Antibody molecule against growth differentiation factor 15 and its application

Abstract

The present invention provides an antibody or a fragment thereof for human growth differentiation factor 15 protein (GDF15). The present invention further provides a use of said antibody or a fragment thereof in the manufacture of a medicament for treating a disease or condition. The antibody or a fragment thereof provided by the present invention can specifically bind to human GDF15 with high affinity, block the interaction of GDF15 with its receptor GFRAL, and has a longer in vivo half-life than its homologous antibody.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine, and in particular to an antibody molecule and an antigen-binding fragment thereof that specifically binds to growth differentiation factor 15 (GDF15), and applications of the antibody molecule and the antigen-binding fragment thereof. [Background technology]

[0002] Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor β (TGFβ) superfamily, which is responsible for regulating food intake, energy expenditure, and body weight in vivo, and responding to metabolic and toxic stress. The receptor of GDF15 is the glial cell line derived neurotrophic factor (GDNF) family receptor α-like (GFRAL) protein, and when GDF15 binds to its receptor GFRAL, it can activate neurons in the area postrema and nucleus tractus solitarius of the brainstem, because the receptor GFRAL is expressed in these areas, and trigger the activation of neurons in the parabrachial nucleus and central amygdala nucleus to form feeding responses to stress conditions. These functions have highlighted GDF15 as a potential therapeutic target for the treatment of obesity and related metabolic diseases.

[0003] Previously, the level of circulating GDF15 in vivo has also been associated with low body mass index (BMI) and cachexia in patients with cancer, heart failure or chronic kidney disease. Recent research data shows that it binds to GFRAL-expressing neurons in the brainstem and triggers the activation of neurons in the parabrachial nucleus and central amygdala, which then suppresses appetite and ultimately reduces body weight. Therefore, it is a potential target for treating diseases such as weight loss and obesity associated with cachexia. Summary of the Invention

[0004] The present invention provides novel antibodies, e.g., monoclonal antibodies, that specifically bind with high affinity to human growth differentiation factor 15 protein (GDF15) as novel therapeutic agents for treating diseases and conditions, e.g., cachexia.

[0005] In view of the above technical problems, the present invention aims to provide an anti-GDF15 antibody or a fragment thereof, and to provide uses based on the antibody or the fragment thereof. The "fragment" of the antibody molecule of the present invention includes various functional fragments of an antibody, such as an antigen-binding fragment thereof.

[0006] The present invention provides the following:

[0007] According to one aspect, the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to GDF15, particularly human GDF15. According to a specific embodiment of the present invention, the antibody of the present invention is a mouse antibody obtained using the extracellular domain of human GDF15 as an immunogen, and a humanized antibody obtained based on the mouse antibody.

[0008] Specifically, the present invention provides an antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of complementarity determining regions (CDRs) selected from the following (H-CDR1, H-CDR2, H-CDR3; and L-CDR1, L-CDR2, L-CDR3): (1) H-CDR1, H-CDR2, and H-CDR3 shown in SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 24 (HILWDDVKRYNPALKS), and SEQ ID NO: 25 (MAWDWFAY), in that order; and L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO: 26 (KASQNVDTNVA), SEQ ID NO: 27 (SASYRSS), and SEQ ID NO: 28 (QQYHSYPT), in that order; (2) H-CDR1, H-CDR2, and H-CDR3 shown in SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 29 (HIRWDDVKRYNPALKS), and SEQ ID NO: 25 (MAWDWFAY), in that order; and L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO: 30 (KASQNVDTDVA), SEQ ID NO: 31 (SASYRYS), and SEQ ID NO: 32 (HQYNSYPT), in that order; (3) H-CDR1, H-CDR2, and H-CDR3 shown in sequence in SEQ ID NO: 33 (TAGMTVG), SEQ ID NO: 34 (HIWWNDDKYYNPALKS), and SEQ ID NO: 35 (IATMNYAMDY); and L-CDR1, L-CDR2, and L-CDR3 shown in sequence in SEQ ID NO: 36 (RASQSVSTSSFSYMH), SEQ ID NO: 37 (YASNLES), and SEQ ID NO: 38 (QHSWEIPYT); (4) H-CDR1, H-CDR2, and H-CDR3 shown in SEQ ID NO: 39 (TSGMGVD), SEQ ID NO: 40 (HIYWDDDKRYNPSLKS), and SEQ ID NO: 41 (RAWDAMDY), in that order; and L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO: 42 (KARQNVGTNVA), SEQ ID NO: 31 (SASYRYS), and SEQ ID NO: 43 (QQYNSYPYT), in that order; (5) H-CDR1, H-CDR2, and H-CDR3 shown in SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 44 (HIWWNDDKYYNPSLKS), and SEQ ID NO: 45 (GAYDFFDY), in that order; and L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO: 26 (KASQNVDTNVA), SEQ ID NO: 31 (SASYRYS), and SEQ ID NO: 46 (QQYNTYPYT), in that order; and (6) H-CDR1, H-CDR2, and H-CDR3 shown in sequence number 47 (TSGIGIT), sequence number 48 (TIWWDDDNRYNPSLKS), and sequence number 49 (SAWDWFAY), respectively; and L-CDR1, L-CDR2, and L-CDR3 shown in sequence number 50 (KASQNVGTNVA), sequence number 51 (SASYRNS), and sequence number 52 (QQYNSHPVT), respectively.

[0009] The antibody or fragment thereof provided by the present invention is an anti-GDF15, particularly an antibody or fragment thereof against human GDF15, and can specifically bind to the GDF15 with high affinity. The combination of light and heavy chain CDRs contained in the antibody or fragment thereof is derived from a specific antibody of the present invention (see the Examples section), and a person skilled in the art can generally determine the CDRs contained therein based on the variable region amino acid sequence contained in the specific antibody. According to a specific embodiment of the present invention, the CDRs in the variable region amino acid sequence of the specific antibody can be classified using Kabat and any other definition method, such as IMGT, ABM, Chotia, etc. Therefore, the light and heavy chain CDRs and their combinations classified by other methods known in the art are also included in the scope of the present invention.

[0010] Preferably, in an antibody or fragment thereof provided by the invention, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21, or an amino acid sequence with at least 75% identity thereto, and / or the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22, or an amino acid sequence with at least 75% identity thereto. "At least 75% identity" as used in the context of the present invention for a sequence refers to any percentage identity of "≧75%", such as for example at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity.

[0011] More preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region and the light chain variable region each comprise: (1) an amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity thereto; (2) an amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity thereto; (3) an amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity thereto; (4) an amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity thereto; (5) an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity thereto; (6) an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having at least 75% identity thereto; (7) an amino acid sequence as set forth in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity thereto; (8) An amino acid sequence as set forth in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity thereto.

[0012] In particular, the antibody or fragment thereof of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which comprise the above-mentioned CDRs and intervening framework regions, and the arrangement of the domains involved is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, optionally, the difference of up to 25% in the amino acid sequence according to the "at least 75% identity" may be present in any framework region in the heavy chain variable region or the light chain variable region, or may be present in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibody or fragment thereof of the present invention. The difference may be caused by deletion, addition or substitution of amino acids at any position.

[0013] The antibodies provided by the present invention are murine, chimeric or fully or partially humanized antibodies that bind to GDF15, in particular human GDF15; the fragments may be scFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or a half antibody or antigen-binding fragment of said antibody comprising an Fv fragment. Preferably, said antibody is a monoclonal antibody or a single chain antibody.

[0014] Besides the variable region, said antibody or fragment thereof comprises a human or mouse constant region, preferably a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL); preferably, said antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, said antibody or fragment thereof comprises a heavy chain constant region and / or a κ or λ type light chain constant region selected from IgG, IgA, IgM, IgD or IgE. According to a specific embodiment of the invention, said antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. According to a specific embodiment of the invention, said monoclonal antibody is an IgG, in particular an IgG1.

[0015] More preferably, the antibody or fragment thereof provided by the present invention comprises a heavy chain constant region, said heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or the antibody or fragment thereof provided by the present invention comprises a light chain constant region, said light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with said amino acid sequence. Or, said heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or the antibody or fragment thereof provided by the present invention comprises a light chain constant region, said light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0016] In another aspect, the present invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the present invention or a fragment thereof, or comprising a nucleotide sequence encoding a heavy chain CDR, a light chain CDR, a light chain variable region, a heavy chain variable region, a heavy chain or a light chain contained in the antibody or fragment thereof.

[0017] According to yet another aspect, the present invention provides a vector comprising the nucleic acid molecule described above, said vector being capable of being a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like.

[0018] According to yet another aspect, the present invention provides a host cell comprising or transformed or transfected with a nucleic acid molecule or vector as described above. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

[0019] The antibodies or fragments thereof provided by the present invention may be obtained by any method known in the art, for example by culturing a host cell provided by the present invention, where the host cell is allowed to express the heavy and / or light chain variable regions of the antibody or the heavy and / or light chains of the antibody to assemble the antibody. Optionally, the method further comprises the step of recovering the antibody produced.

[0020] According to another aspect, the present invention further provides a composition comprising said antibody or fragment thereof, nucleic acid molecule, vector and / or host cell of the invention, preferably said composition is a pharmaceutical composition, optionally comprising a pharma- ceutically acceptable vector, adjuvant or excipient.

[0021] The antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition provided by the present invention enhances one or more activities of a subject's appetite, food intake, body weight, muscle mass, etc., and has an anti-tumor effect. Therefore, the present invention further provides the following.

[0022] According to yet another aspect, the invention further provides the use of said antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the manufacture of a medicament for treating a disease or condition, wherein the disease or condition is mediated by or associated with expression of GDF15; and / or wherein the disease or condition is a tumor (e.g. cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia.

[0023] Therefore, accordingly, the present invention further provides a method of treating a disease or condition, said method comprising the step of administering to a subject in need thereof an antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition provided by the present invention, wherein said disease or condition is mediated by or associated with expression of GDF15; and / or said disease or condition is a tumor (e.g. cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia.

[0024] Preferably, the subject is a mammal; more preferably, the subject is a human.

[0025] The present invention further provides a method for detecting or diagnosing a disease or condition, said method comprising the step of contacting an antibody molecule or fragment thereof, a nucleic acid molecule, a vector, a host cell and / or a composition of the invention with a sample from a subject, wherein the disease or condition is mediated by or associated with expression of GDF15; or wherein the disease or condition is a tumor (e.g. cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia. Preferably, the subject is a mammal; more preferably, the subject is a human.

[0026] The present invention also provides a kit comprising the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition, which is used for the treatment, detection or diagnosis described above. Optionally, the kit may further comprise an instruction manual for use.

[0027] The present invention provides a novel antibody against human GDF15, which can specifically bind to human GDF15 with high affinity and block the interaction of GDF15 with its receptor GFRAL. Experiments have demonstrated that the antibody provided by the present invention has higher GDF15 affinity and specificity, longer in vivo half-life, and better blocking effect than known homologous antibodies. [Brief description of the drawings]

[0028] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. [Figure 1] 1 shows the results of ELISA detection of binding between the culture supernatant of hybridoma cells and human GDF15. [Diagram 2] 1 shows the results of ELISA detection of binding between the culture supernatant of hybridoma cells and monkey GDF15. [Diagram 3] 1 shows the results of ELISA detection of blocking of the binding of GDF15 to its receptor GFRAL by the culture supernatant of hybridoma cells. [Figure 4] 1 shows the results of ELISA detection of antibody binding to human GDF15. [Diagram 5] 1 shows the results of ELISA detection of antibody binding to monkey GDF15. [Figure 6] 1 shows the results of ELISA detection of blocking of the binding of GDF15 to its receptor GFRAL by antibodies. [Figure 7] ELISA detection of antibody binding to human GDF15 after exposure to PBS and plasma. [Figure 8] 1 shows the results of competitive ELISA detection of antibody blocking of the binding of GDF15 to its receptor GFRAL. [Figure 9] The results of detection of the blocking function of GDF15 by antibodies in reporter cells expressing GFRAL are shown. [Figure 10]The results show that antibodies neutralize GDF15 in reporter cells expressing GFRAL, with 10-1: Ponsegromab; 10-2: 53E5(hz). [Figure 11] 1 shows the results of detecting the binding specificity of antibodies to GDF15, GDF1, and GDF3. [Figure 12] 1 shows the results of detecting the binding specificity between an antibody and PBMC. [Figure 13] The results of in vivo PK experiments of antibodies in mice are shown, including 13-1: AV380-hIgG1; 13-2: Ponsegromab; 13-3: 53E5(hz). [Figure 14] 1 shows the suppressive effect of the antibodies detected in mice on cachexia. [Figure 15] 1 shows the neutralizing effect of antibodies detected in mice against GDF15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention will be described below with reference to specific examples. Those skilled in the art should understand that these examples are only used to explain the present invention and do not limit the scope of the present invention in any manner.

[0030] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are all commercially available products unless otherwise specified.

[0031] Example 1 Preparation of Hybridoma Cells Mice were immunized with a fusion protein of the extracellular domain containing GDF15 protein (Genbank Accession No. NM_004864.3) and mouse IgG2a-FC (Genbank Accession No. AAH31470.1) as an immunogen. Quick Antibody-Mouse 5W water-soluble adjuvant was used as an adjuvant. Two weeks after the booster immunization, the antibody titer was measured, and mice with high antibody titers were selected and subjected to immune shock. After three days, the mouse serum was collected, and the mice were dissected, their spleens were collected, and splenocytes were isolated.

[0032] Spleen cells and cultured myeloma cells were fused in a 96-well plate, and selection medium was added at the same time for screening. The culture medium was replaced after 7 days, and ELISA detection was performed after 10 days. Cells with OD values ​​10 times higher than the negative control were selected and further detected by flow cytometer.

[0033] Double-positive cells were selected, and subcloning was performed by limiting dilution to select monoclonal cells. Culture supernatants were taken from the selected monoclonal cells, and detection was performed by ELISA and flow cytometry. Double-positive cells were selected and expanded. The cell supernatants were purified by affinity chromatography using a protein G gravity column to obtain hybridoma antibodies.

[0034] Example 2 ELISA detection of binding of hybridoma antibodies to human GDF15 A fusion protein of the extracellular domain containing human GDF15 protein (Genbank Accession No. NM_004864.3) and human IgG1-FC (Genbank Accession No. CAC20454.1) was diluted to 1 μg / mL with coating solution, then added to the wells of the ELISA plate at 50 μL / well and left overnight at 4°C. The liquid in the wells was discarded, and the wells were washed three times with washing solution, each time for 3 to 5 minutes, and then dried. 200 μL of blocking solution was added to each well and left overnight at 4°C. The plate was washed three times with washing solution, and the coated plate could be prepared by storing it at -20°C or 4°C.

[0035] The test antibody (purified antibody from hybridoma, see Table 1 for naming), positive control antibody AV380-mIgG1 (sequence from Aveo Oncology; heavy and light chain variable regions are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; heavy and light chain constant regions are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively) and blank control (PBS) were added to the wells, and the antibody was diluted 3.16-fold in 10 steps from 20 μg / mL. The wells were incubated for 1 hour at 37°C, washed and dried. Then, 100 μL of enzyme-labeled secondary antibody, horseradish peroxidase-labeled sheep anti-mouse IgG (SIGMA, catalog number A9044-2mL) diluted 1:10000 was added to each well, incubated for 2 hours at 37°C, washed and dried. 100 μL of freshly prepared substrate color development solution TMB was added to each well, and incubated for 30 minutes at 37°C.

[0036] The AV380-mIgG1 sequence is as follows: Heavy chain variable region VH (SEQ ID NO:1):

[0037] [ka] Light chain variable region VL (SEQ ID NO:2):

[0038] [ka] Heavy chain constant region (SEQ ID NO: 3):

[0039] [ka] Light Chain Constant Region (SEQ ID NO:4):

[0040] [ka] 2 mol / L H 2 SO 4 The reaction was stopped by adding 100% ethanol, and the OD value was read using an ELISA (enzyme-linked immunosorbent assay) reader. The results are shown in Tables 1-1 and 1-2 and in 1-1 and 1-2 in Figure 1.

[0041] [Table 1-1]

[0042] [Table 1-2] Example 3 ELISA detection of binding of hybridoma antibodies to monkey GDF15 (cynoGDF15) The experimental procedure was the same as in Example 2, except that the fusion protein was replaced with a fusion protein of the extracellular domain containing monkey GDF15 protein (Genbank Accession No. EHH29815.1) and human IgG1-FC (Genbank Accession No. CAC20454.1). The results are shown in Tables 2-1 and 2-2 and in Figures 2-1 and 2-2.

[0043] [Table 2-1]

[0044] [Table 2-2] Example 4ELISA detection of blocking of the binding of GDF15 to its receptor GFRAL by hybridoma antibodies. A fusion protein of the extracellular domain containing the GFRAL protein (Genbank Accession No. NM_207410.2) and human IgG1-FC (Genbank Accession No. CAC20454.1) was diluted to 1 μg / mL with the coating solution, then added to the wells of the ELISA plate at 50 μL / well and left overnight at 4°C. The liquid in the wells was discarded, and the wells were washed three times with the washing solution, each time for 3 to 5 minutes, and then dried. 200 μL of blocking solution was added to each well and left overnight at 4°C. After washing three times with the washing solution, the coated plate can be prepared by storing it at -20°C or 4°C.

[0045] The test antibody, positive control antibody AV380-mIgG1, and blank control (PBS) were added to the wells, and the antibody was diluted 3.16-fold in 10 steps starting from 20 μg / mL. At the same time, GDF15-mFC protein (GDF15: Genbank Accession No. NM_004864.3; mFC: Genbank Accession No. AAH31470.1) was added at 20 ng / well and mixed uniformly with the test antibody. The wells were incubated at 37°C for 1 hour, washed, and dried. Then, 50-100 μL of horseradish peroxidase-labeled sheep anti-mouse IgG (SIGMA, catalog number A9044-2mL), which is an enzyme-labeled secondary antibody, was added to each well, incubated at 37°C for 2 hours, washed, and dried. 100 μL of freshly prepared substrate coloring solution TMB was added to each well and incubated at 37°C for 30 minutes. 2 SO 4 The reaction was stopped by adding and the OD value was read using an ELISA (enzyme-linked immunosorbent assay) reader. The results are shown in Tables 3-1 and 3-2 and in 3-1 and 3-2 in FIG.

[0046] [Table 3-1]

[0047] [Table 3-2] The variable region sequences of the above murine antibodies are as follows (heavy and light chain CDRs are shown in bold and underlined, and were obtained according to the Kabat definition method): A1 (mouse anti-53E5)

[0048] [ka] A2 (mouse anti-53A8)

[0049] [ka] A3 (mouse anti-34G9)

[0050] [ka] A4 (mouse anti-2A2)

[0051] [ka] A5 (mouse anti-50G3)

[0052] [ka] A6 (mouse anti-53D10)

[0053] [ka] Example 5 Detecting the binding affinity of mouse antibodies to human GDF15 Testing Method: 1. Selected the same type of Capture Surface Dip.

[0054] 2. Remove the 96 well plate and add 200 μL of SD buffer (1×PBS+0.02% Tween20+0.1% BSA) to each well and place in the ForteBio Octet and precirculate.

[0055] 3. The antibody was immobilized at a concentration of 10 μg / mL. 4. GDF15 (ACROBiosystems, GD5-H5149) was diluted in six steps to 200 nm, 100 nm, 50 nm, 25 nm, 12.5 nm, and 6.25 nm, and added to the corresponding wells. 5. Detected by equipment. The results are shown in Table 4.

[0056] [Table 4] Example 6 Humanizing mouse antibodies For A1 (mouse anti-53E5), human-derived sequences IGHV4-39 and IGKV1-39 were selected as templates for humanizing the heavy and light chains, respectively. Homology modeling was performed on the A1 monoclonal antibody, and a structural simulation of the Fab region was performed. The final predicted Fab structure of A1 was obtained by calculation using homology modeling.

[0057] By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, it was found that the original mouse amino acids were retained in the CDR regions and 2V, 24F, 50L, 51A, 69L, 73K, 75S, 78S, 80I, 98V, and 99Q in this VH, but all other mouse amino acids were replaced with the corresponding human amino acids using IGHV4-39 as a template.

[0058] By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that except for the CDR regions and 42Q, 43S, and 46A in this VL, the original mouse amino acids were retained, and all other mouse amino acids were replaced with the corresponding human amino acids using IGKV1-39 as the template.

[0059] The resulting humanized sequences are as follows (in which the heavy and light chain CDRs are shown in bold and underlined, and were obtained according to the KABAT definition method): >53E5-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO: 19 / 23 / 24 / 25)

[0060] [ka] >53E5-L humanization sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO: 20 / 26 / 27 / 28)

[0061] [ka] For A2 (mouse anti-53A8), human-derived sequences IGHV4-39 and IGKV1-39 were selected as templates for humanizing the heavy and light chains, respectively. Homology modeling was performed on the A2 monoclonal antibody, and a structural simulation of the Fab region was performed. The final predicted Fab structure of A2 was obtained by calculation using homology modeling.

[0062] By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, in this VH, except for the CDR regions and 2V, 24F, 50L, 51A, 69L, 73K, 78S, 80I, and 99Q, which were the original mouse amino acids, all other mouse amino acids were replaced with the corresponding human amino acids based on the IGHV4-39 template.

[0063] By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that except for the CDR regions and 42Q, 43S, and 46A in this VL, the original mouse amino acids were retained, and all other mouse amino acids were replaced with the corresponding human amino acids using IGKV1-39 as the template.

[0064] The resulting humanized sequences are as follows (in which the heavy and light chain CDRs are shown in bold and underlined, and were obtained according to the KABAT definition method): >53A8-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO: 21 / 23 / 29 / 25)

[0065] [ka] >53A8-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO: 22 / 30 / 31 / 32)

[0066] [ka] The sequence shown in SEQ ID NO:5 was used as the heavy chain constant region, and the sequence shown in SEQ ID NO:6 was used as the light chain constant region. Primers were redesigned for the above humanized sequences, and genes encoding the heavy and light chains of the corresponding antibodies were synthesized and introduced into a eukaryotic expression vector. Competent cells of E. coli were transformed with the recombinant vector and cultured overnight at 37°C. A monoclonal strain was selected and identified by sequencing. A strain with the correct sequence was selected, and the recombinant vector was obtained and transfected into mammalian expression cells 293F, and incubated at 37°C and 5% CO. 2 The cells were cultured for 7 days for expression. The supernatant was taken and purified to obtain a humanized antibody, which was named "mouse anti-human antibody (hz)."

[0067] >CH1-CH3 heavy chain constant region (SEQ ID NO: 5)

[0068] [ka]

[0069] [ka] >CL1 light chain constant region (SEQ ID NO:6)

[0070] [ka] Example 7 ELISA detection of binding of humanized antibodies to human GDF15 The experimental procedure was the same as in Example 2, except that the coating protein was GDF15-HIS (ACROBiosystems, CAT#: GD5-H5149), and the test antibody (humanized antibody), negative control antibody (hIgG1), positive control antibody Ponsegromab (Pfizer; Drug Bank: D11909), and AV380-hIgG1 (AV380-mIgG1 in which the heavy and light chain constant regions were replaced with the human IgG constant region, i.e., SEQ ID NO: 5 and SEQ ID NO: 6) were added to each well of the coated plate. The results are shown in Table 5 and Figure 4.

[0071] [Table 5] Example 8 ELISA detection of binding of humanized antibodies to monkey GDF15 (cynoGDF15) The experimental procedure was the same as in Example 2, except that the coating protein was cynoGDF15-mFC (GDF15: Genbank Accession No. EHH29815.1; mFC: Genbank Accession No. AAH31470.1), and the test antibody (humanized antibody), negative control antibody (hIgG1), and positive control antibody Ponsegromab and AV380-hIgG1 were added to each well of the coated plate. The results are shown in Table 6 and Figure 5.

[0072] [Table 6] Example 9 ELISA detection of blocking of the binding of GDF15 protein to its receptor GFRAL by humanized antibodies The experimental procedure was the same as in Example 4, except that the test antibody (humanized antibody), negative control antibody (hIgG1), and positive control antibodies Ponsegromab and AV380-hIgG1 were added to each well of the coated plate. The results are shown in Table 7 and Figure 6.

[0073] [Table 7] Example 10 Detection of the binding affinity of humanized antibodies to human GDF15 The experimental procedure was the same as in Example 5, except that the GDF15 antibody was replaced with the test antibody (humanized antibody), the negative control antibody (hIgG1), the positive control antibody Ponsegromab and AV380-hIgG1. The results are shown in Table 8.

[0074] [Table 8] Example 11 Detection of humanized antibody stability 1. Freeze-thaw An appropriate amount of antibody was taken, the concentration was adjusted to 10mg / mL, and frozen at -80℃ for 10min, then it was taken out and placed in ice-water mixture to slowly melt. After repeating the above steps five times, the degree of protein aggregation was detected by SEC-HPLC, and the change in protein binding activity was detected by ELISA.

[0075] 2. Low pH treatment An appropriate amount of antibody was taken, and the antibody storage buffer was adjusted to pH 3.6 using an ultrafiltration tube, and the concentration was adjusted to 5 mg / mL. After treatment at room temperature for 2-4 hours, the degree of protein aggregation was detected by SEC-HPLC, and the change in protein binding activity was detected by ELISA. 3. 40℃ Acceleration Experiment An appropriate amount of antibody was taken, the concentration was adjusted to 10 mg / mL, and the antibody was treated at 40°C for 1 week and 2 weeks. The degree of protein aggregation was detected by SEC-HPLC, and the change in protein binding activity was detected by ELISA. The results are shown in Table 9.

[0076] [Table 9] 4. Plasma stability experiment 200 μL of plasma and PBS were taken, and 200 μg of the test antibody (final concentration 1 mg / mL) was added to each, blocked, and then left at 37° C. for 2 weeks. Changes in protein binding activity were detected by ELISA. The results are shown in Table 10 and Figure 7.

[0077] [Table 10] Example 12 ELISA detection of blocking of GDF15 binding to its receptor GFRAL by humanized antibodies The GFRAL extracellular domain was stably expressed in a HEK293 luciferase reporter cell line, in which expression of the luciferase gene was controlled by an SRE expression element, to construct the HEK293SRE-LUC2-cRET-GFRAL reporter cell line.

[0078] Blockage Test 1: GFRAL-HIS protein (ACROBiosystems, GFA-H52H3) was dissolved in PBS at a concentration of 1 μg / mL and coated on an ELISA 96-well plate one day before. The next day, the plate was blocked with PBS containing 2% BSA. 53E5(hz), control antibody Ponsegromab and AV380-hIgG1 were serially diluted 3.16-fold from an initial concentration of 20 μg / mL to 10 concentrations, and at the same time, GDF15-mFc protein was added to the final concentration of 0.3 μg / mL and mixed, and incubated at room temperature for 1 hour, then added to the 96-well plate coated with GFRAL-HIS, incubated at room temperature for 1 hour, washed, and then HRP anti-mouse IgG was added, incubated at room temperature for 1 hour, and the HRP enzyme activity signal (OD450) was detected. The HRP enzyme activity signal represents the relative content of GDF15-mFc protein bound to GFRAL. The correlation curve between OD450 value and antibody concentration was plotted, and the dose-effect curve was fitted using log(agonist) vs. response-variable slope (4 parameters) logistic model to calculate IC50. The experimental results showed that 53E5(hz) had a stronger blocking ability against GDF15 than the control antibody. The results are shown in Table 11 and Figure 8.

[0079] [Table 11] Blockage Test 2: HEK293 SRE-LUC2-cRET-GFRAL reporter cells were cultured in a 96-well plate one day before. The next day, 53E5(hz), control antibodies Ponsegromab and AV380-hIgG1 were serially diluted 3.16-fold in medium from an initial concentration of 1 μg / mL to a concentration of 10, and at the same time, GDF15-His (ACROBiosystems, GD5-H5149) was added to the final concentration of 5 ng / mL. After incubation at room temperature for 1 hour, the reporter cells were added to the culture wells to stimulate luciferase expression. After 6 hours of culture, 50 μL of Bright-Glo reagent solution was added to each well, and the culture plate was shaken for 3 to 5 min to destroy the cells. The cold light value was read using a multi-mode microplate reader. The correlation curve between the fold increase in luciferase activity (fold increase over the enzyme activity in wells without GDF15 protein) and the antibody concentration was plotted, and the dose-effect curve was fitted using a log(agonist) vs. response-variable slope (4-parameter) logistic model to calculate IC50. The experimental results showed that 53E5(hz) and the control antibody Ponsegromab had similar inhibitory effects on the function of GDF15. The results are shown in Table 12 and Figure 9.

[0080] [Table 12] Blockage Test 3: HEK293 SRE-LUC2-cRET-GFRAL reporter cells were cultured in a 96-well plate one day before. The next day, 53E5(hz) and control antibody Ponsegromab solutions were prepared in medium at concentrations of 4, 3, 2, 0.5, and 0.1 μg / mL. Five portions of each antibody were taken, and different concentrations of GDF15-His (ACROBiosystems, GD5-H5149) were added to the final concentrations of 100, 31.6, 10, 3.16, and 1 ng / mL, respectively. After incubation at room temperature for 1 hour, the wells were added to stimulate the expression of luciferase. After 6 hours of incubation, 50 μL of Bright-Glo reagent solution was added to each well, and the culture plate was shaken for 3 to 5 min to destroy the cells. The cold light value was read using a multi-mode microplate reader. The correlation curve between the fold increase in luciferase activity (fold increase over enzyme activity in wells to which no GDF15 protein was added) and antibody concentration was plotted, and the dose-effect curve was fitted using a log(agonist) vs. response-variable slope (4-parameter) logistic model to calculate the EC50.

[0081] The experimental results showed that 53E5(hz) has a stronger neutralizing effect on GDF15 than the control antibody Ponsegromab. The results are shown in Figure 10, 10-1 and 10-2. In this experiment, the antibody concentration was kept constant, and then incubated with different concentrations of GDF15 protein to detect the signal that non-neutralized GDF15 stimulates the reporter cells. The EC50 shown in the figure is the value when the antibody is 0.1 μg / mL.

[0082] Example 13 Detection of binding specificity with humanized antibodies Binding specificity experiment 1: GDF15 (ACROBiosystems, GD5-H5149), GDF1 (R&D, 6937-GD-010), and GDF3 (R&D, 5754-G3-010) proteins were each dissolved in PBS at a concentration of 1 μg / mL and coated on an ELISA 96-well plate one day in advance. The next day, the 96-well plate was blocked with PBS containing 2% BSA for 1 hour, and after removing the blocking solution, GDF15 antibody 53E5 (hz), control antibody Ponsegromab and unrelated hIgG1 were added at 4 μg / mL, incubated at room temperature for 1 hour, washed, and then HRP anti-human IgG was added and incubated at room temperature for 1 hour, after which the HRP enzyme activity signal (OD450) was detected. The OD450 value is a relative signal of the amount of antibody binding. The experimental results showed that the antibody 53E5(hz) did not specifically bind to GDF1 or GDF3, and the control antibody Ponsegromab weakly bound to GDF1 and GDF3. The results are shown in Figure 11.

[0083] Binding specificity experiment 2: Cryopreserved PBMCs were resuscitated, and 10, 1, and 0.1 μg / mL of GDF15 antibody 53E5(hz), control antibodies Ponsegromab and AV380-hIgG1, or CD3 antibody OKT3 as a positive control were added to the PBMCs, and one tube without antibody was taken as a negative control. After incubation at 4°C for 30 minutes, the antibody was removed, and PE anti-human IgG flow analysis antibody was added, and after incubation at 4°C for 30 minutes, centrifugation was performed, and then antibody dilution was added, 7AAD was added to remove dead cells, and the antibody binding signal was analyzed by flow analysis. In PBMCs, the mean fluorescence intensity (MFI) of PE represents the signal that the antibody binds to the cells. The curves of antibody concentration and MFI were plotted. The experimental results showed that antibody 53E5(hz) does not specifically bind to PBMCs. The results are shown in Figure 12.

[0084] Example 14 In vivo PK studies in animals GDF15 antibody 53E5(hz), control antibody AV380-hIgG1 and Ponsegromab were injected into the tail vein of 10-12 week old BALB / c female mice, with the injection doses of 10mg / kg and 1mg / kg of mouse body weight. Blood was collected at 10 minutes, 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days and 14 days, and serum was produced. After collecting serum at all time points, the concentration of antibodies in the serum was detected by ELISA.

[0085] The specific process is as follows: 50 ng of GDF15-HIS antigen was coated overnight at 4 ° C. per well, and blocked with 2% BSA blocking solution for 2 h. The serum samples were diluted 50 times and 500 times, respectively. The standard (i.e., the injected antibody) was diluted 3.16 times from 1 μg / mL to obtain 12 concentrations. The blocking solution was removed, and the prepared serum samples and standards were added and incubated at room temperature for 1 hour. After washing, HRP anti-human IgG was added and incubated at room temperature for 1 hour. After washing, the HRP enzyme activity signal (OD450) was detected. The curves of OD450 value and standard concentration were plotted, and the concentration of the antibody in the serum was calculated from the fitted standard curve. The correlation curve between the blood collection time and the concentration of the antibody in the serum was plotted, and the half-life of the antibody in the mouse was calculated. The results are shown in Figure 13.

[0086] Example 15 In vivo functional studies of GDF15 antibodies Experiment 1. Detection of the suppressive effect of GDF15 antibody on cachexia 5x10 mice were injected with 5x10 PBS into 8-10 week old female SCID mice. 6 HT1080 cells (containing 50% Matrigel) were injected subcutaneously, and the mice were weighed daily. The size of the tumor (length and width) was measured daily with a caliper, and the tumor volume (=0.5 x length x width x height) was calculated. The tumor-free weight of the mice was calculated by subtracting the tumor weight from the tumor-bearing weight (tumor weight = volume x 1 mg / mm). 3When the mouse weight reached 93% of the weight on day 0 (day 18 in this experiment), the mice were randomly divided into groups of 10 mice each, and administered hIgG (negative control) or humanized antibody 53E5(hz), control antibody AV380-hIgG1 and Ponsegromab by intraperitoneal injection once every 3 days at an injection dose of 10 mg / kg of mouse weight. The correlation curve between the tumor-free weight of the mice and the number of days after tumor inoculation was plotted, and the results are shown in Figure 14. The experimental results showed that 53E5(hz) and the control antibody Ponsegromab had similar inhibitory effects on cachexia.

[0087] Experiment 2. The efficiency of GDF-15 antibodies to neutralize GDF15 in vivo was detected.

[0088] BALB / c normal mice were administered GDF15-mFc fusion protein (0.25 mg / kg body weight) by intraperitoneal injection (IP) on days 0 and 8, and administered hIgG (3 mg / kg body weight, negative control) or different doses of humanized antibody 53E5(hz) and control antibody Ponsegromab (3, 1, 0.3 mg / kg body weight) by intraperitoneal injection on day 2, and weighed daily. After the experiment, the correlation curve between the weight of the mice and the number of days after injection of GDF15-mFc fusion protein was plotted, and the results are shown in Figure 15. The experimental results showed that 53E5(hz) and the control antibody Ponsegromab had similar inhibitory effects on GDF15 function in vivo.

[0089] The above describes the specific embodiments of the present invention, but does not limit the present invention. Those skilled in the art may make various modifications and variations based on the present invention without departing from the spirit of the present invention, and all such corresponding modifications and variations should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region (VH) and the light chain variable region (VL) each include a combination of complementarity determining regions (CDRs) selected from the following: H-CDR1, H-CDR2, H-CDR3; and L-CDR1, L-CDR2, L-CDR3. An antibody or fragment thereof characterized in that: (1) H-CDR1, H-CDR2, and H-CDR3 shown in the order of SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 24 (HILWDDVKRYNPALKS), and SEQ ID NO: 25 (MAWDWFAY); and L-CDR1, L-CDR2, and L-CDR3 shown in the order of SEQ ID NO: 26 (KASQNVDTNVA), SEQ ID NO: 27 (SASYRSS), and SEQ ID NO: 28 (QQYHSYPT); (2) H-CDR1, H-CDR2, and H-CDR3 shown in the order of SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 29 (HIRWDDVKRYNPALKS), and SEQ ID NO: 25 (MAWDWFAY); and L-CDR1, L-CDR2, and L-CDR3 shown in the order of SEQ ID NO: 30 (KASQNVDTDVA), SEQ ID NO: 31 (SASYRYS), and SEQ ID NO: 32 (HQYNSYPT); (3) H-CDR1, H-CDR2, and H-CDR3 shown in the order of SEQ ID NO: 33 (TAGMTVG), SEQ ID NO: 34 (HIWWNDDKYYNPALKS), and SEQ ID NO: 35 (IATMNYAMDY); and L-CDR1, L-CDR2, and L-CDR3 shown in the order of SEQ ID NO: 36 (RASQSVSTSSFSYMH), SEQ ID NO: 37 (YASNLES), and SEQ ID NO: 38 (QHSWEIPYT); (4) H-CDR1, H-CDR2, and H-CDR3 shown in the order of SEQ ID NO: 39 (TSGMGVD), SEQ ID NO: 40 (HIYWDDDKRYNPSLKS), and SEQ ID NO: 41 (RAWDAMDY); and L-CDR1, L-CDR2, and L-CDR3 shown in the order of SEQ ID NO: 42 (KARQNVGTNVA), SEQ ID NO: 31 (SASYRYS), and SEQ ID NO: 43 (QQYNSYPYT); (5) H-CDR1, H-CDR2, and H-CDR3 shown in the order of SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 44 (HIWWNDDKYYNPSLKS), and SEQ ID NO: 45 (GAYDFFDY); and L-CDR1, L-CDR2, and L-CDR3 shown in the order of SEQ ID NO: 26 (KASQNVDTNVA), SEQ ID NO: 31 (SASYRYS), and SEQ ID NO: 46 (QQYNTYPYT); and (6) H-CDR1, H-CDR2, and H-CDR3 shown in the order of SEQ ID NO: 47 (TSGIGIT), SEQ ID NO: 48 (TIWWDDDNRYNPSLKS), and SEQ ID NO: 49 (SAWDWFAY); and L-CDR1, L-CDR2, and L-CDR3 shown in the order of SEQ ID NO: 50 (KASQNVGTNVA), SEQ ID NO: 51 (SASYRNS), and SEQ ID NO: 52 (QQYNSHPVT).

2. The heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21, or an amino acid sequence having at least 75% identity thereto; and / or the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or an amino acid sequence having at least 75% identity thereto. The antibody or fragment thereof according to claim 1.

3. The heavy chain variable region and the light chain variable region each comprise: The antibody or fragment thereof according to claim 1, (1) an amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity thereto; (2) an amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity thereto; (3) an amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity thereto; (4) an amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity thereto; (5) an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity thereto; (6) an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having at least 75% identity thereto; (7) an amino acid sequence as set forth in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity thereto; and an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity thereto; (8) An amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to said amino acid sequence; and an amino acid sequence as shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to said amino acid sequence.

4. The antibody may be a murine antibody, a chimeric antibody or a fully or partially humanized antibody; the antigen-binding fragment may be a half antibody or a scFv, dsFv, (dsFv) of the antibody. ２ , Fab, Fab', F(ab') ２ or an Fv fragment; Preferably, the antibody is a monoclonal antibody or a single chain antibody; Preferably, the antibody or fragment thereof further comprises a human or mouse constant region, Preferably, it comprises a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL); Preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; More preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region. The antibody or fragment thereof according to claim 1.

5. the antibody is a monoclonal antibody, Preferably, it is a murine, chimeric or humanized monoclonal antibody; More preferably, said monoclonal antibody is an IgG, in particular an IgG1. An antibody or fragment thereof according to any one of claims 1 to 4.

6. The antibody or fragment thereof according to claim 1 comprises a nucleotide sequence encoding a heavy chain CDR, a light chain CDR, a light chain variable region, a heavy chain variable region, a heavy chain or a light chain. A nucleic acid molecule comprising:

7. The nucleic acid molecule according to claim 6 A vector characterized in that

8. The nucleic acid molecule according to claim 6 A host cell characterized in that

9. A composition comprising the antibody or fragment thereof of claim 1, Preferably, the composition is a pharmaceutical composition, Optionally, further comprising a pharma- ceutically acceptable adjuvant. A composition comprising:

10. Use of an antibody or fragment thereof according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8 and / or a composition according to claim 9 in the manufacture of a medicament for the treatment of a disease or condition, comprising The disease or condition is mediated by or associated with expression of GDF15; and / or the disease or condition is a tumor (e.g. cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia.

2. The use according to claim 1 ,

11. 10. A method for treating a disease or condition comprising administering to a subject in need thereof an antibody or fragment thereof according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8 and / or a composition according to claim 9, the disease or condition is mediated by or associated with expression of GDF15; and / or the disease or condition is a tumor (e.g., cancer), heart failure, chronic kidney disease, anorexia, sarcopenia, or cachexia; Preferably, the subject is a mammal; More preferably, the subject is a human.

20. A method for treating a disease or condition, comprising:

12. 10. A method for detecting or diagnosing a disease or condition comprising the step of contacting a sample from a subject with an antibody or fragment thereof according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8 and / or a composition according to claim 9, the disease or condition is mediated by or associated with expression of GDF15; or the disease or condition is a tumor (e.g., cancer), heart failure, chronic kidney disease, anorexia, sarcopenia, or cachexia; Preferably, the subject is a mammal; More preferably, the subject is a human. A method for detecting or diagnosing a disease or condition characterized by:

13. A kit comprising an antibody or fragment thereof according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8 and / or a composition according to claim 9, Optionally, the kit further comprises instructions for use. A kit characterized by:

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