Anti- GDF15 antibodies

Abstract

To provide anti- GDF15 antibodies useful for the treatment of diseases or symptoms associated with GDF15.SOLUTION: The present invention includes anti- hGDF15 antibodies that bind to an epitope of hGDF15 comprising the amino acid sequence of DHCPLGPGRCCRLH (SEQ ID NO: 3) and uses thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to International Application No. PCT / JP2019 / 047956, the entire contents of which are incorporated herein by reference. The present disclosure relates to anti-GDF15 antibodies and uses thereof. [Background technology]

[0002] Cachexia is a complex metabolic disorder associated with chronic illness, characterized by weight loss and muscle weakness. Cancer cachexia, in particular, is seen in many patients with advanced cancer and requires aggressive treatment because it worsens the patient's prognosis and quality of life. However, the pathogenesis of cachexia is complex, and many aspects of its pathogenesis remain unknown, so there are currently no effective treatments.

[0003] GDF15 (Growth Differentiation Factor 15) is a secreted protein of the TGF-β superfamily and has been reported to be involved in various diseases such as cancer and diabetes. Numerous clinical studies have reported on blood GDF15 concentrations, including high GDF15 levels in the blood and cancer tissues of cancer patients and a correlation between blood GDF15 levels and prognosis in cancer patients. GDF15 is also known to act on the feeding center in the brain, inducing anorexia and contributing to the development of cachexia. Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure aims to provide anti-GDF15 antibodies that are useful for treating diseases or conditions associated with GDF15. [Means for solving the problem]

[0005] In some embodiments, the present disclosure provides anti-hGDF15 antibodies that bind to an epitope of hGDF15 comprising the amino acid sequence of DHCPLGPGRCCRLH (SEQ ID NO: 3).

[0006] The present disclosure provides, in a further aspect, a CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 and a heavy chain variable region comprising: a CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 a light chain variable region comprising The present invention provides an anti-hGDF15 antibody comprising:

[0007] In a further embodiment, the present disclosure provides a method for producing a pharmaceutical composition comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising The present invention provides an anti-hGDF15 antibody comprising:

[0008] The present disclosure provides, in a further aspect, A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 8; and A light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 9. The present invention provides an anti-hGDF15 antibody comprising:

[0009] The present disclosure provides, in a further aspect, CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising The present invention provides an anti-hGDF15 antibody comprising:

[0010] The present disclosure provides, in a further aspect, CDR1 comprising the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising The present invention provides an anti-hGDF15 antibody comprising:

[0011] The present disclosure provides, in a further aspect, CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR3 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising The present invention provides an anti-hGDF15 antibody comprising:

[0012] The present disclosure provides, in a further aspect, CDR1 comprising the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising The present invention provides an anti-hGDF15 antibody comprising:

[0013] In a further aspect, the present disclosure provides an anti-hGDF15 antibody that competes with any of the above antibodies for binding to hGDF15.

[0014] In a further aspect, the present disclosure provides a polynucleotide encoding any of the above antibodies, an expression vector containing the polynucleotide, or a transformed cell containing the polynucleotide.

[0015] In a further aspect, the present disclosure provides a pharmaceutical composition comprising any of the above antibodies. [Effects of the Invention]

[0016] The anti-hGDF15 antibodies of the present disclosure recognize epitopes that are distinct from existing anti-hGDF15 antibodies and are useful for treating diseases or conditions associated with GDF15. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows the results of a competition study using four anti-hGDF15 monoclonal antibodies (HuMAB2, MAB17, Hu01G06-127, and MAB957). [Figure 2A]Figure 2A shows the results of the three-dimensional structure analysis of the cocrystal of HuMAB2 Fab and hGDF15. [Figure 2B] Figure 2B shows the results of epitope and paratope analysis by three-dimensional structural analysis of the cocrystal of HuMAB2 Fab and hGDF15. [Figure 3] Figure 3 shows the results of the three-dimensional structural analysis of the binding between HuMAB2 Fab and hGDF15. hGDF15 forms a homodimer, and among the amino acids to which HuMAB2 binds, those present in one of the hGDF15 monomers (Monomer 1) are underlined, and those present in the other monomer (Monomer 2) (71D72T) are double underlined. Amino acids important for binding are shown in bold, and amino acids with particularly strong binding are indicated by arrows. [Figure 4] FIG. 4 shows the results of a binding test between hGDF15 or a synthetic peptide of DHCPLGPGRCCRLH (SEQ ID NO: 3) and HuMAB2. [Figure 5A] Figure 5A shows binding of HuMAB2 mutants to hGDF15 (H chain-modified antibody 1 / 2). [Figure 5B] Figure 5B shows binding of HuMAB2 mutants to hGDF15 (H chain-modified antibody 2 / 2). [Figure 5C] Figure 5C shows binding of HuMAB2 mutants to hGDF15 (light chain modified antibody 1 / 2). [Figure 5D] Figure 5D shows binding of HuMAB2 mutants to hGDF15 (light chain engineered antibody 2 / 2). [Figure 6] Figure 6 shows the capture of circulating hGDF15 by HuMAB2 and MAB17. [Figure 7] FIG. 7 shows the effect of anti-hGDF15 antibodies on the concentration of unbound hGDF15 in the blood in a tumor-bearing mouse model. [Figure 8] Figure 8 shows the effects of HuMAB2 on weight loss, cumulative food intake, and blood unbound hGDF15 concentrations in a tumor-bearing mouse model. [Figure 9] Figure 9 shows the effect of HuMAB2 on activity (circadian rhythm) in a tumor-bearing mouse model. [Figure 10] FIG. 10 shows the binding of HuMAB2 mutants to hGDF15. [Figure 11] Figure 11 shows the binding of HuMAB2 mutants composed of mutated H chains (H49R, H49D, H48S, H71Y, H83R, or H120F) and L chains (L48K, L112D, L72F, or L24H) to hGDF15. [Figure 12] FIG. 12 shows a schematic diagram of the evaluation of the inhibitory activity of anti-GDF15 antibodies against the complex formation of GDF15 and GFRAL. [Figure 13] FIG. 13 shows a schematic diagram of the evaluation of the inhibitory activity of HuMAB2 against the complex formation of GDF15, GFRAL, and RET, and the evaluation results. [Figure 14] FIG. 14 shows the effects of MAB1 on weight loss, cumulative food intake, and blood unbound hGDF15 concentration in a tumor-bearing mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0019] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0020] In this specification, amino acid residues are represented by the following abbreviations. Ala or A: Alanine Arg or R: arginine Asn or N: asparagine Asp or D: aspartic acid Cys or C: cysteine Gln or Q: glutamine Glu or E: glutamic acid Gly or G: glycine His or H: histidine Ile or I: Isoleucine Leu or L: leucine Lys or K: Lysine Met or M: methionine Phe or F: phenylalanine Pro or P: proline Ser or S: serine Thr or T: threonine Trp or W: Tryptophan Tyr or Y: Tyrosine Val or V: Valine In this specification, amino acid residues in a certain amino acid sequence may be indicated by a number representing their position and an abbreviation representing the amino acid residue (for example, the 13th arginine residue is represented as "13R").

[0021] GDF15 (Growth Differentiation Factor 15), also known as MIC-1, PLAB, PDF, and NAG-1, is a secreted protein of the TGF-β superfamily. The GDF15 gene expresses a precursor, pro-GDF15, which is cleaved by a membrane-type metalloendoprotease to generate mature GDF15. GDF15 is soluble and is thought to form a dimer that is recognized by its receptor, GFRAL. Unless otherwise specified, the term GDF15 in this specification refers to mature GDF15.

[0022] As used herein, the term GDF15 encompasses GDF15 from any species. In one embodiment, GDF15 is human GDF15 (hGDF15). A representative amino acid sequence of pro-hGDF15 is set forth in SEQ ID NO: 1. Pro-hGDF15 is a 308-amino acid polypeptide consisting of a 29-amino acid signal peptide (underlined), a 167-amino acid propeptide, and a 112-amino acid mature peptide (double underlined, hGDF15). A representative amino acid sequence of hGDF15 is set forth in SEQ ID NO: 2, although hGDF15 in the present disclosure is not limited to those containing the amino acid sequence of SEQ ID NO: 2. TIFF2026004412000001.tif123123

[0023] The term "antibody" as used herein refers to a molecule comprising an immunoglobulin or a portion thereof that has antigen-binding ability, and is used to encompass not only molecules in the form of natural immunoglobulins, but also molecules of various structures, such as chimeric antibodies, humanized antibodies, multispecific antibodies, and antibody fragments. The term "monoclonal" as used herein is used to distinguish it from "polyclonal antibodies," which are mixtures of multiple types of antibodies each directed against a different epitope, and means that it is obtained from a population of a single type of antibody. Therefore, the term "monoclonal antibody" can refer to, for example, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, and antibody fragments. An antibody fragment refers to a molecule that comprises a portion of an immunoglobulin as a component, for example, but is not limited to, antibody heavy and light chain variable regions (V H and V L ), F(ab')2, Fab', Fab, Fv, disulphide-linked Fv (sdFv), single-chain Fv (scFv), and polymers thereof. The species of antibody is not particularly limited, and examples include antibodies derived from mouse, rat, rabbit, goat, and human.

[0024] As used herein, the term "isolated" means that a biological molecule (eg, an antibody or polynucleotide) is substantially separated from other components of its natural environment.

[0025] The immunoglobulin class of an antibody is determined based on the heavy chain constant region. Immunoglobulin classes include IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chains are called α chains, δ chains, ε chains, γ chains, and μ chains, respectively. Immunoglobulin classes can be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The immunoglobulin class and subclass of antibodies herein are not particularly limited. In one embodiment, the immunoglobulin class is IgG. The light chains of antibodies can be divided into κ chains and λ chains based on their constant regions, and the antibodies herein may have either κ chains or λ chains.

[0026] The variable region of an antibody typically consists of three complementarity determining regions (CDRs) sandwiched between four framework regions (FRs). The FRs and CDRs are generally arranged in the following order for both the light chain and the heavy chain: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Several methods for defining antibody variable regions and CDRs have been reported, including, for example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. 1991), the Chothia definition (Chothia et al., J. Mol. Biol., 1987; 196: 901-917), the AbM definition (Martin et al., Proc. Natl. Acad. Sci. USA, 1989; 86: 9268-9272), the Contact definition (MacCallum et al., J. Mol. Biol. 1996; 262: 732-745), and the IMGT definition (Lefranc et al., Dev Comp Immunol. 2003; 27(1): 55-77). In this specification, Kabat definitions are used unless otherwise stated.

[0027] As used herein, an anti-hGDF15 antibody refers to an antibody that binds to hGDF15 with sufficient affinity to exert the desired effect. The anti-hGDF15 antibody of the present disclosure can be obtained by a conventional method using hGDF15 or a portion thereof as an immunogen. The immunogen can be produced by conventional peptide synthesis methods, such as genetic engineering or chemical synthesis. Alternatively, the antibody of the present disclosure can be obtained by using genetic engineering to create an expression vector containing an antibody gene and expressing it in cells.

[0028] Polyclonal antibodies can be produced by general methods such as those described in "Antibodies: A Laboratory Manual, Lane, H.D. et al. eds., Cold Spring Harbor Laboratory Press, New York, 1989." Specifically, they can be produced by immunizing mammals such as rats, mice, rabbits, goats, and horses with the immunogens described above.

[0029] Monoclonal antibodies can be obtained by known methods, such as producing hybridomas that produce antibodies, or using genetic engineering techniques to produce expression vectors containing antibody genes and expressing them in cells.

[0030] Hybridomas secreting monoclonal antibodies can be prepared according to the method described by Kohler et al., Nature 256:495, 1975. First, an immunogen is mixed with an appropriate substance for enhancing antigenicity (e.g., keyhole limpet hemocyanin or bovine serum albumin) and, if necessary, an immunostimulant (e.g., Freund's complete or incomplete adjuvant), and the mixture is used to immunize non-human mammals such as rats, mice, rabbits, goats, and horses. Typically, immunized animals are immunized multiple times at intervals of 3 to 10 days, with 1 to 100 μg of the immunogen peptide administered. Next, immunocompetent cells (cells capable of producing antibodies in the immunized animal) are collected from the immunized animal after multiple immunizations and fused with myeloma cells (e.g., cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits, or humans) that are not capable of producing autoantibodies. Cell fusion can be achieved using polyethylene glycol, electrofusion, or other methods. Furthermore, cells that have successfully undergone cell fusion are selected based on the selection marker possessed by the fused cells, and the reactivity of the antibodies produced by the selected cells to the immunogen is confirmed by immunoassay methods described below, thereby obtaining hybridomas that produce the desired monoclonal antibodies. The resulting hybridomas can be cultured in vitro, and then isolated from the culture supernatant. Alternatively, the monoclonal antibodies can be cultured in vivo, such as in ascites fluid of mice, rats, guinea pigs, hamsters, or rabbits, and then isolated from the ascites fluid.

[0031] Monoclonal antibodies can also be obtained by constructing an expression vector containing an antibody gene and expressing it in a host cell (P.J. Delves, ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES, 1997 WILEY; P. Shepherd and C. Dean, Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; J.W. Goding, Monoclonal Antibodies: Principles and Practice, 1993 ACADEMIC PRESS). Furthermore, transgenic animal production techniques can be used to produce transgenic animals (e.g., cows, goats, sheep, or pigs) in which the gene for the antibody of interest has been integrated into their endogenous genes, and monoclonal antibodies derived from the antibody gene can be obtained from the milk of the transgenic animals.

[0032] The obtained monoclonal antibody can be purified by an appropriate combination of methods well known in the art, such as chromatography on a protein A column, ion exchange chromatography, hydrophobic chromatography, ammonium sulfate precipitation, gel filtration, affinity chromatography, etc.

[0033] A chimeric antibody is an antibody containing sequences that are derived from different sources, for example, an antibody in which variable and constant regions that are derived from different sources are linked. In one embodiment, a chimeric antibody is composed of a variable region of an antibody derived from a mammal other than human and a constant region derived from a human antibody. A chimeric antibody can be obtained, for example, by linking a polynucleotide encoding the variable region of an antibody derived from a mammal other than human with a polynucleotide encoding the constant region of a human antibody, incorporating the resulting polynucleotide into an expression vector, and introducing the expression vector into a host for expression.

[0034] CDRs are regions that essentially determine the binding specificity of an antibody, and their amino acid sequences are highly diverse. On the other hand, the amino acid sequences constituting FRs show high homology even among antibodies with different binding specificities. Therefore, CDR grafting can transfer the binding specificity of one antibody to another.

[0035] Various methods for CDR grafting are known and are described, for example, in the following documents: U.S. Pat. Nos. 7,022,500, 6,982,321, 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332; 5,585,089, 5,530,101, 5,225,539; Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332: 323-327; Queen, et al. (1989) Proc. Natl. Acad. USA 86:10029-10033; Verhoeyen et al. (1988) Science 239:1534-1536; Winter (1998) FEBS lett 430:92-94.

[0036] A humanized antibody generally consists of a CDR from an antibody derived from a nonhuman animal, a FR from a human antibody, and a constant region from a human antibody. Humanized antibodies can be obtained by grafting the CDR from an antibody derived from a nonhuman animal onto a human antibody. Humanized antibodies can be produced by various methods, including overlap extension PCR (Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)). In this method, PCR is performed using oligonucleotides as primers that have overlapping portions between the CDRs of an antibody derived from a nonhuman animal (e.g., a mouse antibody) and the FRs of a human antibody, synthesizing a polynucleotide in which the CDRs of the antibody derived from a nonhuman animal and the FRs of a human antibody are linked. The resulting polynucleotide is then ligated to a polynucleotide encoding the constant region of a human antibody, incorporated into an expression vector, and the expression vector is introduced into a host for expression, thereby obtaining a humanized antibody.

[0037] The sequence of the FR can be determined, for example, based on a database disclosing germline antibody gene sequences (e.g., VBase, https: / / www2.mrc-lmb.cam.ac.uk / vbase / ) or references (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. 1991); Tomlinson, IM et al. (1992) J. Mol. Biol. 227:776-798; Cox, JPL et al. (1994) Eur. J. Immunol. 24:827-836). The FR may contain one or more amino acid mutations compared to the germline sequence. Methods for selecting suitable FRs are known, and for example, FRs selected by the best-fit method (Sims et al. J. Immunol. 151:2296 (1993)) or FRs derived from consensus sequences of specific subgroups of light or heavy chain variable regions of human antibodies (Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al. J. Immunol. 151:2623 (1993)) can be used.

[0038] Human antibodies can be obtained, for example, by sensitizing human lymphocytes in vitro with a desired antigen and then fusing the sensitized lymphocytes with human myeloma cells (Japanese Patent Publication No. 1-59878). U266 and other types of human myeloma cells can be used as the fusion partner. Human antibodies can also be obtained by immunizing transgenic animals carrying a full repertoire of human antibody genes with a desired antigen (Lonberg, Nat. Biotech. 23: 1117-1125, 2005). Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known (Antibody Phage Display: Methods and Protocols, Methods in Molecular Biology 178, 2001). For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain fragments (scFvs) using phage display, phages that bind to the antigen are selected, and the DNA sequences encoding the variable regions of the antigen-binding human antibodies can be determined by analyzing the genes of the selected phages. Next, this variable region sequence is linked in frame to the sequence of a human antibody constant region, inserted into an appropriate expression vector, and this expression vector is introduced into a host for expression, thereby obtaining a human antibody.

[0039] A multispecific antibody is an antibody that binds to at least two different sites. Examples of multispecific antibodies include bispecific antibodies and trispecific antibodies. In one embodiment, a multispecific antibody binds to hGDF15 and one or more other antigens. Multispecific antibodies can be produced, for example, by genetic engineering techniques or by combining two or more antibodies that recognize different antigens.

[0040] Antibody fragments can be obtained, for example, by digesting antibodies with proteases such as papain and pepsin. Alternatively, antibody fragments can be obtained by introducing an expression vector containing a polynucleotide encoding the antibody fragment into a host cell and expressing the polynucleotide (e.g., Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137; Hudson et al., Nat. Med., (2003) 9, 129-134).

[0041] As described above, antibodies can be obtained by introducing an expression vector containing a polynucleotide encoding the antibody into cells and expressing it. Specifically, an expression vector is constructed so that a sequence encoding the antibody is expressed under the control of an expression control region such as an enhancer or promoter, and host cells are transformed with this expression vector to express the antibody.

[0042] That is, the present disclosure also provides polynucleotides encoding anti-hGDF15 antibodies, expression vectors containing the polynucleotides, and transformed cells containing the polynucleotides capable of expressing the antibodies.

[0043] Eukaryotic cells such as animal cells, plant cells, and fungal cells can be used as host cells. Animal cells include mammalian cells (e.g., CHO, COS, NIH3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero), amphibian cells (e.g., Xenopus oocytes), and insect cells (e.g., Sf9, Sf21, and Tn5). Fungal cells include yeast (e.g., Saccharomyces, e.g., Saccharomyces cerevisiae) and filamentous fungi (e.g., Aspergillus, e.g., Aspergillus niger). Prokaryotic cells such as Escherichia coli (e.g., JM109, DH5α, and HB101) and Bacillus subtilis can also be used as host cells. Vectors can be introduced into host cells by, for example, the calcium phosphate method, the DEAE-dextran method, electroporation, lipofection, or the like.

[0044] The binding of the obtained antibody to the antigen can be confirmed by immunoassays such as enzyme immunoassay (EIA) (including ELISA), radioimmunoassay (RIA), chemiluminescent immunoassay (CIA), and fluorescent immunoassay (FIA), as well as BIACORE® surface plasmon resonance assays. The binding of the antibody to the antigen can also be confirmed by competitive assays. For example, this can be confirmed by examining whether the obtained antibody competes with an anti-hGDF15 antibody whose binding to hGDF15 has been confirmed.

[0045] As used herein, an antibody that competes with a given anti-hGDF15 antibody (i.e., a reference antibody) refers to an antibody that significantly reduces the binding of the reference antibody to hGDF15 when measured under the conditions described in the Examples. In some embodiments, an antibody of the present disclosure reduces the binding of the reference antibody to hGDF15 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more.

[0046] In certain embodiments, an anti-hGDF15 antibody of the present disclosure binds to an epitope of hGDF15 comprising the amino acid sequence DHCPLGPGRCCRLH (SEQ ID NO: 3). The amino acid sequence of SEQ ID NO: 3 corresponds to amino acid residues 5 to 18 of SEQ ID NO: 2. Binding to an epitope comprising the amino acid sequence DHCPLGPGRCCRLH (SEQ ID NO: 3) can be confirmed by examining whether the antibody competes for binding to hGDF15 with an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.

[0047] In further embodiments, an anti-hGDF15 antibody of the present disclosure competes with a given anti-hGDF15 antibody for binding to hGDF15. In certain embodiments, an antibody of the present disclosure competes with an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9 for binding to hGDF15.

[0048] In further embodiments, the anti-hGDF15 antibodies of the disclosure: a CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 and a heavy chain variable region comprising: a CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 a light chain variable region comprising: CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising: CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR3 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and The antibody comprises a CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR3 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. In this embodiment, CDRs may be identified by any method. A CDR contained in a predetermined heavy or light chain variable region refers to a CDR consisting of an amino acid sequence identified from the predetermined heavy or light chain variable region by any method, and the heavy or light chain variable region containing the CDR may differ from the predetermined heavy or light chain variable region in the sequence other than the CDR. Those skilled in the art can identify CDRs by a suitable method, taking various factors into consideration. In one embodiment, CDRs are identified by any definition selected from Kabat, Chothia, AbM, Contact, and IMGT.

[0049] In further embodiments, the anti-hGDF15 antibodies of the disclosure: CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising Includes.

[0050] In further embodiments, the anti-hGDF15 antibodies of the disclosure: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 44, and 149 to 150; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 153; and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, 100 to 104, and 154; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106 to 115, and 155 to 157; and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 a light chain variable region comprising Includes.

[0051] In further embodiments, the anti-hGDF15 antibodies of the disclosure: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 42, and 149 to 150; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 19, 46 to 48, 52 to 66, and 151 to 152; and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, 100 to 104, and 154; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, 113 to 115, and 155 to 157; and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 a light chain variable region comprising Includes.

[0052] In a further embodiment, the anti-hGDF15 antibody of the present disclosure comprises a heavy chain variable region selected from the following (1) to (3) and a light chain variable region selected from the following (4) to (6): (1) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 44, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 153; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 a heavy chain variable region comprising: (4) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 A light chain variable region comprising:

[0053] In a further embodiment, the anti-hGDF15 antibody of the present disclosure comprises a heavy chain variable region selected from the following (1) to (3) and a light chain variable region selected from the following (4) to (6): (1) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 42, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 19, 46 to 48, 52 to 66, and 151 to 152; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 a heavy chain variable region comprising: (4) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, 113 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 A light chain variable region comprising:

[0054] In a further embodiment, the anti-hGDF15 antibody of the present disclosure comprises a heavy chain variable region selected from the following (1) to (3) and a light chain variable region selected from the following (4) to (6): (1) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38, and 39; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any amino acid sequence selected from SEQ ID NOs: 19, 52, and 66; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20 and 77 a heavy chain variable region comprising: (4) CDR1 comprising any amino acid sequence selected from SEQ ID NOs: 21 and 85; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any amino acid sequence selected from SEQ ID NOs: 22, 155, and 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 23 and 95 A light chain variable region comprising:

[0055] In further embodiments, the anti-hGDF15 antibodies of the disclosure: (i) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 44, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 153; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or a light chain variable region comprising (vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 The light chain variable region comprises:

[0056] In further embodiments, the anti-hGDF15 antibodies of the disclosure: (i) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 42, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 152; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, 113 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or a light chain variable region comprising (vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 The light chain variable region comprises:

[0057] In further embodiments, the anti-hGDF15 antibodies of the disclosure: CDR1 comprising the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising Includes.

[0058] In further embodiments, the anti-hGDF15 antibodies of the disclosure: CDR1 comprising the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising Includes.

[0059] In further embodiments, the anti-hGDF15 antibodies of the disclosure: A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 8; and A light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 9. Includes.

[0060] In further embodiments, the anti-hGDF15 antibodies of the disclosure: The amino acid sequence of SEQ ID NO: 8, or H48R, H49R, H49D, H50R, H50S, H50F, H52R, H52Q, H72R, H73D, H73R, H73Y, H119R, H119F, H48S, H71Y, H72A, H72L, H72N, H72T, H72W, H75H, H75L, H75N, H75Q, H79H, H79 a heavy chain variable region comprising an amino acid sequence comprising any amino acid substitution selected from K, H79Q, H79R, H83R, H117Q, H119E, H119H, H119K, H119N, H119Q, H119S, H119T, H120A, H120D, H120F, H120N, H120Q, H122F, H54T, H54N, H71R, H71H, and H77I; and The amino acid sequence of SEQ ID NO: 9, or L47R, L48E, L48R, L48S, L48K, L50D, L50R, L50F, L50Y, L73R, L111F, L111Y, L112E, L112R, L112D, L112F, L113D, L113R, L113F, L48H, L48Y, L50Q, L50W, L51Q, L69Y, L70F, L70H, L72D, L72E, L a light chain variable region comprising an amino acid sequence containing any amino acid substitution selected from 72R, L72Y, L73K, L73N, L73Y, L75Q, L87K, L87N, L111A, L111N, L111S, L112H, L112Q, L112T, L112Y, L113S, L114F, L114H, L114I, L114N, L114Y, L116H, L116Y, L51Y, L72F, L73Q, and L74H; Includes.

[0061] In further embodiments, the anti-hGDF15 antibodies of the disclosure: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence containing any amino acid substitution in the amino acid sequence of SEQ ID NO: 8 selected from H49R, H49D, H50R, H50S, H50F, H73D, H73R, H73Y, H48S, H71Y, H72A, H72L, H72N, H72T, H72W, H75H, H75L, H75N, H75Q, H79H, H79K, H79Q, H79R, H83R, H119N, H119S, H120F, H120Q, H54T, H54N, H71R, and H71H; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence containing any one of the amino acid substitutions selected from L47R, L48E, L48R, L48K, L50D, L50R, L50F, L50Y, L73R, L112E, L112D, L112F, L113D, L113R, L113F, L48H, L48Y, L50Q, L50W, L51Q, L70F, L72D, L72E, L72R, L72Y, L73N, L73Y, L75Q, L112H, L112Q, L112Y, L113S, L116H, L116Y, L51Y, L72F, L73Q, and L74H in the amino acid sequence of SEQ ID NO:9. Includes.

[0062] In further embodiments, the anti-hGDF15 antibodies of the disclosure: A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO: 8 containing any one of amino acid substitutions selected from H49R, H49D, H48S, H71Y, H83R, and H120F; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence of SEQ ID NO: 9 containing any one of amino acid substitutions selected from L48K, L112D, L72F, and L74H. Includes.

[0063] In these embodiments, amino acid substitutions are represented by an abbreviation of the amino acid residue before substitution, a number representing its position, and an abbreviation of the amino acid residue after substitution, e.g., "H48R" means that the 48th histidine residue is replaced with an arginine residue.

[0064] In further embodiments, the anti-hGDF15 antibodies of the present disclosure comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[0065] In further embodiments, the anti-hGDF15 antibodies of the disclosure: A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 5; A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues in the amino acid sequence of SEQ ID NO: 6 have been modified; and a light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 7; A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 133; and a light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 134; or A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 135, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 135; and A light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 136. Includes.

[0066] As used herein, amino acid modifications include deletion, substitution, insertion, and addition of amino acids. The modifications may be any one of deletion, substitution, insertion, and addition, or a combination of two or more of these. The number of modifications is not limited, and may be, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3. In one embodiment, the amino acid modification is an amino acid substitution. In a further embodiment, the amino acid modification is a modification of 1 to 3 amino acids. In a further embodiment, the amino acid modification is a substitution of one amino acid.

[0067] As used herein, an amino acid sequence that "comprises" a given amino acid sequence includes an amino acid sequence in which one or more amino acid residues are added to the given amino acid sequence, and a sequence that consists of the given amino acid sequence.

[0068] As used herein, a heavy chain variable region or light chain variable region comprising an amino acid sequence that has 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of a predetermined heavy chain variable region or light chain variable region, and a heavy chain variable region or light chain variable region comprising an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been modified in the amino acid sequence of the predetermined heavy chain variable region or light chain variable region, include heavy chain variable regions or light chain variable regions in which no modifications have been made to the CDRs in the amino acid sequence of the predetermined heavy chain variable region or light chain variable region.

[0069] As used herein, "sequence identity" in reference to amino acid sequences refers to the percentage of amino acid residues that match between two sequences optimally aligned (maximum identity) across the entire region of the sequences being compared. The sequences being compared may contain additions or deletions (e.g., gaps) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W available from public databases (e.g., DDBJ (http: / / www.ddbj.nig.ac.jp)). Alternatively, it can be determined using commercially available sequence analysis software (e.g., Vector NTI® software, GENETYX® ver. 12).

[0070] Various methods are known for modifying amino acid sequences to obtain antibodies with desired properties. For example, mutants with improved binding affinity can be obtained by phage display-based methods. In this method, the site for mutation is determined by, for example, alanine scanning mutagenesis to identify amino acid residues that affect the antibody-antigen interaction, or by analyzing the crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Mutants with modified amino acids at these sites are generated by error-prone PCR or site-directed mutagenesis, and the resulting mutant library can be screened to obtain mutants with desired properties.

[0071] The antibodies of the present disclosure have the effect of reducing blood GDF15 levels. Whether or not they have such an effect can be confirmed by evaluating whether or not they significantly reduce blood GDF15 levels compared to control antibodies in animals transplanted with GDF15-expressing cancer cells, as described in the Examples. Without being bound by any theory, it is believed that the antibodies of the present disclosure reduce blood GDF15 levels by inhibiting the cleavage of pro-GDF15. The antibodies of the present disclosure may also have the effect of binding to mature GDF15 and inhibiting signal transduction from GDF15.

[0072] The antibodies of the present disclosure can act to improve symptoms such as weight loss, loss of appetite, and circadian rhythm disorders by reducing blood GDF15 concentrations (and optionally by inhibiting GDF15 signaling).

[0073] The antibody may be selected from an immunoglobulin subclass to modulate antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, or pharmacokinetics, and the amino acid sequence or glycosylation of the Fc region may be modified. For example, IgG4 may be selected to reduce complement activation ability. Furthermore, modifications may be made to reduce or enhance binding to Fc receptors or C1q, or to increase binding affinity to FcRn to extend blood half-life.

[0074] The antibody may also be conjugated to a polymer such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol, for example to extend the half-life of the antibody in blood or to improve its stability.

[0075] The antibody may also be conjugated to a chemotherapeutic agent, a toxic peptide, a radioisotope, etc. Chemotherapeutic agents include alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide; antimetabolites such as azathioprine and mercaptopurine; alkaloids such as vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vindesine), taxanes (e.g., paclitaxel, docetaxel), etoposide, and teniposide; topoisomerase inhibitors such as camptothecins (e.g., irinotecan and topotecan); cytotoxic antibiotics such as actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, and mitomycin;

[0076] The antibody of the present disclosure can be used as an active ingredient in a pharmaceutical composition. The antibody of the present disclosure has the effect of reducing blood GDF15 concentration (and optionally, the effect of inhibiting GDF15 signal transduction) and is useful for treating diseases or symptoms associated with GDF15. Diseases or symptoms associated with GDF15 include weight loss, anorexia, muscle loss, decreased activity, circadian rhythm disorders, abnormal pituitary hormone secretion, thermoregulation disorder, cachexia, cancer, diabetes, renal failure, heart failure, AIDS, COPD, multiple sclerosis, rheumatoid arthritis, sepsis, tuberculosis, sarcopenia, bone metastasis of cancer, anticancer drug-induced nausea and vomiting, hyperemesis gravidarum, chronic myeloproliferative disorders (e.g., myelofibrosis, polycythemia vera, essential thrombocythemia), anorexia nervosa, bipolar disorder, mitochondrial disease, and ICU-associated muscle weakness.

[0077] In one embodiment, the cachexia is associated with cancer, diabetes, renal failure, heart failure, AIDS, COPD, multiple sclerosis, rheumatoid arthritis, sepsis, or tuberculosis. In a further embodiment, the cachexia is cancer cachexia. Treatment of cachexia includes amelioration of one or more symptoms selected from weight loss, anorexia, muscle loss, decreased activity, circadian rhythm disorders, abnormal pituitary hormone secretion, and thermoregulation disorders.

[0078] Cancers include, but are not limited to, gastric cancer, esophageal cancer, colon cancer, lung cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, thyroid cancer, hepatocellular carcinoma, intrahepatic bile duct cancer, head and neck cancer, leukemia, multiple myeloma, lymphoma, brain tumor, glioma, melanoma, and the like.

[0079] The antibody of the present disclosure is administered to a subject in an amount (herein referred to as an effective amount) capable of exerting the desired effect (e.g., treatment of a disease or symptom associated with GDF15). The dose of the antibody is appropriately selected depending on the administration method, the age, weight, health condition, etc. of the subject. For example, but not limited to, 10 μg / kg to 100 mg / kg, 100 μg / kg to 10 mg / kg, or 1 mg / kg to 10 mg / kg per day for an adult can be administered daily, or once every few days, once a week, once a week, once a month, or once every several months. The method of antibody administration is also appropriately selected depending on the age, weight, health condition, etc. of the subject. The administration method may be oral or parenteral, with parenteral administration being preferred. Parenteral administration includes subcutaneous administration, intradermal administration, intramuscular administration, and intravenous administration, with intravenous administration being preferred.

[0080] As used herein, a "subject" is a mammal. Mammals include, but are not limited to, mice, rats, rabbits, cats, dogs, sheep, pigs, horses, cows, monkeys, and humans. In some embodiments, the subject is a human.

[0081] Pharmaceutical compositions can be formulated by conventional methods and may contain, in addition to the antibody, pharmaceutically acceptable carriers or additives such as sterilized water, physiological saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc.

[0082] The antibody of the present disclosure may be used in combination with other therapeutic agents. When two or more active ingredients are used in combination, all or some of the active ingredients may be contained in the same composition, or all of the active ingredients may be contained in separate compositions. The administration schedules of the two or more active ingredients may be the same or different.

[0083] In some embodiments, the antibodies of the present disclosure are used in combination with a cancer therapeutic agent, including, but not limited to, alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide; antimetabolites such as azathioprine and mercaptopurine; alkaloids such as vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vindesine), taxanes (e.g., paclitaxel, docetaxel), etoposide, and teniposide; These include topoisomerase inhibitors such as amputeecin (e.g., irinotecan and topotecan); cytotoxic antibiotics such as actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, and mitomycin; molecularly targeted drugs such as EGFR inhibitors, HER2 inhibitors, ALK inhibitors, and VEGFR inhibitors; and immune checkpoint inhibitors such as anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0084] Exemplary embodiments of the present disclosure are described below.

[0085] [1] An anti-hGDF15 antibody that binds to an epitope of hGDF15 comprising the amino acid sequence DHCPLGPGRCCRLH (SEQ ID NO: 3). [2] a CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 and a heavy chain variable region comprising: a CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 a light chain variable region comprising The antibody described in 1 above, comprising: [3] CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising 3. The antibody according to 1 or 2 above, comprising: [4] a CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 and a heavy chain variable region comprising: a CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3 a light chain variable region comprising An anti-hGDF15 antibody comprising: [5] CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising An anti-hGDF15 antibody comprising: [6] 6. The antibody according to any one of 2 to 5 above, wherein the alterations of the 1 to 3 amino acid residues are alterations of one amino acid residue. [7] 7. The antibody according to any one of 2 to 6 above, wherein the amino acid residue modification is an amino acid residue substitution. [8] 8. The antibody according to any one of 2 to 7 above, wherein the modification of the 1 to 3 amino acid residues is substitution of one amino acid residue. [9] 9. The antibody according to any one of 1 to 8 above, which comprises a heavy chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO:20.

[10] 10. The antibody according to any one of 1 to 9 above, which comprises a light chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[11] CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 44, and 149 to 150; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 153; and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, 100 to 104, and 154; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106 to 115, and 155 to 157; and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 a light chain variable region comprising 11. The antibody according to any one of 1 to 10 above, comprising:

[12] CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 42, and 149 to 150; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 19, 46 to 48, 52 to 66, and 151 to 152; and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, 100 to 104, and 154; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, 113 to 115, and 155 to 157; and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 a light chain variable region comprising 12. The antibody according to any one of 1 to 11 above, comprising:

[13] CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18 and 38 to 44; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, and 52 to 66; and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, and 100 to 104; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, and 106 to 115; and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 a light chain variable region comprising 13. The antibody according to any one of 1 to 12 above, comprising:

[14] CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18 and 38 to 42; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 46 to 48, and 52 to 66; and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, and 100 to 104; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, and 113 to 115; and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 a light chain variable region comprising 14. The antibody according to any one of 1 to 13, comprising:

[15] 15. The antibody according to any one of 1 to 14 above, comprising a heavy chain variable region selected from the following (1) to (3) and a light chain variable region selected from the following (4) to (6): (1) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 44, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 153; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 a heavy chain variable region comprising: (4) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 A light chain variable region comprising:

[16] The antibody according to any one of 1 to 15 above, comprising a heavy chain variable region selected from the following (1) to (3) and a light chain variable region selected from the following (4) to (6): (1) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 42, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 19, 46 to 48, 52 to 66, and 151 to 152; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 a heavy chain variable region comprising: (4) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, 113 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 A light chain variable region comprising:

[17] 17. The antibody according to any one of 1 to 16 above, comprising a heavy chain variable region selected from the following (1) to (3) and a light chain variable region selected from the following (4) to (6): (1) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38, and 39; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any amino acid sequence selected from SEQ ID NOs: 19, 52, and 66; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 a heavy chain variable region comprising: (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20 and 77 a heavy chain variable region comprising: (4) CDR1 comprising any amino acid sequence selected from SEQ ID NOs: 21 and 85; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any amino acid sequence selected from SEQ ID NOs: 22, 155, and 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 23 and 95 A light chain variable region comprising:

[18] (i) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 44, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 153; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or a light chain variable region comprising (vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 a light chain variable region comprising The antibody according to any one of 1 to 17 above.

[19] (i) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18, 38 to 42, and 149 to 150; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, 52 to 66, and 151 to 152; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, 100 to 104, and 154; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, 113 to 115, and 155 to 157; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or a light chain variable region comprising (vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 a light chain variable region comprising 19. The antibody according to any one of 1 to 18 above.

[20] (i) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18 and 38 to 44; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 45 to 48, and 52 to 66; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any one of amino acid sequences selected from SEQ ID NOs: 20, 49, 50, and 67 to 80 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 89, and 100 to 104; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, and 106 to 115; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or a light chain variable region comprising (vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 91 to 99, and 118 to 132 a light chain variable region comprising 20. The antibody according to any one of 1 to 19 above. [twenty one] (i) CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 18 and 38 to 42; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising any one of the amino acid sequences selected from SEQ ID NOs: 19, 46 to 48, and 52 to 66; and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising any amino acid sequence selected from SEQ ID NOs: 20, 71, 73, 77, and 79 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising any one of amino acid sequences selected from SEQ ID NOs: 21, 81 to 83, 85 to 89, and 100 to 104; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising: (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising any one of amino acid sequences selected from SEQ ID NOs: 22, 90, 106, 108 to 111, and 113 to 115; and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or a light chain variable region comprising (vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising any one of the amino acid sequences selected from SEQ ID NOs: 23, 93, 95 to 99, 121, 122, 124, 125, 131, and 132 a light chain variable region comprising The antibody described in any one of 1 to 20 above. [twenty two] CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 21; CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 a light chain variable region comprising 22. The antibody according to any one of 1 to 21 above, comprising: [twenty three] An antibody described in any one of 1 to 22 above, comprising CDR1, CDR2, and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and CDR1, CDR2, and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. [twenty four] CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising The antibody described in 1 above, comprising: [twenty five] CDR1 comprising the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising 25. The antibody according to 1 or 24, comprising:

[26] CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising An anti-hGDF15 antibody comprising:

[27] CDR1 comprising the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising An anti-hGDF15 antibody comprising:

[28] 28. The antibody according to any one of 24 to 27 above, wherein the alterations of the 1 to 3 amino acid residues are alterations of one amino acid residue.

[29] 29. The antibody according to 28, wherein the amino acid residue modification is an amino acid residue substitution.

[30] 30. The antibody according to 28 or 29 above, wherein the modification of the 1 to 3 amino acid residues is a substitution of one amino acid residue.

[31] The antibody according to any one of 1 and 24 to 30 above, which comprises a heavy chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO:139.

[32] 32. The antibody according to any one of 1 and 24 to 31 above, which comprises a light chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO: 142.

[33] CDR1 comprising the amino acid sequence of SEQ ID NO: 137; CDR2 comprising the amino acid sequence of SEQ ID NO: 138, and CDR3 comprising the amino acid sequence of SEQ ID NO: 139 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 140; CDR2 comprising the amino acid sequence of SEQ ID NO: 141, and CDR3 comprising the amino acid sequence of SEQ ID NO: 142 a light chain variable region comprising The antibody according to any one of 1 and 24 to 32, comprising:

[34] An antibody described in any one of 1 and 24 to 33, comprising CDR1, CDR2, and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, and CDR1, CDR2, and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 134.

[35] CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR3 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising The antibody described in 1 above, comprising:

[36] CDR1 comprising the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising 36. The antibody according to claim 1 or 35, comprising:

[37] CDR1 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or a CDR3 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. and a heavy chain variable region comprising: CDR1 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or CDR1 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR1; CDR2 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR2 consisting of an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR2; and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136, or a CDR3 comprising an amino acid sequence in which 1 to 3 amino acid residues have been altered in the amino acid sequence of the CDR3. a light chain variable region comprising An anti-hGDF15 antibody comprising:

[38] CDR1 comprising the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence in which 1 to 3 amino acid residues have been modified and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; CDR2 comprising the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence in which 1 to 3 amino acid residues have been modified in the amino acid sequence; and CDR3 comprising the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence in which 1 to 3 amino acid residues have been modified a light chain variable region comprising An anti-hGDF15 antibody comprising:

[39] 39. The antibody according to any one of 35 to 38, wherein the alterations of the 1 to 3 amino acid residues are alterations of one amino acid residue.

[40] 40. The antibody according to 39, wherein the amino acid residue modification is an amino acid residue substitution.

[41] 41. The antibody according to 39 or 40, wherein the modification of the 1 to 3 amino acid residues is substitution of one amino acid residue.

[42] 42. The antibody according to any one of 1 and 35 to 41 above, which comprises a heavy chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO:145.

[43] 43. The antibody according to any one of 1 and 35 to 42 above, which comprises a light chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO:148.

[44] CDR1 comprising the amino acid sequence of SEQ ID NO: 143; CDR2 comprising the amino acid sequence of SEQ ID NO: 144, and CDR3 comprising the amino acid sequence of SEQ ID NO: 145 and a heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 146; CDR2 comprising the amino acid sequence of SEQ ID NO: 147, and CDR3 comprising the amino acid sequence of SEQ ID NO: 148 a light chain variable region comprising 44. The antibody according to any one of 1 and 35 to 43, comprising:

[45] An antibody described in any one of 1 and 35 to 44, comprising CDR1, CDR2, and CDR3 contained in a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, and CDR1, CDR2, and CDR3 contained in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136.

[46] A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 8; and A light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 9. The antibody according to any one of 1 to 45, comprising:

[47] A heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 8; and A light chain variable region comprising an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been altered in the amino acid sequence of SEQ ID NO: 9. An anti-hGDF15 antibody comprising:

[48] 48. The antibody according to any one of 1 to 47 above, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[49] The amino acid sequence of SEQ ID NO: 8, or H48R, H49R, H49D, H50R, H50S, H50F, H52R, H52Q, H72R, H73D, H73R, H73Y, H119R, H119F, H48S, H71Y, H72A, H72L, H72N, H72T, H72W, H75H, H75L, H75N, H75Q, H79H, H79 a heavy chain variable region comprising an amino acid sequence comprising any amino acid substitution selected from K, H79Q, H79R, H83R, H117Q, H119E, H119H, H119K, H119N, H119Q, H119S, H119T, H120A, H120D, H120F, H120N, H120Q, H122F, H54T, H54N, H71R, H71H, and H77I; and The amino acid sequence of SEQ ID NO: 9, or L47R, L48E, L48R, L48S, L48K, L50D, L50R, L50F, L50Y, L73R, L111F, L111Y, L112E, L112R, L112D, L112F, L113D, L113R, L113F, L48H, L48Y, L50Q, L50W, L51Q, L69Y, L70F, L70H, L72D, L72E, L a light chain variable region comprising an amino acid sequence containing any amino acid substitution selected from 72R, L72Y, L73K, L73N, L73Y, L75Q, L87K, L87N, L111A, L111N, L111S, L112H, L112Q, L112T, L112Y, L113S, L114F, L114H, L114I, L114N, L114Y, L116H, L116Y, L51Y, L72F, L73Q, and L74H; The antibody according to any one of 1 to 48, comprising:

[50] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence containing any amino acid substitution in the amino acid sequence of SEQ ID NO: 8 selected from H49R, H49D, H50R, H50S, H50F, H73D, H73R, H73Y, H48S, H71Y, H72A, H72L, H72N, H72T, H72W, H75H, H75L, H75N, H75Q, H79H, H79K, H79Q, H79R, H83R, H119N, H119S, H120F, H120Q, H54T, H54N, H71R, and H71H; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence containing any one of the amino acid substitutions selected from L47R, L48E, L48R, L48K, L50D, L50R, L50F, L50Y, L73R, L112E, L112D, L112F, L113D, L113R, L113F, L48H, L48Y, L50Q, L50W, L51Q, L70F, L72D, L72E, L72R, L72Y, L73N, L73Y, L75Q, L112H, L112Q, L112Y, L113S, L116H, L116Y, L51Y, L72F, L73Q, and L74H in the amino acid sequence of SEQ ID NO:9. The antibody according to any one of 1 to 49, comprising:

[51] A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO: 8 containing any one of amino acid substitutions selected from H49R, H49D, H48S, H71Y, H83R, and H120F; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence of SEQ ID NO: 9 containing any one of amino acid substitutions selected from L48K, L112D, L72F, and L74H. The antibody according to any one of 1 to 50 above, comprising:

[52] 52. The antibody described in any one of 1 to 51 above, which competes with an anti-hGDF15 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 for binding to hGDF15.

[53] 53. The antibody according to any one of 1 to 52 above, which is a monoclonal antibody.

[0086]

[54] An anti-hGDF15 antibody that competes with any one of the antibodies described in 1 to 53 above for binding to hGDF15.

[55] 55. The antibody according to 54, which competes with an anti-hGDF15 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.

[56] 56. The antibody according to 54 or 55, which reduces blood GDF15 concentration.

[57] 57. The antibody according to any one of 54 to 56 above, which inhibits the cleavage of pro-GDF15.

[58] 57. The antibody according to any one of 54 to 56, which is a monoclonal antibody.

[0087]

[59] A polynucleotide encoding the antibody according to any one of 1 to 58 above.

[60] 59. The polynucleotide according to 59, comprising the nucleic acid sequence of SEQ ID NO: 24 and / or 25.

[61] 61. An expression vector comprising the polynucleotide according to 59 or 60.

[62] A transformed cell comprising the polynucleotide according to 59 or 60.

[0088]

[63] A pharmaceutical composition comprising the antibody described in any one of 1 to 58.

[64] 64. The pharmaceutical composition according to 63, for treating a disease or condition associated with GDF15.

[65] 65. The pharmaceutical composition according to 64, wherein the disease or condition associated with GDF15 is cancer.

[66] 65. The pharmaceutical composition according to 64, wherein the disease or condition associated with GDF15 is cachexia.

[67] 67. The pharmaceutical composition according to claim 66, wherein the cachexia is cancer cachexia.

[68] 64. The pharmaceutical composition according to 63, for lowering blood GDF15 concentration.

[0089]

[69] 59. The antibody according to any one of 1 to 58, for use in treating a disease or symptom associated with GDF15.

[70] 59. The antibody according to any one of 1 to 58, for use in reducing blood GDF15 concentration.

[71] A method for treating a disease or symptom associated with GDF15, comprising administering an effective amount of an antibody according to any one of 1 to 58 to a subject in need of treatment.

[72] A method for reducing blood GDF15 concentration, comprising administering an effective amount of the antibody according to any one of 1 to 58 to a subject in need of said reduction.

[73] Use of the antibody according to any one of 1 to 58 above for the manufacture of a medicament for treating a disease or symptom associated with GDF15.

[74] Use of the antibody according to any one of 1 to 58 above for the manufacture of a medicament for reducing blood GDF15 concentration.

[0090] The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the present invention in any way. [Example]

[0091] I. Antibody Production (1) A. Generation of anti-hGDF15 antibodies 1. Immunization of animals schedule Day1; First immunization (25 μg / shot / body, Abisco-100, ip) Day 10: Second immunization (25 μg / shot / body, Abisco-100, ip) Day 17: Third immunization (25 μg / shot / body, Abisco-100, ip) Day 21; Titer check Day24; Final boost(25 μg / shot / body, Abisco-100, ip) Day 27: Spleen removal and cell fusion reagent ·Recombinant Human GDF15(rhGDF15), R&D, 957-GD-025 / CF, 25 μg Abisco-100 (ISCONOVA) animal 3H / HeJ Jms Slc-lpr / lpr (5W, Japanese SLC, female)

[0092] procedure 1. 50 μL of hGDF15 (0.5 mg / mL), 25 μL of Abisco-100, and 175 μL of PBS were mixed and administered intraperitoneally to mice (primary immunization). 2. 10 and 17 days after the primary immunization, the hGDF15 solution prepared in the same manner as for the primary immunization was administered intraperitoneally. 3. 21 days after the first immunization, blood was collected from the tail of each mouse using a capillary collection tube, and plasma was obtained by centrifugation. 4. The plasma was diluted 100-fold, 1000-fold, and 10,000-fold, and ELISA was performed using a 96-well plate coated with hGDF15. An increase in antibody titer to hGDF15 was confirmed. 5. 24 days after the primary immunization, an hGDF15 solution prepared in the same manner as in the primary immunization was administered intraperitoneally. 6. 27 days after the first immunization, the spleen was removed under anesthesia and cell fusion was performed.

[0093] 2. Cell fusion and HAT selection cell · Sp2 / o-14Ag (hereafter Sp2 / o cells, ATCC # CRL-1581) 3×10 5 Subculture so as not to exceed 100 cells / mL reagent DMEM (GIBCO, 10313-021) FBS (heat-inactivated at 56°C for 30 minutes) · Penicillin-Streptomycin-Glutamine (Invitrogen, 10378-016) Insulin (Funakoshi, BT-243) prepared at 10 mg / mL · 55mM 2-Mercaptoethanol(1000x) (Invitrogen, 21985-023) rIL-6 PBS Turkic fluid Trypan blue · Red blood cell lysing buffer (SIGMA, R7757) PEG1500 (Roche, 108014) · HAT Media Supplement (50×) Hybri-Max (SIGMA, H0262) · HT Media Supplement (50×) Hybri-Max (SIGMA, H0137) Equipment 6-well plate (BD, 353046) Large flask for suspension cell culture Surgical forceps (3 pieces) Surgical scissors (3 pairs) 50 mL Falcon tube 1 mL syringe · 23G needle Cell strainer 40μm (BD, 352340) Medium composition Sp2 / o culture medium DMEM 500 mL FBS 55 mL (final 10%) Penicillin-Streptomycin-Glutamine (100×) 5 mL (final 1×) Cell fusion medium DMEM Hybridoma medium DMEM 500 mL FBS 55 mL (final 10%) Penicillin-Streptomycin-Glutamine (100×) 5 mL (final 1×) 2ME 500 μL (final 0.1%) Insulin (10 mg / mL) 250 μL (final 5 μg / mL) rIL-6 as appropriate (final 5 ng / mL) HAT selective medium Hybridoma medium 50× HAT as appropriate (final 1×)

[0094] procedure Myeloma preparation (1 week before cell fusion) 1. Cryopreserved Sp2 / o cells (approximately 1 × 10 6 The cells (200 cells / vial) were thawed, suspended in 10 mL of Sp2 / o medium that had been dispensed into 15 mL Falcon tubes in advance, washed by centrifugation at 1500 rpm for 3 minutes, and the supernatant was removed. 2. The pellet (Sp2 / o cells) was suspended in 4 ml of Sp2 / o medium. 3. 4 mL of Sp2 / o medium was placed in each well of a 6-well plate, and the Sp2 / o cells suspended in step 2 were diluted two-fold. 4. The cells were cultured at 37°C under 5.5% CO2 for 1-2 days. 5. Cells from wells in good condition were collected under a microscope and expanded in a large flask (or medium flask) for suspension cell culture. Subculture was performed two days before cell fusion. (On the day of cell fusion, 5 × 10 5 The cell density was adjusted to approximately 100 cells / mL.

[0095] Removal of spleens from mice 1. Mice were anesthetized, thoroughly sprayed with 70% ethanol, and then the abdomen was opened in a safety cabinet. Sterile forceps and scissors for external and intraperitoneal use were prepared to aseptically remove the spleen. 2. Under anesthesia, exsanguination was performed via the abdominal aorta, and the spleen was then removed. 3. The spleen was placed in a 50 mL Falcon tube containing PBS and placed on ice until cell fusion.

[0096] Cell fusion (creation of hybridomas) DMEM (serum-free) and PEG were pre-warmed in a 37°C incubator, and the following procedure was carried out. 1. Sp2 / o cells were collected and the cell number was counted. 1 x 10 6 Only cells from flasks containing less than 100 cells / mL were used. 2. Approximately 10 mL of PBS was prepared in one well of a 6-well Falcon plate, the excised spleen was placed therein, and any remaining adipose tissue around it was removed. 3. 10 mL of PBS was added to one of the remaining wells, and the spleen was transferred thereto. The spleen was then cut in half using sterile forceps and scissors. 4. A 23G needle was attached to a 1 mL syringe, and PBS was drawn into the halved spleen, pushing out the splenocytes into the PBS. This process was repeated until the spleen turned white. 5. A sterilized mesh was placed on top of a 50 mL Falcon tube, and the PBS containing splenocytes was passed through it to remove tissues other than splenocytes. Cell fusion medium was added to bring the total volume to 40 mL. 6. The mixture was centrifuged at 1500 rpm for 3 minutes and the supernatant was removed. 7. The splenocytes were suspended in 20 mL of cell fusion medium, stained with Turk's solution (Turk's solution:suspension = 10:1), and the white blood cells were counted (generally 1 x 10 8 cells, 5 × 10 6 cells / mL.) 8. The mixture was centrifuged at 1500 rpm for 3 minutes and the supernatant was removed. 9. The cells were suspended in 3 mL of red blood cell lysing buffer and allowed to stand on ice for 3 minutes. 10. 17 mL of cell fusion medium was added, and the mixture was centrifuged at 1500 rpm for 3 minutes to collect the splenocytes. 11. A medium containing Sp2 / o cells was added to the cells so that the cell number became 5:1 in terms of splenocytes:Sp2 / o cells, and the cells were suspended. 12. Centrifuge at 1500 rpm for 3 minutes and remove the supernatant. 13. The precipitated cells were resuspended by tapping. 14. PEG1500 was added dropwise, 1 mL, over 1 minute with slow stirring. 15. Stirring was continued for another minute. 16. As in 14, 1 mL of cell fusion medium was added dropwise. 17. 3 mL of cell fusion medium was added over an additional 3 minutes. 18. An additional 10 mL of cell fusion medium was added over 1 minute. 19. Allow to stand at 37°C for 10 minutes. 20. Cell fusion medium was added to bring the total volume to 40 mL. 21. Centrifuge at 1500 rpm for 3 minutes and remove the supernatant. 22. Splenocyte count: 1 × 10 per mL 6 The hybridoma cells were suspended in hybridoma medium and plated in a 96-well flat-bottom plate at 100 μL per well. 23. Incubated overnight at 37°C and CO2 5.5%. 24. 100 μL / well of 2×HAT selection medium was added, and the cells were then cultured. 25. Every other day, 100 μL of the supernatant was removed and 100 μL of 1×HAT selection medium was added to replace the medium.

[0097] 3. Antibody Screening Hybridomas producing anti-hGDF15 antibodies were selected as follows, using the binding strength to hGDF15 measured by ELISA using hybridoma supernatants 5 days after the start of culture in a 96-well plate.

[0098] 100 μL of culture supernatant was added to each well of a 96-well plate coated with hGDF15 (R&D Systems, 957-GD-025 / CF) and incubated for 2 hours at room temperature. After washing the wells, a secondary antibody for detection (HRP-labeled anti-mouse IgG antibody (Promega, W402B)) was added and incubated for 1 hour at room temperature. After washing the wells, color development was performed using TMB solution (Sigma, T2885), and the absorbance at 450 nm was measured using a plate reader.

[0099] Hybridomas in wells with an absorbance of 1.0 or higher at 450 nm were further isolated into single clones by limiting dilution. Ultimately, 16 clones were obtained. Of these, 13 clones were cultured, excluding one clone that was IgM and two clones that showed nonspecific binding to multiple proteins.

[0100] Antibodies were purified from the culture supernatant using HiTrap rProtein A FF (GE Healthcare Life Sciences, 17508001). The concentration of purified antibodies was measured using a NanoDrop (Thermo Scientific) at absorbance 280 nm.

[0101] The binding affinity of each purified antibody to hGDF15 was measured by ELISA as follows: 200 ng / mL hGDF15 was added to each well at 100 μL / well. Serial dilutions of purified antibodies from 0.39 ng / mL to 25 ng / mL were added at 100 μL / well to a 96-well plate coated with hGDF15. The hGDF15-anti-hGDF15 antibody complex was detected with an HRP-conjugated secondary antibody. Absorbance at 450 nm was measured for color development, and a four-parameter logistic curve was used to calculate the antibody amount at a concentration of 5 ng / mL. The binding value of each antibody was calculated relative to the value of clone MAB17, which was set at 100.

[0102] The affinity results of the obtained anti-hGDF15 antibodies to hGDF15 are shown in Table 1. MAB2, which showed much stronger binding than MAB17, was humanized. [Table 1]

[0103] 4. Production of humanized antibodies ·cell Cell line name: ExpiCHO Origin: Chinese hamster Cell source: Thermo Fisher Scientific (A29127) Culture medium: ExpiCHO expression medium Culture solution source: Thermo Fisher Scientific (A2910001)

[0104] Expression vector Vector name: pcDNA-3.1 cDNA origin: Totally synthetic Available from: Thermo Fisher Scientific

[0105] Recombinant proteins Protein name: Human GDF15 Origin: CHO cells Available from: R&D systems (957-GD-025 / CF)

[0106] Chimeric antibody sequence design The variable region containing the CDR sequence of MAB2, a mouse-derived antibody, was joined with the constant region of bevacizumab to create the sequence of a chimeric antibody.

[0107] Humanized antibody sequence design A Blast search was performed to find human antibodies with high similarity to MAB2. The CDR sequences of these human antibodies were replaced to create variable regions, and the constant region of bevacizumab was then added to create full-length humanized antibodies. Furthermore, for amino acids that were deemed important for maintaining the structure based on structural predictions, the mouse antibody amino acids were left intact and used as part of the humanized antibody.

[0108] Antibody production and purification The heavy and light chain genes were synthesized and then inserted into expression vectors. ExpiCHO cells were seeded onto 24-well plates, and the next day, the heavy and light chain expression vectors were co-transfected using the TransIT-CHO kit (Takara, V2170) to allow co-expression of the heavy and light chains. After five days, the culture supernatant containing the produced antibody was collected, and the antibody was purified using a Hitrap rProtein A FF column (GE Healthcare, 17-5080-01).

[0109] Antibody concentration measurement The antibody concentration in the supernatant was measured using a Human IgG EIA kit (Takara, MK136) according to the attached protocol. The absorbance of the purified antibody was measured at 280 nm using a NanoDrop (Thermo Fisher Scientific) to determine the concentration.

[0110] Measurement of binding to hGDF15 (ELISA) 100 μL of human GDF15 solution (0.4 μg / mL) was added to a 96-well plate and left to stand until the next morning to prepare an ELISA plate. The plate was blocked with 1% BSA for 1 hour. A 3-fold dilution series of antibody-containing culture supernatant was prepared and added in 100 μL portions to the ELISA plate and left to stand for 2 hours. For the secondary antibody reaction, 100 μL of 20,000-fold diluted HRP-conjugated anti-human IgG antibody (GeneTex, GTX26759) was added and allowed to react for 1 hour. TMB (Sigma, T-0440) was used for the color reaction, and 2N sulfuric acid was used as the stop solution. Absorbance was measured at 450 nm.

[0111] Measurement of binding affinity using BLITZ The binding affinity of the antibody was measured using the BLITZ (Pall ForteBio) biomolecular interaction analysis system. Anti-HIS (Pall ForteBio, 18-5114) was used for the sensor chip, and ForteBio Sample Diluent (Pall ForteBio, 18-1048) was used for the wash solution. The measurement program was set to 60 seconds for hGDF15 adsorption to the sensor chip, 120 seconds for antibody binding, and 120 seconds for dissociation.

[0112] B. Sequencing 1. Test Method Total RNA was prepared from hybridomas cultured in 10 cm dishes, and cDNA was synthesized using the SMARTer RACE 5' / 3' Kit (Takara, 634858). The synthesized cDNA was inserted into the pCR4Blunt-TOPO vector, amplified in E. coli, and the plasmid DNA was purified. DNA sequencing was performed using BigDye. TM The sequencing was performed using the Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, 4337454) and M13 primers on an ABI 3130xl Genetic Analyzer (Applied Biosystems).

[0113] 2 Results The amino acid sequences of MAB17, MAB2, and HuMAB2 are shown in Table 2, the CDR sequences in Table 3, and the cDNA sequence of HuMAB2 in Table 4. CDRs were identified according to the Kabat definition. [Table 2-1] [Table 2-2] [Table 3] [Table 4-1] [Table 4-2] [Table 4-3]

[0114] C. Comparison with existing anti-hGDF15 antibodies 1. Test materials and methods Anti-hGDF15 antibody Table 5 shows the antibodies used for comparison of reactivity with HuMAB2. [Table 5]

[0115] Test Method HuMAB2 and the anti-GDF15 monoclonal antibodies (2 μg / mL) listed in Table 5 were added to a 96-well plate at 100 μL / well. Serial dilutions of hGDF15 (R&D Systems, 957-GD-025 / CF) starting from 1 ng / mL were added to each plate at 100 μL / well. HuMAB2 was biotinylated using the EZ-Link Micro NHS-PEG4-Bitinylation Kit (Thermo Fisher Scientific, 21955) and used as the detection antibody (1 μg / mL, 100 μL / well). The sandwich ELISA was performed using the streptavidin-HRP conjugate included with the Human GDF15 ELISA Kit (R&D Systems, DY957). The detection antibody (Table 5) included in the Human GDF15 ELISA Kit (R&D Systems, DY957), which can be sandwiched with any of the monoclonal antibodies, was used as the positive control antibody (PC) for sandwich ELISA.

[0116] 2 Results If the immobilized antibody and the biotinylated antibody recognize similar epitopes, competitive inhibition occurs, but if they recognize different epitopes, competitive inhibition is not expected. As shown in Figure 1, HuMAB2 was able to recognize hGDF15 simultaneously with the other antibodies. This suggests that HuMAB2 recognizes a different epitope from the other antibodies.

[0117] As described above, it was revealed that HuMAB2 does not compete with existing antibodies. Therefore, we tested whether other antibodies obtained this time compete with HuMAB2 using the same method as above. The results were calculated as % inhibition and are shown in Table 6. Of the eight clones tested, four clones (MAB1, MAB11, MAB12, and MAB13) were found to inhibit the binding of HuMAB2 to hGDF15 by 70% or more. [Table 6]

[0118] D. Crystallization of the hGDF15 and HuMAB2 Fab Complex 1. Test materials and methods A complex protein solution was prepared by mixing hGDF15 (R&D Systems, 9279-GD-050) with HuMAB2 Fab (containing a polypeptide consisting of the amino acid sequence of SEQ ID NO:8 and a polypeptide consisting of the amino acid sequence of SEQ ID NO:9) (20 ml Tris-HCl, 200 mM NaCl, pH 7.2) at a molar ratio of 1:1 to a concentration of 5 mg / mL. HuMAB2 crystallization was performed using the sitting drop vapor diffusion method. The protein solution and reservoir solution were mixed at a 1:1 ratio. The crystal structure of the complex was determined at 2.8 Å resolution using crystals obtained using the Crystal Screen 2 screening kit (Hampton Research, HR2-112). Phase determination was performed using molecular replacement.

[0119] 2 Results Structural analysis revealed that hGDF15 forms a homodimer, with two HuMAB2 Fab molecules bound to the hGDF15 dimer (Fig. 2A). The amino acids lining the binding interface between HuMAB2 and hGDF15 were identified as the paratope and epitope, respectively (Fig. 2B). Table 7 shows the identified hGDF15 epitope and the underlined amino acids on HuMAB2 that are important for binding. [Table 7]

[0120] The epitopes of hGDF15 analyzed using MOE (Molecular Operating Environment) are shown in Figure 3. HuMAB2 was found to bind to the 14 amino acids from aspartic acid 5 to histidine 18 on the N-terminus of one of the hGDF15 monomers (Monomer 1), including isoleucine 45 (45I) and glycine 46 (46G), and to aspartic acid 71 (71D) and threonine 72 (72T) on the other monomer (Monomer 2).

[0121] The amino acid residues in the epitope predicted to be particularly strongly involved in the interaction with HuMAB2 were calculated using MOE Protein Contacts (Table 8). The results showed that the amino acids shown in Figure 3 bind particularly strongly to HuMAB2. [Table 8]

[0122] E. Reactivity of anti-hGDF15 antibodies to hGDF15 and mutant hGDF15 1. Test materials and methods Test materials Table 9 shows the antibodies used for reactivity comparison with HuMAB2 antibody. [Table 9]

[0123] Test Method Test Overview Mutant hGDF15 expression constructs were transfected into ExpiCHO-S cells, and the culture supernatants were collected after incubation. Expression of each mutant hGDF15 in the culture supernatant was confirmed using a commercially available GDF15 ELISA kit. The reactivity of HuMAB2 with each mutant hGDF15 was then assessed by ELISA. To identify particularly important amino acid residues within the epitope, the reactivity of HuMAB2 with the mutated GDF15 was assessed by both ELISA and a biomolecular interaction analysis system. The differentiation of HuMAB2 from other anti-hGDF15 antibodies due to epitope differences was verified by comparing the reactivity of HuMAB2 with other anti-hGDF15 antibodies against the mutant hGDF15 using sandwich ELISA.

[0124] Preparation of mutant hGDF15 The pcDNA-3.1 vector (Thermo Fisher Scientific) containing each mutant hGDF15 gene was used for the ExpiCHO TM ExpiCHO-S (Thermo Fisher Scientific) cells were transfected using the Expression System Kit (Thermo Fisher Scientific, A29133). Ten days after transfection, the culture supernatant was centrifuged at 8000 rpm for 30 minutes at 4°C to recover the culture supernatant. The recovered culture supernatant was stored frozen (set temperature: -20°C).

[0125] Confirmation of mutant hGDF15 expression The mutant hGDF15 in the collected culture supernatant was measured using a Human GDF15 ELISA Kit (R&D Systems, DY957) according to the kit's instructions, to confirm expression and measure the concentration.

[0126] Evaluation of GDF15 antibody reactivity against mutant hGDF15 Wild-type (Wt) hGDF15 was prepared using Recombinant Human GDF15 (R&D Systems, 957-GD-025 / CF). Serially diluted Wt and mutant hGDF15 were incubated with each hGDF15 antibody on a plate, and bound Wt and mutant hGDF15 were detected using the detection antibodies (Table 9) in the Human GDF15 ELISA Kit (R&D Systems, DY957).

[0127] Evaluation of HuMAB2 reactivity against mutated GDF15 using a biomolecular interaction analysis system Each mutant hGDF15 was purified using an affinity column with MAB17 immobilized on Sepharose (GE Healthcare, 17-0906-01). Evaluation was performed using an OCTET QKe (ForteBio) biomolecular interaction analysis system. Wild-type GDF15 was analyzed using Recombinant Human GDF15 (R&D Systems, 957-GD-025 / CF). Wild-type and mutant hGDF15 were bound to the Ni-NTA biosensor (ForteBio, 18-5101), followed by serial dilutions of HuMAB2 to evaluate the interaction between mutant hGDF15 and HuMAB2. Analysis was performed using the kinetic method.

[0128] 2 Results The amino acid sequences of wild-type (WT) hGDF15 and mutant hGDF15 are shown in Table 10. The sites where amino acids in wild-type hGDF15 were substituted with alanine are underlined. [Table 10]

[0129] Table 11 shows the binding of four anti-hGDF15 antibodies to hGDF15 and mutant hGDF15. HuMAB2 did not bind to mutant hGDF15 or bound to it weaker than to wild-type hGDF15, whereas the binding of other anti-hGDF15 antibodies to hGDF15 was not affected by the mutations. This indicates that the N-terminal 5DHCPLGPGRCCRLH18 (SEQ ID NO: 3) epitope of hGDF15 for HuMAB2 is important for binding, with 13R and 16R being particularly important for HuMAB2 binding. This result also supports the idea that MAB17, Hu01G06-127, and MAB957 have epitopes distinct from those of HuMAB2. [Table 11]

[0130] F. Evaluation of HuMAB2 antibody reactivity against epitope region peptides 1. Test Method Since the 14 residues from the 5th asparagine residue to the 18th histidine residue of hGDF15 (DHCPLGPGRCCRLH, SEQ ID NO: 3) were found to be important for HuMAB2 binding, a peptide with this sequence was synthesized by GenScript (Table 12). Peptide 1 was unmodified at both ends, while Peptide 2 was synthesized with an acetylated amino terminus and amidated carboxyl terminus to mimic the internal structure of hGDF15. A 10 μg / mL peptide solution was added at 100 μL / well to a 96-well plate, where it was immobilized. HuMAB2 antibody (10 μg / mL) was then added at 100 μL / well and incubated for 3 hours at 25°C. The bound HuMAB2 antibody was detected by adding 100 μL / well of 5000-fold diluted anti-human IgG-HRP (Jackson ImmunoResearch, 109-035-008) and incubating at 25°C for 1 hour. A plate with immobilized hGDF15 (R&D Systems, 957-GD-025 / CF) was used as a plate control. The detection antibody (Table 9) of the Human GDF15 ELISA Kit (R&D Systems, DY957) was used as a positive control antibody reacting with the peptide, and the Human IgG1 Isotype Control (Abcam, ab206198) was used as a negative control antibody. [Table 12]

[0131] 2 Results As shown in Figure 4, the positive control anti-hGDF15 polyclonal antibody reacted with both the two peptides and hGDF15. In contrast, HuMAB2 reacted only with hGDF15 and did not react with the two synthetic peptides, similar to the negative control antibody, Human IgG1 Isotype Control (hIgG1). These results suggest that the surrounding sequence or three-dimensional structure is important for HuMAB2 binding to the hGDF15 epitope.

[0132] G. Binding of HuMAB2 mutants with amino acid substitutions in the CDRs to hGDF15 1. Test materials and methods Test system cell 1) Cell line name: ExpiCHO 2) Origin: Chinese hamster 3) Cell source: Thermo Fisher Scientific (A29127) 4) Culture medium: ExpiCHO expression medium 5) Source of culture solution: Thermo Fisher Scientific (A2910001) antibody expression vector 1) Vector name: pcDNA-3.1 2) cDNA origin: Totally synthetic 3) Available from: Thermo Fisher Scientific Recombinant Proteins 1) Protein name: human GDF15 (hGDF15) 2) Origin: CHO cells 3) Available from: R&D systems (957-GD-025 / CF)

[0133] Test Method Expression vector design Based on information obtained from the crystal structure analysis of HuMAB2 and hGDF15, we designated the amino acids at the antibody-antigen binding interface as the paratope and epitope, and designed expression vectors to insert amino acid mutations into the heavy or light chain of HuMAB2. Table 13 lists the mutant antibodies (altered amino acids are underlined). For both the heavy and light chains, the amino acids are arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus.

[0134] Cellular transfection with expression vectors CHO cells were seeded in a 24-well plate, and the next day, the heavy and light chains of the mutant antibody expression vectors in Table 13 were combined and transfected using a TransIT-CHO kit (Takara, V2170). After 5 days, all supernatants were collected and stored in a freezer set at -20°C until use.

[0135] Measurement of binding amount to hGDF15 100 μL of hGDF15 solution (0.4 μg / ml) was added to a 96-well plate and left at 4°C until the next morning to prepare an ELISA plate. 1% BSA was added for 1 hour to block the plate. A dilution series (common dilution ratio: 3) of the culture supernatant containing the mutant antibody was prepared starting from a 30-fold dilution, and 100 μL of each was added to the ELISA plate and left to stand for 2 hours. HRP-conjugated anti-human IgG antibody (GeneTex, GTX26759) (20,000-fold dilution) was added as the secondary antibody and left to stand for 1 hour. TMB (Sigma, T-0440) was used for the color reaction, and 2N sulfuric acid was used as the stop solution. Absorbance was measured at 450 nm.

[0136] Measurement of antibody concentration using OCTET Antibody concentrations in the culture supernatants were measured using an OCTET QKe (Pall ForteBio) biomolecular interaction analysis system. Protein L (Pall ForteBio, 18-5085) was used for the sensor chip, 10 mM glycine (pH 1.1) was used for the regeneration solution, and ForteBio Sample Diluent (Pall ForteBio, 18-1048) was used for the cleaning solution. IgG from human serum reagent grade (Sigma-Aldrich, I12511-10MG) was used as the standard at 50 mg / mL, 10 mg / mL, 1 mg / mL, and 0.1 mg / mL. The culture supernatants to be measured were diluted 2-fold with Sample Diluent (Pall ForteBio, 18-1104). The measurement program consisted of three cycles of 5 seconds for regeneration of the sensor chip and 5 seconds for washing, followed by 120 seconds for antibody concentration measurement.

[0137] Comparison of binding amount with HuMAB2 The binding amount of the antibody to human hGDF15 at 5 ng / mL was calculated using Excel, and the binding amount of each mutant antibody was expressed as a relative value, with the binding amount of HuMAB2 to hGDF15 set to 100. Three independent experiments were performed, and the mean ± standard error was calculated. The binding amount of antibodies that were no longer expressed due to the mutation was set to 0. The results are shown in Figures 5A-D.

[0138] 2 Results Five antibodies were found to have relative binding levels 1.5 times or higher than HuMAB2. Meanwhile, 30 antibodies showed a decrease in binding levels of 50% or less. Figure 5A shows two results for "H50S-L7" in "H-Chain Modified Antibody 1 / 2." These results were obtained using the same vector, which was used to confirm well-to-well variation in culture and ELISA. The similarity of both values ​​indicated a stable test system.

[0139] The heavy chain mutant antibodies H48S-L7, H83R-L7, and H120F-L7, and the light chain mutant antibodies H3-L48K and H3-L112D, showed approximately 1.5-fold increased binding compared to HuMAB2. On the other hand, the glycine 111 (111G) and leucine 114 (114L) mutants in the HuMAB2 light chain both showed decreased binding, suggesting that these amino acids are important sites for binding to hGDF15 and maintaining the structure of the HuMAB2 CDR sequence.

[0140] [Table 13-1] [Table 13-2] [Table 13-3]

[0141] II. Pharmacological Testing (1) A. Circulating hGDF15 1. Test materials and methods animal 1) Species / Strain:mouse / BALB-c Slc-nu / nu 2) Microbiological grade: specific pathogen-free (SPF) 3) Available from: Japan SLC 4) Gender: Female 5) Age: 6 weeks (at the time of hGDF15 antibody administration) 6) Rearing conditions Animals will be kept in an environment that meets the following conditions: a) Temperature: 23±2℃ b) Humidity: 60±10% c) Lighting: Lighting hours: 7am - 7pm Lights out time: 7pm to 7am d) Food and water: free access, CRF-1 (Oriental Yeast Co., Ltd.), tap water

[0142] Exam Schedule Table 14 shows the test schedule, with the day of antibody administration set as day 0. [Table 14]

[0143] Animal care methods Three days after delivery, the mice were separated into individual cages. To allow them to adapt to individual rearing, the acclimation period before group allocation was set at 5 days.

[0144] Grouping Based on body weight, the animals were divided into six groups, six animals per group, using an experimental data collection and processing system (SAS Institute Japan, R9.3).

[0145] Administration of anti-hGDF15 antibody rhGDF15, HuMAB2 antibody, and MAB17 antibody were administered intraperitoneally at 10 mL / kg. The control group received Dulbecco's Phosphate-Buffered Saline (DPBS) at 10 mL / kg. The group composition is shown in Table 15. [Table 15]

[0146] Measurement of body weight and food intake Body weight and food intake were measured on the day of grouping and on days 0, 1, 2 and 3 after administration.

[0147] Blood collection / dissection Three days after antibody administration, blood samples were collected from the inferior vena cava of mice anesthetized with isoflurane inhalation. Plasma was collected using EDTA2K blood collection tubes and stored in a freezer set at -80°C until use. After blood collection, mice were euthanized by exsanguination via abdominal incision of the inferior vena cava while still under anesthesia.

[0148] Blood unbound hGDF15 concentration To measure unbound hGDF15 in the blood, the antigen-antibody complex was removed using the following procedure. 5 μL of Protein A / G agarose beads (Thermo Scientific, 20421) were mixed with 50 μL of mouse plasma and 145 μL of PBS and incubated at 4°C for 2 hours. After centrifugation, the supernatant was transferred to a new Eppendorf tube and stored in a freezer at -80°C until use. Subsequent blood GDF15 measurements were performed using a GDF15 ELISA kit (R&D Systems, DY957) according to the kit's instructions. For samples below the detection limit, the blood GDF15 concentration was set to 0 pg / mL.

[0149] Statistical analysis method The plasma unbound GDF15 concentration was analyzed using the Wilcoxon rank-sum test. Statistical analysis was performed using SAS software (SAS Institute Japan, R9.3).

[0150] 2 Results The results are shown in Figure 6. The blood concentration of hGDF15 increased depending on the dose of rhGDF15, and both MAB17 and HuMAB2 captured GDF15 in the blood.

[0151] B. Reduction of blood GDF15 levels in tumor-bearing mouse models 1. Test materials and methods Test substance HuMAB2 MAB1 MAB13 Control substance MAB17 MAB957 (R&D Systems) Hu01G06-127 (WO2014 / 100689)

[0152] Preparation of test and control substances All antibodies were prepared at 1 mg / mL in DPBS and stored in a freezer set at -80°C until the day of administration.

[0153] Test system cell 1) Cell line name: MKN45 2) Source: Human gastric cancer 3) Source: JCRB (Japanese Collection of Research Bioresources) cell bank 4) Culture conditions: 37℃, 5% CO2 a) Culture medium: RPMI1640 i) Available from: life technologies b) Serum:Fetal bovine serum i) Concentration: 10% ii) Available from: Tissue Culture Biology animal 5) Species / Strain:mouse / BALB-c Slc-nu / nu 6) Microbiological grade: specific pathogen-free (SPF) 7) Available from: Japan SLC 8) Gender:Female 9) Age: 7 weeks (at the time of cell transplantation) 10) Animals were raised under the conditions described in "A. Capture of hGDF15 in the blood" above.

[0154] Group composition [Table 16] [Table 17]

[0155] Test Method Animal care methods Three days after delivery, the mice were separated into individual cages, and the acclimation period before MKN45 transplantation was set at 5 days to allow them to adapt to individual rearing.

[0156] Cell preparation and cell transplantation into animals MKN45 cells were cultured in 150 mm dishes. On the day of transplantation, the cells were harvested by treatment with 0.25% trypsin-EDTA (SIGMA, T6689) and centrifuged (1200 rpm, 5 min) to remove the supernatant. 5 × 10 cells were cultured in DPBS. 6 The cells were suspended at a concentration of 1 × 10 cells / mL and injected subcutaneously into the abdomen of mice anesthetized with isoflurane inhalation at 0.2 mL / body (1 × 10 6 The transplant was performed using a 2x1000 WT / body (cells / body).

[0157] Grouping In Experiment 1, 42 mice were randomly selected for the Normal (vehicle) group before MKN45 transplantation, and the remaining 37 mice were transplanted with MKN45. Twelve days after MKN45 transplantation, mice were divided into six groups (5 mice per group) based on their body weight using SAS software (SAS Institute Japan, R9.4). These groups were assigned to the MKN45 (vehicle), MKN45 (MAB17), MKN45 (MAB957), MKN45 (Hu01G06-127), and MKN45 (HuMAB2) groups. Similarly, in Experiment 2, mice were assigned to the Normal (vehicle), MKN45 (vehicle), MKN45 (MAB17), MKN45 (MAB1), and MKN45 (13) groups (5 mice per group). After grouping, the remaining animals were euthanized by exsanguination under isoflurane anesthesia.

[0158] Administration of DPBS and antibodies Fourteen days after MKN45 transplantation (two days after group allocation), DPBS and antibody (10 mL / kg) were administered intraperitoneally. In Experiment 1, DPBS and antibody were also administered on day 7 after the start of antibody administration.

[0159] Blood collection Blood samples were collected from the inferior vena cava of mice anesthetized with isoflurane inhalation 14 days (Test 1) or 7 days (Test 2) after the start of antibody administration. Plasma was collected using EDTA2K blood collection tubes and stored in a freezer set at -80°C until use. After blood collection, the mice were euthanized by exsanguination via abdominal incision of the inferior vena cava while still under anesthesia.

[0160] Blood unbound hGDF15 concentration To measure unbound hGDF15 in the blood, the antigen-antibody complex was removed using the following procedure. 5 μL of Protein A / G agarose beads (Thermo Scientific, 20421) were mixed with 50 μL of mouse plasma and 145 μL of PBS and incubated at 4°C for 2 hours. After centrifugation, the supernatant was transferred to a new Eppendorf tube and stored in a freezer at -80°C until use. Subsequent blood GDF15 measurements were performed using a GDF15 ELISA kit (R&D Systems, DY957) according to the kit's instructions. For samples with concentrations below the detection limit (62.5 pg / mL or less), the blood GDF15 concentration was set to 0 pg / mL.

[0161] Statistical analysis method The Wilcoxon rank-sum test was performed on the blood unbound hGDF15 concentration 14 days (Test 1) or 7 days (Test 2) after the start of antibody administration. Statistical analysis was performed using SAS software (SAS Institute Japan, R9.4).

[0162] 2 Results In Experiment 1, the blood hGDF15 concentration in the MKN45 group transplanted with MKN45 cells was approximately 2000 pg / mL. In the groups in which antibodies were administered to MKN45 cell-transplanted model mice, the MAB17, MAB957, and Hu01G06-127 antibody-administered groups showed elevated blood hGDF15 concentrations compared to the vehicle-administered group. On the other hand, the HuMAB2-administered group showed reduced blood hGDF15 concentrations (Figure 7).

[0163] In Test 2, the blood hGDF15 concentration in the vehicle-treated group was set at 100, and the relative values ​​are shown in Table 18. As with HuMAB2, a decrease in blood hGDF15 concentration was observed with MAB1 and MAB13. [Table 18]

[0164] These results differ from those of MAB17 in the study using recombinant hGDF15 (Figure 6). One possible explanation for this is the difference in epitopes between HuMAB2 and the other three antibodies used in the study. hGDF15 is produced by proteolytic cleavage of pro-hGDF15 (AR Bauson et al. Cancer Res. 2005; 65 (6):2320-2336). HuMAB2 has an epitope near this cleavage site, and it is possible that binding to this epitope inhibits hGDF15 production. Previously, it has been reported that a certain anti-CCL-2 neutralizing antibody (ABN912) worsened the condition of rheumatoid arthritis patients without improving their condition. This was thought to be due in part to a dose-dependent increase in serum CCL-2 levels, suggesting the importance of reducing serum antigen levels (JJ Haringman et al. Arthritis & Rheumatism 2006;54 (8):2387-2392). Therefore, anti-GDF15 antibodies such as HuMAB2 that reduce serum GDF15 levels are expected to be more clinically useful than antibodies that increase serum hGDF15 levels.

[0165] C. Improvement of cachexia in tumor-bearing mouse models (1) 1. Test materials and methods 1.1 Preparation of test substance HuMAB2 was prepared at 1 mg / mL with DPBS and stored in a freezer set at -80°C until the day of administration.

[0166] 1.2 Test system 1.2.1 Cells 1) Cell line name: MKN45 2) Source: Human gastric cancer 3) Source: JCRB (Japanese Collection of Research Bioresources) cell bank 4) Culture conditions: 37℃, 5% CO2 a) Culture medium: RPMI 1640 i) Available from: life technologies b) Serum:Fetal bovine serum i) Concentration: 10% ii) Available from: Tissue Culture Biology 1.2.2 Animals 1) Species / Strain:mouse / BALB-c Slc-nu / nu 2) Microbiological grade: specific pathogen-free (SPF) 3) Available from: Japan SLC 4) Gender: Female 5) Age: 7 weeks (at the time of cell transplantation) 6) Rearing conditions Animals will be kept in an environment that meets the following conditions: a) Temperature: 23 ± 2℃ b) Humidity: 60 ± 10% c) Lighting: Lighting hours: 7:00 AM - 7:00 PM Lights out time: 7pm - 7am d) Food and water: free access, CRF-1 (Oriental Yeast Co., Ltd.), tap water

[0167] 1.2.3 Group composition [Table 19]

[0168] 1.3 Test Method 1.3.1 Examination Schedule Table 20 shows the test schedule, with the day of antibody administration set as day 0. [Table 20]

[0169] 1.3.2 Animal husbandry methods Three days after delivery, the mice were separated into individual cages, and the acclimation period before MKN45 transplantation was set at 5 days to allow them to adapt to individual rearing.

[0170] 1.3.3 Cell preparation and cell transplantation into animals MKN45 cells were cultured in 150 mm dishes, and on the day of transplantation, the cells were harvested by treatment with 0.25% trypsin-EDTA (SIGMA, T6689) and centrifuged (1200 rpm, 5 min) to remove the supernatant. 6 The cells were suspended at a concentration of 1 × 10 cells / mL and injected subcutaneously into the abdomen of mice anesthetized with isoflurane inhalation at 0.2 mL / body (1 × 10 6 The transplant was performed using a 2x1000 WT / body (cells / body).

[0171] 1.3.4 Grouping Before MKN45 transplantation, 8 mice were randomly selected from 32 mice to form the normal group, and the remaining 24 mice were transplanted with MKN45. 12 days after MKN45 transplantation, mice were divided into two groups (8 mice per group) based on their body weight using the Experimental Data Collection and Processing System (EDCS, ver. 2.1), and assigned to the MKN45 group and the HuMAB2 group. After grouping, the remaining mice were euthanized by exsanguination under isoflurane anesthesia.

[0172] 1.3.5 Administration of DPBS and HuMAB2 DPBS and HuMAB2 were administered intraperitoneally at 10 mL / kg 14 days after MKN45 transplantation (2 days after group allocation).

[0173] 1.3.6 Measurement of body weight and food intake Body weight and food intake were measured every 3 days from the day of MKN45 transplantation to the day of antibody administration, and from the day of antibody administration onwards, measurements were taken on days 0, 2, 4 and 7 after administration.

[0174] 1.3.7 Blood sampling / dissection Seven days after antibody administration, blood samples were collected from the inferior vena cava of mice anesthetized with isoflurane inhalation. Plasma was collected using EDTA2K blood collection tubes and stored in a freezer set at -80°C until use. After blood collection, mice were euthanized by exsanguination through incision of the inferior vena cava while still under anesthesia. The skin over the tumor area was incised, and the tumor was removed with surgical scissors and weighed.

[0175] 1.4 Statistical analysis method Body weight and tumor weight 7 days after antibody administration, and cumulative food intake from the day of antibody administration to day 7 were compared using unpaired t-tests between the normal and MKN45 groups, and between the MKN45 and HuMAB2 groups. Plasma unbound GDF15 concentrations were compared using Wilcoxon rank-sum tests. Statistical analysis was performed using SAS software (SAS Institute Japan, R9.3).

[0176] 2 Results Using tumor-bearing (MKN45 cell-transplanted) mice, we investigated the effects of HuMAB2 on changes in body weight and food intake, which are symptoms of cancer cachexia, and also examined the effect on blood unbound GDF15 levels. Figure 8 shows the progression of body weight, cumulative food intake over 7 days after antibody administration, and blood unbound GDF15 levels 7 days after antibody administration. Compared to the normal group, body weight in the MKN45 group decreased by approximately 4 g 7 days after antibody administration, demonstrating significant weight loss due to MKN45 cell transplantation (P<0.01). Meanwhile, in the HuMAB2 group, weight loss was reversed after antibody administration, and a significant weight increase was observed compared to the MKN45 group (P<0.01). Similarly, cumulative food intake over 7 days after antibody administration was significantly decreased in the MKN45 group (P<0.01), whereas a significant increase was observed in the HuMAB2 group compared to the MKN45 group (P<0.01). The blood concentration of unbound GDF15 was not detectable in the normal group, but was significantly elevated to approximately 1500 pg / mL in the MKN45 group (P<0.01), and was significantly reduced to approximately one-twentieth of the normal level in the HuMAB2 group (P<0.01), as shown in Figure 7. These results confirmed that the cachexia-like symptoms (weight loss, food intake) of MKN45-implanted mice were reversed by administration of HuMAB2.

[0177] D. Improvement of cachexia in tumor-bearing mouse models (2) 1. Test materials and methods 1.1 Preparation of test substance HuMAB2 was prepared at 1 mg / mL with DPBS and stored in a freezer set at -80°C until the day of administration.

[0178] 1.2 Test system 1.2.1 Cells 1) Cell line name: MKN45 1) Source: Human (human gastric cancer tissue) 2) Available from: JCRB (Japanese Collection of Research Bioresources) 3) Culture conditions: 37℃ 5% CO2 Pre-culture conditions: 37℃ 5% CO2 a) Culture medium: RPMI1640 i) Available from: life technologies b) Serum:Fetal bovine serum i) Concentration: 10% ii) Available from: Tissue Culture Biology 1.2.2 Animals 1) Species / Strain: Mouse / BALB-c Slc-nu / nu 2) Microbiological grade: specific pathogen-free (SPF) 3) Available from: Japan SLC 4) Gender: Female 5) Age: 6-12 weeks 6) Rearing conditions The animals were kept under the following conditions: a) Temperature: 23 ± 2℃ b) Humidity: 60 ± 10% c) Lighting: Lighting hours: 7:00 AM - 7:00 PM Lights out time: 7pm - 7am d) Food and water: Ad libitum intake (the pair-feeding group in Test 1 was fed a specified amount daily), CRF-1 (Oriental Yeast Co., Ltd.), tap water

[0179] 1.2.3 Group composition [Table 21]

[0180] 1.3 Test Method 1.3.1 Model creation, group division, and antibody administration First, five mice were randomly selected as the normal group. The cachexia model was created as described in C above, and the antibody was administered intraperitoneally (Figure 9).

[0181] 1.3.2 Measurement of spontaneous motor activity A wireless running wheel (Brain Science Idea, ENV-044) was placed inside the cage, and the number of revolutions of the wheel was measured as the amount of spontaneous locomotion. Measurements were collected every 12 hours during the light period (7:00 AM to 7:00 PM) and the dark period (7:00 PM to 7:00 AM).

[0182] 1.4 Evaluation items Weight Locomotor activity

[0183] 1.5 Statistical analysis method Grouping was performed unidimensionally using body weight as an index. SAS software (SAS Institute Japan, R9.3) was used for grouping and testing.

[0184] 2 Results To evaluate GDF15-induced cachexia-like symptoms, a running wheel was placed in the cage and spontaneous locomotor activity was measured. Figure 9 shows the test schedule. Compared to the normal group, activity in the MKN45 group was reduced to approximately one-tenth of that in the normal group. This reduction in activity was found to recover to approximately the same level as the normal group approximately three days after HuMAB2 administration. Furthermore, differences in the MKN45 group were evident not only in the reduction in activity but also in the activity pattern. The normal group exhibited characteristics of nocturnal animals, becoming active during the dark period and decreasing during the light period. However, in the MKN45 group, this activity pattern was clearly reversed. Furthermore, in the antibody-administered HuMAB2 group, this pattern also recovered to the same level as the normal group.

[0185] III. Antibody Production (2) A. Sequencing The amino acid sequences of MAB1 and MAB13 (Table 1) were determined in the same manner as for MAB2. The amino acid sequences of the H-chain and L-chain variable regions are shown in Table 22, and the CDR sequences are shown in Table 23.

[0186] [Table 22]

[0187] [Table 23]

[0188] B. Generation of Mutant Antibodies Further HuMAB2 mutants shown in Table 24 were produced in the same manner as described above in "I. Antibody production (1) G. Binding of HuMAB2 mutants to hGDF15 by amino acid substitution in the CDR." The binding amount of each mutant antibody to hGDF15, relative to the binding amount of HuMAB2 binding to 100, is shown in Figure 10. Several antibodies were obtained that showed binding amounts equal to or greater than that of HuMAB2. [Table 24]

[0189] Furthermore, the heavy chains (H49R, H49D, H48S, H71Y, H83R, or H120F) and light chains (L48K, L112D, L72F, or L74H) of HuMAB2 mutants that exhibited high binding affinity to hGDF15 (Table 25) were combined, and the binding of the resulting antibodies to hGDF15 was examined. The results are shown in Figure 11. Many antibodies were obtained that exhibited binding levels equal to or greater than those of HuMAB2. [Table 25]

[0190] C. Evaluation of inhibitory activity against GDF15, GFRAL, and RET complex formation GDF15 forms a complex with its receptor GFRAL and co-receptor RET to transmit intracellular signals. Therefore, we investigated the effect of HuMAB2 on the formation of the GDF15, GFRAL, and RET complex.

[0191] First, we examined the effect of GDF15 on the complex formation between GFRAL and GDF15 (Figure 12). GDF15 (R&D Systems, 957-GD / CF) biotinylated using the EZ-Link Micro NHS-PEG4-Bitinylation Kit (Thermo Fisher Scientific, 21955) was reacted with serial dilutions of anti-GDF15 monoclonal antibodies (Table 26) on a plate coated with GFRAL (R&D Systems, 9697-GR). Human IgG1, kappa-Isotype Control (Abcam) was used as a negative control antibody. GDF15 bound to GFRAL was detected using Pierce High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific, 21130). TMB 1-Component Microwell Peroxidase Substrate, SureBlue (SeraCare Life Sciences) was used for color development, and the reaction was stopped by adding stop solution (Wako). Absorbance was measured at 450 nm (OD450) using Emax (Molecular Devices). The % of control value was calculated using Equation 1 from the OD450 value when each antibody was added, with the OD450 value without antibody as the control. The IC50 value was calculated from the % of control value using Equation 2 and an Excel IC50 calculation macro. % of control = OD450 value of antibody-added sample ÷ OD450 value without antibody addition × 100 (Equation 1) Inhibition rate = 100 - % of control (Equation 2)

[0192] As shown in Table 26, all anti-GDF15 antibodies except HuMAB2 inhibited the complex formation between GDF15 and GFRAL, but HuMAB2 did not. This difference was thought to be due to the difference in the epitopes of these antibodies. [Table 26]

[0193] Next, we examined the effect of GDF15, GFRAL, and RET on complex formation. A mixture of 50 nM GDF15 (R&D Systems, 9279-GD), 30 nM RET (R&D Systems, 1168-CR), and serial dilutions of HuMAB2 (0-500 nM) was added to an ELISA plate coated with 30 nM GFRAL (R&D Systems). Human IgG1, kappa-Isotype Control (Abcam) was used as a negative control antibody. The formed GDF15 / GFRAL / RET complex was detected with an anti-His peroxidase-conjugated antibody (R&D Systems, MAB050H). TMB 1-Component Microwell Peroxidase Substrate, SureBlue (SeraCare Life Sciences) was used for color development, and the reaction was stopped by adding stop solution (Wako). Absorbance was measured at 450 nm using Emax (Molecular Devices). The % of control value was calculated using Formula 1 from the OD450 value at each antibody addition, with the OD450 value at an antibody concentration (0 nM) (when no antibody was added) used as the control. The IC50 value was calculated from the % of control value using Formula 2 to determine the inhibition rate, and then calculated using an Excel IC50 calculation macro.

[0194] HuMAB2 did not exhibit inhibitory activity against GDF15 / GFRAL complex formation (Table 26), but did exhibit inhibitory activity against GDF15 / GFRAL / RET complex formation (Figure 13). These results suggest that HuMAB2 exerts its inhibitory activity against GDF15 function by inhibiting complex formation with RET, which has the function of transmitting signals into cells.

[0195] VI. Pharmacological Testing (2) A. Improvement of cachexia in tumor-bearing mouse models (3) Similar to the above "II. Pharmacological Test (1) C. Improvement of Cachexia Symptoms in a Tumor-Bearing Mouse Model (1)," the effects of MAB1 on body weight, food intake, and blood unbound GDF15 levels were examined using tumor-bearing (MKN45 cell transplanted) mice. The group composition is shown in Table 27. [Table 27]

[0196] Figure 14 shows the changes in body weight, cumulative food intake over 7 days after antibody administration, and blood unbound GDF15 concentration 7 days after antibody administration. While weight loss was observed in the MKN45 group, recovery from weight loss was observed in the MAB1 group after antibody administration. Cumulative food intake over 7 days after antibody administration also tended to be higher in the MAB1 group compared to the MKN45 group. The blood unbound GDF15 concentration, which increased with MKN45 transplantation, was reduced by MAB1, as with HuMAB2 (Figure 8). These results confirmed that the cachexia-like symptoms (weight loss, food intake) of MKN45-transplanted mice were reversed by MAB1 administration.

Claims

[Claim 1] The invention described in the present specification and drawings.

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