An anti-GDF15 antibody and its preparation method and application

Abstract

The present invention discloses an anti-GDF15 antibody and a preparation method and application thereof. The present invention uses prokaryotic host cells to recombinantly express human GDF15 in the immunogen, and by fusing a thioredoxin tag at the N-terminus, the recombinant GDF15 antigen has stronger immunogenicity, more dominant epitopes are exposed, and a monoclonal antibody with higher affinity is prepared. In addition, the present invention was verified using multiple negative and positive clinical samples, and the results showed that the monoclonal antibody screened by the present invention has higher sensitivity and specificity for cancer detection. The antibody of the present invention has broad application prospects in the early diagnosis and prognosis of cancer.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and immunology, and in particular to an anti-GDF15 antibody and a preparation method and application thereof. Background Art

[0002] Growth differentiation factor 15 or macrophage inhibitory cytokine-1 (GDF15 / MIC-1) is a member of the TGF-β superfamily and has different pathophysiological roles in cancer, cardiometabolic disorders, and other diseases. GDF15 can control hematopoietic growth, energy homeostasis, adipose tissue metabolism, body growth, bone remodeling, and response to stress signals. The role of GDF15 in cancer development and progression is complex and depends on cancer type, stage, and tumor microenvironment. Bauskin et al. in 2006 showed that GDF15 levels in the blood circulation of several malignancies (including melanoma, prostate cancer, breast cancer, gastrointestinal cancer, pancreatic cancer, and colorectal cancer) can be nearly 200 times higher and are associated with poor survival. Therefore, GDF15 can serve as a marker for cancer progression.

[0003] There is an urgent need to develop an anti-GDF15 antibody for early diagnosis and prognosis of cancer.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] In order to solve at least some of the technical problems in the prior art, the present invention uses prokaryotic host cells to recombinantly express human GDF15 in the immunogen, and by fusing a thioredoxin tag at the N-terminus, the recombinant GDF15 antigen has stronger immunogenicity, more dominant epitopes are exposed, and a monoclonal antibody with higher affinity is prepared. Specifically, the present invention includes the following contents.

[0006] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, which can target GDF15, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

[0007] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention comprises a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment comprises Fab, Fab', F(ab) 2 、F(ab') 2 , scFv or scFv Fc fragment.

[0009] The second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0010] The third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0011] The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule or vector molecule described in the present invention.

[0012] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which is prepared by artificial synthesis or genetic engineering.

[0013] In a sixth aspect, the present invention provides a detection product for GDF15, which comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0014] In certain embodiments, the detection product according to the present invention comprises a kit, a test paper or a protein chip.

[0015] The seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the present invention in the preparation of a GDF15 detection product.

[0016] The present invention uses prokaryotic host cells to recombinantly express human GDF15 in the immunogen, and by fusing the thioredoxin tag at the N-terminus, the recombinant GDF15 antigen has stronger immunogenicity, more dominant epitopes are exposed, and a monoclonal antibody with higher affinity is prepared. In addition, the present invention is verified using multiple negative and positive clinical samples, and the results show that the monoclonal antibody of the present invention has higher sensitivity and stronger specificity for cancer detection. The antibody of the present invention has broad application prospects in the early diagnosis and prognosis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The recombinant human GDF15 expression plasmid map of the present invention is shown, which contains T7 promoter, N-terminal fusion expression of TrxA protein, 6×His purification tag, human GDF15 Ala 197-Ile 308 target gene and other elements.

[0018] Figure 2 The SDS-PAGE identification results of the purified GDF15 antigen of the present invention are shown, wherein M is the full-form gold BluePlus V protein Marker; S is the supernatant of the expression bacterial lysis; FT is the flow-through of the Ni-NTA affinity chromatography column; W is 40 mM imidazole washing; and E is 250 mM imidazole elution.

[0019] Figure 3 The results of activity tests of the antibodies of the present invention are shown.

[0020] Figure 4 The results of using the antibodies of the present invention to detect serum samples from healthy people and people with clinical backgrounds are shown. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] Antibodies or antigen-binding fragments thereof In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can bind to GDF15 with high affinity and targeting. Without being bound by any theory, the antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

[0025] In a specific embodiment, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0026] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody portion that binds to the antigen.

[0027] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention consists of a "framework" region interspersed with three "complementarity determining regions (CDRs)". The framework region is used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0028] As used herein, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. In order to determine sequence identity, sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required for achieving optimal alignment in the full-length sequence being compared.

[0029] Herein, the antibody sequences obtained by modification also belong to the protection scope of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0030] In the present invention, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a mouse antibody.

[0031] The term "monoclonal antibody" as used herein, sometimes also referred to as "monoantibody" or Ab, refers to an immunoglobulin obtained from a pure cell line, having the same structure and chemical properties, and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies against different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture and are not mixed with other immunoglobulins. This characteristic is in contrast to polyclonal antibody preparations, which generally include antibodies against different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0032] Herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy chain or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, while the remaining segments of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light chain and the heavy chain are all from the variable regions of antibodies of one animal species (such as mice, rats, etc.), while the constant portion is homologous to the antibody sequence from another species (such as humans). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, while the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined therewith. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response during injection is lower than that of using antibodies with non-human constant regions.

[0033] Herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.

[0034] Herein, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, the murine antibody may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo.

[0035] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by: including Fv, scFv, dsFv, Fab, Fab' or F(ab') 2 or a heavy chain of a fragment thereof, or, including Fv, scFv, dsFv, Fab, Fab' or F(ab') 2 The light chain of the fragment.

[0036] In the present invention, the terms "directed against", "binding", "immunobinding", "specific binding" and "targeting" are used interchangeably and generally refer to the non-covalent interaction that occurs between an immunoglobulin molecule and an antigen that is specific for the immunoglobulin. The strength or affinity of the immunological binding interaction can be expressed in terms of a dissociation constant (Kd), with a smaller Kd representing a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be described in terms of a binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the art and described herein.

[0037] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to an antibody or antigen-binding fragment thereof that has been extracted from its natural environment. An antibody or fragment thereof that has been "isolated" thus includes an antibody or fragment thereof that has been purified by standard purification methods. The term also includes antibodies or fragments thereof prepared by recombinant expression in a host cell and chemically synthesized antibodies or fragments thereof.

[0038] Nucleic acid molecules In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0039] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0040] Once the coding sequence of the antibody of the present invention is isolated, the antibody can be obtained in large quantities by recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then separate it from the propagated host cells by conventional methods.

[0041] Carrier molecules One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0042] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0043] Host cells In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule described in the present invention.

[0044] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. A host cell includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0045] Preparation method In one aspect of the present invention, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0046] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention or antigen-binding fragments thereof are obtained by direct amino acid synthesis.

[0047] In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. The genetic engineering expression system used for genetic engineering expression includes, but is not limited to, a prokaryotic cell expression system, a eukaryotic cell expression system, and a cell-free expression system. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems. In a preferred embodiment, the antibodies of the present invention can be prepared by the following steps: (1) constructing a recombinant vector expressing the antibody heavy chain of the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector expressing the antibody light chain of the amino acid sequence shown in SEQ ID NO.8; (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression; (3) Collect and purify antibodies.

[0048] In another preferred embodiment, the antibody of the present invention is prepared by immunization with a recombinant antigen, wherein the recombinant antigen has the amino acid sequence shown in SEQ ID No. 9. In a specific embodiment, the method for preparing the antibody of the present invention comprises: (1') constructing a recombinant vector expressing the antigen sequence shown in SEQ ID No.9, and obtaining a recombinant antigen after expression; (2') inoculating the antigen into mice to prepare monoclonal antibodies; (3') Using the antigen to screen positive hybridoma cells.

[0049] In the step (1') of the present invention, the full-length GDF15 gene is inserted into vectors such as pET28a, pET32a, pET22b, pET30a, etc., preferably pET32a. The nucleotide sequence of the constructed GDF15 prokaryotic expression vector is shown in SEQ ID NO.10.

[0050] In the step (1') of the present invention, the host bacteria used to express the recombinant antigen include BL21 (DE3), BL21Star, Rosetta, etc., preferably Rosetta.

[0051] In the step (2') of the present invention, the amount of antigen inoculated into the experimental mice is 250 μg, and the immune adjuvant used for the antigen is Freund's complete adjuvant.

[0052] In the steps (1') and (3') of the present invention, the amino acid sequence of the full-length antigen used is shown in SEQ ID NO.9.

[0053] Testing products The present invention further provides a detection product for detecting (or quantifying) GDF15, which comprises the antibody or antigen-binding fragment thereof described in the present invention, and instructions on how to implement the detection method of the present invention for GDF15.

[0054] In a preferred embodiment, the detection product includes a kit, a test paper or a protein chip.

[0055] Herein, the term "kit" refers to a combination of reagents and other materials. The kit is expected to include reagents, such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies or their antigen binding fragments, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials, for example, the kit may also include instructions for using the reagents. The kit may be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and instructions for performing detection instructions. The kit may be prepared by a variety of methods known in the art.

[0056] In certain embodiments, the kit may further include at least one of a cleaning solution, a substrate solution, a diluent, and a calibration solution. The composition of the cleaning solution is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and a preservative. The substrate solution may use a known substrate, and examples thereof include, but are not limited to, a chromogenic substrate, a fluorescent substrate, a luminescent substrate, and the like. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and the like. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, a BSA solution, a trehalose solution, an animal serum, and the like.

[0057] use The present invention further provides the use of the antibody or antigen-binding fragment thereof described in the present invention in the preparation of a GDF15 detection product, wherein the detection product includes a kit, a test paper or a protein chip. The detection product is used for the prediction, diagnosis and / or prognosis of GDF15-related diseases, and "GDF15-related diseases" refer to abnormal expression of GDF15 in the subject, or an increase in the amount or activity of GDF15 in the sample to be tested (including but not limited to blood, cell lysate, etc.). For example, in some cases, compared with the normal expression of GDF15 in healthy people, the GDF15 in the sample to be tested of the subject has a significantly upregulated expression.

[0058] In the present invention, GDF15-related diseases include cancers, examples of which include but are not limited to melanoma, prostate cancer, breast cancer, gastrointestinal cancer, pancreatic cancer, rectal cancer, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, glioblastoma, glioma, brain tumor, head and neck cancer, kidney cancer, lung cancer, oral cancer, ovarian cancer, liver cancer, uterine cancer, bone cancer, leukemia, lymphoma, sarcoma, blood cancer, thyroid cancer, thymic cancer, eye cancer, skin cancer, etc.

[0059] Example 1 This example demonstrates the synthesis of a monoclonal antibody.

[0060] 1. Immunogen Preparation In the present invention, the immunogen of GDF15 uses prokaryotic E. coli as a host to recombinantly express human GDF15, and the thioredoxin TrxA tag is fused to the N-terminus, so that the recombinant GDF15 antigen has stronger immunogenicity, more dominant epitopes are exposed, and the affinity of the monoclonal antibody prepared by immunization will also be higher. The constructed GDF15-pET32a recombinant vector is as follows Figure 1 shown.

[0061] The correctly sequenced GDF15-pET32a-Rosetta (DE3) strain was inoculated in 10 ml of LB+kan medium at 1:100. After overnight culture, it was inoculated in 1 L of LB+kan medium at 1:100. When OD600≈0.6, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added. After overnight culture at 220 rpm and 20°C, the bacteria were collected and the fermentation product was added to 250 ml of 50 mM Tris-HCl pH8.0+500 mM NaCl to resuspend completely. After being precooled on ice for 10 min, it was lysed by ultrasound at 300 W, 1 s, 1 s interval, and a total ultrasound time of 30 min. It was identified that GDF15 was expressed as semi-inclusion body and semi-soluble in prokaryotic expression.

[0062] After the bacteria are broken, the lysate is loaded onto the Ni-NTA affinity chromatography column. After loading, it is washed with 50 mM Tris-HCl pH8.0 + 500 mM NaCl + 20 mM imidazole. During the washing period, the absorbance of the A280 band is continuously monitored. When it no longer changes, the target protein is eluted with 50 mM Tris-HCl pH8.0 + 500 mM NaCl + 250 mM imidazole elution solution. During the elution period, the absorbance of the A280 band is continuously monitored. When the absorbance drops below 0.2, the collection is terminated. All components are sampled for SDS-PAGE identification. The identification results are as follows: Figure 2 As shown, the target protein is located around 30 kDa, which is consistent with the predicted molecular weight.

[0063] 2. Mouse Immunization Purebred BALB / C mice were selected for the first immunization with 50 μg of GDF15 antigen, which was added with Freund's complete adjuvant and shaken in a silver-mercury mixer. The mixture was then sucked into a 1 ml sterile syringe and immunized at multiple subcutaneous points and intraperitoneally in the mice.

[0064] ↓3 weeks later The second immunization dose was the same as above, with Freund's incomplete adjuvant added, and the mice were immunized subcutaneously at multiple points and intraperitoneally; ↓2 weeks later The third immunization dose is the same as above, with Freund's incomplete adjuvant added, and the mice are immunized subcutaneously at multiple points and intraperitoneally, and blood is collected 5-7 days later to measure the titer; ↓2-3 weeks later For booster immunization, 100 μg of GDF15 antigen was used, injected intraperitoneally; ↓3 days later The spleen was removed and fused.

[0065] The antibody titer of GDF15 experimental mice was tested, and the experimental mouse serum was tested, where the coating conditions were: cold pack and heat seal, GDF15-Ag, coating concentration: 1 μg / ml, 10 μg / plate, coating solution: 0.05 M CB, 100 μl / well, blocking solution: CEA blocking solution, 130 μl / well, reactant concentration: as shown in Table 1, enzyme concentration: 1:1000, reaction mode: sample addition 30 min + secondary antibody 30 min + color development 10 min + termination.

[0066] Table 1 Results of three immune titer determinations of GDF15 The results are shown in Table 1. The average serum titer of mice #1-3 was about 400,000.

[0067] 3. Cell Fusion On the day of fusion, the immunized BALB / C mice were killed by pulling their necks, soaked in 75% ethanol, taken out and placed in a clean bench, and the spleen of the mouse was removed with ophthalmic scissors and forceps. Place it in a culture dish containing culture medium and remove the connective tissue. Prepare a culture dish with culture medium and place a cell screen in the culture dish. Place the washed spleen in the cell screen and grind it into single cells with the cell screen. Transfer the cell solution to a centrifuge tube, centrifuge, and discard the supernatant. Puff the precipitate, remove the connective tissue, place it in a new centrifuge tube, add culture medium, centrifuge, discard the supernatant, mix it with culture medium, and make up to 20 ml.

[0068] On the day of fusion, discard all the supernatant from 10 dishes of SP2 / 0, mix well with fresh culture medium, put into a centrifuge tube, centrifuge, and discard the supernatant. Mix well with pipetting, centrifuge. Discard the supernatant. Mix well with pipetting, and make up to 20 ml.

[0069] Mix myeloma cells SP2 / 0 and spleen cells in proportion, wash once with serum-free incomplete culture medium in a centrifuge tube, centrifuge, and discard the supernatant. Gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly, centrifuge, and discard the supernatant. Add PEG solution to the centrifuge tube at a uniform speed within 1 minute. Let it stand for 90 seconds, then slowly add the stop solution to terminate the reaction. Centrifuge, add the fused cells to the complete culture medium, and plate them in a 96-well plate with feeder layer cells cultured the day before.

[0070] 4. Cloning and screening of hybridoma cells The OD value of the cell supernatant was detected by indirect ELISA method in about seven days. The hybridoma cells in the wells with higher positive values ​​were cloned, and the hybridoma cells were cloned by limiting dilution method until the monoclonal positive wells were screened, and the cells in the positive wells were transferred to 24-well plates for expansion culture until they were expanded to 10 cm dishes and the cells were frozen.

[0071] 5. Activity verification of GDF15 antibody BALB / C mice were intraperitoneally injected with liquid paraffin, and hybridoma cells were intraperitoneally injected 1-2 weeks later. The ascites was collected 7-10 days after the cells were inoculated and the antibodies were purified. The activity of the antibodies was tested, where the coating conditions were: cold pack heat seal, GDF15-Ag, coating concentration: 1 μg / ml, 10 μg / plate, coating solution: 0.05 M CB, 100 μl / well, blocking solution: CEA blocking solution, 130 μl / well, reactant concentration: as shown in Table 2, enzyme concentration: 1:1000, reaction mode: sample addition 30 min + secondary antibody 30 min + color development 10 min + termination.

[0072] Table 2 GDF15 antibody activity detection The results are shown in Table 2 and Figure 3 As shown, the EC50 value of antibody #12 is the lowest, indicating that it has the strongest ability to bind to the antigen.

[0073] Example 2 This example shows the detection of serum samples of healthy and cancer patients using the anti-GDF15 antibodies screened in Example 1 by enzyme-linked immunosorbent assay.

[0074] The prepared GDF15 antibody was diluted to 2 μg / ml with coating buffer, 100 μl was added to each well, placed at 4℃ overnight, 200 μl of blocking solution was added and placed at 37℃ for 2 h, GDF15 antigen was diluted to 1 ng / ml, three-fold gradient dilution, 7 gradients were diluted, placed at 37℃ for 1 h, the plate was washed, 100 μl of GDF15-HRP secondary antibody with a dilution ratio of 1000 times was added to each well, the plate was washed, the color development solution was added for 5 min, the stop solution was added, and the ELISA plate was used to read and the standard curve was established. The serum GDF15 content of healthy controls (HC) and people with clinical background (patients) was detected, and the sample number was 30 people.

[0075] Test results such as Figure 4As shown, the results indicate that the antibody of the present invention has a high recognition ability for the GDF15 protein, and is capable of detecting the level of GDF15 in serum, thereby being used for scientific research or clinical immunohistochemistry to detect GDF15 expression, and further for early diagnosis and prognosis of cancer patients.

[0076] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that It can target GDF15, and the antibody or its antigen-binding fragment comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies or murine antibodies, and the antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, scFv or scFv Fc fragments.

4. A nucleic acid molecule, characterized in that It comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A carrier molecule, characterized in that It comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 4 or the vector molecule according to claim 5.

7. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: Prepared by artificial synthesis or genetic engineering.

8. A detection product for GDF15, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. The detection product according to claim 8, characterized in that: The detection product includes a test kit, a test paper or a protein chip.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a GDF15 detection product.

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