences from other amino acid residues whose side chains are even partially in contact with solvent molecules. One of ordinary skill in the art can prepare a homology model or a machine learning-based three-dimensional molecular model of a polypeptide or antibody using commercially available or publicly available software, such as homology modeling or machine learning. Alternatively, a three-dimensional molecular model may be generated using methods such as X-ray crystallography. Amino acid residues that can be exposed on the surface can be, for example, identified using software such as MOE (Chemical Computing Group) or Bioluminate The surface exposed sites can be determined using computer programs such as , using coordinates from a three-dimensional molecular model of the antibody. The surface exposed sites can be determined using algorithms known in the art (e.g., Lee and Richards (1971) J. Mol. Biol. 55: 379-400; Connolly, J. Appl. Cryst. (1983) 16: 548-558). The surface exposed sites can be determined using software suitable for protein modeling and analysis of three-dimensional structural information obtained from antibodies. Software that can be used for such purposes includes, for example, MOE (Chemical Computing Group) or Bioluminate Solvent accessible surface (unit: ) area using a probe radius of The water probe can be used to calculate. In addition, Pacios (Pacios, Comput. Chem. 18 (4): 377-386 (1994); J. Mol. Model. 1: 46-53 (1995)) has described a method for determining the surface exposed regions and areas using personal computer software. Based on the information described above, appropriate amino acid residues located on the antibody surface in contact with the solvent can be selected.

[0139] As used herein, the term "treatment" refers to medical treatment of any human subject in need. The subject is expected to have undergone a physical examination by a medical practitioner who has given a preliminary or definitive diagnosis indicating that the use of the specific treatment is beneficial to the health of the human subject. The timing and purpose of the treatment may vary from individual to individual, depending on the current status of the subject's health. Therefore, the treatment may be preventive, palliative, symptomatic and / or curative. For the purposes of the present invention, preventive, palliative, symptomatic and / or curative treatments may represent separate aspects of the present invention.

[0140] As used herein, the term "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, "treat" is not necessarily a cure.

[0141] As used herein, the term "preventing" or variations thereof refers to protecting a subject from developing at least one symptom of a disease or reducing the severity of a symptom of a disorder.

[0142] As used herein, the term "truncated heavy chain" refers to a heavy chain that lacks at least a portion of the variable domain, thereby no longer being able to bind hANGPTL3, and optionally lacks the first constant IgG domain (C H 1) or a portion thereof, thereby not comprising a cysteine ​​residue suitable for heavy chain / light chain pairing (such as IgG1 Cys220 EU residue). In one embodiment, a "truncated heavy chain" refers to a heavy chain lacking the entire variable domain, CH1 and part of the hinge region, such that 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 therefore not considered a "truncated heavy chain".

[0143] 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 in SEQ ID NO: 8 are present (SEQ ID NO: 8) or absent (SEQ ID NO: 7), respectively.

[0144] In one such embodiment, the monovalent antibody disclosed herein comprises a heavy chain and a truncated heavy chain, wherein both chains are of or based on the IgG1 isotype, and wherein the Fab region and the Gly-Lys C-terminal residues at positions 446 and 447 (EU residues), respectively, are not present in the truncated heavy chain, and wherein the Gly-Lys C-terminal residues at positions 446 and 447 (EU residues) of the heavy chain are not present.

[0145] In a preferred embodiment, the truncated heavy chain comprises SEQ ID NO:7.

[0146] The term "high concentration" as used herein in connection with a pharmaceutical composition refers to a concentration of 50 mg / ml or more.

[0147] Production of monovalent antibodies

[0148] The monovalent antibodies or antigen-binding fragments thereof disclosed herein can be produced in a host cell line expressing three different polypeptide chains (particularly a heavy chain, a truncated heavy chain, and a light chain). For non-limiting descriptions of such antibodies, see, e.g. Figure 2 No further arm swapping or in vitro refolding of the antibodies was required to obtain fully functional molecules.

[0149] In one embodiment, the monovalent antibody or antigen-binding fragment thereof comprises a so-called knob-in-hole replacement, thereby promoting heterodimerization of two different heavy chains, such as a full-length heavy chain and a truncated heavy chain. There are multiple sets of knob-in-hole replacements, and in a preferred embodiment, the heavy chain comprises T366W (EU residues), and the truncated heavy chain comprises T366S, L368A and Y407V (EU residues). See also WO98 / 50431, which is incorporated herein by reference.

[0150] In a preferred embodiment, the monovalent antibody contains 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.

[0151] In another preferred embodiment, the monovalent antibody contains 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 producing antibodies.

[0152] Complexes comprising hANGPTL3 and the antibodies disclosed herein were observed at neutral pH (pH 7.4), and thus the avidity of the monovalent antibodies was sufficient to effectively bind hANGPTL3. Binding of 2-3 monovalent antibodies to the trimeric hANGPTL3 antigen has been demonstrated to produce more homogeneous and less heterogeneous hANGPTL3 / antibody immune complexes compared to the ivizumab SIA antibody / hANGPTL3 immune complexes (see Example 6).

[0153] In one embodiment, the antibodies or antigen-binding fragments thereof disclosed herein provide smaller and less heterogeneous antibody / hANGPTL3 immune complexes compared to ivizumab SIA antibody / hANGPTL3 immune complexes. Smaller and / or less heterogeneous immune complexes are expected to result in reduced immunogenicity compared to situations where the antibody / hANGPTL3 complexes are larger and / or more heterogeneous.

[0154] Increased cellular uptake of antibody-antigen complexes

[0155] The monovalent antibodies disclosed herein are capable of pH-dependent binding to hANGPTL3 and comprise substitutions that increase cellular uptake of the antibody-antigen complex.

[0156] pH-dependent antibodies

[0157] The monovalent antibodies or antigen-binding fragments thereof disclosed herein have lower antigen-binding activity in the acidic pH range than in the neutral pH range. Thus, the reduced affinity for hANGPTL3 at lower pH (see Example 7) results in complete or almost complete dissociation of the hANGPTL3 / antibody complex at low pH (pH 6.0).

[0158] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 20 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0159] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 30 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0160] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 40 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0161] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 50 or greater. D (pH6) / K D(pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0162] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 60 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0163] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 70 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0164] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 80 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0165] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 90 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0166] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 95 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0167] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 100 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 Dratio.

[0168] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 110 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0169] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 120 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0170] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 130 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0171] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 140 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0172] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 150 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0173] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 160 or higher, such as 166 or higher. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0174] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 170 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0175] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 180 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0176] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 190 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0177] In one embodiment, the antibody or antigen-binding fragment thereof has a K of 200 or greater. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0178] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 40 to 500. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0179] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 40 to 400. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0180] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 40 to 300. D(pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0181] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 40 to 200. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0182] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 500. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0183] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 400. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0184] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 300. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0185] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 200. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0186] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 80 to 250. D (pH6) / K D(pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0187] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 80 to 200. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0188] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 80 to 170. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0189] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 250. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0190] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 200. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0191] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 170. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0192] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 90 to 166. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6.D The K of the antigen at pH 7.4 D ratio.

[0193] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 95 to 250. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0194] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 95 to 200. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0195] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 95 to 170. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0196] In one embodiment, the antibody or antigen-binding fragment thereof has a K in the range of 95 to 166. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0197] Improved Fc receptor binding

[0198] In the context of the present invention, to reduce the levels of circulating hANGPTL3, it is desirable to increase the cellular uptake of the antibody-antigen complex.

[0199] In some embodiments, increased cellular uptake of antibody-antigen complexes is promoted by increasing the affinity of the monovalent antibody for the cell surface. The introduction of additional positive charges in the Fc portion of the antibody leads to increased uptake due to increased interactions with negatively charged cell surfaces, thereby generally increasing the cellular uptake of the antibody and antibody-antigen complex.

[0200] In some embodiments, the monovalent antibody is directed against human Fc γThe affinity for human FcγRIIb (CD32) was increased to promote increased cellular uptake of antibody-antigen complexes. The affinity for human FcγRIIb (CD32) was increased at pH 7.4 by amino acid substitutions in the Fc portion of the antibody, while the substitutions did not affect the affinity of the antibody for other Fc receptors on the cell surface.

[0201] In some embodiments, the cellular uptake of the antibody-antigen complex is increased by increasing the affinity of the antibody-antigen immune complex to human FcγRIIb (CD32). The affinity of the antibody-antigen immune complex to human FcγRIIb (CD32) is increased at pH 7.4 by amino acid substitutions in the Fc portion of the antibody, while the substitutions do not affect the affinity for other Fc receptors on the cell surface.

[0202] In one embodiment, the monovalent antibody or antigen-binding fragment thereof enhances 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% relative to no administration of the monovalent antibody or antigen-binding fragment thereof. The circulating level of hANGPTL3 can be measured by in vitro assays known in the art and described herein (see Example 5).

[0203] Inhibitory effect of monovalent antibodies

[0204] The inhibitory activity of the monovalent antibodies or antigen-binding fragments thereof described herein on hANGPTL3-mediated LPL inhibition can be determined by methods such as those described in Example 4 herein.

[0205] In one embodiment, the monovalent antibody or antigen-binding fragment thereof described herein is capable of reducing triglyceride concentration in human plasma.

[0206] In one embodiment, the monovalent antibody or antigen-binding fragment thereof described herein is capable of inhibiting an increase in triglyceride concentration in human plasma.

[0207] Indications

[0208] A disease or condition treatable by administration of a monovalent antibody or antigen-binding fragment thereof disclosed herein is any disease or condition (e.g., a hANGPTL3-mediated disease or condition) that is ameliorated, alleviated, inhibited, or prevented, or whose incidence is reduced, by removing, inhibiting, reducing, or otherwise interfering with hANGPTL3 activity, compared to not receiving anti-hANGPTL3 antibody treatment.

[0209] Examples of treatable diseases or conditions include, but are not limited to, diseases or conditions involving lipid metabolism, such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including atherogenic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), etc., which are caused by, for example, reduced LDL receptor (LDLR) activity and / or LDL receptor deficiency (e.g., homozygous familial hypercholesterolemia with LDLR- / -), ApoC2 changes, ApoE deficiency, ApoB increase, increased production and / or decreased elimination of very low density lipoprotein (VLDL), etc.

[0210] The monovalent antibodies or antigen-binding fragments thereof disclosed herein can also be used to prevent or treat diseases or conditions associated with or caused by hyperlipidemia, hyperlipoproteinemia and / or dyslipidemia, including but not limited to cardiovascular diseases or conditions, such as atherosclerosis, atherosclerotic cardiovascular disease, angina pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, etc.

[0211] Pharmaceutical composition

[0212] In another aspect, the present invention provides a composition comprising a compound of the present invention, such as a monovalent antibody or an antigen-binding fragment thereof as described herein. For example, the present invention provides a pharmaceutical composition comprising one or more monovalent antibodies of the present invention formulated with a pharmaceutically acceptable carrier.

[0213] Accordingly, one aspect of the present invention is to provide a pharmaceutical composition comprising such a monovalent antibody or Fab 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 wherein the pH of the composition is 4.0 to 9.0. The composition may further comprise one or more of a buffer system, a preservative, a tonicity 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 the technician. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0214] In one embodiment, the pharmaceutical composition is an aqueous composition. Such compositions are generally solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous composition" is defined as a composition comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0215] In another embodiment, the pharmaceutical composition is a freeze-dried composition, to which solvents and / or diluents are added prior to use.

[0216] In a further aspect, a pharmaceutical composition comprises an aqueous solution of a monovalent antibody or antigen-binding fragment thereof and a buffer, wherein the antibody is present at a concentration of 1 mg / ml or more, and wherein the pH of the composition is about 5.0 to about 8.0.

[0217] In a further aspect, a pharmaceutical composition comprises an aqueous solution of a monovalent antibody or antigen-binding fragment thereof and a buffer, wherein the antibody is present at a concentration of 1 mg / ml to 150 mg / ml, and wherein the pH of the composition is about 5.0 to about 8.0.

[0218] In one such embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 50 mg / ml or higher. In one embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 60 mg / ml or higher. In one embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 70 mg / ml or higher. In one embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 80 mg / ml or higher. In one embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 90 mg / ml or higher. In one embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 100 mg / ml or higher. In one embodiment, a monovalent antibody or antigen binding fragment thereof can be prepared into a composition, wherein the concentration of the antibody or antigen binding fragment thereof is 110 mg / ml or higher. In one embodiment, the monovalent antibody or its antigen binding fragment can be prepared into a composition, wherein the concentration of the antibody or its antigen binding fragment is 120 mg / ml or higher. In one embodiment, the monovalent antibody or its antigen binding fragment can be prepared into a composition, wherein the concentration of the antibody or its antigen binding fragment is 130 mg / ml or higher. In one embodiment, the monovalent antibody or its antigen binding fragment can be prepared into a composition, wherein the concentration of the antibody or its antigen binding fragment is 140 mg / ml or higher.

[0219] In one embodiment, a monovalent antibody or antigen-binding fragment thereof can be prepared into a composition wherein the concentration of the antibody or antigen-binding fragment thereof is 150 mg / ml or higher.

[0220] In one embodiment, the present invention relates to an injection device having the composition as content. In some embodiments, the pharmaceutical composition of the present invention is intended for use in and / or contained in an injection device. In some embodiments, the injection device is A disposable, pre-filled, multi-dose pen of the type ANTIQLO® (supplier Novo Nordisk A / S, Denmark). In some embodiments, the injection device is a single shot device.

[0221] In some embodiments, the injection device is a fixed dose device, such as a device configured to deliver multiple predetermined doses of a drug, sometimes referred to as a multiple fixed dose device or a fixed dose, multi-shot device.

[0222] Administration and Dosage

[0223] The monovalent antibodies or antigen-binding fragments thereof disclosed herein can be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously. In a preferred embodiment, the antibody or its antigen-binding fragment is administered subcutaneously. The monovalent antibody or its antigen-binding fragment can be administered prophylactically. The monovalent antibody or its antigen-binding fragment can be administered therapeutically (on demand).

[0224] The dosage of the compound to be delivered may be from about 0.01 mg to 500 mg of the compound per day.

[0225] Appropriate dosages may also be adjusted for a particular compound based on the properties of the antibody, including its in vivo plasma half-life or mean residence time and its biological activity.

[0226] Compositions containing monovalent antibodies or antigen-binding fragments thereof disclosed herein can be administered for preventive treatment and / or in some embodiments for therapeutic treatment. In therapeutic applications, pharmaceutical compositions such as those disclosed herein are administered to subjects who have suffered from a disease, such as any disease described herein, in an amount sufficient to cure, alleviate or partially prevent the disease and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount". The effective amount for this purpose will depend on the severity of the disease or injury, as well as the weight and general state of the subject, as understood by those skilled in the art.

[0227] Implementation

[0228] In one embodiment, a monovalent antibody or antigen-binding fragment thereof disclosed herein provides a viscosity in solution that is lower than the viscosity of a corresponding bivalent antibody (comprising the same heavy chain).

[0229] In one embodiment, a monovalent antibody or antigen-binding fragment thereof disclosed herein provides a physical stability in solution that is greater than the physical stability of a corresponding bivalent antibody (comprising the same heavy chain).

[0230] In one embodiment, the monovalent antibody or antigen-binding fragment thereof provides a viscosity and a self-association tendency across a wide pH range that is lower than that of a corresponding bivalent antibody (comprising the same heavy chain).

[0231] In one embodiment, the monovalent antibody is an antibody referred to 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.

[0232] The present invention is further described by the following embodiments:

[0233] 1. A monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1),

[0234] wherein the antibody or antigen-binding fragment thereof has a K of 40 or more, such as 50, 60, 70, 80, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 165, 166, 170, 180, 190, 200 or more. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio, and

[0235] The antibody or antigen-binding fragment thereof is capable of reducing the concentration of hANGPTL3 in human plasma.

[0236] 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody or antigen-binding fragment thereof has a K in the range of 90-105, such as 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105. D (pH6) / K D (pH 7.4) value, which is defined as the K of the antigen at pH 6. D The K of the antigen at pH 7.4 D ratio.

[0237] 3. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises a region capable of interacting with a cell surface Fc receptor.

[0238] 4. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises an Fc region, such as one or two Fc regions.

[0239] 5. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody isotype is IgG, and wherein C H 2 or C H At least one negatively charged or neutral surface exposed amino acid residue in the 3 domain is replaced with a positively charged amino acid residue, thereby increasing the isoelectric point of the antibody or antigen-binding fragment thereof as determined using isoelectric focusing.

[0240] 6. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody isotype is IgG and comprises arginine (R) at position 311 and / or 343 (EU residues) in the heavy chain.

[0241] 7. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody isotype is IgG and comprises arginine (R) at positions 311 and 343 (EU residues) in the heavy chain.

[0242] 8. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody isotype is IgG, and wherein the Fc region is modified to enhance binding to combination.

[0243] 9. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises one or more of the following in the heavy chain:

[0244] Tyrosine (Y) at position 234,

[0245] Aspartic acid (D) at position 238,

[0246] Valine (V) at position 250,

[0247] Isoleucine (I) at position 264,

[0248] Proline (P) at position 307, and / or

[0249] Lysine (K) at position 330 (EU residue).

[0250] 10. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises in the heavy chain

[0251] Tyrosine (Y) at position 234,

[0252] Aspartic acid (D) at position 238,

[0253] Valine (V) at position 250,

[0254] Isoleucine (I) at position 264,

[0255] Proline (P) at position 307, and

[0256] Lysine (K) at position 330 (EU residue).

[0257] 11. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody isotype is IgG, and wherein the Fc region is modified to enhance binding to FcRn.

[0258] 12. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises in the heavy chain

[0259] Tyrosine (Y) at position 252,

[0260] Threonine (T) at position 254, and

[0261] Glutamic acid (E) at position 256 (EU residue).

[0262] 13. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of reducing hANGPTL3-mediated lipoprotein lipase (LPL) inhibition.

[0263] 14. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is an antagonist capable of inhibiting hANGPTL3-mediated lipoprotein lipase (LPL) inhibition.

[0264] 15. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of reducing hANGPTL3 concentration in human plasma.

[0265] 16. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is an antagonist capable of inhibiting hANGPTL3-mediated lipoprotein lipase (LPL) inhibition and capable of reducing hANGPTL3 concentration in human plasma.

[0266] 17. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein

[0267] a) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0268] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 2), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0269] a CDR2 sequence of the amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0270] a CDR3 sequence of the amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues; or

[0271] b) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0272] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 24), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0273] a CDR2 sequence of the amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 25), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0274] A CDR3 sequence of the amino acid residues EGWYDNWNDP (SEQ ID NO: 26), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues;

[0275] And wherein the light chain of the antibody or antigen-binding fragment thereof comprises:

[0276] a CDR1 sequence of the amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0277] a CDR2 sequence of the amino acid residues GVSSRAA (SEQ ID NO: 10), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues, and

[0278] - A CDR3 sequence of the amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues.

[0279] 18. The antibody or antigen-binding fragment thereof of embodiment 17, wherein the substitution is a conservative substitution.

[0280] 19. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0281] a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), and

[0282] a CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), and

[0283] a CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4); and

[0284] The light chain of the antibody or antigen-binding fragment thereof comprises:

[0285] a CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), and

[0286] a CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), and

[0287] - CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11).

[0288] 20. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the heavy chain variable domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, and the light chain variable domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12.

[0289] 21. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein

[0290] a) the heavy chain variable domain comprises SEQ ID NO: 5 and the light chain variable domain comprises SEQ ID NO: 12, or

[0291] b) the heavy chain variable domain comprises SEQ ID NO: 17 and the light chain variable domain comprises SEQ ID NO: 12, or

[0292] c) the heavy chain variable domain comprises SEQ ID NO: 22 and the light chain variable domain comprises SEQ ID NO: 12, or

[0293] d) the heavy chain variable domain comprises SEQ ID NO:27 and the light chain variable domain comprises SEQ ID NO:12.

[0294] 22. A monovalent antibody or antigen-binding fragment thereof capable of binding to hANGPTL3 (SEQ ID NO: 1),

[0295] in

[0296] a) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0297] a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 2), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0298] a CDR2 sequence of the amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0299] a CDR3 sequence of the amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues; or

[0300] b) the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0301] a CDR1 sequence of amino acid residues 31 to 35 (SYWMT) of SEQ ID NO: 24, wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and

[0302] a CDR2 sequence of amino acid residues 50 to 66 (SISSHSTYIYYADSVKG) of SEQ ID NO:25, wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0303] A CDR3 sequence of amino acid residues 99 to 110 (EGWYDNWNDP) of SEQ ID NO: 26, wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues;

[0304] And wherein the light chain of the antibody or antigen-binding fragment thereof comprises:

[0305] a CDR1 sequence of the amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and

[0306] a CDR2 sequence of the amino acid residues GVSSRAA (SEQ ID NO: 10), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues, and

[0307] - A CDR3 sequence of the amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues.

[0308] 23. An antibody or antigen-binding fragment thereof according to embodiment 22, wherein the substitution is a conservative substitution.

[0309] 24. An antibody or antigen-binding fragment thereof according to any one of embodiments 17-23, wherein the antibody or antigen-binding fragment thereof comprises a region capable of interacting with a cell surface Fc receptor.

[0310] 25. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-24, wherein the antibody or antigen-binding fragment thereof comprises an Fc region.

[0311] 26. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-24, wherein the antibody or antigen-binding fragment thereof is capable of reducing hANGPTL3-mediated lipoprotein lipase (LPL) inhibition.

[0312] 27. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-24, wherein the antibody or antigen-binding fragment thereof is an antagonist capable of inhibiting hANGPTL3-mediated lipoprotein lipase (LPL) inhibition.

[0313] 28. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-24, wherein the antibody or antigen-binding fragment thereof is capable of reducing hANGPTL3 concentration in human plasma.

[0314] 29. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-24, wherein the antibody or antigen-binding fragment thereof is an antagonist capable of inhibiting hANGPTL3-mediated lipoprotein lipase (LPL) inhibition and capable of reducing hANGPTL3 concentration in human plasma.

[0315] 30. The monovalent antibody or antigen-binding fragment thereof according to any one of the preceding embodiments,

[0316] The heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0317] a CDR1 sequence of amino acid residues SYWMT (SEQ ID NO: 2), and

[0318] a CDR2 sequence of amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), and

[0319] a CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4); and

[0320] The light chain of the antibody or antigen-binding fragment thereof comprises:

[0321] a CDR1 sequence of amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), and

[0322] a CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), and

[0323] - CDR3 sequence of amino acid residues QQYDDHPYT (SEQ ID NO: 11).

[0324] 31. An antibody or antigen-binding fragment thereof according to any one of embodiments 22-30, wherein the heavy chain variable domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, 17, 22, 27 or 32, and the light chain variable domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12.

[0325] 32. The antibody or antigen-binding fragment thereof of any one of embodiments 22-31, wherein the heavy chain variable domain comprises SEQ ID NO:5 and the light chain variable domain comprises SEQ ID NO:12.

[0326] 33. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the isotype of the antibody is IgG1, IgG2, IgG3 or IgG4.

[0327] 34. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the isotype of the antibody is based on IgG1.

[0328] 35. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the isotype of the antibody is based on IgG1, wherein the glycine and / or lysine (EU residues) at positions 446 and / or 447, respectively, in the heavy chain are deleted.

[0329] 36. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the isotype of the truncated heavy chain of the antibody is based on IgG1, wherein the glycine and lysine (EU residues) at positions 446 and / or 447, respectively, in the heavy chain are deleted.

[0330] 37. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-36, wherein the antibody isotype is IgG, and wherein C H 2 or C H At least one negatively charged or neutral surface exposed amino acid residue in the 3 domain is replaced with a positively charged amino acid residue, thereby increasing the isoelectric point of the antibody or antigen-binding fragment thereof as determined using isoelectric focusing.

[0331] 38. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-37, wherein the antibody or antigen-binding fragment thereof comprises arginine (R) at position 311 and / or 343 (EU residues) in the heavy chain.

[0332] 39. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-38, wherein the antibody or antigen-binding fragment thereof comprises arginine (R) at positions 311 and 343 (EU residues) in the heavy chain.

[0333] 40. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-39, wherein the antibody isotype is IgG, and wherein the Fc region is modified to enhance binding to combination.

[0334] 41. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-40, wherein the antibody or antigen-binding fragment thereof comprises one or more of the following in the heavy chain:

[0335] Tyrosine (Y) at position 234,

[0336] Aspartic acid (D) at position 238,

[0337] Valine (V) at position 250,

[0338] Isoleucine (I) at position 264,

[0339] Proline (P) at position 307, and

[0340] Lysine (K) at position 330 (EU residue).

[0341] 42. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-41, wherein the antibody or antigen-binding fragment thereof comprises in the heavy chain

[0342] Tyrosine (Y) at position 234,

[0343] Aspartic acid (D) at position 238,

[0344] Valine (V) at position 250,

[0345] Isoleucine (I) at position 264,

[0346] Proline (P) at position 307, and

[0347] Lysine (K) at position 330 (EU residue).

[0348] 43. The antibody or antigen-binding fragment thereof of any one of embodiments 17-42, wherein the antibody isotype is IgG, and wherein the Fc region is modified to enhance binding to FcRn.

[0349] 44. The antibody or antigen-binding fragment thereof according to any one of embodiments 17-43, wherein the antibody or antigen-binding fragment thereof comprises in the heavy chain

[0350] Tyrosine (Y) at position 252,

[0351] Threonine (T) at position 254, and

[0352] Glutamic acid (E) at position 256 (EU residue).

[0353] 45. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, comprising a first and a second heavy chain each comprising a multimerization domain, wherein the sequences of the heavy chains are different, and wherein the heavy chains are multimerized through interaction of the multimerization domains.

[0354] 46. ​​The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody comprises a first and a second heavy chain, wherein the first heavy chain comprises

[0355] Tryptophan (W) at position 366, and

[0356] wherein the second heavy chain comprises

[0357] Serine (S) at position 366,

[0358] Alanine (A) at position 368, and

[0359] Valine (V) at position 407 (EU residue).

[0360] 47. An antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody comprises a first and a second heavy chain, wherein the first and the second heavy chain comprise lysine (K) at position 214 (EU residue).

[0361] 48. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody comprises a first and a second heavy chain, wherein the first and the second heavy chain comprise

[0362] Lysine (K) at position 214,

[0363] Arginine (R) at positions 311 and 343,

[0364] Tyrosine (Y) at position 234,

[0365] Aspartic acid (D) at position 238,

[0366] Valine (V) at position 250,

[0367] Isoleucine (I) at position 264,

[0368] Proline (P) at position 307,

[0369] Lysine (K) at position 330,

[0370] Tyrosine (Y) at position 252,

[0371] Threonine (T) at position 254,

[0372] Glutamic acid (E) at position 256, and

[0373] wherein the first heavy chain further comprises

[0374] Tryptophan at position 366, and

[0375] wherein the second heavy chain further comprises

[0376] Serine at position 366,

[0377] Alanine at position 368,

[0378] Valine at position 407, and

[0379] Optionally wherein the glycine and lysine at positions 446 and / or 447, respectively, are deleted in the first and / or second heavy chain (EU residues).

[0380] 49. The antibody according to any of the preceding embodiments, wherein the antibody comprises a first heavy chain according to SEQ ID NO:6 and a second heavy chain according to SEQ ID NO:7 or 8, and a single light chain according to SEQ ID NO:13.

[0381] 50. A monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), comprising a first heavy chain, a second heavy chain and a light chain,

[0382] 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.

[0383] 51. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of pH-dependent binding to hANGPTL3.

[0384] 52. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of reducing the size of an immune complex formed between the antibody or antigen-binding fragment thereof and hANGPTL3.

[0385] 53. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of increasing the homogeneity of the immune complex formed between the antibody or antigen-binding fragment thereof and hANGPTL3.

[0386] 54. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of reducing hANGPTL3 plasma concentration.

[0387] 55. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is capable of reducing triglyceride concentrations in plasma without increasing hANGPTL3 plasma concentrations.

[0388] 56. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is humanized or human.

[0389] 57. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is a one-armed antibody.

[0390] 58. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof has cross-species reactivity.

[0391] 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.

[0392] 60. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, and one or more pharmaceutically acceptable carriers.

[0393] 61. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60, for use in medicine.

[0394] 62. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60, for use in treating or preventing elevated triglyceride concentrations in human plasma.

[0395] 63. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60, for use in inhibiting an increase in triglyceride concentration in human plasma.

[0396] 64. A method for treating a patient having elevated triglyceride concentrations in human plasma, comprising administering to the patient the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60.

[0397] 65. A method for treating a patient having highly elevated triglyceride concentrations in human plasma, comprising administering to the patient the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60.

[0398] 66. A method of reducing triglyceride concentration in human plasma, comprising administering to a patient in need thereof the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60.

[0399] 67. A method for inhibiting an increase in triglyceride concentration in plasma, comprising administering the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60 to a patient in need thereof.

[0400] 68. Use of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60 in the preparation of a medicament for treating a patient with elevated triglycerides.

[0401] 69. Use of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60 in the preparation of a medicament for treating patients with highly elevated triglycerides.

[0402] 70. The use or method of any one of embodiments 61-69 or the composition of embodiment 60, wherein the patient is diagnosed with atherosclerotic cardiovascular disease (ASCVD) and / or type 2 diabetes.

[0403] 71. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60, for use in treating or preventing cardiovascular disease.

[0404] 72. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60, for use in treating or preventing atherosclerotic cardiovascular disease (ASCVD).

[0405] 73. A method of treating or preventing cardiovascular disease in a patient, comprising administering to the patient the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60.

[0406] 74. A method of treating or preventing atherosclerotic cardiovascular disease (ASCVD) in a patient, comprising administering to the patient the antibody or antigen-binding fragment thereof of any one of embodiments 1-59 or the composition of embodiment 60.

[0407] 75. Use of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60 in the preparation of a medicament for treating or preventing cardiovascular disease.

[0408] 76. Use of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60 in the preparation of a medicament for treating or preventing atherosclerotic cardiovascular disease (ASCVD).

[0409] 77. A kit comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1-59 or the composition according to embodiment 60, and instructions for use.

[0410] 78. An antibody or antigen-binding fragment thereof according to any one of embodiments 45-48, wherein the first heavy chain is a truncated heavy chain.

[0411] 79. An antibody or antigen-binding fragment thereof according to any one of embodiments 45-50, wherein the second heavy chain is a truncated heavy chain.

[0412] Example

[0413] Abbreviations

[0414] BSA: bovine serum albumin

[0415] ELISA enzyme-linked immunosorbent assay

[0416] hIgG human immunoglobulin G

[0417] HPC4: 12 amino acid sequence of protein C

[0418] HPLC: High Performance Liquid Chromatography

[0419] LPL: Lipoprotein lipase

[0420] LC (Example 2): light chain

[0421] LC (Example 3): Liquid chromatography

[0422] LLOQ: Lower limit of quantitation

[0423] LOCI: Luminescent Oxygen Channeling Immunoassay

[0424] MRT: Mean retention time

[0425] MS: mass spectrometry

[0426] OD 600 : Optical density at 600nm

[0427] QC: Quality Control

[0428] hANGPTL3: human ANGPTL3

[0429] rhANGPTL3: recombinant human ANGPTL3

[0430] P20: Polysorbate 20

[0431] PBS: Phosphate buffered saline

[0432] PEG: polyethylene glycol

[0433] RT: retention time

[0434] SE-HPLC: Size Exclusion High Performance Liquid Chromatography

[0435] SE-UPLC: Size Exclusion Ultra-Performance Liquid Chromatography

[0436] SIA: sequence identical analogs

[0437] SPR: Surface Plasmon Resonance

[0438] ULOQ: Upper limit of quantitation

[0439] UV-VIS: Ultraviolet-visible spectroscopy

[0440] Example 1: Generation of pH-dependent antibody variants

[0441] Initial human antibody library

[0442] A human Fab library was constructed with a highly stable framework and more than 10 diversity in CDR3 10 :

[0443] Phage panning initial screening :

[0444] Biotinylated hANGPTL3 was coated on M280-streptavidin Dynabeads (Thermo Fisher Scientific #11205D). Phage display libraries (>10 13 pfu / mL) of nonspecific binders, followed by the addition of hANGPTL3-coated beads for binding at pH 7.4. After washing ten times with PBS, 0.05% Tween 20, and three times with PBS, the beads with ANGPLT3-bound phage were suspended in 1 mL PBS and incubated with 9 mL of an actively growing E. coli XL-Blue culture (OD 2.0) in 2×YT medium (2% glucose, 25 ug / ml chloramphenicol, 10 ug / ml tetracycline). 600 =0.6) and incubated at 37°C for 30 minutes. All cells were centrifuged and resuspended in 2×YT medium in a 24 cm x 24 cm plate at 37°C overnight. E. coli cells were infected with M13K07 helper phage to amplify and produce phage. After 24 hours, the E. coli cells were removed and phages were harvested by precipitation with PEG / NaCl solution and then resuspended in PBS buffer for the following rounds of panning.

[0445] Antibody identification :

[0446] After 4 rounds of panning, single colonies were picked for Sanger sequencing and ELISA. The top hits were selected for gene synthesis in the form of complete antibodies. After expression and purification (see Example 2), the complete antibodies were tested for ANGPTL3 binding at pH 7.4 and pH 6 by SPR analysis (see Example 7). Antibody mAb0 was determined to have the most ideal binding to hANGPTL3.

[0447] Construction of libraries for incorporating pH-dependent binding

[0448] To incorporate a pH switch into the variable domain of mAb0 for binding to ANGPTL3, a computational analysis of the homology model was performed. Briefly, all mAb0 residues within 7.5 angstroms of a positively charged residue at 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. Amino acid substitution stereochemistry and intramolecular interactions were evaluated computationally from the homology model. Next, a library was constructed using a degenerate codon with 16 mutation sites, with a diversity of approximately 10 7 .

[0449] Phage panning for identification of pH-dependent variants of mAb0

[0450] Biotinylated hANGPTL3 was coated on M280-streptavidin Dynabeads. The generated phage library (10 12 pfu / mL) was depleted with uncoated streptavidin Dynabeads. The depleted phage library was added to hANGPTL3 (17-460) coated beads for binding at pH 7.4. After washing three times with PBT and three times with PB, bound phages were eluted twice with elution buffer at pH 5.5 followed by incubation for 15 min. The eluted phage particles were then amplified and subjected to additional rounds of screening.

[0451] 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 E. coli NEB 5-alpha F′ (OD 600 =0.8) and incubated at 37°C for 1 hour. 10 pfu of M13K07 helper phage and incubated for 1 hour. The infected E. coli were 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, phages were harvested by precipitation with PEG / NaCl solution and resuspended in PBS buffer for the following rounds of panning.

[0452] Identification of antibodies showing pH-dependent hANGPTL3 binding

[0453] Phage pools from 3 rounds of panning were collected for next generation sequencing. The most enriched Ab sequences were selected for gene synthesis, followed by expression and purification (see Example 2). The prepared antibodies were analyzed by SPR essentially according to Example 7. Full-length His6-tagged hANGPTL3 was captured by anti-His antibody immobilized on an SPR sensor chip, while anti-hANGPTL3 antibodies flowed through 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 method. These antibodies retained inhibitory activity against hANGPTL3.

[0454] Example 2: Recombinant expression of antibodies or fragments thereof

[0455] Essentially following the manufacturer's instructions, transient transfection of HEK293 suspension cells (293Expi, Invitrogen) was used to express antibodies or fragments thereof. 293Expi cells were routinely 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 million / mL using Expifectamine. For each liter of Expi293F cells, a total of 1 mg of plasmid DNA (1:1 ratio of V H -C H 1 (for Fab) or (for mAb HC) and LC plasmids) were diluted into 50 mL Optimem (GIBCO, catalog number 51985-026, diluent A) and 2.7 mL Expifectamine was diluted into 50 mL Optimem (diluent B) for transfection. For co-transfection of Fab and mAb production, V H -C H 1 and LC plasmid (Fab) and HC and LC plasmid (mAb). Diluent A and B are mixed and incubated at room temperature for 10-20 minutes. Afterwards, the transfection mixture is added to Expi293F cells, and the cells are incubated at 37°C in a humid incubator under orbital rotation (85-125rpm). One day after transfection, 5ml ExpiFectamine 293 transfection enhancer 1 and 50ml ExpiFectamine 293 transfection enhancer 2 are supplemented to the transfected cells. Cell culture supernatant is generally harvested by centrifugation and subsequent filtration 4-5 days after transfection.

[0456] Basically as above, but using co-transfection of 3 different plasmids for monovalent antibody expression. The one-armed antibody consists of one heavy chain (HC), one truncated heavy chain (tHC) and one LC, therefore, a 1:1:1 ratio DNA mixture of HC, tHC and LC plasmids was used to set up the transfection.

[0457] The monovalent antibodies referred to as tmAb1, tmAb2, tmAb3 and tmAb4 comprise HC, tHC and LC based on human IgG1kappa isotype. The constant regions of tmAb1-4 HC and tHC are engineered to comprise the following substitutions compared to wild-type IgG1: L234Y, P238D, T250V, M252Y, S254T, T256E, V264I, T307P, Q311R, A330K and P343R (EU residues). In order to ensure the correct HC and tHC heterodimerization to effectively produce tmAb1-4, a knob-and-hole technique (U.S. Patent Application 20030078385) was used, i.e., T366W mutation (knob) was inserted into HC, and T366S, L368A, Y407V mutation (hole) were inserted into tHC, and vice versa.

[0458] Example 3: Purification and characterization of antibodies or fragments thereof

[0459] All purification steps were performed at 4°C. For laboratory scale, Milli-Q water was used for buffer preparation. The HPLC system used for SE-HPLC analysis was Agilent 1100. Aggregation and LC / MS were evaluated for QC.

[0460] The Fab was captured by HiTrap KappaSelect (Cytiva) affinity chromatography using 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 with 0.1 M glycine (pH 2.8). The eluted Fab was further purified by size exclusion chromatography on a 320 mL Superdex 200 26 / 60 column (Cytiva) and buffer exchanged to a formulation buffer (20 mM HEPES, 150 mM NaCl) at pH 7.4 and concentrated by a centrifugal ultrafilter (30 KD CO) for storage at -80°C.

[0461] To evaluate the quality of the purified Fab, SDS-PAGE and high performance size exclusion chromatography (SE-HPLC) analysis were performed. LC / MS was performed 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 chain and light chain, respectively.

[0462] Antibody purification and characterization

[0463] 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-scale production, Chromatographic system (Cytiva) wherein an equilibrium buffer consisting of 20 mM sodium phosphate pH 7.2, 150 mM NaCl and an elution buffer consisting of 10 mM formic acid pH 3.5 are employed. Cell supernatant is applied directly to a pre-equilibrated MabSelect SuRe column without any adjustment. The column is washed with approximately 5 column volumes of equilibrium buffer, and the antibody is isocratically eluted with approximately 2-5 column volumes of elution buffer. The eluted antibody is applied directly 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.

[0464] Different methods, such as SDS-PAGE / Coomassie, size exclusion high performance liquid chromatography (SE-HPLC) and liquid chromatography mass spectrometry (LC-MS) analysis, characterize the purified antibodies. NuPage 4-12% Bis-Tris gel (Invitrogen, catalog number NP0321BOX) is used to perform SDS-PAGE / Coomassie analysis. Here, all antibodies show expected light chain and heavy chain components. Liquid chromatography electrospray ionization time-of-flight mass spectrometry is used to set up complete molecular weight determination on an Agilent 6210 instrument equipped with a desalting column MassPREP (Waters, catalog number USRM10008656). The buffer system used is an equilibrium buffer composed of 0.1% formic acid in LC-MS grade-H2O and an elution buffer composed of 0.1% formic acid in LC-MS grade acetonitrile (ACN). The analysis was performed with and without N-Glycosidase F (Roche Diagnostics, catalog number 11365177001) and reducing agents (i.e., mercaptoethanol or DTT). All antibodies showed the expected intact molecular weight consistent with the sequence and one heavy chain N-glycan. Purity was determined based on SE-HPLC. Final protein purity was analyzed based on the 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 consisting of 200 mM sodium phosphate pH 6.9, 300 mM NaCl, and 10% isopropanol. UV280 and fluorescence (excitation 280 nm / emission 354 nm) detectors were used for detection. The antibodies eluted as a single symmetrical peak, and the retention time reflected the size of the antibody. The purity estimates for the different antibodies were all between 95% and 99%. To determine the final protein concentration, a NanoDrop spectrophotometer (Thermo Scientific) and the specific extinction coefficient for each antibody were used.

[0465] Example 4: Assay for Determining Inhibition of rhANGPTL3-Mediated LPL Inhibition

[0466] Inhibition of hANGPTL3-mediated LPL inhibition was measured in a recombinant hANGPTL3-LPL assay. In this assay, EnzChek TM The in vitro enzymatic activity of lipoprotein lipase (LPL) was measured using lipase as a substrate. The activity of bovine LPL was inhibited by adding recombinant hANGPTL3 (rhANGPTL3). This inhibition could then be blocked by rhANGPTL3 inhibitory compounds such as the anti-hANGPTL3 antibodies disclosed herein. The apparent affinity of the anti-hANGPTL3 antibodies relative to the ivizumab SIA was measured.

[0467] rhANGPTL3-LPL enzyme activity procedure

[0468] In addition to EnzChek made with ultrapure water TM Except for the Zwittergent reagent, all solutions were prepared with assay buffer (DPBS (1×) (Gibco 14190-144) + 1% fatty acid-free BSA (MP Biomedicals 05033)).

[0469] A dilution series of anti-rhANGPTL3 antibody was prepared as follows. The starting concentration was 3 μM and 3-fold dilutions were performed in assay buffer. The dilution series was repeated 7 times. The mix of compounds used for the analysis is described below.

[0470] The assay buffer and 10 μL of each dilution were mixed with 10 μL 1.2 μM rhANGPTL3 (SEQ ID NO: 1) and 10 μL 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 1.8 μM human ApoCII (chemically synthesized residues 59-79) and 10 μL 60 nM bovine LPL (Sigma L2254) were added to each well and incubated for another 15 minutes at room temperature. Finally, 10 μL of EnzChek TM / Zwittergent substrate (4.8μM EnzChek TM Lipase (Thermo Fisher Scientific E33955), 0.03% Zwittergent 3-18 (Merck 41570), and fluorescence was measured kinetically (once every minute for 20 minutes) in an Envision multimode plate reader (PerkinElmer) using an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0471] In addition to the diluted compounds, the plate also contained 6 wells in which the compound was replaced by 10 μL of assay buffer, and 6 wells in which both the compound and rhANGPTL3 were replaced by 10 μL of assay buffer. These wells were used to establish fully inhibited and non-inhibited levels of LPL activity, respectively. In addition, the plate contained a well for each compound in which the second highest compound concentration was used but without rhANGPTL3 to assess the effect of the anti-ANGPTL3 antibody directly on LPL activity in the absence of rhANGPTL3.

[0472] Each plate contained a dilution series of the ivizumab SIA in duplicate.

[0473] Statistical analysis

[0474] LPL activity in each well of the assay plate was calculated by performing a set of linear regressions from the kinetic data over a window of 5 consecutive time points, selecting the fit with the highest slope (Vmax) and an assay correlation above 0.95, and calculating the slope of the selected line. The Vmax values ​​were then normalized using the average of the Vmax from the six fully inhibited LPL activity control wells as the value 0 and the average of the Vmax from the six non-inhibited LPL activity control wells as the value 1. The apparent affinity of the tested compounds for rhANGPTL3 was calculated by performing a nonlinear 4-parameter logistic (4PL) curve fit of the normalized Vmax-concentration data from the dilution series of the compounds. The fit 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 six fully inhibited LPL activity level control wells) was used as the common lower asymptote in the fit. The apparent affinity of the compound was extracted from the 4PL fit as the "e" parameter, named IC 50 and use the on-board evoximab SIA IC 50 The relative apparent affinity was calculated using the average of the values ​​of as the denominator. In addition, for each compound, it was determined that the normalized Vmax of the control wells assessing 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 non-inhibited LPL activity control wells).

[0475] result

[0476] Table 1 shows the average relative IC of rhANGPTL3-mediated LPL inhibition of the five compounds 50 and standard deviation. For completeness, the number of replicates that formed the basis for the mean is also included. For the ivizumab SIA, by definition, the mean relative IC 50 =1.0, but the standard deviation of 58 replicates was calculated from individual results. Since only one analysis was performed for tmAb4, the standard deviation could not be calculated. The monovalent antibodies showed similar or slightly reduced activity against rhANGPTL3 relative to the ivizumab SIA, which is consistent with the relative IC 50 This is demonstrated by the ratio being greater than 1 when the values ​​are compared.

[0477] Table 1: Average relative IC 50

[0478]

[0479] Example 5: In vivo evaluation of anti-ANGPTL3 antibodies in normal mice

[0480] As described below, studies measuring the effects of ANGPTL3 inhibition on plasma triglyceride levels and levels of endogenous ANGPLT3 (250-300 μg / ml at T=0 h) were performed in healthy male C57bl6 / 6J mice from Janvier Labs, France, using evizumab SIA and monovalent anti-ANGPTL3 antibodies termed tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4.

[0481] The tested monovalent antibodies differ from tmAb1-4 in that positions 234, 238, 250, 252, 254, 256, 264, 307, 311, 330, 343 in the Fc region are not modified, ie comprise wild-type human IgG1 residues at said positions.

[0482] Since Fc substitutions lack functionality in mice, wild-type human IgG1 residues (particularly 234L, 238P, 250T, 252M, 254S, 256T, 264V, 307T, 311Q, 330A, 343P) were used at the indicated positions. The effects of the Fc substitutions introduced into tmAb1-4 are specific for the human receptor (see Example 2).

[0483] Blood sampling

[0484] Blood samples were collected from 12 mice in each group at 0 h, 48 h, 168 h, 240 h, and 336 h. Blood samples (tongue blood) (120 μL) were collected by sublingual venipuncture and transferred to EDTA-coated tubes ( VetMed200K3E, Sarstedt nr 09.1293.100). Blood was centrifuged at 6000G, 4°C for 5 minutes within 20 minutes. Plasma samples were transferred to micronic tubes, 25 μl for triglyceride measurement and 25 μl for mANGPTL3 and hIgG measurement (antibody plasma exposure measurement).

[0485] Triglycerides were measured using a COBAS 6000 multianalyzer following the manufacturer's recommendations (Roche Diagnostics).

[0486] Mouse ANGPTL3 in mouse plasma samples was analyzed using an ELISA from R&D Systems (Mouse Angiopoietin-Like 3 ELISA Kit - QuantikineELISA, Catalog No. MANL30) following the kit protocol. To avoid matrix effects, samples were diluted at least 250-fold in the calibrator diluent from the kit. The assay range spanned 62.5–4000 pg / ml. Samples that measured above the ULOQ were further diluted in the calibrator diluent. The LLOQ was 15.625 pg / ml (250 x 62.5 pg / ml).

[0487] Analysis of mouse ANGPTL3 in mouse plasma

[0488] Mouse ANGPTL3 in mouse plasma samples was analyzed using an ELISA from R&D Systems (Mouse Angiopoietin-Like 3 ELISA Kit - QuantikineELISA, Catalog No. MANL30) following the kit protocol. To avoid matrix effects, samples were diluted at least 250-fold in the calibrator diluent from the kit. The assay range spanned 62.5–4000 pg / ml. Samples measured above the ULOQ were further diluted in the calibrator diluent. The LLOQ was 15.625 pg / ml (250 x 62.5 pg / ml).

[0489] Principle of the LOCI assay for determination of anti-ANGPTL3 antibody concentrations

[0490] Anti-ANGPTL3 antibodies (hIgG) in plasma samples were analyzed using a luminescent oxygen channeling immunoassay (LOCI). Donor beads were coated with streptavidin, while acceptor beads were conjugated with a first monoclonal mouse anti-human IgG Fc antibody specific for the Fc region of the anti-ANGPTL3 antibody (Southern Biotech, catalog number 9040-01). The other antibody used was a polyclonal antibody from goat specific for the human IgG Fc region (Jackson ImmunoResearch, code: 109-005-098). The polyclonal antibody was biotinylated in-house. The three reactants (donor beads with streptavidin, acceptor beads conjugated with the target-specific antibody, and another biotinylated target-specific antibody) were combined with the anti-ANGPTL3 antibody (hIgG) and formed a two-site immune complex. Illumination of the complex released singlet oxygen atoms from the donor beads. They were directed to the acceptor beads and triggered chemiluminescence, which was measured by the EnVision plate reader. The amount of light is proportional to the concentration of anti-ANGPTL3 antibody (tmAb0.1).

[0491] Analysis of hIgG in mouse plasma

[0492] 1 μL of plasma sample / calibrator / control was applied to the wells of a 384-well LOCI plate, followed by 15 μL of a mixture of acceptor beads coated with mAb anti-hIgG and biotinylated pAb anti-hIgG (0.5 μg / well). The plate was incubated for 1 h at room temperature (RT). Then 30 μL of streptavidin-coated donor beads (2 μg / well) were added to each well and incubated for 30 min at RT. The plate was read with 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 for each well was 210 ms, including 70 ms of excitation time. The calibrator material was an anti-ANGPTL3 antibody diluted in mouse plasma (untreated K2EDTA plasma, 0.22 μm filtered from Bioreclamation). Since the calibrator varied between different antibodies, the lower limit of quantification was estimated between different runs (usually between 100-500 pM).

[0493] The assay range spanned 41–30.000 pM. Mouse samples were tested undiluted and diluted 20-fold in mouse plasma. Samples that measured above the ULOQ were further diluted in mouse plasma.

[0494] In vivo study 1:

[0495] Group 1: 11 mice were given vehicle (iv)

[0496] Group 2: 11 mice were given ivizumab SIA 40nmol / kg (intravenous)

[0497] Group 3: 11 mice were given tmAb 0.1 40nmol / kg (intravenously)

[0498] Group 4: 11 mice were given tmAb 0.2 40nmol / kg (intravenously)

[0499] Group 5: 11 mice were given tmAb0.3 40nmol / kg (intravenously)

[0500] Group 6: 11 mice were given tmAb 0.440 nmol / kg (intravenously)

[0501] The vehicle was 20 mM HEPES; 150 mM sodium chloride, pH = 7.4; the injection volume was 5 ml / kg, and the dosing solution concentration was 8 nmol / ml.

[0502] result

[0503] TG levels

[0504] All antibodies reduced triglyceride (TG) levels by 50%-60% after 48 hours, with ivsumab SIA and tmAb0.1 showing the highest TG-lowering ability. ivsumab SIA, tmAb0.1, and tmAb0.3 were still able to reduce TG by 58%-67% after 168h, while the efficacy of tmAb0.2 and tmAb0.4 was less obvious (31%-38%). After 336 hours, only ivsumab SIA and tmAb0.1 were observed to significantly reduce TG levels compared with the vehicle group, both of which were 40%-45% lower than baseline. See Figure 3 .

[0505] ANGPTL3 levels

[0506] After 48 hours, ivizumab SIA increased plasma total ANGPTL3 levels by approximately 10-fold from baseline, and then slowly decreased toward baseline. In contrast, tmAb0.1, tmAb0.2, and tmAb0.3 all reduced plasma total ANGPTL3 levels to or below baseline throughout the study. Figure 4A and Figure 4B .

[0507] Antibody levels

[0508] All antibodies were present in plasma in the nanomolar range, with ivizumab SIA and tmAb0.1 showing the highest exposure levels, which remained above 200 nM throughout the study. Figure 5 .

[0509] in conclusion

[0510] From this study, tmAb0.1 appears to be on par with ivizumab SIA in terms of efficacy in lowering TG.

[0511] The results are impressive given that tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4 are monovalent antibodies (i.e., each antibody has only one antagonist ANGPTL3 binding site). In addition, tmAb0.1, tmAb0.2, tmAb0.3, and tmAb0.4 had lower exposure compared to ivizumab SIA ( Figure 5 ), indicating that despite the lower plasma concentrations of the monovalent antibodies, they were able to reduce plasma triglyceride levels to levels similar to those achieved with ivizumab SIA.

[0512] In addition, tmAb0.1, tmAb0.2, tmAb0.3 and tmAb0.4 also showed the ability to remove target ANGPTL3 in plasma. In contrast, ivizumab SIA administration resulted in the accumulation of total ANGPTL3 in plasma. By combining the Fc replacement described herein with the Fabs of tmAb0.1-0.4 (generating tmAb1-4), the inventors expect that the level of circulating hANGPTL3 will be significantly reduced in the human setting.

[0513] Example 6: Determination of ANGPTL3 Antibody Complex by SE-UPLC Chromatography

[0514] The immune complex was analyzed by SE-UPLC on a Waters ACQUITY UPLC system. The complex was formed by mixing the antibody at a concentration range of 0.2-2.0 mg / ml with rhANGTPL3 at a molar ratio of 1:3. The antibody-antigen mixture was equilibrated for one hour and then eluted on an ACQUITY UPLC Protein BEH SEC column. Analyses were performed on 2.5 μm. Each sample was injected twice, using an injection volume of 20 uL, a flow rate of 0.3 mL / min, and a running buffer consisting of 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 refrigerated at 4°C, while the column temperature was kept constant at 22°C + / - 2°C. The elution profile was monitored by UV-VIS absorbance at 276 nm.

[0515] Quantification of immune complex size and pH-dependent target binding

[0516] The size of the complex was assessed by its retention time and the mean retention time (MRT), i.e. the mass midpoint of the chromatographic trace. On the other hand, the pH responsiveness of target binding was assessed by subtracting the mean retention time at pH 6.0 from the mean retention time at pH 7.4.

[0517] result

[0518] The results of SE-UPLC analysis were Figure 6A-6E and are shown in Table 2 below. Obviously, Ivsuomab SIA and tmAb1, tmAb2, tmAb3 and tmAb4 formed immune complexes with the target antigen rhANGTPL3. However, compared with tmAb1-4, Ivsuomab SIA formed larger and more polydisperse immune complexes (see Figure 6A-6E ).

[0519] In addition, as can be seen by comparing the MRT shifts in Table 2, the formation of tmAb1 immune complexes is particularly sensitive to changes in pH. Compared with the complex formed by Ivesu monoclonal antibody SIA, the complex shows lower heterogeneity (more uniform) using tmAb1. Well-defined antibody / antigen complexes are essential for increasing target clearance and antibody circulation by cellular uptake of the complex. Having more than one binding site for Fc receptors on the cell surface can increase affinity, thereby increasing the uptake of the complex. In addition, the size and complexity of the complex are related to the immune response, and it is believed that smaller antibody / antigen complexes lead to less obvious immune responses. Almost complete dissociation was observed at pH 6.0. The higher the degree of dissociation at pH 6.0, the better the clearance potential of the antibody is believed to be. Being able to completely release the antigen when the antibody-antigen complex is absorbed by the cell is essential for the clearance behavior of the antibody. For antibodies that can release antigens and recirculate into the circulation, continue to function / antigen binding (Igawa et al. (2016) Immun. Rev. 270: 132-151).

[0520] Table 2: Retention time

[0521]

[0522] Retention time (RT) and mean retention time (MRT) of immune complexes eluted at pH 6.0 and pH 7.4. The rightmost column lists the change in mean retention time from pH 7.4 to pH 6.0.

[0523] Example 7: Characterization of the interaction kinetics between monovalent antibodies and ANGPTL3 protein

[0524] Binding studies were performed on a Biacore 8K (GE Healthcare), an instrument that measures molecular interactions in real time by surface plasmon resonance. Experiments were run at 37°C, while samples were stored at 15°C in the sample chamber. The signal (RU, response unit) reported by Biacore is directly related to the mass on the surface of each sensor chip in four serial flow cells. Anti-HPC4 monoclonal antibodies were immobilized on two flow cells in the active channel of the C1 sensor chip according to the manufacturer's instructions. Purified trimeric target protein, human ANGPTL3 (SEQ ID NO: 1 residues 17-220 with a C-terminal HPC4 tag), was captured as follows: the protein was diluted into running buffer (20 mM tris-HCl, 150 mM NaCl, 1 mM CaCl2, 0.05% v / v P20, 0.1% BSA pH 7.4, or 20 mM histidine, 150 mM NaCl, 1 mM CaCl2, 0.05% v / v P20, 0.1% BSA, pH 6.0) and injected on flow cell 2, creating a reference surface in flow cell 1 where only anti-HPC4 antibody was immobilized. Binding of monovalent antibodies to captured targets was performed by injecting analyte (monovalent antibody) on both flow cells to allow comparative analysis of binding of different monovalent antibodies to captured targets relative to binding to the reference surface. The monovalent antibody was serially diluted into running buffer, injected at 30 μl / min for 240 s, and then allowed to dissociate for 300 s. After each injection cycle of analyte, the C1 surface was regenerated by injecting 20 mM Tris, 150 mM NaCl, 50 mM EDTA, 0.05% P20, pH 7.4. This regeneration step removes the captured target and any bound monovalent antibody from the immobilized capture antibody surface and allows the subsequent binding of the next interacting sample pair. This regeneration procedure does not remove the directly immobilized anti-HPC4 capture antibody from the chip surface.

[0525] The equilibrium dissociation constant (K) was determined by measuring the kinetics of complex formation and dissociation. D ), thereby quantifying the binding affinity between the monovalent antibody and the target. Using the Biacore evaluation software for data analysis, the rate constants corresponding to the association and dissociation of the monovalent complex, such as k, were retrieved by globally fitting the data to a 1:1 Langmuir model with a local Rmax. a (association rate) and k d (dissociation rate). D =k d / k a , K D With k a and k d Associated.

[0526] Binding curves were processed by double referencing (subtraction of reference surface signal as well as blank buffer injection on captured bispecific protein prior to data analysis). This allowed correction for instrument noise, volume shifts and drift during sample injection.

[0527] Table 3 shows the binding constants K for the interaction between human ANGPTL3 and tmAb1-4 D The measurement result of (equilibrium dissociation constant). K D The calculated ratio is expressed as the value at pH 6.0 / the value at pH 7.4.

[0528] When the HC G26D mutation was introduced into tmAb1 (generating tmAb2), the slower association rate drove the K D A 2-fold increase. The weaker binding at pH 7.4 correlates with the weaker binding at pH 6.0. All listed variants have characteristics that are either too low to measure or have low affinity (μM range) at pH 6.0.

[0529] Table 3: K D Values ​​and ratios

[0530]

[0531] nd the affinity could not be determined by SPR assay.

[0532] Example 8: Monovalent Form Reduces Self-Association

[0533] It is important that antibodies have a low tendency to self-associate over a wide pH range to ensure successful bioprocessing steps and formulation development.Herein, the self-association of tmAb1 and its bivalent form, referred to as "mAb1," was evaluated to investigate the impact of changing the antibody format from bivalent to monovalent.

[0534] Materials and Methods

[0535] Initial protein stock solutions of tmAb1 and mAb1 were prepared in 10 mM L-histidine, 10 mM NaCl (pH 6.0) at a concentration of 40 mg / ml. The protein stock solution was mixed with the pH screening stock solution at a 1:1 ratio (10 μl protein stock solution: 10 μl pH screening stock solution) to achieve final pH values ​​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 screening stock solution consisted of 70 mM succinic acid, 70 mM L-histidine and was adjusted to the target pH. The protein stock solution and the pH screening stock solution were mixed in a 384-well plate (corning 3540), sealed with clear plastic film, and incubated overnight at room temperature in the dark. Afterwards, the plate was centrifuged at 1400 rpm (equivalent to 169 x g) for 5 min and then analyzed using a dynamic light scattering (DLS) plate reader (Wyatt DynaPro) with a collection time of 5 s and 40 acquisitions per sample. The hydrodynamic radius (R h ) and plotted it as a function of pH (see also Dingfelder et al. (2022) In: Houen G. (ed.) Therapeutic Antibodies. Methods in Molecular Biology, vol 2313. Humana, New York, NY).

[0536] Results and Discussion

[0537] The hydrodynamic radius (R) of mAb1 h ) suggests that it has extremely high self-association, which may affect the further development of this molecule. In particular, in the pH range of pH 6.4 to 8.4, R h By changing the molecular form from divalent to monovalent, a sharp decrease in the self-association tendency was observed, with only a slight increase at pH 6.4 to 8.4. The slight increase in self-association of tmAb1 is considered low and acceptable. See Tables 4 and Figure 7 The data support that highly concentrated compositions (formulations) suitable for subcutaneous administration can be prepared.

[0538] Table 4: Hydrodynamic radius (R) of tmAb1 and mAb at different pH values h )

[0539] pH <![CDATA[tmAb1R h (nm)]]> <![CDATA[mAb1R h (nm)]]> 3.3 5.6 5.5 3.7 3.9 4.2 4.2 4.4 4.8 5.2 5.9 6.9 5.6 6.0 7.8 6.0 6.4 8.3 6.4 6.9 10.1 6.9 7.8 12.5 7.2 7.9 15.4 7.5 8.2 17.8 8.1 8.7 13.9 8.4 7.9 15.6

[0540] Example 9: Monovalent Form Reduces Gel Formation

[0541] High physical stability at high protein concentrations (50-175 mg / ml) is often critical for successful development of antibodies as drugs. This is required for many steps in the development process, including the final steps of the purification process, the formulation process, and is required for subcutaneous administration. Here we evaluated physical stability at low and high ionic strengths, physiological pH, and high protein concentrations.

[0542] Materials and Methods

[0543] tmAb1 and its bivalent form referred to as "mAb1" were buffer exchanged to high ionic strength formulations (20mM Hepes, 150mM NaCl, pH 7.4) and low ionic strength formulations (20mM Hepes, pH 7.4). The samples were then concentrated at room temperature in an Amicon 30kDa centrifugal spin filter with a target of 70mg / ml. Protein concentration was determined by absorbance at 280nm.

[0544] Results and Discussion

[0545] tmAb1 was easily concentrated to 70 mg / ml, but for mAb1 (divalent) it was not possible to concentrate above 30 mg / ml in the high ionic strength formulation. For mAb1, gel formation in the high ionic strength formulation resulted in phase separation as visually observed, preventing concentrations from reaching formulation-relevant concentrations. Figure 8 .

[0546] The data support that highly concentrated formulations suitable for subcutaneous administration can be prepared, in which the concentration of monovalent antibody tmAb1 or antigen-binding fragment thereof can be 70 mg / ml or higher.

[0547] Example 10: Monovalent Form Reduces Viscosity

[0548] Low solution viscosity of antibody solutions is crucial for the final production steps, formulation and subcutaneous administration.Here we evaluated the effect of changing from a bivalent format (mAb1) to a monovalent format (tmAb1) on solution viscosity.

[0549] Materials and Methods

[0550] tmAbl and mAbl were buffer exchanged to high ionic strength formulation (20 mM Hepes, 150 mM NaCl, pH 7.4) and low ionic strength formulation (20 mM Hepes, pH 7.4) Samples were then concentrated in Amicon 30 kDa centrifugal spin filters at room temperature with a target of 70 mg / ml.

[0551] Viscosity was measured using a Rheosense Initium instrument using 50 μl samples at 5° C. No shear effects were observed when testing at 1.000-10.000 1 / s shear, suggesting a Newtonian solution, so a single shear rate of 5.000 1 / s was used for comparison.

[0552] Results and Discussion

[0553] In the low ionic strength formulation, mAbl had a high viscosity of 10.2 mPa*s, while tmAbl had a significantly lower viscosity of 6.6 mPa*s (see Table 5). In the high ionic strength formulation, tmAbl had a similar viscosity of 5.8 mPa*s, suggesting that the ionic strength of the formulation had a limited effect. However, for mAbl, it was not possible to concentrate above 30 mg / ml in the high ionic strength formulation because gel formation resulted in phase separation as visually observed.

[0554] The results demonstrate how the poor properties of highly concentrated mAb1 solutions can be rescued by changing the bivalent antibody format to a monovalent antibody format. mAb1 was considered undruggable, whereas tmAb1 has good and acceptable biophysical properties, supporting the preparation of highly concentrated formulations suitable for subcutaneous administration.

[0555] Table 5: Viscosity of tmAb1 and mAb at 5 °C, using a shear force of 5000 1 / s

[0556]

[0557] Example 11: In vivo evaluation of tmAb1 in healthy cynomolgus monkeys

[0558] The aim of this study was to determine the pharmacokinetics and effects on triglycerides of the anti-ANGPTL3 monoclonal antibody tmAb1. tmAb1 was administered as a single intravenous bolus to normolipidemic cynomolgus monkeys at three different dose levels (0.3, 3, and 30 mg / kg). The injection volume was 0.3 mL / kg. All animals were naive to therapeutic antibodies and other protein-based therapeutics.

[0559] Blood sampling and sample analysis:

[0560] Blood samples (0.5 mL) were collected from all animals at the following time points: before administration, 10 minutes, 30 minutes, 1, 2, 4, 8, 14, 24, 48, 72, 96, 120, 144, 168, 192 and 240 hours (h) after administration. Blood was collected from the femoral vein and collected in Teklab K3EDTA tubes (product number 3K200PP), containing 1.75 mg of EDTA / mL blood. Samples were fully mixed by inversion, and immediately placed on wet ice for up to 30 minutes, then centrifuged. Samples were centrifuged at 2-8 ° C for 10 minutes with approximately 2000 g, and plasma aliquots were transferred to Micronic tubes (catalog number MP32022).

[0561] Samples were analyzed to quantify tmAbl and triglyceride concentrations.

[0562] Triglycerides

[0563] Triglycerides were measured using a COBAS 6000 multianalyzer following the manufacturer's recommendations (Roche Diagnostics).

[0564] Human IgG measurement (tmAb1), assay principle :

[0565] Anti-ANGPTL3 hIgG (tmAb1) in plasma samples was analyzed using a luminescent oxygen channeling immunoassay (LOCI). Donor beads were coated with streptavidin, while acceptor beads were conjugated with an in-house monoclonal mouse anti-human IgG that does not cross-bind to cynomolgus monkey IgG. Biotinylated recombinant antibody fragments (Nanobody / V H H) from ThermoFisher (CaptureSelect, catalog number: 7103322500). The three reactants (donor beads with streptavidin, acceptor beads conjugated to target-specific antibody, and biotinylated target-specific antibody fragment) are combined with anti-ANGPTL3 antibody (hIgG) to form a two-site immune complex. Illumination of the complex releases singlet oxygen atoms from the donor beads. They are directed to the acceptor beads and trigger chemiluminescence, which is measured by the EnVision plate reader (PerkinElmer) according to the manufacturer's instructions. The amount of light is proportional to the concentration of anti-ANGPTL3 antibody (tmAb1).

[0566] Human IgG measurement (tmAb1), assay procedure :

[0567] 2 μL plasma samples / calibrators / controls were applied to the wells of a 384-well LOCI plate, followed by 15 μL of a mixture of mAb anti-hIgG and biotinylated antibody fragment-coated acceptor beads (0.5 μg / well). The plate was incubated for 1 h at room temperature (RT). 30 μL of streptavidin-coated donor beads (2 μg / well) were then added to each well and incubated for 30 min at room temperature. After excitation with a 680 nm laser, a filter with a bandwidth of 520-645 nm was used to read the plate with an Envision plate reader at 21-22 ° C. The total measurement time for each well was 210 ms, including an excitation time of 70 ms. The calibrator material was tmAb1 diluted in PBS + 1% cynomolgus monkey plasma. The assay range spanned 11-20.000 pM. The cynomolgus monkey samples were tested after being diluted 100 times in PBS plasma. Samples measuring above the upper limit of quantitation (ULOQ) were further diluted in PBS + 1% cynomolgus monkey plasma.

[0568] result

[0569] PK

[0570] The pharmacokinetics (PK) of tmAb1 Fig. 9 PK was linear at all dose levels through 240 h, with evidence of broad nonlinearity between all dose levels.

[0571] Half-lives calculated using non-compartmental analysis and the "linear up log down" method were in the range of 90.9-171 for all animals, with geometric means of 119, 107 and 107 hours for the 0.3, 3 and 30 mg / kg groups, respectively, based on 240 h of observation.

[0572] TG

[0573] The effects on TG were evaluated during 0-240 h. Fig.10 ). 3mg / kg and 30mg / kg resulted in a rapid decrease in TG levels, with a decrease of approximately 40-50% after 4 hours. The maximum decrease observed during the study ranged from 56% to 78%. In general, the effects on TG lasted for at least 240 hours. At the 0.3mg / kg dose level, a transient effect on TG levels of 28%-44% was observed, with TG levels returning to baseline levels after 4 hours and 12 hours.

[0574] Example 12: Generation of tmAb2, tmAb3, and tmAb4

[0575] A CDR single-site mutagenesis library of the variable domain sequence of tmAb1 was generated to explore the possibility of further modulating pH-dependent target binding (see Examples 6 and 7) and LPL activity (Example 4). The best performing molecules were evaluated as tmAbs, resulting in the selection of tmAb2, tmAb3, and tmAb4, considering 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).

Claims

1. A monovalent antibody or antigen-binding fragment thereof capable of binding to human ANGPTL3 (hANGPTL3) (SEQ ID NO: 1), in a) the heavy chain of the antibody or antigen-binding fragment thereof comprises: a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 2), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and a CDR2 sequence of the amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 3), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and a CDR3 sequence of amino acid residues EGWYDNWFDP (SEQ ID NO: 4), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues; or b) the heavy chain of the antibody or antigen-binding fragment thereof comprises: a CDR1 sequence of the amino acid residues SYWMT (SEQ ID NO: 24), wherein zero or one of these amino acid residues may be replaced by a different amino acid residue, and a CDR2 sequence of the amino acid residues SISSHSTYIYYADSVKG (SEQ ID NO: 25), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and A CDR3 sequence of the amino acid residues EGWYDNWNDP (SEQ ID NO: 26), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues; and The light chain of the antibody or antigen-binding fragment thereof comprises: a CDR1 sequence of the amino acid residues RASQNIRSPYLA (SEQ ID NO: 9), wherein zero, one, two or three of these amino acid residues may be replaced by different amino acid residues, and The CDR2 sequence of amino acid residues GVSSRAA (SEQ ID NO: 10), wherein None, one or two of these amino acid residues may be replaced by different amino acid residues, and - A CDR3 sequence of the amino acid residues QQYDDHPYT (SEQ ID NO: 11), wherein none, one or two of these amino acid residues may be replaced by different amino acid residues.

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

3. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the heavy chain variable domain is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and the light chain variable domain is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein 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.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises in the heavy chain Tyrosine (Y) at position 252, Threonine (T) at position 254, and Glutamic acid (E) at position 256 (EU residue).

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, 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 / or Lysine (K) at position 330 (EU residue).

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

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

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

11. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, a truncated heavy chain, and a light chain, wherein the heavy chain comprises SEQ ID NO: 6, the truncated 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 truncated heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO:6, the truncated 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 the preceding claims, 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 treating or preventing elevated 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 treating or preventing cardiovascular diseases, such as atherosclerotic cardiovascular disease (ASCVD).

17. The method or use according to any one of claims 14-16, wherein the antibody or antigen-binding fragment thereof or the composition 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 instructions for use.

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