Separated anti-GDF15 antibody as well as preparation and application thereof

Abstract

The present invention provides an isolated anti-GDF15 antibody for use in the treatment of GDF15 mediated diseases and disorders. The invention further provides an anti-GDF15 antibody preparation, the preparation is high in stability under the conditions of freeze thawing, oscillation, high temperature, refrigeration and acceleration, it can be guaranteed that the preparation keeps good stability in the preparation, transportation and storage processes, and clinical medication safety and quality controllability are guaranteed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an isolated anti-GDF15 antibody and a preparation and application thereof. Background Art

[0002] Growth differentiation factor-15 (GDF15), also known as macrophage inhibitory cytokine 1 (MIC-1), is an atypical member of the transforming growth factor β (TGF-β) superfamily. The maturation of human GDF15 undergoes a series of post-translational modifications. The unprocessed GDF15 protein (pre-pro-GDF15) after translation has 308 amino acids, including a signal peptide sequence (29aa), a propeptide (167aa) and a mature protein (112aa). The synthesized precursor form of pro-GDF15 protein forms a pro-GDF15 homodimer through a cysteine ​​residue; it is then cleaved at the RXXR site, releasing the mature dimeric GDF15 protein and a propeptide at the C-terminus (Wang X et al., Biochem Pharmacol, 2013).

[0003] The mature dimeric GDF15 protein is secreted into the extracellular matrix, and the normal circulating concentration in healthy people ranges from about 0.15-1.15 ng / ml. The concentration of GDF15 increases with age, but there is no gender difference in healthy elderly people. The concentration of GDF15 is related to the concentration of cystatin C and C-reactive protein (Brown, DA et al. Lancet, 2002.; Kempf, T. et al. Clin. Chem, 2007.). In humans, the circulating GDF15 increases during exercise and post-exercise recovery. GDF15 is expressed in most tissues, including skeletal muscle. Studies have shown that GDF15 is secreted from muscle during cellular stress or damage, acting locally in an autocrine or paracrine manner (Kleinert, M. et al. Mol. Metab, 2018). GDF15 is not usually expressed in adult myocardium, but it is significantly induced in damaged or failing hearts. During myocardial injury, GDF15 is expressed in cardiomyocytes, adipocytes, macrophages, endothelial cells, and vascular smooth muscle cells (Wollert, KCet al. Clin Chem. 2017; Planavila, Aet al. Adv. Protein Chem. 2017). In short, under physiological conditions, the production of GDF15 in most tissues is weak, but it is strongly induced during inflammation and tissue injury.

[0004] Although GDF15 is classified as a member of the TGFβ family, current studies have not confirmed the TGFβ receptor that GDF15 directly binds to. Four different research teams simultaneously confirmed GFRAL as the receptor of GDF15 (Hsu et al., Nature, 2017; Yang, L. et al., Nat Med, 2017; Mullican, SE et al., Nat Med, 2017; Emmerson, PJ et al., Nat Med, 2017.). Hsu et al.'s study found that GDF15 does not bind to any known TGFβ receptor. By analyzing the crystal structure of the GDF15 protein, it was found that there is an additional disulfide bond in the protein that is different from the TGFβ family members, and the glial cell-derived neurotrophic factor (GDNF) receptor α-like protein GFRAL was screened and confirmed as the brainstem-restricted receptor of the GDF15 protein (Hsu et al., Nature, 2017). This receptor mediates downstream signal transduction through the tyrosine kinase co-receptor RET. In the GDF15-GFRAL signaling pathway, the GDF15 dimer protein binds to the GFRAL receptor, and the GDF15-GFRAL complex recruits the co-receptor RET, causing dimerization and phosphorylation of RET and phosphorylation of intracellular signals AKT, ERK1 / 2, and phospholipase C (PLCγ) (Shannon E. Mullican & Rangwala, 2018; L. Yang, et al., 2017). Although the GDF15 / GFRAL signaling pathway is still not fully understood, and more research is needed to link the GDF15 receptor to the biological activity of mature GDF15, it can be confirmed that the molecular basis of GDF15 activity is through GFRAL / RET, rather than through TGFβ signal transduction (Hsu et al., Nature, 2017).

[0005] GDF15 protein is associated with a variety of diseases such as obesity and insulin resistance (SNBreit et al., Growth Factors, 2011.). Knockout of GDF15 in genetically engineered mice leads to weight gain; while overexpression of GDF15 leads to reduced weight and fat content in mice, and altered metabolic parameters (L. Maccia et al., PLOS ONE, 2012; VWTsai et al., PLOS ONE, 2013). Subsequent studies in transgenic mice further confirmed the reduction in weight and confirmed these phenotypes, including: widespread expression of hGDF15 in mice did not reduce food intake, but improved glucose tolerance, insulin sensitivity, and reduced body weight, insulin, glucose, serum IGF-1 levels, leptin levels and adipose tissue (Chrysovergis, et al., 2014). These findings indicate that GDF15 is an important regulator of body weight and energy balance.

[0006] GDF15 plays an important role in the occurrence of cancer cachexia. Cachexia is a medical symptom caused by cancer, usually involving weight loss, muscle atrophy, fatigue, weakness and severe loss of appetite in people who do not actively lose weight. It is one of the main causes of morbidity and mortality in the late stage of chronic diseases such as AIDS, chronic obstructive pulmonary disease (COPD), congestive heart failure, multiple sclerosis, tuberculosis and cancer. Among them, malignant tumors are the main factor inducing refractory cachexia. It is conservatively estimated that the number of new cases of tumor cachexia in my country exceeds 1.5 million each year (accounting for about 36.4% of new tumor patients), and about 20% of cancer patients die from cachexia itself (Murphy KT. et al., Expert Opin Emerg Drugs. 2009). Many malignant cancers, especially invasive brain cancer, melanoma, lung cancer, gastrointestinal tumors, colon cancer, pancreatic cancer, prostate cancer and breast cancer patients, show a significant increase in GDF15 levels in their tumors and serum (Huang CY et al., Clin Cancer Res. 2009.). The study by L. Lerner et al. showed that compared with cancer patients and non-cancer patient controls who did not lose weight, the circulating levels of GDF15 in cancer patients who lost weight were significantly increased (Lerner L et al., J Cachexia Sarcopenia Muscle. 2015.). In addition, GDF15 levels are associated with weight loss, reduced fat mass, muscle mass, strength, physical performance scores and survival in cancer patients, while a decrease in lean body mass (LBM) is a negative and independent prognostic factor in cancer patients (Reuben DB, et al., Arch Intern Med, 1988.), and decreased grip strength is associated with reduced survival in this population (Gale CR. et al., Int J Epidemiol. 2007).

[0007] Patent application WO2014100689A1 discloses monoclonal antibodies that bind to human GDF15 and inhibit the activity of human GDF15, and discloses that these antibodies can be used to treat weight loss associated with overexpression of human GDF15, including cachexia, etc. WO2015144855A1 relates to a monoclonal antibody against human GDF15 and an antigen-binding portion thereof, which can inhibit the growth of cancer cachexia and / or cancer, and is used to treat weight loss and / or cancer cachexia caused by cancer, as well as cancer.

[0008] Therefore, there is still a need in the art for therapeutic antibodies and stable high-concentration preparations that effectively inhibit or otherwise antagonize GDF15, as well as related methods for treating diseases or conditions mediated by GDF15, such as cancer, cancer cachexia, cancer-induced weight loss, and the like.

[0009] The present invention provides an isolated anti-GDF15 antibody and a high-concentration preparation thereof with strong stability, which are used for treating diseases or disorders mediated by GDF15. Summary of the invention

[0010] In one aspect, the present invention provides an isolated anti-GDF15 antibody comprising V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:4, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:5, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6.

[0011] In some embodiments, the antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, and V L , which comprises the amino acid sequence of SEQ ID NO: 11; or

[0012] The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:9, and V L , which comprises the amino acid sequence SEQ ID NO: 11; or

[0013] The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, and V L , which comprises the amino acid sequence SEQID NO:12.

[0014] In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:15.

[0015] In some embodiments, the antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, V L , which comprises the amino acid sequence of SEQ ID NO: 11, and a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 13, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15; or

[0016] The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:9, V L , which comprises the amino acid sequence of SEQ ID NO: 11, and a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15; or

[0017] The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, V L , which comprises the amino acid sequence of SEQ ID NO:12, and a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:15.

[0018] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 20; or,

[0019] The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 20; or,

[0020] The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:22, and a light chain comprising the amino acid sequence of SEQ ID NO:23.

[0021] In another aspect, the present invention provides a high-concentration preparation of an anti-GDF15 antibody, wherein the preparation comprises the anti-GDF15 antibody isolated as described in any one of the above items, a stabilizer, a surfactant, and a buffer.

[0022] In some embodiments, the antibody concentration is 145 mg / ml-175 mg / ml; in some specific embodiments, the antibody concentration is 145 mg / ml, 150 mg / ml, 155 mg / ml, 160 mg / ml, 165 mg / ml, 170 mg / ml or 175 mg / ml; preferably, the antibody concentration is 150 mg / ml-170 mg / ml.

[0023] In some embodiments, the stabilizer is sucrose. In some preferred embodiments, the stabilizer concentration is 50mg / ml-80mg / ml, more preferably 50mg / ml, 55mg / ml, 60mg / ml, 65mg / ml, 70mg / ml, 75mg / ml or 80mg / ml. In some more preferred embodiments, the stabilizer concentration is 55mg / ml-65mg / ml.

[0024] In some embodiments, the buffer is a histidine-acetate buffer. In some preferred embodiments, the concentration of the buffer is 5mM-40mM, more preferably 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM. In some more preferred embodiments, the concentration of the buffer is 10mM-30mM; more preferably, the concentration of the buffer is 15mM-25mM.

[0025] In some embodiments, the pH value of the formulation is 5.0-6.0, more preferably 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 or 6.0. In some more preferred embodiments, the pH value of the formulation is 5.2-5.8; more preferably, the pH value of the formulation is 5.4-5.6.

[0026] In some embodiments, the surfactant is a polysorbate or a poloxamer; preferably, the polysorbate is Tween-20 or Tween-80, and the poloxamer is P188. In some preferred embodiments, the concentration of the surfactant is 0.05mgml-0.4mg / ml; more preferably 0.05mg / ml, 0.10mg / ml, 0.15mg / ml, 0.2mg / ml, 0.25mg / ml, 0.3mg / ml or 0.4mg / ml. In some more preferred embodiments, the concentration of the surfactant is 0.1mg / ml-0.3mg / ml.

[0027] In some embodiments, the high-concentration GDF15 antibody preparation of the present invention comprises the anti-GDF15 antibody isolated as described in any of the above items, a stabilizer, a surfactant, and a buffer, wherein:

[0028] (1) The antibody concentration is 150 mg / ml, 155 mg / ml, 160 mg / ml, 165 mg / ml, 170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM-25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188;

[0029] (2) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml sucrose; the buffer is 15 mM-25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188;

[0030] (3) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM, 20 mM or 25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188;

[0031] (4) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM, 20 mM or 25 mM histidine-acetate buffer; the pH value of the preparation is 5.2, 5.4, 5.5, 5.6 or 5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188;

[0032] (5) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM-25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml or 0.3 mg / ml of Tween-20, Tween-80 and / or P188.

[0033] In some preferred embodiments, according to the high concentration formulation of the GDF15 antibody of the present invention, wherein:

[0034] (1) The preparation comprises 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.5;

[0035] (2) The preparation contains 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.4;

[0036] (3) The preparation contains 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.6;

[0037] (4) The preparation contains 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80 or Tween-20 and 0.1 mg / ml of P188; the pH of the preparation is 5.6;

[0038] (5) The preparation contains 150 mg / ml of anti-GDF15 antibody, the stabilizer is 65 mg / ml of sucrose; the buffer is 15 mM histidine-acetate buffer; the surfactant is 0.3 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.2;

[0039] (6) The preparation contains 170 mg / ml of anti-GDF15 antibody, the stabilizer is 55 mg / ml of sucrose; the buffer is 25 mM histidine-acetate buffer; the surfactant is 0.1 mg / ml of Tween-80, Tween-20 or P188; and the pH of the preparation is 5.8.

[0040] In some embodiments, the formulation may further comprise an antioxidant, preferably, the antioxidant is ethylenediaminetetraacetic acid (EDTA); preferably, the concentration of the antioxidant is 0-1 mM. In some specific embodiments, the concentration of the antioxidant is 0, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM.

[0041] In some embodiments, the antibody preparation described above is a liquid preparation or a powder for injection.

[0042] In another aspect, the present invention provides the use of any of the antibodies described above, or any of the antibody preparations described above, in the preparation of a medicament for treating a disease. In some embodiments, the disease is a disease and / or condition caused by dysregulation of the GDF15 signaling pathway, such as cancer, cachexia, and / or metabolic disease.

[0043] In some embodiments, the disease or condition is selected from cancer (such as brain cancer, melanoma, lung cancer, gastrointestinal tumors, colon cancer, pancreatic cancer, prostate cancer, breast cancer, oral cancer, liver cancer, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, bladder cancer, cervical cancer, uterine body cancer, testicular cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharyngeal cancer, laryngeal cancer, pharyngeal cancer, esophageal cancer, etc.), cachexia, renal failure, sepsis, AIDS, cachexia caused by chronic obstructive pulmonary disease, cachexia-related weight loss, nausea and vomiting caused by radiotherapy and chemotherapy, advanced chronic tubulointerstitial nephritis, morning sickness, cirrhosis, sarcopenia, metabolic diseases (such as imbalance of fat and energy metabolism, regulation of appetite, regulation of weight).

[0044] In another aspect, the present invention provides a method for treating a disease or condition in an individual of interest, comprising administering to the individual an effective amount of any anti-GDF15 antibody or antibody preparation as described above. The administration method includes: intravenous injection, intra-arterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration or transdermal administration. In some embodiments, the preparation is administered intravenously. In some embodiments, the preparation is administered intramuscularly. In some preferred embodiments, the preparation can be administered subcutaneously.

[0045] In some embodiments, any of the anti-GDF15 antibodies separated as described above, the anti-GDF15 antibody separated comprises an Fc fragment. In some embodiments, the anti-GDF15 antibody separated is a full-length IgG antibody. In some embodiments, the anti-GDF15 antibody separated is a full-length IgG1 or IgG4 antibody. In some embodiments, the anti-GDF15 antibody separated is a full-length IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, the anti-GDF15 antibody separated is a chimeric, murine, fully human or humanized antibody. In some embodiments, the anti-GDF15 antibody separated is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody) and linear antibody.

[0046] The "antibodies" of the present invention include full-length antibodies and antigen-binding fragments thereof, preferably full-length antibodies. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three highly variable loops, known as complementary determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conservative than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of the full-length antibodies described in the present invention are IgA, IgD, IgE, IgG and IgM, characterized by having α, δ, ε, γ and μ heavy chains, respectively. Further, the several major antibody classes described in the present invention can be further divided into subclasses, including: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0047] As used herein, the term "antigen-binding fragment" includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized double-chain antibodies (dsdiabodies), single-chain antibodies (scFv), scFv dimers (divalent double-chain antibodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure.

[0048] The anti-GDF15 antibodies and preparations thereof provided by the present invention have the following excellent effects:

[0049] 1. The anti-GDF15 antibody of the present invention is a highly effective antibody molecule that can bind to human GDF15 and inhibit the interaction between human GDF15 and its receptor GFRAL. Compared with the known anti-GDF15 antibody Hu01G06-127 (AVEO), the antibody of the present invention is even more effective than or equivalent to Hu01G06-127 in various biological experiments.

[0050] 2. The anti-GDF15 antibody and its preparation of the present invention also have good viscosity at an antibody concentration of up to 170 mg / ml, which has a great advantage when administered subcutaneously. In addition, the GDF15 antibody preparation of the present invention has high stability under freeze-thaw, oscillation, high temperature, refrigeration, and accelerated conditions, which can ensure that the preparation maintains good stability during preparation, transportation, and storage, and ensures clinical drug safety and quality controllability. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shown is the effect of an exemplary lead anti-GDF15 antibody GDF-R37-rabIg1 on body weight changes in mice in the tumor cachexia animal model HT1080. DETAILED DESCRIPTION

[0052] In order to make the technical problems to be solved, the technical solutions and advantages adopted by the present invention clearer, the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0053] Unless otherwise specified, the reagents used in the following examples were prepared by conventional methods or obtained from commercial sources; the experimental methods used were conventional methods unless otherwise specified; the materials, instruments, etc. used were obtained from commercial sources unless otherwise specified.

[0054] In the following disclosed embodiments, the following abbreviations are used: GDF15 (Growth Differentiation Factor-15); GFRAL (GDNF family receptor α-like); RET (Tyrosine kinase receptor)

[0055] Example 1: Screening and preparation of antibodies

[0056] Human GDF15-his fusion protein (SEQ ID NO: 16) and human GDF15-hFc (SEQ ID NO: 17) were expressed and purified using a mammalian cell expression system.

[0057] Human GDF15-his was used as an antigen and immunized with an equal amount (v / v) of adjuvant in New Zealand rabbits. The rabbit serum was collected after immunization and the total IgG titer in the rabbit serum was detected by ELISA. After several rounds of immunization, the rabbit spleen was collected, B cells were sorted, RNA was extracted, cDNA was obtained by reverse transcription, and V H and V K Fragment, V H and V K The antibody was linked to a lentiviral shuttle vector with a constant region to construct a complete antibody and package it into a lentivirus, which was then infected with 293T cells to display the antibody on the surface of 293T cells to obtain a cell display library.

[0058] Using a flow cytometer, positive single cells binding to human GDF15-hFc were sorted and cultured in a 96-well plate. Human GDF15-his protein was coated in an ELISA plate, and 293T cell expression supernatant was added to screen positive clones binding to human GDF15-His, and the positive clones were sequenced to obtain the sequence of the lead antibody. The lead antibody was identified by ELISA and evaluated for in vitro biological activity to obtain the preferred lead antibody GDF-R37-rabIg1, and then the human germline kappa light chain (Gene ID-V Gene: IGKV1-39*01) and heavy chain (Gene ID-V Gene: IGHV3-15) were used as the skeleton, and the lead antibody was humanized by CDR transplantation. All antibodies were recombined with the human IgG1 constant region to obtain full-length humanized antibody molecules hum_R37-2, hum_R37-5, and hum_R37-8. The amino acid sequence of the antibody is shown in the following table.

[0059] Table 1 Anti-GDF15 antibody CDR sequences

[0060]

[0061] Table 2 Anti-GDF15 antibody sequences

[0062]

[0063]

[0064] Example 2: Affinity and biological activity of anti-GDF15 antibodies

[0065] 1. ELISA method to detect the affinity of anti-GDF15 antibodies

[0066] Detection of the ability of humanized antibodies to bind to GDF15 protein:

[0067] ELISA binding test: The purified monoclonal antibody was subjected to a binding test with the human GDF15-his protein. This test was used to identify the binding activity of the anti-GDF15 antibody. In brief, the anti-GDF15 antibody to be tested was coated into a 96-well plate (200 ng / well), incubated at 37°C for 1 hour, washed with PBST and blocked with 1% BSA at room temperature for 1 hour. Subsequently, 100 μl / well of gradient diluted human GDF15-his solution (dose range 0-50 μg / ml) was added, and after incubation, 100 μl of anti-his tag secondary antibody (anti-his-HRP (1:2000), Borsi, catalog number BHR790) was added to each well. Finally, TMB solution was used for color development (100 μl / well), and the reaction was terminated with 2MH2SO4. OD450 was measured, and a binding curve was generated using PRISM software to analyze the affinity of each candidate antibody to the antigen human GDF15, and the EC was calculated. 50 value.

[0068] The results of the neutralization activity of the humanized antibody molecules are shown in Table 3. The results show that the antibodies of the present invention have higher GDF15 binding activity, and compared with the lead antibody GDF-R37-rabIg1, the humanized antibodies all show comparable GDF15 binding activity.

[0069] Table 3: Affinity activity of anti-GDF15 antibodies after humanization

[0070] Antibody Name EC50(nM) hum-R37-2-Ig1 57.52 hum-R37-5-Ig1 72.72 hum-R37-8-Ig1 79.17 GDF-R37-rabIg1 63.77

[0071] 2. ELISA to detect the neutralizing activity of anti-GDF15 antibodies

[0072] Detection of the inhibitory ability of humanized antibodies on the binding of GDF15 and GFRAL:

[0073] The inhibitory activity of anti-GDF15 antibodies at different concentrations on the binding of GDF15 and GFRAL receptors was detected by ELISA, and the IC 50 The value is used as a standard to measure the neutralizing activity of candidate molecules. Briefly, 96-well plates were coated with 100 ng / well of GFRAL-his protein (SEQID NO: 18), incubated at 37°C for 1 hour, washed with PBST solution and blocked with 1% BSA at 37°C for 1 hour. Subsequently, a gradient dilution of the antibody to be tested (dose range 0-10 μg / ml) was added to each well, and an equal volume of human GDF15-hFc protein (0.5 μg / ml) was immediately added. After incubation and washing, 100 μl of anti-human IgG secondary antibody (goat anti-human IgG-HRP, Sigma, A8667) was added to each well. Finally, TMB solution was used for color development (100 μl / well), and the reaction was terminated with 2M H2SO4. OD450 was measured, and a binding curve was generated by PRISM to calculate IC50 value.

[0074] The results of the neutralizing activity of the humanized antibody molecules are shown in Table 4, which show that the antibodies of the present invention can effectively inhibit the binding of GDF15 and GFRAL, and compared with the lead antibody GDF-R37-rabIg1, the humanized anti-GDF15 antibodies all exhibited comparable neutralizing activity.

[0075] Table 4: Neutralizing activity of humanized anti-GDF15 antibodies

[0076] Antibody Name <![CDATA[IC 50 (nM)]]> hum-R37-2 1.72 hum-R37-5 1.90 hum-R37-8 1.59 GDF-R37-rabIg1-EF 2.49

[0077] 3. Detection of biological activity of anti-GDF15 antibodies

[0078] In the GDF15-GFRAL / RET signaling pathway, after the mature GDF15 protein binds to two GFRAL proteins on the cell surface, it recruits two other co-receptor RET proteins to form a complex, which induces the activation of RET intracellular signal peptides, and further causes key signal transduction such as RET-Ras-Erk-SRF. GFRAL and RET proteins were overexpressed in 293T cells, and the reporter gene plasmid of SRE, a key factor downstream of RET protein, was transferred at the same time to complete cell transfection and monoclonal cell line screening. The inhibitory activity of antibodies on GFRAL and RET downstream signal activation can be evaluated by the expression level of reporter genes, thereby reflecting the biological activity of anti-GDF15 antibodies at the cellular level.

[0079] In brief, the plasmid carrying the human GFRAL / RET protein encoding gene and the plasmid pGL4.33[luc2p / SRE / Hygro] (E1340, Promega) carrying the RET downstream protein reporter gene were co-transfected into 293T cells to screen and obtain the target monoclonal cell line 293T-GFRAL / RET-Luc. The 293T-GFRAL / RET-Luc cells were inoculated in a black light-proof 96-well plate. After overnight culture, human GDF15-his protein dilution (15 ng / ml, cell culture medium containing 0.1% BSA) was added, and an equal volume of gradient dilutions of anti-GDF15 antibody (concentration range 0-0.5 μg / ml) were added at the same time, and culture was continued for 6 hours at 37°C and 5% CO2. Finally, the luciferase substrate was used for color development and the signal luminescence value was read. The inhibition curve was generated by PRISM to calculate the IC 50 Values ​​are used to analyze the biological activity of each candidate antibody.

[0080] The biological activity results of the humanized antibodies are shown in Table 5, which show high biological activity and comparable inhibitory effects to the lead antibody GDF-R37-rabIg1.

[0081] Table 5: Biological activity of humanized anti-GDF15 antibodies I

[0082] Antibody Name IC50(nM) hum-R37-2-Ig1 0.13 hum-R37-5-Ig1 0.11 hum-R37-8-Ig1 0.12 GDF-R37-rabIg1-EF 0.16

[0083] 4. Biacore method to detect the affinity of anti-GDF15 antibodies

[0084] Biacore 8K (GE) was used to characterize the affinity of anti-GDF15 antibodies to human GDF15, rhesus monkey GDF15, and mouse GDF15. Anti-GDF15 antibodies were immobilized on the sensor chip CM5, and the affinity of antibodies at different concentrations to GDF15 proteins of different species was detected. The binding rate and dissociation rate of the antibodies were measured using SPR technology, and the binding affinity was determined. The Kon, Koff, and Kd values ​​of the anti-GDF15 antibodies are shown in Table 6.

[0085] The results are shown in Table 6. The humanized antibodies hum-R37-2, hum-R37-5, and hum-R37-8 of the present invention can bind to human GDF15. In addition, the humanized antibodies hum-R37-2, hum-R37-5, and hum-R37-8 of the present invention also bind to rhesus monkey GDF15, and weakly bind to mouse GDF15.

[0086] Table 6: Biacore method to detect the affinity activity of humanized molecules

[0087] Antibody Ligand Kon(1 / Ms) Koff(1 / s) Kd hum-R37-2 Human GDF15-hFc 1.29E+05 1.37E-04 1.07E-09 hum-R37-5 Human GDF15-hFc 1.19E+05 6.95E-05 5.84E-10 hum-R37-8 Human GDF15-hFc 1.39E+05 8.46E-05 6.07E-10

[0088] Example 3: Activity detection of anti-GDF15 antibody in in vivo disease model

[0089] The rapid weight loss model of mice (GDF15-hFc modeling) was used to verify the in vivo biological activity of anti-GDF15 antibodies. In brief, the modeling was performed by subcutaneously injecting human GDF15-hFc protein (modeling dose of 2 mpk) into C57 / BL6 mice. After a significant decrease in body weight compared with normal mice, a single intravenous injection of exemplary candidate antibodies Hum-R37-2, Hum-R37-5, Hum-R37-8, positive control antibody Hu01G06-127-IgG1, and negative control antibody (from Shutaishen (Beijing) Biopharmaceutical Co., Ltd.) was performed, and the dosage was 10 mpk; the normal mouse group was injected with normal saline. The biological activity of the candidate antibody was detected by drawing a curve of the change rate of mouse body weight.

[0090] The results are as follows Figure 1As shown, the anti-GDF15 antibodies Hum-R37-2, Hum-R37-5 and Hum-R37-8 of the present invention can effectively restore the body weight of mice in the model group, and their in vivo activities are better than or equivalent to the positive control antibody Hu01G06-127-IgG1.

[0091] Example 4: Preparation of high concentration anti-GDF15 antibody preparation

[0092] The high concentration antibody preparations of hum_R37-2, hum_R37-5 (L234A / L235A), and hum_R37-8 (L234A / L235A) were prepared respectively with reference to the prescription in Table 7. Among them, the difference between hum_R37-5 (L234A / L235A) and hum_R37-8 (L234A / L235A) relative to hum_R37-5 and hum_R37-8, respectively, is that the heavy chain constant region of the former has L234A and L235A mutations (i.e., LALA mutations, EU numbering, and the heavy chain constant region sequence is shown in SEQ ID NO: 14), and the rest are exactly the same.

[0093] Specifically, the heavy chain amino acid sequence of hum_R37-2 antibody is shown in SEQ ID NO: 19, and the light chain amino acid sequence is shown in SEQ ID NO: 20; the heavy chain amino acid sequence of hum_R37-5(L234A / L235A) antibody is shown in SEQ ID NO: 21, and the light chain amino acid sequence is shown in SEQ ID NO: 20; the heavy chain amino acid sequence of hum_R37-8(L234A / L235A) antibody is shown in SEQ ID NO: 22, and the light chain amino acid sequence is shown in SEQ ID NO: 23.

[0094] Table 7: Anti-GDF15 Antibody Preparation Prescription

[0095]

[0096]

[0097] Example 5: Stability test of high concentration preparation of anti-GDF15 antibody

[0098] Freeze-thaw, oscillation, high temperature, refrigeration, acceleration and illumination conditions were set to investigate the stability of different antibody preparations prepared in Example 4. The test items included visible foreign matter, concentration, pH, osmotic pressure, viscosity, insoluble particles, degradation characteristics and charge heterogeneity of the antibody preparation. Among them, the polymer characteristics were detected by SEC method, and the charge heterogeneity was detected by SCX method.

[0099] 1. Viscosity detection of anti-GDF15 antibody preparations

[0100] After the preparation of the anti-GDF15 antibody was completed, the viscosity of the antibody preparation was tested with a viscometer. The results are shown in Table 8:

[0101] Table 8: Viscosity test results of anti-GDF15 antibody preparations

[0102]

[0103] Public documents indicate that the existing anti-GDF15 antibodies reach the acceptable viscosity limit (20 cP) at about 140 mg / ml, making it impossible to prepare preparations with higher concentrations. However, as shown in the results in Table 8, under the above formulation, the antibodies of the present invention still have a lower viscosity at a concentration of 150 mg / ml-170 mg / ml, which is more advantageous when injected subcutaneously.

[0104] 2. Stability test under freeze-thaw conditions

[0105] The stability of each antibody preparation was tested under freeze-thaw conditions (freeze: -70°C ± 10°C; thaw: room temperature, freeze-thaw 0, 5, and 10 times).

[0106] (1) The test results of each preparation of hum_R37-5 (L234A / L235A) are shown in Table 9: After 10 freeze-thaw cycles, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged compared to 0 freeze-thaw cycles; the change of aggregates detected by SEC was very small, all within 0.20%. The above results show the excellent stability of the preparation of hum_R37-5 (L234A / L235A) antibody under freeze-thaw conditions.

[0107] Table 9: Freeze-thaw test results of hum_R37-5 (L234A / L235A) antibody preparation

[0108]

[0109]

[0110] (2) The various preparations of anti-GDF15 antibodies hum_R37-2 and hum_R37-8 (L234A / L235A) also showed excellent stability under freeze-thaw conditions: after 10 freeze-thaw cycles, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged compared with 0 freeze-thaw cycles; the changes in aggregates detected by SEC were very small, all within 0.22% (specific values ​​not shown).

[0111] The results of the freeze-thaw test indicate that the degradation characteristics of the anti-GDF15 antibody of the present invention in each preparation are almost unchanged under freeze-thaw conditions, indicating that each antibody preparation has excellent stability.

[0112] 3. Stability test under oscillation conditions

[0113] The stability of the antibody preparation was tested under shaking conditions (5°C ± 3°C, 300 rpm shaking for 0 days, 10 days, 15 days, and 28 days).

[0114] (1) The test results of each preparation of hum_R37-8 (L234A / L235A) are shown in Table 10: After 28 days of oscillation, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged compared with the 0 day of oscillation; the change of aggregates detected by SEC was very small, all within 0.28%. The above results show the excellent stability of the preparation of hum_R37-8 (L234A / L235A) antibody under oscillation conditions.

[0115] Table 10: Oscillation test results of hum_R37-8 (L234A / L235A) antibody preparation

[0116]

[0117] (2) The various preparations of anti-GDF15 antibodies hum_R37-2 and hum_R37-5 (L234A / L235A) also showed excellent stability under oscillation conditions: compared with 0 days of oscillation, after 28 days of oscillation, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged; the SEC detection of aggregates changed very little, all within 0.30% (specific values ​​not shown).

[0118] The results of the above-mentioned oscillation test for each test item indicate that the degradation characteristics of the anti-GDF15 antibody of the present invention in each preparation are almost unchanged under oscillation conditions, indicating that each antibody preparation has excellent stability.

[0119] 3. Stability test under high temperature conditions

[0120] The stability of each formulation of each antibody was tested under high temperature conditions (40°C ± 2°C, 75% ± 5% RH (relative humidity), 0 days, 10 days, 15 days, 28 days).

[0121] (1) The test results of each preparation of hum_R37-8 (L234A / L235A) are shown in Table 11: After 28 days of high temperature, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged compared with the day 0; the main peak of SEC detection was slightly reduced, and the change range was within 4.06%. The above results show that the preparation of hum_R37-8 (L234A / L235A) antibody can maintain basic stability under high temperature conditions.

[0122] Table 11: hum_R37-8 (L234A / L235A) antibody high temperature test results

[0123]

[0124] (2) Each preparation of anti-GDF15 antibody hum_R37-2 and hum_R37-5 (L234A / L235A) can also maintain basic stability under high temperature conditions: after 28 days at high temperature, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged compared to day 0; the changes in aggregates detected by SEC were small, all within 4.50% (specific data not shown).

[0125] The above results show that the degradation characteristics of the anti-GDF15 antibody of the present invention in each preparation did not change significantly under high temperature conditions, and the main peak detected by SEC after 28 days could still reach more than 95%, indicating that each antibody preparation can maintain basic stability under high temperature conditions.

[0126] 4. Stability test under refrigerated conditions

[0127] Stability of antibody preparations under long-term conditions (0 days, 10 days, 15 days, and 28 days at 5°C ± 3°C).

[0128] (1) The test results of each preparation of hum_R37-2 are shown in Table 12: After 28 days under refrigerated conditions, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged compared with day 0; the change range of the main peak detected by SEC was within 0.25%, and there was almost no change. After 15 days under refrigerated conditions, compared with day 0, the acidic peak, main peak, and alkaline peak detected by SCX were almost unchanged, with the main peak changing within 0.02%, the acidic peak changing within 1.08%, and the alkaline peak changing within 1.10%. The above results show the excellent stability of the preparation of hum_R37-2 antibody under refrigerated conditions.

[0129] Table 12: Refrigerated stability results of hum_R37-2 antibody formulations

[0130]

[0131]

[0132] (2) Anti-GDF15 antibodies hum_R37-8 (L234A / L235A) and hum_R37-5 (L234A / L235A) also showed excellent stability under refrigerated conditions: after 28 days under refrigerated conditions, compared with day 0, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged; the change range of the main peak detected by SEC was within 0.29%, with almost no change. After 15 days under refrigerated conditions, compared with day 0, the acidic peak, main peak, and alkaline peak detected by SCX showed almost no change, with the main peak changing within 0.04%, the acidic peak changing within 1.20%, and the alkaline peak changing within 1.15% (specific data not shown).

[0133] The above-mentioned long-term test results show that the degradation characteristics and charge heterogeneity of the anti-GDF15 of the present invention in each antibody preparation are almost unchanged under refrigerated conditions, indicating that each antibody preparation has excellent stability.

[0134] 5. Stability test under accelerated conditions

[0135] The stability of the antibody formulation was tested under accelerated conditions (25°C ± 2°C, 60% ± 10% RH (relative humidity) for 0 days, 10 days, 15 days, and 28 days).

[0136] (1) The test results of each preparation of hum_R37-8 (L234A / L235A) are shown in Table 13: After 28 days under accelerated conditions, compared with day 0, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged; the change range of the main peak detected by SEC was within 0.75%, and there was no significant change. After 15 days under accelerated conditions, compared with day 0, the acidic peak, main peak, and alkaline peak detected by SCX did not change significantly, the main peak changed within 1.16%, the acidic peak changed within 0.15%, and the alkaline peak changed within 1.18%. The above results show the excellent stability of the preparation of hum_R37-8 (L234A / L235A) antibody under accelerated conditions.

[0137] Table 13: Accelerated test results of hum_R37-8 (L234A / L235A) antibody formulation

[0138]

[0139] (2) Anti-GDF15 antibodies hum_R37-2 and hum_R37-5 (L234A / L235A) also showed excellent stability under refrigerated conditions: after 28 days under refrigerated conditions, compared with day 0, the appearance, visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of each antibody preparation were basically unchanged; the change range of the main peak detected by SEC was within 0.80%, with no significant change. After 15 days under accelerated conditions, compared with day 0, the acidic peak, main peak, and alkaline peak detected by SCX showed no significant change, the main peak changed within 1.20%, the acidic peak changed within 0.18%, and the alkaline peak changed within 1.20% (specific data not shown).

[0140] The results of the accelerated test indicate that the degradation characteristics and charge heterogeneity of the anti-GDF15 antibody of the present invention in each preparation do not change significantly under accelerated conditions, indicating that each antibody preparation has excellent stability.

[0141] 6. Stability test under light conditions

[0142] Under lighting conditions (5℃±3℃, 4500±500lx, 70mW*hr / cm 2 , illumination for 0 days, 10 days, and 15 days) to detect the stability of the antibody preparation.

[0143] The test results of various preparations of hum_R37-8 (L234A / L235A) are shown in Table 14 as an example: after 28 days under light conditions, compared with day 0, the visible foreign matter, concentration, pH, osmotic pressure, viscosity, and insoluble particles of various antibody preparations were basically unchanged, and the color of the solution deepened to a dark yellow solution; the main peak of SEC detection decreased, with a change range of 14.32%-27.01%, indicating that the preparations of hum_R37-8 (L234A / L235A) antibodies should be stored in a dark environment.

[0144] Table 14: Results of illumination test of hum_R37-8 (L234A / L235A) antibody preparation

[0145]

[0146] In summary, the anti-GDF15 antibody preparation of the present invention has strong stability under freeze-thaw, oscillation, high temperature, refrigeration and accelerated conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, and ensures clinical drug safety and quality controllability, but it should be stored away from light.

Claims

1. An isolated anti-GDF15 antibody, characterized in that The antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:4, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:5, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO: 8, and V L , which comprises the amino acid sequence of SEQ ID NO: 11; or The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:9, and V L , which comprises the amino acid sequence of SEQ ID NO: 11; or The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, and V L , which comprises the amino acid sequence SEQ ID NO:

12.

3. The antibody according to claim 1, characterized in that The antibody further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

15.

4. The antibody according to claim 1, characterized in that The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, V L , which comprises the amino acid sequence of SEQ ID NO: 11, and a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 13, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15; or The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:9, V L , which comprises the amino acid sequence of SEQ ID NO: 11, and a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15; or The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:8, V L , which comprises the amino acid sequence of SEQ ID NO:12, and a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

15.

5. The antibody according to claim 1, characterized in that The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 20; or, The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 20; or, The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:22, and a light chain comprising the amino acid sequence of SEQ ID NO:

23.

6. An anti-GDF15 antibody preparation, characterized in that The preparation comprises the isolated anti-GDF15 antibody according to any one of claims 1 to 5, a stabilizer, a surfactant, and a buffer.

7. The preparation according to claim 6, characterized in that The antibody concentration is 145 mg / ml-175 mg / ml; preferably, the antibody concentration is 150 mg / ml-170 mg / ml.

8. The preparation according to claim 6 or 7, characterized in that The stabilizer is sucrose; preferably, the stabilizer concentration is 50 mg / ml-80 mg / ml, preferably 55 mg / ml-65 mg / ml.

9. The preparation according to any one of claims 6 to 8, characterized in that The buffer is a histidine-acetate buffer; preferably, the concentration of the buffer is 5mM-40mM; preferably, the concentration of the buffer is 10mM-30mM; more preferably, the concentration of the buffer is 15mM-25mM.

10. The preparation according to any one of claims 6 to 9, characterized in that The pH value of the preparation is 5.0-6.0; preferably, the pH value of the preparation is 5.2-5.8; more preferably, the pH value of the preparation is 5.4-5.

6.

11. The preparation according to any one of claims 6 to 10, characterized in that The surfactant is polysorbate or poloxamer; preferably, the polysorbate is Tween-20 or Tween-80, and the poloxamer is P188; preferably, the concentration of the surfactant is 0.05mg / ml-0.4mg / ml; more preferably, the concentration of the surfactant is 0.1mg / ml-0.3mg / ml.

12. The preparation according to claim 6, characterized in that The preparation is any one of the following preparations: (1) The antibody concentration is 150 mg / ml, 155 mg / ml, 160 mg / ml, 165 mg / ml, 170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM-25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188; (2) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml sucrose; the buffer is 15 mM-25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188; (3) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM, 20 mM or 25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188; (4) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM, 20 mM or 25 mM histidine-acetate buffer; the pH value of the preparation is 5.2, 5.4, 5.5, 5.6 or 5.8; the surfactant is 0.1 mg / ml-0.3 mg / ml Tween-20, Tween-80 and / or P188; (5) The antibody concentration is 150 mg / ml-170 mg / ml; the stabilizer is 55 mg / ml-65 mg / ml sucrose; the buffer is 15 mM-25 mM histidine-acetate buffer; the pH value of the preparation is 5.2-5.8; the surfactant is 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml or 0.3 mg / ml of Tween-20, Tween-80 and / or P188.

13. The preparation according to claim 6, characterized in that The preparation is any one of the following preparations: (1) The preparation comprises 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.5; (2) The preparation contains 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.4; (3) The preparation contains 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.6; (4) The preparation contains 160 mg / ml of anti-GDF15 antibody, the stabilizer is 60 mg / ml of sucrose; the buffer is 20 mM histidine-acetate buffer; the surfactant is 0.2 mg / ml of Tween-80 or Tween-20 and 0.1 mg / ml of P188; the pH of the preparation is 5.6; (5) The preparation contains 150 mg / ml of anti-GDF15 antibody, the stabilizer is 65 mg / ml of sucrose; the buffer is 15 mM histidine-acetate buffer; the surfactant is 0.3 mg / ml of Tween-80, Tween-20 or P188; the pH of the preparation is 5.2; (6) The preparation contains 170 mg / ml of anti-GDF15 antibody, the stabilizer is 55 mg / ml of sucrose; the buffer is 25 mM histidine-acetate buffer; the surfactant is 0.1 mg / ml of Tween-80, Tween-20 or P188; and the pH of the preparation is 5.

8.

14. The preparation according to any one of claims 6 to 13, characterized in that The preparation may further comprise an antioxidant. Preferably, the antioxidant is ethylenediaminetetraacetic acid. Preferably, the concentration of the antioxidant is 0-1 mM.

15. The preparation according to any one of claims 6 to 14, characterized in that The preparation is a liquid preparation or a powder for injection.

16. Use of the antibody according to any one of claims 1 to 5 or the preparation according to any one of claims 6 to 15 in the preparation of a medicament for treating a disease; preferably, the disease is a disease and / or condition caused by dysregulation of the GDF15 signaling pathway, such as cancer, cachexia and / or metabolic disease.

17. The use according to claim 16, characterized in that The disease or condition is selected from cancer (such as brain cancer, melanoma, lung cancer, gastrointestinal tumors, colon cancer, pancreatic cancer, prostate cancer, breast cancer, oral cancer, liver cancer, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, bladder cancer, cervical cancer, uterine body cancer, testicular cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharyngeal cancer, laryngeal cancer, pharyngeal cancer, esophageal cancer, etc.), cachexia, renal failure, sepsis, AIDS, cachexia caused by chronic obstructive pulmonary disease, cachexia-related weight loss, nausea and vomiting caused by radiotherapy and chemotherapy, advanced chronic tubulointerstitial nephritis, morning sickness, cirrhosis, sarcopenia, metabolic diseases (such as imbalance of fat and energy metabolism, regulation of appetite, and regulation of weight).

Images