Monoclonal antibody of anti-AQP3 protein and application of monoclonal antibody in detection of various cancers

Abstract

The invention discloses an anti-AQP3 protein monoclonal antibody and application thereof in detection of various cancers, and particularly relates to the technical field of biology, the anti-AQP3 protein monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2. According to the invention, a complementary determining region (CDR) sequence is designed, so that the CDR sequence is efficiently combined with a target epitope, and compared with an unoptimized anti-AQP3 protein monoclonal antibody, the antibody has high specificity and high affinity. Meanwhile, the screening and culture conditions of the hybridoma cells are improved, the batch-to-batch consistency of the antibodies is improved, the high quality and reliability of the antibodies are ensured, and the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against AQP3 protein and its application in the detection of various cancers. Background Technology

[0002] AQP3 (Aquaporin-3) is an aquaporin expressed in various tissues. Studies have shown that AQP3 is strongly associated with tumor development and progression, especially affecting tumor cell migration and invasion. Its expression level is significantly increased in various cancers (such as lung cancer, breast cancer, and ovarian cancer). If a monoclonal antibody specifically binding to AQP3 is designed, its mediated signaling pathway can be blocked, thereby inhibiting tumor cell migration and invasion.

[0003] Currently, anti-AQP3 monoclonal antibodies are widely used in the detection of various cancers, but some drawbacks still exist, limiting their applicability in certain tumor types or early-stage cancers. For example, anti-AQP3 protein monoclonal antibodies are prone to cross-reactivity, meaning that the antibody may bind not only to the target AQP3 protein but also to other similar or structurally related proteins. This non-specific binding can lead to high background staining or mislabeling in immunoassays, thus affecting the accuracy of the results. The root cause of this problem is that AQP3 shares some structural similarities with other aquaporins, and the design of the variable regions of existing antibodies is not precise enough, making it easy for anti-AQP3 protein monoclonal antibodies to label other aquaporins. Simultaneously, some existing anti-AQP3 monoclonal antibodies may exhibit low affinity when binding to the AQP3 protein, meaning that the antibody-antigen binding is not tight enough, resulting in insufficient detection sensitivity in tumors with low expression levels and a high risk of missed diagnoses. Low affinity is mainly due to the less-than-ideal matching between the antibody variable region and the binding site of the AQP3 protein, failing to effectively optimize the interaction between the antibody and the target antigen.

[0004] Therefore, the heterogeneity of AQP3 expression in specific cancer types and the inadequacy of antibodies in recognizing low expression levels result in less than ideal detection sensitivity and accuracy for some cancers in clinical practice. These shortcomings are primarily attributed to limitations in antibody design and manufacturing processes. In antibody design, current technologies often fail to adequately optimize the sequence of variable regions, limiting the precision and strength of antibody binding to the target protein. In manufacturing processes, minor variations during cell culture and antibody purification can lead to insufficient consistency and quality control of antibody products, thus limiting their application scope. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly specific and affinity-rich anti-AQP3 monoclonal antibody by optimizing the antibody variable region sequence and preparation process. This antibody can be used for the detection or treatment of various cancers, such as lung cancer, breast cancer, or esophageal cancer.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem: In a first aspect, the present invention discloses an anti-AQP3 protein monoclonal antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:1; and the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:2.

[0007] Secondly, this invention discloses the application of an anti-AQP3 protein monoclonal antibody in the preparation of a drug for detecting cancer, wherein the cancer is lung cancer.

[0008] Thirdly, this invention discloses the application of an anti-AQP3 protein monoclonal antibody in the preparation of a drug for detecting cancer, wherein the cancer is breast cancer.

[0009] Fourthly, this invention discloses the application of an anti-AQP3 protein monoclonal antibody in the preparation of a drug for detecting cancer, wherein the cancer is esophageal cancer.

[0010] In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0011] In some embodiments, the pharmaceutical composition is an injectable dosage form.

[0012] Fifthly, the present invention also discloses a method for preparing a monoclonal antibody against AQP3 protein, the method comprising the following steps: S1. Animals were immunized with recombinant AQP3 protein to prepare a spleen cell suspension. The spleen cell suspension was mixed with myeloma cells at a ratio of 5:1, a fusion promoter was added, positive hybridoma cells were screened, and cloned and amplified to obtain cloned and purified hybridoma cells. S2. The cloned and purified hybridoma cells were injected into the peritoneal cavity of an animal for amplification. The hybridoma cells were then extracted and the secreted monoclonal antibody against AQP3 protein was collected.

[0013] In a sixth aspect, the present invention also discloses a kit for detecting AQP3 content, the kit comprising an anti-AQP3 protein monoclonal antibody.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a monoclonal antibody against AQP3 protein. By optimizing the design of the antibody's variable region sequence, the monoclonal antibody against AQP3 protein can more accurately recognize AQP3 protein and reduce cross-reactivity with similar proteins such as AQP1 and AQP4. At the same time, the antibody also has high affinity and still shows high sensitivity when detecting low levels of AQP3 protein.

[0015] Furthermore, the anti-AQP3 protein monoclonal antibody of the present invention also exhibits batch-to-batch high efficiency and stability. Attached Figure Description

[0016] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a diagram showing the identification results of recombinant AQP3 protein.

[0018] Figure 2 This is a graph showing the identification results of the anti-AQP3 monoclonal antibody.

[0019] Figure 3 This is an image of the immunohistochemical staining results for AQP3 in lung cancer. In the image: A is the commercially available monoclonal antibody AQP3 (clone number: EPR28053-11); B is the anti-AQP3 protein monoclonal antibody.

[0020] Figure 4 This is an image showing the immunohistochemical staining results of AQP3 in breast cancer. In the image: A is the commercially available monoclonal antibody AQP3 (clone number: EPR28053-11); B is the anti-AQP3 protein monoclonal antibody.

[0021] Figure 5 This is an image showing the immunohistochemical staining results of AQP3 in esophageal cancer. In the image: A is the commercially available monoclonal antibody AQP3 (clone number: EPR28053-11); B is the anti-AQP3 protein monoclonal antibody.

[0022] Figure 6 This is a Western blot result of the anti-AQP3 monoclonal antibody. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0025] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0026] Example 1 I. A monoclonal antibody against AQP3 protein, composed of two heavy chains and two light chains, each with a variable region (VH, Variable Heavy Chain and VL, Variable Light Chain), the specific amino acid sequence of which is as follows: VH---EVQLEQSGAELMKPGASVKLSCKATGYTFTGYWIEWVKQRPGHGLEWIGEFLPGSGSTIYNEKFKGKATFTADTSSNTAYMQLSGLTTEDSAIYYCARKYGGYYDPYAMDYWGQGTSVTVSS (SEQ ID NO.1); VL---DIVLTQTPSSLTVASGEKVTMSCKSSQSLLNSGNQRNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISNVQAEDLAVYYCQNDYNYPFTFGTGTKLEIK (SEQ ID NO. 2).

[0027] Preparation of recombinant AQP3 protein: 1. The human AQP3 protein sequence with ID Q92482 from the Uniprot database (http: / / www.uniprot.org) was selected as the standard sequence. Based on analysis of immunoreactivity, amino acid hydrophilicity / hydrophobicity, secondary / tertiary structure, and species specificity, amino acids 120-135 of the AQP3 extracellular region were selected as the core antigenic epitope, with the corresponding amino acid sequence being GVPEVQKPGTELSVNV (SEQ ID NO.3).

[0028] 2. The target protein fragment was ligated into the plasmid vector pET-28a(+) (Novagen) to synthesize the AQP3 recombinant protein granule. The Histidine tag containing His was used as a purification tag for the fusion protein.

[0029] 3. Transform the recombinant plasmid into E. coli competent cells DH5α and culture overnight. Then, pick single clones from the plate for inoculation and amplification, extract plasmid DNA, and perform PCR identification.

[0030] 4. Perform sequencing analysis on clones that show positive results for the target gene by PCR, and select clones with completely correct sequences for recombinant protein expression and purification.

[0031] Recombinant protein expression and purification: 1. Transform the recombinant plasmid into E. coli competent cells DH5α, pick clones from the plate for inoculation and amplification, and maintain the bacteria.

[0032] 2. Take an appropriate amount of bacterial culture into 500 mL of LB liquid medium containing 80 μg / mL kanamycin, and culture it with shaking at 37℃ and 250 rpm for 12-16 hours. Collect the bacterial cells.

[0033] 3. Centrifuge the bacterial culture at 6000×g for 10 min, discard the supernatant culture medium, retain the bacterial cells, resuspend and wash with an appropriate amount of PBS buffer, add nickel column equilibration buffer to the bacterial cells for resuspending, and sonicate to disrupt. After sonication until the bacterial culture is clear, centrifuge and collect the precipitate and supernatant separately.

[0034] 4. The supernatant, precipitate, and induced bacterial cells were subjected to SDS-PAGE electrophoresis to analyze the location of the recombinant protein. The recombinant protein was purified using QIAGEN nickel column affinity chromatography.

[0035] 5. The purified recombinant protein was desalted by dialysis (the protein sample was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in pre-cooled PBS buffer at 4°C for 24 hours, changing the buffer three times during this period to thoroughly remove salts and small molecule impurities). After dialysis, the protein was concentrated using an ultrafiltration tube (molecular weight cutoff of 30 kDa) at 4°C and 4000g. Finally, the protein concentration was determined by a UV micro-spectrophotometer, and the recombinant AQP3 protein concentration was found to be 1.2 mg / mL. Protein purity was assessed by SDS-PAGE electrophoresis. Figure 1 Electrophoresis showed that the recombinant AQP3 protein had a single clear band at approximately 28 kDa.

[0036] II. The preparation of a monoclonal antibody against AQP3 protein mainly includes steps such as immunizing mice, cell fusion, hybridoma screening, and antibody purification, as detailed below: 1. Immunization of Mice: Healthy BALB / c mice aged 6-8 weeks were selected, and recombinant AQP3 protein was used as the immunogen. For the first immunization, 100 μg of protein was thoroughly emulsified with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion. This emulsion was administered via subcutaneous injection at multiple sites on the back, with 50 μg injected at each site, for a total of 4-6 injection sites. A booster immunization was performed 2 weeks later, using Freund's incomplete adjuvant at half the dose (50 μg), for a total of 3 booster immunizations. On the 3rd day after the last immunization, a small serum sample was obtained via orbital blood sampling. The titer of anti-AQP3 antibodies in the serum was detected using immunoprecipitation to ensure it met the requirements for subsequent experiments.

[0037] 2. Cell Fusion: After euthanizing immunized mice, the spleen was aseptically removed in a laminar flow hood and placed in pre-cooled RPMI-1640 medium. The spleen tissue was gently ground using the syringe plunger to pass through a 100-mesh cell sieve to obtain a single-cell suspension. After treatment with erythrocyte lysis buffer for 5 minutes, the spleen cells were collected by centrifugation, resuspended in complete medium, and counted. Simultaneously, SP2 / 0 myeloma cells in logarithmic growth phase were prepared, and the cell concentration was adjusted to 1×10^7 cells / mL. The spleen cells and myeloma cells were mixed at a 5:1 ratio, centrifuged to remove the supernatant, and 1 mL of 50% PEG-1500 solution preheated to 37°C was slowly added while gently stirring. After 90 seconds, the fusion reaction was terminated with preheated serum-free medium.

[0038] 3. Hybridoma screening: The fused cells were seeded into 96-well plates containing HAT selective medium, with 1 × 10⁶ cells per well. 5Cells were cultured at 37°C in a 5% CO2 incubator. Cell growth was observed starting on day 3, and half of the medium was replaced on day 7. After 10-14 days of culture, hybridoma cell colony formation was observed using an inverted microscope, and single-cell clones were selected for further culture. Positive clones that secrete anti-AQP3 antibodies were screened using immunofluorescence staining. Specifically, the cell culture supernatant was incubated with AQP3-expressing cells, and the binding was detected using fluorescently labeled secondary antibodies.

[0039] 4. Monoclonalization: Positive hybridoma cells obtained from the initial screening are subcloned using a limiting dilution method. Cells are diluted to a concentration of 0.5 cells / well and seeded into 96-well plates, then cultured for 10-14 days. After confirming that only one cell is aggregated in each well using an inverted microscope, the supernatant is collected, and the screening step is repeated. This process is repeated at least three times to ensure that the obtained hybridoma cell line has stable monoclonal activity and antibody secretion capacity.

[0040] 5. Antibody Production: Stable antibody-secreting hybridoma cells were cultured in T75 culture flasks and mass-produced using serum-free medium. Simultaneously, an ascites preparation system was established: BALB / c mice were pre-sensitized by intraperitoneal injection of 0.5 mL of norphytane, and 7 days later, they were inoculated with 5 × 10⁶ cells / mL of the ascites solution. 6 Hybridoma cells were collected, and ascites fluid was collected 10-14 days later. After centrifugation to remove cell debris, the ascites fluid was initially purified by ammonium sulfate precipitation, followed by fine purification using a Protein A affinity chromatography column to finally obtain high-purity monoclonal antibodies.

[0041] 6. Antibody Identification: Antibody purity was detected by SDS-PAGE electrophoresis. Under reducing conditions, bands of approximately 50 kDa heavy chain and 25 kDa light chain should appear. Figure 2 As shown in the figure. Immunoblot was used to verify antibody specificity, confirming that it specifically binds only to the AQP3 protein. Further immunohistochemistry was used to examine the staining characteristics of the antibody in different tumor tissue sections, and its sensitivity and specificity were evaluated by comparing it with known commercial antibodies. Strict quality control standards were established, including protein concentration determination and endotoxin detection, to ensure the quality stability of each batch of antibody product.

[0042] Example 2 I. Specificity verification of anti-AQP3 protein monoclonal antibody: 1. Using the indirect ELISA method, human AQP1 (MyBioSource, MBS212491), AQP3 (MyBioSource, MBS540662), and AQP4 (MyBioSource, MBS540995) (1 μg / mL, diluted with PBS) were coated onto 96-well plates and incubated overnight at 4°C.

[0043] 2. After blocking with 5% skim milk-PBS, add serially diluted anti-AQP3 protein monoclonal antibody (using concentration gradients of 0.1, 1, 10, 100, 1000 ng / mL) or commercially available antibody (clone number EPR28053-11) and incubate at room temperature for 1 hour.

[0044] 3. After washing, add HRP-labeled anti-human IgG secondary antibody (1:5000), TMB color development, and measure the OD450 value after terminating the reaction.

[0045] Experimental results: Affinity (EC) 50 ): EC2 binding of the antibody of this invention to human AQP3 50 The concentration was 15.4 ± 1.8 ng / mL (n=3), which was significantly better than the 52.3 ± 4.9 ng / mL of commercially available antibodies.

[0046] Cross-reactivity: The OD of the anti-AQP3 protein monoclonal antibody of the present invention against AQP1 / AQP4... 450 The values ​​were all <0.1 (consistent with background), and the cross-reactivity rate was <5%; while the OD values ​​of commercially available antibodies against AQP1 / AQP4 were <0.1 (consistent with background). 450 The values ​​ranged from 0.18 to 0.21, with a cross-reactivity rate of 10 to 12% (Table 1).

[0047] Table 1. Cross-reactivity analysis of anti-AQP3 monoclonal antibodies II. Experimental methods for antibody titer determination: During the hybridoma cell screening stage, cell supernatant was collected, and antibody titers were determined using the ELISA method described above. A positive threshold was defined as an OD450 value ≥ 2.1 times that of the negative control.

[0048] Experimental results: The final cloned hybridoma cell supernatant titer was 1:11800, and the antibody titer after purification of ascites fluid reached 1:45200.

[0049] In practical applications, the anti-AQP3 monoclonal antibody of this invention specifically marks cancer cells by binding to the AQP3 protein. Immunohistochemical experiments were performed using an immunohistochemical kit (CAT#RK05872, Ibotek). In this experiment, the antibody first binds to the AQP3 protein in the tissue section. Following incubation with a secondary antibody and DAB staining, the AQP3 protein in the cancer cells is stained and visualized. Figure 3-5 As shown, brown represents DAB staining, indicating AQP3 protein expression. In Western blot, the antibody binds to the protein separated by electrophoresis, and the target protein band is displayed through a chemical staining process, as shown in the image. Figure 6As shown.

[0050] In summary, this invention provides a highly specific and affinity anti-AQP3 monoclonal antibody through structural optimization and process improvement. Through detailed technical implementation and experimental verification, this invention demonstrates its broad application prospects in cancer detection and treatment, and can provide a more accurate and reliable tool for various detection methods.

[0051] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A monoclonal antibody against AQP3 protein, characterized in that, It comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:1; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:

2.

2. The application of the anti-AQP3 protein monoclonal antibody as described in claim 1 in the preparation of a drug for detecting cancer, characterized in that, The cancer in question is lung cancer.

3. The application of the anti-AQP3 protein monoclonal antibody as described in claim 1 in the preparation of a drug for detecting cancer, characterized in that, The cancer in question is breast cancer.

4. The application of the anti-AQP3 protein monoclonal antibody as described in claim 1 in the preparation of a drug for detecting cancer, characterized in that, The cancer in question is esophageal cancer.

5. The application as described in any one of claims 2-4, characterized in that, The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

6. The application as described in any one of claims 2-4, characterized in that, The pharmaceutical composition is an injectable dosage form.

7. A method for preparing a monoclonal antibody against AQP3 protein, characterized in that, The preparation method includes the following steps: S1. Animals were immunized with recombinant AQP3 protein to prepare a spleen cell suspension. The spleen cell suspension was mixed with myeloma cells at a ratio of 5:1, a fusion promoter was added, positive hybridoma cells were screened, and cloned and amplified to obtain cloned and purified hybridoma cells. S2. The cloned and purified hybridoma cells were injected into the peritoneal cavity of an animal for amplification. The hybridoma cells were then extracted and the secreted monoclonal antibody against AQP3 protein was collected.

8. A reagent kit for detecting AQP3 content, characterized in that, The kit includes a monoclonal antibody against the AQP3 protein.

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