Monoclonal antibody against IL13Ralpha2 and application thereof

Abstract

The invention provides an anti-IL13R alpha 2 monoclonal antibody. The amino acid sequences of three heavy chain complementary determining regions H-CDR1, H-CDR2 and H-CDR3 of the monoclonal antibody are respectively as shown in SEQ ID NO. 11 to SEQ ID NO. 13; the amino acid sequences of three light chain complementary determining regions (L-CDR1, L-CDR2 and L-CDR3) of the monoclonal antibody are respectively as shown in SEQ ID NO. 23 to SEQ ID NO. 25. The invention also provides application of the monoclonal antibody in preparation of drugs for treating triple negative breast tumor or triple negative breast tumor pulmonary metastasis. Tests find that the IL13Ralpha2 monoclonal antibody 1-3C-1 can inhibit the multiplication capacity of triple-negative breast cancer cells in vitro, the migration and invasion capacity of the triple-negative breast cancer cells in vitro, the growth of triple-negative breast cancer in vivo and the lung metastasis of triple-negative breast cancer in vivo.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a monoclonal antibody, in particular to an anti-IL13Rα2 monoclonal antibody and uses thereof. BACKGROUND

[0002] The Global Cancer Statistics Report 2022 shows that breast cancer is the second largest disease in the global cancer incidence, with an incidence of 2.3 million, accounting for 11.6% of all cancer cases. In recent years, the incidence and mortality of breast cancer have shown an increasing trend, and it is also the main cause of death in women. At present, the early diagnosis of breast cancer and the surgical and radiotherapy technology have made great progress, which has reduced the overall mortality, but the risk of metastasis to organs such as lung, bone and brain is still high. The risk factors for distant metastasis in breast cancer patients include tumor size, lymph node metastasis, histological grade, ER, PR, HER2 expression, etc. According to the expression of estrogen receptor (estrogen receptor, ER), progesterone receptor (progesterone receptor, PR), human epidermal growth factor receptor (human epidermal growth factor receptor 2, HER2) and cell proliferation (evaluated by Ki67), breast cancer is clinically classified into four types: luminal A (ER+ / PR+), luminal B (ER+ / PR+ / HER2- / + / Ki67+), HER2 overexpression (ER- / PR- / HER2+) and triple-negative breast cancer (triple-negative breast cancer, TNBC). Each clinical subtype of breast cancer has different clinical characteristics of metastasis. Bone metastasis often occurs in luminal breast cancer, 32% of triple-negative breast cancer patients have lung metastasis, which is also the most common type of lung metastasis.

[0003] Metastasis is the main cause of death of breast cancer, and more than 90% of breast cancer deaths are caused by complications caused by metastasis. According to statistics, about 500,000 breast cancer patients die from distant metastasis every year, and about 70% have lung metastasis, 60% have bone and liver metastasis. Lung metastasis often occurs within 5 years of initial diagnosis, and the average survival time of breast cancer patients with single lung metastasis is only 25 months. Although various schemes such as surgery, chemotherapy, radiotherapy and endocrine therapy have made certain breakthroughs, which can control the growth of primary tumors, the prognosis of lung metastasis patients is often difficult: for lesions that cannot be surgically removed, the treatment measures are very limited. Therefore, lung metastasis is still the biggest challenge in the treatment of breast cancer, and it is very important to explore genes and signaling pathways related to metastasis for the development of more effective diagnosis and treatment strategies and intervention measures.

[0004] Metastasis is a complex biological process involving multiple genes and multiple steps. In this dynamic environment, signaling generated by ligand-receptor complexes coordinates a wide range of cellular responses, significantly affecting tumor behavior. Interleukin 13 receptor subunit alpha 2 (IL13Rα2), also known as CT19, IL-13R, IL13BP, CD213A2, is a specific cell surface receptor composed of 380 amino acids, with the encoding gene located at Xq24. It was initially considered to be a second decoy receptor for interleukin 13 (IL13). However, recent studies have gradually revised this view, with increasing evidence that IL13Rα2 can signal through IL13, promoting tumor progression and immune escape. In ovarian cancer, up to 83% of tumor tissues express IL13Rα2. Clinical data show that breast cancer patients with high expression of IL13Rα2 have significantly poorer prognosis in terms of metastasis-free survival. Similarly, in renal clear cell carcinoma, high expression of IL13Rα2 is also closely related to poor prognosis. In the field of colorectal cancer, immunohistochemical detection shows that about 66% of cases have high expression of IL13Rα2, and there is a statistically significant association with T3 / T4 staging. In summary, IL13Rα2 has become a promising tumor-associated antigen and biomarker in various cancers.

[0005] Monoclonal antibody therapy is a highly precise targeted therapy that specifically recognizes and binds to specific antigens (such as tumor cell surface proteins, pathogens, or inflammatory factors) to exert therapeutic effects. Its main advantages are: 1) precise targeting, reducing damage to normal tissues; 2) significant therapeutic effect, which can act through multiple mechanisms such as blocking signaling pathways, activating immune killing, or directly neutralizing pathogens; 3) higher safety, with generally mild side effects (such as infusion reactions or mild allergies), providing a safety advantage over traditional chemotherapy or broad-spectrum drugs. Currently, this technology has been widely used in the treatment of cancer, autoimmune diseases, and infectious diseases.

[0006] The present invention is directed to the abnormal high expression of IL13Rα2 in triple-negative breast tumors and lung metastatic breast cancer, so we prepared an IL13Rα2 monoclonal antibody (IL13Rα2 mAb) 1-3C-1, which can bind to the specific epitope of the IL13Rα2 antigen. In vivo treatment with this antibody can significantly reduce breast tumor growth and lung metastasis, prolonging survival. These results suggest that IL13Rα2 monoclonal antibody is a potential cancer treatment drug. There is currently no preclinical study of IL13Rα2 monoclonal antibody treatment for triple-negative breast cancer growth and metastasis. SUMMARY

[0007] In view of the above-mentioned technical problems in the prior art, the present invention provides a monoclonal antibody against IL13Rα2 and its use, which aims to solve the technical problem of poor efficacy of drug treatment for the growth and metastasis of triple-negative breast cancer in the prior art.

[0008] This invention provides a monoclonal antibody against IL13Rα2, wherein the heavy chain variable region of the monoclonal antibody includes three complementarity-determining regions H-CDR1, H-CDR2 and H-CDR3, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.11-SEQ ID NO.13, respectively. The light chain variable region of the monoclonal antibody includes three complementarity-determining regions, L-CDR1, L-CDR2, and L-CDR3, the amino acid sequences of which are shown in SEQ ID NO.23-SEQ ID NO.25, respectively.

[0009] Furthermore, the heavy chain variable region of the monoclonal antibody also includes a leader sequence, the amino acid sequence of which is shown in SEQ ID NO.9; the light chain variable region of the monoclonal antibody also includes a leader sequence, the amino acid sequence of which is shown in SEQ ID NO.21.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.10; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.22.

[0011] Furthermore, the complete amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.8; the complete amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.20.

[0012] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.4; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.16.

[0013] Furthermore, the complete nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; the complete nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.14.

[0014] The present invention also provides the use of any of the above-described monoclonal antibodies in the preparation of medicaments for treating triple-negative breast tumors or lung metastases of triple-negative breast tumors.

[0015] The present invention also provides a pharmaceutical composition comprising the antibody described above and a pharmaceutically acceptable carrier.

[0016] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating triple-negative breast tumors or lung metastases of triple-negative breast tumors.

[0017] Furthermore, the antibody is administered in combination with chemotherapy agents, radiotherapy, and / or other agents used for cancer treatment.

[0018] This invention provides an anti-IL13Rα2 monoclonal antibody 1-3C-1 and its effect on inhibiting the growth and metastasis of triple-negative breast cancer.

[0019] Compared with existing technologies, the technical effects of this invention are positive and significant, mainly including: 1. The IL13Rα2 monoclonal antibody 1-3C-1 can inhibit the proliferation of triple-negative breast cancer cells in vitro.

[0020] 2. The IL13Rα2 monoclonal antibody 1-3C-1 can inhibit the migration and invasion of triple-negative breast cancer cells in vitro.

[0021] 3. The IL13Rα2 monoclonal antibody 1-3C-1 can inhibit the growth of triple-negative breast cancer in vivo.

[0022] 4. The IL13Rα2 monoclonal antibody 1-3C-1 can inhibit lung metastasis of triple-negative breast cancer in vivo. Attached Figure Description

[0023] Figure 1 IL13Rα2 is significantly overexpressed in triple-negative breast cancer cells.

[0024] Figure 2 IL13Rα2 was significantly overexpressed in the triple-negative breast cancer lung metastasis cell line (LM2-4175).

[0025] Figure 3 Preparation process of IL13Rα2 monoclonal antibody.

[0026] Figure 4 Eleven IL13Rα2 monoclonal antibodies were obtained from the purified ascites fluid.

[0027] Figure 5 Specificity verification of IL13Rα2 monoclonal antibody 1-3C-1.

[0028] Figure 6 Verification of the inhibitory effect of IL13Rα2 monoclonal antibody 1-3C-1 on the proliferation of triple-negative breast cancer cells.

[0029] Figure 7Validation of the inhibitory effect of IL13Rα2 monoclonal antibody 1-3C-1 on the migration of triple-negative breast cancer cells.

[0030] Figure 8 Validation of a mouse model of IL13Rα2 monoclonal antibody 1-3C-1 inhibiting the growth of triple-negative breast cancer tumors.

[0031] Figure 9 Validation of a mouse model of IL13Rα2 monoclonal antibody 1-3C-1 inhibiting lung metastasis of triple-negative breast cancer tumors. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0033] Unless otherwise specified, all materials used in the embodiments of this invention are commercially available products. Example 1

[0034] 1. Using the R programming language, download the sequencing dataset of different breast cancer cell subtypes (GSE2603) from the GEO database. After preprocessing the data (removing batch effects and standardizing), perform gene annotation, extract IL13Rα2 expression data, and group the samples (Luminal breast cancer group and triple-negative breast cancer group). Use the t-test to statistically analyze the expression of IL13Rα2 in Luminal and triple-negative breast cancer cells. P < 0.05 was considered statistically significant. Use a scatter plot to visualize the expression differences between the two groups.

[0035] 2. Using the R language tool, download the sequencing dataset (GSE124380) of triple-negative breast cancer maternal low lung metastasis cells (MDA-MB-231) and progeny high lung metastasis cells (LM2-4175) from the GEO database. After preprocessing the data (removing batch effects and standardizing), gene annotation was performed to obtain the expression matrices of the two cell lines, MDA-MB-231 and LM2-4175, and then heatmaps were generated.

[0036] The above experiments revealed that IL13Rα2 is abnormally highly expressed in breast cancer, especially in metastatic breast cancer (see appendix). Figure 1 -Appendix Figure 2). Example 2

[0037] To promote the clinical application of IL13Rα2 targeting in the treatment of triple-negative breast cancer, we prepared an IL13Rα2 monoclonal antibody (see attached image). Figure 3 -Appendix Figure 4 (As shown).

[0038] The original sequence of recombinant human IL13Rα2 protein is as follows (SEQ ID NO.1): GACACCGAGATAAAAGTTAACCCTCCTCAGGATTTTGAGATAGTGGATCCCGGATACTTAGGTTATCTCTATTTGCAATGGCAACCCCCACTGTCTCTGGATCATTTTAAGGAATGCACAGTGGAATATGAACTAAAATACCGAAACATTGGTAGTGAAACATGGAAGACCATCATTACTAAGAATCTACATTACAAAGATGGGTTTGATCTTAACAAGGGCATTGAAGCGAAGATACACACGCTTTTACCATGGCAATGCACAAATGGATCAGAAGTTCAAAGTTCCTGGGCAGAAACTACTTATTGGATATCACCACAAGGAATTCCAGAAACTAAAGTTCAGGATATGGATTGCGTATATTACAATTGGCAATATTTACTCTGTTCTTGGAAACCTGGCATAGGTGTACTTCTTGATACCAATTACAACTTGTTTTACTGGTATGAGGGCTTGGATCATGCATTACAGTGTGTTGATTACATCAAGGCTGATGGACAAAATATAGGATGCAGATTTCCCTATTTGGAGGCATCAGACTATAAAGATTTCTATATTTGTGTTAATGGATCATCAGAGAACAAGCCTATCAGATCCAGTTATTTCACTTTTCAGCTTCAAAATATAGTTAAACCTTTGCCGCCAGTCTATCTTACTTTTACTCGGGAGAGTTCATGTGAAATTAAGCTGAAATGGAGCATACCTTTGGGACCTATTCCAGCAAGGTGTTTTGATTATGAAATTGAGATCAGAGAAGATGATACTACCTTGGTGACTGCTACAGTTGAAAATGAAACATACACCTTGAAAACAACAAATGAAACCCGACAATTATGCTTTGTAGTAAGAAGCAAAGTGAATATTTATTGCTCAGATGACGGAATTTGGAGTGAGTGGAGTGATAAACAATGCTGGGAAGGTGAAGACCTATCGAAGAAAACTTTGCTACGTTTCTGGCTACCATTTGGTTTCTGA Materials for monoclonal antibody preparation:

[0039] Name Source Balb / c mice Shandong Poney Biological Technology Co., Ltd. 1640 culture medium GIBCO Fetal bovine serum FBS GIBCO TMB Thermo Glutamine GIBCO Serum-free cell freezing solution Xin Saimei NEAA GBICO HAT sigma Protein G agarose purification resin Yixing PEG sigma Pristane sigma TRIZOL Thermo Complete and incomplete adjuvant sigma Penicillin-streptomycin Xin Saimei IL13Rα2 antigen Thermo Poly-Prep® Chromatography Columns bio-rad Monoclonal antibody preparation experimental instruments

[0040] Biosafety cabinets, CO2 incubators, plate washers, ELISA readers, centrifuges, microscopes, spectrometers, pH meters, -20℃ refrigerators, -80℃ refrigerators, 4℃ refrigerators, water baths, autoclaves, drying ovens, microsyringes, and other instruments.

[0041] Experimental methods for preparing monoclonal antibodies.

[0042] 1. Preparation of mouse myeloma cells

[0043] (1) Resuscitation of mouse myeloma cells SP2 / 0 The mice were rapidly anesthetized, dissected, and the bilateral femurs, tibias, and humeri were removed, along with the removal of muscles and connective tissue. The bones were then temporarily stored in pre-cooled PBS.

[0044] (2) Irrigate the bone marrow cavity Cut the bone at both ends with scissors (exposing the bone marrow cavity). Using a 25 G syringe, draw pre-cooled PBS and flush the bone marrow from one end into a centrifuge tube (repeat 3-5 times until the bone turns white). Collect the flushing fluid and filter it through a 70 μm cell sieve to remove bone fragments and clumps.

[0045] (3) Centrifugation and resuspension Centrifuge at 300 g for 5 minutes (4 ℃) and discard the supernatant. Resuspend the cells in PBS or culture medium and count them (assess viability by trypan blue staining).

[0046] 2. Preparation of mouse spleen cells

[0047] (1) Take Balb / c mice that have been immunized with enhanced IL13Rα2 antigen, collect blood from their orbital fossa and separate the serum as positive control serum for antibody detection; (2) Anesthetize the mice, disinfect them with 75% alcohol, fix them on the dissection table, lift the skin of the left abdomen to see the spleen, replace the ophthalmic forceps, cut the peritoneum with sterile surgical scissors in the laminar flow hood, remove the spleen, place it in a culture dish containing 10 mL of serum-free 1640+P / S, wash it gently, and carefully peel off the surrounding connective tissue. Use a sterile 2 mL syringe to draw serum-free 1640 culture medium to rinse the spleen cells into the culture dish; (3) Harvest the spleen cell suspension, centrifuge at 1000 rpm / min for 10 minutes, discard the supernatant, wash 1-2 times with serum-free 1640 medium, then resuspend the cells in 20 mL of serum-free 1640 medium and mix well. Take the above suspension and count the cells. Usually, 1-2.5 × 10⁶ cells can be obtained per mouse. 8 One spleen cell; 3. Cell fusion (1) Resuspend all spleen cells and myeloma cells, centrifuge at 1000 rpm for 5-10 minutes, and use a dropper to remove the supernatant as much as possible to avoid affecting the concentration of PEG; (2) Gently tap the bottom of the centrifuge tube with your palm or your fingers to loosen and evenly disperse the precipitated cells; preheat in a 37 ℃ water bath; (3) Add 1 mL of 50% PEG (pH=8.0) preheated to 37 ℃ while stirring gently, and let stand for 90 s; (4) Add 10 mL of 1640 culture medium preheated to 37 °C; (5) Centrifuge at 1000 rpm for 7 minutes and discard the supernatant; (6) Add 80-100 mL of HAT medium (containing IL6) and suspend and mix well; (7) Dispense 0.1-0.15 mL into each well of a 96-well cell culture plate; (8) Incubate at 37 ℃ in a 5% CO2 incubator, and supplement with HAT medium after 3 days; (10) Observe the growth of hybridoma cells, and when they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for ELISA detection.

[0048] 4. Semi-solid screening of monoclonal antibodies (1) Based on the ELISA results, select the positive wells, blow the cells in the wells evenly, and put 10 μL into a cell counting plate for counting. (2) Take 30 μL of the diluted cells and add them to 500 μL of complete culture medium; (3) Add 2-3 mL of semi-solid culture medium to the 6-well plate; (4) Add the cells from the complete culture medium to the semi-solid culture medium and mix gently. After examining the single clones under a microscope, transfer the single clones to 96-well plates using a 10 μL pipette and incubate them in an incubator. (5) Expand the culture when the cells in the well reach the logarithmic growth phase, and freeze a portion of them in liquid nitrogen.

[0049] 5. Purification treatment of ascites fluid obtained from strongly positive cell lines

[0050] (1) Load the protein A agarose gel medium into a nickel ion affinity chromatography column, and slowly load the ascites fluid and PBS into a 1:1 volume ratio. (2) After the antibody binds, it is eluted with glycine elution buffer to obtain the purified monoclonal antibody.

[0051] like Figure 4As shown, 11 monoclonal antibodies were finally purified (monoclonal antibodies 1-2H-1, 1-2H-2, 1-3C-1, 1-3C-3, 1-10C-1, 2-1B-3, 2-8A-1, 3-5H-1, 4-11B-3, 4-12E-1, and 4-12E-2).

[0052] Example 3

[0053] The 11 monoclonal antibodies obtained in Example 2 were screened and verified by Western blot to identify and bind to IL13Rα2. (See attached image) Figure 5 ).

[0054] Experimental process

[0055] Western blot screening method for single clones: Total protein was extracted from three groups of cells (LM2-4175-shIL13Rα2-1 and -2 and their control), separated by molecular weight by SDS-PAGE, and then electrotransferred onto a membrane. Non-specific sites were blocked with 5% skim milk powder, and specific primary antibodies were added sequentially and incubated overnight at 4°C. The next day, the membrane was washed with TBST, incubated with enzyme-labeled secondary antibody at room temperature for 1 hour, washed again with TBST, and then chemiluminescent substrate was added for development. The target protein can then be detected and quantified.

[0056] Conclusion: The IL13Rα2 monoclonal antibody 1-3C-1 has the best specificity.

[0057] Example 4

[0058] Further sequence and structural analysis of the IL13Rα2 monoclonal antibody 1-3C-1 was performed using the classic Sanger sequencing method.

[0059] Experimental process

[0060] Sanger sequencing workflow: 1. Sample preparation: Extract DNA from hybridoma cells, design specific primers, and amplify the antibody variable region gene (including heavy and light chains) by PCR. 2. Sequencing reaction: Four common deoxynucleotides (dATP, dTTP, dCTP, dGTP) and a small amount of dideoxynucleotides (ddATP, ddTTP, ddCTP, ddGTP) are added; the dideoxynucleotides pair with the template DNA, causing chain synthesis to terminate at a specific position; 3. Capillary electrophoresis separation: DNA fragments are separated by capillary electrophoresis. Shorter fragments move faster and longer fragments move slower, thus separating DNA fragments of different lengths. 4. Signal detection and sequence analysis: Detect the fluorescence signal at the end of each fragment; analyze the peak positions of the electrophoresis pattern to infer the complete sequence of the DNA fragment.

[0061] Sequencing results showed that the IL13Rα2 monoclonal antibody 1-3C-1 contains heavy chain variable regions and light chain variable regions. The heavy chain variable regions include H-CDR1, H-CDR2, and H-CDR3, while the light chain variable regions include L-CDR1, L-CDR2, and L-CDR3. The sequence is as follows: H-DNA Full sequence (SEQ ID NO.2): ATGAAATGCAGCTGGGTTATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCTGAGTTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCATCAACATTAAAGACACCTATATGCACTGGGTGAAACAGAGGCTTGAACAGGGCCTGGAGTGGA TTGGAAGGATTGATCCTGCGAATGGTAATACTAAATTTGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTACTCTCTACTATGATTCCCCTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA H-leader-DNA sequence (SEQ ID NO.3): ATGAAATGCAGCTGGGTTATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCA H-Variable Domain DNA-sequence(SEQ ID NO.4):GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGTTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCATCAACATTAAAGACACCTATATGCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATTTGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTACTCTCTACTATGATTCCCCTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA H-CDR1 DNA-sequence(SEQ ID NO.5):GACACCTATATGCAC H-CDR2 DNA-sequence(SEQ ID NO.6):AGGATTGATCCTGCGAATGGTAATACTAAATTTGACCCGAAGTTCCAGGGC H-CDR3 DNA-sequence(SEQ ID NO.7):TACTATGATTCCCCTGTTTAC H- Full amino acid(SEQ ID NO.8):MKCSWVIFFLMAVVTGVNSEVQLQQSGAEFVKPGASVKLSCTASGINIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKFDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTLYYDSPVYWGQGTLVTVSA H-leader-AA sequence(SEQ ID NO.9):MKCSWVIFFLMAVVTGVNS H-Variable Domain AA-sequence(SEQ ID NO.10):EVQLQQSGAEFVKPGASVKLSCTASGINIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKFDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTLYYDSPVYWGQGTLVTVSA H-CDR1 AA-sequence(SEQ ID NO.11):DTYMH H-CDR2 AA-sequence(SEQ ID NO.12):RIDPANGNTKFDPKFQG H-CDR3 AA-sequence(SEQ ID NO.13):YYDSPVY L-DNA Full sequence(SEQ ID NO.14):ATGGAATCACAGACTCAGGTCTTCCTCTCCCTGCTGCTCTGGGTATCTGGTACCTGTGGGAACATTATGCTGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACATTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAAGTGCTGATCTACTGGGCATCCACTAGGGCATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGGAGTGTACAAGCTGAAGACCTGGCAGTTTATTATTGTCATCAATACATCTCCTCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA L-leader-DNA sequence(SEQ ID NO.15):ATGGAATCACAGACTCAGGTCTTCCTCTCCCTGCTGCTCTGGGTATCTGGTACCTGTGGG L-Variable Domain DNA-sequence(SEQ ID NO.16):AACATTATGCTGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACATTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAAGTGCTGATCTACTGGGCATCCACTAGGGCATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGGAGTGTACAAGCTGAAGACCTGGCAGTTTATTATTGTCATCAATACATCTCCTCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA L-CDR1 DNA-sequence(SEQ ID NO.17):AAGTCCAGTCAAAGTGTTTTATACATTTCAAATCAGAAGAACTACTTGGCC L-CDR2 DNA-sequence(SEQ ID NO.18):TGGGCATCCACTAGGGCATCT L-CDR3 DNA-sequence(SEQ ID NO.19):CATCAATACATCTCCTCGCTCACG L- Full amino acid(SEQ ID NO.20):MESQTQVFLSLLLWVSGTCGNIMLTQSPSSLAVSAGEKVTMSCKSSQSVLYISNQKNYLAWYQQKPGQSPKVLIYWASTRASGVPDRFTGSGSGTDFTLTIRSVQAEDLAVYYCHQYISSLTFGAGTKLELK L-leader-Aasequence(SEQ ID NO.21):MESQTQVFLSLLLWVSGTCG L-Variable Domain AA-sequence (SEQ ID NO.22): NIMLTQSPSSLAVSAGEKVTMSCKSSQSVLYISNQKNYLAWYQQKPGQSPKVLIYWASTRASGVPDRFTGSGSGTDFTLTIRSVQAEDLAVYYCHQYISSLTFGAGTKLELK L-CDR1 AA-sequence (SEQ ID NO.23): KSSQSVLYISNQKNYLA L-CDR2 AA-sequence (SEQ ID NO.24): WASTRAS L-CDR3 AA-sequence (SEQ ID NO.25): HQYISSLT Table 1. Amino acid sequence comparison of the IL13Rα2 antibody heavy chain CDR in this patent with other patents. Clone number SEQ ID NO. H-CDR1 SEQ ID NO. H-CDR2 SEQ ID NO. H-CDR3 1-3C-1 11 DTYMH 12 RIDPANGNTKFDPKFQG 13 YYDSPVY CN 114805581 B patent 5 SYSMN SISSSSSYIYYADSVKG AGGSLGAFDY WO2019178078A1 patent H08 RNGMS TVSSGGSYIYYADSVKG QGTTALATRFFDV Table 1. Amino acid sequence comparison of the IL13Rα2 antibody light chain CDR in this patent with other patents. Clone number SEQ ID NO. L-CDR1 SEQ ID NO. L-CDR2 SEQ ID NO. L-CDR3 1-3C-1 23 KSSQSVLYISNQKNYLA 24 WASTRAS 25 HQYISSLT CN 114805581 B patent 5 RASQDIRSYLA AASTLQS QQLNSFPAT WO2019178078A1 patent H08 KASQDVGTAVA SASYRST QHHYSAPWT Conclusion: The IL13Rα2 monoclonal antibody 1-3C-1 obtained in this invention has specific heavy chain and light chain CDR regions, which are significantly different from those of previously reported monoclonal antibodies.

[0062] Example 5 This invention also verified the effect of L13Rα2 monoclonal antibody in inhibiting the proliferation, migration, and invasion of triple-negative breast cancer cells through in vitro experiments (see appendix). Figure 6 -Appendix Figure 7 ).

[0063] Experimental methods 1. Cell proliferation capacity assay (CCK8) 1) The IL13Rα2 monoclonal antibody was serially diluted in DMEM complete medium and co-cultured with well-growing breast cancer cells (MDA-MB-231, purchased from ATCC) for 48 h. The cells were then digested and counted. 2) Adjust the cell suspension concentration by adding 100 μL of cell suspension to each well and seeding the plate to achieve a cell density of 1000 cells / well. Seed the plate for a total of 4 96-well plates (0 h, 24 h, 48 h, 72 h). 3) Group according to the above time, incubate in a 5% CO2, 37 ℃ cell culture incubator, add 10 µL of CCK8 reagent to each well, gently shake to mix, and continue to culture for 1~4 hours; 4) Measure the absorbance of each well at the OD450nm wavelength of the enzyme-linked immunosorbent assay (ELISA) instrument.

[0064] Conclusion: The IL13Rα2 monoclonal antibody 1-3C-1 significantly inhibited the proliferation of triple-negative breast cancer cells.

[0065] 2. Transwell method for detecting cell migration and invasion capabilities 1) Cell preparation The IL13Rα2 monoclonal antibody was serially diluted in DMEM complete medium and co-cultured with well-grown breast cancer cells (MDA-MB-231, purchased from ATCC) for 48 h. The cells were then digested and counted. 2) Transwell chamber treatment Migration assay: Place the Transwell chamber into a culture plate, add 600 μL of complete culture medium (chemotactic agent) containing 10% FBS to the lower chamber; add 100-200 μL of cell suspension (prepared with serum-free culture medium) to the upper chamber. Invasion test: Matrigel coating (melted at 4°C in advance): Dilute Matrigel with serum-free medium (1:8~1:10) using a pre-cooled pipette tip; add 50~100 μL to each chamber, cure at 37°C for 1~2 h, and follow the same steps as migration test; 3) Incubation Incubate at 37 ℃ in a 5% CO2 incubator for 12-48 h (adjust the time according to the cell migration ability). Invasion experiments are usually 6-12 h longer than migration experiments. 4) Termination of experiment and staining Cell fixation: Gently wipe away unmigrated cells from the upper chamber with a cotton swab, fix with 4% paraformaldehyde for 30 minutes, and wash with PBS; Staining: Stain with 0.1% crystal violet for 20 minutes, wash off excess dye with PBS, and observe and photograph under a fluorescence microscope.

[0066] Conclusion: The IL13Rα2 monoclonal antibody 1-3C-1 significantly inhibited the migration and invasion of triple-negative breast cancer cells.

[0067] Example 6 In vivo experiments verified the inhibitory effect of IL13Rα2 monoclonal antibody on the growth of triple-negative breast cancer tumors and lung metastasis (see appendix). Figure 8 -Appendix Figure 9 ).

[0068] animal experiments 1) Establishment of a breast cancer tumor-bearing animal model: 4-6 week old BALB / c female mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. 1×10⁻⁶ mc²⁻¹ of mammary fat pads were subcutaneously injected into the fourth pair of mammary fat pads of the mice. 5A mouse model of breast cancer bearing was established using 14 breast cancer cells (n=14). Five days later, mice were injected intraperitoneally with IL13Rα2 monoclonal antibody and its control IgM (10 mg / kg) twice weekly. 2) Tumor Growth Recording and Subsequent Analysis: One week after inoculation with breast tumor cells, mice were observed daily. Their health, diet, and the exact date of tumor initiation were recorded. Tumor size was measured every other day using calipers. This observation and recording continued for four weeks, and a tumor growth curve was plotted. Finally, the mice were euthanized, and tumor tissue, along with lung tissue, was collected. Tumor volume and weight were measured. Tumor and lung tissue were fixed in 4% paraformaldehyde. Ki67 staining was performed on breast tumor tissue sections, and HE staining was performed on lung tissue sections. The proliferation and metastasis of breast tumor cells were assessed.

[0069] Conclusion: The IL13Rα2 monoclonal antibody 1-3C-1 significantly inhibited the growth of mammary tumors and lung metastasis in triple-negative breast cancer-bearing mice.

[0070] The above-described methods for preparing and screening IL13Rα2 monoclonal antibody 1-3C-1, and for verifying its anti-breast cancer function, are merely preferred embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A monoclonal antibody against IL13Rα2, characterized in that, The amino acid sequences of the three heavy chain complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3 of the monoclonal antibody are shown in SEQ ID NO.11-SEQ ID NO.13, respectively. The three light chain complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3 of the monoclonal antibody have amino acid sequences as shown in SEQ ID NO.23-SEQ ID NO.25, respectively.

2. The monoclonal antibody against IL13Rα2 according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody further includes a leader sequence, the amino acid sequence of which is shown in SEQ ID NO.9; the light chain variable region of the monoclonal antibody further includes a leader sequence, the amino acid sequence of which is shown in SEQ ID NO.

21.

3. The monoclonal antibody against IL13Rα2 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.10; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

22.

4. The monoclonal antibody 1-3C-1 against IL13Rα2 according to claim 1, characterized in that, The complete amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.8; the complete amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

20.

5. The monoclonal antibody against IL13Rα2 according to claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.4; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

16.

6. The monoclonal antibody against IL13Rα2 according to claim 1, characterized in that, The complete nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; the complete nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

14.

7. The use of the monoclonal antibody according to any one of claims 1-6 in the preparation of a medicament for treating triple-negative breast tumors or lung metastases of triple-negative breast tumors.

8. A pharmaceutical composition, characterized in that, It comprises the monoclonal antibody as described in any one of claims 1-6 above, and a pharmaceutically acceptable vector.

9. Use of the pharmaceutical composition of claim 8 in the preparation of a medicament for treating triple-negative breast tumors or lung metastases of triple-negative breast tumors.

10. A pharmaceutical composition according to claim 9, characterized in that, The antibody is administered in combination with chemotherapy agents, radiotherapy, and / or other agents used for cancer treatment.

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