Low viscosity antigen-binding proteins and methods for producing them

Abstract

This invention provides low-viscosity antigen-binding proteins and methods for producing them. [Solution] The present invention relates to a method for reducing the viscosity of an antigen-binding protein by modifying the sequence in the framework region and / or Fc domain, which has been shown to be associated with high viscosity. The present invention provides antigen-binding proteins, particularly antibodies, that have been mutated to reduce viscosity. Preferred antigen-binding proteins according to the present invention include antibodies, as shown in Figure 1B, having one or more, preferably all, of the following: VH1|1-18 germline subfamily substitution; VH3|3-33 germline subfamily substitution; VK3|L16 germline subfamily substitution; VK3|L6 germline subfamily substitution; or Fc substitution.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 546,469 filed on 16 August 2017, U.S. Provisional Patent Application No. 62 / 430,773 filed on 06 December 2016, and U.S. Provisional Patent Application No. 62 / 401,770 filed on 29 September 2016, each of which is incorporated herein by reference in whole for all purposes.

[0002] This invention relates to biopharmaceuticals, particularly therapeutic antigen-binding proteins, methods of use thereof, pharmaceutical compositions thereof, and methods for producing them. In particular, this invention relates to antigen-binding proteins, especially antibodies, that have been mutated to reduce viscosity.

[0003] A brief explanation of sequence listings The sequence listing, titled "A-2063-US-PSP3_SeqList_ST25.txt," containing sequence numbers 1 through 383, is incorporated herein by reference in its entirety and contains the nucleic acid sequences and / or amino acid sequences disclosed herein. The sequence listing is submitted herein in ASCII text format via EFS and thus constitutes both paper and computer-readable format. The sequence listing was first created on August 16, 2017, using PatentIn and is 4.32 MB in size. [Background technology]

[0004] Currently, monoclonal antibodies (mAbs) are the most common therapeutic agents among the latest therapeutic proteins on the market and under development. Differences between antibodies lie primarily in the antigen-binding domain or complementarity-determining region (CDR). These differences in CDR are thought to lead to differences in transient protein-protein interaction tendencies, which manifest as bulk solution viscosity. Several groups have described the presence of reversible clusters (mainly dimers) of antibodies in viscous antibody solutions. To explain the interactions of these clusters as a mechanism of bulk solution viscosity behavior, several theoretical descriptions of polymer viscosity have been proposed.

[0005] Antibodies typically act as antagonists and therefore require large doses (often exceeding 100 mg per dose) to block undesirable interactions. For patient comfort, a single subcutaneous injection of 1 mL volume is the most preferred mode of administration. The need to administer large amounts of mAbs in relatively small volumes necessitated high-concentration formulations of 100 mg / ml or higher. Antibodies are large biomolecules with a molecular weight of approximately 150 kDa, and their high concentrations result in high shear stress and high viscosity due to protein-protein and protein-wall interactions during filtration, passage through the injection needle, and in the subcutaneous space. High viscosity presents challenges in the production of therapeutic antigen-binding proteins and their administration to patients, such as malfunction of injection devices, difficulty in manual administration, reduced bioavailability, and extremely high back pressure during injection, leading to patient discomfort.

[0006] The development and use of high-concentration therapeutic monoclonal antibody solutions have accelerated as the cost of biopharmaceutical manufacturing has decreased. In some cases, these antibody solutions have viscous solution properties that can make manufacturing and administering the intended dose difficult. The difference in CDR that seems to determine whether an antibody is "viscous" or "non-viscous" is likely related to the CDR tendency that drives protein-protein interactions.

[0007] The industry is making considerable efforts to understand the nature of the interactions that lead to high viscosity and to reduce the viscosity of high-viscosity antibody formulations. The most important parameters that affect the viscosity of antibody formulations include the following: • Intermolecular interactions determined by the protein's pI and the solution's pH. Cheng et al. (2013), “Linking the solution viscosity of an IgG2 monoclonal antibody to its structure as a function of pH and temperature”, J. Pharm Sci. 102:4291-4304. ·Charge interaction. Yadav et al. (2012), “Viscosity behavior of high-concentration monoclonal antibody solutions:correlation with interaction parameter and electroviscous effects”, J.Pharm Sci.101:998-1011; Yadav et al. (2012), “The influence of charge distribution on self-association and viscosity behavior of monoclonal antibody solutions”.Mol Pharm 9(4):791-802;Singh et al.(2014),“Dipole-Dipole Interaction in Antibody Solutions:Correlation with Viscosity Behavior at High Concentration”,Pharm Res.31(9):2549-2558; Chaudhri et al. (2013), “The role of amino acid sequence in the self-association of therapeutic monoclonal antibodies: insights from coarse-grained modeling”, J. Phys. Chem. B 117:1269-1279。 · Hydrophobic interaction. Guo et al. (2012), “Structure-activity relationship for hydrophobic salts as viscosity-lowering excipients for concentrated solutions of monoclonal antibodies”, Pharm Res 29:3102-3109。

[0008] The highest solution viscosity was observed under conditions of the most negative diffusion interaction parameter kD, highest apparent radius, and lowest effective charge. Neergaard et al. (2013), “Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - prediction of viscosity through protein-protein interaction measurements”, Eur.J.Pharm Sci.49:400-410. The diffusion interaction parameter (kD), a component of the osmotic pressure second virial coefficient (B(2)), correlated well with the viscosity of concentrated mAb solutions (R>0.9), while the mAb effective charge correlated weakly (R<0.6), indicating that weak intermolecular interactions are important in governing the viscoelastic behavior of concentrated mAb solutions. Connolly, et al. (2012), “Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter”, Biophys.J.103:69-78. The studies reported herein utilized primary sequences linked to 3D structures. See Honegger et al. (2001), “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, J.Mol.Biol.309:657-670. The viscosity values ​​of several mAb molecules were measured, and a viscosity prediction model for mAbs was developed using a machine learning algorithm. Structural position, charge, and hydrophobicity were the main parameters of the amino acids used in the model.

[0009] The viscosity of monoclonal antibodies was evaluated using molecular information from the following papers: Li, L. et al. (2014), “Concentration dependent viscosity of monoclonal antibody solutions: explaining experimental behavior in terms of molecular properties”, Pharm. Res. 31:3161-3178; and Sharma et al. (2014), “In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability”, Proc. Natl. Acad. Sci. USA 111:18601-6.

[0010] The ultimate outcome of antibody-antibody interactions is either the extension of a transient network of interactions (osmotic network) that produces a viscous solution, or the formation of larger oligomers as larger structures that have some effect on the solution rheology. In the studies reported herein, a small number of viscous antibodies were used as subjects for biochemical and biophysical analysis in an attempt to deduce specific protein-protein interactions that may lead to viscous antibody solutions. In the past, the Aho numbering approach was used to improve stability and other biophysical properties. Ewert et al. (2003), “Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach”, Biochemistry 42:1517-1528; Ewert et al. (2003), “Biophysical properties of human antibody variable domains”, J.Mol.Biol.325:531-553; al. (2004), “Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering”, Methods 34:184-199; and Rothlisberger et al. (2005), “Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability”, J Mol.Biol.347:773-789. The Aho numbering system has also been used in the past to reduce the tendency for aggregation. Borras et al. (2013), U.S. Patent No. 8,545,849. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Cheng et al. (2013), “Linking the solution viscosity of an IgG2 monoclonal antibody to its structure as a function of pH and temperature”, J. Pharm Sci. 102:4291 - 4304 [Non - Patent Document 2] Yadav et al. (2012), “Viscosity behavior of high - concentration monoclonal antibody solutions: correlation with interaction parameter and electroviscous effects”, J. Pharm Sci. 101:998 - 1011 [Non - Patent Document 3] Yadav et al. (2012), ”The influence of charge distribution on self - association and viscosity behavior of monoclonal antibody solutions”. Mol Pharm 9(4):791 - 802 [Non - Patent Document 4] Singh et al. (2014), “Dipole - Dipole Interaction in Antibody Solutions: Correlation with Viscosity Behavior at High Concentration”, Pharm Res. 3(9):2549 - 2558 [Non - Patent Document 5] Chaudhri et al. (2013), “The role of amino acid sequence in the self - association of therapeutic monoclonal antibodies: insights from coarse - grained modeling”, J. Phys. Chem. B 117:1269 - 1279 [Non - Patent Document 6] Guo et al.(2012),“Structure-activity relationship for hydrophobic salts as viscosity-lowering excipients for concentrated solutions of monoclonal antibodies”,Pharm Res 29:3102-3109

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[0012] The present invention relates to a method for reducing the viscosity of an antigen-binding protein by modifying the sequence in a framework region and / or Fc domain, which has been shown to be associated with high viscosity.

[0013] In the details of the following process, all variable region amino acids are identified by Aho numbering, and all conserved region amino acids are identified by EU numbering. Aho numbering is a counterpart to other major numbering schemes, e.g., EU (Edelman et al. (1969), “The covalent structure of an entire gamma immunoglobulin molecule,”). Aligned and correlated with Proc. Natl. Acad. Sci. U.S.A. 63, 78 - 85), Kabat (Kabat et al. (1991), Sequences of proteins of immunological interest, Fifth Edition. NIH Publication No. 91 - 3242), Chothia (Chothia et al., (1992), “Structural repertoire of the human VH segments”, J. Mol. Biol. 227:799 - 817); (Tomlinson et al., (1995), “The structural repertoire of the human V kappa domain”, EMBO J 14:4628 - 4638), etc. Any of the four numbering systems can be used interchangeably to identify the preferred amino acid substitutions described herein.

[0014] When the antigen - binding protein contains the VH1|1 - 18 germline sub - family, this method involves modifying the VH1 sequence to include one or more substitutions selected from 82X 1 , 94X 2 and 95X 3 (where X 1 is a basic residue (R, K or H), X 2 is a polar uncharged residue (S, T, N or Q), and X 3 is a basic residue (R, K or H)). All residues are identified by the Aho numbering system. Preferred mutations in the VH1|1 - 18 germline sub - family are 82R, 94S and 95R. This method applied to the VH1|1 - 18 germline sub - family may further include the substitution 59X 20 (where X 20 is a basic residue (R, K or H)), preferably the mutation 59K.

[0015] When the antigen - binding protein contains the VH3|3 - 33 germline sub - family, this method involves modifying the VH3 sequence with 1X 4 , 17X5 and 85X 6 This includes modifying to include one or more substitutions selected from (where X 4 is a negatively charged residue (D or E), X 5 These are small hydrophobic residues (G, A, V, I, L, or M), and X 6 These are small hydrophobic residues (G, A, V, I, L, or M). All residues are identified by the Aho numbering system. Preferred variants of the VH3|3-33 germline subfamily are 1E, 17G, and 85A.

[0016] If the antigen-binding protein contains the VK3|L16 germline subfamily, this method will convert the VK3 sequence to 4X 10 , 13X 11 , 76X 12 78F, 95X 13 , 97X 14 and modify to include one or more substitutions selected from 98P (where X 10 The following are selected from G, A, V, I, L, and M, and X 11 The following are selected from G, A, V, I, L, and M, and X 12 The choice is between D and E, and X 13 is selected from R, K, and H, and X 14 (The residue is selected from D and E). All residues are identified by the Aho numbering system. Preferred variants of the VK3|L16 germline subfamily are 4L, 13L, 76D, 95R, 97E, and 98P.

[0017] If the antigen-binding protein contains the VK3|L6 germline subfamily, this method converts the VK3 sequence to 76X 12 and 95X 13 This includes modifying the formula to include one or more substitutions selected from the following. Preferred mutations in the VK3|L6 germline subfamily are 76D and 95R.

[0018] The method of the present invention further involves the Fc domain being 253X 15 , 440X 16 and 439X17 This includes modifying to include one or more substitutions selected from (where X 15 These are small hydrophobic residues (G, A, V, I, L, or M), and X 16 is a basic residue (R, K, or H), and X 17 This is a negatively charged residue (D or E), and the Fc domain sequence is 440X 16 and 439X 17 (Containing only one of the above). All residues are identified by the EU numbering system. Preferred mutations in the Fc domain are 253A, 440K, and 439E.

[0019] The method of the present invention further involves modifying the C-terminus of the Fc domain sequence to X 18 X 19 This includes modifying to include (where X 18 is 1-4 amino acids selected from D and E, or H, K and R, and X 19 is selected from P, M, G, A, V, I, L, S, T, N, Q, F, Y, and W, and X 18 It does not exist when it contains D or E, X 18 It exists when it contains K or R at its C-terminus, X 18 (Present or absent when it contains H at its C-terminus). Preferred Fc C-terminus modifications include KP, KKP, KKKP (SEQ ID NO: 380), E, ​​or EE at its C-terminus.

[0020] The methods described above are preferably applied to the high viscosity antibodies shown in Figures 1A and 1B below. Parts of the methods including the VH1|1-18 sequence are preferably applied to antibodies AF, AK, AL, AN, and AO in Figure 1B. Parts of the methods including the VH3|3-33 sequence are preferably applied to antibodies AQ, AM, AI, and AG in Figure 1B. Parts of the methods including the VK3|L16 sequence are preferably applied to antibodies AF and AQ in Figure 1B. Parts of the methods including the VK3|L6 sequence are preferably applied to antibody AJ.

[0021] The present invention further includes a method for preparing an antigen-binding protein that reaches a maximum serum concentration faster than the parent antibody when administered at the same concentration, wherein the antigen-binding protein and the parent antibody are sequence-modified 440X. 16 (Here, X 16 This involves introducing (selected from R, K, and H). In a preferred method, the sequence-modified antigen-binding protein reaches its maximum serum concentration at least twice as fast as the parent antibody after subcutaneous injection. Methods for preparing an antigen-binding protein that reaches a higher maximum serum concentration than the parent antibody when administered at the same concentration by subcutaneous injection are also within the scope of the present invention, and this method involves introducing the sequence-modified 440X into the parent antibody. 16 (Here, X 16 This involves introducing (selected from R, K, and H). In the preferred method, the sequence-modified antigen-binding protein reaches a maximum serum concentration at least about 25% higher than that of the parent antibody. In each of these methods, the preferred X 16 The antibody is K, and the preferred parent antibody is the PCSK9 polypeptide (antibody AK is the most preferred).

[0022] The present invention further relates to a mutant antigen-binding protein comprising one or more sequences selected from the following. a.82X 1 , 94X 2 , and 95X 3 VH1|1-18 germline subfamily sequences containing one or more substitutions selected from (where X 1 is selected from R, K, and H, and X 2 is selected from S, T, N, and Q, X 3 (This is selected from R, K, and H); b.1X 4 , 17X 5 and 85X 6 VH3|3-33 germline subfamily sequences containing one or more substitutions selected from (where X 4 The choice is between D and E, and X 5 The following are selected from G, A, V, I, L, and M, and X 6 (This is selected from GA, V, I, L, and M); c.4X 10 , 13X 11 , 76X 12 78F, 95X 13 , 97X 14 and the VK3|L16 germline subfamily including one or more substitutions selected from 98P (where X 10 The following are selected from G, A, V, I, L, and M, and X 11 The following are selected from G, A, V, I, L, and M, and X 12 The choice is between D and E, and X 13 is selected from R, K, and H, and X 14 (The mutant antigen-binding protein is selected from D and E, and does not contain only substitution 78F.) d.76X 12 and 95X 13 The VK3|IL6 germline subfamily, which includes one or more substitutions selected from; e.253X 10 , 440X 11 and 439X 12 Fc domain sequence containing one or more substitutions selected from (where X 10 The following are selected from G, A, V, I, L, and M, and X 11 is selected from R, K, and H, and X 12 The choice is between D and E, and X 16 If K and X 17 When E, the antigen-binding protein is 253X 15 , or comprising at least one modification selected from subparagraphs a, b, c, d and f, wherein the antigen-binding protein specifically binds to CD20); and X at the fC terminus 18 X 19 Fc domain sequence containing (where X 18 is 1-4 amino acids selected from D and E, or H, K and R, and X 19 is selected from P, M, G, A, V, I, L, S, T, N, Q, F, Y, and W, and X 18 It does not exist when it contains D or E, X 18 It exists when it contains K or R at its C-terminus, X 18When it contains H at its C-terminus, it is present or absent, and when PGKP (SEQ ID NO: 381), PGKKP (SEQ ID NO: 382), PGKKKP (SEQ ID NO: 383), or PGE appears at its C-terminus, the antigen-binding protein is 253X 15 , or comprising at least one substitution selected from subparagraphs a-e, and the antigen-binding protein specifically binds to CD20 or CD38). Preferred Fc C-terminal modifications include KP, KKP, KKKP (SEQ ID NO: 380), E, ​​or EE at the C-terminus. Here, the amino acids in the variable region are numbered using the Aho numbering system, while the amino acids in the conserved region, including Fc, are numbered using the EU numbering system.

[0023] A preferred antigen-binding protein according to the present invention is one in which the above modifications are applied to the antibodies shown in Figures 1A and 1B below. The following antigen-binding proteins are also preferred:

[0024] The VH1|1-18 germline subfamily sequence contains one or more substitutions selected from 82R, 94S, and 95R, and antigen-binding proteins having all of such substitutions are most preferred;

[0025] The VH3|3-33 germline subfamily sequence contains one or more substitutions selected from 1E, 17G, and 85A, and antigen-binding proteins having all such substitutions are most preferred;

[0026] The VK3|L16 germline subfamily sequence contains one or more substitutions selected from 4L, 13L, 76D, 95R, 97E, and 98P, and antigen-binding proteins having all of such substitutions are most preferred;

[0027] The VK3|L6 germline subfamily sequence contains one or more substitutions selected from 76D and 95R, and antigen-binding proteins having all such substitutions are most preferred;

[0028] The Fc domain sequence contains one or more substitutions selected from 253A, 440K, and 439E, and antigen-binding proteins having all such substitutions are most preferred; and

[0029] The C-terminus of the Fc domain contains a sequence selected from KP, KKP, KKKP (sequence number 380), and E.

[0030] All of the above preferred amino acid substitutions in the variable region are identified by the Aho numbering system. All residues in the conserved region, including Fc, are identified by the EU numbering system.

[0031] Preferred antigen-binding proteins according to the present invention include antibodies AF, AK, AL, AN, and AO as shown in Figure 1B, having one or more, most preferably all, of the above VH1|1-18 germline subfamily substitutions; antibodies AQ, AM, AI, and AG as shown in Figure 1B, having one or more, most preferably all, of the above VH3|3-33 germline subfamily substitutions; antibodies AF and AQ as shown in Figure 1B, having one or more, most preferably all, of the above VK3|L16 germline subfamily substitutions; antibody AJ as shown in Figure 1B, having one or more, most preferably all, of the VK3|L6 germline subfamily substitutions; and antibodies BA, AH, and AN as shown in Figure 1B, having one or more, preferably all, of the above Fc substitutions.

[0032] With the above sequence modifications, the present invention further relates to an antigen-binding protein that specifically binds to PCSK9, comprising a heavy chain sequence selected from SEQ ID NOs. 352, 353, 354, 366, and 368, and preferably also comprising the light chain sequence of SEQ ID NO. 351.

[0033] Through the sequence modifications described above, the present invention also relates to an antigen-binding protein that specifically binds to c-fms, comprising a heavy chain sequence selected from SEQ ID NOs. 356, 357, and 358, and preferably further comprising the light chain sequence of SEQ ID NO. 355.

[0034] By the above-described array modification, the present invention also relates to an antigen-binding protein that specifically binds to GIPR and comprises a heavy-chain sequence selected from SEQ ID NOs: 359, 361, 362, 364, and 368, and preferably further comprises a light-chain sequence selected from SEQ ID NOs: 360, 363, 365, and 367.

[0035] All modified antigen-binding proteins are useful for the same indications as previously described for unmodified antibodies.

[0036] Each antigen-binding protein from FIGS. 1A and 1B having a mutated heavy chain preferably further comprises a light-chain sequence as described for the unmodified parental antibodies of FIGS. 1A and 1B. Each of the aforementioned antigen-binding proteins having a mutated light chain preferably further comprises a heavy-chain sequence as described above or as found in the unmodified parental antibodies of FIGS. 1A and 1B.

[0037] The present invention further comprises the above-described antigen-binding protein having improved pharmacokinetic properties. The present invention is an antigen-binding protein optionally having any of the above-described array modifications, a. 440X 16 comprising the array modification 440X relative to the parental antibody lacking the array modification 16 and b. reaching the maximum serum concentration faster than the parental antibody and c. reaching a higher maximum serum concentration than the parental antibody when the antigen-binding protein and the parental antibody are administered at the same concentration by subcutaneous injection comprises an antigen-binding protein. A preferred parental antibody for such an antigen-binding protein is a PCSK9-binding polypeptide, and antibody AK is most preferred. The preferred substituent X 16 in such an antigen-binding protein is K. Furthermore, a method for treating hypercholesterolemia with such an antigen-binding protein is also within the scope of the present invention.

[0038] The present invention also relates to isolated nucleic acids encoding the antigen-binding proteins of the present invention, as well as vectors containing nucleic acids, host cells containing vectors, and methods for producing and using antigen-binding proteins.

[0039] In other embodiments, the present invention provides compositions comprising antigen-binding proteins, kits comprising antigen-binding proteins, and products comprising antigen-binding proteins. [Brief explanation of the drawing]

[0040] [Figure 1A] Figures 1A and 1B show tables of viscosity values ​​measured for IgG1 and IgG2 monoclonal antibodies formulated at 150 mg / mL in a formulation buffer (without polysorbate) containing 20 mM acetate and 9% sucrose at pH 5.2. Figures 1A and 1B show the targets of the tested antibodies, as well as their light and heavy chain types, germline subfamily, concentration, pI, and viscosity. Each antibody in Figures 1A and 1B has the amino acid sequence shown in the figure and sequence listing. The amino acid sequences of the heavy and light chains are encoded by the nucleic acid having the sequence number immediately preceding them in the sequence listing. [Figure 1B] Figures 1A and 1B show tables of viscosity values ​​measured for IgG1 and IgG2 monoclonal antibodies formulated at 150 mg / mL in a formulation buffer (without polysorbate) containing 20 mM acetate and 9% sucrose at pH 5.2. Figures 1A and 1B show the targets of the tested antibodies, as well as their light and heavy chain types, germline subfamily, concentration, pI, and viscosity. Each antibody in Figures 1A and 1B has the amino acid sequence shown in the figure and sequence listing. The amino acid sequences of the heavy and light chains are encoded by the nucleic acid having the sequence number immediately preceding them in the sequence listing. [Figure 1C] Figures 1C and 1D show the sequence identification numbers (SEQ ID NOs) of the framework region and Fc region of the antibody shown in Figures 1A and 1B. Figure 1D also shows antibody BA, which is explained in Figure 17. [Figure 1D]Figures 1C and 1D show the sequence identification numbers (SEQ ID NOs) of the framework region and Fc region of the antibody shown in Figures 1A and 1B. Figure 1D also shows antibody BA, which is explained in Figure 17. [Figure 2] Figure 2 shows the high-viscosity and low-viscosity subtype pairs identified herein. [Figure 3] Figure 3 shows the expression results of AK and AO antibody molecules and their variants, including titer, terminal viable cell density (VCD), and relative viability at harvesting 7 days after cell culture. An AK value of 100% is shown. [Figure 4] Figure 4 shows the efficacy of AK parent antibodies (AK control, AK) and their variants. [Figure 5] Figure 5 shows the viscosity of mutants relative to the parental AK antibody and parental AO antibody. The average viscosity values ​​for high viscosity VH1|1-18 and low viscosity VH1|1-02 from a set of 43 antibodies are also shown for comparison. [Figure 6] Figure 6 shows the measured viscosity values ​​of mAbs for the high-viscosity VH1|1-18 subtype and the low-viscosity VH1|1-02 subtype, as well as the calculated total molecular pI values. Low-viscosity variants of AK and AO antibodies are also shown. [Figure 7] Figure 7 shows the viscosity values ​​obtained by mAb measurements versus the calculated total molecular pI values ​​for the high-viscosity VH3|3-33 subtype and the low-viscosity VH3|3-07 subtype. The low-viscosity mutant AQ(1, 17, 85) is also shown. [Figure 8] Figure 8 shows the total molecular pI values ​​calculated from viscosity values ​​obtained by measurement of mAbs containing the high viscosity VK3|L16 and VK3|L6 subfamilies and the low viscosity VK3|A27 subfamilies. The low viscosity mutant AQ (4 13 76 95 97 98) is also shown. [Figure 9]Figure 9 is a table showing the global sequence parameters for the high-viscosity VH1|1-18 subtype and low-viscosity VH1|1-02 subtype (germline) of mAbs. The mAbs in Figure 9 are classified by viscosity. The table includes their mAb symbols, measured viscosity values, calculated pI values, and VL and VH germline values. For the VH germline, the higher viscosity VH1|1-18 is shown in bold and underlined. The heavy chain framework 3 sequence is shown. Residues correlated with high viscosity are shown in bold and underlined. For the VH1|1-18 and VH1|1-02 germline subfamilies, V-base sequences are added for comparison to show that different residues are typical of the subfamily. [Figure 10] Figure 10 is a table showing the variants that produced and characterized AK and AO antibodies. [Figure 11] Figure 11 is a table showing the global sequence parameters of 13 mAbs containing the VH3 heavy chain. Figure 11 includes the mAb symbol, measured viscosity value, calculated pI value, HC and LC type, and VL and VH subtype (germline). The VH3|3-33 subfamily with higher viscosity is shown in bold and underlined. Residues correlated with high viscosity are shown in bold and underlined. For the VH3|3-33 and VH3|3-07 germline subfamilies, V-base sequences have been added for comparison to show that different residues are typical of those subfamilies. [Figure 12]Figure 12 is a table showing the global sequence parameters of 14 mAbs with the VH3 light chain. The mAbs in Figure 12 are classified by viscosity and include the mAb symbol, measured viscosity value, calculated pI value, HC and LC type, and VL and VH subtype (germline). The higher viscosity VK3|L16 and VK3|L6 subfamilies are shown in bold and underlined. Light chain residues that consistently differ between the VK3|L16 and VK3|L6 subfamilies compared to the VK3|A27 subfamily are shown on the right. Residues associated with high viscosity in the VK3|L16 and VK3|L6 subfamilies are shown in bold and underlined. For the VK3|L16 and VK3|A27 germline subfamilies, V-base sequences have been added for comparison to show that the different residues are typical of the subfamily. [Figure 13] Figures 13A and 13B show the mean measured viscosity and calculated pI values ​​for intact antibody molecules containing specific heavy and light chain germline families, as well as the germline subfamilies VH1, VH3, and VK3. The X-axis includes the family and the number of members in each family and subfamily. [Figure 14A] Figure 14A shows that mutations on the Fc-Fc interaction surface can reduce solution viscosity. Viscosity at concentrations of antibody AK and antibody AK variants I253A and S440K is shown. [Figure 14B] Figure 14B shows that double mutants that restore wild-type complement activity also restore wild-type viscosity. The figure shows the viscosity at concentrations of antibody AK, antibody AK mutant I253A, antibody AK mutant S440K, and antibody AK double mutant K439E / S440K. This figure also shows the viscosity of antibody AK mutant K439E, antibody AK mutant H433A, and antibody AK mutant N434A, which do not decrease viscosity compared to the antibody AK parent. [Figure 15] Figure 15 is a table showing the absolute and relative viscosity values ​​of the parent antibody AK and various mutants. The parent antibody AK and its mutants are used in non-human primate studies of the pharmacokinetics and pharmacodynamics of antibody AK and its low-viscosity mutants. [Figure 16] Figure 16 is a schematic diagram of EDC chemical crosslinking (see Example 2). [Figure 17] Figure 17 is a table showing the viscosity of proteins selected for Fc mutations against low-viscosity mutants. [Figure 18] Figure 18 shows the concentration-dependent generation of antibody oligomers by EDC chemical crosslinking of antibody AH. [Figure 19] Figure 19A shows that the S440K mutation in the Fc region reduces the viscosity of antibody AQ. (Note that the mutant concentration is actually 150 mg / mL.) Figure 19B is a scatter plot of the same data with exponential fitting. In Figure 19B, the diamonds represent unmodified antibody AQ at the indicated concentrations, and the squares represent the S440K mutant at 150 mg / mL. [Figure 20] Figure 20A is a table showing variants that produced and characterized antibody AQ, including measured concentrations and viscosities. Figure 20B shows the cAMP response of 293 / huGIPR cells expressing the human GIP receptor, which is activated by GIP and blocked by the anti-GIPR antibody. In vitro cAMP activity was similarly unaffected by viscosity variants. The potency was the same within the assay's tolerance. [Figure 21] Figure 21 shows an outline of the experimental design for a single-dose subcutaneous bolus pharmacokinetic study in male cynomolgus monkeys, which will be described in more detail in the following examples. [Figure 22] Figure 22 shows the estimated mean pharmacokinetic parameters of the antibody AK or low-viscosity mutant homolog after subcutaneous administration of 10 mg / kg to male cynomolgus monkeys (N=4 males). Introducing mutations in the viscosity-reducing Fc region lowers Tmax and increases Cmax. [Figure 23] Figure 23 shows the percentage of LDL-C compared to the preliminary study (Day CLAB). *The percentage change is expressed by dividing the value of each animal after administration by the preliminary study value on day 1. All four antibodies (parent antibody AK, AK Fc variant, AK Fab variant, and AK Fc / Fab double variant) induce a decrease in LDL-C. [Figure 24] Figures 24A and 24B show the pharmacokinetic profiles (μg / mL) in plasma, along with the corresponding low-density lipoprotein (LDL) concentration (mg / dL) profiles. LDL and serum concentrations of the test product are shown as mean values ​​from four animals. Black circles with solid lines indicate serum concentrations of the parent antibody AK. White squares with dashed lines indicate serum concentrations of antibodies with the Fab mutation. Black triangles with solid lines indicate serum concentrations of antibodies with the Fc mutation. White diamonds with dashed lines indicate serum concentrations of antibodies with both Fab and Fc mutations. These data demonstrate that the presence of mutations in the Fc region, which reduces the viscosity of the antibody formulation, results in a shorter time to Tmax and a higher Cmax. All variant forms of the parent antibody retain their ability to reduce serum LDL-C (lower panel). [Modes for carrying out the invention]

[0041] Definition of Terms Many terms are used broadly in the following description. To facilitate understanding of the present invention, the following definitions are provided.

[0042] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more.

[0043] An "antigen-binding protein" refers to a protein or polypeptide that contains an antigen-binding region or part of the molecule to which it binds (antigen), and has a strong affinity for that region. Antigen-binding proteins include antibodies, peptide bodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, single-domain antibodies), antibody derivatives, antibody analogs, fusion proteins, and antigen receptors, including chimeric antigen receptors (CARs).

[0044] "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins with the same structural properties. Antibodies exhibit binding specificity to specific antigens, while immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced at low levels in some lymphoid systems and at high levels in some myelomas, for example. Therefore, as used herein, the terms "antibody" or "antibody peptide" refer to an intact antibody, an antibody that competes for specific binding with an antibody disclosed herein, or its antigen-binding fragment that competes for specific binding with an intact antibody (e.g., Fab, Fab', F(ab')2, Fv, single-domain antibodies), and include chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. In certain embodiments, the antigen-binding fragment is prepared, for example, by recombinant DNA technology. In further embodiments, the antigen-binding fragment is prepared by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments include Fab, Fab', F(ab) 2 , F(ab') 2Examples of antibodies suitable for use in the present invention include, but are not limited to, those listed in Figures 1A and 1B, as well as avagovomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ALD518, alemtuzumab, alirocumab, alemtuzumab, altumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, alsitumomab, aselizumab, atinumab, atolizumab, atrolimumab, tocilizumab, bapinuzumab, basiliximab, and babitu Ximab, Vectumomab, Belimumab, Benralizumab, Vertilimumab, Besilezumab, Bevacizumab, Bezlotoxumab, Bisilomab, Vibatuzumab, Vibatuzumab Meltansine, Blinatumomab, Brosozumab, Brentuximab Vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab Meltansine, Caplacizumab, Capromab Pendetide, Carlumab, Katsumakisomab, CC49, Sedelizumab, Certolizumab Pegol, Cetuximab, Sitatuzumab Bogatox, Sixtumumab Clazakizumab, crenoliximab, cribatuzumab tetraxetan, conatumumab, crenezumab, CR6261, dasetuzumab, dacrizumab, darotuzumab, daratumumab, demicizumab, denosumab, detumomab, dorlimomab aritox, dorozizumab, duligotuzumab, dupilumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elcilimomab, enabatuzumab, enlimomab pegor, enokizumab, enokizumab, enochikuma, enochikuma B, encituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, erzumaxomab, etalacizumab, etrolizumab, evolocumab, excivivirumab, excivivirumab, fanolesomab, falalimomab, falletuzumab, facinumab, FBTA05, felbizumab, fezakinumab, ficratuzumab, figitumumab, frambotumab, fontrizumab, foralumab, folavirumab, fresolimmab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab,Gabirimomab, gemtuzumab ozogamicin, gevokizumab, gylenetuximab, glembatumumab vedotin, golimumab, gomiliximab, GS6624, ibalizumab, ibritumomab tiuxetan, iclucumab, igovomab, imusilomab, imugatuzumab, incrakumab, indatuximab tansine, infliximab, inorimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, itorizumab, ixekizumab, keriximab, rabetsumab, lebrikizumab, remalesomab, reldelimumab, lex Saturumab, rivivirumab, rigerizumab, lintuzumab, lirirumab, lorbotuzumab meltansine, lucatumumab, lumiliximab, mapatumumab, masurimomab, mapurilimumab, matsuzumab, mepolizumab, meterimumab, milatuzumab, minretumomab, mitomomab, mogamulizumab, morolimmab, motabizumab, moxetumomab pasdotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumamab, natalizumab, nevacumab, nesitumumab, nererimomab, nesbakumab, nimotsu Zumab, nivolumab, nofetumomab, merpentan, okalatuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onarutuzumab, oporutuzumab, monatox, olegobomab, orticumab, otelixizumab, oxerumab, ozanezumab, ozoralizumab, padibaximab, palivizumab, panitumumab, panobacumab, pulsatuzumab, pascolizumab, pateclizumab, patrizumab, pemtumomab, perakizumab, pertuzumab, pexerizumab, pizilizumab, pintumomab, plac Lumab, ponezumab, prezalumab, priliximab, pritumumab, PRO140, quilizumab, lacosumomab, radrezumab, rafivirumab, ramucirumab, ranibizumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendecide, secukinumab, sevilumab, cibrotuzumab, cifalimumab, siltuximab, simtuzumab, ciprizumab, silucumab, solanezumab,Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Thresomab, Subizumab, Tavarumab, Takatuzumab Tetraxetan, Tadocizumab, Talizumab, Tanezumab, Tapritumomab Paptox, Tefibazumab, Terimomab Aritox, Tenatumomab, Tefibazumab, Terimomab Aritox, Tenatumomab, Teneriximab, Teprizumab, Teprotumumab, Tezeperumab, TGN1412, Tremerimumab, Ticilimumab, Childurakizumab, Tigatuzumab, TNX-650, Tocilizumab, To This includes larizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tucotzumab cermoloukin, tubilumab, ubrituximab, urerumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, bertuzumab, bepalimomab, besenkumab, bicilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanorimumab, zatuximab, diralimumab, and zolimomab aritox.

[0045] The term “isolated antibody,” as used herein, refers to an antibody identified, isolated, and / or recovered from components of its natural environment. Contaminating components of the natural environment are substances that interfere with the antibody’s diagnostic or therapeutic use and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to a concentration of over 95% by weight, most preferably over 99% by weight, by Lowry determination; (2) to a concentration sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by spinning cup sequencing; or (3) to a homogenized antibody by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. Since isolated antibodies do not contain at least one component of their natural environment, they may contain antibodies present in situ within recombinant cells. However, isolated antibodies are typically prepared by at least one purification step.

[0046] The term "binding" of an antigen or other polypeptide includes, but is not limited to, the binding of the ligand polypeptide of the present invention to a receptor; the binding of the receptor polypeptide of the present invention to a ligand; the binding of the antibody of the present invention to an antigen or epitope; the binding of the antigen or epitope of the present invention to an antibody; the binding of the antigen of the present invention to an anti-idiotype antibody of the present invention; the binding of the anti-idiotype antibody of the present invention to a ligand; the binding of the anti-idiotype antibody of the present invention to a receptor; the binding of the anti-anti-idiotype antibody of the present invention to a ligand, receptor or antibody, etc.

[0047] The term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer, consisting of two identical immunoglobulin chain pairs, each having one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains work together to bind to the antigen, while the constant region performs antibody effector function.

[0048] The "light chain" of full-length immunoglobulin (approximately 25 kD or approximately 214 amino acids) is encoded by a variable region gene at the NH2 terminus (approximately 110 amino acids) and a kappa or lambda constant region gene at the COOH terminus. The "heavy chain" of full-length immunoglobulin (approximately 50 kD or approximately 446 amino acids) is similarly encoded by a variable region gene (approximately 116 amino acids) and one of the other aforementioned constant region genes (approximately 330 amino acids). The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, defining the antibody isotypes as IgG (IgG1, IgG2, IgG3, and IgG4, etc.), IgM, IgA, IgD, and IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region consisting of approximately 10 or more amino acids. (Generally, please refer to Fundamental Immunology (Paul, W., ed., 2nd Edition, Raven Press, NY (1989)), Chapter 7 (the entire text is incorporated by reference for all purposes).

[0049] The variable domains or regions of the light or heavy chains of immunoglobulins include "framework regions" (FRs) that are interrupted by "complementarity-determining regions" (CDRs). Kabat et al. al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Chothia et al. (1987), J.Mol.Biol.196:901-917 (both incorporated herein by reference). FR residues are variable domain residues other than CDR region residues as defined herein. The sequences of different light chain or heavy chain framework regions are relatively conserved within species. Therefore, the “human framework region” is substantially identical (approximately 85% or more, usually 90-95% or more) to the framework region of naturally occurring human immunoglobulins. The antibody framework region, i.e., the combined framework region of the constituent light and heavy chains, has the function of positioning and aligning the CDR. The CDR is primarily involved in the binding of the antigen to the epitope. Therefore, the term “humanized” immunoglobulin refers to an immunoglobulin that contains a human framework region and one or more CDRs derived from non-human (usually mouse or rat) immunoglobulins. The non-human immunoglobulin providing the CDRs is called the “donor,” and the human immunoglobulin providing the framework is called the “acceptor.” A constant region is not required, but if present, it must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85–90%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin are substantially identical to the corresponding parts of the natural human immunoglobulin sequence, except perhaps the CDRs. Furthermore, one or more residues in the human framework region may be reverse-mutated to the parent sequence in order to maintain optimal antigen-binding affinity and specificity. In this way, several framework residues derived from the non-human parent antibody are retained in the humanized antibody to maintain the binding properties of the parent antibody while minimizing their immunogenicity. The term “human framework region,” as used herein, includes regions having such reverse mutations.A "humanized antibody" is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. For example, a humanized antibody does not include typical chimeric antibodies, such as those defined below, because, for instance, the entire variable region of a chimeric antibody is non-human.

[0050] The monoclonal antibodies and antibody constructs of the present invention include, in particular, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain originates from a particular species or is identical or homologous to a corresponding sequence in an antibody belonging to a particular class or subclass of antibodies, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody belonging to a different species or class or subclass of antibodies, insofar as it exhibits the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al. (1984), Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include “primitized” antibodies containing variable domain antigen-binding sequences and human constant region sequences derived from non-human primates (e.g., Old World monkeys, Apes, etc.). Various methods for producing chimeric antibodies are described. For example, see Morrison et al. (1985), Proc. Natl. Acad. Sci. USA 81:6851; Takeda et al. (1985), Nature 314:452; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent No. 0171496; European Patent No. 0173494; and British Patent No. 2177096.

[0051] The terms "human antibody" and "fully human antibody" each refer to an antibody having the amino acid sequence of a human immunoglobulin, which is isolated from a human immunoglobulin library or from an animal into which one or more human immunoglobulin genes have been introduced, and which does not express endogenous immunoglobulins, such as Xenomouse® antibodies, and antibodies such as those described in U.S. Patent No. 5,939,598 by Kucherlapati et al.

[0052] The term "genetically modified antibody" means an antibody whose amino acid sequence has been modified from that of a natural antibody. Due to the relevance of recombinant DNA technology in antibody production, the amino acid sequences found in natural antibodies need not be limiting, and antibodies can be redesigned to obtain desired properties. There are many possible variations, ranging from changes to just one or a few amino acids to, for example, a complete redesign of the variable and / or constant regions. Changes to the constant region are generally made to improve or alter properties such as complement binding, membrane interaction, and other effector functions, as well as manufacturability and viscosity. Changes in the variable region are made to improve antigen-binding properties.

[0053] A "Fab fragment" consists of one light chain and the C H1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0054] A "Fab' fragment" consists of one light chain and one heavy chain that contains more of the constant region between the C H1 domain and the C H2 domain such that an interchain disulfide bond can be formed between the two heavy chains to form an F(ab')2 molecule.

[0055] An "F(ab')2 fragment" consists of two light chains and two heavy chains such that an interchain disulfide bond is formed between the C H1 domain and the C H2It includes two heavy chains, each containing a portion of the constant region between the domains.

[0056] The terms "Fv fragment" and "single-chain antibody" refer to polypeptides that contain both heavy and light chain variable regions of the antibody but lack a constant region. Like the whole antibody, it can selectively bind to a specific antigen. With a molecular weight of only about 25 kDa, Fv fragments are much smaller than typical antibodies (150-160 kD), which consist of two heavy protein chains and two light chains, and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain).

[0057] A "single-domain antibody" is a single-domain Fv unit, for example, V H or V L It is an antibody fragment consisting of the following: Like the whole antibody, it can selectively bind to a specific antigen. Single-domain antibodies, with a molecular weight of only 12-15 kDa, are much smaller than typical antibodies (150-160 kDa) which consist of two heavy protein chains and two light chains, and even smaller than Fab fragments (approximately 50 kDa, one light chain and half heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable domains, one from the light chain and one from the heavy chain). The first single-domain antibody was genetically engineered from a heavy chain antibody found in camelid animals. Most research on single-domain antibodies currently focuses on the heavy chain variable domain, but light chain variable domains and nanobodies derived from the light chain have also been shown to specifically bind to target epitopes.

[0058] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryote, prokaryote, or phage clone, and does not refer to a method for producing such an antibody.

[0059] In some embodiments, the antigen-binding proteins of the present invention selectively inhibit the human antigen of the antibody from which it is derived. For example, an antigen-binding protein having the sequence of antibody AF substituted as described herein selectively inhibits the antigen of antibody AF as shown in Figure 1B. An antibody or its functional fragment "selectively inhibits" its specific receptor or ligand compared to another receptor or ligand if the IC50 of the antibody in a specific receptor inhibition assay is at least 50 times lower than the IC50 in an inhibition assay of another "reference" ligand or receptor. "IC50" is the dose / concentration required to achieve 50% inhibition of a biological or biochemical function. In the case of radioligands, IC50 is the concentration of a competing ligand that substitutes 50% of the specific binding of the radioligand. The IC50 of any particular substance or antagonist can be determined by constructing dose-response curves and testing the effect of different concentrations of the drug or antagonist on reversing agonist activity in a particular functional assay. The IC50 value for a given antagonist or substance can be calculated by determining the concentration required to inhibit half of the maximum biological response of the agonist. Therefore, for example, the IC50 value of an anti-PCSK9 antibody or its functional fragment can be calculated by determining the concentration of the antibody or fragment required to inhibit half of the maximum biological response of PCSK9 in the activation of the human PCSK9 receptor in a functional assay. An antibody or its functional fragment that selectively inhibits a particular ligand or receptor is understood to be a neutralizing antibody or neutralizing fragment with respect to that ligand or receptor. Therefore, in some embodiments, the anti-PCSK9 antibody or functional fragment is a neutralizing antibody or neutralizing fragment of human PCSK9.

[0060] The substituted antigen-binding proteins of the present invention can cross-block the non-substituted antibodies from which they are derived. The terms “cross-blocking,” “cross-blocked,” and “cross-blocking” are used interchangeably herein and refer to the ability of an antigen-binding protein to interfere with the binding of other antigen-binding proteins (e.g., antibodies or binding fragments) to a target (e.g., human PCSK9). The extent to which an antibody or binding fragment can interfere with the binding of another to a target, and therefore whether it can be said to cross-block, can be determined by a competitive binding assay. In some embodiments, the cross-blocking antigen-binding proteins of the present invention reduce the binding of a reference antibody to a target antigen by about 40% to 100%, for example, about 60% to about 100%, particularly preferably about 70% to 100%, and more preferably about 80% to 100%. A particularly preferred quantitative assay for detecting cross-blocking is to use a Biacore instrument that measures the degree of interaction using surface plasmon resonance technology. Another preferred cross-blocking quantitative assay uses a FACS-based approach to measure competition between antibodies with respect to the binding of antibodies to the target antigen.

[0061] The term “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may consist of monomers of naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., alpha-enantioma forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have alterations to the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar may be modified with functional groups as ethers or esters. Furthermore, the entire sugar moiety may be replaced with sterically and electronically similar structures, such as aza sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutions. Nucleic acid monomers may be linked by phosphodiester bonds or analogues of such bonds. Analogues of phosphodiester bonds include phosphorothioates, phosphorodioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilidates, and phosphoramidates. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded.

[0062] The term "complementary nucleic acid molecule" refers to a nucleic acid molecule that has a complementary nucleotide sequence in the opposite direction to a reference nucleotide sequence.

[0063] The term "degenerate nucleotide sequence" refers to a sequence of nucleotides that contains one or more degenerate codons compared to a reference nucleic acid molecule that codes for a polypeptide. Degenerate codons contain different triplets of nucleotides but code for the same amino acid residue (i.e., the GAU and GAC triplets code for Asp, respectively).

[0064] An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated into the genomic DNA of an organism. For example, the DNA molecule encoding the heavy chain of an antibody, isolated from the genomic DNA of a cell, is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. A nucleic acid molecule isolated from a particular species is smaller than the complete DNA molecule of that species' chromosome.

[0065] A "nucleic acid molecule construct" is a single- or double-stranded nucleic acid molecule that has been modified by human intervention to contain juxtaposed nucleic acid fragments arranged in configurations not found in nature.

[0066] Complementary DNA (cDNA) is a single-stranded DNA molecule formed from an mRNA template by the enzyme reverse transcriptase. Generally, a primer complementary to a portion of the mRNA is used to initiate reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template.

[0067] A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Generally, promoters are located in the 5' non-coding region of a gene, close to the transcription start site of the structural gene. The sequence elements within the promoter that function in the initiation of transcription are often characterized by a consensus nucleotide sequence. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSE; McGehee et al. (1993), Mol. Endocrinol., 7:551), cyclic AMP reaction elements (CRE), serum reaction elements (SRE; Treisman (1990), Seminars in Cancer Biol., 1:47), glucocorticoid reaction elements (GRE), and binding sites for other transcription factors, such as CRE / ATF (O'Reilly et al. (1992), J. Biol. Chem., 267:19938), AP2 (Ye et al. (1994), J. Biol. Chem., 269:25728), SP1, cAMP reaction element binding protein (CREB; Loeken (1993), Gene Expr., 3:253), and octameric factors (generally, Watson et al. See al. (1987), eds., Molecular Biology of the Gene, 4th Edition, The Benjamin / Cummings Publishing Company, Inc., and Lemaigre et al. (1994), Biochem. J., 303:1). When the promoter is an inductive promoter, the transcription rate increases in response to the inducer. In contrast, when the promoter is a constitutive promoter, the transcription rate is not regulated by the inducer. Repressive promoters are also known.

[0068] A "regulatory element" is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may include a nucleic acid sequence that binds to a cytofactor that enables transcription exclusively or preferentially in a particular cell, tissue, or organelle. Such types of regulatory elements are typically associated with genes that are expressed in a "cell-specific," "tissue-specific," or "organelle-specific" manner.

[0069] An "enhancer" is a type of regulatory element that can increase transcription efficiency regardless of the distance or direction of the enhancer relative to the transcription initiation site.

[0070] "Heterogeneous DNA" refers to a DNA molecule or a collection of DNA molecules that do not naturally exist within a given host cell. A DNA molecule that is heterogeneous to a particular host cell may include DNA derived from the host cell type (endogenous DNA), insofar as the host DNA is bound to non-host DNA (i.e., exogenous DNA). For example, a DNA molecule containing a non-host DNA fragment encoding a polypeptide operably ligated to a host DNA fragment containing a transcription promoter is considered heterogeneous DNA. Conversely, heterogeneous DNA molecules may contain endogenous genes operably ligated to an exogenous promoter. Alternatively, a DNA molecule containing genes derived from wild-type cells is considered heterogeneous DNA if that DNA molecule is introduced into a mutant cell lacking the wild-type gene.

[0071] An expression vector is a nucleic acid molecule that encodes a gene to be expressed in a host cell. Typically, an expression vector contains a transcription promoter, the gene, and a transcription terminator. Gene expression is usually under the control of a promoter, and such a gene is said to be "operably ligated to" the promoter. Similarly, regulatory elements and core promoters are operably ligated if the regulatory element modulates the activity of the core promoter.

[0072] A "recombinant host" is a cell containing a heterogeneous nucleic acid molecule, such as a cloning vector or an expression vector. In this context, an example of a recombinant host is a cell that produces the antagonist of the present invention from an expression vector. In contrast, such an antagonist is a "natural source" of the antagonist and can be produced by a cell lacking the expression vector.

[0073] The terms “amino terminus” and “carboxyl terminus” are used herein to indicate locations within a polypeptide. Where possible, these terms are used in reference to specific sequences or portions of a polypeptide to indicate proximity or relative position. For example, a particular sequence located carboxyl-terminal to a reference sequence within a polypeptide is located in proximity to the carboxyl terminus of the reference sequence, but not necessarily to the carboxyl terminus of the complete polypeptide.

[0074] A "fusion protein" is a hybrid protein expressed by a nucleic acid molecule containing the nucleotide sequences of at least two genes. For example, a fusion protein may include at least a portion of an antibody heavy chain fused with an affinity matrix or a polypeptide that binds to another target of interest.

[0075] The term "receptor" refers to a cell-associated protein that binds to a bioactive molecule called a "ligand." This interaction mediates the effect of the ligand on the cell. Receptors can be membrane-bound, cytosolic, or karyotype; monomeric (e.g., thyroid-stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor, and IL-6 receptor). Membrane-bound receptors are characterized by a multi-domain structure that includes an extracellular ligand-binding domain and an intracellular effector domain, usually involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides containing the fully functional receptor. Generally, ligand binding to a receptor results in a structural change of the receptor that triggers interactions between the effector domain and other molecules in the cell, which in turn results in changes in the cell's metabolism. Metabolic events often associated with receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, cellular calcium recruitment, membrane lipid recruitment, cell adhesion, inositol lipid hydrolysis, and phospholipid hydrolysis.

[0076] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression includes the transcription of the structural gene into mRNA and the translation of the mRNA into one or more polypeptides.

[0077] The term "complement / anti-complement pair" refers to different parts that form a stable, non-covalently bonded pair under appropriate conditions. For example, biotin and avidin (or streptavidin) are archetypal members of complement / anti-complement pairs. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, etc. If subsequent dissociation of the complement / anti-complement pair is desired, the complement / anti-complement pair is 10 9 M -1 It is preferable to have a binding affinity of less than 1.

[0078] A "detectable label" is a molecule or atom that binds to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelating agents, photoactivators, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.

[0079] The term "affinity tag" is used herein to refer to a polypeptide fragment that can bind to a second polypeptide for the purpose of providing a site for the purification or detection of the second polypeptide, or for attaching the second polypeptide to a substrate. In principle, any peptide or protein for which an antibody or other specific binder is available can be used as an affinity tag. Affinity tags include polyhistidine tracts, protein A (Nilsson et al. (1985), EMBO J.4:1075; Nilsson et al. (1991), Methods Enzymol., 198:3), glutathione S transferase (Smith et al. (1988), Gene, 67:31), Glu-Glu affinity tag (Grussenmeyer et al. (1985), Proc. Natl. Acad. Sci. USA 82:7952), substance P, FLAG® peptide (Hopp et al. (1988), Biotechnology 6:1204), streptavidin-binding peptide, or other antigenic epitopes or binding domains. For general information, see Ford et al. (1991), Protein Expression and Purification, 2:95. DNA molecules encoding affinity tags are available from commercial suppliers (e.g., Pharmacia). It is available from Biotech (Piscataway, NJ).

[0080] The terms "acidic residue" and "negatively charged residue" refer to amino acid residues that have a side chain containing an acidic group. Exemplary acidic or negatively charged residues include D and E.

[0081] The term "amide residue" refers to an amino acid that has a side chain containing an amide derivative with an acidic group. Exemplary amide residues include N and Q.

[0082] The term "aromatic residue" refers to an amino acid residue that has a side chain containing an aromatic group. Exemplary aromatic residues include F, Y, and W.

[0083] The terms "basic residue" and "positively charged residue" refer to amino acid residues that have a side chain containing a basic group. Exemplary basic or positively charged residues include H, K, and R.

[0084] The terms "hydrophilic residue" and "polar uncharged residue" refer to amino acid residues that have a side chain containing a polar group. Exemplary hydrophilic or polar uncharged residues include C, S, T, N, and Q.

[0085] The terms "non-functional residue" and "small hydrophobic residue" refer to amino acid residues that have side chains that are acidic, basic, or lack aromatic groups. Exemplary non-functional small hydrophobic residues include M, G, A, V, I, L, and norleucine (Nle).

[0086] One aspect of the present invention relates to a PCSK9-binding polypeptide. “PCSK9-binding polypeptide” means a polypeptide that binds to the proprotein convertase subtilisin / kexin type 9 (PCSK9) protein. In some cases, the PCSK9-binding polypeptide blocks the binding of PCSK9 to the low-density lipid receptor (LDLR). Such a blocking PCSK9-binding polypeptide may be a monoclonal antibody (mAb) and may be one of the following: a. mAbs (antibody AK, evolocumab) or antigen-binding fragments thereof, comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 136 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 134; b. mAbs that compete with evolocumab for binding to PCSK9; c. i. Heavy chain polypeptides containing the following complementarity-determining regions (CDRs): heavy chain CDR1, which is CDR1 of SEQ ID NO: 376 or 378; heavy chain CDR2, which is CDR2 of SEQ ID NO: 376 or 378; heavy chain CDR3, which is CDR3 of SEQ ID NO: 376 or 378; and ii. Light chain polypeptides containing the following complementarity-determining regions (CDRs): Light chain CDR1, which is CDR1 of SEQ ID NO: 377 or 379; Light chain CDR2, which is CDR2 of SEQ ID NO: 377 or 379; Light chain CDR3, which is CDR3 of SEQ ID NO: 377 or 379 mAb containing; d. mAb that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains the amino acid sequence of SEQ ID NO: 369: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238; e. An mAb that binds to PCSK9 at an epitope that overlaps with the epitope bound by the antibody containing the following: i. The heavy chain variable region of the amino acid sequence of SEQ ID NO: 136; and ii. Light chain variable region of the amino acid sequence of Sequence ID No. 134, iii. The mAb epitope further overlaps with the binding site of the epidermal growth factor-like repeat A (EGF-A) domain of the low-density lipoprotein receptor (LDLR) protein (Horton, Cohen, & Hobbs (2007), Trends Biochem Sci, 32(2), 71-77. doi:10.1016 / j.tibs.2006.12.008; Seidah & Prat (2007), J Mol Med (Berl), 85(7), 685~696) mAb; f. mAbs containing heavy chain polypeptides with the following complementarity-determining regions (CDRs): i. Heavy chain CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs. 373, 374, and 375, respectively; and ii. Light chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 369, 370, and 371, respectively; or g. An mAb containing the heavy chain variant sequence of SEQ ID NO: 378 and the light chain variant sequence of SEQ ID NO: 379.

[0087] Preferred Embodiment Correlation between global sequence characteristics and viscosity The primary objective of the study reported in Example 1 of this specification was to clarify the relationship between viscosity and the amino acid sequence, or global sequence features, of IgG monoclonal antibodies, with the aim of reducing the viscosity of high-concentration monoclonal antibody preparations. To this end, the viscosity values ​​of 43 different monoclonal antibodies were measured at 150 mg / ml, yielding a wide range of values ​​from 5 to 33 cP (Figures 1A and 1B). Major global sequence features of monoclonal antibodies, such as light and heavy chain types, their subtypes (germline), and pI, were calculated and correlated with viscosity, but no immediately significant correlation was revealed (Figures 1A and 1B). Polymorphisms within IgG isotypes (known as allotypes) have been described using human-derived serological reagents (Ropartz, C., Schanfield, MS, and Steinberg, AG (1976), “Review of the notation for the allotypic and related markers of human immunoglobulins,” WHO meeting on human immunoglobulin allotypic markers, held 16-19 July 1974, Rouen, France; report amended June 1976, J Immunogenet. 3, 357-362), and have correlated with specific amino acid residues at several specific positions in the conserved regions of the heavy and light chains (Jefferis and Lefranc (2009) Human immunoglobulin allotypes: possible implications for immunogenicity, mAbs 1, 332-338.) (Vidarsson, G., Dekkers, G., and Rispens, T. (2014) IgG subclasses and allotypes: From Structure to effector functions (Front Immunol. 5, 1-17). Allotypes introduce several different residues (described below) into other conserved regions of the light and heavy chains. All kappa light chains used in this study were of the same (3) allotype (characterized by residues A153 and V191 in EU numbering). All IgG2 heavy chains were of the same (n-) allotype (characterized by P189). Four IgG1 heavy chain allotypes were described (including the following related residues in EU numbering): f(R214); z(K214); a(D356, L358) and x(G431) (Jefferis & Lefranc, 2009) (Vidarsson et al., 2014). IgG1 heavy chains with alternative residues at positions E356, M358 and A431 do not constitute an allotype because these amino acid residues are present in other IgG subclasses. The IgG1 allotype (x) was not present in this study, and all IgG1 heavy chains had A431. IgG1 heavy chain allotypes (f), (z), (a) and associated residues are shown in Figures 1A and 1B.

[0088] The antibodies in Figures 1A and 1B are classified by viscosity. The tables in Figures 1A and 1B contain the monoclonal antibody name, measured concentration, measured viscosity value, and global sequence parameters including type, subtype, and calculated pI. IgG1 type, lambda light chain, and VH1 heavy chain subtypes are shown in bold.

[0089] IgG1 and IgG2 heavy chains, as well as kappa and lambda light chains, were fairly uniformly distributed across the viscosity range. Subtype sequence evaluation revealed several high-viscosity and low-viscosity subtype pairs: VH1|1-18 and VH1|1-02; VH3|3-33 and VH3|3-07; VK3|L16 and VK3|A27, which correlated with viscosity residues with random correlation probabilities of 0.0002, 0.076, and 0.031, respectively (Figure 2). This study also explored correlations of viscosity with D and J region sequences, but no significant correlations were found.

[0090] Figure 2 shows the p-values ​​indicating the random correlation probability with viscosity. Figure 2 also shows the residues in the high viscosity subtype, their positions in Aho numbering, and the residues in the low viscosity subtype.

[0091] Of the 14 IgG molecules of the VH1 subtype, high viscosity molecules were strongly associated with the VH1 1-18 subtype, and low viscosity molecules were strongly associated with the VH1 1-01 subtype, making it highly unlikely that this was a random coincidence (Figure 9).

[0092] To assess the correlation between two subtypes and viscosity, the probability of the same population mean was calculated using Student's t-tests with two-sided distributions for VH1|1-02 vs. VH1|1-18, VH3|3-33 vs. VH3|3-07, and VK3|L16 vs. VK3|A27, using two-sample equal variances.

[0093] A correlation (t-test, p=0.031) was detected between the high-viscosity light chain VK3|L16 and the low-viscosity VK3|A27 (Figure 2, Figure 12, left side). VK3|L16 vs. VK3|A27 and VH3|3-33 vs. VH3|3-07 will be discussed further in this specification. Due to their strong correlation with viscosity, VH1|1-18 and VH1|1-02 were further evaluated as follows. In the next step, 43 antibody chain sequences were aligned and evaluated as follows.

[0094] Sequence alignment and numbering system Several IgG numbering systems exist, including the following: ·EU--Edelman et al. (1969), “The covalent structure of an entire gamma immunoglobulin molecule,” Proc. Natl. Acad. Sci. USA63, 78-85; ·Kabat--Kabat et al.(1991), Sequences of proteins of immunological interest,Fifth Edition.NIH Publication No.91-3242; ·Chothia--Chothia et al.(1992),“Structural repertoire of the human VH segments”,J.Mol.Biol.227:799-817;Tomlinson et al.,(1995),“The structural repertoire of the human V kappa domain.EMBO J.14:4628-4638; ·Aho--Hoenegger et al.(2001),“Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool,”J.Mol.Biol.309,657-670; For example, all four numbering systems described above are shown on the right side of Figure 9 for frame region 3 of the heavy chain of the VH1 subtype. The Aho scheme was constructed by utilizing the spatial positions of amino acid residues derived from the crystal structures of over 400 variable domains of different antibodies. As it is a 3D structure-based numbering system, we used the Aho numbering system defined by A. Honegger (see above) in this study. This is particularly significant for residues in the CDR: the same number of residues are located in similar spatial regions and are equivalent across different IgG sequences. Since the positions of residues in the Aho numbering scheme are related to the tertiary structure, they should be more relevant to biophysical and biochemical properties, and possibly viscosity. As shown in several tables herein, Aho numbering aligns and correlates with other major numbering schemes. Any of the four numbering systems can be used interchangeably to identify preferred amino acid substitutions. The heavy chain variable regions end at the following residues in different numbering systems: 149 Aho, 117 EU, 113 Kabat, and 113 Chothia. The light chain variable regions end at 149 Aho, 107 EU, 107 Kabat, and 107 Chothia. The Aho numbering assigns more numbers to the CDR region instead of using letters for the CDR residues, as in Kabat and Chothia (e.g., 82b for Kabat and Chothia). As a result, the number of Aho for the same residue is often larger. Each variable region contains three complementarity-determining regions (CDRs) and four framework regions (FRs) in the following sequences: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. While the CDRs offer great sequence diversity for antigen binding (the CDR3 region binds most frequently), the FR sequences are more conserved and contain only minor differences, some of which are subtype-specific.

[0095] Correlation of arrangement with viscosity In addition to evaluating global sequence features, the sequences of the variable region were aligned to identify residues responsible for viscosity differences. Beyond visual observation, a software machine learning algorithm was developed and applied to identify the residues most influencing viscosity and to predict antibody viscosity values ​​from their sequences. The predictive model was constructed using the charge and hydrophobicity of residues at the Aho alignment positions.

[0096] Evaluation of heavy chain VH1 sequence alignment and the five sequences of the high-viscosity VH1 1-18 subtype and the five sequences of the low-viscosity VH1 1-02 subtype molecule revealed that only four residues differed between the two subtypes in the frame, and all four were located in frame 3 (Figure 9, right). More sequence differences were observed in the CDR, but these were excluded from the study because CDRs are often involved in antigen binding, and manipulating CDRs (mutating residues) to reduce viscosity carries a high risk of losing efficacy. The differences in viscosity and FR3 residues associated with the subtypes indicated that the following amino acid substitutions in Aho numbering may potentially reduce viscosity: T82R, T86I, R94S, and S95R. The software algorithm supported four substitutions and suggested that the following two substitutions correlated with viscosity reduction: G13V / L at FR1 of the light chain and S59R / K from the end of CDR2 of the heavy chain (the latter interphase was observed in the VH3 subtype) (Figure 9, right).

[0097] In silico evaluation of suggested amino acid substitutions VH T82R. R82 occurs frequently in VH1-02. IgG structural modeling has shown that heavy chain Aho position 82 is part of the upper core of the globulin fold and does not normally come into contact with the antigen, but according to Ewert et al. (2003), “Biophysical properties of human antibody variable domains”, J.Mol.Biol.325:531-553, it can come into direct contact with the CDR backbone. HC82 has a very conserved main-chain-side-chain H bond interaction with the CDR1 and CDR2 backbone amides (Honegger et al., supra). R82 can also modulate the backbone oxygen atom of the CDR2 loop.

[0098] VH R94S and S95R. S94 and R95 occur frequently in VH1-02. These locations are on the surface, away from the antigen-binding domain, and are thought to be part of the lower core.

[0099] T86I. I86 occurs frequently in VH1-02. The data suggest substitution of hydrophilic surface residues (T) with hydrophobic residues (I), which may potentially lead to aggregation.

[0100] In VH1 and VH3, the hydrophilic 59th position is associated with lower viscosity. The VH1 S59R / K position exhibits high structural and sequence variability, is considerably exposed to solvents, and is directly located between residues 58 and 60, which are part of the upper core and can affect binding. The structure is likely to depend directly on the differences between residues 58 and 60 (especially 58 if it is embedded). The frequency of R / K59 occurrences is low (less than 2%) and, according to amino acid residue frequency analysis, was not observed in VH1. All R / K59s are present in the VH3 dataset, with the exception of one (VH4).

[0101] VL G13V / L - This position is structurally embedded, as described above by Ewert et According to al., it is part of the lower core of the variable domain. From this perspective, the G13V mutation to a more hydrophobic residue should strengthen the core.

[0102] In summary, in silico sequence analysis showed that the proposed mutations do not introduce further glycosylation sites or sites susceptible to rapid degradation under physiological or mild acidic formulation conditions (NG, NS, NT, DG, DH). The VH T82R, T86I, R94S, and S95R mutations provide a switch from subtype VH1-18 to VH1-02 in frame region 3, and therefore they do not introduce any unusual or rare motifs. The addition of the VH S59R and VL G13V mutations was suggested by software from non-VH1 VH3 subtypes. Except for S59R at the end of HC CDR2, none of the mutation sites are located near the binding region and therefore pose a low risk of interfering with potency / binding. Arginine is a very low-frequency residue at position 59 (R59), so its effect is difficult to predict. T86I was identified as having a high risk of aggregation and was removed from the list of mutations.

[0103] The generated mutants and their expression, potency, chemical modification, glycosylation, and viscosity. Considering the above considerations, several variants were generated for two IgG2 antibodies, AK and AO, of the high-viscosity VH1-18 subtype, with the aim of reducing viscosity while maintaining potency (Figure 9B). Figure 9B includes monoclonal antibody variant symbols as well as the relevant mutations on the heavy and light chains.

[0104] Two AK mutants containing the S59R substitution showed very low expression levels (marked with * in Figure 3). Survival rates and live cell density were also lower in one of them, AK(59 82 The titer was low in 94 95 13). On the other hand, antibody AO variants containing the S59R substitution produced titers comparable to those of the parent AO molecule. Although statistics were not sufficient to draw general conclusions about heavy chain position 59, this example suggests that a single amino acid substitution can dramatically alter expression. Chemical modifications, including oxidation, deamidation, isomerization, and glycosylation patterns, were similar between the two parents and their variants, as measured by peptide mapping LC-MS analysis.

[0105] The potency values ​​of the parental AK antibody and two well-expressed mutants were measured by binding to PCSK9 and were similar (Figure 4). Finally, the viscosity measurements of the AK and AO mutants were significantly lower than the parent, as predicted (Figure 5). For example, the AK(82 94 95) mutant had only 39% of the parental viscosity. The viscosity of the two AO mutants containing the S59R substitution was even lower, at approximately 28% of the parental viscosity (Figure 5). The S59R mutant was not well-expressed with the AK antibody, and its viscosity could not be measured. For comparison, the mean viscosity values ​​of the VH1|1-18 and VH1|1-02 germline subfamilies were added. A total of 12 consistent sequence differences were identified between the VH1|1-18 and VH1|1-02 subfamilies, including 8 in the CDR and 4 in the frame region (Figure 9). For amino acid substitutions from high-viscosity VH1|1-18 to low-viscosity VH1|1-02, three sites (all located in frame region 3) were selected. The three mutations within the frame were introduced into two mAbs (AK and AO) of the VH1|1-18 subfamily, converting only these residues to those present in VH1|1-02. While the opportunity to achieve a possible twofold viscosity reduction was theoretically low (3 / 12), these substitutions yielded unexpectedly desirable results, with approximately a twofold viscosity reduction in both antibody molecules with just three substitutions.

[0106] Viscosity vs. pI The dependence of viscosity on pI was not clear from the full set of 43 mAbs, but the VH1 subset clearly showed that the viscosity of the mAbs steadily increased as the pI value of the mAbs decreased from pI 8.5 to pI 6.5 in a formulation at pH 5.2 (Figure 6). A shift was also seen between the high-viscosity VH1|1-18 mAb and the low-viscosity VH1|1-02 mAb. As predicted, the T82R, R94S, and S95R variants of the AK and AO antibodies shifted approximately twofold downward along the viscosity scale from the VH1|1-18 region to the VH1|1-02 region on the plot (Figure 6). The variants AO(59 82 94 95) and AO(59 82 94 95 13) shifted to even lower viscosities and slightly higher pIs, indicating that the S59R substitution adopted from outside the VH1 group was effective in further reducing the viscosity. Unfortunately, the AK variants containing R59 were not sufficiently expressed, suggesting that the presence of the low-frequency arginine residue at position 59 could affect expression.

[0107] An increase in the pI of an antibody in a formulation where pH < pI (e.g., the mildly acidic formulation used in this study) generally results in a decrease in viscosity. This result can be explained by the Columbian repulsive force of the positively charged antibody molecules. Proteins such as antibodies exhibit low solubility and high precipitation, which are known to affect viscosity at high concentrations. It is interesting that the substitution from VH1|1-18 to VH1|1-02 in framework region 3 resulted in a twofold decrease in viscosity and a slight increase in the antibody pI value, suggesting that rather than an increase in charge, some structural changes could be the cause of the dramatic decrease in viscosity.

[0108] After superimposing the crystal structures of hundreds of Fab domains, hydrogen bonding interactions were confirmed for all VH and VL positions (Honegger et al. (2001), “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, J.Mol.Biol.309:657-670). The data show that the confirmed positions can bind to other residues via back-chain and side-chain hydrogen bonding interactions (for type III AK and AO antibody molecules). For example, in some VH type III immunoglobulin structures, 94 bound to 77, 95 to 18, 59 to 67, 66, 65, 61, and 60, and VL13 to 146 and 148. Therefore, residue substitutions at these positions can alter interactions and immunoglobulin folds. Crystal structure of AK antibody (Jackson et al., 2007), “The The "Crystal Structure of PCSK9: a Regulator of Plasma LDL-Cholesterol" (Structure 15: 545-52) suggests that all three positions 82, 94, and 95 are located precisely on the periphery of the Fab region, exposed to the solvent and other antibody molecules. Changes in the positions of VH3 (Figure 11) and VK3 (Figure 12) in FR3 also correlate with viscosity. One explanation for their role in viscosity is that these positions in FR3 are on the periphery of the molecule and actively participate in intermolecular interactions between motion within the injection needle and shear stresses related to viscosity measurement.

[0109] Lower viscosity VK3|A27 and higher viscosity VK3|L16 antibodies showed an inverse correlation between viscosity and pI (Figures 8 and 12).

[0110] It should be noted that pI is not a very strong predictor of viscosity, but in some cases it is a useful predictor of viscosity. Generally, viscosity decreases with increasing pI, and pI should be taken into consideration. For example, the viscosity value of VH3|3-33 is lower, and for antibody molecules with higher pI, it becomes similar to that of VH3|3-07 (Figure 7). Therefore, pI should be considered when predicting the viscosity of VH3|3-33.

[0111] Viscosity reduction proposed for the germline families VH3 and VK3 High-viscosity VH3|3-33 and low-viscosity VH3|3-07 antibodies, on average, exhibit a significant difference in viscosity but occupy similar pI ranges (Figure 7). Unexpectedly, on average, VH3|3-07 has a lower viscosity and a lower pI, which contradicts the general behavior reported in the literature. The viscosity values ​​of the following VH3|3-33 molecules can be reduced by mutation: AQ, AM, AI, AG.

[0112] Monoclonal antibodies with high-viscosity VK3|L16 and low-viscosity VK3|A27 light chains also showed a large viscosity difference, but the pI values ​​showed a relatively small difference, which also suggested structural differences between subfamilies (Figure 8). The viscosity values ​​of the following VK3|L16 molecules can be reduced by the mutations described in Figure 11: antibodies AQ and AF.

[0113] Global sequence feature analysis revealed the following high / low viscosity pairs: VH1|1-18 / VH1|1-02; VK3|L16 / VK3|A27; and VH3|3-33 / VH3|3-07, with p-values ​​of correlation of 0.0002; 0.031 and 0.076, respectively (Figure 2). Sequence locations and residues correlated with viscosity differences can be identified and considered as candidates for viscosity reduction point mutations (Figure 2). The correlations of the VH and VL families with respect to viscosity performed above theoretically indicate that antibodies with the following VH and VL combinations have the lowest viscosity (Figures 13A and B): VH2, VH3|3-07, VH1|1-02, and VK3, VL1, VK3|A27 for VL. Three antibodies with the structures of VH1|1-02 and VK3|A27 were indeed produced in our set, and they actually showed low viscosity values: B (5.6 cP), J (8.2 cP), and Y (12.1 cP).

[0114] Research on chemical crosslinking We attempted a comprehensive investigation of potential protein-protein interactions in viscous antibody solutions using chemical crosslinking at high protein concentrations. Chemical crosslinking is a classic biochemical technique used to demonstrate that specific portions of proteins interact with each other. This specification describes the use of zero-length chemical crosslinking reagents to elucidate potential protein-protein interactions in high-concentration viscous antibody solutions. Using the results of chemical crosslinking of viscous and non-viscous antibodies, we construct a model of potential protein-protein interactions in solution.

[0115] Chemical crosslinking by EDC reveals the existence of a common chemically crosslinkable oligomer distribution. Surprisingly, the chemical crosslinks that give rise to the oligomer pattern are intramolecular rather than intermolecular. Intramolecular crosslinking between the apex of Fc and the base of Fab results in an antibody conformation that may be favorable for the formation of Fc-Fc-mediated antibody oligomers. The 1HZH antibody crystal structure contains an antibody hexamer in a chiral unit with one Fab arm pinned to the Fc domain to facilitate the Fc-Fc interaction crucial for the formation of the IgG1 hexamer. It is unclear whether the appearance of this conformation is important for hexamer formation in the protein crystal. The increased tendency to form hexamers in solution in the presence of Fab-Fc intramolecular chemical crosslinking suggests that the Fab arm pinned to Fc may increase the tendency to form Fc-Fc-based antibody oligomers.

[0116] A decrease in Fc-Fc interaction can reduce solution viscosity. The scientific literature includes studies on antibody hexamerization related to interactions with Clq1 and CDC activity. Diebolder et al. found that anti-CD20 antibodies with specific Fc point mutations, including K439E and S440K, inhibit CDC activity, while the associated K439E / S440K double mutant restores CDC activity. Additionally, the I253A mutation reduced CDC activity. Diebolder et al. (2014), “Complement is Activated by IgG Hexamers Assembled at the Cell Surface”, Science 343:1260-3. Diebolder et al. did not associate the mutants they disclosed with an effect on antibody viscosity. Similarly, van den Bremer et al. (2015) found that a charged residue at the C-terminus of an antibody may reduce Clq1 interaction by decreasing its ability to form IgG hexamer structures. The authors did not associate the presence of a charged residue at the C-terminus of IgG with an effect on the viscosity of the antibody solution.

[0117] Observation of antibody hexamers in viscous antibody solutions suggested that Fc-Fc interactions present in the antibody hexamer crystal structure, and structures thought to be formed before Clq1 (complement) replenishment, would exist in viscous antibody solutions in the absence of the chemical crosslinking agent EDC. To test whether Fc-Fc interactions can contribute to solution viscosity, Fc mutants were prepared in Amgen based on a study by Diebolder et al. These materials were then evaluated in anti-PCSK9 formulation buffer by cone plate rheology. Comparison of the parental anti-PCSK9 antibody AK and the Fc mutant anti-PCSK9 antibody showed that a decrease in the affinity of Fc to Fc reduced the solution viscosity of the antibody. Point mutants retained FcRn binding ability and showed no change in biological activity. The ability of the double mutant with restored wild-type complement activity to return to wild-type viscosity suggests that the decrease in viscosity can be reversed when the ability of Fc-Fc interactions is restored to wild-type levels. In conjunction with the observation of Fc-Fc-mediated oligomer species that increase solution viscosity, a common Fc-mediated protein-protein interaction exists that contributes to antibody solution viscosity. Of the five mutants—S440K, I253A, K439E, H433A, and N434A—identified by Diebolder et al. as reducing CDC activity and tested for viscosity in this study, only the first two showed a decrease in viscosity in high-concentration anti-PCSK9 formulations. However, K439E, H433A, and N343A did not decrease viscosity in high-concentration anti-PCSK9 formulations, indicating no direct correlation and suggesting that those skilled in the art cannot accurately predict lower viscosities from the information provided by Diebolder et al. The K439E mutant was also evaluated in high-protein-concentration sucrose formulations and found to have lower viscosity than the parent anti-PCSK9 mutant at the same concentration. The presence of arginine in anti-PCSK9 formulations may have contributed to charge screening, potentially reducing the effectiveness of the negative charge introduced into the K439E variant to decrease Fc-Fc interactions. The H433A and N434A variants do not exhibit the same overt sensitivity to charge screening as the K439E variant.

[0118] Fc-Fc interactions can affect solution viscosity by increasing the number of possible potential interactions in two potential ways. It can increase the number of interactions per antibody from two CDR-mediated interactions per antibody to two CDR-mediated interactions + two Fc-mediated interactions per antibody, or it can alter the number of free CDR ends available in the oligomer present in solution. Given the fact that the Fc domains of IgG1, IgG2, IgG3, and IgG4 antibodies are very similar, it is likely that Fc-Fc-mediated interactions are present in all antibodies. Analysis of non-viscous antibodies shows that intramolecular crosslinks indicating the presence of Fc-Fc interactions are absent, in contrast to viscous antibodies at the same concentration. This suggests that Fc-Fc interactions, while theoretically possible, are not present in non-viscous antibodies. The next nearest neighbor interaction of a CDR can influence the relative distance and relative orientation between Fc to enhance Fc-Fc interactions. This may explain why viscous antibodies have Fc-Fc interactions at room temperature, while non-viscous antibodies do not.

[0119] The presence of Fc-Fc interactions also increases the likelihood that antibody oligomers (dimers, trimers, tetramers, etc.) contain the maximum number of free CDR ends. A greater number of free CDR ends leads to a greater number of the next nearest neighbor interactions of the CDR. This, in turn, can increase the tendency to form networks and raise the viscosity of the solution, as a result of more efficient "percolation".

[0120] C-terminal modification that reduces viscosity Specific modifications to the antibody C-terminus interfere with C1q binding and complement-dependent cytotoxicity (CDC). Van den Bremer et al. (2015), “Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation”,mAbs 7(4):672-80. The authors found that C-terminal lysine and C-terminal glutamate may reduce the tendency for Fc-Fc interactions, resulting in antibody hexamers that can most efficiently interact with Clq1. The authors constructed variants of CD20 and CD38 antibodies with PGKP (SEQ ID NO: 381), PGKKP (SEQ ID NO: 382), PGKKKP (SEQ ID NO: 383), and PGE at the C-terminus. They found that these variants significantly reduced or completely lost CDC activity. Thus, it can be concluded that the mutations blocked hexameration, which the authors previously found to correlate with CDC activity. Given the current correlation between hexameration and viscosity, such mutations should also reduce the viscosity of antigen-binding proteins. Therefore, it is reasonable to conclude that placing a positively or negatively charged amino acid at the C-terminus, whether it is an addition or substitution of the C-terminal amino acid present, reduces the viscosity of antigen-binding proteins.

[0121] Sequence modification to improve pharmacokinetic parameters The present invention also includes the discovery of improved pharmacokinetic properties of antigen-binding proteins having mutations that can reduce viscosity. In particular, the S440K mutation was found to improve both Tmax (time after administration at which the maximum concentration was observed) and Cmax (maximum observed concentration measured after administration). Variants of antibody AK having S440K, optionally possessing other mutations, were found to have a Tmax more than half that of the parent antibody AK after subcutaneous injection of the variant and the parent antibody at the same concentrations. Such variants were also found to have a Cmax 28% or 42% higher after subcutaneous injection of the variant and the parent antibody. See Figure 22.

[0122] nucleic acids, vectors, host cells The present invention also includes isolated nucleic acids encoding the bispecific antibodies of the present invention, for example, the light chain, light chain variable region, light chain constant region, heavy chain, heavy chain variable region, heavy chain constant region, linker, and some and all of these components, and combinations thereof, of the bispecific antibodies disclosed herein. The nucleic acids of the present invention include nucleic acids having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% homology to the nucleic acids of the present invention. When referring to specific sequences, the terms “similarity percentage,” “identity percentage,” and “homology percentage” are used as described in the University of Wisconsin GCG® software program. The nucleic acids of the present invention also include complementary nucleic acids. In some examples, the sequences, when aligned, are perfectly complementary (no mismatch). In other examples, there may be up to about 20% mismatch in the sequences. In some embodiments of the present invention, nucleic acids encoding both the heavy chain and the light chain of the antibody of the present invention are provided.

[0123] The nucleic acids of the present invention can be cloned into vectors, such as plasmids, cosmids, bacmids, phages, artificial chromosomes (BAC, YAC), or viruses, into which another gene sequence or element (DNA or RNA) can be inserted to result in replication of the linked sequence or element. In some embodiments, the expression vector contains a constitutively active promoter fragment (such as CMV, SV40, elongation factor, or LTR sequence, but not limited to these), or an inducible promoter sequence such as a steroid-inducible pIND vector (Invitrogen), and the expression of this nucleic acid can be regulated. The expression vector of the present invention may further include a regulatory sequence, such as an internal ribosome entry site. The expression vector can be introduced into cells, for example, by transfection.

[0124] In another embodiment, the present invention provides an expression vector comprising the following operably linked elements; a transcription promoter; a first nucleic acid molecule encoding the heavy chain of the bispecific antigen-binding protein, antibody, or antigen-binding fragment of the present invention; a second nucleic acid molecule encoding the light chain of the bispecific antigen-binding protein, antibody, or antigen-binding fragment of the present invention; and a transcription terminator. In another embodiment, the present invention provides an expression vector comprising the following operably linked elements; a first transcription promoter; a first nucleic acid molecule encoding the heavy chain of the bispecific antigen-binding protein, antibody, or antigen-binding fragment of the present invention; a first transcription terminator; a second transcription promoter; a second nucleic acid molecule encoding the light chain of the bispecific antigen-binding protein, antibody, or antigen-binding fragment of the present invention; and a second transcription terminator.

[0125] A secretion signal peptide sequence operably linked to the target coding sequence can also be optionally encoded by the expression vector, thereby causing recombinant host cells to secrete the expressed polypeptide, which can be more easily isolated from the cells if desired.

[0126] Recombinant host cells containing such vectors and expressing heavy and light chains are also provided.

[0127] purification Methods for purifying antibodies are known in the art and can be used in conjunction with the production of the antibodies and bispecific antibodies of the present invention. In some embodiments of the present invention, the antibody purification method includes filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. The filtration step preferably includes ultrafiltration, more preferably ultrafiltration and dialysis filtration. Filtration is preferably performed at least about 5 to 50 times, more preferably 10 to 30 times, and most preferably 14 to 27 times. Affinity column chromatography can be performed, for example, using PROSEP® affinity chromatography (Millipore, Billerica, MA). In preferred embodiments, the affinity chromatography step includes PROSEP®-vA column chromatography. The eluate can be washed in a solvent detergent. Cation exchange chromatography may include, for example, SP-Sepharose cation exchange chromatography. Anion exchange chromatography may include, but is not limited to, Q-Sepharose fast-flow anion exchange. The anion exchange step is preferably unbound, thereby allowing for the removal of contaminants including DNA and BSA. The antibody product is preferably nanofiltered, for example, using a Pall DV 20 nanofilter. The antibody product can be concentrated by, for example, ultrafiltration and dialysfiltration. This method may further include a step of size exclusion chromatography to remove aggregates. Further parameters of purification are described in the following examples.

[0128] Bispecific antibodies, antibody-or antigen-binding fragments can also be produced by other methods known in the art, such as the chemical binding of an antibody and an antibody fragment.

[0129] Each manuscript, research paper, review, abstract, patent application, patent, or other publication cited herein is incorporated herein by reference in its entirety. [Examples]

[0130] The present invention will be further clarified by the following embodiments, but these will not limit the scope of the invention.

[0131] Example 1 Fab mutation material Sets of 43 human and humanized recombinant monoclonal antibody molecules with different targets and sequences were prepared and purified according to standard procedures (Figures 1A and 1B). Sets with equivalent purity >98% were recovered by size exclusion chromatography (SEC). Samples were concentrated to 3 mL at a maximum pressure of 30 ± 10 psi at 2–8°C using an Amicon Ultrafiltration Stirred Cell Model 8003 (Millipore, Billerica, MA). They were then concentrated to 150 mg / mL in a pH 5.2 formulation buffer containing 20 mM acetate and 9% sucrose (without polysorbate) by approximate volume reduction, and the final concentration was determined (±10%) using protein absorbance at 280 nm (after dilution to within 0.1–1 absorbance unit (AU)) and protein-specific extinction coefficient.

[0132] Following similar standard procedures, several low-viscosity variants of the two mAbs were prepared, purified, and formulated. These included antibodies against proprotein convertase subtilisin / kexin type 9 (PCSK9, antibody AK) and against macrophage colony-stimulating factor (M-CSF, AO).

[0133] Viscosity measurement Viscosity analysis was performed using a Brookfield LV-DVIII cone plate instrument (Brookfield Engineering, Middleboro, MA, USA) with a CP-40 spindle and sample cup. All measurements were performed at 25°C, controlled by a water bath attached to the sample cup. Numerous viscosity measurements were collected manually within the specified torque range (10–90%) by increasing the spindle RPM. To simplify the resulting comparison chart, measurements were averaged to report one viscosity value per sample.

[0134] Array alignment Structural-based sequence alignment was performed using the Ab Initio software tool, which was developed using an Excel macro downloaded from the Department of Biochemistry at Zuerich University.

[0135] Example 2 Fc mutation Mutant expression and purification material and method • Anti-C-kit antibody (antibody BA, SEQ ID NOs. 174 and 176, encoded by the nucleic acids of SEQ ID NOs. 173 and 175, respectively), anti-sclerostin antibody AH, and anti-PCSK9 antibody AK • Anti-streptavidin IgG1 and IgG2. • 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) Solubilization by n-methyl-2-pyrrolidone • Size exclusion by light scattering (LS) in high-performance liquid chromatography (SE-HPLC) • Reverse-phase high-performance liquid chromatography (RA RP-HPLC) for reduction and alkylation • Trypsin peptide mapping by electrospray ionization mass spectrometry (ESI-MS) • Cone-plate viscometer

[0136] result SE-HPLC of EDC crosslinking of high-concentration monoclonal antibody solutions shows a tendency to form oligomers. The compound 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) has been used in other studies to chemically crosslink acidic residues in antibodies to primary amines (N-terminal and / or Lys residues) and to determine the region of protein-protein interaction. The proximity of the carboxyl group to the primary amine is important because an amide bond is formed between the two groups (Figure 16). The crosslinks formed are likely salt bridges present in solution. Carraway and Koshland, Jr. (1972). "Carbodiimide modification of proteins". Methods Enzymol 25:616-623. Antibody panels were chemically crosslinked with EDC under identical solution conditions. Previous rheological studies have shown that some antibodies in the panel were viscous and some were not (Figure 17). Non-viscous antibodies showed a slight increase in dimer content but did not contain large amounts of higher-order dimers. In contrast, viscous antibodies contained a large number of dimers and higher-order oligomers. A summary is shown in Figure 17. All antibodies confirmed to be viscous contained EDC crosslinked species that appeared to be larger than dimers. The appearance of larger oligomer species was concentration-dependent (antibody AH is shown as an example in Figure 18). To facilitate further analysis, the chemical crosslinking conditions were modified to complete the crosslinking reaction. After chemical crosslinking at 200 mg / mL, the solution became solid. The solid was resolubilized with buffer or 3% buffered NMP solution. SE-HPLC analysis of both samples showed similarity. The 3% buffered NMP solution significantly solubilized the protein more rapidly as more of the substance became solution. Antibody AH was resolubilized and further analyzed as an example.

[0137] Size analysis using SE-HPLC with online LS The crosslinked antibody solution was analyzed by SE-HPLC with online light scattering to determine the size of the eluted species. SE-HPLC was performed using online light scattering analysis of antibody AH after EDC chemical crosslinking and resolubilization with 3% NMP. SE-HPLC showed three peaks present in UV and RI. The first peak was identified as a species with a mass of 840.5 kD, which is close to the expected mass for the hexamer of antibody AH, which is 873.2 kD. Another species had a mass of 494.6 kD, which is close to the predicted mass of 436.6 kD for the trimer of antibody AH. A third species had a mass of 139.9 kD, which is close to the predicted mass of 145.5 kD for the monomer of antibody AH.

[0138] Reduction and alkylation of EDC crosslinked antibody AH in reverse-phase HPLC Crosslinked antibody solutions were analyzed by reductive and alkylation reverse-phase high-performance liquid chromatography. The recovery of the majority of both light chains (LC) and heavy chains (HC) was unexpected, as it was presumed that the HC or LC crosslinked with each other to form unnatural LC-HC peptides or unnatural LC-LC or HC-HC peptides reflecting the crosslinked oligomers observed by SEC analysis. In the case of antibody AH, the LC eluted at the exact same mass and location as the uncrosslinked antibody AH LC. There was a concentration-dependent change in the HC in antibody AH. A small amount of HC-HC crosslinking material with a confirmed mass of 100 kD (eluted at approximately 33 minutes, Figure 21) was present. At 150 mg / mL and 200 mg / mL, the distribution of HC species was similar. At lower protein concentrations, the distribution contained more species, eluting at approximately 28.5 minutes. The distribution of HC species correlates with the amount of oligomers present in each sample, as analyzed by SEC using a 200 mg / mL sample containing the maximum amount of hexamers and a 10 mg / mL sample containing very few oligomers. This pattern was observed in other viscous antibody solutions.

[0139] Example 3 Pharmacokinetic and pharmacodynamic (PKPD) studies of anti-PCSK9 parental antibody AK and low-viscosity variants in non-human primates. Materials: Antibody AK and its variants. All mutations in the heavy chain.

[0140] Fab variants: T82(72)R, R94(84)S, S95(85)R; Aho numbering (actual numbering)

[0141] Fc mutant S(434)K (S440K in EU numbering):

[0142] Double mutants: T82(72)R, R94(84)S, S95(85)R; S(434)K:

[0143] Four groups of four male cynomolgus monkeys were used in this study. Each group received a single subcutaneous (SC) dose of 10 mg / kg as follows: Group 1 received the parent antibody AK (140 mg / ml); Group 2 received the Fab variant (210 mg / ml); Group 3 received the Fc variant (210 mg / ml); and Group 4 received the Fab / Fc variant (210 mg / ml). Groups 1 and 2 also received SC doses as diluent controls. The Fab variant included substitutions at the following positions: T82(72)R, R94(84)S, and S95(85)R. The Fc variant included a substitution at the S(434)K position.

[0144] To measure viscosity at 210 mg / ml, the parent and all mutants were formulated at 210 mg / ml in 10 mM acetate, 155 mM N-acetylarginine (NAR), 70 mM ArgHCl, pH 5.4, and 0.01% polysorbate 80. Viscosity was measured using an ARG2 cone / plate at 1000 sec-1 and 25C. See Figure 15.

[0145] Overview of the study design: Four groups of four male crab-eating macaques Each group received a single dose of SC (10 mg / kg) as follows: Group 1: Parent antibody AK (140mg / ml) Group 2: Fab variant (210 mg / ml) Group 3: Fc variant (210 mg / ml) Group 4: Fab / Fc variant (210 mg / ml) • Groups 1 and 2 also received SC doses as a diluent control. • A skin biopsy was performed at the injection site three days after administration. • Conducting histopathological analysis Plasma LDL, HDL, total cholesterol, and PK were tracked for 6 weeks after administration.

[0146] Exam conclusions All four homologs produced a significant decrease in LDL cholesterol. • Maximum reduction 2 weeks after administration: 1 week later than previously observed in parents. • The bottom of the effect was not very deep with the Fab / Fc variant (approximately 78% vs. approximately 90%). • The return to baseline appears to be slightly more accelerated in the Fc mutant. • PK: Mean exposure levels (based on Cmax and AUClast) were similar across all treatment groups (within 1.4 times). Mutations in Fab and / or Fc of the anti-PCSK9 antibody AK did not significantly affect injection site response (ISR) or pharmacokinetic and pharmacodynamic (PKPD) profiles in non-human primates (NHPs) (cynomolgus monkeys). Fab variants: T82(72)R, R94(84)S, S95(85)R; Aho numbering (actual numbering). Fc variant: S(434)K (S440K in EU numbering).

[0147] Example 4 Generation and Characterization of Low-Viscosity Mutants of the GIPR(2G10.006) Antibody AQ Cloning, expression, purification, and high-concentration formulation of low-viscosity variants of antibody AQ. The GIPR(2G10.006) AQ parent is described as 2G10_LC1.006 (SEQ ID NO: 74 of the cited patent application) in U.S. Provisional Patent Application No. 62 / 387,486, which is incorporated herein by reference. A heavy chain variant AQ (HC 1, 17, 85) having mutation sites Q1(1)E, R17(16)G, S85(75)A, and a light chain variant AQ (LC 4 13 76 95 97 98) having mutation sites M4(4)L, V13(13)L, A76(60)D, S95(77)R, Q97(79)E, S98(80)P were prepared as follows. A synthetic gene for the GIPR(2G10.006) (antibody AQ) low viscosity variant was prepared, digested, and ligated into a plasmid expression vector. The construct was confirmed by DNA sequencing. A stable cell pool was prepared by electroporation of a clonal CHO host cell line. The pool was cultured under selection until the viability exceeded 85%. The pool was seeded into a fed-batch production culture for 10 days, and the centrifuged medium was collected.

[0148] The collected supernatant was sterile filtered and purified by three-column chromatography consisting of Protein A, cation exchange, and anion exchange, similar to the method described above (Shukla et al. (2007), “Downstream processing of monoclonal antibodies―Application of platform approaches”, J. Chrom. B 848:28-39). The resulting purified pool was dialyzed into a formulation buffer (without polysorbate) at pH 5.2 containing 20 mM acetate and 9% sucrose to achieve a final pH of approximately 5.2 and concentrated to approximately 150 mg / mL on a 30 kDa cut-off filter by tangential ultrafiltration (Figure 20A).

[0149] Measurement of Potency The potency was measured by an assay using mammalian cells 293 / huGIPR that express the glucose-dependent insulinotropic polypeptide receptor (GIPR). Increases in the concentrations of anti-GIPR parental AQ and the low-viscosity variants blocked the interaction between GIP and GIPR that induces cAMP changes monitored during the assay. The application of the assay was previously described in Tseng C.C. et al. (1996), “Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat”, J. Clin.. Invest. 98:2440-2445.

[0150] Measurement of viscosity Viscosity analysis of AQ and the two low-viscosity variants was performed on an Anton Paar rheometer using a CP25-1 / TG spindle. All measurements were made at 25 °C and controlled by a water bath attached to the sample cup. Viscosity measurements were collected manually while increasing the shear rate from 0 to 2000 rpm. Ten viscosity measurements at a shear rate of 10,00 1 / s and ten viscosity measurements at a shear rate of 2000 1 / s were collected for each sample and averaged, and one viscosity value per sample was reported.

[0151] Note that the accuracy of viscosity measurement is much better than the precision because viscosity measurement is sensitive to slight changes in several parameters such as the condition of the viscometer, the temperature in the room, and several other small parameters during measurement. Therefore, it is important to measure all the target samples in one setting or, if measuring the target samples in two settings, to have the same reference standard in the two settings.

[0152] Results The anti-GIPR(2G10.006) antibody AQ belongs to the high-viscosity germline subfamily of heavy chain VH3|3-33 and light chain VK3|L16. Several mutations derived from Figures 11 and 12 were generated in frame to reduce the viscosity of AQ. The viscosities of the parent AQ and the two mutants, measured at one viscometer setting, were as follows: AQ - 19.1 cP, AQ(HC 1, 17, 85) - 15.8 cP, AQ(LC 4 13 76 95 97 98) - 12.7 cP (Figure 20). Compared to the parent AQ, the heavy chain mutant AQ(HC 1, 17, 85) was 83%, and the light chain mutant AQ(LC 4 13 76 95 97 98) was 67%. Figures 7 and 8 show the positions of the mutants on viscosity vs. pI plots for VH3 and VK3 family members. In vitro cAMP activity was similarly unaffected by the viscosity mutations. Potency remained the same within the tolerance of the in vitro cell-based assay (see Figure 20B). In summary, the introduced mutations reduced viscosity without loss of potency.

[0153] Example 5 GIPR low viscosity mutant light chain V78F (LC V78F in Aho numbering, LC V62F in linear numbering) The GIPR(2G10.006) antibody AQ exhibited a high viscosity of 23 cP at 150 mg / mL in the A52Su formulation. This antibody was characterized by the low-frequency residue V78 (Aho numbering) (V62 linear numbering) in the kappa light chain (LC V78 Aho). In light chain sequences associated with the kappa germline, the occurrence frequency of V78 is <1%, while that of F78 is >98%. The residue LC V78 attracted attention because it deviates from the covariance. Analysis of covariance allows for the establishment of pairwise conserved residue positions based on the physiological and chemical characteristics of residues in the variable region of the antibody and identifies inaccurately placed residues (often non-germline residues). Covariance analysis may further suggest that the deviations may be due to amino acid substitutions at the deviant positions in more common germline sequences, which result in large conformational changes revealed by molecular dynamics simulations (Kannan G., "Method of correlated mutational analysis to improve therapeutic antibodies," U.S. Patent Application No. 61 / 451929, International Application PCT / US2012 / 028596, International Publication No. 2012 / 125495). To eliminate covariance deviations and increase the proportion of human sequences, the LC V78F mutation was introduced into the GIPR(2G10.006) antibody AQ.

[0154] Unexpectedly, the viscosity of the mutant decreased by 25%, but it maintained similar efficacy against human GIPR as measured by a cAMP (cell-based) assay. The sequences of both the GIPR(2G10.006)AQ parent and its LC V78F mutant are described in U.S. Provisional Patent Application No. 62 / 387,486 as 2G10_LC1.006 (SEQ ID NO: 74 of the cited application) and 2G10_LC1.003 (SEQ ID NO: 71 of the cited application), respectively. This U.S. patent application is incorporated herein by reference. What is newly discovered in this invention is that such substitution by the LC V78F mutation resulted in approximately 25% reduction in viscosity. Viscosity analysis of GIPR_2G10.006AQ and its V78F variant was performed at 150 mg / ml using an AR-G2 magnetic bearing cone-plate rheometer from TA Instruments-Waters LLC at a pH 5.2 formulation containing 20 mM acetate, 9% sucrose, and 0.01% polysorbate 80, at a shear rate of 1000 and 25C. The cone-plate size was 20 mm in diameter, with a cone angle of 1.988°, and equipped with a steel-990918 Peltier plate. The operation was performed using a flow sweep procedure. The measured viscosity values ​​were 21 cP for GIPR_2G10.006 and 15.3 cP for the GIPR(2G10.003)LC V78F variant, representing a 25% decrease in viscosity.

[0155] As mentioned in the previous example, the accuracy of viscosity measurement is far better than the precision. The viscosity at 150 mg / mL with 0.01% polysorbate is typically 10% lower than without polysorbate, as observed in the case of GIPR(2G10.006). Its viscosity was 23 cP without polysorbate (for all 43 antibodies) and 21 cP with polysorbate.

[0156] Example 6 Tests using cynomolgus monkeys The antibody referred to as AK (also known as control, AMG145, and evolocumab), as well as the Fab, Fc, and double mutants shown in Example 3, were generated using the methodology disclosed in Examples 1 and 2. The pharmacokinetic properties of these antibodies were tested in vivo by single subcutaneous bolus injection into male cynomolgus monkeys.

[0157] Test design The study was conducted using male cynomolgus monkeys. The animals were 2.7–3.8 years old and weighed 2.9–3.8 kg. For 7 days prior to the start of treatment, the animals were adapted to laboratory life. Inclusion criteria included acceptable outcomes from pre-treatment cholesterol levels (including LDL and HDL). Before the start of treatment, all animals were randomized and assigned to groups using a computer-based randomization procedure.

[0158] The test and control samples were administered subcutaneously once on day 1 into the central dorsal region of appropriate animals. The injection sites were shaved and marked with indelible ink before administration. Animals were temporarily restrained for dose administration and no sedatives were administered. The dose volume for each animal was based on the most recent body weight measurement. Groups 1 and 2 received the dose solution by two subcutaneous injections into the back of each animal (one dose of the test substance and one dose of the diluent). The injection sites were separated by at least 5–6 cm. The test substance was administered to the right side of the spine of each animal. The diluent was administered to the left side of the spine of each animal. Groups 3 and 4 received the dose solution by a single subcutaneous injection into the back of each animal. The dose levels and dose volumes for each group are summarized in Figure 21.

[0159] Blood samples were collected by venous puncture into tubes containing potassium (K2)EDTA at various time points during the survival period of this study (43 days). The animals were not fasted before serum chemical blood collection.

[0160] The samples were cooled after blood collection and then divided for serum or plasma preparation. The samples were gently mixed and centrifuged. Blood samples were kept on ice immediately after collection until centrifuged at approximately 4°C (1500-2000 × g for approximately 10 minutes). The resulting plasma or serum was separated, divided into two aliquots (primary and backup), transferred to appropriately labeled polypropylene tubes, and stored in a freezer set to maintain -80°C until analysis. Using plasma samples, test substance concentrations for pharmacokinetic evaluation were determined, and serum samples were analyzed for cholesterol, HDL, and LDL.

[0161] Pharmacokinetic evaluation Plasma samples were analyzed for the concentrations of each test antibody (antibody AK, AK Fab variant, AK S440K Fc variant, and AK Fab / S440K double variant) using enzyme-linked immunosorbent assay (ELISA). The assay used recombinant human PCSK9 as the capture reagent and horseradish peroxidase-labeled antibody against human IgG1 as the detection reagent. Standards and quality control (QC) samples were prepared by spiking antibody AK or its low-viscosity homolog into a 100% cynomolgus monkey K2-EDTA pool. Costar 9 6-well microplate wells (Corning Incorporated) were coated with recombinant human PCSK9. After a blocking step, the samples were pretreated at a dilution factor of 100 in Blocker® BLOTTO in TBS (Thermo Scientific) before loading the standards, matrix blanks (NSB), QC, and test samples into the microplate wells. Antibody AK in the samples was captured by the immobilized recombinant human PCSK9 coated on the microplate. Unbound material is removed by washing the microplate wells. After washing, mouse anti-human IgG, Ab35, HRP-binding detection antibody is added to the microplate wells to bind the captured antibody AK. Unbound detection antibody is removed by washing the microplate wells. A one-component TMB solution is added to the microplate wells for detection of the bound mouse anti-human IgG Ab35 HRP complex. The TMB substrate solution reacts with peroxide and, in the presence of HRP, produces a colorimetric signal proportional to the amount of antibody AK or low-viscosity mutant homolog bound by the capture reagent. Color development is stopped using 2N sulfuric acid, and the color intensity (optical density or OD) is measured at 450 nm - 650 nm. The data is reduced using the Watson version 7.4 SP3 (or later) data reduction package with a 4-parameter (Marquardt) regression model with a weighting coefficient of 1.

[0162] Pharmacokinetic parameters were estimated using WinNonlin pharmacokinetic software. A non-compartmental approach, consistent with the subcutaneous administration route, was used for parameter estimation. All parameters were generated from individual plasma concentrations from day 1. The following parameters were determined: Tmax (time after administration when the maximum observed concentration was observed), Cmax (maximum observed concentration measured after administration), AUC(0-t) (area under the concentration-time curve from the start of administration to the time after administration of the last quantifiable concentration observed, using a linear or linear / logarithmic trapezoidal method). AUC(0-t) / D (AUC(0-t) divided by the administered dose), and RAUC (area under the curve from T1 to T2 in the steady state divided by the area under the curve from T1 to T2 during the first dosing interval).

[0163] Results and Discussion The concentrations of the test samples plotted against time are shown in Figures 24A and 24B (mean concentration of each test sample, n=4 at each time point). The pharmacokinetic parameters of the four test samples are summarized in Figure 22. Antibodies containing the Fc mutation S440K (both antibody AK (Fc variant) and antibody AK (Fc and Fab double variant)) showed a decrease in Tmax (0.81 and 1 day vs. 2.5 days, respectively) and an increase in Cmax (125 μg / mL and 112 μg / mL vs. 87.8 μg / mL, respectively) compared to antibody AK (control), and antibody AK (Fab variant) showed that antibodies containing the viscosity-reducing Fc mutation were rapidly distributed into the bloodstream after subcutaneous injection.

[0164] Administration of all low-viscosity homologs of antibody AK resulted in a predicted pharmacological mild to moderate decrease in low-density lipoprotein (LDL) associated with a decrease in total cholesterol concentration compared to baseline (-6 days). The magnitude of the decrease in total lipoprotein cholesterol and low-density lipoprotein cholesterol after administration of the AK Fab variant and the AK S440K Fc variant was generally similar to that of control animals with a tendency to recover to baseline at day 25 for the AK S440K Fc variant and at day 29 for AK (control) and the AK Fab variant. The magnitude of the decrease in total lipoprotein cholesterol and low-density lipoprotein cholesterol for the AK Fab / S440K double variant was generally not as significant compared to the control (antibody AK at 10 mg / kg). There was no change in high-density lipoprotein for any of the AK low-viscosity homologs. The rate of change of LDL-C relative to baseline is presented in tabular form in Figure 23 and plotted against time in Figures 24A and 24B.

[0165] Abbreviations The abbreviations used throughout this specification are defined as follows. AEI Allelic expression imbalance ANOVA Analysis of variance AUC Area under the curve BSA Bovine serum albumin DMEM Dulbecco's modified Eagle's medium DMSO Dimethyl sulfoxide EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay eQTL Expression quantitative trait locus ESI-TOF Electrospray ionization time of flight [[ID=3o]]FACS Fluorescence-activated cell sorting FBS Fetal bovine serum FPLC Fast protein liquid chromatography FVB A mouse strain congenic for the Friend leukemia virus 1b (Fv1b) allele H&E Hematoxylin-Eosin HA Hypoxanthine HIC Hydrophobic Interaction Chromatography HPLC (High-Performance Liquid Chromatography) HRP (Horseradish Peroxidase) HUVEC Human Umbilical Vein Epithelial Cells IBD inflammatory bowel disease IDMEM (Glutamine-free DMEM) IFN (Interferon) IL Interleukin MCP (Monocyte Chemotropic Protein) MSD Polymer Structure Database PBMC peripheral blood mononuclear cells PBS (phosphate-buffered saline) PCR (polymerase chain reaction) PEG polyethylene glycol PEI Polyethyleneimine QTL (Quantitative Trait Locus) Culture medium developed at RPMI Roswell Park Memorial Institute SNP (Single Nucleotide Polymorphism) TFA (Trifluoroacetic Acid) TMB 3,3',5,5'-Tetramethylbenzidine

[0166] The present invention provides, for example, the following: (Item 1) A method for reducing the viscosity of an antigen-binding protein, a. If the antigen-binding protein includes the VH1|1-18 germline subfamily, the VH1 sequence is 82X 1 , 94X 2 , and 95X 3 Modify to include one or more substitutions selected from (where X 1 is selected from R, K, and H, and X 2 is selected from S, T, N, and Q, X 3 (This is selected from R, K, and H); b. If the antigen-binding protein includes the VH3|3-33 germline subfamily, the VH3 sequence is 1X 4 , 17X 5 , and 85X 6 Modify to include one or more substitutions selected from (where X 4 The choice is between D and E, and X 5 is selected from G, A, V, I, L, and M and W, X 6 (This is selected from G, A, V, I, L, and M); c. If the antigen-binding protein includes the VK3|L16 germline subfamily, the VK3 sequence is 4X 10 , 13X 11 , 76X 12 78F, 95X 13 , 97X 14 , and modify to include one or more substitutions selected from 98P (where X 10 The following are selected from G, A, V, I, L, and M, and X 11 The following are selected from G, A, V, I, L, and M, and X 12 The choice is between D and E, and X 13 is selected from R, K, and H, and X 14 (This is selected from D and E); d. If the antigen-binding protein includes the VK3|L6 germline subfamily, the VK3 sequence is 76X 12 and 95X 13 Modify to include one or more substitutions selected from; e.Fc domain sequence 253X 15 , 440X 16 , and 439X 17 Modify to include one or more substitutions selected from (where X 15 The following are selected from G, A, V, I, L, and M, and X 16 is selected from R, K, and H, and X 17 The Fc domain sequence is selected from D and E, and the Fc domain sequence is 440X 16 and 439X 17 It contains only one of the following; and f. The C-terminus of the Fc domain sequence is X18 X 19 Modify to include (where X 18 is 1-4 amino acids selected from D and E, or H, K and R, and X 19 is selected from P, M, G, A, V, I, L, S, T, N, Q, F, Y and W, X 18 It does not exist when it contains D or E, X 18 It exists when it contains K or R at its C-terminus, X 18 (Whether it exists or not when it contains H at its C-terminus) This includes making one or more modifications to the sequence of the antigen-binding protein selected from, The amino acids in subparagraphs a, b, c, and d are numbered using the Aho numbering system, while the amino acids in subparagraph e are numbered using the EU numbering system. (Item 2) The antigen-binding protein is selected from the sequence of the antibody (SEQ ID NOs. 166 and 168; 2 and 4; 178 and 180; 170 and 172; 6 and 8; 10 and 12; 14 and 16; 18 and 20; 22 and 24; 26 and 28; 30 and 32; 34 and 36; 38 and 40; 43 and 44; 46 and 48; 50 and 52; 54 and 56; 58 and 60; 62 and 64; 66 and 68; 70 and 72; 74 and The method described in item 1, including 76; 78 and 80; 82 and 84; 86 and 88; 90 and 92; 94 and 96; 98 and 100; 102 and 104; 106 and 108; 110 and 112; 114 and 116; 118 and 120; 122 and 124; 126 and 128; 130 and 132; 134 and 136; 158 and 160; 138 and 140; 142 and 144; 146 and 148; 150 and 152; and 154 and 156). (Item 3) The method according to item 1, comprising modifying the VH1 sequence of an antigen-binding protein comprising the VH1|1-18 germline subfamily to include one or more substitutions selected from 82R, 94S, and 95R. (Item 4) The method according to item 2, comprising modifying the VH1 sequence of an antigen-binding protein comprising the VH1|1-18 germline subfamily to include 82R, 94S, and 95R substitutions. (Item 5) The VH1 sequence of the antigen-binding protein containing the VH1|1-18 germline subfamily is 59X 20 (Here, X 20 The method of item 1, further comprising modifying it to include the substitution of (selected from R, K, and H). (Item 6) The method according to item 1, further comprising modifying the VH1 sequence of an antigen-binding protein comprising the VH1|1-18 germline subfamily to include a 59K substitution. (Item 7) The method according to item 2, further comprising modifying the VH1 sequence of an antigen-binding protein comprising the VH1|1-18 germline subfamily to include a 59K substitution. (Item 8) The method according to item 3, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies AF, AK, AL, AN, and AO from Figure 1B, excluding substitutions (SEQ ID NOs: 114 and 116; 134 and 136; 138 and 140; 146 and 148; and 150 and 152). (Item 9) The method according to item 1, comprising modifying the VH3 sequence of an antigen-binding protein comprising the VH3|3-33 germline subfamily to include one or more substitutions selected from 1E, 17G, and 85A. (Item 10) The method according to item 2, comprising modifying the VH3 sequence of an antigen-binding protein comprising the VH3|3-33 germline subfamily to include substitutions of 1E, 17G, and 85A. (Item 11) The method according to item 10, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies AQ, AM, AI, and AG from Figure 1B, excluding substitutions (SEQ ID NOs. 158 and 160; 142 and 144; 126 and 128; and 118 and 120). (Item 12) The method according to item 1, comprising modifying the VK3|L16 sequence of the antigen-binding protein to include one or more substitutions selected from 4L, 13L, 76D, 78F, 95R, 97E, and 98P. (Item 13) The method according to item 2, comprising modifying the VK3|L16 sequence of the antigen-binding protein to include substitutions of 4L, 13L, 76D, 78F, 95R, 97E, and 98P. (Item 14) The method according to item 13, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies AF and AQ from Figure 1B, excluding substitutions (SEQ ID NOs: 114 and 116; and 158 and 160). (Item 15) The method according to item 1, comprising modifying the VK3|L6 sequence of the antigen-binding protein to include one or more substitutions selected from 76D and 95R. (Item 16) The method according to item 2, comprising modifying the VK3|L6 sequence of an antigen-binding protein to include substitutions at 76D and 95R. (Item 17) The antigen-binding protein is the method described in item 16, comprising the sequences of antibody AJ from Figure 1B (SEQ ID NOs. 130 and 132), excluding substitutions. (Item 18) The method according to item 1, comprising modifying the Fc domain sequence to include one or more substitutions selected from 253A, 440K, and 439E. (Item 19) The method according to item 2, comprising modifying the Fc domain sequence to include substitutions at 253A, 440K, and 439E. (Item 20) The method according to item 18, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies BA, AH, and AN (SEQ ID NOs: 174 and 176; 122 and 124; and 146 and 148), excluding substitutions. (Item 21) The method according to item 1, comprising modifying the C-terminus of the Fc domain to include an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 22) The method according to item 2, comprising modifying the C-terminus of the Fc domain to include an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 23) The method according to item 18, comprising modifying the C-terminus of the Fc domain to include an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 24) a.82X 1 , 94X 2 , and 95X 3 VH1|1-18 germline subfamily sequences containing one or more substitutions selected from (where X 1 is selected from R, K, and H, and X 2 is selected from S, T, N, and Q, X 3 (This is selected from R, K, and H); b.1X 4 , 17X 5 , and 85X 6 VH3|3-33 germline subfamily sequences containing one or more substitutions selected from (where X 4 The choice is between D and E, and X 5 The following are selected from G, A, V, I, L, and M, and X 6 (This is selected from GA, V, I, L, and M); c.4X 10 , 13X 11 , 76X 12 , 95X 13 , 97X 14, and VK3|L16 germline subfamily sequences containing one or more substitutions selected from 98P (where X 10 The following are selected from G, A, V, I, L, and M, and X 11 The following are selected from G, A, V, I, L, and M, and X 12 The choice is between D and E, and X 13 is selected from R, K, and H, and X 14 (The antigen-binding protein is selected from D and E, and does not contain only substitution 78F); d.76X 12 and 95X 13 VK3|L6 germline subfamily sequences containing one or more substitutions selected from; e.253X 15 , 440X 16 , and 439X 17 Fc domain sequence containing one or more substitutions selected from (where X 15 The following are selected from G, A, V, I, L, and M, and X 16 is selected from R, K, and H, and X 17 The Fc domain sequence is selected from D and E, and the Fc domain sequence is 440X 16 and 439X 17 It includes only one of X 16 Is K or X 17 When is E, the antigen-binding protein is 253X 15 , or comprising at least one modification selected from subparagraphs a, b, c, d and f, wherein the antigen-binding protein specifically binds to CD20); and f. X at its C-terminus 18 X 19 Fc domain sequence containing (where X 18 is 1-4 amino acids selected from D and E, or H, K and R, and X 19 is selected from P, M, G, A, V, I, L, S, T, N, Q, F, Y, and W, and X 18 It does not exist when it contains D or E, X 18 It exists when it contains K or R at its C-terminus, X 18When H is present at its C-terminus, it is present or absent, and when PGKP (SEQ ID NO: 381), PGKKP (SEQ ID NO: 382), PGKKKP (SEQ ID NO: 383), or PGE appears at the C-terminus of the Fc domain, the antigen-binding protein is 253X 15 (or comprising at least one substitution selected from subparagraphs a-e, wherein the antigen-binding protein specifically binds to CD20 or CD38.) An antigen-binding protein comprising one or more sequence modifications selected from, The amino acids in subparagraphs a, b, c, and d are numbered using the Aho numbering system, and the amino acids in subparagraph e are numbered using the EU numbering system for antigen-binding proteins. (Item 25) The antigen-binding protein is selected from the sequence of the antibody (SEQ ID NOs: 166 and 168; 2 and 4; 178 and 180; 170 and 172; 6 and 8; 10 and 12; 14 and 16; 18 and 20; 22 and 24; 26 and 28; 30 and 32; 34 and 36; 38 and 40; 43 and 44; 46 and 48; 50 and 52; 54 and 56; 58 and 60; 62 and 64; 66 and 68; 70 and 72; 74 and 76; 7 Antigen-binding proteins as described in item 24, including 8 and 80; 82 and 84; 86 and 88; 90 and 92; 94 and 96; 98 and 100; 102 and 104; 106 and 108; 110 and 112; 114 and 116; 118 and 120; 122 and 124; 126 and 128; 130 and 132; 134 and 136; 158 and 160; 138 and 140; 142 and 144; 146 and 148; 150 and 152; and 154 and 156). (Item 26) The VH1|1-18 germline subfamily sequence comprises one or more substitutions selected from 82R, 94S, and 95R, as described in item 24, for the antigen-binding protein. (Item 27) The VH1|1-18 germline subfamily sequence is the antigen-binding protein described in item 25, including the 82R, 94S, and 95R substitutions. (Item 28) The aforementioned VH1|1-18 germline subfamily sequence is 59X 20 (Here, X 20 Antigen-binding proteins as described in item 24, including substitutions (selected from R, K, and H). (Item 29) The VH1|1-18 germline subfamily sequence is the antigen-binding protein described in item 24, including a 59K substitution. (Item 30) The VH1|1-18 germline subfamily sequence is the antigen-binding protein described in item 25, including a 59K substitution. (Item 31) The antigen-binding protein is the antigen-binding protein described in item 30, comprising the sequence of an antibody selected from the antibodies AF, AK, AL, AN, and AO from Figure 1B, excluding substitutions (SEQ ID NOs: 114 and 116; 134 and 136; 138 and 140; 146 and 148; and 150 and 152). (Item 32) The VH3|3-33 germline subfamily sequence comprises one or more substitutions selected from 1E, 17G, and 85A, as described in item 24, for the antigen-binding protein. (Item 33) The VH3|3-33 germline subfamily sequence is the antigen-binding protein described in item 25, including the substitutions 1E, 17G, and 85A. (Item 34) The antigen-binding protein is the antigen-binding protein described in item 33, comprising the sequence of an antibody selected from antibodies AQ, AM, AI, and AG from Figure 1B, excluding substitutions (SEQ ID NOs. 158 and 160; 142 and 144; 126 and 128; and 118 and 120). (Item 35) The VK3|L16 sequence is an antigen-binding protein as described in item 24, comprising one or more substitutions selected from 4L, 13L, 76D, 78F, 95R, 97E, and 98P. (Item 36) The VK3|L16 sequence is the antigen-binding protein described in item 25, comprising the substitutions 4L, 13L, 76D, 95R, 97E, and 98P. (Item 37) The VK3|L16 sequence comprises one or more substitutions selected from 4L, 13L, 76D, 95R, 97E, and 98P, as described in item 26, for the antigen-binding protein. (Item 38) The VK3|L16 sequence comprises one or more substitutions selected from 4L, 13L, 76D, 95R, 97E, and 98P, as described in item 29, for the antigen-binding protein. (Item 39) The VK3|L16 sequence comprises one or more substitutions selected from 4L, 13L, 76D, 95R, 97E, and 98P, as described in item 32, for the antigen-binding protein. (Item 40) The antigen-binding protein is the antigen-binding protein described in item 36, comprising the sequence of an antibody selected from antibodies AF and AQ from Figure 1B, excluding substitutions (SEQ ID NOs: 114 and 116; and 158 and 160). (Item 41) The VK3|L6 sequence is an antigen-binding protein as described in item 24, comprising one or more substitutions selected from 76D and 95R. (Item 42) The VK3|L6 sequence is the antigen-binding protein described in item 25, including the substitutions 76D and 95R. (Item 43) The VK3|L6 sequence is the antigen-binding protein described in item 26, including the substitutions 76D and 95R. (Item 44) The VK3|L6 sequence is the antigen-binding protein described in item 29, including the substitutions 76D and 95R. (Item 45) The VK3|L6 sequence is the antigen-binding protein described in item 32, including the substitutions 76D and 95R. (Item 46) The antigen-binding protein is the antigen-binding protein described in item 39, comprising the sequence of antibody AJ from Figure 1B (SEQ ID NOs. 130 and 132), excluding substitutions. (Item 47) An antigen-binding protein as described in item 24, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 48) An antigen-binding protein as described in item 25, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 49) The antigen-binding protein is the antigen-binding protein described in item 42, comprising the sequence of an antibody selected from antibodies BA, AH, and AN (SEQ ID NOs. 174 and 176; 122 and 124; and 146 and 148), excluding substitutions. (Item 50) An antigen-binding protein as described in item 24, comprising an Fc domain sequence containing one or more substitutions selected from the 253A, 440K, and 439E substitutions. (Item 51) An antigen-binding protein as described in item 26, comprising an Fc domain sequence containing one or more substitutions selected from the 253A, 440K, and 439E substitutions. (Item 52) An antigen-binding protein as described in item 29, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 53) An antigen-binding protein as described in item 32, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 54) An antigen-binding protein as described in item 35, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 55) An antigen-binding protein as described in item 37, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 56) An antigen-binding protein as described in item 38, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 57) An antigen-binding protein as described in item 39, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 58) An antigen-binding protein as described in item 41, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 59) An antigen-binding protein as described in item 42, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 60) An antigen-binding protein as described in item 43, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 61) An antigen-binding protein as described in item 44, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 62) An antigen-binding protein as described in item 45, comprising an Fc domain sequence containing one or more substitutions selected from 253A, 440K, and 439E. (Item 63) The antigen-binding protein described in item 24, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 64) The antigen-binding protein described in item 26, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 65) The antigen-binding protein described in item 29, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 66) The antigen-binding protein described in item 32, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 67) The antigen-binding protein described in item 35, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 68) The antigen-binding protein described in item 37, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 69) The antigen-binding protein described in item 38, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 70) The antigen-binding protein described in item 39, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 71) The antigen-binding protein described in item 41, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 72) The antigen-binding protein described in item 42, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 73) The antigen-binding protein described in item 43, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 74) The antigen-binding protein described in item 44, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 75) The antigen-binding protein described in item 45, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 76) The antigen-binding protein described in item 47, wherein the C-terminus of the Fc domain contains an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item 77) A series of substitutions selected from Figure 10: a. T82R, R94S, and S95R in heavy chains, b. S59K, T82R, R94S, and S95R in heavy chains c. T82R, R94S, and S95R in the heavy chain, and G13L in the light chain, d. S59K, T82R, R94S, and S95R in heavy chains, and G13L in light chains. Except for including the heavy and light chain sequences of the PCSK-9-binding polypeptide, The aforementioned amino acids are numbered using the Aho numbering system. An antigen-binding protein that specifically binds to PCSK9. (Item 78) The PCSK-9-binding polypeptide is the antigen-binding protein described in item 77, comprising the heavy and light chain sequences of antibody AK (SEQ ID NOs. 134 and 136) from Figure 1B. (Item 79) An antigen-binding protein that specifically binds to PCSK9, comprising the heavy chain sequence of antibody AK (SEQ ID NO: 136) in Figure 1B, except that it contains one or more substitutions of I253A, S440K, or K439E, wherein the amino acids are numbered according to the EU numbering system. (Item 80) The antigen-binding protein described in item 77, wherein the heavy chain further comprises one or more substitutions of I253A, S440K, or K439E, and the amino acids are numbered according to the EU numbering system. (Item 81) An antigen-binding protein that specifically binds to PCSK9, containing an amino acid sequence selected from SEQ ID NOs. 352, 353, and 354. (Item 82) The antigen-binding protein described in item 81 further comprises amino acid sequences selected from SEQ ID NOs. 351 and 367. (Item 83) A series of substitutions selected from Figure 10: a. T82R, R94S, and S95R in heavy chains, b. S59K, T82R, R94S, and S95R in heavy chains c. T82R, R94S, and S95R in the heavy chain, and V13L in the light chain, d. S59K, T82R, R94S, and S95R in heavy chains, and V13L in light chains. Except for including the heavy and light chain sequences of antibody AO in Figure 1B (SEQ ID NOs: 150 and 152), The aforementioned amino acids are numbered using the Aho numbering system. An antigen-binding protein that specifically binds to c-fms. (Item 84) An antigen-binding protein that specifically binds to c-fms, containing an amino acid sequence selected from SEQ ID NOs. 356, 357, and 358. (Item 85) The antigen-binding protein described in item 84, further containing the amino acid sequence of sequence number 355. (Item 86) An antigen-binding protein that specifically binds to GIPR, containing an amino acid sequence selected from SEQ ID NOs. 359, 361, 362, 364, and 368. (Item 87) The antigen-binding protein described in item 86 further comprises amino acid sequences selected from SEQ ID NOs. 360, 363, 365, and 367. (Item 88) a. The antigen-binding protein is 440X 16 Substitution 440X for parent antibodies lacking substitution 16 Includes, b. When the antigen-binding protein and the parent antibody are administered by subcutaneous injection at the same concentration, the antigen-binding protein reaches its maximum serum concentration faster than the parent antibody, and c. When the antigen-binding protein and the parent antibody are administered by subcutaneous injection at the same concentration, the antigen-binding protein reaches a higher maximum serum concentration than the parent antibody. The antigen-binding protein described in item 24. (Item 89) The antigen-binding protein described in item 88 is the antigen-binding protein described in item 88 that reaches its maximum serum concentration at a rate at least about twice as fast as the parent antibody. (Item 90) The antigen-binding protein described in item 88 is the antigen-binding protein described in item 88 that reaches a maximum serum concentration at least about 25% higher than that of the parent antibody. (Item 91) The parent antibody is the antigen-binding protein described in item 88, which is a PCSK-9 conjugated polypeptide. (Item 92) The parent antibody is antibody AK (SEQ ID NOs. 134 and 136) from Figure 1B, the antigen-binding protein described in item 88. (Item 93) X 16 It is K, the antigen-binding protein described in item 88. (Item 94) X 16 It is K, the antigen-binding protein described in item 89. (Item 95) X 16 It is K, the antigen-binding protein described in item 90. (Item 96) X 16 It is K, the antigen-binding protein described in item 91. (Item 97) X 16 It is K, the antigen-binding protein described in item 92. (Item 98) A method for preparing an antigen-binding protein that, when administered at the same concentration, reaches a higher maximum serum concentration than the parent antibody and reaches the maximum serum concentration faster than the parent antibody, wherein the parent antibody is sequence-modified 440X 16 (Here, X 16 A method that involves introducing (which is selected from R, K, and H). (Item 99) The method according to item 98, wherein the antigen-binding protein reaches its maximum serum concentration at least twice as fast as the parent antibody. (Item 100) X 16 The method described in item 98, where K is the same. (Item 101) X 16 The method described in item 99, where K is the same. (Item 102) The method according to item 98, wherein the parent antibody is a PCSK9-conjugated polypeptide. (Item 103) The method according to item 99, wherein the parent antibody is a PCSK9-conjugated polypeptide. (Item 104) The method according to item 100, wherein the parent antibody is a PCSK9-conjugated polypeptide. (Item 105) The aforementioned parent antigen-binding protein is the antibody AK (SEQ ID NOs. 134 and 136) from Figure 1B, the antigen-binding protein described in item 98. (Item 106) The aforementioned parent antigen-binding protein is the antibody AK (SEQ ID NOs. 134 and 136) from Figure 1B, the antigen-binding protein described in item 99. (Item 107) The aforementioned parent antigen-binding protein is the antibody AK (SEQ ID NOs. 134 and 136) from Figure 1B, as described in item 100. (Item 108) A method for treating PCSK9-related symptoms in a patient, comprising administering an antigen-binding protein as described in item 81. (Item 109) The symptom associated with PCSK9 is hypercholesterolemia, as described in item 108. (Item 110) A method for treating PCSK9-related symptoms in a patient, comprising administering an antigen-binding protein as described in item 82. (Item 111) The symptom associated with PCSK9 is hypercholesterolemia, as described in item 110. (Item 112) A method for treating PCSK9-related symptoms in a patient, comprising administering an antigen-binding protein as described in item 88. (Item 113) The symptom associated with PCSK9 is hypercholesterolemia, as described in item 112. (Item A1) A method for reducing the viscosity of an antigen-binding protein, a. If the antigen-binding protein includes the VH1|1-18 germline subfamily, the VH1 sequence is 82X 1 , 94X 2 , and 95X 3 Modify to include one or more substitutions selected from (where X 1 is selected from R, K, and H, and X 2 is selected from S, T, N, and Q, X 3 (This is selected from R, K, and H); b. If the antigen-binding protein includes the VH3|3-33 germline subfamily, the VH3 sequence is 1X 4 , 17X 5 , and 85X 6 Modify to include one or more substitutions selected from (where X 4 The choice is between D and E, and X 5 is selected from G, A, V, I, L, and M and W, X 6 It is selected from G, A, V, I, L, and M; c. If the antigen-binding protein includes the VK3|L16 germline subfamily, the VK3 sequence is 4X 10 , 13X11 , 76X 12 78F, 95X 13 , 97X 14 , and modify to include one or more substitutions selected from 98P (where X 10 The following are selected from G, A, V, I, L, and M, and X 11 The following are selected from G, A, V, I, L, and M, and X 12 The choice is between D and E, and X 13 is selected from R, K, and H, and X 14 (This is selected from D and E); d. If the antigen-binding protein includes the VK3|L6 germline subfamily, the VK3 sequence is 76X 12 and 95X 13 Modify to include one or more substitutions selected from; e.Fc domain sequence 253X 15 , 440X 16 , and 439X 17 Modify to include one or more substitutions selected from (where X 15 The following are selected from G, A, V, I, L, and M, and X 16 is selected from R, K, and H, and X 17 The Fc domain sequence is selected from D and E, and the Fc domain sequence is 440X 16 and 439X 17 It contains only one of the following; and f. The C-terminus of the Fc domain sequence is X 18 X 19 Modify to include (where X 18 is 1-4 amino acids selected from D and E, or H, K and R, and X 19 is selected from P, M, G, A, V, I, L, S, T, N, Q, F, Y and W, X 18 It does not exist when it contains D or E, X 18 It exists when it contains K or R at its C-terminus, X 18 (Whether it exists or not when it contains H at its C-terminus) This includes making one or more modifications to the sequence of the antigen-binding protein selected from, The amino acids in subparagraphs a, b, c, and d are numbered using the Aho numbering system, while the amino acids in subparagraph e are numbered using the EU numbering system. (Item A2) The antigen-binding protein is selected from the sequence of the antibody (SEQ ID NOs. 166 and 168; 2 and 4; 178 and 180; 170 and 172; 6 and 8; 10 and 12; 14 and 16; 18 and 20; 22 and 24; 26 and 28; 30 and 32; 34 and 36; 38 and 40; 43 and 44; 46 and 48; 50 and 52; 54 and 56; 58 and 60; 62 and 64; 66 and 68; 70 and 72; 74 and The method described in item A1, including 76; 78 and 80; 82 and 84; 86 and 88; 90 and 92; 94 and 96; 98 and 100; 102 and 104; 106 and 108; 110 and 112; 114 and 116; 118 and 120; 122 and 124; 126 and 128; 130 and 132; 134 and 136; 158 and 160; 138 and 140; 142 and 144; 146 and 148; 150 and 152; and 154 and 156). (Item A3) The method according to item A1 or 2, comprising modifying the VH1 sequence of an antigen-binding protein comprising the VH1|1-18 germline subfamily to include one or more substitutions selected from 82R, 94S, and 95R. (Item A4) The VH1 sequence of the antigen-binding protein containing the VH1|1-18 germline subfamily is 59X 20 (Here, X 20 The method of item A1 or 2, further comprising modifying to include the substitution of (selected from R, K, and H). (Item A5) The method according to item A3, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies AF, AK, AL, AN, and AO from Figure 1B, excluding substitutions (SEQ ID NOs: 114 and 116; 134 and 136; 138 and 140; 146 and 148; and 150 and 152). (Item A6) The method according to item A1 or 2, comprising modifying the VH3 sequence of an antigen-binding protein comprising the VH3|3-33 germline subfamily to include one or more substitutions selected from 1E, 17G, and 85A. (Item A7) The method according to item A6, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies AQ, AM, AI, and AG from Figure 1B, excluding substitutions (SEQ ID NOs: 158 and 160; 142 and 144; 126 and 128; and 118 and 120). (Item A8) The method according to item A1 or 2, comprising modifying the VK3|L16 sequence of the antigen-binding protein to include one or more substitutions selected from 4L, 13L, 76D, 78F, 95R, 97E, and 98P. (Item A9) The method according to item A8, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies AF and AQ from Figure 1B, excluding substitutions (SEQ ID NOs: 114 and 116; and 158 and 160). (Item A10) The method according to item A1 or 2, comprising modifying the VK3|L6 sequence of the antigen-binding protein to include one or more substitutions selected from 76D and 95R. (Item A11) The antigen-binding protein is the method described in item A10, comprising the sequences of antibody AJ from Figure 1B (SEQ ID NOs. 130 and 132), excluding substitutions. (Item A12) The method according to item A1 or 2, comprising modifying the Fc domain sequence to include one or more substitutions selected from 253A, 440K, and 439E. (Item A13) The method according to item A12, wherein the antigen-binding protein comprises the sequence of an antibody selected from antibodies BA, AH, and AN (SEQ ID NOs: 174 and 176; 122 and 124; and 146 and 148), excluding substitutions. (Item A14) The method according to item A1 or 2, comprising modifying the C-terminus of the Fc domain to include an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E. (Item A15) The method according to item A14, comprising modifying the C-terminus of the Fc domain to include an amino acid sequence selected from KP, KKP, KKKP (SEQ ID NO: 380), and E.

Claims

[Claim 1] The method described in the specification or an antigen-binding protein.

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