Preparation method of anti-CD98 antibody as well as related antigen and application of anti-CD98 antibody

Abstract

The invention discloses a preparation method of an anti-CD98 antibody as well as a related antigen and application thereof, and relates to the field of antibodies. A new mRNA is obtained through screening, the mRNA is used as an immunogen immune animal, a high-titer anti-CD98 antibody can be obtained, and the mRNA can be applied to construction of multispecific antibodies and preparation of products for inhibiting tumor cell growth or preventing and treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a method for preparing an anti-CD98 antibody and related antigens and applications thereof. Background Art

[0002] CD98 (also known as CD98 heavy chain, CD98hc, 42F heavy chain, SLC3A2) is a type II transmembrane glycoprotein composed of 529 amino acid residues. The protein contains an N-terminal intracellular cytoplasmic domain of 75 amino acids, a single transmembrane domain, and a C-terminal extracellular domain of 426 amino acids. CD98 is covalently linked to one of several light chains (SLC7A5, 6, 7, 8, 10 or 11) via a disulfide bond, which are L-type amino acid transporters. This interaction is required for the cell surface expression and amino acid transport function of the light chain. CD98 also binds to the integrin β subunit, thereby regulating integrin signaling that controls cell proliferation, survival, migration, and epithelial cell adhesion / polarity.

[0003] CD98 was originally identified as a cell surface antigen associated with lymphocyte activation. Since then, CD98 has been identified in all cell types except platelets, and the expression level of CD98 is highest in the gastrointestinal (GI) tract and renal tubules. Upregulation of CD98 has been observed in enteritis. Recently, intestinal CD98 expression has been shown to play an important role in controlling the homeostasis and innate immune response in the intestine. Therefore, regulation of CD98 expression in intestinal epithelial cells has been shown to be a promising therapeutic strategy for the treatment and prevention of inflammatory bowel diseases, such as inflammatory bowel disease (IBD) and colitis-related cancers. Regardless of tissue origin, CD98 is also overexpressed on the cell surface of almost all tumor cells.

[0004] Increased expression of one of the CD98-binding light chains, L-type amino acid transporter 1 (LAT 1; also known as SLC7A5), has also been observed in a variety of human cancer cell types, including breast cancer, colon cancer, oral cancer, ovarian cancer, esophageal cancer, gliomas, and leukemias. Increased amino acid supply may be required to support the high growth rate of cancer cells, which is achieved by providing amino acid building blocks for protein synthesis and stimulating growth through the mammalian target of rapamycin (mTOR). The expression of LAT1 and CD98 is significantly higher in metastatic sites of human cancer compared to primary sites, suggesting that overexpression of LAT1 / CD98 is necessary for the development and metastasis of human cancer. Specifically, LAT1 / CD98 overexpression appears to be necessary for tumor metastasis in patients with colon cancer.

[0005] Currently, anti-CD98 monoclonal antibodies have been found to have an inhibitory effect on tumor growth. Analysis of new primary acute myeloid leukemia (AML) tumor samples from patients using the surface marker antigen spectrum of the cell surface proteome identified the transmembrane protein CD98 as being present at high density on the surface of AML tumor cells. Therefore, CD98 is a target for treating AML by using a binding agent, such as an antibody that specifically binds to CD98. Binding agents specific for CD98, such as anti-CD98 antibodies, have also been shown in multiple in vivo xenograft models to be useful not only in the treatment of AML, but also in the treatment of a variety of cancers, such as sarcomas, lymphomas, non-small cell lung cancer (NSCLC) and colorectal cancer.

[0006] Currently, there is a lack of antigens for preparing high-titer anti-CD98 monoclonal antibodies.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a method for preparing an anti-CD98 antibody and its related antigens and applications.

[0009] The present invention is achieved in that:

[0010] In a first aspect, an embodiment of the present invention provides an antigen, which includes: an mRNA whose nucleotide sequence is at least 80% identical to the sequence shown in SEQ ID NO:1.

[0011] In a second aspect, an embodiment of the present invention provides a composition comprising the antigen described in the preceding embodiment.

[0012] In a third aspect, an embodiment of the present invention provides a vaccine comprising the antigen described in the preceding embodiment.

[0013] In a fourth aspect, an embodiment of the present invention provides a method for preparing an anti-CD98 antibody, comprising: immunizing an animal with the antigen described in the preceding embodiment, the composition described in the preceding embodiment, or the vaccine described in the preceding embodiment.

[0014] In a fifth aspect, the embodiments of the present invention provide the use of the antigen described in the preceding embodiments, or the composition described in the preceding embodiments, or the vaccine described in the preceding embodiments in any of the following items: (1) preparing a product for inhibiting the growth of tumor cells; (2) a product for preventing, treating or assisting in the treatment of tumors; (3) a product for detecting CD98; (4) a multispecific antibody.

[0015] The present invention has the following beneficial effects:

[0016] The present invention screened and obtained a new mRNA, which was used as an immunogen to immunize animals, and a high-titer anti-CD98 antibody could be obtained, which could be applied to construct multi-specific antibodies and prepare products that inhibit tumor cell growth or prevent and treat tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 Schematic diagram of the preparation process of monoclonal antibodies;

[0019] Figure 2 Schematic diagram of Human CD98 plasmid construction;

[0020] Figure 3 Schematic diagram of Cyno CD98 plasmid construction;

[0021] Figure 4 Schematic diagram of using mRNA as an immunogen to collect serum from mice after the second immunization and to detect serum titer using the FACS method;

[0022] Figure 5 Schematic diagram of using protein as immunogen, collecting serum from mice after second immunization and detecting serum titer by FACS method;

[0023] Figure 6 Schematic diagram of the comparison of serum titers after the second immunization of mice using mRNA and protein as immunogens;

[0024] Figure 7 Schematic diagram of anti-CD98 subclone binding ELISA (Cell-Based ELISA); HuCD98 FS1 st is the subclone cell supernatant, PC is the mouse serum before fusion, and NC is the blank culture medium;

[0025] Figure 8 A in the figure is a schematic diagram of anti-CD98 cell-based combination with FACS (CHO-K1-HuCD98-Cell), and B is a schematic diagram of anti-CD98 cell-based combination with FACS (CHO-K1-CynoCD98-Cell); wherein, HuCD98 FS single Clone Sup is the supernatant of cell-based combination, BMK is the mouse serum before fusion, CIC is the IgG isotype control, APC is the APC single-stained tube, and NC is the empty cell;

[0026] Fig. 9 7 anti-CD98 chimeric antibodies EC 50 Schematic diagram of Cell-Based ELISA (CHO-K1-HuCD98); BMK-hlgG is a positive control, Isotype-hlgG is an isotype control, and Ab1 to 5 are CD98 chimeric antibodies 1, 2, 3, 4, and 5, respectively;

[0027] Fig.10 7 anti-CD98 chimeric antibodies EC 50 Schematic diagram of FACS (CHO-K1-HuCD98-Cell); wherein BMK-hlgG is a positive control, Isotype-hlgG is an isotype control, and Ab1 to 5 are CD98 chimeric antibodies 1, 2, 3, 4, and 5, respectively;

[0028] Fig.11 Schematic diagram of the amino acid transport function assay of 7 anti-CD98 chimeric antibodies;

[0029] Fig.12 Schematic diagram of receptor detection of HCMEC cells at 0h and 24h using 7 anti-CD98 chimeric antibodies. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0031] In one aspect, an embodiment of the present invention provides an antigen, comprising: an mRNA having a nucleotide sequence that is at least 80% identical to the sequence shown in SEQ ID NO:1.

[0032] Using the above mRNA as an immunogen to prepare anti-CD98 antibodies can effectively prepare anti-CD98 antibodies with high specificity and affinity, and the preparation cost is lower.

[0033] In some embodiments, having at least 80% identity can be having at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% identity, or a range between any two of these.

[0034] In some embodiments, the antigen is an mRNA with a nucleotide sequence as shown in SEQ ID NO:1.

[0035] Based on the disclosure of the nucleotide sequence of the antigen in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the mRNA.

[0036] On the other hand, an embodiment of the present invention provides a composition comprising the antigen of any of the aforementioned embodiments.

[0037] In some embodiments, the composition further comprises: any one or more of a carrier, an adjuvant, and a diluent.

[0038] In some embodiments, the diluent includes any one of water and an injection solvent.

[0039] In some embodiments, the carrier includes any one or more of lipid nanoparticles, polymer nanoparticles, composite carriers of lipids and polymers, and inorganic carriers. There is no particular limitation on the specific preparation method of the carrier, and it can be implemented based on the existing carriers used in mRNA vaccines.

[0040] On the other hand, an embodiment of the present invention further provides a vaccine, which includes the antigen of any of the aforementioned embodiments.

[0041] In some embodiments, the vaccine further comprises a vector, and the mRNA is encapsulated in the vector or connected to the vector.

[0042] In some embodiments, the carrier comprises any one or more of lipid nanoparticles, polymer nanoparticles, composite carriers of lipids and polymers, and inorganic carriers.

[0043] In some embodiments, the vaccine further comprises: any one or more of an adjuvant and a diluent.

[0044] On the other hand, an embodiment of the present invention further provides a method for preparing an anti-CD98 antibody, comprising: immunizing an animal with the antigen of any of the foregoing embodiments, the composition of any of the foregoing embodiments, or the vaccine of any of the foregoing embodiments, thereby obtaining an anti-CD98 antibody.

[0045] In some embodiments, after immunizing the target animal, the method for preparing the anti-CD98 antibody further comprises: fusing spleen cells of the immunized animal with myeloma cells to obtain hybridoma cells.

[0046] In some embodiments, after obtaining the hybridoma cells, the method for preparing anti-CD98 antibodies further comprises: screening the hybridoma cells to obtain positive hybridoma cells.

[0047] In some embodiments, the screening method includes ELISA (enzyme-linked immunosorbent assay) and / or FACS (fluorescence activated cell sorting).

[0048] In some embodiments, the ELISA method includes a Cell based ELISA method. Using the Cell based ELISA method to screen hybridomas can reduce costs.

[0049] In some embodiments, after obtaining high-titer hybridoma cells through screening, the method for preparing anti-CD98 antibodies further comprises: producing monoclonal antibodies using the high-titer hybridoma cells obtained through screening.

[0050] In some embodiments, the animal is a mammal, preferably a rodent, including but not limited to a mouse or a rat. Optionally, the aforementioned animal is a humanized mouse.

[0051] The monoclonal antibody prepared in the embodiment of the present invention can specifically bind to CD98 on the cancer cell membrane and form a complex with a protein with amino acid transport activity, thereby helping macromolecular drugs to cross the blood-brain barrier. This antibody has the function of helping drugs to be better delivered to the lesions and effectively inhibiting the growth of cancer cells, and can be used as an active ingredient of a drug for preventing and / or treating tumors.

[0052] In addition, the embodiments of the present invention also provide the use of the antigen of any of the foregoing embodiments, the composition of any of the foregoing embodiments, or the vaccine of any of the foregoing embodiments in any of the following items: (1) preparing a product for inhibiting the growth of tumor cells; (2) a product for preventing, treating or assisting in the treatment of tumors; (3) a product for detecting CD98; (4) a multispecific antibody.

[0053] In some embodiments, the tumor comprises a CD98 high expressing tumor.

[0054] In some embodiments, the tumor includes any one or more of sarcoma, lymphoma, liver cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, bile duct cancer, colon cancer and leukemia. The leukemia may specifically be primary acute myeloid leukemia.

[0055] In some embodiments, the multispecific antibody includes any one or more of a bispecific antibody, a trispecific antibody, and a tetraspecific antibody.

[0056] "Treatment" as used herein includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0057] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0058] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0059] Example 1

[0060] A method for preparing a monoclonal antibody against CD98, the schematic diagram of the process can be referred to Figure 1 , specifically including the following steps.

[0061] 1. Experimental Materials

[0062] Immunogen: HuCD98-mRNA-LNP was synthesized in Suzhou Jinan Protein Technology Co., Ltd. First, the nucleotide sequence was subjected to gene synthesis, plasmid linearization, in vitro transcription of mRNA, and finally LNP encapsulation (synthetic mRNA-LNP is a milky white liquid). The nucleotide sequence of HuCD98-mRNA is shown in SEQ ID NO: 1; HuCD98 Protein, Histag were purchased from ACRO.

[0063] Adjuvant: CPG ODN was purchased from InvivoGen, and Freund's Adjuvant was purchased from Sigma.

[0064] Immunized animals: Humanized mice (KB1-ssBAC2 / H-sLBAC1) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0065] Cells: Detection cells (CHO-K1-Human CD98); Detection cells (CHO-K1-Cyno CD98). The preparation methods of CHO-K1-Human CD98 and CHO-K1-Cyno CD98 are as follows:

[0066] a. Plasmid construction: Construction of Human CD98 plasmid (see Figure 2 ) and Cyno CD98 plasmid (see Figure 3 );

[0067] b. Cell plating: 16 hours before transfection, CHO-K1 cells 5E6 were seeded into a 10 cm culture dish, so that the cell confluence reached 80-90% on the day of transfection, and cultured in a 37°C, 5% CO2 incubator;

[0068] c. Transfection: Add the diluted PEI to the diluted plasmid DNA solution. The order of adding samples cannot be reversed. Mix the solution thoroughly and incubate at room temperature for 15 to 20 minutes. After the incubation, discard the old culture medium in the CHO-K1 cell culture dish, add 9.5mL Opti-MEM serum-free culture medium, and then evenly and slowly add the PEI-plasmid complex to the culture dish (to avoid the cells floating when adding the PEI-plasmid complex, the PEI-plasmid complex can be mixed with the Opti-MEM serum-free culture medium and added to the culture dish together). After 2 hours of transfection, add FBS to the culture medium to a concentration of 10% (1mL). After 6 hours, replace the culture medium in the culture dish with 10mL of fresh complete culture medium (DMEM+10%) and place it in a 37°C, 5% CO2 incubator for continued culture;

[0069] d. Plate laying: The day before infection, the host cells were inoculated in a 6-well plate with a cell density of 4E5 / 2mL, and the cell confluence was ensured to be about 20-30% on the day of infection. The cells were cultured in a 37°C, 5% CO2 incubator. If a 24-well plate or other vessel is used for plating, the cell volume should be appropriately increased or decreased according to the size of the bottom area;

[0070] e. Lentivirus infection: On the day of infection, dilute the viral supernatant with complete culture medium (without PS) to 30-50% of the initial concentration. For example, if the viral supernatant needs to be diluted to 40% of the original, 0.8 mL of viral supernatant and 1.2 mL of complete culture medium should be added to each well. Add the transduction aid Polybrene at a final concentration of 10 ug / mL, place in a centrifuge and centrifuge at 1000g for one hour at room temperature. After centrifugation, place in a 37°C, 5% CO2 incubator for culture. After 6 hours of infection or overnight, discard the supernatant and replace with complete culture medium (basal culture medium + 10% FBS) and place in the incubator for continued culture;

[0071] f. Drug screening: 48 to 72 hours after infection, the cells are subjected to flow cytometry verification to verify whether the target gene is expressed and its positive rate, and drug screening is performed at the same time. The cells are transferred from the 6-well plate to a 10 cm or 15 cm culture dish for expansion, and a certain concentration of antibiotics are added. Before drug screening, the host cells need to be tested for the drug screening working concentration of the corresponding antibiotics;

[0072] g. Single clone selection: dilute and culture the mixed clone stable strains after infection and screening, pick out the cell clones grown from single cells, and then expand the culture to obtain a cell strain with a single trait and stable expression.

[0073] 2. Animal immunization

[0074] Table 1 Immunization strategy

[0075]

[0076] G1 group: HuCD98-mRNA-LNP dosage and immunization method: HuCD98-mRNA-LNP concentration is 0.35μg / μL, and 4μg+4μg is injected into each hind leg muscle without dilution; in addition, the immune adjuvant CpG ODN concentration is 1μg / μL, 4μg CpG ODN per mouse, and mice are subjected to shock immunization 3 days before fusion (using the same dose of 8μg / 36μL / mouse + 4μg / 4μL / mouse CpG ODN);

[0077] G2 group: Immunization antigen HuCD98 Protein, His tag dosage and immunization method: subcutaneous and intraperitoneal injection, the immunogen is evenly distributed during each immunization. Subcutaneous injection is at 5 points on the back and one point in the abdominal cavity. The dosage for each immunization is 30μg / mouse / time (double for the first immunization), dissolved in 100-300μL, generally 200μL. Freund's complete adjuvant (CFA) is used for the first immunization, and Freund's incomplete adjuvant (IFA) is used for booster immunization. Mice are immunized with shock immunization 3 days before fusion (the dosage is 60μg / 100μL / mouse, without adjuvant);

[0078] Immunization: G1 group mRNA immunization and G2 group protein immunization were carried out simultaneously. S0, i.e. blank serum, was collected 3 days before immunization. Each immunization was separated by 14 days. S1 was collected on Day 7 after the second immunization. When the serum titer reached 5W or above, hybridoma fusion could be performed. The results of the two groups were as follows: Figure 4 and Figure 5 shown.

[0079] 3. Preparation of hybridoma cells

[0080] (1) Serum titer comparison was performed, and the results are shown in Figure 6 The mRNA immunization group performed well, while the protein immunization group could not perform hybridoma fusion. Finally, the mice in the mRNA immunization group were used for hybridoma fusion. Before fusion, the mice were subjected to shock immunization to maximize the number of B lymphocytes in the mice. After shock immunization, cell fusion should be completed within 96 hours, otherwise the corresponding number of B cells will drop to the level before shock.

[0081] (2) preparing hybridoma cells by electroporation to obtain polyclonal cell lines;

[0082] a. The mice were killed by dislocating the neck and soaked in 75% alcohol for 3-5 minutes for disinfection. The skin was cut open with sterile scissors to expose the peritoneum. The spleen of the mice was removed and gently ground into a single cell suspension.

[0083] b. Place mouse spleen cells in a 50 mL centrifuge tube and wash them three times with basal medium at 4°C × 1200 rpm × 5 min. Replace the basal medium with electrolysis buffer for the last time and resuspend them for later use;

[0084] c. Preparation of myeloma cells: Myeloma cell lines should belong to the same strain as the immunized animal, and the hybridization fusion rate will be very high; myeloma cells should be revived two weeks before fusion to ensure that myeloma cells are in the logarithmic growth phase, with good morphology and viable cell counts higher than 95%; ① Resuscitate 1 S / P20 cell (purchased from ATCC); ② Add the revived cells to 9 ml of complete culture medium, centrifuge at 4°C × 1200rpm × 5min, discard the supernatant, add 10 ml of complete culture medium and resuspend; ③ Count using a cell counter, and according to the counting results, perform cell passaging according to 1E6 cells / 15 square centimeter DISH; ④ Observe the cell state, and perform cell counting and passaging when the fusion rate reaches 80%-90%;

[0085] d. Cell fusion: After counting, perform electrofusion (electrofusion instrument parameters) at a cell ratio of 2:1 (spleen cells: myeloma cells). After standing for 10 min, add 2.5E4 / 150 μL of HAT medium to each well and culture in a 5% CO2 incubator.

[0086] 4. Preparation of monoclonal antibodies

[0087] Preparation of monoclonal antibody against CD98

[0088] (1) After culturing the fused hybridoma cells in HAT medium for 4 days, the cells were replaced with HT medium and cultured for another 3 days before the main clone positive strain was detected (Cell-Based ELISA method);

[0089] (2) Cell-Based ELISA was used as the primary clone screening method to screen the hybridoma supernatant;

[0090] The Cell-Based ELISA method includes: a. Coating the bottom of the microplate with poly-lysine for 20 minutes and washing three times; b. After resuspending CHO-K1 HuCD98 cells at the required concentration, 1E5 cells per well are plated on the bottom of the microplate and incubated in a CO2 incubator overnight; c. Fixation: remove the cells on the microplate, add 5% paraformaldehyde to fix for 30 minutes, and wash three times; d. Blocking: add 2 / 3 of 2% BSA to the microplate, block in a 37°C incubator for 1 hour, and wash once; e. Add hybridoma supernatant to the microplate and incubate for 1 hour, wash three times; add mouse secondary antibody to the microplate and incubate for 1 hour, wash three times; color development, read the absorbance value at 450nm.

[0091] (3) After positive strains were screened from the hybridoma master clones, they were selected into 24-well plates and cultured for 3 days. Positive strains were rescreened by FACS and subcloned in HT medium for 7 days (200 μL / well);

[0092] (4) Using FACS as a secondary screening method to screen and identify hybridoma supernatants;

[0093] The FACS method includes: a. Resuspend CHO-K1-Human CD98 cells and CHO-K1-Cyno CD98 cells to 2E5 / well and spread them on the bottom of a microplate, centrifuge at 4℃×1400rpm×5min, and discard the supernatant; b. Add 50μL of hybridoma supernatant to the microplate, incubate at 4℃ for 30min, centrifuge at 4℃×1400rpm×5min, and discard the supernatant*3; c. Add 50μL of fluorescent secondary antibody to the microplate, incubate at 4℃ in the dark for 30min, centrifuge at 4℃×1400rpm×5min, and discard the supernatant*3; d. Detect on a flow cytometer.

[0094] (5) Detect subcloned cells using the Cell-Based ELISA method, screen out positive cell lines, and then expand them into 24-well plates. Perform FACS cell identification after 3 days.

[0095] (6) Obtain monoclonal antibody cell lines and freeze them for sequencing.

[0096] 5. Antibody performance test

[0097] Test method 1: Detect hybridoma supernatant and screen the supernatant using Cell-based ELISA and FACS.

[0098] Test result 1: Figure 7 , Figure 8 As shown, more than 100 BGD-positive cell lines with a BGD positive rate greater than 3 times were screened, which proved that CD98 mRNA immunization could obtain a large number of anti-CD98 antibody molecules and that the anti-CD98 molecules had good specific binding at the hybridoma stage.

[0099] Test method 2: EC50 test for chimeric antibodies: Chimeric antibodies were verified by Cell-based ELISA and FACS methods;

[0100] Test result 2: Fig. 9 , Fig.10 and as shown in the table below.

[0101] Table 2 Fig. 9 Test data

[0102] Bottom Hillslope Top EC50 BMK-hlgG 0.4373 1.038 2.276 0.006398 lsotype-hlgG 0.3320 1.093 0.4545 7.751 Ab1 0.3215 0.6651 2.065 0.01609 Ab2 0.3523 0.8740 2.082 0.02996 Ab3 0.4354 1.005 1.976 0.01115 Ab4 0.3729 0.9687 1.939 0.01916 Ab5 0.4020 1.223 2.065 0.006950

[0103] Table 3 Fig.10 Test data

[0104] Bottom Hillslope Top EC50 BMK-hlgG 11216 1.367 971474 0.7132 lsotype-hlgG 843.4 ～20.68 916.6 ～0.4021 Ab1 17268 1.508 1057864 0.4814 Ab2 14448 1.361 967889 1.756 Ab3 11829 1.197 993530 1.078 Ab4 15827 1.214 877263 2.907 Ab5 22556 1.470 919820 0.5533

[0105] The anti-CD98 monoclonal antibody obtained by the hybridoma method has a good EC50 effect (proving that the anti-CD98 antibody molecule has good binding ability and is highly specific to the targeted antigen epitope).

[0106] Test method 3: The amino acid transport function of CD98hc is determined as follows: a. Prepare cells, plate them in a 6-well plate, 1E6 / well, overnight; b. Remove the supernatant from each well, add PBS to wash, and incubate at 37°C for 30 minutes; c. After 30 minutes, resuspend in PBS, collect in a 15mL centrifuge tube and centrifuge; resuspend the cells with PBS and transfer to a 96V well plate, 100μL per well; d. Add diluted BCH and antibody molecules, 25μL / well, resuspend the cells, and incubate at 37°C for 30 minutes; e. Dilute leucine, leucine concentration in each well is 20mM, prepare and use immediately, 25μL / well, incubate at 37°C, incubate for 2 hours; at the same time, set up a blank cell well without adding leucine; f. After 2 hours, discard the supernatant in the well, wash once, and add 200μl of Assay Resuspend the cells in buffer and lyse for 5 minutes; after sufficient pipetting, take out and put into Eppendorf tube, 15000g, 10min, then take out 50μL and put into new well; g. Add the detection system into the cell experiment well and the standard sample well, 50μL per well, incubate at room temperature for 30min; then use microplate reader to detect OD 450 value.

[0107] The difference between the amount of cell surface receptors at 0 and 24 hours includes: a. HCMEC, 5E4 / we 11,100 μ l / we ll plating; b. Antibody dilution: the final concentration of the first well of the antibody is 20μg / mL, 5-fold gradient dilution, 8 concentration points; c. Add the diluted antibodies to the cell plate and mix well. Note that each sample corresponds to 2 columns (1 and 2 column cell wells). The diluted samples on the first day are added to the cell wells in the first column and incubated with the cells overnight. The samples need to be diluted again on the next day and added to the cell wells in the second column. The purpose is to compare the difference in 24h and 0h endocytosis; antibody dilution, the dilution method is the same as Day1, and 2 dilutions are required (1 part is added to the cell wells in the first column to replenish the antibody again and bind to the undegraded receptors), and the other part of the sample is added to the cell wells in the second column, and both are placed at 4℃ for incubation for 30min; d. Blow up the cells, collect the cells into a 96V well plate, centrifuge and wash; e. Add secondary antibody and incubate at 4℃ for 30min; f. Detect on flow cytometer.

[0108] Test result 3: Figures 11-12 As shown, the anti-CD98 monoclonal antibody obtained by the hybridoma method did not change the amino acid transport function mediated by CD98hc, and would not affect the normal operation of cell functions; according to the detection of receptor content at different times, it was found that after the receptor brought the antibody into the cell to complete the transport, it returned to the outside of the cell and continued to perform the transport function, proving that the anti-CD98 antibody molecule will not affect the normal cell operation and transport function when treating diseases.

[0109] The sequence information involved in the present invention is shown in the table below.

[0110]

[0111]

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antigen, characterized in that It includes: An mRNA whose nucleotide sequence is at least 80% identical to the sequence shown in SEQ ID NO:

1.

2. A composition, characterized in that It comprises the antigen according to claim 1.

3. The composition according to claim 2, characterized in that The composition further comprises: any one or more of a carrier, an adjuvant and a diluent; Optionally, the carrier includes any one or more of lipid nanoparticles, polymer nanoparticles, composite carriers of lipids and polymers, and inorganic carriers.

4. A vaccine, characterized in that It comprises the antigen according to claim 1.

5. The vaccine according to claim 4, characterized in that The vaccine further comprises a carrier, wherein the mRNA is encapsulated in the carrier or connected to the carrier; Optionally, the carrier includes any one or more of lipid nanoparticles, polymer nanoparticles, composite carriers of lipids and polymers, and inorganic carriers.

6. The vaccine according to claim 4 or 5, characterized in that The vaccine further comprises: any one or more of an adjuvant and a diluent.

7. A method for preparing an anti-CD98 antibody, characterized in that: It includes: An animal is immunized with the antigen according to claim 1, the composition according to claim 2 or 3, or the vaccine according to any one of claims 4 to 6.

8. The preparation method according to claim 7, characterized in that: After immunizing the target animal, the preparation method further comprises: taking spleen cells of the immunized animal and fusing them with myeloma cells to obtain hybridoma cells.

9. The preparation method according to claim 7 or 8, characterized in that: After obtaining the hybridoma cells, the preparation method further comprises: screening the hybridoma cells to obtain hybridoma cells with high titer; Optionally, the screening method includes ELISA method and / or FACS method; Optionally, the ELISA method includes Cellbased ELISA method; Optionally, after obtaining high-titer hybridoma cells through screening, the preparation method further comprises: producing monoclonal antibodies using the high-titer hybridoma cells obtained through screening.

10. Use of the antigen according to claim 1, the composition according to claim 2 or 3, or the vaccine according to any one of claims 4 to 6 in any of the following; (1) Preparation of products that inhibit tumor cell growth; (2) Products for the prevention, treatment or adjuvant treatment of tumors; (3) Products for detecting CD98; (4) Multispecific antibodies; Optionally, the tumor includes a CD98 high-expressing tumor; Optionally, the tumor includes any one or more of sarcoma, lymphoma, liver cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, bile duct cancer, colon cancer and leukemia.

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