Anti-MerTK agonist antibody, anti-MerTK bispecific antibody and application thereof

Abstract

The present invention addresses the problem of providing an anti-MerTK antibody and an anti-MerTK bispecific antibody which can be used in the prevention or treatment of diseases associated with phagocytosis. According to the invention, people explore an anti-MerTK agonist antibody which is combined with MerTK and activates a downstream signal, and a new anti-MerTK agonist antibody is obtained. The anti-MerTK agonist antibody activates a downstream signal by means of MerTK expressed on the surface of an RPE cell, thereby activating the phagocytosis clearance function of the RPE cell. Further, an anti-MerTK-anti-misfolded protein bispecific antibody, which is produced on the basis of the sequence of the anti-MerTK agonist antibody, activates the misfolded protein phagocytosis removal function of macrophages. Therefore, an anti-MerTK antibody or an anti-MerTK-anti-misfolded protein bispecific antibody can be expected to be used in the prevention or treatment of diseases associated with phagocytosis.

Description

Technical Field

[0001] This invention relates to novel anti-MerTK agonist antibodies, anti-MerTK bispecific antibodies, and their applications. Background Technology

[0002] The rapid phagocytosis / clearance mechanism of macrophages and other phagocytes of apoptotic cells and cellular debris is known as endocytosis, and is considered a crucial mechanism for maintaining tissue and immune homeostasis (Nat. Rev. Mol. Cell Biol., 2020, 21: 398-414). For example, in atherosclerotic lesions, macrophages help suppress inflammatory responses and inhibit disease progression by recognizing and rapidly clearing autologous apoptotic cells (Nature, 2016, 536: 86-90).

[0003] Mer tyrosine kinase (MerTK), a member of the TAM (Tyro3, ​​Axl, and MerTK) receptor tyrosine kinase family, is known to be expressed in phagocytes such as macrophages and plays a key role in cell burial. MerTK is a single-transmembrane protein with two immunoglobulin-like domains and two fibronectin type III domains extracellularly, and one tyrosine kinase domain intracellularly. Ligands identified as ligands include growth arrest specific gene 6 (Gas6), protein S (ProS), Tubby, and Tubby-like protein 1 (TULP-1). Gas6 and ProS are ligands that bridge the binding of phosphatidylserine residues (PtdSer) exposed on the surface of apoptotic cells to the immunoglobulin-like domain of MerTK on phagocytes. Through these ligands, MerTK binds to apoptotic cells, transmitting downstream activation signals (tyrosine autophosphorylation) required for cell demise and other processes (Nat. Rev. Mol. Cell Biol., 2020, 21: 398-414; Cold Spring Harb. Perspect. Biol., 2013, 5: a009076; EMBO J., 2010, 29: 3898-3910). Furthermore, it is suggested that MerTK promotes anti-inflammatory function by inhibiting the production of inflammatory cytokines by dendritic cells via Toll-like receptors (Cell, 2007, 131: 1124-1136) and inducing the synthesis of inflammatory remission mediators by macrophages (Sci. Signal., 2018, 11: eaar3721).

[0004] MerTK is also expressed in retinal pigment epithelium (RPE) cells. By activating downstream intracellular signaling via MerTK, RPE cells engulf senescent and detached photoreceptor outer segments (POS). RPE cells are located at the point of contact with the POS. PtdSer (photoreceptor detachment cells) are exposed at the anterior end of the POS, and RPE cells recognize PtdSer using MerTK and its ligands, thereby engulfing and clearing the POS. This phagocytic function is crucial for the homeostasis and functional maintenance of photoreceptor cells and the retina (Front. Immunol., 2020, 11: 604-605).

[0005] Retinitis pigmentosa is a hereditary disease caused by gene mutations affecting the function and homeostasis of photoreceptor cells or RPE. To date, more than 80 pathogenic genes have been identified. Most of these pathogenic genes originate in photoreceptor cells, and their mutations induce cell death and lead to retinal tissue degeneration. Subsequently, dead cells or their debris induce an inflammatory state, exacerbating the symptoms. Rapid phagocytic clearance of dead cells or debris induced by MerTK activation may be a potential treatment for retinitis pigmentosa (Prog. Retin. Eye Res., 2017, 63: 107-131; Adv. Exp. Med. Biol., 2023, 1415: 365-370; PNAS Nexus, 2022, 1: pgac003).

[0006] As a treatment for retinal degeneration caused by debris accumulation (such as retinitis pigmentosa and age-related macular degeneration), gene therapy targeting pathogenic genes and treatments that inhibit retinal disorders by using pharmacological agents such as angiogenesis inhibitors and complement inhibitors are being developed (Prog. Retin. Eye Res., 2018, 63: 107-130). However, there are no sufficiently effective treatments yet, and further research and development of new drugs are needed.

[0007] MerTK activation requires tyrosine autophosphorylation of the intracellular kinase domain. This suggests that anti-MerTK antibodies selected based on phosphorylation of the intracellular kinase domain of MerTK and its downstream molecule AKT could potentially activate downstream intracellular signaling and debris clearance via MerTK, thus potentially curing retinal degeneration (Patent Document 4). However, to date, while anti-MerTK antibodies that activate phagocytosis via MerTK on macrophages have been suggested (Patent Documents 1-4, Non-Patent Document 1), no activated anti-MerTK antibodies demonstrating phagocytosis in RPE cells have been reported.

[0008] Furthermore, in recent years, in order to activate efficient phagocytic clearance of phagocytosed substances by bringing the physical distance between phagocytes and phagocytosed substances closer, bispecific antibodies that bind to MerTK and phagocytosed substances are being investigated. To date, bispecific antibodies that bind to MerTK and CD20, bispecific antibodies that bind to MerTK and amyloid-beta (Aβ) (Non-Patent Literature 2), and bispecific antibodies that bind to MerTK and epidermal growth factor receptor (EGFR) on cancer cells as a tumor-associated antigen (TAA) (Non-Patent Literature 3) have been reported, demonstrating that these bispecific antibodies can activate phagocytic clearance function using MerTK.

[0009] Transthyretin (TTR) is a 14 kDa protein composed of 127 amino acids. The primary site of TTR production is the liver, but it can also be produced in the choroid plexus of the brain, retinal pigment epithelial cells of the retina, and pancreatic α cells. TTR typically forms a stable tetrameric structure in the blood, primarily functioning as a transporter of the vitamin A-retinol-binding protein complex and thyroid hormone T4 in the blood and cerebrospinal fluid (Patent Document 5). TTR is known to be one of the proteins contributing to systemic amyloidosis, primarily caused by genetic mutations and aging. Misfolded TTR deposits throughout the body, causing functional impairment. Antibodies targeting misfolded TTR activate phagocytosis of misfolded TTR by phagocytes via the Fc region, suggesting the potential to inhibit its deposition (Amyloid, 2016, 23: 86-97). However, in a Phase I clinical trial of NI006, a monoclonal anti-TTR IgG1 antibody that binds to misfolded TTRs, several subjects reportedly developed cytokine release syndrome (non-patent literature 4), in addition to its effect of inhibiting the deposition of misfolded TTRs. Further drug development is expected to treat systemic amyloidosis such as TTR-type amyloidosis.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: International Publication No. 2016 / 106221

[0013] Patent Document 2: International Publication No. 2019 / 005756

[0014] Patent Document 3: International Publication No. 2020 / 176497

[0015] Patent Document 4: International Publication No. 2021 / 202590

[0016] Patent Document 5: International Publication No. 2015 / 115332

[0017] Non-patent literature

[0018] Non-patent literature 1: PLoS ONE, 2015, 10: e0145078

[0019] Non-patent literature 2: mAbs, 2020, 12: 1685832

[0020] Non-patent literature 3: Int. J. Mol. Sci., 2022, 23: 15673

[0021] Non-patent literature 4: N. Engl. J. Med., 2023, 389: 239-250 Summary of the Invention

[0022] The problem that the invention aims to solve

[0023] The objective of this invention is to provide anti-MerTK agonist antibodies and anti-MerTK bispecific antibodies that can be used to prevent or treat phagocytosis-related diseases.

[0024] Methods for solving problems

[0025] The inventors conducted numerous innovative studies in the preparation of anti-MerTK antibodies that bind to MerTK and activate the phagocytic clearance function of phagocytes via MerTK, resulting in the development of a novel anti-MerTK agonist antibody, tA3-20 (Example 1). tA3-20 activated the phagocytic clearance function of RPE cells in vitro (Example 2), and subsequently activated the phosphorylation of the intracellular kinase domain of MerTK in RPE and the phosphorylation signaling of its downstream AKT in vivo (Example 3). Furthermore, an anti-MerTK-anti-TTR bispecific antibody containing a single-arm antibody with both the heavy chain and light chain variable regions of tA3-20 can activate the phagocytosis of misfolded TTRs via MerTK (Example 6), and further significantly activated the phagocytic clearance function against the same target in aged mice (Example 7). An anti-MerTK-anti-immunoglobulin light chain (IgL) bispecific antibody containing a single-arm antibody with both the heavy chain variable region and the light chain variable region of tA3-20 binds to human MerTK and misfolded IgL in an antibody concentration-dependent manner and activates phagocytic clearance function (Example 10). Thus, the inventors have provided an anti-MerTK agonist antibody and an anti-MerTK bispecific antibody that bind to MerTK and activate the phagocytic clearance function of phagocytes with the aid of MerTK, thereby completing the present invention.

[0026] That is, the present invention relates to the following [1] to

[86] , but is not limited thereto.

[0027] [1] An anti-MerTK antibody or its antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1 composed of an amino acid sequence of sequence number 1, CDR2 composed of an amino acid sequence of sequence number 2 and CDR3 composed of an amino acid sequence of sequence number 3, and the light chain variable region comprises CDR1 composed of an amino acid sequence of sequence number 4, CDR2 composed of an amino acid sequence of sequence number 5 and CDR3 composed of an amino acid sequence of sequence number 6.

[0028] [2] The anti-MerTK antibody or its antigen-binding fragment as described in [1] contains a heavy chain variable region and a light chain variable region selected from the group consisting of (1) to (6) below: (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; and (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0029] [3] The anti-MerTK antibody according to [1] or [2], wherein it comprises a heavy chain containing a heavy chain variable region and a light chain containing a light chain variable region.

[0030] [4] The anti-MerTK antibody according to [3] contains an amino acid mutation (LALA mutation) of L234A and L235A in the heavy chain (here, the above mutation position is the amino acid position according to the EU index in the human Igγ1 constant region).

[0031] [5] The anti-MerTK antibody according to [3] contains a P331G mutation in the heavy chain (here, the mutation is located at the amino acid position according to the EU index in the human Igγ1 constant region).

[0032] [6] The anti-MerTK antibody according to [3] contains amino acid mutations (LALA mutations) of L234A and L235A and P331G mutation in the heavy chain (here, the above mutations are located at the amino acid positions according to the EU index in the human Igγ1 constant region).

[0033] [7] The anti-MerTK antibody according to [6] is selected from the group consisting of the following (1) to (6): (1) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 10. (2) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 12. (3) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 14. (4) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 16. (5) An anti-MerTK antibody containing a heavy chain consisting of the amino acid sequence of sequence number 8 and a light chain consisting of the amino acid sequence of sequence number 18; and (6) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 20.

[0034] [8] The anti-MerTK antibody according to any one of [1] to [7] is an IgG antibody (anti-MerTK IgG antibody).

[0035] [9] The anti-MerTK antibody or its antigen-binding fragment as described in [1] or [2], wherein the antigen-binding fragment is a single-chain variable region fragment (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

[0036]

[10] The anti-MerTK antibody or its antigen-binding fragment according to any one of [1] to [9] has undergone post-translational modification.

[0037]

[11] A fusion or complex of any one of the anti-MerTK antibodies or antigen-binding fragments thereof as described in any one of [1] to

[10] , or a cell on which the anti-MerTK antibody or antigen-binding fragment of any one of [1] to

[10] is presented on its cell surface.

[0038]

[12] A polynucleotide containing a base sequence encoding a heavy chain variable region or a light chain variable region of an anti-MerTK antibody or an antigen-binding fragment thereof, said polynucleotide being selected from the group consisting of (1) to (7): (1) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8. (2) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (3) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (4) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (5) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (6) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (7) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0039]

[13] A polynucleotide containing a heavy or light chain base sequence encoding an anti-MerTK antibody, said polynucleotide being selected from the group consisting of (1) to (7): (1) A polynucleotide containing a base sequence encoding a heavy chain consisting of the amino acid sequence of sequence number 8; (2) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 10; (3) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 12; (4) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 14; (5) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 16; (6) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 18; and (7) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 20.

[0040]

[14] An expression vector containing the polynucleotides described in

[12] or

[13] .

[0041]

[15] A host cell that has been transformed with the expression vector described in

[14] .

[0042]

[16] A host cell containing polynucleotides selected from the group consisting of (1) to (13) below: (1) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8. (2) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (3) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (4) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (5) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (6) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18. (7) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20. (8) Polynucleotides containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and polynucleotides containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (9) Polynucleotides containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and polynucleotides containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (10) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (11) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (12) A polynucleotide containing a base sequence encoding the heavy chain variable region consisting of amino acids numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding the light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (13) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0043]

[17] A method for producing an anti-MerTK antibody or an antigen-binding fragment thereof, comprising the steps of culturing the host cells described in

[15] or

[16] to express the anti-MerTK antibody or an antigen-binding fragment thereof.

[0044]

[18] A pharmaceutical composition comprising any one of [1] to

[10] an anti-MerTK antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient.

[0045]

[19] The pharmaceutical composition according to

[18] is used for the prevention and / or treatment of eye diseases.

[0046]

[20] The pharmaceutical composition according to

[19] , wherein the eye disease is a retinal disease.

[0047]

[21] The pharmaceutical composition according to

[20] , wherein the retinal disease is a retinal degenerative disease.

[0048]

[22] The pharmaceutical composition according to

[21] wherein the retinal degenerative disease is retinitis pigmentosa or age-related macular degeneration.

[0049]

[23] The anti-MerTK antibody or its antigen-binding fragment according to any one of [1] to

[10] is used for the prevention and / or treatment of eye diseases.

[0050]

[24] A method for preventing or treating an eye disease, comprising administering a therapeutically effective amount of any one of [1] to

[10] an anti-MerTK antibody or an antigen-binding fragment thereof to a subject.

[0051] The use of any one of the anti-MerTK antibodies or antigen-binding fragments thereof described in

[25] [1] to

[10] in the manufacture of pharmaceutical compositions for the prevention and / or treatment of eye diseases.

[0052]

[26] A bispecific antibody that binds to MerTK and misfolded proteins and / or amyloid proteins, comprising a heavy chain variable region and a light chain variable region of an anti-MerTK antibody, and a heavy chain variable region and a light chain variable region of an antibody that binds to misfolded proteins and / or amyloid proteins, wherein the bispecific antibody, The heavy chain variable region of the anti-MerTK antibody contains CDR1, which consists of the amino acid sequence of sequence number 1; CDR2, which consists of the amino acid sequence of sequence number 2; and CDR3, which consists of the amino acid sequence of sequence number 3. The light chain variable region of the anti-MerTK antibody contains CDR1, which consists of the amino acid sequence of sequence number 4, CDR2, which consists of the amino acid sequence of sequence number 5, and CDR3, which consists of the amino acid sequence of sequence number 6.

[0053]

[27] According to the bispecific antibody described in

[26] , wherein the heavy chain variable region and the light chain variable region of the anti-MerTK antibody are selected from the group consisting of the following (1) to (6): (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; and (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0054]

[28] The bispecific antibody according to

[27] includes a single-arm antibody (single-arm anti-MerTK antibody), the single-arm antibody comprising a heavy chain fragment containing a heavy chain variable region of anti-MerTK antibody and a light chain containing a light chain variable region of anti-MerTK antibody, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide.

[0055]

[29] According to the bispecific antibody described in

[28] , the heavy chain fragment and light chain of the single-arm anti-MerTK antibody are selected from the group consisting of (1) to (6) below: (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

[0056]

[30] According to the bispecific antibody described in

[29] , wherein the C-terminus of the scFv or Fab region containing the heavy chain variable region and the light chain variable region of the antibody that binds to misfolded proteins and / or amyloid proteins is connected by means of a hinge region to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody.

[0057]

[31] The bispecific antibody according to any one of

[26] to

[30] , wherein the antibody that binds to misfolded protein and / or amyloid protein is an anti-TTR antibody that binds to misfolded TTR.

[0058]

[32] According to the bispecific antibody described in

[31] , wherein, The heavy chain variable region of the anti-TTR antibody contains CDR1, consisting of the amino acid sequence of sequence number 27, CDR2, consisting of the amino acid sequence of sequence number 28, and CDR3, consisting of the amino acid sequence of sequence number 29. The light chain variable region of the anti-TTR antibody contains CDR1, which consists of the amino acid sequence of sequence number 30, CDR2, which consists of the amino acid sequence of sequence number 31, and CDR3, which consists of the amino acid sequence of sequence number 32.

[0059]

[33] According to the bispecific antibody described in

[32] , the heavy chain variable region of the anti-TTR antibody is a heavy chain variable region composed of amino acid sequences numbered 1 to 119 of sequence number 43. The light chain variable region of the anti-TTR antibody is a light chain variable region consisting of amino acid sequences numbered 135 to 245 of sequence number 43.

[0060]

[34] The bispecific antibody according to

[33] , wherein the scFv (anti-TTR-scFv) contains a heavy chain variable region and a light chain variable region containing an anti-TTR antibody.

[0061]

[35] The bispecific antibody according to

[34] , wherein the anti-TTR-scFv is composed of the amino acid sequence of sequence number 43.

[0062]

[36] The bispecific antibody according to

[34] or

[35] , wherein it comprises a single-arm antibody (single-arm anti-MerTK antibody), the single-arm antibody comprising a heavy chain fragment containing a heavy chain variable region of anti-MerTK antibody and a light chain containing a light chain variable region of anti-MerTK antibody, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide, wherein a C-terminus of anti-TTR-scFv is connected to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody via a hinge region.

[0063]

[37] The bispecific antibody according to any one of

[28] to

[36] , wherein the Fc region contains amino acid mutations (LALA mutation) containing L234A and L235A and P331G mutation (here, the above mutations are located at the amino acid positions according to the EU index in the human Igγ1 constant region).

[0064]

[38] According to the bispecific antibody described in

[37] , wherein the first Fc polypeptide and the second Fc polypeptide contain an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 39 or has 1 to 10 substituted amino acid sequences in the amino acid sequence of sequence number 39.

[0065]

[39] The bispecific antibody according to

[38] contains an Fc region containing a knocks into holes mutation.

[0066]

[40] The bispecific antibody according to

[38] contains an Fc region containing LALA mutation, P331G mutation and mortis mutation.

[0067]

[41] According to the bispecific antibody described in

[39] or

[40] , wherein the mortis mutation is a T366W mutation in one Fc polypeptide that forms the Fc region and a T366S, L368A and Y407V mutation in another Fc polypeptide that forms the Fc region (here, the above mutations are located at the amino acid positions according to the EU index in the human Igγ1 constant region).

[0068]

[42] According to the bispecific antibody of

[38] , wherein the sequences of the first Fc polypeptide and the second Fc polypeptide are polypeptides consisting of any one of the sequences of (1) or (2) below: (1) A first Fc polypeptide consisting of the amino acid sequence of sequence number 40 and a second Fc polypeptide consisting of the amino acid sequence of sequence number 41; or (2) The first Fc polypeptide consisting of the amino acid sequence of sequence number 41 and the second Fc polypeptide consisting of the amino acid sequence of sequence number 40.

[0069]

[43] The bispecific antibody according to

[42] contains a hinge region consisting of the amino acid sequence shown in sequence number 42 and / or a hinge region consisting of the amino acid sequence shown in sequence number 70.

[0070]

[44] The bispecific antibody according to any one of

[26] to

[43] has undergone post-translational modification.

[0071]

[45] A fusion or complex of any one of the bispecific antibodies described in any one of

[26] to

[44] , or a cell on which the bispecific antibody described in any one of

[26] to

[44] is presented on its cell surface.

[0072]

[46] A polynucleotide for producing any one of

[26] to

[44] , said polynucleotide being selected from the group consisting of (1) to (9): (1) A polynucleotide containing a base sequence of the heavy chain variable region encoding an amino acid sequence consisting of amino acids numbered 1 to 119 of sequence number 8 for an anti-MerTK antibody. (2) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 10. (3) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 12. (4) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of amino acid sequence number 14. (5) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 16. (6) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 18. (7) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid 20. (8) A polynucleotide containing a base sequence encoding the base sequence of the heavy chain variable region of an anti-TTR antibody consisting of amino acids numbered 1 to 119 of sequence number 43; and (9) A polynucleotide containing a base sequence of the light chain variable region encoding an anti-TTR antibody consisting of an amino acid sequence consisting of amino acid numbers 135 to 245 of sequence number 43.

[0073]

[47] An expression vector containing the polynucleotide described in

[46] .

[0074]

[48] ​​A host cell that has been transformed with the expression vector described in

[47] .

[0075]

[49] A host cell for producing the bispecific antibody described in any one of

[27] to

[44] , said host cell containing polynucleotides selected from the group consisting of (1) to (3) below: (1) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 16, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 43, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 135 to 245 of sequence number 43; (2) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 119 of sequence number 8; a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 110 of sequence number 18; a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acids numbered 1 to 119 of sequence number 43; and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acids numbered 135 to 245 of sequence number 43; and (3) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 20, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 43, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 135 to 245 of sequence number 43.

[0076]

[50] A method for producing bispecific antibodies, comprising the steps of culturing the host cells described in

[48] or

[49] to express bispecific antibodies that bind to MerTK and TTR.

[0077]

[51] A pharmaceutical composition comprising any one of

[26] to

[44] a bispecific antibody and a pharmaceutically acceptable excipient.

[0078]

[52] The pharmaceutical composition according to

[51] is used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0079]

[53] The pharmaceutical composition according to

[52] wherein the disease caused by the accumulation of misfolded proteins is amyloidosis.

[0080]

[54] The pharmaceutical composition according to

[53] wherein the amyloidosis is systemic amyloidosis.

[0081]

[55] According to the pharmaceutical composition described in

[54] , the systemic amyloidosis is TTR type amyloidosis or polyneuropathy amyloidosis.

[0082]

[56] A bispecific antibody according to any one of

[26] to

[44] , used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0083]

[57] A method for preventing and / or treating a disease caused by the accumulation of misfolded proteins, comprising the step of administering a therapeutically effective amount of any one of

[26] to

[44] of a bispecific antibody to a subject.

[0084] The use of any one of the bispecific antibodies described in

[58]

[26] to

[44] in the manufacture of a pharmaceutical composition for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0085]

[59] The bispecific antibody according to any one of

[26] to

[30] , wherein the antibody binding to misfolded protein and / or amyloid protein is an anti-IgL antibody binding to misfolded IgL.

[0086]

[60] According to the bispecific antibody described in

[59] , wherein, The heavy chain variable region of the anti-IgL antibody contains CDR1, consisting of amino acid sequence 26 to 35 of sequence number 57; CDR2, consisting of amino acid sequence 50 to 68 of sequence number 57; and CDR3, consisting of amino acid sequence 101 to 108 of sequence number 57. The light chain variable region of the anti-IgL antibody contains CDR1, which consists of amino acid sequence number 24 to 39 of sequence number 59; CDR2, which consists of amino acid sequence number 55 to 61 of sequence number 59; and CDR3, which consists of amino acid sequence number 94 to 103 of sequence number 59.

[0087]

[61] According to the bispecific antibody described in

[60] , the heavy chain variable region of the anti-IgL antibody is a heavy chain variable region composed of amino acid sequences numbered 1 to 119 of sequence number 57. The light chain variable region of the anti-IgL antibody is a light chain variable region consisting of amino acid sequences numbered 1 to 112 of sequence number 59.

[0088]

[62] The bispecific antibody according to

[61] , wherein it comprises a Fab fragment (anti-IgL-Fab) containing a heavy chain variable region and a light chain variable region containing an anti-IgL antibody.

[0089]

[63] According to the bispecific antibody described in

[62] , the anti-IgL-Fab contains a fragment consisting of a heavy chain variable region (VH) and a light chain constant region (CL) consisting of amino acid sequences numbered 1 to 226 of sequence number 57, and a fragment consisting of a light chain variable region (VL) and a CH1 domain consisting of amino acid sequences numbered 59.

[0090]

[64] The bispecific antibody according to

[62] or

[63] , wherein it comprises a single-arm antibody (single-arm anti-MerTK antibody), the single-arm antibody comprising a heavy chain fragment containing a heavy chain variable region of anti-MerTK antibody and a light chain containing a light chain variable region of anti-MerTK antibody, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide, wherein an anti-IgL-Fab C-terminus is connected to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody via a hinge region.

[0091]

[65] The bispecific antibody according to any one of

[59] to

[64] , wherein the Fc region contains an amino acid mutation (LALA mutation) containing L234A and L235A and a P331G mutation (here, the above mutation is located at the amino acid position according to the EU index in the human Igγ1 constant region).

[0092]

[66] According to the bispecific antibody described in

[65] , wherein the first Fc polypeptide and the second Fc polypeptide contain an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 39 or has 1 to 10 substituted amino acid sequences in the amino acid sequence of sequence number 39.

[0093]

[67] The bispecific antibody according to

[66] contains an Fc region containing a knocks into holes mutation.

[0094]

[68] The bispecific antibody according to

[66] contains an Fc region containing LALA mutation, P331G mutation and mortis mutation.

[0095]

[69] According to the bispecific antibody described in

[67] or

[68] , wherein the mortis mutation is a T366W mutation in one Fc polypeptide that forms the Fc region, and a T366S, L368A and Y407V mutation in another Fc polypeptide that forms the Fc region (here, the above mutations are located at the amino acid positions according to the EU index in the human Igγ1 constant region).

[0096]

[70] According to the bispecific antibody of

[66] , wherein the sequences of the first Fc polypeptide and the second Fc polypeptide are polypeptides consisting of any one of the sequences of (1) or (2) below: (1) A first Fc polypeptide consisting of the amino acid sequence of sequence number 40 and a second Fc polypeptide consisting of the amino acid sequence of sequence number 41; or (2) The first Fc polypeptide consisting of the amino acid sequence of sequence number 41 and the second Fc polypeptide consisting of the amino acid sequence of sequence number 40.

[0097]

[71] The bispecific antibody according to

[70] contains a hinge region consisting of the amino acid sequence shown in sequence number 42.

[0098]

[72] The bispecific antibody according to any one of

[59] to

[71] has undergone post-translational modification.

[0099]

[73] A fusion or complex of any one of the bispecific antibodies described in any one of

[59] to

[72] , or a cell on which the bispecific antibody described in any one of

[59] to

[72] is presented on its cell surface.

[0100]

[74] A polynucleotide for producing any one of

[59] to

[72] of a bispecific antibody, said polynucleotide being selected from the group consisting of (1) to (9) below: (1) A polynucleotide containing a base sequence of the heavy chain variable region encoding an amino acid sequence consisting of amino acids numbered 1 to 119 of sequence number 8 for an anti-MerTK antibody. (2) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 10. (3) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 12. (4) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of amino acid sequence number 14. (5) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 16. (6) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 18. (7) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid 20. (8) A polynucleotide containing a base sequence encoding the base sequence of the heavy chain variable region of an anti-IgL antibody consisting of amino acids numbered 1 to 119 of sequence number 57; and (9) A polynucleotide containing a base sequence of the light chain variable region encoding an anti-IgL antibody consisting of an amino acid sequence numbered 1 to 112 of amino acid sequence number 59.

[0101]

[75] An expression vector containing the polynucleotide described in

[74] .

[0102]

[76] A host cell that has been transformed with the expression vector described in

[75] .

[0103]

[77] A host cell for producing the bispecific antibody described in any one of

[59] to

[72] , said host cell containing polynucleotides selected from the group consisting of (1) to (4) below: (1) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 109 of sequence number 12, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 57, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 112 of sequence number 59; (2) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 16, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 57, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 112 of sequence number 59; (3) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 119 of sequence number 8; a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 110 of sequence number 18; a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acids numbered 1 to 119 of sequence number 57; and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acids numbered 1 to 112 of sequence number 59; and (4) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 20, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 57, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 112 of sequence number 59.

[0104]

[78] A method for producing bispecific antibodies, comprising the steps of culturing the host cells described in

[76] or

[77] to express bispecific antibodies that bind to MerTK and IgL.

[0105]

[79] A pharmaceutical composition comprising any one of the bispecific antibodies described in

[59] to

[72] and a pharmaceutically acceptable excipient.

[0106]

[80] The pharmaceutical composition according to

[79] is used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0107]

[81] The pharmaceutical composition according to

[80] , wherein the disease caused by the accumulation of misfolded proteins is amyloidosis.

[0108]

[82] The pharmaceutical composition according to

[81] , wherein the amyloidosis is systemic amyloidosis.

[0109]

[83] The pharmaceutical composition according to

[82] , wherein the systemic amyloidosis is AL amyloidosis.

[0110]

[84] A bispecific antibody according to any one of

[59] to

[72] , used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0111]

[85] A method for preventing and / or treating a disease caused by the accumulation of misfolded proteins, comprising the step of administering a therapeutically effective amount of any one of

[59] to

[72] of a bispecific antibody to a subject.

[0112] The use of any one of the bispecific antibodies described in

[86]

[59] to

[72] in the manufacture of a pharmaceutical composition for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0113] Invention Effects

[0114] The anti-MerTK antibody or its antigen-binding fragment, as well as the anti-MerTK bispecific antibody of the present invention, bind to MerTK expressed in phagocytes, thereby activating the phagocytic clearance function of phagocytes by activating downstream intracellular signaling via MerTK. The anti-MerTK antibody or its antigen-binding fragment, the anti-MerTK bispecific antibody, or pharmaceutical compositions containing the thereof of the present invention can be used to prevent or treat diseases (e.g., eye diseases) by activating downstream intracellular signaling via MerTK. Attached Figure Description

[0115] Figure 1 The graph shows the binding activity of the test antibodies to human MerTK-His protein, as determined by ELISA. The vertical axis represents the binding activity (obtained by subtracting the background absorbance at 570 nm from the absorbance at 450 nm), and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average absorbance of each test antibody.

[0116] Figure 2-1 The effect of the tested antibody on the phagocytosis of apoptotic cells by iPS-RPE cells is shown. The vertical axis represents fluorescence intensity as an indicator of the amount of apoptotic cells phagocytosed. The horizontal axis represents the elapsed time since the start of the evaluation. Each point shows the average value of four wells.

[0117] Figure 2-2 The effect of the tested antibody on the phagocytosis of porcine POS by iPS-RPE cells is shown. The vertical axis represents the cumulative fluorescence intensity values ​​used as an indicator of POS phagocytosis from the start of the assay up to 60 hours later. Each point represents the value for each well. The values ​​in the bar chart represent the average values.

[0118] Figure 3-1 The graph shows the phosphorylated MerTK protein level in the retinocyte plasma (RPE) 1 hour after intravitreal administration of tA3-20 to mice. The vertical axis represents the phosphorylated MerTK protein level corrected for total MerTK protein levels, and the horizontal axis represents the dose of the test antibody. Each point represents the value for each individual. Bars represent the mean. Error bars represent the standard deviation.

[0119] Figure 3-2 The graph shows the phosphorylated AKT protein level in the retinocyte plasma (RPE) 1 hour after intravitreal administration of tA3-20 to mice. The vertical axis represents the phosphorylated AKT protein level corrected for total AKT protein levels, and the horizontal axis represents the dose of the test antibody. Each point represents the value for each individual. Bars represent the mean. Error bars represent the standard deviation.

[0120] Figure 4-1The graph shows the binding activity of the test antibody to misTTR as determined by ELISA. The vertical axis represents the binding activity (obtained by subtracting the background absorbance at 570 nm from the absorbance at 450 nm), and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average absorbance of each test antibody.

[0121] Figure 4-2 The graph shows the binding activity of the test antibodies to human MerTK-Fc protein, as determined by ELISA. The vertical axis represents the binding activity (obtained by subtracting the background absorbance at 570 nm from the absorbance at 450 nm), and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average absorbance of each test antibody.

[0122] Figure 5 The MerTK-dependent phagocytosis of pHrodo-misTTR by tA-009 is shown. The vertical axis of the figure represents the total phagocytosis ([p / s]·h) during the observation period. Values ​​represent the mean plus standard error. Student's t-tests were used to compare the solvent group (as a control) with the tA-009 group, and to compare the tA-009 group (as a control) with the tA-009+ONO7475 group. A p-value less than 0.05 was considered statistically significant. The asterisks in the figure (…) The numbers () and (#) indicate significant differences between the solvent group and the tA-009 group, and between the tA-009 group and the tA-009+ONO7475 group, respectively.

[0123] Figure 6-1 The figure shows the activation of phagocytic clearance function of DL800-misTTR by tA-009 or mouse 371M. The vertical axis of the figure represents the total residual amount of misTTR ([p / s]·h) during the observation period. Values ​​represent the mean plus standard error. The comparison between the solvent group (as a control) and the drug (mouse 371M or tA-009) group was performed using the Dunnett test, and a p-value less than 0.05 was considered statistically significant. This indicates a significant difference between the solvent group and the drug group.

[0124] Figure 6-2 The figure illustrates the activation of phagocytic clearance function of DL800-misTTR by tA-009 or mouse-type 371M administered subcutaneously to aged mice. The vertical axis of the figure represents the total residual amount of misTTR ([p / s]·h) during the observation period. Values ​​represent the mean plus standard error. Student's t-test was used to compare the young mouse-solvent group (control group) with the aged mouse-solvent group, and Dunnett's test was used to compare the aged mouse-solvent group (control group) with the aged mouse-drug (mouse-type 371M or tA-009) group. Statistical significance was considered to be less than 0.05. The figures in the figure... # and # represent significant differences, respectively, between the young mouse solvent group and the aged mouse solvent group, and between the aged mouse solvent group and the aged mouse drug group.

[0125] Figure 7 The graph shows the binding activity of the test antibodies against human MerTK and misfolded IgL proteins, as determined by ELISA. The vertical axis represents the binding activity (obtained by subtracting the background absorbance at 570 nm from the absorbance at 450 nm), and the horizontal axis represents the concentration of the test antibody. The symbols indicate the absorbance of each test antibody. Detailed Implementation

[0126] The present invention will now be described in detail.

[0127] <Definitions> Unless otherwise specifically defined below, the terms used in this specification shall be used in the sense commonly understood by those skilled in the art.

[0128] Antibodies (or immunoglobulins) are glycoproteins with a basic structure consisting of four chains in a Y-shape, characterized by symmetry. These chains are composed of two heavy chains with a single sequence and two light chains with a single sequence. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of antibody molecules is common across all classes: two heavy chains with a molecular weight of 50,000–70,000 and two light chains with a molecular weight of 20,000–30,000 are linked by disulfide bonds and non-covalent bonds, forming an antibody molecule with a molecular weight of 150,000–190,000, consisting of four Y-shaped chains. The heavy chains are typically composed of polypeptide chains containing approximately 440 amino acids and have characteristic structures in each class, corresponding to Igγ, Igμ, Igα, Igδ, and Igε, respectively. Furthermore, IgG contains subclasses of IgG1, IgG2, IgG3, and IgG4, with their corresponding heavy chains designated as Igγ1, Igγ2, Igγ3, and Igγ4, respectively. Light chains typically consist of polypeptide chains containing approximately 220 amino acids, and are known to exist in two types: λ and κ, referred to as Igλ and Igκ, respectively. These two types of light chains can pair with any type of heavy chain.

[0129] Regarding the intrachain disulfide bonds in antibody molecules, there are 4 in the heavy chain (5 in Igμ and Igε) and 2 in the light chain, forming a ring every 100-110 amino acid residues. These three-dimensional structures are similar between the rings and are called structural units or domains. The domain located at the amino terminus (also referred to as the "N-terminus" in this specification) in both the heavy and light chains is called the variable region. It is known that even antibodies produced from the same class (or subclass) of the same animal species have diverse amino acid sequences, which are involved in the specificity of antibody-antigen binding. The amino acid sequence of the domain located downstream of the variable region at the carboxyl terminus (also referred to as the "C-terminus" in this specification) is roughly constant in each class or subclass and is called the constant region. In the heavy chain, from the N-terminus to the C-terminus, there are heavy chain variable regions (VH) and heavy chain constant regions (CH). In the CH, it is further divided into three domains from the N-terminus side: CH1 domain, CH2 domain, and CH3 domain. In a light chain, there are two regions from the N end to the C end: the variable region (VL) and the constant region (CL).

[0130] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL are highly variable, contributing to the variability of the variable region. Each CDR is a region of approximately 5–10 amino acid residues located at the N-terminus of the heavy and light chains, in the order CDR1, CDR2, and CDR3, forming the antigen-binding site. On the other hand, the portion of the variable region outside the CDRs is called the framework region (FR), composed of FR1–4, and its amino acid sequence varies less.

[0131] When the antibody is treated with papain, a proteolytic enzyme, three antibody fragments are obtained. The two fragments at the N-terminus are called Fab (antigen-binding fragment) regions. In this specification, "Fab region" refers to the region composed of the VH and CH1 domains of the heavy chain and the light chains (VL and CL), and the antigen-binding site at the front end of this Fab region binds to the antigen. In this specification, "heavy chain fragment" refers to the fragment composed of the VH and CH1 domains of the heavy chain constituting the Fab region. Additionally, the fragment at the C-terminus is called the Fc (crystallizable fragment) region. In this specification, "Fc polypeptide" refers to a polypeptide composed of the CH2 and CH3 domains of the heavy chain, and "Fc region" refers to a complex composed of the first Fc polypeptide and the second Fc polypeptide. The terms "first" or "second" used for Fc polypeptides are used here for ease of distinction and are not intended to assign a specific order or meaning.

[0132] In this specification, the "hinge region" refers to a highly mobile peptide region located between the CH1 and CH2 domains of the heavy chain, where the heavy chain fragment and the Fc polypeptide are linked. Additionally, the two heavy chains of the antibody are linked in the hinge region by disulfide bonds.

[0133] In this specification, "antigen" is used in its usual sense, as a term referring to a molecule or part of a molecule that can specifically bind to an antibody or antigen-binding fragment. Antigens can be molecules such as proteins and nucleic acids. An antigen may sometimes have one or more epitopes capable of interacting with different antibodies or other similar substances.

[0134] In this specification, "IgG antibody" refers to an antibody having a Y-shaped structure consisting of two Fab regions and an Fc region. That is, an IgG antibody has a structure consisting of two heavy chains and two light chains. In one embodiment, the two Fab regions of the IgG antibody contain the same VH and VL sequences.

[0135] In this specification, "antigen-binding fragment" refers to a molecule containing at least one polypeptide chain with antigen-binding activity, comprising both a heavy chain variable region and a light chain variable region of an immunoglobulin molecule. Representative antigen-binding fragments include single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, and F(ab')2 fragments. scFv is a monovalent antigen-binding fragment consisting of VH and VL linked by a linker. Fab fragments are monovalent antigen-binding fragments consisting of a light chain and a fragment containing the VH and CH1 domains of the heavy chain. Fab fragments contain a Fab region. Fab' fragments are monovalent antigen-binding fragments consisting of a light chain and a fragment containing the VH and CH1 domains of the heavy chain and a portion of a hinge region, the hinge region containing cysteine ​​residues that form the SS bond between the heavy chains. F(ab')2 fragments are divalent molecules formed by linking Fab' fragments with disulfide bonds. Monovalent means containing one antigen-binding site, and divalent means containing two antigen-binding sites. In addition, Fab fragments, Fab' fragments, and F(ab')2 fragments may also contain fragments formed by crossing all or part of the VH1 and CH1 domains of the heavy chain and the VL and CL domains of the light chain (mAbs, 2016, 8(6): 1010-1020). Furthermore, "antigen-binding fragments" may also contain heavy chain antibodies such as VHH antibodies (mAbs, 2017, 9(2): 182-212).

[0136] In this specification, "Half-Ig" refers to a monovalent antibody containing one VH, one VL, and one Fc polypeptide. In one embodiment, Half-Ig contains one VH, one CH1 domain, one VL, and one Fc polypeptide. In another embodiment, Half-Ig contains one heavy chain fragment (VH and CH1 domains), one Fc polypeptide (CH2 and CH3 domains), and one light chain (VL and CL). In yet another embodiment, Half-Ig contains one heavy chain and one light chain.

[0137] In this specification, a "single-arm antibody" refers to a monovalent antibody containing one VH, one CH1 domain, one VL, one CL, and one Fc region composed of a first Fc polypeptide and a second Fc polypeptide. In one embodiment, the single-arm antibody contains one heavy chain fragment (VH and CH1 domains), one Fc region (first and second Fc polypeptides (CH2 and CH3 domains)), and one light chain (VL and CL). In one embodiment, the single-arm antibody contains one heavy chain fragment, a hinge region, one Fc region, and one light chain. In one embodiment, the single-arm antibody contains one Fab region, a hinge region, and one Fc region. In one embodiment, the single-arm antibody has a structure containing one Fab region, a hinge region, and one Fc region, with the heavy chain fragment of the Fab region linked to a first Fc polypeptide in the Fc region via the hinge region. In one embodiment, the single-arm antibody has a structure (one heavy chain) comprising a Fab region, a hinge region, and an Fc region, wherein a heavy chain fragment of the Fab region is linked to the N-terminus of a first Fc polypeptide in the Fc region via the hinge region.

[0138] In this specification, "bispecific antibody" refers to an antibody that can specifically bind to two different antigens. Bispecific antibodies consist of two antibodies or antigen-binding fragments that can bind to different antigens respectively. Various structures of bispecific antibodies are well known to those skilled in the art (mAbs, 2017, 9(2): 182-212).

[0139] In this specification, "multispecific antibody" refers to an antibody capable of specifically binding to three or more different antigens. Depending on the number of antigens bound, it may be called, for example, a trispecific antibody or a tetraspecific antibody. A multispecific antibody consists of three or more antibodies and / or antigen-binding fragments capable of binding to different antigens respectively.

[0140] In this specification, unless the context otherwise requires, the term "antibody" is used as a term that also includes IgG antibodies, Half-Ig, single-arm antibodies, bispecific antibodies, and multispecific antibodies.

[0141] In this specification, "human antibody" refers to an antibody containing the amino acid sequence of human immunoglobulins. "Fully human antibody" refers to an antibody composed solely of the amino acid sequence of human immunoglobulins. "Mouse-human chimeric antibody" refers to an antibody containing both the antigen-binding variable region of the mouse immunoglobulin amino acid sequence and the constant region of the human immunoglobulin amino acid sequence.

[0142] The amino acid residue numbers of the antibodies used in this specification may be specified according to these numbering systems by specifying the Kabat number or the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, NIHP Publication: No. 91-3242).

[0143] In this specification, "object" refers to a person or other animal that needs to prevent or treat a disease. In one embodiment, the object is a person who needs to prevent or treat a disease. In another embodiment, the object is a person who needs to prevent or treat various eye diseases, lung diseases, autoimmune diseases, neurological diseases, heart diseases, endocrine / metabolic diseases, cancer, etc.

[0144] In this instruction manual, "diagnosis" is not limited to determining whether one has the disease, but includes all diagnoses after diagnosis, such as determining the severity (progression) of the disease and determining the likelihood of future occurrence of the disease.

[0145] In this manual, "prevention" refers to any intervention undertaken to prevent or delay the occurrence of disease.

[0146] In this instruction manual, "treatment" refers to any intervention, treatment, or administration of an active ingredient to a subject in order to restore, alleviate, improve, suppress, or delay the progression, occurrence, aggravation, or recurrence of the symptoms, condition, or biochemical indicators related to the disease.

[0147] In this specification, "active ingredient" refers to a substance that exhibits any physiological activity in a pharmaceutical composition, medicine, or other substance used in the prevention or treatment of a disease. In one embodiment, the active ingredient is an antibody or an antigen-binding fragment. In another embodiment, the active ingredient is a fusion / complex of an antibody or an antigen-binding fragment. In yet another embodiment, the active ingredient is a bispecific antibody.

[0148] In this specification, "pharmaceutical composition" means a pharmaceutical preparation containing an active ingredient and a pharmaceutically acceptable excipient (e.g., pharmaceutical excipients, pharmaceutical carriers, etc., but not limited thereto) formulated for the prevention or treatment of a subject.

[0149] In this instruction manual, the “effective amount” of a drug refers to the dosage required to induce physiological changes in a subject by acting on the subject’s tissues or cells when the drug is administered to the subject.

[0150] <The anti-MerTK antibody of the present invention>

[0151] In this specification, anti-MerTK antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment capable of binding to human MerTK. Whether or not it binds to human MerTK can be confirmed using known methods for determining binding activity. For example, the enzyme-linked immunosorbent assay (ELISA) described in Examples 1-2 or 1-3 can be used.

[0152] This invention provides the following anti-MerTK antibody or antigen-binding fragment thereof: An anti-MerTK antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1 consisting of an amino acid sequence of sequence number 1, CDR2 consisting of an amino acid sequence of sequence number 2, and CDR3 consisting of an amino acid sequence of sequence number 3, and the light chain variable region comprises CDR1 consisting of an amino acid sequence of sequence number 4, CDR2 consisting of an amino acid sequence of sequence number 5, and CDR3 consisting of an amino acid sequence of sequence number 6.

[0153] In this specification, the anti-MerTK antibody is also referred to as "the anti-MerTK antibody of the present invention", and the anti-MerTK antibody or its antigen-binding fragment is also referred to as "the anti-MerTK antibody or its antigen-binding fragment of the present invention".

[0154] In one embodiment, the anti-MerTK antibody or its antigen-binding fragment of the present invention is an anti-MerTK antibody or its antigen-binding fragment containing a heavy chain variable region and a light chain variable region selected from the group consisting of (1) to (6) below: (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; and (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0155] In one embodiment, the anti-MerTK antibody of the present invention is an antibody containing both a heavy chain and a light chain. Antibodies containing both heavy and light chains include IgG antibodies, Half-Ig, and single-arm antibodies. In one embodiment, the anti-MerTK antibody of the present invention is an IgG antibody (anti-MerTK IgG antibody).

[0156] In one embodiment, the antigen-binding fragment of the present invention is scFv, Fab fragment, Fab' fragment, or F(ab')2 fragment or VHH antibody.

[0157] The heavy chain constant region contained in the anti-MerTK antibody or its antigen-binding fragment of the present invention may be selected from any of Igγ, Igμ, Igα, Igδ, or Igε. For example, Igγ can be selected from Igγ1, Igγ2, Igγ3, or Igγ4. The light chain constant region contained in the anti-MerTK antibody or its antigen-binding fragment of the present invention may be selected from any of Igλ or Igκ. In one embodiment, the heavy chain constant region and the light chain constant region of the anti-MerTK antibody or its antigen-binding fragment are constant regions of human Igγ1 and Igλ, respectively.

[0158] The anti-MerTK antibody of the present invention may contain mutations that reduce antibody-dependent cell killing (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent killing (CDC) activity. L234A refers to the substitution of leucine at position 234 of the human Igγ1 constant region with alanine. L235A refers to the substitution of leucine at position 235 of the human Igγ1 constant region with alanine. The amino acid mutations L234A and L235A in the human Igγ1 constant region are referred to as "LALA mutations." This mutation is known to reduce the antibody's ADCC, ADCP, and CDC by decreasing the binding activity of the antibody's Fc region to the Fcγ receptor and its binding activity to C1q (Mol. Immunol., 1992, 29: 633-639; J. Immunol., 2000, 164: 4178-4184; Front. Immunol., 2019, 10: 1296). P329A refers to the substitution of proline to alanine at position 329 of the human Igγ1 constant region. P331G or P331S refers to the substitution of proline to glycine or serine at position 331 of the human Igγ1 constant region. This mutation is known to reduce antibody CDC (J. Immunol., 2000, 164: 4178-4184).

[0159] In one embodiment, the anti-MerTK antibody of the present invention contains amino acid mutations (LALA mutations) of L234A and L235A in the heavy chain. In one embodiment, the anti-MerTK antibody of the present invention contains any one of the P329A, P331G, or P331S mutations in the heavy chain. In one embodiment, the anti-MerTK antibody of the present invention contains both the LALA mutation and the P329A mutation in the heavy chain. In one embodiment, the anti-MerTK antibody of the present invention contains both the LALA mutation and the P331G mutation in the heavy chain.

[0160] It should be noted that in this specification, the descriptions of amino acid mutations such as LALA mutation, P329A, P331G, or P331S mutation are based on the amino acid positions in the human Igγ1 constant region according to the EU index. For example, as mentioned above, L234A refers to the substitution of leucine for alanine at position 234 of the human Igγ1 constant region according to the EU index.

[0161] The anti-MerTK antibody of the present invention may also contain other mutations based on known technologies. For example, the anti-MerTK antibody of the present invention may contain the N297G mutation (Protein cell, 2018, 9: 63-73), a mutation based on the mortar and pestle (kih) technology (hereinafter also referred to as "mortar and pestle mutation"), or an electrostatic orientation mutation.

[0162] The KIH technique involves replacing the amino acid side chain in the CH3 region of one heavy chain with a larger side chain (knob; protrusion), and replacing the amino acid side chain in the CH3 region of another heavy chain with a smaller side chain (hole; gap). This allows the protrusion to be positioned within the gap, promoting heterodimerization of the heavy chain and efficiently obtaining the target heterodimerized antibody molecule (Nature, 1994, 372: 379-383; Nature Biotech., 1998, 16: 677-681; J. Mol. Biol., 1997, 270: 26-35; Proc. Natl. Acad. Sci. USA, 2013, 110: E2987-E2996, International Publication No. 1998 / 050431).

[0163] The electrostatic switching technique involves replacing the amino acid side chain in the CH3 domain of one heavy chain with a negatively charged amino acid side chain, and replacing the amino acid side chain in the CH3 domain of another heavy chain with a positively charged amino acid side chain (referred to as "electrostatic switching mutation" in this specification). This promotes the heterodimerization of the heavy chain through electrostatic force, thereby efficiently obtaining the target heterodimerized antibody molecule (J. Biol. Chem., 2010, 285: 19637-19646). As mutations used to stabilize electrostatic shifting and heterodimerization antibody molecules in mouse-derived antibodies, mutations containing E356K, T364S, M368L, T370K, D399K, R411T in the CH3 domain of one heavy chain and T364S, M368L, T370K, K409E, R411T, K439D in the CH3 region of another heavy chain are known (mAbs, 2019, 12: 1-12).

[0164] In one embodiment, the anti-MerTK antibody of the present invention is an anti-MerTK antibody selected from the group consisting of (1) to (6) below: (1) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 10. (2) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 12. (3) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 14. (4) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 16. (5) An anti-MerTK antibody containing a heavy chain consisting of the amino acid sequence of sequence number 8 and a light chain consisting of the amino acid sequence of sequence number 18; and (6) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 20.

[0165] The anti-MerTK antibody or antigen-binding fragment thereof of the present invention also includes antibodies or antigen-binding fragments thereof that bind to mouse, monkey, and / or rat-derived MerTK in addition to binding to human MerTK. In one embodiment, the anti-MerTK antibody or antigen-binding fragment thereof of the present invention is an antibody or antigen-binding fragment thereof that binds to human, mouse, monkey, and / or rat-derived MerTK. In one embodiment, the anti-MerTK antibody or antigen-binding fragment thereof of the present invention binds to human MerTK (gene number: NM_006343). Binding to various MerTKs can be confirmed using known methods for measuring binding activity. Examples of methods for measuring binding activity include, for example, ELISA and flow cytometry. When using ELISA or flow cytometry, for example, the methods described in Example 3 or 5 of International Publication No. 2022 / 224997 can be used.

[0166] As a method for evaluating the agonist activity of the anti-MerTK antibody or its antigen-binding fragment of the present invention, the AKT phosphorylation-inducible activity, which is a downstream signal of MerTK, can be evaluated. For example, known methods can be used as methods for evaluating AKT phosphorylation-inducible activity. In this specification, anti-MerTK antibodies having AKT phosphorylation-inducible activity are also referred to as "anti-MerTK agonist antibodies".

[0167] In this specification, "post-translational modification" refers to modifications that antibodies undergo after translation during intracellular expression. Examples of post-translational modifications include the addition of N- or O-bound glycans, N-terminal or C-terminal processing, deamidation, isomerization of aspartic acid, and oxidation of methionine. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci., 2008, 97: 2426-2447).

[0168] In one embodiment, the anti-MerTK antibody or its antigen-binding fragment of the present invention may be post-translational modified. In one embodiment, the post-translational modification includes the addition of an N-binding or O-binding glycan, processing of the N-terminus or C-terminus, deamidation, isomerization of aspartic acid, and / or oxidation of methionine.

[0169] The anti-MerTK antibody or its antigen-binding fragment of the present invention can be readily prepared by those skilled in the art using methods known in the art, based on the sequence information of the heavy chain variable region and light chain variable region of the MerTK antibody of the present invention disclosed in this specification. The anti-MerTK antibody or its antigen-binding fragment of the present invention is not particularly limited; for example, it can be manufactured according to the method described later in the section "Method for Producing the Anti-MerTK Antibody of the Present Invention".

[0170] <The polynucleotide of this invention>

[0171] The present invention further provides a polynucleotide containing a base sequence encoding the heavy chain variable region of the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof, and a polynucleotide containing a base sequence encoding the light chain variable region of the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof (also referred to as "the polynucleotide of the present invention").

[0172] In one embodiment, the polynucleotide containing the base sequence encoding the anti-MerTK antibody or its antigen-binding fragment of the present invention is a polynucleotide containing the base sequence encoding the heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8.

[0173] In one embodiment, the polynucleotide containing the base sequence of the heavy chain encoding the anti-MerTK antibody of the present invention is a polynucleotide containing the base sequence encoding the heavy chain consisting of the amino acid sequence shown in Serial No. 8.

[0174] In one embodiment, the polynucleotide containing the base sequence of the light chain variable region encoding the anti-MerTK antibody or its antigen-binding fragment of the present invention is a polynucleotide selected from the group consisting of (a) to (f): (a) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (b) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (c) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acids numbered 1 to 109 of sequence number 14. (d) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 16. (e) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (f) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0175] In one embodiment, the polynucleotide containing the base sequence of the light chain encoding the anti-MerTK antibody of the present invention is a polynucleotide selected from the group consisting of (a) to (f): (a) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 10; (b) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 12; (c) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 14; (d) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 16; (e) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 18; and (f) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 20.

[0176] The polynucleotides of the present invention can be readily produced by those skilled in the art based on their base sequences using methods known in the art. For example, the polynucleotides of the present invention can be synthesized using gene synthesis methods known in the art. As such gene synthesis methods, various methods known to those skilled in the art, such as the antibody gene synthesis method described in International Publication No. 90 / 07861, can be used.

[0177] <Expression Vector of the Invention>

[0178] The present invention further provides expression vectors (also referred to as "inventive vectors of the present invention") comprising a polynucleotide containing a base sequence encoding the heavy chain variable region of the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof and / or a polynucleotide containing a base sequence encoding the light chain variable region of the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof.

[0179] In one embodiment, the expression vector of the present invention may be an expression vector comprising a polynucleotide containing a base sequence encoding the heavy chain variable region of the anti-MerTK antibody of the present invention, an expression vector comprising a polynucleotide containing a base sequence encoding the light chain variable region of the anti-MerTK antibody of the present invention, or an expression vector comprising a polynucleotide containing a base sequence encoding the heavy chain variable region of the anti-MerTK antibody of the present invention and a polynucleotide containing a base sequence encoding the light chain variable region.

[0180] In one embodiment, the expression vector of the present invention may be an expression vector comprising a polynucleotide containing a base sequence encoding the heavy chain of the anti-MerTK antibody of the present invention, an expression vector comprising a polynucleotide containing a base sequence encoding the light chain of the anti-MerTK antibody of the present invention, or an expression vector comprising a polynucleotide containing a base sequence encoding the heavy chain of the anti-MerTK antibody of the present invention and a polynucleotide containing a base sequence encoding the light chain.

[0181] In one embodiment, the expression vector of the present invention contains polynucleotides selected from the group consisting of (1) to (7) below: (1) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8. (2) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (3) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (4) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (5) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (6) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (7) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0182] In one embodiment, the expression vector of the present invention contains polynucleotides selected from the group consisting of (1) to (7) below: (1) A polynucleotide containing a base sequence encoding a heavy chain consisting of the amino acid sequence of sequence number 8; (2) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 10; (3) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 12; (4) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 14; (5) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 16; (6) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 18; and (7) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 20.

[0183] As the expression vector of the present invention, there are no particular limitations as long as it can express polynucleotides containing the base sequence of the heavy chain variable region encoding the anti-MerTK antibody or its antigen-binding fragment of the present invention and / or polynucleotides containing the base sequence of the light chain variable region encoding the anti-MerTK antibody or its antigen-binding fragment of the present invention in various host cells of eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (e.g., Escherichia coli), and can produce polypeptides encoded by these, there are no particular limitations. Examples of such expression vectors include plasmid vectors, viral vectors (e.g., adenovirus, retrovirus), such as pcDNA3.4 TOPO vector, pEE6.4, and pEE12.4. In addition, the antibody gene can also be expressed by introducing the variable region gene fragment into expression vectors such as AG-γ1 and AG-κ (e.g., refer to International Publication No. 94 / 20632) that pre-contain the human Ig constant region gene.

[0184] The expression vector of the present invention may contain a promoter operably linked to the polynucleotide of the present invention. Examples of promoters for expressing the polynucleotide of the present invention in animal cells include viral promoters such as CMV, RSV, and SV40, actin promoters, EF (elongation factor) 1α promoters, and heat shock promoters. Examples of promoters for expression in bacteria (e.g., Escherichia coli) include trp promoters, lac promoters, λPL promoters, and tac promoters. Furthermore, examples of promoters for expression in yeast include GAL1 promoters, GAL10 promoters, PH05 promoters, PGK promoters, GAP promoters, and ADH promoters.

[0185] When using animal cells, insect cells, or yeast as host cells, the expression vector of the present invention may contain a start codon and a stop codon. In this case, the expression vector of the present invention may contain an enhancer sequence, untranslated regions on the 5' and 3' sides of a gene encoding the antibody of the present invention or its heavy chain variable region or light chain variable region, a secretion signal sequence, a splice junction, a polyadenylation site, or a reproducible unit, etc. When using *E. coli* as a host cell, the expression vector of the present invention may contain a start codon, a stop codon, a stop region, and a reproducible unit. In this case, the expression vector of the present invention may contain commonly used selection markers (e.g., tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, dihydrofolate reductase genes) depending on the purpose.

[0186] <Transformed host cells of the present invention>

[0187] The present invention further provides host cells (also referred to as "host cells of the present invention") transformed using the polynucleotides of the present invention or the expression vectors of the present invention (collectively referred to as "polynucleotides of the present invention"). The host cells of the present invention contain the polynucleotides of the present invention. The introduced polynucleotides of the present invention may or may not be integrated into the genomic DNA of the host cell. The cell used as the host cell can be any of a cell capable of being cultured in vitro or a cell in vivo. When the host cell is a cell capable of being cultured in vitro, the host cell of the present invention can be created by introducing the polynucleotides of the present invention into the cell in vitro. The method of transforming the host cell is not particularly limited, and methods commonly used by those skilled in the art, such as the calcium phosphate method, electroporation method, or liposome transfection method, can be used. When the host cell is a cell in vivo, the method of introducing the polynucleotides of the present invention into the host cell is not particularly limited, and nucleic acid delivery vectors (including cationic vectors or non-cationic vectors (e.g., liposomes, lipid nanoparticles (LNPs), but not limited thereto)) can be used.

[0188] The cells that can be cultured in vitro are not particularly limited to any cells that can be transformed and express antibodies or peptides using methods such as expression vectors or electroporation. Examples of cells that can be cultured in vitro include various conventional cells or artificially created cells commonly used in the technical field of this invention (e.g., animal cells such as CHO-K1 cells, ExpiCHO-S (registered trademark) cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, Expi293F cells, NSO cells), insect cells (e.g., Sf9), bacteria (Escherichia coli, etc.), yeast (Saccharomyces, Pichia pastoris, etc.)). In one embodiment, the host cells of this invention are Expi293F cells, CHO-K1 cells, or ExpiCHO-S cells.

[0189] Screening of host cells after in vitro transformation can be performed using methods commonly employed by those skilled in the art. Screening methods may include, for example, agent selection using marker genes and agents such as tetracycline, ampicillin, neomycin, or hygromycin, limiting dilution methods, single-cell sorting, or colony picking, and other cell isolation methods.

[0190] In one embodiment, the host cell of the present invention contains polynucleotides selected from the group consisting of (a) to (c): (a) A polynucleotide containing a base sequence encoding the heavy chain variable region of the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof, and a polynucleotide containing a base sequence encoding the light chain variable region of the antibody or an antigen-binding fragment thereof. (b) A polynucleotide containing the base sequence of the heavy chain variable region encoding the anti-MerTK antibody or its antigen-binding fragment of the present invention; and (c) A polynucleotide containing a base sequence of a light chain variable region encoding the anti-MerTK antibody of the present invention or its antigen-binding fragment.

[0191] In one embodiment, the host cell of the present invention contains polynucleotides selected from the group consisting of (a) to (c): (a) A polynucleotide containing the base sequence of the heavy chain encoding the anti-MerTK antibody of the present invention and a polynucleotide containing the base sequence of the light chain encoding the antibody. (b) A polynucleotide containing the base sequence of the heavy chain encoding the anti-MerTK antibody of the present invention; and (c) A polynucleotide containing the base sequence of the light chain encoding the anti-MerTK antibody of the present invention.

[0192] In one embodiment, the host cell of the present invention contains polynucleotides selected from the group consisting of (1) to (13): (1) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8. (2) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (3) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (4) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (5) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (6) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18. (7) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20. (8) Polynucleotides containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and polynucleotides containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (9) Polynucleotides containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and polynucleotides containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (10) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (11) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (12) A polynucleotide containing a base sequence encoding the heavy chain variable region consisting of amino acids numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding the light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (13) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0193] <Method for producing the anti-MerTK antibody of the present invention>

[0194] The present invention further provides a method for producing the anti-MerTK antibody or its antigen-binding fragment of the present invention (also referred to as "the production method of the present invention"). The production method of the present invention may include the above-described method for producing the polynucleotide, the method for producing the expression vector, and the method for producing the host cell. Furthermore, the production method of the present invention may also include the steps of culturing the host cell described in the above-described <transformed host cell of the present invention> and expressing the antibody in the cell or the culture supernatant, and methods for recovering, separating, and purifying the antibody. However, the production method of the present invention is not limited to these methods as long as it produces the anti-MerTK antibody or its antigen-binding fragment of the present invention.

[0195] In one embodiment, the production method of the present invention includes the following steps: culturing host cells containing a polynucleotide containing a base sequence of a heavy chain variable region encoding the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof, and a polynucleotide containing a base sequence of a light chain variable region encoding the antibody or an antigen-binding fragment thereof, to express the anti-MerTK antibody.

[0196] The culture of transformed host cells can be performed using known methods. Culture conditions, such as temperature, pH of the medium, and culture time, can be appropriately selected. When the host cells are animal cells, suitable culture media include, for example, MEM medium (Science, 1959, 130: 432-437) containing approximately 5–20% fetal bovine serum, DMEM medium (Virology, 1959, 8: 396-397), RPMI 1640 medium (J. Am. Med. Assoc., 1967, 199: 519), and 199 medium (Proc. Soc. Exp. Biol. Med., 1950, 73: 1-8). The preferred pH of the medium is approximately 6–8, and culture should be performed at approximately 30–40°C for approximately 15–336 hours, with aeration and stirring as needed. When the host cell is an insect cell, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 1985, 82: 8404-8408) can be used as the culture medium. The pH of the medium is preferably about 5 to 8, and culture should be carried out for about 15 to 100 hours at a temperature of about 20 to 40°C, with aeration and stirring as needed. When the host cell is *Escherichia coli* or yeast, a liquid medium containing nutrients is suitable as the culture medium. The nutrient medium preferably contains the carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of the transformed host cell. Examples of carbon sources include glucose, dextran, soluble starch, and sucrose; examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. The culture medium may contain other nutrients (e.g., inorganic salts (e.g., calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins), and antibiotics (e.g., tetracycline, neomycin, ampicillin, kanamycin), depending on preference. The pH of the culture medium is preferably about 5 to 8. When the host cell is *Escherichia coli*, preferred culture media include, for example, LB medium or M9 medium (Cold Spring Harb. Laboratory, 2001, 3: A2.2). Culture is carried out at approximately 14 to 39°C for about 3 to 24 hours, with aeration and stirring as needed. When the host cell is yeast, Burkholder minimal medium (Proc. Natl. Acad. Sci. USA, 1980, 77: 4504-4508) can be used as the culture medium. Culture is carried out at approximately 20 to 35°C for about 14 to 144 hours, with aeration and stirring as needed. Through the above-described culture, the anti-MerTK antibody or its antigen-binding fragment of the present invention can be expressed.

[0197] The production method of the present invention, in addition to the steps of culturing the transformed host cells of the present invention and expressing the anti-MerTK antibody or its antigen-binding fragment, may also include the steps of recovering, for example, separating or purifying the anti-MerTK antibody or its antigen-binding fragment from the transformed host cells. Examples of separation or purification methods include, for instance, methods utilizing solubility such as salting out and solvent precipitation; methods utilizing molecular weight differences such as dialysis, ultrafiltration, and gel filtration; methods utilizing charged charges such as ion exchange chromatography and hydroxyapatite chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing hydrophobic differences such as reversed-phase high-performance liquid chromatography; and methods utilizing isoelectric point differences such as isoelectric point electrophoresis. For example, antibodies accumulated in the culture supernatant can be purified by various chromatography methods, such as column chromatography using protein A columns or protein G columns.

[0198] The anti-MerTK antibody or its antigen-binding fragment of the present invention also includes the anti-MerTK antibody or its antigen-binding fragment produced by the production method of the present invention.

[0199] <The fusion body of the present invention, the complex of the present invention, and cells whose cell surfaces are presented with the anti-MerTK antibody of the present invention or its antigen-binding fragment>

[0200] This invention further provides an anti-MerTK antibody or its antigen-binding fragment thereof linked to a protein (including an antibody) or polypeptide other than MerTK (also referred to as "the fusion body of this invention"). That is, in this specification, "the fusion body of this invention" refers to a substance formed by linking the anti-MerTK antibody or its antigen-binding fragment of this invention to a protein (including an antibody) or polypeptide other than MerTK. The proteins and polypeptides used in the fusion body of this invention are not particularly limited, and can include, for example, antibodies, antigen-binding fragments, ligands, receptors, cytokines, chemokines, growth factors, physiologically active substances, human serum albumin, various tag peptides, artificial helical motif peptides, maltose-binding proteins, glutathione S-transferase, and other peptides or proteins that can promote polymerization. In one embodiment, the fusion body of this invention can be a fusion body formed by linking a protein or polypeptide to the anti-MerTK antibody or its antigen-binding fragment of this invention via a linker (e.g., a peptide linker). In one embodiment, the proteins and peptides used in the fusion body of the present invention may be, for example, antibodies or antigen-binding fragments thereof targeting cell surface antigens of immune cells such as T cells, B cells, natural killer (NK) cells, dendritic cells, granulocytes, macrophages, mast cells, epithelial cells, fibroblasts, or RPE cells, or peptides that activate immune cells such as various interleukins (e.g., IL-2, IL-7, IL-12, IL-15). In this case, the proteins and peptides used in the fusion body of the present invention can be directly linked to the anti-MerTK antibody or its antigen-binding fragment of the present invention, or they can be linked using any linker (e.g., peptide linker).

[0201] This invention further provides the anti-MerTK antibody or its antigen-binding fragment thereof (also referred to as the "complex of this invention") formed by combining with glycosides, lipids, metals (including radioactive isotopes), organic compounds (including toxins, near-infrared fluorescent dyes, chelating agents, etc.) (also referred to as "modifiers"). That is, in this specification, "complex of this invention" refers to a complex formed by linking the anti-MerTK antibody or its antigen-binding fragment of this invention with substances other than proteins and peptides. In this specification, "modifier" refers to a non-peptide substance that binds directly or with the aid of a linker or the like to the antibody or its antigen-binding fragment. The modifiers used in the complex of this invention are not particularly limited, and examples include polyethylene glycol, sugar chains, phospholipids, radioactive isotopes (e.g., zirconium-89 (89Zr), yttrium-90 (90Y), indium-111 (111In), astatine-211 (211At), actinium-225 (225Ac)), organic compounds, toxins, near-infrared fluorescent dyes (e.g., IRDye (registered trademark)), chelating agents, etc. The substances used in this complex can bind directly to the anti-MerTK antibody or its antigen-binding fragment of the present invention, or they can bind using any adapter. In one embodiment, the complex of the present invention is a drug complex of the anti-MerTK antibody or its antigen-binding fragment (antibody-drug conjugate, ADC). The pharmaceutical agent and adapter used in the ADC can be selected by those skilled in the art from commonly used pharmaceutical agents and adapters. In one embodiment, the complex of the present invention is a radioisotope-labeled antibody to which a radioisotope is bound.

[0202] The composite of this invention can also be used to detect soluble or membrane-based MerTK. In this specification, "detection" includes quantitative detection or qualitative detection.

[0203] The present invention further provides cells (e.g., chimeric antigen receptor-T cells (CAR-T cells)) whose surface is presented with the anti-MerTK antibody or its antigen-binding fragment of the present invention (hereinafter also referred to as "the presenting cell of the present invention"). The method for producing the presenting cell of the present invention is not particularly limited; for example, it can be produced by expressing the anti-MerTK antibody or its antigen-binding fragment on the cell surface using a polynucleotide encoding the anti-MerTK antibody or its antigen-binding fragment of the present invention. Various cells (macrophages, alveolar macrophages, T cells, and NK cells, etc.) can be used as the cells presenting the anti-MerTK antibody or its antigen-binding fragment of the present invention.

[0204] The fusion body, the composite body, and the presenting cell of the present invention are also collectively referred to as "fusion body, etc." of the present invention.

[0205] In one embodiment, the antibody or antigen-binding fragment used in the fusion composite of the present invention may be post-translational modified. In one embodiment, the post-translational modification includes the addition of N-binding or O-binding glycans, processing of the N-terminus or C-terminus, deamidation, isomerization of aspartic acid, and / or oxidation of methionine.

[0206] The anti-MerTK antibody or its antigen-binding fragment of the present invention, the fusion complex of the present invention, etc., can be readily prepared by those skilled in the art using methods known in the art, based on the VH and VL sequence information of the anti-MerTK antibody or its antigen-binding fragment of the present invention disclosed in this specification, other peptides or proteins (e.g., antibodies) used in the fusion complex of the present invention, and information on the modifiers used in the complex of the present invention. The anti-MerTK antibody or its antigen-binding fragment of the present invention is not particularly limited; for example, it can be manufactured according to the method described in the section "Method for producing the anti-MerTK antibody of the present invention".

[0207] <Pharmaceutical Compositions of the Invention>

[0208] The present invention further provides pharmaceutical compositions (also referred to as "pharmaceutical compositions of the present invention") containing the anti-MerTK antibody of the present invention, its antigen-binding fragment or the fusion compound of the present invention, and pharmaceutically acceptable excipients. The pharmaceutical compositions of the present invention can be prepared using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc., by commonly used methods. Examples of dosage forms for these pharmaceutical compositions include non-oral dosage forms such as injections, infusions, and eye drops, which can be administered via intraocular, intravitreal, retrobulbar, subconjunctival, subfascial, subretinal, inhalation, intramedullary, intraperitoneal, intravenous, subcutaneous, intra-articular, nasal, and intramuscular administration. During formulation, excipients, carriers, additives, etc., corresponding to these dosage forms can be used within pharmaceutically acceptable limits.

[0209] The pharmaceutical compositions of the present invention may contain various anti-MerTK antibodies of the present invention, their antigen-binding fragments or fusion bodies of the present invention, and their post-translational modifications. For example, pharmaceutical compositions containing antibodies that have undergone the addition of N-binding or O-binding glycans, N-terminal or C-terminal processing, deamidation, isomerization of aspartic acid and / or oxidation of methionine are also included in the present invention.

[0210] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising an anti-MerTK antibody of the present invention or an antigen-binding fragment thereof or a fusion compound thereof containing any one of the following (a) to (f), and / or a post-translational modified version thereof of the antibody or an antigen-binding fragment thereof or a fusion compound thereof: (a) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (b) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (c) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (d) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (e) The heavy chain variable region consisting of amino acid sequences 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences 1 to 110 of sequence number 18; and (f) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0211] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising the anti-MerTK antibody or fusion compound of the present invention containing any one of the heavy and light chains of (a) to (f) below, and / or a post-translational modified version of the antibody or fusion compound of the present invention: (a) The heavy chain consisting of the amino acid sequence of sequence number 8 and the light chain consisting of the amino acid sequence shown in sequence number 10. (b) The heavy chain consisting of the amino acid sequence of sequence number 8 and the light chain consisting of the amino acid sequence shown in sequence number 12. (c) The heavy chain consisting of the amino acid sequence of sequence number 8 and the light chain consisting of the amino acid sequence shown in sequence number 14. (d) The heavy chain consisting of the amino acid sequence of sequence number 8 and the light chain consisting of the amino acid sequence shown in sequence number 16. (e) The heavy chain consisting of the amino acid sequence of sequence number 8 and the light chain consisting of the amino acid sequence shown in sequence number 18; and (f) The heavy chain consisting of the amino acid sequence of sequence number 8 and the light chain consisting of the amino acid sequence shown in sequence number 20.

[0212] During the formulation process described above, the amount of the anti-MerTK antibody or its antigen-binding fragment of the present invention added varies depending on the severity of the patient's symptoms, age, dosage form of the formulation used, or antibody binding titer, etc. For example, it may be used from about 0.0001 mg / kg to about 100 mg / kg.

[0213] <Pharmaceutical Uses of the Anti-MerTK Antibody of the Invention>

[0214] The anti-MerTK antibody, its antigen-binding fragment, or the fusion compound of the present invention, as well as pharmaceutical compositions containing them, can be used to prevent or treat diseases caused by the accumulation of apoptotic cells, fragments from apoptotic cells, and / or cells, tissues, or fragments that highly express PtdSer. In this specification, "fragment" is a term distinct from undamaged cells, referring to cell fragments and / or mixtures containing substances not needed by the organism, such as building blocks. In this specification, "fragment from cells" refers to cell fragments derived from undamaged cell fragmentation and / or mixtures containing substances not needed by the organism, such as building blocks. In this specification, "cells, tissues, or fragments that highly express PtdSer" refers to cells, tissues, or fragments containing PtdSer other than apoptotic cells and fragments from apoptotic cells.

[0215] In one embodiment, the anti-MerTK antibody of the present invention, its antigen-binding fragment or the fusion body of the present invention, or a pharmaceutical composition containing them, can activate phagocytes by means of activating MerTK, thereby phagocytosing the phagocytosed material.

[0216] In this manual, "phagocyte" refers to a cell with phagocytic and clearance functions, "phagocytosis" refers to the function of phagocytes in taking in phagocytosed material, and "phagocytic and clearance functions" refers to the functions that begin with phagocytosis and continue until the ingested phagocytosed material is digested and broken down. Examples of phagocytic cells that enhance or are induced to phagocytosis and / or phagocytic clearance function through the anti-MerTK antibody of the present invention, its antigen-binding fragment or the fusion body of the present invention, or pharmaceutical compositions containing them include, for example, macrophages, alveolar macrophages, dendritic cells, microglia, oligodendrocytes, epithelial cells, RPE cells, airway epithelial cells, Köpper cells, hepatocytes, endothelial cells, neutrophils, monocytes, megakaryocytes, astrocytes, Langerhans cells, satellite cells, astrocytes, neural progenitor cells, podocytes, mesangial cells, CD103+ dendritic cells, CD11b+ macrophages, CD11b+CD103+ macrophages, intestinal epithelial cells and Sertoli cells (Nat. Rev. Mol. Cell Biol., 2020, 23: 398-414; Cells, 2021, 10: 1443). It should be noted that, in this specification, the enhancement or induction of phagocytosis and phagocytic clearance functions are sometimes collectively referred to as the "activation" of phagocytosis and phagocytic clearance functions. In one embodiment, the phagocytes activated by the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion composite of the present invention, or pharmaceutical compositions containing them, are epithelial cells (e.g., RPE cells, airway epithelial cells, intestinal epithelial cells). In one embodiment, the phagocytes activated by the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion composite of the present invention, or pharmaceutical compositions containing them, are RPE cells. In this specification, "RPE cells" refers to cells that form the retinal pigment epithelium (RPE). In one embodiment, the phagocytes activated by the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion composite of the present invention, or pharmaceutical compositions containing them, are macrophages or tissue-resident macrophages (e.g., alveolar macrophages, microglia, Kupffer cells). In one embodiment, the phagocytes activated by the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion variant of the present invention, or a pharmaceutical composition containing the thereof, are alveolar macrophages.

[0217] In this specification, "phagocytosed material" refers to the object phagocytosed by phagocytes. The anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion compound of the present invention, or pharmaceutical compositions containing them, can enhance the clearance of phagocytosed material by activating the phagocytic clearance function of phagocytes. Phagocytosed material includes, but is not limited to, apoptotic cells, cancer cells, neutrophils, mast cells, myofibroblasts, adipocytes, debris from these cells, POS and myelin fragments.

[0218] In one embodiment, the phagocytosed material is apoptotic cells. In one embodiment, the phagocytosed material is debris from apoptotic cells. In one embodiment, the phagocytosed material is cells, tissues, or debris highly expressing PtdSer. In one embodiment, the phagocytosed material is POS (Positive Pathogen). In one embodiment, the phagocytosed material is myelin debris. In one embodiment, the phagocytosed material is neutrophils. In one embodiment, the phagocytosed material is debris from neutrophils.

[0219] Diseases that are the target of prevention or treatment according to this invention include diseases caused by the accumulation of apoptotic cells, fragments from apoptotic cells, or cells, tissues, or fragments highly expressing PtdSer, or diseases caused by reduced phagocytic clearance function. The diseases caused by the accumulation of apoptotic cells, fragments from apoptotic cells, or cells, tissues, or fragments highly expressing PtdSer, or diseases caused by reduced phagocytic clearance function, are not particularly limited, and examples include various eye diseases, lung diseases, autoimmune diseases, neurological diseases, fibrotic diseases, circulatory system diseases, and cancer, etc.

[0220] In one embodiment, the disease that is the object of prevention or treatment according to the present invention is an eye disease. In one embodiment, the eye disease that is the object of prevention or treatment according to the present invention is a retinal disease. In one embodiment, the retinal disease that is the object of prevention or treatment according to the present invention is a retinal degenerative disease. In one embodiment, the retinal degenerative disease that is the object of prevention or treatment according to the present invention is retinitis pigmentosa, age-related macular degeneration, and / or diabetic retinopathy.

[0221] In one embodiment, the disease that is the object of prevention or treatment according to the present invention is a lung disease. In one embodiment, the lung disease that is the object of prevention or treatment according to the present invention is an acute lung disease. In one embodiment, the acute lung disease that is the object of prevention or treatment according to the present invention is acute respiratory distress syndrome and / or acute lung injury.

[0222] In one embodiment, the disease that is the object of prevention or treatment according to the present invention is an autoimmune disease. In one embodiment, the autoimmune disease that is the object of prevention or treatment according to the present invention is systemic lupus erythematosus. In one embodiment, the autoimmune disease that is the object of prevention or treatment according to the present invention is multiple sclerosis.

[0223] In one embodiment, the disease that is the object of prevention or treatment of the present invention is rejection reaction during various organ transplants.

[0224] In one embodiment, the disease that is the object of prevention or treatment according to the present invention is a circulatory system disease. In one embodiment, the circulatory system disease that is the object of prevention or treatment according to the present invention is atherosclerosis. In one embodiment, the circulatory system disease that is the object of prevention or treatment according to the present invention is an ischemic disease.

[0225] In one embodiment, the disease that is the target of prevention or treatment according to the present invention is cancer. There is no particular limitation on the type of cancer that is the target of prevention or treatment; examples include peritoneal implantation of various cancer cells, gastric cancer, lung cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, T-cell lymphoma and other hematologic malignancies, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, etc. Solid cancers including cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, small bowel cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, and brain tumors; as well as cancers of bone, cartilage, adipose, muscle, vascular, and hematopoietic tissues; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, and soft tissue sarcoma; and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleural pulmonary blastoma, and retinoblastoma.

[0226] <Diagnostic Uses of the Anti-MerTK Antibody of the Invention>

[0227] The anti-MerTK antibody, its antigen-binding fragment, or the fusion complex of the present invention, or a diagnostic test kit containing them (hereinafter also referred to as "the test kit of the present invention"), can be used to detect MerTK. The test kit of the present invention may contain detectable markers, such as indicator enzymes, radiolabels, fluorescent dye molecules, or magnetic particles, as needed. The test kit of the present invention may contain an information brochure, such as a brochure instructing on the steps of using the reagents to perform the methods described in this specification. In one embodiment, the anti-MerTK antibody of the present invention can diagnose an inflammatory state by detecting soluble MerTK in bodily fluids (blood, etc.). In one embodiment, the complex of the present invention can diagnose an inflammatory state and the glycan modification state of MerTK by detecting and analyzing soluble MerTK in blood.

[0228] Furthermore, the present invention includes methods for diagnosing, preventing, or treating diseases caused by the accumulation of apoptotic cells, fragments from apoptotic cells, or cells, tissues, or fragments highly expressing PtdSer, or diseases caused by reduced phagocytic clearance function. These methods include using the anti-MerTK antibody of the present invention, its antigen-binding fragment, and the fusion composite of the present invention, and administering a diagnostically, preventively, or therapeutically effective amount to a subject. Additionally, the present invention includes the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion composite of the present invention for diagnosing, preventing, or treating diseases caused by the accumulation of apoptotic cells, fragments from apoptotic cells, or cells, tissues, or fragments highly expressing PtdSer, or diseases caused by reduced phagocytic clearance function. Furthermore, the present invention includes the use of the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion composite of the present invention in the manufacture of pharmaceutical compositions for the diagnosis, prevention, or treatment of diseases caused by the accumulation of apoptotic cells, fragments from apoptotic cells, or cells, tissues, or fragments highly expressing PtdSer, or diseases caused by reduced phagocytic clearance function.

[0229] <The anti-MerTK bispecific antibody of the present invention>

[0230] The present invention further provides a bispecific antibody containing the anti-MerTK antibody or its antigen-binding fragment of the present invention (hereinafter also referred to as "the anti-MerTK bispecific antibody of the present invention") by linking an anti-MerTK antibody or its antigen-binding fragment to any antibody or its antigen-binding fragment against an antigen other than MerTK (hereinafter collectively referred to as "other antibodies"). The anti-MerTK bispecific antibody of the present invention contains the heavy chain variable region and the light chain variable region of the anti-MerTK antibody of the present invention, as well as the heavy chain variable region and the light chain variable region of other antibodies.

[0231] The anti-MerTK bispecific antibody of the present invention contains the heavy chain variable region and light chain variable region of the anti-MerTK antibody as shown below: The heavy chain variable region contains CDR1, composed of the amino acid sequence of sequence number 1; CDR2, composed of the amino acid sequence of sequence number 2; and CDR3, composed of the amino acid sequence of sequence number 3; and It contains light chain variable regions consisting of CDR1 (composed of amino acid sequence number 4), CDR2 (composed of amino acid sequence number 5), and CDR3 (composed of amino acid sequence number 6).

[0232] In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region of an anti-MerTK antibody selected from the group consisting of (1) to (6) below: (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; or (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0233] The anti-MerTK bispecific antibody of the present invention can be any structure described in the literature (mAbs, 2017, 9: 182-212, Figure 2). In one embodiment, the anti-MerTK bispecific antibody of the present invention can have a symmetrical or asymmetrical structure. In one embodiment, the anti-MerTK bispecific antibody of the present invention is an antibody fusion type (e.g., IgG2 type), a variable region-only type (e.g., DART type, Diabody type (dual antibody type)), a CH1 / CL fusion protein type (e.g., scFv2-CH1 / CL type), a Fab fusion protein type (e.g., Fab-scFv type), a non-immunoglobulin fusion protein type (e.g., scFv2-alubumin type), an Fc region modified IgG type (e.g., IgG(kih) type, CrossMab(IgG-kih) type, DuetMab type), or an IgG with added and Fc region modified Ig. Type G (e.g., IgG(kih)-scFab, CrossMab-Fab), modified Fc region and CH3 fusion protein type (e.g., scFv-Fc(kih), Fab-scFv-Fc(kih)), added IgGs-heavy chain fusion type (e.g., IgG-HC-scFv), added IgGs-light chain fusion type, added IgGs-heavy chain and light chain fusion type, Fc region fusion type, CH3 fusion type, IgE / IgM and CH2 fusion type, F(ab')2 fusion type, CH1 / CL fusion protein type, modified IgG type, or non-immunoglobulin fusion type. In one embodiment, the anti-MerTK bispecific antibody of the present invention has an asymmetric structure.

[0234] In one embodiment, the anti-MerTK bispecific antibody of the present invention may have a structure in which other antibodies or antigen-binding fragments thereof are attached to the N-terminus or C-terminus of the heavy chain or light chain of the anti-MerTK antibody of the present invention, which is an IgG antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention has a structure in which other antibodies or antigen-binding fragments thereof are attached to the N-terminus of the heavy chain or the N-terminus of the second Fc polypeptide, the C-terminus of the Fc region, or the N-terminus or C-terminus of the light chain of the anti-MerTK antibody of the present invention, which is a single-arm antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention has a structure in which other antibodies or antigen-binding fragments thereof are attached to the N-terminus of the second Fc polypeptide of the anti-MerTK antibody of the present invention, which is a single-arm antibody, via a hinge region.

[0235] In one embodiment, the anti-MerTK bispecific antibody of the present invention can be DuetMab type, CrossMab type (IgG-kih) type, Fab-scFv-Fc(kih) type, IgG(kih)-scFab type, CrossMab-Fab type or IgG-HC-scFv type.

[0236] In one embodiment, the anti-MerTK bispecific antibody of the present invention can be F(ab')2 fusion type, Diabody type, DART type, and Fab-scFv type.

[0237] In one embodiment, the antigen-binding fragment of the anti-MerTK antibody contained in the anti-MerTK bispecific antibody of the present invention can be scFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or VHH antibody.

[0238] In one embodiment, the antigen-binding fragments of other antibodies contained in the anti-MerTK bispecific antibody of the present invention may be scFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or VHH antibody.

[0239] In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody and a light chain containing a light chain variable region of an anti-MerTK antibody. In another embodiment, the anti-MerTK bispecific antibody of the present invention comprises a single-arm antibody (hereinafter also referred to as "single-arm anti-MerTK antibody") and the heavy chain variable regions and light chain variable regions of other antibodies, wherein the single-arm antibody comprises a heavy chain fragment containing the heavy chain variable region of an anti-MerTK antibody, a light chain containing the light chain variable region, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide. In another embodiment, the anti-MerTK bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody and an scFv or Fab region containing the heavy chain variable region and light chain variable region of other antibodies. In yet another embodiment, the anti-MerTK bispecific antibody of the present invention is a bispecific antibody in which the C-terminus of the scFv or Fab region containing the heavy chain variable region and light chain variable region of other antibodies is linked to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody via a hinge region. In one embodiment, the anti-MerTK bispecific antibody of the present invention is of the Fab-scFv-Fc(kih) type. In another embodiment, the anti-MerTK bispecific antibody of the present invention is of the CrossMab(IgG-kih) type.

[0240] In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody containing a heavy chain fragment and a light chain selected from the group consisting of (1) to (6) below: (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

[0241] The anti-MerTK bispecific antibody of the present invention is not limited to the structure described above, and may also be a bispecific antibody containing monovalent or polyvalent anti-MerTK antibody or its antigen-binding fragment, and monovalent or polyvalent antibodies or their antigen-binding fragments against antigens other than MerTK. Furthermore, by further combining the anti-MerTK bispecific antibody of the present invention with antibodies against a third antigen and a fourth antigen, multispecific antibodies such as trispecific or tetraspecific antibodies can be provided.

[0242] The type and length of the linker used in the bispecific antibody of the present invention are not particularly limited, and those skilled in the art can choose appropriately. For example, when the anti-MerTK antibody or other antibody of the present invention is scFv, the length of the peptide linker connecting the heavy chain variable region and the light chain variable region of the antibody is preferably 5 amino acids or more (the upper limit is not particularly limited, usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. As peptide linkers, for example, glycine-serine linkers (GS linkers) and glycine-lysine-proline-glycine-serine linkers (GKPGS linkers) can be used. Examples of such linkers include the following.

[0243] Ser

[0244] Gly-Ser

[0245] Gly-Gly-Ser

[0246] Ser-Gly-Gly

[0247] Gly-Gly-Gly-Ser (Serial Number 46)

[0248] Ser-Gly-Gly-Gly (Serial Number 47)

[0249] Gly-Gly-Gly-Gly-Ser (serial number 48)

[0250] Ser-Gly-Gly-Gly-Gly (serial number 49)

[0251] Gly-Gly-Gly-Gly-Gly-Ser (serial number 50)

[0252] Ser-Gly-Gly-Gly-Gly-Gly (serial number 51)

[0253] Gly-Gly-Gly-Gly-Gly-Gly-Ser (serial number 52)

[0254] Ser-Gly-Gly-Gly-Gly-Gly-Gly (serial number 53)

[0255] (Gly-Gly-Gly-Gly-Ser)n (when n=3, the sequence number is 54)

[0256] (Ser-Gly-Gly-Gly-Gly)n

[0257] Gly-Lys-Pro-Gly-Ser (Serial Number 55)

[0258] (Gly-Lys-Pro-Gly-Ser)n

[0259] The n above represents an integer greater than or equal to 1. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to their purpose.

[0260] In one embodiment, when the anti-MerTK bispecific antibody of the present invention contains the scFv of other antibodies, the peptide linker connecting the heavy chain variable region and the light chain variable region of the other antibody has the amino acid sequence described in Serial No. 48. In one embodiment, the peptide linker connecting the heavy chain variable region and the light chain variable region of the other antibody has the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n. In one embodiment, the peptide linker connecting the heavy chain variable region and the light chain variable region of the other antibody has the amino acid sequence described in Serial No. 54.

[0261] In the case where the anti-MerTK bispecific antibody of the present invention contains an Fc region, the Fc region of the bispecific antibody may contain mutations that reduce antibody-dependent cell killing activity (ADCC), antibody-dependent cell phagocytosis activity (ADCP), and complement-dependent killing activity (CDC). In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises an Fc region containing amino acid mutations of L234A and L235A (LALA mutation) (here, the mutation positions are located at the amino acid positions according to the EU index in the human Igγ1 constant region). In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises an Fc region containing a P329A mutation or a P331G mutation (here, the mutation positions are located at the amino acid positions according to the EU index in the human Igγ1 constant region). In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises an Fc region containing amino acid mutations of L234A and L235A (LALA mutation) and a P331G mutation (here, the mutation positions are located at the amino acid positions according to the EU index in the human Igγ1 constant region). In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises an Fc region containing a knocks-in-holes mutation. In another embodiment, the anti-MerTK bispecific antibody of the present invention comprises an Fc region containing an LALA mutation, a P331G mutation, and a knocks-in-holes mutation. In one embodiment, the knocks-in-holes mutation in the anti-MerTK bispecific antibody of the present invention is a T366W mutation in one Fc polypeptide forming the Fc region and T366S, L368A, and Y407V mutations in another Fc polypeptide forming the Fc region (here, the above mutations are located at the amino acid positions according to the EU index in the human Igγ1 constant region). The mortise and tenon mutation contained in the anti-MerTK bispecific antibody of the present invention may be the introduction of the T366W mutation in the first Fc polypeptide and the introduction of the T366S, L368A and Y407V mutations in the second Fc polypeptide, or the introduction of the T366S, L368A and Y407V mutations in the first Fc polypeptide and the introduction of the T366W mutation in the second Fc polypeptide.

[0262] Other antibody or antigen-binding fragments, without particular limitation, may include, for example, misfolded proteins, amyloid proteins, myelin fragments, Aβ, immune cells (e.g., T cells), receptors expressed on immune cells, or antibodies or antigen-binding fragments targeting tumor-associated antigens on cancer cells.

[0263] In one embodiment, the anti-MerTK bispecific antibody of the present invention is a bispecific antibody that binds to MerTK as well as misfolded proteins and / or amyloid proteins (hereinafter also referred to as "the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention"). In this specification, "misfolded" or "misfolded" means that it has not folded correctly in vivo. Additionally, "misfolded protein" refers to a misfolded protein that does not possess its intended function, and "amyloid protein" is a protein formed by the coagulation of such a misfolded protein.

[0264] Proteins known to form misfolded proteins include, for example, immunoglobulin light chain (IgL), IgH, SAA, TTR, β2-microglobulin, apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, apolipoprotein CII, apolipoprotein CIII, coagulin, lysozyme, LECT2, fibrinogen α chain, cystatin C, ABri (precursor) protein, ADan (precursor) protein, Aβ precursor protein, α-synuclein, tau protein, prion protein, TMEM106B, calcitonin (procalcitonin), amylin, ANP, prolactin, somatostatin (procalcitonin), and hypertensive disorders. Glycosin, PTH, insulin, enfuvritide, GLP1, IL1RAP, pulmonary surfactant, corneal lockstreptin, MFG-E8, corneal epithelial protein, lactoferrin, OAAP, protamine 1, cathepsin K, EFEMP1, huntingtin, myelin, TDP43, and SOD1, etc. (Amyloid, 2022, 4: 213-219; J. Neurosci., 2006, 26: 328-332; Neurobiology of Disease, 2023, 184: 106218). In one embodiment, the protein forming the misfolded protein is TTR. In another embodiment, the protein forming the misfolded protein is IgL.

[0265] The anti-MerTK-anti-misfolded protein bispecific antibody of the present invention contains the heavy chain variable region and light chain variable region of the anti-MerTK antibody of the present invention, as well as the heavy chain variable region and light chain variable region of the antibody that binds to misfolded proteins and / or amyloid proteins. The anti-MerTK-anti-misfolded protein bispecific antibody of the present invention contains the heavy chain variable region and light chain variable region of the anti-MerTK antibody as shown below: The heavy chain variable region contains CDR1, composed of the amino acid sequence of sequence number 1; CDR2, composed of the amino acid sequence of sequence number 2; and CDR3, composed of the amino acid sequence of sequence number 3; and It contains light chain variable regions consisting of CDR1 (composed of amino acid sequence number 4), CDR2 (composed of amino acid sequence number 5), and CDR3 (composed of amino acid sequence number 6).

[0266] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-MerTK antibody selected from the group consisting of (1) to (6) below: (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; or (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0267] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody and a light chain containing a light chain variable region of an anti-MerTK antibody. In another embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody comprising a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody, a light chain containing a light chain variable region, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide. In a further embodiment, in the single-arm anti-MerTK antibody contained in the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention, the heavy chain fragment containing the heavy chain variable region of the anti-MerTK antibody comprises a heavy chain linked to the N-terminus of the first Fc polypeptide via a hinge region. In a further embodiment, the heavy chain fragment containing the heavy chain variable region of the anti-MerTK antibody is linked to the N-terminus of the first Fc polypeptide via a hinge region. In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody comprising a single-arm anti-MerTK antibody, wherein the single-arm anti-MerTK antibody comprises a heavy chain fragment containing a heavy chain variable region of the anti-MerTK antibody, a light chain containing a light chain variable region, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide, wherein the heavy chain fragment of the single-arm anti-MerTK antibody contains a heavy chain linked to the N-terminus of the first Fc polypeptide via a hinge region. In another embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody comprising a single-arm anti-MerTK antibody, wherein the single-arm anti-MerTK antibody comprises a heavy chain fragment containing a heavy chain variable region of the anti-MerTK antibody, a light chain containing a light chain variable region, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide, wherein the heavy chain fragment is linked to the N-terminus of the first Fc polypeptide via a hinge region.

[0268] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody containing a heavy chain fragment and a light chain selected from the group consisting of (1) to (6) below: (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

[0269] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody and an scFv or Fab region containing a heavy chain variable region and a light chain variable region of an antibody that binds to misfolded proteins and / or amyloid proteins. In another embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody in which an scFv or Fab region containing an antibody that binds to misfolded proteins and / or amyloid proteins is further linked via a hinge region to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody.

[0270] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention contains an antibody or antigen-binding fragment thereof that binds to the misfolded protein, myelin fragment, or Aβ or its amyloid protein. Examples of proteins that form misfolded proteins, myelin fragments, or Aβ or its amyloid protein include, for example, IgL, IgH, SAA, TTR, β2 microglobulin, apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, apolipoprotein CII, apolipoprotein CIII, coagulin, lysozyme, LECT2, fibrinogen α chain, cystatin C, ABri (precursor) protein, ADan (precursor) protein, Aβ, Aβ precursor protein, α-synuclein, and ta u protein, prions, TMEM106B, calcitonin (progenitor), amylin, ANP, prolactin, somatostatin (progenitor), glucagon, PTH, insulin, enfuvritide, GLP1, IL1RAP, pulmonary surfactant, corneal lock-chain protein, MFG-E8, corneal epithelial protein, lactoferrin, OAAP, protamine 1, cathepsin K, EFEMP1, huntingtin, myelin fragments, TDP43 or SOD1, etc.

[0271] In one embodiment, the other antibodies used in the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention may be anti-SAA antibody, anti-amylin antibody, anti-Aβ antibody, anti-huntington protein antibody, anti-TTR antibody, or anti-IgL antibody or antigen-binding fragments thereof. In another embodiment, the other antibodies used in the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention may be anti-TTR antibody selectively binding to misfolded TTR or anti-IgL antibody selectively binding to misfolded IgL.

[0272] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody containing the anti-MerTK antibody or its antigen-binding fragment of the present invention and an anti-TTR antibody or its antigen-binding fragment, and having binding activity against MerTK and misfolded TTR (referred to herein as "anti-MerTK-anti-TTR bispecific antibody"). In another embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody containing the anti-MerTK antibody or its antigen-binding fragment of the present invention and an anti-IgL antibody or its antigen-binding fragment, and having binding activity against MerTK and misfolded IgL (referred to herein as "anti-MerTK-anti-IgL bispecific antibody").

[0273] In one embodiment, the anti-MerTK bispecific antibody of the present invention contains an antibody or antigen-binding fragment targeting a T-cell receptor.

[0274] In one embodiment, the anti-MerTK bispecific antibody of the present invention may be a bispecific antibody containing an antibody or antigen-binding fragment that binds to MerTK and tumor-associated antigen (TAA) (hereinafter also referred to as "the anti-MerTK-anti-TAA bispecific antibody of the present invention").

[0275] Examples of antibodies or antigen-binding fragments thereof used in the anti-MerTK-anti-TAA bispecific antibody of the present invention include, for example, HER2, HER3, HER4, CD19, CD20, CD22, CD33, CD38, CD40, CD44, CD70, CD123, CD138, CXCR3, CXCR5, CCR3, CCR4, CCR9, CRTH2, PMCH, CD4, CD25, CD200, endoplasmin, BCMA, CD276, TSPAN8, CLDN4, CLDN6, CLDN18.2, ENPP3, SLC34A2, TROP2, Nectin-4, ASGR1, mesothelin, PSMA, LIV- 1. MUC1, MUC2, MUC4, MUC16, CDH6, CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21, URLC10, NY-ESO-1, GAA, OFA, cyclin B1, WT-1, CEF, VEGRR1, VEGFR2, TTK, HPV16, HPV16E7, HPV18, CEAIMA910, KOC1, SL-701, MART-1, gp100, tyrosinase, survival protein, MAGE-3.1, MAGE-10.A2, OVA BiP, gp209-2M, melan-A, NA17.A2, KOC1, CO16, DEPDC1, MPHOSPH1, MAGE12, ONT-10, GD2L, GD3L, URLC10, CDCA1, TF , PSA, TERT, STF-II, G17DT, ICT-107, Dex2, hTERT, PAP, TRP2, LRRC15, c-Met, PD-L1, FAP, EGFR, B3-H3, GPC3, 5T4.

[0276] The anti-MerTK bispecific antibody of the present invention may contain mutations that reduce ADCC, CDC, ADCP and / or promote heterodimer formation. Such mutations are as described above in the <Anti-MerTK Antibody of the Present Invention>.

[0277] In one embodiment, the anti-MerTK bispecific antibody of the present invention may be post-translational modified. This post-translational modification is as described above in the <Anti-MerTK Antibody of the Present Invention>. In one embodiment, the post-translational modification includes the addition of an N-binding or O-binding glycan, processing of the N-terminus or C-terminus, deamidation, isomerization of aspartic acid, and / or oxidation of methionine.

[0278] The anti-MerTK bispecific antibody of the present invention can be prepared by those skilled in the art using methods known in the art, based on the sequence information of the heavy chain variable region and light chain variable region of the anti-MerTK antibody of the present invention disclosed in this specification, as well as the sequence information of other known antibodies or antigen-binding fragments.

[0279] <Fusion and complex of the anti-MerTK bispecific antibody of the present invention, and cells on the cell surface presenting the anti-MerTK bispecific antibody of the present invention>

[0280] For the anti-MerTK bispecific antibody of the present invention, a fusion composite of the anti-MerTK bispecific antibody of the present invention is provided, which is formed by linking a protein (including polypeptides) other than the antigen bound to MerTK and other antibodies, and an antibody (including antigen-binding fragments) other than the anti-MerTK antibody and other antibodies. Additionally, for the anti-MerTK bispecific antibody of the present invention, a complex of the anti-MerTK bispecific antibody of the present invention is provided, which is formed by binding a modifying agent. Furthermore, for the anti-MerTK bispecific antibody of the present invention, cells (e.g., Tandem CARs) whose surface is presented with the anti-MerTK bispecific antibody of the present invention are provided (e.g., immune cells or effector cells) (referred to in this specification as "cells whose cell surface is presented with the anti-MerTK bispecific antibody of the present invention") (Oncology Reports, 2019, 42: 2183-2195). The fusion composite, the complex, and the cells with the presented antibody on their cell surface are as described above in <The fusion composite, the complex, and the cells whose cell surface is presented with the anti-MerTK antibody of the present invention or its antigen-binding fragment>.

[0281] <The polynucleotide, expression vector, transformed host cells, and production method of the anti-MerTK bispecific antibody of the present invention>

[0282] The present invention further provides a polynucleotide for producing the anti-MerTK bispecific antibody of the present invention. This polynucleotide can be implemented by those skilled in the art based on the descriptions above in "Polynucleotides of the Present Invention" and below in "Polynucleotides of the Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention".

[0283] The present invention further provides an expression vector containing the anti-MerTK bispecific antibody of the present invention and a method for producing the anti-MerTK bispecific antibody of the present invention. These can be implemented by those skilled in the art based on the above-described "Expression Vector of the Present Invention" and "Method for Producing the Anti-MerTK Antibody of the Present Invention".

[0284] The present invention further provides host cells transformed with the anti-MerTK bispecific antibody of the present invention. These host cells can be implemented by those skilled in the art based on the above-described <Transformed Host Cells of the Present Invention> and the following <Transformed Host Cells with the MerTK-Anti-TTR Bispecific Antibody of the Present Invention>.

[0285] <Pharmaceutical Uses of the Anti-MerTK Bispecific Antibody of the Invention>

[0286] The present invention further provides pharmaceutical compositions comprising the anti-MerTK bispecific antibody of the present invention and pharmaceutically acceptable excipients. These pharmaceutical compositions can be implemented by those skilled in the art based on the foregoing description of "Pharmaceutical Compositions of the Invention".

[0287] The anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody can be used to prevent or treat diseases caused by the accumulation of misfolded proteins.

[0288] The anti-MerTK anti-bispecific antibodies of the present invention or pharmaceutical compositions containing them can bring phagocytes and phagocytosed substances closer together by binding to them and activating phagocytes by activating MerTK, thereby efficiently taking up phagocytosed substances and digesting them.

[0289] Phagocytes activated by the anti-MerTK bispecific antibody of the present invention can be used according to the above-described "Pharmaceutical Use of the Anti-MerTK Antibody of the Present Invention". Examples of phagocytosed material that can be cleared with enhanced phagocytic clearance function due to activation of the phagocytic clearance function of phagocytes by the anti-MerTK bispecific antibody of the present invention include misfolded proteins, amyloid proteins, and / or cancer cells. In one embodiment, the phagocytosed material is a misfolded protein. In one embodiment, the phagocytosed material is amyloid protein. In one embodiment, the phagocytosed material is a misfolded TTR (hereinafter also referred to as "misfolded TTR"). In one embodiment, the phagocytosed material is a misfolded IgL (hereinafter also referred to as "misfolded IgL"). In one embodiment, the phagocytosed material is myelin fragments. In one embodiment, the phagocytosed material is Aβ.

[0290] The anti-MerTK bispecific antibody of the present invention, or a pharmaceutical composition containing the antibody, can be used to prevent or treat diseases caused by the accumulation of misfolded proteins, diseases caused by the accumulation of amyloid proteins, or diseases caused by reduced phagocytic clearance function. Furthermore, the present invention includes a method for preventing or treating diseases caused by the accumulation of misfolded proteins, diseases caused by the accumulation of amyloid proteins, or diseases caused by reduced phagocytic clearance function, comprising the step of administering a therapeutically effective amount of the anti-MerTK bispecific antibody of the present invention to a subject. Additionally, the present invention includes the anti-MerTK bispecific antibody of the present invention for preventing or treating diseases caused by the accumulation of misfolded proteins, diseases caused by the accumulation of amyloid proteins, or diseases caused by reduced phagocytic clearance function. Furthermore, the present invention includes the use of the anti-MerTK bispecific antibody of the present invention in the manufacture of pharmaceutical compositions for the prevention or treatment of diseases caused by the accumulation of misfolded proteins, diseases caused by the accumulation of amyloid proteins, or diseases caused by reduced phagocytic clearance function. The diseases that are the target of prevention or treatment according to the present invention, including those caused by the accumulation of misfolded proteins, the accumulation of amyloid proteins, or reduced phagocytic clearance function, are not particularly limited. Examples include various types of amyloidosis, heart disease, neurological diseases, eye diseases, or endocrine / metabolic diseases. In one embodiment, the diseases that are the target of prevention or treatment of the anti-MerTK bispecific antibody and the pharmaceutical composition containing it according to the present invention are amyloidosis, heart disease, neurological diseases, eye diseases, or endocrine / metabolic diseases.

[0291] In one embodiment, the disease that is the target of prevention or treatment for the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is amyloidosis (e.g., localized amyloidosis or systemic amyloidosis). In one embodiment, the amyloidosis that is the target of prevention or treatment for the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is localized amyloidosis. Localized amyloidosis that is the target of prevention or treatment for the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention includes Alzheimer's disease, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease, hereditary cerebral amyloid angiopathy, familial British dementia, familial Danish dementia, localized atrial amyloidosis, prolactinoma, insulinoid amyloidosis, corneal amyloidosis, aortic medial amyloidosis, or localized nodular amyloidosis (Circ.J., 2020, 84: 1610-1671). In one embodiment, the amyloidosis that is the target of prevention or treatment for the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is systemic amyloidosis (e.g., transthyretin amyloid cardiomyopathy (ATTR-CM) or polyneuropathy amyloidosis (ATTR-PN), AL amyloidosis, AH amyloidosis, Aβ2M amyloidosis (dialysis-associated amyloidosis), AA amyloidosis, AApoAI amyloidosis, AApoAII amyloidosis, AApoAIV amyloidosis, AApoCII amyloidosis, AApoCIII amyloidosis, AGel amyloidosis, ALys amyloidosis, ALECT2 amyloidosis, AFib amyloidosis, ACys amyloidosis, ABri amyloidosis, APrP amyloidosis, ACal amyloidosis) (Heart) Int., 2023, 17: 27-35; Amyloid, 2022, 4: 213-219; Lancet, 2016, 387: 2641-2654). In one embodiment, systemic amyloidosis that is the target of prevention or treatment for the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is ATTR-CM and ATTR-PN. In one embodiment, systemic amyloidosis that is the target of prevention or treatment for the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is AL amyloidosis.

[0292] In one embodiment, the disease targeted for prevention or treatment by the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is a neurological disease. In another embodiment, the neurological disease targeted for prevention or treatment by the anti-MerTK bispecific antibody or pharmaceutical composition containing the antibody of the present invention is multiple sclerosis and / or Alzheimer's disease.

[0293] The anti-MerTK bispecific antibody of the present invention, or a pharmaceutical composition containing such antibody, can also be used for the prevention or treatment of cancer, and can be practiced by those skilled in the art based on the description in "Pharmaceutical Uses of the Invention" above. Furthermore, the cancers that are the target of prevention or treatment for the anti-MerTK bispecific antibody of the present invention, or a pharmaceutical composition containing such antibody, are listed in the "Pharmaceutical Uses of the Invention" above.

[0294] <The anti-MerTK-anti-TTR bispecific antibody of the present invention>

[0295] This invention provides an anti-MerTK-anti-TTR bispecific antibody that binds to MerTK and misfolded TTR.

[0296] The anti-MerTK-anti-TTR bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-MerTK antibody, as well as a heavy chain variable region and a light chain variable region of an antibody that binds to a misfolded TTR. The anti-MerTK-anti-TTR bispecific antibody of the present invention contains the heavy chain variable region and light chain variable region of an anti-MerTK antibody as shown below: The heavy chain variable region contains CDR1, composed of the amino acid sequence of sequence number 1; CDR2, composed of the amino acid sequence of sequence number 2; and CDR3, composed of the amino acid sequence of sequence number 3; and It contains light chain variable regions consisting of CDR1 (composed of amino acid sequence number 4), CDR2 (composed of amino acid sequence number 5), and CDR3 (composed of amino acid sequence number 6).

[0297] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-MerTK antibody selected from the group consisting of (1) to (6) below: (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; or (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0298] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-MerTK antibody selected from the group consisting of (4) to (6) below: (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; or (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0299] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody and a light chain containing a light chain variable region of an anti-MerTK antibody. In another embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody comprising a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody, a light chain containing a light chain variable region, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide.

[0300] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody containing a heavy chain fragment and a light chain selected from the group consisting of (1) to (6) below: (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

[0301] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody containing a heavy chain fragment and a light chain selected from the group consisting of (4) to (6) below: (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

[0302] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody and an scFv or Fab region containing the heavy chain variable region and light chain variable region of an anti-TTR antibody that binds to misfolded TTRs. In another embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention is a bispecific antibody with an scFv or Fab region containing the heavy chain variable region and light chain variable region of an anti-TTR antibody that binds to misfolded TTRs linked via a hinge region to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody.

[0303] The anti-MerTK-anti-TTR bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-TTR antibody that binds to misfolded TTRs. The anti-TTR antibody is not particularly limited to any antibody that binds to misfolded TTRs; for example, the 371M antibody (International Publication No. 2015 / 115332) can be used.

[0304] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region of an anti-TTR antibody that binds to misfolded TTRs, as shown below: The heavy chain variable region contains CDR1, consisting of the amino acid sequence of sequence number 27; CDR2, consisting of the amino acid sequence of sequence number 28; and CDR3, consisting of the amino acid sequence of sequence number 29; and It contains a light chain variable region consisting of CDR1 (composed of amino acid sequence number 30), CDR2 (composed of amino acid sequence number 31), and CDR3 (composed of amino acid sequence number 32).

[0305] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises the following heavy chain variable region and light chain variable region of an anti-TTR antibody that binds to misfolded TTRs: The heavy chain variable region consisting of amino acids numbered 1 to 119 of sequence number 43; and The light chain variable region consists of amino acid sequences numbered 135 to 245 of sequence number 43.

[0306] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention contains an antigen-binding fragment of an anti-TTR antibody that binds to misfolded TTRs. In one embodiment, the antigen-binding fragment of the anti-TTR antibody that binds to misfolded TTRs contained in the anti-MerTK-anti-TTR bispecific antibody of the present invention can be an scFv, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a VHH antibody. In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an scFv (referred to herein as "anti-TTR-scFv") containing a heavy chain variable region and a light chain variable region of an anti-TTR antibody that binds to misfolded TTRs.

[0307] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention contains anti-TTR-scFv consisting of the amino acid sequence of sequence number 43.

[0308] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention is of the Fab-scFv-Fc(kih) type. In another embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention is a bispecific antibody in which the C-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody is further linked by a hinge region to the N-terminus of the anti-TTR-scFv.

[0309] In one embodiment, the peptide linker connecting the heavy chain variable region and the light chain variable region of the anti-TTR antibody in the anti-TTR-scFv has the amino acid sequence of sequence number 48. In one embodiment, the peptide linker connecting the heavy chain variable region and the light chain variable region of the anti-TTR antibody has the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n. In one embodiment, the peptide linker connecting the heavy chain variable region and the light chain variable region of the anti-TTR antibody has the amino acid sequence of sequence number 54.

[0310] In the case where the anti-MerTK-anti-TTR bispecific antibody of the present invention contains an Fc region, the Fc region of the bispecific antibody may contain mutations that reduce antibody-dependent cell killing activity (ADCC), antibody-dependent cell phagocytosis activity (ADCP), and complement-dependent killing activity (CDC). In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region containing amino acid mutations of L234A and L235A (LALA mutation) (here, the mutation position is at the amino acid position according to the EU index in the human Igγ1 constant region). In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region containing a P331G mutation or a P329A mutation (here, the mutation position is at the amino acid position according to the EU index in the human Igγ1 constant region). In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region containing one or more mutations selected from LALA mutation, P331G mutation, and P329A mutation. In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region containing both LALA and P331G mutations.

[0311] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region composed of a first Fc polypeptide and a second Fc polypeptide. In another embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a first Fc polypeptide and a second Fc polypeptide composed of an amino acid sequence having more than 90% identity with the amino acid sequence of sequence number 39 or having 1 to 10 substituted amino acid sequences in the amino acid sequence of sequence number 39.

[0312] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region containing a club-and-mortise mutation. In another embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region containing one or more mutations selected from LALA mutation, P331G mutation, P329A mutation, and club-and-mortise mutation.

[0313] In one embodiment, the mortis mutation contained in the anti-MerTK-anti-TTR bispecific antibody of the present invention is the T366W mutation in one Fc polypeptide forming the Fc region and the T366S, L368A and Y407V mutations in another Fc polypeptide forming the Fc region (here, the above mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[0314] In one embodiment, the first and second polypeptides contained in the Fc region of the anti-MerTK-anti-TTR bispecific antibody of the present invention comprise polypeptides selected from (1) or (2) below: (1) A first Fc polypeptide consisting of the amino acid sequence of sequence number 40 and a second Fc polypeptide consisting of the amino acid sequence of sequence number 41; or (2) The first Fc polypeptide consisting of the amino acid sequence of sequence number 41 and the second Fc polypeptide consisting of the amino acid sequence of sequence number 40.

[0315] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention may contain a hinge region. The hinge region is not particularly limited to any sequence commonly used by those skilled in the art. In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention contains a hinge region consisting of the amino acid sequence shown in Serial Number 42 and / or a hinge region consisting of the amino acid sequence shown in Serial Number 70.

[0316] Those skilled in the art can easily prepare a full-length Fab-scFv-Fc(kih) type anti-MerTK-anti-TTR bispecific antibody by combining the sequences of the anti-MerTK antibody, anti-TTR-scFv, hinge region, Fc region, etc., described in the above-mentioned <Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention>.

[0317] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention may have undergone post-translational modification. This post-translational modification is as described above in the description of the anti-MerTK bispecific antibody of the present invention.

[0318] <Fusion and complex of the anti-MerTK-anti-TTR bispecific antibody of the present invention, and cells on the cell surface presenting the anti-MerTK-anti-TTR bispecific antibody of the present invention>

[0319] For the anti-MerTK-anti-TTR bispecific antibody of the present invention, a fusion composite of the anti-MerTK-anti-TTR bispecific antibody of the present invention is provided, which is formed by linking a protein (including peptides) other than MerTK and misfolded TTR and an antibody (including antigen-binding fragments) other than anti-MerTK antibody and anti-TTR antibody. Additionally, for the anti-MerTK-anti-TTR bispecific antibody of the present invention, a complex of the anti-MerTK-anti-TTR bispecific antibody of the present invention is provided, which is formed by binding a modifying agent. Furthermore, for the anti-MerTK-anti-TTR bispecific antibody of the present invention, cells (e.g., immune cells or effector cells) whose surface is presented with the anti-MerTK-anti-TTR bispecific antibody of the present invention are provided (e.g., Tandem CARs) (referred to in this specification as "cells whose cell surface is presented with the anti-MerTK-anti-TTR bispecific antibody of the present invention") (Oncology Reports, 2019, 42: 2183-2195). The fusion, the complex, and the cells with presenting antibodies on their cell surfaces can be implemented based on the above description of the fusion, the complex, and the cells with presenting anti-MerTK antibodies or antigen-binding fragments of the present invention on their cell surfaces.

[0320] <The polynucleotide of the anti-MerTK-anti-TTR bispecific antibody of the present invention>

[0321] The present invention further provides a polynucleotide for the production of anti-MerTK-anti-TTR bispecific antibodies. Those skilled in the art can implement this based on the above descriptions of the <Polynucleotides of the present invention> and <Polynucleotides of the present invention for anti-MerTK bispecific antibodies>.

[0322] In one embodiment, the polynucleotide used to produce the anti-MerTK-anti-TTR bispecific antibody of the present invention is a polynucleotide selected from the group consisting of (1) to (9) below: (1) A polynucleotide containing a base sequence of the heavy chain variable region encoding an amino acid sequence consisting of amino acids numbered 1 to 119 of sequence number 8 for an anti-MerTK antibody. (2) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 10. (3) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 12. (4) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of amino acid sequence number 14. (5) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 16. (6) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 18. (7) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid 20. (8) A polynucleotide containing a base sequence encoding the base sequence of the heavy chain variable region of an anti-TTR antibody consisting of amino acids numbered 1 to 119 of sequence number 43; and (9) A polynucleotide containing a base sequence of the light chain variable region encoding an anti-TTR antibody consisting of an amino acid sequence consisting of amino acid numbers 135 to 245 of sequence number 43.

[0323] The polynucleotides described herein can be produced by those skilled in the art based on their base sequences using methods known in the art.

[0324] <Expression vector and production method of the anti-MerTK-anti-TTR bispecific antibody of the present invention>

[0325] The present invention further provides an expression vector and a method for producing the anti-MerTK-anti-TTR bispecific antibody of the present invention. This vector and method can be implemented by those skilled in the art based on the descriptions above regarding <the polynucleotide of the present invention>, <the expression vector of the present invention>, <the method for producing the anti-MerTK antibody of the present invention>, and <the polynucleotide, expression vector, transformed host cell, and production method of the anti-MerTK bispecific antibody of the present invention>.

[0326] <Host cells transformed with the anti-MerTK-anti-TTR bispecific antibody of the present invention>

[0327] The present invention further provides a host cell transformed with the anti-MerTK-anti-TTR bispecific antibody of the present invention (hereinafter referred to as "host cell of the anti-MerTK-anti-TTR bispecific antibody of the present invention"). Except for the polynucleotide contained in the host cell of the anti-MerTK-anti-TTR bispecific antibody of the present invention, it can be implemented by those skilled in the art based on the above description of the "transformed host cell of the present invention".

[0328] In one embodiment, the host cell of the anti-MerTK-anti-TTR bispecific antibody of the present invention contains polynucleotides selected from the group consisting of (1) to (3) below: (1) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 16, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 43, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 135 to 245 of sequence number 43; (2) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 119 of sequence number 8; a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 110 of sequence number 18; a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acids numbered 1 to 119 of sequence number 43; and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acids numbered 135 to 245 of sequence number 43; and (3) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 20, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 43, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 135 to 245 of sequence number 43.

[0329] <Pharmaceutical Uses of the Anti-MerTK-Anti-TTR Bispecific Antibody of the Invention>

[0330] The present invention further provides pharmaceutical compositions comprising the anti-MerTK-anti-TTR bispecific antibody of the present invention and pharmaceutically acceptable excipients (hereinafter referred to as "the anti-MerTK-anti-TTR bispecific antibody of the present invention or pharmaceutical compositions comprising the thereof"). Such pharmaceutical compositions may be implemented based on the description in "Pharmaceutical Uses of the Anti-MerTK Bispecific Antibody of the Present Invention" above.

[0331] The anti-MerTK-anti-TTR bispecific antibodies of the present invention, or pharmaceutical compositions containing them, can be used to prevent and / or treat diseases caused by the accumulation of misfolded proteins.

[0332] In one embodiment, the disease targeted for prevention or treatment by the anti-MerTK-anti-TTR bispecific antibody or pharmaceutical composition containing the present invention is a neurological disease, heart disease, eye disease, or endocrine / metabolic disease. In one embodiment, the disease targeted for prevention or treatment by the anti-MerTK-anti-TTR bispecific antibody or pharmaceutical composition containing the present invention is amyloidosis (e.g., localized amyloidosis or systemic amyloidosis). In one embodiment, the amyloidosis targeted for prevention or treatment by the anti-MerTK-anti-TTR bispecific antibody or pharmaceutical composition containing the present invention is systemic amyloidosis. In one embodiment, the systemic amyloidosis targeted for prevention or treatment by the anti-MerTK-anti-TTR bispecific antibody or pharmaceutical composition containing the present invention is ATTR-CM and ATTR-PN.

[0333] <The anti-MerTK-anti-IgL bispecific antibody of the present invention>

[0334] This invention provides a bispecific anti-MerTK-anti-IgL antibody that binds to both MerTK and misfolded IgL.

[0335] IgL is a 25 kDa protein composed of 210 amino acids. It is produced by plasma cells, a type of white blood cell found in the bone marrow. Normally, it forms a heterodimer with the immunoglobulin heavy chain (IgH) also produced by plasma cells, and is responsible for humoral immunity in the adaptive immune system. However, excessive production of misfolded IgL due to abnormal plasma cells can lead to the deposition of IgL-derived amyloid proteins in various organs, including the heart, kidneys, digestive tract, and nervous system (Drugs, 2023, 83: 203-216). Misfolded IgL is a protein that contributes to AL amyloidosis, a type of systemic amyloidosis. Antibodies against misfolded IgL inhibit its deposition, suggesting the potential for improved function (Br. J. Haematol., 2020, 189: 228-238), but these antibodies, like monoclonal anti-TTR IgG1 antibodies, activate phagocytosis by targeting the Fc region, potentially leading to cytokine release syndrome.

[0336] The anti-MerTK-anti-IgL bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-MerTK antibody, as well as a heavy chain variable region and a light chain variable region of an antibody that binds to misfolded IgL. The anti-MerTK-anti-IgL bispecific antibody of the present invention contains the heavy chain variable region and light chain variable region of an anti-MerTK antibody as shown below: The heavy chain variable region contains CDR1, composed of the amino acid sequence of sequence number 1; CDR2, composed of the amino acid sequence of sequence number 2; and CDR3, composed of the amino acid sequence of sequence number 3; and It contains light chain variable regions consisting of CDR1 (composed of amino acid sequence number 4), CDR2 (composed of amino acid sequence number 5), and CDR3 (composed of amino acid sequence number 6).

[0337] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-MerTK antibody selected from the group consisting of (1) to (6) below: (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; or (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

[0338] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody and a light chain containing a light chain variable region of an anti-MerTK antibody. In another embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody comprising a heavy chain fragment containing a heavy chain variable region of an anti-MerTK antibody, a light chain containing a light chain variable region, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide.

[0339] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody, said single-arm anti-MerTK antibody containing a heavy chain fragment and a light chain selected from the group consisting of (1) to (6) below: (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

[0340] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody and an scFv or Fab region containing a heavy chain variable region and a light chain variable region of an anti-IgL antibody that binds to misfolded IgL. In another embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention is a bispecific antibody in which an scFv or Fab region containing a heavy chain variable region and a light chain variable region of an anti-IgL antibody that binds to misfolded IgL is linked via a hinge region to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody.

[0341] The anti-MerTK-anti-IgL bispecific antibody of the present invention contains a heavy chain variable region and a light chain variable region of an anti-IgL antibody that binds to misfolded IgL. This anti-IgL antibody is not particularly limited to any antibody that binds to misfolded IgL; for example, the 2A4 antibody (Japanese Patent No. 5730020) can be used.

[0342] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises the heavy chain variable region and light chain variable region of an anti-IgL antibody that binds to misfolded IgL, as shown below: The heavy chain variable region contains CDR1, consisting of amino acid sequences numbered 26 to 35 of sequence number 57; CDR2, consisting of amino acid sequences numbered 50 to 68 of sequence number 57; and CDR3, consisting of amino acid sequences numbered 101 to 108 of sequence number 57; and The light chain variable region contains CDR1, which consists of amino acid sequences numbered 24 to 39 of sequence number 59; CDR2, which consists of amino acid sequences numbered 55 to 61 of sequence number 59; and CDR3, which consists of amino acid sequences numbered 94 to 103 of sequence number 59.

[0343] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises the following heavy chain variable region and light chain variable region of an anti-IgL antibody that binds to misfolded IgL: The heavy chain variable region consisting of amino acids numbered 1 to 119, starting with sequence number 57; and The light chain variable region consists of amino acid sequences numbered 1 to 112, which are part of sequence number 59.

[0344] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention contains an antigen-binding fragment of an anti-IgL antibody that binds to misfolded IgL. In one embodiment, the antigen-binding fragment of the anti-IgL antibody that binds to misfolded IgL contained in the anti-MerTK-anti-IgL bispecific antibody of the present invention can be scFv, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a VHH antibody. In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a Fab fragment (referred to herein as "anti-IgL-Fab") containing a heavy chain variable region and a light chain variable region of an anti-IgL antibody that binds to misfolded IgL.

[0345] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention is of the CrossMab (IgG-kih) type. In another embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention is a bispecific antibody in which the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody is further linked to the C-terminus of the anti-IgL-Fab via a hinge region.

[0346] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention contains anti-IgL-Fab, wherein the anti-IgL-Fab contains a fragment consisting of a heavy chain variable region and a light chain constant region consisting of amino acid sequences numbered 1 to 226 of sequence number 57, and a fragment consisting of a light chain variable region and a CH1 domain consisting of sequence number 59.

[0347] When the anti-MerTK-anti-IgL bispecific antibody of the present invention contains an Fc region, the Fc region of the bispecific antibody may contain mutations that reduce ADCC, CDC, ADCP, and / or mutations that promote heterodimer formation. Such mutations are as described above in the <Anti-MerTK-anti-TTR Bispecific Antibody of the Present Invention>.

[0348] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention contains an Fc region composed of a first Fc polypeptide and a second Fc polypeptide. In another embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention contains a first Fc polypeptide and a second Fc polypeptide composed of an amino acid sequence having more than 90% identity with the amino acid sequence of sequence number 39 or having 1 to 10 substituted amino acid sequences in the amino acid sequence of sequence number 39.

[0349] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an Fc region containing a club-and-mortise mutation. In another embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an Fc region containing one or more mutations selected from LALA mutation, P331G mutation, P329A mutation, and club-and-mortise mutation. The club-and-mortise mutation is as described above in the <Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention>.

[0350] In one embodiment, the first and second polypeptides contained in the Fc region of the anti-MerTK-anti-IgL bispecific antibody of the present invention contain polypeptides selected from (1) or (2) below: (1) A first Fc polypeptide consisting of the amino acid sequence of sequence number 40 and a second Fc polypeptide consisting of the amino acid sequence of sequence number 41; or (2) The first Fc polypeptide consisting of the amino acid sequence of sequence number 41 and the second Fc polypeptide consisting of the amino acid sequence of sequence number 40.

[0351] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention may contain a hinge region. In another embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention contains a hinge region consisting of the amino acid sequence of sequence number 42.

[0352] Those skilled in the art can easily prepare full-length anti-MerTK-anti-IgL bispecific antibodies (e.g., CrossMab(IgG-kih) type anti-MerTK-anti-IgL bispecific antibodies) by combining the sequences of anti-MerTK antibody, anti-IgL-Fab, hinge region, Fc region, etc., described in the above-mentioned <Anti-MerTK-Anti-IgL Bispecific Antibody of the Invention>.

[0353] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention may have undergone post-translational modification. This post-translational modification is as described above in the <Anti-MerTK Bispecific Antibody of the Present Invention>.

[0354] <The fusion and complex of the anti-MerTK-anti-IgL bispecific antibody of the present invention, and cells on the cell surface presenting the anti-MerTK-anti-IgL bispecific antibody of the present invention>

[0355] For the anti-MerTK-anti-IgL bispecific antibody of the present invention, a fusion composite of the anti-MerTK-anti-IgL bispecific antibody of the present invention is provided, which is formed by linking a protein (including peptides) other than MerTK and misfolded IgL and an antibody (including antigen-binding fragments) other than anti-MerTK antibody and anti-IgL antibody. Additionally, for the anti-MerTK-anti-IgL bispecific antibody of the present invention, a complex of the anti-MerTK-anti-IgL bispecific antibody of the present invention is provided, which is formed by binding a modifying agent. Furthermore, for the anti-MerTK-anti-IgL bispecific antibody of the present invention, cells (e.g., immune cells or effector cells) whose surface is presented with the anti-MerTK-anti-IgL bispecific antibody of the present invention are provided (e.g., Tandem CARs) (referred to in this specification as "cells whose cell surface is presented with the anti-MerTK-anti-IgL bispecific antibody of the present invention") (Oncology Reports, 2019, 42: 2183-2195). The fusion, the complex, and the cells with presenting antibodies on their cell surfaces can be implemented based on the above description of the fusion, the complex, and the cells with presenting anti-MerTK antibodies or antigen-binding fragments of the present invention on their cell surfaces.

[0356] <The polynucleotide of the anti-MerTK-anti-IgL bispecific antibody of the present invention>

[0357] The present invention further provides a polynucleotide for producing anti-MerTK-anti-IgL bispecific antibodies. Those skilled in the art can implement this based on the above descriptions of the <Polynucleotides of the Present Invention> and <Polynucleotides of the Present Invention for Anti-MerTK Bispecific Antibodies>.

[0358] In one embodiment, the polynucleotide used to produce the anti-MerTK-anti-IgL bispecific antibody of the present invention is a polynucleotide selected from the group consisting of (1) to (9) below: (1) A polynucleotide containing a base sequence of the heavy chain variable region encoding an amino acid sequence consisting of amino acids numbered 1 to 119 of sequence number 8 for an anti-MerTK antibody. (2) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 10. (3) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 12. (4) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of amino acid sequence number 14. (5) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 16. (6) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 18. (7) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid 20. (8) A polynucleotide containing a base sequence encoding the base sequence of the heavy chain variable region of an anti-IgL antibody consisting of amino acids numbered 1 to 119 of sequence number 57; and (9) A polynucleotide containing a base sequence of the light chain variable region encoding an anti-IgL antibody consisting of an amino acid sequence numbered 1 to 112 of amino acid sequence number 59.

[0359] The polynucleotides described herein can be produced by those skilled in the art based on their base sequences using methods known in the art.

[0360] <Expression vector and production method of the anti-MerTK-anti-IgL bispecific antibody of the present invention>

[0361] The present invention further provides an expression vector and a method for producing the anti-MerTK-anti-IgL bispecific antibody of the present invention. This vector and method can be practiced by those skilled in the art in accordance with the descriptions above in <The Polynucleotide of the Present Invention>, <The Expression Vector of the Present Invention>, <The Method for Producing the Anti-MerTK Antibody of the Present Invention>, and <The Polynucleotide, Expression Vector, Transformed Host Cell, and Production Method of the Anti-MerTK Bispecific Antibody of the Present Invention>.

[0362] <Host cells transformed with the anti-MerTK-anti-IgL bispecific antibody of the present invention>

[0363] The present invention further provides a host cell transformed with the anti-MerTK-anti-IgL bispecific antibody of the present invention (hereinafter referred to as "host cell of the anti-MerTK-anti-IgL bispecific antibody of the present invention"). Except for the polynucleotide contained in the host cell of the anti-MerTK-anti-IgL bispecific antibody of the present invention, it can be implemented by those skilled in the art based on the above description of the "transformed host cell of the present invention".

[0364] In one embodiment, the host cell of the anti-MerTK-anti-IgL bispecific antibody of the present invention contains polynucleotides selected from the group consisting of (1) to (4) below: (1) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 109 of sequence number 12, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 57, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 112 of sequence number 59; (2) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 16, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 57, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 112 of sequence number 59; (3) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 119 of sequence number 8; a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 110 of sequence number 18; a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acids numbered 1 to 119 of sequence number 57; and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acids numbered 1 to 112 of sequence number 59; and (4) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 20, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 57, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-IgL antibody consisting of amino acid sequences numbered 1 to 112 of sequence number 59.

[0365] <Pharmaceutical Uses of the Anti-MerTK-Anti-IgL Bispecific Antibody of the Invention>

[0366] The present invention further provides pharmaceutical compositions comprising the anti-MerTK-anti-IgL bispecific antibody of the present invention and pharmaceutically acceptable excipients (hereinafter referred to as "the anti-MerTK-anti-IgL bispecific antibody of the present invention or pharmaceutical compositions comprising them"). Such pharmaceutical compositions may be implemented based on the above description of the "Pharmaceutical Uses of the Anti-MerTK Bispecific Antibody of the Present Invention".

[0367] The anti-MerTK-anti-IgL bispecific antibodies of the present invention, or pharmaceutical compositions containing them, can be used to prevent and / or treat diseases caused by the accumulation of misfolded proteins.

[0368] In one embodiment, the disease targeted for prevention or treatment by the anti-MerTK-anti-IgL bispecific antibody or pharmaceutical composition containing the present invention is kidney disease. In one embodiment, the disease targeted for prevention or treatment by the anti-MerTK-anti-IgL bispecific antibody or pharmaceutical composition containing the present invention is amyloidosis (e.g., localized amyloidosis or systemic amyloidosis). In one embodiment, the amyloidosis targeted for prevention or treatment by the anti-MerTK-anti-IgL bispecific antibody or pharmaceutical composition containing the present invention is systemic amyloidosis. In one embodiment, the systemic amyloidosis targeted for prevention or treatment by the anti-MerTK-anti-IgL bispecific antibody or pharmaceutical composition containing the present invention is AL amyloidosis.

[0369] Specific embodiments are provided herein for further understanding of the invention, but these are for illustrative purposes only and are not intended to limit the invention.

[0370] Example

[0371] [Example 1: Preparation of anti-MerTK antibody]

[0372] [Example 1-1: Obtaining anti-MerTK antibody]

[0373] (1) Preparation of MerTK-Fc fusion protein and MerTK-His protein

[0374] Polynucleotides encoding the extracellular domain of human MerTK (composed of amino acid sequences 21 to 505 of UniProt: Q12866) or the extracellular domain of mouse MerTK (composed of amino acid sequences 19 to 97 of UniProt: Q60805) were linked to a polynucleotide encoding an amino acid sequence cleavable by factor Xa protease (New England Biolabs, P8010L). These polynucleotides were then inserted into the pFUSE-hIgG1-Fc1 vector (InvivoGen, pfuse-hg1fc1) to create expression vectors encoding proteins fused with human Fc on human MerTK (hereinafter referred to as "human MerTK-human Fc fusion protein") and proteins fused with human Fc on mouse MerTK (hereinafter referred to as "mouse MerTK-human Fc fusion protein"). Subsequently, the polynucleotide encoding the extracellular domain of human MerTK was tandemly linked to a polynucleotide encoding the His tag (Sequence No. 21) and inserted into the pcDNA3.4 TOPO (registered trademark) vector (Thermo Fisher Scientific) to create an expression vector encoding a protein with a His tag bound to human MerTK (hereinafter referred to as "human MerTK-His protein"). The created expression vector was transfected into ExpiCHO-S cells (Thermo Fisher Scientific, A29127) to obtain recombinant proteins using conventional methods.

[0375] (2) Obtaining fully human anti-MerTK agonist antibodies

[0376] AlivaMab mice (Ablexis, US Patent 9346873) were immunized several times with human MerTK / Mer protein, His tag (ACROBiosystems, MEK-H52H6), and mouse MerTK protein obtained by cleaving and purifying the mouse MerTK-human Fc fusion protein with factor Xa protease. Lymphocytes were recovered from the lymph nodes of the immunized mice using standard methods. Based on the paper (BMC Biol., 2012, 10:80), fluorescently labeled B cells binding to human MerTK-His protein and mouse MerTK-human Fc fusion protein were sorted from the recovered mouse lymphocytes using a cell sorter (Becton Dickinson, FACSMelody). Nucleic acids encoding the heavy chain variable region and light chain variable region of the antibody extracted from the sorted B cells were cloned using standard methods. Monoclonal antibodies were prepared based on their sequence information using standard methods. The antibodies produced were evaluated based on their binding activity against recombinant human and mouse MerTK protein, their binding activity against MerTK on the membrane of mouse bone marrow-derived macrophages, their ability to induce MerTK internalization into cells, and their AKT phosphorylation induction activity as a downstream signal of MerTK. The results yielded a fully human anti-MerTK agonist antibody exhibiting binding activity against recombinant human and mouse MerTK protein, binding activity against MerTK on the membrane of mouse bone marrow-derived macrophages, ability to induce MerTK internalization into cells, and AKT phosphorylation induction activity. The sequence of the heavy chain variable region of this anti-MerTK agonist antibody is identical to amino acid numbers 1 to 119 of sequence number 8, and the sequence of the light chain variable region is identical to amino acid numbers 1 to 110 of sequence number 16.

[0377] (3) Preparation and acquisition of fully human anti-MerTK antibody with Fc mutation

[0378] Based on the amino acid sequences of the heavy chain variable region and light chain variable region of the fully human anti-MerTK agonist antibody obtained in (2), the amino acid sequences of the heavy chain and light chain were designed. Specifically, a peptide was designed with the amino acid sequence of the human λ chain constant region linked to the C-terminus of the light chain variable region, and a peptide was designed with the amino acid sequence of the human Igγ1 constant region linked to the C-terminus of the heavy chain variable region. Furthermore, amino acid mutations of L234A, L235A, and P331G were introduced into the heavy chain constant region. Thus, a fully human anti-MerTK antibody tA3-20 composed of the heavy chain of sequence number 8 and the light chain of sequence number 10 was designed.

[0379] Typically, N-binding glycan modifications in the variable region of an antibody can be a factor that destabilizes antibody quality during manufacturing. Therefore, to avoid potential glycan modifications on the framework of the light chain variable region of tA3-20, mutations are introduced into the amino acid sequence of tA3-20. Specifically, for tA3-20, a light chain (Sequence No. 12) is designed to replace the asparagine amino acid at position 18 of Sequence No. 10 (the framework amino acid) with threonine, and a light chain (Sequence No. 14) is designed to replace the asparagine amino acid at position 18 of Sequence No. 10 with serine. Furthermore, light chains (Sequence Nos. 16, 18, and 20) with asparagine are designed to be added to the N-terminus of the light chains of Sequence Nos. 10, 12, and 14. Thus, a human anti-MerTK antibody is designed consisting of the heavy chain of Sequence No. 8 and the light chains of Sequence Nos. 12, 14, 16, 18, or 20. The fully human anti-MerTK antibodies composed of the heavy chain (serial number 8) and the light chains (serial numbers 12, 14, 16, 18, or 20) are designated as tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, or tA3-20.5, respectively. The amino acid sequence information of the variable regions of the heavy and light chains of the fully human anti-MerTK antibodies is shown in Table 1.

[0380]

[0381] The amino acid sequences of CDR1-3 of sequence number 8 are shown in sequence numbers 1-3, and the amino acid sequences of CDR1-3 of sequence numbers 10, 12, 14, 16, 18 and 20 are shown in sequence numbers 4-6.

[0382] Based on the amino acid sequences of the heavy chain variable region of sequence number 5 and the light chain variable region of sequence number 34 in Patent Document 4, a mouse-human chimeric anti-MerTK antibody (hereinafter also referred to as "MTK201") was designed in the same manner as in (3). Based on the amino acid sequences of the heavy chain variable region of sequence number 49 and the light chain variable region of sequence number 50 in International Publication No. 2016 / 106221, a mouse-human chimeric anti-MerTK antibody (hereinafter also referred to as "M6") was designed in the same manner as in (3).

[0383] Polynucleotides encoding the heavy or light chains of tA3-20, tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, tA3-20.5, MTK201, and M6 were prepared. Each polynucleotide was inserted into the pcDNA3.4 TOPO vector using standard methods to create expression vectors for the heavy and light chains of tA3-20, tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, tA3-20.5, MTK201, or M6. The combinations of heavy and light chain polynucleotide sequences encoding the antibodies tA3-20, tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, tA3-20.5, MTK201, and M6 are shown in Table 2. Using a transfection kit (ExpiFectamine CHO Transfection Kit, Thermo Fisher Scientific, A29129), equal volumes of the heavy and light chain expression vectors were introduced into ExpiCHO-S cells, and the cells were incubated at 37°C in a 5% CO2 incubator. The following day, the reagents supplied with the kit (ExpiCHO Enhancer and ExpiCHO Feed) were added according to the protocol, and the cells were incubated again, thereby causing the antibody to be secreted into the culture supernatant.

[0384]

[0385] Antibodies were purified from the culture supernatant using affinity purification with MabSelect SuRe (Cytiva, 175438) or a combination of affinity purification with MabSelect SuRe pcc (Cytiva, 17549102) and anion exchange chromatography with Sartobind Lab Q100 (sartorius stedim, 93IEXQ42BC-12). The purified antibodies were then confirmed by size exclusion chromatography under reducing and non-reducing conditions using SDS-PAGE or an ACQUITY UPLC protein BEH SEC column (Waters, 186005225).

[0386] [Examples 1-2: Evaluation of the binding activity of anti-MerTK antibodies against human MerTK]

[0387] The binding affinity of tA3-20, tA3-20.1, and tA3-20.2 to human MerTK was evaluated using ELISA.

[0388] The human MerTK-His protein prepared in Example 1-1(1) was prepared to a concentration of 1 μg / mL using PBS. 20 μL was added to each well of a 384-well transparent flat-bottomed immunosorbent assay (IRISA) plate (Thermo Fisher Scientific, 464718), and the plate was incubated overnight at 4°C to immobilize the protein. The human MerTK-His protein was then removed. A blocking solution containing 0.05% Tween-20 in TBS (TBS containing 0.5% Tween-20, prepared by diluting 310-07375 10-fold, hereinafter referred to as "TBS-T") and 20% Blocking One (Nacalitesk, 03953-95) was added to each well, and the plate was incubated for 1 hour to block the protein. The wells were then washed once with TBS-T. Various anti-MerTK antibodies and anti-egg white lysozyme antibody (LYS_1c3-m1_h1i, prepared by our company) as an isotype control antibody were serially diluted from 10000 ng / mL to 0.006 ng / mL. TBS-T containing 5% Blocking One was used for antibody dilution. 20 μL of each diluted antibody was added to each well, and the mixture was incubated at room temperature for 1 hour. After removing the antibodies, the wells were washed three times with TBS-T. Then, 15 μL of secondary antibody (goat anti-human IgG Fc, prepared by diluting Multi-Species SP ads-HRP (SouthernBiotech, 2014-05) 4000-fold with TBS-T containing 5% Blocking One) was added to each well, and the mixture was incubated at room temperature for 1 hour. The wells were cleaned three times with TBS-T, and then 15 μL of TMB-Plus Substrate-Chromogen (Dako Pharmaceuticals, S1599) was added to each well. The mixture was allowed to stand at room temperature for 5 minutes. The reaction was stopped by adding 1 mol / L sulfuric acid (Fujifilm Wako Pure Chemicals, 198-09595), and the absorbance at 450 nm and 570 nm was measured using an Infinite M200 PRO (Tecan). The binding affinity of tA3-20.3, tA3-20.4, and tA3-20.5 to human MerTK was evaluated using the same method as described above.

[0389] The results of the measurement are as follows: Figure 1 As shown, tA3-20, tA3-20.1, and tA3-20.2 all exhibit concentration-dependent binding activity to human MerTK, and their binding activities are equivalent. Furthermore, tA3-20.3, tA3-20.4, and tA3-20.5 were also confirmed to have equivalent binding activity to human MerTK as tA3-20.

[0390] [Examples 1-3: Analysis of species cross-reactivity of anti-MerTK agonist antibody tA3-20]

[0391] The binding activity of tA3-20 to MerTK proteins in various animals (human, mouse, rat, and monkey) was evaluated by ELISA. The human MerTK-human Fc fusion protein, mouse MerTK-human Fc fusion protein, recombinant rat MerTK protein (His tag) (abcam, ab267966), and recombinant cynomolgus monkey Mer Fc chimeric protein CF (R&D Systems, 10576-MR-050) obtained in Examples 1-1 were diluted to 1 μg / mL with PBS. 20 μL of the dilution was added to each well of a 384-well immunoassay plate (transparent flat-bottomed non-sterile immunoassay plate), and the protein was immobilized overnight at 4°C. The next day, the immobilized plates were washed three times with TBS-T (Thermo Fisher Scientific, 28360). 100 μL of Blocking One was added to each well, and the plates were blocked for 1 hour at room temperature. The blocked plates were then washed three times with TBS-T. Using TBS-T containing 10% Blocking One (hereinafter also referred to as "10% dilution"), serially dilute tA3-20 from a maximum concentration of 20 μg / mL in 4-fold series, adding 20 μL to each well. After incubating the antibody-added plate at room temperature for 1 hour, wash three times with TBS-T. Add 20 μL of HRP-labeled anti-human IgG F(ab')2 (abcam, ab98535) diluted 10,000 times with 10% dilution as the secondary antibody to each well. After incubating at room temperature for 1 hour, wash three times with TBS-T, and add 20 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) to each well. After incubating at room temperature for 15 minutes, stop the reaction by adding 1 mol / L sulfuric acid, and measure the absorbance at 450 nm using a SpectraMax (Molecular Devices).

[0392] The results confirmed that tA3-20 exhibits concentration-dependent binding activity to human, mouse, rat, and monkey MerTK proteins. It was also found that tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, and tA3-20.5, which share the same CDR sequence as tA3-20, also possess binding activity not only for human MerTK but also for mouse, rat, and monkey MerTK proteins.

[0393] [Example 2: Confirmation of the phagocytic activity of tA3-20 on human retinal pigment epithelial (RPE) cells]

[0394] [Example 2-1: Confirmation of the activity of tA3-20 in phagocytosis and apoptosis of human RPE cells]

[0395] The activation effect of tA3-20 on the phagocytic clearance function of phagocytes was evaluated by using the activation effect of RPE cells, one of the phagocytes, on the phagocytosis of apoptotic cells.

[0396] Human RPE cells use iCell (registered trademark) retinal pigment epithelial cells-01279 (Fujifilm and Kazumitsu Pure Chemical Industries, Inc., C1046), which are RPE cells derived from iPS cells. The culture medium used was MEMα (Thermo Fisher Scientific, 12571-063) containing gentamicin (Thermo Fisher Scientific, 15750-060) at a final concentration of 25 μg / mL, N-2 supplement (Thermo Fisher Scientific, 17502-048) at a 1-fold concentration, B-27 supplement (Thermo Fisher Scientific, 17504-044) at a 1-fold concentration, hydrocortisone (Merck, H0888-1G) at 55 nM, taurine (Merck, T8691-25G) at 0.25 mg / mL, and 3,3',5-triiodo-L-thyroxine sodium salt (Merck, T5516-1MG) at 14 pg / mL. One flask of iCell (registered trademark) retinal pigment epithelial cell-01279 was evenly seeded into 96 wells of a CellCarrier-96 (PerkinElmer, 6055300) plate and cultured for 5 weeks at 37°C and 5% CO2. The resulting material was used as an iPS-RPE cell culture plate for future research. Jurkat cells (ATCC, TIB-152) were prepared into 1×10⁶ cells using RPMI 1640 (Merck, R8758-500ML) culture medium supplemented with 10% FBS (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Merck, P4458-100ML). 6Jurkat cells were induced to apoptosis by treating them at a final concentration of 100 ng / mL with SUPERFASLIGAND (registered trademark) protein (soluble) (human), (recombinant) (Enzo Life Sciences, ALX-522-020-3005) at 37°C for 3 hours. The apoptotic Jurkat cells (hereinafter referred to as "apoptotic Jurkat cells") were then prepared into 2×10⁻⁶ cells / mL buffer (20 mM CHES / PBS, pH 9.0, prepared by our company). 6 After achieving a cell / mL concentration, cells were labeled with pHrodo Red SE (ThermoFisher Scientific, P36600) at a final concentration of 400 ng / mL for 20 minutes at room temperature. The labeled apoptotic Jurkat cells were then cultured in medium to prepare 6 × 10⁶ cells / mL solutions. 6 Cells / mL concentration.

[0397] tA3-20, MTK201, and LYS_1c3-m1_h1i (isotype control) were used as test antibodies. These antibodies were prepared to a concentration of 10 μg / mL using culture medium.

[0398] Various test antibodies and pHrodo-labeled apoptotic Jurkat cells were added at 50 μL per well to iPS-RPE cell culture plates washed once with RPMI 1640. The iPS-RPE cell culture plates were placed in an Incucyte S3 live cell analyzer (Sartorius) in an incubator at 37°C and 5% CO2. Image measurements were taken every hour using bright field and red laser for 24 hours. The measured images were analyzed using the analysis tools and algorithms in the Incucyte system software, and the total red fluorescence intensity (red calibration units [RCU] × μm) was calculated. 2 (Image) is used as an indicator of the amount of food consumed.

[0399] The results of the evaluation of phagocytic activation are shown in Figure 2-1The analysis showed that tA3-20 peaked approximately 16 hours after the start of evaluation, with an increase in fluorescence intensity, confirming significant activation of phagocytosis. This result indicates that tA3-20 activates phagocytosis in RPE cells via MerTK binding. Furthermore, the activation of phagocytosis by tA3-20 was significantly higher than that by MTK201. This suggests that tA3-20 has a stronger phagocytic clearance activation effect compared to MTK201. There are reports suggesting that anti-MerTK agonist antibodies can activate the phagocytic clearance function of phagocytes such as macrophages (Patent Document 4, Non-Patent Document 1), therefore, it is expected that tA3-20 can activate phagocytic clearance function not only in RPE cells but also in other phagocytes (e.g., macrophages).

[0400] [Example 2-2: Confirmation of tA3-20 activity against POS phagocytosis in human RPE cells]

[0401] The activation of phagocytic activity in porcine POS cells was used as an indicator to evaluate the phagocytic clearance function of RPE cells by tA3-20.

[0402] Porcine POS was prepared based on the method of S. Alemedawar et al. (Stem Cell Reports, 2020, 14: 374-389). The prepared POS was diluted with 100 mM sodium bicarbonate (pH 8.2, prepared by our company) to a concentration of 3 × 10⁻⁶. 7 After reaching a POS / mL concentration, labeling was performed using pHrodo Red, SE to a final concentration of 1 μM, while stirring at room temperature for 1 hour. The labeled POS was then prepared into 6 × 10⁶ mL culture medium. 6 POS / mL concentration.

[0403] tA3-20, MTK201, M6, and LYS_1c3-m1_h1i (isotype control) were used as test antibodies. These antibodies were prepared to a concentration of 10 μg / mL using culture medium.

[0404] Various test antibodies and pHrodo-labeled POS were added at 50 μL per well to iPS-RPE cell culture plates washed once with DMEM. The iPS-RPE cell culture plates were placed in an Incucyte S3 live cell analyzer in a 5% CO2, 37°C incubator. Images were measured every hour using bright field and red laser for 60 hours. The measured images were analyzed using the analysis tools and algorithms built into the Incucyte system software, and the total red fluorescence intensity (red calibration units [RCU] × μm) was calculated. 2 (Image), used as an indicator of the amount of food consumed.

[0405] The results of the evaluation of POS phagocytosis activation are shown in Figure 2-2 The analysis showed that tA3-20 also exhibited activated phagocytosis in POS. This result suggests that phagocytic clearance is also activated in POS.

[0406] Based on the results of Example 2-2, tA3-20 was found to have a stronger phagocytic activation compared to MTK201 and M6, suggesting that it has a stronger phagocytic and clearance function compared to MTK201 and M6.

[0407] [Example 3: Confirmation of the phosphorylation induction effect of intravitreal tA3-20 on MerTK and AKT in RPE]

[0408] Using mice, this study evaluated whether tA3-20 can act as an agonist for MerTK on the RPE in vivo, using the activation of the downstream signaling pathway of MerTK as an indicator.

[0409] [Example 3-1: Intravitreal administration of the test antibody and collection of RPE]

[0410] In 8-week-old male C57BL6 / J mice (Jackson Laboratories, Japan), mydriasis was achieved by instilling Mydrin P eye drops (Santen Pharmaceutical) and then general anesthesia was achieved by intraperitoneal administration of a combination of three anesthesia agents (Domitor, Nippon Zenya Kogyo; Dormicum injection 10mg, Maruishi Pharmaceutical; Vetofaril 5mg, Meiji Animaru Health). Local anesthesia was then achieved by instilling Xylocaine 4% eye drops (Sand Co., Ltd.). The fundus was examined using a microscope (Zeiss OPMI LUMERA 300), and 1 μL of the test antibody was injected into the vitreous humor using a microinjector (Ito Manufacturing Co., Ltd., MS-N05) and a 37G needle (Ito Manufacturing Co., Ltd.). As test antibodies (using PBS as solvent), tA3-20 (0.1447 mg / mL, 1.447 mg / mL, and 14.47 mg / mL) and LYS_1c3-m1_h1i (isotype control, 14.47 mg / mL) were used. After intravitreal administration, 0.5% CRAVIT (registered trademark) eye drops (Santen Pharmaceutical) were instilled into the treated eye, 0.3% TARIVID (registered trademark) eye ointment (Santen Pharmaceutical) was applied, and ANTISEDAN (registered trademark) (Nippon Zenya Kogyo) was injected subcutaneously to awaken the patient.

[0411] One hour after administering the test antibody intravitreally, mice were euthanized, their eyeballs were removed, and the refractive epithelial cells (RPEs) were collected, frozen, and stored until use. It should be noted that this experiment was conducted with N=4 per group.

[0412] [Example 3-2: Preparation of samples for SDS-PAGE electrophoresis]

[0413] The collected mouse RPE samples were homogenized in 10 μL of RIPA buffer (Merck, R0278) containing a mixture of EDTA-free Halt protease and phosphatase inhibitor (100×) (Thermo Fisher Scientific, 78441). The homogenate was centrifuged at 9200×g for 1 minute, and the supernatant was collected into a sample tube. 7.8 μL of the collected supernatant was mixed with 3 μL of NuPAGE LDS sample buffer (4×) (Thermo Fisher Scientific, NP0007) and 1.2 μL of NuPAGE sample reducing agent (10×) (Thermo Fisher Scientific, NP0004), and heat-treated at 70°C for 10 minutes to obtain the sample for SDS-PAGE electrophoresis.

[0414] 4 μL of sample for SDS-PAGE electrophoresis was applied to an SDS-PAGE membrane using NuPAGE 4-12% Bis-Tris Gel (Thermo Fisher Scientific, NP0329BOX). The gel was then transferred to a PVDF membrane (Thermo Fisher Scientific, IB24002) using an iBlot2 gel transfer apparatus (Thermo Fisher Scientific, IB21001). The transferred PVDF membrane (hereinafter referred to as the "membrane") was blocked with a blocking reagent (PVDF Blocking Reagent for Can Get Signal, TOYOBO, NYPBR01) at room temperature for at least 1 hour. The membrane was then reacted overnight at 4°C with an antiphosphorylated MerTK antibody (Merck, SAB4504621-100UG) or an antiphosphorylated AKT antibody (Cell Signaling Technology, #4060) diluted 1000-fold with Can Get Signal Solution 1 (TOYOBO, NKB-201). After antibody reaction, the membrane was washed with TBS-T (hereinafter referred to as "washing buffer") containing 1% BlockAce (Snow Brand Megamilk, UKB40), and then reacted with HRP-labeled anti-rabbit antibody (Cytiva, NA9340V) diluted 10,000 times with Can Get Signal Solution 2 (TOYOBO, NKB-301) at room temperature for 1 hour. The membrane was then washed with the washing buffer. The membrane was immersed in ECL Select (Cytiva, RPN2235) solution, and phosphorylated MerTK or phosphorylated AKT was detected using a ChemiDoc imaging system (BioRad). To detect total MerTK or total AKT in the same membrane, the antibody was stripped from the membrane using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430), and then blocked using PVDF Blocking Reagent for Can Get Signal Solution 2. The signal was blocked at room temperature for 1 hour. Then it was reacted overnight at 4°C with anti-MerTK antibody (Cell Signaling Technology, #38102) or anti-AKT antibody (Cell Signaling Technology, #4691) diluted 3000 times with CanGet Signal Solution 1.Total MerTK or total AKT testing was performed using ECL Prime (Cytiva, RPN2236) via the same method described above.

[0415] [Example 3-3: Analysis]

[0416] The detected bands were numerically converted using Image Lab software version 6.0.0 build 25 (BioRad). The value of each phosphorylated protein band was divided by the value of the total protein bands to obtain a correction value.

[0417] The results of MerTK phosphorylation are shown in... Figure 3-1 Compared to the value of the isotype control antibody LYS_1c3-m1_h1i administered at 14.47 mg / mL (1 μL / eye), tA3-20 showed enhanced activity of MerTK phosphorylation starting from administration of 0.1447 mg / mL (1 μL / eye).

[0418] The results of AKT phosphorylation are shown in Figure 3-2 Similar to the results of MerTK phosphorylation, tA3-20 showed enhanced AKT phosphorylation activity starting from 0.1447 mg / mL (1 μL / eye) relative to the LYS_1c3-m1_h1i value at a dose of 14.47 mg / mL (1 μL / eye), and this activity was dose-dependent. This indicates that tA3-20 has enhanced phosphorylation activity against MerTK expressed in RPE and downstream AKT under in vivo conditions.

[0419] Based on the results of Examples 2 and 3, it is expected that tA3-20 will also enhance the phagocytic clearance function of RPE in vivo via the downstream signaling pathway of MerTK, suggesting its usefulness in preventing or treating diseases associated with reduced phagocytic clearance function of RPE.

[0420] [Example 4: Preparation of mouse-type anti-MerTK-anti-TTR bispecific antibody, mouse-type anti-TTR antibody, and TTR protein]

[0421] The mouse-type anti-MerTK-anti-TTR bispecific antibody, mouse-type anti-TTR antibody, and misfolded recombinant human TTR V30M protein were prepared.

[0422] [Example 4-1: Preparation of recombinant human TTR V30M protein]

[0423] The recombinant human TTR V30M protein was purified and prepared according to the method of Matsubara et al. (Protein Expr. Purif., 2003, 30: 55-61). The gene sequence encoding human TTR V30M (a mutant composed of the mature peptide region of human TTR with valine replaced by methionine at position 30) was inserted into the expression vector pQE-30 (QIAGEN, N-Terminus pQE Vector Set, 32915) to obtain pQE30_GOI_02. pQE30_GOI_02 was then transformed into Escherichia coli JM109 competent cells (Takara Bio, 9052). Transformed *E. coli* were cultured in LB liquid medium (prepared by our company) with a final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (Nacalitesk Co., 19742-94) at 37°C for approximately 16 hours. The recovered *E. coli* were lysed using the xTractor buffer kit (Clontech, 635623). The lysate was centrifuged at 10000 × g for 10 minutes, and only the supernatant was collected.

[0424] From the recovered supernatant, His-tagged human TTR V30M protein was purified using TALON (registered trademark) metal affinity resin (Clontech, 635502) and a His buffer kit (Cytiva, 11-0034-00). For the eluent containing recombinant human TTR V30M protein, the eluent was replaced with 20 mM phosphate buffer (pH 7.0, prepared by our company) using an Amicon Ultra-4 (Merck, C7719) or PD-10 desalting column (Cytiva, 17-0851-01) to obtain purified recombinant human TTR V30M protein. For a fraction of the purified recombinant human TTR V30M protein used for administration to mice, endotoxin was further removed using a Pierce high-volume endotoxin removal centrifuge column (Thermo Fisher Scientific, 88276) according to the accompanying guidelines.

[0425] The concentration of purified recombinant human TTR V30M protein, after buffer replacement and / or endotoxin removal, was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) or the NanoDrop A280 assay (Thermo Fisher Scientific).

[0426] [Example 4-2: Preparation of misfolded recombinant human TTR V30M protein]

[0427] To prepare misfolded recombinant human TTR V30M protein (referred to as "misfolded recombinant human TTR V30M protein" in this specification), 200 μL of purified recombinant human TTR V30M protein (10 mg / mL) was mixed with 800 μL of 50 mM phosphate buffer (pH 2.7, prepared by our company) and incubated at 37°C for at least 4 days (hereinafter also referred to as "misfolding treatment") to prepare a misfolded recombinant human TTR V30M protein solution (referred to as "misTTR" in this specification). Using a portion of the prepared misTTR solution, a mixture of the misTTR solution and 4M sodium hydroxide at a ratio of 100:1 was prepared (hereinafter referred to as "neutralized misTTR").

[0428] To confirm that both misTTR and neutralized misTTR formed misfolds, purified recombinant human TTR V30M protein before misfolding was used as a control, and thioflavone T, a specific fluorescent dye for amyloid protein, was used for detection. The protein and thioflavone T (prepared by our company) were mixed at a ratio of 1:5, incubated at room temperature for 15 minutes, and then fluorescence was measured at excitation / emission of 430 / 480 nm. The results showed a significant increase in fluorescence intensity for both misTTR and neutralized misTTR, indicating that these TTRs formed misfolds. Based on the above experiments, misTTR and neutralized misTTR were used as misfolded TTRs in subsequent studies.

[0429] [Example 4-3: Labeling of misfolded recombinant human TTR V30M protein]

[0430] Mix 50 μL of neutralized misTTR (2 mg / mL), 950 μL of 100 mM sodium bicarbonate (pH 8.2, prepared by our company), and 5 μL of 10 mM pHrodo, and incubate at 37°C for 15 minutes to prepare pHrodo-labeled misTTR (referred to as "pHrodo-misTTR" in this specification). After removing the supernatant containing excess pHrodo by centrifugation, resuspend in PBS and remove the supernatant again by centrifugation. Resuspend the precipitated pHrodo-misTTR in PBS to prepare the specified concentration.

[0431] 250 μL of neutralized misTTR (2 mg / mL), 250 μL of 50 mM borate (pH 8.5, Thermo Fisher Scientific, 28341), and 16.75 μL of 10 mM DyLight800 NHS Ester (Thermo Fisher Scientific, 46421) were mixed and incubated at 25 °C for 1 hour to prepare DyLight800-labeled misTTR (referred to as "DL800-misTTR" in this specification). After removing the supernatant containing excess DyLight800 by centrifugation, the precipitate was resuspended in PBS and the supernatant was removed again by centrifugation. The precipitated DL800-misTTR was resuspended in PBS to prepare the specified concentration.

[0432] [Example 4-4: Preparation of mouse-type anti-MerTK-anti-TTR bispecific antibody]

[0433] The following method was used to prepare a bispecific antibody (hereinafter also referred to as "mouse-type anti-MerTK-anti-TTR bispecific antibody") consisting of a heavy chain fragment containing the variable region of the heavy chain containing anti-MerTK antibody, a hinge region and a mouse first Fc polypeptide, a light chain containing the variable region of the light chain containing anti-MerTK antibody and anti-TTR-scFv, and a polypeptide with a mouse second Fc polypeptide linked to the hinge region.

[0434] (1) Preparation of vectors encoding the heavy and light chains of mouse-type anti-MerTK antibodies

[0435] The amino acid sequences of the heavy and light chains of the mouse anti-MerTK antibody were designed. The heavy chain (Sequence No. 34) was designed by linking the variable region and hinge region of the heavy chain of tA3-20 with the constant region of the mouse heavy chain IgG2a (EU index: 215 to 447). The light chain (Sequence No. 36) was designed by linking the variable region of the light chain of tA3-20.3 with the constant region of the mouse light chain Lambda chain (EU index: 108 to 213). The design proceeds in a manner that results in an amino acid sequence containing the following mutations in the aforementioned heavy chain: (i) LALA mutations (L234A and L235A) in which amino acids 236 and 237 (EU index: 234 and 235) are replaced with alanine (A), respectively; (ii) glutamic acid (E) in which amino acids 358, 366, 370, 372, 401 and 413 (EU index: 356, 364, 368, 370, 399 and 411) are replaced with lysine (A), respectively. Electrostatic shift mutations (K) replacing threonine (T) with serine (S), methionine (M) with leucine (L), threonine (T) with lysine (K), aspartic acid (D) with lysine (K), and arginine (R) with threonine (T) to stabilize heterodimerized antibody molecules (mAbs, 2019, 12: 1-12), and (iii) a mutation replacing proline (P) with alanine (A) at amino acid number 331 (EU index: 329). The base sequences (sequence numbers 33 and 35) encoding the heavy and light chains of the designed mouse-type anti-MerTK antibody were artificially synthesized and inserted into the pcDNA3.4TOPO vector to create vectors MHC and MLC, respectively.

[0436] (2) Preparation of vector encoding mouse anti-TTR-scFv-Fc

[0437] A polypeptide (hereinafter referred to as "mouse-type anti-TTR-scFv-Fc") (Sequence No. 38) with a hinge region and anti-TTR-scFv linked to the N-terminus of the mouse second Fc polypeptide was designed to consist of the following amino acid sequence, which is the heavy chain variable region of the anti-TTR antibody 371M antibody (Sequence No. 13 of International Publication No. 2015 / 115332) that binds to misfolded TTRs, the GS linker (Sequence No. 54), the light chain variable region of the 371M antibody (Sequence No. 14 of International Publication No. 2015 / 115332), the hinge region, and the constant region of mouse heavy chain IgG2a (EU index: 215 to 447) linked sequentially. To stabilize (i) the anti-TTR-scFv located on the N-terminal side (Protein Engineering, Design and Selection, 2019, 25: 321-329), the mouse anti-TTR-scFv-Fc was further designed to have an amino acid sequence containing the following mutations (sequence number 38): (ii) replacing glycine (G) at amino acid number 44 (EU index: 44) of sequence number 38 with cysteine ​​(C), (iii) replacing glycine (G) at amino acid number 237 (EU index: 100) with cysteine ​​(C), (ii) LALA mutations (L234A and L235A) replacing leucine (L) with alanine (A) at amino acids number 265 and 266 (EU index: 234 and 235) of sequence number 38, respectively; (iii) replacing amino acids number 38 with leucine (L) at amino acids number 265 and 266 (EU index: 234 and 235) of sequence number 38, respectively. The amino acid numbers 395, 399, 401, 440, 442, and 470 (EU index: 364, 368, 370, 409, 411, and 439) are electrostatic reversal mutations that replace threonine (T) with serine (S), methionine (M) with leucine (L), threonine (T) with lysine (K), lysine (K) with glutamic acid (E), arginine (R) with threonine (T), and lysine (K) with aspartic acid (D), respectively, and mutations used to stabilize heterodimerized antibody molecules, and (iv) a mutation that replaces proline (P) with alanine (A) in amino acid number 360 (EU index: 329) of sequence number 38. The base sequence encoding the designed mouse-type anti-TTR-scFv-Fc amino acid sequence was artificially synthesized and inserted into the pcDNA3.4 TOPO vector to create the vector TscFvFc.

[0438] (3) Preparation of mouse-type anti-MerTK-anti-TTR bispecific antibody

[0439] Mouse-type anti-MerTK-anti-TTR bispecific antibodies were prepared using the vectors described in (1) and (2) above. Specifically, vectors MHC, MLC, and TscFvFc were co-introduced into ExpiCHO-S cells using the ExpiFectamine CHO transfection kit, and the mouse-type anti-MerTK-anti-TTR bispecific antibodies were secreted into the culture supernatant using standard methods. The antibodies were purified from the obtained culture supernatant by a combination of affinity purification using MabSelect SuRe pcc and ion exchange chromatography using HiTrapSP HP (Cytiva, 17115201) or size exclusion chromatography using Superdex 200 Increase 10 / 300 GL (Cytiva, 28990944).

[0440] The mouse-type anti-MerTK-anti-TTR bispecific antibody produced hereafter will be referred to as tA-009.

[0441] [Examples 4-5: Preparation of mouse-type anti-TTR antibodies]

[0442] The 371M antibody (referred to as "mouse-type 371M" in this specification) is prepared as an anti-TTR antibody having a variable region of human anti-TTR antibody and a constant region of mouse anti-TTR antibody by the following method.

[0443] As the heavy chain, the variable region of the heavy chain of the anti-TTR antibody 371M antibody (Sequence No. 13, International Publication No. 2015 / 115332) was linked with the amino acid sequence of the constant region (EU index: 118 to 447) of the mouse heavy chain IgG2a (Sequence No. 44). As the light chain, the variable region of the light chain of the 371M antibody (Sequence No. 14, International Publication No. 2015 / 115332) was linked with the amino acid sequence of the mouse light chain Lambda1 (EU index: 108 to 213) (Sequence No. 45). The base sequence encoding the designed amino acid sequence was artificially synthesized and inserted into the pcDNA3.4 TOPO vector. Mouse-type 371M was created using the prepared vector. Specifically, the vector was introduced into ExpiCHO-S cells using the ExpiFectamine CHO transfection kit, and the antibody was secreted into the culture supernatant using standard methods. The antibody was purified from the culture supernatant by a combination of affinity purification using MabSelect SuRe pcc and size exclusion chromatography using HiLoad 26 / 600 superdex (registered trademark) 200 pg (Cytiva, 28-9893-36).

[0444] [Example 5: In vitro activity evaluation of tA-009]

[0445] [Example 5-1: Evaluation of the binding activity of tA-009 on human MerTK and misTTR]

[0446] The binding activity of tA-009 to human MerTK and misTTR was evaluated by ELISA.

[0447] The human MerTK-Fc protein prepared in Example 1-1(1) and the misTTR prepared in Example 4-2 were prepared to concentrations of 1 μg / mL and 2 μg / mL respectively using PBS. 20 μL was added to each well of a clear, flat-bottomed, non-sterile 384-well immunochromatographic plate, and the plate was incubated at room temperature for 1 hour to immobilize it. The human MerTK-Fc protein and misTTR were then removed. A blocking solution of 20% Blocking One was added to TBS-T to prepare a blocking solution, 50 μL was added to each well, and the plate was incubated for 1 hour to block. The wells were then washed once with TBS-T. As tA-009 and isotype control antibodies, anti-keyhole hemocyanin (KLH) antibody (KLH_173A1_ma0, manufactured by our company) was prepared from 2 × 10⁻⁶ ppm. 4 Continuous dilution to 1×10 -2 ng / mL. When diluting the antibodies, use TBS-T containing 5% Blocking One. Add 15 μL of each diluted antibody to each well and react at room temperature for 1 hour. Remove the antibodies, wash the wells three times with TBS-T, then add 15 μL of secondary antibody (prepared by diluting goat anti-mouse IgG human ads-HRP (Southern Biotech, 1030-05) 4000-fold with TBS-T containing 5% Blocking One) to each well and react at room temperature for 1 hour. Wash the wells three times with TBS-T, then add 15 μL of TMB-Plus Substrate-Chromogen to each well and incubate at room temperature for 5 minutes. Stop the reaction by adding 1 mol / L sulfuric acid, and measure the absorbance at 450 nm and 570 nm using an Infinite M200 PRO.

[0448] The assay results showed that tA-009 exhibited concentration-dependent binding activity to human MerTK-Fc protein and misTTR. Figure 4-1 and 4-2 ).

[0449] [Example 5-2: Activation of phagocytic clearance function of mouse bone marrow-derived macrophages by tA-009]

[0450] The phagocytic effect of tA-009 on phagocytic cells was evaluated using the phagocytic effect of mouse bone marrow-derived macrophages (one of the phagocytic cells) on misTTR.

[0451] Mouse bone marrow-derived macrophages were prepared using the following method. Bone marrow cells were collected from the femur and tibia of male C57BL6 / J mice (Jackson Laboratories, Japan). The cells were cultured for 3 days at 37°C and 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, R8758-500ML) containing 10% FBS (Cytiva, SH30070.03) and 40 ng / ml of recombinant mouse M-CSF protein (R&D Systems, 416-ML-010) (hereinafter also referred to as "RPMI-1640 containing FBS, etc."). The medium was then replaced with RPMI-1640 containing 0.1 μM dexamethasone (Sigma-Aldrich, D4902-100MG) and cultured at 1 × 10⁻⁶ ppm. 4 Mouse bone marrow-derived macrophage culture plates were prepared by culturing cells at a concentration of 10 cells / well at 37°C and 5% CO2 for 3 days, followed by washing once with RPMI-1640 medium. tA-009 was used as the test antibody, and tA3-20.3 was used as the control antibody. 50 μL of RPMI-1640 medium containing a final concentration of 5 μg / mL pHrodo-misTTR and a final concentration of 0.7 μmol / L of tA-009 or tA3-20.3 (serial numbers 8 and 16) as the control antibody was added to each well of the mouse bone marrow-derived macrophage culture plate. The mouse bone marrow-derived macrophage culture plates were placed in an Incucyte S3 live cell analyzer in a 5% CO2, 37°C incubator for assays and analysis, as in Example 2.

[0452] The analysis showed that tA-009, as an anti-MerTK-anti-TTR bispecific antibody, significantly activated the phagocytic activity of mouse bone marrow-derived macrophages (MDM) against misTTR compared to tA3-20.3, an anti-MerTK antibody. This result suggests that tA-009 efficiently activates the phagocytic clearance function of MBM macrophages against misTTR by bringing misTTR closer to them.

[0453] [Example 6: Activation of phagocytic clearance function of misTTR by phagocytes induced by tA-009 in mice]

[0454] pHrodo-misTTR was administered subcutaneously to mice, and its fluorescence intensity was measured over time using in vivo imaging to evaluate whether tA-009 could activate phagocytosis of misTTR by phagocytes. pHrodo-misTTR emitted fluorescence when phagocytosed by phagocytes. Furthermore, to confirm that tA-009-activated phagocytosis was MerTK-dependent, a combination assay with a MerTK inhibitor was conducted.

[0455] 40 μL of a solution containing a mixture of pHrodo-misTTR prepared according to Examples 4-3 and neutralized misTTR at a protein mass ratio of 1:4 was administered subcutaneously to the dorsal side of 13-week-old female BALB / c-nu (nu / nu) mice (Jackson Laboratories, Japan). Mice were anesthetized with isoflurane (Mira Pharmaceutical) at 1, 6, 12, 24, 48, 72, 120, 168, 240, and 336 hours after administration, and the fluorescence intensity (photons / second, [p / s]) around the administration site was measured using an IVIS imaging system (Lumina II; PerkinElmer) to quantify the amount of pHrodo-misTTR phagocytosed by the aggregated phagocytes. The cumulative value of fluorescence intensity at the above measurement time points was calculated as the total phagocytosis ([p / s]·h) during the observation period (14 days), thereby evaluating phagocytosis. One hour before subcutaneous administration of pHrodo-misTTR, the test antibody tA-009 (100 mg / kg) or the control solvent (PBS) was administered intraperitoneally. From day 0 to day 5, ONO7475 (10 mg / kg, MedChemExpress, HY-114358), a MerTK inhibitor, was administered orally once daily for a total of 6 times. It should be noted that on the trial start day (day 0), ONO7475 was administered orally 0.5 hours before tA-009 administration. This trial was conducted in groups of 5 patients. The mean and standard error of the total phagocytosis of pHrodo-misTTR and the test for significance were performed using GraphPad Prism (GraphPad Software, ver 8.0.2). The comparison between the solvent group and the tA-009 group, and between the tA-009 group and the tA-009+ONO7475 group, was conducted using the Student's t-test. A p-value less than 0.05 was considered statistically significant.

[0456] The MerTK-dependent phagocytosis induced by tA-009 in mice was evaluated using the fluorescence intensity of pHrodo-misTTR as an indicator. The results showed that the fluorescence intensity of pHrodo-misTTR was significantly increased in the tA-009-treated group compared to the solvent-treated group. Figure 5These results indicate that phagocytosis of pHrodo-misTTR by phagocytes was activated in the tA-009-treated group. On the other hand, the activation of pHrodo-misTTR phagocytosis induced by tA-009 administration was almost entirely inhibited by the MerTK inhibitor ONO7475. These results suggest that tA-009-dependent MerTK-dependent activation of phagocytic clearance function was observed.

[0457] [Example 7: Activation of phagocytic clearance function of misTTR by phagocytes induced by tA-009 or mouse-type 371M in aged mice]

[0458] As is well known, TTR-type amyloidosis, caused by the deposition of misfolded TTRs in organs throughout the body, is an age-related disease (Front. Cardiovasc. Med., 2022, 9: 863179). Furthermore, it has been reported that aging reduces the phagocytic clearance function of phagocytes such as macrophages, and that this dysfunction is one of the causes of various diseases (Immunol. Lett., 2021, 230: 1-10). To evaluate whether the activation of MerTK-dependent and Fcγ receptor-dependent phagocytic clearance functions under age-related reduced phagocytic clearance conditions is effective in clearing misfolded TTRs, the phagocytic clearance activity of DL800-misTTR in aged mice was used as an indicator.

[0459] [Example 7-1: Dosage setting using tA-009 or mouse-type 371M from young mice]

[0460] To compare the phagocytic clearance function of misTTR in tA-009, which relies on MerTK to activate phagocytes, and that of mouse-type 371M (Fc being wild-type mouse IgG2a (Immunol Rev., 2015, 268: 25-51)) which relies on Fcγ receptor activation, DL800-misTTR was administered subcutaneously to young mice with normal phagocytic clearance function, and changes in fluorescence intensity were measured over time using in vivo imaging. The fluorescence intensity of DL800-misTTR was proportional to its residual amount.

[0461] 40 μL of a solution containing DL800-misTTR prepared according to Examples 4-3 and neutralized misTTR at a protein mass ratio of 1:4 was subcutaneously administered to the dorsal side of 6-week-old (young) female BALB / c-nu (nu / nu) mice (Jackson Laboratories, Japan). Mice were anesthetized with isoflurane (Mira Pharmaceutical) at 1, 6, 12, 22, 46, 71, 118, 168, 240, and 336 hours after administration, and the fluorescence intensity (photons / second, [p / s]) around the administration site was measured using an IVIS imaging system (Lumina II; PerkinElmer) to quantify the residual amount of misTTR. The cumulative value of fluorescence intensity at the above measurement time points was calculated as the total residual amount ([p / s]·h) during the observation period (14 days), thereby evaluating phagocytic clearance function. One hour prior to subcutaneous administration of DL800-misTTR, the test antibody tA-009 (100 mg / kg) or mouse 371M (30 mg / kg), along with the control solvent (PBS), were administered intraperitoneally. This study was conducted in groups of 4. The mean and standard error of the total residual DL800-misTTR, as well as the significance test, were performed using GraphPad Prism (GraphPad Software, ver 8.0.2). Comparisons between the solvent group and the test antibody (mouse 371M or tA-009) group were performed using the Dunnett test; a p-value less than 0.05 was considered statistically significant.

[0462] The evaluation results of the phagocytic clearance function of DL800-misTTR by tA-009 or mouse-type 371M in mice are shown in the figure. Figure 6-1 The results showed a significant and nearly identical reduction in the total residual amount of DL800-misTTR in the tA-009 (100 mg / kg) or mouse-type 371M (30 mg / kg) groups compared to the solvent-administered group. This result indicates that, in vivo, 100 mg / kg of tA-009 and 30 mg / kg of mouse-type 371M possess substantially equivalent phagocytic clearance capabilities for DL800-misTTR.

[0463] Based on the above results, in young mice where the phagocytic clearance function of macrophages and other phagocytes was maintained, 100 mg / kg of tA-009 and 30 mg / kg of mouse-type 371M induced approximately equivalent phagocytic clearance function in phagocytes, and the following experiments were conducted.

[0464] [Example 7-2: Activation of phagocytic clearance function of DL800-misTTR by tA-009 or mouse-type 371M in aged mice]

[0465] In aged mice, it has been taught that aging leads to a decline in the phagocytic clearance function of phagocytes such as macrophages (Clin. Exp. Immunol. 2008, 152(3): 448-455, Immunol. Lett. 2021, 230: 1-10). The present inventors hypothesize that MerTK-dependent agents, which activate the phagocytic clearance function of phagocytes, may be more useful in the treatment of diseases than agents that activate via Fcγ receptors. Studies evaluating the activation of phagocytic clearance function were conducted using doses of tA-009 and mouse-type 371M, which have shown equivalent effects in young mice.

[0466] Following the results of Example 7-1, male C57BL / 6J mice (Jackson Laboratories, Japan) aged 20 weeks (young) or 103 weeks (old) were intraperitoneally administered tA-009 (100 mg / kg) as the test antibody, mouse-type 371M (30 mg / kg) or PBS as a control. Subsequently, 40 μL of a solution prepared according to Example 4-3 and neutralized misTTR mixed at a protein mass ratio of 1:4 was subcutaneously administered to the dorsal side of the mice. At 1, 6, 12, 24, 48, 72, 120, and 168 hours after administration, the mice were anesthetized with isoflurane (Mirai Pharmaceutical), and the fluorescence intensity (photons / second, [p / s]) around the administration site was measured using an IVIS imaging system (Lumina II; PerkinElmer) to quantify the residual amount of DL800-misTTR. This experiment was conducted with 5–6 mice per group. The mean and standard error of the total residual DL800-misTTR were calculated, and the significance test was performed using GraphPad Prism (GraphPad Software, ver8.0.2). The comparison between the young mouse solvent group and the aged mouse solvent group was performed using the Student's t-test, and the comparison between the aged mouse solvent group and the aged mouse test antibody (mouse type 371M or tA-009) group was performed using the Dunnett test. A p-value less than 0.05 was considered statistically significant.

[0467] The results of evaluating the activation of phagocytic clearance function of DL800-misTTR by tA-009 and mouse-type 371M in aged mice are shown in the figure. Figure 6-2 Compared with young mice, a significant increase in the total residual amount of DL800-misTTR was observed in aged mice, confirming that the phagocytic clearance function of phagocytes for foreign bodies is reduced in aged mice.

[0468] In this study using aged mice, a significant reduction in the total residual amount of DL800-misTTR was observed in the tA-009 (100 mg / kg) administration group compared to the solvent administration group. On the other hand, no effect was observed in the mouse-type 371M (30 mg / kg) administration group.

[0469] These results indicate that tA-009, which can activate phagocytic clearance function in a MerTK-dependent manner, significantly activates phagocytic clearance function in phagocytic cells with reduced phagocytic clearance function due to age, compared to mouse 371M, which can activate phagocytic clearance function in a Fcγ receptor-dependent manner.

[0470] This study found that tA-009, an anti-MerTK-anti-TTR bispecific antibody that activates phagocytic clearance function in MerTK-dependent mice, significantly activated phagocytic clearance function in aged mice compared to mouse-type 371M, which activates phagocytic clearance function in response to Fcγ receptors. This result suggests that activating phagocytic clearance function using anti-MerTK-anti-misfolded protein bispecific antibodies may be very useful in diseases caused by the accumulation of age-related misfolded proteins.

[0471] [Example 8: Design of a fully human anti-MerTK-anti-TTR bispecific antibody]

[0472] (1) Design of fully human anti-MerTK single-arm antibody

[0473] Based on the sequence of the mouse-type anti-MerTK-anti-TTR bispecific antibody obtained in Example 4, a fully human anti-MerTK-anti-TTR bispecific antibody (hereinafter also referred to as "fully human anti-MerTK-anti-TTR bispecific antibody") was designed. First, a fully human MerTK single-arm antibody (hereinafter also referred to as "fully human MerTK single-arm antibody") was designed. As the heavy chain fragment of the fully human anti-MerTK single-arm antibody, amino acid sequences numbered 1 to 217 of sequence number 8 were used. Furthermore, an LALA mutation and a P331G amino acid mutation (sequence number 39) were introduced into the first or second Fc peptide of the fully human anti-MerTK single-arm antibody. A mortar-and-hollow mutation was introduced into the first Fc peptide and the second Fc peptide based on existing patent literature (International Publication No. 1998 / 050431). Specifically, in one of the first or second Fc peptides, a sequence (Sequence No. 40) is designed to replace amino acid number 136 (EU index: 366) of sequence No. 39 with tryptophan (W) to form knots. Additionally, a sequence (Sequence No. 41) is designed to replace amino acids numbered 136, 138, and 177 (EU index: 366, 368, and 407) of sequence No. 39 with serine (S), L (leucine) with alanine, and tyrosine (Y) with valine (V) to form holes in the other Fc peptide. The light chain of the fully human anti-MerTK single-arm antibody uses amino acid sequences numbered 16, 18, or 20. The fully human anti-MerTK single-arm antibody was designed by combining the heavy chain fragment of the fully human anti-MerTK single-arm antibody, the light chain of the fully human anti-MerTK single-arm antibody, the hinge region (serial number 42), and the first and second Fc peptides (serial numbers 40 and 41).

[0474] (2) Design of fully human anti-MerTK-anti-TTR bispecific antibodies

[0475] The amino acid sequence (Sequence No. 43) and hinge region (Sequence No. 70) of the anti-TTR-scFv were linked to the N-terminus of the second Fc polypeptide of the fully human anti-MerTK single-arm antibody designed in (1), thereby designing a fully human anti-MerTK-anti-TTR bispecific antibody. The combination of amino acid sequences of each part of the designed fully human anti-MerTK-anti-TTR bispecific antibody is shown in Table 3.

[0476]

[0477] The anti-MerTK antibody of the present invention is expected to be useful in the prevention or treatment of various diseases caused by reduced phagocytic function.

[0478] [Example 9: Preparation of anti-MerTK-anti-IgL bispecific antibody, anti-IgL antibody and misfolded IgL protein]

[0479] Preparation of misfolded IgL protein, anti-MerTK-anti-IgL bispecific antibody, and anti-IgL antibody.

[0480] [Example 9-1: Preparation of recombinant human IgL protein]

[0481] Referring to the gene sequence encoding the human IgL Vλ6Wil amino acid sequence described in the report by J. Wall et al. (Biochemistry, 1999, 38: 14101-14108, Figure 1), the gene sequence encoding 6×histidine (serial number 22) was inserted into the expression vector pcDNA3.4 TOPO to obtain the Vlambda6Wil vector. The Vlambda6Wil vector was introduced into ExpiCHO-S cells using the ExpiFectamine CHO transfection kit, and the recombinant protein was secreted into the culture supernatant using standard methods. His-labeled human IgL protein was purified from the culture supernatant using TALON (registered trademark) metal affinity resin (Clontech, 635502) and a His Buffer kit (Cytiva, 11-0034-00). The elution fraction containing recombinant human IgL protein was replaced with PBS using a NAP-25 column (Cytiva, 17-0852-01) or an Amicon Ultra-15 column (Merck, UFC901096), and concentrated to obtain purified recombinant human IgL protein.

[0482] [Example 9-2: Preparation of misfolded IgL protein]

[0483] To prepare misfolded recombinant human IgL protein (referred to as "misfolded IgL protein" in this specification), 200 μL of purified recombinant human IgL protein (1.3 mg / mL) was mixed with 800 μL of 50 mM phosphate buffer (pH 2.7, prepared by our company) and incubated at 37°C for at least 4 days to prepare a misfolded recombinant human IgL protein solution.

[0484] [Example 9-3: Preparation of anti-MerTK-anti-IgL bispecific antibody]

[0485] A bispecific antibody (hereinafter also referred to as "anti-MerTK-anti-IgL bispecific antibody") consisting of a heavy chain fragment containing the variable region of the heavy chain containing an anti-MerTK antibody, a hinge region and a human first Fc polypeptide, a light chain containing the variable region of the light chain containing an anti-MerTK antibody, a heavy chain consisting of a polypeptide linking the variable region of the heavy chain containing an anti-IgL antibody, the hinge region and a human second Fc polypeptide, and a light chain containing the variable region of the light chain containing an anti-IgL antibody is prepared by the following method.

[0486] (1) Preparation of vectors encoding the heavy and light chains of anti-IgL antibodies

[0487] The fabrication of the vectors encoding the heavy and light chains of the anti-IgL antibody was based on the description in the paper (mAbs, 2016, 8(6): 1010-1020). A mouse-human chimeric heavy chain (serial number 57) (hereinafter referred to as the "anti-IgL antibody heavy chain") was designed, consisting of the VH and CL regions of the mouse-derived anti-IgL antibody 2A4 antibody described in Japanese Patent No. 5730020, the hinge region, and a human Fc polypeptide with amino acid mutations of mortis, LALA, and P331G. In addition, a mouse-human chimeric light chain (serial number 59) (hereinafter referred to as the "anti-IgL antibody light chain") with the VL of the 2A4 antibody and the human CH1 domain was designed. The base sequences (Sequence No. 56 and Sequence No. 58) encoding the heavy chain and light chain of the designed anti-IgL antibody were synthesized artificially and inserted into the pcDNA3.4 TOPO vector, respectively, thereby creating vectors encoding the heavy chain and light chain of the anti-IgL antibody.

[0488] (2) Preparation of vectors encoding the heavy and light chains of fully human anti-MerTK antibodies

[0489] The heavy chain fragment of the human MerTK single-arm antibody, consisting of amino acid sequences numbered 1 to 217 (Sequence No. 8), the hinge region (Sequence No. 42), and the first Fc polypeptide (Sequence No. 41) were sequentially linked to design the heavy chain (Sequence No. 61) of the fully human anti-MerTK single-arm antibody. The base sequence encoding the amino acid sequence of the designed fully human anti-MerTK single-arm antibody heavy chain was artificially synthesized and inserted into the pcDNA3.4 TOPO vector to create a vector encoding the heavy chain of the fully human anti-MerTK antibody.

[0490] The base sequence of the light chain (serial number 12) encoding tA3-20.1 designed in Example 1-1 was artificially synthesized and inserted into the pcDNA3.4 TOPO vector, thereby creating a vector encoding the light chain of a fully human anti-MerTK antibody.

[0491] (3) Preparation of anti-MerTK-anti-IgL bispecific antibody

[0492] Anti-MerTK-anti-IgL bispecific antibodies were prepared using the four vectors described in (1) and (2) above. Specifically, using the ExpiFectamine CHO transfection kit, vectors encoding the heavy chain of anti-IgL antibody, light chain of anti-IgL antibody, fully human anti-MerTK antibody, and light chain of fully human anti-MerTK antibody were introduced into ExpiCHO-S cells. The anti-MerTK-anti-IgL bispecific antibody was then secreted into the culture supernatant using standard methods. The antibody was purified from the obtained culture supernatant using affinity purification with MabSelect SuRe. The prepared anti-MerTK-anti-IgL bispecific antibody will be referred to as tA3-20_2A4_h1x.

[0493] [Example 9-4: Preparation of anti-IgL antibody]

[0494] The variable region of mouse-derived anti-IgL antibodies and anti-IgL antibodies with human constant regions (referred to as "mouse-human chimeric 2A4" in this specification) of serial numbers 152 and 154 of existing patent documents (Japanese Patent No. 5730020) are prepared by the following method.

[0495] The heavy chain described in Serial No. 63 and the light chain described in Serial No. 65 were designed with reference to the sequence of the 2A4 antibody described in Japanese Patent No. 5730020. The base sequences encoding the designed two amino acid sequences (Serial Nos. 62 and 64) were artificially synthesized using conventional methods, and mouse-human chimeric 2A4 was prepared using the same method as in Examples 1-1.

[0496] [Example 10: Evaluation of the binding activity of the anti-MerTK-anti-IgL bispecific antibody against human MerTK and misfolded IgL proteins]

[0497] The binding activity of tA3-20_2A4_h1x to human MerTK and misfolded IgL proteins was evaluated by ELISA.

[0498] The misfolded IgL protein prepared in Examples 9-2 was prepared to a concentration of 2.5 μg / mL using PBS. 50 μL was added to each well of Ni-NTA HisSorb Plates (QIAGEN, 35061) and incubated overnight at 4°C to immobilize the protein on the plates. The misfolded IgL protein was then removed, and the wells were washed three times with PBST. tA3-20_2A4_h1x was used as the evaluation antibody, and tA3-20.1, mouse-human chimeric 2A4, and LYS_1c3-m1_h1i (isotype control) were used as control antibodies. PBS containing 0.05% Tween-20 and 0.5% Blocking One (prepared by diluting Thermo Scientific 28352 20-fold, hereinafter referred to as "PBST") was serially diluted from 20 μg / mL to 6.4 ng / mL, and 50 μL was added to each well. The reaction was carried out at room temperature for 1 hour. The wells were then washed three times with PBST. The MerTK-Fc fusion protein prepared in Example 1 was biotinylated using a biotinylation kit (Dōjin Chemical Research Institute, LK03). A 2 μg / mL solution of PBST containing 0.5% Blocking One was added to each well, and the reaction was carried out at room temperature for 1 hour. The wells were washed three times with PBST. Then, 50 μL of a 200-fold diluted Streptavidin-HRP solution (R&D Systems, DY998) was added to each well, and the reaction was carried out at room temperature for 1 hour. The wells were washed three times with PBST. Then, 50 μL of TMB-Plus Substrate-Chromogen was added to each well, and the reaction was allowed to stand at room temperature for 5 minutes. The reaction was stopped by adding 50 μL of 1 mol / L sulfuric acid to each well, and the absorbance at 450 nm and 570 nm was measured using an Infinite M200 PRO.

[0499] The results showed that tA3-20_2A4_h1x could bind to both human MerTK and misfolded IgL proteins simultaneously, and this activity was concentration-dependent (i.e., the antibody concentration required to bind to both). Figure 7 ).

[0500] Furthermore, using pHrodo misfolded IgL protein labeled in the same manner as in Examples 4-3, the phagocytic activity of mouse bone marrow-derived macrophages was confirmed in the same manner as in Examples 5-2. The results showed that tA3-20_2A4_h1x activates phagocytosis in a MerTK-dependent manner. This result suggests that tA3-20_2A4_h1x can activate the phagocytic clearance function of mouse bone marrow-derived macrophages for misfolded IgL protein.

[0501] Sequence List Free Text

[0502] Serial numbers 1-3 represent the amino acid sequences of CDR1-3 of the heavy chain tA3-20 to tA3-20.5, respectively; serial numbers 4-6 represent the amino acid sequences of CDR1-3 of the light chain tA3-20 to tA3-20.5, respectively. Serial number 8 represents the amino acid sequence of the heavy chain tA3-20 to tA3-20.5, and serial number 7 represents the base sequence encoding the amino acid sequence of the heavy chain tA3-20 to tA3-20.5 shown in serial number 8. Serial number 10 represents the amino acid sequence of the light chain tA3-20, and serial number 9 represents the base sequence encoding the amino acid sequence of the light chain tA3-20 shown in serial number 10. Serial number 12 represents the amino acid sequence of the light chain tA3-20.1, and serial number 11 represents the base sequence encoding the amino acid sequence of the light chain tA3-20.1 shown in serial number 12. Serial number 14 is the amino acid sequence of the light chain tA3-20.2, and serial number 13 is the base sequence encoding the amino acid sequence of the light chain tA3-20.2 shown in serial number 14. Serial number 16 is the amino acid sequence of the light chain tA3-20.3, and serial number 15 is the base sequence encoding the amino acid sequence of the light chain tA3-20.3 shown in serial number 16. Serial number 18 is the amino acid sequence of the light chain tA3-20.4, and serial number 17 is the base sequence encoding the amino acid sequence of the light chain tA3-20.4 shown in serial number 18. Serial number 20 is the amino acid sequence of the light chain tA3-20.5, and serial number 19 is the base sequence encoding the amino acid sequence of the light chain tA3-20.5 shown in serial number 20. Serial number 22 is the amino acid sequence with a histidine tag, and serial number 21 is the base sequence encoding the amino acid sequence with a histidine tag shown in serial number 22. Serial number 24 is the amino acid sequence of the heavy chain of MTK201, and serial number 23 is the base sequence encoding the amino acid sequence of the heavy chain of MTK201 shown in serial number 24. Serial number 26 is the amino acid sequence of the light chain of MTK201, and serial number 25 is the base sequence encoding the amino acid sequence of the light chain of MTK201 shown in serial number 26. Serial numbers 27-29 are the amino acid sequences of CDR1-3 of the heavy chain of the 371M antibody, respectively, and serial numbers 30-32 are the amino acid sequences of CDR1-3 of the light chain of the 371M antibody, respectively. Serial number 34 is the amino acid sequence of the heavy chain of the mouse-type anti-MerTK antibody tA3-20.3, and serial number 33 is the base sequence encoding the amino acid sequence of the heavy chain of the mouse-type anti-MerTK antibody tA3-20.3 shown in serial number 34. Serial number 36 is the amino acid sequence of the light chain of mouse-type anti-MerTK antibody tA3-20.3, and serial number 35 is the base sequence encoding the amino acid sequence of the light chain of mouse-type tA3-20.3 shown in serial number 36.Sequence number 38 is the amino acid sequence of a polypeptide containing the mouse second Fc polypeptide and anti-TTR-scFv. Sequence number 37 is the base sequence encoding the polypeptide containing the mouse second Fc polypeptide and anti-TTR-scFv shown in sequence number 38. Sequence number 39 is the amino acid sequence of the first or second Fc polypeptide of tA3-20 to tA3-20.5. Sequence numbers 40 and / or 41 are the amino acid sequences of the first and / or second Fc polypeptides of the anti-MerTK-anti-TTR bispecific antibody. Sequence number 42 is the amino acid sequence of the hinge region linked to anti-MerTK-Fab in tA3-20 to tA3-20.5 and the anti-MerTK-anti-TTR bispecific antibody. Sequence number 43 is the amino acid sequence of anti-TTR-scFv. Serial number 44 is the amino acid sequence of the heavy chain of the 371M antibody containing the constant region of the mouse heavy chain IgG2a. Serial number 45 is the amino acid sequence of the light chain of the 371M antibody containing the mouse light chain Lambda1. Serial numbers 46-55 are the amino acid sequences of various linkers. Serial numbers 57 and 59 are the amino acid sequences of the heavy and light chains of the mouse-human chimeric anti-MerTK-anti-IgL bispecific antibody, respectively. Serial numbers 56 and 58 are the base sequences encoding the amino acid sequences of the heavy and light chains of the mouse-human chimeric anti-MerTK-anti-IgL bispecific antibody shown in serial numbers 57 and 59, respectively. Serial number 61 is the amino acid sequence of the heavy chain of the human anti-MerTK single-arm antibody. Serial number 60 is the base sequence encoding the amino acid sequence of the heavy chain of the human anti-MerTK single-arm antibody shown in serial number 61. Serial number 63 is the amino acid sequence of the heavy chain of the mouse-human chimeric 2A4. Serial number 62 is the base sequence encoding the amino acid sequence of the heavy chain of the mouse-human chimeric 2A4 shown in serial number 63. Serial number 65 is the amino acid sequence of the light chain of the mouse-human chimeric 2A4. Serial number 64 is the base sequence encoding the amino acid sequence of the light chain of the 2A4 antibody shown in serial number 65. Serial numbers 67 and 69 are the amino acid sequences of the heavy and light chains of M6, respectively. Serial numbers 66 and 68 are the base sequences encoding the amino acid sequences of the heavy and light chains of M6 shown in serial numbers 67 and 69, respectively. Serial number 70 is the amino acid sequence of the hinge region linked to anti-TTR-scFv in the anti-MerTK-anti-TTR bispecific antibody.

Claims

1. An anti-MerTK antibody or its antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1 consisting of an amino acid sequence of sequence number 1, CDR2 consisting of an amino acid sequence of sequence number 2, and CDR3 consisting of an amino acid sequence of sequence number 3, and the light chain variable region comprises CDR1 consisting of an amino acid sequence of sequence number 4, CDR2 consisting of an amino acid sequence of sequence number 5, and CDR3 consisting of an amino acid sequence of sequence number 6.

2. The anti-MerTK antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of (1) to (6): (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; and (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

3. The anti-MerTK antibody according to claim 1 or 2, comprising a heavy chain containing a heavy chain variable region and a light chain containing a light chain variable region.

4. The anti-MerTK antibody according to claim 3, wherein, The heavy chain contains an amino acid mutation of L234A and L235A (LALA mutation) (here, the mutation is located at the amino acid position according to the EU index in the human Igγ1 constant region).

5. The anti-MerTK antibody according to claim 3, wherein, The heavy chain contains the P331G mutation (here, the mutation is located at the amino acid position according to the EU index in the human Igγ1 constant region).

6. The anti-MerTK antibody according to claim 3, wherein, The heavy chain contains amino acid mutations of L234A and L235A (LALA mutation) and P331G mutation (here, the mutation location is the amino acid position according to the EU index in the human Igγ1 constant region).

7. The anti-MerTK antibody according to claim 6, wherein it is selected from the group consisting of (1) to (6): (1) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 10. (2) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 12. (3) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 14. (4) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 16. (5) An anti-MerTK antibody containing a heavy chain consisting of the amino acid sequence of sequence number 8 and a light chain consisting of the amino acid sequence of sequence number 18; and (6) Anti-MerTK antibody containing a heavy chain consisting of an amino acid sequence of sequence number 8 and a light chain consisting of an amino acid sequence of sequence number 20.

8. The anti-MerTK antibody according to any one of claims 1 to 7, wherein it is an IgG antibody (anti-MerTK IgG antibody).

9. The anti-MerTK antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antigen-binding fragment is a single-stranded variable region fragment (scFv), Fab fragment, Fab' fragment, or F(ab')2 fragment.

10. The anti-MerTK antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein it has undergone post-translational modification.

11. A fusion or complex of any anti-MerTK antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, or a cell on which the anti-MerTK antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10 is presented on its cell surface.

12. A polynucleotide containing a base sequence encoding a heavy chain variable region or a light chain variable region of an anti-MerTK antibody or an antigen-binding fragment thereof, said polynucleotide being selected from the group consisting of (1) to (7): (1) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8. (2) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (3) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (4) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (5) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (6) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (7) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

13. A polynucleotide containing a base sequence encoding a heavy or light chain of an anti-MerTK antibody, said polynucleotide being selected from the group consisting of (1) to (7): (1) A polynucleotide containing a base sequence encoding a heavy chain consisting of the amino acid sequence of sequence number 8; (2) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 10; (3) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 12; (4) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 14; (5) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 16; (6) A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence of sequence number 18; and (7) A polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence of sequence number 20.

14. An expression vector containing the polynucleotide of claim 12 or 13.

15. A host cell that has been transformed with the expression vector of claim 14.

16. A host cell containing polynucleotides selected from the group consisting of (1) to (13) below: (1) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8. (2) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (3) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (4) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (5) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (6) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18. (7) A polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20. (8) Polynucleotides containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and polynucleotides containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (9) Polynucleotides containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and polynucleotides containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (10) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (11) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (12) A polynucleotide containing a base sequence encoding the heavy chain variable region consisting of amino acids numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding the light chain variable region consisting of amino acids numbered 1 to 110 of sequence number 18; and (13) A polynucleotide containing a base sequence encoding a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and a polynucleotide containing a base sequence encoding a light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

17. A method for producing an anti-MerTK antibody or an antigen-binding fragment thereof, comprising the steps of culturing a host cell as described in claim 15 or 16 and expressing the anti-MerTK antibody or an antigen-binding fragment thereof.

18. A pharmaceutical composition comprising the anti-MerTK antibody or its antigen-binding fragment as described in any one of claims 1 to 10, and a pharmaceutically acceptable excipient.

19. The pharmaceutical composition according to claim 18, for the prevention and / or treatment of eye diseases.

20. The pharmaceutical composition according to claim 19, wherein, The eye disease is a retinal disease.

21. The pharmaceutical composition according to claim 20, wherein, Retinal diseases are degenerative diseases of the retina.

22. The pharmaceutical composition according to claim 21, wherein, Degenerative retinal diseases include retinitis pigmentosa or age-related macular degeneration.

23. The anti-MerTK antibody or its antigen-binding fragment according to any one of claims 1 to 10, for the prevention and / or treatment of eye diseases.

24. A method for preventing and / or treating an eye disease, comprising the step of administering a therapeutically effective amount of the anti-MerTK antibody or its antigen-binding fragment as described in any one of claims 1 to 10 to a subject.

25. The use of the anti-MerTK antibody or its antigen-binding fragment as described in any one of claims 1 to 10 in the manufacture of a pharmaceutical composition for the prevention and / or treatment of eye diseases.

26. A bispecific antibody that binds to MerTK and misfolded proteins and / or amyloid proteins, comprising a heavy chain variable region and a light chain variable region of an anti-MerTK antibody, and a heavy chain variable region and a light chain variable region of an antibody that binds to misfolded proteins and / or amyloid proteins, wherein the bispecific antibody, The heavy chain variable region of the anti-MerTK antibody contains CDR1, which consists of the amino acid sequence of sequence number 1; CDR2, which consists of the amino acid sequence of sequence number 2; and CDR3, which consists of the amino acid sequence of sequence number 3. The light chain variable region of the anti-MerTK antibody contains CDR1, which consists of the amino acid sequence of sequence number 4, CDR2, which consists of the amino acid sequence of sequence number 5, and CDR3, which consists of the amino acid sequence of sequence number 6.

27. The bispecific antibody according to claim 26, wherein, The heavy chain variable region and light chain variable region of the anti-MerTK antibody are selected from the following groups (1) to (6): (1) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 10. (2) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 12. (3) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 109 of sequence number 14. (4) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 16. (5) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 18; and (6) The heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 8 and the light chain variable region consisting of amino acid sequences numbered 1 to 110 of sequence number 20.

28. The bispecific antibody according to claim 27, wherein, The antibody comprises a single-armed antibody (single-armed anti-MerTK antibody), the single-armed antibody comprising a heavy chain fragment containing a heavy chain variable region of the anti-MerTK antibody, a light chain containing a light chain variable region of the anti-MerTK antibody, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide.

29. The bispecific antibody according to claim 28, wherein, The heavy chain fragment and light chain of the single-arm anti-MerTK antibody are selected from the following groups (1) to (6): (1) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 10. (2) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 12. (3) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 14. (4) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 16. (5) A heavy chain segment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 18; and (6) A heavy chain fragment consisting of amino acid sequences numbered 1 to 217 of sequence number 8 and a light chain consisting of amino acid sequences numbered 20.

30. The bispecific antibody according to claim 29, wherein, The second Fc polypeptide of the single-arm anti-MerTK antibody has a C-terminus of the heavy chain variable region and light chain variable region of the antibody that binds to misfolded proteins and / or amyloid proteins connected by a hinge region to the N-terminus.

31. The bispecific antibody according to any one of claims 26 to 30, wherein, Antibodies that bind to misfolded proteins and / or amyloid proteins are anti-TTR antibodies that bind to misfolded TTRs.

32. The bispecific antibody according to claim 31, wherein, The heavy chain variable region of the anti-TTR antibody contains CDR1, consisting of the amino acid sequence of sequence number 27, CDR2, consisting of the amino acid sequence of sequence number 28, and CDR3, consisting of the amino acid sequence of sequence number 29. The light chain variable region of the anti-TTR antibody contains CDR1, which consists of the amino acid sequence of sequence number 30, CDR2, which consists of the amino acid sequence of sequence number 31, and CDR3, which consists of the amino acid sequence of sequence number 32.

33. The bispecific antibody according to claim 32, wherein, The heavy chain variable region of the anti-TTR antibody is a heavy chain variable region consisting of amino acid sequences numbered 1 to 119 of sequence number 43. The light chain variable region of the anti-TTR antibody is a light chain variable region consisting of amino acid sequences numbered 135 to 245 of sequence number 43.

34. The bispecific antibody according to claim 33, wherein, scFv containing heavy chain variable regions and light chain variable regions with anti-TTR antibodies (anti-TTR-scFv).

35. The bispecific antibody according to claim 34, wherein, Anti-TTR-scFv is composed of the amino acid sequence of sequence number 43.

36. The bispecific antibody according to claim 34 or 35, wherein, The antibody comprises a single-armed antibody (single-armed anti-MerTK antibody), the single-armed antibody comprising a heavy chain fragment containing a heavy chain variable region of the anti-MerTK antibody and a light chain containing a light chain variable region of the anti-MerTK antibody, and an Fc region composed of a first Fc polypeptide and a second Fc polypeptide, wherein the C-terminus of the anti-TTR-scFv is connected to the N-terminus of the second Fc polypeptide of the single-armed anti-MerTK antibody via a hinge region.

37. The bispecific antibody according to any one of claims 28 to 36, wherein, The Fc region contains amino acid mutations containing L234A and L235A (LALA mutation) and P331G mutation (here, the mutation location is the amino acid position according to the EU index in the human Igγ1 constant region).

38. The bispecific antibody according to claim 37, wherein, The first Fc polypeptide and the second Fc polypeptide contain an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 39, or have 1 to 10 substituted amino acid sequences in the amino acid sequence of sequence number 39.

39. The bispecific antibody according to claim 38, wherein, It contains the Fc region with the club-and-mortar mutation.

40. The bispecific antibody according to claim 38, wherein, It contains the Fc region containing LALA mutation, P331G mutation and mortis mutation.

41. The bispecific antibody according to claim 39 or 40, wherein, The mortar mutation is the T366W mutation in one Fc polypeptide that forms the Fc region and the T366S, L368A and Y407V mutations in another Fc polypeptide that forms the Fc region (here, the mutation positions are the amino acid positions according to the EU index in the human Igγ1 constant region).

42. The bispecific antibody according to claim 38, wherein, The sequences of the first Fc polypeptide and the second Fc polypeptide are polypeptides composed of any one of the following sequences (1) or (2): (1) A first Fc polypeptide consisting of the amino acid sequence of sequence number 40 and a second Fc polypeptide consisting of the amino acid sequence of sequence number 41; or (2) The first Fc polypeptide consisting of the amino acid sequence of sequence number 41 and the second Fc polypeptide consisting of the amino acid sequence of sequence number 40.

43. The bispecific antibody according to claim 42, wherein, It contains a hinge region consisting of the amino acid sequence shown in sequence number 42 and / or a hinge region consisting of the amino acid sequence shown in sequence number 70.

44. The bispecific antibody according to any one of claims 26 to 43, wherein it has undergone post-translational modification.

45. A fusion or complex of any bispecific antibody according to any one of claims 26 to 44, or a cell on which the bispecific antibody according to any one of claims 26 to 44 is presented on its cell surface.

46. ​​A polynucleotide for producing the bispecific antibody according to any one of claims 26 to 44, said polynucleotide being selected from the group consisting of (1) to (9): (1) A polynucleotide containing a base sequence of the heavy chain variable region encoding an amino acid sequence consisting of amino acids numbered 1 to 119 of sequence number 8 for an anti-MerTK antibody. (2) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 10. (3) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of the amino acid sequence number 12. (4) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 109 of amino acid sequence number 14. (5) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 16. (6) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid sequence number 18. (7) A polynucleotide containing a base sequence encoding an anti-MerTK antibody consisting of an amino acid sequence numbered 1 to 110 of amino acid 20. (8) A polynucleotide containing a base sequence encoding the base sequence of the heavy chain variable region of an anti-TTR antibody consisting of amino acids numbered 1 to 119 of sequence number 43; and (9) A polynucleotide containing a base sequence of the light chain variable region encoding an anti-TTR antibody consisting of an amino acid sequence consisting of amino acid numbers 135 to 245 of sequence number 43.

47. An expression vector comprising the polynucleotide of claim 46.

48. A host cell that has been transformed with the expression vector of claim 47.

49. A host cell for producing the bispecific antibody according to any one of claims 27 to 44, said host cell containing a polynucleotide selected from the group consisting of (1) to (3): (1) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 16, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 43, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 135 to 245 of sequence number 43; (2) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 119 of sequence number 8; a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acids numbered 1 to 110 of sequence number 18; a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acids numbered 1 to 119 of sequence number 43; and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acids numbered 135 to 245 of sequence number 43; and (3) A polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 8, a polynucleotide containing a base sequence encoding the light chain variable region of an anti-MerTK antibody consisting of amino acid sequences numbered 1 to 110 of sequence number 20, a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 1 to 119 of sequence number 43, and a polynucleotide containing a base sequence encoding the light chain variable region of an anti-TTR antibody consisting of amino acid sequences numbered 135 to 245 of sequence number 43.

50. A method for producing a bispecific antibody, comprising the steps of culturing a host cell as described in claim 48 or 49 to express a bispecific antibody that binds to MerTK and TTR.

51. A pharmaceutical composition comprising the bispecific antibody as described in any one of claims 26 to 44 and a pharmaceutically acceptable excipient.

52. The pharmaceutical composition according to claim 51, for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

53. The pharmaceutical composition according to claim 52, wherein, Amyloidosis is a disease caused by the accumulation of misfolded proteins.

54. The pharmaceutical composition according to claim 53, wherein, Amyloidosis is a systemic form of amyloidosis.

55. The pharmaceutical composition according to claim 54, wherein, Systemic amyloidosis is classified as TTR amyloidosis or polyneuropathy amyloidosis.

56. The bispecific antibody according to any one of claims 26 to 44, used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

57. A method for preventing and / or treating a disease caused by the accumulation of misfolded proteins, comprising the step of administering a therapeutically effective amount of the bispecific antibody according to any one of claims 26 to 44 to a subject.

58. Use of the bispecific antibody according to any one of claims 26 to 44 in the manufacture of a pharmaceutical composition for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

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