Antibody molecules against growth differentiation factor 15 and their applications

Abstract

This invention provides an antibody or fragment thereof comprising a protein targeting human growth differentiation factor 15 (GDF15). This invention also provides the use of said antibody or fragment thereof in the preparation of a medicament for treating a disease or condition. The antibody or fragment thereof provided by this invention can bind to human GDF15 with high affinity and specificity, blocking the interaction between GDF15 and its receptor GFRAL, and has a longer in vivo half-life compared to similar antibodies.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to antibody molecules that specifically bind to growth differentiation factor 15 (GDF15) and their antigen-binding fragments, as well as the applications of said antibody molecules and their antigen-binding fragments. Background Technology

[0002] Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor β (TGFβ) superfamily and is responsible for regulating food intake, energy expenditure, and body weight in response to metabolic and toxin-induced stress in the human body. GDF15's receptor is a GDNF-family receptor α-like (GFRAL) protein. When it binds to its receptor GFRAL, it activates neurons in the posterior pole of the brainstem and the nucleus of the solitary tract, as these areas express GFRAL. It then triggers activation of neurons in the parabrachial nucleus and central amygdala, forming a feeding response to stress conditions. Due to these functions, GDF15 has attracted attention as a potential therapeutic target for obesity and related metabolic diseases.

[0003] Circulating GDF15 levels have long been associated with lower body mass index (BMI) and cachexia in patients with cancer, heart failure, or chronic kidney disease. Recent data suggest that when GDF15 binds to neurons expressing GFRAL in the brainstem and triggers activation of neurons in the parabrachial nucleus and central amygdala, it leads to appetite suppression and ultimately weight loss. Therefore, it appears to be a potential target for treating cachexia-related weight loss and obesity. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new antibody, such as a monoclonal antibody, that binds to human growth differentiation factor 15 protein (GDF15) with high affinity and specificity as a new therapeutic agent for treating diseases or symptoms such as cachexia.

[0005] To address the aforementioned technical problems, the object of this invention is to provide an antibody against GDF15 or a fragment thereof, and based on this antibody or fragment thereof, to provide its uses. The term "fraction" in the antibody molecule described in this invention encompasses various functional fragments of the antibody, such as its antigen-binding fragment.

[0006] The present invention provides the following technical solutions.

[0007] On one hand, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or fragment thereof being 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 region of human GDF15 as an immunogen, and a humanized antibody based on said mouse antibody.

[0008] Specifically, the present invention provides an antibody or a fragment thereof, the 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):

[0009] (1) H-CDR1, H-CDR2, and H-CDR3, sequentially shown in SEQ ID NO.23 (TSGMGVG), SEQ ID NO.24 (HILWDDVKRYNPALKS), and SEQ ID NO.25 (MAWDWFAY); and L-CDR1, L-CDR2, and L-CDR3, sequentially shown in SEQ ID NO.26 (KASQNVDTNVA), SEQ ID NO.27 (SASYRSS), and SEQ ID NO.28 (QQYHSYPT);

[0010] (2) H-CDR1, H-CDR2, and H-CDR3, sequentially shown in SEQ ID NO.23 (TSGMGVG), SEQ ID NO.29 (HIRWDDVKRYNPALKS), and SEQ ID NO.25 (MAWDWFAY); and L-CDR1, L-CDR2, and L-CDR3, sequentially shown in SEQ ID NO.30 (KASQNVDTDVA), SEQ ID NO.31 (SASYRYS), and SEQ ID NO.32 (HQYNSYPT);

[0011] (3) H-CDR1, H-CDR2, and H-CDR3, sequentially shown 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, sequentially shown in SEQ ID NO.36 (RASQSVSTSSFSYMH), SEQ ID NO.37 (YASNLES), and SEQ ID NO.38 (QHSWEIPYT);

[0012] (4) H-CDR1, H-CDR2, and H-CDR3, sequentially shown in SEQ ID NO.39 (TSGMGVD), SEQ ID NO.40 (HIYWDDDKRYNPSLKS), and SEQ ID NO.41 (RAWDAMDY); and L-CDR1, L-CDR2, and L-CDR3, sequentially shown in SEQ ID NO.42 (KARQNVGTNVA), SEQ ID NO.31 (SASYRYS), and SEQ ID NO.43 (QQYNSYPYT);

[0013] (5) H-CDR1, H-CDR2, and H-CDR3, sequentially shown in SEQ ID NO.23 (TSGMGVG), SEQ ID NO.44 (HIWWNDDKYYNPSLKS), and SEQ ID NO.45 (GAYDFFDY); and L-CDR1, L-CDR2, and L-CDR3, sequentially shown in SEQ ID NO.26 (KASQNVDTNVA), SEQ ID NO.31 (SASYRYS), and SEQ ID NO.46 (QQYNTYPYT); and

[0014] (6) H-CDR1, H-CDR2, and H-CDR3 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 of SEQ ID NO.50 (KASQNVGTNVA), SEQ ID NO.51 (SASYRNS), and SEQ ID NO.52 (QQYNSHPVT).

[0015] The antibody or fragment thereof provided by this invention is an antibody or fragment against GDF15, particularly human GDF15, which can bind to GDF15 with high affinity and specificity. The combination of light and heavy chain CDRs contained in the antibody or fragment thereof is derived from a specific antibody of this invention (see the Examples section). Based on the variable region amino acid sequence contained in this specific antibody, those skilled in the art can conventionally determine the CDRs contained therein. According to specific embodiments of this invention, the CDRs in the variable region amino acid sequence of this specific antibody can be partitioned using Kabat and any other definition method such as IMGT, ABM, Chotia, etc. Therefore, light and heavy chain CDRs and their combinations obtained by other methods known in the art are also covered within the scope of this invention.

[0016] Preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region may comprise the amino acid sequence shown 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 with said amino acid sequence; and / or, the light chain variable region may comprise the amino acid sequence shown 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 with said amino acid sequence. In the context of this invention, “at least 75% identity” for the sequence means any percentage of identity of ≥75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%.

[0017] More preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region and the light chain variable region respectively comprise:

[0018] (1) The amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:8;

[0019] (2) The amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10;

[0020] (3) The amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12;

[0021] (4) The amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14;

[0022] (5) The amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16;

[0023] (6) The amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18;

[0024] (7) The amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20;

[0025] (8) The amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:22.

[0026] Specifically, 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 include the CDRs described above and spaced framework regions, with the included domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the maximum 25% difference in amino acid sequence resulting from the "at least 75% identity" may exist in any framework region of the heavy chain variable region or the light chain variable region, or 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. This difference may be caused by amino acid deletions, additions, or substitutions at any position.

[0027] The antibodies provided by this invention are mouse anti-GDF15, particularly human GDF15, chimeric antibodies, or fully or partially humanized antibodies; the fragments are haptens or antigen-binding fragments of the antibodies, including scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv fragments. Preferably, the antibodies are monoclonal antibodies or single-chain antibodies.

[0028] In addition to the variable region, the antibody or fragment thereof also contains 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 contains a heavy chain and a light chain; more preferably, the antibody or fragment thereof contains a heavy chain constant region selected from IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region. According to a specific embodiment of the invention, the antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the invention, the monoclonal antibody is IgG, particularly IgG1.

[0029] 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 shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:4. Alternatively, the 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 the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:6.

[0030] On the other hand, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or a fragment thereof described in the present invention, 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 a fragment thereof.

[0031] Furthermore, the present invention provides a vector comprising the nucleic acid molecules described above. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0032] In another aspect, the present invention provides a host cell comprising the nucleic acid molecules or vectors described above, or a host cell transformed or transfected by the nucleic acid molecules or vectors described above. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant or animal cells.

[0033] The antibodies or fragments thereof provided by this invention can be obtained using any method known in the art. For example, the host cells are cultured in a manner that allows host cells provided by this invention to express the heavy chain variable region and / or the light chain variable region of the antibody, or the heavy chain and / or light chain of the antibody, to assemble the antibody. Optionally, the method further includes the step of recovering the generated antibody.

[0034] On the other hand, the present invention also provides a composition comprising the antibody or a fragment thereof described in the present invention, a nucleic acid molecule, a carrier, and / or a host cell. Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, excipient, or excipient.

[0035] The antibodies or their fragments, nucleic acid molecules, vectors, host cells, and / or compositions provided by this invention have one or more activities that increase appetite, food intake, weight, muscle mass, etc. in subjects, and also have anti-tumor effects. Therefore, this invention also provides the following technical solutions.

[0036] In another aspect, the present invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for treating a disease or condition mediated by or related to the expression of GDF15; and / or, the disease or condition being a tumor (e.g., cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia.

[0037] Accordingly, the present invention also provides a method for treating a disease or condition, the method comprising administering to a subject in need an antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition provided by the present invention, the disease or condition being mediated by or related to the expression of GDF15; and / or, the disease or condition being a tumor (e.g., cancer), heart failure, chronic kidney disease, anorexia, sarcopenia or cachexia.

[0038] Preferably, the subject is a mammal; more preferably, the subject is a human.

[0039] The present invention also provides a method for detecting or diagnosing a disease or condition, the method comprising contacting an antibody molecule or fragment thereof, a nucleic acid molecule, a vector, a host cell, and / or a composition thereof of the present invention with a sample from a subject, wherein the disease or condition is mediated by or related to the 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.

[0040] Furthermore, the present invention provides a kit comprising the antibody or a fragment thereof, a nucleic acid molecule, a vector, a host cell, and / or a composition thereof. The kit is used for the treatment, detection, or diagnosis described above. Optionally, the kit may also include instructions for use.

[0041] This invention provides a novel antibody against human GDF15, which can bind to human GDF15 with high affinity and specificity, blocking the interaction between GDF15 and its receptor GFRAL. Experiments have shown that the antibody provided by this invention has higher GDF15 affinity and specificity, a longer in vivo half-life, and a superior blocking effect compared to known similar antibodies. Attached Figure Description

[0042] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0043] Figure 1 The results of ELISA detection of the binding of hybridoma cell culture supernatant to human GDF15 are shown.

[0044] Figure 2 The results of ELISA detection of the binding of hybridoma cell culture supernatant to monkey GDF15 are shown.

[0045] Figure 3 The results of ELISA detection show that the supernatant of hybridoma cell culture blocked the binding of GDF15 to its receptor GFRAL.

[0046] Figure 4The results of ELISA detection show the binding of the antibody to human GDF15.

[0047] Figure 5 The results of ELISA detection show the binding of the antibody to monkey GDF15.

[0048] Figure 6 The results of ELISA assays showed that the antibody blocked the binding of GDF15 to its receptor GFRAL.

[0049] Figure 7 The results of ELISA detection showing antibody binding to human GDF15 after incubation in PBS and plasma are shown.

[0050] Figure 8 The results of a competitive ELISA assay show that the antibody blocks the binding of GDF15 to its receptor GFRAL.

[0051] Figure 9 The results of detecting antibody blocking of GDF15 function on reporter cells expressing GFRAL are shown.

[0052] Figure 10 The results of antibody neutralization of GDF15 are shown on reporter cells expressing GFRAL; where 10⁻¹: Ponsegromab; 10⁻²: 53E5(hz).

[0053] Figure 11 The results show the detection results of the specificity of antibody binding to GDF15, GDF1, and GDF3.

[0054] Figure 12 The results show the detection results of the antibody's specificity in binding to PBMCs.

[0055] Figure 13 The results of the PK experiment of the antibody in mice are shown; where 13-1: AV380-hIgG1; 13-2: Ponsegromab; 13-3: 53E5(hz).

[0056] Figure 14 The study demonstrated the cachexia-inhibiting effect of the antibody as detected in mice.

[0057] Figure 15 The effect of the antibody on neutralizing GDF15, as detected in mice, was demonstrated. Detailed Implementation

[0058] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0060] Example 1 Preparation of hybridoma cells

[0061] Mice were immunized with a fusion protein containing the extracellular region of GDF15 protein (Genbank accession number NM_004864.3) and mouse IgG2a-FC (Genbank accession number AAH31470.1) as the immunogen. Quick Antibody-Mouse 5W water-soluble adjuvant was used. Titer was measured two weeks after booster immunization. Mice with high titers were selected for immunization shock, and serum was collected three days later. Spleens were harvested from the dissected mice, and spleen cells were isolated.

[0062] Spleen cells were fused with cultured myeloma cells, plated in 96-well plates, and selected using selective culture medium. The medium was changed after 7 days, and ELISA was performed after 10 days. Cells with OD values ​​greater than 10 times that of the negative control were then analyzed by flow cytometry.

[0063] Double-positive cells were selected and subcloned using a cell limiting dilution method to select monoclonal cells. The culture supernatant from the selected monoclonal cells was collected and subjected to ELISA and flow cytometry analysis. Double-positive cells were then selected for large-scale culture. The cell supernatant was purified by Protein G gravity column affinity chromatography to obtain hybridoma antibodies.

[0064] Example 2 ELISA detection of hybridoma antibody binding to human GDF15

[0065] The fusion protein containing the extracellular region of human GDF15 protein (Genbank accession number NM_004864.3) and human IgG1-FC (Genbank accession number CAC20454.1) was diluted to 1 μg / ml with coating buffer, and then added to 50 μl / well of an ELISA plate and incubated overnight at 4°C. The liquid in the wells was discarded, and the plate was washed three times with washing buffer for 3-5 minutes each time, then patted dry. 200 μl of blocking buffer was added to each well and incubated overnight at 4°C. The plate was washed three times with washing buffer. The coated plate can then be stored at -20°C or 4°C for later use.

[0066] Add the test antibody (purified antibody from hybridoma, nomenclature shown in Table 1), positive reference 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) to each well. Antibodies were added at a starting concentration of 20 μg / mL, diluted 3.16-fold, in 10 gradients. Incubate at 37°C for 1 hour, wash, and blot dry. Then add 100 μl of enzyme-labeled secondary antibody (1:10000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG (SIGMA, catalog number A9044-2ml) per well, incubate at 37°C for 2 hours, wash, and blot dry. Add 100 μl of freshly prepared substrate chromogenic solution (TMB) to each well, and incubate at 37°C for 30 minutes.

[0067] The AV380-mIgG1 sequence is as follows:

[0068] Heavy chain variable region VH (SEQ ID NO.1):

[0069] EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGQINPNNGGIFFNQKFKGKATLTVDKSSNTAFMEVRSLTSEDTAVYYCAREAITTVGAMDYWGQGTSVTVSS

[0070] Light chain variable region VL (SEQ ID NO.2):

[0071] DIQMTQSPASLSSASVGETVTITCRTSENLHNYLAWYQQKQGKSPQLLVYDAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSSPYTFGGGTKLEIK

[0072] Heavy chain constant region (SEQ ID NO.3):

[0073] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0074] Light chain constant region (SEQ ID NO.4):

[0075] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0076] The reaction was terminated by adding 2 mol / L H2SO4, and the OD values ​​were read using an ELISA reader. The results are shown in Tables 1-1 and 1-2. Figure 1 1-1 and 1-2 in the example.

[0077] Table 1-1 ELISA results of hybridoma antibody binding to human GDF15

[0078] AV380-mIgG1 0.03134 50G3 0.05563 53E5 0.04546 53D10 0.06025 53A8 0.03780

[0079] Table 1-2 ELISA results of hybridoma antibody binding to human GDF15

[0080] AV380-mIgG1 0.02958 2A2 0.04602 34G9 0.1094

[0081] Example 3 ELISA detection of hybridoma antibody binding to monkey GDF15 (cynoGDF15)

[0082] The experimental procedure was the same as in Example 2, except that the fusion protein was replaced with a fusion protein containing the extracellular region of monkey GDF15 protein (Genbank accession number EHH29815.1) and human IgG1-FC (Genbank accession number CAC20454.1). The results are shown in Tables 2-1 and 2-2. Figure 2 2-1 and 2-2 in the example.

[0083] Table 2-1 ELISA results of hybridoma antibody binding to monkey GDF15

[0084]

[0085]

[0086] Table 2-2 ELISA results of hybridoma antibody binding to monkey GDF15

[0087] AV380-mIgG1 0.02577 2A2 0.08271 34G9 0.1574

[0088] Example 4 ELISA detection of hybridoma antibody blocking the binding of GDF15 to its receptor GFRAL

[0089] The fusion protein containing the extracellular region of GFRAL protein (Genbank accession number NM_207410.2) and human IgG1-FC (Genbank accession number CAC20454.1) was diluted to 1 μg / ml with coating buffer, and then added 50 μl / well to each well of an ELISA plate and incubated overnight at 4°C. The liquid in the wells was discarded, and the plates were washed three times with washing buffer for 3-5 minutes each time, then patted dry. 200 μl of blocking buffer was added to each well and incubated overnight at 4°C. The plates were then washed three times with washing buffer. The coated plates can now be stored at -20°C or 4°C for later use.

[0090] Add the test antibody, positive reference antibody AV380-mIgG1, and blank control (PBS) to each well, starting at 20 μg / mL and diluting 3.16-fold in 10 gradients. Simultaneously add GDF15-mFC protein (GDF15: Genbank accession number NM_004864.3; mFC: Genbank accession number AAH31470.1) at 20 ng / well and mix well with the test antibody. Incubate at 37°C for 1 hour, wash, and blot dry. Then add enzyme-labeled secondary antibody (1:10000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG (SIGMA, catalog number A9044-2ml)) to each well (50-100 μl per well), incubate at 37°C for 2 hours, wash, and blot dry. Add 100 μl of freshly prepared substrate development solution (TMB) to each well and incubate at 37°C for 30 minutes. The reaction was terminated by adding 2 mol / L H2SO4, and the OD value was read on an enzyme-linked immunosorbent assay (ELISA) reader.

[0091] The results are shown in Tables 3-1 and 3-2. Figure 3 3-1 and 3-2 in the example.

[0092] Table 3-1. ELISA results of hybridoma antibody blocking the binding of GDF15 to its receptor GFRAL

[0093]

[0094]

[0095] Table 3-2. ELISA results of hybridoma antibody blocking the binding of GDF15 to its receptor GFRAL

[0096] AV380-mIgG1 0.1395 2A2 0.08984 34G9 0.09157

[0097] The variable region sequence of the above mouse antibody is as follows (where the heavy chain and light chain CDRs are shown in bold and underlined, and are obtained according to the Kabat definition method):

[0098] A1 (Rat Anti-53E5)

[0099] >53E5-H(VH / HCDR-1 / HCDR-2 / HCDR-3:SEQ ID NO.7 / 23 / 24 / 25)

[0100]

[0101] >53E5-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO.8 / 26 / 27 / 28)

[0102]

[0103] A2 (Rat Anti-53A8)

[0104] >53A8-H(VH / HCDR-1 / HCDR-2 / HCDR-3:SEQ ID NO.9 / 23 / 29 / 25)

[0105]

[0106] >53A8-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO.10 / 30 / 31 / 32)

[0107]

[0108] A3 (Mouse Anti-34G9)

[0109] >34G9-H(VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO.11 / 33 / 34 / 35)

[0110]

[0111] >34G9-L(VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO.12 / 36 / 37 / 38)

[0112]

[0113] A4 (Rat Anti-A2)

[0114] >2A2-H(VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO.13 / 39 / 40 / 41)

[0115]

[0116] >2A2-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO.14 / 42 / 31 / 43)

[0117]

[0118] A5 (Rat Antibody 50G3)

[0119] >50G3-H(VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO.15 / 23 / 44 / 45)

[0120]

[0121] >50G3-L(VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO.16 / 26 / 31 / 46)

[0122]

[0123] A6 (Mouse Anti-53D10)

[0124] >53D10-H(VH / HCDR-1 / HCDR-2 / HCDR-3:SEQ ID NO.17 / 47 / 48 / 49)

[0125]

[0126] >53D10-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO.18 / 50 / 51 / 52)

[0127]

[0128] Example 5 Affinity detection of mouse antibody binding to human GDF15

[0129] Experimental methods:

[0130] 1. Select a Capture Surface Dip of the same species.

[0131] 2. Take a 96-well plate, add 200 μl of SD buffer (1×PBS + 0.02% Tween 20 + 0.1% BSA) to each well, and place it in a ForteBio Octet for pre-circulation.

[0132] 3. Fix the antibody at a concentration of 10 μg / ml.

[0133] 4. GDF15 (Bepsys, GD5-H5149) was diluted to 200nm, 100nm, 50nm, 25nm, 12.5nm and 6.25nm respectively, and added to the corresponding wells.

[0134] 5. On-machine testing.

[0135] The results are shown in Table 4.

[0136] Table 4. Affinity test results of mouse antibody binding to human GDF15

[0137]

[0138]

[0139] Example 6 Humanization of mouse antibodies

[0140] Human sequences IGHV4-39 and IGKV1-39 were selected for A1 (mouse anti-53E5) as templates for heavy and light chain humanization, respectively. Homology modeling was performed on the A1 monoclonal antibody, and the structure of the Fab region was simulated. Homology modeling calculations were performed, and the predicted Fab structure of A1 was finally obtained.

[0141] By comparing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, it was found that all mouse amino acids in the VH were replaced with the corresponding human amino acids from the IGHV4-39 template, except for the CDR region and 2V, 24F, 50L, 51A, 69L, 73K, 75S, 78S, 80I, 98V, and 99Q, which retained the original mouse amino acids.

[0142] By comparing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that all mouse amino acids in the VL except for the CDR region and 42Q, 43S, and 46A, which retained the original mouse amino acids, were replaced with the corresponding human amino acids from the IGKV1-39 template.

[0143] The obtained humanized sequences are as follows (where the heavy and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition method):

[0144] >53E5-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO.19 / 23 / 24 / 25)

[0145]

[0146] >53E5-L humanized sequences (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO.20 / 26 / 27 / 28)

[0147]

[0148] Human sequences IGHV4-39 and IGKV1-39 were selected for A2 (mouse anti-53A8) as templates for heavy and light chain humanization, respectively. Homology modeling was performed on the A2 monoclonal antibody, and the structure of the Fab region was simulated. Homology modeling calculations were performed, and the predicted Fab structure of A2 was finally obtained.

[0149] By comparing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, it was found that all mouse amino acids in the VH, except for the CDR region and 2V, 24F, 50L, 51A, 69L, 73K, 78S, 80I, and 99Q which are original mouse amino acids, were replaced with the corresponding human amino acids from the IGHV4-39 template.

[0150] By comparing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that all mouse amino acids in the VL except for the CDR region and 42Q, 43S, and 46A, which retained the original mouse amino acids, were replaced with the corresponding human amino acids from the IGKV1-39 template.

[0151] The obtained humanized sequences are as follows (where the heavy and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition method):

[0152] >53A8-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO.21 / 23 / 29 / 25)

[0153]

[0154] >53A8-L humanized sequences (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO.22 / 30 / 31 / 32)

[0155]

[0156] Using the sequence shown in SEQ ID NO.5 as the heavy chain constant region and the sequence shown in SEQ ID NO.6 as the light chain constant region, primers were redesigned for the above humanized sequences, and the corresponding antibody heavy and light chain encoding genes were synthesized and ligated into a eukaryotic expression vector. The recombinant vector was transformed into competent E. coli cells and cultured overnight at 37°C. Single clones were selected for sequencing identification. Strains with correct sequences were selected, and recombinant vectors were obtained and transfected into mammalian expression cells 293F. The cells were cultured at 37°C and 5% CO2 for 7 days. The supernatant was collected, and the humanized antibody was purified and named "mouse antibody (hz)".

[0157] >CH1-CH3 heavy chain constant region (SEQ ID NO.5)

[0158] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0159] >CL1 light chain constant region (SEQ ID NO.6)

[0160] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0161] Example 7 ELISA detection of humanized antibody binding to human GDF15

[0162] The experimental procedure was the same as in Example 2, except that the coating protein was GDF15-HIS (Bepsys, CAT#: GD5-H5149), and the test antibody (humanized antibody), negative reference antibody (hIgG1), and positive reference antibody Ponsegromab (Pfizer; Drug Bank: D11909) and AV380-hIgG1 (the heavy and light chain constant regions of AV380-mIgG1 were replaced with the constant regions of human IgG, 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 .

[0163] Table 5. ELISA detection results of humanized antibody binding to human GDF15

[0164] 53E5(hz) 0.08331 1.053 53A8(hz) 0.08266 1.051 AV380-hIgG1 0.08233 1.066 Ponsegromab 0.09599 1.100

[0165] Example 8 ELISA detection of humanized antibody binding to monkey GDF15 (cynoGDF15)

[0166] The experimental procedure was the same as in Example 2, except that the coating protein was cynoGDF15-mFC (GDF15: Genbank accession number EHH29815.1; mFC: Genbank accession number AAH31470.1), and the test antibody (humanized antibody), negative reference antibody (hIgG1), and positive reference antibodies Ponsegromab and AV380-hIgG1 were added to each well of the coated plate. The results are shown in Table 6. Figure 5 .

[0167] Table 6. ELISA results of humanized antibody binding to monkey GDF15

[0168] 53E5(hz) 0.08159 1.032 53A8(hz) 0.07749 1.03 AV380-hIgG1 0.07407 1.000 Ponsegromab 0.06973 1.067

[0169] Example 9 ELISA detection of humanized antibody blocking the binding of GDF15 protein to its receptor GFRAL

[0170] The experimental procedure was the same as in Example 4, except that the test antibody (humanized antibody), negative reference antibody (hIgG1), and positive reference antibodies Ponsegromab and AV380-hIgG1 were added to each well of the coated plate. The results are shown in Table 7. Figure 6 .

[0171] Table 7. ELISA results of humanized antibody blocking the binding of GDF15 to its receptor GFRAL

[0172] 53E5(hz) 0.2525 1.049 53A8(hz) 0.2831 1.104 AV380-hIgG1 0.2311 10.965 Ponsegromab 0.5037 1.019

[0173] Example 10 Affinity detection of humanized antibody binding to human GDF15

[0174] The experimental procedure was the same as in Example 5, except that the GDF15 antibody was replaced with the antibody to be tested (humanized antibody), the negative reference antibody (hIgG1), and the positive reference antibodies Ponsegromab and AV380-hIgG1. The results are shown in Table 8.

[0175] Table 8. Affinity test results of humanized antibodies binding to human GDF15

[0176] 53E5(hz) 2.93E-10 9.23E+05 2.71E-04 53A8(hz) 2.66E-10 9.67E+05 2.57E-04 AV380-hIgG1 8.45E-11 8.17E+05 6.91E-05 Ponsegromab <1.0E-12 7.11E+05 <1.0E-07

[0177] Example 11 Stability testing of humanized antibodies

[0178] 1. Freeze-thaw

[0179] Take an appropriate amount of antibody, adjust the concentration to 10 mg / mL, freeze at -80℃ for 10 min, then remove and place in an ice-water mixture to thaw slowly. Repeat the above steps 5 times, then use SEC-HPLC to detect the degree of protein aggregation and ELISA to detect changes in protein binding activity.

[0180] 2. Low pH treatment

[0181] Take an appropriate amount of antibody, adjust the antibody storage buffer to pH 3.6 using an ultrafiltration tube, and adjust the concentration to 5 mg / mL. After incubation at room temperature for 2-4 hours, detect the degree of protein aggregation using SEC-HPLC and detect changes in protein binding activity using ELISA.

[0182] 3. Accelerated test at 40℃

[0183] Take an appropriate amount of antibody, adjust the concentration to 10 mg / mL, and treat at 40℃ for 1 and 2 weeks. Protein aggregation was detected by SEC-HPLC, and changes in protein binding activity were detected by ELISA. The results are shown in Table 9.

[0184] Table 9. Stability test results of humanized antibodies

[0185]

[0186] 4. Plasma stability test

[0187] Take 200 μL of plasma and PBS, add 200 μg of the antibody to be tested (final concentration 1 mg / mL) to each, block well, and incubate at 37°C for 2 weeks. ELISA is used to detect changes in protein binding activity. Results are shown in Table 10. Figure 7 .

[0188] Table 10. ELISA detection results of humanized antibody binding to human GDF15

[0189] 53E5(hz)(PBS) 0.1272 53E5(hz)(plasma) 0.03725 53A8(hz)(PBS) 0.1125 53A8(hz)(plasma) 0.04129

[0190] Example 12 ELISA detection of humanized antibody blocking the binding of GDF15 to receptor GFRAL

[0191] The extracellular region of GFRAL was stably expressed in the HEK293 luciferase reporter cell line, where the expression of the luciferase gene was controlled by the SRE expression element, thus constructing the HEK293 SRE-LUC2-cRET-GFRAL reporter cell line.

[0192] Blocking Experiment 1:

[0193] GFRAL-HIS protein (Bepsys, GFA-H52H3) was dissolved in PBS at a concentration of 1 μg / ml and used to coat ELISA 96-well plates one day in advance. The plates were blocked the next day with PBS containing 2% BSA. 53E5 (Hz), control antibody Ponsegromab, and AV380-hIgG1 were serially diluted 3.16-fold at an initial concentration of 20 μg / ml to 10 concentrations. GDF15-mFc protein was added simultaneously to bring the final concentration to 0.3 μg / ml. After incubation at room temperature for 1 hour, the mixture was added to the 96-well plates coated with GFRAL-HIS and incubated at room temperature for 1 hour. After washing, HRP-anti-mouse IgG was added, and the plates were 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. OD450 values ​​and antibody concentration correlation curves were plotted. A dose-response model was used to fit the log(agonist) vs. response-variable slope (four-parameter) logistic model to calculate the IC50. Experimental results showed that 53E5(Hz) exhibited stronger blocking ability against GDF15 than the control antibody. Results are shown in Table 11 and... Figure 8 .

[0194] Table 11. Competitive ELISA results of humanized antibodies blocking the binding of GDF15 to its receptor GFRAL

[0195]

[0196]

[0197] Blocking Experiment 2:

[0198] HEK293 SRE-LUC2-cRET-GFRAL reporter cells were cultured in 96-well plates one day in advance. The next day, 53E5 (Hz), the control antibody Ponsegromab, and AV380-hIgG1 were serially diluted 3.16-fold with culture medium at an initial concentration of 1 μg / ml to obtain 10 different concentrations. GDF15-His (Bepsys, GD5-H5149) was added to bring the final concentration to 5 ng / ml. After incubation at room temperature for 1 hour, the cultured cells were added to the wells containing the reporter cells to stimulate luciferase expression. After culturing for another 6 hours, 50 μL of Bright-Glo reagent solution was added to each well, and the plate was shaken for 3-5 minutes to lyse the cells. The fluorescence values ​​were read using a Multi-mode Microplate Reader. A fold increase in luciferase activity (the fold increase in enzyme activity in wells without GDF15 protein) was plotted against antibody concentration. A dose-response model was fitted using a log(agonist) vs. response-variable slope (four-parameter) logistic model, and the IC50 was calculated. The 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 .

[0199] Table 12. Results of detecting the function of humanized antibodies in blocking GDF15 on reporter cells expressing GFRAL.

[0200] 53E5(hz) 0.02254 1.096 AV380-hIgG1 0.04471 1.033 Ponsegromab 0.03211 1.066

[0201] Blocking Experiment 3:

[0202] HEK293 SRE-LUC2-cRET-GFRAL reporter cells were cultured in 96-well plates one day in advance. The next day, solutions of 53E5(Hz) and the control antibody Ponsegromab were prepared in culture medium at concentrations of 4, 3, 2, 0.5, and 0.1 μg / ml, respectively. Five aliquots of each antibody concentration were taken and added to different concentrations of GDF15-His (Pypsy, GD5-H5149) to achieve final concentrations of 100, 31.6, 10, 3.16, and 1 ng / ml, respectively. After incubation at room temperature for 1 hour, the aliquots were added to the wells containing reporter cells to stimulate luciferase expression. After culturing for another 6 hours, 50 μL of Bright-Glo reagent solution was added to each well, and the culture plate was shaken for 3-5 minutes to induce cell lysis. The fluorescence values ​​were read using a Multi-mode Microplate Reader. Plot the fold increase in luciferase activity (fold increase in enzyme activity in wells without GDF15 protein) versus antibody concentration curves. Fit the dose-response curves using a log(agonist) vs. response-variable slope (four-parameter) logistic model and calculate EC50.

[0203] Experimental results showed that 53E5(hz) had a stronger neutralizing effect on GDF15 than the control antibody Ponsegromab. See results below. Figure 10 The values ​​are 10⁻¹ and 10⁻². In this experiment, the antibody concentration was fixed, and then co-incubated with different concentrations of GDF15 protein. The signal of unneutralized GDF15-stimulated reporter cells was detected. The EC50 shown in the figure represents the value when the antibody concentration is 0.1 μg / ml.

[0204] Example 13 Detection of binding specificity of humanized antibodies

[0205] Combined with specificity experiment 1:

[0206] GDF15 (Bepsys, GD5-H5149), GDF1 (R&D, 6937-GD-010), and GDF3 (R&D, 5754-G3-010) proteins were dissolved in PBS at a concentration of 1 μg / ml and used to coat 96-well ELISA plates one day in advance. The next day, the plates were blocked with PBS containing 2% BSA for 1 hour. After removing the blocking solution, GDF15 antibody 53E5(hz), control antibody Ponsegromab, and irrelevant hIgG1 were added at 4 μg / ml. After incubation at room temperature for 1 hour, the plates were 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 the relative signal of antibody binding. The results showed that antibody 53E5(hz) did not specifically bind to GDF1 and GDF3, while the control antibody Ponsegromab weakly bound to GDF1 and GDF3. (See attached table for details.) Figure 11 .

[0207] Combined with specificity experiment 2:

[0208] Frozen PBMCs were resuscitated, and 10, 1, and 0.1 μg / ml of GDF15 antibody 53E5 (hz), control antibody Ponsegromab, and AV380-hIgG1, or CD3 antibody OKT3 as positive controls, were added to the PBMCs. One 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 was added for flow cytometry analysis. After incubation at 4°C for 30 minutes, centrifugation was performed, antibody dilution buffer was added, and 7AAD was added to remove dead cells. The antibody binding signal was analyzed by flow cytometry. The mean fluorescence intensity (MFI) of PE on PBMCs represents the antibody binding signal on cells; a curve of antibody concentration versus MFI was plotted. The experimental results showed that antibody 53E5 (hz) did not specifically bind to PBMCs. See [details omitted]. Figure 12 .

[0209] Example 14 Animal in vivo PK experiment

[0210] Female BALB / c mice aged 10-12 weeks were injected intravenously via the tail vein with GDF15 antibody 53E5(hz), control antibody AV380-hIgG1, and Ponsegromab at doses of 10 mg / kg and 1 mg / kg body weight, respectively. Blood was collected at 10 minutes, 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days, and 14 days to prepare serum. After collecting serum at all time points, antibody concentrations in the serum were detected by ELISA.

[0211] The specific procedure is as follows: GDF15-HIS antigen was coated overnight at 4℃, 50 ng per well, and blocked with 2% BSA blocking solution for 2 h. Serum samples were diluted 50-fold and 500-fold, respectively; standards (i.e., injected antibodies) were started at 1 μg / mL, diluted 3.16-fold, and 12 concentrations were obtained. The blocking solution was removed, and the prepared serum samples and standards were added, and incubated at room temperature for 1 h; after washing, HRP-anti-human IgG was added, and incubated at room temperature for 1 h; after washing, the HRP enzyme activity signal (OD450) was detected. An OD450 value versus standard concentration curve was plotted, and the antibody concentration in serum was calculated based on the fitted standard curve. A correlation curve between blood collection time and antibody concentration in serum was plotted, and the half-life of the antibody in mice was calculated. The results are shown below. Figure 13 .

[0212] Example 15 GDF15 antibody in vivo functional assay

[0213] Experiment 1. Detect the efficiency of GDF15 antibody in inhibiting cachexia.

[0214] Female SCID mice aged 8–10 weeks were subcutaneously injected with 5x10 6 HT1080 cells (containing 50% matrigel) were used. Mouse body weight was measured daily, and tumor size (length and width) was measured daily using calipers. Tumor volume was calculated as 0.5 x length x width x height. Tumor-free body weight was calculated as the mouse's body weight with tumor minus the tumor weight (tumor weight calculated as volume * 1 mg / mm²). 3 When the mice's body weight reached 93% of their day 0 body weight (day 18 in this experiment), they were randomly divided into groups of 10 mice each. Each group received an intraperitoneal injection of hIgG (negative control) or humanized antibody 53E5(hz) and control antibody AV380-hIgG1 with Ponsegromab at a dose of 10 mg / kg body weight, administered every 3 days. A correlation curve was plotted between the tumor-free body weight and the number of days after tumor inoculation. The results are shown in […]. Figure 14 Experimental results showed that 53E5(hz) and the control antibody Ponsegromab had similar cachexia-inhibiting effects.

[0215] Experiment 2. Detect the efficiency of GDF-15 antibody in neutralizing GDF15 in vivo.

[0216] BALB / c normal mice were intraperitoneally injected with GDF15-mFc fusion protein (0.25 mg / kg body weight) on days 0 and 8, respectively. On day 2, they were intraperitoneally injected with hIgG (3 mg / kg body weight, negative control) or different doses of humanized antibody 53E5(hz) and control antibody Ponsegromab (3, 1, and 0.3 mg / kg body weight). Body weight was measured daily. At the end of the experiment, a correlation curve between mouse body weight and the number of days after GDF15-mFc fusion protein injection was plotted. The results are shown in [Figure 1]. Figure 15 Experimental results showed that 53E5(hz) and the control antibody Ponsegromab had similar effects in inhibiting GDF15 function in vivo.

[0217] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims. sequence list <110> Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. <120> Antibody molecules against growth differentiation factor 15 and their applications <130> LC21110127 <160> 52 <170> PatentIn version 3.3 <210> 1 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH <400> 1 Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gln Ile Asn Pro Asn Asn Gly Gly Ile Phe Phe Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Phe 65 70 75 80 Met Glu Val Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Leu His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 324 <212> PRT <213> artificial sequence <220> <223> CH1-CH3 <400> 3 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115 120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 225 230 235 240 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro Gly Lys <210> 4 <211> 107 <212> PRT <213> Artificial sequence <220> <223> CL1 <400> 4 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 5 <211> 330 <212> PRT <213> artificial sequence <220> <223> CH1-CH3 <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 6 <211> 107 <212> PRT <213> artificial sequence <220> <223> CL1 <400> 6 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 7 <211> 118 <212> PRT <213> artificial sequence <220> <223> VH <400> 7 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Leu Trp Asp Asp Val Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Ser Ser Asn Ser Gln Ile 65 70 75 80 Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Val Gln Met Ala Trp Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 8 <211> 106 <212> PRT <213> artificial sequence <220> <223> VL <400> 8 Asp Ile Val Met Thr Gln Ala Gln Lys Phe Met Ser Thr Ser Val Glu 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr His Ser Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 118 <212> PRT <213> artificial sequence <220> <223> VH <400> 9 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Arg Trp Asp Asp Val Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Ser Gln Ile 65 70 75 80 Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Gln Met Ala Trp Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 10 <211> 106 <212> PRT <213> artificial sequence <220> <223> VL <400> 10 Asp Ile Val Met Thr Gln Ala Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys His Gln Tyr Asn Ser Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 120 <212> PRT <213> artificial sequence <220> <223> VH <400> 11 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr Ala 20 25 30 Gly Met Thr Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asn Asp Asp Lys Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Ile 65 70 75 80 Phe Leu Glu Ile Ala Ser Val Val Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Thr Arg Ile Ala Thr Met Asn Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 12​<210> 12 <211> 111 <212> PRT <213> Artificial sequence <220> <223> VL <400> 12 Asp Ile Val Met Thr Gln Ala Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 13 <211> 118 <212> PRT <213> Artificial sequence <220> <223> VH <400> 13 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Met Gly Val Asp Trp Ile Arg Gln Ser Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Ala Trp Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Phe Thr Val Ser Ser 115 <210> 14 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 14 Asp Ile Val Met Thr Gln Ala Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Arg Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 118 <212> PRT[[ID= / / ]] <213> artificial sequence <220> <223> VH <400> 15 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asn Asp Asp Lys Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser His Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Met Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Gly Ala Tyr Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> `16` <211> `107` <212> `PRT` <213> `artificial sequence` <220> <223> `VL` <400> `16` Asp Ile Val Met Thr Gln Ala Gln Asn Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Asp Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 118 <212> PRT <213> artificial sequence <220> <223> VH <400> 17 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Val Gln Pro Ser Gln 1 5 10 15 Pro Phe Arg Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Ile Gly Ile Thr Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Thr Ile Trp Trp Asp Asp Asp Asn Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Asn Asn Gln Ala 65 70 75 80 Phe Leu Asn Ile Ile Thr Val Glu Thr Ala Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Gln Ser Ala Trp Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 18 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 18 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Ser Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Glu Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser His Pro Val 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 118 <212> PRT <213> Artificial sequence <220> <223> VH <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Leu Trp Asp Asp Val Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Ser Ser Lys Ser Gln Ile 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Gln Met Ala Trp Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 20 <211> 106 <212> PRT <213> artificial sequence <220> <223> VL <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 21 <211> 118 <212> PRT <213> Artificial sequence <220> <223> VH <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 4​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <223> VL <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Asn Ser Tyr Pro Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys [[ID=??]] 100 105 <210> 23 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HCDR-1 <400> 23 Thr Ser Gly Met Gly Val Gly 1 5 <210> 24 <211> 16<00??1160><212> PRT It should be noted that there are some tags in the original text that seem to be incomplete or have incorrect formatting (such as "??" in some tag numbers), which may cause problems in a more formal context. This translation is based on the best understanding of the provided text.<213> Artificial sequence <220> <223> HCDR-2 <400> twenty four His Ile Leu Trp Asp Asp Val Lys Arg Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR-3 <400> 25 Met Ala Trp Asp Trp Phe Ala Tyr 1 5 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR-1 <400> 26 Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR-2 <400> 27 Ser Ala Ser Tyr Arg Ser Ser 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial sequence <220> <223> LCDR-3 <400> 28 Gln Gln Tyr His Ser Tyr Pro Thr 1 5 <210> 29 <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR-2 <400> 29 His Ile Arg Trp Asp Asp Val Lys Arg Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 30 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR-1 <400> 30 Lys Ala Ser Gln Asn Val Asp Thr Asp Val Ala 1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR-2 <400> 31 Ser Ala Ser Tyr Arg Tyr Ser 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <223> LCDR-3 <400> 32 His Gln Tyr Asn Ser Tyr Pro Thr 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HCDR-1 <400> 33 Thr Ala Gly Met Thr Val Gly 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR-2 <400> 34 His Ile Trp Trp Asn Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> HCDR-3 <400> 35 Ile Ala Thr Met Asn Tyr Ala Met Asp Tyr 1 5 10 <210> 36 <211> 15 <212> PRT <213> Artificial sequence <220> <223> LCDR-1 <400> 36 Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Phe Ser Tyr Met His 1 5 10 15 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR-2 <400> 37 Tyr Ala Ser Asn Leu Glu Ser 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR-3 <400> 38 Gln His Ser Trp Glu Ile Pro Tyr Thr 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HCDR-1 <400> 39 Thr Ser Gly Met Gly Val Asp 1 5 <210> 40 <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR-2 <400> 40 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 41 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR-3 <400> 41 Arg Ala Trp Asp Ala Met Asp Tyr 1 5 <210> 42 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR-1 <400> 42 Lys Ala Arg Gln Asn Val Gly Thr Asn Val Ala 1 5 10 <210> 43 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR-3 <400> 43 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 44 <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR-2 <400> 44 His Ile Trp Trp Asn Asp Asp Lys Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 45 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR-3 <400> 45 Gly Ala Tyr Asp Phe Phe Asp Tyr 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR-3 <400> 46 Gln Gln Tyr Asn Thr Tyr Pro Tyr Thr 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HCDR-1 <400> 47 Thr Ser Gly Ile Gly Ile Thr 1 5 <210> 48 <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR-2 <400> 48 Thr Ile Trp Trp Asp Asp Asp Asn Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 49 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR-3 <400> 49 Ser Ala Trp Asp Trp Phe Ala Tyr 1 5 <210> 50 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR-1 <400> 50 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR-2 <400> 51 Ser Ala Ser Tyr Arg Asn Ser 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR-3 <400> 52 Gln Gln Tyr Asn Ser His Pro Val Thr 1 5

Claims

1. An antibody against growth differentiation factor 15 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) comprise a combination of complementarity-determining regions (CDRs) (H-CDR1, H-CDR2, H-CDR3; and L-CDR1, L-CDR2, L-CDR3): (1) H-CDR1, H-CDR2, and H-CDR3, respectively, are shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25; And, respectively shown as L-CDR1, L-CDR2, L-CDR3 in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28; or (2) H-CDR1, H-CDR2, and H-CDR3, respectively, are shown in SEQ ID NO.23, SEQ ID NO.29, and SEQ ID NO.25; And, respectively, L-CDR1, L-CDR2, and L-CDR3 of SEQ ID NO.30, SEQ ID NO.31, and SEQ ID NO.

32.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that: (1) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, or the amino acid sequence of the heavy chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8, or the amino acid sequence of the light chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 8; (2) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 19, or the amino acid sequence of the heavy chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 19; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 20, or the amino acid sequence of the light chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

20. (3) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 9, or the amino acid sequence of the heavy chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 10, or the amino acid sequence of the light chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 10; or (4) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 21, or the amino acid sequence of the heavy chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 21; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 22, or the amino acid sequence of the light chain variable region is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

22.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The growth differentiation factor 15 mentioned is human growth differentiation factor 15.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a mouse antibody, a chimeric antibody, or a humanized antibody; the antigen-binding fragment is a fragment of the antibody such as scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a monoclonal antibody or a single-chain antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment also contains a human or mouse constant region.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The constant region of the human or mouse includes the heavy chain constant region (CH) of the human or mouse.

8. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The constant region of the human or mouse includes the light chain constant region (CL) of the human or mouse.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE, and / or contains a κ or λ type light chain constant region.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a monoclonal antibody.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The antibody is a mouse, chimeric, or humanized monoclonal antibody.

13. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The monoclonal antibody is IgG.

14. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The monoclonal antibody is IgG1.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 3, or the amino acid sequence of which is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

3.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment comprises a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 4, or the amino acid sequence of which is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

4.

17. The antibody or antigen-binding fragment thereof according to claim 15, characterized in that, The antibody or its antigen-binding fragment comprises a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 4, or the amino acid sequence of which is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

4.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 5, or the amino acid sequence of which is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

5.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment comprises a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 6, or the amino acid sequence of which is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

6.

20. The antibody or antigen-binding fragment thereof according to claim 18, characterized in that, The antibody or its antigen-binding fragment comprises a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 6, or the amino acid sequence of which is an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

6.

21. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 20.

22. A vector comprising the nucleic acid molecule of claim 21.

23. A host cell comprising the nucleic acid molecule of claim 21 or the vector of claim 22.

24. A composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 20, a nucleic acid molecule as described in claim 21, a vector as described in claim 22, or a host cell as described in claim 23.

25. The composition according to claim 24, characterized in that, The composition is a pharmaceutical composition and further comprises pharmaceutically acceptable excipients.

26. The composition according to claim 25, characterized in that, The composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

27. The composition according to claim 25, characterized in that, The composition is a pharmaceutical composition and further comprises pharmaceutically acceptable excipients.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 20, the nucleic acid molecule of claim 21, the vector of claim 22, the host cell of claim 23, or the composition of any one of claims 24 to 27 in the preparation of a medicament for treating a disease or condition, said disease or condition being cachexia.

29. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 20, a nucleic acid molecule as claimed in claim 21, a vector as claimed in claim 22, a host cell as claimed in claim 23, or a composition as claimed in any one of claims 24 to 27.

30. The reagent kit according to claim 29, characterized in that, The kit also includes an instruction manual.

Images