GDF15 single-domain antibody and its use

Abstract

The present invention relates to the field of biotechnology, and more specifically, to an antibody against GDF15 or its use. The present invention provides a heavy-chain antibody that targets GDF15, which specifically binds to GDF15, inhibits the interaction between GDF15 and GFRAL, and can be used to treat diseases associated with GDF15 overexpression or to diagnose a patient's GDF15 level. The antibody provided by the present invention exhibits superior inhibitory activity against the binding of GDF15 to GFRAL compared to the positive control CTL002, superior specificity compared to the positive control PF-06946860, and superior hydrophilicity compared to the positive control PF-06946860.

Description

Priority Information

[0001] This application claims the priority and rights of the patent application with patent application number 202310318320.0, which was filed with the China National Intellectual Property Administration on March 24, 2023, and incorporates the entire content thereof by reference herein.

Technical Field

[0002] The present invention relates to the field of biotechnology. Specifically, the present invention relates to an antibody against GDF15 or its use. More specifically, the present invention relates to an antibody or an antigen-binding fragment thereof that can specifically recognize GDF15, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, a pharmaceutical use, and a kit for detecting GDF15.

Background Art

[0003] Growth differentiation factor 15 (GDF15), also known as macrophage inhibitory cytokine-1 (MIC-1), was identified and discovered in 1997 from an activated macrophage cell line clone by Bootcov et al. and is a member of the transforming growth factor β (TGFβ) superfamily. GDF15 is a secreted protein that circulates as a 25 kDa dimer and consists of two polypeptide chains containing 112 amino acids, with the single chains linked by disulfide bonds. Its receptor is the glial cell line-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL) protein. GFRAL, which is a GDNF family α-like receptor, binds to GDF15 and then binds to the co-receptor RET to form a complex, further activating an intracellular signaling pathway that is consistent with GDNF signaling.

[0004] GDF15 and Disease Treatment: GDF15 is a circulating protein that, under normal physiological conditions, is expressed in many tissues and organs such as the liver, kidneys, muscles, fat, and placenta, but at relatively low levels. On the other hand, under conditions of aging, pregnancy, tissue damage, and various diseases (e.g., cancer, cardiovascular disease, kidney disease, etc.), GDF15 expression levels increase significantly. Numerous studies have shown that elevated GDF15 levels are associated with cardiovascular diseases such as myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction, as well as obesity, diabetes, cancer, and cachexia (Spanopoulou A et al., Clin Exp Metastasis, 2020, 37:451-464; Adolph TE et al., J Obes, 2018, 2018:7108075). Therefore, inhibiting or suppressing the activity of GDF15 (i.e., neutralizing GDF15) can treat the aforementioned GDF15-related diseases.

[0005] GDF15 and Disease Diagnosis: GDF15 levels are elevated in multiple solid tumors (Traeger L et al., BMC Cancer. 2019 Jan 15;19(1):74). It is a diagnostic biomarker for pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, and lung cancer. It is associated with disease progression, prognosis, and overall survival in multiple cancers, including colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, ovarian cancer, and breast cancer, but is unrelated to hematological malignancies. Studies have shown that serum GDF15 levels in patients with epithelial ovarian cancer resistant to platinum-based first-line chemotherapy (carboplatin or cisplatin) are significantly higher than in patients sensitive to chemotherapy, and that patients with high GDF15 expression have relatively shorter progression-free survival (Zhao D et al., BMC Cancer, 2018, 18:328). In short, GDF15 can be used as a relatively clear biomarker for cancer diagnosis, prognosis, and chemotherapy resistance. Furthermore, a two-year follow-up study of patients with acute heart failure (Adela R et al., J Diabetes Res, 2015, 2015:490842) found that elevated GDF15 was associated with a poor prognosis in acute heart failure, suggesting that GDF15 can also be used as a potential biomarker for assessing the severity and treatment response of cardiovascular disease.

[0006] Research on GDF15 monoclonal antibodies includes (1) CTL-002 (WO2022101263A1) developed by CatalYm (a humanized monoclonal antibody aimed at neutralizing tumor-produced GDF-15 to counteract immunosuppression, enhance immune cell infiltration into tumors, improve dendritic cell-mediated T cell activation, and increase the tumor-killing effects of T cells and NK cells); and (2) PF-06946860 (CN112912395A) developed by Pfizer, Inc. (a GDF15 monoclonal antibody primarily being studied for anorexia nervosa, aiming to improve anorexic reactions in cancer patients undergoing chemotherapy through drug intervention, ultimately improving cachexia).

[0007] However, the number of GDF antibodies disclosed so far is relatively small, and there is room for improvement in their specificity. [Overview of the project]

[0008] This application is based on the inventor's discoveries and knowledge regarding the following facts and problems.

[0009] The advantages of heavy chain single-domain antibodies are: (1) they have a small molecular weight and strong tissue penetration, making them widely used in tumor diagnosis and treatment, as well as molecular imaging; (2) they have a simple structure, making them easy to modify through genetic engineering; (3) they are easy to prepare, resulting in low production costs and short cycles as the industry scales up; (4) they are highly stable, easy to store, and can withstand mutagenic conditions such as high temperatures, strong acids, and strong alkalis; and (5) they have excellent specificity. The number of GDF antibodies disclosed to date is relatively small, and no heavy chain single-domain antibodies targeting GDF15 have yet been reported.

[0010] The inventors immunized alpacas with human GDF15 protein, collected peripheral blood cells from the immunized alpacas, isolated GDF15-affinity lymphocytes, extracted total RNA, cloned the heavy chain antibody variable region fragment of the alpaca heavy chain antibody using nested PCR technology, and inserted the fragment into a phage plasmid to construct a phage display library. Next, GDF15 antibodies were screened multiple times using phage display technology, and finally, the high-affinity antibodies obtained from the screening were expressed in large quantities in prokaryotic cells and purified. The binding activity of the obtained single-domain antibodies was verified by enzyme-linked immunosorbent assay (ELISA), and the inventors successfully screened for GDF15 single-domain antibodies with high affinity activity.

[0011] The present invention provides a heavy chain antibody that targets GDF15, which specifically binds to GDF15, inhibits the interaction between GDF15 and GFRAL, and can be used to treat diseases associated with GDF15 overexpression or to diagnose a patient's GDF15 level. The antibody provided by the present invention exhibits superior inhibitory activity against the binding of GDF15 to GFRAL compared to the positive control CTL002, superior specificity compared to the positive control PF-06946860, and superior hydrophilicity compared to the positive control PF-06946860.

[0012] To achieve the above objective, in a first aspect of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically identify GDF15. According to embodiments of the present invention, the antibody comprises the following amino acid sequence. (i) an amino acid sequence having the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in SEQ ID NOs: 1, 2, 4-6 (wherein CDR1, CDR2, and CDR3 are determined according to the IMGT, Kabat, Chothia, AbM, or Contact definitions); or (i) An amino acid sequence having one or more conservative amino acid substitutions.

[0013] In a second aspect of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically identify GDF15. According to an embodiment of the present invention, the antibody comprises at least one CDR sequence selected from the following or an amino acid sequence having at least 90% identity thereto. Heavy chain variable region CDR sequences: SEQ ID NOs: 7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21, 22, 23, and 24. YYAIG (Sequence ID 7) CISSNDGSTYYSDSVKG (Sequence ID 8) DLTPCPVNPSVDHY (Sequence ID 9) DYAIG (Sequence No. 10) CISSSDESTYYADSVKG (Sequence ID 11) DTECPVQVTSMA (Sequence ID 12) YYAIG (Sequence No. 16) CISSSDGSTYYSDSVKG (Sequence ID 17) GEYGSDCPVQVGS (Sequence ID 18) AYAVG (Sequence ID 19) CISSSDGSAYYADSVKG (Sequence ID 20) DLSCPVQIATFHS(Sequence ID 21) DLSIG (Sequence ID 22) CISSSDGSTYYADSVKG (Sequence ID 23) DREDCPVQPWGIVAGTS ​​(Sequence ID 24)

[0014] The antibody according to the embodiment of the present invention can specifically bind to GDF15 and suppress the activity of GDF15.

[0015] According to the embodiments of the present invention, the antibody may further include at least one of the following additional technical features.

[0016] According to embodiments of the present invention, the antibody includes the following: The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively.

[0017] According to an embodiment of the present invention, the antibody contains a heavy chain framework region sequence, and at least a part of the heavy chain framework region sequence is derived from at least one of an antibody derived from a camel, an antibody derived from an alpaca, an antibody derived from a mouse, an antibody derived from a primate, or a variant thereof.

[0018] According to an embodiment of the present invention, the heavy chain framework region sequence is derived from an antibody derived from an alpaca.

[0019] According to an embodiment of the present invention, the antibody comprises a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 4 to 6. QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGREGVSCISSNDGSTYYSDSVKGRFTTSRDNAKNTVYLQMNSLKPEDTAVYYCAADLTPCPVNPSVDHYWGQGTQVTVSS (SEQ ID NO: 1) QLQLVESGGGLVQPGGSLRLSCVASGFTLDDYAIGWFRQAPGKGLEGVSCISSSDESTYYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAIYYCAADTECPVQVTSMAWGQGTQVTVSS (SEQ ID NO: 2) QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGPEAVSCISSSDGSTYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGEYGSDCPVQVGSWGQGTQVTVSS (SEQ ID NO: 4) QVQLVESGGGLVQPGGSLRLACAASGFTLDAYAVGWFRQAPGKNPEGVSCISSSDGSAYYADSVKGRFTISRDNAKNTVYLQMNSLKPGDTAVYYCAADLSCPVQIATFHSRGQGTQVTVSS (SEQ ID NO: 5) QLQLVESGGGEVQPGGSLRLSCAASGATLDDLSIGWFRQAPGKGLEGVSCISSSDGSTYYADSVKGRFTISRDNAKNTAYLQMNSLRPEDTAVYYCAADREDCPVQPWGIVAGTSRGQGTQVTVSS (SEQ ID NO: 6)

[0020] According to an embodiment of the present invention, the antibody contains a heavy chain constant region, and at least a part of the heavy chain constant region is derived from at least one of a mouse-derived antibody, a primate-derived antibody or a variant thereof. Thereby, the in vivo half-life of the antibody is further extended, and the stability of the antibody is improved.

[0021] According to an embodiment of the present invention, the heavy chain constant region of the antibody is derived from a human IgG antibody or a variant thereof.

[0022] According to an embodiment of the present invention, the heavy chain constant region of the antibody is derived from human IgG1.

[0023] According to an embodiment of the present invention, the sequence of Fc in the heavy chain constant region of the antibody is as shown in SEQ ID NO: 25. Amino acid sequence of IgG1 Fc region: EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 25)

[0024] According to embodiments of the present invention, the antibody comprises a heavy chain (the full length of a heavy chain single-domain antibody) having the amino acid sequence shown in any one of SEQ ID NOs: 26 to 30. Full amino acid sequence of LN05: QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGREGVSCISSNDGSTYYSDSVKGRFTTSRDNAKNTVYLQMNSLKPEDTAVYYCAADLTPCPVNPSVDHYWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 26) Full amino acid sequence of LN06: QLQLVESGGGLVQPGGSLRLSCVASGFTLDDYAIGWFRQAPGKGLEGVSCISSSDESTYYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAIYYCAADTECPVQVTSMAWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 27) Full amino acid sequence of LN13: QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKGPEAVSCISSSDGSTYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGEYGSDCPVQVGSWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 28) Full amino acid sequence of LN14: QVQLVESGGGLVQPGGSLRLACAASGFTLDAYAVGWFRQAPGKNPEGVSCISSSDGSAYYADSVKGRFTISRDNAKNTVYLQMNSLKPGDTAVYYCAADLSCPVQIATFHSRGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 29) Full amino acid sequence of LN25: QLQLVESGGGEVQPGGSLRLSCAASGATLDDLSIGWFRQAPGKGLEGVSCISSSDGSTYYADSVKGRFTISRDNAKNTAYLQMNSLRPEDTAVYYCAADREDCPVQPWGIVAGTSRGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 30)

[0025] An antibody comprising a heavy chain having the amino acid sequence shown in any one of the above SEQ ID NOs: 26 to 30, according to an embodiment of the present invention, has high affinity activity for GDF15, an extended half-life in the body, and higher stability.

[0026] According to embodiments of the present invention, the antibody is a low-molecular-weight antibody.

[0027] According to embodiments of the present invention, the small molecule antibody comprises at least one of a single-domain antibody, a Fab antibody, an Fv antibody, and a minimum recognition unit.

[0028] According to embodiments of the present invention, the antibody is a single-domain antibody.

[0029] The present invention provides a heavy chain single-domain antibody that targets GDF15, which specifically binds to GDF15, inhibits the interaction between GDF15 and GFRAL, and can be used to treat diseases associated with GDF15 overexpression or to diagnose a patient's GDF15 level. The antibody provided by the present invention exhibits superior inhibitory activity against the binding of GDF15 to GFRAL compared to the positive control CTL002, superior specificity compared to the positive control PF-06946860, and superior hydrophilicity compared to the positive control PF-06946860.

[0030] In a third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in the first or second aspect.

[0031] In a fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect.

[0032] According to embodiments of the present invention, the expression vector is a prokaryotic expression vector, a eukaryotic expression vector, or a virus.

[0033] After introducing the expression vector according to the embodiment of the present invention into receptor cells, the aforementioned antibody is expressed under conditions suitable for protein expression to obtain an antibody with high GDF15 affinity activity.

[0034] In a fifth aspect of the present invention, the present invention provides recombinant cells. According to an embodiment of the present invention, the recombinant cells carry a nucleic acid molecule as described in the third aspect or an expression vector as described in the fourth aspect, or express an antibody or its antigen-binding fragment as described in the first or second aspect.

[0035] According to embodiments of the present invention, the recombinant cells are obtained by introducing the expression vector described in the fourth embodiment into host cells. According to embodiments of the present invention, the expression vector is introduced into the host cells by electroporation.

[0036] In a sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition contains the following: An antibody or its antigen-binding fragment according to the first or second embodiment; Nucleic acid molecule according to the third embodiment; The expression vector according to the fourth embodiment; or Recombinant cells according to the fifth embodiment.

[0037] The pharmaceutical compositions according to the embodiments of the present invention can specifically inhibit GDF15 and inhibit the binding of GDF15 to GFRAL, and can be used for the effective treatment or prevention of GDF15-related diseases.

[0038] In a seventh aspect of the present invention, the present invention provides the use of an antibody or antigen-binding fragment thereof as described in the first or second aspect, a nucleic acid molecule as described in the third aspect, an expression vector as described in the fourth aspect, recombinant cells as described in the fifth aspect, or a pharmaceutical composition as described in the sixth aspect, in the preparation of a pharmaceutical, said pharmaceutical used for the diagnosis, treatment, or prevention of GDF15-related disease.

[0039] According to embodiments of the present invention, the GDF15-related diseases include cardiovascular diseases, kidney diseases, obesity, diabetes, tumors, and cachexia.

[0040] According to embodiments of the present invention, the cardiovascular diseases include myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction.

[0041] According to embodiments of the present invention, the tumor is a solid tumor, and the solid tumor includes pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, and breast cancer.

[0042] In an eighth aspect of the present invention, the present invention provides a kit for detecting GDF15. According to an embodiment of the present invention, the kit comprises an antibody or an antigen-binding fragment thereof as described in the first or second aspect.

[0043] In a ninth aspect of the present invention, the present invention provides the use of an antibody or antigen-binding fragment thereof according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, or recombinant cells according to the fifth aspect in the preparation of a kit, the kit being used for the detection of GDF15 or the diagnosis of GDF15-related diseases.

[0044] In a tenth aspect of the present invention, the present invention provides a method for diagnosing, treating or preventing GDF15-related diseases. According to embodiments of the present invention, the method comprises administering to a subject at least one of the following: An antibody or its antigen-binding fragment according to the first or second embodiment; Nucleic acid molecule according to the third embodiment; An expression vector according to the fourth embodiment; Recombinant cells according to the fifth embodiment; The pharmaceutical composition according to the sixth embodiment.

[0045] According to embodiments of the present invention, the GDF15-related diseases include cardiovascular diseases, kidney diseases, obesity, diabetes, tumors, and cachexia.

[0046] According to embodiments of the present invention, the cardiovascular diseases include myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction.

[0047] According to embodiments of the present invention, the tumor is a solid tumor, and the solid tumor includes pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, and breast cancer.

[0048] In an eleventh aspect of the present invention, the present invention provides the use of an antibody or antigen-binding fragment thereof described in the first or second aspect, a nucleic acid molecule described in the third aspect, an expression vector described in the fourth aspect, a recombinant cell described in the fifth aspect, or a pharmaceutical composition described in the sixth aspect, in the diagnosis, treatment, or prevention of GDF15-related diseases.

[0049] According to embodiments of the present invention, the GDF15-related diseases include cardiovascular diseases, kidney diseases, obesity, diabetes, tumors, and cachexia.

[0050] According to embodiments of the present invention, the cardiovascular diseases include myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction.

[0051] According to embodiments of the present invention, the tumor is a solid tumor, and the solid tumor includes pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, and breast cancer.

[0052] Some further aspects and advantages of the present invention are described below, but other aspects will become clear from the following description or can be understood through the implementation of the present invention. [Brief explanation of the drawing]

[0053] The above and / or further aspects and advantages of the present invention will become clearer and easier to understand by describing the embodiments in combination with the following drawings. [Figure 1] The results of detecting the affinity of the GDF15 heavy chain single-domain antibody in Example 3 are shown. [Figure 2] The results of detecting the inhibitory activity at the protein level of the GDF15 heavy chain single-domain antibody in Example 4 are shown. [Figure 3] The results of detecting the inhibitory activity of the GDF15 heavy chain single-domain antibody at the cellular level in Example 5 are shown. [Figure 4] The results of detecting the specificity of the GDF15 heavy chain single-domain antibody in Example 6 are shown. [Modes for carrying out the invention]

[0054] The following describes in detail embodiments of the present invention. The embodiments shown below are illustrative and are used solely to illustrate the present invention and should not be understood as limiting the invention.

[0055] Furthermore, the terms “first” and “second” are used solely for descriptive purposes and should not be understood as indicating or implying relative importance, or implicitly specifying the number of technical features being described. Therefore, features designated as “first” or “second” may explicitly or implicitly include one or more features. Moreover, in the description of this invention, unless otherwise specified, “multiple” means two or more.

[0056] The endpoints of the ranges and any values ​​disclosed herein are not limited to those precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to them. For numerical ranges, one or more new numerical ranges can be obtained by combining the endpoint values ​​of each range, the endpoint values ​​of each range with individual values, and individual values ​​with each other, and these numerical ranges should be considered as specifically disclosed herein.

[0057] To facilitate understanding of the present invention, several technical and scientific terms are defined below. Unless otherwise explicitly defined elsewhere in this specification, other technical and scientific terms used herein have meanings that are generally understood by a person skilled in the art to which the present invention pertains.

[0058] In this specification, the terms “includes” or “contains” are open-ended expressions, meaning they include the contents of this invention but do not exclude the contents of other embodiments.

[0059] In this specification, the terms “optional,” “optional,” and “optional” generally mean that the events or circumstances described below may occur, but do not necessarily occur, and such descriptions include cases in which such events or circumstances occur and cases in which such events or circumstances do not occur.

[0060] term The term "antibody" is used in its broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments capable of binding to antigens (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, bi-antibodies, Fab), and recombinant peptides containing them, as long as they exhibit the desired biological activity. "Immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein consisting of two heavy chains and two light chains, each containing a variable region and a constant region. The antigen-binding region can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. The antibody fragment or antigen-binding region particularly includes Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR-transplant antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, bipolar antibody, triple antibody, quadruple antibody, mini-antibody, linear antibody; chelated recombinant antibody, triple-chain antibody (tribody) or double-chain antibody (bibody), intracellular antibody, nano-antibody, small modular immunotherapy (SMIP), antigen-binding domain immunoglobulin fusion protein, camelized antibody, VHH-containing antibody, or variants or derivatives thereof, as well as peptides containing at least a portion of an immunoglobulin (sufficient to give a specific antigen that binds to the peptide, e.g., 1, 2, 3, 4, 5, or 6 CDR sequences), provided that the antibody can retain the desired biological activity.

[0061] As used herein, “heavy chain variable region” refers to a region of an antibody molecule that includes at least one complementarity-determining region (CDR) of the heavy chain variable domain. The heavy chain variable region may include one, two, or three CDRs of the antibody heavy chain.

[0062] In this specification, the term "single-domain antibody" refers to heavy-chain antibodies found in camel / alpaca blood that naturally lack a light chain, and these antibodies contain only the heavy chain (H) with a relatively large molecular weight. Of these, the amino acid sequence at the amino terminus (N terminus) of the peptide chain changes significantly and is therefore called the variable region (V region), while the carboxyl terminus (C terminus) is relatively stable and changes very little, and is therefore called the constant region (C region). The V region of the H chain is called VH. A specific region within the variable region has a high degree of variation in amino acid composition and sequence order and is called the hypervariable region (HVR). The hypervariable region is the binding site between the antigen and the antibody, and is therefore also called the complementarity-determining region (CDR). The heavy-chain variable region has three CDR regions. CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure, so it can be inferred that single-domain antibodies have higher antigen-binding specificity and affinity than conventional antibodies.

[0063] The variable domain of a single-domain antibody exhibits the same general structure as a relatively conserved framework region (FR) linked by three hypervariable regions or CDRs. The heavy chain contains the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The amino acid assignment of each domain conforms to the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991), or Chothia and Lesk, J. Mol. Biol., 196:901-917, 1987; Chothia et al., Nature, 342:878-883, 1989).

[0064] The hypervariable region of an antibody refers to the CDR amino acid residues responsible for antigen binding. The hypervariable region includes amino acid residues derived from the CDR, such as 31-35(H1), 50-65(H2), and 95-102(H3) of the heavy chain variable domain (e.g., described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md (1991)); and / or amino acid residues derived from the hypervariable loop, such as 26-32(H1), 53-55(H2), and 96-101(H3) of the heavy chain variable domain (described in Chothia et al., J.Mol.Biol., 196:901-917 (1987)).

[0065] Monoclonal antibodies refer to antibodies obtained from a substantially homogeneous group of antibodies, where all antibodies in the mixture have a single amino acid sequence derived from a single clone. Monoclonal antibodies are usually highly specific, targeting a single antigenic site or epitope. Polyclonal antibody preparations, on the other hand, typically contain a mixture of antibodies with different amino acid sequences that target the same or different antigenic determinants (epitopes). In addition to their specificity, monoclonal antibodies also have the advantage of not being contaminated by other immunoglobulins with different specificities and characteristics because they are synthesized in a homogeneous culture.

[0066] As used herein, the term “hinge region” refers to the region between the CH1 and CH2 regions of the antibody heavy chain. This region contains inter-H chain disulfide bonds, is rich in proline, does not form an α-helix, and is prone to stretching and some degree of twisting, thus favoring complementary binding between the antibody’s antigen-binding site and the antigen epitope. In the heavy chain single-domain antibody of the present invention, the heavy chain variable region is linked to the Fc region via the hinge region.

[0067] As used herein, the term “Fc region” refers to a protein containing the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, and the protein does not contain the heavy chain variable region and heavy chain constant region 1 (CH1). In the present invention, the Fc fragment includes not only the native amino acid sequence but also its mutant sequence. The Fc region of an immunoglobulin may be derived from a human or animal, such as a cattle, goat, pig, mouse, rabbit, hamster, rat, or guinea pig.

[0068] As used herein, the term “constant region” refers to the carboxyl terminus (C-terminus) of a polypeptide chain, 3 / 4 or 4 / 5 of the H chain, and 1 / 2 of the L chain. This region is called the “constant region,” or C region, because the number, types, sequence order, arrangement, and glycan content of the amino acids contained therein are relatively stable. The C regions of the H and L chains are denoted by CH and CL, respectively. The length of CH varies depending on the class of immunoglobulin; IgG, IgA, and IgD have three CHs, including CH1, CH2, and CH3, while IgM and IgE have four CHs, including CH1, CH2, CH3, and CH4. Each heavy chain consists of one variable region (VH) and first, second, third, and fourth (optional) constant regions (which are CH1, CH2, CH3, and CH4, respectively). Naturally occurring complete antibodies typically exhibit a "Y" shape, with the stem of the "Y" structure consisting of second and third constant regions of two heavy chains linked by disulfide bonds. Each arm of the "Y" structure contains the VH and CH1 (VH-CH1) of the heavy chain and the light chain (VL-CL).

[0069] As used herein, the term “nucleic acid” is interchangeable with the term “polynucleotide” and refers to single-stranded or double-stranded deoxyribonucleotides or ribonucleotides, and polymers thereof, encompassing nucleic acids containing known nucleotide analogs or modified skeletal residues or bindings. Such nucleic acids are synthetic, native, and non-native nucleic acids, possessing similar binding properties to the reference nucleic acid and being metabolized in a similar manner to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including one or more nucleic acid molecules contained in a single vector or separate vectors, and one or more nucleic acid molecules present at one or more locations within a host cell. Unless otherwise noted, a particular nucleic acid sequence implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions) and complementary sequences, in addition to the explicitly stated sequence.

[0070] As used herein, the term “vector” refers to a transport tool capable of functionally inserting a genetic factor (e.g., the nucleic acid molecule described above) and expressing said genetic factor, for example, by generating a protein, RNA, or DNA encoded by the genetic factor, or by replicating said genetic factor. Vectors can be used for transformation, transduction, or transfection of host cells to cause them to express the genetic factor they contain within the host cell. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection sequences, and reporter genes. Vectors may also contain replication origins. Vectors may contain components that assist in cell entry, such as, but are not limited to, viral particles, liposomes, or protein shells. Vectors may be expression vectors or cloning vectors. In some embodiments, the vector provided by the present invention (e.g., an expression vector) comprises a nucleic acid sequence encoding a fusion protein described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0071] As used herein, the term “host cell” refers to a cell into which an exogenous polynucleotide and / or vector can be introduced, or a cell into which an exogenous polynucleotide and / or vector has already been introduced. The host cell contains the vector, and after introducing the vector into mammalian cells and constructing the host cells, these host cells can be used to express the antibodies or antigen-binding fragments provided by the present invention. Culturing the host cells yields the corresponding antibodies or fusion proteins. The mammalian cells that can be used may include CHO cells, for example.

[0072] As used herein, the term “composition” allows for the effective presence of the biological activity of the active ingredient and does not contain any other ingredients that are unacceptably toxic to the recipient of the composition.

[0073] As used herein, the term “treatment” means to partially or completely eliminate, reduce, suppress or improve, temporarily or permanently, the clinical symptoms, manifestations or progression of an event, disease, or medical condition.

[0074] As used herein, the term “diagnosis” refers to the definition of identifying, elucidating, confirming, and / or localizing a pathological condition, disease, or situation. In some embodiments, the pharmaceutical compositions of the present invention are useful in diagnosing cancer, tumorigenesis, or a situation when administered to a subject or when in contact with a sample from a subject.

[0075] In many embodiments, the terms “subject” and “patient” can be used interchangeably regardless of the form of treatment the subject has received or is currently receiving. As used herein, the terms “subject” or “patient” refer to a mammalian subject or patient. Unless otherwise noted, the terms “patient” or “subject” can be used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep. In some embodiments, the subject is human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or a human who is infected.

[0076] Single-domain antibody In this invention, alpacas were immunized using human GDF15 protein as an antigen to produce a highly specific and affinity anti-GDF15 single-domain antibody (Nanobody, Nb). Because this antibody can specifically bind to the GDF15 antigen, it can be used for targeted treatment or prevention of diseases such as cardiovascular disease, renal disease, obesity, diabetes, tumors, and cachexia.

[0077] In some embodiments, the present invention provides an antibody or antigen-binding fragment that can specifically identify GDF15, wherein the antibody comprises at least one CDR sequence selected from the following or an amino acid sequence having at least 95% identity thereto. Heavy chain variable region CDR sequences: SEQ ID NOs: 7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

[0078] In another embodiment, the antibody or antigen-binding fragment provided by the present invention has a conserved amino acid substitution compared to the heavy chain described above. “Antigen-binding fragment” refers to an antibody fragment that retains the ability to specifically bind to the antigen (GDF15). “Conserved amino acid substitution” refers to the substitution of an amino acid with a biologically, chemically, or structurally similar residue of another amino acid. Biological similarity means that the substitution does not interfere with the biological activity of the GDF15 antibody or its binding to the GDF15 antigen. Structural similarity means that the amino acids have side chains of similar length, e.g., alanine, glycine, or serine, or side chains of similar size. Chemical similarity means that the amino acids have the same charge, or are both hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine can be mutually substituted. Alternatively, polar amino acids can be mutually substituted; for example, lysine can be substituted with arginine, aspartic acid with glutamic acid, asparagine with glutamine, and threonine with serine.

[0079] In some embodiments, the present invention provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having the following characteristics: The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 16, 17, and 18, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 16, 17, and 18, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 19, 20, and 21, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 19, 20, and 21, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 22, 23, and 24, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 22, 23, and 24, respectively.

[0080] In some embodiments, the present invention provides an anti-GDF15 single-domain antibody having a heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 4-6, or a heavy chain of the amino acid sequence shown in any one of SEQ ID NOs: 26-30.

[0081] In this invention, the antibody against GDF15 is a variable domain of heavy chain (VHH), which is commonly referred to as a "nanobody." The inventors immunized alpacas with recombinant GDF15 protein, constructed and screened a phage library, and obtained a heavy chain single-domain antibody that binds to GDF15. The GDF15 heavy chain single-domain antibody of this invention can be obtained by constructing an expression vector using a GDF15 monoclonal antibody sequence, transiently introducing it into CHO cells, and purifying it by affinity chromatography using a protein A packing material.

[0082] The inventors evaluated the affinity of the obtained GDF15 monoclonal antibody protein using ELISA, assessed the inhibitory activity of the GDF15 heavy chain single-domain antibody against GDF15 using GDF-GFRAL ligand receptor ELISA binding experiments, evaluated the inhibitory activity of the GDF15 heavy chain single-domain antibody against GDF15 using reporter gene methods, detected the binding of the GDF15 heavy chain single-domain antibody to proteins belonging to the same family as GDF15 (GDNF, ARTN, NRTN) using ELISA, investigated whether the GDF15 heavy chain single-domain antibody binds to proteins belonging to the same family as GDF15, and detected the hydrophilicity of the GDF15 heavy chain single-domain antibody using hydrophobic interaction chromatography (HIC). As a result, the inventors demonstrated that the novel GDF15 heavy chain single-domain antibody provided by the present invention specifically binds to GDF15, inhibits the interaction between GDF15 and GFRAL, and can be used to treat diseases associated with GDF15 overexpression or to diagnose a patient's GDF15 level.

[0083] nucleic acid molecules, expression vectors, recombinant cells In the process of preparing or acquiring these antibodies, the nucleic acid molecules expressing these antibodies can be linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0084] For this purpose, the present invention also provides isolated nucleic acid molecules encoding the antibody or antigen-binding fragments described above.

[0085] The present invention also provides an expression vector containing the isolated nucleic acid molecule. When ligating the isolated polynucleotide into a vector, the polynucleotide may be directly or indirectly ligated to a control element on the vector, provided that these control elements can control the translation and expression of the polynucleotide. Of course, these control elements may be directly derived from the vector itself, or they may be exogenous, i.e., not derived from the vector itself. Of course, the polynucleotide and the control element only need to be operably ligated. In this specification, "operably ligated" means ligating the foreign gene to the vector so that the control elements in the vector, such as transcriptional control sequences and translational control sequences, can perform the expected function of controlling the transcription and translation of the foreign gene. Of course, polynucleotides encoding antibody heavy chains may be inserted independently into different vectors, but it is common for them to be inserted into the same vector. Commonly used vectors may be plasmids, phages, etc. For example, the pcDNA3.4 plasmid.

[0086] The present invention also provides recombinant cells, which include the expression vector. After introducing the expression vector into prokaryotic cells to construct recombinant cells, these recombinant cells can be used to express antibodies or antigen-binding fragments provided by the present invention. When these recombinant cells are cultured, the corresponding antibodies can be obtained.

[0087] Pharmaceutical compositions, kits, and pharmaceutical applications, as well as their use in the preparation of kits. The present invention also provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0088] The anti-GDF15 antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to subjects. Generally, these pharmaceutical compositions comprise the anti-GDF15 antibodies provided herein and a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may include any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial agent, antifungal agent, isotonic agent, and absorption retarder. Specific examples include water, physiological saline, phosphate-buffered saline, glucose, glycerin, ethanol, and one or more of these compositions. Often, the pharmaceutical composition contains sugars, polyols (e.g., mannitol, sorbitol), or isotonic agents such as sodium chloride. Of course, the pharmaceutically acceptable carrier may also contain small amounts of auxiliary substances, such as wetting agents, emulsifiers, preservatives, or buffers, to extend the shelf life or potency of the antibody.

[0089] For example, the antibodies of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquid, semi-solid, and solid, and include, but are not limited to, liquid solutions (e.g., injections and infusions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injections or infusions. The antibodies can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0090] Of course, the anti-GDF15 antibody described herein can also be prepared as part of a kit or other diagnostic reagent, if necessary. According to the embodiments of the present invention, the present invention also provides a kit, which comprises the above-mentioned GDF15 antibody. The kits provided by the present invention can be used for applications that utilize the specific binding properties of the GDF15 antigen and antibody for detection, such as immunoblotting and immunoprecipitation. These kits may include one or more of the following: an antagonist, an anti-GDF15 antibody, or a pharmaceutical reference material; a protein purification column; an immunoglobulin affinity purification buffer; a cell assay diluent; instructions or literature. The anti-GDF15 antibody can be used in various types of diagnostic tests, for example, to detect the presence of various diseases in vitro or in vivo, or the presence of pharmaceuticals, toxins, or other proteins. For example, related diseases can be detected by examining the serum or blood of a subject. Such related diseases may include GDF15-related diseases, such as cardiovascular disease, kidney disease, obesity, diabetes, tumors, cachexia, etc.

[0091] When treating the above-mentioned disease using the anti-GDF15 antibody provided by the present invention, the anti-GDF15 antibody provided by the present invention should be provided to the subject. For this purpose, the present invention provides a method for treating the above-mentioned disease, which includes administering the antibody or antigen-binding fragment provided by the present invention to a subject in need.

[0092] The technical proposals of this disclosure will be explained below by combining examples. Those skilled in the art will understand that the following examples are used solely to illustrate this disclosure and should not be considered to limit the scope of this disclosure. Where no specific technical or condition is specified in the examples, the technical or condition described in the relevant art literature or in the product description shall apply. Unless the manufacturer of the reagents or equipment used is specified, they are all commercially available conventional products.

[0093] Example 1: Screening of heavy chain single-domain antibodies targeting GDF15 1. Construction of a phage library Two alpacas were selected and immunized five times with human GDF15 protein (Novoprotein, product number: DRA26). After the fifth immunization, 50 mL of peripheral blood was collected from the alpacas, and lymphocytes were separated using lymphocyte isolation solution (manufacturer: GE, product number: 17-1440-03). Total RNA was extracted using RNA extraction reagent (RNAiso Plus), and the extracted RNA was reverse transcribed into cDNA using the PrimeScript II kit (TAKARA 6210A). Subsequently, the VHH target gene fragment, i.e., the variable region fragment of the heavy chain antibody, was amplified using nested PCR. The target gene VHH and vector pComb3xss were digested with SfiI, ligated with T4 DNA ligase, and then transformed into TG1 electroporation-competent cells to construct a VHH gene library. Simultaneously, 0.1 μL, 0.01 μL, 0.001 μL, and 0.0001 μL of the mixed transformation solution were collected and uniformly spread onto 90 mm culture dishes. The calculated library volume was 2.2 × 10⁶. 9 The result was CFU. After randomly selecting 100 colonies, amplifying them, and then sequencing, the insertion efficiency was 100%. From the above gene library, 10 to 100 times the library volume of live cells were collected, inoculated, and cultured. After culturing until the logarithmic growth phase, the cells were rescued using the M13K07 phage, rescue culture was performed, and the phages were recovered by centrifugation. The phages were purified with PEG-NaCl, and the titer was 2.0 × 10⁶. 13 A phage display library with cfu / mL was obtained. This was directly used for subsequent affinity screening of specific phages.

[0094] 2. Phage library screening Human GDF15 protein (Novoprotein, product number: DRA26) was coated onto plates at 100 μg / mL and 50 μg / mL concentrations and incubated overnight at 4°C. The following day, the plates were blocked with 1% OVA at 37°C for 1 hour. 100 μL of phage (2.0 × 10⁶) was then added. 13 After adding cfu / mL and incubating, the mixture was washed five times with PBST (PBS containing 0.05% Tween20) to remove unbound phages. Finally, phages specifically bound to human GDF15 protein were eluted with 100 μL of Gly-HCl (pH 2.2) and used to infect logarithmically growing E. coli TG1 to generate and purify phages for the next screening. After repeating the same screening process twice, the obtained phages were used to infect E. coli TG1, seeded onto plates, and single clones were taken from the plates and sequenced. Based on the sequence alignment results, the protein sequences of each clone were analyzed, and clones with different CDR1, CDR2, and CDR3 sequences were considered different antibody strains. Ultimately, a total of six different antibody strains were obtained, and the antibody sequences are shown in Table 1. TIFF2026511269000001.tif230153 TIFF2026511269000002.tif218153

[0095] Example 2: Preparation of a single-domain heavy chain antibody of GDF15 The variable region sequence of the GDF15 heavy chain single-domain antibody (see Table 1) was ligated to the human IgG1 Fc sequence to synthesize the expression plasmid pcDNA3.4. After transforming E. coli, the plasmid was extracted. The expression plasmid was filtered through a 0.22 μm filter, homogeneously mixed with CHO cells and electroporation buffer, and added to a 1 mL electroporation cuvette. After electroporation, the cells in the electroporation cuvette were dispensed into a shaking flask containing 20 mL of pre-prepared culture medium and incubated for 40 minutes. After incubation, the shaking flask was cultured at 37°C, 270 rpm, and 8% CO2. After 24 hours, supplement / sodium butyrate / double antibody (manufacturer: Gibco, product number: 15140-122) was added, and the cells were cultured for 3-6 days before the cell supernatant was collected. The cell supernatant was added to a protein A affinity column, the column was washed with 20 mL of PBS buffer, then 5 mL of sodium acetate buffer (pH 3.4) was added, and elution was performed on the column. The eluate was collected in a dialysis bag and dialyzed to PBS buffer to obtain the target product, a single heavy-chain antibody of GDF15. Table 2 below shows the Fc region of the GDF15 single heavy-chain antibody and the full-length sequence of each antibody. TIFF2026511269000003.tif209153 TIFF2026511269000004.tif223153

[0096] Example 3: Affinity detection of GDF15 heavy chain single-domain antibody Human GDF15 protein (Novoprotein, product number: DRA26) was diluted to obtain a 1 μg / mL coating buffer, which was added to an ELISA plate at 100 μL / well and coated at 2-8°C for at least 12 hours. The remaining coating buffer was discarded, 200 μL / well of 2% BSA-PBS was added, and the plates were blocked at 37°C for 2 hours. Then, 300 μL of PBST was added to each well, and the plates were washed three times. Test samples (LN05, LN06, LN11, LN13, LN14, LN25, PF-06946860, CTL-002) were diluted to 20 nM with PBS, and then further diluted 3-fold in eight steps. These samples were added to an ELISA plate at 100 μL / well. After incubation at 37°C for 1 hour, 300 μL of PBST was added to each well, and after washing three times, 100 μL / well of 10,000-fold diluted goat anti-human Fc-HRP (abcam, ab97225) was added. After incubation at 37°C for 1 hour, 300 μL of PBST was added to each well, and after washing three times, the wells were gently tapped and dried. 100 μL of TMB chromogenic solution was added to each well. After reacting at room temperature for 5 minutes, the reaction was stopped by adding 50 μL / well of 2 M H2SO4. The stopped ELISA plate was placed in a microplate reader, and the absorbance OD450 value at a wavelength of 450 nm was read. The data was analyzed using GraphPad Prism 9.0 software, and binding activity fitting was performed to obtain the EC50 value of GDF15 binding activity. The smaller the EC50 value, the stronger the binding activity. As shown in Table 3 and Figure 1, LN05, LN06, LN13, LN14, and LN25 showed significant binding activity to human GDF15, comparable to PF-06946860 (Pfizer) and CTL002 (CatalYM), while LN11 also showed significant binding activity to human GDF15, but weaker than PF-06946860 and CTL002. TIFF2026511269000005.tif76153

[0097] Example 4: Study on the inhibitory activity of a GDF15 heavy chain single-domain antibody at the protein level. GFRAL protein (KACTUS, GFL-HM401) was diluted to obtain a 1 μg / mL coating buffer, which was added to an ELISA plate at 100 μL / well and coated at 2-8°C for at least 12 hours. The remaining coating buffer was discarded, 200 μL / well of 2% BSA-PBS was added, and the plates were blocked at 37°C for 2 hours. Then, 300 μL of PBST was added to each well, and the plates were washed three times. Sample dilutions containing 20 ng / mL of GDF15-biotin protein (KACTUS, GDF-HM215B) were prepared, and the test samples (LN05, LN06, LN13, LN14, LN25, PF-06946860, CTL-002) were diluted to 20 nM. Further 3-fold dilutions were performed 12 times, and the dilutions were added to an ELISA plate at 100 μL / well. After incubation at 37°C for 1 hour, 300 μL of PBST was added to each well, and after washing three times, 100 μL / well of 30,000-fold diluted streptavidin (HRP) (abcam, ab7403) was added. After incubation at 37°C for 1 hour, 300 μL of PBST was added to each well, and after washing three times, the wells were gently tapped to dry. 100 μL of TMB chromogenic solution was added to each well. After reacting at room temperature for 5 minutes, the reaction was stopped by adding 50 μL / well of 2 M H2SO4. The stopped ELISA plate was placed in a microplate reader, the absorbance OD450 value at a wavelength of 450 nm was read, and the data was analyzed using GraphPad Prism 9.0 software. Inhibitory activity fitting was performed to obtain the IC50 value of ELISA inhibitory activity. The lower the IC50 value, the stronger the inhibitory activity. As shown in Table 4 and Figure 2, LN05, LN06, LN13, LN14, and LN25 exhibit significant dose-dependent inhibitory activity against the binding of human GDF15 to its receptor GFRAL, and their inhibitory activity is comparable to that of PF-06946860 and superior to that of CTL-002. TIFF2026511269000006.tif71153

[0098] Example 5: Study on the cellular-level inhibitory activity of a GDF15 heavy chain single-domain antibody. Place 1.3 × 10⁶ H-GDF15 reporter 293 cell suspension (Manufacturer: Genomeditech, Product No.: GM-C06718) into a 96-well microplate (BeyoGold, FCP968). 4 Cells were added at a concentration of 50 μL / well and incubated overnight in a CO2 incubator. The microplate was removed, and 50 μL of mixed medium containing GDF15 (final concentration 5 ng / mL, Novoprotein, DRA26) and serially diluted test samples (LN05, LN06, LN13, LN14, LN25, PF-06946860, CTL002) (starting working concentration: 5.13 nM, 3-fold dilution, a total of 9 concentrations) was added to each well. Culture was then maintained in a 37°C, 5% CO2 incubator. After 17 hours, 100 μL of luciferase reporter gene detection reagent (Vazyme DD1203) was added to each well, incubated for 15 minutes, and then the chemiluminescence RLU was read. The data was analyzed using GraphPad Prism 8.0 software, and inhibitory activity fitting was performed to obtain the IC50 value for GDF15 inhibitory activity (GDF15 heavy chain single-domain antibody inhibits the binding of GDF15 to GFRAL). The lower the IC50 value, the stronger the inhibitory activity. As shown in Table 5 and Figure 3, LN05, LN06, LN13, LN14, and LN25 exhibit significant dose-dependent inhibitory activity against reporter gene expression via GDF15, and their inhibitory activity is comparable to PF-06946860 and superior to CTL-002. TIFF2026511269000007.tif71153

[0099] Example 6: Study on the specificity of GDF heavy chain single-domain antibodies Human GDNF protein (Novoprotein, C226), ARTN protein (Novoprotein, CR34), and NRTN protein (Novoprotein, CE27) were diluted to obtain a 1 μg / mL coating buffer. This buffer was added to an ELISA plate at 100 μL / well and coated at 2-8°C for at least 12 hours. The remaining coating buffer was discarded, 200 μL / well of 2% BSA-PBS was added, and the plates were blocked at 37°C for 2 hours. Then, 300 μL of PBST was added to each well, and the plates were washed three times. Using PBS, the test samples (LN05, LN06, LN13, LN14, LN25, PF-06946860) were diluted to 2000 nM, and then further diluted 3-fold in 12 steps. These dilutions were added to an ELISA plate at 100 μL / well. After incubation at 37°C for 1 hour, 300 μL of PBST was added to each well, and after washing three times, 100 μL / well of 10,000-fold diluted goat anti-human Fc-HRP (abcam, ab97225) was added. After incubation at 37°C for 1 hour, 300 μL of PBST was added to each well, and after washing three times, the wells were gently tapped to dry. 100 μL of TMB chromogenic solution was added to each well. After reacting at room temperature for 5 minutes, the reaction was stopped by adding 50 μL / well of 2 M H2SO4. The stopped ELISA plate was placed in a microplate reader, and the absorbance OD450 value at a wavelength of 450 nm was read. The data was analyzed using GraphPad Prism 9.0 software, and binding activity fitting was performed to obtain the EC50 values ​​of GFRA1, GFRA2, and GFRA3 binding activity. A smaller EC50 value indicates stronger binding activity and weaker specificity. As shown in Table 6 and Figure 4, LN05, LN06, LN13, LN14, and LN25 exhibited binding activity with GDNF, NRTN, and ARTN only at high concentrations, while PF-06946860 exhibited dose-dependent binding activity with GDNF and ARTN. At the highest concentration, the binding activity of PF-06946860 with NRTN was higher than that of LN05, LN06, LN13, LN14, and LN25. This suggests that LN05, LN06, LN13, LN14, and LN25 have superior specificity to GDNF, NRTN, and ARTN compared to PF-06946860. TIFF2026511269000008.tif86153

[0100] Example 7: Study on the hydrophilicity of GDF heavy chain single-domain antibodies The test sample was diluted with mobile phase A (1.8M ammonium sulfate + 0.1M sodium dihydrogen phosphate solution, pH 6.5) to obtain a final test solution containing 1.0M ammonium sulfate. The mobile phases were mobile phase A (1.8M ammonium sulfate + 0.1M sodium dihydrogen phosphate solution, pH 6.5) and mobile phase B (0.1M sodium dihydrogen phosphate solution, pH 6.5). A chromatography column (manufacturer: Sepax Technologies, model: Protemix HIC Butyl-NP5 4.6*10mm, 5μm) was used with a wavelength of 214nm, flow rate of 1.0mL / min, injection volume of 5μg, column temperature of 25℃, and sample chamber temperature of 8℃. The gradient elution method was performed as follows: 0-5 min, 44%B; 5-15 min, 44%B → 100%B; 15-20 min, 100%B; 20.1-25 min, 44%B. The hydrophilicity of the target protein was determined by eluting and comparing the retention times of the target protein's peak. The shorter the peak retention time, the stronger the hydrophilicity, and the better the solubility of highly hydrophilic proteins. As shown in Table 7, the peak retention times for LN05, LN06, LN13, LN14, and LN25 were shorter than those for PF-06946860, suggesting that LN05, LN06, LN13, LN14, and LN25 have superior hydrophilicity compared to PF-06946860. TIFF2026511269000009.tif62153

[0101] In this specification, reference terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” “several embodiments,” or “several examples” mean that the specific features, structures, materials, or properties described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the general expressions of the above terms do not necessarily refer to the same embodiment or example. The specific features, structures, materials, or properties described may be combined as appropriate in one or more embodiments or examples. Furthermore, a person skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described herein, as long as they do not conflict with each other.

[0102] Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are illustrative and do not limit the present invention. Those skilled in the art can change, modify, substitute, and alter the above embodiments within the scope of the present invention.

Claims

1. (i) an amino acid sequence having the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in SEQ ID NOs: 1, 2, 4-6 (wherein CDR1, CDR2, and CDR3 are determined according to the IMGT definition, Kabat definition, Chothia definition, AbM definition, or Contact definition); or (i) an amino acid sequence having one or more conservative amino acid substitutions An antibody or its antigen-binding fragment that can specifically identify GDF15, including the above.

2. An antibody or its antigen-binding fragment that can specifically identify GDF15, comprising at least one CDR sequence selected from the following or an amino acid sequence having at least 90% identity thereto. CDR sequences of the heavy chain variable region: SEQ ID NOs: 7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

3. The aforementioned antibody The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 16, 17, and 18, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 16, 17, and 18, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 19, 20, and 21, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 19, 20, and 21, respectively; or The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in the amino acid sequences of SEQ ID NOs. 22, 23, and 24, respectively, or the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region having at least 90% identity with the amino acid sequences of SEQ ID NOs. 22, 23, and 24, respectively. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

4. The antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody contains a heavy chain framework region sequence, and at least a portion of the heavy chain framework region sequence is derived from at least one of a camel-derived antibody, an alpaca-derived antibody, a mouse-derived antibody, a primate-derived antibody, or a variant thereof.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain framework region sequence is derived from an alpaca-derived antibody.

6. The antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 4 to 6.

7. The antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody contains a heavy chain constant region, and at least a portion of the heavy chain constant region is derived from at least one of a mouse-derived antibody, a primate-derived antibody, or a variant thereof.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain constant region of the antibody is derived from a human IgG antibody or a variant thereof.

9. The antibody or its antigen-binding fragment according to claim 7 or 8, wherein the heavy chain constant region of the antibody is derived from human IgG1.

10. The antibody or its antigen-binding fragment according to any one of claims 7 to 9, wherein the Fc sequence in the heavy chain constant region of the antibody is as shown in SEQ ID NO:

25.

11. The antibody or its antigen-binding fragment according to any one of claims 1 to 10, wherein the antibody comprises a heavy chain having the amino acid sequence shown in any one of SEQ ID NOs. 26 to 30.

12. The antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody is a low molecular weight antibody.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the small molecule antibody comprises at least one of a single-domain antibody, a Fab antibody, an Fv antibody, and a minimum recognition unit.

14. The antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody is a single-domain antibody.

15. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14.

16. An expression vector for transporting nucleic acid molecules according to claim 15.

17. The expression vector according to claim 16, wherein the expression vector is a prokaryotic expression vector, a eukaryotic expression vector, or a virus.

18. Recombinant cells that carry a nucleic acid molecule according to claim 15, or an expression vector according to claim 16 or 17, or express an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

19. Recombinant cells according to claim 18, obtained by introducing the expression vector according to claim 16 or 17 into host cells.

20. A pharmaceutical composition containing at least one of the following: an antibody or antigen-binding fragment according to any one of claims 1 to 14, a nucleic acid molecule according to claim 15, an expression vector according to claim 16 or 17, or a recombinant cell according to claim 18 or 19.

21. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, a nucleic acid molecule according to claim 15, an expression vector according to claim 16 or 17, a recombinant cell according to claim 18 or 19, or a pharmaceutical composition according to claim 20 in the preparation of a pharmaceutical, wherein the pharmaceutical is used for the diagnosis, treatment or prevention of a GDF15-related disease.

22. The aforementioned GDF15-related diseases include cardiovascular disease, renal disease, obesity, diabetes, tumors, and cachexia. Optionally, the cardiovascular disease includes myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction. The use according to claim 21, optionally wherein the tumor is a solid tumor, and the solid tumor includes pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, and breast cancer.

23. A kit for detecting GDF15, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14.

24. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, a nucleic acid molecule according to claim 15, an expression vector according to claim 16 or 17, or a recombinant cell according to claim 18 or 19 in the preparation of a kit, wherein the kit is used for the detection of GDF15 or for the diagnosis of GDF15-related disease.

25. A method for diagnosing, treating, or preventing a GDF15-related disease, comprising administering to a subject at least one of the following: an antibody or antigen-binding fragment according to any one of claims 1 to 14, a nucleic acid molecule according to claim 15, an expression vector according to claim 16 or 17, recombinant cells according to claim 18 or 19, and a pharmaceutical composition according to claim 20.

26. The aforementioned GDF15-related diseases include cardiovascular disease, renal disease, obesity, diabetes, tumors, and cachexia. Optionally, the cardiovascular disease includes myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction. The method according to claim 25, optionally wherein the tumor is a solid tumor, and the solid tumor includes pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, and breast cancer.

27. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, a nucleic acid molecule according to claim 15, an expression vector according to claim 16 or 17, a recombinant cell according to claim 18 or 19, or a pharmaceutical composition according to claim 20 in the diagnosis, treatment, or prevention of GDF15-related disease.

28. The aforementioned GDF15-related diseases include cardiovascular disease, renal disease, obesity, diabetes, tumors, and cachexia. Optionally, the cardiovascular disease includes myocardial hypertrophy, heart failure, atherosclerosis, and endothelial dysfunction. The use according to claim 27, optionally wherein the tumor is a solid tumor, and the solid tumor includes pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, primary liver cancer, lung cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, urothelial carcinoma / renal cell carcinoma, and breast cancer.

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