WO2025223473A1 - Anti-gdf-15 antibody and use thereof - Google Patents

Abstract

Provided are an anti-GDF-15 antibody and the use thereof. The anti-GDF-15 antibody contains a heavy chain variable region. The heavy chain variable region contains CDR1: GFTX1X2X3X4X5; CDR2: IX6X7X8X9X10X11X12; and CDR3: X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30X31X32X33X34. The provided antibody has a good affinity and cross-reactivity for GDF-15, effectively blocks the GDF-15 pathway, and can significantly improve cachexia in a subject. The provided bispecific antibody has a good affinity for both GDF-15 and IL-6, significantly improves cachexia in tumor-bearing mice, and alleviates an inflammatory response.

Description

Anti-GDF-15 antibody and its application

This application claims priority to Chinese Patent Application No. 2024104978483, filed on April 23, 2024, and Chinese Patent Application No. 2025104881035, filed on April 17, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference.

Technical Field

This invention belongs to the field of biomedicine, specifically relating to an anti-GDF-15 antibody and its application.

Background Technology

Growth differentiation factor 15 (GDF-15) is a pluripotent cytokine with a molecular weight of about 25 kDa. It has the common structural features of TGFβ family proteins, including an N-terminal signal peptide, a propeptide containing a glycosylation site, a protease hydrolysis site, and a C-terminal domain containing multiple cysteine residues. Various cell types, such as cardiomyocytes, adipocytes, macrophages, endothelial cells, and vascular smooth muscle cells, can express GDF-15. After translation, the GDF-15 precursor forms a homodimer through disulfide bonds, and after cleavage, it is secreted into the extracellular space to form a biologically active soluble protein ^[1] .

In normal human tissues and organs, constitutive expression of GDF-15 is mainly found in the placenta, followed by the prostate. However, it is expressed at low levels in other organs and tissues, such as the bladder, kidney, colon, stomach, liver, gallbladder, pancreas, and endometrium. The expression of GDF-15 is affected by factors such as cellular metabolic stress and disease status. Under conditions of tissue damage and chronic inflammation, the expression level of GDF-15 will be upregulated, such as in malignant tumors, viral infections, cardiovascular diseases, and chronic kidney disease. Furthermore, the level of GDF-15 can be used as a prognostic factor for diseases. The level of GDF-15 in tumor patients treated with chemotherapy and radiotherapy will also increase, and the level is related to tumor metastasis and poor prognosis of patients ^[2] .

Multiple studies have shown a negative correlation between plasma GDF-15 levels and activity levels and muscle strength, while high levels of GDF-15 are commonly seen in patients with cancer cachexia, including those with gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer ^[3,4] . Cachexia is a metabolic syndrome, clinically characterized by weight loss, decreased appetite or anorexia, and may be accompanied by anemia, fatigue, chronic inflammation, insulin resistance, and accelerated protein catabolism. Up to one-third of deaths in patients with advanced cancer are related to cachexia, and the deterioration of physical condition caused by cachexia reduces the tolerance of patients with advanced cancer to radiotherapy and chemotherapy, affecting the efficacy of treatment. In addition to malignant tumors, chronic kidney disease, heart failure, and AIDS can also lead to cachexia.

GFRAL is the only known GDF-15 receptor, which is mainly expressed in the brainstem and hypothalamus of the central nervous system. After GDF-15 binds to GFRAL, it recruits RET to form a complex, which further activates the downstream AKT, Erk1/2, and PLCγ signaling pathways. GDF-15 secreted by tumor tissue can regulate food intake, energy metabolism and body weight by acting on GFRAL/RET receptors. Overactivation of this pathway is an important cause of tumor cachexia ^[2,5] . At present, there are still no safe and effective drugs for the treatment of cachexia in clinical practice. The main drugs used to promote appetite and improve body weight are glucocorticoids and progestins, which only improve symptoms such as inflammation and fatigue in patients within a few weeks. Long-term use does not have a significant benefit on body weight or patient survival ^[6] . Therefore, developing antagonists targeting the GDF-15/GFRAL/RET signaling pathway is a potential technical approach for the prevention and treatment of cachexia. To date, monoclonal antibodies targeting GDF-15, such as PF-06946860 (Ponsegromab) and CTL002 (Visugromab), as well as monoclonal antibody targeting GFRAL, such as NGM120, have entered clinical development and have shown preliminary efficacy in cancer cachexia and anti-tumor therapy ^[7,8,9] .

Cancer cachexia is associated with a variety of inflammatory cytokines, such as IL-1, IL-6, TNFα, IFNγ and GDF-15, as well as various hormones such as parathyroid hormone PTHrP ^[10] . These complex factors can act directly or indirectly on the central nervous system, muscle and adipose tissue, and regulate appetite, synthesis and catabolism. Therefore, the efficacy of antagonizing a single cytokine for cachexia may be limited. Developing bispecific or multispecific antibodies against two or more inflammatory cytokines to reduce the inflammatory response while improving cachexia symptoms may be a potentially more effective treatment strategy.

IL-6 is a pluripotent cytokine that plays a role in various physiological processes such as acute immune response, tissue regeneration, and lipid metabolism. The serum concentrations of IL-6 and GDF-15 in tumor patients with reduced weight are significantly higher than those in tumor patients with normal weight, and there is a positive correlation between the concentrations of IL-6 and GDF-15 ^[4] . Non-clinical studies have shown that IL-6 secreted by tumor cells causes nude mice to exhibit a cachexia-like weight loss phenotype, while the IL-6 antibody CNTO 328 can effectively prevent weight loss in mice bearing melanoma and prostate cancer cells ^[11] . In the clinical study of the IL-6 antibody ALD518, the 12-week lean body mass of non-small cell lung cancer patients in the ALD518 treatment group was also higher than that in the placebo control group, and there was no significant decrease compared with before treatment ^[12] . The above preclinical and clinical study data suggest that IL-6 antibodies have the effect of preventing and improving tumor cachexia.

Summary of the Invention

The technical problem this invention aims to solve is the lack of effective antibody drugs for treating tumor cachexia in current technology. This invention provides an anti-GDF-15 antibody and its applications, specifically an anti-GDF-15 antibody and a bispecific antibody against GDF-15 and IL-6. The antibody of this invention exhibits good affinity and cross-reactivity with GDF-15, effectively blocking the GDF-15 pathway and significantly improving cachexia in subjects. The bispecific antibody of this invention exhibits good affinity for both GDF-15 and IL-6, significantly improving cachexia in tumor-bearing mice.

The present invention solves the above-mentioned technical problems through the following technical solutions.

A first aspect of the present invention provides an anti-GDF-15 antibody, the anti-GDF-15 antibody comprising a heavy chain variable region, the heavy chain variable region comprising:

CDR1: GFTX ₁ X ₂ X ₃ X ₄ X ₅ ; where X ₁ is L or F, X ₂ is D or S, X ₃ is G, Y or S, X ₄ is Y or F, and X ₅ is W, A or D;

CDR2: IX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ ; where X ₆ is S or N, X ₇ is T, S or N, X ₈ is G or S, X ₉ is S, D or G, X ₁₀ is S, D or G, X ₁₁ is S or N, and X ₁₂ is S or T; and

CDR3: X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ X ₃₂ X ₃₃ X ₃₄ ; where X ₁₃ is C, A, or G; X ₁₄ is A or R; X ₁₅ is A, D, or S; X ₁₆ is D, L, or T; X ₁₇ is P, S, Q, or T; X ₁₈ is S, C, T, or D; X ₁₉ is A, P, S, or F; X ₂₀ is V, W, or H; X ₂₁ is P, Q, G, or I; X ₂₂ is G, P, I, or R; X ₂₃ is P, M, or T; X ₂₄ is S, A, N, or V; X _X25 is F, P, D, or Q; _X26 is Q, Y, N, or S; _X27 is Y, D, M, or none; _X28 is R, Y, W, or none; _X29 is Y, D, G, or none; _X30 is Y or none; _X31 is H or none; _X32 is F or none; _X33 is D or none; _X34 is Y or none.

In some implementations, the heavy chain variable region comprises:

CDR1: GFTX ₁ X ₂ X ₃ X ₄ X ₅ ; where X ₁ is L or F, X ₂ is D or S, X ₃ is Y or S, X ₄ is Y or F, and X ₅ is A or D;

CDR2: IX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ T; where X ₆ is S or N, X ₇ is T or S, X ₈ is G or S, X ₉ is D or G, X ₁₀ is G, and X ₁₁ is S or N; and

CDR3: X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ YX ₃₁ X ₃₂ X ₃₃ X ₃₄ ; where X ₁₃ is A or G, X ₁₄ is A or R, X ₁₅ is A or S, X ₁₆ is L or T, X ₁₇ is P or T, X ₁₈ is T or D, X ₁₉ is S or F, X ₂₀ is W or H, X ₂₁ is G or I, X ₂₂ is I or R, X ₂₃ is P or T, X ₂₄ is N or V, X ₂₅ is P or Q, X ₂₆ is N or S, X ₂₇ is D or M, X ₂₈ is Y or W, X ₂₉ is D or G, X ₃₁ is H or none, X ₃₂ is F or none, X ₃₃ is D or none, X ₃₄ is Y or none.

In other embodiments, the heavy chain variable region includes:

CDR1: GFTX ₁ X ₂ X ₃ X ₄ X ₅ ; where X ₁ is L, X ₂ is D, X ₃ is Y, X ₄ is Y, and X ₅ is A;

CDR2: IX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ ; where X ₆ is S, X ₇ is S or N, X ₈ is S, X ₉ is D or G, X ₁₀ is S or D, X ₁₁ is S, and X ₁₂ is T; and

CDR3: X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ X ₃₂ X ₃₃ X ₃₄ ; where X ₁₃ is A, X ₁₄ is A, X ₁₅ is D, X ₁₆ is L, X ₁₇ is S or Q, X ₁₈ is C, X ₁₉ is P, X ₂₀ is V, X ₂₁ is Q, X ₂₂ is P, X ₂₃ is M or T, X ₂₄ is S or A, X ₂₅ is P or D, X ₂₆ is Y, X ₂₇ is none, X ₂₈ is none, X ₂₉ is none, X ₃₀ is none, X ₃₁ is none, X ₃₂ is none, X ₃₃ is non-existent, X ₃₄ is non-existent.

In some embodiments, the anti-GDF-15 antibody includes a heavy chain variable region comprising CDR1 with an amino acid sequence as shown in SEQ ID NO:10, CDR2 with an amino acid sequence as shown in SEQ ID NO:11, and CDR3 with an amino acid sequence as shown in SEQ ID NO:12.

In other embodiments, the heavy chain variable region comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:18, CDR2 with an amino acid sequence as shown in SEQ ID NO:19, and CDR3 with an amino acid sequence as shown in SEQ ID NO:20.

In other embodiments, the heavy chain variable region comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:2, CDR2 with an amino acid sequence as shown in SEQ ID NO:3, and CDR3 with an amino acid sequence as shown in SEQ ID NO:4.

In other embodiments, the heavy chain variable region comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:6, CDR2 with an amino acid sequence as shown in SEQ ID NO:7, and CDR3 with an amino acid sequence as shown in SEQ ID NO:8.

In other embodiments, the heavy chain variable region comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:6, CDR2 with an amino acid sequence as shown in SEQ ID NO:15, and CDR3 with an amino acid sequence as shown in SEQ ID NO:16.

In this invention, CDR1, CDR2 and CDR3 are defined using IMGT.

In some embodiments, the heavy chain variable region comprises amino acids 1-125 of any of SEQ ID NO:9 and SEQ ID NO:33-35, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with amino acids 1-125 of any of SEQ ID NO:9 and SEQ ID NO:33-35.

In other embodiments, the heavy chain variable region comprises amino acids 1-129 of any of SEQ ID NO:17 or SEQ ID NO:39-43, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with amino acids 1-129 of any of SEQ ID NO:17 or SEQ ID NO:39-43.

In other embodiments, the heavy chain variable region comprises amino acids 1-124 of any of SEQ ID NO:1 or SEQ ID NO:27-29, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with amino acids 1-124 of any of SEQ ID NO:1 or SEQ ID NO:27-29.

In other embodiments, the heavy chain variable region comprises amino acids 1-121 of any of SEQ ID NO:5 or SEQ ID NO:30-32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with amino acids 1-121 of any of SEQ ID NO:5 or SEQ ID NO:30-32.

In other embodiments, the heavy chain variable region comprises amino acids 1-121 of any of SEQ ID NO:13 or SEQ ID NO:36-38, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with amino acids 1-121 of any of SEQ ID NO:13 or SEQ ID NO:36-38.

In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO:9, 74-76, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:9, 74-76.

In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO:17, 80-84, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:17, 80-84.

In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO:1, 68-70, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:1, 68-70.

In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO:5, 71-73, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:5, 71-73.

In other embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO:13, 77-79, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:13, 77-79.

In some implementations, the anti-GDF-15 antibody also includes a constant region.

In some implementations, the constant region is the Fc region.

In some implementations, the Fc region is selected from the Fc region of human IgG; for example, the Fc region of human IgG1.

In some embodiments, the Fc region comprises an amino acid sequence as shown in any of SEQ ID NO:21, 44-45 and 85 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:21, 44-45 and 85.

In some embodiments, the anti-GDF-15 antibody has an amino acid sequence as shown in any of SEQ ID NO:22-43 and 86 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:22-43 and 86.

In some implementations, the GDF-15 is human GDF-15 and/or monkey GDF-15.

A second aspect of the present invention provides a bispecific recombinant protein comprising a first binding domain for binding GDF-15 and a second binding domain for binding IL-6.

In some implementations, the first binding domain includes a heavy-chain variable region.

In some implementations, the first binding domain includes a heavy chain variable region and/or a light chain variable region.

In some implementations, the heavy chain variable region is as described in the first aspect.

In some implementations, the second binding domain includes a heavy chain variable region and a light chain variable region.

In some embodiments, the second binding domain includes a heavy chain variable region and a light chain variable region of ALD518 or MEDI-5117.

A third aspect of the present invention provides a bispecific recombinant protein comprising a first binding domain for binding GDF-15 and a second binding domain for binding IL-6; the first binding domain comprising a heavy chain variable region and a light chain variable region, and the second binding domain comprising a heavy chain variable region and a light chain variable region.

In some implementations, the first binding domain includes a heavy chain variable region and a light chain variable region of Ponsegromab or Visugromab.

In some embodiments, the second binding domain includes a heavy chain variable region and a light chain variable region of ALD518 or MEDI-5117.

In some implementations, the second binding domain is an antigen-binding fragment that binds to IL-6.

In some embodiments, the antigen-binding fragment is selected from Fab, Fab'-SH, Fv, or (Fab') ₂ ; the Fv is, for example, scFv.

In some implementations, the antigen-binding fragment is Fab.

In some embodiments, the antigen-binding fragment is Fab, and the antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains HCDR1 with the amino acid sequence shown in SEQ ID NO:14, HCDR2 with the amino acid sequence shown in SEQ ID NO:58, and HCDR3 with the amino acid sequence shown in SEQ ID NO:59. The light chain variable region contains LCDR1 with the amino acid sequence shown in SEQ ID NO:60, LCDR2 with the amino acid sequence shown in KAS, and LCDR3 with the amino acid sequence shown in SEQ ID NO:61.

In other embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:62, HCDR2 as shown in SEQ ID NO:63, and HCDR3 as shown in SEQ ID NO:64; the light chain variable region comprises LCDR1 as shown in SEQ ID NO:65, LCDR2 as shown in RAS, and LCDR3 as shown in SEQ ID NO:66.

In this invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined using IMGT.

In some embodiments, the heavy chain variable region of the antigen-binding fragment comprises an amino acid sequence as shown in amino acids 1-120 of SEQ ID NO:49 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it, and the light chain variable region of the antigen-binding fragment comprises an amino acid sequence as shown in amino acids 1-106 of SEQ ID NO:50 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it.

In other embodiments, the heavy chain variable region of the antigen-binding fragment comprises an amino acid sequence as shown in amino acids 1-120 of SEQ ID NO:51 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it, and the light chain variable region of the antigen-binding fragment comprises an amino acid sequence as shown in amino acids 1-110 of SEQ ID NO:52 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it.

In some embodiments, the bispecific recombinant protein further includes an Fc region.

In this invention, the Fc region is selected from the Fc region of human IgG; for example, the Fc region of human IgG1.

In some embodiments, the Fc region comprises an amino acid sequence as shown in any of SEQ ID NO:21, 44-45 and 85 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with any of SEQ ID NO:21, 44-45 and 85.

In some implementations, the first binding domain is connected to the N-terminus of the Fc region, and the second binding domain is connected to the C-terminus of the Fc region or the N-terminus of the first binding domain.

In other embodiments, the second bonding domain is connected to the N-terminus of the Fc region, and the first bonding domain is connected to the N-terminus of the second bonding domain; the connection is a direct connection or a connection via a linker.

In some implementations, the connector is (G4S)n, where n is any integer from 1 to 6.

In some embodiments, the linker has an amino acid sequence as shown in any of SEQ ID NO:46-48.

In some embodiments, the bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2; the polypeptide chain 2, from the N-terminus to the C-terminus, consists of a light chain variable region of a second binding domain and a light chain constant region.

The polypeptide chain 1, from the N-terminus to the C-terminus, is as follows:

First binding domain - second binding domain heavy chain variable region - heavy chain constant region 1 (CH1) - Fc region;

Alternatively, the heavy chain variable region of the second binding domain - the heavy chain constant region 1 (CH1) - the first binding domain - the Fc region;

Alternatively, the first binding domain - Fc region - heavy chain variable region of the second binding domain - heavy chain constant region 1 (CH1).

In some implementations, the light chain constant region is a kappa chain.

In some implementations, the heavy chain variable region of the second binding domain is connected to the Fc region or the first binding domain via a connector.

The connector is as described above.

In some embodiments, when the heavy chain variable region of the second binding domain is linked to the Fc region or the first binding domain via a linker, the polypeptide chain 1 from the N-terminus to the C-terminus is:

First binding domain - Linker - second binding domain heavy chain variable region - heavy chain constant region 1 (CH1) - Fc region;

Alternatively, the heavy chain variable region of the second binding domain - heavy chain constant region 1 (CH1) - Linker - first binding domain - Fc region;

Alternatively, the first binding domain - Fc region - Linker - heavy chain variable region of the second binding domain - heavy chain constant region 1 (CH1).

In some embodiments, the polypeptide chain 2 comprises an amino acid sequence as shown in SEQ ID NO: 50 or 52 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it.

In some embodiments, the heavy chain variable region-heavy chain constant region 1 (CH1) of the second binding domain in the polypeptide chain 1 comprises an amino acid sequence as shown in SEQ ID NO:49 or 51 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it.

In some embodiments, the polypeptide chain 1 comprises an amino acid sequence as shown in any of SEQ ID NO:53-57 or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity with it.

A fourth aspect of the invention provides a polynucleotide encoding an anti-GDF-15 antibody as described in the first aspect, or a bispecific recombinant protein as described in the second or third aspect.

A fifth aspect of the present invention provides a recombinant expression vector comprising the polynucleotides described in the fourth aspect.

A sixth aspect of the present invention provides a recombinant cell comprising a polynucleotide as described in the fourth aspect or a recombinant expression vector as described in the fifth aspect, or expressing an anti-GDF-15 antibody as described in the first aspect or a bispecific recombinant protein as described in the second or third aspect.

A seventh aspect of the present invention provides a method for preparing an anti-GDF-15 antibody or a bispecific recombinant protein, the method comprising culturing recombinant cells as described in the sixth aspect and obtaining the anti-GDF-15 antibody or the bispecific recombinant protein from the culture.

An eighth aspect of the present invention provides a pharmaceutical composition comprising an anti-GDF-15 antibody as described in the first aspect, a bispecific recombinant protein as described in the second or third aspect, a polynucleotide as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, and/or a recombinant cell as described in the sixth aspect.

And pharmaceutically acceptable carriers and/or excipients.

The ninth aspect of the present invention provides the use of an anti-GDF-15 antibody as described in the first aspect, a bispecific recombinant protein as described in the second or third aspect, a polynucleotide as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, a recombinant cell as described in the sixth aspect, or a pharmaceutical composition as described in the eighth aspect in the preparation of a reagent for detecting GDF-15 or a medicament for preventing and/or treating diseases and/or symptoms caused by GDF-15 signaling pathway dysregulation.

In some implementations, diseases and/or conditions resulting from dysregulation of the GDF-15 signaling pathway include: cachexia, weight loss due to anorexia, chronic inflammation, malignancy, viral infection, cardiovascular disease, liver fibrosis, neurodegenerative diseases, COVID-19, and chronic kidney disease.

In some implementations, the malignant tumor includes gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer; the viral infection includes HIV infection; and the cardiovascular disease includes heart failure.

The tenth aspect of the present invention provides a method for preventing and/or treating diseases and/or symptoms caused by GDF-15 signaling pathway dysregulation, the method comprising administering to a subject in need an effective amount of an anti-GDF-15 antibody as described in the first aspect, a bispecific recombinant protein as described in the second or third aspect, a polynucleotide as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, a recombinant cell as described in the sixth aspect, or a pharmaceutical composition as described in the eighth aspect.

In some implementations, diseases and/or conditions resulting from dysregulation of the GDF-15 signaling pathway include: cachexia, weight loss due to anorexia, chronic inflammation, malignancy, viral infection, cardiovascular disease, liver fibrosis, neurodegenerative diseases, COVID-19, and chronic kidney disease.

In some implementations, the malignant tumor includes gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer; the viral infection includes HIV infection; and the cardiovascular disease includes heart failure.

The eleventh aspect of the present invention provides the use of an anti-GDF-15 antibody as described in the first aspect, a bispecific recombinant protein as described in the second or third aspect, a polynucleotide as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, a recombinant cell as described in the sixth aspect, or a pharmaceutical composition as described in the eighth aspect for the prevention and/or treatment of diseases and/or symptoms caused by GDF-15 signaling pathway dysregulation.

Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

The reagents and raw materials used in this invention are all commercially available.

The positive and progressive effects of this invention are as follows:

The antibody of the present invention has good affinity and cross-reactivity with GDF-15 and has an extended half-life; the antibody of the present invention effectively blocks the GDF-15 pathway and can significantly improve cachexia in subjects; after forming a bispecific recombinant protein with cytokines, it still has good affinity for GDF-15 and cytokines, significantly improves cachexia in tumor-bearing mice, and alleviates inflammatory response.

Attached Figure Description

Figure 1A and Figure 1B: SEC-HPLC purity test results of VHH-Fc recombinant antibody.

Figure 2: Blocking activity of recombinant GDF-15 antibody against GDF-15-GFRAL ligand receptor.

Figure 3: Blocking activity of E12 and G8 VHH-Fc antibodies against GDF-15-GFRAL ligand receptor.

Figure 4: Effects of B6, B3, E12, and A5 VHH-Fc antibodies on body weight in HT-1080 tumor-bearing mice.

Figure 5: Effect of G8 VHH-Fc antibody on body weight of HT-1080 tumor-bearing mice.

Figure 6: Effects of G8 VHH-Fc antibody on body weight, subcutaneous fat weight, and gastrocnemius muscle weight in LNCAP tumor-bearing mice.

Figure 7: Binding activity of humanized E12 VHH-Fc antibody to human GDF-15 protein.

Figure 8: Binding activity of humanized G8 VHH-Fc antibody to human GDF-15 protein.

Figure 9: Inhibition of downstream GFRAL/RET signaling by humanized E12 VHH-Fc antibody.

Figure 10: Inhibition of downstream GFRAL/RET signaling by humanized G8 VHH-Fc antibody.

Figure 11: Effect of humanized E12 VHH-Fc antibody on body weight of HT-1080 tumor-bearing mice.

Figure 12: The activity of bispecific antibodies simultaneously binding to GDF-15 and IL-6.

Figure 13: Effect of bispecific antibody GF-001 on body weight of TOV21g tumor-bearing mice.

Figure 14: Effect of bispecific antibody GF-001 on net body weight of TOV21g mice.

Figure 15: Effects of bispecific antibodies GF-002 to GF-005 on body weight of TOV21g tumor-bearing mice.

Figure 16: Effects of bispecific antibodies GF-002 to GF-005 on net body weight of TOV21g cachectic mice.

Figure 17: Effects of bispecific antibodies GF-002 to GF-005 on subcutaneous fat weight in TOV21g mice.

Figure 18: Changes in C-reactive protein concentration in mouse serum after administration of bispecific antibodies.

Figure 19: Inhibition of downstream GFRAL/RET signaling by humanized E12 VHH-Fc antibody.

Figure 20: Binding activity of humanized E12 VHH-Fc antibody to human GDF-15 protein.

In each figure, some antibody names can be used interchangeably. For example, E12 VHH-Fc can be used interchangeably with E12-Fc, A5 VHH-Fc can be used interchangeably with A5-Fc, B6 VHH-Fc can be used interchangeably with B6-Fc, B3 VHH-Fc can be used interchangeably with B3-Fc, and G8 VHH-Fc can be used interchangeably with G8-Fc.

Detailed Implementation

definition

As used herein, the terms "antibody" or "immunoglobulin" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains. The amino acid composition and sequence of the constant regions of the immunoglobulin heavy chains differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

In this article, "antibody" can be derived from any animal, including but not limited to humans and non-human animals. Non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

In this article, "heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen binding specificity. The terms "complementarity-determining region" and "CDR" are used interchangeably in this article, typically referring to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form precise complementarity with the antigen epitope in its spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR.

As used herein, a "complementarity-determining region" or "CDR region" or "CDR" is a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and/or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. Within a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of many well-known antibody CDR assignment systems or combinations thereof. It is well known to those skilled in the art that antibody CDRs can be defined using various methods, such as Chothia based on the antibody's three-dimensional structure and the topology of the CDR loop; Kabat, AbM, and the international ImMunoGeneTics database (IMGT) based on antibody sequence variability; and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

As used herein, an "antigen-binding fragment" does not possess the complete structure of a full antibody, but only contains a portion or a variant of the full antibody, which has the ability to bind antigens. Exemplarily, "antigen-binding fragments" herein include, but are not limited to, Fab, F(ab') ₂ , Fab', Fab'-SH, Fd, Fv, scFv, diabody, and single-domain antibody. The terms "single-domain antibody (sdAb),""VHHdomain," and "nanobody" herein have the same meaning and are used interchangeably. They refer to the cloning of the variable region of a heavy chain antibody to construct a single-domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Typically, a naturally occurring heavy chain antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region. The instructions require that nanobodies can be used to form other forms of antibodies, such as antibodies containing VH-CH2-CH3 or VH-CH1-CH2-CH3 from the N-terminus to the C-terminus; they can also form homodimers, such as heavy chain dimer antibodies that do not have a light chain.

In this article, "identity" or "sequence identity" is used interchangeably, referring to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

As used herein, the term "antigen-binding fragment" or "Fab" comprises a variable region (VL) of the light chain, a constant region (CL) of the light chain, a variable region (VH) of the heavy chain, and a constant region 1 (CH1) domain of the heavy chain, which can bind to an antigen. When referring to the direct connection or linkage between the variable and constant regions of the antigen-binding fragment via a linker sequence, the constant region refers to either the constant region (CL) of the light chain or the constant region 1 (CH1) of the heavy chain.

As used herein, the term "Fab'" comprises a portion of a light chain and a heavy chain containing the VH and CH1 domains, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab' segments to form the F(ab') ₂ molecule. "F(ab') ₂ " comprises two light chains and two heavy chains containing the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab') ₂ segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

As used in this article, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

In this invention, the scFv (single chain antibody fragment) can be a conventional single chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids. The VL and VH domains enable linker pairing to form a monovalent molecule as a single polypeptide chain. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating _G4S amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence ( _G4S ) ₄ (SEQ ID NO:67) or ( _G4S ) ₃ (SEQ ID NO:47) can be used, but variants thereof may also be used.

As used herein, the term “Fc” (fragment crystallizable) consists of the constant CH2 and CH3 domains and hinge region of an immunoglobulin such as IgG.

As used herein, the term "recombinant protein" refers to an artificially designed/constructed protein, rather than a naturally occurring protein. The "recombinant" in "recombinant protein" of this invention does not represent its production method; it is used only to indicate that the "recombinant protein" does not exist naturally. The recombinant protein of this invention can be an expressed protein or an assembled protein.

As used herein, the term “linker” refers to an amino acid sequence that connects different functional binding segments (such as a first binding domain and a second binding domain, a first binding domain or a second binding domain and Fc), or connects different domains within the same functional binding segment.

As used herein, the term "recombinant cell" includes "host cell" used to prepare a transformant, and typically comprises a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, containing the polynucleotides disclosed in this application, or expressing a protein heterodimer (e.g., a heterodimeric protein) of this application. The host cell may include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (morphologically or in terms of total genomic DNA complementarity). The host cell may include cells transfected in vitro with the vectors disclosed in this application. The host cell may be a bacterial cell (e.g., *Escherichia coli*), yeast cell, or other eukaryotic cell, such as HEK293 cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, or myeloma cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a CHO cell.

As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and/or between host cells. This term may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for the replication of DNA or RNA, and expression vectors for the transcription and/or translation of DNA or RNA. It also includes vectors that provide more than one of the aforementioned functions. An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide.

As used herein, the terms "treatment" and "therapeutic method" are used interchangeably. The term "treatment" includes controlling the progression of a disease, symptom, condition, and associated symptoms, preferably reducing the impact of the disease, symptom, condition, or alleviating one or more symptoms of the disease, symptom, condition. This term includes curing the disease or completely eliminating the symptoms. This term includes symptom relief. This term also includes, but is not limited to, non-curative palliative treatment. The term "treatment" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein or fusion protein of the present invention to prevent or delay, reduce or alleviate the progression of a disease, symptom, condition, or the impact of one or more symptoms of the disease, symptom, condition.

As used herein, the term "administration" refers to the delivery of a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein or fusion protein of the present invention to a subject. Administration can be systemic or local. Administration can be performed using an administration device, such as a syringe. Methods of administration include, but are not limited to, implantation, nasal inhalation, spraying, and injection. Routes of administration include inhalation, intranasal administration, oral administration, intravenous administration, subcutaneous administration, or intramuscular administration.

Example

Example 1: Construction of an anti-human GDF-15 single-domain antibody library

Two four-year-old alpacas were selected and immunized initially with recombinant human GDF-15-His protein (Bepsys, catalog number: GD5-H5149) containing Freund's complete adjuvant. Booster immunizations were performed every three weeks alternately with either human GDF-15-His protein (Bepsys, catalog number: GD5-C5148) containing Freund's incomplete adjuvant. 50 mL of peripheral blood was collected from the alpacas after the third and fourth immunizations to obtain serum and peripheral blood mononuclear cells. After measuring the antibody titer in the serum using ELISA, RNA was extracted from peripheral blood mononuclear cells using RNAiso Plus reagent, and the RNA was reverse transcribed into cDNA using a PrimeScript II kit. The VHH gene fragment was amplified using PCR primers. The target gene and the vector pCAN were digested with SfiI enzyme, ligated with T4 DNA ligase, and then electroporated into TG1 competent cells to construct a VHH library.

The cryopreserved glycerol-containing bacteria of the library were inoculated into 250 mL of culture medium. When the OD ₆₀₀ value reached 0.6, helper phage M13KO7 (phage:bacteria ratio of 1:500) was added. After incubation, the culture was centrifuged and purified using PEG solution (20% PEG6000, 2.5 M NaCl) to obtain the phage display library, which was used for subsequent screening of specific binding phages.

In centrifuge tubes blocked with MPBS (Milk in 1×PBS) for 1 h, the above-mentioned phages were added. After removing non-specifically bound phages with magnetic beads, biotinylated human GDF-15 protein was added and incubated for 1 h. Phages bound to GDF-15 were then separated using magnetic beads. After centrifugation, the magnetic beads were collected and washed five times with PBST (PBS containing 0.05% Tween 20) to remove unbound phages. Eluting was performed with trypsin and 500 nM GFRAL solution (ACRO Biosystems) to obtain GDF-15-bound phages, which were then transformed into TG1 cells.

Example 2: Phage Library Screening

During the initial screening stage of VHH supernatant, the binding activity of VHH to human and cynomolgus monkey GDF-15 protein and its activity in blocking the interaction between human GDF-15-GFRAL ligand receptor were determined by ELISA.

GDF-15 binding assay: Add 50 μL of 1 μg/mL streptavidin to each well of a 96-well plate and incubate overnight at 4°C. After washing, block with 2% BSA PBS at 37°C for 1 h. Wash three times with PBST buffer, then add 50 μL of 1 μg/mL biotinylated human GDF-15 or cynomolgus monkey GDF-15 and incubate at 37°C for 1 h, followed by three washes with PBST buffer. Then add 50 μL of IPTG-induced VHH supernatant and incubate for 1 h, followed by three washes with PBST. Add 50 μL of anti-c-myc-HRP antibody (Sigma) to each well and incubate for 0.5 h, followed by nine washes with PBST buffer. After drying, add 100 μL of TMB chromogenic solution to each well. Incubate at room temperature for 5 minutes, then stop the reaction with 1M HCl solution (50 μL/well). Read the OD ₄₅₀ absorbance using a microplate reader at 450 nm.

Ligand receptor blocking assay: Add 50 μL of 1 μg/mL human GFRAL-hFc (ACRO Biosystems) to each well of a 96-well plate and incubate overnight at 4°C. After washing, block with 2% BSA PBS at 37°C for 1 h, and wash three times with PBST buffer. Mix IPTG-induced VHH supernatant with biotinylated human GDF-15 and pre-incubate for 0.5 h. Add 50 μL of this mixture to each well of a 96-well plate and incubate for 1 h. Wash three times with PBST, then add 50 μL of SA HRP antibody (Sino Biological, 1:5000 dilution) to each well and incubate for 0.5 h. Wash nine times with PBST buffer. After drying the 96-well plate, add 100 μL of TMB chromogenic solution to each well. Incubate at room temperature for 5 minutes, then stop the reaction with 1M HCl solution (50 μL/well). Read the OD ₄₅₀ absorbance using a microplate reader at 450 nm.

A total of 8 96-well plates were used for initial screening, from which 70 VHH clones exhibiting human and cynomolgus monkey GDF-15 binding activity and GDF-15-GFRAL blocking activity were selected for secondary screening. ELISA was used to verify that the VHH clones did not specifically bind to human TGFβ1, GDF8, or BMP3, and the dissociation rate of the VHH supernatant was measured using the Octet method. The Octet assay used a streptavidin biosensor loaded with biotinylated human GDF-15, with a five-fold dilution of the VHH supernatant as the analyte. Selected single clones were sequenced, and clones with different CDR1/2/3 sequences were considered as different antibody clones.

Example 3: Preparation of GDF-15 recombinant antibody

The 70 VHH clones were sorted according to their dissociation rates as determined by Octet. The top 40 VHH clones with the lowest dissociation rates were selected, and their DNA sequences were synthesized and inserted into the pcDNA3.4-hIgG1 Fc expression plasmid to construct the full-length VHH-Fc. The expression plasmid was amplified and extracted by *E. coli*, and then transiently transfected into Expi293 cells using PEI transfection reagent for recombinant expression. After 5-7 days of culture, the cell culture medium was collected by centrifugation, added to a Protein A affinity chromatography column for capture, and then eluted with 1M glycine (pH 3) to obtain the VHH-Fc recombinant antibody. The antibody was dialyzed against pH 7.4 PBS buffer. The monomer purity of the obtained antibody was >95% as determined by SEC-HPLC, as shown in Figures 1A and 1B. The antibody sequences are listed in Tables 1 and 1A.

Table 1. Amino acid sequence of the variable region of GDF-15 nanobody (Numbering Scheme IMGT)

Table 1A. Amino acid sequence of GDF-15VHH-Fc recombinant antibody

Example 4: In vitro activity of GDF-15 recombinant antibody

The in vitro activity of the above-mentioned GDF-15 recombinant antibody was evaluated using ELISA and reporter gene assays.

The blocking activity of the above-mentioned VHH-Fc recombinant antibody against the GDF-15-GFRAL ligand receptor was tested using ELISA. 96-well plates were coated with 1 μg/mL GFRAL-hFc protein overnight at 4°C. After washing, PBST buffer containing 1% BSA was added, and the plates were blocked at 37°C for 1 h. After washing three times with PBST buffer, the antibody was prepared as a 500 nM initial solution, diluted 10-fold in seven gradients, and 50 μL was added to each well. Simultaneously, 50 μL of 2 nM human GDF-15 solution (ACRO Biosystems) was added to each well, and the plates were incubated together at 37°C for 1 h. After washing three times with PBST buffer, 5000-fold diluted anti-His-HRP (Proteintech) was added, and the plates were incubated at 37°C for 0.5 h, 100 μL/well. Then, 100 μL of TMB chromogenic solution was added to each well, and the reaction was stopped by adding 50 μL of 1M HCl solution to each well after reacting at room temperature for 5 minutes. The absorbance OD ₄₅₀ value was read using an ELISA reader, and the data was analyzed using GraphPad Prism 8.0.1 software. Curve fitting was performed and the IC ₅₀ value was calculated. The results are shown in Figures 2 and 3. The B6, B3, A5 and E12 VHH-Fc recombinant antibodies can effectively block the interaction between GDF-15 and GFRAL, while the G8 VHH-Fc recombinant antibody does not block the interaction between GDF-15 and GFRAL ligand receptor.

The effect of the VHH-Fc recombinant antibody on downstream signal transduction of GFRAL/RET was detected using a reporter gene assay. 293T-SRE-Luc2-RET-GFRAL cells (Kangyuan Bochuang Biotechnology) were seeded at 2.0 × ^10⁴ cells/well in 96-well plates and cultured at 37℃ _CO₂ for 24 h. The antibody was diluted with 10% FBSDMEM medium, with eight 2-fold serial dilutions from 5 nM, each containing a final concentration of 0.02 μg/mL human GDF-15. 20 μL of the mixture was added to each well, and the plates were cultured for another 16 h. After equilibration at room temperature for 15 minutes, 100 μL of Bright-Lite reagent was added to each well, and the plates were shaken for 10 minutes. The fluorescence value was measured using the chemiluminescent reaction of luciferase binding to the substrate. Data were analyzed using GraphPad Prism 8.0.1 software, curve fitting was performed, and the _IC₅₀ value for antibody blocking GDF-15 was calculated.

The binding activity, GDF-15 binding specificity, and species cross-reactivity with cynomolgus monkey GDF-15 of the five antibodies that effectively block downstream GFRAL signal transduction during the VHH supernatant screening stage (Example 2) are shown in Table 2. The blocking activity against downstream GFRAL/RET signal transduction in reporter gene assays is shown in Table 3. The B6, B3, A5, and E12 VHH-Fc recombinant antibodies all effectively inhibited downstream GFRAL/RET receptor signal transduction. Although the G8VHH-Fc recombinant antibody did not directly block the interaction between GDF-15 and GFRAL, it still effectively inhibited downstream receptor signal transduction.

Table 2. Binding activity and specificity of VHH clone with human GDF-15, and species cross-reactivity with cynomolgus monkey GDF-15.

Table 3. Reporter gene activity (5 VHH-Fc cells)

Example 5: Effect of GDF-15 recombinant antibody on body weight of HT-1080 tumor-bearing mice

After HT-1080 human fibrosarcoma cells were inoculated into immunodeficient mice to form tumors, the tumor tissue continuously secreted human GDF-15 into the mouse circulatory system. Human GDF-15 can bind to mouse GFRAL, activate the GFRAL/RET signaling pathway, and cause rapid weight loss in mice. This model was used to evaluate the effect of the above-mentioned recombinant GDF-15 antibody on tumor cachexia. HT-1080 cells (2 × ^10⁶ cells/mouse) were subcutaneously injected into the back of 10-12 week old female CB17/SCID mice (Vitallix). When the mice lost about 5% of their body weight on day 14 post-inoculation, serum was collected and the human GDF-15 content was detected by ELISA. Mice were randomly divided into groups of 5 according to body weight and administered GDF-15 antibody or a vehicle control via intraperitoneal injection (ip) once a week. Tumor volume and body weight changes were recorded twice a week, and graphs were generated using GraphPad Prism 8.0.1 and statistical analysis was performed.

The results are shown in Figure 4. Fourteen days after HT-1080 cell inoculation, mice were administered Vehicle or recombinant antibody via intraperitoneal injection. The body weight of mice in each antibody-treated group increased rapidly within two days of administration. At the experimental endpoint, the body weight of the 10 mg/kg B3, E12, and A5 treatment groups increased by 20.17%, 11.06%, and 9.45% respectively compared to pre-inoculation levels. At the same dose, the PF-06946860 control group showed a 13.79% increase in body weight, while the Vehicle control group showed a 9.88% decrease in body weight compared to pre-inoculation levels. At a 5 mg/kg dose, the E12 and A5 treatment groups showed increases in body weight of 16.3% and 12.45% respectively. This experiment demonstrates that GDF-15 antibodies B3, E12, and A5 can effectively improve the GDF-15-induced weight loss in HT-1080 tumor-bearing mice.

The effect of G8 treatment on the body weight of HT-1080 tumor-bearing mice is shown in Figure 5. The antibody does not block the interaction between GDF-15 and GFRAL, but the G8 treatment group, administered at a dose of 5 mg/kg, can still rapidly and effectively reverse the trend of weight loss. At the end of the experiment, the body weight of the mice in this group increased by 17.48%, while the body weight of the mice in the Vehicle control group decreased by 5.93% compared with that before tumor inoculation.

In the LNCAP tumor cachexia model, the G8 treatment group effectively reversed the trend of weight loss in mice, and its effect on weight gain in subcutaneous fat and gastrocnemius muscle was also superior to that of the control antibody PF-06946860 (Figure 6).

Example 6: Humanization of GDF-15 recombinant antibody

To reduce the immunogenicity of alpaca-derived antibodies in humans, the camel-derived GDF-15 single-domain antibody was humanized using a CDR transplantation method. First, based on sequence homology, the 10 germline sequences with the highest homology to the camel-derived VHH were searched on the IMGT website, and CDR regions were transplanted. The VH frames used were IGHV3-NL1*01/JH4, IGHV3-23*01/IGHJ5*02, and IGHV3-23*04/IGHJ4*01. The corresponding CDR regions were replaced with the CDR regions of the selected sequences. Through post-translational modification sites and structural analysis, individual amino acids in the FR region were reverse-mutated using camel-derived amino acids. At least three humanization schemes were developed for each sequence. The antibody preparation process was the same as above. The antibody sequences are listed in Tables 4 and 4A. The humanized antibody sequence was synthesized, plasmid constructed, expressed, and purified according to Example 3. The binding activity of the humanized antibody to GDF-15 protein and the ligand receptor blocking activity were detected by ELISA, and the affinity of the humanized antibody to GDF-15 protein was determined by SPR.

The plate was coated overnight at 4°C with 1 μg/mL human GDF-15 protein. After washing, PBST buffer containing 1% BSA was added, and the plate was blocked at 37°C for 1 h. The plate was then washed three times with PBST buffer. The antibody was prepared as an initial 200 nM solution, diluted 10-fold in seven gradients, and 100 μL was added to each well. The plate was incubated at 37°C for 1 h, washed three times with PBST buffer, and then 100 μL/well of a 5000-fold diluted anti-Human Fc-HRP (Sigma) solution was added, and the plate was incubated at 37°C for 0.5 h. 100 μL of TMB chromogenic buffer was added to each well, and the reaction was stopped at room temperature for 5 minutes. 50 μL of 1M HCl solution was added to terminate the reaction. The absorbance ( _OD450) at 450 nm was read using a microplate reader. Data were analyzed using GraphPad Prism 8.0.1 software, and a concentration- _OD450 curve was fitted and the _EC50 was calculated. The binding activities of humanized E12 and G8 antibodies to GDF-15 protein are shown in Figures 7, 20, and 8, respectively. Compared with their camel-derived parent antibody or control antibody PF-06946860, humanized E12 and G8 antibodies have similar strong binding activities to GDF-15.

The binding constant, dissociation constant, and affinity between the humanized antibody and GDF-15 protein were further determined using the surface plasmon resonance (SPR) method. Antibody (6 μg/mL concentration, buffer: 10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4) was captured using a Protein A chip, and multiple concentrations of human GDF-15 protein were used as analytes for detection. The binding time was 120 s, and the dissociation time was 300 s. The binding data between the antibody and the analyte GDF-15 protein were calculated using Data Analysis 10.0 software. The ka, kd, and _KD of the E12, G8 camel-derived parental antibodies and the humanized antibody are shown in Tables 5 and 6.

Table 4. Amino acid sequence of GDF-15-Fc antibody

Table 4A. Amino acid sequence of the variable region of GDF-15 nanobody (Numbering Scheme IMGT)

Table 5. SPR results of antibody E12-Fc and its humanized sequence

Table 6. SPR results of cloned G8 and its humanized sequences

Example 7: Effects of GDF-15 humanized antibody on downstream signal transduction of receptor

The cellular activity of the humanized antibodies was tested using the reporter gene assay method described in Example 4, and the results are shown in Figures 9, 10, and 19. In the reporter gene system, the _{IC50 values} of the E12 humanized antibodies Hu02 and Hu03 for inhibiting downstream GFRAL/RET signaling were 0.341 nM, 0.349 nM, and 0.8468 nM, respectively, comparable to their parental antibodies. The G8 humanized antibody effectively blocked downstream GFRAL/RET signal transduction, and the difference in _IC50 between the G8 humanized antibody and its parental antibody was less than three-fold.

Example 8: Efficacy of GDF-15 humanized antibody in HT-1080 fibrosarcoma-bearing mouse cachexia model

The in vivo efficacy of the humanized GDF-15 antibody was evaluated using HT-1080 tumor-bearing mice, and its effect on body weight was observed. Ten- to twelve-week-old female CB17/SCID mice (Vitol) were subcutaneously inoculated with HT-1080 cells (2 × ^10⁶ cells/mouse) on their backs. Around day 14 post-inoculation, when the mice had lost approximately 5% of their body weight, serum was collected and human GDF-15 concentration was measured using ELISA. Mice were randomly assigned to groups of five, receiving either the antibody or a control via intraperitoneal injection once weekly. Tumor volume and body weight changes were recorded twice weekly, and graphs were generated using GraphPad Prism 8.0.1 for statistical analysis.

The experimental results are shown in Figure 11. On day 14, after drug administration, the body weight of mice in the Vehicle control group showed a decreasing trend, while the decreasing trend in the E12-Fc antibody and humanized E12-Fc antibody treatment groups was rapidly reversed. At the experimental endpoint, the body weight of mice in the 5 mg/kg E12-Fc, E12-Hu02-Fc, and E12-Hu03-Fc treatment groups increased by 16.74%, 5.42%, and 15.48% respectively compared to before vaccination (Day 0), while the body weight of mice in the Vehicle group decreased by 3.26%. The control group treated with PF-06946860 showed an increase of 17.43%.

Example 9: Preparation of anti-GDF-15 and IL-6 bispecific antibodies

The above-mentioned humanized single-domain antibody sequence against GDF-15 was ligated with anti-IL-6Fab (WO 2008/065378; WO 2008/144757) and IgG1 Fc in different forms using the (G4S)n linker (Table 7). Each fragment was synthesized and spliced into the pcDNA3.4 expression plasmid. Antibodies were generated using transient transfection in Expi293 or CHO cells. The transfection plasmid and PEI were added to Expi293 medium at a ratio of 1:4, resulting in a cell concentration of (3.0 × 10⁶). 1/10 volume of protein-free feed was added 24 h after transfection. After 5-7 days of culture, the supernatant was harvested and purified. Subsequent affinity purification was performed using protein A, followed by elution with glycine at pH 3.0. The protein was collected and neutralized with Tris-HCl at pH 8.0. If the purity is less than 90%, further purification is performed using gel size exclusion chromatography to achieve a purity of over 96%. Once the purity meets the standard, the protein is transferred to PBS buffer for subsequent use. The bispecific antibody is then expressed and purified according to the method in Example 3.

Table 7. Amino acid sequences of IgG1 Fc and anti-IL-6 Fab

Table 8. Structure of Bispecific Antibodies

Example 10: Binding activity of bispecific antibody with human GDF-15 and IL-6

After obtaining a high-purity bispecific antibody according to the production method in Example 9, the binding of the bispecific antibody to the two antigens was simultaneously detected by an experiment.

Human IL-6 protein was added to a high-adsorption 96-well plate and incubated overnight at 4°C. After washing, the plate was blocked at 37°C for 1 hour with PBST buffer containing 1% BSA. After washing the plate three times with PBST buffer, the antibody was prepared as an initial 200 nM solution and diluted 10-fold in seven gradients. 100 μL of each solution was added to each well and incubated at 37°C for 1 hour. The plate was washed three times with PBST buffer. 2 nM GDF-15-His solution was added to each well and incubated at 37°C for 1 hour. After washing three times with PBST buffer, 100 μL/well of 5000-fold diluted anti-His-HRP (Proteintech) was added and incubated at 37°C for 0.5 hours. 100 μL of TMB chromogenic solution was added to each well. After reacting for 5 minutes at room temperature, 1 M HCl was added to stop the reaction, 50 μL/well. The absorbance OD ₄₅₀ value was read using a microplate reader, and the data was analyzed using GraphPad Prism 8.0.1 software to fit the binding activity and obtain the GDF-15 binding activity EC ₅₀ value.

The experimental results are shown in Figure 12. The bispecific antibodies GF-001 to GF-005 can bind to GDF-15 and IL-6 simultaneously, and their _EC50 values are all <1 nM.

Example 11: Efficacy of bispecific antibodies in the TOV21g cachexia model

Ovarian cancer cells TOV21g can simultaneously secrete GDF-15 and IL-6. When inoculated into immunodeficient mice, they act on receptors, inducing cachexia phenotypes such as weight loss, accelerated catabolism of muscle and adipose tissue, and reduced activity. ^Five × 10⁶ TOV21g cells were mixed with matrix gel and inoculated into the backs of CB17/SCID mice (Vitallix). Approximately two weeks after inoculation, when the mice had lost about 8%–10% of their body weight, they were randomly assigned to different groups for drug administration. Bispecific antibodies or controls were administered via intraperitoneal injection twice weekly, and mouse body weight and tumor volume were measured. Blood samples were collected 24 hours after the third and fourth administrations, and serum was separated. CRP levels in mouse serum were measured according to the instructions in the CRP ELISA kit (QuantiCyto, catalog number EMC028). At the end of the study, the tumor-free body weight, subcutaneous fat tumor weight, and gastrocnemius muscle weight of the mice were measured.

The in vivo efficacy results of the bispecific antibody GF-001 are shown in Figures 13 and 14. When mice lost 10% of their body weight, they were administered the drug in groups. During the study period, the body weight of the animals in the Vehicle group decreased by 15% to 20% compared with that before vaccination. The antibody GF-001 could effectively reverse the trend of body weight loss at all doses, increasing the body weight of mice by 6.36%, 7.67%, and 5.96% at doses of 6, 12, and 24 mg/kg, respectively.

The in vivo efficacy of bispecific antibodies GF-002–GF-005 in the TOV21g model is shown in Figures 15–18. The body weight of each antibody treatment group increased rapidly within 3 days after the first administration. The net body weight of the animals at the study endpoint showed the same trend, and compared with the Vehicle group, each antibody treatment group effectively increased the net body weight of mice by more than 35% (Figure 16). Compared with the Vehicle group, the subcutaneous fat weight of mice in each antibody treatment group was significantly increased. The bispecific antibodies were significantly more effective than the control antibody PF-06946860 in increasing subcutaneous fat weight. The subcutaneous fat weight of the GF-002–GF-005 groups increased by 52.84%, 46.20%, 28.62%, and 29.58% respectively compared with PF-06946860 (Figure 17). After treatment with bispecific antibodies GF-002–GF-004, the serum CRP level of mice decreased significantly (Figure 18).

While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

References

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8.Ignacio Melero Bermejo et al.,Initial results from the phase 2A trial of visugromab(CTL-002)+nivolumab in advanced/metastatic anti-PD1/-L1 relapsed/refractory solid tumors(The GDFATHER-TRIAL).JCO 41,2501-2501(2023).

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Claims (21)

An anti-GDF-15 antibody, characterized in that the anti-GDF-15 antibody comprises a heavy chain variable region, the heavy chain variable region comprising: CDR1: GFTX ₁ X ₂ X ₃ X ₄ X ₅ ; where X ₁ is L or F, X ₂ is D or S, X ₃ is G, Y or S, X ₄ is Y or F, and X ₅ is W, A or D; CDR2: IX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ ; where X ₆ is S or N, X ₇ is T, S or N, X ₈ is G or S, X ₉ is S, D or G, X ₁₀ is S, D or G, X ₁₁ is S or N, and X ₁₂ is S or T; and CDR3: X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ X ₃₂ X ₃₃ X ₃₄ ; where X ₁₃ is C, A, or G; X ₁₄ is A or R; X ₁₅ is A, D, or S; X ₁₆ is D, L, or T; X ₁₇ is P, S, Q, or T; X ₁₈ is S, C, T, or D; X ₁₉ is A, P, S, or F; X ₂₀ is V, W, or H; X ₂₁ is P, Q, G, or I; X ₂₂ is G, P, I, or R; X ₂₃ is P, M, or T; X ₂₄ is S, A, N, or V; X _X25 is F, P, D or Q; _X26 is Q, Y, N or S; _X27 is Y, D, M or none; _X28 is R, Y, W or none; _X29 is Y, D, G or none; _X30 is Y or none; _X31 is H or none; _X32 is F or none; _X33 is D or none; _X34 is Y or none. Preferably, the heavy chain variable region comprises: CDR1: GFTX ₁ X ₂ X ₃ X ₄ X ₅ ; where X ₁ is L or F, X ₂ is D or S, X ₃ is Y or S, X ₄ is Y or F, and X ₅ is A or D; CDR2: IX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ T; where X ₆ is S or N, X ₇ is T or S, X ₈ is G or S, X ₉ is D or G, X ₁₀ is G, and X ₁₁ is S or N; and CDR3: X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ YX ₃₁ X ₃₂ X ₃₃ X ₃₄ ; where X ₁₃ is A or G, X ₁₄ is A or R, X ₁₅ is A or S, X ₁₆ is L or T, X ₁₇ is P or T, X ₁₈ is T or D, X ₁₉ is S or F, X ₂₀ is W or H, X ₂₁ is G or I, X ₂₂ is I or R, X ₂₃ is P or T, X ₂₄ is N or V, X ₂₅ is P or Q, X ₂₆ is N or S, X ₂₇ is D or M, X ₂₈ is Y or W, X ₂₉ is D or G, X ₃₁ is H or none, X ₃₂ is F or none, X ₃₃ is D or none, X ₃₄ is Y or none; Alternatively, the heavy chain variable region may include: CDR1: GFTX ₁ X ₂ X ₃ X ₄ X ₅ ; where X ₁ is L, X ₂ is D, X ₃ is Y, X ₄ is Y, and X ₅ is A; CDR2: IX ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ ; where X ₆ is S, X ₇ is S or N, X ₈ is S, X ₉ is D or G, X ₁₀ is S or D, X ₁₁ is S, and X ₁₂ is T; and CDR3: X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ X ₃₂ X ₃₃ X ₃₄ ; where X ₁₃ is A, X ₁₄ is A, X ₁₅ is D, X ₁₆ is L, X ₁₇ is S or Q, X ₁₈ is C, X ₁₉ is P, X ₂₀ is V, X ₂₁ is Q, X ₂₂ is P, X ₂₃ is M or T, X ₂₄ is S or A, X ₂₅ is P or D, X ₂₆ is Y, X ₂₇ is none, X ₂₈ is none, X ₂₉ is none, X ₃₀ is none, X ₃₁ is none, X ₃₂ is none, X ₃₃ is non-existent, X ₃₄ is non-existent. The anti-GDF-15 antibody according to claim 1, characterized in that the anti-GDF-15 antibody comprises a heavy chain variable region, the heavy chain variable region comprising CDR1 with an amino acid sequence as shown in SEQ ID NO:10, CDR2 with an amino acid sequence as shown in SEQ ID NO:11, and CDR3 with an amino acid sequence as shown in SEQ ID NO:12; Alternatively, the heavy chain variable region comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:18, CDR2 with an amino acid sequence as shown in SEQ ID NO:19, and CDR3 with an amino acid sequence as shown in SEQ ID NO:20; Alternatively, the heavy chain variable region may contain amino acid sequences such as CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4; Alternatively, the heavy chain variable region comprises CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:7, and CDR3 as shown in SEQ ID NO:8; Alternatively, the heavy chain variable region comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:6, CDR2 with an amino acid sequence as shown in SEQ ID NO:15, and CDR3 with an amino acid sequence as shown in SEQ ID NO:16; CDR1, CDR2 and CDR3 are defined using IMGT. The anti-GDF-15 antibody as described in claim 1 or 2, characterized in that the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 9, 74-76 or comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: 9, 74-76; Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in any of SEQ ID NO:17, 80-84 or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO:17, 80-84; Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in any of SEQ ID NO:1, 68-70 or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO:1, 68-70. Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in any of SEQ ID NO:5, 71-73 or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO:5, 71-73; Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in any of SEQ ID NO:13, 77-79, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO:13, 77-79. The anti-GDF-15 antibody according to any one of claims 1-3 is characterized in that the anti-GDF-15 antibody further comprises a constant region; Preferably, the constant region is an Fc region; the Fc region is preferably selected from the Fc region of human IgG; for example, the Fc region of human IgG1; More preferably, the Fc region comprises an amino acid sequence as shown in any of SEQ ID NO:21, 44-45 and 85 or having at least 80% sequence identity with any of SEQ ID NO:21, 44-45 and 85. The anti-GDF-15 antibody according to any one of claims 1-4, characterized in that the anti-GDF-15 antibody has an amino acid sequence as shown in any one of SEQ ID NO:22-43 and 86 or having at least 80% sequence identity with any one of SEQ ID NO:22-43 and 86; And/or, the GDF-15 is human GDF-15 and/or monkey GDF-15. A bispecific recombinant protein, characterized in that the bispecific recombinant protein comprises a first binding domain for binding GDF-15 and a second binding domain for binding IL-6. The bispecific recombinant protein of claim 6, wherein the first binding domain comprises a heavy chain variable region; and/or the second binding domain comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region is as described in any one of claims 1-3; and/or, the second binding structural domain comprises a heavy chain variable region and a light chain variable region of ALD518 or MEDI-5117. A bispecific recombinant protein, characterized in that the bispecific recombinant protein comprises a first binding domain for binding GDF-15 and a second binding domain for binding IL-6; the first binding domain comprises a heavy chain variable region and/or a light chain variable region, and the second binding domain comprises a heavy chain variable region and a light chain variable region. Preferably, the first binding domain comprises a heavy chain variable region and a light chain variable region of Ponsegromab or Visugromab; and/or, the second binding domain comprises a heavy chain variable region and a light chain variable region of ALD518 or MEDI-5117. The bispecific recombinant protein according to any one of claims 6-8 is characterized in that the second binding domain is an antigen-binding fragment that binds to IL-6; Preferably, the antigen-binding fragment is selected from Fab, Fab'-SH, Fv, or (Fab') ₂ ; the Fv is, for example, scFv; More preferably, the antigen-binding fragment is Fab. The bispecific recombinant protein of claim 9, characterized in that the antigen-binding fragment is Fab, the antigen-binding fragment includes a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:14, HCDR2 as shown in SEQ ID NO:58, and HCDR3 as shown in SEQ ID NO:59; the light chain variable region comprising LCDR1 as shown in SEQ ID NO:60, LCDR2 as shown in KAS, and LCDR3 as shown in SEQ ID NO:61; Alternatively, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:62, HCDR2 as shown in SEQ ID NO:63, and HCDR3 as shown in SEQ ID NO:64; the light chain variable region comprises LCDR1 as shown in SEQ ID NO:65, LCDR2 as shown in RAS, and LCDR3 as shown in SEQ ID NO:66. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined using IMGT. The bispecific recombinant protein according to any one of claims 6-10, characterized in that the heavy chain variable region of the antigen-binding fragment comprises an amino acid sequence as shown in amino acids 1-120 of SEQ ID NO:49 or having at least 80% sequence identity with it, and the light chain variable region of the antigen-binding fragment comprises an amino acid sequence as shown in amino acids 1-106 of SEQ ID NO:50 or having at least 80% sequence identity with it. Alternatively, the heavy chain variable region of the antigen-binding fragment may contain an amino acid sequence as shown in amino acids 1-120 of SEQ ID NO:51 or having at least 80% sequence identity with it, and the light chain variable region of the antigen-binding fragment may contain an amino acid sequence as shown in amino acids 1-110 of SEQ ID NO:52 or having at least 80% sequence identity with it. The bispecific recombinant protein according to any one of claims 6-11, characterized in that the bispecific recombinant protein further comprises an Fc region; Preferably, the Fc region is selected from the Fc region of human IgG; for example, the Fc region of human IgG1; the Fc region preferably contains an amino acid sequence as shown in any of SEQ ID NO:21, 44-45 and 85 or having at least 80% sequence identity with any of SEQ ID NO:21, 44-45 and 85; and/or, the first binding domain is connected to the N-terminus of the Fc region, and the second binding domain is connected to the C-terminus of the Fc region or the N-terminus of the first binding domain; or, the second binding domain is connected to the N-terminus of the Fc region, and the first binding domain is connected to the N-terminus of the second binding domain; the connection is a direct connection or a connection via a linker; More preferably, the linker is (G4S)n, where n is any integer from 1 to 6; the linker preferably has an amino acid sequence as shown in any of SEQ ID NO:46-48. The bispecific recombinant protein according to any one of claims 6-12 is characterized in that the bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2; the polypeptide chain 2, from the N-terminus to the C-terminus, is: a light chain variable region of a second binding domain and a light chain constant region. The polypeptide chain 1, from the N-terminus to the C-terminus, is as follows: First binding domain - second binding domain heavy chain variable region - heavy chain constant region 1 (CH1) - Fc region; Alternatively, the heavy chain variable region of the second binding domain - the heavy chain constant region 1 (CH1) - the first binding domain - the Fc region; Alternatively, the first binding domain - Fc region - heavy chain variable region of the second binding domain - heavy chain constant region 1 (CH1); Preferably, the light chain constant region is a kappa chain; and/or, the heavy chain variable region of the second binding structural domain is connected to the Fc region or the first binding structural domain via a connector. The bispecific recombinant protein of claim 13 is characterized in that the polypeptide chain 2 comprises an amino acid sequence as shown in SEQ ID NO: 50 or 52 or having at least 80% sequence identity with it; And/or, in the polypeptide chain 1, the heavy chain variable region-heavy chain constant region 1 (CH1) of the second binding domain contains an amino acid sequence as shown in SEQ ID NO:49 or 51 or having at least 80% sequence identity with it; Preferably, the polypeptide chain 1 comprises an amino acid sequence as shown in any of SEQ ID NO:53-57 or having at least 80% sequence identity with it. A polynucleotide, characterized in that the polynucleotide encodes an anti-GDF-15 antibody as described in any one of claims 1-5 or a bispecific recombinant protein as described in any one of claims 6-14. A recombinant expression vector, characterized in that the recombinant expression vector comprises the polynucleotide as described in claim 15. A recombinant cell, characterized in that the recombinant cell comprises the polynucleotide as described in claim 15 or the recombinant expression vector as described in claim 16, or expresses the anti-GDF-15 antibody as described in any one of claims 1-5 or the bispecific recombinant protein as described in any one of claims 6-14. A method for preparing an anti-GDF-15 antibody or a bispecific recombinant protein, characterized in that the method comprises culturing recombinant cells as described in claim 17, and obtaining the anti-GDF-15 antibody or the bispecific recombinant protein from the culture. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises an anti-GDF-15 antibody as described in any one of claims 1-5, a bispecific recombinant protein as described in any one of claims 6-14, a polynucleotide as described in claim 15, a recombinant expression vector as described in claim 16, and/or a recombinant cell as described in claim 17; And pharmaceutically acceptable carriers and/or excipients. The use of an anti-GDF-15 antibody as described in any one of claims 1-5, a bispecific recombinant protein as described in any one of claims 6-14, a polynucleotide as described in claim 15, a recombinant expression vector as described in claim 16, a recombinant cell as described in claim 17, or a pharmaceutical composition as described in claim 19 in the preparation of a reagent for detecting GDF-15 or a medicament for preventing and/or treating diseases and/or symptoms caused by GDF-15 signaling pathway dysregulation; Preferably, the diseases and/or conditions caused by the dysregulation of the GDF-15 signaling pathway include: cachexia, weight loss due to anorexia, chronic inflammation, malignant tumors, viral infections, cardiovascular diseases, liver fibrosis, neurodegenerative diseases, COVID-19, and chronic kidney disease; More preferably, the malignant tumor includes gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer; the viral infection includes HIV infection; and the cardiovascular disease includes heart failure. A method for preventing and/or treating diseases and/or symptoms caused by GDF-15 signaling pathway dysregulation, characterized in that the method comprises administering to a subject in need an effective amount of an anti-GDF-15 antibody as described in any one of claims 1-5, a bispecific recombinant protein as described in any one of claims 6-14, a polynucleotide as described in claim 15, a recombinant expression vector as described in claim 16, a recombinant cell as described in claim 17, or a pharmaceutical composition as described in claim 19; Preferably, the diseases and/or conditions caused by the dysregulation of the GDF-15 signaling pathway include: cachexia, weight loss due to anorexia, chronic inflammation, malignant tumors, viral infections, cardiovascular diseases, liver fibrosis, neurodegenerative diseases, COVID-19, and chronic kidney disease; More preferably, the malignant tumor includes gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer; the viral infection includes HIV infection; and the cardiovascular disease includes heart failure.