Diagnostic antibody suitable for immunohistochemical detection of CD228

Abstract

The invention relates to a specific antibody aiming at CD228 protein and an antigen binding fragment thereof, which are used for detecting and treating various tumors, such as melanoma, mesothelioma, colorectal cancer and the like. The antibody binding fragment is helpful for diagnosis and treatment of cancers through specific binding with CD228. The invention further comprises antibody drug conjugates (ADCs) which bind a drug to an antibody through a linker, so as to improve the therapeutic effect and reduce the influence on normal cells. Meanwhile, nucleic acids encoding the antibodies or antigen-binding fragments thereof are provided to produce recombinant antibodies or fragments. Besides, the invention also provides a kit which is used for laboratory research, clinical diagnosis and treatment effect monitoring, is beneficial to determining whether tissues or cells express CD228 or not, and provides a new strategy for personalized and precise treatment of cancers.

Description

A diagnostic antibody suitable for immunohistochemical detection of CD228 Technical field:

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to novel anti-CD228 antibodies, as well as methods for diagnosing cancer using such anti-CD228 antibodies and the use of such antibodies in preparing cancer diagnostic reagents. Background technology:

[0002] CD228, also known as melanotransferrin (MTf), p97, and MFI2, is an iron-binding glycoprotein belonging to the transferrin (Tf) family of molecules. MTf shares 40% protein sequence identity with human lactoferrin and transferrin and is highly conserved across species. CD228 plays an essential role in iron metabolism.

[0003] CD228 was first identified as a surface marker of malignant melanoma cells and is overexpressed in the majority of clinically isolated melanomas. It has also been observed in many other human cancers. CD228 is now a broad-spectrum anti-tumor target, with high expression in melanoma, mesothelioma, pancreatic cancer, cervical cancer, gallbladder cancer, colon cancer, and other cancers. Anti-CD228 antibody drugs, when bound to CD228, can inhibit tumor cell proliferation and improve the survival rate of cancer patients.

[0004] In clinical practice, patients with malignant solid tumors who need to use anti-CD228 therapeutic drugs must undergo immunohistochemistry (IHC) testing of tumor tissue in advance to determine whether the tumor tissue expresses CD228 molecules and the proportion of cells expressing CD228, so as to use anti-CD228 drugs more specifically and assist doctors and patients in making rational choices of treatment methods.

[0005] Immunohistochemistry is a method of localizing, qualitatively analyzing, and relatively quantitatively analyzing antigens within tissues based on the principle of specific binding between antigens and antibodies. Tissue sections used in immunohistochemistry include paraffin sections and frozen sections. Both types of tissue sections have their own advantages and disadvantages: The advantage of paraffin sections is that the tissue structure is well preserved, continuous thin sections can be cut, the tissue structure is clear, the antigen localization is accurate, and they can be preserved for a long time; the disadvantage is that during the production process, they must be treated with organic solvents such as alcohol and xylene, which causes a significant loss of antigen activity within the tissue, affecting the conformation of the antigen and the exposure of the antigen epitope; the advantage of frozen sections is that they can better preserve the immune activity of multiple antigens, especially cell surface antigens, which should be frozen sections; the disadvantage is that the tissue is fragile and easy to rot, and it is difficult to obtain a complete section structure. When frozen, water in the tissue easily forms ice crystals, which often affects antigen localization. Therefore, frozen sections are often used for rapid pathological testing during surgery and are only suitable for short-term low-temperature storage.

[0006] There are currently two major drawbacks in the development of immunohistochemical detection methods: one is that the antigenic epitopes exposed in tissue sections may differ from those in their natural state, resulting in insufficient affinity and specificity of the detection antibody and low detection sensitivity; the other is that various types of normal and tumor tissues in the human body bind nonspecifically to the detection antibody, leading to false-positive results. For these reasons, current clinical immunohistochemical testing requires first screening for indications and control antibodies, then exploring and optimizing the experimental system, using a large number of experiments to screen for suitable diagnostic antibodies from a large number of candidate antibodies, and then evaluating the sensitivity, specificity, accuracy and other indicators of the candidate antibodies in immunohistochemical detection of paraffin sections or frozen sections of different tissues.

[0007] Therefore, an effective, rapid, and low-cost diagnostic test to detect CD228 on tissues is highly necessary for clinical diagnosis and targeted therapy.

[0008] Summary of the invention:

[0009] The present invention provides an anti-CD228 antibody or antigen-binding fragment thereof. The anti-CD228 antibody provided by the present invention has a high binding ability to CD228 and is suitable for diagnosis by immunohistochemical testing. In immunohistochemical testing, the anti-CD228 antibody provided by the present invention can achieve staining effects similar to existing commercial anti-CD228 antibodies at working concentrations and can replace existing commercial anti-CD228 monoclonal antibodies used in immunohistochemical testing.

[0010] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD228 protein, wherein the antibody or antigen-binding fragment thereof comprises the following three light chain complementary determining regions and three heavy chain complementary determining regions:

[0011] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 as shown in SEQ ID NO: 2, LCDR2 as shown in SEQ ID NO: 3, and LCDR3 as shown in SEQ ID NO: 4; and the three heavy chain complementary determining regions comprise HCDR1 as shown in SEQ ID NO: 6, HCDR2 as shown in SEQ ID NO: 7, and HCDR3 as shown in SEQ ID NO: 8;

[0012] The antibody or antigen-binding fragment thereof comprises three light chain complementary determining regions comprising LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO: 12; and / or three heavy chain complementary determining regions comprising HCDR1 as shown in SEQ ID NO: 14, HCDR2 as shown in SEQ ID NO: 15, and HCDR3 as shown in SEQ ID NO: 16;

[0013] Or the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof include LCDR1 as shown in SEQ ID NO: 18, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20; and the three heavy chain complementary determining regions include HCDR1 as shown in SEQ ID NO: 22, HCDR2 as shown in SEQ ID: 23, and HCDR3 as shown in SEQ ID NO: 24.

[0014] Furthermore, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5;

[0015] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13;

[0016] Or the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 17, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 21.

[0017] Further, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:25, and a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:26.

[0018] In the embodiments of the present invention, the antibodies or antigen-binding fragments thereof include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, scFv or dsFv fragments, etc.

[0019] The second aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0020] The third aspect of the present invention provides a vector comprising the nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0021] The vector can be used to express the antibody or antigen-binding fragment thereof. Preferably, the vector can be a viral vector; preferably, the viral vector includes but is not limited to a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector; preferably, the vector can be a non-viral vector; preferably, the vector can be a mammalian cell expression vector; preferably, the expression vector can be a bacterial expression vector; preferably, the expression vector can be a fungal expression vector.

[0022] The fourth aspect of the present invention provides a cell, comprising the nucleic acid or the vector, and capable of expressing the antibody or antigen-binding fragment thereof. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is an Escherichia coli cell, etc.; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cell is a Pichia pastoris cell, etc.; preferably, the cell is a mammalian cell; preferably, the mammalian cell is a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (293), a B cell, a T cell, a DC cell, or a NK cell, etc.

[0023] The fifth aspect of the present invention provides an antibody conjugate, which is composed of the antibody or antigen-binding fragment thereof of the present invention coupled with at least one detectable label.

[0024] The sixth aspect of the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention, the conjugate of the present invention, or a nucleic acid encoding the antibody or antigen-binding fragment thereof; optionally, the kit comprises a detection buffer reagent.

[0025] A seventh aspect of the present invention provides a kit for detecting or quantifying CD228, the kit comprising the anti-CD228 antibody or antigen-binding fragment thereof or the conjugate of the present invention; optionally, the kit further comprises a buffer solution related to detection or quantification.

[0026] An eighth aspect of the present invention provides a kit for diagnosing a disease or evaluating the efficacy of a disease treatment, the kit comprising the antibody or antigen-binding fragment thereof that specifically binds to the CD228 protein according to the present invention, wherein the disease is a CD228-related disease.

[0027] A ninth aspect of the present invention provides use of the anti-CD228 antibody or antigen-binding fragment thereof, encoding nucleic acid, vector, or cell of the present invention in the preparation of a kit for detecting and / or quantifying whether cells or tissues express CD228, wherein the use is for non-diagnostic purposes and / or non-disease treatment effect prediction purposes.

[0028] Preferably, the kit is an immunohistochemistry detection kit.

[0029] The tenth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof, encoding nucleic acid, vector, and cell of the present invention that specifically binds to the CD228 protein in the preparation of a disease diagnostic kit, wherein the disease is a CD228-related disease;

[0030] Preferably, the kit is an immunohistochemistry detection kit;

[0031] Preferably, the CD228-related diseases include one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

[0032] In an eleventh aspect, the present invention provides use of the anti-CD228 antibody or antigen-binding fragment thereof, encoding nucleic acid, vector, or cell of the present invention in preparing a kit for evaluating the therapeutic effect of a CD228-related disease, wherein the disease is a CD228-related disease;

[0033] Preferably, the kit is an immunohistochemistry detection kit;

[0034] Preferably, the CD228-related diseases include one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

[0035] In a twelfth aspect, the present invention provides a method for detecting or quantifying anti-CD228 antibodies in a sample, the method being for diagnostic or non-diagnostic purposes, the method comprising: step (i) contacting the anti-CD228 antibody or antigen-binding fragment thereof, encoding nucleic acid, vector, cell or antibody conjugate of the present invention with a sample to be tested; and (ii) detecting a complex formed by the antibody or antigen-binding fragment thereof and CD228 expressed in the sample, or detecting a complex formed by the antibody conjugate and CD228 expressed in the sample.

[0036] Furthermore, the detection and / or quantification method is an immunohistochemical method, which includes the step of performing immunohistochemical detection on the sample using the anti-CD228 antibody or antigen-binding fragment thereof, encoding nucleic acid, vector, and cells described in the present invention.

[0037] In a thirteenth aspect, the present invention provides a method for diagnosing and monitoring cancer, comprising the steps of: (i) contacting a sample with the anti-CD228 antibody or antigen-binding fragment thereof or the antibody conjugate of the present invention, and (ii) detecting a complex bound to CD228 expressed in the sample by the antibody or antigen-binding fragment thereof, or detecting a complex bound to CD228 expressed in the sample by the antibody conjugate.

[0038] The disease is a CD228-related disease, and preferably the CD228-related disease includes one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

[0039] A fourteenth aspect of the present invention provides a method for evaluating the therapeutic effect of a disease, wherein the disease is a CD228-related disease, comprising the steps of: (i) contacting a sample with the anti-CD228 antibody or antigen-binding fragment thereof or the antibody conjugate of the present invention, and (ii) detecting a complex bound to the antibody or antigen-binding fragment thereof and CD228 expressed in the sample, or detecting a complex bound to the antibody conjugate and CD228 expressed in the sample;

[0040] Furthermore, the CD228-related diseases include one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

[0041] Furthermore, the evaluation method is a method for determining whether the disease can be treated by cancer therapy targeting CD228.

[0042] A fifteenth aspect of the present invention provides an immunohistochemical detection method using the anti-CD228 antibody of the present invention, comprising: 1) preparing tissue sections; 2) applying the anti-CD228 antibody as a primary antibody to the prepared tissue sections and incubating for 30 minutes; 3) adding a secondary antibody that binds to the primary antibody, labeling, developing, washing, and mounting the sections; and 4) observing under a microscope and detecting. Furthermore, the tissue sections are selected from one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer, and bile duct cancer.

[0043] Furthermore, the diagnostic antibody - anti-CD228 antibody is used in the detection of human cancer tissue at a concentration of 1 μg / mL-4 μg / mL; preferably 1 μg / mL, 2 μg / mL, 3 μg / mL or 4 μg / mL.

[0044] Beneficial effects of the present invention:

[0045] 1. Sensitivity and affinity: Through ELISA and biomembrane interference (BLI) experiments, the anti-CD228 antibodies of the present invention exhibited high sensitivity and strong affinity to CD228 protein.

[0046] 2. Specific binding: The anti-CD228 antibody of the present invention specifically binds only to the CD228 protein and has little binding to transferrin (TF) and lactoferrin (LF) of the same family, showing good selectivity and specificity.

[0047] 3. Staining Efficacy: In immunohistochemical (IHC) staining of various cancer tissues, the anti-CD228 antibodies of the present invention demonstrated comparable or superior staining efficacy to the commercially available antibody NBP1-85777. In particular, in the detection of certain specific cancer types, the anti-CD228 antibodies of the present invention demonstrated significantly higher positive rates and staining sensitivity than NBP1-85777.

[0048] 4. Applicability: The anti-CD228 antibody of the present invention showed consistent staining intensity and positive rate in tissue microarrays of various cancer types (melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer), indicating its wide applicability.

[0049] 5. Stability: The anti-CD228 antibodies of the present invention exhibit good stability under different environmental conditions.

[0050] 6. Substitutability: Based on the above experimental results, the anti-CD228 antibody of the present invention can be used as an effective substitute for the commercial antibody NBP1-85777 for immunohistochemical detection of CD228 targets.

[0051] Early detection and treatment selection: Due to the high positive rate and specificity of the anti-CD228 antibodies of the present invention, they can be used for early detection of cancer and more accurate treatment selection. Description of the drawings:

[0052] Figure 1: Serum titers of CD228-immunized mice.

[0053] Figure 2: Serum titers of CD228-immunized rabbits.

[0054] Figure 3: IHC detection of CD228 immune rabbit serum.

[0055] Figure 4: ELISA results for detecting the binding sensitivity of various rabbit anti-CD228 monoclonal antibodies to CD228 protein.

[0056] Figure 5: Biomembrane interferometry (BLI) analysis of the affinity between CA-2204 and CD228.

[0057] Figure 6: Biomembrane interferometry (BLI) analysis of the affinity between R546 and CD228.

[0058] Figure 7: Biomembrane interferometry (BLI) analysis of the affinity between R526 and CD228.

[0059] Figure 8: Western blotting to detect the specificity of the binding of rabbit anti-CD228 monoclonal antibody to CD228 protein.

[0060] Figure 9: Leica BOND TM Flowchart of the fully automated IHC and ISH staining system.

[0061] Figure 10: Screening of positive and negative tissues using a CD228 control antibody.

[0062] Figure 11: Comparison of the staining effects of different commercial antibodies in different tissues.

[0063] Figure 12: Comparison of staining results of different concentrations of candidate antibodies in the same mesothelioma tissue.

[0064] Figure 13: DAB staining results of R546 antibody in mesothelioma tissues for 1 min and 5 min.

[0065] Figure 14: Comparison of staining effects of anti-CD228 candidate antibodies and commercial antibodies in mesothelioma tissues.

[0066] Figure 15: Comparison of the staining effects of R546 and commercial antibodies on paraffin-embedded tissue sections.

[0067] Figure 16: Comparison of staining sensitivity and specificity of R546 and NBP1-85777 in colon cancer tissue microarray.

[0068] Figure 17: Comparison of staining sensitivity and specificity of R546 and NBP1-85777 in cervical cancer tissue microarray.

[0069] Figure 18: Comparison of staining sensitivity and specificity of R546 and NBP1-85777 in esophageal cancer tissue microarray.

[0070] Figure 19: Comparison of staining sensitivity and specificity of R546 and NBP1-85777 in pancreatic cancer tissue microarray.

[0071] Figure 20: Comparison of staining sensitivity and specificity of R546 and NBP1-85777 in gastric cancer tissue microarray.

[0072] Figure 21: Comparison of staining sensitivity and specificity of R546 and NBP1-85777 in extrahepatic cholangiocarcinoma tissue microarray.

[0073] Figure 22: Comparison of staining of R546 and NBP1-85777 in gallbladder cancer tissue microarray.

[0074] Figure 23: Comparison of staining of R546 and NBP1-85777 in single sections of gallbladder cancer tissue.

[0075] Figure 24: Comparison of staining of R546 and NBP1-85777 in single sections of cholangiocarcinoma tissue.

[0076] Figure 25: Comparison of staining of R546 and NBP1-85777 in single sections of esophageal cancer tissue.

[0077] Figure 26: Comparison of staining of R546 and NBP1-85777 in single sections of pancreatic cancer tissue.

[0078] Figure 27: Comparison of staining of R546 and NBP1-85777 in single sections of gastric cancer tissue.

[0079] Figure 28: ELISA detection of the binding sensitivity of R546 and NBP1-85777 to CD228 and other proteins in the same family.

[0080] Figure 29: Biomembrane interference detection of affinity detection charts of R546 and NBP1-85777 with CD228 and others in the same family. Specific implementation method:

[0081] definition

[0082] To better understand this disclosure, some terms are first defined. As used in this application, unless otherwise indicated herein, the following terms shall have the meanings set forth below. Additional definitions are provided throughout this application.

[0083] The terms used in this article: antibodies and their antigen-binding fragments, which can be immunoglobulins (Igs), monoclonal antibodies (mAbs), polyclonal antibodies (pAbs), antibody fragments (scFvs), single-chain variable regions (scFvs), etc. Antibodies and their antigen-binding fragments refer to antibodies or their derivatives with antigen-binding ability, which have important applications in biomedical research, diagnosis, and treatment. Monoclonal antibodies and polyclonal antibodies are the two main types of antibodies. The former are produced by a single clone and are highly specific for a single antigen epitope; the latter are produced by multiple clones and can recognize multiple antigen epitopes. Antibody fragments, such as scFvs, are smaller parts of antibodies that still have specific antigen-binding ability but have a simpler structure.

[0084] The terms used in this article: immunohistochemistry, which can be immunohistochemistry (IHC), immunofluorescence (IF), immunoperoxidase staining (IHC), immunogold staining (Immunogold staining), etc. Immunohistochemistry is a technique that uses the principle of specific binding of antibodies to specific antigens to locate, qualitatively and quantitatively identify antigens through tissue sections or cell samples. Immunofluorescence and immunoperoxidase staining are commonly used methods in immunohistochemistry, which use fluorescent labeling and enzyme labeling to visualize the location and expression level of antigens, respectively. Immunogold staining uses gold nanoparticles as labels to provide high-resolution antigen localization.

[0085] The term cancer, as used in this article, refers to a group of diseases involving abnormal and uncontrolled cell proliferation and growth. These abnormal cells have the ability to invade surrounding tissues and metastasize to distant sites through the blood and lymphatic systems. Malignant tumors are a type of cancer that grow rapidly, are resistant to treatment, and have a high risk of recurrence and metastasis. Cancer lesions and tumor pathology refer to the manifestations and characteristics of cancer at the tissue and cellular levels.

[0086] The terms "treatment efficacy assessment" used in this article include therapeutic efficacy assessment, treatment response monitoring, clinical efficacy analysis, and treatment outcome determination. Treatment efficacy assessment involves evaluating the impact of medical interventions on the disease process through a series of biomarkers, imaging studies, clinical symptoms, and laboratory indicators. This includes comparative analysis before and after treatment and long-term follow-up of treatment effects. Treatment efficacy assessment is crucial for guiding clinical treatment decisions, optimizing treatment plans, and improving patients' quality of life.

[0087] Example 1: Generation of anti-CD228 monoclonal antibodies

[0088] 1. Experimental Methods

[0089] (1) Preparation of immunogen

[0090] Table 1: Amino acid sequence of human CD228 protein

[0091] (2) Animal immunization

[0092] A) Immunization of mice

[0093] Wild-type Balb / c mice were used for immunization experiments. CD228 antigen protein (0.23 mg / mL, Boan, 20200924, SEQ ID NO: 27) produced by Shandong Boan Biotechnology Co., Ltd. was used for immunization.

[0094] The immunization method adopted multiple injections at the subcutaneous and inguinal points of the abdomen, and the antigen protein immunization dose was 20 μg / mouse. Freund's complete adjuvant was used to emulsify the antigen for the first immunization, and Freund's incomplete adjuvant was used to emulsify the antigen for the second to fourth immunizations. The first batch of mice underwent 3 immunizations and one booster immunization; the second batch of mice underwent 4 immunizations and one booster immunization. Each immunization was 14 days apart. Starting from the second immunization, peripheral blood serum was collected on the 7th day after each immunization to detect the antibody titer and mice with unqualified titers were eliminated. After immunization of mice, the results of mouse serum titer detection are shown in Figure 1, where 2500X, 12500X, and 62500X represent the dilution ratios. Three days after the booster immunization, the mice were euthanized and the spleens were taken to make single cells for bank construction.

[0095] B) Rabbit Immunization:

[0096] Beijing Yiqiao Shenzhou Science and Technology Co., Ltd. was commissioned to conduct a rabbit immunization experiment. Two Japanese white rabbits were used and immunized with CD228 (0.23 mg / mL, Boan, 20200924, SEQ ID NO: 27) antigen protein produced by Shandong Boan Biotechnology Co., Ltd. Each rabbit was immunized with 500 μg of antigen. For the first immunization, the immunogen was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple points on the back. The same dose of immunogen was emulsified with an equal volume of incomplete Freund's adjuvant at intervals of 2 weeks. Blood was collected one week after the three immunizations and serum titers were determined by ELISA. Rabbits with qualified serum titers had their spleens and bone marrow removed and frozen in liquid nitrogen for later use. The serum was sent for IHC testing, and animals with good IHC results were selected for library construction.

[0097] Serum was diluted at ratios of 1:22,000, 1:64,000, and 1:128,000, and the serum titer was detected by ELISA, as shown in Figure 2 . The serum was also detected by IHC (melanoma tissue), as shown in Figure 3 . The rabbit with the best titer and IHC test results was selected for library construction and screening.

[0098] C) Library construction and screening

[0099] Based on the serum titer and IHC test results, one rabbit and one mouse with the best immune response were selected for library construction, and the ELISA method was used to screen positive clones for subsequent recombinant expression production.

[0100] 1) Phage library construction:

[0101] Based on the serum titer test results, the spleens of one rabbit and one mouse with the best results were selected for RNA extraction. The antibody variable region genes were amplified by RT-PCR. The heavy and light chain fragments of interest were then spliced ​​together to create a fused scFv fragment, which was inserted into an expression vector and electroporated into competent cells to generate a phage antibody library. The library capacity data for the constructed library are shown in Tables 2 and 3.

[0102] Table 2: Phage library capacity constructed from immunized mice

[0103] Table 3: Phage library capacity constructed by immunizing rabbits

[0104] 2) Phage library screening:

[0105] Plates were coated with CD228-His protein (homemade). The next day, the phage library was added and incubated for 2 hours. After washing 4-10 times, the specifically bound phage was eluted with elution buffer. The antibody clones obtained by screening and their sources are shown in Tables 4 and 5.

[0106] Table 4: Sources of anti-CD228 antibodies obtained through screening

[0107] Table 5: Sources of anti-CD228 antibodies obtained through screening

[0108] 3) Positive clone detection:

[0109] The positive library is plated and a single clone is selected for expression. The expression supernatant is tested by ELISA. The clone that binds to the antigen is selected as the positive clone. PCR and enzyme digestion typing are performed on the positive clones. The clones with different banding patterns are selected for ELISA retest. After confirmation of positive results, they are sent for sequencing.

[0110] 4) Sequence confirmation and expression vector construction:

[0111] Select positive clones with different sequences to construct full-length antibody expression vectors. After sequencing is correct, transfer expression and production. D) Molecular construction and production of complete antibodies

[0112] Antibody variable region genes were amplified using standard molecular biology PCR (2× Phanta Max Master Mix, Vazyme, Catalog No. P515-P1-AA). The heavy chain variable region gene was ligated into the pCDNA3.4 vector (Life Technology) containing the nucleotide sequence of the rabbit heavy chain constant region. The light chain variable region gene was ligated into the pCDNA3.4 vector (Life Technology) containing the nucleotide sequence of the rabbit light chain constant region. Plasmids from sequenced positive clones were extracted and co-transfected into HEK293 cells and cultured at 37°C, 8% CO₂, and a shaker at 125 rpm. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography. Antibody concentrations were determined by UV280 combined with theoretical extinction coefficients. The amino acid sequences of the heavy and light chain constant regions of the three antibodies are shown in Table 6, and the amino acid sequences of the variable regions of the three antibodies are shown in Table 7 (CDR regions are underlined, and the analysis system was the IMGT system).

[0113] 2. Experimental Results

[0114] FIG1 shows the results of serum titer experiments on mice immunized using the method of this embodiment;

[0115] FIG2 shows the results of the serum titer test of rabbits immunized using the method of this embodiment;

[0116] FIG3 shows the IHC detection of CD228 immune rabbit serum using the method of this embodiment;

[0117] The amino acid sequences of the heavy and light chain constant regions of the three antibodies screened in this example are shown in Table 6, and the amino acid sequences of the variable regions of the three antibodies are shown in Table 7 (the CDR regions are underlined, and the analysis system is the IMGT system).

[0118] Table 6: Amino acid sequences of heavy and light chain constant regions of three antibodies

[0119] Table 7: Amino acid sequences of the variable regions of three antibodies

[0120] Example 2: Sensitivity Detection of Binding of Rabbit Anti-CD228 Monoclonal Antibody to CD228 Protein

[0121] 1. Experimental Methods

[0122] The CD228 protein was diluted to 0.2 μg / mL with CBS, 100 μL / well, and incubated at 4°C overnight; blocking solution was added to block at 37°C for 1 hour, and the plate was washed; the CD228 rabbit antibody was diluted fourfold from 0.2 μg / mL with PBST in 8 gradients, 100 μL / well, and incubated at 37°C for 1 hour, and the plate was washed; the enzyme-labeled secondary antibody Goat pAb to Rb IgG (HRP) was added, 100 μL / well, and incubated at 37°C for 1 hour, and the plate was washed; 100 μL TMB color development solution was added, color was developed for 10 minutes, and 2 M sulfuric acid was added to stop the color development; the OD450 nm value was read on a microplate reader.

[0123] 2. Experimental Results

[0124] The OD450 values ​​were plotted against the antibody concentrations, and the results are shown in Figure 4. Based on the data in the figure, the EC50 (half-maximal effective concentration, Effective Concentration 50) values ​​of each monoclonal antibody were calculated and are shown in Table 8.

[0125] Table 8: EC50 of rabbit anti-CD228 monoclonal antibodies binding to CD228 protein

[0126] It can be seen from the data in Table 8 that antibodies 546, 526, and 2204 can all bind to the CD228 protein.

[0127] FIG4 shows the results of the ELISA assay of the present invention for detecting the binding sensitivity of various rabbit anti-CD228 monoclonal antibodies to CD228 protein.

[0128] Example 3: Biomembrane Interference (BLI) Detection of the Affinity of Rabbit Anti-CD228 Monoclonal Antibody to CD228

[0129] 1. Experimental Methods

[0130] Rabbit antibody was coupled to a ProA chip (Fortebio Cat. 18-5010) on a Fortebio RED96 instrument at a concentration of 4 μg / mL. The threshold was set at 1.5 nM. CD228 protein was serially diluted two-fold in PBST over four concentrations, starting at 100 nM. Binding assays were performed for 150 s, dissociation assays for 300 s, equilibration for 60 s, and probe regeneration for 15 s. Kinetic parameters, including KD values, were calculated using the instrument's accompanying software.

[0131] 2. Experimental Results

[0132] In this example, kinetic parameters such as KD values ​​were calculated using the instrument's supporting software. The test data are shown in Table 9. The affinity detection profiles of the anti-CD228 antibodies numbered CA2204, R546, and R526 screened and obtained in this project are shown in Figures 5, 6, and 7. Figure 5 shows the affinity detection profile of CA-2204 and CD228 detected by biomembrane interferometry (BLI) in this example, Figure 6 shows the affinity detection profile of R546 and CD228 detected by biomembrane interferometry (BLI) in this example, and Figure 7 shows the affinity detection profile of R526 and CD228 detected by biomembrane interferometry (BLI) in this example.

[0133] Table 9: KD values ​​of rabbit anti-CD228 monoclonal antibodies binding to CD228 protein

[0134] The graphs shown in Figures 5-7 and the data in Table 9 indicate that only R546 and CA2204 antibodies can bind to the CD228 protein with high affinity.

[0135] Example 4: Western Blot Detection of Protein Levels Specificity of Rabbit Anti-CD228 Monoclonal Antibody Binding to CD228 Protein

[0136] Experimental methods:

[0137] Proteins were extracted from A375 cell lines using PierceTM RIPA Buffer (purchased from thermo scientific, catalog number WD321069) as experimental samples, and CD228-his protein was used as a control sample. Samples were loaded at 6 μg and 600 ng, respectively, for electrophoresis. After electrophoresis, the membrane was transferred to a 400 mA wet-cooled incubator for 20 minutes. The membrane was then blocked with 5% skim milk powder for 1 hour and incubated with R546, R526, and CA2204 antibodies at 4°C overnight. The next day, the membrane was incubated with the corresponding secondary antibody for 1 hour. The membrane was then incubated with a chemiluminescent substrate and photographed using a gel imager.

[0138] Experimental results:

[0139] The results are shown in FIG8 . R546, R526, and CA2204 antibodies can accurately bind to the band at the same position as the control CD228-his protein in A375 cells, indicating that the three antibodies have good specificity.

[0140] Example 5: Immunohistochemical assay for detecting CD228 in paraffin-embedded tissue sections using rabbit anti-CD228 monoclonal antibodies

[0141] 1. Experimental Methods

[0142] 1) Preparation of paraffin sections:

[0143] The obtained target tissue was placed in 4% paraformaldehyde and fixed at 4°C overnight. It was first dehydrated with gradient ethanol, and then immersed in xylene to make the dehydration more complete. Then it was immersed in a mixed solution of xylene and paraffin at 65°C for 30 minutes to allow the wax to penetrate the tissue. Finally, the tissue was completely placed in paraffin at 65°C for 45 minutes and embedded.

[0144] 2) The immunohistochemistry procedure is as follows:

[0145] Using Leica BOND TM Fully automated IHC and ISH staining system (model: BOND-MAX). The staining process is shown in Figure 9. Figure 9 shows the Leica BOND TM Flowchart of the fully automated IHC and ISH staining system.

[0146] 3) Result determination method:

[0147] Negative control tissue: The experiment uses normal human spleen tissue sections that theoretically have no CD228 expression as negative control tissue. This tissue should have no specific binding reaction with irrelevant antibodies or anti-CD228 antibodies.

[0148] Positive control tissue: Human mesothelioma tissue sections with high CD228 expression were used as positive control tissue. This tissue should have no specific binding reaction with irrelevant antibodies and specific binding reaction with anti-CD228 antibodies. The use of these two tissues in the experiment can serve as quality control standards in the system to ensure the authenticity and reliability of the results of batch experiments.

[0149] 4) Result judgment criteria:

[0150] Step 1: Analyze control tissue

[0151] First, stain and analyze the positive and negative control tissue samples.

[0152] Ensure that the positive control shows the expected specific staining and that the negative control shows no staining or only very weak nonspecific staining.

[0153] Step 2: Verify staining specificity

[0154] When observing human tissue sections treated with irrelevant antibodies, theoretically there should be no staining.

[0155] If staining occurs, compare it with samples treated with the specific antibody.

[0156] The result was considered specific positive only if the stained area in the specific antibody treatment group was not stained in the irrelevant antibody group.

[0157] Step 3: Quantify CD228 expression levels

[0158] Assess the staining intensity on tumor cell membranes.

[0159] Calculate the proportion of tumor cell membrane staining within the tumor area.

[0160] The CD228 expression level score was obtained by multiplying the staining intensity by the tumor cell membrane staining ratio.

[0161] During this procedure, ignore the staining of immune cells and focus on the evaluation of tumor cells.

[0162] Example 6: Exploration of experimental conditions for immunohistochemical detection of rabbit anti-CD228 monoclonal antibodies

[0163] 1. Experimental methods:

[0164] 1) Screening of CD228 positive and negative tissues

[0165] A commercial CD228 / MFI2 antibody (Novμsbio NBP1-85777) was selected as a control antibody at a concentration of 1 μg / mL for screening of positive and negative tissues; tissue sections were purchased from Shanghai Xinchao.

[0166] 2) Selection of commercial antibodies

[0167] Under the experimental conditions described in Example 5, the staining effects of three commercial antibodies were compared. The three commercial antibodies are: ① Abcam, product number ab236732, working concentration 7 μg / ml; ② R&D system, product number MAB8175, working concentrations 1 μg / ml and 2 μg / ml; ③ Novusbio, product number NBP1-85777, 1 μg / ml.

[0168] 3) Exploration of immunohistochemistry experimental conditions

[0169] The optimal staining conditions were explored to obtain clear staining effects on mesothelioma tissue. The three anti-CD228 candidate antibodies (R546, R526, CA2204) prepared in Example 1 of the present application were used to perform LEICA BOND on human mesothelioma tissue (purchased from Shanghai Xinchao). TM The conditions of the fully automatic IHC and ISH staining system (model: BOND-MAX) were explored. The primary antibody of the negative control group was added with an irrelevant antibody, and the antibody concentration and DAB staining time were used as exploration variables.

[0170] Antibody concentration gradient setting: Set three different antibody working concentrations, namely low concentration (e.g., 1 μg / mL), medium concentration (e.g., 5 μg / mL), and high concentration (e.g., 10 μg / mL). Ensure that each concentration gradient of antibody is stained on the same batch of mesothelioma tissue samples to reduce the impact of batch differences on experimental results.

[0171] Adjustment of DAB staining time: For each concentration gradient of antibody, perform DAB staining twice, one for 1 minute and the other for 5 minutes, and observe the staining effect.

[0172] 2. Experimental Results

[0173] Result 1: Normal spleen tissue sections were selected as negative tissues, as these tissues had no specific binding reaction with anti-CD228 antibodies; human mesothelioma tissue sections were selected as positive tissues, as these tissues had a specific binding reaction with anti-CD228 antibodies. The screening results of positive and negative tissues are shown in Figure 10.

[0174] Result 2: As shown in FIG11 , the staining effect comparison of different commercial antibodies in different tissues shows that the commercial antibody NBP1-85777 has a better staining effect and can be used as a subsequent control antibody.

[0175] Result 3: The staining results are shown in Figure 12. The primary antibody concentration was determined to be 1 μg / mL for the experiment. The DAB staining results are shown in Figure 13. The R546 antibody was selected and the DAB staining time of 5 minutes was the best in mesothelioma tissue.

[0176] Example 7: Comparison of staining effects of anti-CD228 candidate antibodies and commercial antibodies in different tissues

[0177] 1. Experimental methods:

[0178] The experimental group was set up with an anti-CD228 candidate antibody, and the control group was set up with a commercial CD228 / MFI2 antibody (purchased from Novusbio, NBP1-85777) screened in Example 6; the positive and negative controls used the tissues screened in Example 6: normal human spleen tissue sections as negative tissues, and human mesothelioma tissue sections as positive tissues; the immunohistochemical detection method was similar to that in Example 5, wherein the DAB staining time was 5 min and the primary antibody concentration was 1-4 μg / mL.

[0179] 2. Experimental results:

[0180] Result 1: Comparison of staining effects of anti-CD228 candidate antibodies and commercial antibody NBP1-85777 in mesothelioma tissue

[0181] The antibody was stained at 1 μg / mL in four different mesothelioma tissues (purchased from Shanghai Xinchao), and the immunohistochemical results are shown in Figure 14. The results showed that the staining intensity of the R546 antibody was more consistent with that of the commercial antibody NBP1-85777.

[0182] Result 2: Comparison of staining of R546 and NBP1-85777 in single sections of mesothelioma, colon cancer, and cervical cancer tissues.

[0183] Candidate antibody R546 (working concentration 1 μg / mL), commercial anti-CD228 monoclonal antibody NBP1-85777 (working concentration 1 μg / mL) and normal unrelated antibody (working concentration 1 μg / mL) were used as the first antibody to perform LEICA BOND on normal human spleen tissue (CD228 negative tissue), human mesothelioma tissue (CD228 positive tissue), colon cancer tissue, and cervical cancer tissue (all tissues were purchased from Shanghai Xinchao Biological). TM The fully automatic IHC and ISH staining system (model: BOND-MAX) was used for detection, and acid retrieval was selected for antigen retrieval. The immunohistochemical results are shown in Figure 15, and the staining results are shown in Table 10.

[0184] Table 10: Comparison of the staining effects of CD228-2204 and commercial antibodies on paraffin-embedded tissue sections

[0185] According to the observation of the staining results, the results are as follows:

[0186] (1) Specificity Verification: In CD228-negative spleen tissue sections, the homemade antibody R546, the commercial antibody NBP1-85777, and unrelated antibodies all failed to induce staining reactions. This result indicates that the antibody R546 has a high specificity for CD228-negative samples and can effectively distinguish tissues that express or do not express CD228.

[0187] (2) Consistency of staining effects: The homemade antibody R546 specifically stained CD228-positive mesothelioma tissue at a working concentration of 1 μg / mL, and its staining intensity matched that of the commercial antibody NBP1-85777 at the same concentration. This indicates that the homemade antibody R546 has the same staining effect as the commercial antibody NBP1-85777 in practical applications and can serve as an effective alternative to it.

[0188] (3) Staining performance in other cancer tissues: In colon cancer tissues and cervical cancer tissues, both the homemade antibody R546 and the commercial antibody NBP1-85777 showed varying degrees of staining. This indicates that both antibodies have good staining effects in these cancer types, and the staining intensity is comparable, further confirming the applicability and reliability of the homemade antibody R546.

[0189] Result 3: Comparison of staining of R546 and NBP1-85777 in colon cancer tissue microarray

[0190] A colon cancer tissue chip containing 28 patient tissue samples (purchased from Shanghai Xinchao, catalog number Hcol-ade060CS-01) was subjected to LEICA BOND with R546 (4 μg / mL) and NBP1-85777 (1 μg / mL). TM The immunohistochemistry results are shown in Figure 16 and the staining results are shown in Table 11. The staining results show that the staining intensity and positive rate of R546 and NBP1-85777 in colon cancer tissue microarray are consistent. Result 4: Comparison of staining of R546 and NBP1-85777 in cervical cancer tissue microarray

[0191] A cervical cancer tissue chip containing 15 patient tissue samples (purchased from Shanghai Xinchao, HUteS30PG01-1) was subjected to LEICA BOND with R546 (4 μg / mL) and NBP1-85777 (1 μg / mL). TM The fully automated IHC and ISH staining system (model: BOND-MAX) was used for detection. The immunohistochemistry results are shown in Figure 17 , and the staining results are shown in Table 11 . The staining results showed that the staining intensity and positive rate of R546 and NBP1-85777 in the cervical cancer tissue chip were consistent.

[0192] Result 5: Comparison of staining of R546 and NBP1-85777 in esophageal cancer tissue microarray

[0193] An esophageal cancer tissue chip containing 30 patient tissue samples (purchased from Shanghai Xinchao, HEsoS030PG02) was subjected to LEICA BOND with R546 (3 μg / mL) and NBP1-85777 (1 μg / mL). TM The fully automated IHC and ISH staining system (model: BOND-MAX) was used for detection. The immunohistochemistry results are shown in Figure 18 , and the staining results are shown in Table 11 . The staining results showed that the staining intensity and positive rate of R546 and NBP1-85777 on the esophageal cancer tissue chip were consistent.

[0194] Result 6: Comparison of staining of R546 and NBP1-85777 in pancreatic cancer tissue microarray

[0195] Pancreatic cancer tissue microarray containing 20 patient tissue samples (purchased from Shanghai Xinchao, HPanA020PG01) was subjected to LEICA BOND with R546 (1.5 μg / mL) and NBP1-85777 (1 μg / mL). TM The fully automated IHC and ISH staining system (model: BOND-MAX) was used for detection. The immunohistochemistry results are shown in Figure 19 , and the staining results are shown in Table 11 . The staining results showed that the staining intensity and positive rate of R546 and NBP1-85777 on the esophageal cancer tissue chip were consistent.

[0196] Result 7: Comparison of staining of R546 and NBP1-85777 in gastric cancer tissue microarray

[0197] A gastric cancer tissue chip containing 40 patient tissue samples (purchased from Shanghai Xinchao, HStm-Ade040PG-02-2) was subjected to LEICA BOND with R546 (3 μg / mL) and NBP1-85777 (1 μg / mL). TM The fully automated IHC and ISH staining system (model: BOND-MAX) was used for detection. The immunohistochemistry results are shown in Figure 20 , and the staining results are shown in Table 11 . The staining results showed that the staining intensity and positive rate of R546 and NBP1-85777 on the gastric cancer tissue chip were consistent.

[0198] Result 8: Comparison of staining of R546 and NBP1-85777 in extrahepatic cholangiocarcinoma tissue microarray

[0199] Extrahepatic bile duct carcinoma tissue array containing 40 patient tissue samples (purchased from Shanghai Xinchao, HEBD-ade036PG-01) was subjected to LEICA BOND with R546 (3 μg / mL) and NBP1-85777 (1 μg / mL). TMThe fully automated IHC and ISH staining system (model: BOND-MAX) was used for detection. The immunohistochemistry results are shown in Figure 21 , and the staining results are shown in Table 11 . The staining results showed that the staining intensity and positive rate of R546 and NBP1-85777 in the extrahepatic cholangiocarcinoma tissue chip were consistent.

[0200] Result 9: Comparison of staining of R546 and NBP1-85777 in gallbladder cancer tissue microarray

[0201] A gallbladder cancer tissue chip containing 80 patient tissue samples (purchased from Shanghai Xinchao, HGal-Ade100PG-01) was subjected to LEICA BOND with R546 (3 μg / mL) and NBP1-85777 (1 μg / mL). TM The fully automated IHC and ISH staining system (model: BOND-MAX) was used for detection. The immunohistochemistry results are shown in Figure 22 , and the staining results are shown in Table 11 . The staining results showed that the staining intensity and positive rate of R546 and NBP1-85777 in the gallbladder cancer tissue chip were consistent.

[0202] Table 11: Comparison of staining intensity and proportion of R546 and NBP1-85777 in different cancer tissue microarrays

[0203] The data in Table 11 show that the staining intensity and positive rate of R546 and NBP1-85777 in the above 7 cancer types (colon cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, extrahepatic bile duct cancer, and cholangiocarcinoma) are consistent.

[0204] Result 10: The staining of R546 and NBP1-85777 antibodies in different cancer tissues was verified using the same experimental methods as in Example 5. Specifically, the company performed immunohistochemical staining on single sections of gallbladder cancer, bile duct cancer, esophageal cancer, pancreatic cancer, and gastric cancer tissues and compared the staining effects of the two antibodies.

[0205] The candidate antibody R546 (working concentration 1 μg / mL) and the commercial anti-CD228 monoclonal antibody NBP1-85777 (working concentration 1 μg / mL) were used as the primary antibodies to perform LEICA BOND on 10 single tissue samples of gallbladder cancer, bile duct cancer, esophageal cancer, pancreatic cancer, and gastric cancer (all tissues were obtained from ACRODiagnostics). TMImmunohistochemistry results, using a fully automated IHC and ISH staining system (model: BOND-III), are shown in Figures 23-27, and the staining results are summarized in Table 12. The staining results demonstrate that R546 significantly outperforms NBP1-85777 in staining intensity and sensitivity across tissue sections from the five cancer types (gallbladder cancer, bile duct cancer, esophageal cancer, pancreatic cancer, and gastric cancer).

[0206] Table 12: Comparison of staining results of R546 and NBP1-85777 in single sections of gallbladder cancer, bile duct cancer, esophageal cancer, pancreatic cancer, and gastric cancer tissues

[0207] The data in the table show that in the five cancer types (gallbladder cancer, bile duct cancer, esophageal cancer, pancreatic cancer, and gastric cancer) tissue sections, the positive rate of R546 was significantly higher than that of NBP1-85777, demonstrating that the R546 antibody of the present invention can replace existing commercial antibodies for immunohistochemical detection of the CD228 target.

[0208] Based on the above experiments, it can be seen that the R546 antibody has higher sensitivity and potentially better diagnostic efficacy in immunohistochemical detection of these specific cancer types. The high positive rate of the R546 antibody may also mean that it has better specificity and can more accurately distinguish cancer cells from normal cells, which is crucial for early detection of cancer and treatment selection. In addition, the high sensitivity of the R546 antibody may help identify cases with low or heterogeneous expression of NBP1-85777 that may be missed by the NBP1-85777 antibody.

[0209] Example 8: Detection of the binding specificity of R546 by ELISA.

[0210] CD228 protein, transferrin (TF), and lactoferrin (LF) were diluted with CBS to 0.2 μg / mL, 100 μL / well, and incubated at 4°C overnight; blocking solution was added and blocked at 37°C for 1 hour, and the plate was washed; R546 and NBP1-85777 were diluted fourfold from 10 μg / mL with PBST in 8 gradients, 100 μL / well, and incubated at 37°C for 1 hour, and the plate was washed; enzyme-labeled secondary antibody Goat pAb to Rb IgG (HRP) was added, 100 μL / well, and incubated at 37°C for 1 hour, and the plate was washed; 100 μL TMB color development solution was added, color was developed for 10 minutes, and 2 M sulfuric acid was added to terminate the color development; the OD450 nm value was read with a microplate reader. The OD450 value was plotted against the antibody concentration, and the results are shown in Figure 28. The results show that both R546 and NBP1-85777 only bind to the CD228 protein, and have little binding to transferrin and lactoferrin in the same family, indicating that the antibodies have good selectivity and specificity.

[0211] Example 9: Detection of the binding specificity of R546 by biomembrane interferometry.

[0212] R546 and NBP1-85777 antibodies were coupled to a ProA chip (Fortebio Cat. 18-5010) on a Fortebio RED96 instrument at a concentration of 4 μg / mL. The threshold was set to 1.5 nm. CD228 protein, transferrin (TF), and lactoferrin (LF) were diluted two-fold in PBST over four concentrations, starting at 100 nm. The binding assay was performed for 150 s, the dissociation assay for 300 s, the equilibrium for 60 s, and the probe regeneration for 15 s. Kinetic parameters such as KD values ​​were calculated using the instrument's accompanying software. The assay data are shown in Table 13, and the corresponding affinity assay profile is shown in Figure 29.

[0213] Table 13: KD of R546 and NBP1-85777 binding to CD228 and other proteins in the same family

[0214] The data in the table show that both R546 and NBP1-85777 bind only to the CD228 protein and do not bind to transferrin and lactoferrin, which are members of the same family, indicating that the antibodies have good selectivity and specificity.

[0215] Example 10: Storage stability test of R546 antibody.

[0216] The R546 antibody was stored at 25°C and removed on day 30. The R546 antibody was stored at 40°C and removed on days 0, 7, and 14. The stability of the samples was assessed by size / size exclusion chromatography (SEC), capillary electrophoresis with reduced sodium dodecyl sulfate (rCE-SDS), and capillary electrophoresis with isoelectric focusing (icIEF). SEC assesses changes in antibody multimers and small molecules by molecular size separation, rCE-SDS assesses structural stability by analyzing the percentage of light and heavy chains, and icIEF characterizes charge heterogeneity by analyzing the distribution of isoelectric points. The numerical trends and peak characteristics of these techniques provide a comprehensive assessment of antibody stability. The test data are shown in Tables 14, 15, and 16.

[0217] Table 14: Changes in icIEF of R546 antibody after storage at different temperatures for different days

[0218] Table 15: Changes in rCE-SDS of R546 antibody after storage at different temperatures for different days

[0219] Table 16: Changes in SEC of R546 antibody at different temperatures for different days

[0220] Under high temperature (40°C) conditions, icIEF (isoelectric focusing) results showed that the main peak content was slightly decreased, the acidic peak content was slightly increased, and the alkaline peak content was slightly increased; rCE-SDS (capillary electrophoresis-sodium dodecyl sulfate polyacrylamide gel electrophoresis) results showed that the light chain %, heavy chain % and (light chain + heavy chain) % contents did not change significantly; SEC (size exclusion chromatography) purity did not change significantly. Under high temperature (25°C) conditions, all test results of the R546 antibody did not show significant changes; the above results indicate that the antibody has good stability.

[0221] In summary, the homemade antibody R546 monoclonal antibody has the highest sensitivity and strong affinity for binding to the CD228 protein. It also specifically binds to the CD228 protein and does not bind to other proteins in the same family. At the same time, according to the immunohistochemical results, the combined staining effects on single slides and chips of different cancer tissues showed that the staining sensitivity and positive rate of the homemade antibody R546 were better than those of the commercial antibody NBP1-85777. Therefore, we determined that R546 can replace commercial antibodies as a diagnostic antibody suitable for immunohistochemical detection.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD228 protein, characterized in that: The antibody or antigen-binding fragment thereof comprises the following three light chain complementary determining regions and three heavy chain complementary determining regions: The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof include LCDR1 as shown in SEQ ID NO:2, LCDR2 as shown in SEQ ID NO:3, and LCDR3 as shown in SEQ ID NO:4; and the three heavy chain complementary determining regions include HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:8; The antibody or antigen-binding fragment thereof has three light chain complementary determining regions comprising LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO: 12; and / or three heavy chain complementary determining regions comprising HCDR1 as shown in SEQ ID NO: 14, HCDR2 as shown in SEQ ID NO: 15, and HCDR3 as shown in SEQ ID NO: 16; Or the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof include LCDR1 as shown in SEQ ID NO:18, LCDR2 as shown in SEQ ID NO:19, and LCDR3 as shown in SEQ ID NO:20; and the three heavy chain complementary determining regions include HCDR1 as shown in SEQ ID NO:22, HCDR2 as shown in SEQ ID:23, and HCDR3 as shown in SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:9, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:13; Or the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:17, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

21.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:25, and a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

26.

4. An antibody conjugate, characterized in that The antibody conjugate is composed of the antibody or antigen-binding fragment thereof that specifically binds to the CD228 protein according to any one of claims 1 to 3 and at least one detectable marker.

5. A nucleic acid, characterized in that Encoding the antibody or antigen-binding fragment thereof that specifically binds to the CD228 protein according to any one of claims 1 to 3.

6. A kit for detecting or quantifying CD228, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof that specifically binds to the CD228 protein according to any one of claims 1 to 3, or the antibody conjugate according to claim 4; optionally, the kit further comprises a detection or quantification-related buffer solution.

7. A kit for diagnosing a disease or evaluating the therapeutic effect of a disease, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof that specifically binds to the CD228 protein according to any one of claims 1 to 3, or the antibody conjugate according to claim 4, and the disease is a CD228-related disease; Preferably, the CD228-related diseases include one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

8. Use of the antibody or antigen-binding fragment thereof that specifically binds to CD228 protein according to any one of claims 1 to 3, the antibody conjugate according to claim 4, or the nucleic acid according to claim 5 in preparing a kit for detecting and / or quantifying whether a cell or tissue expresses CD228; Preferably, the kit is an immunohistochemistry detection kit.

9. Use of the antibody or antigen-binding fragment thereof that specifically binds to CD228 protein according to any one of claims 1 to 3 in the preparation of a disease diagnosis kit, wherein the disease is a CD228-related disease; Preferably, the kit is an immunohistochemistry detection kit; Preferably, the CD228-related diseases include one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

10. Use of the antibody or antigen-binding fragment thereof that specifically binds to CD228 protein according to any one of claims 1 to 3, the antibody conjugate according to claim 4, or the nucleic acid according to claim 5 in preparing a kit for evaluating the therapeutic effect of a CD228-related disease, wherein the disease is a CD228-related disease; Preferably, the CD228-related diseases include one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, gallbladder cancer and bile duct cancer.

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