Antibodies specific for fgfr2b, methods of making and use thereof

Abstract

The application provides an FGFR2b specific antibody (ADx018 monoclonal antibody), a preparation method and application thereof. The ADx018 monoclonal antibody has excellent immunogen binding performance to specific epitopes of FGFR2b protein, and has higher sensitivity and specificity compared with antibodies in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of immunohistochemistry, and more specifically, this invention relates to an FGFR2b-specific antibody, its preparation method and application. Background Technology

[0002] The fibroblast growth factor receptor (FGFR) family includes FGFR1, FGFR2, FGFR3, and FGFR4. They are all composed of three extracellular immunoglobulin (Ig)-like domains (D1, D2, and D3), a transmembrane domain, and an intracellular tyrosine kinase domain. FGFR2 contains two isoforms, FGFR2b and FGFR2c, which are generated by alternative splicing within the C-terminal half of the third Ig loop (D3) in the extracellular FGF-binding domain. The FGFR2b isoform is generated when the C-terminal half of D3 is encoded by exon 8, and the FGFR2c isoform is generated when the C-terminal half of D3 is encoded by exon 9. FGFR2b is a high-affinity receptor for FGF1, -3, -7, -10, and -22, while FGFR2c binds to FGF1, -2, -4, -6, -8, -9, -17, and -18.

[0003] Due to the expression patterns of FGFR2 isoforms and their ligands, FGFR2 plays a role in epithelial-mesenchymal interactions. FGFR2 contributes to carcinogenesis in various cancers through different mechanisms (mutation, amplification, fusion, and isoform switching), including breast cancer, endometrial cancer, esophageal cancer, gastric cancer, and colorectal cancer. Studies have found that 30% of HER2-negative gastric cancer patients have high expression of FGFR2b.

[0004] Currently, there are no commercially available antibodies in this field capable of detecting FGFR2b with high sensitivity and specificity, especially anti-FGFR2b monoclonal antibodies applicable to immunohistochemistry (IHC). Given the complexity of FGFR family proteins, existing detection reagents inevitably suffer from cross-reactivity issues.

[0005] Therefore, the preparation of anti-FGFR2b monoclonal antibodies that minimize cross-reactivity, have high specificity, high sensitivity, and wide application range has important practical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to provide an FGFR2b-specific antibody, its preparation method, and its application.

[0007] In a first aspect of the present invention, an FGFR2b specific antibody (including its antigen-binding fragment) is provided, the antibody comprising a light chain variable region and a heavy chain variable region; wherein in the light chain variable region, the amino acid sequence of light chain CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of light chain CDR2 is as shown in SEQ ID NO:7, and the amino acid sequence of light chain CDR3 is as shown in SEQ ID NO:8; wherein in the heavy chain variable region, the amino acid sequence of heavy chain CDR1 is as shown in SEQ ID NO:2, the amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO:4.

[0008] In one or more preferred embodiments, the FGFR2b-specific antibody comprises: an amino acid sequence of the light chain variable region as shown in SEQ ID NO:5, or having more than 80% (e.g., 85%, 90%, 93%, 95%, 97%, or 99%) identity with the sequence shown in SEQ ID NO:5; and an antibody of the heavy chain variable region amino acid sequence as shown in SEQ ID NO:1, or having more than 80% (e.g., 85%, 90%, 93%, 95%, 97%, or 99%) identity with the sequence shown in SEQ ID NO:1, and retaining specific FGFR2b binding activity.

[0009] In one or more preferred embodiments, the antibody comprises: rabbit anti-human FGFR2b monoclonal antibody, humanized antibody or chimeric antibody; or, the antibody comprises (but is not limited to): Fab, Fv, Fd, Fab'-SH, scFv or (Fab')2 fragment.

[0010] In another aspect of the invention, a separated polynucleotide is provided that encodes the FGFR2b-specific antibody described in the preceding aspect.

[0011] In another aspect of the invention, an expression construct containing the said polynucleotide is provided, or an antibody expression system including the expression construct (including the polynucleotide integrated into the genome); preferably, the construct is an expression vector; preferably, the expression system is a cell (expression system).

[0012] In one or more preferred embodiments, the cell (host cell) includes a eukaryotic cell or a prokaryotic cell.

[0013] In one or more preferred embodiments, the eukaryotic cells include yeast cells, mammalian cells, etc.

[0014] In one or more preferred embodiments, the prokaryotic cells are, for example (but not limited to), Escherichia coli cells, Bacillus subtilis cells, etc.

[0015] In one or more preferred embodiments, the mammalian cells include (but are not limited to): HEK-293 cells, Chinese hamster ovary (CHO) cells, Vero cells, NSO cells, BHK cells, PER-C6 cells, etc.

[0016] In another aspect of the present invention, a method for preparing the FGFR2b specific antibody is provided, comprising: expressing the antibody using the antibody expression system under suitable conditions for expressing the antibody, thereby obtaining the antibody; preferably, the method further comprises: purifying and separating the antibody.

[0017] In another aspect of the invention, the use of the antibody in the preparation of a targeted detection reagent that specifically targets cells expressing FGFR2b is provided; preferably, in the targeted detection reagent, the antibody is further modified with a conjugate.

[0018] In one or more preferred embodiments, the conjugate includes a detectable marker; more preferably, the detectable marker includes (but is not limited to) one or more of the following: alkaline phosphatase, horseradish peroxidase, biotin, fluorescent dyes (such as fluorescein isothiocyanate, Cy3 or Cy5), radioisotopes, ferritin, colloidal gold.

[0019] In one or more preferred embodiments, the conjugate comprises: a cytotoxin or a bioactive protein (including a second antibody or multiple antibodies).

[0020] In another aspect of the invention, a targeted detection reagent is provided that specifically targets cells expressing FGFR2b, comprising the antibody and a conjugate operatively linked thereto.

[0021] In one or more preferred embodiments, the conjugate includes a detectable marker; more preferably, the detectable marker includes (but is not limited to) one or more of the following: alkaline phosphatase, horseradish peroxidase, biotin, fluorescent dyes (such as fluorescein isothiocyanate, Cy3 or Cy5), radioisotopes, ferritin, colloidal gold.

[0022] In one or more preferred embodiments, the conjugate comprises: a cytotoxin or a bioactive protein (including a second antibody or multiple antibodies).

[0023] In one or more preferred embodiments, the antibody and the conjugate can be operatively linked by means of covalent linkage, cross-linking, coupling, attachment, etc.

[0024] In one or more preferred embodiments, when the conjugate is a cytotoxin, a radioactive isotope, or a bioactive protein, the antibody has the function of specifically targeting cells expressing FGFR2b and carrying the cytotoxin or bioactive protein to the cells.

[0025] In one or more preferred embodiments, the cytotoxicant is an antitumor toxin, including (but not limited to): methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, lalotaxel, tescetaxel or oxatataxel, orlistatine, monomethylaurestatin, maytansine, maytansine alkaloids, dolalastatin, cazithromycin, trichothecotoxin, CC1065, DXd, Duocarmycin, Calicacamicin, Pyrrolobenzodiazepines or SN-38, etc.

[0026] In another aspect of the invention, the use of the aforementioned FGFR2b-specific antibody and the aforementioned targeted detection reagent in the preparation of formulations, kits, or reagent kits for detecting diseases related to FGFR2b expression / overexpression is provided; preferably, the diseases related to FGFR2b expression / overexpression include: tumors expressing / overexpressing FGFR2b; preferably, the tumors include: gastric cancer, lung cancer (non-small cell lung cancer), ovarian cancer, endometrial cancer, and colorectal cancer (colorectal adenocarcinoma).

[0027] In another aspect of the invention, a kit or reagent kit is provided, comprising the aforementioned FGFR2b specific antibody, the aforementioned targeted detection reagent, or a composition or formulation containing the thereof.

[0028] In one or more preferred embodiments, the kit may also include (but is not limited to) any or more of the following reagents: diluent (such as sample and antibody diluent), secondary antibody (such as Polymer), chromogenic agent (such as DAB chromogenic reagent), washing solution, calibrator, quality control, stop solution, luminescent solution, antigen retrieval agent, dewaxing agent, and blocking reagent.

[0029] In one or more preferred embodiments, the kit or reagent kit further contains a solid-phase carrier, or the antibody is immobilized (coated) on the solid-phase carrier.

[0030] In one or more preferred embodiments, the solid-phase carrier includes (but is not limited to): a chip (including a liquid-phase chip, a microfluidic chip), a test pad (such as a colloidal gold test pad), a test strip (including a test strip, such as a colloidal gold test strip), a glass slide, a well plate (such as a coated plate), microspheres, and magnetic beads.

[0031] In one or more preferred embodiments, the kit or reagent kit further includes a container for containing the antibody, reagent, or composition, wherein the container may be one or more.

[0032] In one or more preferred embodiments, the kit may further include: sample collection tubes, instructions for use, etc.

[0033] In one or more preferred embodiments, the kit or reagent kit further includes instructions for use (packaging insert) describing how to use the antibody, reagent, or composition.

[0034] In another aspect of the present invention, a method for in vitro specific detection of FGFR2b protein or cells carrying the protein is provided, the method comprising: analyzing the presence of FGFR2b protein in a sample to be tested using the FGFR2b specific antibody or the targeted detection reagent; preferably, the method comprises (but is not limited to): immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry.

[0035] In one or more preferred embodiments, the method for detecting FGFR2b protein or cells carrying the protein is a non-diagnostic method (e.g., performing protein expression analysis on cells without the direct purpose of obtaining disease diagnosis results).

[0036] In one or more preferred embodiments, the method is an immunohistochemistry method, comprising: providing a sample (e.g., a slide) and labeling and staining it with the antibodies described above in this invention.

[0037] In one or more preferred embodiments, the method is an enzyme-linked immunosorbent assay (ELISA) based on the antibody or bispecific antibody of the present invention; for example, it includes: (a) contacting the sample to be tested with an FGFR2b specific antibody (first antibody), thereby causing the FGFR2b protein in the sample to bind to the FGFR2b specific antibody on a solid-phase carrier, forming a solid-phase carrier with a binary complex of "FGFR2b protein-FGFR2b specific antibody"; (b) adding a second antibody (an antibody carrying a detectable marker and binding to the first antibody) to the system of (a), thereby forming a solid-phase carrier with a ternary complex of "second antibody-FGFR2b protein-FGFR2b specific antibody"; and the second antibody carrying a marker; (c) detecting the marker in the ternary complex, thereby determining the presence and amount of FGFR2b protein in the sample to be tested; with the additional condition that steps (a) and (b) can be performed sequentially or simultaneously. Attached Figure Description

[0038] Figure 1 Analysis of the position of the purified FGFR2b protein band.

[0039] Figure 2 1. Immunoblotting analysis using ADx018 monoclonal antibody. Lane 1: FGFR1b recombinant protein; Lane 2: FGFR2b recombinant protein; Lane 3: FGFR2c recombinant protein; Lane 4: FGFR3b recombinant protein; Lane 5: FGFR4 recombinant protein.

[0040] Figure 3 The results of immunohistochemical detection using the ADx018 monoclonal antibody of the present invention were obtained for cell line samples HEK293T, FGFR1b plasmid transiently overexpressing HEK293T, FGFR2b plasmid transiently overexpressing HEK293T, FGFR2c plasmid transiently overexpressing HEK293T, and FGFR3b plasmid transiently overexpressing HEK293T. The commercially available monoclonal antibodies FGFR2b (SDT-423-33) and FGFR2b (D4L2V) were used as comparative antibodies.

[0041] Figure 4 Immunological analysis of human cell lines. Cell line samples included: human gastric cancer cells KATO III, human colorectal adenocarcinoma cells H716, and human colorectal adenocarcinoma cells HCT-15. Immunohistochemical analysis of the ADx018 monoclonal antibody was performed; commercially available monoclonal antibodies FGFR2b (SDT-423-33) and FGFR2b (D4L2V) were used as comparative antibodies.

[0042] Figure 5 The results of immunohistochemical detection using the ADx018 monoclonal antibody of the present invention on normal human gastric, lung, and ovarian paraffin tissues were analyzed; commercially available monoclonal antibodies FGFR2b (SDT-423-33) and FGFR2 (D4L2V) were used as comparative antibodies.

[0043] Figure 6 Staining of gastric cancer and endometrial cancer samples with FGFR2b (ADx018) monoclonal antibody was performed, with commercially available monoclonal antibodies FGFR2b (SDT-423-33) and FGFR2 (D4L2V) used as comparative antibodies.

[0044] Figure 7 The staining of ADx018 monoclonal antibody in lung cancer and ovarian cancer samples was performed, with commercially available monoclonal antibodies FGFR2b (SDT-423-33) and FGFR2 (D4L2V) used as comparative antibodies.

[0045] Figure 8 Staining of ADx018 monoclonal antibody on gastric cancer tissue microarray.

[0046] Figure 9 Staining of ADx018 monoclonal antibody on lung cancer tissue microarray.

[0047] Figure 10 Staining of ADx018 monoclonal antibody on ovarian cancer tissue microarray.

[0048] Figure 11 Staining of ADx018 monoclonal antibody on endometrial cancer tissue microarray. Detailed Implementation

[0049] Through in-depth research, the inventors have disclosed a monoclonal antibody against FGFR2b, called ADx018 monoclonal antibody. The ADx018 monoclonal antibody has excellent immunogen binding performance against a specific epitope of the FGFR2b protein and has higher sensitivity and specificity compared with existing antibodies in the field.

[0050] Those skilled in the art will understand that an antigen may contain a variety of epitopes (antigenic determinants). Therefore, more than one antibody can be obtained against the same antigen, and these antibodies generally have different binding properties (such as specificity). Therefore, those skilled in the art need to compare and screen for the same antigen to find a monoclonal antibody that specifically binds to and meets specific laboratory or clinical needs. Because antigens have a three-dimensional spatial structure, many epitopes are contained within this structure, making it difficult to find antibodies that specifically bind to preferred epitopes that are stably located outside the spatial structure. The inventors have conducted in-depth research and isolated a monoclonal antibody with high specificity and sensitivity to FGFR2b, particularly suitable for immunohistochemistry.

[0051] In this invention, the target of detection is the FGFR2b protein, whose expression indicates specific indications (such as cancer, including gastric cancer, lung cancer, ovarian cancer, endometrial cancer, and colorectal cancer). Existing anti-FGFR2b monoclonal antibodies suffer from non-specific reactions and cross-reactivity, making it difficult to overcome the challenges of non-specificity and low sensitivity. This invention provides a novel detection reagent to address these technical difficulties.

[0052] As used in this invention, "recombinant" refers to proteins, genetic engineering vectors, or cells obtained (or mass-produced) through genetic engineering.

[0053] As used in this invention, "sample to be tested" encompasses a variety of sample types, including various objects for which FGFR2b content needs to be detected.

[0054] As used in this invention, "specificity" means that the antibody can bind to FGFR2b or a fragment thereof. Specifically, it refers to an antibody that can bind to FGFR2b or a fragment but does not recognize or bind to other unrelated molecules. More specifically, it refers to an antibody that can bind to cell membrane FGFR2b or a fragment but does not recognize or bind to other molecules.

[0055] As used in this invention, "sample to be tested" can cover a variety of sample types, including cells (including ex vivo), tissues, and body fluids.

[0056] As used in this invention, the terms "first monoclonal antibody," "first antibody," and "primary antibody" are interchangeable and refer to the anti-FGFR2b monoclonal antibody of this invention.

[0057] As used in this invention, the terms "detection antibody," "second monoclonal antibody," "second antibody," "enzyme-labeled antibody," and "secondary antibody" are used interchangeably and refer to antibodies that specifically target FGFR2b and correspond to the first antibody. For the antigen FGFR2b, the corresponding first and second antibodies are different and can simultaneously bind to different epitopes (antigenic determinants) of the FGFR2b.

[0058] As used in this invention, the term "marker" or "detectable marker" refers to a marker located on or linked to (indirectly linked to) the monoclonal antibody or second monoclonal antibody of this invention, used to determine the presence and amount of FGFR2b in a sample to be tested. For example, the marker may be selected from: alkaline phosphatase (AP), horseradish peroxidase (HRP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0059] In this invention, a human FGFR2b protein fragment (positions 154-358) is selected as the immunogen. The amino acid sequence of the immunogen is shown as positions 1-205 in SEQ ID NO:9. The FGFR2b protein fragment provides suitable epitopes, which facilitates the screening of antibodies in this invention. The immunogen is fused with a linker and a tag to facilitate recombinant operations such as purification. The FGFR2b protein fragment can be a chemically synthesized product or generated from a prokaryotic or eukaryotic host (e.g., mammalian cells, yeast, bacteria, higher plants, and insects) using recombinant technology. The polynucleotide encoding the polypeptide can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods.

[0060] The anti-FGFR2b protein monoclonal antibody of the present invention comprises a binding domain containing a complementary determining region (CDR) selected from the following: heavy chain CDR1 as shown in SEQ ID NO:2, heavy chain CDR2 as shown in SEQ ID NO:3, heavy chain CDR3 as shown in SEQ ID NO:4; light chain CDR1 as shown in SEQ ID NO:6, light chain CDR2 as shown in SEQ ID NO:7, and light chain CDR3 as shown in SEQ ID NO:8.

[0061] In a preferred embodiment, the anti-FGFR2b antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO:5; and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1.

[0062] In some implementations, the antibodies provided herein are antibody fragments. Antibody structures can be modified to construct molecules with similar antigen affinity but different structures, such as Fab, Fv, Fd, Fab'-SH, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies, antibody conjugates, bifunctional antibodies, etc. The Fab' fragment refers to a Fab with a small number of amino acid residues (including one or more cysteine ​​residues from the antibody hinge region) added to the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH refers to a Fab' with free thiol groups on the cysteine ​​residues in the constant region.

[0063] In some embodiments, the invented antibody may be modified into a chimeric antibody or further shaped into a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, humanized antibodies may also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues derived from the non-human antibody, for example, to repair or improve antibody specificity or affinity.

[0064] This invention also includes variants of the aforementioned anti-FGFR2b antibody. For example, antibody variants prepared to further improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, residue deletions and / or insertions and / or substitutions within the antibody's amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain a final construct that can possess desired characteristics, such as antigen binding.

[0065] Amino acids can be grouped according to their common side chain properties for preferred amino acid substitution, for example, substitution with amino acids of the same / property: (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0066] After the antibodies are disclosed in this invention, those skilled in the art can artificially prepare them using bioengineering techniques known in the art. The monoclonal antibodies of this invention can be obtained using conventional immunoassay techniques, utilizing FGFR2b or its fragments or functional regions. Alternatively, they can be prepared using recombinant methods or synthesized using a peptide synthesizer.

[0067] To recombinantly produce anti-FGFR2b antibodies, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding both the heavy and light chains of the antibody.

[0068] Suitable host cells for cloning or expressing antibody-encoding vectors include the eukaryotic or prokaryotic cells listed herein. After expression, the antibody in the soluble fraction can be isolated from the cells and further purified.

[0069] In practical applications, known antibody modification schemes can be used to modify the structure and sequence of the monoclonal antibody FGFR2b as needed to obtain the required properties.

[0070] In a preferred embodiment, the anti-FGFR2b monoclonal antibody further comprises a modifying conjugate, which includes several detectable markers. A variety of detectable markers conventionally used in the art for binding with detection antibodies can be employed. Any marker capable of binding to the monoclonal antibody of the present invention and, after appropriate processing, accurately indicating the presence and amount of the target protein in the sample to be tested is acceptable.

[0071] When antibodies are labeled for detection, the labeling methods can include fluorescent labeling, chemiluminescent labeling, radioactive labeling, enzyme-linked labeling, biotin / avidin labeling, magnetic bead labeling, or nanoparticle labeling.

[0072] The detectable markers described in this invention may include, but are not limited to: fluorescent markers, chromogenic markers, colloidal gold; such as: enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. More specifically, examples include alkaline phosphatase, horseradish peroxidase, glucose oxidase, biotin, β-D-galactosidase, urease, catalase, glucosyl amylase, fluorescein isothiocyanate, radioactive isotopes, Cy3, or Cy5. More than one marker may also be included.

[0073] When using some of the enzyme markers shown above, it is also necessary to use some substrates that bind to the corresponding enzymes, so that the presence or amount of the markers can be reported by means of color development, etc.

[0074] The substrate corresponding to the label, such as the enzyme, can be catalyzed by the label to produce a colorimetric reaction, displaying a recognition signal indicating the binding of the secondary antibody to the target protein. Examples of such substrates include: p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase; o-phenylenediamine (OPD), tetramethylbenzidine (MB), and ABTS for horseradish peroxidase; phycoerythrin (streptavidin-phycoerythrin, also known as PE-labeled streptavidin, SA-R-PE) for biotin; and so on. Those skilled in the art can select a suitable substrate based on the type and characteristics of the label used.

[0075] The marker can be directly applied to the detection antibody; or, the marker can be applied to the anti-antibody of the specific anti-second antibody. Those skilled in the art can select a suitable marker according to the type and characteristics of the antibody used.

[0076] For detection and / or analytical purposes, the labeling used to label antibodies depends on the specific detection / analytical technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labeling for detection / analytical techniques and / or methods known in the art is well known to those skilled in the art.

[0077] As a specific example, the detectable marker is HRP, which acts as a colorimetric marker and generates a signal when combined with OPD, MB, or ABTS.

[0078] After obtaining the anti-FGFR2b antibody of the present invention, those skilled in the art can sensitively detect the presence or concentration of FGFR2b in a sample through various methods, using techniques commonly used in the field of immunology. These include qualitative and quantitative detection methods. Preferably, the qualitative detection includes: identifying the presence of FGFR2b by immunoblotting or immunofluorescence methods; for example, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, and immunofluorescence; more specifically, methods such as ELISA, immunogold immunofluorescence strips, immunofluorescence strips, and homogeneous enzyme immunoassay to measure FGFR2b. The application results are ideal.

[0079] Compared to some existing anti-FGFR2b antibodies, the anti-FGFR2b antibody of this invention exhibits highly ideal specificity, specifically binding to cell membrane FGFR2b without nuclear staining. Nuclear staining is considered non-specific. Many cell membrane targets are primarily detected through cell membrane staining using IHC. For example, FGFR2b ADC drugs target cell membrane FGFR2b. If the FGFR2b IHC antibody cannot accurately assess cell membrane FGFR2b staining, it cannot be used for retrospective clinical trials, CTA enrollment, or companion assays.

[0080] The anti-FGFR2b antibody described in this invention can also be linked to one or more cytotoxins, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof), or radioisotopes. For example, antibody-drug conjugates (ADCs) can be formed, wherein the antibody is bound to one or more drugs, including but not limited to maytansine; orlistatine, such as the monomethyl orlistatine drug portions DE and DF (MMAE and MMAF); dolalastatin; cazithromycin or its derivatives; methotrexate; vinca disine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; or trichothecene, etc.

[0081] The anti-FGFR2b antibody of the present invention can also be linked to one or more enzyme-active toxins or fragments thereof, including but not limited to diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain, and trichothecotoxin.

[0082] The anti-FGFR2b antibody described in this invention can also bind to one or more radioactive atoms to form radioactive conjugates. Examples of various radioactive isotopes include At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 And radioactive isotopes of Lu.

[0083] This invention also provides a kit for detecting FGFR2b, the kit containing the monoclonal antibody described in this invention. In some embodiments, one of the monoclonal antibodies is immobilized on a solid-phase support, and the other serves as the detection antibody. As an example, the solid-phase support used may be a microtiter plate (ELISA plate).

[0084] To eliminate false positives and false negatives, quality controls (restrictions) can be set up during the testing process. Furthermore, to obtain quantitative results, multiple FGFR2b standards with known concentrations can be set up during the testing process. Conventional methods can be used to set up the standards. For quantitative detection, a standard curve can be constructed using the aforementioned standards, including plotting the OD values ​​of the standards on the ordinate (Y-axis) and the standard concentrations on the abscissa (X-axis) to create a quantitative standard curve for the FGFR2b kit. Therefore, based on the OD values ​​obtained from the test sample, the concentration of FGFR2b in the test sample can be calculated using the standard curve.

[0085] Furthermore, to facilitate testing, the kit preferably includes other auxiliary reagents, such as those commonly used in immunohistochemical methods. The properties and preparation methods of these reagents are well known to those skilled in the art. These reagents include, but are not limited to, chromogenic agents, washing buffers, stop solutions, and sensitizing diluents. These reagents can be adjusted according to different detection methods. The kit may also include instructions for use.

[0086] The main beneficial effects of this invention are:

[0087] The anti-FGFR2b antibody described in this invention has high affinity and high specificity in recognizing FGFR2b, especially cell membrane FGFR2b, exhibiting very high specificity, sensitivity, and accuracy. Currently, there is a lack in the art of antibodies that can detect FGFR2b with high specificity, high sensitivity, and strict avoidance of cross-reactivity, as described in this invention, especially anti-FGFR2b monoclonal antibodies that can be applied to immunohistochemistry (IHC).

[0088] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0089] Example 1: Preparation of Immunogen

[0090] First, the inventors analyzed human FGFR2b (UNIPROT ID: P21802-3). After analysis and experiments targeting different sequence segments, the inventors selected amino acids 154-358 of human FGFR2b (i.e., positions 1-205 in SEQ ID NO: 9 below) as the immunogen.

[0091] When used for recombinant expression, the inventors added a tag consisting of 6 histidines to the C-terminus of the protein (SEQ ID NO:9; the 6 histidines are indicated by underscores).

[0092] PYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTYHLDVVERSPHR PILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQWIKHVEKNGSKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQSAWLTVL HHHHHH

[0093] The preparation process of the recombinant human FGFR2b protein fragment is as follows:

[0094] Recombinant human FGFR2b protein fragment was expressed using the HEK293 human embryonic kidney cell system. The protein gene was synthesized chemically, and a six-histidine tag was added to the C-terminus. Overexpression was performed via transient transfection in the HEK293 system, and the overexpression supernatant was purified using a Ni column. The purified recombinant protein was evaluated for purity and endotoxin was detected using SDS-PAGE.

[0095] Test results as follows Figure 1 The purified FGFR2b protein band was correctly positioned, with a purity >95%. Meeting these requirements, it was used as an immunogen for immunization of experimental animals.

[0096] Example 2: Immunoassay and ELISA Detection

[0097] Healthy New Zealand white rabbits were selected and immunized using standard experimental injection methods. Four to five injections were administered, with an interval of 3+2+2+2 weeks. Before each injection, the immunogen was thoroughly emulsified with Freund's adjuvant to ensure effectiveness. Small amounts of serum were collected during this period for ELISA titer testing to assess immunization efficacy until the serum titer met expectations. After the fourth and fifth immunizations, 1 ml of rabbit blood was collected for serum titer testing using ELISA. The indirect ELISA procedure was as follows: the rabbits were coated with recombinant immunogen protein and incubated overnight at 4°C. The next day, the plate was dried and blocked with BSA for one hour before use. Serum was diluted 1:250 with buffer, and then serially diluted 1:4 (6-8 serial dilutions as usual). The diluted serum was added to the wells of coated and sealed ELISA plates, incubated for 1-2 hours, then dried and washed 3-5 times. Alkaline phosphatase-labeled goat anti-rabbit serum was then added for the reaction. Color development was performed using p-nitrophenyl phosphate (p-NPP). After stopping the color development, the OD value at 405 nm was measured. At a 1:64000 dilution, a reading >0.2 was considered a strong immunoreaction, and the next step could proceed.

[0098] All three rabbits showed good immune responses. Blood was collected after the fourth immunization; the serum titer was slightly low, close to 0.2 at a 1:64000 dilution. Further steps could be attempted. The overall titer decreased slightly after the fifth immunization, indicating that immunization could be discontinued. Spleen cells from the three rabbits were mixed and fused, resulting in a doubling of the number of fused spleen cells.

[0099] Example 3: Fusion and Hybridoma Screening

[0100] Three rabbits were euthanized, and their immunized spleens were immediately isolated. Viable spleen cells were then fused with rabbit myeloma cells to develop hybridoma cells.

[0101] After the cell fusion operation was completed, the cells were plated into 12 96-well cell culture plates after adjusting the density, and then placed in a cell culture incubator for 2-3 weeks. After observing the growth of hybridoma cells and the formation of cell clones (colonies), the hybridoma culture supernatant was taken for ELISA detection. 153 positive hybridoma clones were initially screened from 1152 wells.

[0102] The hybridoma clones that were initially screened were transferred into 24-well cell culture plates for expansion culture for about one week. The culture supernatant of the hybridoma was then used for ELISA and IHC validation.

[0103] Of the 153 selected hybridomas, 86 clones were qualified by ELISA after screening by the inventors.

[0104] Eighty-six ELISA-positive clones were tested with IHC. After repeated experiments, it was found that only 11 clones recognized FGFR2IIIb but not FGFR2IIIc, FGFR3IIIb, or FGFR1IIIb overexpressing 293T cell paraffin sections.

[0105] The inventors performed immunoblotting on these 11 clones, and the results showed that only 6 clones stained the KATO III cell lysate, indicating that obtaining ideal detection antibodies is difficult.

[0106] Example 4: Recombination, Production and Purification

[0107] 1. Reorganization and small-scale transfer

[0108] Six positive hybridoma cells were cultured normally for 2-3 days. After collecting an appropriate amount of hybridoma cells, antibody mRNA was prepared by lysis. The nucleic acids encoding the heavy and light chains of B cells secreting specific antibodies were amplified by RT-PCR. The obtained nucleic acids encoding the heavy and light chains were constructed into the pcDNA3.4 vector by homologous recombination. The vector was transformed into TOP10 competent cells and cultured overnight at 37°C. Single colonies were picked and sequenced to obtain the nucleic acid sequences encoding the heavy and light chains.

[0109] Using this template, the heavy and light chain genes of the antibody were amplified by RT-PCR using specially designed primers. After sequencing confirmation, the amplified genes were constructed into an expression vector.

[0110] The constructed antibody expression plasmid was transfected into HEK293 cells to express a small amount of recombinant antibody. The transfection supernatant was subjected to IHC and Western blot staining. The clone with the best sensitivity and specificity in gastric cancer tissue was selected as ADx018, and the antibody was purified.

[0111] 2. Structural analysis of monoclonal antibody ADx018

[0112] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows (SEQ ID NO:1; underlined portion indicates CDR region) (Kabat):

[0113]

[0114] In the heavy chain of the anti-FGFR2b monoclonal antibody, each CDR includes:

[0115] CDR1: RYAMS (SEQ ID NO:2);

[0116] CDR2: YITYSGKLYYATWAKG (SEQ ID NO:3);

[0117] CDR3: AGATYSGDIYSNI(SEQ ID NO:4);

[0118] The amino acid sequence of the light chain variable region of the monoclonal antibody is as follows (SEQ ID NO:5; underlined portion indicates CDR region) (Kabat):

[0119]

[0120] In the light chain of the anti-FGFR2b monoclonal antibody, each CDR includes:

[0121] CDR1: QASQSISYYLA (SEQ ID NO:6);

[0122] CDR2: EASKLAS (SEQ ID NO:7);

[0123] CDR3: QNTGYISSSYNG (SEQ ID NO:8).

[0124] 3. Production purification

[0125] HEK293 cells were co-transfected with pcDNA3.4 plasmid containing the heavy and light chains of ADx018 using transfection reagents to produce recombinant antibodies. The HEK293 cell culture supernatant was collected, centrifuged to remove cells and debris, and then purified using a protein A column for affinity purification. The appropriate protein A column size was selected based on the total amount of recombinant antibody expressed. Before purification, the protein A column was equilibrated with PBS buffer, and then loaded at a suitable flow rate (generally 0.5 ml / min–5 ml / min). After loading, non-specific proteins and other impurities were washed away with PBS buffer. Finally, the antibody specifically bound to protein A was eluted with acidic elution buffer (pH 2.5–4.0), and the eluted antibody peak was collected based on the A280 absorption peak. The acidic elution buffer was then neutralized to a neutral pH using neutral buffer (pH 7.0–8.5).

[0126] After purification, the antibody was replaced with PBS buffer. The antibody purity was determined by size exclusion chromatography (SEC), and verified by immunohistochemistry and Western blotting. The purified antibody was then stored at -20°C.

[0127] Example 5: Immunoblot detection based on monoclonal antibody ADx018

[0128] (1) Experimental steps

[0129] The ADx018 monoclonal antibody was subjected to Western blotting reactions with recombinant FGFR1b protein (Univitamin, UA020008), recombinant FGFR2b protein (Example 1), recombinant FGFR2c protein (Nearshore Protein, C13P), recombinant FGFR3b protein (Nearshore Protein, CS68), and recombinant FGFR4 protein (Univitamin, S0A1103) to verify the specificity of the monoclonal antibody. Specifically, each protein was added to 4× Loading Buffer, denatured at 95°C for 10 min, and 20 ng was loaded into each lane.

[0130] After SDS-PAGE, the protein was transferred to a PVDF membrane; after blocking with 5% skim milk powder (prepared with 1×TBST) at room temperature for 2 hours, it was incubated overnight at 4°C with the ADx018 monoclonal antibody (0.2ug / ml).

[0131] The following day, after washing, HRP-labeled goat anti-rabbit IgG (1:2000) was added and incubated at room temperature for 1 hour, followed by ECL chemiluminescence color development.

[0132] (2) Test results

[0133] like Figure 2 As shown, the ADx018 monoclonal antibody can be used in immunoblotting experiments and specifically recognizes the FGFR2b recombinant protein, but does not recognize FGFR1b, FGFR2c, FGFR3, and FGFR4 without any cross-reactivity.

[0134] Example 6: Immunological analysis of overexpressing cell lines

[0135] 1. Experimental Procedure

[0136] In this embodiment, the cell line samples were FGFR1b (Uniprot ID: P11362-19), FGFR2b (Uniprot ID: P21802-3), FGFR2c (Uniprot ID: P21802), and FGFR3b (Uniprot ID: P22607-2) transiently transfected and overexpressing 293T cells, which were then embedded in paraffin-embedded cell blocks (source: Abcam). After the cell lines were paraffin-embedded and baked, immunohistochemical staining was performed using the ADx018 monoclonal antibody (1.4 ug / ml) described in this invention, the commercially available FGFR2b antibody (clone number SDT-423-33, catalog number S0B2232, purchased from Unimicron, 1.4 ug / ml), and the FGFR2 antibody (clone number D4L2V, catalog number 23328, purchased from CST, 0.2 ug / ml) on a Leica BOND-MAX fully automated immunostaining system. The specific procedures are shown in Table 1.

[0137] Table 1

[0138] step reagents time temperature Dewaxing Bond Dewax Solution default RT Antigen repair Bond Epitope Retrieval ER2 Solution 40min 100℃ Closed Peroxide Block 5min RT Primary Antibody Incubation FGFR2b 60min RT Secondary antibody incubation Polymer 15min RT DAB colorimetric assay Mixed DAB Refine 5min RT Hematoxylin counterstaining Hematoxylin 5min RT

[0139] 2. Test Results

[0140] As shown in Table 2 and Figure 3 As shown, the immunohistochemical results are as follows:

[0141] FGFR2b(ADx018): Specific strong cell membrane staining was observed on 293T cells transiently overexpressing FGFR2b, but no staining was observed on 293T cells transiently overexpressing FGFR1b, FGFR2c, and FGFR3b.

[0142] FGFR2b (SDT-423-33): Strong cell membrane staining was observed on 293T cells transiently overexpressing FGFR2b, but background staining was observed on 293T cells transiently overexpressing FGFR1b and FGFR3b, indicating that the antibody recognizes FGFR2b but not FGFR2c, but cross-recognizes FGFR1b and FGFR3b.

[0143] FGFR2(D4L2V): Strong cell membrane staining was observed in 293T cells transiently overexpressing FGFR2b and FGFR2c, but background staining was observed in 293T cells transiently overexpressing FGFR1b and FGFR3b, indicating that the antibody recognizes both FGFR2b and FGFR2c subtypes simultaneously, and there is significant cross-recognition for FGFR1b and FGFR3b.

[0144] Therefore, the ADx018 monoclonal antibody obtained in this invention has better specificity than commercially available SDT-423-33 and D4L2V in recognizing the FGFR2b antigen.

[0145] Table 2

[0146]

[0147] Example 7: Immunological analysis of cell lines

[0148] 1. Experimental Procedure

[0149] The cell line samples in this embodiment are: human gastric cancer cells KATO III, human colorectal adenocarcinoma cells H716, and human colorectal adenocarcinoma cells HCT-15, with paraffin-embedded cell blocks (source: Haixing Biotechnology).

[0150] After baking paraffin sections of cell lines, the FGFR2b mRNA in situ hybridization technique (company: ACD, trade name: BaseScope Probe-BA-Hs-FGFR2-tv2-E7E8, catalog number: 715151) recommended in the literature (PMID: 35866380) was used to determine whether the samples expressed FGFR2b, and the FGFR2c mRNA in situ hybridization technique (company: ACD, trade name: BaseScopeProbe-BA-Hs-FGFR2-tv1-E7E8, catalog number: 710031) was used to determine whether the samples expressed FGFR2c.

[0151] Next, immunohistochemical detection was performed using the ADx018 monoclonal antibody (1.4 ug / ml) described in this invention, commercially available FGFR2b antibody (clone number SDT-423-33, catalog number S0B2232, purchased from Unimicron, 1.4 ug / ml), and FGFR2 antibody (clone number D4L2V, catalog number 23328, purchased from CST, 0.2 ug / ml) on a Leica BOND-MAX fully automated immunostaining system. The specific procedure is shown in Table 1.

[0152] 2. Test Results

[0153] According to the Basescope semi-quantitative scoring guide provided by the manufacturer, the scoring method for FGFR2b and FGFR2c mRNA in situ hybridization is as follows:

[0154] 0 means that there are 0 or fewer than 1 signal points per 20 cells;

[0155] 1+ means that there is one signal point in each cell;

[0156] 2+ means that there are 2-3 signal points or very few clusters of points in each cell;

[0157] 3+ means that there are 4-10 signal points per cell or less than 10% of cells have clusters of signal points;

[0158] 4+ means that each cell has more than 10 signal points or more than 10% of the cells have clusters of signal points.

[0159] In this embodiment, staining scores of 1 / 2 / 3 / 4+ are used as positive expression of FGFR2b and FGFR2c mRNA.

[0160] In situ hybridization results showed that KATO III cells simultaneously expressed FGFR2b and FGFR2c mRNA, H716 cells expressed only FGFR2c mRNA and not FGFR2b, and HCT-15 cells did not express either subtype.

[0161] Immunohistochemical results (Table 3) showed that in KATO III cells, FGFR2b (ADx018), FGFR2b (SDT-423-33), and FGFR2 (D4L2V) were all strongly positive staining; in H716 cells, FGFR2b (ADx018) and FGFR2b (SDT-423-33) were negative, while FGFR2 (D4L2V) was positive; in HCT15 cells, all three antibodies were negative. The immunohistochemical results of the three antibodies were consistent with the results of mRNA in situ hybridization. FGFR2b (ADx018) and FGFR2b (SDT-423-33) only recognized FGFR2b and not FGFR2c. FGFR2 (D4L2V) could recognize both FGFR2b and FGFR2c subtypes simultaneously. See Figure 4 .

[0162] Table 3

[0163]

[0164] Example 8: Immunohistochemical staining of normal tissue microarrays

[0165] 1. Experimental Procedure

[0166] In this embodiment, normal tissue chips are used for staining. The normal tissues to be tested include mammary gland, cerebellum, cerebrum, stomach, small intestine, colon, heart, kidney, liver, lung, ovary, pancreas, prostate, testis, thyroid gland, tonsils, adrenal gland, spleen, and bladder.

[0167] After baking the normal tissue microarray, firstly, FGFR2b mRNA in situ hybridization (company: ACD, BaseScope Probe-BA-Hs-FGFR2-tv2-E7E8, catalog number 715151) was used to determine whether the sample expressed FGFR2b. Secondly, immunohistochemical detection was performed on the Leica BOND-MAX fully automated immunostaining system using the ADx018 monoclonal antibody (1.4 ug / ml) described in this invention, the commercially available SDT-423-33 (1.4 ug / ml), and the commercially available D4L2V (0.2 ug / ml), as shown in Table 1.

[0168] 2. Test Results

[0169] The immunohistochemical results showed that FGFR2b mRNA in situ hybridization, FGFR2b(ADx018), FGFR2b(SDT-423-33), and FGFR2(D4L2V) monoclonal antibodies were all unstained, indicating that the staining results of FGFR2b(ADx018) monoclonal antibody and FGFR2(D4L2V) were consistent with those of FGFR2b mRNA in situ hybridization, demonstrating good specificity. FGFR2b(SDT-423-33) showed a yellow background staining in normal stomach. Figure 5 As shown.

[0170] Example 9: Immunohistochemical staining of tumor tissue

[0171] 1. Experimental Procedure

[0172] This embodiment uses various cancer tissue samples for immunohistochemical detection, including paraffin sections of gastric cancer tissue, lung cancer tissue, ovarian cancer tissue, and endometrial cancer tissue.

[0173] After collecting tissue samples and baking the samples, FGFR2b mRNA in situ hybridization technology (company: ACD, BaseScope Probe-BA-Hs-FGFR2-tv2-E7E8, catalog number 715151) was first used to determine whether the samples expressed FGFR2b. Then, immunohistochemical detection was performed on the Leica BOND-MAX fully automated immunostaining system using the ADx018 monoclonal antibody (1.4ug / ml) described in this invention, the commercially available SDT-423-33 monoclonal antibody (1.4ug / ml), and the commercially available D4L2V monoclonal antibody (0.2ug / ml). The specific procedure is shown in Table 1.

[0174] 2. Test Results

[0175] When using BaseScope Probe-BA-Hs-FGFR2-tv2-E7E8 to detect FGFR2b mRNA in situ hybridization, staining scores of 1 / 2 / 3 / 4+ were considered as positive expression of FGFR2b mRNA.

[0176] Immunohistochemical results showed that, when staining intensity 1 / 2 / 3+ was considered positive, the IHC staining of ADx018 monoclonal antibody and D4L2V monoclonal antibody in gastric cancer, lung cancer, ovarian cancer, and endometrial cancer tissues described in this invention was consistent with the in situ hybridization staining of FGFR2b mRNA. However, SDT-423-33 exhibited insufficient sensitivity due to failure to stain in endometrial cancer, lung cancer, and ovarian cancer, and also suffered from non-specific nuclear staining. Figure 6 and Figure 7 .

[0177] Therefore, ADx018 exhibits excellent sensitivity and specificity.

[0178] Example 10: Immunohistochemical staining of tumor tissue microarray

[0179] 1. Experimental Procedure

[0180] This embodiment uses tissue microarrays of gastric cancer, lung cancer, ovarian cancer, and endometrial cancer. The gastric cancer tissue microarray is sourced from Haixing Biotechnology, while the lung cancer, ovarian cancer, and endometrial cancer tissue microarrays are sourced from a standardized tissue sample bank.

[0181] After collecting tissue samples and baking the samples, the FGFR2b mRNA in situ hybridization technology (company: ACD, BaseScope Probe-BA-Hs-FGFR2-tv2-E7E8, catalog number 715151) was first used to determine whether the sample expressed FGFR2b. Then, the ADx018 monoclonal antibody (1.4ug / ml) described in this invention was used to perform immunohistochemical detection on the Leica BOND-MAX fully automated immunostaining system. The specific procedure is shown in Table 1.

[0182] 2. Test Results

[0183] When using BaseScope Probe-BA-Hs-FGFR2-tv2-E7E8 to detect FGFR2b mRNA in situ hybridization, staining scores of 1 / 2 / 3 / 4+ were considered as positive expression of FGFR2b mRNA.

[0184] Immunohistochemical results showed that, when staining intensity 1 / 2 / 3+ was considered positive, the in situ hybridization of ADx018 monoclonal antibody IHC with FGFR2b mRNA in gastric cancer, lung cancer, ovarian cancer, and endometrial cancer tissue microarrays was greater than 85%, indicating that ADx018 has excellent sensitivity and accuracy. Figure 8-11 As shown in the figure, and in Table 4.

[0185] Furthermore, it is clear that immunohistochemistry is more convenient and easier to scale up for detection compared to mRNA in situ hybridization.

[0186] Table 4

[0187]

[0188]

[0189] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An FGFR2b-specific antibody, said antibody comprising a light chain variable region and a heavy chain variable region; In the light chain variable region, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:6, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:7, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:

8. In the heavy chain variable region, the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:2, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:

4.

2. The FGFR2b specific antibody as described in claim 1, characterized in that, The antibodies include: The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5, or has more than 80% identity with the sequence shown in SEQ ID NO:5; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, or has more than 80% identity with the sequence shown in SEQ ID NO:1, and retains the antibody that specifically binds to FGFR2b activity. Preferably, the antibody comprises: rabbit anti-human FGFR2b monoclonal antibody, humanized antibody or chimeric antibody; or, the antibody comprises: Fab, Fv, Fd, Fab'-SH, scFv or (Fab')2 fragment.

3. An isolated polynucleotide encoding the FGFR2b-specific antibody as described in either claim 1 or 2.

4. An expression construct containing the polynucleotide of claim 3, or an antibody expression system including the expression construct; preferably, the construct is an expression vector; preferably, the expression system is a cell.

5. A method for preparing the FGFR2b specific antibody according to any one of claims 1 or 2, comprising: The antibody is obtained by expressing it using the antibody expression system of claim 4 under suitable conditions for expression. Preferably, the method further includes: purifying and separating the antibody.

6. The use of the antibody according to claim 1 or 2 in the preparation of a targeted detection reagent, wherein the targeted detection reagent specifically targets cells expressing FGFR2b; preferably, in the targeted detection reagent, the antibody is further modified with a conjugate; Preferably, the conjugate includes a detectable marker; more preferably, the detectable marker includes one or more of the following: alkaline phosphatase, horseradish peroxidase, biotin, fluorescent dye, radioactive isotope, ferritin, colloidal gold; Preferably, the conjugate comprises: Cytotoxins or bioactive proteins.

7. A targeted detection reagent that specifically targets cells expressing FGFR2b, comprising the antibody as described in any one of claims 1 or 2, and a conjugate operatively linked thereto; Preferably, the conjugate includes a detectable marker; more preferably, the detectable marker includes one or more of the following: alkaline phosphatase, horseradish peroxidase, biotin, fluorescent dye, radioactive isotope, ferritin, colloidal gold; Preferably, the conjugate comprises: Cytotoxins or bioactive proteins.

8. Use of the FGFR2b specific antibody of claim 1 or 2, or the targeted detection reagent of claim 7, in the preparation of formulations, kits, or reagent kits for detecting FGFR2b expression / overexpression-related diseases; preferably, the FGFR2b expression / overexpression-related diseases include: Tumors expressing / overexpressing FGFR2b; preferably, the tumors include: gastric cancer, lung cancer, ovarian cancer, endometrial cancer, and colorectal cancer.

9. A kit or reagent kit comprising the FGFR2b specific antibody of claim 1 or 2, the targeted detection reagent of claim 7, or a composition or formulation containing the like.

10. A method for in vitro specific detection of FGFR2b protein or cells carrying the protein, the method comprising: The presence of FGFR2b protein in the sample to be tested is analyzed using the FGFR2b specific antibody as described in claim 1 or 2 or the targeted detection reagent as described in claim 7; preferably, the method includes: immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry.

Images