Anti-growth differentiation factor 15 antibody molecule and its applications

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), a member of the transforming growth factor β (TGFβ) superfamily, regulates food intake, energy expenditure, and body weight in vivo, and responds to metabolic and toxic stress. The receptor for GDF15 is the glial cell line-derived neurotrophic factor (GDNF)-family receptor α-like (GFRAL) protein. Upon binding to its receptor GFRAL, GDF15 activates neurons in the area postrema and nucleus tractus solitarius in the brainstem. This triggers the activation of neurons in the parabrachial nucleus and central amygdala, which are expressed in these areas, thereby forming feeding responses to stressful conditions. These functions have made GDF15 an attractive potential therapeutic target for treating obesity and related metabolic diseases.

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

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

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

[0006] The present invention provides the following:

[0007] In one aspect, the present invention provides an antibody or 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, or 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 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 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 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 sequence numbers 47 (TSGIGIT), 48 (TIWWDDDNRYNPSLKS), and 49 (SAWDWFAY), respectively; and L-CDR1, L-CDR2, and L-CDR3 shown in sequence numbers 50 (KASQNVGTNVA), 51 (SASYRNS), and 52 (QQYNSHPVT).

[0009] The antibody or fragment thereof provided by the present invention is an anti-GDF15 antibody, particularly an antibody or fragment thereof against human GDF15, and is capable of specifically binding to 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 those skilled in the art can generally determine the CDRs contained therein based on the amino acid sequence of the variable region 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 defined using Kabat or any other definition method, such as IMGT, ABM, Chotia, etc. Therefore, light and heavy chain CDRs and combinations thereof defined by other methods known in the art are also within the scope of the present invention.

[0010] Preferably, in the antibodies or fragments thereof provided by the present 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 having 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 having at least 75% identity thereto. "At least 75% identity" as used in the context of the present invention in reference to 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%, and 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) the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity thereto; (2) the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity thereto; (3) the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity thereto; (4) the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity thereto; (5) the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity thereto; (6) the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least 75% identity thereto; (7) the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity thereto; (8) The amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity thereto; and the amino acid sequence shown 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 contain the above-mentioned CDRs and intervening framework regions, and the arrangement of the domains is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, optionally, up to 25% of the difference in amino acid sequence implied by the "at least 75% identity" may be present in any framework region in the heavy chain variable region or light chain variable region, or may be present in any domain or sequence other than the heavy chain variable region or 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 antibody provided by the present invention is a murine antibody, a chimeric antibody, or a fully or partially humanized antibody that binds to GDF15, particularly human GDF15; the fragment is a half antibody or an antigen-binding fragment of the antibody, including an scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv fragment. Preferably, the antibody is a monoclonal antibody or a single-chain antibody.

[0014] In addition to the variable region, the 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, 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 and / or a kappa or lambda light chain constant region selected from IgG, IgA, IgM, IgD, or IgE. According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a murine, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody is an IgG, particularly an IgG1.

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

[0016] In another aspect, the present invention further provides a nucleic acid molecule, the 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, or a heavy chain or a light chain contained in the antibody or a fragment thereof.

[0017] In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule described above. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, or the like.

[0018] In 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 permitted to express the heavy chain variable region and / or the light chain variable region of the antibody or the heavy and / or light chain 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 the antibody or fragment thereof, nucleic acid molecule, vector and / or host cell of the invention, preferably a pharmaceutical composition, optionally comprising a pharmaceutically 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 also has an anti-tumor effect. Therefore, the present invention further provides the following.

[0022] According to yet another aspect, the present invention further provides the use of the 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 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, nucleic acid molecule, vector, host cell and / or composition of the present invention with a sample from a subject, wherein the disease or condition is mediated by or associated with expression of GDF15; or the disease or condition is tumor (e.g., cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia. Preferably, the subject is a mammal; more preferably, the subject is human.

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

[0027] The present invention provides a novel antibody against human GDF15, which specifically binds to human GDF15 with high affinity and can 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, a longer in vivo half-life, and a more effective blocking effect than known homologous antibodies. [Brief explanation of the drawings]

[0028] Hereinafter, embodiments 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. [Figure 2] 1 shows the results of ELISA detection of binding between the culture supernatant of hybridoma cells and monkey GDF15. [Figure 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 binding of antibodies to human GDF15. [Figure 5] 1 shows the results of ELISA detection of antibody binding to monkey GDF15. [Figure 6] 1 shows the results of ELISA detection of antibody-mediated blocking of the binding of GDF15 to its receptor GFRAL. [Figure 7] 1 shows the results of 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] 1 shows the results of detecting the blocking function of GDF15 by an antibody in reporter cells expressing GFRAL. [Figure 10]The results show that antibodies neutralizing GDF15 were detected using reporter cells expressing GFRAL, with 10-1 being Ponsegromab and 10-2 being 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 the antibodies in mice are shown, including 13-1: AV380-hIgG1; 13-2: Ponsegromab; and 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 on GDF15 detected in mice. DETAILED DESCRIPTION OF THE INVENTION

[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 illustrate the present invention and are not intended to limit the scope of the present invention in any manner.

[0030] Unless otherwise specified, the experimental methods used 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.

[0031] Example 1 Preparation of hybridoma cells Mice were immunized with a fusion protein of the extracellular domain of GDF15 protein (Genbank Accession No. NM_004864.3) and mouse IgG2a-FC (Genbank Accession No. AAH31470.1) as the immunogen. The adjuvant was Quick Antibody-Mouse 5W water-soluble adjuvant. Two weeks after the booster immunization, antibody titers were measured, and mice with high antibody titers were selected for immune shock. Three days later, serum was collected from the mice. The mice were then dissected, their spleens were collected, and splenocytes were isolated.

[0032] Spleen cells and cultured myeloma cells were fused in a 96-well plate, and selective medium was added at the same time for screening. After 7 days, the medium was replaced, 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 cytometry.

[0033] Double-positive cells were selected and subcloned by limiting dilution plating to select monoclonal cells. Culture supernatants were collected from the selected monoclonal cells and analyzed by ELISA and flow cytometry. Double-positive cells were selected and expanded. 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 in coating solution, then added to the wells of an 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 3-5 minutes, and then allowed to dry. 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 then be stored at -20°C or 4°C.

[0035] Test antibodies (purified antibodies derived from hybridomas; see Table 1 for nomenclature), positive control antibody AV380-mIgG1 (sequence from Aveo Oncology; heavy and light chain variable regions are shown in SEQ ID NOs: 1 and 2, respectively; heavy and light chain constant regions are shown in SEQ ID NOs: 3 and 4, respectively), and blank control (PBS) were added to the wells. The antibodies were diluted 3.16-fold in 10 increments starting from 20 μg / mL. The wells were incubated at 37°C for 1 hour, washed, and dried. Subsequently, 100 μL of an enzyme-conjugated secondary antibody, horseradish peroxidase-conjugated sheep anti-mouse IgG (SIGMA, catalog no. A9044-2mL) diluted 1:10,000, was added to each well, and the wells were incubated at 37°C for 2 hours, washed, and dried. 100 μL of freshly prepared substrate solution TMB was added to each well and incubated at 37°C for 30 minutes.

[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] The reaction was stopped by adding 2 mol / L H2SO4, 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 Figure 1, 1-1 and 1-2.

[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 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 GFRAL protein (Genbank Accession No. NM_207410.2) and human IgG1-FC (Genbank Accession No. CAC20454.1) was diluted to 1 μg / mL in coating solution, then added to the wells of an ELISA plate at 50 μL / well and left overnight at 4°C. The liquid in the wells was discarded, and the plate was washed three times with washing solution, each time for 3-5 minutes, and then allowed to dry. 200 μL of blocking solution was added to each well and left overnight at 4°C. After washing three times with washing solution, the coated plate could be stored at -20°C or 4°C.

[0045] Test antibodies, positive control antibody AV380-mIgG1, and blank control (PBS) were added to wells. The antibodies were diluted 3.16-fold in 10-fold increments starting from 20 μg / mL. Simultaneously, GDF15-mFC protein (GDF15: GenBank Accession No. NM_004864.3; mFC: GenBank Accession No. AAH31470.1) was added at 20 ng / well and uniformly mixed with the test antibodies. The wells were incubated at 37°C for 1 hour, washed, and dried. Subsequently, 50–100 μL of an enzyme-conjugated secondary antibody, horseradish peroxidase-conjugated sheep anti-mouse IgG (Sigma, Catalog No. A9044-2mL) diluted 1:10,000, 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 for 30 minutes at 37°C. The reaction was stopped by adding 2 mol / L H2SO4, 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 Detection of binding affinity of mouse antibodies to human GDF15 Experimental Method: 1. Selected the same type of Capture Surface Dip.

[0054] 2. The 96-well plate was removed and 200 μL of SD buffer (1×PBS+0.02% Tween 20+0.1% BSA) was added to each well, placed in a ForteBio Octet, and pre-circulated.

[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 Humanization of 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 structural simulations of the Fab region were 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 determined that the original mouse amino acids were retained in the CDR regions of this VH and in 2V, 24F, 50L, 51A, 69L, 73K, 75S, 78S, 80I, 98V, and 99Q, but all other mouse amino acids were replaced with the corresponding human amino acids using IGHV4-39 as the template.

[0058] By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that the original mouse amino acids in the CDR regions and 42Q, 43S, and 46A in this VL were retained, but 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 (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 NOs: 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 structural simulations of the Fab region were 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, it was determined that 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 using IGHV4-39 as the template.

[0063] By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that the original mouse amino acids in the CDR regions and 42Q, 43S, and 46A in this VL were retained, but 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 (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 NOs: 21 / 23 / 29 / 25)

[0065] [ka] >53A8-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NOs: 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 using the above humanized sequences to synthesize genes encoding the corresponding antibody heavy and light chains, which were then introduced into a eukaryotic expression vector. Competent E. coli cells 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 then transfected into mammalian expression cells 293F and cultured at 37°C and 5% CO2 for 7 days. The supernatant was collected and purified to obtain a humanized antibody, designated "Mouse 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 human IgG constant regions, 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 antibodies 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), negative control antibody (hIgG1), and positive control antibodies 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, adjusted to a concentration of 10 mg / mL, and frozen at -80°C for 10 minutes. After that, it was removed and placed in an ice-water mixture to slowly thaw. After repeating the above steps five times, the degree of protein aggregation was detected by SEC-HPLC, and changes in protein binding activity were 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 incubation at room temperature for 2-4 hours, the degree of protein aggregation was detected by SEC-HPLC, and changes in protein binding activity were detected by ELISA. 3.40℃ acceleration experiment An appropriate amount of antibody was taken, adjusted to a concentration of 10 mg / mL, and incubated at 40°C for 1 and 2 weeks. The degree of protein aggregation was detected by SEC-HPLC, and changes in protein binding activity were 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. After blocking, the mixture was 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 luciferase gene expression 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 onto a 96-well ELISA plate one day in advance. The next day, the plate was blocked with PBS containing 2% BSA. 53E5(hz), the control antibody Ponsegromab, and AV380-hIgG1 were serially diluted 3.16-fold from an initial concentration of 20 μg / mL to 10 concentrations. GDF15-mFc protein was added to the plate at a final concentration of 0.3 μg / mL. The mixture was then incubated at room temperature for 1 hour, then added to the GFRAL-HIS-coated 96-well plate. After incubation at room temperature for 1 hour, the plate was washed. HRP anti-mouse IgG was then added and incubated at room temperature for 1 hour. The HRP enzyme activity signal (OD450) was then detected. The HRP enzyme activity signal represents the relative amount of GDF15-mFc protein bound to GFRAL. The correlation curve between OD450 values ​​and antibody concentrations 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) 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 prior to the experiment. The following day, 53E5(hz), the control antibody Ponsegromab, and AV380-hIgG1 were serially diluted 3.16-fold in medium from an initial concentration of 1 μg / mL to 10 concentrations. GDF15-His (ACROBiosystems, GD5-H5149) was simultaneously added to the wells containing the reporter cells to stimulate luciferase expression. After 1 hour of incubation at room temperature, 50 μL of Bright-Glo reagent solution was added to each well, and the plate was shaken for 3–5 minutes to disrupt the cells. Cold light readings were measured 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 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 IC50. The experimental results showed that 53E5(hz) and the control antibody ponsegromab had similar inhibitory effects on GDF15 function. The results are shown in Table 12 and Figure 9.

[0080] [Table 12] Blocking Test 3: HEK293 SRE-LUC2-cRET-GFRAL reporter cells were cultured in a 96-well plate one day in advance. The next day, 53E5(hz) and the control antibody ponsegromab were prepared in medium at concentrations of 4, 3, 2, 0.5, and 0.1 μg / mL. Five aliquots of each antibody concentration were added to the wells containing GDF15-His (ACROBiosystems, GD5-H5149) at final concentrations of 100, 31.6, 10, 3.16, and 1 ng / mL, respectively. After 1 hour of incubation at room temperature, the cells were added to the wells containing the reporter cells to stimulate luciferase expression. After 6 hours of incubation, 50 μL of Bright-Glo reagent solution was added to each well, and the plate was shaken for 3–5 minutes to disrupt the cells. Cold light readings were 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 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) had a stronger neutralizing effect on GDF15 than the control antibody Ponsegromab. The results are shown in Figures 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 signals induced by non-neutralized GDF15 stimulating reporter cells. The EC50 values ​​shown in the figure are those at 0.1 μg / mL of antibody.

[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 dissolved in PBS at 1 μg / mL each and coated onto a 96-well ELISA plate one day in advance. The following day, the plate was blocked with 2% BSA in PBS for 1 hour. After removing the blocking solution, the GDF15 antibody 53E5 (hz), control antibody Ponsegromab, and unrelated hIgG1 were added at 4 μg / mL. The plate was then incubated at room temperature for 1 hour, washed, and then HRP anti-human IgG was added. After incubation at room temperature for 1 hour, the HRP enzyme activity signal (OD450) was detected. The OD450 value is a relative signal of antibody binding. The experimental results showed that 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 treated with 10, 1, or 0.1 μg / mL of the GDF15 antibody 53E5(hz), control antibodies Ponsegromab and AV380-hIgG1, or the CD3 antibody OKT3 as a positive control. A separate tube without antibody was used 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. After incubation at 4°C for 30 minutes, the mixture was centrifuged. Antibody dilutions were then added, and 7AAD was added to remove dead cells. The antibody binding signal was analyzed by flow analysis. The mean fluorescence intensity (MFI) of PE in PBMCs represents the signal of antibody binding to the cells. A curve of antibody concentration versus MFI was plotted. The experimental results demonstrated that antibody 53E5(hz) did 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 female BALB / c mice at doses of 10 mg / kg and 1 mg / kg of body weight, respectively. Blood samples were collected at 10 min, 1 h, 6 h, 1 day, 3 days, 5 days, 7 days, and 14 days, and serum samples were prepared. After serum was collected at all time points, serum antibody concentrations were measured by ELISA.

[0085] The specific procedure was as follows: 50 ng of GDF15-HIS antigen per well was coated overnight at 4°C and blocked for 2 hours with 2% BSA blocking solution. Serum samples were diluted 50-fold and 500-fold, respectively. The standard (i.e., the injected antibody) was diluted 3.16-fold from 1 μg / mL to give 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. A curve of OD450 values ​​versus standard concentration was plotted, and the serum antibody concentration was calculated from the fitted standard curve. A correlation curve between blood collection time and serum antibody concentration was plotted, and the half-life of the antibody in mice 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 5 x 10 female SCID mice aged 8-10 weeks 6 HT1080 cells (containing 50% Matrigel) were injected subcutaneously, and mice were weighed daily. Tumor size (length and width) was measured daily with a vernier caliper to calculate tumor volume (= 0.5 x length x width x height). 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 mice's body weight reached 93% of their 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), humanized antibody 53E5(hz), control antibody AV380-hIgG1, or ponsegromab via intraperitoneal injection at a dose of 10 mg / kg of mouse body weight once every 3 days. A correlation curve between the tumor-free body 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 demonstrated that 53E5(hz) and the control antibody ponsegromab had similar inhibitory effects on cachexia.

[0087] Experiment 2: The efficacy of GDF-15 antibodies in neutralizing GDF15 in vivo was determined.

[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. On day 2, they were administered hIgG (3 mg / kg body weight, negative control) or different doses of the humanized antibody 53E5(hz) and the control antibody ponsegromab (3, 1, or 0.3 mg / kg body weight) by IP injection. Body weights were measured daily. After the experiment, a correlation curve was plotted between the mouse weight and the number of days after injection of the GDF15-mFc fusion protein. The results are shown in Figure 15. The experimental results demonstrated that 53E5(hz) and the control antibody ponsegromab had similar inhibitory effects on GDF15 function in vivo.

[0089] Although the specific embodiments of the present invention have been described above, they are not intended to 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 shall fall within the scope of protection of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human GDF15, 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 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. An antibody or antigen-binding fragment thereof, characterized in that: (1) H-CDR1, H-CDR2, and H-CDR3 are 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 are shown in the order of SEQ ID NO: 26 (KASQNVDTNVA), SEQ ID NO: 27 (SASYRSS), and SEQ ID NO: 28 (QQYHSYPT); and (2) H-CDR1, H-CDR2, H-CDR3 shown in the order of SEQ ID NO: 23 (TSGMGVG), SEQ ID NO: 29 (HIRWDDVKRYNPALKS), SEQ ID NO: 25 (MAWDWFAY); and L-CDR1, L-CDR2, L-CDR3 shown in the order of SEQ ID NO: 30 (KASQNVDTDVA), SEQ ID NO: 31 (SASYRYS), SEQ ID NO: 32 (HQYNSYPT).

2. The heavy chain variable region and the light chain variable region each comprise: The antibody or antigen-binding fragment thereof of claim 1, (1) an amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 90% identity thereto; and an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto; (2) the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% identity thereto; and the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 90% identity thereto; (7) The amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 90% identity thereto; and the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 90% identity thereto; or (8) An amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90% identity with said amino acid sequence; and an amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 90% identity with said amino acid sequence.

3. 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, or (dsFv) of the antibody. ２ , Fab, Fab', F(ab') ２ or an Fv fragment 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The antibody or antigen-binding 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 antigen-binding fragment thereof of claim 1.

5. the antibody is a monoclonal antibody, The monoclonal antibody is IgG1. The antibody or antigen-binding fragment thereof according to claim 4 .

6. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising a nucleic acid molecule according to claim 6 and / or a vector according to claim 7.

9. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7 and / or the host cell of claim 8, the composition is a pharmaceutical composition, Optionally, further comprising a pharmaceutically acceptable adjuvant. , composition.

10. 10. Use of an antibody or antigen-binding 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 treating 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.

11. A kit comprising an antibody or antigen-binding 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.

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