A monoclonal antibody against glypican-3, polynucleotide, and preparation method and application thereof

Abstract

This application relates to the field of tumor immunodiagnostic technology, and particularly to a monoclonal antibody, polynucleotide, preparation method, and application of antiphosphatidylinositol proteoglycan-3; the monoclonal antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes heavy chain region CDR1 with amino acid sequence as shown in SEQ ID NO:1, heavy chain region CDR2 with amino acid sequence as shown in SEQ ID NO:2, and heavy chain region CDR3 with amino acid sequence as shown in SEQ ID NO:3; the light chain variable region includes light chain region CDR1 with amino acid sequence as shown in SEQ ID NO:4, light chain region CDR2 with amino acid sequence as shown in SEQ ID NO:5, and light chain region CDR3 with amino acid sequence as shown in SEQ ID NO:6; the dissociation constant KD(M) of the monoclonal antibody against GPC3 antigen is 1.21E-11, which is a high-affinity N-terminal antibody, suitable for the detection of GPC3 in clinical serum samples.

Description

Technical Field

[0001] This application relates to the field of tumor immunodiagnostic technology, and in particular to a monoclonal antibody against phosphatidylinositol proteoglycan-3, a polynucleotide, its preparation method and application. Background Technology

[0002] Primary liver cancer is one of the most common malignant tumors in the world, ranking fifth among all malignant tumors globally. Hepatocellular carcinoma (HCC) is the most common primary liver tumor. Because liver cancer often presents with symptoms at an advanced stage, and recurrence and metastasis rates are high after resection, early diagnosis of liver cancer is crucial for prolonging patient survival and reducing mortality.

[0003] Currently, early diagnosis and screening for liver cancer mainly rely on two methods: imaging examinations and tumor marker detection. However, imaging examinations have a certain time lag and are dependent on the operator's skill and experience, making them unsuitable for large-scale application and hindering early diagnosis and treatment. Therefore, highly sensitive serum liver cancer-specific markers are still needed for early diagnosis. The most commonly used tumor marker for primary liver cancer is alpha-fetoprotein (AFP), but its positive rate for diagnosing HCC is only 50%. Its sensitivity is significantly reduced in small liver cancers (<3cm in diameter), with a positive rate of less than 40%, easily leading to missed diagnoses. Furthermore, elevated levels can also be found in patients with benign liver diseases, reproductive teratomas, and lung cancer, easily causing misdiagnosis. Therefore, finding new, highly specific, sensitive, and easily detectable liver cancer diagnostic markers has profound clinical significance.

[0004] Glypican-3 (GPC3) is a cell surface protein belonging to the heparan sulfate proteoglycan family. GPC3 is highly expressed in fetal liver but not in normal adult liver tissue. However, its expression is restored in hepatocellular carcinoma (HCC) and it is closely related to the occurrence and development of HCC. Its detection rate is high not only in the early stages of HCC but also increases with the progression of the disease. Therefore, GPC3 can replace AFP as a diagnostic marker for HCC. However, due to the significant differences in affinity and specificity of existing antibodies against cancer markers, most are insufficient for practical applications. For example, in HCC, if GPC3 is used to replace AFP as a diagnostic marker, the low affinity between GPC3 monoclonal antibodies and the target antigen results in low specificity, affecting detection accuracy. Therefore, providing GPC3 monoclonal antibodies with high affinity for the target antigen is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] This application provides a monoclonal antibody against phosphatidylinositol proteoglycan-3, a polynucleotide, a method for preparing the same, and its application, in order to solve the technical problem of low affinity between GPC3 monoclonal antibodies and target antigens in the prior art.

[0006] In a first aspect, this application provides a monoclonal antibody against phosphatidylinositol proteoglycan-3, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain region CDR1, heavy chain region CDR2 and heavy chain region CDR3, the amino acid sequence of heavy chain region CDR1 is shown in SEQ ID NO:1, the amino acid sequence of heavy chain region CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain region CDR3 is shown in SEQ ID NO:3;

[0007] The light chain variable region includes light chain region CDR1, light chain region CDR2 and light chain region CDR3. The amino acid sequence of light chain region CDR1 is shown in SEQ ID NO:4, the amino acid sequence of light chain region CDR2 is shown in SEQ ID NO:5 and the amino acid sequence of light chain region CDR3 is shown in SEQ ID NO:6.

[0008] Optionally, the amino acid sequence of the heavy chain region is shown in SEQ ID NO:7.

[0009] Optionally, the amino acid sequence of the light chain region is shown in SEQ ID NO:8.

[0010] Secondly, this application provides a polynucleotide for encoding the monoclonal antibody described in the first aspect.

[0011] Optionally, the polynucleotide includes a first coding region to encode the heavy chain variable region of the monoclonal antibody, the nucleotide sequence of the first coding region being shown in SEQ ID NO:9.

[0012] Optionally, the polynucleotide further includes a second coding region encoding a light chain variable region of the monoclonal antibody, the nucleotide sequence of which is shown in SEQ ID NO:10.

[0013] Thirdly, this application provides an expression vector comprising the polynucleotide described in the second aspect.

[0014] Fourthly, this application provides a host cell containing the polynucleotide described in the second aspect or the expression vector described in the third aspect.

[0015] Fifthly, this application provides a method for preparing a monoclonal antibody against phosphatidylinositol proteoglycan-3, the method comprising:

[0016] The host cells described in the third aspect are cultured and then expressed to obtain the fusion protein;

[0017] Hybridoma cell lines were constructed using the fusion protein;

[0018] The hybridoma cell line was cultured in vivo to obtain a monoclonal antibody against phosphatidylinositol proteoglycan-3.

[0019] In a sixth aspect, this application provides the application of a monoclonal antibody against phosphatidylinositol proteoglycan-3, the application including the use of the monoclonal antibody described in the first aspect in the preparation of a detection reagent for liver cancer detection.

[0020] The technical solutions provided in this application have the following advantages compared with the prior art:

[0021] This application provides a monoclonal antibody against phosphatidylinositol proteoglycan-3. By designing the complementarity-determining regions (CDRs) of the heavy chain variable region and the light chain variable region of the monoclonal antibody, respectively, the binding characteristics of the antibody and antigen can be described by the CDRs. Using the amino acid sequences of the six designed CDRs, the heavy chain variable region or the light chain variable region can be divided into four frame regions. During the stage where the β-sheets formed in the frame regions are spatially close to each other, the six designed CDRs constitute the antigen-binding sites, enhancing the binding strength between the antibody parasite and the antigen epitope, thereby improving the affinity between the antibody and the antigen. Therefore, by designing the CDRs of the heavy chain variable region and the light chain variable region, the affinity between the GPC3 monoclonal antibody and the target antigen can be improved. Attached Figure Description

[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A detailed flowchart illustrating the method provided in the embodiments of this application;

[0025] Figure 2The SDS-PAGE image of the expression and purification of GPC3 25-554A protein provided in the embodiments of this application shows that 1 represents the whole sample of cell expression supernatant, 2 represents the effluent of the whole sample, and 3 represents the eluted target protein.

[0026] Figure 3 This is a comparison of flow cytometry results between four antibodies and the HepG2 liver cancer cell line provided in the embodiments of this application;

[0027] Figure 4 A schematic diagram of the standard curve provided for the embodiments of this application. Detailed Implementation

[0028] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0029] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0031] The creative thinking behind this application is:

[0032] Because the GPC3 gene encodes a precursor core protein of about 70 kDa, this precursor protein can be cleaved by furin to produce a soluble N-terminal peptide of about 40 kDa that can enter the bloodstream and a membrane-bound C-terminal peptide of about 30 kDa containing two heparan sulfate (HS) sugar chains. The GPC3 protein is anchored to the cell membrane via glycosylphosphatidylinositol (GPI). GPC3 is highly expressed in fetal liver but not in normal adult liver tissue, but its expression is restored in hepatocellular carcinoma. Therefore, GPC3 is closely related to the occurrence and development of liver cancer. It has a high detection rate not only in the early stage of liver cancer but also increases with the development of liver cancer.

[0033] Because existing antibodies targeting cancer biomarkers vary greatly in affinity and specificity, most fail to meet the needs of practical applications. For example, reports from abroad indicate that GPC3 levels in peripheral blood are low, and the increase in GPC3 concentration in liver cancer patients is relatively small, making it difficult to distinguish from normal controls, especially those with cirrhosis. This is because the specificity of GPC3 monoclonal antibodies is low, and their affinity for the target antigen is low, resulting in unsatisfactory detection accuracy and limiting their application in tumor detection.

[0034] The technical solution provided by the embodiments of the present invention is to solve the above-mentioned technical problems, and the general idea is as follows:

[0035] In one embodiment of this application, a monoclonal antibody against phosphatidylinositol proteoglycan-3 is provided. The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain region CDR1, heavy chain region CDR2, and heavy chain region CDR3. The amino acid sequence of heavy chain region CDR1 is shown in SEQ ID NO:1, the amino acid sequence of heavy chain region CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain region CDR3 is shown in SEQ ID NO:3.

[0036] The light chain variable region includes light chain region CDR1, light chain region CDR2 and light chain region CDR3. The amino acid sequence of light chain region CDR1 is shown in SEQ ID NO:4, the amino acid sequence of light chain region CDR2 is shown in SEQ ID NO:5 and the amino acid sequence of light chain region CDR3 is shown in SEQ ID NO:6.

[0037] In some alternative embodiments, the amino acid sequence of the heavy chain region is as shown in SEQ ID NO:7.

[0038] In this embodiment, the amino acid sequence of the heavy chain region is controlled so that, based on the complementarity-determining region of the heavy chain variable region, a portion of the monoclonal antibody can be successfully expressed in the entire heavy chain variable region.

[0039] In some alternative embodiments, the amino acid sequence of the light chain region is as shown in SEQ ID NO:8.

[0040] In this embodiment, the amino acid sequence of the light chain region is controlled so that, based on the complementarity-determining region of the light chain variable region, a portion of the monoclonal antibody can be successfully expressed in the entire light chain variable region.

[0041] Next, a polynucleotide provided in an embodiment of this application is described, the polynucleotide being used to encode the monoclonal antibody described in the first aspect.

[0042] Since the polynucleotides described in the embodiments of this application encode the monoclonal antibodies provided in the aforementioned embodiments of this application, the amino acid sequence and composition information of the monoclonal antibodies will not be repeated here. All polynucleotides encoding the monoclonal antibodies of the embodiments of this application fall within the scope of protection of this application.

[0043] In some alternative embodiments, the polynucleotide includes a first coding region to encode the heavy chain variable region of the monoclonal antibody, the nucleotide sequence of the first coding region being shown in SEQ ID NO:9.

[0044] In this embodiment of the application, controlling the first coding region to encode the heavy chain variable region allows the nucleotides corresponding to the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region to be expressed simultaneously with the expression of the first coding region, thereby enabling the smooth expression of the heavy chain variable region.

[0045] In some alternative embodiments, the polynucleotide further includes a second coding region encoding a light chain variable region of the monoclonal antibody, the nucleotide sequence of which is shown in SEQ ID NO:10.

[0046] In this embodiment of the application, controlling the second coding region to encode the light chain variable region allows the nucleotides corresponding to the amino acid sequences of the three complementarity-determining regions in the light chain variable region to be expressed simultaneously with the expression of the second coding region, thereby enabling the smooth expression of the light chain variable region.

[0047] Next, an expression vector provided by an embodiment of this application is described, the expression vector comprising the polynucleotide.

[0048] Since the expression vectors described in the embodiments of this application include the polynucleotides provided in the aforementioned embodiments of this application, the nucleotide sequences and composition information of the polynucleotides will not be repeated here. All expression vectors including the polynucleotides described in the embodiments of this application fall within the scope of protection of this application.

[0049] Next, a host cell provided by an embodiment of this application is described, wherein the host cell contains the polynucleotide or the expression vector.

[0050] Since the host cells described in the embodiments of this application include the polynucleotides or expression vectors provided in the aforementioned embodiments of this application, the nucleotide sequences and composition information of the polynucleotides or expression vectors will not be repeated here. All host cells including the polynucleotides or expression vectors described in the embodiments of this application fall within the scope of protection of this application.

[0051] Next, a method for preparing a monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in the embodiments of this application will be described, the preparation method comprising:

[0052] S1. Culture the host cells described in the third aspect, and then express them to obtain the fusion protein;

[0053] S2. Construct a hybridoma cell line using the fusion protein;

[0054] S3. The hybridoma cell line was cultured in vivo to obtain a monoclonal antibody against phosphatidylinositol proteoglycan-3.

[0055] Since the preparation method described in the embodiments of this application prepares the monoclonal antibody provided in the aforementioned embodiments of this application, the amino acid sequence and composition information of the monoclonal antibody will not be repeated here. All methods for preparing monoclonal antibodies, including those described in the embodiments of this application, fall within the scope of protection of this application.

[0056] Next, the application of a monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in the embodiments of this application is described, the application including the use of the monoclonal antibody in the preparation of a detection reagent for liver cancer detection.

[0057] Since the applications described in this application's embodiments include monoclonal antibodies as provided in the aforementioned embodiments of this application, the amino acid sequences and compositional information of the monoclonal antibodies will not be repeated here. All methods for preparing monoclonal antibodies, including those described in the embodiments of this application, fall within the scope of protection of this application.

[0058] Example 1

[0059] like Figure 1 As shown, the carrier is constructed as follows:

[0060] 1. Gene synthesis NM_004484 218-1807bp (refer to NP_001158089.1, glypican-3isoform1, 25-554AA), wherein the synthesized gene includes the addition of V5H vector cloning restriction sites NheI / BamHI at both ends of the synthesized gene.

[0061] 2. The synthesized gene sequence and vector V5H were then double-digested with the restriction enzymes NheI / BamHI. The products were then recovered by agarose gel electrophoresis to obtain the double-digested products.

[0062] 3. The double digestion products were ligated overnight in a 16°C water bath using T4 ligase, and then transformed into E. coli DH5α strain for screening of positive clones.

[0063] Sequencing confirmed that the inserted fragment was completely identical to the fragment in the published full sequence and was inserted into the cloning site of the expression vector in the correct orientation. The recombinant plasmid V5H-25-554AA was amplified and extracted.

[0064] Example 2

[0065] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0066] like Figure 1 As shown, the expression and purification of the fusion protein:

[0067] 1. Select qualified HEK293 cells at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell per well in 6-well plates and cultured for 24 hours. Transfection was then performed when the cell confluence reached 80%–90%.

[0068] 2. Wash the cell suspension three times with PBS and add serum-free DMEM high-glucose medium. Transfect the recombinant expression plasmid V5H-25-554AA according to the Lipofectamine™ 2000 transfection reagent instructions and incubate at 37°C and 5% CO2 at 120 rpm for 72 h.

[0069] 3. Collect the cell culture medium, centrifuge at 4500g for 15 minutes to remove the cells, and take the supernatant.

[0070] 4. Pack 1 mL of Ni-NTA Agarose affinity packing material into a column. Equilibrate the Ni-NTA affinity column with equilibration buffer (50 mM PB, 0.3 M NaCl, 10 mM imidazole, pH 8.0) for 10 column volumes. Then, pass the cell culture supernatant after centrifugation through the Ni-NTA affinity column at 1 mL / min and collect the flow-through. Store at 4°C.

[0071] 5. Wash 10 column volumes with washing buffer (50 mM PB, 0.3 M NaCl, 20 mM imidazole, pH 8.0), collect the flow-through and store at 4°C. Wash 4–5 column volumes with elution buffer (50 mM PB, 0.3 M NaCl, 250 mM imidazole, pH 8.0), collect the eluent, and dialyze overnight at 4°C in dialysis buffer (50 mM PB, pH 7.8, 0.3 M NaCl, 5% glycerol) to obtain GPC3 25-554A protein. Perform SDS-PAGE electrophoresis on a small amount; the results are as follows. Figure 2 As shown.

[0072] Example 3

[0073] Comparing Example 3 and Example 2, the differences between Example 3 and Example 2 are as follows:

[0074] like Figure 1 As shown, hybridoma cell line establishment:

[0075] 1. The GPC3 25-554A protein prepared in Example 2 at a concentration of 1.0 mg / mL was used as an antigen and thoroughly emulsified with 1 mL of complete Freund's adjuvant (purchased from Sigma-Aldrich). The mixture was then subcutaneously immunized with 100 μg of GPC3 25-554A protein antigen per mouse.

[0076] 2. After 3 weeks, the GPC3 25-554A protein antigen was emulsified and mixed with incomplete Freund's adjuvant, and the mice were subcutaneously immunized with 50 μg of GPC3 25-554A protein antigen per mouse. Two weeks later, a booster immunization of 50 μg of GPC3 25-554A protein antigen was administered subcutaneously.

[0077] 3. One week after the fourth booster immunization, mice were coated with GPC3 25-554A protein, and the antiserum titer was measured by ELISA. Booster immunizations were continued until the antiserum titer reached >10. 5 Three weeks after the final booster immunization, administer 20 μg of GPC3 25-554A protein antigen intraspleurally for later use.

[0078] 4. Four days after the mice were boosted with intrasplenic immunization, the spleen was harvested under sterile conditions, and lymphocytes were separated by filtering through a 100-mesh filter. The cells were then fused with the myeloma cell line SP2 / 0 and selectively cultured for 3 days with hypoxanthine, aminopterin and thymidine (HAT). HT medium was then added, and the cells were cultured for another week.

[0079] 5. The cells were coated with GPC3 25-554A protein antigen, and positive clones were screened by ELISA. Subcloning was performed three times using the limiting dilution method. The cells were then cultured continuously for two months to obtain stable hybridoma cell lines (clone numbers were named 1G3, 4C8, 10F9 and 9D5, respectively).

[0080] 6. The indirect ELISA screening results for hybridoma cell lines with different clone numbers are shown in Table 1.

[0081] Table 1. ELISA screening results of hybridoma cell lines with different clone numbers.

[0082] Coated antibody strain 1G3 4C8 10F9 9D5 GPC3 25-554AA antigen 2.892 1.9 1.932 1.512 blank 0.057 0.089 0.076 0.078

[0083] Example 4

[0084] Comparing Example 4 and Example 3, the differences between Example 4 and Example 3 are as follows:

[0085] like Figure 1 As shown, ascites production and antibody purification:

[0086] 1. Intraperitoneally inject 500 μL of norperidine (purchased from Sigma-Aldrich) into 8–10 week old F1 mice. Culture four hybridoma cell lines separately, and collect fresh, viable cells. Follow the 1×10⁻⁶ regimen. 6 Cells / mouse doses were injected into the peritoneal cavity of mice. Ascites fluid was collected 7 to 10 days later and centrifuged at 10,000g for 10 min. The supernatant was collected for later use. A protein G affinity column (purchased from GE) was equilibrated with 5 column volumes of PBS (0.01M PB, 0.15M NaCl, pH 7.4).

[0087] 2. Mix the ascites supernatant with 2 volumes of PBS (0.01M PB, 0.15M NaCl, pH 7.4), filter with a 0.22μM filter, and load the filtered ascites supernatant onto a protein G affinity column. Wash with 5 column volumes of PBS.

[0088] 3. Elute with elution buffer (0.1M Glycine HCl, pH 2.8), and neutralize the eluent with 1 / 10 volume of neutralization buffer (1M NaH2PO4, pH 9.0).

[0089] 4. Dialyze the solution with PBS (0.01M PB, 0.15M NaCl, pH 7.4), changing the solution twice, with an interval of more than 5 hours between the two solutions, to obtain the dialysate.

[0090] 5. Centrifuge the dialysis solution at 10000g for 10 min, filter the supernatant through a 0.22μm filter membrane and store it to obtain the purified anti-GPC3 monoclonal antibody solution corresponding to each clone.

[0091] Example 5

[0092] Comparing Example 5 and Example 4, the differences between Example 5 and Example 4 are as follows:

[0093] Antibody epitope classification:

[0094] 1. Antigen coating: Carbonate buffer was used as the coating solution. The concentration of the original GPC3 recombinant protein was 0.5 μg / mL. 100 μL of coating solution and original GPC3 protein was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing, the plate was allowed to return to room temperature. The coating solution was then discarded, and 300 μL of washing buffer was added to each well. The plate was shaken for 1 min each time and washed 3-4 times. The plate was then patted dry.

[0095] 2. Blocking: Add 200 μL of 10% fetal bovine serum to each well as blocking solution and incubate at 37°C for 1 hour; then wash, wait to return to room temperature, discard the blocking solution, wash three more times, shaking for 1 minute each time, and pat dry;

[0096] 3. Dilute GPC3 antibody with buffer solution to a concentration of 5 μg / mL, add 100 μL to each well, and set up blank control wells (PBS) and negative wells (negative serum). Incubate at 37°C for 30 min; wash 3 times, shaking for 1 min each time, and pat dry.

[0097] 4. Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:10000 to each well, incubate at 37°C for 30 min; wash 3 times, shaking for 1 min each time, and pat dry.

[0098] 5. For color development, add 100 μL of substrate and colorimetric solution to each well and incubate at 37°C in the dark for 15 min. To terminate the reaction, add 50 μL of stop solution to each well.

[0099] 6. Measure the OD450nm value of the solution after termination. Read the optical density value of each well using an ELISA reader with a detection wavelength of 450nm. Set the OD450nm value of the negative control well as N and the positive well as P. A positive result is defined as P / N≥2.1. The results are shown in Table 2.

[0100] Table 2. Results of Indirect ELISA Epitope Classification

[0101] Coated antibody strain 1G3 4C8 10F9 9D5 GPC3 25-554AA antigen 2.892 1.9 1.932 1.512 Purchased GPC3 N-terminal antigen (25-358AA) 2.413 2.318 0.783 0.115 Purchased GPC3 C-terminal antigen (359-554AA) 0.033 0.115 0.096 1.661

[0102] In the table, antibodies 1G3, 4C8, and 10F9 are antibodies against the N-terminal antigenic epitope of GPC3, and antibody 9D5 is an antibody against the C-terminal antigenic epitope of GPC3.

[0103] Example 6

[0104] Comparing Example 6 and Example 5, the differences between Example 6 and Example 5 are as follows:

[0105] Affinity assay of anti-GPC3 monoclonal antibody:

[0106] The affinity of each anti-GPC3 monoclonal antibody for recombinant human GPC3 was determined using a BIACORE 3000 biomacromolecule interaction analyzer (purchased from GE), and the results are shown in Table 3. AW mab-5, with the highest affinity, was selected as the anti-GPC3 monoclonal antibody for subsequent detection and further research.

[0107] Table 3. Dissociation constants of anti-GPC3 monoclonal antibodies with target antigens.

[0108] Antibody strain Dissociation constant KD(M) 9D5 8.66E-09 10F9 7.58E-08 4C8 2.78E-10 1G3 1.21E-11

[0109] Example 7

[0110] Comparing Example 7 and Example 6, the differences between Example 7 and Example 6 are as follows:

[0111] Evaluation of binding activity:

[0112] 1. HepG2, HuH-7, and LO2 cell lines were injected at a rate of 1 × 10⁻⁶ cells / mL. 6 Cells were suspended in FACS buffer (1% FBS / PBS). The suspension was aliquoted into 100 μL wells using a Multiscreen–HV Filter Plate (Millopre), and the supernatant was removed after centrifugation. 2.

[0113] 2. Add anti-GPC3 antibody diluted to an appropriate concentration and react on ice for 30 minutes.

[0114] 3. Rinse the cells once with FACS buffer.

[0115] 4. Add FITC-labeled anti-mouse IgG antibody and react on ice for 30 min.

[0116] 5. After the reaction, centrifuge the cells at 500 rpm for 1 minute and remove the supernatant.

[0117] 6. Resuspend the cells in 400 μL of FACS buffer and use for flow cell counting.

[0118] The results showed that GPC3 antibodies 1G3, 4C8, and 10F9 strongly bound to the hepatocellular carcinoma cell lines HepG2 and HuH-7, while the other two 9D5 antibodies bound to the same hepatocellular carcinoma cell lines HepG2 and HuH-7 to a weaker degree. None of the four cell lines bound to the normal hepatocyte cell line LO2, indicating that these antibodies can specifically recognize hepatocellular carcinoma.

[0119] The results of flow cytometry analysis of four antibodies with the HepG2 liver cancer cell line are shown in the figure. Figure 3 .

[0120] Example 8

[0121] Comparing Example 8 and Example 7, the differences between Example 8 and Example 7 are as follows:

[0122] Cloning of the variable region of monoclonal antibody 1G3:

[0123] 1. RNA was extracted from hybridomas containing GPC3 antibody 1G3 using the RNeasy Plus Universal Mini Kit (QIAGEN), and the variable region of the anti-GPC3 antibody was amplified by RT-PCR.

[0124] 2. Using 1 μg of total RNA, the 5'-terminal gene fragment was amplified using the SMART RACE cDNA amplification kit (CLONTECH) and synthetic oligonucleotides. Reverse transcription was performed at 42°C for 1 h and 30 min to obtain the PCR mixture. The synthetic oligonucleotides were as follows:

[0125] The synthesized oligonucleotides complementary to the mouse IgG1 constant region sequence are shown in SEQ ID NO:11: GGG CCAGTG GAT AGACAG ATG;

[0126] The synthesized oligonucleotides complementary to the mouse κ chain constant region sequence are shown in SEQ ID NO:12: GCT CAC TGGATG GTG GGA AGA TG.

[0127] 3. Using a PCR mixture (50 μL) containing 5 μL of 10×Advantage 2 PCR buffer, 5 μL of 10× universal primer A Mix, 0.2 mM of dNTPs (dATP, dGTP, dCTP, and dTTP), 1 μL of Advantage 2 Polymerase Mix (all from CLONTECH), 2.5 μL of reverse transcription product, and 10 pmol of synthetic oligonucleotides, perform PCR. The PCR program includes: 5 cycles of 94℃ for 30 s, 94℃ for 5 s, and 72℃ for 3 min; 5 cycles of 94℃ for 5 s, 70℃ for 10 s, and 72℃ for 3 min; and 25 cycles of 94℃ for 5 s, 68℃ for 10 s, and 72℃ for 3 min to obtain the reaction product.

[0128] 4. The reaction products were heated at 72℃ for 7 min, and then each PCR product was purified by agarose gel extraction using the QIAquick Gel Extraction Kit (QIAGEN), cloned into the pGEM-T Easy vector (Promega), and the nucleotide sequence was determined.

[0129] The results showed that the nucleotide sequence of the first coding region (heavy chain variable region) of monoclonal antibody 1G3 is shown in SEQ ID NO:9, while its translated amino acid sequence is shown in SEQ ID NO:7.

[0130] The nucleotide sequence of the second coding region (light chain variable region) of monoclonal antibody 1G3 is shown in SEQ ID NO:10, and its translated amino acid sequence is shown in SEQ ID NO:8.

[0131] Example 9

[0132] Comparing Example 9 and Example 8, the differences between Example 9 and Example 8 are as follows:

[0133] Antibody-magnetic bead conjugation:

[0134] 1. Washing: Take 300 μL (1 mg / mL) of magnetic beads, add 1 mL of reaction buffer (0.05 M MES, 0.5 M NaCl, pH 5.5) and shake to wash three times, 1 min each time.

[0135] 2. Activation: After the magnetic beads have been washed, remove the reaction buffer, then add 200 μL of activator EDC (1 mg / mL) and 200 μL of NHS (1 mg / mL). Prepare fresh before use, shake and react for 60 min to complete the activation.

[0136] 3. Protein conjugation: After activation, take 1 mL of conjugation solution (0.15 M sodium phosphate, 0.15 M NaCl, pH 7.5) and wash with shaking for 1 min each time. After washing, add 200 μL of conjugation solution and add 30 μg of monoclonal antibody 1G3. Incubate at room temperature with shaking for 2 h.

[0137] 4. Blocking: Remove the coating solution, add 1 mL of blocking solution (a mixture of BSA with a mass concentration of 20 mg / mL, glycine with a mass concentration of 20 mg / mL and coupling solution), and shake at room temperature for 1 h.

[0138] 5. Storage: After sealing, wash three times with 100 μL of blocking solution, shaking for 5 min each time. After washing, bring the volume to 300 μL with stock solution (20 mg / mL BSA, 20 mg / mL glycine, coupling solution and 0.05% Proclin 300) and store at 4°C.

[0139] Example 10

[0140] Comparing Example 10 and Example 9, the differences between Example 10 and Example 9 are as follows:

[0141] Chemiluminescence detection of clinical samples:

[0142] Standards S1-S6 were prepared using GPC3 antigen 25-554AA as raw material. Antibody 4C8, labeled with alkaline phosphatase-based GPC3 antibody, and magnetic beads conjugated with GPC3 antibody 1G3 were used as the main components of the kit. A chemiluminescent double-antibody sandwich method was established. The standard curve is shown in [Figure number missing]. Figure 4 .

[0143] A total of 500 serum samples were collected from different individuals through multiple hospitals in different regions. Among them, there were 200 samples from healthy individuals, 250 samples from patients with liver disease (including 220 samples from patients with liver cancer and 30 samples from patients with hepatitis and cirrhosis), and 50 samples from patients with other cancers.

[0144] The sample was tested according to the established chemiluminescence method using the following steps:

[0145] 1. Add 30 μL of serum sample to be tested and high and low concentration calibrators to the reaction vessel;

[0146] 2. Add 50 μL of the magnetic microsphere solution conjugated with monoclonal antibody 1G3 from the kit;

[0147] 3. Add 50 μL of the alkaline phosphatase-labeled antibody 4C8 solution from the kit;

[0148] 4. Incubate each group at 37℃ for 15 minutes, then wash them three times in a magnetic environment;

[0149] 5. Add luminescent substrate to each group and detect the light signal intensity;

[0150] 6. The working curve corrected by the calibrator can automatically calculate the GPC3 concentration of the sample to be tested based on the light intensity detected by the sample.

[0151] Clinical test results showed that the concentration of GPC3 in the serum of 220 HCC samples was 4.0±20.01 ng / mL, of which 15 samples had a concentration higher than 20 ng / mL; the concentration of GPC3 in the serum of 30 hepatitis and cirrhosis samples was 0.5±2 ng / mL; the concentration in other cancer samples was 0.4±0.5 ng / mL; and the concentration in normal samples was 0.3±0.4 ng / mL.

[0152] The concentrations of GPC3 in each group showed a non-normal distribution. According to SPSS software calculations, when the cutoff value was 1.1 ng / mL, the detection specificity was 96% and the sensitivity was 65.6%.

[0153] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0154] (1) The monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in this application embodiment has a dissociation constant KD(M) of 1.21E-11 against the GPC3 antigen, as determined by the BIACORE3000 biomolecular interaction analyzer, which is a high affinity antibody.

[0155] (2) The monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in this application, as determined by epitope ELISA, belongs to the N-terminal antibody of GPC3 protein. It has been reported that GPC-3 protein mainly enters the bloodstream in its N-terminal form; therefore, the antibody of this invention is suitable for the detection of GPC3 in clinical serum samples.

[0156] (3) The monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in the embodiments of this application can effectively identify liver cancer cell lines by combining it with flow cytometry detection technology.

[0157] (4) The monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in this application embodiment, by coupling the monoclonal antibody to magnetic beads and then using a chemiluminescence assay to detect clinical samples, has a sensitivity of 65.6% and a specificity of 96% in the detection of liver cancer samples, which is superior to similar antibodies or other liver cancer markers.

[0158] (5) The monoclonal antibody against phosphatidylinositol proteoglycan-3 provided in this application has high sensitivity and good specificity, can achieve rapid detection in large quantities, has low cost, and is easier to promote and apply. It can play a significant role in the diagnosis and treatment of tumors in clinical applications.

[0159] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0160] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0161] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A monoclonal antibody against phosphatidylinositol proteoglycan-3, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain region CDR1, heavy chain region CDR2 and heavy chain region CDR3. The amino acid sequence of heavy chain region CDR1 is shown in SEQ ID NO:1, the amino acid sequence of heavy chain region CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain region CDR3 is shown in SEQ ID NO:

3. The light chain variable region includes light chain region CDR1, light chain region CDR2 and light chain region CDR3. The amino acid sequence of light chain region CDR1 is shown in SEQ ID NO:4, the amino acid sequence of light chain region CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of light chain region CDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

7.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

4. A polynucleotide, characterized in that, The polynucleotide is used to encode the monoclonal antibody as described in any one of claims 1-3.

5. The polynucleotide according to claim 4, characterized in that, The polynucleotide includes a first coding region encoding the heavy chain variable region of the monoclonal antibody, the nucleotide sequence of which is shown in SEQ ID NO:

9.

6. The polynucleotide according to claim 4, characterized in that, The polynucleotide also includes a second coding region encoding a light chain variable region of the monoclonal antibody, the nucleotide sequence of which is shown in SEQ ID NO:

10.

7. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in any one of claims 4-6.

8. A host cell, characterized in that, The host cell contains the polynucleotide as described in any one of claims 4-6 or the expression vector as described in claim 7.

9. The application of a monoclonal antibody against phosphatidylinositol proteoglycan-3, characterized in that, The application is in the preparation of a detection reagent for liver cancer detection using the monoclonal antibody according to any one of claims 1-3.

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