Antibodies targeting gfralpha3, methods of making and use in anticancer

Abstract

Provided in the present application are antibodies targeting GFRa3, methods for preparing the same and uses thereof in anti-cancer. Specifically, the present application relates to an antibody or antigen-binding fragment thereof targeting GFRa3, wherein the antibody comprises heavy chain complementarity determining regions (VH CDRs) and light chain complementarity determining regions (VL CDRs), which have sequences selected from the group consisting of SEQ ID NOs: 1-6, respectively. The present application also relates to nucleic acid molecules, vectors or host cells, products associated with the antibody or antigen-binding fragment thereof, and uses thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine. In particular, the present application relates to antibodies targeting GFR3a and methods of making the same and use thereof in anticancer. BACKGROUND

[0002] Tumor is one of the major diseases that seriously endanger the health and life of human beings and other mammals. For example, liver cancer is one of the most common malignant tumors and is one of the major factors of global cancer death. At present, the main methods for treating liver cancer are still mainly surgery, radiotherapy, chemotherapy, etc., but the effects of these traditional treatments are often unsatisfactory. Most patients are in the advanced stage of liver cancer when they seek medical treatment, which does not meet the indications for surgical treatment and has high postoperative recurrence rate, etc.; and most liver cancer patients have abnormal liver function and poor tolerance to radiotherapy and chemotherapy.

[0003] In recent years, biological therapy of tumor, such as targeted therapy and immunotherapy, has achieved remarkable effects in tumor treatment. Among them, immunotherapy (such as immune checkpoint inhibitors, cellular immunotherapy, antibody immunotherapy, non-specific immunomodulators, etc.) has achieved good effects in clinical trials of tumor. However, these biological therapy methods also face key bottleneck problems, such as toxic side effects, low clinical response rate, etc. For example, the effective rate of targeted checkpoint antibody therapy in the immunotherapy of liver cancer is only 15-20% (Lancet 2017, 389:2492-2502). Therefore, the development of new immunotherapy drugs and the search for new therapeutic targets are of great significance for the treatment of tumor and the reduction of toxic side effects of drugs.

[0004] GFRa3 is a receptor of Artemin (ARTN), a glial cell line-derived neurotrophic factor ligand, which activates RET signaling through receptor-ligand binding, promotes tumor progression and metastasis. In acute myeloid leukemia (AML), the ARTN / GFRa3 complex inhibits autophagy of AML cells by activating the RET-mTORC1 signaling pathway, promoting the progression of AML (Leukemia, 2018, 32(10): 2189-2202). ARTN-GFRa3-ERK signaling can promote the proliferation and invasion of pancreatic ductal adenocarcinoma (PDAC) cells and can be used as a diagnostic marker for PDAC (Int J Cancer, 2021, 148(7): 1756-1767). GFRa3 promotes the migration and invasion of urothelial carcinoma (UC) cells, and its expression is negatively correlated with the overall survival rate of UC (Minerva Urol Nefrol, 2018, 70(1): 79-86). In hepatocellular carcinoma, GFRa3 expression and the signals mediated thereby are negatively correlated with the prognosis of hepatocellular carcinoma, and the receptor GFRa3 expressed by hepatocellular carcinoma cells binds to a large amount of ARTN produced by Ter cells, promoting the growth and metastasis of hepatocellular carcinoma (Cell, 2018, 173(3): 634-648), suggesting that GFRa3 can be an important target for hepatocellular carcinoma.

[0005] In summary, antibody immunotherapy targeting GFRa3 has important application prospects and significance in the clinical treatment of tumors such as hepatocellular carcinoma. SUMMARY

[0006] The present application provides an antibody and antigen-binding fragment thereof targeting GFRa3, which has important application prospects and significance in the clinical prevention, treatment and / or detection of tumors.

[0007] In a first aspect of the present application, an antibody or antigen-binding fragment thereof targeting GFRa3 is provided, wherein the antibody comprises heavy chain complementarity determining regions (VH CDRs) and light chain complementarity determining regions (VL CDRs), and the VH CDRs and VL CDRs have amino acid sequences selected from the group consisting of: VH CDR1: SEQ ID NO: 1; VH CDR2: SEQ ID NO: 2; VH CDR3: SEQ ID NO: 3; VL CDR1: SEQ ID NO: 4; VL CDR2: SEQ ID NO: 5; and VL CDR3: SEQ ID NO: 6.

[0008] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which has an amino acid sequence selected from the group consisting of: VH: (a) the sequence set forth in SEQ ID NO: 7; or (b) a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 7; and VL: (a') the sequence set forth in SEQ ID NO: 8; or (b') a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 8.

[0009] In some embodiments, the antibody of the present application comprises a combination of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the antibody of the present application comprises a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8. In some embodiments, the VH of the antibody of the present application has at least 80% sequence identity to the sequence set forth in SEQ ID NO: 7 and / or the VL of the antibody has at least 80% sequence identity to the sequence set forth in SEQ ID NO: 8, and the difference in amino acid residues is not in the CDRs, and the antibody has the activity of targeting and blocking GFRa3. In some embodiments, the VH and / or VL of the present application comprises one or more substitutions of amino acid residues in its non-CDR regions (e.g., humanization design and substitutions).

[0010] The antibody sequences can be numbered using antibody numbering systems known in the art, such as the Kabat, Chothia, IMGT, AHo, etc. numbering systems. The IMGT numbering system is used in the present application. It is understood that other numbering systems can also be used to number the antibodies of the present application without departing from the spirit of the present application.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof of the present application is selected from the group consisting of: a monoclonal antibody, a polyclonal antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a disulfide bonded Fv, a V-NAR domain, an IgNar, an intrabody, an IgGACFh, a minibody, a F(ab')3, a tetrabody, a triabody, a bispecific antibody, a single domain antibody, a DVD-Ig, a Fcab, a mAb2, a (scFv)2, or a scFv-Fc.

[0012] In some embodiments, the antibody of the present application has one or more features selected from the group consisting of:

[0013] (i) it specifically binds to GFRa3, for example, has a binding affinity K D of 0.1 nM to 0.2 nM as determined by surface plasmon resonance (SPR);

[0014] (ii) specifically blocks the binding of ARTN to GFRa3, for example, by ELISA assay, its IC50 for blocking the binding of ARTN to GFRa3 is less than 200 nM; 50 is 200 nM to 400 nM;

[0015] (iii) it can inhibit the proliferation and / or invasion of cancer cells, especially cancer cells (such as liver cancer cells) whose proliferation and / or invasion are associated with the activation of ARTN-GFRa3 pathway.

[0016] In some aspects of the application, there is provided a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the application.

[0017] In some embodiments, the nucleic acid molecule of the application comprises one or more sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, the nucleic acid molecule of the application comprises the sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or, SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, the nucleic acid molecule of the application is codon-optimized.

[0018] In some aspects of the application, there is provided a vector or host cell comprising a nucleic acid molecule of the application.

[0019] In some aspects of the application, there is provided a product comprising an antibody or antigen-binding fragment thereof, a nucleic acid molecule, and / or a vector or host cell of the application.

[0020] In some aspects of the application, there is provided the use of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector or host cell of the application in the manufacture of a product, wherein the product:

[0021] (a) is for preventing and / or treating an ARTN-GFRa related disease (e.g. solid tumor such as liver cancer, pancreatic cancer (e.g. pancreatic ductal adenocarcinoma), urothelial cancer (e.g. bladder cancer, renal pelvis cancer and ureter cancer); non-solid tumor such as leukemia (e.g. acute myelocytic leukemia)); and / or

[0022] (b) is for detecting the presence, level or activity of a binding product of ARTN, GFRa3 or both.

[0023] In some embodiments, the product of the present application is a medicament or a kit.

[0024] In some aspects of the present application, a method for treating a tumor is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, and / or a vector or host cell, or a product of the present application.

[0025] The foregoing technical solutions and technical features can be combined by those skilled in the art without departing from the inventive concept and protection scope of the present application. Other aspects of the present application are apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0026] The present application is further described below in conjunction with the accompanying drawings, which are shown only for the purpose of illustrating embodiments of the present application and not for limiting the scope of the present application.

[0027] Figure 1 Flow sorting of antigen (GFRa3)-specific memory single B cells.

[0028] Figure 2 Dot blot detection of GFRa3 antibody specificity.

[0029] Figure 3 Polyacrylamide gel electrophoresis detection of GFRa3 #119 antibody purity and molecular weight size.

[0030] Figure 4 SPR detection of GFRa3 #119 antibody affinity.

[0031] Figure 5 Detection of GFRa3 #119 antibody blocking effect.

[0032] Figure 6 Detection of GFRa3 #119 antibody-mediated ADCC effect; left panel: test against 7721 cells; right panel: test against Hepal-6-hGFRa3 cells.

[0033] Figure 7 CCK8 detection of GFRa3 #119 antibody in vitro inhibition of HepG2 cell proliferation.

[0034] Figure 8 CCK8 detection of GFRa3 #119 antibody in vitro inhibition of 7721 cell proliferation.

[0035] Figure 9 Blocking effect of GFRa3 #119 antibody on ARTN-mediated promotion of liver cancer invasion. DETAILED DESCRIPTION

[0036] The present inventors have obtained a monoclonal antibody against GFRa3 through long-term and in-depth research. The preparation and identification of the novel anti-GFRa3 antibody can include antigen immunization, single B cell sorting, single cell sequencing, vector construction, antibody screening, CHO-S expression and purification, affinity detection, antibody blocking effect, functional GFRa3 antibody screening, and antibody anti-tumor function detection, etc.

[0037] It is proved in the examples in the present application that the antibody targeting GFRa3 of the present application can specifically bind to GFRa3 and block the binding of GFRa3 to ARTN, thereby inhibiting the proliferation and invasion of tumor cells. Therefore, the antibody of the present application has a wide prospect in the prevention, treatment and / or detection of tumors, especially tumors related to the ARTN-GFRa3 signaling pathway.

[0038] All numerical ranges expressed in the present application in terms of "from X to Y" are intended to include each and every number and range falling within the range expressed. The disclosure of any document, reference, or article herein is not an admission that any of the foregoing is prior art. All references disclosed herein are expressly incorporated by reference. All document, reference, or article disclosures are expressly incorporated by reference. All combinations of the items disclosed herein can also be encompassed.

[0039] As used herein, "containing," "having," or "including" comprises "comprising," "consisting essentially of," "consisting substantially of," and "consisting of"; "consisting essentially of," "consisting substantially of," and "consisting of" are subsumed by "containing," "having," or "including."

[0040] Specific antibodies of the present application and preparation thereof

[0041] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150 kD with the same structural characteristics, which is usually composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regular interval intrachain disulfide bonds. Each heavy chain can comprise a heavy chain variable region (VH); each light chain can comprise a light chain variable region (VL).

[0042] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and the heavy chain variable regions. The more conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely β-sheet in structure, connected by three CDRs, which form loops that connect, and in some cases pack against, the FRs of the same chain and the CDRs of the other chain. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, form the antibody's antigen binding site (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)).

[0043] The antibodies of the present application comprise heavy chain complementarity determining regions (VH CDRs) and light chain complementarity determining regions (VL CDRs), which can be selected from the group consisting of: a VH CDR1 of SEQ ID NO: 1; a VH CDR2 of SEQ ID NO: 2; a VH CDR3 of SEQ ID NO: 3; a VL CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 5; and a VL CDR3 of SEQ ID NO: 6, respectively. In some embodiments, the antibodies of the present application have a combination of the six CDRs set forth in SEQ ID NOs: 1-6.

[0044] In some embodiments, the antibodies of the present application comprise a heavy chain variable region (VH) and a light chain variable region (VL), which can have sequences selected from the group consisting of: a VH having a sequence as set forth in SEQ ID NO: 7 or at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to SEQ ID NO: 7; and a VL having a sequence as set forth in SEQ ID NO: 8 or at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to SEQ ID NO: 8. Those of ordinary skill in the art will appreciate that, with the CDRs being unchanged, a small number of amino acid residue changes in the VH and / or VL can be tolerated without affecting or significantly affecting the overall performance of the antibody, e.g., one or more amino acid residues in the non-CDR regions of the VH and / or VL can be substituted to achieve humanization, etc.

[0045] As used herein, the term "sequence identity" or "% identity" refers to the percentage of identical residues (e.g., amino acids or nucleic acids) in a candidate sequence to a reference sequence after aligning the sequences and, if necessary, introducing gaps to obtain the maximum percentage of sequence identity. For example, as used herein, "at least 80% sequence identity" means that the sequence identity between a candidate sequence and a reference sequence is at least 80%, such as 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any numerical point or range therein.

[0046] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two major types, called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chains of different classes of immunoglobulins have different constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally (see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Meade E. Terr, and Richard R. Rich, eds., Appleton & Lange, 1994, Chapter 10). In some embodiments of the application, the light chain of an antibody of the application is of the kappa or lambda type, preferably the kappa type. In some embodiments of the application, the heavy chain of an antibody of the application is of the alpha, delta, epsilon, gamma, or mu type, preferably the gamma type. In some embodiments of the application, an antibody of the application is of the IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 class, preferably the IgG1 class.

[0047] The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, unfettered by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Preferably, the antibodies of the application are monoclonal antibodies.

[0048] Monoclonal antibodies can be produced by a variety of methods well known to those skilled in the art. For example, methods for producing monoclonal antibodies include, but are not limited to, the hybridoma method (Kohler et al., Nature, 256:495 (1975)); recombinant DNA methods (U.S. Patent No. 4,816,567); phage display technologies (Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)); and, single B cell antibody production technology (Chu et al., Biotechnol. Prog., 2012, 28(6):651-660). In some embodiments of the present application, it is preferred to use single B cell antibody production technology to produce the antibody library. This technology retains the natural pairing of light and heavy chain variable regions, has the advantages of good genetic diversity, high efficiency, fully human, and less required cell amount.

[0049] The antibodies or antigen-binding fragments thereof in the present application can include, but are not limited to, monoclonal antibodies, polyclonal antibodies, Fab, Fab', F(ab')2, Fd, scFv, disulfide-bond linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, bispecific antibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0050] In some embodiments, the antibodies of the present application are monoclonal antibodies, humanized antibodies, such as selected from the group consisting of chimeric antibodies, CDR-grafted antibodies, homologous substitution antibodies, resurfaced antibodies, complementation changed antibodies, and site-directed conserved humanization antibodies. In some embodiments, the constant region (C region) of the antibodies of the present application is human or non-human mammal (e.g., murine) constant region.

[0051] In some embodiments, the antibodies of the present application have one or more characteristics selected from the group consisting of:

[0052] (i) it specifically binds to GFRa3, for example, with a binding affinity K D of 0.1 nM to 0.2 nM;

[0053] (ii) it specifically blocks the binding of ARTN to GFRa3, for example, with an IC 50 of 200 nM to 400 nM;

[0054] (iii) it can inhibit the proliferation and / or invasion of cancer cells, especially cancer cells (such as liver cancer cells) activated by the GFRa3-ARTN pathway.

[0055] Encoding molecules of the antibodies or functional fragments thereof, expression vectors containing the molecules and host cells

[0056] Also provided in the present application are nucleic acid molecules encoding the anti-GFRa3 antibody or fragment thereof. The sequences of these nucleic acid molecules can be obtained using conventional techniques, such as PCR amplification or screening of genomic libraries. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single chain antibody.

[0057] In some embodiments, the nucleic acid molecules of the present application can comprise a nucleotide sequence encoding a VH CDR and / or a VL CDR. In some embodiments, the nucleic acid molecules of the present application can comprise a nucleotide sequence as set forth in SEQ ID NO: 9, 10, 11, 12, 13, and / or 14, or a nucleotide sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the sequence. In some embodiments, the nucleic acid molecules of the present application can comprise a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 15, or a nucleotide sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to SEQ ID NO: 15; and / or a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 16, or a nucleotide sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to SEQ ID NO: 16. Those of ordinary skill in the art will appreciate that the coding sequences can be codon-optimized, or otherwise manipulated, to introduce small changes in nucleotide residues without affecting or significantly affecting the overall performance of the encoded antibody.

[0058] In some embodiments, the present application also discloses expression vectors containing the above-mentioned nucleotide sequences. In some embodiments, the expression vectors can be selected from, but not limited to, pFUSE-CHIg-mGl, pFUSEss-CHIg-mGl, pcDNA3.1, pPICZa, pPIC9K.

[0059] In some embodiments, the present application also discloses host cells transformed with the expression vectors of the present application. The host cells can be prokaryotic cells, such as bacterial cells, for example, E. coli, B. subtilis, etc.; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. In some embodiments, the host cells can be selected from the group consisting of COS cells, Chinese hamster ovary (CHO) cells, Vero cells, HeLa cells, baby hamster kidney (BHK) cells, human hepatocarcinoma cells (e.g., HepG2), and other many cell lines. They provide post-translational modifications for the protein molecules, including correct folding, correct disulfide bond formation, and correct site glycosylation.

[0060] There are many methods for transforming host cells with expression vectors, and the transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, Polybrene (1,5-dimethyl-1,5-diazolidinylonyl) -mediated transfection, electroporation, liposome-mediated transfection, and direct microinjection of the DNA into the nucleus. In the present application, the preferred methods are electroporation or liposome-mediated methods, etc. For example, the liposome method kit from Invitrogen can be used to transfect host cells such as COS, CHO cells, etc. Then, the transformed host cells are cultured under conditions suitable for expression of the antibody. The antibody of the present application is then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography, etc., which are well known to those skilled in the art.

[0061] Medicaments or pharmaceutical compositions

[0062] The present application also provides a pharmaceutical or pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof of the present application, the nucleic acid molecule, the vector, or the host cell. As used herein, the term "active substance" or "active substance of the present application" is used interchangeably to refer to the GFRa3 antibody or antigen-binding fragment thereof of the present application, the nucleic acid molecule encoding the same, the vector, or the host cell.

[0063] As used herein, the terms "comprising" or "including," or "having" include "including," "in combination with," "consisting essentially of," and "consisting of." As used herein, the term "pharmaceutically acceptable" means an ingredient that is appropriate for use with humans and / or animals without undue adverse side effects such as toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio. As used herein, the term "effective amount" means an amount that is functional or active for humans and / or animals and is acceptable to humans and / or animals.

[0064] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent including various excipients and diluents. The term refers to a carrier that is nontoxic to the subject in which it is administered and that does not substantially interfere with the effectiveness of the active ingredient. Suitable carriers are well known to persons of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.

[0065] The pharmaceutically acceptable carrier in the composition can contain a liquid such as water, saline, glycerol and ethanol. In addition, there can be present auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, and the like. Generally, these substances are formulated into a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, the pH of which is generally about 5 to 8, preferably about 6 to 8.

[0066] As used herein, the term "unit dosage form" refers to a dosage form of the active substance of the present application prepared for the convenience of administration in a single administration, including but not limited to various solid agents (such as tablets), liquid agents, capsules, sustained-release agents.

[0067] It should be understood that the effective dose of the active substance used, such as an antibody or its coding sequence, can vary depending on the severity of the subject to be administered or treated. The specific case is determined according to the individual condition of the subject (for example, the weight, age, physical condition of the subject, the effect desired to be achieved), which is within the range that can be judged by a skilled physician.

[0068] The active substance of the present application can be used to inhibit the proliferation and / or invasion of tumor cells, especially the proliferation and / or invasion of tumors associated with abnormal ARTN-GFRa3 signaling pathway. Therefore, the active substance of the present application can be used to treat tumors including but not limited to: solid tumors such as liver cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), urothelial carcinoma (e.g., bladder cancer, renal pelvis cancer and ureter cancer); non-solid tumors such as leukemia (e.g., acute myeloid leukemia).

[0069] The pharmaceutical composition of the present application can also comprise or be used in combination with other anti-tumor active substances to achieve better therapeutic effect. The other anti-tumor active substances include, but are not limited to, TNF-α, TGF-β, IFN-α, angiostatins, endothelial statins, phosphoramide mustard, hematoporphyrin, lycoramine, bulbus luffi lactis, etoposide, anhydroagrostinol, doxorubicin, tamoxifen, 5-fluorouracil, norcantharidin, bifuridine, cucurbitacin, harringtonine, 2-epi-oxymatrine, krestin, flutamide, ifosfamide, ubenimex, leuprolide acetate, doxifluridine, lobaplatin, irinotecan, roglethimide, or teniposide, etc.

[0070] When two or more drugs are administered in combination, the effect is generally superior to that of the two drugs administered separately. Preferably, the drugs or other agents administered in combination do not interfere with the therapeutic activity of the active substance of the present application.

[0071] Kits

[0072] Also provided in the present application is a kit for detecting the presence, level or activity of ARTN, GFRα3 or both, comprising the antibody or active fragment thereof of the present application.

[0073] In some embodiments, the kit of the present application can be used to detect the presence or amount of GFRα3 in a biological sample, comprising the steps of: (a) contacting the sample with the antibody or active fragment thereof in the kit; and (b) detecting the formation of antigen-antibody complex, wherein the formation of the complex indicates the presence of GFRα3 in the sample or the amount of the formed antigen-antibody complex is quantitatively detected to reflect the amount of GFRα3 in the sample. The sample can be pre-treated or not, for example, can be extracted, purified or concentrated, etc.

[0074] The kit contains a container and the antibody or antigen-binding fragment thereof of the present application in the container, or a detection plate with the antibody or antigen-binding fragment thereof, and instructions for use. The kit can also contain other reagents required for detection, such as buffers, indicators, etc. The skilled person can adjust the contents of the kit according to specific needs.

[0075] Examples

[0076] The present application is further illustrated by the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art can make appropriate modifications and variations to the present application, and these modifications and variations are within the scope of the present application.

[0077] The experimental methods in the following examples, unless otherwise specified, can use conventional methods in the art, for example, refer to Molecular Cloning: A Laboratory Manual (3rd Edition, New York: Cold Spring Harbor Laboratory Press, 1989) or follow the conditions suggested by the supplier. The DNA sequencing method is a conventional method in the art, which can also be provided by a commercial company.

[0078] Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all of the professional and scientific terms used herein have the same meanings as familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied to the methods of the present application. The preferred implementation methods and materials described herein are only for demonstration.

[0079] Example I, immunization of mice with human GFRa3 protein and preparation of single B cells and sequencing

[0080] (I) Experimental materials

[0081] Balb / c mice were purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd., Freund's adjuvant was purchased from Sigma-Aldrich Company, GFRα3 protein (cat: 10213-H08H) was purchased from Beijing Yiqiao God State Technology Co., Ltd., Mouse memory B cell isolation kit, QuadroMACS Starting kit, and sorting buffer were purchased from Miltenyi Company, PBS and 0.4% trypan blue were purchased from Shengong Biological Engineering Co., Ltd.

[0082] (II) Experimental methods

[0083] Mouse immunization: mix equal volumes of complete Freund's adjuvant and 50 μg of antigen protein, and subcutaneously inject 6-8 week old female Balb / c mice for the first time; on the 21st day and the 42nd day, mix equal volumes of incomplete Freund's adjuvant and 50 μg of antigen, and respectively inject for the second time and the third time; on the 70th day, use 50 μg of antigen protein for the fourth time.

[0084] GFRα3-specific memory B cell sorting: 3 days after the fourth immunization, the mice were sacrificed by decapitation, and fresh spleen and lymph nodes were taken and ground on a 70 μm screen to filter out obvious tissue blocks; resuspend the cells with PBS and centrifuge. Add the corresponding sorting buffer (Miltenyi Company, cat: 130-091-221) at a ratio of 380 μl / 10 8 cells; add 10 μl of anti-CD43 antibody (Miltenyi Company, cat: 130-091-221) per 10 8cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The negative cells were collected and resuspended in 500 μl sorting buffer. The cells were then resuspended in 400 μl sorting buffer and 100 μl Anti-APC MicroBeads were added. The cells were incubated at 4°C for 15 min. The cells were then resuspended in 10x volume of sorting buffer and centrifuged (300 g, 10 min) to remove the supernatant. The cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The IgG1 + cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The negative cells were collected and resuspended in 500 μl sorting buffer. The cells were then resuspended in 400 μl sorting buffer and 100 μl Anti-APC MicroBeads were added. The cells were incubated at 4°C for 15 min. The cells were then resuspended in 10x volume of sorting buffer and centrifuged (300 g, 10 min) to remove the supernatant. The cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The IgG1 7 cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The negative cells were collected and resuspended in 500 μl sorting buffer. The cells were then resuspended in 400 μl sorting buffer and 100 μl Anti-APC MicroBeads were added. The cells were incubated at 4°C for 15 min. The cells were then resuspended in 10x volume of sorting buffer and centrifuged (300 g, 10 min) to remove the supernatant. The cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The IgG1 5 -10 6 cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The negative cells were collected and resuspended in 500 μl sorting buffer. The cells were then resuspended in 400 μl sorting buffer and 100 μl Anti-APC MicroBeads were added. The cells were incubated at 4°C for 15 min. The cells were then resuspended in 10x volume of sorting buffer and centrifuged (300 g, 10 min) to remove the supernatant. The cells were resuspended in 500 μl sorting buffer and passed through a MACS column. The IgG1

[0085] (Three) Experimental results and analysis

[0086] We immunized Balb / c mice with GFRa3 protein by subcutaneous injection. Four immunizations were performed according to the plan. After the experiment, the mice were euthanized, and the spleen and lymph nodes were dissociated to obtain a single cell suspension. GFRa3 specific IgG1 + GFRa3 + memory B cells Figure 1 .

[0087] Shanghai Jingneng Biotechnology Co., Ltd. was commissioned to perform single B cell 10x Genomics BCR gene sequencing. GFRa3 antibody sequences with a selection frequency of 2 or more were selected for subsequent cloning and expression purification.

[0088] Further identification showed that the #119 antibody has the following characteristics: the light chain is of the kappa type, and the heavy chain is of the IgG1 antibody of the gamma type; the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 according to the IMGT numbering system are shown in SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively (the coding sequences thereof can be shown in SEQ ID NO: 9, 10, 11, 12, 13 and 14 respectively); the VH and VL amino acid sequences according to the IMGT numbering system are shown in SEQ ID NO: 7 and 8 respectively (the coding sequences thereof can be shown in SEQ ID NO: 15 and 16 respectively).

[0089] Example II, construction and primary screening of GFRa3 antibody recombinant vectors

[0090] (I) Experimental materials

[0091] The light and heavy chain expression plasmids of the antibody were purchased from InvivoGen Company, the antibody light and heavy genes were synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd., the gel recovery kit was purchased from Takara Company, the homologous recombinase was purchased from Nanjing Novozyme Biotech Co., Ltd., the jetPEI plasmid transfection reagent was purchased from Polyplus, the nitrocellulose membrane was purchased from GE Company, the GFRα3 protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., the primary antibody and the secondary antibody were purchased from Abeam Company, and the color developing solution was purchased from Thermo Fisher Scientific Company.

[0092] (II) Experimental methods

[0093] Construction of recombinant vectors: the VH and VL plasmids synthesized by the company were used as templates for subcloning, and the PCR fragments were purified by the method of gel recovery; the PCR fragments were connected to the VH (pFUSEss-CHIg-hG1) and VL (pFUSE2ss-CLIg-hk) expression vectors by homologous recombination; transformation was performed by DH5a competent cells, and the single colony was selected and cultured by shaking and sequencing. The correct sequencing results were used for large-scale plasmid extraction; 3x10 5 The 293t cells were plated in a 12-well plate at a density of 3x10

[0094] GFRα3 antibody screening: the antibody binding to GFRα3 protein was preliminarily screened by Dot blot method, and the specific method was as follows: 0.1 μg of GFRα3 protein diluted with PBS was added to the nitrocellulose membrane, and was placed at room temperature for 10 min, and was blocked with 5% skim milk at room temperature for 1 h; the blocked membrane was added with the supernatant of transfected 293t cells, positive control antibody (anti-his) and negative control antibody (IgG) respectively, and was placed at 4°C overnight. The membrane was washed with TBST for 3 times, each time for 15 min; the secondary antibody (HRP-human, HRP-mouse, HRP-rabbit) diluted with 5% skim milk was added to the membrane, and was incubated at room temperature for 1 h, the membrane was washed with TBST for 3 times, each time for 15 min, color developing solution was used for color development, and chemiluminescence instrument was used for luminescence detection.

[0095] (III) Experimental results and analysis

[0096] The plasmid containing the GFRα3 antibody VH and VL sequences was synthesized by the entrusted company, the antibody sequence was amplified and purified by using the plasmid as a template, the GFRα3 antibody sequence fragment was connected to the VH and VL expression vectors by homologous recombination, the large-scale plasmid extraction was performed on the sequencing results, and the 293t cells were transfected, and the supernatant was collected after 72 h. The GFRα3 antibody was preliminarily screened by Dot blot.

[0097] Through the above experiments and tests, it was found that a plurality of GFRα3 antibodies had strong binding capacity with GFRα3 antigen, and the #119 antibody had the strongest binding capacity with human GFRα3 antigen. Figure 2 ).

[0098] The above results show that we have prepared GFRα3 antibodies with good specific binding to GFRα3 protein through screening.

[0099] Example III, expression and purification of GFRa3 #119 antibody

[0100] (I) Experimental materials

[0101] The expiCHO-S cell line and transfection reagent were purchased from Thermo Fisher Scientific Company, and Prism A column was purchased from Cytiva Company.

[0102] (II) Experimental method

[0103] The GFRα3 antibody was expressed by using CHO-S cell expression system, and the specific method was as follows:

[0104] 1. Preparation of reaction mixture 1: 1.84ml OptiPro-SFM + 160μl ExpiFectamine™ CHO Reagent; Preparation of reaction mixture 2: 2ml OptiPro-SFM + plasmids (VH, VL expression plasmid 50μg each);

[0105] 2. Shake reaction mixtures 1 and 2 respectively. After 5min of room temperature standing, reaction mixture 1 is added to reaction mixture 2 and mixed, and left to stand at room temperature for 20min;

[0106] 3. Slowly add the prepared reagent to the 50ml CHO-S cell system. After 18-22h, add 300μl ExpiCHO TM Enhancer, 12ml ExpiCHO TM Feed to the cell culture system and incubate at 37℃ for 7 days. Centrifuge at 12000rpm, 20min, 4℃ (remove cell debris in supernatant), collect cell culture supernatant.

[0107] Antibody purification is carried out by AKTA protein purification system, and the steps are as follows: wash Prism A column with 1xPBS until UV line is flat, load the treated cell culture supernatant on AKTA, after completion, wash the column again with 1xPBS to remove unbound impurities, elute the antibody with glycine-hydrochloric acid eluent (0.1M, pH2.8), add neutralizing solution (1M Tris, pH9.0) to the antibody to neutralize the pH to neutral, and finally replace the eluted antibody into PBS with ultrafiltration tube (4600g, 20min, 4℃).

[0108] (III) Experimental results and analysis

[0109] We expressed GFRα3 antibody by transfecting CHO-S cells, and purified it by AKTA protein purification system. The purity and molecular weight of the purified GFRα3 antibody were detected by gel electrophoresis and staining. The results showed that the GFRα3 antibody presented two bands with molecular weights of about 55kDa and 30kDa, which were the heavy chain and light chain bands of the antibody, respectively. Figure 3 ).

[0110] The above results suggest that the molecular weight of the GFRα3 antibody obtained by construction is consistent with the theoretical value, and the antibody structure is correct.

[0111] Example IV, affinity determination of GFRa3 #119 antibody

[0112] (I) Experimental materials

[0113] S series CM5 sensor chip was purchased from Cytiva company.

[0114] (II) Experimental Methods

[0115] SPR was used to detect the affinity of the antibody: the chip was placed on the Biacore T200 (Cytiva) at room temperature 25°C, and the buffer was HBSEP (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20); the antigen protein GFRa3 was covalently linked to the experimental channel by amino coupling method, and the antibody was diluted by the ratio as the analyte, which was flowed through the control channel and the experimental channel at the rate of 30 mΐ / min, the binding time and the dissociation time were 120 seconds and 400 seconds respectively, the regeneration buffer was Glycine 2.0, and the Biacore T200 Evaluation Software 3.1 (Cytiva) software was used to analyze the affinity (K D ) of the experimental results, and the action mode of 1:1 binding was used.

[0116] (III) Experimental Results and Analysis

[0117] We detected the affinity of the #119 antibody by the SPR method, and the results showed that the K D of the GFRa3 #119 antibody to its target GFRa3 could reach 1.35 x 10 -10 M( Figure 4 ).

[0118] The results proved that the GFRa3 #119 antibody had very high affinity to its target GFRa3.

[0119] Example V, detection of blocking effect of the antibody

[0120] (I) Experimental Materials

[0121] The biotin labeling kit was purchased from Genemore Company; the consumables for ELISA were purchased from Thermo Fisher Scientific Company; the IgG antibody, TMB developing solution, and ELISA termination solution were purchased from Abeam Company; PBS and Tween were purchased from Shenguo Bioengineering Co., Ltd.; and BSA was purchased from Shanghai Boyue Biotechnology Co., Ltd.

[0122] (II) Experimental Methods

[0123] GFRa3 protein was biotin-labeled, and ARTN protein was coated on a plate and incubated in a refrigerator at 4°C overnight. The next day, the plate was washed with PBST (PBS+0.05% Tween) four times, blocking solution (PBS+3% BSA) was added, and the plate was incubated at room temperature for 1 h. The plate was washed with PBST four times, and control IgG or different concentrations of GFRa3 antibody to be tested and biotin-labeled GFRa3 protein were added, and the plate was incubated in an incubator at 37°C for 1 h. The plate was washed with PBST four times, and diluted horseradish peroxidase was added, and the plate was incubated in an incubator at 37°C for 1 h. The plate was washed with PBST four times, TMB color developing solution was added to each well, and after the color changed, stop solution was immediately added to each well, and the OD value was detected at a single wave of 450 nm.

[0124] (III) Experimental results and analysis

[0125] We detected the blocking function of GFRa3 antibody by antibody blocking experiment. First, GFRa3 protein was biotin-labeled, and control IgG antibody or different concentrations of GFRa3 antibody to be tested were added to the GFRa3 labeled protein and ARTN co-incubation system, and the OD value of the reaction system was detected.

[0126] The test results show that the #119 antibody can significantly block the binding of ARTN and GFRa3, IC 50 = 350.2 nM (as shown in Figure 5 ).

[0127] The above results prove that the obtained #119 antibody has high blocking effect.

[0128] Example VI, detection of ADCC effect

[0129] (I) Experimental materials

[0130] PBS was purchased from Shenguo Bioengineering Co., Ltd., Accutase buffer was purchased from Gibco Company, fetal bovine serum (FBS) was purchased from Biowest Company, trypsin digestion solution was purchased from Bio Basic Inc (BBI) Company, and IgG1 was purchased from Abeam Company.

[0131] (II) Experimental methods

[0132] The specific method of antibody-dependent cell-mediated cytotoxicity (ADCC) detection is as follows:

[0133] Cell preparation:

[0134] Target cells 7721 and humanized Hepa1-6-hGFRa3 cells (purchased from Shanghai Nanmo Biology) were collected, washed with PBS twice, centrifuged at 1500 rpm for 5 min, resuspended with fresh culture medium, and counted. The target cell density was adjusted to 5x10 4cells / ml. 100 μl cell suspension was added to each well of 96-well plate, and the number of cells was 5 x 105cells / well. The outermost of the well plate was filled with culture medium, and the plate was placed in a 37°C, 5% CO2incubator overnight. The next day, 95 μl of cell culture supernatant was aspirated from each well, and 25 μl of detection reaction medium was added. 3 6 cells / ml. 100 μl cell suspension was added to each well of 96-well plate, and the number of cells was 5 x 105cells / well. The outermost of the well plate was filled with culture medium, and the plate was placed in a 37°C, 5% CO2incubator overnight. The next day, 95 μl of cell culture supernatant was aspirated from each well, and 25 μl of detection reaction medium was added.

[0135] Co-incubation of effector cells and antibodies:

[0136] a. Preparation of antibodies: the target antibodies or control antibodies hlgG1 were serially diluted with culture medium, and 25 μl of the serially diluted antibodies were added to the culture dishes containing target cells, 3 replicates;

[0137] b. Collection of effector cells Jurkat / NFAT-luc, adjustment of cell density to 3 x 105cells / ml, and addition of 25 μl / well to the detection plate (the ratio of effector cells to target cells was 15:1), and the total system was 75 μl (background wells were set, and 75 μl of culture medium was added); 6

[0138] c. The well plate was placed in a 37°C, 5% CO2incubator for 6 h.

[0139] Chemiluminescence detection:

[0140] The detection plate was taken out of the 37°C incubator and placed at room temperature for 15 min, and the substrate solution was mixed in advance and equilibrated to room temperature. 75 μl of detection substrate was added to each of the above detection plates, and 75 μl of detection substrate was added to each of the background wells. Incubation at room temperature for 5-30 min, and detection was performed on the chemiluminescence module of the enzyme marker.

[0141] (Three) Experimental results and analysis

[0142] We used 7721, Hepa1-6-hGFRa3 as target cells, and Jurkat / NFAT-luc as effector cells, and found that GFRa3#119 antibody could mediate significant ADCC effect Figure 6 ) through ADCC effect detection.

[0143] Example VII, inhibitory effect of anti-human GFRa3 antibody on proliferation of liver cancer cells

[0144] (One) Experimental materials

[0145] ARTN protein was purchased from R&D company, and CCK8 reagent was purchased from Dakewe Biotechnology Co., Ltd.

[0146] (Two) Experimental methods

[0147] 7721、HepG2 cells were plated: cells were washed once with PBS, trypsin was added, after 1 minute complete medium was added to stop the digestion and centrifuged (1000 rpm, 5 min), the medium resuspended the cells and counted, cells were plated in 96-well plates at a density of 6 x 10 3 After the cells adhered, ARTN (50 ng / ml), ARTN + GFRa3 antibody (50 pg / ml), ARTN + IgG1 antibody (50 pg / ml) were added, respectively, at 48 h, 72 h, 96 h, 10 pl of CCK8 reagent was added to each well, incubated in the incubator for 1-4 h, and the absorbance at 450 nm was measured by a microplate reader.

[0148] (III) Experimental results and discussion

[0149] To further study the effect of GFRa3 antibody on the proliferation of hepatoma cells, we detected the effect of GFRa3 antibody on cell proliferation after co-incubation with hepatoma cells at 48 h, 72 h, 96 h. First, 7721, HepG2 cells were plated, after the cells adhered, ARTN, ARTN + GFRa3 antibody, ARTN + IgG1 antibody were added, respectively, by CCK-8 experiment, it was found that ARTN promoted the proliferation of hepatoma cells HepG2 ( Figure 7 ), and further addition of GFRa3 #119 antibody eliminated the effect of ARTN on promoting the proliferation of hepatoma cells HepG2, we used 7721 cells and got similar results ( Figure 8 ).

[0150] The above results show that anti-GFRa3 #119 antibody can significantly inhibit or reverse the effect of ARTN on promoting the proliferation of hepatoma cells, and can be used for the inhibition and treatment of ARTN / GFRa3 related tumors.

[0151] Example VIII, inhibition of ARTN / GFRa3-mediated promotion of liver cancer cell invasion by GFRa3 antibody

[0152] (I) Experimental materials

[0153] Matrigel, Transwell chamber purchased from Corning company, rapid Wright-Giemsa staining solution purchased from Nanjing Jiancheng Technology Co., Ltd.

[0154] (II) Experimental method

[0155] Dissolve Matrigel at 4°C overnight. The next day, dilute Matrigel with serum-free medium at a ratio of 1:10, add 100 μl of the diluted Matrigel to the upper chamber of the Transwell chamber, and incubate in the incubator for 1-5 hours. Wash the gel with serum-free medium to hydrate it fully. Collect HepG2 cells in a 12-well plate, resuspend the cells in serum-free medium, and count them. Take 200 μl (1.2 x 10 5 cells / well) of the cell suspension and add it to the Matrigel-precoated upper chamber of the Transwell chamber. Add 600 μl of complete medium to the lower chamber, and incubate in the incubator for 24 hours. Remove the medium in the upper chamber of the Transwell chamber, and gently wipe the inner membrane of the chamber with a cotton swab. Stain with Giemsa solution reagent 1 for 6 minutes at room temperature, and stop the staining with reagent 2 for 8 minutes at room temperature. Wash with PBS for 3 times, and air dry at room temperature. Randomly select 5-10 fields of view, take pictures under a microscope at 200x, and count the number of cells that have invaded.

[0156] (III) Experimental results and analysis

[0157] We further studied the effect of GFRa3 antibody on the invasion of liver cancer cells. Collect HepG2 cells in a 12-well plate, and add 1.2 x 10 5 cells / well to the upper chamber of the Transwell chamber. After 24 hours, fix and stain the cells, take pictures, and count the number of invasive cells. The results are shown in Figure 9 ARTN can promote the invasion of liver cancer cells, and GFRa3 #119 antibody can block the ARTN-mediated effect of promoting liver cancer invasion.

[0158] The above results show that the anti-GFRa3 #119 antibody can significantly inhibit or reverse the ARTN-mediated effect of promoting liver cancer invasion, and can be used for the inhibition and treatment of ARTN / GFRa3-related tumors.

[0159] In summary, based on the results in the examples, we found that the GFRa3 #119 antibody has a significant effect in blocking the binding of the protein ARTN to GFRa3, and in inhibiting the growth and invasion of tumor cells.

[0160] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications can be made to the present application by those skilled in the art after reading the above description of the present application, and these equivalent forms also fall within the scope of the appended claims.

[0161] Appendix: Sequence information

[0162]

[0163]

Claims

1. An antibody or antigen-binding fragment thereof targeting GFRα3, wherein, The antibody comprises a heavy chain complementarity-determining region (VHCDR) and a light chain complementarity-determining region (VL CDR), the amino acid sequences of which are shown below: VH CDR1: SEQ ID NO: 1; VH CDR2: SEQ ID NO: 2; VH CDR3: SEQ ID NO: 3; VL CDR1: SEQ ID NO: 4; VL CDR2: SEQ ID NO: 5; and VL CDR3: SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL each have an amino acid sequence selected from the group consisting of: VH: (a) the sequence shown in SEQ ID NO: 7; or (b) a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO: 7; and VL: (a') the sequence shown in SEQ ID NO: 8; or (b') a sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO:

8.

3. The antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody comprises a combination of VH CDR1 (amino acid sequence as shown in SEQ ID NO: 1), VH CDR2 (amino acid sequence as shown in SEQ ID NO: 2), VH CDR3 (amino acid sequence as shown in SEQ ID NO: 3), VL CDR1 (amino acid sequence as shown in SEQ ID NO: 4), VL CDR2 (amino acid sequence as shown in SEQ ID NO: 5), and VL CDR3 (amino acid sequence as shown in SEQ ID NO: 6); and / or The antibody comprises a VH sequence as shown in SEQ ID NO: 7 and a VL sequence as shown in SEQ ID NO: 8; and / or The antibody has at least 80% sequence identity with the sequence shown in SEQ ID NO: 7 and / or the antibody has at least 80% sequence identity with the sequence shown in SEQ ID NO: 8, and the difference in amino acid residues is not located in the CDR, wherein the antibody has the activity of targeting and blocking GFRα3.

4. The antibody or its antigen-binding fragment as described in claim 3, wherein, The VH and / or VL contain substitutions of one or more amino acid residues in their non-CDR regions.

5. The antibody or its antigen-binding fragment as described in claim 4, wherein, The VH and / or VL contain humanized designs and alternatives in their non-CDR regions.

6. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is selected from: monoclonal antibody, polyclonal antibody, tetraclonal antibody, triclonal antibody and bispecific antibody.

7. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is an intracellular antibody.

8. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is selected from: Fab, Fab', F(ab')2, F(ab')3, scFv, disulfide-linked Fv, IgGΔCH2, DVD-Ig, Fcab, mAb2, (scFv)2, and scFv-Fc.

9. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, wherein, The antibody has one or more characteristics selected from the group consisting of: (i) It specifically binds to GFRα3; (ii) It specifically blocks ARTN and GFR The combination of 3; (iii) It can inhibit the proliferation and / or invasion of cancer cells.

10. The antibody or antigen-binding fragment thereof as described in claim 9, wherein, (i) The binding affinity K of the antibody to GFRα3 was determined by surface plasmon resonance (SPR). D The range is 0.1 nM to 0.2 nM; (ii) The antibody was detected by ELISA against ARTN and GFR. 3-binding blocking IC 50 The range is 200 nM to 400 nM; (iii) The antibody can inhibit ARTN and GFR The proliferation and / or invasion of cancer cells associated with the activation of the 3rd pathway.

11. The antibody or antigen-binding fragment thereof as described in claim 9, wherein, The antibody can inhibit ARTN and GFR The proliferation and / or invasion of hepatocellular carcinoma cells associated with the activation of the 3rd pathway.

12. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8.

13. The nucleic acid molecule of claim 12, wherein, The nucleic acid molecule contains the following nucleotide sequence: VH CDR1 encoded sequence: SEQ ID NO: 9; VH CDR2 encoded sequence: SEQ ID NO: 10; VH CDR3 encoded sequence: SEQ ID NO: 11; VL CDR1 encoded sequence: SEQ ID NO: 12; VL CDR2 encoded sequence: SEQ ID NO: 13; and VL CDR3 encoded sequence: SEQ ID NO:

14.

14. The nucleic acid molecule of claim 12, wherein, The nucleic acid molecule comprises: VH encoded sequence: SEQ ID NO: 15; and VL encoded sequence: SEQ ID NO:

16.

15. The nucleic acid molecule of claim 12, wherein, The nucleic acid molecules were codon-optimized.

16. A vector or host cell comprising a nucleic acid molecule as described in any one of claims 12-15.

17. A composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 11, a nucleic acid molecule as described in any one of claims 12 to 15, and / or a vector or host cell as described in claim 16.

18. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-11, the nucleic acid molecule as described in any one of claims 12-15, the vector or host cell as described in claim 16, in the preparation of a product, wherein the product is used for: (a) Prevention and / or treatment of liver cancer; and / or (b) Detection of GFR 3. Presence or absence, level or activity.

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