Hybridoma cell strain S1-C56-ab of monoclonal antibody secreting anti-p53R175H / HLA new antigen and application of hybridoma cell strain S1-C56-ab

Abstract

The invention provides a hybridoma cell strain S1-C56-ab for secreting an anti-p53R175H / HLA new antigen monoclonal antibody, the hybridoma cell strain S1-C56-ab can secrete an anti-p53R175H / HLA 02: 01 monoclonal antibody, and the anti-p53R175H / HLA 02: 01 monoclonal antibody has a variable region amino acid sequence and a CDR sequence of a light chain and a heavy chain. By adopting the technical scheme provided by the invention, the hybridoma cell strain S1-C56-ab capable of stably and continuously secreting the monoclonal antibody resisting p53R175H / HLA 02: 01 is constructed; according to the present invention, the p53R175H / HLA 02: 01 monoclonal antibody provided by the hybridoma cell strain S1-C56-ab is combined with the cancer cell specific protein generated by target point p53 gene mutation, and the p53R175H / HLA 02: 01 presented on the cancer cell surface has characteristics of absolute cancer cell specificity, no normal cell killing, and no side effect;

Description

Technical Field

[0001] This application relates to the field of biological preparation technology, in particular to anti-p53 R175H Hybridoma cell line S1-C56-ab producing monoclonal antibodies against HLA neoantigens and its application. Background Art

[0002] In the field of cancer treatment, antibody drugs have become an important therapeutic approach due to their high specificity and targeted properties. Currently, commonly used antibody drugs in clinical practice primarily target key targets such as human epidermal growth factor receptor-2 (HER-2), fibroblast growth factor receptor (FGFR), and anaplastic lymphoma kinase (ALK). Trastuzumab, for example, specifically binds to the HER-2 receptor, blocking the binding of EGFR to HER-2, thereby inhibiting cancer cell proliferation and survival. These targets are typically proteins expressed at low levels in normal cells but highly in cancer cells, also known as tumor-associated antigens (TAAs). While these commonly used antibody drugs in clinical practice exhibit a certain degree of cancer cell specificity, minimizing damage to surrounding normal tissues, their specificity remains limited, making true precision therapy difficult to achieve.

[0003] In recent years, the concept of neoantigens has provided new insights into precision cancer therapy. Neoantigens are immunogenic, abnormal proteins produced by sense mutations in cancer cell genes. Due to their mutational origin, neoantigens are highly cancer cell-specific and are also known as tumor-specific antigens (TSAs). Unlike traditional tumor-associated antigens, neoantigens are expressed exclusively in cancer cells and are completely absent in normal cells. Therefore, targeting neoantigens holds the promise of achieving true precision cancer therapy while minimizing damage to normal tissue. Approximately 500 genes in the cancer cell transcriptome are involved in the transformation of normal cells into cancer cells. Of these, only about 10% encode cell membrane proteins, while the majority encode proteins within the cell. This means that mutations within the cell's interior can generate a vast array of potential neoantigens, significantly expanding the range of available targets for cancer therapy. However, traditional antibody drugs have limited cell membrane penetration, making them difficult to directly target regulatory factors within the cell. However, once proteins within the cell mutate and produce abnormal proteins (neoantigens), these neoantigens can be presented on the cell surface in the form of antigen peptide / HLA complexes through the antigen presentation process, making them ideal therapeutic targets. The p53 gene is a key tumor suppressor gene in the human body, and its mutations are closely associated with the development and progression of over 50% of malignant tumors. The abnormal protein produced by p53 gene mutations is highly cancer cell-specific, making it an ideal target for cancer therapy. The development of antibody drugs targeting neoantigens not only further enhances the antibody's specificity for cancer cells but also significantly expands the range of targets available for cancer therapy, providing more personalized treatment options for cancer patients.

[0004] Commonly used cancer treatment antibody drugs, such as trastuzumab and rituximab, primarily target tumor-associated antigens on the cell membrane surface. While these drugs have achieved some clinical efficacy, they still suffer from issues such as insufficient target specificity and a limited therapeutic range. Therefore, the development of antibody drugs targeting neoantigens has become a key research direction in cancer treatment, promising breakthroughs in precision cancer treatment. Summary of the Invention

[0005] In view of the technical problems existing in the prior art, the purpose of this application is to provide a secretory anti-p53 R175H / HLA neoantigen monoclonal antibody hybridoma cell line S1-C56-ab, which can specifically target p53 on the surface of specific cancer cells R175H / HLA, which can specifically bind to p53 presented on the surface of cancer cells R175H / HLA-A 02:01, has strong specificity and absolute cancer cell specificity, will not kill normal cells, has no side effects, and can be used in a variety of treatment methods.

[0006] The purpose of this application can be achieved through the following technical solutions:

[0007] A hybridoma cell line S1-C56-ab secreting a monoclonal antibody against p53R175H / HLA neoantigen, wherein the hybridoma cell line S1-C56-ab is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C202578; the hybridoma cell line S1-C56-ab can secrete a monoclonal antibody against p53R175H / HLA neoantigen. R175H / HLA-A 02:01 monoclonal antibody.

[0008] Preferably, the anti-p53 R175H / HLA-A 02:01 monoclonal antibody has a variable region amino acid sequence and possesses the CDR sequences of the light and heavy chains.

[0009] Preferably, the screening method of the hybridoma cell line S1-C56-ab comprises: using human p53 R175H Female BALB / c mice were immunized with HLA-A 02:01 recombinant protein as the immunogen, and spleen cells from the immunized mice were fused with SP2 / 0 myeloma cells. R175H The monoclonal antibody-positive fusion cell of the HLA-A 02:01 complex is the hybridoma cell.

[0010] Using the p53 provided by this application R175H / HLA-A 02:01 antigen or other forms such as monomers, dimers, tetramers, etc. can be used as immunogens to immunize animals. Other experimental animals besides BALB / c mice can also be used for antigen immunization.

[0011] An anti-p53 R175H / HLA-A 02:01 monoclonal antibody, secreted by the hybridoma cell line S1-C56-ab.

[0012] Preferably, the amino acid sequences of the heavy chain variable regions of monoclonal antibody S1-C56-ab are shown as SEQ ID NO: 1 to SEQ ID NO: 3, respectively; and the amino acid sequences of the light chain variable regions are shown as SEQ ID NO: 4 to SEQ ID NO: 6, respectively.

[0013] This application is not limited to p53 antibodies of the same sequence obtained by other antibody preparation methods. R175H / HLA-A02:01 monoclonal antibody.

[0014] Preferably, the monoclonal antibody can specifically bind to p53 expressed on the surface of cancer cells. R175H / HLA-A 02:01.

[0015] Preferably, the anti-p53 antibody is prepared by the mouse ascites method using the hybridoma cell line S1-C56-ab. R175H / HLA-A 02:01 monoclonal antibody.

[0016] Preferably, the target that the monoclonal antibody can bind to is the cancer cell-specific p53 produced by p53 gene mutation. R175H / HLA-A 02:01 recombinant protein.

[0017] Preferably, the drug is used to treat p53 R175H / HLA-A 02:01-induced tumors can trigger ADCC effect to kill tumor cells in vitro.

[0018] A cancer treatment drug comprising the p53 R175H / HLA-A 02:01 monoclonal antibody.

[0019] The technical effects of the technical solution of this application are:

[0020] 1. By adopting the technical solution of this application, we have successfully constructed a drug that can stably and continuously secrete anti-p53 R175H / HLA-A02:01 monoclonal antibody hybridoma cell line S1-C56-ab; anti-p53 produced by hybridoma cell line S1-C56-ab secretion R175H The target of the monoclonal antibody against HLA-A 02:01 is the cancer cell-specific protein produced by p53 gene mutation. R175H / HLA-A 02:01, has absolute cancer cell specificity, will not kill normal cells, and has no side effects.

[0021] 2. p53 provided by the technical solution of this application R175H / HLA-A 02:01 monoclonal antibody can trigger ADCC effect to kill tumor cells in vitro; it can be prepared by methods such as the ascites method, with high yield, good stability, and low production cost. After purification, the obtained antibody has high titer and strong specificity, and also has specific antibody variable region sequences and CDR1 / 2 / 3 sequences of the antibody light and heavy chains.

[0022] 3. p53 provided by the technical solution of this application R175H / HLA-A 02:01 monoclonal antibody can be combined with a variety of immunotherapies, has a wide range of application scenarios, and has strong application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart for the preparation of the hybridoma cell line S1-C56-ab of Example 1 of the present application.

[0024] Figure 2 These are the 216 positive hybridoma clones screened in Example 1 of this application.

[0025] Figure 3 These are the 7 antigenic peptides generated from the antigens screened for antigenic peptide responsiveness in Example 1 of the present application.

[0026] Figure 4 This is a confocal fluorescence imaging diagram of the specificity of the S1-C56-ab antibody identified by KMS26 and AU565 in Example 1 of the present application.

[0027] Figure 5 In Example 1 of the present application, the affinity of the S1-C56-ab antibody was analyzed by diluting the recombinant protein to different concentrations. DETAILED DESCRIPTION

[0028] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0030] Explanation of relevant terms in this application:

[0031] p53: A tumor suppressor gene in the human body. p53 inactivation is closely related to tumor formation.

[0032] p53 R175H : A mutation of the p53 gene found in cancer cells, in which the 175th amino acid of its expression product mutates from R (arginine) to H (histidine).

[0033] Neoantigens: Abnormal proteins with immunogenicity produced by cancer cells due to gene mutations, which are specific to cancer cells.

[0034] HLA: human major histocompatibility complex.

[0035] p / HLA: The human major histocompatibility complex is related to antigen presentation and forms a complex with the antigen peptide on the cell membrane. T cells recognize this complex through the T cell receptor (TCR) and kill tumor cells.

[0036] TCRm antibody: TCR mimic antibody, which simulates the HLA restriction of TCR when recognizing antigen peptides and can recognize p / HLA complex.

[0037] The hybridoma cell line S1-C56-ab prepared by the present invention is deposited in the China Center for Type Culture Collection and classified as Hybridoma cell line S1-C56-ab. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: C202578 and a deposit date of March 26, 2025. Example 1

[0038] This embodiment provides a secretion of anti-p53 R175H / HLA neoantigen monoclonal antibody preparation method of hybridoma cell line S1-C56-ab, refer to Figure 1 , including the following steps:

[0039] 1. Preparation of Hybridoma Cell Lines

[0040] 1.1 Immunization of mice

[0041] See Figure 1 , using purified p53 R175H 7-8 week old female BALB / c mice were immunized with HLA-A 02:01 recombinant protein (novoprotein Cat. No.: C16S) as the antigen.

[0042] Specifically: p53 R175H / HLA was dissolved in PBS to a concentration of 1 μg / μL.

[0043] Use a pipette to draw 100 μL of Freund's complete adjuvant into a 1.5 mL centrifuge tube; vortex the centrifuge tube and dropwise add 100 μL of p53 R175HAfter emulsification, the antigen was injected intraperitoneally into mice. Mice were subsequently immunized with the antigen every two weeks. The second and third immunizations were performed using the same dose of antigen emulsified in Freund's incomplete adjuvant. Blood was collected from the orbital vein three days after each immunization to measure serum antibody titers. Three days after the third immunization, 100 μL of antigen (without adjuvant) was injected intravenously into the mice. Three days later, the mice were sacrificed, and spleen cells were harvested for cell fusion.

[0044] 1.2 Hybridoma Preparation

[0045] See Figure 1 Mouse spleens were ground and lysed to prepare a single cell suspension for counting. Pre-thawed and stable SP2 / 0 myeloma cells were harvested, centrifuged, resuspended, and counted. Splenocytes and SP2 / 0 myeloma cells were adjusted to an appropriate density and mixed at a ratio of 5:1 (splenocyte:myeloma cells). The cells were centrifuged at 1200 rpm for 5 minutes to collect the cells, and the supernatant was discarded. The bottom of the centrifuge tube was gently tapped to loosen cell clumps and form a dense cell homogenate. The homogenate was incubated in a 37°C water bath. 1 mL of preheated PEG1500 was slowly added along the side of the centrifuge tube, rotating the tube dropwise and gently shaking to ensure uniform distribution. The mixture was allowed to stand for 1 minute. Preheated blank RPMI medium was slowly added dropwise, rotating the tube gently to ensure adequate contact between the cells, until the total volume reached 20 mL. Finally, the cells were centrifuged at 1300 rpm for 5 minutes to collect the cells. Discard the cell supernatant, prepare RPMI medium containing HAT, and resuspend the cells in a certain volume of medium to make the cell suspension density approximately 1*10 6 Cells were inoculated into culture dishes or six-well plates according to the total volume and cultured in a 37°C, 5% CO2 incubator.

[0046] 1.3 Hybridoma Cell Monocloning

[0047] Using fluorescently labeled p53 R175H Hybridoma cells were labeled with the HLA-A 02:01 recombinant protein tetramer and sorted by flow cytometry. Single positive cells were isolated and placed in a 96-well plate. The 96-well plate was incubated at 37°C in a 5% CO2 incubator to allow single cells to form colonies.

[0048] 2. Screening of hybridoma cell monoclones

[0049] Furthermore, the hybridoma cell monoclones obtained after the screening in the above steps are screened, and the screening process includes:

[0050] 2.1 Preliminary screening of positive hybridoma monoclones by ELISA

[0051] p53 was added using PBS R175H The recombinant protein of HLA-A 02:01 and the control antigen (control antigen peptide / HLA-A 02:01) were adjusted to a concentration of 1 μg / mL and added to the ELISA plate for coating. 100 μL was added to each well and the plate was placed in a 4°C refrigerator for overnight coating.

[0052] Prepare sufficient ELISA wash buffer (10 mM PBS, pH 7.2, 0.05% Tween-80). Shake dry the overnight coated plate, invert it on absorbent paper to prevent backflow, and wash it three times with ELISA wash buffer for 5 minutes each. After the final wash, add 100 μL of prepared blocking buffer (10 mM PBS, pH 7.2, 3% BSA) to each well and incubate the plate at 37°C. After 2 hours, remove the plate and wash it three times with ELISA wash buffer for 5 minutes each. After washing, allow to stand at room temperature until ready to use.

[0053] Remove 100 μL of the monoclonal hybridoma supernatant from step 1.3 and add it to the blocked ELISA plate. Incubate the plate at 37°C for 2 hours. Remove the plate and wash it three times with ELISA wash buffer for 5 minutes each. Add 100 μL of the prepared goat anti-mouse HRP antibody (1:1000 dilution) to the ELISA plate and incubate it at 37°C for 2 hours. After incubation, wash it three times with ELISA wash buffer for 5 minutes each. Add 100 μL of TMB to each well and incubate it at 37°C for 5-15 minutes (depending on the degree of color development). Finally, stop the color development by adding 50 μL of 2 mol / L sulfuric acid to each well. Measure the OD value at 450 nm using a microplate reader.

[0054] Compare the OD value of each monoclonal supernatant with that of the control group and R175H The OD value of the HLA-A 02:01 group was twice that of the control group and was considered as a positive monoclonal clone and selected for the next screening.

[0055] 2.2 Screening of positive hybridoma monoclones by T2 cell binding assay

[0056] T2 cells are a special cell line with TAP gene defects. Since TAP protein plays an essential role in the antigen presentation process, T2 cells cannot present endogenous antigen peptides. Only empty HLA molecules exist on the surface of T2 cells. The added β2m and antigen peptide can form a complex with the empty HLA molecules to stabilize the antigen peptide / HLA structure. Therefore, the T2 cell binding experiment can effectively determine whether the antibodies secreted by hybridoma cells can specifically bind to p53 R175H / HLA-A 02:01.

[0057] Stable T2 cells were collected by centrifugation, resuspended in 1 mL of RPMI complete medium, and counted. An appropriate volume of medium was then added to adjust the T2 cell density to 1*10 6 Use PBS to dissolve β2m and dilute to 1 mg / mL; use PBS to dissolve p53 R175H Dissolve the antigen peptide and other control antigen peptides and adjust the concentration to 1 mg / mL for use. 6 T2 cells were seeded into 48-well plates with 250 μL per well, and 2.5 μL of β2m, p53 R175H Antigen peptide or control antigen peptide was cultured overnight in a 37°C 5% CO2 incubator. The next day, cells in the 48-well plate were collected into a centrifuge tube, centrifuged, and the supernatant was discarded.

[0058] Antibodies secreted by screened positive clones were purified and quantified. 1 μg of antibody was added to each well of a 48-well plate and incubated at room temperature for 1 hour. Cells were harvested by centrifugation, the supernatant discarded, and the cells were washed with 500 μL of FACS buffer (PBS containing 1% FBS). The cells were centrifuged again at 1200 rpm for 3 minutes. The supernatant was discarded, and 100 μL of the prepared anti-mouse fluorescent secondary antibody was added to each well of the 48-well plate. The cells were incubated at room temperature for 1 hour and centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and 300 μL of FACS buffer was added to the cells and transferred to flow cytometry tubes for analysis.

[0059] like Figure 2 As shown, 216 positive hybridoma clones were screened in this experiment.

[0060] 2.3 Screening of response to control antigen peptides

[0061] Using the experimental method provided in step 2.2, the antibodies secreted by 96 hybridoma clones were further screened. R175H Based on the characteristics of antigenic peptides, seven different antigenic peptides produced by the antigen presentation pathway were selected as control peptides. The purpose was to exclude antibodies that could bind to other control antigenic peptide / HLA complexes from the 96 antibodies. The selected peptides included seven antigenic peptides produced by antigens such as Survivin, Flu, and WT-1. The experimental results are as follows: Figure 3 Finally, we selected S1-C56-ab clone for the next experiment.

[0062] 3. Monoclonal Antibody Preparation Process

[0063] 3.1 Ascites method for large-scale antibody preparation

[0064] Eight-week-old BALB / c mice were used as model animals. They were first immunized with Freund's incomplete adjuvant via intraperitoneal injection, followed by injection of 0.5 mL of liquid paraffin per mouse to induce ascites.

[0065] On the 7th day after immunization, the hybridoma cells were collected by centrifugation and washed once with serum-free medium. The hybridoma cells were resuspended in serum-free medium and the cell density was adjusted to 1*10 7 Each mouse was then intraperitoneally injected with 100 μL of the cell suspension. Three days after injection, when the mouse abdomen was noticeably enlarged, ascites fluid was collected multiple times. The ascites was centrifuged at 4000 rpm for 20 minutes. After removing the precipitate, the supernatant was collected and stored at -20°C until further use.

[0066] 3.2 Protein A purified antibodies

[0067] The collected ascites was filtered once through a 0.22 μm filter. The filtered ascites was diluted fivefold with a prepared diluent (10 mM PBS, 0.1 M sodium chloride, adjusted to pH 7.5). Protein A-coupled agarose microspheres were washed three times with PBS and centrifuged at 3000 rpm for later use. Each mL of ascites was incubated with the protein A-coupled agarose microspheres for 2 hours. After the incubation period, the mixed suspension was loaded onto an adsorption column. The column was washed with approximately three column volumes of the diluent. After washing, the prepared eluent (0.1 M glycine-HCl buffer, adjusted to pH 4) was added to the column for elution. Immediately after collecting the eluate, a volume of 2 M Tris-HCl buffer (adjusted to pH 7.5) was added, along with 0.01% NaN3 to prevent contamination. This yielded the S1-C56-ab antibody. The obtained antibodies were quantified using BCA and their functions were verified using SDS-PAGE, ELISA and other experimental techniques.

[0068] 4. Antibody Characterization

[0069] 4.1 S1-C56-ab antibody specificity

[0070] The S1-C56-ab hybridoma cell clone was prepared in large quantities using the method in 3.1 and purified and quantified using the method in 3.2. The specificity of the S1-C56-ab antibody was determined using two cancer cell lines: KMS26 and AU565. KMS26 is a human lymphoblastoid tumor with HLA typing of HLA-A 02:01 and carries p53. R175HThis mutation is the target cell for identifying the specificity of S1-C56-ab antibody. AU565 is a human breast cancer cell line that carries p53 R175H This mutation does not have HLA typing of HLA-A 02:01, so AU565 cells were used as the control group. The specific experimental steps are as follows: Stable KMS26 and AU565 cells were centrifuged and collected, and appropriate amount of culture medium was added to adjust the cell density to 1*10 4 Cells were fixed at 100 μg / mL in a 20 mm glass-bottomed dish. 2 mL of the cell suspension was added to a 20 mm glass-bottomed dish and incubated overnight to allow the cells to settle and adhere. The next day, the supernatant was aspirated and the cells were washed with PBS. 1 mL of 4% paraformaldehyde was added to the dish and fixed for 10 min. The cells were washed three times with PBS and then 200 μL of 10 μg / mL S1-C56-ab antibody was added and incubated at room temperature for 1 h. The cells were washed three times with PBS and then incubated with an anti-mouse fluorescent-labeled secondary antibody at room temperature for 1 h. The cells were washed three times with PBS and then mounted with Hoechst-containing anti-fluorescence quenching mounting medium and coverslips. Images were obtained using a confocal fluorescence microscope.

[0071] like Figure 4 As shown in the figure, confocal fluorescence imaging results show that there is green fluorescence on the KMS26 cell membrane, but no green fluorescence on the AU565-VC cell membrane. The experimental results show that S1-C56-ab can specifically bind to its cellular target.

[0072] 4.2 S1-C56-ab antibody affinity

[0073] The affinity of the S1-C56-ab antibody was determined using a Biacore T200 (Cytiva). The specific steps were as follows: S1-C56-ab was used as the ligand and diluted to 20 μg / mL in 10 mM NaAc (pH 5.0). HBS-EP buffer was used as the running buffer for pre-enrichment, and HBS-EP buffer was flowed through the chip at a flow rate of 10 μL / min. The ligand was coupled to the chip using an amino coupling kit (Cytiva, BR100050). The specific steps were as follows: HBS-EP buffer was used as the running buffer for the instrument; NHS and EDC were dissolved and diluted to 11.5 mg / mL and 75 mg / mL, respectively, and a 1:1 mixture of NHS and EDC was used to activate the Fc2 channel for 420 s. S1-C56-ab was diluted to a concentration of 5 μg / mL in 10 mM Acetate solution and injected into the activated Fc2 channel. The flow rate was adjusted to 10 μL / min, and the target coupling amount was set to 250 RU. After coupling, the channel was blocked with 1 M ethanolamine for 420 s.

[0074] p53 R175H HLA-A 02:01 recombinant protein was used as the analyte; HBS-EP buffer was used as the machine's running buffer, and the analytes were diluted to concentrations of 50 μg / mL, 25.0 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, and 1.56 μg / mL. Binding analysis was performed by injecting the six analytes into the flow channel at a flow rate of 30 μL / min; the association time was set to 180 s, and the dissociation time was set to 300 s. Data were analyzed using the Biacore T200 evaluation software, selecting a 1:1 binding model. See the attached data. Figure 5 According to the experimental results, the equilibrium dissociation constant (KD) was calculated to be 6.260×10 -8 .

[0075] 4.3 Confirmation of the variable region sequence of the S1-C56 antibody

[0076] Hybridoma cells secreting the aforementioned antibodies were expanded and cultured in 10 cm dishes. The culture medium was changed until the hybridoma cells reached the logarithmic growth phase. Total RNA was extracted using the Trizol method. The procedure was as follows: 1 mL of Trizol reagent was added to the 10 cm dish containing the hybridoma cells. The mixture was allowed to stand for 2 minutes and repeatedly pipetted until viscous. The cell lysate was transferred to a 1.5 mL centrifuge tube and allowed to stand at room temperature for 5 minutes. 200 μL of chloroform was added to the 1.5 mL centrifuge tube and the mixture was mixed by inverting the tube 15 times. The tube was then centrifuged at 12,000 g for 15 minutes at 4°C. After centrifugation, the tube was removed. The liquid in the centrifuge tube separated into three layers: the first clear liquid layer was the RNA lysis solution, the middle white film layer was protein, and the bottom layer was organic matter. 500 μL of the first clear layer was transferred to a new centrifuge tube. 500 μL of isopropanol was added and mixed thoroughly. The tube was allowed to stand for 3 minutes. After the stand, the tube was centrifuged at 7,500 g for 10 minutes at 4°C. Remove the centrifuge tube. A small white precipitate will be visible at the bottom of the tube, representing the RNA. Discard the supernatant and invert the tube onto absorbent paper. Air-dry for 3 minutes. Then, add 1 mL of 75% ethanol to rinse the RNA pellet. Briefly vortex and centrifuge the tube at 7500 g in a refrigerated centrifuge at 4°C for 5 minutes. Discard the supernatant and invert the tube onto absorbent paper. After the ethanol evaporates, dissolve the RNA in 55°C DEPC-free water and measure the RNA concentration using an instrument. Design primers for the antibody variable regions, clone the heavy and light chains of the antibody by PCR, and send for sequencing to obtain the CDR sequences.

[0077] The p53 R175HThe heavy chain VH CDR1, VH CDR2, and VH CDR3 sequences of the monoclonal antibody S1-C56-ab targeting HLA-A 02:01 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain VK CDR1, VK CDR2, and VK CDR3 sequences are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Among them, the sequence of SEQ ID NO:1 is GFSLSTSGMG; the sequence of SEQ ID NO:2 is IWWNDVK; the sequence of SEQ ID NO:3 is ARMGGPWFAY; the sequence of SEQ ID NO:4 is KSLLHSNGITY; the sequence of SEQ ID NO:5 is QMS; and the sequence of SEQ ID NO:6 is AQNLELPYT.

[0078] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A secretory anti-p53 R175H / HLA neoantigen monoclonal antibody hybridoma cell line S1-C56-ab, characterized in that The hybridoma cell line S1-C56-ab is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C202578; the hybridoma cell line S1-C56-ab can secrete anti-p53 R175H / HLA-A 02:01 monoclonal antibody.

2. The secretory anti-p53 according to claim 1 R175H / HLA neoantigen monoclonal antibody hybridoma cell line S1-C56-ab, characterized in that The anti-p53 R175H / HLA-A 02:01 monoclonal antibody has a variable region amino acid sequence and possesses the CDR sequences of the light and heavy chains.

3. The secretory anti-p53 according to claim 1 R175H / HLA neoantigen monoclonal antibody hybridoma cell line S1-C56-ab, characterized in that The screening method of the hybridoma cell line S1-C56-ab comprises: using human p53 R175H Female BALB / c mice were immunized with HLA-A02:01 recombinant protein as the immunogen, and spleen cells from the immunized mice were fused with SP2 / 0 myeloma cells. R175H The monoclonal antibody-positive fusion cell of the HLA-A 02:01 complex is the hybridoma cell. 4.An anti-p53 R175H / HLA-A 02:01 monoclonal antibody, characterized in that The protein is secreted by the hybridoma cell line S1-C56-ab according to any one of claims 1 to 3.

5. The anti-p53 according to claim 4 R175H / HLA-A 02:01 monoclonal antibody, characterized in that The amino acid sequences of the heavy chain variable regions of monoclonal antibody S1-C56-ab are shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively; and the amino acid sequences of the light chain variable regions are shown in SEQ ID NO: 4 to SEQ ID NO: 6, respectively.

6. The anti-p53 according to claim 4 R175H / HLA-A 02:01 monoclonal antibody, characterized in that The monoclonal antibody can specifically bind to p53 presented on the surface of cancer cells R175H / HLA-A 02:

01.

7. The anti-p53 according to claim 4 R175H / HLA-A 02:01 monoclonal antibody, characterized in that The anti-p53 was prepared by the mouse ascites method using the hybridoma cell line S1-C56-ab. R175H / HLA-A 02:01 monoclonal antibody.

8. Use of the monoclonal antibody according to any one of claims 5 to 7 in cancer treatment, characterized in that: The target that the monoclonal antibody can bind to is the cancer cell-specific p53 produced by p53 gene mutation. R175H / HLA-A 02:01 recombinant protein.

9. Use of the monoclonal antibody according to any one of claims 5 to 7 in the preparation of anticancer drugs, characterized in that: The drug is used to treat p53 R175H / HLA-A 02:01-induced tumors can trigger ADCC effect to kill tumor cells in vitro.

10. A cancer treatment drug, characterized in that: Comprising the anti-p53 according to any one of claims 5 to 7 R175H / HLA-A 02:01 monoclonal antibody.

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