GDF-15 nanoantibody and use thereof

Abstract

The present invention provides a GDF15-binding nanoantibody, and a use thereof in preparation of a drug for treating tumor cachexia and in detection of GDF15. The nanoantibody provided in the present invention exhibits high affinity for GDF-15 and is capable of blocking the binding of GDF-15 to GFRAL.

Description

A GDF-15 nanobody and application thereof

[0001] This application claims priority to the prior application filed on July 19, 2024 with the China National Intellectual Property Office and with the patent application number 2024109687268, and with the invention title of "A GDF-15 nanobody and application thereof". The prior application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and relates to a nanobody molecule capable of neutralizing GDF-15 and the application thereof in anti-tumor crisis. BACKGROUND

[0003] GDF-15 (Growth / differentiation factor 15) is also known as Macrophage inhibitory cytokine 1 (MIC-1), and belongs to the TGF-β superfamily. The molecule is expressed in the form of an inactive precursor and is secreted into the extracellular matrix. After the removal of the propeptide by protease, the active ingredient is released. Mature GDF-15 is a disulfide-bonded homodimer. In 2017, research teams from Novo Nordisc, Eli Lilly and other pharmaceutical companies identified GFRAL (Glial-derived neurotrophic factor receptor alpha-like) as the functional receptor of GDF-15 (Nat. Med 2017; 23: 1158-1166). GFRAL belongs to type I transmembrane proteins. Based on the existing structural information, GFRAL binds to GDF-15 using the D2 domain of its extracellular region. Subsequently, it recruits the RET protein as a co-receptor, and then transduces signals by activating Erk1 / 2 in the cell. Histological studies have shown that GFRAL is specifically distributed in the nucleus of the solitary tract and the area postrema of the brainstem.

[0004] The expression of GDF-15 is strictly regulated under physiological conditions (blood concentration is less than 1 ng / ml), and significant expression can only be detected in tissues such as placenta and fetal membranes. During pregnancy, the expression of GDF-15 gradually increases with the progress of pregnancy and reaches a peak in the late pregnancy; a retrospective study on spontaneous abortion found that the expression level of GDF-15 in pregnant women with abortion during pregnancy was lower (Lancet 2004; 363: 129-30). Therefore, GDF-15 may play an important role in the process of pregnancy, and it is speculated that its significance lies in acting as an immunosuppressive factor to cause the immune system to tolerate the semi-allogeneic embryo, thereby improving the success rate of pregnancy.

[0005] Although the expression level is low under physiological conditions, the expression and secretion of GDF-15 are significantly increased in many malignant tumor cells (such as oral cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, kidney cancer, melanoma, non-small cell lung cancer, etc.). Studies have shown that GDF-15 is one of the diagnostic markers of various malignant tumors, and its blood level is closely related to the development of tumors, poor prognosis and low survival rate (Frontiers in Immunology 2020; 11: Article 951). In terms of pathological mechanism, high levels of GDF-15 may play an immunosuppressive role in the tumor microenvironment, thereby enhancing the survival and immune escape of tumor cells. Studies have shown that the introduction of GDF-15 into the culture system can significantly inhibit the maturation of dendritic cells (DC), manifested as a decrease in cell surface protrusions and membrane surface costimulatory factors; and inhibit the activation of DC to T cells and cytotoxic T lymphocytes (PLoS ONE 2013; 8: e78618). In addition, after down-regulating GDF-15 by RNAi, the sensitivity of glioma to natural killer cells increased, and the glioma showed a higher level of T cell infiltration in vivo (Clin Cancer Res 2010; 16: 3851-9). In addition to immune regulation, GDF-15 is also closely involved in the development of tumor anaplasia by causing anorexia.

[0006] Cancer cachexia is a secondary syndrome that occurs during the development of tumors, and its clinical manifestations are reduced appetite / eating, persistent weight loss (with skeletal muscle loss as the core feature, possibly accompanied by fat loss), and cannot be reversed by nutritional support treatment, eventually leading to physical function impairment and exhaustion. According to statistics, 80% of patients with advanced tumors are accompanied by the occurrence of cachexia, which seriously affects the quality of life, survival time and the implementation of treatment options. From the pathological point of view, cancer cachexia is mainly caused by the negative balance of body energy and material metabolism due to low intake and metabolic changes (increased resting energy consumption, accelerated fat and protein decomposition, etc.); and the abnormal secretion of cytokines from the tumor microenvironment plays an important role in the regulation of dietary and metabolic changes (Nature Reviews Disease Primers 2018; 4: 17105).

[0007] Previously in the study of healthy / obese animal models, the role of GDF-15 in down-regulating diet and causing weight loss through GFRAL has been repeatedly confirmed (Nat Med 2017; 23: 1215-9); at the same time, statistical studies on tumor patients have shown that the blood GDF-15 level of tumor patients rises, and is closely related to weight loss, reduction of limb muscle and fat, and shortening of survival time (J Cachexia Sarcopenia Muscle 2015; 6: 317-24). On this basis, a study on prostate cancer animal models showed that GDF-15 secreted by tumors mediated the cachexia symptoms of mice, and neutralizing GDF-15 could effectively reverse the weight loss of mice (Nat Med 2007; 13: 1333-40). Similarly, blocking the signal transduction of GDF-15 through antibodies targeting GFRAL can also reverse the weight loss of tumor-bearing mice; not only that, the antibody is still effective under the condition of dietary restriction, which suggests that the mechanism of weight recovery may also include metabolic improvement (Nature medicine 2020; 26: 1264-1270). Based on numerous studies, blocking the GDF-15 / GFRAL signaling pathway has become an important strategy for the development of anti-cancer cachexia drugs.

[0008] Currently, several antibody-based inhibitors targeting the GDF-15 / GFRAL signaling pathway have entered clinical research stages, including NGM120 from NGM-bio, Ponsegromab from Pfizer, and Visugromab (CTL-002) from CatalYm. NGM120 inhibits GDF-15 signaling by targeting GFRAL and blocking the recruitment of RET. This molecule has completed clinical phase I research and is indicated for tumor cachexia and solid tumors. Ponsegromab and Visugromab are neutralizing antibodies targeting GDF-15, both of which have entered clinical phase II trials. The former aims to test the improvement of Ponsegromab monotherapy in the symptoms of tumor cachexia, while the latter combines Visugromab with Nivolumab to treat patients with advanced metastatic solid tumors who have failed to respond to PD1 antibody therapy, in order to test the potential improvement of neutralizing GDF-15 on PD1 antibody therapy.

[0009] From the perspective of molecular type, the GDF-15 / GFRAL inhibitors under research internationally are all complete antibodies, which have large molecular weights, low tissue permeability, and relatively complex genetic engineering modification. From the perspective of production, complete antibodies rely on mammalian cell expression systems, which are costly.

[0010] Nanobodies are the smallest known antigen-binding units, with simple molecular structures, strong tissue permeability, and low immunogenicity. In addition, nanobodies are easy to genetically engineer and can be multivalent by simple fusion with other protein drugs. Whether used for developing GDF-15-GFRAL signal blockers alone or combined with other targets to develop multivalent anti-cachexia drugs, nanobodies are a very promising molecular type. Furthermore, nanobodies can be produced through various expression systems, including eukaryotic expression systems, E. coli, and yeast, which have stronger production process flexibility. SUMMARY

[0011] To improve the above technical problems, in one aspect, the present application provides a nanobody binding to GDF15, comprising CDR1-3 selected from the following group:

[0012] (1) SEQ ID NO: 7, 8, 9;

[0013] (2) SEQ ID NO: 10, 8, 11;

[0014] (3) SEQ ID NO: 12, 13, 14;

[0015] (4) SEQ ID NO: 15, 8, 16;

[0016] (5) SEQ ID NO: 17, 8, 18;

[0017] (6) SEQ ID NO: 12, 13, 19.

[0018] In one embodiment of the present application, the amino acid sequence of the aforementioned nanobody has 98% or more identity with any one of SEQ ID NOs: 1-6.

[0019] In one embodiment of the present application, the amino acid sequence of the aforementioned nanobody is as set forth in any one of SEQ ID NOs: 1-6.

[0020] On the other hand, the present application provides a fusion protein fused with the aforementioned nanobody and an Fc fragment.

[0021] In one embodiment of the present application, the aforementioned Fc fragment is derived from hIgG1, and the amino acid sequence thereof is as set forth in SEQ ID NO: 20.

[0022] In one embodiment of the present application, the amino acid sequence of the aforementioned fusion protein has 98% or more identity with any one of SEQ ID NOs: 21-26.

[0023] In one embodiment of the present application, the amino acid sequence of the aforementioned fusion protein is as set forth in any one of SEQ ID NOs: 21-26.

[0024] On the other hand, the present application provides a nucleic acid molecule encoding the aforementioned nanobody or fusion protein.

[0025] On the other hand, the present application provides an expression vector comprising the aforementioned nucleic acid molecule.

[0026] In one embodiment of the present application, the aforementioned expression vector is a pTT5 vector.

[0027] On the other hand, the present application provides a host cell expressing the aforementioned expression vector.

[0028] On the other hand, the present application provides a pharmaceutical composition comprising the aforementioned nanobody, fusion protein, nucleic acid molecule, expression vector, and host cell, and a pharmaceutically acceptable carrier or excipient.

[0029] On the other hand, the present application provides the aforementioned nanobody, fusion protein, nucleic acid molecule, expression vector, and host cell for use in the manufacture of a medicament for treating multiple myeloma, oral cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, esophageal adenocarcinoma, prostate cancer, renal cancer, hepatocellular carcinoma, melanoma, urothelial carcinoma, non-small cell lung cancer, and tumor anaplasia.

[0030] In another aspect, the present application provides use of the aforementioned nanobody or fusion protein in the preparation of a reagent for detecting GDF-15.

[0031] In one embodiment of the present application, the aforementioned reagent is used for detecting the amount of GDF-15 expression in a subject suffering from or suspected of suffering from multiple myeloma, oral cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, esophageal adenocarcinoma, prostate cancer, renal cancer, hepatocellular carcinoma, melanoma, urothelial carcinoma, non-small cell lung cancer, and tumor anaplasia.

[0032] In another aspect, the present application provides a kit for detecting GDF-15, comprising the aforementioned nanobody, fusion protein, or reagent. Advantages

[0033] The nanobody provided by the present application is a nanobody molecule having high affinity to GDF-15 and capable of blocking the binding of GDF-15 to GFRAL; the molecule has comparable anti-tumor anaplasia activity to Ponsegromab in a mouse model. BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1 shows the results of detecting the antigen binding specificity of the nanobody-Fc fusion protein by SPR.

[0035] FIG. 2 shows the results of detecting the anti-anaplasia efficacy of the nanobody-Fc fusion protein.

[0036] FIG. 3 shows the results of detecting the PK of the nanobody-Fc fusion protein in rat serum. DETAILED DESCRIPTION

[0037] Definitions

[0038] Antibody proteins obtained from camels and dromedary family members, including New World members such as the llama species (Lama paco, Lama glama, and Lama vicugna), have been characterized with respect to size, structural complexity, and antigenicity in human subjects. Certain IgG antibodies from this family of mammals lack light chains, and thus exhibit a significantly different structure from the typical four-chain structure of antibodies from other animals, which have two heavy chains and two light chains. See PCT / EP93 / 02214 (WO94 / 04678 published March 3, 1994).

[0039] The single variable region domain of a camelid heavy chain antibody, i.e., VHH, can be genetically engineered to obtain a small protein with high affinity to a target, is the smallest unit known to bind an antigen of interest, and is referred to as a "camelid nanobody." See U.S. Patent 5,759,808, issued June 2, 1998; also see Stijlemans, B., et al., 2004, J Biol Chem, 279: 1256-1261; Dumoulin, M., et al., 2003, Nature, 424: 783-788; Pleschberger, M., et al., 2003, Bioconjugate Chem, 14: 440-448; Cortez-Retamozo, V., et al., 2002, Int J Cancer, 89: 456-62; and Lauwereys, M., et al., 1998, EMBO J, 17: 3512-3520. Libraries of camelid antibodies and antibody fragments can be purchased, for example, from Ablynx, Ghent, Belgium. The amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that is more similar to a human sequence, i.e., the nanobody can be humanized. Thus the naturally low antigenicity of a camelid antibody to a human can be further reduced.

[0040] A camelid nanobody has a molecular weight that is about one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is that a nanobody can bind to an antigenic site that is not recognized by a larger antibody protein, i.e., a camelid nanobody can be used as a reagent to detect an antigen that is hidden from a classical immunization technique. A camelid nanobody can also be used as a potential therapeutic agent. Another consequence of the small size is that a camelid nanobody can bind to a specific site in a groove or cleft of a target protein structure to produce an inhibitory or agonistic effect that more closely mimics the function of a classical low molecular weight drug than a classical high molecular weight antibody.

[0041] A camelid antibody or nanobody with high affinity to the target protein GDF15 is provided in the present application. In certain embodiments herein, a camelid antibody or nanobody is naturally produced in a camelid, i.e., produced in a camelid after immunization with the target protein GDF15 or a peptide fragment thereof using techniques described herein. On this basis, a camelid nanobody against GDF15 is screened, i.e., produced by selection from a phage library using a panning method, with GDF15 as the target, using phage displaying appropriately mutagenized camelid nanobody proteins as described in the Examples herein. The nanobody can be further engineered by protein engineering to have a half-life of 45 minutes to one week in a recipient subject.

[0042] As used herein, the sequence "identity" has the art-recognized meaning and the percent of sequence identity between two nucleic acid or polypeptide molecules can be calculated using published techniques. Sequence identity can be aligned over the full length or a portion of the nucleic acid or polypeptide. While there are a variety of methods of aligning nucleic acid or polypeptide sequences, the term "identity" is art-recognized (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0043] It will be appreciated that the foregoing nanobodies or fusion proteins will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated to provide improved transfer, delivery, tolerance, etc. Numerous appropriate formulations can be found in the formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th Ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMS M102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous abs, oil-in-water and water-in-oil emulsions, emulsions in polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the foregoing mixtures can be suitable for use in the treatment or therapy according to the present application, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerable to the route of administration.

[0044] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively and explain the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[0045] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0046] Example 1: Construction of a llama immunization and nanobody phage display library

[0047] Human GDF-15 mature fragment (Uniprot ID: Q99988, Ala197-Ile308) was expressed and purified as reported in the literature (Nature 2017; 550: 255-259) using the antigen, and the final product purity was detected by HPLC-SEC to reach 99.8%. The present application entrusted Jiangsu Aber Biotechnology Co., Ltd. to carry out alpaca immunization, phage display library construction and screening. Immunization was carried out according to the following method: 2 alpacas (about 2 years old) that had not been immunized with other antigens were selected, and immunization was carried out 4 times at a frequency of 2 weeks. The immunization dose was 1 mg / time / one, and the alpacas were immunized subcutaneously on the back with GDF-15 protein and adjuvant emulsified uniformly. The first immunization used Freund's complete adjuvant, and the subsequent ones used Freund's incomplete adjuvant.

[0048] After 4 immunizations, 100 mL of peripheral blood was collected from each of the 2 alpacas for total RNA extraction, and the product RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions (Kangwei Century, HiFiScript cDNA Synthesis Kit). Then, the cDNA was used as a template to amplify the nanobody gene fragment. The amplified fragment and the pcomb3X vector were respectively digested with sfiI enzyme, gel recovered, and then connected with T4 DNA ligase. The connection product was incubated with XL1-Blue competent cells, then electroporated, and then the transformed product was recovered in 200 ml of 2YT medium containing ampicillin and tetracycline for 1 h (37°C, 250 rpm) to obtain a nanobody bacterial library (titer 9.8 x 10 8 ).

[0049] The bacterial library was centrifuged after adding VCSM13 helper phage and continuing to culture for 1 h, resuspended in 100 ml of 2YT-ATK medium (containing ampicillin, tetracycline and kanamycin), and cultured at 30°C, 225 rpm overnight. The next day, the supernatant was collected, 4% PEG800 and 3% NaCl were added to the ice bath, and the phage precipitate was collected by centrifugation. The obtained phage was resuspended with PBS and filtered to remove bacteria, and then frozen at -80°C after adding 7% DMSO to obtain a nanobody phage display library (phage concentration 3.02 x 10 12 / mL).

[0050] Example 2: Screening of nanobody phage display library

[0051] The ELISA plate was coated with 10 μg / ml GDF-15 antigen overnight, washed with PBS containing 0.1% Tween20 (PBST) after coating, and blocked with 3% skim milk powder. 5 x 10 12The library of phage particles was incubated at 37°C for 2 h; after washing with PBST, the bound phage was eluted with Glycine-HCl (pH 2.2) and neutralized to pH 7.4 with Tris-HCl. After the eluate infected E. coli XL1-Blue, helper phage was added for rescue culture, and the library after the first round of selection was obtained. The same method was repeated for the second round of selection.

[0052] Single clones were selected from the library after two rounds of selection, and the supernatant after phage expression was used for ELISA detection: the enzyme-labeled plate was coated with 0.5 μg / ml GDF-15 at 4°C overnight, and then milk powder blocking was performed (the enzyme-labeled plate without coating antigen was used as a negative control); the phage expression supernatant was added for incubation at room temperature for 1 h, and then PBST washing was performed, followed by the addition of horseradish peroxidase-labeled anti-M13 antibody for incubation at room temperature for 1 h; after washing, TMB (3,3',5,5'-Tetramethylbenzidine) was used for color development, and the enzyme-labeled instrument was read at 450 nm. Strong positive clones (signal more than 40 times higher than the negative control) were selected for sequencing, and the gene sequence of the GDF-15 nanobody was obtained. By sequence alignment of the intermolecular CDR1-3 segments, 6 nanobody molecules with large differences were selected for subsequent analysis and identification, and the amino acid sequences are shown in Table 1.

[0053] Table 1 Amino acid sequences of the nanobody and CDR in the application (CDR is defined according to the alignment results in the www.ncbi.nlm.nih.gov / igblast / database in the IMGT manner)

[0054] Example 3: Construction of expression vector and expression of nanobody-Fc fusion protein

[0055] The application entrusted Suzhou Jinyuizhi Biotechnology Co., Ltd. to synthesize the coding sequences of the GDF-15 nanobody (SEQ ID NO. 1-6), the GFRAL-D2 domain (Uniprot ID: Q6UXV0, Trp129-Asn213), and the human IgG1-Fc region (SEQ ID NO. 20), respectively. According to the operation method in the Guide to Molecular Cloning, the nanobody or the GFRAL-D2 fragment was spliced with the Fc, respectively, and then connected to the pTT5 vector to form an expression vector carrying the coding sequence of the nanobody-Fc or GFRAL-D2-Fc. The nanobody molecule fused with the Fc was numbered as PM1176-PM1181 (SEQ ID NO. 21-26), and the amino acid sequences are shown in Table 2.

[0056] Table 2 Amino acid sequences of the nanobody-Fc fusion protein in the application

[0057] The constructed expression vector is transformed into DH10B competent cells, and the bacterial solution is cultured for preparing transient transfection expression of the required plasmid; and the plasmid is extracted according to the method in the Qiagen Endofree Maxi-prep Kit. The transient transfection expression is performed according to the standard operation procedure: the Expi293 cell suspension with a density of 2.0-2.5 x 10 6 / mL is prepared according to the required volume of expression; the input amount of plasmid and PEI is calculated according to the working concentration of 1 μg / mL of plasmid and 2 μg / mL of PEI in the final culture system, and the plasmid and PEI are diluted with 1 / 30 volume of Opti-MEM medium (Gibco) of the culture system at room temperature for 10 minutes; then the transfection reagent is added to the plasmid reagent. After incubation at room temperature for 10 min, the mixed solution is added to the cell suspension, and the culture is cultured in a shaker at 115 rpm, 36.8°C, and 5% CO2. After 24 hours, 50x KT Feed is added to a final concentration of 1x, and then the culture is continued in the shaker for 5-7 days, and the cell supernatant is collected by centrifugation at 8500 rpm for 15 min for purification.

[0058] Example 4: Purification and analysis of nanobody-Fc fusion proteins

[0059] All nanobody-Fc fusion proteins and GFRAL-D2-Fc in the present application are purified by two-step purification of Protein A affinity chromatography and molecular sieve. The specific operation steps are as follows: the centrifuged and clarified cell culture supernatant is added with sodium chloride to a final concentration of 0.3 M, and adjusted to pH 7.5 with 1 M Tris-HCl buffer. The AT Protein A Diamond purification column (Bio-Rad) is equilibrated with 2x PBS pH 7.4 for 5 column volumes, and then the sample is flowed through to capture the target protein; then 5 column volumes of equilibration buffer are used to rinse the purification column to remove host proteins, DNA and other non-specifically bound impurities. Finally, the target protein is eluted with a buffer containing 100 mM glycine, 100 mM arginine, 150 mM sodium chloride, pH 3.0, and the eluate is quickly neutralized to pH 8.0 with 1 M Tris pH 9.0.

[0060] Molecular sieve (Hiload Superdex 200 16 / 600 pg, Cytiva) was equilibrated with 1x PBS pH 7.4 for 1 column volume. Eluate of target protein was concentrated to 3 ml by 30 kDa Amicon centrifugal filter device (Millipore) and then purified by molecular sieve at a flow rate of 1.0 ml / min, while removing the target protein oligomers and replacing the sample buffer with 1x PBS pH 7.4. The purified target protein was filtered through a 0.22 pm filter to remove bacteria, and then the purity was determined by HPLC-SEC, and the endotoxin content was determined by the Limulus reagent method according to the reagent instruction (Zhanjiang Andus Biological Co., Ltd., item number KC-125). The protein purity of all nanobody-Fc was greater than 95%, and the endotoxin was less than 0.1 EU / mg.

[0061] Example 5: Test of affinity of nanobody-Fc to GDF-15 and the ability to block the interaction of GDF-15-GFRAL

[0062] To test the affinity level of nanobody to antigen, the present application used SPR (Surface Plasma Resonance. Equipment: Biacore SPR 8K, Cytiva) to determine the affinity of nanobody-Fc fusion protein to human, mouse, and cynomolgus monkey GDF-15 of three species; among them, mouse and cynomolgus monkey GDF-15 were purchased from Peprotech (item number GD5-M5149, GD5-C5148). The specific detection method is as follows: different species of GDF-15 were respectively immobilized on the surface of CM5 chip (Cytiva, BR100530) at a concentration of 2.5 pg / ml, and the immobilization amount was about 200 RU. The flow phase of the experiment was PBS containing 0.05% Tween 20, pH 7.4. For the affinity detection of human and cynomolgus monkey derived GDF15, the highest detection concentration of the tested antibody was 2 nM; for mouse derived GDF15, the highest concentration of the tested antibody was 20 nM. Each tested molecule was diluted 3 times in series with the flow phase buffer, and there were 6 concentrations including zero concentration. Each dilution sample was sequentially flowed through the chip surface to collect the binding-dissociation signal, and the equilibrium dissociation constant was obtained by kinetic fitting (1:1 binding) by the analysis software of the equipment.

[0063] The blocking experiment of the nano antibody-Fc on the GDF-15-GFRAL interaction was also performed by the SPR method, and the specific method was as follows: the nano antibody to be tested was adjusted to a concentration of 50 nM with a mobile phase buffer, and flowed through a CM5 chip so that the surface-fixed human GDF-15 was saturated with the antibody; then 50 nM nano antibody and 50 nM GFRAL-D2-Fc were mixed to form an analysis object, and the binding signal of GFRAL-D2 to GDF-15 in the presence of the antibody was obtained. Then the response signal generated by the interaction of 50 nM GFRAL-D2 with GDF-15 in the same channel was combined to obtain the signal inhibition rate of the nano antibody to the GDF-15-GFRAL interaction; the molecules with an inhibition rate of more than 50% were determined to have the blocking ability to the GDF-15-GFRAL interaction. The SPR detection results of each nano antibody-Fc molecule are shown in Table 3. The results show that each nano antibody-Fc has a strong affinity for human GDF15, and is equivalent to the positive control molecule (Ponsegromab, Pfizer company); each molecule has a significant competitive effect on the interaction of human GDF15 and GFRAL-D2.

[0064] Table 3: Affinity of nano antibody-Fc to human GDF-15 and blocking ability to GDF-15-GFRAL binding

[0065] Example 6: Antigen binding specificity detection of nano antibody-Fc

[0066] In order to analyze the binding specificity of the nano antibody-Fc molecule to GDF-15, the present application used the SPR method to test the interaction of the target antibody with TGF-β family proteins TGF-β1, BMP-2 and Activin A (Pfizer company, TG1-H4212, BM2-H4117, ACA-H421b) in parallel. The specific method was as follows: the nano antibody-Fc molecule was flowed through the Protein A chip for ligand capture, and then 2 nM of different types of TGF-β protein was flowed through, and the binding time was 250 s; the signals generated at the end of the binding were selected to form a column chart (Figure 1). The experimental results show that each nano antibody-Fc molecule only binds to GDF-15, and has good antigen specificity.

[0067] Example 7: Stability test of nano antibody-Fc fusion protein

[0068] In order to compare the drugability of each nano antibody-Fc molecule, the present application evaluated the stability of the target molecule from three aspects of acid treatment, freeze-thaw and thermal stability. The specific method was as follows:

[0069] 1. Acid treatment stability test

[0070] The concentration of the molecule to be tested was adjusted to 2.0 mg / ml with PBS pH 7.4 as the background buffer. The sample was adjusted to pH 3.6 with 1M glycine, pH 2.5, and incubated at room temperature for 4 hours. The pH was then adjusted to neutral with 1M Tris pH 9.0, and the changes in the target molecule aggregates (HMW) before and after acid treatment were detected by HPLC-SEC (the analysis column was Superdex200 increase, 10 / 300 GL, and the buffer was PBS pH 7.4). The results are shown in Table 4.

[0071] 2. Freeze-thaw stability test

[0072] The sample concentration and background buffer were consistent with the acid treatment experiment. 200 μl of sample was placed in a -80°C freezer for 60 minutes, then taken out and placed at room temperature for 30 minutes to fully thaw. After repeating the freeze-thawing 3 times, the degree of aggregation of the target molecule (HMW) was detected by HPLC-SEC. The results are shown in Table 4.

[0073] Table 4. Results of acid treatment and freeze-thaw stability test of nanobody-Fc

[0074] 3. Accelerated thermal stability test at 37°C

[0075] Each nanobody-Fc and control antibody was replaced into 20 mM histidine pH 6.6, 150 mM sodium chloride buffer, and the concentration was adjusted to 14.0 mg / ml. The sample was incubated at 37°C for 28 days, and the changes in aggregates (HMW) and degradation products (LMW) before and after treatment were detected by HPLC-SEC. The affinity of the sample after incubation for human GDF-15 was detected by the SPR method in Example 5. The results are shown in Table 5.

[0076] Based on the above stability test results, PM1177-PM1180 showed good stability and could tolerate acid treatment and repeated freeze-thawing. Their performance in the accelerated thermal stability test was comparable to that of the positive molecule.

[0077] Table 5. Results of thermal stability test of nanobody-Fc

[0078] Example 8: Anti-cachexia efficacy experiment of nanobody-Fc fusion protein

[0079] The anti-cachexia efficacy of the nanobody was evaluated using a HT-1080 (human fibrosarcoma cell) mouse model. This cell grows rapidly in vivo and secretes a large amount of human GDF-15, causing a decrease in body weight and food intake in mice, thereby simulating the symptoms of tumor cachexia. The specific method is as follows: 5 x 10 6BALB / c female nude mice of 7 weeks old were inoculated subcutaneously with HT-1080 cells on the back; the mice were weighed at regular time intervals and the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume was calculated. The calculation formula is: tumor volume TV (mm 3 ) = 0.5 x long diameter (mm) x short diameter (mm) 2 ; the tumor-free body weight of the mouse was the total body weight minus the tumor weight (the tumor weight was calculated as volume x 1 g / cm 3 ). After 14 days, the average tumor-free body weight of the mice decreased to 90% of the initial body weight (the body weight on the day of inoculation of tumor cells was taken as the initial body weight); at this time, the mice were randomly divided into groups (6 mice per group), and PBS (negative control group), 10 mg / kg Ponsegromab (positive control group) or 5 mg / kg nanoantibody-Fc were injected intraperitoneally at a frequency of twice a week, for a total of 3 doses. The body weight of the mice, the tumor size and the tumor-free body weight were measured every day after the start of administration. The ratio of tumor-free body weight to initial body weight was plotted against the number of days after inoculation, and the results are shown in Figure 2. The experimental results show that the efficacy of each nanoantibody-Fc molecule is comparable to that of Ponsegromab.

[0080] Example 9: Rat PK experiment

[0081] Female Sprague-Dawley rats were divided into groups of 6, and a single intravenous injection of nanoantibody-Fc was administered at a dose of 5 mg / kg; at the same time as administration, a zero-time blood sample was collected, and then blood samples were collected at 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h in sequence, and the concentration of nanoantibody-Fc in the serum was detected by ELISA. The results are shown in Figure 3.

[0082] The above describes embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A Nanobody binding to GDF15, comprising CDR1-3 selected from the following group: (1) SEQ ID NO: 7, 8, 9; (2) SEQ ID NO: 10, 8, 11; (3) SEQ ID NO: 12, 13, 14; (4) SEQ ID NO: 15, 8, 16; (5) SEQ ID NO: 17, 8, 18; (6) SEQ ID NO: 12, 13, 19. 2.The Nanobody of claim 2, having an amino acid sequence of 98% or more identical to any one of SEQ ID NO: 1-6. Preferably, the Nanobody has an amino acid sequence as set forth in any one of SEQ ID NO: 1-6. 3.A fusion protein fused with the Nanobody of claim 1 or 2 with an Fc fragment. Preferably, the Fc fragment is derived from hIgG1, and has an amino acid sequence as set forth in SEQ ID NO:

20. 4.The fusion protein of claim 3, having an amino acid sequence of 98% or more identical to any one of SEQ ID NO: 21-26. Preferably, the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NO: 21-26. 5.A nucleic acid molecule encoding the Nanobody of claim 1 or 2, or the fusion protein of claim 3 or 4. 6.An expression vector comprising the nucleic acid molecule of claim 5. Preferably, the expression vector is pTT5 vector. 7.A host cell expressing the expression vector of claim 6. 8.A pharmaceutical composition comprising the Nanobody of claim 1 or 2, the fusion protein of claim 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7, and a pharmaceutically acceptable carrier or excipient. 9.Use of the Nanobody of claim 1 or 2, the fusion protein of claim 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7 in the preparation of a medicament for treating multiple myeloma, oral cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, esophageal adenocarcinoma, prostate cancer, renal cancer, hepatocellular carcinoma, melanoma, urothelial carcinoma, non-small cell lung cancer, and tumor anaplasia. 10.Use of the Nanobody of claim 1 or 2, or the fusion protein of claim 3 or 4 in the preparation of a reagent for detecting GDF-15. Preferably, the reagent is used for detecting the expression amount of GDF-15 in a subject suffering from or suspected to suffer from multiple myeloma, oral cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, esophageal adenocarcinoma, prostate cancer, renal cancer, hepatocellular carcinoma, melanoma, urothelial carcinoma, non-small cell lung cancer, and tumor anaplasia.

11. A kit for detecting GDF-15 comprising the Nanobody of claim 1 or 2 or the fusion protein of claim 3 or 4.

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