Artificial protein and pharmaceutical composition

Abstract

Provided is a novel substance suitable for functioning as a Met agonist. Provided is an artificial protein comprising an Fc region of an antibody, in which an amino acid sequence represented by SEQ ID NO: 1 is inserted (in SEQ ID NO: 1, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, and X11 each independently represent an amino acid, and n1 and n2 each independently represent an integer of 0-6). (X1) n1YX3X4FYYNYX5X6X7WX8X9X10X11 (X2) n2 (serial number 1).

Description

Technical Field

[0001] The present invention relates to artificial proteins and pharmaceutical compositions. Background Art

[0002] The maintenance and recovery of liver function contribute to the treatment of hepatitis, but the current situation is that there is no extremely effective treatment for the maintenance and recovery of liver function, and it is difficult to improve liver fibrosis in particular. Regarding hepatocytes, stellate cells, and liver macrophages, in mice with Met (hepatocyte growth factor (HGF) receptor) deficiency in each cell specificity, the hepatitis condition worsens. On the other hand, in various liver disease models, it is shown that HGF promotes regeneration, inhibits inflammation, and improves fibrosis through Met activation. It is known that Met-HGF is corely involved in morphogenesis in development, wound repair, and organ homeostasis maintenance by stimulating cell growth, survival, and migration. Recombinant HGF protein shows therapeutic effects in preclinical models and patients with specific diseases (non-patent literature 1-20).

[0003] Prior art literature

[0004] Non-patent literature

[0005] Non-patent literature 1: Trusolino, L., Bertotti, A., & Comoglio, PMMET signaling: principles and functions in development, organ regeneration and cancer. Nat. Rev. Mol. Cell. Biol. 11, 834-848 (2010).

[0006] Non-patent literature 2: Sakai, K., Aoki, S., & Matsumoto, K. Hepatocyte growth factor and Met in drug discovery. J. Biochem. 157, 271-284 (2015).

[0007] Non-patent document 3: Desole, C. et al., HGF and MET: From brain development to neurological disorders. Front Cell Dev Biol. 9, 683609 (2021).

[0008] Non-Patent Document 4: Hirano, S. et al., A phase I / II exploratory clinical trial for intracordal injection of recombinant hepatocyte growth factor for vocal fold scar and sulcus. J Tissue Eng Regen Med. 12, 1031 - 1038 (2018).

[0009] Non-Patent Document 5: Nagoshi, N. et al., Phase I / II Study of intrathecal administration of recombinant human hepatocyte growth factor in patients with acute spinal cord injury: A double-blind, randomized clinical trial of safety and efficacy. J Neurotrauma. 37, 1752 - 1758 (2020).

[0010] Non-Patent Document 6: Ido, A. et al., Safety and pharmacokinetics of recombinant human hepatocyte growth factor (rh-HGF) in patients with fulminant hepatitis: A phase I / II clinical trial, following preclinical studies to ensure safety. J Transl Med. 9, 55 (2011).

[0011] Non-Patent Document 7: Li, N. et al., Therapeutic effect of HGF on NASH mice through HGF / c-Met and JAK2-STAT3 signalling pathway. Ann Hepatol. 17, 501 - 510 (2018).

[0012] Non-patent document 8: Yang, YM et al., Interventional potential of recombinant feline hepatocyte growth factor in a mouse model of non-alcoholic steatohepatitis. Front Endocrinol. 9, 378 (2018).

[0013] Non-patent literature 9: Matsuda, Y. et al., Hepatocyte growth factor prevents liver cirrhosis caused by dimethylnitrosamine in rats. J Biochem. 118, 643-649 (1995).

[0014] Non-patent literature 10: Mizuno, S. et al., Hepatocyte growth factor prevents renal fibrosis and dysfunction in a mouse model of chronic renal disease. J Clin Invest. 101, 1827-1834 (1998).

[0015] Non-patent literature 11: Kawaida, K. et al., Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proc Natl Acad Sci USA. 91, 4357-4361 (1994).

[0016] Non-patent literature 12: Kosai, K. Hepatocyte growth factor abrogates Fas-inducedlethal liver apoptosis in mice. Biochem Biophys Res Commun. 244: 683-690 (1998).

[0017] Non-patent literature 13: Tahara, M. et al., Hepatocyte growth factor leads to recovery from alcohol-induced fatty liver in rats. J Clin Invest. 103: 313-320 (1999).

[0018] Non-patent literature 14: Yaekashiwa, M. et al., Simultaneous or delayed administration of hepatocyte growth factor (HGF) equally repress the fibrotic change in murinelung injury induced by bleomycin: Amorphologic study. Am J Resp&Crit CareMed. 156: 1937-1944 (1997).

[0019] Non-patent document 15: Nakamura, T. et al., Hepatocyte growth factor prevents tissue fibrosis, remodeling, and dysfunction in cardiomyopathic hamster hearts. Am J Physiol. 288: H2131-H2139 (2005).

[0020] Non-patent literature 16: Azuma, H. et al., Hepatocyte growth factor prevents development of chronic allograft nephropathy in an experimental rat transplant model. J Am Soc Nephrol. 12: 1280-1292 (2001).

[0021] Non-patent document 17: Ito, W. et al., Growth factors temporally associate with airway responsiveness and inflammation in allergen-exposed mice. Int Arch Allergy Immunol. 145, 324-339 (2008).

[0022] Non-patent literature 18: Ohmichi, H. Matsumoto, K. & Nakamura, T. In vivo mitogenic action of hepatocyte growth factor on lung epithelial cells: pulmotrophic role in lung regeneration. Am J Physiol. 270, L1031-L1039 (1996).

[0023] Non-patent document 19: Date, I. et al., Hepatocyte growth factor attenuates cerebralischemia-induced learning dysfunction. Biochem Biophys Res Commun. 319, 1152-1158 (2004).

[0024] Non-patent literature 20: Inampudi, C. et al., Angiogenesis in peripheral arterial disease. Curr Opin Pharmacol. 39, 60-67 (2018). Summary of the invention

[0025] Problems to be solved by the invention

[0026] However, although some cytokines and cell growth factors, not limited to HGF, are used as drugs, it is difficult to develop technologies related to the improvement of physicochemical stability and / or pharmacokinetics, especially the significant extension of half-life and / or the realization of blood-brain barrier permeability. Therefore, it is difficult to use HGF in therapeutic drugs including therapeutic drugs for chronic hepatitis.

[0027] Therefore, an object of the present invention is to provide a novel substance that functions suitably as a Met agonist.

[0028] Means for solving problems

[0029] The inventors of the present application have conducted intensive studies to solve the above problems and have found that an artificial protein having a portion in which a specific amino acid sequence is inserted into the Fc region of an antibody is suitable for functioning as a Met agonist, thereby completing the present invention.

[0030] That is, the present invention is as follows. [1]

[0032] An artificial protein comprising an Fc region of an antibody, wherein the amino acid sequence represented by SEQ ID NO: 1 is inserted into the Fc region,

[0033] (X 1 ) n1 YX 3 X 4 FYYNYX 5 X 6 X 7 WX 8 X 9 X 10 X 11 (X 2 ) n2 (Serial No. 1)

[0034] (In sequence number 1, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 Each independently represents an amino acid, and n1 and n2 each independently represent an integer of 0 to 6). [2]

[0036] The artificial protein according to [1], wherein the amino acid sequence represented by SEQ ID NO: 1 is inserted into the loop portion on the molecular surface exposed in the Fc region of an antibody.

[0037] [2-1]

[0038] The artificial protein according to [1], wherein the amino acid sequence represented by SEQ ID NO: 1 is inserted into the following (10) or (11),

[0039] (10) In the case where the Fc region of the aforementioned antibody is represented by SEQ ID NO: 2, 3, 4 or 12, Figure 1 The parts represented as T1 to T3, M1 to M3, and B1 to B3,

[0040] (11) In the case where the Fc region of the aforementioned antibody has an amino acid sequence other than SEQ ID NO: 2, 3, 4 or 12, when the amino acid sequence of the Fc region is aligned with the amino acid sequence represented by SEQ ID NO: 4, Figure 1 The portion corresponding to the portion indicated as T1 to T3, M1 to M3, and B1 to B3. [3]

[0042] The artificial protein according to any one of [1], [2] and [2-1], wherein the amino acid sequence inserted into the Fc region is represented by SEQ ID NO: 28,

[0043] (X 1 ) n1 YPTFYYNYRSEWVLTS(X 2 ) n2 (Serial No. 28)

[0044] (In sequence number 28, X 1 , X 2 , n1, and n2 are defined the same as those for sequence number 1). [4]

[0046] The artificial protein according to any one of [1], [2], [2-1], and [3], wherein the Fc region of the antibody is derived from the Fc region of IgG.

[0047] [4-1]

[0048] The artificial protein according to any one of [1], [2], [2-1], and [3], wherein the Fc region of the antibody is an Fc region of an antibody derived from any one of human, mouse, rat, rabbit, horse, and dog, preferably human.

[0049] [4-2]

[0050] The artificial protein according to any one of [1], [2], [2-1], [3] and [4-1], wherein the Fc region of the antibody is an Fc region of an antibody derived from any one of IgG, IgM, IgA, IgD and IgE. [5]

[0052] The artificial protein according to any one of [4], [4-1] and [4-2], wherein the Fc region of the antibody is derived from IgG1 or IgG4, preferably from the Fc region of IgG1. [6]

[0054] The artificial protein as described in any one of [1] to [5], [2-1] and [4-1] to [4-2], wherein the Fc region of the antibody is an Fc region of the antibody in which one or more amino acids are deleted, added and / or substituted with respect to the Fc region of the wild-type antibody.

[0055] [6-1]

[0056] An artificial protein as described in any one of [1] to [5], [2-1] and [4-1] to [4-2], wherein the Fc region of the antibody is an antibody Fc region in which one or more amino acids are deleted, added and / or substituted (which reduces or deletes Fc receptor binding and / or effector function of the Fc region) relative to the Fc region of a wild-type antibody. [7]

[0058] A Met protein agonist comprising the artificial protein described in any one of [1] to [6], [2-1], [4-1] to [4-2], and [6-1]. [8]

[0060] A pharmaceutical composition comprising the artificial protein according to any one of [1] to [6], [2-1], [4-1] to [4-2], and [6-1]. [9]

[0062] A pharmaceutical composition as described in [8], which is used for the treatment or prevention of a disease selected from the group consisting of acute hepatitis, acute severe hepatitis, cirrhosis, liver fibrosis, biliary atresia, fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, acute renal failure, chronic kidney disease, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcer, cerebral infarction, amyotrophic lateral sclerosis, glomerulonephritis, chronic allograft nephropathy, occlusive arteriosclerosis, severe lower limb ischemia, emphysema, asthma, and vocal cord scar.

[10]

[0064] A method for treating a disease, comprising administering a therapeutically effective amount of the artificial protein described in any one of [1] to [6], [2-1], [4-1] to [4-2], and [6-1] to a patient in need thereof.

[11]

[0066] The artificial protein according to any one of [1] to [6], [2-1], [4-1] to [4-2] and [6-1], which is used for the treatment or prevention of a disease.

[12]

[0068] Use of the artificial protein described in any one of [1] to [6], [2-1], [4-1] to [4-2] and [6-1] for producing a pharmaceutical composition for use in the treatment or prevention of a disease.

[13]

[0070] A therapeutic or preventive agent for a disease, comprising the artificial protein described in any one of [1] to [6], [2-1], [4-1] to [4-2], and [6-1],

[0071] Among them, in

[10] to

[13] , the disease is preferably selected from the group consisting of acute hepatitis, acute severe hepatitis, cirrhosis, liver fibrosis, biliary atresia, fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, acute renal failure, chronic kidney disease, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcer, cerebral infarction, amyotrophic lateral sclerosis, glomerulonephritis, chronic transplant nephropathy, occlusive arteriosclerosis, severe lower limb ischemia, emphysema, asthma, and vocal cord scar.

[0072] Effects of the Invention

[0073] According to the present invention, a novel substance that functions suitably as a Met agonist can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] [ Figure 1 ] shows the three-dimensional structure of the Fc region from human IgG (PDB ID: 1FC1). One heavy chain constituting the Fc region is shown as a surface model (inside), and the other heavy chain is shown as a ribbon model (front side). In the heavy chain shown in the ribbon model, the loop part exposed on the molecular surface is represented as 9 black Cα spheres.

[0075] [ Figure 2 ] shows the amino acid sequence of the hinge region and Fc region of human IgG1 (SEQ ID NO: 3). The black background shows the loop structure suitable for inserting the insert sequence. The amino acid numbering uses the numbering method of Chothia et al. The underlined portion shows the hinge region connecting the Fc region and the Fab region in the IgG molecule.

[0076] [ Figure 3 ] shows the relationship between the Met phosphorylation level in each artificial protein and the protein concentration.

[0077] [ Figure 4 ] shows the results of alanine scanning in artificial proteins.

[0078] [ Figure 5 ] shows the results of deep mutation scanning of human Met by artificial proteins.

[0079] [ Figure 6 ] shows the relationship between the Met phosphorylation level in each artificial antibody or artificial protein and the antibody concentration or artificial protein concentration. DETAILED DESCRIPTION

[0080] Hereinafter, a specific embodiment of the present invention (hereinafter referred to as “the present embodiment”) will be described in detail, but the present invention is not limited thereto and various modifications can be made without departing from the gist of the invention.

[0081] [Artificial protein]

[0082] The artificial protein of the present invention comprises an antibody Fc region, in which the amino acid sequence represented by SEQ ID NO: 1 (hereinafter also referred to as “insertion sequence”) is inserted.

[0083] (X 1 ) n1 YX 3 X 4 FYYNYX 5 X 6 X 7 WX 8 X 9 X 10 X 11 (X 2 ) n2 (Serial No. 1)

[0084] Among them, in the above sequence number 1, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 Each independently represents an amino acid, and n1 and n2 each independently represent an integer of 0 to 6.

[0085] In the present specification, "insertion" is used as a term meaning that, in the amino acid sequence of a protein before the insertion of the insertion sequence, the insertion sequence appears as if it has been inserted into the amino acid sequence.

[0086] Therefore, in the artificial protein of the present invention, a specific site of the amino acid sequence in the protein before the insertion of the insertion sequence is cut, and the two cut fragments are combined in a manner such that the insertion sequence is connected thereto, or a part of the amino acid sequence in the protein before the insertion of the insertion sequence is deleted, and the insertion sequence is combined in a manner such that it replaces the deleted part of the sequence.

[0087] The artificial protein of the present invention is a protein obtained by inserting the above-mentioned insertion sequence into the Fc region of a protein comprising the Fc region of an antibody (hereinafter also referred to as a "scaffold protein").

[0088] (Scaffold Protein)

[0089] In the artificial protein of the present invention, the scaffold protein for inserting the insertion sequence is not particularly limited as long as it contains the Fc region of an antibody, and examples thereof include the Fc fragment of an antibody, a protein containing the Fc fragment of an antibody, a protein containing the Fc region and hinge region of an antibody, a protein containing the Fc region and antigen-binding domain of an antibody, and an antibody, etc. The scaffold protein may be a wild-type protein or an artificially modified artificial protein. The scaffold protein may be a protein or an antibody containing the wild-type or artificially modified Fc fragment of an antibody, the Fc region and hinge region of an antibody.

[0090] That is, the amino acid sequence of the artificial protein of the present invention has at least an amino acid sequence corresponding to the Fc region of an antibody and has an inserted sequence.

[0091] The amino acid sequence of the artificial protein of the present invention is not particularly limited as long as the amino acid sequence other than the inserted sequence is derived from a protein containing the Fc region of an antibody, and an amino acid sequence corresponding to any Fc region can be used.

[0092] In this specification, the term "Fc region" refers to the region containing the heavy chain constant region 2 (C H 2) and heavy chain constant region 3 (C H 3) region. The Fc region contains C H 2 and C H 3, for example, it may be a region including a fragment on the C-terminal side obtained by digesting an antibody with papain, or it may be a region including C-terminal side such as scFc (single-chain Fc). H 2 and C H 3. In addition, "Fc fragment" refers to a protein containing an Fc region.

[0093] In this specification, "antigen-binding domain" refers to the part of an antibody that specifically binds to a part or all of an antigen. The antigen-binding domain may include one or more variable regions of an antibody, and may include a light chain variable region (V L ) and the heavy chain variable region (V H ). Examples of antigen-binding domains include Fv, Fab, Fab', F(ab')2, scFv (single-chain Fv), scFv2 (single-chain FV2), bispecific antibodies (Diabody), and taFv.

[0094] The Fc region of the scaffold protein is not particularly limited, but is preferably derived from any one of human, mouse, rat, rabbit, horse and dog, more preferably from human. In addition, the Fc region may also be derived from any one of IgG, IgM, IgA, IgD and IgE, preferably from IgG, more preferably from human IgG.

[0095] As IgG, multiple subclasses are known. As subclasses of IgG, for example, it is known that there are 4 subclasses of IgG1, IgG2, IgG3, and IgG4 in humans, 4 subclasses of IgG1, IgG2a, IgG2b, and IgG3 in mice, and 4 subclasses of IgG1, IgG2a, IgG2b, and IgG2c in rats. Any subclass can be used. The Fc region of the scaffold protein can be derived from IgG1 or IgG4, can be derived from IgG1, can be derived from human IgG1 or human IgG4, can be derived from human IgG1.

[0096] In the present specification, the Fc region of the scaffold protein being derived from a specific antibody means that the Fc region has any one of the following amino acid sequences (1) to (3).

[0097] (1) Amino acid sequence of the Fc region of a specific antibody

[0098] (2) An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (1)

[0099] (3) An amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (1)

[0100] The Fc region of a specific antibody may be an Fc fragment of an antibody, and when it has the amino acid sequence of (2) or (3) above, it may be an artificially modified Fc fragment of an antibody.

[0101] In the present specification, when one or more amino acids are deleted, substituted and / or added in an amino acid sequence, the one or more may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, may be within the range of 1 to 10, or may be 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0102] In the case where the artificial protein includes an antigen binding domain, the antigen recognized by the antigen binding domain is not particularly limited, and for example, it may be an antigen specifically expressed in an organ or tissue to which the artificial protein of the present invention is desired to be directed, or an antigen that is hyperexpressed in the organ or tissue. For example, the antigen binding domain of the artificial protein may bind to PD-L1, PD-1, TfR, or CD3. The antigen binding domain of the artificial protein may be the antigen binding domain of the antibody used in the examples or an antigen binding fragment thereof.

[0103] The Fc region of the antibody included in the scaffold protein is preferably a Fc region of an antibody having more than one amino acid deleted, added and / or replaced relative to the Fc region of a wild-type antibody. The deletion, addition and / or replacement of more than one amino acid can be a mutation that reduces or deletes the effector function of Fc receptor binding and / or Fc region, a mutation that improves the effector function of Fc receptor binding and / or Fc region, a mutation that prolongs the biological half-life, or a mutation that stabilizes the antibody. As such a mutation, U.S. Patent Application Publication No. 2012 / 0251531 specification, U.S. Patent No. 9505826 specification, U.S. Patent Application Publication No. 2014 / 0056879 specification, U.S. Patent Application Publication No. 2012 / 0107871 specification, and U.S. Patent Application Publication No. 2019 / 0300621 specification can be cited.

[0104] From the viewpoint of improving the stability of the artificial protein in vivo and / or improving the Met agonist activity of the artificial protein, the Fc region of the antibody contained in the scaffold protein preferably comprises a deletion, addition and / or substitution of one or more amino acids that reduce or delete the Fc receptor binding and / or the effector function of the Fc region, more preferably comprises a substitution of P329G in the wild-type human IgG1 Fc region, and at least two substitutions corresponding to substitutions of at least one other amino acid, and even more preferably comprises substitutions corresponding to substitutions of P329G, L234A, and L235A in the wild-type human IgG1 Fc region, respectively. The residues in the substitutions of P329G, L234A, and L235A are numbered according to the EU index of Kabat.

[0105] The antibody Fc region contained in the scaffold protein preferably has an amino acid sequence of any one of (4) to (6) and (4′) to (6′) below, and more preferably has an amino acid sequence of any one of (4) to (6).

[0106] (4) The amino acid sequence represented by SEQ ID NO: 2

[0107] (5) An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (4)

[0108] (6) An amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (4)

[0109] (4') The amino acid sequence represented by SEQ ID NO: 4 (from Figure 2 The amino acid sequence represented by SEQ ID NO: 3 excluding the sequence from D at position 221 to S at position 239)

[0110] (5') An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (4')

[0111] (6') an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (4')

[0112] [Chemical formula 1]

[0113]

[0114] In the amino acid sequences of (5) and (6) above, the deletion, substitution and / or addition of one or more amino acids is preferably performed in an amino acid other than G at position 90 of SEQ ID NO: 2.

[0115] When the scaffold protein is an antibody, the antibody is not particularly limited, and examples thereof include mouse antibodies, rat antibodies, rabbit antibodies, horse antibodies, dog antibodies, chimeric antibodies, humanized antibodies, and fully humanized antibodies, etc. These antibodies may be wild-type proteins or artificially modified proteins.

[0116] The scaffold protein preferably has, for example, any one of the following amino acid sequences (7) to (9) and (7′) to (9′), and more preferably has any one of the following amino acid sequences (7) to (9).

[0117] (7) The amino acid sequence represented by SEQ ID NO: 12

[0118] (8) An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (7)

[0119] (9) An amino acid sequence (7′) having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity with the amino acid sequence of (7) The amino acid sequence represented by SEQ ID NO: 3 ( Figure 2 The amino acid sequence of

[0120] (8') An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (7')

[0121] (9') an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (7')

[0122] It should be noted that the amino acid sequence of SEQ ID NO: 12 will be described later in the Examples.

[0123] In the amino acid sequences of (8) and (9) above, the deletion, substitution and / or addition of one or more amino acids is preferably performed in an amino acid other than A at positions 14 and 15 and G at position 109 of SEQ ID NO: 12.

[0124] (Insertion of Insertion Sequence)

[0125] In the present invention, the insertion sequence is inserted into the Fc region of the antibody in the scaffold protein. The insertion sequence is represented by SEQ ID NO:1.

[0126] (X 1 ) n1 YX 3 X 4 FYYNYX 5 X 6 X 7 WX 8 X 9 X 10 X 11 (X 2 ) n2 (Serial No. 1)

[0127] Among them, in the above sequence number 1, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 Each independently represents an amino acid, and n1 and n2 each independently represent an integer of 0 to 6.

[0128] In this specification, the term "amino acid" includes not only natural amino acids, but also artificial amino acid mutants and derivatives. As amino acids, for example, proteinaceous L-amino acids, non-proteinaceous amino acids, and chemically synthesized compounds having properties known in the art as characteristics of amino acids can be cited.

[0129] Proteinogenic amino acids are Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val when represented by three letters known in the art. In addition, proteinogenic amino acids are R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, and V when represented by one letter known in the art.

[0130] Non-proteinogenic amino acids include natural or unnatural amino acids other than proteinogenic amino acids.

[0131] Examples of non-natural amino acids include amino acids whose main chain structures are different from those of natural ones (α,α-disubstituted amino acids (α-methylalanine, etc.), N-alkyl amino acids, D-amino acids, β-amino acids, γ-amino acids, δ-amino acids, and α-hydroxy acids, etc.); amino acids whose side chain structures are different from those of natural ones (norleucine, homohistidine, etc.); amino acids having extra methylene groups in the side chains (“homo” amino acids, homophenylalanine, and homohistidine, etc.); and amino acids whose carboxylic acid functional groups in the side chains are substituted with sulfonic acid groups (cysteic acid, etc.).

[0132] In sequence number 1, (X 1 ) n1 and (X 2 ) n2 The amino acid sequence portion represented by is equivalent to a linker and may not exist in the inserted sequence. 1 ) n1 and (X 2 ) n2 The amino acid sequence portion represented is not particularly limited as long as it can link the inserted sequence to the scaffold protein, but preferably has a physiologically inactive amino acid sequence.

[0133] X 1 and X 2 Preferably, they are each independently selected from Ser, Gly and Cys, more preferably selected from Ser and Gly.

[0134] n1 and n2 are each independently greater than 0 and less than 6, and may be greater than 0 and less than 5, greater than 0 and less than 4, greater than 0 and less than 3, greater than 1 and less than 6, greater than 1 and less than 5, greater than 1 and less than 4, greater than 1 and less than 3, greater than 2 and less than 6, greater than 2 and less than 5, greater than 2 and less than 4, or greater than 2 and less than 3.

[0135] The sum of n1 and n2 is not particularly limited as long as n1 and n2 are within the above range, and can be, for example, 0 to 10, 0 to 8, 2 to 10, 2 to 8, 4 to 10, or 4 to 8.

[0136] Therefore, the linker can be, for example, an amino acid sequence selected from Ser and Gly with 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0137] In sequence number 1,

[0138] X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 Each independently is an amino acid, which may be a natural amino acid. In addition, X 3 ~X 11 Each independently may be the following amino acids.

[0139] X 3 Amino acids other than G, D, and C are preferred, and P is particularly preferred.

[0140] X 4 Amino acids other than G, P, D, and W are preferred, and T is particularly preferred.

[0141] X 5 An amino acid other than G, P, C, and Y is preferred, A, E, or R is more preferred, and R is particularly preferred.

[0142] X 6 Preferred are amino acids other than V, I, P, E, C, and W, more preferably G, A, N, Q, R, or S, and particularly preferably S.

[0143] X 7 Preferred are amino acids other than G, P, C, and W, more preferred are A, L, V, I, D, E, Q, R, S, or T, and particularly preferred is E.

[0144] X 8 Preferred are amino acids other than G, P, D, N, F, Y, and W, more preferably A, V, I, E, K, or T, and particularly preferably V.

[0145] X 9Amino acids other than D are preferred, and L is particularly preferred.

[0146] X 10 Preferred are amino acids other than G, P, D, N, H, K, R, and S, more preferred are A, L, V, I, or T, and particularly preferred is T.

[0147] X 11 Preferred are amino acids other than V and C, more preferably A, I, P, D, E, N, Q, H, K, R, S, F, Y, or W, and particularly preferably S.

[0148] The amino acid of sequence number 1 is preferably represented by sequence number 28.

[0149] (X 1 ) n1 YPTFYYNYRSEWVLTS(X 2 ) n2 (Serial No. 28)

[0150] Among them, in the above sequence number 28, X 1 , X 2 , n1, and n2 are defined in the same manner as in sequence number 1, and the preferred method is also the same.

[0151] In the present invention, the site where the insertion sequence of the scaffold protein is inserted is not particularly limited, but is preferably a loop portion exposed on the molecular surface of the Fc region of the antibody. The loop portion is preferably a polypeptide chain region connecting two β chains in the Fc region and has the following structure: having a folded structure and exposed on the surface of the antibody molecule.

[0152] When inserting the insertion sequence into the scaffold protein, the insertion sequence can also be inserted between two adjacent amino acid residues of the scaffold protein, preferably between two non-adjacent amino acid residues of the scaffold protein. Inserting the insertion sequence between two non-adjacent amino acid residues of the scaffold protein means that the amino acid residues between the two amino acid residues selected for inserting the insertion sequence are deleted from the scaffold protein, so that the selected two amino acid residues are directly bound to the insertion sequence. The number of amino acid residues deleted from the scaffold protein for inserting the insertion sequence is not particularly limited, preferably 0 or more and 15 or less, more preferably 0 or more and 10 or less, 0 or more and 8 or less, 0 or more and 5 or less, 0 or more and 3 or less, 1 or more and 3 or less, or 1 or more and 2 or less, and further preferably 1. The amino acid residue deleted from the scaffold protein for inserting the insertion sequence is 1, which means that the n+1th amino acid residue from the N-terminus of the scaffold protein is deleted, and the insertion sequence is inserted between the nth amino acid residue and the n+2th amino acid residue. Wherein, n is an arbitrary natural number.

[0153] When the insertion sequence is inserted into the scaffold protein, the insertion sequence is preferably bound to the scaffold protein by covalent bonds.

[0154] The loop portion into which the insert sequence is inserted is not particularly limited in the loop structure in the Fc region of the antibody, and any site in the loop structure can be selected.

[0155] An arbitrary site in the loop structure means that the two amino acid residues where the insert sequence is inserted are selected from sites within the loop structure.

[0156] The insertion site of the insertion sequence in the scaffold protein is not particularly limited. Figure 1 and Figure 2 A specific example will be described.

[0157] Figure 1 The three-dimensional structure of the Fc region derived from human IgG is shown. One heavy chain constituting Fc is shown as a surface model (inside), and the other heavy chain is shown as a ribbon model (front side).

[0158] exist Figure 1 The loop portion preferably selected as the insertion site of the insertion sequence is shown in . The portions indicated as T1 to T3, M1 to M3, and B1 to B3 are such portions.

[0159] Figure 2 The amino acid sequence derived from the Fc region of human IgG1 is shown, and the black background shows a loop portion preferably selected as the insertion site of the insert sequence.

[0160] The loop portion preferably selected includes the one derived from the Fc region of human IgG1 represented by SEQ ID NO: 4

[0161] T1 (SHEDP, sequence number 5): 267 to 271 bits

[0162] T2 (YNST, sequence number 6): 296 to 299 bits

[0163] T3 (NKALPAP, sequence number 7): 325 to 331

[0164] M1 (VDGV, sequence number 8): 279 to 282

[0165] M2(KGQ): 340-342 bits

[0166] M3 (SDGS, serial number 9): 400 to 403 digits

[0167] B1 (TKNQ, serial number 10): 359 to 362 bits

[0168] B2 (SNG): 383-385

[0169] B3 (QQGNV, serial number 11): 418 to 422 bits.

[0170] Among them, B2 and B3 are more preferred. The amino acid sequence represented by SEQ ID NO: 4 is derived from Figure 2 The amino acid sequence represented by SEQ ID NO: 3 excluding the sequence from D at position 221 to S at position 239.

[0171] Taking T1 as an example, positions 267 to 271 correspond to positions 47 to 51 in SEQ ID NO. 3, and positions 28 to 32 in SEQ ID NO. 4. That is, the portions represented as T2 to T3, M1 to M3, and B1 to B3 correspond to the amino acids at the positions after subtracting 220 in SEQ ID NO. 3, and correspond to the amino acids at the positions after subtracting 239 in SEQ ID NO. 4.

[0172] In addition, the insertion site of the insertion sequence can be a site selected from the loop structure represented by the position of the above-mentioned amino acid residues. Taking T1 as an example, as the site selected from the loop structure, positions 267 and 268, 268 and 269, 269 and 270, 270 and 271, 267 and 269, 268 and 270, 269 and 271, 267 and 270, 268 and 271, and 267 and 271 can be selected, and the insertion sequence can be inserted between these selected amino acid residues. Wherein, when the two selected amino acid residues are not adjacent, the amino acid residue therebetween is deleted.

[0173] In the case of antibodies other than human IgG1, or when human IgG1 is modified, the portion corresponding to the portions represented by T1 to T3, M1 to M3, and B1 to B3 by aligning with the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 can be used as the insertion site of the insertion sequence, among which the portion corresponding to the portion represented by B2 and B3 is more preferred.

[0174] Among them, in the loop portion of the Fc region derived from human IgG1 represented by SEQ ID NO: 4,

[0175] T1 (SH*EDP): 267 to 271

[0176] T2 (Y*NST): 296 to 299 bits

[0177] T3 (NKALP*AP): 325-331

[0178] M1 (VD*GV): 279 to 282 bits

[0179] M2(K*GQ): 340-342 bits

[0180] M3 (SD*GS): 400 to 403 digits

[0181] B1(TK*NQ): 359 to 362

[0182] B2(S*NG): 383-385

[0183] B3 (QQ*GNV): 418 to 422

[0184] The position between the amino acid residues with the * symbol is a more preferred position for inserting the insertion sequence. The position between the amino acid residues with the * symbol in B2 and B3 is further preferred. When inserting the insertion sequence, any of the adjacent amino acid residues with the * symbol may be deleted.

[0185] In the case of an antibody other than human IgG1, or a modified human IgG1, the portion corresponding to the portion indicated by the * symbol in T1 to T3, M1 to M3, and B1 to B3 can be used as the insertion site of the insertion sequence by aligning with the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.

[0186] The artificial protein of the present invention can delete N at position 384 in the above-mentioned B2 and insert the insertion sequence between S at position 383 and G at position 385, and can delete G at position 420 in the above-mentioned B3 and insert the insertion sequence between Q at position 419 and N at position 421.

[0187] In the artificial protein of the present invention, the scaffold protein comprises an Fc region of an antibody derived from any one of human, mouse, rat, rabbit, horse and dog, an Fc region of an antibody derived from any one of IgG, IgM, IgA, IgD and IgE, an Fc region of an antibody derived from human, an Fc region of an antibody derived from human IgG, or an Fc region of an antibody derived from human IgG1, and the site for inserting the insert sequence is preferably the following (10) or (11). In this embodiment, the insertion site of the scaffold protein and the insert sequence may be any of the embodiments exemplified above.

[0188] (10) When the Fc region in the scaffold protein is represented by SEQ ID NO: 2, 3, 4 or 12, the above-mentioned parts represented by T1 to T3, M1 to M3, B1 to B3

[0189] (11) When the Fc region in the scaffold protein has an amino acid sequence other than SEQ ID NO: 2, 3, 4 or 12, the portion corresponding to the portion represented by T1 to T3, M1 to M3, B1 to B3 when the amino acid sequence of the Fc region is aligned with the amino acid sequence represented by SEQ ID NO: 4

[0190] It should be noted that, in the case where the artificial protein includes a portion other than the Fc region as a scaffold protein, the portion of the artificial protein corresponding to the amino acid sequence represented by SEQ ID NO: 4 when the artificial protein is aligned with the amino acid sequence represented by SEQ ID NO: 4 can also be regarded as the Fc region portion of the artificial protein. It should be noted that, when aligning, the insertion sequence is deleted from the amino acid sequence of the artificial protein. When deleting the insertion sequence, the sequence represented by (X) in SEQ ID NO: 1 can be modified in such a way that the homology with the amino acid sequence represented by SEQ ID NO: 4 obtained by the alignment is maximized. 1 ) n1 and (X 2 ) n2 In the amino acid sequence portion shown, n1 and n2 are appropriately set so as to fall within the above ranges.

[0191] The artificial protein of the present invention may have, for example, any one of the following amino acid sequences (12) to (17) and (12′) to (17′).

[0192] (12) The amino acid sequence represented by SEQ ID NO: 13

[0193] (13) An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (12)

[0194] (14) An amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (12)

[0195] (15) The amino acid sequence represented by SEQ ID NO: 14

[0196] (16) An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (15)

[0197] (17) An amino acid sequence (12′) having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity to the amino acid sequence of (15) The amino acid sequence represented by SEQ ID NO: 15

[0198] (13') An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (12')

[0199] (14') an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (12')

[0200] (15') Amino acid sequence represented by SEQ ID NO: 16

[0201] (16') An amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence of (15')

[0202] (17') an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of (15')

[0203] It should be noted that the amino acid sequences of SEQ ID NOs: 13 and 15 to 16 will be described later in the Examples.

[0204] [Chemical formula 2]

[0205]

[0206] In the amino acid sequences of (13) and (14) above, the deletion, substitution and / or addition of one or more amino acids is preferably carried out in the amino acids other than A at positions 14 and 15, G at position 109, and S from position 164 to position 185 of sequence number 13.

[0207] In the amino acid sequences of (16) and (17) above, the deletion, substitution and / or addition of one or more amino acids is preferably performed in the amino acids other than A at positions 14 and 15, G at position 109, and S from position 200 to position 221 of sequence number 14.

[0208] In the amino acid sequences of (13') and (14') above, the deletion, substitution and / or addition of one or more amino acids is preferably performed in the amino acids other than S from positions 164 to 185 of SEQ ID NO: 15.

[0209] In the amino acid sequences of (16') and (17') above, the deletion, substitution and / or addition of one or more amino acids is preferably performed in the amino acids other than S from position 200 to position 221 of SEQ ID NO: 16.

[0210] The artificial protein of the present invention may have an amino acid sequence obtained by replacing the portion "SGGYPTFYYNYRSEWVLTSGGS" (SEQ ID NO: 17) with the amino acid sequence of SEQ ID NO: 1 in any of the amino acid sequences of (12) to (17) and (12') to (17').

[0211] The artificial protein of the present invention may have an amino acid sequence obtained by replacing the portion "SGGYPTFYYNYRSEWVLTSGGS" (SEQ ID NO: 17) with the amino acid sequence of SEQ ID NO: 28 in the amino acid sequence of any one of (12) to (17) and (12') to (17').

[0212] The artificial protein of the present invention may have the amino acid sequence of any one of (12) to (17) and (12') to (17') above, wherein "SGGYPTFYYNYRSEWVLTSGGS" (SEQ ID NO: 17) is replaced by "SGGYPTFYYNYRSEWVLTSGGS" (SEQ ID NO: 17). 3 X 4 FYYNYX 5 X 6 X 7 WX 8 X 9 X 10 X 11 GGS" (SEQ ID NO: 29) 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 The definition and preferred embodiment of are as described above).

[0213] The artificial protein of the present invention may be an artificial antibody in which an antigen-binding domain is bound to a fragment having the amino acid sequence of any one of (12) to (17) and (12′) to (17′).

[0214] The artificial protein of the present invention may be an artificial antibody having an antigen-binding domain combined with a fragment of an amino acid sequence obtained by replacing the portion of "SGGYPTFYYNYRSEWVLTSGGS" (sequence number 17) in the amino acid sequence of any one of (12) to (17) and (12') to (17') above with the amino acid sequence of sequence number 1.

[0215] The artificial protein of the present invention may be an artificial antibody having an antigen-binding domain coupled to a fragment of an amino acid sequence having a portion of "SGGYPTFYYNYRSEWVLTSGGS" (sequence number 17) in the amino acid sequence of any one of (12) to (17) and (12') to (17') above replaced with the amino acid sequence of sequence number 28.

[0216] The artificial protein of the present invention may be a protein having an amino acid sequence of any one of (12) to (17) and (12') to (17') above, wherein "SGGYPTFYYNYRSEWVLTSGGS" (SEQ ID NO: 17) is replaced by "SGGYPTFYYNYRSEWVLTSGGS" (SEQ ID NO: 17). 3 X 4 FYYNYX 5 X 6 X 7 WX 8 X 9 X 10 X 11 GGS" (SEQ ID NO: 29) and an artificial antibody (X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 The definition and preferred embodiment of are as described above).

[0217] The antigen-binding domain in the artificial antibody described above may be the antigen-binding domain of the antibody used in the Examples or an antigen-binding fragment thereof.

[0218] The method for inserting the insertion sequence into the scaffold protein is not particularly limited, and examples thereof include the methods described in International Publication No. 2019 / 026920, International Publication No. 2020 / 027224, and International Publication No. 2019 / 216436. In addition, the methods described in the Examples can also be used.

[0219] [Pharmaceutical compositions, etc.]

[0220] The artificial protein of the present invention can promote the autophosphorylation of Met protein, which is a hepatocyte growth factor (HGF) receptor. Therefore, the artificial protein of the present invention is useful as a Met protein agonist and a pharmaceutical composition. In addition, it can be used in vitro and in vivo as an organ protector and regeneration promoter during organ transplantation.

[0221] The pharmaceutical composition of the present invention comprises the artificial protein of the present invention as an active ingredient. Since it has HGF-like functions, it is useful for promoting cell proliferation, promoting cell migration, inhibiting cell apoptosis, inducing morphogenesis, angiogenesis, and regeneration and / or protection of tissues and organs, and is useful as a therapeutic or preventive agent for diseases related to these.

[0222] Examples of such diseases include diseases that can be prevented or treated using Met protein. In addition, diseases in the present invention may be diseases for which therapeutic effects or preventive effects can be expected using HGF.

[0223] Examples of such diseases include diseases of the liver, kidney, lung, blood vessel, skin, brain, muscle, and nerve. Among them, examples of liver diseases include hepatitis and diseases caused by hepatitis.

[0224] Specifically, examples include acute hepatitis, acute severe hepatitis, cirrhosis, liver fibrosis, biliary atresia, fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, acute renal failure, chronic kidney disease, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcers, cerebral infarction, amyotrophic lateral sclerosis, glomerulonephritis, chronic transplant kidney disease, occlusive arteriosclerosis, severe lower limb ischemia, emphysema, asthma, and vocal cord scar, but are not limited to these.

[0225] The administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be oral administration or parenteral administration. Examples of parenteral administration include intramuscular injection, intravenous injection, subcutaneous injection and other injections, transdermal administration, and mucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0226] The pharmaceutical composition may be directly used as an active ingredient, or may be formulated by adding a pharmaceutically acceptable carrier, excipient, and / or additive. As dosage forms, for example, liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nose drops, ear drops, and cataplasms may be cited.

[0227] Formulation can be carried out by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption accelerators, surfactants, pH adjusters, preservatives, and antioxidants as appropriate.

[0228] Examples of ingredients used for formulation include purified water, saline, phosphate buffer, dextrose, pharmaceutically acceptable organic solvents such as glycerol and ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin, but are not limited thereto.

[0229] As absorption promoters for improving absorption, surfactants such as polyoxyethylene lauryl ether, sodium lauryl sulfate, and saponin can be used; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylates; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, carbon monoxide donors, etc. can be used.

[0230] Pills or tablets can be coated with sugar coating, gastric soluble or enteric soluble substances.

[0231] The injection may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. In addition, a wetting agent, an emulsifier, a dispersant, a stabilizer, a solubilizing agent, a solubilizing agent, and a preservative, etc. may also be added.

[0232] The pharmaceutical composition of the present invention can be administered in combination with other drugs or treatments useful for the above-mentioned diseases.

[0233] The dosage of the pharmaceutical composition of the present invention when administered to mammals (e.g., humans, monkeys, etc.), especially humans, varies depending on the symptoms, age, sex, weight, sensitivity difference of the patient, administration method, administration interval, type of active ingredient, and type of preparation, and is not particularly limited. For example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or multiple times. In the case of injection administration, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg can also be administered once or multiple times according to the patient's weight.

[0234] The present invention also provides:

[0235] A method of treating comprising administering an effective amount of the artificial protein of the present invention to a patient suffering from the above-mentioned disease;

[0236] The artificial protein of the present invention for use in the treatment or prevention of the above-mentioned diseases;

[0237] Use of the artificial protein of the present invention for preparing a pharmaceutical composition for use in the treatment or prevention of the above-mentioned diseases;

[0238] A therapeutic or preventive agent for the above-mentioned diseases comprising the artificial protein of the present invention.

[0239] However, the artificial protein of the present invention may be in any of the above-mentioned forms.

[0240] Among the above-mentioned aspects described in this specification, all aspects including aspects described in this specification and aspects described as preferred aspects in this specification can be arbitrarily combined.

[0241] Example

[0242] Hereinafter, the present invention will be described in more detail using Examples and Comparative Examples. The present invention is not limited to the following Examples.

[0243] [Preparation of artificial proteins]

[0244] As scaffold proteins, the following proteins were used: Figure 2 The hinge region and Fc region of human IgG1 (SEQ ID NO: 3) having the amino acid sequence shown, or the amino acid sequence shown in SEQ ID NO: 12 in which mutations that reduce or delete Fc receptor binding and / or the effector function of the Fc region are introduced into the hinge region and Fc region of human IgG1.

[0245] In addition, as the insertion sequence, the amino acid sequences represented by the following sequence numbers 17 to 20 were used. It should be noted that sequence number 17 corresponds to the amino acid sequence represented by sequence number 1, and sequence numbers 18 and 20 are partial sequences of cyclic peptides that function as human Met agonists and are inserted into proteins in International Publication No. 2019 / 026920, International Publication No. 2020 / 027224, and International Publication No. 2019 / 216436. Sequence number 19 is an amino acid sequence in which Cys is introduced at both ends of the amino acid sequence of sequence number 18, and in sequence number 19, the Cys at both ends are bound by disulfide bonds.

[0246] [Chemical formula 3]

[0247]

[0248] In the case of the amino acid sequences represented by SEQ ID NOs: 17 to 20, appropriate linkers are bound to the N-terminal and C-terminal ends of the amino acid sequences, and the amino acid sequences represented by SEQ ID NOs: 3 and 12 are inserted. Figure 1 The site corresponding to the B2 site or the B3 site.

[0249] The amino acid sequences of the artificial proteins used in this example are shown in the table below. In the table below, "0D09", "aMD4", "aMD4ds", and "aMD5" represent amino acid sequences into which sequence numbers 17 to 20 are inserted, respectively. "B2" and "B3" represent sites into which amino acid sequences represented by sequence numbers 17 to 20 are inserted. "-Variant" represents that as a scaffold protein, the protein represented by sequence number 12 is used, and the other proteins represented by sequence number 3 are used.

[0250] [Table 1-1]

[0251]

[0252] [Table 1-2]

[0253]

[0254] Specifically, the artificial proteins in Table 1 were prepared as follows.

[0255] Protein expression was performed using the Expi293 expression system (Thermo Fisher Scientific). Secreted proteins were purified using HiTrap Protein A HP columns (Cytiva), eluted with 2 mM dilute hydrochloric acid (pH 3.0), and neutralized with 10× PBS. Dialysis was performed using Slide-A-Lyzer, 2K MWCO (Thermo Fisher Scientific), and the buffer was replaced with PBS. Amicon Ultra 10K (Millipore) was used for concentration as needed. Size exclusion chromatography was performed using a Superdex 200Increase 10 / 300GL column (Cytiva) equilibrated with PBS to purify the monodisperse fraction.

[0256] [Quantification of Met phosphorylation level in situ]

[0257] In a 96-well black μClear culture plate (Greiner Bio-One), EHMES-1 human mesothelioma cells were seeded at 8,000 cells per well and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) for 24 hours. Each artificial protein prepared above was added to RPMI1640 medium supplemented with 10% FBS and allowed to contact the cells for 10 minutes. Then, the cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde (in PBS) for 30 minutes, washed with PBS, and blocked with 5% goat serum and 0.02% Triton X-100 in PBS for 30 minutes.

[0258] Phosphorylation of Met was detected using anti-phospho-Met (Tyr1234 / 1235) antibody (1:1,000 dilution, D26, Cell Signaling Technologies) and horseradish peroxidase (HRP)-conjugated anti-rabbit goat antibody (1:1,000 dilution, Dako), and then chemiluminescence was measured using ImmunoStar LD reagent (Wako) using an ARVO MX microplate reader (Perkin Elmer).

[0259] The relationship between each protein concentration and the measured Met phosphorylation level is shown in Figure 3 Each phosphorylation level was normalized to the value of 100 nM of Fc(aMD4ds)B3.

[0260] [Alanine Scan]

[0261] Alanine scanning was performed on the portion "YPTFYYNYRSEWVLTS" (SEQ ID NO: 30) in Fc(0D09)B2 having the amino acid sequence of SEQ ID NO: 15.

[0262] First, an artificial protein was prepared by the same method as above, in which a portion of "YPTFYYNYRSEWVLTS" (SEQ ID NO: 30) was substituted with any of the proteins shown in the following table.

[0263] [Table 2]

[0264]

[0265] Next, in order to investigate whether these artificial proteins have the ability to bind to human Met, a pull-down binding assay was performed.

[0266] Purified biotinylated human Met extracellular region protein was used in the pull-down binding test. The protein containing the extracellular region of human Met was added to Dynabeads M-280Streptavidin (Thermo Fisher Scientific) for immobilization, and the beads were washed with HBS (20mM Hepes-NaOH, 150mM NaCl) to prepare Met-immobilized beads. After the culture supernatant containing artificial protein was reacted with the prepared Met-immobilized beads, the beads were washed with HBS, and after rinsing the non-bound proteins, SDS sample buffer was added and heated at 95°C for 5 minutes as a sample. The eluted sample was subjected to electrophoresis under non-reducing conditions and stained with Coomassie Brilliant Blue. In this way, the artificial protein that specifically binds to the extracellular region of human Met was detected. The results are shown in Figure 4 (It should be noted that lane B represents a negative control.) Figure 4 It was found that in "YPTFYYNYRSEWVLTS" (SEQ ID NO: 30), the amino acids at positions 2 to 3, positions 9 to 11, and positions 13 to 16 can be substituted.

[0267] [Deep mutation scan]

[0268] The insertion sequence "YPTFYYNYRSEWVLTS" (SEQ ID NO: 30) was subjected to a deep mutation scan.

[0269] Specifically, the method was reported using the RaPID method (PNAS, 2018; 115(43): 10959-64) in the form of a macrocyclic peptide. A DNA library was prepared in which the appropriate codons were replaced with NNK (N is independently A, T, G or C, and K is T or G) in a manner that allows the synthesis of a peptide sequence group in which only one amino acid in sequence number 30 is replaced with an arbitrary amino acid. A sequence for adding an HA tag for purification at the C-terminus was introduced into the synthesized DNA.

[0270] After making the display library of macrocyclic peptides from the mixture of the above-mentioned DNA by the RaPID method, in order to remove the RNA of unbound peptides, purification based on the HA tag is performed. After purification, a portion of the recovered peptide solution is separated (hereinafter referred to as "INPUT A"). After the remainder is reacted with the above-mentioned beads bound to biotinylated human Met, the beads are washed and heat-treated, thereby recovering the library of peptides bound to the target (hereinafter referred to as "INPUT B"). The libraries of INPUT A and INPUTB are amplified by PCR, and the index sequence (index sequence) for sequencing is added by PCR. The sequences of INPUT A and INPUT B are analyzed by the Miseq sequencer of illumina. Next, the enrichment score is calculated as shown below.

[0271] First, for the number of reads in INPUT A and INPUT B of each mutant, in order to correct the difference in readings, the ratio is calculated by dividing the total readings of each INPUT according to the following formula (I). Next, for each mutant, the ratio before and after the reaction relative to the target is calculated according to the following formula (II). In addition, for each mutant, a comparison with the sequence before mutation is performed according to the following formula (III), and for the calculated ratio, as shown in the following formula (IV), the logarithm with base 2 is taken to calculate the enrichment score. A heat map is made based on the enrichment score for each mutant. The results are shown in Figure 5 .

[0272] [Mathematical formula 1]

[0273]

[0274] Enrichment score = log2δ 变体X (IV)

[0275] [Changes in Scaffold Protein]

[0276] In the above-mentioned method for producing artificial proteins, the scaffold protein was changed from the hinge region and Fc region of human IgG1 to the following antibody, and an artificial antibody was produced by inserting "SGGYPTFYYNYRSEWVLTSGGS" (sequence number 17) into the Fc region of the antibody. The Met phosphorylation ability of each artificial antibody produced was evaluated by the same method as above. The Met phosphorylation ability was also measured in the same manner for the antibody without "SGGYPTFYYNYRSEWVLTSGGS" (sequence number 17). The results are shown in Figure 6. It should be noted that in Table 3, "B2MB_0D09" is a positive control, which is an artificial protein having the same amino acid sequence as Fc(0D09)B2 in Table 1 above. In addition, in the following table, "Insertion Position" indicates the position at which the insertion sequence is inserted in each artificial antibody, and indicates the corresponding portion of the Fc region derived from human IgG1 when each antibody before the insertion of the insertion sequence is compared with the Fc region derived from human IgG1 represented by Sequence No. 4. That is, in each artificial antibody, the insertion sequence is inserted at the position corresponding to the B2 (SNG: 383 to 385) site of the Fc region derived from human IgG1 when each antibody before the insertion of the insertion sequence is compared with the Fc region derived from human IgG1 represented by Sequence No. 4.

[0277] [Table 3]

[0278]

Claims

1. An artificial protein comprising an Fc region of an antibody, wherein the amino acid sequence represented by SEQ ID NO: 1 is inserted into the Fc region, (X 1 ) n1 YX 3 X 4 FYYNYX 5 X 6 X 7 WX 8 X 9 X 10 X 11 (X 2 ) n2 (Serial No. 1) In sequence number 1, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 Each independently represents an amino acid, and n1 and n2 each independently represent an integer of 0 to 6.

2. The artificial protein according to claim 1, wherein The amino acid sequence represented by SEQ ID NO: 1 is inserted into the loop portion exposed on the molecular surface of the Fc region of the antibody.

3. The artificial protein according to claim 1 or 2, wherein The amino acid sequence inserted into the Fc region is represented by SEQ ID NO:

28. (X 1 ) n1 YPTFYYNYRSEWVLTS(X 2 ) n2 (Serial No. 28) In sequence number 28, X 1 , X 2 , n1, and n2 are defined in the same way as sequence number 1.

4. The artificial protein according to any one of claims 1 to 3, wherein The Fc region of the antibody is derived from the Fc region of IgG.

5. The artificial protein according to any one of claims 1 to 3, wherein The Fc region of the antibody is derived from the Fc region of IgG1.

6. The artificial protein according to any one of claims 1 to 5, wherein The antibody Fc region is an antibody Fc region in which one or more amino acids are deleted, added and / or substituted with respect to the wild-type antibody Fc region.

7. A Met protein agonist comprising the artificial protein according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising the artificial protein according to any one of claims 1 to 6.

9. The pharmaceutical composition of claim 8, which is used for the treatment or prevention of a disease selected from the group consisting of acute hepatitis, acute severe hepatitis, liver cirrhosis, liver fibrosis, biliary atresia, fatty liver, nonalcoholic steatohepatitis, alcoholic steatohepatitis, acute renal failure, chronic kidney disease, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcer, cerebral infarction, amyotrophic lateral sclerosis, glomerulonephritis, chronic transplant nephropathy, occlusive arteriosclerosis, severe lower limb ischemia, emphysema, asthma, and vocal cord scar.

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