An anti-GDF15 antibody, its preparation method and applications

Abstract

The present invention discloses an anti-GDF15 antibody, a preparation method thereof and an application thereof. In the present invention, a prokaryotic host cell is used to recombinantly express human GDF15 in an immunogen. By fusing a thioredoxin tag at the N-terminus, the recombinant GDF15 antigen has stronger immunogenicity and more exposed dominant epitopes, and a monoclonal antibody with higher affinity is prepared. In addition, the present invention uses multiple negative and positive clinical samples for verification, and the results show that the monoclonal antibody screened in the present invention has higher detection sensitivity and stronger specificity for cancer. 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 particularly to an anti-GDF15 antibody, a preparation method thereof, and an application thereof. Background Art

[0002] Growth differentiation factor 15 or macrophage inhibitory cytokine-1 (GDF15 / MIC-1) belongs to the TGF-β superfamily members 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 responses to stress signals. The role of GDF15 in cancer occurrence and development is complex, and its role depends on cancer type, stage, and tumor microenvironment. The research by Bauskin et al. in 2006 showed that the level of GDF15 in the blood circulation of several malignant tumors (including melanoma, prostate cancer, breast cancer, gastrointestinal cancer, pancreatic cancer, and colorectal cancer) can be nearly 200 times higher and is associated with low survival rate. 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 art is only for explaining the general background of the present invention and should not be regarded as admitting or implying in any form that this information constitutes the prior art known to those of ordinary skill in the art. Summary of the Invention

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

[0006] In a first aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof that can target GDF15, and the antibody or the antigen-binding fragment thereof includes 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, for the antibody or the antigen-binding fragment thereof according to the present invention, wherein the antibody or the antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0008] (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;

[0009] (II) An amino acid sequence having at least 90% homology with the amino acid sequence shown in (I) and having the same function;

[0010] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment includes Fab, Fab’, F(ab) 2 、F(ab’) 2 、scFv or scFv Fc fragment.

[0012] A second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to the present invention.

[0013] A third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule according to the present invention.

[0014] A fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule or vector molecule according to the present invention.

[0015] A fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the present invention, which is prepared by artificial synthesis or genetic engineering methods.

[0016] A sixth aspect of the present invention provides a detection product for GDF15, which comprises the antibody or antigen-binding fragment thereof according to the present invention.

[0017] In certain embodiments, the detection product according to the present invention, wherein the detection product includes a kit, a test strip or a protein chip.

[0018] A seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof according to the present invention in the preparation of a GDF15 detection product.

[0019] In the present invention, a prokaryotic host cell is used for recombinant expression of human GDF15 in an immunogen. By fusing a thioredoxin tag at the N-terminus, the recombinant GDF15 antigen has stronger immunogenicity and more exposed dominant epitopes, and a monoclonal antibody with higher affinity is prepared. In addition, multiple negative and positive clinical samples are used for verification in the present invention, and the results show that the monoclonal antibody of the present invention has higher detection sensitivity and stronger specificity for cancer. The antibody of the present invention has broad application prospects in the early diagnosis and prognosis of cancer. Brief Description of the Drawings

[0020] Figure 1 Shows the recombinant human GDF15 expression plasmid map of the present invention, which contains elements such as the T7 promoter, the TrxA protein expressed by N-terminal fusion, the 6×His purification tag, and the human GDF15 Ala 197-Ile 308 target gene, etc.

[0021] Figure 2 Shows the SDS-PAGE identification results of the purified GDF15 antigen of the present invention. Among them, M is the TransGen Biotech BluePlus V Protein Marker; S is the supernatant of the lysed expression bacteria; FT is the flow-through of the Ni-NTA affinity chromatography column; W is the washing with 40 mM imidazole; E is the elution with 250 mM imidazole.

[0022] Figure 3 Shows the activity detection results of the antibody of the present invention.

[0023] Figure 4 Shows the results of detecting serum samples of healthy and clinically-background populations using the antibody of the present invention. Detailed Description of the Invention

[0024] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Antibody or its antigen-binding fragment

[0028] In one aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof that can bind to GDF15 with high affinity and targeting specificity. Without being bound by any theory, the antibody or antigen-binding fragment 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.

[0029] In a specific embodiment, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0030] (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;

[0031] (II) An amino acid sequence having at least 90% homology with the amino acid sequence shown in (I) and having the same function;

[0032] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0033] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each heavy and light chain. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors. Thus, 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.

[0034] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention is composed of "framework" regions interspersed with three "complementary determining regions (CDR)". The framework regions are used to orient the CDRs for specific binding to an antigenic epitope. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxy terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0035] 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. To determine sequence identity, sequence alignment can be performed, which can be carried out in various ways known to those skilled in the art, for example, using software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment in the full-length sequences being compared.

[0036] As used herein, antibody sequences obtained by modification also fall within the scope of protection of the present invention. The term "modification" refers to any chemical modification of an 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 the addition of one or more amino acids compared to the naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in regions other than the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional characteristics of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding characteristics.

[0037] In the present invention, the antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies or murine antibodies.

[0038] As used herein, the term "monoclonal antibody", sometimes also referred to as "mAb" or Ab, refers to an immunoglobulin obtained from a pure line of cells, having the same structure and chemical properties, and being specific for a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies against different determinants), and each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained by culturing hybridomas or recombinant engineered cells and are not contaminated with other immunoglobulins. This property contrasts with polyclonal antibody preparations, which generally include antibodies against different antigenic determinants. The modifier "monoclonal" indicates the property of the antibody, which is obtained from a homogeneous population of antibodies, but this should not be construed as requiring any particular method for producing the antibody.

[0039] In this text, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species or belongs to a particular class, while the remaining segments of that chain are homologous to the corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are from the variable regions of an antibody of an animal species (such as a mouse, rat, etc.), while the constant portions are homologous to the antibody sequences from another species (such as a human). For example, to obtain a chimeric antibody, non-human B cells or hybridoma cells can be used to generate the variable regions, and the constant regions combined with them are from humans. The variable regions have the advantage of being easy to prepare, and their specificity is not affected by the source of the constant regions combined with them. At the same time, since the constant regions of chimeric antibodies can be derived from humans, the likelihood of the chimeric antibody triggering an immune response upon injection is lower than that of an antibody with non-human-derived constant regions.

[0040] In this text, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from non-human immunoglobulins, thereby reducing the immunogenicity when the xenogeneic antibody is introduced into humans, while maintaining the full antigen-binding affinity and specificity of the antibody.

[0041] In this text, the term "murine antibody" generally refers to an antibody in which the variable region framework and CDR regions are derived from murine germline immunoglobulin sequences. Additionally, if the antibody contains constant regions, they are also derived from murine germline immunoglobulin sequences. In the present invention, murine antibodies can contain amino acid residues not encoded by murine germline immunoglobulin sequences, for example, they can include mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or by in vivo somatic mutagenesis.

[0042] In this text, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that perform the function of specifically binding to an antigen. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by: including the heavy chain of a fragment such as Fv, scFv, dsFv, Fab, Fab' or F(ab') 2 or, including the light chain of a fragment such as Fv, scFv, dsFv, Fab, Fab' or F(ab') 2

[0043] ​In the present invention, the terms "directed against", "bind to", "immunobind", "specifically bind", and "target" are used interchangeably and generally refer to non-covalent interactions that occur between an immunoglobulin molecule and an antigen specific for the immunoglobulin. The strength or affinity of an immunobinding interaction can be expressed as a dissociation constant (Kd), where a smaller Kd represents a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, when used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (such as an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed using the binding dissociation equilibrium constant. Affinity can be measured by common methods well known in the art, including those known in the prior art and described herein.

[0044] Unless otherwise specified, 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 removed from its natural environment. An antibody or fragment thereof that has been "isolated" thus includes antibodies or fragments thereof purified by standard purification methods. The term also includes antibodies or fragments thereof prepared by recombinant expression in host cells and chemically synthesized antibodies or fragments thereof.

[0045] Nucleic acid molecule

[0046] In one aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof described in the present invention.

[0047] The term "nucleic acid" as used in the present invention is intended to include polymeric forms of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and / or their analogs, which include DNA, RNA, and DNA / RNA hybrids, and which also include DNA or RNA analogs, such as those containing modified backbones (such as peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Thus, the nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0048] Once the coding sequence of the antibody described in the present invention is isolated, the antibody can be obtained in large quantities using recombinant techniques. An exemplary method is to clone its coding gene into a vector, transfer it into cells, and then isolate it from the proliferated host cells by conventional methods.

[0049] Carrier molecule

[0050] In one aspect of the present invention, there is provided a vector molecule comprising the nucleic acid molecule described in the present invention.

[0051] The vector of the present invention refers to an artificially constructed entity 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 contains a target gene encoding the antibody of the present invention, a promoter, a terminator, or optionally further contains a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0052] Host cell

[0053] In one aspect of the present invention, there is provided a host cell comprising the nucleic acid molecule or the vector molecule of the present invention.

[0054] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or an expression vector containing the nucleic acid molecule of the present invention. The host cell includes any progeny of the parental cell that is different from the parental cell due to mutations occurring during replication.

[0055] Preparation method

[0056] In one aspect of the present invention, there is provided a method for preparing the antibody or its antigen-binding fragment of the present invention. The preparation method is not particularly limited and includes preparation by artificial synthesis or genetic engineering methods.

[0057] In certain embodiments, the antibody of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art. For example, the antibody or its antigen-binding fragment of the present invention can be obtained by direct amino acid synthesis.

[0058] In certain embodiments, the antibody of the present invention is obtained by genetic engineering expression. Genetic engineering expression systems for genetic engineering expression include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include the Escherichia coli expression system. Eukaryotic cell expression systems include yeast expression systems, insect cell expression systems, and mammalian cell expression systems. In a preferred embodiment, the antibody of the present invention can be prepared by the following steps:

[0059] (1) Construct a recombinant vector for the heavy chain of the antibody expressing the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector for the light chain of the antibody expressing the amino acid sequence shown in SEQ ID NO.8;

[0060] (2) Transform the vector into a host cell and culture it under conditions suitable for antibody expression;

[0061] (3) Collect the antibody and purify it.

[0062] In another preferred embodiment, the antibody of the present invention is prepared by immunizing 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 includes:

[0063] (1’) Construct a recombinant vector expressing the antigen sequence shown in SEQ ID No. 9, and obtain the recombinant antigen after expression;

[0064] (2’) Inoculate the antigen into mice to prepare monoclonal antibodies;

[0065] (3’) Use the antigen to screen for positive hybridoma cells.

[0066] 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, and the nucleotide sequence of the constructed prokaryotic expression vector of GDF15 is as shown in SEQ ID NO. 10.

[0067] In the step (1’) of the present invention, the host bacteria used for expressing the recombinant antigen include BL21(DE3), BL21Star, Rosetta, etc., preferably Rosetta.

[0068] In the step (2’) of the present invention, the amount of antigen inoculated into the experimental mice is 250 μg, and the immunoadjuvant used for the antigen is Freund's complete adjuvant.

[0069] In the steps (1’) and (3’) of the present invention, the amino acid sequence of the full-length antigen is as shown in SEQ ID NO. 9.

[0070] Detection product

[0071] The present invention further provides a detection product, which is used to detect (or quantify) GDF15, and contains the antibody of the present invention or its antigen-binding fragment, and instructions on how to implement the detection method of the present invention for GDF15.

[0072] In a preferred embodiment, the detection product includes a kit, a test strip or a protein chip.

[0073] As used herein, the term "kit" refers to a combination of reagents and other materials. A kit is expected to contain reagents such as buffers, protein stabilizing reagents, 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, a kit may also include instructions for using the reagents. A kit can be packaged in any suitable manner. Generally, it has components in a single container or (if necessary) in multiple containers and an instruction manual for performing the detection. Kits can be prepared by a variety of methods known in the art.

[0074] In certain embodiments, the kit may further include at least one of a washing solution, a substrate solution, a diluent, and a calibration solution. Among them, the composition of the washing solution is not particularly limited, and examples thereof include, but are not limited to, buffers, surfactants, and preservatives. Known substrates can be used for the substrate solution, and examples thereof include, but are not limited to, chromogenic substrates, fluorescent substrates, chemiluminescent substrates, etc. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, buffers, surfactants, etc. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, BSA solutions, trehalose solutions, animal sera, etc.

[0075] Use

[0076] The present invention further provides the use of the antibody or its antigen-binding fragment of the present invention in the preparation of a GDF15 detection product, wherein the detection product includes a kit, a test strip, or a protein chip. The detection product is used for the prediction, diagnosis, and / or prognosis of GDF15-related diseases. "GDF15-related diseases" refers to a subject having abnormal expression of GDF15, or an increase in the amount or activity of GDF15 in a test sample (including but not limited to blood, cell lysates, etc.). For example, in some cases, compared with the normal expression of GDF15 in healthy individuals, the expression level of GDF15 in the test sample of the subject is significantly upregulated.

[0077] In the present invention, GDF15-related diseases include cancers, and examples of the cancers 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.

[0078] Example 1

[0079] This example shows the synthesis of monoclonal antibodies.

[0080] 1. Immunogen Preparation

[0081] In the present invention, the immunogen of GDF15 uses prokaryotic E. Coli as the host to recombinantly express human GDF15, with a thioredoxin TrxA tag fused at the N-terminus, making the recombinant GDF15 antigen have stronger immunogenicity and more exposed dominant epitopes. The monoclonal antibodies prepared by immunization will also have higher affinity. The constructed GDF15-pET32a recombinant vector is as Figure 1 shown.

[0082] The GDF15-pET32a-Rosetta(DE3) strain with correct sequencing was inoculated into 10 ml of LB+kan medium at a ratio of 1:100. After overnight culture, it was inoculated into 1 L of LB+kan medium at a ratio of 1:100. When the OD600 reached approximately 0.6, 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG) was added, and the culture was continued overnight at 220 rpm and 20 °C. The cells were collected, and the fermentation product was resuspended completely in 250 ml of 50 mM Tris-HCl pH8.0 + 500 mM NaCl, pre-cooled on ice for 10 min, and then lysed by sonication at a sonication intensity of 300 W, sonication for 1 s, interval of 1 s, and a total sonication time of 30 min. After identification, GDF15 was expressed as semi-inclusion body and semi-soluble in prokaryotic expression.

[0083] After the cells were disrupted, the lysate supernatant was loaded onto a Ni-NTA affinity chromatography column. After loading, it was washed with 50 mM Tris-HCl pH8.0 + 500 mM NaCl + 20 mM imidazole. During the washing process, the absorbance at the A280 wavelength was continuously monitored. After it no longer changed, the target protein was eluted with an eluent of 50 mM Tris-HCl pH8.0 + 500 mM NaCl + 250 mM imidazole. During the elution process, the absorbance at the A280 wavelength was continuously monitored. After the absorbance decreased below 0.2, the collection was ended. Samples of all components were taken for SDS-PAGE identification, and the identification results are as Figure 2 shown, and the target protein is located near 30 kDa, which is consistent with the predicted molecular weight.

[0084] 2. Mouse Immunization

[0085] Purebred BALB / C mice were selected. For the primary immunization, 50 μg of GDF15 antigen was used, mixed with Freund's complete adjuvant and shaken in an amalgamator, then aspirated into a 1 ml sterile syringe and immunized at multiple subcutaneous points and intraperitoneally in the mice.

[0086] ↓ 3 weeks later

[0087] For the second immunization, the dose was the same as above, with Freund's incomplete adjuvant added, and immunized at multiple subcutaneous points and intraperitoneally in the mice;

[0088] ↓ 2 weeks later

[0089] The third immunization dose is the same as above, with Freund's incomplete adjuvant added, and immunize the mice subcutaneously at multiple points and intraperitoneally. Blood is collected 5 - 7 days later to measure its titer;

[0090] ↓ 2 - 3 weeks later

[0091] Booster immunization, using 100 μg of GDF15 antigen, intraperitoneal injection;

[0092] ↓ 3 days later

[0093] Take the spleen for fusion.

[0094] Detect the antibody titer of the GDF15 experimental mice, detect the serum of the experimental mice. Among them, the coating conditions: cold coating and heat sealing, GDF15 - Ag, coating concentration: 1 μg / ml, 10 μg / well, 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 for 30 min + secondary antibody for 30 min + color development for 10 min + termination.

[0095] Table 1 Results of the three - immunization titer determination of GDF15

[0096]

[0097] The results are shown in Table 1. The average serum titer of mice No. 1 - 3 is about 400,000.

[0098] 3. Cell fusion

[0099] On the day of fusion, the immunized BALB / C mice are sacrificed by cervical dislocation, soaked in 75% ethanol, then taken out and placed in a laminar flow hood. The spleens of the mice are taken out with ophthalmic scissors and forceps. Put them into a culture dish containing medium and remove the connective tissue. Prepare a culture dish with medium and place a cell sieve in the culture dish. Put the washed spleen into the cell sieve and grind it into single cells with the cell sieve. Transfer the cell suspension to a centrifuge tube, centrifuge, and discard the supernatant. Pipette the precipitate to remove the connective tissue, put it into a new centrifuge tube, add medium, centrifuge, discard the supernatant, pipette and mix well with the medium, and make up the volume to 20 ml.

[0100] On the day of fusion, discard the supernatant from 10 dishes of SP2 / 0, pipette and mix well with fresh medium, put it into a centrifuge tube, centrifuge, and discard the supernatant. Pipette and mix well, centrifuge. Discard the supernatant. Pipette and mix well, and make up the volume to 20 ml.

[0101] Mix myeloma cells SP2 / 0 and splenocytes in proportion, wash them 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 slightly loosen the cell pellet, centrifuge, and remove the supernatant. Add PEG solution to the centrifuge tube uniformly within 1 minute. Let it stand for 90 s, then slowly add the termination solution to terminate the reaction. Centrifuge, add the fused cells to the complete medium, and plate them in a 96-well plate pre-cultured with feeder cells the previous day.

[0102] 4. Cloning and screening of hybridoma cells

[0103] Detect the OD value of the cell supernatant by indirect ELISA method around seven days. Clone the hybridoma cells in the wells with higher positive values. Use the limiting dilution method to clone the hybridoma cells until monoclonal positive wells are screened out. Transfer the cells in the positive wells to a 24-well plate for expansion culture until it is expanded to a 10 cm dish, and freeze the cells.

[0104] 5. Activity verification of GDF15 antibody

[0105] Inject liquid paraffin intraperitoneally into BALB / C mice. Inject hybridoma cells intraperitoneally 1 - 2 weeks later. Collect ascites and purify the antibody 7 - 10 days after cell inoculation. Detect the activity of the antibody. Among them, the coating conditions are: cold coating and heat sealing, GDF15-Ag, coating concentration: 1 μg / ml, 10 μg / well, 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 for 30 min + secondary antibody for 30 min + color development for 10 min + termination.

[0106] Table 2 Activity detection of GDF15 antibody

[0107]

[0108] The results are as shown in Table 2 and Figure 3 as follows. The EC50 value of antibody 12# is the lowest, indicating that its binding ability to the antigen is the strongest.

[0109] Example 2

[0110] This example shows the detection of serum samples from healthy and cancer patients using the enzyme-linked immunosorbent assay method with the anti-GDF15 antibody screened in Example 1.

[0111] The prepared GDF15 antibody was diluted to 2 μg / ml with coating buffer, 100 μl of the sample was added to each well, and it was placed overnight at 4°C. Then, 200 μl of blocking solution was added and placed at 37°C for 2 h. The GDF15 antigen was diluted to 1 ng / ml and serially diluted in three-fold dilutions for 7 gradients, and then placed at 37°C for 1 h. The plate was washed, 100 μl of GDF15-HRP secondary antibody diluted 1000-fold was added to each well, the plate was washed again, chromogenic solution was added for color development for 5 min, stop solution was added, and the microplate reader was used to establish a standard curve. The serum GDF15 levels of healthy controls (HC) and individuals with clinical backgrounds (patients) were detected, with 30 individuals in each group.

[0112] The detection results are as Figure 4 shown. The results indicate that the antibody of the present invention has a high recognition ability for GDF15 protein, and it can detect the GDF15 level in serum, thus can be used for scientific research or clinical immunohistochemical detection of GDF15 expression, and further can be used for the early diagnosis and prognosis of cancer patients.

[0113] 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. Without departing from the scope or spirit of the present invention, various adjustments or changes can be made to the exemplary embodiments of the present invention specification. The scope of the claims should be interpreted 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 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 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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