WO2025223473A1 - Anticorps anti-gdf-15 et son utilisation - Google Patents

Anticorps anti-gdf-15 et son utilisation

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Publication number
WO2025223473A1
WO2025223473A1 PCT/CN2025/090729 CN2025090729W WO2025223473A1 WO 2025223473 A1 WO2025223473 A1 WO 2025223473A1 CN 2025090729 W CN2025090729 W CN 2025090729W WO 2025223473 A1 WO2025223473 A1 WO 2025223473A1
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WIPO (PCT)
Prior art keywords
seq
variable region
chain variable
gdf
heavy chain
Prior art date
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Pending
Application number
PCT/CN2025/090729
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English (en)
Chinese (zh)
Inventor
何向宇
朱僧
厉玉梅
金磊
周福生
兰炯
吕强
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genfleet Therapeutics Shanghai Inc
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Genfleet Therapeutics Shanghai Inc
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Publication of WO2025223473A1 publication Critical patent/WO2025223473A1/fr
Pending legal-status Critical Current
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • This invention belongs to the field of biomedicine, specifically relating to an anti-GDF-15 antibody and its application.
  • Growth differentiation factor 15 is a pluripotent cytokine with a molecular weight of about 25 kDa. It has the common structural features of TGF ⁇ 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] .
  • GDF-15 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] .
  • 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 PLC ⁇ 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.
  • GDF-15/GFRAL/RET signaling pathway 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.
  • 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, TNF ⁇ , IFN ⁇ and GDF-15, as well as various hormones such as parathyroid hormone PTHrP [10] .
  • inflammatory cytokines such as IL-1, IL-6, TNF ⁇ , IFN ⁇ and GDF-15
  • PTHrP parathyroid hormone
  • 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] .
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anti-GDF-15 antibody also includes a constant region.
  • the constant region is the Fc region.
  • the Fc region is selected from the Fc region of human IgG; for example, the Fc region of human IgG1.
  • 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.
  • 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.
  • 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.
  • the first binding domain includes a heavy-chain variable region.
  • the first binding domain includes a heavy chain variable region and/or a light chain variable region.
  • the heavy chain variable region is as described in the first aspect.
  • the second binding domain includes a heavy chain variable region and a light chain variable region.
  • 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.
  • the first binding domain includes a heavy chain variable region and a light chain variable region of Ponsegromab or Visugromab.
  • the second binding domain includes a heavy chain variable region and a light chain variable region of ALD518 or MEDI-5117.
  • the second binding domain is an antigen-binding fragment that binds to IL-6.
  • the antigen-binding fragment is selected from Fab, Fab'-SH, Fv, or (Fab') 2 ; the Fv is, for example, scFv.
  • the antigen-binding fragment is Fab.
  • 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 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.
  • 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.
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined using IMGT.
  • 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
  • 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.
  • 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
  • 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.
  • the bispecific recombinant protein further includes an Fc region.
  • the Fc region is selected from the Fc region of human IgG; for example, the Fc region of human IgG1.
  • 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.
  • the first binding domain is connected to the N-terminus of the Fc region
  • the second binding domain is connected to the C-terminus of the Fc region or the N-terminus of the first binding domain.
  • 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.
  • the connector is (G4S)n, where n is any integer from 1 to 6.
  • the linker has an amino acid sequence as shown in any of SEQ ID NO:46-48.
  • 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:
  • the heavy chain variable region of the second binding domain - the heavy chain constant region 1 (CH1) - the first binding domain - the Fc region;
  • first binding domain - Fc region - heavy chain variable region of the second binding domain - heavy chain constant region 1 CH1
  • the light chain constant region is a kappa chain.
  • 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.
  • the polypeptide chain 1 from the N-terminus to the C-terminus is:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the malignant tumor includes gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer;
  • the viral infection includes HIV infection;
  • 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.
  • 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.
  • the malignant tumor includes gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer;
  • the viral infection includes HIV infection;
  • 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.
  • the reagents and raw materials used in this invention are all commercially available.
  • 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.
  • 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.
  • FIG. 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.
  • 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
  • G8 VHH-Fc can be used interchangeably with G8-Fc.
  • 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.
  • VH variable region
  • 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.
  • VL variable region
  • 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 ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains, respectively.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as ⁇ or ⁇ chains based on differences in their constant regions.
  • Each of the five classes of Ig can possess either a ⁇ or ⁇ chain.
  • 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).
  • variable domains VH and VL, respectively
  • VH and VL variable domains 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.
  • FRs conserved frame regions
  • HVRs hypervariable regions
  • CDR complementarity-determining region
  • the complementarity-determining 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.
  • CDR region 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.
  • CDR1, CDR2, and CDR3 are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3.
  • 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.
  • 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.
  • IMGT international ImMunoGeneTics database
  • 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.
  • "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.
  • sdAb single-domain antibody
  • VHHdomain single-domain antibody
  • variable region of a heavy chain antibody refers 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.
  • 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.
  • identity or “sequence identity” is used interchangeably, referring to the sequence similarity between two polynucleotide sequences or two polypeptides.
  • 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.
  • 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.
  • VL variable region
  • CL constant region
  • CH1 constant region 1
  • 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.
  • 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.
  • the scFv single chain antibody fragment
  • the scFv 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.
  • 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.
  • Fc fragment crystallizable
  • 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.
  • 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.
  • 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.
  • the host cell is a mammalian cell.
  • the mammalian cell is a CHO cell.
  • 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.
  • 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.
  • 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.
  • 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 1 Construction of an anti-human GDF-15 single-domain antibody library
  • 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.
  • helper phage M13KO7 phage:bacteria ratio of 1:500 was added.
  • 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.
  • GDF-15 binding assay Add 50 ⁇ L of 1 ⁇ g/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 ⁇ L of 1 ⁇ g/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 ⁇ L of IPTG-induced VHH supernatant and incubate for 1 h, followed by three washes with PBST.
  • Ligand receptor blocking assay Add 50 ⁇ L of 1 ⁇ g/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 ⁇ L of this mixture to each well of a 96-well plate and incubate for 1 h.
  • ACRO Biosystems ACRO Biosystems
  • 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 TGF ⁇ 1, 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.
  • 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.
  • 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.
  • VHH-Fc recombinant antibody against the GDF-15-GFRAL ligand receptor was tested using ELISA.
  • 96-well plates were coated with 1 ⁇ g/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 ⁇ L was added to each well.
  • 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°C 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 ⁇ g/mL human GDF-15. 20 ⁇ L of the mixture was added to each well, and the plates were cultured for another 16 h.
  • 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.
  • Example 5 Effect of GDF-15 recombinant antibody on body weight of HT-1080 tumor-bearing mice
  • 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.
  • mice 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 camel-derived GDF-15 single-domain antibody was humanized using a CDR transplantation method.
  • 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.
  • 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 ⁇ g/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 ⁇ L was added to each well. The plate was incubated at 37°C for 1 h, washed three times with PBST buffer, and then 100 ⁇ L/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.
  • 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 ⁇ g/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.
  • 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.
  • 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
  • mice 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.
  • 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 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.
  • Example 10 Binding activity of bispecific antibody with human GDF-15 and IL-6
  • 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 ⁇ L 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.
  • 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.
  • 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.
  • mice lost 10% of their body weight they were administered the drug in groups.
  • 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.
  • Plasma growth differentiation factor 15 is associated with weight loss and mortality in cancer patients. J Cachexia Sarcopenia Muscle. 2015; 6(4): 317-324.

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Abstract

L'invention concerne un anticorps anti-GDF-15 et son utilisation. L'anticorps anti-GDF-15 contient une région variable de chaîne lourde. La région variable de chaîne lourde contient CDR1 : GFTX1X2X3X4X5 ; CDR2 : IX6X7X8X9X10X11X12 ; et CDR3 : X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30X31X32X33X34. L'anticorps selon l'invention présente une bonne affinité et une bonne réactivité croisée pour GDF-15, bloque efficacement la voie de GDF-15, et peut améliorer significativement la cachexie chez un sujet. L'anticorps bispécifique selon l'invention présente une bonne affinité à la fois pour GDF-15 et IL-6, améliore significativement la cachexie chez des souris porteuses de tumeur, et atténue une réponse inflammatoire.
PCT/CN2025/090729 2024-04-23 2025-04-23 Anticorps anti-gdf-15 et son utilisation Pending WO2025223473A1 (fr)

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