Anti-mertk agonist antibody, Anti-mertk bispecific antibody, and use thereof

Abstract

The present invention addresses the problem of providing an anti-MerTK antibody and an anti-MerTK bispecific antibody, capable of being used for preventing or treating diseases associated with phagocytosis.　The present inventors have found a novel anti-MerTK agonist antibody by searching for an anti-MerTK agonist antibody that binds to MerTK and activates a downstream signal. The anti-MerTK agonist antibody activated a function of phagocytic clearance of RPE cells by activating a downstream signal via MerTK expressed on surfaces of the RPE cells. The anti-MerTK-anti-misfolded protein bispecific antibody produced on the basis of the sequence of the anti-MerTK agonist antibody activated a function of phagocytic clearance of a misfolded protein by macrophages. Accordingly, the anti-MerTK antibody or the anti-MerTK-anti-misfolded protein bispecific antibody is expected to be used for preventing or treating diseases associated with phagocytosis.

Description

Anti-MerTK agonist antibodies, anti-MerTK bispecific antibodies, and uses thereof

[0001] The present invention relates to novel anti-MerTK agonist antibodies, anti-MerTK bispecific antibodies, and uses thereof.

[0002] The rapid engulfment and removal of apoptotic cells and cell-derived debris by phagocytes such as macrophages is called efferocytosis, and is recognized as an extremely important mechanism for maintaining tissue and immune homeostasis ( Nat. Rev. Mol. Cell Biol., 2020, 21:398-414 ). For example, in arteriosclerotic lesions, macrophages recognize and rapidly clear their own apoptotic cells, thereby contributing to the suppression of inflammatory responses and the progression of the lesion ( Nature, 2016, 536:86-90 ).

[0003] Mer Tyrosine Kinase (MerTK), a member of the TAM (Tyro3, ​​Axl, and MerTK) receptor tyrosine kinase family, is expressed in phagocytes such as macrophages and is known to function as one of the key molecules in efferocytosis. MerTK is a single-pass transmembrane protein with two extracellular immunoglobulin-like domains and two fibronectin type III domains, and one intracellular tyrosine kinase domain. Growth arrest specific gene 6 (Gas6), Protein S (ProS), Tubby, and Tubby-like protein 1 (TULP-1) have been identified as its ligands. Among these, Gas6 and ProS are ligands that act as bridges between phosphatidylserine (PtdSer) exposed on the surface of apoptotic cells and the immunoglobulin-like domain of MerTK on phagocytes. Through these ligands, MerTK binds to apoptotic cells and transmits downstream activation signals (tyrosine autophosphorylation) required for efferocytosis and other processes (Nat. Rev. Mol. Cell Biol., 2020, 21:398-414; Cold Spring Herb. Perspect. Biol., 2013, 5:a009076; EMBO J., 2010, 29:3898-3910). Furthermore, MerTK has been suggested to promote anti-inflammatory functions through the suppression of inflammatory cytokine production mediated by Toll-like receptors in dendritic cells (Cell, 2007, 131: 1124-1136) and the induction of the synthesis of inflammation-resolving mediators from macrophages (Sci. Signal., 2018, 11: ear3721).

[0004] MerTK is also expressed in retinal pigment epithelium (RPE) cells. Activation of MerTK-mediated downstream signals within the cell causes RPE cells to phagocytose photoreceptor outer segments (POS), which become debris after aging and detachment. RPE cells are located in contact with POS. PtdSer is exposed at the tip of POS, and RPE cells phagocytose and remove POS by recognizing PtdSer via MerTK and its ligand. This phagocytic function is extremely important for maintaining the homeostasis and function of photoreceptors and the retina (Front. Immunol., 2020, 11:604205).

[0005] Retinitis pigmentosa (RPE) is a genetic disease caused by mutations in genes that affect the function and homeostasis of photoreceptors or the RPE. More than 80 causative genes have been identified. The majority of causative genes originate in photoreceptors, and mutations induce cell death, leading to retinal tissue degeneration. Furthermore, dead cells and their debris induce an inflammatory state, worsening symptoms. Rapid phagocytic removal of dead cells and debris by MerTK activation has been suggested as a potential treatment for RPE (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 (for example, retinitis pigmentosa and age-related macular degeneration), development is underway to suppress retinal damage through gene therapy targeting the causative gene and the use of pharmacological agents such as angiogenesis inhibitors and complement inhibitors (Prog. Retin. Eye Res., 2018, 63: 107-130). However, no satisfactory treatment has yet been found, and further research and development into drug discovery is required.

[0007] MerTK activation requires tyrosine autophosphorylation of the intracellular kinase domain. It has been suggested that anti-MerTK antibodies selected based on the phosphorylation of the intracellular kinase domain of MerTK and its downstream molecule, AKT, may be able to cure retinal degeneration by activating MerTK-mediated downstream signal transduction and removing debris (Patent Document 4). However, although the existence of anti-MerTK antibodies that activate phagocytosis via MerTK on macrophages has been suggested (Patent Documents 1 to 4, Non-Patent Document 1), no anti-MerTK antibodies have been reported to date that have actually activated the phagocytosis of RPE cells.

[0008] Furthermore, in recent years, research has been conducted on bispecific antibodies that bind to MerTK and phagocytic food, with the aim of activating efficient phagocytic removal of phagocytic food by shortening the physical distance between phagocytes and phagocytic food. To date, for example, bispecific antibodies that bind to MerTK and CD20, bispecific antibodies that bind to MerTK and amyloid beta (Aβ) (Non-Patent Document 2), and bispecific antibodies that bind to MerTK and epidermal growth factor receptor (EGFR), a tumor-associated antigen (TAA) on cancer cells (Non-Patent Document 3) have been reported, and it has been reported that bispecific antibodies can activate the phagocytic removal function mediated by MerTK.

[0009] Transthyretin (TTR) is a protein consisting of 127 amino acids and having a molecular weight of 14 kDa. TTR is primarily produced in the liver, but is also produced in the ventricular choroid plexus, retinal pigment epithelial cells in the retina, pancreatic α cells, and other organs. TTR normally forms a stable tetrameric structure in blood and functions primarily in blood and cerebrospinal fluid as a transporter of vitamin A-retinol-binding protein complexes and the thyroid hormone T4 (Patent Document 5). TTR is one of the proteins that causes systemic amyloidosis caused by hereditary mutations and aging, and it is known that misfolded TTR deposition in organs throughout the body causes functional disorders. It has been suggested that antibodies against misfolded TTR may activate the phagocytosis of misfolded TTR by phagocytes via their Fc region, thereby inhibiting the deposition of misfolded TTR (Amyloid, 2016, 23:86-97). However, in a phase I clinical trial of NI006, a monoclonal anti-TTR IgG1 antibody that binds to misfolded TTR, in addition to the inhibitory effect on misfolded TTR deposition, the onset of cytokine release syndrome was also reported in several test patients (Non-Patent Document 4). Therefore, the development of additional drugs for the treatment of systemic amyloidosis such as TTR amyloidosis is desired.

[0010] International Publication No. WO 2016 / 106221 International Publication No. WO 2019 / 005756 International Publication No. WO 2020 / 176497 International Publication No. WO 2021 / 202590 International Publication No. WO 2015 / 115332

[0011] PLoS ONE, 2015, 10: e0145078mAbs, 2020, 12: 1685832 Int. J. Mol. Sci. , 2022, 23:15673N. Engl. J. Med. , 2023, 389: 239-250

[0012] An object of the present invention is to provide an anti-MerTK agonist antibody and an anti-MerTK bispecific antibody that can be used for the prevention or treatment of diseases associated with phagocytosis.

[0013] The present inventors conducted extensive research to develop an anti-MerTK antibody that binds to MerTK and activates the phagocytic clearance function of phagocytes via MerTK. As a result, they isolated 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 further activated the phosphorylation of the intracellular kinase domain of MerTK and the downstream phosphorylation signaling of AKT in the RPE in vivo (Example 3). Furthermore, an anti-MerTK-anti-TTR bispecific antibody containing a single-arm antibody containing the heavy and light chain variable regions of tA3-20 activated the phagocytic activity of misfolded TTR via MerTK (Example 6) and significantly activated the phagocytic clearance function of the same target in aged mice (Example 7). An anti-MerTK-anti-immunoglobulin light chain (IgL) bispecific antibody comprising a single-arm antibody containing the heavy chain variable region and light chain variable region of tA3-20 bound to human MerTK and misfolded IgL in an antibody concentration-dependent manner and activated their phagocytic clearance function (Example 10). Thus, the present inventors have provided an anti-MerTK agonist antibody and an anti-MerTK bispecific antibody that bind to MerTK and activate the MerTK-mediated phagocytic clearance function of phagocytes, thereby completing the present invention.

[0014] That is, the present invention relates to the following [1] to

[86] , but is not limited thereto: [1] An anti-MerTK antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6. [2] The anti-MerTK antibody or antigen-binding fragment thereof according to [1], comprising a heavy chain variable region and a light chain variable region selected from the group consisting of the following (1) to (6): (1) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; and (6) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20. [3] The anti-MerTK antibody of [1] or [2], comprising a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region. [4] The anti-MerTK antibody of [3], comprising amino acid mutations L234A and L235A (LALA mutations) in the heavy chain (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).[5] The anti-MerTK antibody according to [3], which comprises a P331G mutation in the heavy chain (wherein the mutation position is an amino acid position according to the EU index in the human Igγ1 constant region). [6] The anti-MerTK antibody according to [3], which comprises L234A and L235A amino acid mutations (LALA mutations) in the heavy chain and a P331G mutation (wherein the mutation position is an amino acid position according to the EU index in the human Igγ1 constant region). [7] An anti-MerTK antibody according to [6], selected from the group consisting of the following (1) to (6): (1) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 20. [8] The anti-MerTK antibody according to any one of [1] to [7], which is an IgG antibody (anti-MerTK IgG antibody). [9] The antigen-binding fragment is a single-chain variable region fragment (scFv), a Fab fragment, a Fab' fragment, or an F(ab'). ２

[10] The anti-MerTK antibody or antigen-binding fragment thereof according to any one of [1] to [9], which is a post-translationally modified antibody.

[11] A fusion or complex of the anti-MerTK antibody or antigen-binding fragment thereof according to any one of [1] to

[10] , or a cell displaying on its surface the anti-MerTK antibody or antigen-binding fragment thereof according to any one of [1] to

[10] .

[12] A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-MerTK antibody or an antigen-binding fragment thereof, selected from the group consisting of (1) to (7) below: (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18; and (7) A polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20.

[13] A polynucleotide comprising a nucleotide sequence encoding a heavy chain or light chain of an anti-MerTK antibody selected from the group consisting of the following (1) to (7): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 14;(5) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (7) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[14] An expression vector comprising the polynucleotide according to

[12] or

[13] .

[15] A host cell transformed with the expression vector according to

[14] .

[16] A host cell comprising a polynucleotide selected from the group consisting of (1) to (13) below: (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18; (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20; (8) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (9) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12;(10) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14; (11) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16; (12) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18; and (13) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20.

[17] A method for producing an anti-MerTK antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cell of

[15] or

[16] and expressing the anti-MerTK antibody or antigen-binding fragment thereof.

[18] A pharmaceutical composition comprising the anti-MerTK antibody or antigen-binding fragment thereof of any one of [1] to

[10] and a pharmaceutically acceptable excipient.

[19] The pharmaceutical composition of

[18] , for use in the prevention and / or treatment of an ocular disease.

[20] The pharmaceutical composition of

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

[21] The pharmaceutical composition of

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

[22] The pharmaceutical composition of

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

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

[10] , for use in the prevention and / or treatment of an ocular disease.

[24] A method for preventing or treating an eye disease, comprising the step of administering a therapeutically effective amount of the anti-MerTK antibody or antigen-binding fragment thereof according to any one of [1] to

[10] to a subject.

[25] Use of the anti-MerTK antibody or antigen-binding fragment thereof according to any one of [1] to

[10] in the manufacture of a pharmaceutical composition for the prevention and / or treatment of an eye disease.

[26] A bispecific antibody that binds to MerTK and misfolded proteins and / or amyloid, comprising the heavy chain variable region and light chain variable region of an anti-MerTK antibody and the heavy chain variable region and light chain variable region of an antibody that binds to misfolded proteins and / or amyloid, wherein the heavy chain variable region of the anti-MerTK antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and the light chain variable region of the anti-MerTK antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

[27] The bispecific antibody according to

[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) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 18; and (6) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20.

[28] The bispecific antibody according to

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

[29] The bispecific antibody according to

[28] , wherein the heavy chain fragment and the light chain of the single-armed anti-MerTK antibody are selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[30] The bispecific antibody according to

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

[31] The bispecific antibody according to any of

[26] to

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

[32] The bispecific antibody according to

[31] , wherein the heavy chain variable region of the anti-TTR antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO: 27, CDR2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 29,A bispecific antibody, wherein the light chain variable region of the anti-FTR antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO: 30, CDR2 consisting of the amino acid sequence of SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32.

[33] The bispecific antibody of

[32] , wherein the heavy chain variable region of the anti-FTR antibody comprises the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43, and the light chain variable region of the anti-FTR antibody comprises the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43.

[34] The bispecific antibody of

[33] , comprising an scFv (anti-FTR-scFv) comprising the heavy chain variable region and light chain variable region of the anti-FTR antibody.

[35] The bispecific antibody of

[34] , wherein the anti-FTR-scFv consists of the amino acid sequence of SEQ ID NO: 43.

[36] The bispecific antibody according to

[34] or

[35] , comprising a single-armed antibody (single-armed anti-MerTK antibody) comprising a heavy-chain fragment comprising the heavy-chain variable region of an anti-MerTK antibody, a light chain comprising the light-chain variable region of an anti-MerTK antibody, and an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide, wherein the C-terminus of the anti-TTR-scFv is linked 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 of

[28] to

[36] , comprising an Fc region comprising amino acid mutations L234A and L235A (LALA mutations) and a P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[38] The bispecific antibody of

[37] , wherein the first Fc polypeptide and the second Fc polypeptide comprise an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having 1 to 10 substitutions in the amino acid sequence of SEQ ID NO: 39.

[39] The bispecific antibody of

[38] , comprising an Fc region comprising knobs-into-holes mutations.

[40] The bispecific antibody of

[38] , comprising an Fc region comprising a LALA mutation, a P331G mutation, and a knobs-into-holes mutation.

[41] The bispecific antibody of

[39] or

[40] , wherein the knobs-into-holes mutations are 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 (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[42] The bispecific antibody of

[38] , wherein the sequences of the first Fc polypeptide and the second Fc polypeptide are polypeptides consisting of either the following sequences (1) or (2): (1) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41; or (2) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40.

[43] The bispecific antibody of

[42] , comprising a hinge region consisting of the amino acid sequence shown in SEQ ID NO: 42 and / or a hinge region consisting of the amino acid sequence shown in SEQ ID NO: 70.

[44] The bispecific antibody according to any one of

[26] to

[43] , which is post-translationally modified.

[45] A fusion or complex of the bispecific antibody according to any one of

[26] to

[44] , or a cell displaying the bispecific antibody according to any one of

[26] to

[44] on its surface.

[46] A polynucleotide selected from the group consisting of the following (1) to (9) for use in producing the bispecific antibody according to any one of

[26] to

[44] : (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14;(5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20; (8) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43; and (9) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43.

[47] An expression vector comprising the polynucleotide of

[46] .

[48] A host cell transformed with the expression vector of

[47] .

[49] A host cell containing a polynucleotide selected from the group consisting of the following (1) to (3), used for producing the bispecific antibody according to any one of

[27] to

[44] : (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43;(2) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43; and (3) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43.

[50] A method for producing a bispecific antibody that binds to MerTK and FTR, comprising the step of culturing the host cell according to

[48] or

[49] to express the bispecific antibody.

[51] A pharmaceutical composition comprising the bispecific antibody according to any of

[26] to

[44] and a pharmaceutically acceptable excipient.

[52] The pharmaceutical composition according to

[51] , for use in the prevention and / or treatment of a disease caused by the accumulation of misfolded proteins.

[53] The pharmaceutical composition according to

[52] , wherein the disease derived from the accumulation of misfolded proteins is amyloidosis.

[54] The pharmaceutical composition according to

[53] , wherein the amyloidosis is systemic amyloidosis.

[55] The pharmaceutical composition according to

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

[56] The bispecific antibody according to any of

[26] to

[44] , for use in the prevention and / or treatment of a disease derived from 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 to a subject a therapeutically effective amount of the bispecific antibody of any of

[26] to

[44] .

[58] Use of the bispecific antibody of any of

[26] to

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

[59] The bispecific antibody of any of

[26] to

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

[60] The bispecific antibody according to

[59] , wherein the heavy chain variable region of the anti-IgL antibody comprises a CDR1 consisting of the amino acid sequence of amino acids 26 to 35 of SEQ ID NO: 57, a CDR2 consisting of the amino acid sequence of amino acids 50 to 68 of SEQ ID NO: 57, and a CDR3 consisting of the amino acid sequence of amino acids 101 to 108 of SEQ ID NO: 57, and the light chain variable region of the anti-IgL antibody comprises a CDR1 consisting of the amino acid sequence of amino acids 24 to 39 of SEQ ID NO: 59, a CDR2 consisting of the amino acid sequence of amino acids 55 to 61 of SEQ ID NO: 59, and a CDR3 consisting of the amino acid sequence of amino acids 94 to 103 of SEQ ID NO: 59.

[61] The bispecific antibody according to

[60] , wherein the heavy chain variable region of the anti-IgL antibody consists of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 57, and the light chain variable region of the anti-IgL antibody consists of the amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 59.

[62] The bispecific antibody according to

[61] , which comprises a Fab fragment (anti-IgL-Fab) comprising the heavy chain variable region and light chain variable region of an anti-IgL antibody.

[63] The bispecific antibody according to

[62] , wherein the anti-IgL-Fab comprises a fragment consisting of a heavy chain variable region (VH) and a light chain constant region (CL) comprising the amino acid sequence of amino acids 1 to 226 of SEQ ID NO: 57, and a fragment consisting of a light chain variable region (VL) and a CH1 domain comprising the amino acids of SEQ ID NO: 59.

[64] The bispecific antibody according to

[62] or

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

[65] The bispecific antibody according to any of

[59] to

[64] , comprising an Fc region comprising amino acid mutations L234A and L235A (LALA mutations) and a P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[66] The bispecific antibody of

[65] , wherein the first Fc polypeptide and the second Fc polypeptide comprise an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having 1 to 10 substitutions in the amino acid sequence of SEQ ID NO: 39.

[67] The bispecific antibody of

[66] , comprising an Fc region comprising knobs-into-holes mutations.

[68] The bispecific antibody of

[66] , comprising an Fc region comprising a LALA mutation, a P331G mutation, and knobs-into-holes mutations.

[69] The bispecific antibody of

[67] or

[68] , wherein the knobs-into-holes mutations are 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 (wherein the mutation positions are amino acid positions according to the EU index in a human Igγ1 constant region).

[70] The bispecific antibody according to

[66] , wherein the sequences of the first Fc polypeptide and the second Fc polypeptide are polypeptides consisting of either the following sequences (1) or (2): (1) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41; or (2) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40.

[71] The bispecific antibody according to

[70] , which comprises a hinge region consisting of the amino acid sequence shown in SEQ ID NO: 42.

[72] The bispecific antibody according to any one of

[59] to

[71] , which is post-translationally modified.

[73] A fusion or complex of the bispecific antibody according to any one of

[59] to

[72] , or a cell displaying the bispecific antibody according to any one of

[59] to

[72] on its surface.

[74] A polynucleotide selected from the group consisting of (1) to (9) below, used for producing the bispecific antibody according to any one of

[59] to

[72] : (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20; (8) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 57; and (9) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 59.

[75] An expression vector comprising the polynucleotide according to

[74] .

[76] A host cell transformed with the expression vector according to

[75] .

[77] A host cell used for producing the bispecific antibody according to any one of

[59] to

[72] , comprising a polynucleotide selected from the group consisting of the following (1) to (4): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59; (2) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59; (3) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59; and(4) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 59.

[78] A method for producing a bispecific antibody that binds to MerTK and IgL, comprising the step of culturing the host cell according to

[76] or

[77] to express the bispecific antibody.

[79] A pharmaceutical composition comprising the bispecific antibody according to any of

[59] to

[72] and a pharmaceutically acceptable excipient.

[80] The pharmaceutical composition according to

[79] , for use in the prevention and / or treatment of a disease caused by the accumulation of misfolded proteins.

[81] The pharmaceutical composition according to

[80] , wherein the disease resulting from the accumulation of misfolded proteins is amyloidosis.

[82] The pharmaceutical composition according to

[81] , wherein the amyloidosis is systemic amyloidosis.

[83] The pharmaceutical composition according to

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

[84] The bispecific antibody according to any of

[59] to

[72] , for use in the prevention and / or treatment of a disease resulting from the accumulation of misfolded proteins.

[85] A method for the prevention and / or treatment of a disease resulting from the accumulation of misfolded proteins, comprising the step of administering a therapeutically effective amount of the bispecific antibody according to any of

[59] to

[72] to a subject.

[86] Use of the bispecific antibody according to any of

[59] to

[72] in the manufacture of a pharmaceutical composition for the prevention and / or treatment of a disease resulting from the accumulation of misfolded proteins.

[0015] The anti-MerTK antibody or antigen-binding fragment thereof, and anti-MerTK bispecific antibody of the present invention bind to MerTK expressed in phagocytes and activate MerTK-mediated intracellular downstream signaling, thereby activating the phagocytic removal function of phagocytes. The anti-MerTK antibody or antigen-binding fragment thereof, anti-MerTK bispecific antibody of the present invention, or pharmaceutical compositions comprising them, can be used for the prevention or treatment of diseases that can be prevented or treated by MerTK-mediated activation of intracellular downstream signaling (e.g., eye diseases).

[0016] Figure 1 shows the binding activity of test antibodies to human MerTK-His protein, measured by ELISA. The vertical axis of the figure represents binding activity (the value 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. Symbols indicate the average absorbance for each test antibody. Figure 2-1 shows the effect of test antibodies on the phagocytosis of apoptotic cells by iPS-RPE cells. The vertical axis represents fluorescence intensity, which is an indicator of the amount of apoptotic cells phagocytosed. The horizontal axis represents the elapsed time from the start of evaluation. Each dot represents the average value for four wells. Figure 2-2 shows the effect of test antibodies on the phagocytosis of porcine POS by iPS-RPE cells. The vertical axis represents fluorescence intensity, an indicator of the amount of POS phagocytosed, integrated from the start of measurement up to 60 hours. Each dot represents the value for each well. Values ​​in the bar graph represent the average value. Figure 3-1 shows the phosphorylated MerTK protein level in the RPE of mice 1 hour after intravitreal administration of tA3-20. The vertical axis of the figure shows the amount of phosphorylated MerTK protein corrected for the total amount of MerTK protein, and the horizontal axis shows the dose of the test antibody. Each dot shows the value for each individual. The bars show the average value. The error bars show the standard deviation. Figure 3-2 shows the phosphorylated AKT protein level in the RPE of mice 1 hour after intravitreal administration of tA3-20. The vertical axis of the figure shows the amount of phosphorylated AKT protein corrected for the total amount of AKT protein, and the horizontal axis shows the dose of the test antibody. Each dot shows the value for each individual. The bars show the average value. The error bars show the standard deviation. Figure 4-1 shows the binding activity of the test antibodies to misTTR measured by ELISA. The vertical axis of the figure represents binding activity (the value 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 represent the average absorbance for each test antibody. Figure 4-2 shows the binding activity of test antibodies to human MerTK-Fc protein, measured by ELISA. The vertical axis of the figure represents binding activity (the value 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 represent the average absorbance for each test antibody. Figure 5 shows the MerTK-dependent phagocytosis of pHrodo-misTTR by tA-009. The vertical axis of the figure represents the total amount of phagocytosis during the observation period ([p / s]·h). Values ​​represent the mean value + standard error.Comparisons between the vehicle control group and the tA-009 group, and between the vehicle control group and the tA-009 + ONO7475 group, were performed using Student's t-test, with P values ​​of less than 0.05 considered statistically significant. Asterisks (*) and sharp symbols (#) in the figures indicate significant differences between the vehicle group and the tA-009 group and between the tA-009 group and the tA-009 + ONO7475 group, respectively. Figure 6-1 shows the activation of the phagocytic clearance function of DL800-misTTR by tA-009 or mouse 371M. The vertical axis of the figure indicates the total remaining amount of misTTR ([p / s]·h) during the observation period. Values ​​represent the mean + standard error. Comparisons between the control vehicle group and the drug (mouse type 371M or tA-009) groups were performed using Dunnett's test, and P values ​​of less than 0.05 were considered statistically significant. * indicates a significant difference between the vehicle group and the drug. Figure 6-2 shows the activation of phagocytic clearance function by tA-009 or mouse type 371M for DL800-misTTR administered subcutaneously to aged mice. The vertical axis of the figure indicates the total remaining amount of misTTR during the observation period ([p / s]·h). Values ​​represent the mean + standard error. Comparisons between the control young mouse / vehicle group and the old mouse / vehicle group were performed using Student's t-test, and comparisons between the control old mouse / vehicle group and the old mouse / drug (mouse type 371M or tA-009) group were performed using Dunnett's test. P values ​​of less than 0.05 were considered statistically significant. * and # in the figures indicate significant differences between the young mouse / vehicle group and the old mouse / vehicle group, and between the old mouse / vehicle group and the old mouse / drug group, respectively. Figure 7 shows the binding activity of test antibodies to human MerTK and misfolded IgL protein measured by ELISA. The vertical axis of the figure represents binding activity (the value 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.

[0017] The present invention will be described in detail below.

[0018] <Definitions> Terms used in this specification are used in the sense commonly used by those skilled in the art unless otherwise defined below.

[0019] An antibody (or immunoglobulin) is a glycoprotein whose basic structure is a four-chain structure with a symmetric Y-shape, consisting 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 an antibody molecule is common to all classes, consisting of two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000, bound by disulfide bonds and non-covalent bonds to form an antibody molecule with a molecular weight of 150,000 to 190,000, consisting of a Y-shaped four-chain structure. The heavy chain is typically a polypeptide chain containing approximately 440 amino acids, and has a characteristic structure for each class, called Igγ, Igμ, Igα, Igδ, and Igε, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. IgG is further divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4. Light chains are typically polypeptide chains containing approximately 220 amino acids, and two types, λ and κ, are known, called Igλ and Igκ, respectively. The two types of light chains can pair with either type of heavy chain.

[0020] Antibody molecules have four intrachain disulfide bonds in heavy chains (five in Igμ and Igε) and two in light chains, forming a loop every 100-110 amino acid residues. The three-dimensional structures of these disulfide bonds are similar between each loop and are referred to as structural units or domains. The domain located at the amino terminus (also referred to herein as the "N-terminus") of both heavy and light chains is called the variable region. Even antibodies produced from the same class (or subclass) of the same animal species have diverse amino acid sequences, and are known to be involved in the binding specificity of the antibody to the antigen. The amino acid sequence of the domain downstream of the variable region at the carboxy terminus (also referred to herein as the "C-terminus") is nearly constant for each class or subclass and is referred to as the constant region. From the N-terminus to the C-terminus, the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is further divided into three domains from the N-terminus: CH1 domain, CH2 domain, and CH3 domain. Light chains have, from N- to C-terminus, a light chain variable region (VL) and a light chain constant region (CL).

[0021] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL vary greatly, contributing to the variability of the variable regions. The CDRs are regions consisting of approximately 5 to 10 amino acid residues located in the order of CDR1, CDR2, and CDR3 at the N-terminus of each heavy chain and light chain, and form the antigen-binding site. On the other hand, the portions of the variable regions other than the CDRs are called framework regions (FRs), and consist of FR1 to FR4, and show relatively little variation in amino acid sequence.

[0022] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two N-terminal fragments are called Fab (Fragment, antigen binding) regions. As used herein, "Fab region" refers to a region consisting of the VH and CH1 domains of the heavy chain and the light chain (VL and CL), and binds to an antigen at the antigen-binding site at the tip of the Fab region. As used herein, "heavy chain fragment" refers to a fragment consisting of the VH and CH1 domains of the heavy chain that constitute the Fab region. The C-terminal fragment is called Fc (Fragment, crystallizable) region. As used herein, "Fc polypeptide" refers to a polypeptide consisting of the CH2 and CH3 domains of the heavy chain, and "Fc region" refers to a complex consisting of two Fc polypeptides, a first Fc polypeptide and a second Fc polypeptide. The terms "first" and "second" used herein to refer to Fc polypeptides are used for convenience and distinction, and are not intended to confer any particular order or meaning.

[0023] As used herein, the term "hinge region" refers to a highly flexible peptide region located between the CH1 and CH2 domains of a heavy chain, and the heavy chain fragment and the Fc polypeptide are connected via the hinge region. Furthermore, the two heavy chains of an antibody are connected via disulfide bonds at the hinge region.

[0024] As used herein, the term "antigen" is used in its commonly used sense to refer to a molecule or a portion of a molecule to which an antibody or antigen-binding fragment can specifically bind. Antigens can be proteins, nucleic acids, or other molecules. A single antigen may have one or more epitopes that can interact with different antibodies, etc.

[0025] As used herein, the term "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 an IgG antibody contain identical VH and VL sequences.

[0026] As used herein, the term "antigen-binding fragment" refers to a molecule that contains a heavy chain variable region and a light chain variable region of an immunoglobulin molecule and at least one polypeptide chain that has antigen-binding activity. Representative antigen-binding fragments include single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, and F(ab') fragments. ２ Examples of such fragments include F(ab') and F(ab') fragments. An scFv is a monovalent antigen-binding fragment composed of a VH and VL linked by a linker. An Fab fragment is a monovalent antigen-binding fragment composed of a light chain and a fragment containing the VH and CH1 domains of the heavy chain. An Fab fragment contains the Fab region. An Fab' fragment is a monovalent antigen-binding fragment composed of a light chain and a fragment containing the VH and CH1 domains of the heavy chain and a portion of the hinge region, and this hinge region contains cysteine ​​residues that constituted the inter-heavy chain S-S bonds. F(ab') ２ A Fab fragment is a bivalent molecule in which Fab' fragments are linked by disulfide bonds. Monovalent means that it contains one antigen-binding site, and bivalent means that it contains two antigen-binding sites. Fab fragments, Fab' fragments, and F(ab') fragments are also known. ２ Fragments may also include fragments in which all or part of the VH1 and CH1 domains of the heavy chain and the VL and CL domains of the light chain are crossed over (mAbs, 2016, 8(6):1010-1020). Furthermore, "antigen-binding fragments" may also include heavy chain antibodies such as VHH antibodies (mAbs, 2017, 9(2):182-212).

[0027] As used herein, a "Half-Ig" is a monovalent antibody comprising one VH, one VL, and one Fc polypeptide. In one embodiment, a Half-Ig comprises one VH, one CH1 domain, one VL, and one Fc polypeptide. In one embodiment, a Half-Ig comprises one heavy chain fragment (VH and CH1 domain), one Fc polypeptide (CH2 domain and CH3 domain), and one light chain (VL and CL). In one embodiment, a Half-Ig comprises one heavy chain and one light chain.

[0028] As used herein, a "single-armed antibody" refers to a monovalent antibody comprising one VH, one CH1 domain, one VL, one CL, and one Fc region consisting of a first Fc polypeptide and a second Fc polypeptide. In one embodiment, a single-armed antibody comprises one heavy chain fragment (VH and CH1 domain), one Fc region (first and second Fc polypeptides (CH2 domain and CH3 domain)), and one light chain (VL and CL). In one embodiment, a single-armed antibody comprises one heavy chain fragment, a hinge region, one Fc region, and one light chain. In one embodiment, a single-armed antibody comprises one Fab region, a hinge region, and one Fc region. In one embodiment, a single-armed antibody comprises one Fab region, a hinge region, and one Fc region, and has a structure in which the heavy chain fragment of the Fab region is linked to the first Fc polypeptide of the Fc region via the hinge region. In one embodiment, a single-arm antibody has a structure (one heavy chain) comprising one Fab region, a hinge region, and an Fc region, in which the heavy chain fragment of the Fab region is linked via the hinge region to the N-terminus of a first Fc polypeptide of the Fc region.

[0029] As used herein, the term "bispecific antibody" refers to an antibody that can specifically bind to two different antigens. A bispecific antibody is composed of two antibodies or antigen-binding fragments, each capable of binding to a different antigen, and bispecific antibodies with various structures are known to those skilled in the art (mAbs, 2017, 9(2):182-212).

[0030] As used herein, the term "multispecific antibody" refers to an antibody that can specifically bind to three or more different antigens, and is called, for example, a trispecific antibody or a tetraspecific antibody depending on the number of antigens to which it binds. A multispecific antibody is composed of three or more antibodies and / or antigen-binding fragments, each capable of binding to a different antigen.

[0031] As used herein, the term "antibody" includes IgG antibodies, half-Igs, single-arm antibodies, bispecific antibodies, and multispecific antibodies, unless otherwise limited by the context.

[0032] As used herein, the term "human antibody" refers to an antibody having a human immunoglobulin amino acid sequence. As used herein, the term "fully human antibody" refers to an antibody consisting solely of a human immunoglobulin amino acid sequence. As used herein, the term "mouse-human chimeric antibody" refers to an antibody having an antigen-binding variable region of a mouse immunoglobulin amino acid sequence and a constant region of a human immunoglobulin amino acid sequence.

[0033] The amino acid residue numbers of antibodies used herein can be specified according to the Kabat numbering system or the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, NIH Publication: No. 91-3242) by designating the numbering system.

[0034] As used herein, the term "subject" refers to a human or other animal in need of disease prevention or treatment. In one embodiment, the subject is a human in need of disease prevention or treatment. In one embodiment, the subject is a human in need of disease prevention or treatment, such as various eye diseases, lung diseases, autoimmune diseases, neurological disorders, heart diseases, endocrine / metabolic diseases, cancer, etc.

[0035] In this specification, "diagnosis" is not limited to determining whether or not a disease is present, but also encompasses all types of diagnosis, such as determining the severity (progression) after a definitive diagnosis, or determining whether or not there is a high possibility of future disease or onset.

[0036] As used herein, "prophylaxis" refers to any intervention carried out to prevent or delay the onset of a disease.

[0037] As used herein, "treatment" refers to any intervention, procedure performed on a subject or administration of an active ingredient to a subject for the purpose of reversing, alleviating, ameliorating, suppressing or delaying the progression, onset, severity or recurrence of symptoms of a disease, a pathological condition or biochemical signs associated with a disease.

[0038] As used herein, the term "active ingredient" refers to a substance contained in a pharmaceutical composition, drug, etc. used for the prevention or treatment of a disease, which exhibits some kind of physiological activity. In one embodiment, the active ingredient is an antibody or an antigen-binding fragment. In one embodiment, the active ingredient is a fusion or conjugate of an antibody or antigen-binding fragment. In one embodiment, the active ingredient is a bispecific antibody.

[0039] As used herein, the term "pharmaceutical composition" refers to a drug that contains an active ingredient and a pharmaceutically acceptable excipient (including, but not limited to, pharmaceutical excipients and pharmaceutical carriers) and is prescribed for the purpose of preventing or treating a subject.

[0040] As used herein, the term "effective amount" of a drug refers to the amount of drug required to cause a physiological change in a subject by acting on the tissues or cells of the subject when the drug is administered to the subject.

[0041] <Anti-MerTK Antibodies of the Present Invention> As used herein, an anti-MerTK antibody or an antigen-binding fragment thereof refers to an antibody or an antigen-binding fragment thereof that can bind to human MerTK. Whether or not an antibody binds to human MerTK can be confirmed using a known method for measuring binding activity. For example, the enzyme-linked immunosorbent assay (ELISA) method described in Examples 1-2 or 1-3 can be used to measure binding activity.

[0042] The present invention provides the following anti-MerTK antibody or antigen-binding fragment thereof: An anti-MerTK antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6. Herein, this anti-MerTK antibody is also referred to as the "anti-MerTK antibody of the present invention," and this anti-MerTK antibody or antigen-binding fragment thereof is also referred to as the "anti-MerTK antibody of the present invention or antigen-binding fragment thereof."

[0043] In one embodiment, the anti-MerTK antibody or antigen-binding fragment thereof of the present invention is an anti-MerTK antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region selected from the group consisting of the following (1) to (6): (1) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 18; and (6) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20.

[0044] In one embodiment, the anti-MerTK antibody of the present invention is an antibody comprising a heavy chain and a light chain. Antibodies comprising a heavy chain and a light chain 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).

[0045] In one embodiment, the antigen-binding fragment of the present invention is an scFv, a Fab fragment, a Fab' fragment, or a F(ab') ２ It is a fragment or a VHH antibody.

[0046] The heavy chain constant region contained in the anti-MerTK antibody or antigen-binding fragment thereof of the present invention can be any of Igγ, Igμ, Igα, Igδ, or Igε constant regions. Igγ can be selected from, for example, Igγ1, Igγ2, Igγ3, or Igγ4. The light chain constant region contained in the anti-MerTK antibody or antigen-binding fragment thereof of the present invention can be any of Igλ or Igκ constant regions. In one embodiment, the heavy chain constant region and light chain constant region of the anti-MerTK antibody or antigen-binding fragment thereof are human Igγ1 and Igλ constant regions, respectively.

[0047] The anti-MerTK antibodies of the present invention may contain mutations that reduce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). L234A is a substitution of leucine at amino acid position 234 in the human Igγ1 constant region with alanine. L235A is a substitution of leucine at amino acid position 235 in 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 reducing the binding activity of the antibody's Fc region to Fcγ receptors and C1q (Mol. Immunol., 1992, 29: 633-639; J. Immunol., 2000, 164: 4178-4184; Front. Immunol., 2019, 10: 1296). P329A is a substitution of proline at amino acid position 329 of the human Igγ1 constant region with alanine, and P331G or P331S is a substitution of proline at amino acid position 331 of the human Igγ1 constant region with glycine or serine. This mutation is known to reduce the antibody's CDC (J. Immunol., 2000, 164: 4178-4184).

[0048] In one embodiment, an anti-MerTK antibody of the invention comprises amino acid mutations L234A and L235A (LALA mutations) in the heavy chain. In one embodiment, an anti-MerTK antibody of the invention comprises either a P329A, P331G, or P331S mutation in the heavy chain. In one embodiment, an anti-MerTK antibody of the invention comprises an LALA mutation and either a P329A, P331G, or P331S mutation in the heavy chain. In one embodiment, an anti-MerTK antibody of the invention comprises an LALA mutation and a P329A mutation in the heavy chain. In one embodiment, an anti-MerTK antibody of the invention comprises an LALA mutation and a P331G mutation in the heavy chain.

[0049] In this specification, the descriptions of amino acid mutations such as the LALA mutation, P329A, P331G, and P331S mutation are based on the amino acid position in the human Igγ1 constant region according to the EU index. For example, as described above, L234A is a substitution of leucine with alanine at amino acid position 234 in the human Igγ1 constant region according to the EU index.

[0050] The anti-MerTK antibodies of the present invention may further include mutations based on other known techniques, such as the N297G mutation (Protein Cell, 2018, 9:63-73), a mutation based on the Knobs-into-Hole (Kih) technique (hereinafter also referred to as "Knobs-into-Hole mutation"), or an electrostatic steering mutation.

[0051] The kih technology involves replacing the amino acid side chains present in the CH3 region of one heavy chain with a larger side chain (knob) and replacing the amino acid side chains present in the CH3 region of the other heavy chain with a smaller side chain (hole), thereby positioning the knob within the cavity, promoting heterodimerization of the heavy chains and enabling efficient acquisition of the desired heterodimerized antibody molecules (Nature, 1994, 372: 379-383; Nature Biotech., 1998, 16: 677-681; J. Mol. Biol., 1997, 270: 26-35; Proc. Natl. Acad. Sci. U.S.A., 2013, 110: E2987-E2996, WO 1998 / 050431).

[0052] The electrostatic steering technique involves substituting an amino acid side chain present in the CH3 domain of one heavy chain with a negatively charged amino acid side chain and substituting an amino acid side chain present in the CH3 domain of the other heavy chain with a positively charged amino acid side chain (referred to herein as "electrostatic steering mutation"). This technique promotes heterodimerization of heavy chains using electrostatic force, thereby enabling efficient production of the desired heterodimerized antibody molecule (J. Biol. Chem., 2010, 285: 19637-19646). As electrostatic steering mutations in mouse-derived antibodies and mutations for stabilizing heterodimerized antibody molecules, mutations including E356K, T364S, M368L, T370K, D399K, and R411T in the CH3 domain of one heavy chain and T364S, M368L, T370K, K409E, R411T, and K439D in the CH3 region of the other heavy chain are known (mAbs, 2019, 12: 1-12).

[0053] In one embodiment, the anti-MerTK antibody of the present invention is an anti-MerTK antibody selected from the group consisting of the following (1) to (6): (1) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[0054] The anti-MerTK antibodies or antigen-binding fragments thereof of the present invention include antibodies or antigen-binding fragments thereof that bind to human MerTK as well as mouse, monkey, and / or rat MerTK. In one embodiment, the anti-MerTK antibodies or antigen-binding fragments thereof of the present invention are antibodies or antigen-binding fragments thereof that bind to human, mouse, monkey, and / or rat MerTK. In one embodiment, the anti-MerTK antibodies or antigen-binding fragments thereof of the present invention bind to human MerTK (gene ID: NM_006343). Binding to various MerTKs can be confirmed using known binding activity measurement methods. Examples of methods for measuring binding activity include ELISA and flow cytometry. When using ELISA or flow cytometry, the methods described in Examples 3 or 5 of WO 2022 / 224997 can be used.

[0055] A method for evaluating the agonist activity of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention may be to evaluate its activity of inducing AKT phosphorylation, which is a downstream signal of MerTK. For example, a known method can be used to evaluate the activity of inducing AKT phosphorylation. Herein, an anti-MerTK antibody having AKT phosphorylation-inducing activity is also referred to as an "anti-MerTK agonist antibody."

[0056] As used herein, the term "post-translational modification" refers to post-translational modification of an antibody when the antibody is expressed in a cell. Examples of post-translational modifications include the addition of N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, and methionine oxidation. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci., 2008, 97: 2426-2447).

[0057] In one embodiment, the anti-MerTK antibody or antigen-binding fragment thereof of the present invention may be post-translationally modified, in one embodiment, by N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, and / or methionine oxidation.

[0058] The anti-MerTK antibodies or antigen-binding fragments thereof of the present invention can be easily produced 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 antibodies of the present invention disclosed herein. The anti-MerTK antibodies or antigen-binding fragments thereof of the present invention can be produced, for example, by the method described below in "Method for producing anti-MerTK antibodies of the present invention," without particular limitation.

[0059] <Polynucleotides of the Present Invention> The present invention also provides polynucleotides comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention, and polynucleotides comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention (also referred to as "polynucleotides of the present invention").

[0060] In one embodiment, the polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention is a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8.

[0061] In one embodiment, the polynucleotide comprising a nucleotide sequence encoding the heavy chain of the anti-MerTK antibody of the present invention is a polynucleotide comprising a nucleotide sequence encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:8.

[0062] In one embodiment, the polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention is a polynucleotide selected from the group consisting of the following (a) to (f): (a) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 10; (b) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (c) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 14; (d) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (e) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; and (f) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20.

[0063] In one embodiment, the polynucleotide comprising a nucleotide sequence encoding the light chain of the anti-MerTK antibody of the present invention is a polynucleotide selected from the group consisting of the following (a) to (f): (a) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (b) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (c) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (d) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (e) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (f) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[0064] The polynucleotides of the present invention can be easily prepared 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. Various methods known to those skilled in the art can be used for such gene synthesis, such as the antibody gene synthesis method described in WO 90 / 07861.

[0065] <Expression Vector of the Present Invention> The present invention also provides an expression vector (also referred to as the "vector of the present invention") comprising a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention and / or a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention.

[0066] In one embodiment, the expression vector of the present invention may be an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody of the present invention, an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody of the present invention, or an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody of the present invention.

[0067] In one embodiment, the expression vector of the present invention may be an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-MerTK antibody of the present invention, an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-MerTK antibody of the present invention, or an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain and a polynucleotide comprising a base sequence encoding the light chain of the anti-MerTK antibody of the present invention.

[0068] In one embodiment, the expression vector of the present invention comprises a polynucleotide selected from the group consisting of the following (1) to (7): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; and (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20.

[0069] In one embodiment, the expression vector of the present invention comprises a polynucleotide selected from the group consisting of the following (1) to (7): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (7) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[0070] The expression vector of the present invention is not particularly limited, as long as it is capable of expressing a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention and / or a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention in various host cells, such as eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (e.g., Escherichia coli), and producing the polypeptides encoded thereby. Examples of such expression vectors include plasmid vectors and viral vectors (e.g., adenoviruses and retroviruses). For example, pcDNA3.4 TOPO vector, pEE6.4, and pEE12.4 can be used. Alternatively, antibody genes can be expressed by introducing variable region gene fragments into an expression vector already containing a human Ig constant region gene, such as AG-γ1 or AG-κ (see, for example, WO 94 / 20632).

[0071] 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) include the trp promoter, lac promoter, λPL promoter, and tac promoter. Examples of promoters for expression in yeast include the GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.

[0072] When animal cells, insect cells, or yeast cells are used as host cells, the expression vector of the present invention may contain an initiation codon and a termination codon. In this case, the expression vector of the present invention may also contain an enhancer sequence, 5'- and 3'-untranslated regions of the gene encoding the antibody of the present invention or its heavy chain variable region or light chain variable region, a secretion signal sequence, a splicing junction, a polyadenylation site, or a replication unit. When Escherichia coli is used as a host cell, the expression vector of the present invention may also contain an initiation codon, a termination codon, a terminator region, and a replication unit. In this case, the expression vector of the present invention may also contain a commonly used selection marker (e.g., a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, or a dihydrofolate reductase gene) depending on the purpose.

[0073] <Transformed Host Cells of the Present Invention> The present invention also provides host cells (also referred to as "host cells of the present invention") transformed with the polynucleotide of the present invention or the expression vector of the present invention (collectively referred to as "polynucleotides, etc. of the present invention"). The host cells of the present invention contain the polynucleotides, etc. of the present invention intracellularly. The introduced polynucleotides, etc. of the present invention may or may not be integrated into the genomic DNA of the host cell. Cells used as host cells may be either cells that can be cultured in vitro or cells in vivo. When the host cells are cells that can be cultured in vitro, the host cells of the present invention can be produced by introducing the polynucleotides, etc. of the present invention into the cells in vitro. The method for transforming host cells is not particularly limited, and methods commonly used by those skilled in the art, such as the calcium phosphate method, electroporation, or lipofection, can be used. When the host cell is a cell in a living organism, the method for introducing the polynucleotide of the present invention into the host cell is not particularly limited, and a nucleic acid delivery carrier (a cationic carrier or a non-cationic carrier (for example, including but not limited to liposomes and lipid nanoparticles (LNPs))) can be used.

[0074] Cells that can be cultured in vitro are not particularly limited, as long as they can be transformed by the expression vector used or a method such as electroporation, and can express an antibody or polypeptide. Examples of cells that can be cultured in vitro include various cells, such as conventional cells or artificially established cells, that are commonly used in the technical field of the present invention (e.g., animal cells (e.g., CHO-K1 cells, ExpiCHO-S (registered trademark) cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, Expi293F cells, NS0 cells), insect cells (e.g., Sf9), bacteria (e.g., Escherichia spp.), yeast (e.g., Saccharomyces spp., Pichia spp.), etc.). In one embodiment, the host cells of the present invention are Expi293F cells, CHO-K1 cells, or ExpiCHO-S cells.

[0075] In vitro transformed host cells can be selected by methods commonly used by those skilled in the art, such as a drug selection method using a drug selection marker gene and a drug such as tetracycline, ampicillin, neomycin, or hygromycin, or a cell isolation method such as limiting dilution, single cell sorting, or colony picking.

[0076] In one embodiment, the host cell of the present invention comprises polynucleotides selected from the group consisting of (a) to (c) below: (a) a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof; (b) a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention; and (c) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention.

[0077] In one embodiment, the host cell of the present invention comprises polynucleotides selected from the group consisting of the following (a) to (c): (a) a polynucleotide comprising a nucleotide sequence encoding the heavy chain of the anti-MerTK antibody of the present invention, and a polynucleotide comprising a nucleotide sequence encoding the light chain of the antibody; (b) a polynucleotide comprising a nucleotide sequence encoding the heavy chain of the anti-MerTK antibody of the present invention; and (c) a polynucleotide comprising a nucleotide sequence encoding the light chain of the anti-MerTK antibody of the present invention.

[0078] In one embodiment, the host cell of the present invention comprises a polynucleotide selected from the group consisting of the following (1) to (13): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20; (8) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (9) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (10) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14;(11) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16; (12) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18; and (13) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20.

[0079] <Method of Producing Anti-MerTK Antibodies of the Present Invention> The present invention also provides methods for producing anti-MerTK antibodies of the present invention or antigen-binding fragments thereof (also referred to as "production methods of the present invention"). The production methods of the present invention may include the aforementioned methods for producing polynucleotides of the present invention, methods for producing expression vectors of the present invention, and methods for producing host cells of the present invention. Furthermore, the production methods of the present invention may also include the steps of culturing the host cells described above in <Transformed Host Cells of the Present Invention> and expressing the antibody in the cells or the culture supernatant, and methods for recovering, isolating, and purifying the antibody. However, the production methods of the present invention are not limited to these methods, as long as the anti-MerTK antibodies of the present invention or antigen-binding fragments thereof are produced.

[0080] In one embodiment, the production method of the present invention comprises the step of culturing a host cell containing a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof, to express the anti-MerTK antibody.

[0081] Transformed host cells can be cultured by known methods. Culture conditions, such as temperature, medium pH, and culture time, are appropriately selected. When the host cells are animal cells, examples of media that can be used include MEM medium containing about 5 to 20% fetal bovine serum (Science, 1959, 130:432-437), 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 pH of the medium is preferably about 6 to 8, and the culture is typically performed at about 30 to 40°C for about 15 to 336 hours, with aeration and stirring as necessary. When the host cells are insect cells, the culture medium can be, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. U.S.A., 1985, 82:8404-8408). The pH of the medium is preferably about 5 to 8, and the culture is typically carried out at about 20 to 40°C for about 15 to 100 hours, with aeration or stirring as necessary. When the host cells are Escherichia coli or yeast, the culture medium is preferably, for example, a liquid medium containing nutrient sources. The nutrient medium preferably contains a carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of the transformed host cells. Examples of carbon sources include glucose, dextran, soluble starch, and sucrose. Examples of inorganic and organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If desired, the medium may contain other nutrients (for example, inorganic salts (e.g., calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, etc.) and antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.). The pH of the medium is preferably about 5 to 8. When the host cell is Escherichia coli, preferred media include, for example, LB medium and M9 medium (Cold Spring Harb. Laboratory, 2001, 3: A2.2). The culture is usually carried out at about 14 to 39°C for about 3 to 24 hours, with aeration and stirring as necessary.When the host cell is yeast, the culture medium can be, for example, Burkholder's minimal medium (Proc. Natl. Acad. Sci. U.S.A., 1980, 77:4504-4508). Cultivation is typically carried out at about 20 to 35°C for about 14 to 144 hours, with aeration and stirring as necessary. The anti-MerTK antibody or antigen-binding fragment thereof of the present invention can be expressed by culturing as described above.

[0082] The production methods of the present invention can include not only the step of culturing the transformed host cells of the present invention to express the anti-MerTK antibody or antigen-binding fragment thereof, but also the step of recovering, e.g., isolating or purifying, the anti-MerTK antibody or antigen-binding fragment thereof from the transformed host cells. Examples of isolation or purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, and gel filtration; methods that utilize charge, such as ion exchange chromatography and hydroxylapatite chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. For example, antibodies accumulated in the culture supernatant can be purified by various types of chromatography, for example, column chromatography using a protein A column or a protein G column.

[0083] The anti-MerTK antibody or antigen-binding fragment thereof of the present invention also includes an anti-MerTK antibody or antigen-binding fragment thereof produced by the production method of the present invention.

[0084] <Fusions of the Invention, Conjugates of the Invention, and Cells Displaying the Anti-MerTK Antibody of the Invention or an Antigen-Binding Fragment Thereof on the Cell Surface> The present invention also provides anti-MerTK antibodies of the invention or antigen-binding fragments thereof linked to a protein (including antibodies) or polypeptide other than MerTK (also referred to as "fusions of the invention"). Specifically, as used herein, "fusions of the invention" refers to the anti-MerTK antibody of the invention or antigen-binding fragment thereof linked to a protein (including antibodies) or polypeptide other than MerTK. The proteins or polypeptides used in the fusions of the invention are not particularly limited, and examples include antibodies, antigen-binding fragments, ligands, receptors, cytokines, chemokines, growth factors, physiologically active substances, human serum albumin, various tag peptides, artificial helix motif peptides, maltose-binding protein, glutathione S-transferase, and other peptides or proteins that can promote multimerization. In one embodiment, the fusions of the invention may comprise a protein or polypeptide linked to the anti-MerTK antibody of the invention or antigen-binding fragment thereof via a linker (e.g., a peptide linker). In one embodiment, the protein or polypeptide used in the fusion of the present invention may be, for example, an antibody or antigen-binding fragment thereof against a cell surface antigen of immune cells such as T cells, B cells, natural killer (NK) cells, dendritic cells, granulocytes, macrophages, and mast cells, epithelial cells, fibroblasts, or RPE cells, or a polypeptide that activates immune cells such as various interleukins (e.g., IL-2, IL-7, IL-12, and IL-15). In this case, the protein or polypeptide used in the fusion of the present invention may be linked directly to the anti-MerTK antibody or antigen-binding fragment thereof of the present invention, or may be linked via any linker (e.g., a peptide linker).

[0085] The present invention also provides an anti-MerTK antibody of the present invention or an antigen-binding fragment thereof (also referred to as a "conjugate of the present invention") conjugated to a carbohydrate, lipid, metal (including a radioisotope), organic compound (including a toxin, a near-infrared fluorescent dye, a chelating agent), or the like (also referred to as a "modifying agent"). That is, as used herein, a "conjugate of the present invention" refers to an anti-MerTK antibody of the present invention or an antigen-binding fragment thereof linked to a substance other than a protein or polypeptide. As used herein, a "modifying agent" refers to a non-peptide substance that is bound to an antibody or its antigen-binding fragment directly or via a linker, etc. The modifying agent used in the conjugate of the present invention is not particularly limited, and examples thereof include polyethylene glycol, sugar chains, phospholipids, radioisotopes (e.g., zirconium-89 (89Zr), yttrium-90 (90Y), indium-111 (111In), astatine-211 (211At), and actinium-225 (225Ac)), organic compounds, toxins, near-infrared fluorescent dyes (e.g., IRDye (registered trademark)), and chelating agents. The substance used in the conjugate may be directly bound to the anti-MerTK antibody or antigen-binding fragment thereof of the present invention, or may be bound via an optional linker. In one embodiment, the conjugate of the present invention is an anti-MerTK antibody or antigen-binding fragment thereof drug conjugate (antibody drug conjugate: ADC). The drug and linker used in the ADC may be selected from drugs and linkers commonly used by those skilled in the art. In one embodiment, the conjugate of the present invention is a radioisotope-labeled antibody in which a radioisotope is bound to an anti-MerTK antibody or an antigen-binding fragment thereof.

[0086] The complex of the present invention can also be used to detect soluble or membrane-type MerTK. As used herein, the term "detection" includes quantitative and qualitative detection.

[0087] The present invention also provides cells (e.g., chimeric antigen receptor-T cells: CAR-T cells) (hereinafter also referred to as "presenting cells of the present invention") in which the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof is presented on the surface of the cell (e.g., an immunocompetent cell or an immune effector cell). The method for producing the presenting cells of the present invention is not particularly limited, and for example, the presenting cells can be produced by expressing the anti-MerTK antibody or the antigen-binding fragment thereof on the cell surface using a polynucleotide encoding the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof. Various cells (macrophages, alveolar macrophages, T cells, NK cells, etc.) can be used as cells that present the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof.

[0088] The fusion product of the present invention, the complex of the present invention, and the present display cell are collectively referred to as the "fusion product of the present invention, etc."

[0089] In one embodiment, the antibody or antigen-binding fragment used in the fusion or the like of the present invention may be post-translationally modified. In one embodiment, the post-translational modification is addition of N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, and / or methionine oxidation.

[0090] The anti-MerTK antibodies or antigen-binding fragments thereof of the present invention, the fusions of the present invention, etc. can be easily produced by those skilled in the art using methods known in the art, based on the VH and VL sequence information of the anti-MerTK antibodies or antigen-binding fragments thereof of the present invention disclosed herein, other peptides or proteins (e.g., antibodies) used in the fusions of the present invention, and information on modifying agents used in the conjugates of the present invention. The anti-MerTK antibodies or antigen-binding fragments thereof of the present invention can be produced, for example, by the method described in the section <Methods for Producing Anti-MerTK Antibodies of the Present Invention>, without particular limitation.

[0091] <Pharmaceutical Compositions of the Present Invention> The present invention also provides pharmaceutical compositions (also referred to as "pharmaceutical compositions of the present invention") comprising the anti-MerTK antibody of the present invention, its antigen-binding fragment, or the fusion of the present invention, and a pharmaceutically acceptable excipient. The pharmaceutical compositions of the present invention can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients and pharmaceutical carriers. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections, infusions, and eye drops, which can be administered intraocularly, intravitreal, retrobulbar, subconjunctival, subthecal, subretinal, inhalation, intrathecal, intraperitoneal, intravenous, subcutaneous, intraarticular, nasal, and intramuscular. When formulating the pharmaceutical compositions, excipients, carriers, additives, and the like appropriate for these dosage forms can be used within pharmaceutically acceptable limits.

[0092] Pharmaceutical compositions of the present invention may contain multiple types of anti-MerTK antibodies of the present invention, antigen-binding fragments thereof, or fusion products of the present invention, as well as post-translationally modified versions thereof. For example, pharmaceutical compositions containing antibodies that have undergone N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, and / or methionine oxidation are also included in the present invention.

[0093] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing an anti-MerTK antibody of the present invention or an antigen-binding fragment thereof, or a fusion product thereof of the present invention, etc., and / or a post-translationally modified version of the antibody, antigen-binding fragment thereof, or fusion product thereof of the present invention, etc., comprising a heavy chain variable region and a light chain variable region of any of the following (a) to (f): (a) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 10; (b) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (c) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 14; (d) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (e) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 18; and (f) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 20.

[0094] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing the anti-MerTK antibody of the present invention or the fusion product thereof, etc. of the present invention, and / or a post-translationally modified version of the antibody or the fusion product thereof, etc., comprising a heavy chain and a light chain of any of the following (a) to (f): (a) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10; (b) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 12; (c) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 14; (d) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 16; (e) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 18; and (f) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 20.

[0095] The amount of the anti-MerTK antibody or antigen-binding fragment thereof of the present invention to be added when preparing the above-mentioned formulation will vary depending on the severity of symptoms and age of the patient, the dosage form of the formulation used, the binding titer of the antibody, and the like, but can be, for example, about 0.0001 mg / kg to 100 mg / kg.

[0096] <Medicinal Uses of Anti-MerTK Antibodies of the Present Invention> The anti-MerTK antibodies of the present invention, antigen-binding fragments thereof, or fusions of the present invention, etc., and pharmaceutical compositions containing them, can be used for the prevention or treatment of diseases caused by the accumulation of apoptotic cells, apoptotic cell-derived debris, and / or cells, tissues, or debris that highly express PtdSer. As used herein, "debris" is a concept distinct from intact cells, and refers to a mixture containing cell fragments and / or substances no longer needed in the body, such as biological components. As used herein, "cell-derived debris" refers to cell fragments formed by fragmentation of intact cells, or a mixture containing substances no longer needed in the body, such as biological components. As used herein, "cells, tissues, or debris that highly express PtdSer" refers to cells, tissues, or debris that contain PtdSer, other than apoptotic cells and debris derived from apoptotic cells.

[0097] In one embodiment, the anti-MerTK antibody of the present invention, its antigen-binding fragment, or fusion protein of the present invention, or a pharmaceutical composition containing them, can activate phagocytic cells via activation of MerTK, thereby enabling the phagocytosis of target food.

[0098] As used herein, "phagocytes" refers to cells that have the function of phagocytosis and removal, "phagocytosis" refers to the action of phagocytes taking in phagocytosed food, and "phagocytosis and removal function" refers to the function from phagocytosis to subsequently digesting and breaking down the ingested food. Examples of phagocytes whose phagocytosis and / or phagocytic removal function are enhanced or induced by the anti-MerTK antibody of the present invention, the antigen-binding fragment thereof, or the fusion product of the present invention, or a pharmaceutical composition containing them include macrophages, alveolar macrophages, dendritic cells, microglia, oligodendrocytes, epithelial cells, RPE cells, airway epithelial cells, Kupffer 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). In this specification, the enhancement or induction of phagocytosis or phagocytic elimination function may be collectively referred to as "activation" of phagocytosis or phagocytic elimination function. In one embodiment, the phagocytes activated by the anti-MerTK antibody of the present invention, its antigen-binding fragment, or fusion product of the present invention, or a pharmaceutical composition 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 fusion product of the present invention, or a pharmaceutical composition containing them, are RPE cells. As used herein, "RPE cells" refer 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 fusion product of the present invention, or a pharmaceutical composition 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 fusion thereof of the present invention, or the like, or a pharmaceutical composition containing them, are alveolar macrophages.

[0099] As used herein, the term "phagocytosed food" refers to a substance that is subject to phagocytosis by phagocytes. The anti-MerTK antibodies, antigen-binding fragments thereof, fusion proteins, etc. of the present invention, or pharmaceutical compositions containing them, can enhance the removal of phagocytosed food by activating the phagocytic removal function of phagocytes. Examples of phagocytosed food include, but are not limited to, apoptotic cells, cancer cells, neutrophils, mast cells, myofibroblasts, adipocytes, debris derived from these cells, POS, and myelin debris.

[0100] In one embodiment, the phagocytosed food is an apoptotic cell. In one embodiment, the phagocytosed food is debris derived from an apoptotic cell. In one embodiment, the phagocytosed food is a cell, tissue, or debris that highly expresses PtdSer. In one embodiment, the phagocytosed food is POS. In one embodiment, the phagocytosed food is myelin debris. In one embodiment, the phagocytosed food is a neutrophil. In one embodiment, the phagocytosed food is debris derived from a neutrophil.

[0101] Diseases that can be prevented or treated by the present invention include diseases caused by the accumulation of apoptotic cells, apoptotic cell-derived debris, or cells, tissues, or debris that highly express PtdSer, or diseases resulting from impaired phagocytic removal function. Diseases caused by the accumulation of apoptotic cells, apoptotic cell-derived debris, or cells, tissues, or debris that highly express PtdSer, or diseases resulting from impaired phagocytic removal function, include, but are not limited to, various eye diseases, lung diseases, autoimmune diseases, neurological disorders, fibrotic diseases, cardiovascular diseases, and cancer.

[0102] In one embodiment, the disease to be prevented or treated by the present invention is an ocular disease. In one embodiment, the ocular disease to be prevented or treated by the present invention is a retinal disease. In one embodiment, the retinal disease to be prevented or treated by the present invention is a retinal degenerative disease. In one embodiment, the retinal degenerative disease to be prevented or treated by the present invention is retinitis pigmentosa, age-related macular degeneration, and / or diabetic retinopathy.

[0103] In one embodiment, the disease to be prevented or treated by the present invention is a pulmonary disease. In one embodiment, the pulmonary disease to be prevented or treated by the present invention is an acute pulmonary disease. In one embodiment, the acute pulmonary disease to be prevented or treated by the present invention is acute respiratory distress syndrome and / or acute lung injury.

[0104] In one embodiment, the disease to be prevented or treated by the present invention is an autoimmune disease. In one embodiment, the autoimmune disease to be prevented or treated by the present invention is systemic lupus erythematosus. In one embodiment, the autoimmune disease to be prevented or treated by the present invention is multiple sclerosis.

[0105] In one embodiment, the disease to be prevented or treated by the present invention is rejection reaction during various organ transplants.

[0106] In one embodiment, the disease to be prevented or treated by the present invention is a cardiovascular disease. In one embodiment, the cardiovascular disease to be prevented or treated by the present invention is atherosclerosis. In one embodiment, the cardiovascular disease to be prevented or treated by the present invention is an ischemic disease.

[0107] In one embodiment, the disease to be prevented or treated by the present invention is cancer. The cancer to be prevented or treated is not particularly limited, but includes, for example, peritoneal dissemination of various cancer cells, gastric cancer, lung cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), blood cancer such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, 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, Examples of cancers include solid cancers such as head and neck cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, large intestine cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, as well as 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, pleuropulmonary blastoma, and retinoblastoma.

[0108] Diagnostic Uses of Anti-MerTK Antibodies of the Present Invention The anti-MerTK antibodies of the present invention, their antigen-binding fragments, or fusions of the present invention, or diagnostic test kits containing them (hereinafter also referred to as "test kits of the present invention") can be used to detect MerTK. The test kits of the present invention may optionally contain a detectable label, such as an indicator enzyme, a radioactive label, a fluorescent dye molecule, or a paramagnetic particle. The test kits of the present invention may also include an informational booklet, such as a booklet providing instructions for carrying out the methods described herein using the reagents. In one embodiment, the anti-MerTK antibodies of the present invention can diagnose inflammatory conditions by detecting soluble MerTK in body fluids (e.g., blood). In one embodiment, the conjugates of the present invention can diagnose inflammatory conditions or the glycosylation status of MerTK by detecting and analyzing soluble MerTK in blood.

[0109] The present invention also includes methods for diagnosing, preventing, or treating diseases caused by the accumulation of apoptotic cells, apoptotic cell-derived debris, or cells, tissues, or debris that highly express PtdSer, or diseases caused by impaired phagocytic clearance function, comprising the step of administering to a subject an effective diagnostic, prophylactic, or therapeutic amount of the anti-MerTK antibody of the present invention, an antigen-binding fragment thereof, a fusion product of the present invention, or the like. The present invention also includes the anti-MerTK antibody of the present invention, an antigen-binding fragment thereof, or a fusion product of the present invention, for use in the diagnosis, prevention, or treatment of diseases caused by the accumulation of apoptotic cells, apoptotic cell-derived debris, or cells, tissues, or debris that highly express PtdSer, or diseases caused by impaired phagocytic clearance function. The present invention also includes the use of the anti-MerTK antibody of the present invention, an antigen-binding fragment thereof, or a fusion product of the present invention, or the like, in the manufacture of a pharmaceutical composition for the diagnosis, prevention, or treatment of diseases caused by the accumulation of apoptotic cells, apoptotic cell-derived debris, or cells, tissues, or debris that highly express PtdSer, or diseases caused by impaired phagocytic clearance function.

[0110] <Anti-MerTK bispecific antibodies of the present invention> The present invention also provides bispecific antibodies comprising the anti-MerTK antibody of the present invention or an antigen-binding fragment thereof by linking the anti-MerTK antibody or an antigen-binding fragment thereof to an antibody against an antigen other than MerTK (hereinafter collectively referred to as "another antibody") (hereinafter also referred to as "anti-MerTK bispecific antibodies of the present invention"). The anti-MerTK bispecific antibodies of the present invention comprise the heavy chain variable region and light chain variable region of the anti-MerTK antibody of the present invention and the heavy chain variable region and light chain variable region of the other antibody.

[0111] The anti-MerTK bispecific antibody of the present invention comprises the heavy chain variable region and light chain variable region of the anti-MerTK antibody shown below: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

[0112] 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 the following (1) to (6): (1) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 18; or (6) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20.

[0113] The anti-MerTK bispecific antibody of the present invention may have any structure, for example, as described in the literature (mAbs, 2017, 9:182-212, Fig. 2). In one embodiment, the anti-MerTK bispecific antibody of the present invention may have a symmetric or asymmetric structure. In one embodiment, the anti-MerTK bispecific antibody of the present invention is an antibody fusion type (e.g., IgG ２ type), variable region only type (e.g., DART type, diabody type), CH1 / CL fusion protein type (e.g., scFv ２ -CH1 / CL type), Fab fusion protein type (e.g., Fab-scFv type), non-immunoglobulin fusion protein type (e.g., scFv ２-albumin type), Fc region-modified IgG type (e.g., IgG(kih) type, CrossMab(IgG-kih) type, DuetMab type), added and Fc region-modified IgG type (e.g., IgG(kih)-scFab type, CrossMab-Fab type), modified Fc region and CH3 fusion protein type (e.g., scFv-Fc(kih) type, Fab-scFv-Fc(kih) type), added IgGs-heavy chain fusion type (e.g., IgG-HC-scFv type), 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 invention is asymmetric in structure.

[0114] In one embodiment, the anti-MerTK bispecific antibody of the present invention may have a structure in which another antibody or an antigen-binding fragment thereof is linked 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 another antibody or an antigen-binding fragment thereof is linked 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-armed antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention has a structure in which another antibody or an antigen-binding fragment thereof is linked to the N-terminus of the second Fc polypeptide of the anti-MerTK antibody of the present invention, which is a single-armed antibody, via a hinge region.

[0115] In one embodiment, the anti-MerTK bispecific antibody of the present invention may be of the DuetMab type, CrossMab type (IgG-kih) type, Fab-scFv-Fc(kih) type, IgG(kih)-scFab type, CrossMab-Fab type, or IgG-HC-scFv type.

[0116] In one embodiment, the anti-MerTK bispecific antibody of the invention comprises an F(ab') ２They may be of the fusion type, diabody type, DART type, and Fab-scFv type.

[0117] In one embodiment, the antigen-binding fragment of the anti-MerTK antibody of the present invention contained in the anti-MerTK bispecific antibody of the present invention is an scFv, a Fab fragment, a Fab' fragment, an F(ab') fragment, or an F(ab') fragment. ２ The fragment may be a VHH antibody.

[0118] In one embodiment, the antigen-binding fragment of another antibody included in the anti-MerTK bispecific antibody of the present invention is an scFv, a Fab fragment, a Fab' fragment, an F(ab') fragment, or a Fab' fragment. ２ The fragment may be a VHH antibody.

[0119] In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a heavy chain fragment comprising the heavy chain variable region of the anti-MerTK antibody and a light chain comprising the light chain variable region of the anti-MerTK antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a single-arm antibody (hereinafter also referred to as a "single-arm anti-MerTK antibody") comprising a heavy chain fragment comprising the heavy chain variable region and a light chain comprising the light chain variable region of the anti-MerTK antibody, and an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide, as well as the heavy chain variable region and light chain variable region of another antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody and an scFv or Fab region comprising the heavy chain variable region and light chain variable region of the other antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention is a bispecific antibody in which the C-terminus of an scFv or Fab region comprising a heavy chain variable region and a light chain variable region of another antibody is linked via a hinge region to the N-terminus of the second Fc polypeptide of a single-armed anti-MerTK antibody. In one embodiment, the anti-MerTK bispecific antibody of the present invention is of the Fab-scFv-Fc(kih) type. In one embodiment, the anti-MerTK bispecific antibody of the present invention is of the CrossMab (IgG-kih) type.

[0120] In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment and a light chain selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[0121] The anti-MerTK bispecific antibodies of the present invention are not limited to the above structure, and may be bispecific antibodies comprising a monovalent or polyvalent anti-MerTK antibody or antigen-binding fragment thereof and a monovalent or polyvalent antibody against an antigen other than MerTK or antigen-binding fragment thereof. Furthermore, by combining the anti-MerTK bispecific antibodies of the present invention with antibodies against a third or fourth antigen, multispecific antibodies such as trispecific antibodies or tetraspecific antibodies can be provided.

[0122] The type and length of the linker used in the bispecific antibodies of the present invention are not particularly limited and can be appropriately selected by those skilled in the art. For example, when the anti-MerTK antibody or other antibody of the present invention is an scFv, the length of the peptide linker connecting the heavy chain variable region and light chain variable region of the antibody is preferably 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. Examples of peptide linkers that can be used include a glycine-serine linker (GS linker) and a glycine-lysine-proline-glycine-serine linker (GKPGS linker). Examples of such linkers include the following: Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 46) Ser-Gly-Gly-Gly (SEQ ID NO: 47) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 48) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 49) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 50) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 52) Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 53) (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 54 when n = 3) (Ser-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (SEQ ID NO: 55) (Gly-Lys-Pro-Gly-Ser)n wherein n above represents an integer of 1 or more. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0123] In one embodiment, when the anti-MerTK bispecific antibody of the present invention comprises an scFv of another antibody, the peptide linker linking the heavy chain variable region and light chain variable region of the other antibody has the amino acid sequence of SEQ ID NO: 48. In one embodiment, the peptide linker linking the heavy chain variable region and light chain variable region of the other antibody has the amino acid sequence of (Gly-Gly-Gly-Gly-Ser)n. In one embodiment, the peptide linker linking the heavy chain variable region and light chain variable region of the other antibody has the amino acid sequence of SEQ ID NO: 54.

[0124] When the anti-MerTK bispecific antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may comprise a mutation that reduces antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In one embodiment, the anti-MerTK bispecific antibody of the present invention comprises an Fc region comprising the amino acid mutations L234A and L235A (LALA mutations) (wherein the mutation positions are 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 comprising the amino acid mutations P329A or P331G (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region). In one embodiment, an anti-MerTK bispecific antibody of the invention comprises an Fc region comprising the L234A and L235A amino acid mutations (LALA mutations) and a P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region). In one embodiment, an anti-MerTK bispecific antibody of the invention comprises an Fc region comprising knobs-into-holes mutations. In one embodiment, an anti-MerTK bispecific antibody of the invention comprises an Fc region comprising the LALA mutation, a P331G mutation, and a knobs-into-holes mutation. In one embodiment, the knobs-into-holes mutations comprised in the anti-MerTK bispecific antibody of the invention are 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 (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region). The knobs-into-holes mutations comprised in the anti-MerTK bispecific antibody of the invention may be a T366W mutation in the first Fc polypeptide and a T366S, L368A, and Y407V mutation in the second Fc polypeptide, or may be a T366S, L368A, and Y407V mutation in the first Fc polypeptide and a T366W mutation in the second Fc polypeptide.

[0125] Examples of other antibodies or antigen-binding fragments include, but are not limited to, antibodies or antigen-binding fragments against misfolded proteins, amyloid, myelin debris, Aβ, immune cells (e.g., T cells), receptors expressed on immune cells, or tumor-associated antigens on cancer cells.

[0126] In one embodiment, the anti-MerTK bispecific antibody of the present invention is a bispecific antibody that binds to MerTK and misfolded proteins and / or amyloid (hereinafter also referred to as the "anti-MerTK-anti-misfolded protein bispecific antibody of the present invention"). As used herein, "misfolding" or "misfolded" means that the protein is not properly folded in vivo. Furthermore, a "misfolded protein" is a protein that has been misfolded and no longer has its original function, and an "amyloid" is a clump of such misfolded protein.

[0127] Examples of proteins that are known to become misfolded include immunoglobulin light chain (IgL), IgH, SAA, TTR, β2 microglobulin, apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, apolipoprotein CII, apolipoprotein CIII, gelsolin, lysozyme, LECT2, fibrinogen α chain, cystatin C, ABri (precursor) protein, ADan (precursor) protein, Aβ precursor protein, α-synuclein, tau protein, prion protein, TMEM106B, (pro)calcitonin, amylin, ANP, prolactin, and (pro)somatostatin. Known misfolding proteins include statins, glucagon, PTH, insulin, enfuvirtide, GLP1, IL1RAP, pulmonary surfactant proteins, corneodesmosin, MFG-E8, keratoepithelin, lactoferrin, OAAP, semenogelin 1, cathepsin K, EFEMP1, huntingtin, myelin, TDP43, and SOD1 (Amyloid, 2022, 4:213-219; J. Neurosci., 2006, 26:328-332; Neurobiology of Disease, 2023, 184:106218). In one embodiment, the misfolded protein is TTR. In one embodiment, the misfolded protein is IgL.

[0128] The anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises the heavy chain variable region and light chain variable region of the anti-MerTK antibody of the present invention, and the heavy chain variable region and light chain variable region of an antibody that binds to misfolded proteins and / or amyloid. The anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises the heavy chain variable region and light chain variable region of the anti-MerTK antibody shown below: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

[0129] In one embodiment, the anti-MerTK-anti-misfolded protein 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 the following (1) to (6): (1) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 18; or (6) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20.

[0130] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a heavy chain fragment comprising the heavy chain variable region of the anti-MerTK antibody and a light chain comprising the light chain variable region of the anti-MerTK antibody. In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment comprising the heavy chain variable region of the anti-MerTK antibody, a light chain comprising the light chain variable region, and an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide. In a further embodiment, the single-arm anti-MerTK antibody comprised in the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single heavy chain in which a heavy chain fragment comprising 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 a further embodiment, the heavy chain fragment comprising 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 comprising a heavy chain fragment comprising a heavy chain variable region of the anti-MerTK antibody, a light chain comprising a light chain variable region, and an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide, wherein the single-arm anti-MerTK antibody comprises a heavy chain in which the heavy chain fragment 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 comprising a heavy chain fragment comprising a heavy chain variable region of the anti-MerTK antibody, a light chain comprising a light chain variable region, and an Fc region consisting 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.

[0131] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment and a light chain selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) A heavy chain fragment consisting of the amino acid sequence from amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain fragment consisting of the amino acid sequence of SEQ ID NO: 20.

[0132] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises a single-armed anti-MerTK antibody and an scFv or Fab region comprising the heavy and light chain variable regions of an antibody that binds to misfolded proteins and / or amyloid. In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody in which an scFv or Fab region comprising the heavy and light chain variable regions of an antibody that binds to misfolded proteins and / or amyloid is linked to the N-terminus of the second Fc polypeptide of the single-armed anti-MerTK antibody via a hinge region.

[0133] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention comprises an antibody or antigen-binding fragment thereof that binds to misfolded proteins, myelin debris, or Aβ, or amyloid thereof. Examples of proteins that form misfolded proteins, myelin debris, or Aβ, or amyloid thereof, include IgL, IgH, SAA, TTR, β2 microglobulin, apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, apolipoprotein CII, apolipoprotein CIII, gelsolin, lysozyme, LECT2, fibrinogen α chain, cystatin C, ABri (precursor) protein, ADan (precursor) protein, Aβ, Aβ precursor protein, α-synuclein, and tau protein. Examples of the protein include proteins, prion protein, TMEM106B, (pro)calcitonin, amylin, ANP, prolactin, (pro)somatostatin, glucagon, PTH, insulin, enfuvirtide, GLP1, IL1RAP, pulmonary surfactant protein, corneodesmosin, MFG-E8, keratoepithelin, lactoferrin, OAAP, semenogelin 1, cathepsin K, EFEMP1, huntingtin, myelin debris, TDP43, and SOD1.

[0134] In one embodiment, the other antibody used in the anti-MerTK-anti-misfolded protein bispecific antibody of the invention may be an anti-SAA antibody, anti-amylin antibody, anti-Aβ antibody, anti-huntingtin antibody, anti-TTR antibody, or anti-IgL antibody, or an antigen-binding fragment thereof. In one embodiment, the other antibody used in the anti-MerTK-anti-misfolded protein bispecific antibody of the invention may be an anti-TTR antibody that selectively binds to misfolded TTR or an anti-IgL antibody that selectively binds to misfolded IgL.

[0135] In one embodiment, the anti-MerTK-anti-misfolded protein bispecific antibody of the present invention is a bispecific antibody comprising an anti-MerTK antibody or antigen-binding fragment thereof and an anti-TTR antibody or antigen-binding fragment thereof of the present invention, and having binding activity to MerTK and misfolded TTR (herein referred to as an "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 comprising an anti-MerTK antibody or antigen-binding fragment thereof and an anti-IgL antibody or antigen-binding fragment thereof of the present invention, and having binding activity to MerTK and misfolded IgL (herein referred to as an "anti-MerTK-anti-IgL bispecific antibody").

[0136] In one embodiment, the anti-MerTK bispecific antibody of the invention comprises an antibody or antigen-binding fragment directed against a T-cell receptor.

[0137] In one embodiment, the anti-MerTK bispecific antibody of the present invention may be a bispecific antibody comprising an antibody or antigen-binding fragment that binds to MerTK and a tumor-associated antigen (TAA) (hereinafter referred to as the "anti-MerTK-anti-TAA bispecific antibody of the present invention").

[0138] Examples of antibodies or antigen-binding fragments thereof used in the anti-MerTK-anti-TAA bispecific antibodies of the present invention include 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, L IV-1, MUC1, MUC2, MUC4, MUC16, CDH6, CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM 6, CEACAM7, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21, URLC10, NY-ESO- 1, GAA, OFA, cyclinB1, WT-1, CEF, VEGRR1, VEGFR2, TTK, HPV16, HPV16E7, HPV18, CEA IMA910, KOC1, SL-701, MART-1, gp100, tyrosinase, survivin, 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, and 5T4.

[0139] The anti-MerTK bispecific antibodies of the present invention may contain mutations that reduce ADCC, CDC, or ADCP and / or mutations that promote heterodimer formation. The mutations are similar to those described above in <Anti-MerTK antibodies of the present invention>.

[0140] In one embodiment, the anti-MerTK bispecific antibody of the present invention may be post-translationally modified. The post-translational modification is as described above in <Anti-MerTK Antibodies of the Present Invention>. In one embodiment, the post-translational modification is addition of N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, and / or methionine oxidation.

[0141] The anti-MerTK bispecific antibodies 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 antibodies of the present invention disclosed herein, as well as the sequence information of other known antibodies or antigen-binding fragments.

[0142] <Fusions and Conjugates of the Anti-MerTK Bispecific Antibody of the Present Invention, and Cells Displaying the Anti-MerTK Bispecific Antibody of the Present Invention on Their Surface> The anti-MerTK bispecific antibody of the present invention provides fusions of the anti-MerTK bispecific antibody of the present invention linked to proteins (including polypeptides) other than the antigens bound by MerTK and other antibodies, and to antibodies (including antigen-binding fragments) other than the anti-MerTK antibody and other antibodies. The anti-MerTK bispecific antibody of the present invention also provides conjugates of the anti-MerTK bispecific antibody of the present invention bound to a modifying agent. Furthermore, the anti-MerTK bispecific antibody of the present invention provides cells (e.g., tandem CARs) in which the anti-MerTK bispecific antibody of the present invention is displayed on the surface of the cell (e.g., an immunocompetent cell or effector cell) (herein referred to as "cells displaying the anti-MerTK bispecific antibody of the present invention on their surface") (Oncology Reports, 2019, 42: 2183-2195). The fusion product, the complex, and the cells on whose surface the antibody is displayed are similar to those described above in <The fusion product of the present invention, the complex of the present invention, and the cells on whose surface the anti-MerTK antibody or antigen-binding fragment thereof of the present invention is displayed>.

[0143] <Polynucleotides, expression vectors, transformed host cells, and production methods for anti-MerTK bispecific antibodies of the present invention> The present invention also provides polynucleotides used in the production of anti-MerTK bispecific antibodies of the present invention. Those skilled in the art can produce such polynucleotides in accordance with the descriptions in the above section <Polynucleotides of the present invention> and the below section <Polynucleotides of anti-MerTK-anti-TTR bispecific antibodies of the present invention>.

[0144] The present invention also provides expression vectors containing polynucleotides encoding the anti-MerTK bispecific antibodies of the present invention and methods for producing the anti-MerTK bispecific antibodies of the present invention, which can be performed by those skilled in the art according to the above-mentioned <Expression vectors of the present invention> and <Methods for producing anti-MerTK antibodies of the present invention>.

[0145] The present invention also provides host cells transformed with the anti-MerTK bispecific antibodies of the present invention. Those skilled in the art can prepare such host cells according to the above-mentioned "Transformed host cells of the present invention" and the below-mentioned "Transformed host cells of the MerTK-anti-TTR bispecific antibodies of the present invention."

[0146] <Medicinal Uses of the Anti-MerTK Bispecific Antibody of the Present Invention> The present invention also provides a pharmaceutical composition comprising the anti-MerTK bispecific antibody of the present invention and a pharmaceutically acceptable excipient. Those skilled in the art can prepare such a pharmaceutical composition in accordance with the description of the above <Pharmaceutical Composition of the Present Invention>.

[0147] The anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody can be used for the prevention or treatment of diseases caused by the accumulation of misfolded proteins.

[0148] The anti-MerTK anti-bispecific antibodies of the present invention or pharmaceutical compositions containing them bind to phagocytes and phagocytosed food, shorten the physical distance between the phagocytes and the phagocytosed food, and activate the phagocytes via activation of MerTK, thereby allowing the phagocytes to efficiently take up and digest the phagocyted food.

[0149] Phagocytic cells activated by the anti-MerTK bispecific antibody of the present invention are as described above in <Medicinal Uses of the Anti-MerTK Antibody of the Present Invention>. Examples of phagocytic foods whose removal is enhanced by activation of the phagocytic removal function of phagocytes by the anti-MerTK bispecific antibody of the present invention include misfolded proteins, amyloid, and / or cancer cells. In one embodiment, the phagocytic food is a misfolded protein. In one embodiment, the phagocytic food is amyloid. In one embodiment, the phagocytic food is misfolded TTR (hereinafter referred to as "misfolded TTR"). In one embodiment, the phagocytic food is misfolded IgL (hereinafter referred to as "misfolded IgL"). In one embodiment, the phagocytic food is myelin debris. In one embodiment, the phagocytic food is Aβ.

[0150] The anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody can be used for the prevention or treatment of diseases caused by the accumulation of misfolded proteins, diseases caused by amyloid accumulation, or diseases caused by impaired phagocytic clearance. The present invention also includes a method for preventing or treating diseases caused by the accumulation of misfolded proteins, diseases caused by amyloid accumulation, or diseases caused by impaired phagocytic clearance, comprising the step of administering a therapeutically effective amount of the anti-MerTK bispecific antibody of the present invention to a subject. The present invention also includes the anti-MerTK bispecific antibody of the present invention for use in the prevention or treatment of diseases caused by the accumulation of misfolded proteins, diseases caused by amyloid accumulation, or diseases caused by impaired phagocytic clearance. The present invention also includes the use of the anti-MerTK bispecific antibody of the present invention in the manufacture of a pharmaceutical composition for the prevention or treatment of diseases caused by the accumulation of misfolded proteins, diseases caused by amyloid accumulation, or diseases caused by impaired phagocytic clearance. Diseases caused by the accumulation of misfolded proteins, diseases caused by the accumulation of amyloid, or diseases caused by impaired phagocytic clearance function that can be prevented or treated by the present invention are not particularly limited, and examples include various amyloidoses, heart diseases, neurological disorders, eye diseases, endocrine / metabolic diseases, etc. In one embodiment, the diseases that can be prevented or treated by the anti-MerTK bispecific antibodies of the present invention and pharmaceutical compositions containing them are amyloidoses, heart diseases, neurological disorders, eye diseases, or endocrine / metabolic diseases.

[0151] In one embodiment, the disease to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is amyloidosis (e.g., localized amyloidosis or systemic amyloidosis). In one embodiment, the amyloidosis to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is localized amyloidosis. The localized amyloidosis that can be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is Alzheimer's disease, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease, hereditary cerebral amyloid angiopathy, familial British dementia, familial Danish dementia, localized atrial amyloidosis, prolactin-secreting tumor, insulin amyloidosis, corneal amyloidosis, aortic medial amyloidosis, or localized nodular amyloidosis (Circ. J., 2020, 84: 1610-1671). In one embodiment, the amyloidosis to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is systemic amyloidosis (e.g., transthyretin-mediated cardiac amyloidosis (ATTR-CM) or polyneuropathy amyloidosis (ATTR-PN), AL amyloidosis, AH amyloidosis, Aβ2M amyloidosis (dialysis-related amyloidosis), AA amyloidosis, or the like. sis, 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, the systemic amyloidosis to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is ATTR-CM and ATTR-PN.In one embodiment, the systemic amyloidosis to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is AL amyloidosis.

[0152] In one embodiment, the disease to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is a neuropathic disease. In one embodiment, the neuropathic disease to be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody is multiple sclerosis or / and Alzheimer's disease.

[0153] The anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody can also be used for the prevention or treatment of cancer, and this can be done by a person skilled in the art in accordance with the description in the above section <Medicinal Uses of the Present Invention>. Furthermore, cancers that can be prevented or treated by the anti-MerTK bispecific antibody of the present invention or a pharmaceutical composition containing the antibody include those described in the above section <Medicinal Uses of the Present Invention>.

[0154] <Anti-MerTK-anti-TTR bispecific antibodies of the present invention> The present invention provides anti-MerTK-anti-TTR bispecific antibodies that bind to both MerTK and misfolded TTR.

[0155] The anti-MerTK-anti-TTR bispecific antibody of the present invention comprises the heavy and light chain variable regions of an anti-MerTK antibody and the heavy and light chain variable regions of an antibody that binds to misfolded TTR. The anti-MerTK-anti-TTR bispecific antibody of the present invention comprises the heavy and light chain variable regions of the anti-MerTK antibody shown below: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

[0156] 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-MerTK antibody selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (5) a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; or (6) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20.

[0157] 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-MerTK antibody selected from the group consisting of the following (4) to (6): (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 18; or (6) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 20.

[0158] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the invention comprises a heavy chain fragment comprising the heavy chain variable region of an anti-MerTK antibody and a light chain comprising the light chain variable region of an anti-MerTK antibody. In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment comprising the heavy chain variable region and a light chain comprising the light chain variable region of an anti-MerTK antibody, and an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide.

[0159] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment and a light chain selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) A heavy chain fragment consisting of the amino acid sequence from amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain fragment consisting of the amino acid sequence of SEQ ID NO: 20.

[0160] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment and a light chain selected from the group consisting of the following (4) to (6): (4) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) a heavy chain fragment consisting of the amino acid sequence of amino acids 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 20.

[0161] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a single-armed anti-MerTK antibody and an scFv or Fab region comprising the heavy and light chain variable regions of an anti-TTR antibody that binds to misfolded TTR. In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention is a bispecific antibody in which an scFv or Fab region comprising the heavy and light chain variable regions of an anti-TTR antibody that binds to misfolded TTR is linked via a hinge region to the N-terminus of the second Fc polypeptide of the single-armed anti-MerTK antibody.

[0162] The anti-MerTK-anti-TTR bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of an anti-TTR antibody that binds to misfolded TTR. The anti-TTR antibody is not particularly limited as long as it binds to misfolded TTR, and for example, the 371M antibody (WO 2015 / 115332) can be used.

[0163] In one embodiment, the anti-MerTK-anti-FTR bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of the following anti-FTR antibody that binds to misfolded FTR: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO:27, CDR2 consisting of the amino acid sequence of SEQ ID NO:28, and CDR3 consisting of the amino acid sequence of SEQ ID NO:29; and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO:30, CDR2 consisting of the amino acid sequence of SEQ ID NO:31, and CDR3 consisting of the amino acid sequence of SEQ ID NO:32.

[0164] In one embodiment, the anti-MerTK-anti-FTR bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of the following anti-FTR antibody that binds to misfolded FTR: a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 119 of SEQ ID NO:43, and a light chain variable region consisting of the amino acid sequence from amino acid number 135 to amino acid number 245 of SEQ ID NO:43.

[0165] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an antigen-binding fragment of an anti-TTR antibody that binds to misfolded TTR. In one embodiment, the antigen-binding fragment of an anti-TTR antibody that binds to misfolded TTR contained in the anti-MerTK-anti-TTR bispecific antibody of the present invention is an scFv, a Fab fragment, a Fab' fragment, or an F(ab') fragment. ２ In one embodiment, the anti-MerTK-anti-FTR bispecific antibody of the present invention comprises an scFv (herein referred to as "anti-FTR-scFv") comprising the heavy chain variable region and the light chain variable region of an anti-FTR antibody that binds to misfolded FTR.

[0166] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an anti-TTR-scFv consisting of the amino acid sequence of SEQ ID NO:43.

[0167] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention is a Fab-scFv-Fc(kih) type bispecific antibody in which the C-terminus of an anti-TTR-scFv is linked to the N-terminus of the second Fc polypeptide of a single-arm anti-MerTK antibody via a hinge region.

[0168] In one embodiment, in the anti-FTR-scFv, the peptide linker linking the heavy chain variable region and light chain variable region of the anti-FTR antibody has the amino acid sequence of SEQ ID NO: 48. In one embodiment, the peptide linker linking the heavy chain variable region and light chain variable region of the anti-FTR antibody has the amino acid sequence of (Gly-Gly-Gly-Gly-Ser)n. In one embodiment, the peptide linker linking the heavy chain variable region and light chain variable region of the anti-FTR antibody has the amino acid sequence of SEQ ID NO: 54.

[0169] When the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may comprise a mutation that reduces antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises an Fc region comprising the amino acid mutations L234A and L235A (LALA mutations) (wherein the positions of the mutations are amino acid positions 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 comprising the amino acid mutations P331G or P329A (wherein the positions of the mutations are amino acid positions according to the EU index in the human Igγ1 constant region). In one embodiment, an anti-MerTK-anti-TTR bispecific antibody of the invention comprises an Fc region comprising one or more of the following mutations: LALA, P331G, and P329A. In one embodiment, an anti-MerTK-anti-TTR bispecific antibody of the invention comprises an Fc region comprising the LALA and P331G mutations.

[0170] In one embodiment, an anti-MerTK-anti-FTR bispecific antibody of the invention comprises an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide. In one embodiment, an anti-MerTK-anti-FTR bispecific antibody of the invention comprises a first Fc polypeptide and a second Fc polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:39 or an amino acid sequence having 1 to 10 substitutions in the amino acid sequence of SEQ ID NO:39.

[0171] In one embodiment, an anti-MerTK-anti-FTR bispecific antibody of the invention comprises an Fc region comprising knobs-into-holes mutations. In one embodiment, an anti-MerTK-anti-FTR bispecific antibody of the invention comprises an Fc region comprising one or more of the following mutations: LALA, P331G, P329A, and knobs-into-holes mutations.

[0172] In one embodiment, the knobs-into-holes mutations comprised in the anti-MerTK-anti-FTR bispecific antibody of the invention are 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 (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[0173] In one embodiment, the first and second polypeptides comprised in the Fc region of the anti-MerTK-anti-TTR bispecific antibody of the present invention comprise polypeptides selected from the following (1) or (2): (1) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41; or (2) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40.

[0174] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention may comprise a hinge region. The hinge region is not particularly limited as long as it has a sequence that can be commonly used by those skilled in the art. In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a hinge region consisting of the amino acid sequence shown in SEQ ID NO: 42 and / or a hinge region consisting of the amino acid sequence of SEQ ID NO: 70.

[0175] 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, and the like described above in <Anti-MerTK-anti-TTR bispecific antibody of the present invention>.

[0176] In one embodiment, the anti-MerTK-anti-TTR bispecific antibody of the present invention may be post-translationally modified. The post-translational modification is in accordance with the description of <Anti-MerTK bispecific antibody of the present invention> above.

[0177] <Fusions and Conjugates of the Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention, and Cells Displaying the Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention on Their Surface> The anti-MerTK-anti-TTR bispecific antibody of the present invention provides fusions of the anti-MerTK-anti-TTR bispecific antibody of the present invention linked to proteins (including polypeptides) other than MerTK and misfolded TTR, and antibodies (including antigen-binding fragments) other than anti-MerTK antibodies and anti-TTR antibodies. The anti-MerTK-anti-TTR bispecific antibody of the present invention also provides conjugates of the anti-MerTK-anti-TTR bispecific antibody of the present invention linked to a modifying agent. Furthermore, the anti-MerTK-anti-TTR bispecific antibody of the present invention provides cells (e.g., tandem CAR) in which the anti-MerTK-anti-TTR bispecific antibody of the present invention is displayed on the surface of the cell (e.g., immunocompetent cell or effector cell) (herein referred to as "cells in which the anti-MerTK-anti-TTR bispecific antibody of the present invention is displayed on the cell surface") (Oncology Reports, 2019, 42: 2183-2195). The fusion, the complex, and the cell in which the bispecific antibody is displayed on the cell surface can be produced in accordance with the description above in <The fusion of the present invention, the complex of the present invention, and the cell in which the anti-MerTK antibody or antigen-binding fragment thereof is displayed on the cell surface>.

[0178] <Polynucleotides for anti-MerTK-anti-TTR bispecific antibodies of the present invention> The present invention also provides polynucleotides for use in producing anti-MerTK-anti-TTR bispecific antibodies. Those skilled in the art can perform such production in accordance with the descriptions in the above sections <Polynucleotides of the present invention> and <Polynucleotides for anti-MerTK bispecific antibodies of the present invention>.

[0179] 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 the following (1) to (9): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18; (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20; (8) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43; and (9) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43.

[0180] Those skilled in the art can prepare the polynucleotides described in this section based on the nucleotide sequences using methods known in the art.

[0181] <Expression Vector and Production Method for Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention> The present invention also provides an expression vector and production method used for producing the anti-MerTK-anti-TTR bispecific antibody of the present invention. Those skilled in the art can implement the vector and method in accordance with the descriptions in the above sections <Polynucleotide of the Present Invention>, <Expression Vector of the Present Invention>, <Method of Producing Anti-MerTK Antibody of the Present Invention>, and <Polynucleotide, Expression Vector, Transformed Host Cell, and Production Method for Anti-MerTK Bispecific Antibody of the Present Invention>.

[0182] <Host Cells Transformed for the Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention> The present invention also provides host cells transformed for the anti-MerTK-anti-TTR bispecific antibody of the present invention (hereinafter referred to as "host cells for the anti-MerTK-anti-TTR bispecific antibody of the present invention"). A person skilled in the art can carry out the above-mentioned procedure for <Transformed Host Cells of the Present Invention>, except for the polynucleotide contained in the host cells for the anti-MerTK-anti-TTR bispecific antibody of the present invention.

[0183] In one embodiment, the host cell for the anti-MerTK-anti-TTR bispecific antibody of the present invention comprises a polynucleotide selected from the group consisting of the following (1) to (3): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 16, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-TTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-TTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43; (2) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 18, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43; and (3) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 8; a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acids 1 to 110 of SEQ ID NO: 20; a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 1 to 119 of SEQ ID NO: 43; and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acids 135 to 245 of SEQ ID NO: 43.

[0184] <Medicinal Uses of the Anti-MerTK-Anti-TTR Bispecific Antibody of the Present Invention> The present invention also provides a pharmaceutical composition comprising the anti-MerTK-anti-TTR bispecific antibody of the present invention and a pharmaceutically acceptable excipient (hereinafter referred to as the "anti-MerTK-anti-TTR bispecific antibody of the present invention or a pharmaceutical composition containing the same"). The pharmaceutical composition can be prepared in accordance with the description in <Medicinal Uses of the Anti-MerTK Bispecific Antibody of the Present Invention> above.

[0185] The anti-MerTK-anti-TTR bispecific antibodies of the present invention or pharmaceutical compositions containing them can be used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0186] In one embodiment, the disease to be prevented or treated by the anti-MerTK-anti-TTR bispecific antibody of the invention or a pharmaceutical composition containing the same is a neurological disease, a cardiac disease, an ocular disease, or an endocrine / metabolic disease. In one embodiment, the disease to be prevented or treated by the anti-MerTK-anti-TTR bispecific antibody of the invention or a pharmaceutical composition containing the same is amyloidosis (e.g., localized amyloidosis or systemic amyloidosis). In one embodiment, the amyloidosis to be prevented or treated by the anti-MerTK-anti-TTR bispecific antibody of the invention or a pharmaceutical composition containing the same is systemic amyloidosis. In one embodiment, the systemic amyloidosis to be prevented or treated by the anti-MerTK-anti-TTR bispecific antibody of the invention or a pharmaceutical composition containing the same is ATTR-CM and ATTR-PN.

[0187] <Anti-MerTK-anti-IgL Bispecific Antibodies of the Present Invention> The present invention provides anti-MerTK-anti-IgL bispecific antibodies that bind to both MerTK and misfolded IgL.

[0188] IgL is a protein consisting of 210 amino acids with a molecular weight of 25 kDa. It is produced by plasma cells, a type of white blood cell found in the bone marrow. Normally, IgL forms a heterodimer with immunoglobulin heavy chains (IgH), which are also produced by plasma cells, and is responsible for the humoral immunity of the adaptive immune system. However, excessive production of misfolded IgL by abnormal plasma cells leads to the deposition of IgL-derived amyloid in multiple organs, including the heart, kidneys, gastrointestinal tract, and nerves (Drugs, 2023, 83:203-216). Misfolded IgL is the protein responsible for AL amyloidosis, a type of systemic amyloidosis. It has been suggested that antibodies against misfolded IgL may inhibit the deposition of misfolded IgL and improve its function (Br. J. Haematol., 2020, 189: 228-238). However, like the monoclonal anti-TTR IgG1 antibody, these antibodies also activate the phagocytic activity of phagocytes via the Fc region, and therefore may induce the onset of cytokine release syndrome.

[0189] 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-MerTK antibody and the heavy chain variable region and light chain variable region of an antibody that binds to misfolded IgL. The anti-MerTK-anti-IgL bispecific antibody of the present invention comprises the heavy chain variable region and light chain variable region of the anti-MerTK antibody shown below: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

[0190] In one embodiment, the anti-MerTK-anti-IgL 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 the following (1) to (6): (1) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 18; or (6) A heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20.

[0191] In one embodiment, an anti-MerTK-anti-IgL bispecific antibody of the invention comprises a heavy chain fragment comprising the heavy chain variable region of an anti-MerTK antibody, and a light chain comprising the light chain variable region of an anti-MerTK antibody. In one embodiment, an anti-MerTK-anti-IgL bispecific antibody of the invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment comprising the heavy chain variable region and a light chain comprising the light chain variable region of an anti-MerTK antibody, and an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide.

[0192] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a single-arm anti-MerTK antibody comprising a heavy chain fragment and a light chain selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 217 of SEQ ID NO: 8, and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) A heavy chain fragment consisting of the amino acid sequence from amino acid numbers 1 to 217 of SEQ ID NO: 8, and a light chain fragment consisting of the amino acid sequence of SEQ ID NO: 20.

[0193] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a single-armed anti-MerTK antibody and an scFv or Fab region comprising the heavy and light chain variable regions of an anti-IgL antibody that binds to misfolded IgL. In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention is a bispecific antibody in which an scFv or Fab region comprising the heavy and light chain variable regions 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-armed anti-MerTK antibody.

[0194] The anti-MerTK-anti-IgL bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of an anti-IgL antibody that binds to misfolded IgL. The anti-IgL antibody is not particularly limited as long as it binds to misfolded IgL, and for example, the 2A4 antibody (Japanese Patent No. 5730020) can be used.

[0195] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the invention comprises the heavy chain variable region and the light chain variable region of an anti-IgL antibody that binds to misfolded IgL shown below: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence from amino acid number 26 to 35 of SEQ ID NO:57, CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO:57, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 108 of SEQ ID NO:57; and a light chain variable region comprising CDR1 consisting of the amino acid sequence from amino acid number 24 to 39 of SEQ ID NO:59, CDR2 consisting of the amino acid sequence from amino acid number 55 to 61 of SEQ ID NO:59, and CDR3 consisting of the amino acid sequence from amino acid number 94 to 103 of SEQ ID NO:59.

[0196] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the invention comprises the heavy chain variable region and the light chain variable region of the following anti-IgL antibody that binds to misfolded IgL: a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO:57, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 112 of SEQ ID NO:59.

[0197] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an antigen-binding fragment of an anti-IgL antibody that binds to misfolded IgL. In one embodiment, the antigen-binding fragment of an anti-IgL antibody that binds to misfolded IgL comprised in the anti-MerTK-anti-IgL bispecific antibody of the present invention is an scFv, a Fab fragment, a Fab' fragment, or an F(ab') fragment. ２ In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a Fab fragment comprising the heavy chain variable region and the light chain variable region of an anti-IgL antibody that binds to misfolded IgL (herein referred to as "anti-IgL-Fab").

[0198] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention is a crossMab (IgG-kih) type bispecific antibody in which the C-terminus of an anti-IgL-Fab is linked to the N-terminus of the second Fc polypeptide of a single-armed anti-MerTK antibody via a hinge region.

[0199] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an anti-IgL-Fab comprising a fragment consisting of a heavy chain variable region and a light chain constant region comprising the amino acid sequence of amino acids 1 to 226 of SEQ ID NO: 57, and a fragment consisting of a light chain variable region and a CH1 domain comprising SEQ ID NO: 59.

[0200] When the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may contain mutations that reduce ADCC, CDC, or ADCP and / or mutations that promote heterodimer formation. The mutations are similar to those described above in <Anti-MerTK-anti-TTR bispecific antibody of the present invention>.

[0201] In one embodiment, an anti-MerTK-anti-IgL bispecific antibody of the invention comprises an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide. In one embodiment, an anti-MerTK-anti-IgL bispecific antibody of the invention comprises a first Fc polypeptide and a second Fc polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having 1 to 10 substitutions in the amino acid sequence of SEQ ID NO: 39.

[0202] In one embodiment, an anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an Fc region comprising knobs-into-holes mutations. In one embodiment, an anti-MerTK-anti-IgL bispecific antibody of the present invention comprises an Fc region comprising one or more mutations selected from the group consisting of an LALA mutation, a P331G mutation, a P329A mutation, and knobs-into-holes mutations. The knobs-into-holes mutations are as described above in <Anti-MerTK-anti-TTR bispecific antibody of the present invention>.

[0203] In one embodiment, the first polypeptide and the second polypeptide comprised in the Fc region of the anti-MerTK-anti-IgL bispecific antibody of the present invention comprise polypeptides selected from the following (1) or (2): (1) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41; or (2) a first Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 41, and a second Fc polypeptide consisting of the amino acid sequence of SEQ ID NO: 40.

[0204] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention may comprise a hinge region consisting of the amino acid sequence of SEQ ID NO: 42.

[0205] Those skilled in the art can easily prepare full-length anti-MerTK-anti-IgL bispecific antibodies (for example, crossMab (IgG-kih) type anti-MerTK-anti-IgL bispecific antibodies) by combining the sequences of the anti-MerTK antibody, anti-IgL-Fab, hinge region, Fc region, and the like described above in <Anti-MerTK-anti-IgL bispecific antibodies of the present invention>.

[0206] In one embodiment, the anti-MerTK-anti-IgL bispecific antibody of the present invention may be post-translationally modified, in accordance with the description of <Anti-MerTK bispecific antibody of the present invention> above.

[0207] <Fusions and conjugates of the anti-MerTK-anti-IgL bispecific antibody of the present invention, and cells displaying the anti-MerTK-anti-IgL bispecific antibody of the present invention on their surface> The anti-MerTK-anti-IgL bispecific antibody of the present invention provides fusions of the anti-MerTK-anti-IgL bispecific antibody of the present invention linked to proteins (including polypeptides) other than MerTK and misfolded IgL, and antibodies (including antigen-binding fragments) other than anti-MerTK antibodies and anti-IgL antibodies. The anti-MerTK-anti-IgL bispecific antibody of the present invention also provides conjugates of the anti-MerTK-anti-IgL bispecific antibody of the present invention linked to a modifying agent. Furthermore, the anti-MerTK-anti-IgL bispecific antibody of the present invention provides cells (e.g., tandem CAR) in which the anti-MerTK-anti-IgL bispecific antibody of the present invention is displayed on the surface of the cell (e.g., immunocompetent cell or effector cell) (herein referred to as "cells in which the anti-MerTK-anti-IgL bispecific antibody of the present invention is displayed on the cell surface") (Oncology Reports, 2019, 42: 2183-2195). The fusion, the complex, and the cell in which the bispecific antibody is displayed on the cell surface can be produced in accordance with the description above in <The fusion of the present invention, the complex of the present invention, and the cell in which the anti-MerTK antibody or antigen-binding fragment thereof is displayed on the cell surface>.

[0208] <Polynucleotides for anti-MerTK-anti-IgL bispecific antibodies of the present invention> The present invention also provides polynucleotides for use in producing anti-MerTK-anti-IgL bispecific antibodies. Those skilled in the art can produce such polynucleotides in accordance with the descriptions in the above sections <Polynucleotides of the present invention> and <Polynucleotides for anti-MerTK bispecific antibodies of the present invention>.

[0209] In one embodiment, the polynucleotide used for producing the anti-MerTK-anti-IgL bispecific antibody of the present invention is a polynucleotide selected from the group consisting of the following (1) to (9): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16; (6) A polynucleotide comprising a base sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 18; (7) A polynucleotide comprising a base sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence from amino acid numbers 1 to 110 of SEQ ID NO: 20; (8) A polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence from amino acid numbers 1 to 119 of SEQ ID NO: 57; and (9) A polynucleotide comprising a base sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence from amino acid numbers 1 to 112 of SEQ ID NO: 59.

[0210] Those skilled in the art can prepare the polynucleotides described in this section based on the nucleotide sequences using methods known in the art.

[0211] <Expression Vector and Production Method for Anti-MerTK-Anti-IgL Bispecific Antibody of the Present Invention> The present invention also provides an expression vector and production method used for producing the anti-MerTK-anti-IgL bispecific antibody of the present invention. Those skilled in the art can implement the vector and method in accordance with the descriptions in the above sections <Polynucleotide of the Present Invention>, <Expression Vector of the Present Invention>, <Method of Producing Anti-MerTK Antibody of the Present Invention>, and <Polynucleotide, Expression Vector, Transformed Host Cell, and Production Method for Anti-MerTK Bispecific Antibody of the Present Invention>.

[0212] <Transformed host cells for the anti-MerTK-anti-IgL bispecific antibody of the present invention> The present invention also provides host cells transformed for the anti-MerTK-anti-IgL bispecific antibody of the present invention (hereinafter referred to as "host cells for the anti-MerTK-anti-IgL bispecific antibody of the present invention"). A person skilled in the art can carry out transformation of the host cells for the anti-MerTK-anti-IgL bispecific antibody of the present invention in accordance with the description of the above <Transformed host cells of the present invention>, except for the polynucleotide contained in the host cells.

[0213] In one embodiment, the host cell for the anti-MerTK-anti-IgL bispecific antibody of the present invention comprises a polynucleotide selected from the group consisting of the following (1) to (4): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59; (2) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59; (3) A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59;and (4) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 57, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-IgL antibody consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 59;

[0214] <Medicinal Uses of the Anti-MerTK-Anti-IgL Bispecific Antibodies of the Present Invention> The present invention also provides pharmaceutical compositions comprising the anti-MerTK-anti-IgL bispecific antibodies of the present invention and a pharmaceutically acceptable excipient (hereinafter referred to as "anti-MerTK-anti-IgL bispecific antibodies of the present invention or pharmaceutical compositions containing them"). The pharmaceutical compositions can be prepared in accordance with the description above in <Medicinal Uses of the Anti-MerTK Bispecific Antibodies of the Present Invention>.

[0215] The anti-MerTK-anti-IgL bispecific antibodies of the present invention or pharmaceutical compositions containing them can be used for the prevention and / or treatment of diseases caused by the accumulation of misfolded proteins.

[0216] In one embodiment, the disease to be prevented or treated by the anti-MerTK-anti-IgL bispecific antibody of the invention or a pharmaceutical composition containing same is a kidney disease. In one embodiment, the disease to be prevented or treated by the anti-MerTK-anti-IgL bispecific antibody of the invention or a pharmaceutical composition containing same is amyloidosis (e.g., localized amyloidosis or systemic amyloidosis). In one embodiment, the amyloidosis to be prevented or treated by the anti-MerTK-anti-IgL bispecific antibody of the invention or a pharmaceutical composition containing same is systemic amyloidosis. In one embodiment, the systemic amyloidosis to be prevented or treated by the anti-MerTK-anti-IgL bispecific antibody of the invention or a pharmaceutical composition containing same is AL amyloidosis.

[0217] To provide a further understanding of the present invention, reference is now made to specific examples which are provided for purposes of illustration and not limitation.

[0218] Example 1: Preparation of anti-MerTK antibodies Example 1-1: Obtaining anti-MerTK antibodies (1) Preparation of MerTK-Fc fusion protein and MerTK-His protein A polynucleotide encoding the extracellular domain of human MerTK (comprising the amino acid sequence from amino acid numbers 21 to 505 of UniProt Q12866) or a polynucleotide encoding the extracellular domain of mouse MerTK (comprising the amino acid sequence from amino acid numbers 19 to 497 of UniProt Q60805) was ligated with Factor Xa protease (New England Polynucleotides linked to polynucleotides encoding amino acid sequences cleaved by pFUSE-hIgG1-Fc1 (InvivoGen, P8010L) were inserted into the pFUSE-hIgG1-Fc1 vector (InvivoGen, pfuse-hg1fc1) to prepare expression vectors encoding a protein in which human MerTK is fused with human Fc (hereinafter referred to as "human MerTK-human Fc fusion protein") and a protein in which mouse MerTK is fused with human Fc (hereinafter referred to as "mouse MerTK-human Fc fusion protein"). Furthermore, the polynucleotide encoding the extracellular domain of human MerTK was tandemly linked to a polynucleotide encoding a His tag (SEQ ID NO: 21), and inserted into a pcDNA3.4 TOPO (registered trademark) vector (Thermo Fisher Scientific) to prepare an expression vector encoding a protein in which a His tag is linked to human MerTK (hereinafter referred to as "human MerTK-His protein"). The prepared expression vector was transfected into ExpiCHO-S cells (Thermo Fisher Scientific, A29127), and recombinant protein was obtained according to standard methods.

[0219] (2) Obtaining a fully human anti-MerTK agonist antibody AlivaMab mice (Ablexis, U.S. Patent No. 9,346,873) were immunized several times with human MerTK / Mer protein, His Tag (AcroBiosystems, MEK-H52H6) and mouse MerTK protein prepared by cleaving mouse MerTK-human Fc fusion protein with Factor Xa protease and purifying it. Lymphocytes were collected from the lymph nodes of the immunized mice according to standard methods. Based on the description in the paper (BMC Biol., 2012, 10:80), B cells binding to fluorescently labeled human MerTK-His protein and mouse MerTK-human Fc fusion protein were sorted using a cell sorter (Becton Dickinson, FACSMelody) from lymphocytes recovered from mice. Nucleic acids encoding the heavy and light chain variable regions of antibodies extracted from the sorted B cells were cloned according to standard methods. Monoclonal antibodies were produced from the sequence information according to standard methods. The produced antibodies were evaluated for their binding activity to human and mouse MerTK recombinant proteins, their binding activity to MerTK on the cell membrane of mouse bone marrow-derived macrophages, their ability to induce MerTK internalization into cells, and their ability to induce AKT phosphorylation, a downstream signal of MerTK. As a result, a fully human anti-MerTK agonist antibody was obtained that exhibits binding activity to human and mouse MerTK recombinant proteins, binding activity to MerTK on the cell membrane of mouse bone marrow-derived macrophages, the ability to induce MerTK internalization into cells, and the activity to induce AKT phosphorylation. The sequence of the heavy chain variable region of this anti-MerTK agonist antibody corresponds to amino acids 1 to 119 of SEQ ID NO:8, and the sequence of the light chain variable region corresponds to amino acids 1 to 110 of SEQ ID NO:16.

[0220] (3) Preparation and Identification of Fully Human Anti-MerTK Antibodies Introduced with Fc Mutations 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 and light chains were designed. Specifically, a polypeptide was designed in which the amino acid sequence of the human λ chain constant region was linked to the C-terminus of the light chain variable region, and a polypeptide was designed in which the amino acid sequence of the human Igγ1 constant region was linked to the C-terminus of the heavy chain variable region. Furthermore, amino acid mutations L234A, L235A, and P331G were introduced into the heavy chain constant region. This resulted in the design of tA3-20, a fully human anti-MerTK antibody consisting of a heavy chain of SEQ ID NO: 8 and a light chain of SEQ ID NO: 10.

[0221] In general, N-linked glycosylation in antibody variable regions can cause unstable quality during antibody production. Therefore, mutations were introduced into the amino acid sequence of tA3-20 to avoid glycosylation that may occur on the framework of the light chain variable region of tA3-20. Specifically, a light chain (SEQ ID NO: 12) in which the asparagine at the 18th framework amino acid of SEQ ID NO: 10 was substituted with threonine was designed for tA3-20, and a light chain (SEQ ID NO: 14) in which the asparagine at the 18th amino acid of SEQ ID NO: 10 was substituted with serine was designed for tA3-20. Furthermore, light chains (SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20) were designed by adding asparagine to the N-terminus of the light chains (SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14). In this way, human anti-MerTK antibodies consisting of a heavy chain (SEQ ID NO: 8) and a light chain (SEQ ID NO: 12, 14, or 20) were designed. Fully human anti-MerTK antibodies consisting of a heavy chain of SEQ ID NO: 8 and a light chain of SEQ ID NO: 12, 14, 16, 18, or 20 are designated tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, or tA3-20.5, respectively. Information on the amino acid sequences of the heavy chain variable region and light chain variable region of the fully human anti-MerTK antibodies is shown in Table 1.

[0222]

[0223] The amino acid sequences of CDR1 to 3 of SEQ ID NO:8 are shown in SEQ ID NOs:1 to 3, and the amino acid sequences of CDR1 to 3 of SEQ ID NOs:10, 12, 14, 16, 18 and 20 are shown in SEQ ID NOs:4 to 6, respectively.

[0224] A mouse-human chimeric anti-MerTK antibody (hereinafter 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 SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 34 in Patent Document 4. A mouse-human chimeric anti-MerTK antibody (hereinafter referred to as "M6") was designed in the same manner as in (3) based on the amino acid sequences of the heavy chain variable region of SEQ ID NO: 49 and the light chain variable region of SEQ ID NO: 50 in WO 2016 / 106221.

[0225] Polynucleotides encoding the heavy chain or light chain of tA3-20, tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, tA3-20.5, MTK201, and M6 were prepared, and each polynucleotide was inserted into a pcDNA3.4 TOPO vector according to a standard method to prepare expression vectors for the heavy chain and light chain of tA3-20, tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, tA3-20.5, MTK201, or M6. The combinations of polynucleotide sequences encoding the heavy and light chains of each of 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 amounts of heavy and light chain expression vectors were introduced into ExpiCHO-S cells, and the cells were cultured at 37°C, 5% CO ２ Culture was initiated in an incubator. The next day, the reagents included in the kit (ExpiCHO Enhancer and ExpiCHO Feed) were added according to the protocol, and the cells were cultured to secrete the antibody into the culture supernatant.

[0226]

[0227] Antibodies were purified from the resulting culture supernatants by affinity purification using MabSelect SuRe (Cytiva, 175438), or by a combination of affinity purification using MabSelect SuRe pcc (Cytiva, 17549102) and anion exchange chromatography using Sartobind® Lab Q100 (Sartorius Stedim, 93IEXQ42BC-12). The presence or absence of protein expression and the degree of purification of the purified antibodies were confirmed by SDS-PAGE under reducing and non-reducing conditions or size exclusion chromatography using an ACQUITY UPLC Protein BEH SEC Column (Waters, 186005225).

[0228] Example 1-2: Evaluation of binding activity of anti-MerTK antibodies to human MerTK The binding activity of tA3-20, tA3-20.1, and tA3-20.2 to human MerTK was evaluated by ELISA. The human MerTK-His protein prepared in Example 1-1(1) was adjusted to 1 μg / mL with PBS, and 20 μL was added per well to a well plate (Clear Flat-Bottom Immuno Nonsterile 384-Well Plate, Thermo Fisher Scientific, 464718). The protein was immobilized on the plate by leaving it at 4°C overnight. The human MerTK-His protein was then removed. A blocking solution was prepared by adding Blocking One (Nacalai Tesque, 03953-95) to a final concentration of 20% in TBS containing 0.05% Tween-20 (prepared by diluting 0.5% Tween-20-containing TBS, Nippon Gene Co., Ltd., 310-07375, 10-fold; hereafter referred to as "TBS-T"). 50 μL of this solution was added per well and allowed to stand for 1 hour for blocking. The wells were then washed once with TBS-T. Various anti-MerTK antibodies and an isotype control antibody, an anti-hen egg white lysozyme antibody (LYS_1c3-m1_h1i, prepared in-house), were serially diluted from 10,000 ng / mL to 0.006 ng / mL. Antibodies were diluted in TBS-T containing 5% Blocking One. 20 μL of diluted antibodies were added to each well and allowed to react at room temperature for 1 hour. After removing the antibodies, the wells were washed three times with TBS-T, and then 15 μL of secondary antibody (Goat Anti-Human IgG Fc, Multi-Species SP ads-HRP (SouthernBiotech, 2014-05) was prepared by diluting 4,000-fold with TBS-T containing 5% Blocking One) was added to each well and allowed to react for 1 hour at room temperature. After washing the wells three times with TBS-T, 15 μL of TMB-Plus Substrate-Chromogen (Dako, S1599) was added to each well and allowed to stand at room temperature for 5 minutes.The reaction was stopped by adding 1 mol / L sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd., 198-09595), and the absorbance at 450 nm and 570 nm was measured using an Infinite M200 PRO (Tecan). Binding of tA3-20.3, tA3-20.4, and tA3-20.5 to human MerTK was also evaluated using the same method as above.

[0229] As a result of the measurement, tA3-20, tA3-20.1, and tA3-20.2 all had binding activity to human MerTK in a concentration-dependent manner, and the binding activities were equivalent, as shown in Figure 1. Furthermore, it was confirmed that tA3-20.3, tA3-20.4, and tA3-20.5 also had binding activity to human MerTK equivalent to that of tA3-20.

[0230] Example 1-3: Analysis of species cross-reactivity of anti-MerTK agonist antibody tA3-20 The binding activity of tA3-20 to MerTK proteins from 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 Chimera Protein, CF (R&D Systems, 10576-MR-050) obtained in Example 1-1 were diluted to 1 μg / mL with PBS. The diluted solution was added to a well plate (Clear Flat-Bottom Immuno Nonsterile 384-Well Plate) at 20 μL per well, and the protein was immobilized on the plate overnight at 4°C. The next day, the plate with the immobilized protein was washed three times with TBS-T (Thermo Fisher Scientific, 28360). 100 μL of Blocking One was added per well, and the plate was blocked at room temperature for 1 hour. After blocking, the plate was washed three times with TBS-T. tA3-20 was serially diluted 4-fold from a maximum concentration of 20 μg / mL using TBS-T containing 10% Blocking One (hereinafter referred to as "10% diluted solution"), and 20 μL was added per well. The plate to which the antibody was added was incubated at room temperature for 1 hour and then washed three times with TBS-T. The secondary antibody was HRP-labeled anti-human IgG F(ab') diluted 10,000 times with a 10% dilution solution. ２ (abcam, ab98535) was added at 20 μL per well. After incubation at room temperature for 1 hour, the plates were washed three times with TBS-T, and 20 μL per well of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, 34028) was added. After incubation at room temperature for 15 minutes, the reaction was stopped by adding 1 mol / L sulfuric acid, and the absorbance at 450 nm was measured using a SpectraMax (Molecular Devices).

[0231] The measurement results confirmed that tA3-20 has concentration-dependent binding activity to all of the human, mouse, rat, and monkey MerTK proteins. tA3-20.1, tA3-20.2, tA3-20.3, tA3-20.4, and tA3-20.5, which have the same CDR sequences as tA3-20, are also thought to have binding activity not only to human MerTK but also to mouse, rat, and monkey MerTK proteins, similar to tA3-20.

[0232] [Example 2: Confirmation of the activity of tA3-20 on phagocytosis by human retinal pigment epithelial (RPE) cells] [Example 2-1: Confirmation of the activity of tA3-20 on phagocytosis of apoptotic cells by human RPE cells] The activation of the phagocytic removal function of phagocytes by tA3-20 was evaluated using as an indicator the activation of phagocytosis of apoptotic cells by RPE cells, a type of phagocyte.

[0233] The human RPE cells used were iCell® Retinal Pigment Epithelial Cells-01279 (Fujifilm Wako Pure Chemical Industries, Ltd., C1046), which are iPS cell-derived RPE cells. The medium used consisted of MEMα (Thermo Fisher Scientific, 12571-063) supplemented with 25 μg / mL of Gentamicin (Thermo Fisher Scientific, 15750-060), 1x N-2 supplement (Thermo Fisher Scientific, 17502-048), and 1x B-27 supplement (Thermo Fisher Scientific, 17502-048). The following antibodies were used: 55 nM hydrocortisone (Merck, H0888-1G), 0.25 mg / mL taurine (Merck, T8691-25G), and 14 pg / mL 3,3',5-triiodo-L-thyronine sodium salt (Merck, T5516-1MG). One vial of iCell® Retinal Pigment Epithelial Cells-01279 was evenly seeded into a 96-well plate of a CellCarrier-96 (PerkinElmer, 6055300) and incubated at 37°C and 5% CO. ２Jurkat cells (ATCC, TIB-152) were cultured at 1 × 10 in a culture medium (hereinafter referred to as "culture medium") prepared by adding 10% FBS (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Merck, P4458-100ML) to a final concentration of RPMI 1640 (Merck, R8758-500ML) at a concentration of 1 × 10 ６ The Jurkat cells were adjusted to a concentration of 100 ng / mL. The adjusted concentration was treated with SUPERFASLIGAND® Protein (soluble) (human), (recombinant) (Enzo Life Sciences, ALX-522-020-3005) at a final concentration of 100 ng / mL at 37°C for 3 hours to induce apoptosis. The apoptosis-induced Jurkat cells (hereinafter referred to as "apoptotic Jurkat cells") were diluted to 2 x 10 in 20 mM CHES / PBS buffer, pH 9.0 (prepared in-house). ６ The cells were adjusted to a final concentration of 100 ng / mL and then labeled with pHrodo Red, SE (Thermo Fisher Scientific, P36600) at a final concentration of 400 ng / mL for 20 minutes at room temperature. The labeled apoptotic Jurkat cells were diluted to 6 × 10 in culture medium. ６ The concentration was adjusted to 1000 cells / mL.

[0234] The test antibodies used were tA3-20, MTK201, and LYS_1c3-m1_h1i (isotype control). These antibodies were adjusted to a concentration of 10 μg / mL using culture medium. 50 μL of each test antibody and pHrodo-labeled apoptotic Jurkat cells were added per well to an iPS-RPE cell culture plate that had been washed once with RPMI 1640. The iPS-RPE cell culture plate was incubated in a 5% CO atmosphere. ２The cells were placed in an Incucyte S3 live cell analyzer (Sartorius) placed in a 37°C incubator. Images were measured every hour for 24 hours using bright field and red laser. The images were analyzed using the analysis tools and algorithms built into the Incucyte system software to calculate the total red fluorescence intensity (Red Calibrated Unit [RCU] × μm ２ / Image) was used as an index of the amount of phagocytosis.

[0235] The results of the evaluation of phagocytic activation are shown in Figure 2-1. Analysis revealed that tA3-20 exhibited a significant increase in fluorescence intensity, peaking approximately 16 hours after the start of the evaluation, indicating significant activation of phagocytosis. These results indicate that tA3-20 activates phagocytosis by RPE cells via MerTK binding. Furthermore, the activation of phagocytosis by tA3-20 was significantly greater than that of MTK201. These results suggest that tA3-20 has a stronger activation effect on phagocytic clearance function than MTK201. Given 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), tA3-20 is expected to activate the phagocytic clearance function not only in RPE cells but also in other phagocytes (e.g., macrophages, etc.).

[0236] Example 2-2: Confirmation of activity of tA3-20 on phagocytosis of POS by human RPE cells The activation of the phagocytic removal function of RPE cells by tA3-20 was evaluated using activation of phagocytosis of porcine POS as an index.

[0237] Porcine POS was prepared according to the method of S. Alemedawar et al. (Stem Cell Reports, 2020, 14: 374-389). The prepared POS was diluted to 3 × 10 with 100 mM sodium bicarbonate (pH 8.2, prepared in-house). ７ After adjusting the POS concentration to 1000 kJ / mL, the cells were labeled with pHrodo Red, SE at a final concentration of 1 μM for 1 hour at room temperature with stirring. ６ The concentration was adjusted to POS / mL.

[0238] The test antibodies used were tA3-20, MTK201, M6, and LYS_1c3-m1_h1i (isotype control). These antibodies were adjusted to a concentration of 10 μg / mL using culture medium. 50 μL of each test antibody and pHrodo-labeled POS were added per well to an iPS-RPE cell culture plate that had been washed once with DMEM. The iPS-RPE cell culture plate was incubated in a 5% CO atmosphere. ２ The cells were placed in an Incucyte S3 live cell analyzer placed in a 37°C incubator. Images were taken every hour for 60 hours using bright field and red laser. The images were analyzed using the analysis tools and algorithms built into the Incucyte system software to calculate the total red fluorescence intensity (Red Calibrated Unit [RCU] × μm ２ / Image) was calculated and used as an index of the amount of phagocytosis.

[0239] The results of the evaluation of activation of POS phagocytosis are shown in Figure 2-2. As a result of the analysis, tA3-20 was shown to activate phagocytosis in POS as well. This result suggests that tA3-20 also has the effect of activating the phagocytic removal function in POS.

[0240] The results of Example 2-2 showed that tA3-20 activated phagocytosis more potently than MTK201 and M6, suggesting that tA3-20 has a stronger activation effect on phagocytic removal function than MTK201 and M6.

[0241] Example 3: Confirmation of the effect of intravitreally administered tA3-20 on inducing phosphorylation of MerTK and AKT in the RPE. Whether tA3-20 can act as an agonist against MerTK on the RPE in vivo was evaluated using mice, using activation of the downstream signaling pathway of MerTK as an indicator.

[0242] Example 3-1: Intravitreal administration of test antibody and collection of RPE tissue. Eight-week-old male C57BL6 / J mice (Jackson Laboratory Japan) were given Mydrin (registered trademark) P eye drops (Santen Pharmaceuticals) to dilate their eyes, followed by intraperitoneal administration of a triple-anesthesia mixture (Domitor, Nippon Zenyaku Kogyo; Dormicum (registered trademark) injection 10 mg, Maruishi Pharmaceutical; and Betolfar (registered trademark) 5 mg, Meiji Animal Health) to induce general anesthesia. Subsequently, Xylocaine (registered trademark) eye drops 4% (Sandoz Co., Ltd.) were administered to induce local anesthesia. While observing the fundus under a microscope (Zeiss OPMI LUMERA 300), 1 μL of the test antibody was administered intravitreally using a microsyringe (Ito Seisakusho, MS-N05) and a 37-gauge needle (Ito Seisakusho). The test antibodies (PBS was used as the solvent) were 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). After intravitreal administration, Cravit® ophthalmic solution 0.5% (Santen Pharmaceuticals) was instilled into the treated eye, Tarivid® ophthalmic ointment 0.3% (Santen Pharmaceuticals) was applied, and Antisedan® (Nippon Zenyaku Kogyo) was injected subcutaneously to awaken the mice. One hour after intravitreal administration of the test antibodies, the mice were euthanized, the eyeballs were enucleated, and the RPE tissue was collected and cryopreserved until use. This experiment was performed with N=4 per group.

[0243] [Example 3-2: Preparation of SDS-PAGE sample] The collected mouse RPE was homogenized in 10 μL of RIPA Buffer (Merck, R0278) containing Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) (Thermo Fisher Scientific, 78441). The homogenate was centrifuged at 9,200 × g for 1 minute, and the supernatant was collected in a sampling 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 the mixture was heat-treated at 70°C for 10 minutes, after which it was used as a sample for SDS-PAGE electrophoresis. Four microliters of the sample for SDS-PAGE electrophoresis was subjected to SDS-PAGE 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 Device (Thermo Fisher Scientific, IB21001). The PVDF membrane after transfer (hereinafter referred to as "membrane") was blocked for at least 1 hour at room temperature using a blocking reagent (PVDF Blocking Reagent for Can Get Signal, TOYOBO, NYPBR01). Then, the membrane was reacted overnight at 4°C with anti-phosphorylated MerTK antibody (Merck, SAB4504621-100UG) or anti-phosphorylated AKT antibody (Cell Signaling Technology, #4060) diluted 1,000-fold with Can Get Signal Solution 1 (TOYOBO, NKB-201).The antibody-reacted membrane was washed with TBS-T (hereinafter referred to as "washing buffer") containing 1% BlockAce (Megmilk Snow Brand, UKB40) and then reacted with HRP-labeled anti-rabbit antibody (Cytiva, NA9340V) diluted 10,000-fold with Can Get Signal Solution 2 (TOYOBO, NKB-301) at room temperature for 1 hour. The antibody-reacted membrane was 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 on the same membrane, the membrane after detection was washed with stripping buffer (Restore PLUS The antibodies were stripped using Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430), and the cells were blocked with a blocking reagent (PVDF Blocking Reagent for Can Get Signal) at room temperature for 1 hour. Subsequently, the cells were reacted overnight at 4°C with anti-MerTK antibody (Cell Signaling Technology, #38102) or anti-AKT antibody (Cell Signaling Technology, #4691) diluted 3,000-fold with Can Get Signal Solution 1. Total MerTK or total AKT was detected using ECL. This was carried out in the same manner as above using Prime (Cytiva, RPN2236).

[0244] [Example 3-3: Analysis] The detected bands were quantified using Image Lab software version 6.0.0 build 25 (BioRad). The numerical value of each phosphorylated protein band was divided by the numerical value of each total protein band to obtain a corrected value.

[0245] The results of MerTK phosphorylation are shown in Figure 3-1. Compared to the isotype control antibody LYS_1c3-m1_h1i at 14.47 mg / mL (1 μL / eye), tA3-20 demonstrated enhanced MerTK phosphorylation from administration at 0.1447 mg / mL (1 μL / eye). The results of AKT phosphorylation are shown in Figure 3-2. Similar to the results of MerTK phosphorylation, tA3-20 demonstrated enhanced AKT phosphorylation from administration at 0.1447 mg / mL (1 μL / eye), compared to the isotype control antibody LYS_1c3-m1_h1i at 14.47 mg / mL (1 μL / eye), and this activity was dose-dependent. tA3-20 was found to enhance the phosphorylation of MerTK expressed in the RPE and AKT downstream of MerTK under in vivo conditions.

[0246] The results of Examples 2 and 3 suggest that tA3-20 is expected to enhance the phagocytic clearance function of the RPE via the MerTK downstream signaling pathway in vivo, and is useful for preventing or treating diseases associated with a decrease in the phagocytic clearance function of the RPE.

[0247] Example 4 Preparation of Mouse Anti-MerTK-Anti-TTR Bispecific Antibody, Mouse Anti-TTR Antibody, and TTR Protein Mouse anti-MerTK-anti-TTR bispecific antibody, mouse anti-TTR antibody, and misfolded recombinant human TTR V30M protein were prepared.

[0248] Example 4-1: Preparation of recombinant human TTR V30M protein Purified recombinant human TTR V30M protein was prepared with reference to the method of Matsubara et al. (Protein Expr. Purif., 2003, 30:55-61). A gene sequence encoding human TTR V30M (a mutant consisting of the mature peptide region of human TTR in which the 30th valine is changed to methionine) was inserted into the expression vector pQE-30 (QIAGEN, N-Terminus pQE Vector Set, 32915) to obtain pQE30_GOI_02. E. coli JM109 Competent Cells (Takara Bio, 9052) were transformed with pQE30_GOI_02. A final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (Nacalai Tesque, 19742-94) was added to LB liquid medium (prepared in-house), and the transformed E. coli was cultured in liquid at 37°C for approximately 16 hours. The E. coli cells harvested after culture were solubilized using xTractor Buffer Kit (Clontech, 635623). The lysate was centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected. His-tagged human TTR V30M protein was purified from the collected supernatant using TALON® Metal Affinity Resin (Clontech, 635502) and His Buffer Kit (Cytiva, 11-0034-00). The eluted fraction containing recombinant human TTR V30M protein was eluted with 20 mM phosphate buffer (pH 7.0, prepared in-house) using an Amicon Ultra-4 (Merck, C7719) or a PD-10 Desalting Column (Cytiva, 17-0851-01) to obtain purified recombinant human TTR V30M protein. A portion of the purified recombinant human TTR V30M protein to be administered to mice was further subjected to endotoxin removal using Pierce High Capacity Endotoxin Removal Spin Columns (Thermo Fisher Scientific, 88276) according to the attached manual.The concentration of purified recombinant human TTR V30M protein after buffer replacement and / or endotoxin removal was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) or NanoDrop A280 assay (Thermo Fisher Scientific).

[0249] Example 4-2: Preparation of misfolded recombinant human TTR V30M protein To prepare misfolded recombinant human TTR V30M protein (herein referred to as "misfolded recombinant human TTR V30M protein"), 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 in-house) and incubated at 37°C for at least 4 days (hereinafter referred to as "misfolding treatment") to prepare a misfolded recombinant human TTR V30M protein (herein referred to as "misTTR"). A portion of the prepared misTTR solution was used to prepare a 100:1 mixture of the misTTR solution and 4 M sodium hydroxide (hereinafter referred to as "neutralized misTTR"). To confirm that both misTTR and neutralized misTTR were misfolded, an amyloid-specific fluorescent dye, thioflavin T, was assayed using purified recombinant human TTR V30M protein before misfolding as a control. Protein and thioflavin T (prepared in-house) were mixed at a ratio of 1:5, incubated at room temperature for 15 minutes, and then fluorescence measurements were performed under excitation / emission conditions of 430 / 480 nm. Significant increases in fluorescence intensity were observed in misTTR and neutralized misTTR, indicating that these TTRs were misfolded. Based on the above experiments, misTTR and neutralized misTTR were used as misfolded TTRs in subsequent studies.

[0250] Example 4-3: Labeling of misfolded recombinant human TTR V30M protein 50 μL of neutralized misTTR (2 mg / mL), 950 μL of 100 mM sodium bicarbonate (pH 8.2, prepared in-house), and 5 μL of 10 mM pHrodo were mixed and incubated at 37°C for 15 minutes to prepare pHrodo-labeled misTTR (referred to herein as "pHrodo-misTTR"). After removing the supernatant containing excess pHrodo by centrifugation, the sample was suspended in PBS and centrifuged again to remove the supernatant. The precipitated pHrodo-misTTR was resuspended in PBS and adjusted to the desired concentration. DyLight800-labeled misTTR (herein referred to as "DL800-misTTR") was prepared by mixing 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) and incubating at 25°C for 1 hour. After removing the supernatant containing excess DyLight800 by centrifugation, the mixture was suspended in PBS and centrifuged again to remove the supernatant. The precipitated DL800-misTTR was resuspended in PBS to a predetermined concentration.

[0251] Example 4-4: Preparation of mouse anti-MerTK-anti-TTR bispecific antibody A bispecific antibody consisting of a heavy chain fragment containing the heavy chain variable region of an anti-MerTK antibody, a heavy chain consisting of a hinge region and a mouse first Fc polypeptide, a light chain containing the light chain variable region of an anti-MerTK antibody, and anti-TTR-scFv, and a polypeptide in which a mouse second Fc polypeptide is linked to the hinge region (hereinafter referred to as "mouse anti-MerTK-anti-TTR bispecific antibody") was prepared by the following method: (1) Preparation of vectors encoding the heavy and light chains of mouse anti-MerTK antibody The amino acid sequences of the heavy and light chains of a mouse anti-MerTK antibody were designed. The heavy chain (SEQ ID NO: 34) was designed by linking the heavy chain variable region, hinge region, and mouse heavy chain IgG2a constant region (EU index: 215 to 447) of tA3-20, and the light chain (SEQ ID NO: 36) was designed by linking the light chain variable region of tA3-20.3 to the mouse light chain Lambda constant region (EU index: 108 to 213). The heavy chain further contains (i) the LALA mutation (L234A and L235A) in which the amino acids at amino acid numbers 236 and 237 (EU index: 234 and 235) are substituted from leucine (L) to alanine (A), respectively; (ii) the amino acids at amino acid numbers 358, 366, 370, 372, 401, and 413 (EU index: 356, 364, 368, 370, 399, and 411) are substituted from glutamic acid (E) to lysine (K), from threonine (T) to serine (S), and from amino acid (M) to methyl (M). The amino acid sequence was designed to include electrostatic steering mutations substituting thionine (M) with leucine (L), threonine (T) with lysine (K), aspartic acid (D) with lysine (K), and arginine (R) with threonine (T), as well as mutations to stabilize the heterodimerized antibody molecule (mAbs, 2019, 12: 1-12), and (iii) a mutation substituting the amino acid at amino acid number 331 (EU index: 329) from proline (P) to alanine (A). The base sequences (SEQ ID NOs: 33 and 35) encoding the amino acid sequences of the heavy and light chains of the designed mouse anti-MerTK antibody were synthesized using artificial genes and inserted into the pcDNA3.4 TOPO vector to prepare vector MHC and vector MLC, respectively.

[0252] (2) Preparation of a vector encoding mouse anti-TTR-scFv-Fc A polypeptide (hereinafter referred to as "mouse anti-TTR-scFv-Fc") (SEQ ID NO: 38) in which a hinge region and anti-TTR-scFv were linked to the N-terminus of a mouse second Fc polypeptide was designed to comprise an amino acid sequence in which the heavy chain variable region of the 371M antibody, an anti-TTR antibody that binds to misfolded TTR as shown in SEQ ID NO: 13 of WO 2015 / 115332, a GS linker (SEQ ID NO: 54), the light chain variable region of the 371M antibody as shown in SEQ ID NO: 14 of WO 2015 / 115332, a hinge region, and the constant region of mouse heavy chain IgG2a (EU index: 215 to 447) were linked in this order. The mouse anti-TTR-scFv-Fc further has the following properties: (i) stabilization of the anti-TTR-scFv located on the N-terminal side (Protein Engineering, Design and Selection, 2019, 25:321-329), (ii) a substitution of glycine (G) at amino acid number 44 (EU index: 44) of SEQ ID NO: 38 with cysteine ​​(C) and a substitution of glycine (G) at amino acid number 237 (EU index: 100) with cysteine ​​(C), (iii) a substitution of leucine (L) at amino acid numbers 265 and 266 (EU index: 234 and 235) of SEQ ID NO: 38 with alanine (A) (LALA mutations L234A and L235A), (iv) a substitution of leucine (L) at amino acid numbers 395, 399, 401, 440, 442, and 470 (EU index: 395A and 399A) of SEQ ID NO: 38 with alanine (A), (v) a substitution of leucine (L) at amino acid numbers 395, 399, 401, 440, 442, and 470 (EU index: 395A and 399A) of SEQ ID NO: 38 with alanine (A), (vi ... The antibody was designed to have an amino acid sequence (SEQ ID NO: 38) comprising electrostatic steering mutations in which amino acids (amino acids: 364, 368, 370, 409, 411, and 439) are substituted with threonine (T) to serine (S), methionine (M) to leucine (L), threonine (T) to lysine (K), lysine (K) to glutamic acid (E), arginine (R) to threonine (T), and lysine (K) to aspartic acid (D), respectively, as well as mutations for stabilizing the heterodimerized antibody molecule, and (iv) a mutation in the amino acid at amino acid number 360 (EU index: 329) of SEQ ID NO: 38 that substitutes proline (P) with alanine (A).An artificial gene was synthesized from a base sequence encoding the amino acid sequence of the designed mouse anti-TTR-scFv-Fc, and inserted into the pcDNA3.4 TOPO vector to prepare the vector TscFvFc.

[0253] (3) Preparation of Mouse Anti-MerTK-Anti-TTR Bispecific Antibody A mouse anti-MerTK-anti-TTR bispecific antibody was prepared using the vectors prepared in (1) and (2) above. Specifically, vector MHC, vector MLC, and vector TscFvFc were co-transfected into ExpiCHO-S cells using an ExpiFectamine CHO Transfection Kit, and the mouse anti-MerTK-anti-TTR bispecific antibody was secreted into the culture supernatant according to standard methods. The antibody was purified from the resulting culture supernatant by a combination of affinity purification using MabSelect SuRe pcc and ion exchange chromatography using HiTrap SP HP (Cytiva, 17115201) or size exclusion chromatography using Superdex 200 Increase 10 / 300 GL (Cytiva, 28990944). Hereinafter, the mouse anti-MerTK-anti-TTR bispecific antibody produced is referred to as tA-009.

[0254] Example 4-5: Preparation of mouse anti-TTR antibody The 371M antibody (herein referred to as "mouse 371M"), an anti-TTR antibody having a human-derived anti-TTR antibody variable region and a mouse-derived constant region, was prepared by the following method. The heavy chain was designed to have an amino acid sequence in which the heavy chain variable region of the 371M antibody, an anti-TTR antibody of SEQ ID NO: 13 in WO 2015 / 115332, was linked to the mouse heavy chain IgG2a constant region (EU index: 118 to 447) (SEQ ID NO: 44). The light chain was designed to have an amino acid sequence in which the light chain variable region of the 371M antibody of SEQ ID NO: 14 in WO 2015 / 115332, was linked to the mouse light chain Lambda1 (EU index: 108 to 213) (SEQ ID NO: 45). The base sequence encoding the designed amino acid sequence was synthesized as an artificial gene and inserted into the pcDNA3.4 TOPO vector. The mouse 371M was produced 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 according to standard methods. The antibody was purified from the resulting 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).

[0255] [Example 5: Evaluation of in vitro activity of tA-009] [Example 5-1: Evaluation of binding activity of tA-009 to human MerTK and misTTR] The binding activity of tA-009 to human MerTK and misTTR was evaluated by ELISA. The human MerTK-Fc protein prepared in Example 1-1(1) and misTTR prepared in Example 4-2 were adjusted to 1 μg / mL and 2 μg / mL, respectively, in PBS, and added at 20 μL per well to a Clear Flat-Bottom Immuno Nonsterile 384-Well Plate. The plate was immobilized by allowing to stand at room temperature for 1 hour. Thereafter, the human MerTK-Fc protein and misTTR were each removed. A blocking solution was prepared by adding Blocking One to TBS-T at a final concentration of 20%, and 50 μL of this solution was added per well and allowed to stand for 1 hour for blocking. The wells were then washed once with TBS-T. tA-009 and an anti-Keyhole Limpet Hemocyanin (KLH) antibody (KLH_173A1_ma0, produced in-house) were added at 2 × 10 ４ From 1 x 10 －２ The antibodies were serially diluted to 100 ng / mL. TBS-T containing 5% Blocking One was used for antibody dilution. 15 μL of the diluted antibodies were added to each well and allowed to react at room temperature for 1 hour. After removing the antibodies and washing the wells three times with TBS-T, 15 μL of the secondary antibody (Goat Anti-Mouse IgG, Human adz-HRP (SouthernBiotech, 1030-05) prepared by diluting 4,000-fold with TBS-T containing 5% Blocking One) was added to each well and allowed to react at room temperature for 1 hour. After washing the wells three times with TBS-T, 15 μL of TMB-Plus Substrate-Chromogen was added to each well and allowed to stand at room temperature for 5 minutes. The reaction was stopped by adding 1 mol / L sulfuric acid, and the absorbance at 450 nm and 570 nm was measured using an Infinite M200 PRO.

[0256] The measurement results revealed that tA-009 has concentration-dependent binding activity to human MerTK-Fc protein and misTTR (FIGS. 4-1 and 4-2).

[0257] [Example 5-2: Activation of phagocytic clearance function of mouse bone marrow-derived macrophage cells by tA-009] The phagocytic activity of phagocytes by tA-009 was evaluated using the phagocytic activity of misTTR by mouse bone marrow-derived macrophage cells, which are one type of phagocyte, as an indicator.

[0258] Mouse bone marrow-derived macrophage cells were prepared as follows: Myeloid cells were collected from the femur and tibia of male C57BL6 / J mice (Jackson Laboratory Japan) and cultured in RPMI-1640 medium (Sigma-Aldrich, R8758-500ML) containing 10% FBS (Cytiva, SH30070.03) and 40 ng / ml Recombinant Mouse M-CSF Protein (R&D Systems, 416-ML-010) (hereinafter referred to as "FBS-containing RPMI-1640") at 37°C and 5% CO. ２ After that, the medium was replaced with RPMI-1640 containing FBS and 0.1 μM dexamethasone (Sigma-Aldrich, D4902-100MG), and the cells were cultured at 1 × 10 ４ Cells were cultured at a cell density of 1000 cells / well at 37°C and 5% CO ２ The cells were cultured under these conditions for 3 days and then washed once with RPMI-1640 medium to prepare mouse bone marrow-derived macrophage cell culture plates. tA-009 was used as the test antibody, and tA3-20.3 was used as the control antibody. RPMI-1640 medium containing pHrodo-misTTR at a final concentration of 5 μg / mL and tA-009 at a final concentration of 0.7 μmol / L or tA3-20.3 (SEQ ID NOs: 8 and 16) as the control antibody was added to the mouse bone marrow-derived macrophage cell culture plate at a concentration of 50 μL per well. The mouse bone marrow-derived macrophage cell culture plate was incubated at 25°C for 3 days under 5% CO ２ The cells were placed in an Incucyte S3 live cell analyzer placed in an incubator at 37°C, and measurements and analyses were carried out in the same manner as in Example 2.

[0259] The analysis showed that the anti-MerTK-anti-TTR bispecific antibody tA-009 activated the phagocytosis of misTTR by mouse bone marrow-derived macrophages more significantly than the anti-MerTK antibody tA3-20.3. These results suggest that tA-009 efficiently activates the phagocytic removal function of misTTR by mouse bone marrow-derived macrophages by bringing misTTR into close proximity with mouse bone marrow-derived macrophages.

[0260] Example 6: tA-009 activates the phagocytic clearance function of phagocytes against misTTR in mice in vivo. The ability of tA-009 to activate the phagocytic activity of misTTR by phagocytes was evaluated by subcutaneously administering pHrodo-misTTR to mice and measuring its fluorescence intensity over time using in vivo imaging. pHrodo-misTTR emits fluorescence when phagocytes engulf it. Furthermore, to confirm that the activation of phagocytic activity by tA-009 is MerTK-dependent, a combination test with a MerTK inhibitor was conducted. Thirteen-week-old female BALB / c-nu (nu / nu) mice (Jackson Laboratory Japan) were subcutaneously administered 40 μL of a solution containing pHrodo-misTTR and neutralized misTTR at a protein ratio of 1:4, as prepared as in Example 4-3. At 1, 6, 12, 24, 48, 72, 120, 168, 240, and 336 hours after administration, the mice were anesthetized with isoflurane (Mylan Pharmaceuticals), and the amount of pHrodo-misTTR engulfed by accumulated phagocytes was quantified by measuring the fluorescence intensity (photons / second, [p / s]) around the injection site using an IVIS Imaging System (Lumina II; PerkinElmer). The phagocytic activity was evaluated by calculating the integrated value of the fluorescence intensity at the above measurement time points as the total amount of phagocytosis ([p / s]·h) during the observation period (14 days). The test antibody tA-009 (100 mg / kg) or the control vehicle (PBS) was administered intraperitoneally 1 hour before the subcutaneous administration of pHrodo-misTTR. The MerTK inhibitor ONO7475 (10 mg / kg, MedChemExpress, HY-114358) was orally administered once daily from day 0 to day 5, for a total of 6 doses. On the first day of the study (day 0), ONO7475 was orally administered 0.5 hours before the administration of tA-009. This study was conducted with 5 cases per group. The calculation of the mean and standard error of the total amount of pHrodo-misTTR phagocytosis and the significance test were performed using GraphPad Prism (GraphPad Software, ver. 8.0.2).Comparisons between the vehicle group and the tA-009 group, and between the tA-009 group and the tA-009+ONO7475 group were performed using Student's t-test, and a P value of less than 0.05 was considered statistically significant.

[0261] MerTK-dependent phagocytosis by tA-009 in mice was evaluated in vivo using the fluorescence intensity of pHrodo-misTTR as an index. A significant increase in pHrodo-misTTR fluorescence intensity was observed in the tA-009-treated group compared to the vehicle-treated group (Figure 5). This result indicates that activation of phagocytosis of pHrodo-misTTR by phagocytes was observed in the tA-009-treated group. On the other hand, activation of phagocytosis of pHrodo-misTTR by tA-009 administration was almost completely suppressed by the MerTK inhibitor ONO7475. These results suggest that tA-009 activates MerTK-dependent phagocytic clearance function.

[0262] Example 7: Activation of phagocyte phagocytosis of misTTR by tA-009 or mouse 371M in aged mice in vivo. TTR amyloidosis, caused by the deposition of misfolded TTR in organs throughout the body, is widely known as an age-related disease (Front. Cardiovasc. Med., 2022, 9:863-179). It has also been reported that aging reduces the phagocytosis and elimination function of phagocytes such as macrophages, and that this dysfunction contributes to the onset of various diseases (Immunol. Lett., 2021, 230:1-10). To evaluate whether MerTK-dependent or Fcγ receptor-dependent activation of phagocytic clearance is more effective for removing misfolded TTR under conditions where phagocytic clearance function is impaired with aging, we evaluated the phagocytic clearance activity of DL800-misTTR in aged mice.

[0263] Example 7-1: Dose setting of tA-009 or mouse-type 371M using young mice To compare the phagocytic elimination function of misTTR by tA-009, which activates the phagocytic elimination function of phagocytes in a MerTK-dependent manner, and mouse-type 371M, which activates the phagocytic elimination function in an Fcγ receptor-dependent manner (Fc is wild-type mouse IgG2a (Immunol Rev., 2015, 268:25-51)), DL800-misTTR was administered subcutaneously to young mice with normal phagocytic elimination function, and changes in its fluorescence intensity were measured over time by in vivo imaging. The fluorescence intensity of DL800-misTTR is proportional to its remaining amount. Six-week-old (young) female BALB / c-nu (nu / nu) mice (Jackson Laboratory Japan) were subcutaneously injected with 40 μL of a solution containing DL800-misTTR and neutralized misTTR (prepared as in Example 4-3) at a protein ratio of 1:4 into the dorsal flank. At 1, 6, 12, 22, 46, 71, 118, 168, 240, and 336 hours after injection, the mice were anesthetized with isoflurane (Mylan Pharmaceuticals), and the fluorescence intensity (photons / second, [p / s]) around the injection site was measured using an IVIS Imaging System (Lumina II; PerkinElmer) to quantify the remaining amount of misTTR. The phagocytic clearance function was evaluated by calculating the integrated value of the fluorescence intensity at the above measurement time points as the total remaining amount ([p / s]·h) during the observation period (14 days). The test antibody tA-009 (100 mg / kg) or mouse type 371M (30 mg / kg), and the vehicle (PBS) as a control, were administered intraperitoneally 1 hour before the subcutaneous administration of DL800-misTTR. This study was conducted with 4 cases per group. Calculation of the mean and standard error of the total remaining amount of DL800-misTTR and significance tests were performed using GraphPad Prism (GraphPad Software, ver. 8.0.2). Comparison between the vehicle group and the test antibody (mouse type 371M or tA-009) group was performed using Dunnett's test, and a P value of less than 0.05 was considered statistically significant.

[0264] The results of an in vivo mouse study evaluating the phagocytic elimination of DL800-misTTR by tA-009 or mouse-type 371M are shown in Figure 6-1. Compared to the vehicle-treated group, the tA-009 (100 mg / kg)-treated group or mouse-type 371M (30 mg / kg)-treated group showed a significant and nearly identical decrease in the total remaining amount of DL800-misTTR. These results indicate that 100 mg / kg of tA-009 and 30 mg / kg of mouse-type 371M have nearly equivalent in vivo phagocytic elimination of DL800-misTTR.

[0265] Based on the above results, the subsequent experiments were carried out assuming that in young mice in which the phagocytic elimination function of phagocytes such as macrophages is maintained, 100 mg / kg of tA-009 and 30 mg / kg of mouse type 371M induce approximately the same phagocytic elimination function in phagocytes.

[0266] Example 7-2: Activation of phagocytic elimination function by tA-009 or mouse type 371M against DL800-misTTR in aged mice in vivo It has been suggested that the phagocytic elimination function of phagocytes such as macrophages is reduced with age in aged mice (Clin. Exp. Immunol. 2008, 152 (3): 448-455, Immunol. Lett. 2021, 230: 1-10). The present inventors hypothesized that drugs that activate the phagocytic elimination function of phagocytes in a MerTK-dependent manner may be more useful for treating diseases than drugs that activate Fcγ receptor-dependently. We conducted studies to evaluate the activation of phagocytic elimination function at doses of tA-009 and mouse type 371M, which showed equivalent effects in young mice.

[0267] According to the results of Example 7-1, 20-week-old (young) or 103-week-old (old) male C57BL / 6J mice (Jackson Laboratory Japan) were intraperitoneally administered the test antibody tA-009 (100 mg / kg), mouse 371M (30 mg / kg), or a control vehicle (PBS). DL800-misTTR and neutralized misTTR, prepared as in Example 4-3, were mixed at a protein ratio of 1:4, and 40 μL of the mixture was administered subcutaneously to the dorsal side of the mice. At 1, 6, 12, 24, 48, 72, 120, and 168 hours after administration, mice were anesthetized with isoflurane (Mylan Pharmaceuticals), and the remaining amount of DL800-misTTR was quantified by measuring the fluorescence intensity (photons / second, [p / s]) around the administration site using an IVIS Imaging System (Lumina II; PerkinElmer). This study was conducted with 5 to 6 animals per group. Calculation of the mean and standard error of the total remaining amount of DL800-misTTR and significance tests were performed using GraphPad Prism (GraphPad Software, version 8.0.2). Comparisons between young mice / vehicle groups and old mice / vehicle groups were performed using Student's t-test, and comparisons between old mice / vehicle groups and old mice / test antibody (mouse type 371M or tA-009) groups were performed using Dunnett's test, with P values ​​of less than 0.05 considered statistically significant. Figure 6-2 shows the results of evaluating the activation of the phagocytic elimination function of tA-009 and mouse type 371M against DL800-misTTR in old mice in vivo. A significant increase in the total remaining amount of DL800-misTTR was observed in old mice compared to young mice, confirming that the phagocytic elimination function of phagocytes against foreign substances is reduced in old mice. In this study using aged mice, a significant decrease in the total remaining amount of DL800-misTTR was observed in the tA-009 (100 mg / kg) group compared to the vehicle-treated group, whereas no effect was observed in the mouse-type 371M (30 mg / kg) group.The above results show that tA-009, which can activate the phagocytic elimination function in a MerTK-dependent manner, can significantly activate the phagocytic elimination function of phagocytes whose phagocytic elimination function has declined due to aging, compared to mouse type 371M, which can activate the phagocytic elimination function in an Fcγ receptor-dependent manner.

[0268] In this study, we found that tA-009, an anti-MerTK-anti-TTR bispecific antibody that activates phagocytic clearance in a MerTK-dependent manner, significantly activates phagocytic clearance in aged mice compared with murine 371M, which activates phagocytic clearance in an Fcγ receptor-dependent manner. This suggests that activation of phagocytic clearance by anti-MerTK-anti-misfolded protein bispecific antibodies may be very useful in treating diseases caused by the accumulation of age-related misfolded proteins.

[0269] Example 8: Design of fully human anti-MerTK-anti-TTR bispecific antibody (1) Design of fully human anti-MerTK uniarmed antibody A fully human anti-MerTK-anti-TTR bispecific antibody (hereinafter referred to as a "fully human anti-MerTK-anti-TTR bispecific antibody") was designed based on the sequence of the murine anti-MerTK-anti-TTR bispecific antibody obtained in Example 4. First, a fully human MerTK uniarmed antibody (hereinafter referred to as a "fully human MerTK uniarmed antibody") was designed. The heavy chain fragment of the fully human anti-MerTK uniarmed antibody used was the amino acid sequence from amino acid numbers 1 to 217 of SEQ ID NO: 8. In addition, an LALA mutation and a P331G amino acid mutation (SEQ ID NO: 39) were introduced into the first or second Fc polypeptide of the fully human anti-MerTK uniarmed antibody. Knobs-into-holes mutations were introduced into the first Fc polypeptide and the second Fc polypeptide based on a prior patent document (WO 1998 / 050431). Specifically, to form knobs in one of the first or second Fc polypeptides, a sequence (SEQ ID NO: 40) was designed in which threonine (T) at amino acid number 136 (EU index: 366) of SEQ ID NO: 39 was substituted with tryptophan (W). Furthermore, to form holes in the remaining Fc polypeptide, a sequence (SEQ ID NO: 41) was designed in which threonine (T) at amino acid numbers 136, 138, and 177 (EU index: 366, 368, and 407) of SEQ ID NO: 39 was substituted with serine (S), alanine (A), and alanine (Y), respectively. The light chain of the fully human anti-MerTK single-armed antibody used the amino acid sequence of SEQ ID NO: 16, 18, or 20. A fully human anti-MerTK single-armed antibody was designed by combining a heavy chain fragment of the fully human anti-MerTK single-armed antibody, the light chain of the fully human anti-MerTK single-armed antibody, a hinge region (SEQ ID NO: 42), and first and second Fc polypeptides (SEQ ID NOs: 40 and 41).

[0270] (2) Design of a fully human anti-MerTK-anti-TTR bispecific antibody A fully human anti-MerTK-anti-TTR bispecific antibody was designed by linking the amino acid sequence (SEQ ID NO: 43) and hinge region (SEQ ID NO: 70) of anti-TTR-scFv to the N-terminus of the second Fc polypeptide of the fully human anti-MerTK single-armed antibody designed in (1). The combination of amino acid sequences of each part in the designed fully human anti-MerTK-anti-TTR bispecific antibody is shown in Table 3.

[0271]

[0272] The anti-MerTK antibody of the present invention is expected to be useful in preventing or treating various diseases caused by a decrease in phagocytic function.

[0273] Example 9: Preparation of anti-MerTK-anti-IgL bispecific antibody, anti-IgL antibody, and misfolded IgL protein Misfolded IgL protein, anti-MerTK-anti-IgL bispecific antibody, and anti-IgL antibody were prepared.

[0274] Example 9-1: Preparation of recombinant human IgL protein A gene sequence encoding 6x histidine (SEQ ID NO: 22) was inserted into the expression vector pcDNA3.4 TOPO vector with reference to the gene sequence encoding the human IgL Vλ6Wil amino acid sequence reported by J. Wall et al. (Biochemistry, 1999, 38: 14101-14108, Figure 1) to obtain the Vlambda6Wil vector. The Vlambda6Wil vector was introduced into ExpiCHO-S cells using an ExpiFectamine CHO Transfection Kit, and the recombinant protein was secreted into the culture supernatant according to standard methods. His-tagged human IgL protein was purified from the resulting culture supernatant using TALON® Metal Affinity Resin (Clontech, 635502) and His Buffer Kit (Cytiva, 11-0034-00). The eluted fraction containing the recombinant human IgL protein was substituted with PBS using a NAP-25 Column (Cytiva, 17-0852-01) or an Amicon Ultra-15 (Merck, UFC901096) and concentrated to obtain purified recombinant human IgL protein.

[0275] Example 9-2 Preparation of Misfolded IgL Protein To prepare misfolded recombinant human IgL protein (herein referred to as "misfolded IgL protein"), 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 in-house) and incubated at 37°C for at least 4 days to prepare a misfolded recombinant human IgL protein solution.

[0276] Example 9-3 Preparation of anti-MerTK-anti-IgL bispecific antibody A bispecific antibody (hereinafter referred to as "anti-MerTK-anti-IgL bispecific antibody") consisting of a heavy chain comprising a heavy chain fragment containing the heavy chain variable region of an anti-MerTK antibody, a hinge region, and a human first Fc polypeptide, a light chain comprising the light chain variable region of an anti-MerTK antibody, a heavy chain comprising a polypeptide formed by linking a heavy chain fragment containing the heavy chain variable region of an anti-IgL antibody, a hinge region, and a human second Fc polypeptide, and a light chain comprising the light chain variable region of an anti-IgL antibody was prepared by the following method.

[0277] (1) Preparation of vectors encoding heavy and light chains of anti-IgL antibodies. Vectors encoding heavy and light chains of anti-IgL antibodies were prepared based on the description in a paper (mAbs, 2016, 8(6):1010-1020). A mouse-human chimeric heavy chain (SEQ ID NO: 57) (hereinafter referred to as the "anti-IgL antibody heavy chain") was designed, consisting of the VH and CL, hinge region, and human-derived Fc polypeptide containing the knobs-into-holes mutation, LALA mutation, and P331G amino acid mutation of the 2A4 antibody, a mouse-derived anti-IgL antibody described in Japanese Patent No. 5730020. A mouse-human chimeric light chain (SEQ ID NO: 59) (hereinafter referred to as the "anti-IgL antibody light chain") was also designed, consisting of the VL of the 2A4 antibody and a human-derived CH1 domain. Artificial genes were synthesized from the base sequences (SEQ ID NO: 56 and SEQ ID NO: 58) encoding the amino acid sequences of the designed heavy chain and light chain of the anti-IgL antibody, and inserted into the pcDNA3.4 TOPO vector, respectively, to prepare a vector encoding the heavy chain of the anti-IgL antibody and a vector encoding the light chain of the anti-IgL antibody.

[0278] (2) Preparation of Vectors Encoding Heavy and Light Chains of a Fully Human Anti-MerTK Antibody A heavy chain fragment of a human MerTK single-armed antibody consisting of the amino acid sequence from amino acid numbers 1 to 217 of SEQ ID NO: 8, a hinge region (SEQ ID NO: 42), and a first Fc polypeptide (SEQ ID NO: 41) were linked in this order to design a heavy chain (SEQ ID NO: 61) of a fully human anti-MerTK single-armed antibody. A nucleotide sequence encoding the amino acid sequence of the heavy chain of the designed fully human anti-MerTK single-armed antibody was synthesized as an artificial gene and inserted into a pcDNA3.4 TOPO vector to prepare a vector encoding the heavy chain of a fully human anti-MerTK antibody. A nucleotide sequence encoding the light chain (SEQ ID NO: 12) of tA3-20.1 designed in Example 1-1 was synthesized as an artificial gene and inserted into a pcDNA3.4 TOPO vector to prepare a vector encoding the light chain of a fully human anti-MerTK antibody.

[0279] (3) Preparation of anti-MerTK-anti-IgL bispecific antibodies Anti-MerTK-anti-IgL bispecific antibodies were prepared using the four vectors prepared in (1) and (2) above. Specifically, a vector encoding the heavy chain of an anti-IgL antibody, a vector encoding the light chain of an anti-IgL antibody, a vector encoding the heavy chain of a fully human anti-MerTK antibody, and a vector encoding the light chain of a fully human anti-MerTK antibody were co-transfected into ExpiCHO-S cells using the ExpiFectamine CHO Transfection Kit, and the anti-MerTK-anti-IgL bispecific antibody was secreted into the culture supernatant according to standard methods. The antibody was purified from the resulting culture supernatant using affinity purification with MabSelect SuRe. Hereinafter, the prepared anti-MerTK-anti-IgL bispecific antibody is referred to as tA3-20_2A4_h1x.

[0280] Example 9-4: Preparation of anti-IgL antibody An anti-IgL antibody (herein referred to as "mouse-human chimeric 2A4") having the variable regions of the mouse-derived anti-IgL antibodies of SEQ ID NOS: 152 and 154 in a prior patent document (Japanese Patent No. 5730020) and a human-derived constant region was prepared by the following method. The heavy chain of SEQ ID NOS: 63 and the light chain of SEQ ID NOS: 65 were designed with reference to the sequence of the 2A4 antibody described in Japanese Patent No. 5730020. Artificial genes were synthesized from nucleotide sequences (SEQ ID NOS: 62 and 64) encoding the above two designed amino acid sequences, and mouse-human chimeric 2A4 was prepared according to standard methods using the same method as in Example 1-1.

[0281] Example 10: Evaluation of binding activity of anti-MerTK-anti-IgL bispecific antibodies to human MerTK and misfolded IgL protein The binding activity of tA3-20_2A4_h1x to human MerTK and misfolded IgL protein was evaluated by ELISA. The misfolded IgL protein prepared in Example 9-2 was adjusted to 2.5 μg / mL in PBS, and 50 μL was added per well to Ni-NTA HisSorb Plates (QIAGEN, 35061). The plate was then left to stand overnight at 4°C to immobilize the protein. After removing the misfolded IgL protein, the wells were washed three times with PBST. The antibody used for evaluation was tA3-20_2A4_h1x, and the control antibodies were tA3-20.1, mouse-human chimeric 2A4, and LYS_1c3-m1_h1i (isotype control). These antibodies were serially diluted from 20 μg / mL to 6.4 ng / mL in 0.05% Tween-20-containing PBS (prepared by diluting Thermo Scientific 28352 20-fold; hereafter referred to as "PBST") containing 0.5% Blocking One. 50 μL of each solution was added to each well and incubated 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 Biotin Labeling Kit (Dojin Chemical Laboratory, LK03), then adjusted to 2 μg / mL in PBST containing 0.5% Blocking One. 50 μL of this solution was added to each well and incubated at room temperature for 1 hour. After washing the wells three times with PBST, 50 μL of 200-fold diluted Streptavidin-HRP (R&D Systems, DY998) was added to each well and incubated at room temperature for 1 hour. After washing the wells three times with PBST, 50 μL of TMB-Plus Substrate-Chromogen was added to each well and incubated 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 Infinite M200 PRO.

[0282] The results demonstrated that tA3-20_2A4_h1x can simultaneously bind to human MerTK and misfolded IgL protein, and that this activity is antibody concentration-dependent (FIG. 7).

[0283] Furthermore, using pHrodo misfolded IgL protein labeled in the same manner as in Example 4-3, phagocytosis by mouse bone marrow-derived macrophages was confirmed in the same manner as in Example 5-2. The results demonstrated that tA3-20_2A4_h1x activates phagocytosis in a MerTK-dependent manner. These results suggest that tA3-20_2A4_h1x activates the phagocytic removal function of misfolded IgL protein by mouse bone marrow-derived macrophages.

[0284] SEQ ID NOs: 1 to 3 are the amino acid sequences of the heavy chain CDR1 to 3 of tA3-20 to tA3-20.5, respectively, and SEQ ID NOs: 4 to 6 are the amino acid sequences of the light chain CDR1 to 3 of tA3-20 to tA3-20.5, respectively. SEQ ID NO: 8 is the amino acid sequence of the heavy chain of tA3-20 to tA3-20.5, and SEQ ID NO: 7 is a nucleotide sequence encoding the heavy chain amino acid sequence of tA3-20 to tA3-20.5 shown in SEQ ID NO: 8. SEQ ID NO: 10 is the amino acid sequence of the light chain of tA3-20, and SEQ ID NO: 9 is a nucleotide sequence encoding the light chain amino acid sequence of tA3-20 shown in SEQ ID NO: 10. SEQ ID NO: 12 is the amino acid sequence of the light chain of tA3-20.1, and SEQ ID NO: 11 is a nucleotide sequence encoding the light chain amino acid sequence of tA3-20.1 shown in SEQ ID NO: 12. SEQ ID NO: 14 is the amino acid sequence of the light chain of tA3-20.2, and SEQ ID NO: 13 is a nucleotide sequence encoding the light chain amino acid sequence of tA3-20.2 shown in SEQ ID NO: 14. SEQ ID NO: 16 is the amino acid sequence of the light chain of tA3-20.3, and SEQ ID NO: 15 is a nucleotide sequence encoding the light chain amino acid sequence of tA3-20.3 shown in SEQ ID NO: 16. SEQ ID NO: 18 is the amino acid sequence of the light chain of tA3-20.4, and SEQ ID NO: 17 is a nucleotide sequence encoding the light chain amino acid sequence of tA3-20.4 shown in SEQ ID NO: 18. SEQ ID NO: 20 is the amino acid sequence of the light chain of tA3-20.5, and SEQ ID NO: 19 is a nucleotide sequence encoding the light chain amino acid sequence of tA3-20.5 shown in SEQ ID NO: 20. SEQ ID NO: 22 is the amino acid sequence of a histidine tag, and SEQ ID NO: 21 is a nucleotide sequence encoding the amino acid sequence of the histidine tag shown in SEQ ID NO: 22. SEQ ID NO: 24 is the amino acid sequence of the heavy chain of MTK201, and SEQ ID NO: 23 is a nucleotide sequence encoding the heavy chain amino acid sequence of MTK201 shown in SEQ ID NO: 24. SEQ ID NO: 26 is the amino acid sequence of the light chain of MTK201, and SEQ ID NO: 25 is a nucleotide sequence encoding the light chain amino acid sequence of MTK201 shown in SEQ ID NO: 26. SEQ ID NOs: 27 to 29 are the amino acid sequences of the heavy chain CDR1 to 3 of the 371M antibody, respectively, and SEQ ID NOs: 30 to 32 are the amino acid sequences of the light chain CDR1 to 3 of the 371M antibody, respectively.SEQ ID NO: 34 is the heavy chain amino acid sequence of mouse anti-MerTK antibody tA3-20.3, and SEQ ID NO: 33 is a nucleotide sequence encoding the heavy chain amino acid sequence of mouse anti-MerTK antibody tA3-20.3 shown in SEQ ID NO: 34. SEQ ID NO: 36 is the light chain amino acid sequence of mouse anti-MerTK antibody tA3-20.3, and SEQ ID NO: 35 is a nucleotide sequence encoding the light chain amino acid sequence of mouse tA3-20.3 shown in SEQ ID NO: 36. SEQ ID NO: 38 is the amino acid sequence of a polypeptide comprising a mouse second Fc polypeptide and an anti-TTR-scFv, and SEQ ID NO: 37 is a nucleotide sequence encoding a polypeptide comprising a mouse second Fc polypeptide and an anti-TTR-scFv shown in SEQ ID NO: 38. SEQ ID NO: 39 is the amino acid sequence of the first Fc polypeptide or the second Fc polypeptide of tA3-20 to tA3-20.5. SEQ ID NO: 40 and / or 41 are the amino acid sequences of the first Fc polypeptide and / or the second Fc polypeptide of the anti-MerTK-anti-TTR bispecific antibody. SEQ ID NO: 42 is the amino acid sequence of the hinge region linking tA3-20 to tA3-20.5 and the anti-MerTK-Fab in the anti-MerTK-anti-TTR bispecific antibody. SEQ ID NO: 43 is the amino acid sequence of the anti-TTR-scFv. SEQ ID NO: 44 is the amino acid sequence of the heavy chain of the 371M antibody containing the mouse heavy chain IgG2a constant region, and SEQ ID NO: 45 is the amino acid sequence of the light chain of the 371M antibody containing the mouse light chain Lambda1. SEQ ID NOs: 46 to 55 are the amino acid sequences of various linkers. SEQ ID NOs: 57 and 59 are the amino acid sequences of the heavy and light chains, respectively, of a mouse-human chimeric anti-MerTK-anti-IgL bispecific antibody. SEQ ID NOs: 56 and 58 are nucleotide 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 SEQ ID NOs: 57 and 59, respectively. SEQ ID NO: 61 is the amino acid sequence of the heavy chain of a human anti-MerTK single-armed antibody. SEQ ID NO: 60 is a nucleotide sequence encoding the amino acid sequence of the heavy chain of a human anti-MerTK single-armed antibody shown in SEQ ID NO: 61. SEQ ID NO: 63 is the amino acid sequence of the heavy chain of mouse-human chimeric 2A4. SEQ ID NO: 62 is a nucleotide sequence encoding the amino acid sequence of the heavy chain of mouse-human chimeric 2A4 shown in SEQ ID NO: 63.SEQ ID NO: 65 is the amino acid sequence of the light chain of mouse-human chimeric 2A4. SEQ ID NO: 64 is a nucleotide sequence encoding the amino acid sequence of the light chain of the 2A4 antibody shown in SEQ ID NO: 65. SEQ ID NOs: 67 and 69 are the amino acid sequences of the heavy chain and light chain of M6, respectively. SEQ ID NOs: 66 and 68 are nucleotide sequences encoding the amino acid sequences of the heavy chain and light chain of M6 shown in SEQ ID NOs: 67 and 69, respectively. SEQ ID NO: 70 is the amino acid sequence of the hinge region linked to the anti-TTR-scFv in the anti-MerTK-anti-TTR bispecific antibody.

Claims

1. An anti-MerTK antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO:1, CDR2 consisting of the amino acid sequence of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence of SEQ ID NO:3, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO:4, CDR2 consisting of the amino acid sequence of SEQ ID NO:5, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

6.

2. The anti-MerTK antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of the following (1) to (6): (1) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 18; and (6) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO:

20.

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

4. The anti-MerTK antibody of claim 3, comprising amino acid mutations L234A and L235A (LALA mutations) in the heavy chain, wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region.

5. The anti-MerTK antibody of claim 3, comprising a P331G mutation in the heavy chain, wherein the mutation position is an amino acid position according to the EU index in the human Igγ1 constant region.

6. The anti-MerTK antibody of claim 3, comprising amino acid mutations L234A and L235A (LALA mutations) and a P331G mutation in the heavy chain, wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region.

7. The anti-MerTK antibody according to claim 6, selected from the group consisting of the following (1) to (6): (1) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 10; (2) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 12; (3) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 14; (4) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 16; (5) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO: 18; and (6) an anti-MerTK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain consisting of the amino acid sequence of SEQ ID NO:

20.

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

9. The antigen-binding fragment is a single chain variable region fragment (scFv), a Fab fragment, a Fab' fragment, or an F(ab') ２ 3. The anti-MerTK antibody or antigen-binding fragment thereof of claim 1 or 2, which is a fragment.

10. An anti-MerTK antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is post-translationally modified.

11. A fusion or complex of the anti-MerTK antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a cell displaying the anti-MerTK antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 on the cell surface.

12. A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-MerTK antibody or an antigen-binding fragment thereof, selected from the group consisting of (1) to (7) below: (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:18; and (7) A polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 110 of SEQ ID NO:

20.

13. A polynucleotide comprising a nucleotide sequence encoding a heavy chain or light chain of an anti-MerTK antibody selected from the group consisting of (1) to (7) below: (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain consisting of the amino acid sequence of SEQ ID NO:8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO:10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO:12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO:14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO:16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO:18; and (7) a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO:

20.

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

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

16. A host cell comprising a polynucleotide selected from the group consisting of the following (1) to (13): (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:16; (6) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:18; (7) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:20; (8) a polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 10; (9) a polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 12; (10) a polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 14; (11) a polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 16;(12) A polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18; and (13) A polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, and a polynucleotide comprising a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:

20.

17. A method for producing an anti-MerTK antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cell of claim 15 or 16 to express the anti-MerTK antibody or an antigen-binding fragment thereof.

18. A pharmaceutical composition comprising an anti-MerTK antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharma- ceutically acceptable excipient.

19. The pharmaceutical composition according to claim 18 for use in 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 the retinal disease is a retinal degenerative disease.

22. The pharmaceutical composition according to claim 21, wherein the retinal degenerative disease is retinitis pigmentosa or age-related macular degeneration.

23. An anti-MerTK antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 for use in the prevention and / or treatment of an eye disease.

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

25. Use of an anti-MerTK antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10 in the manufacture of a pharmaceutical composition for the prevention and / or treatment of an eye disease.

26. A bispecific antibody that binds to MerTK and misfolded proteins and / or amyloid, comprising the heavy and light chain variable regions of an anti-MerTK antibody and the heavy and light chain variable regions of an antibody that binds to misfolded proteins and / or amyloid, wherein the heavy chain variable region of the anti-MerTK antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO:1, CDR2 consisting of the amino acid sequence of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the anti-MerTK antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO:4, CDR2 consisting of the amino acid sequence of SEQ ID NO:5, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

6.

27. The bispecific antibody according to claim 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) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 10; (2) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 12; (3) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 14; (4) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 16; (5) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO: 18; and (6) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 119 of SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 110 of SEQ ID NO:

20.

28. The bispecific antibody of claim 27, comprising a heavy chain fragment comprising the heavy chain variable region of an anti-MerTK antibody and a light chain comprising the light chain variable region of an anti-MerTK antibody, and a single-arm antibody (single-arm anti-MerTK antibody) comprising an Fc region consisting of a first Fc polypeptide and a second Fc polypeptide.

29. The bispecific antibody according to claim 28, wherein the heavy chain fragment and the light chain of the single-arm anti-MerTK antibody are selected from the group consisting of the following (1) to (6): (1) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO:8, and a light chain consisting of the amino acid sequence of SEQ ID NO:10; (2) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO:8, and a light chain consisting of the amino acid sequence of SEQ ID NO:12; (3) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO:8, and a light chain consisting of the amino acid sequence of SEQ ID NO:14; (4) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO:8, and a light chain consisting of the amino acid sequence of SEQ ID NO:16; (5) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO:8, and a light chain consisting of the amino acid sequence of SEQ ID NO:18; and (6) a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 217 of SEQ ID NO:8, and a light chain consisting of the amino acid sequence of SEQ ID NO:

20.

30. The bispecific antibody described in claim 29, in which the C-terminus of an scFv or Fab region comprising the heavy chain variable region and the light chain variable region of an antibody that binds to misfolded proteins and / or amyloid is linked via a hinge region to the N-terminus of the second Fc polypeptide of the one-armed anti-MerTK antibody.

31. The bispecific antibody according to any one of claims 26 to 30, wherein the antibody that binds to misfolded proteins and / or amyloid is an anti-TTR antibody that binds to misfolded TTR.

32. A bispecific antibody according to claim 31, wherein the heavy chain variable region of the anti-FTR antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO:27, CDR2 consisting of the amino acid sequence of SEQ ID NO:28, and CDR3 consisting of the amino acid sequence of SEQ ID NO:29, and the light chain variable region of the anti-FTR antibody comprises CDR1 consisting of the amino acid sequence of SEQ ID NO:30, CDR2 consisting of the amino acid sequence of SEQ ID NO:31, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

32.

33. The bispecific antibody of claim 32, wherein the heavy chain variable region of the anti-FTR antibody is a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 119 of SEQ ID NO: 43, and the light chain variable region of the anti-FTR antibody is a light chain variable region consisting of the amino acid sequence from amino acid number 135 to amino acid number 245 of SEQ ID NO:

43.

34. The bispecific antibody of claim 33, comprising an scFv (anti-FTR-scFv) comprising the heavy chain variable region and the light chain variable region of an anti-FTR antibody.

35. The bispecific antibody of claim 34, wherein the anti-FTR scFv consists of the amino acid sequence of SEQ ID NO:

43.

36. A bispecific antibody according to claim 34 or 35, comprising a heavy chain fragment comprising the heavy chain variable region of an anti-MerTK antibody, a light chain comprising the light chain variable region of an anti-MerTK antibody, and a single-arm antibody (single-arm anti-MerTK antibody) comprising one Fc region consisting of a first Fc polypeptide and a second Fc polypeptide, in which the C-terminus of the anti-TTR-scFv is linked via a hinge region to the N-terminus of the second Fc polypeptide of the single-arm anti-MerTK antibody.

37. A bispecific antibody according to any one of claims 28 to 36, comprising an Fc region comprising the amino acid mutations L234A and L235A (LALA mutations) and the P331G mutation, wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region.

38. The bispecific antibody of claim 37, wherein the first Fc polypeptide and the second Fc polypeptide comprise an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:39 or an amino acid sequence having 1 to 10 substitutions in the amino acid sequence of SEQ ID NO:

39.

39. The bispecific antibody of claim 38, comprising an Fc region comprising knobs-into-holes mutations.

40. The bispecific antibody of claim 38, comprising an Fc region comprising a LALA mutation, a P331G mutation and a knobs-into-holes mutation.

41. A bispecific antibody according to claim 39 or 40, wherein the knobs-into-holes mutations are 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, wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region.

42. The bispecific antibody of claim 38, wherein the sequences of the first Fc polypeptide and the second Fc polypeptide are either of the following (1) or (2): (1) a first Fc polypeptide having the amino acid sequence of SEQ ID NO: 40, and a second Fc polypeptide having the amino acid sequence of SEQ ID NO: 41; or (2) a first Fc polypeptide having the amino acid sequence of SEQ ID NO: 41, and a second Fc polypeptide having the amino acid sequence of SEQ ID NO:

40.

43. The bispecific antibody of claim 42, comprising a hinge region consisting of the amino acid sequence set forth in SEQ ID NO:42 and / or a hinge region consisting of the amino acid sequence set forth in SEQ ID NO:

70.

44. A bispecific antibody according to any one of claims 26 to 43, which is post-translationally modified.

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

46. ​​A polynucleotide selected from the group consisting of the following (1) to (9), for use in producing the bispecific antibody according to any one of claims 26 to 44: (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:8; (2) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:10; (3) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:12; (4) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO:14; (5) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:16; (6) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18; (7) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20; (8) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 43; and (9) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 135 to 245 of SEQ ID NO:

43.

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

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

49. A host cell comprising a polynucleotide selected from the group consisting of the following (1) to (3), for use in producing the bispecific antibody according to any one of claims 27 to 44: (1) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:8, a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO:16, a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:43, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 135 to 245 of SEQ ID NO:43; (2) A polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8, a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 18, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 43, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 135 to 245 of SEQ ID NO: 43; and (3) A polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 8; a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-MerTK antibody consisting of the amino acid sequence of amino acid numbers 1 to 110 of SEQ ID NO: 20; a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 43; and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-FTR antibody consisting of the amino acid sequence of amino acid numbers 135 to 245 of SEQ ID NO:

43.

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

51. A pharmaceutical composition comprising the bispecific antibody of any one of claims 26 to 44 and a pharma- ceutically acceptable excipient.

52. The pharmaceutical composition according to claim 51 for use in the prevention and / or treatment of a disease resulting from the accumulation of misfolded proteins.

53. The pharmaceutical composition of claim 52, wherein the disease resulting from the accumulation of misfolded proteins is amyloidosis.

54. The pharmaceutical composition of claim 53, wherein the amyloidosis is systemic amyloidosis.

55. The pharmaceutical composition according to claim 54, wherein the systemic amyloidosis is TTR amyloidosis or polyneuropathy amyloidosis.

56. A bispecific antibody according to any one of claims 26 to 44 for use in the prevention and / or treatment of a disease resulting from the accumulation of misfolded proteins.

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

58. Use of a 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 a disease resulting from the accumulation of misfolded proteins.

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