Monoclonal antibody and application thereof in preparation of medicine for treating tumors

Abstract

The invention relates to the technical field of tumor treatment, in particular to a monoclonal antibody and application thereof in preparation of a medicine for treating tumors. The monoclonal antibody comprises a monoclonal antibody B6 or a monoclonal antibody E6. According to the application, the early pregnancy factor monoclonal antibodies and cancer cells are co-cultured in vitro, the influence of the early pregnancy factor monoclonal antibodies on malignant biological behaviors of the tumor cells is studied, and it is found that the two early pregnancy factor monoclonal antibodies B6 and E6 can remarkably inhibit proliferation, migration and clone formation of HeLa cells and Mcf-7 cells and promote apoptosis of the cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to a monoclonal antibody and its application in preparing a drug for treating tumor. Background Art

[0002] Early Pregnancy Factor (EPF) is a trace protein with immunosuppressive and growth regulatory effects detected in the serum of pregnant mice by Australian scholars in 1974. It can protect the embryo from maternal immune attack by inhibiting the immune response of maternal lymphocytes. In addition, it is also closely related to the occurrence and development of tumors. Studies have shown that early pregnancy factor is also expressed in the serum of tumor patients. It not only promotes tumor progression as a growth factor, but also may simulate the maternal-fetal immune tolerance mechanism to help tumor cells evade the body's immune clearance.

[0003] Some scholars have pointed out that EPF or EPF-like active substances are positively expressed in the serum of testicular cancer patients, while the healthy control group is negative, indicating that EPF has the potential value as a specific biomarker for early screening of testicular cancer. Some scholars in my country have measured the serum of 11 tumor patients with high incidence rates and found that EPF exists in high-incidence cancers such as ovarian cancer, cervical cancer, melanoma, colorectal cancer, lymphoma, and liver cancer. This shows that early pregnancy factor does not exist alone in a certain tumor. It is widely present in patients with a variety of tumors and promotes the growth and proliferation of tumors, aggravating the patient's condition. It has a universal harmful effect on tumors. Therefore, it is necessary to use early pregnancy factor to prepare monoclonal antibodies for the treatment of cancer. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a monoclonal antibody and its use in the preparation of a drug for treating tumors. The monoclonal antibodies B6 and E6 provided by the present invention are studied for their inhibitory effects on the growth inhibition, migration and clone formation of human cervical cancer HeLa cells and human breast cancer Mcf-7 cells, and promoting apoptosis, providing a certain theoretical basis for the use of early pregnancy factor monoclonal antibodies to treat cancer.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a monoclonal antibody, including monoclonal antibody B6 or monoclonal antibody E6;

[0007] The amino acid sequence of the heavy chain variable region of the monoclonal antibody B6 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2;

[0008] The amino acid sequence of the heavy chain variable region of the monoclonal antibody E6 is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.4.

[0009] The present invention also provides a drug for treating tumors, comprising the monoclonal antibody described in the above technical solution.

[0010] Preferably, the tumor comprises human breast cancer and / or human cervical cancer.

[0011] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of drugs for treating tumors.

[0012] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of drugs for inhibiting tumor cell growth.

[0013] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for inhibiting tumor cell migration.

[0014] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of drugs for inhibiting tumor cell cloning.

[0015] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for promoting tumor cell apoptosis.

[0016] Preferably, the tumor comprises human breast cancer and / or human cervical cancer.

[0017] Beneficial effects:

[0018] The present invention utilizes early pregnancy factor monoclonal antibodies to co-culture with cancer cells in vitro to explore their effects on the malignant biological behaviors of tumor cells, and finds that both early pregnancy factor monoclonal antibodies B6 and E6 can significantly inhibit the proliferation, migration, cloning of HeLa cells and Mcf-7 cells and promote apoptosis of cancer cells, and compared with previous studies by scholars, these two monoclonal antibodies are more effective in inhibiting tumor proliferation and promoting apoptosis. The present invention further explores the cell cycle and finds that B6 and E6 monoclonal antibodies can significantly block the G0 / G1 phase and G2 / M phase of DNA synthesis in cancer cells, which indicates that B6 and E6 monoclonal antibodies promote apoptosis of cancer cells and are closely related to the early and late stages of DNA synthesis, but their specific mechanisms and pathways still need to be further explored. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1SDS-PAGE analysis of recombinant EPF; A. SDS-PAGE detection of rEPF expression. M: Protein Marker; 1: IPTG induced 15 μL; 2: IPTG induced 10 μL; 3: IPTG not induced. B. SDS-PAGE detection of srEPF expression. M: Protein Marker; 1: PET28a-EPF empty plasmid; 2: IPTG not induced; 3: IPTG induced. C. SDS-PAGE detection of srEPF purification and enzyme digestion. M: Protein Marker; 1: sr EPF whole bacterial protein; 2: srEPF purified protein; 3: srEPF purified protein cleavage; 4: EPF.D. SDS-PAGE detection of srdEPF expression. M: protein marker; 1: uninduced control; 2-4: L-arabinose induced control; 5: lysis precipitate; 6: lysis supernatant. E. SDS-PAGE detection of srdEPF purification. M: protein marker; 1: filtrate; 2: elution 1; 3: elution 2; 4: elution 3; 5: elution 4; 6: enzyme cleavage product dEPF;

[0021] Figure 2 The indirect ELISA method was used to detect the OD value of the hybridoma cell culture supernatant;

[0022] Figure 3 This is the SDS electrophoresis verification result of EPF monoclonal antibody B6 and E6 antibody purification. The light chain of the monoclonal antibody is about 25kD and the heavy chain is about 50kD. Relatively pure monoclonal antibodies are obtained and can be used for subsequent experiments.

[0023] Figure 4 is the affinity curve of EPF mAb;

[0024] Figure 5 This is the result of EPF mAb subtype identification;

[0025] Figure 6 For the specific identification of monoclonal antibody WB, A and B respectively show the results of the reaction between B6 antibody and E6 antibody and recombinant protein and EPF after enzyme digestion. M: protein marker.1: srEPF.2: EPF; C and D respectively show the results of the reaction between B6 antibody and E6 antibody and natural protein in the supernatant of tumor cell culture; M: protein marker.1: human ovarian cancer cell A2780.2: human prostate cancer cell DU145.3: human liver cancer cell Hepg-2.4: human breast cancer cell Mcf-7.5: human endometrial cancer cell Ishiwaka3-H-12.6: human cervical cancer cell HeLa;

[0026] Figure 7The results of the prediction analysis of the mutual binding between EPF mAb and EPF protein; A is the B6 monoclonal antibody structure predicted by AlphaFold, B is the E6 monoclonal antibody structure predicted by AlphaFold, C is the EPF protein structure predicted by AlphaFold, D is the scoring result of the complex formed by B6 and EPF predicted by AlphaFold, E is the visualization result of the complex predicted by AlphaFold, F is the visualization result of the complex predicted by AlphaFold, and G is the stable binding mode of the formed complex, showing a typical immunoglobulin folding structure;

[0027] Figure 8 is the growth inhibition rate of B6 and E6 monoclonal antibodies on HeLa cells and Mcf-7 cells;

[0028] Fig. 9 B6 monoclonal antibody inhibits the migration of HeLa cells and Mcf-7 cells. A: The migration distance of HeLa cells inhibited by B6 monoclonal antibody. B: The migration distance of Mcf-7 cells inhibited by B6 monoclonal antibody. C: The migration inhibition rate of HeLa cells and Mcf-7 cells by B6 monoclonal antibody.

[0029] Fig.10 E6 monoclonal antibody inhibits the migration of HeLa cells and Mcf-7 cells. A: E6 monoclonal antibody inhibits the migration distance of HeLa cells. B: E6 monoclonal antibody inhibits the migration distance of Mcf-7 cells. C: E6 monoclonal antibody inhibits the migration rate of HeLa cells and Mcf-7 cells.

[0030] Fig.11 B6 monoclonal antibody inhibits the clone formation of HeLa cells and Mcf-7 cells. A: The number of clone formation of HeLa cells inhibited by B6 monoclonal antibody. B: The number of clone formation of Mcf-7 cells inhibited by B6 monoclonal antibody. C: The inhibition rate of clone formation of HeLa cells and Mcf-7 cells by B6 monoclonal antibody.

[0031] Fig.12 E6 monoclonal antibody inhibits the clone formation of HeLa cells and Mcf-7 cells. A: The number of clone formation of HeLa cells inhibited by E6 monoclonal antibody. B: The number of clone formation of Mcf-7 cells inhibited by E6 monoclonal antibody. C: The inhibition rate of clone formation of HeLa cells and Mcf-7 cells by E6 monoclonal antibody.

[0032] Fig.13 B6 monoclonal antibody promotes apoptosis of HeLa cells and Mcf-7 cells. A: B6 monoclonal antibody promotes apoptosis of HeLa cells. B: B6 monoclonal antibody promotes apoptosis of Mcf-7 cells. C: The apoptosis rate of HeLa cells and Mcf-7 cells induced by B6 monoclonal antibody.

[0033] Fig.14E6 monoclonal antibody promotes apoptosis of HeLa cells and Mcf-7 cells. A: E6 monoclonal antibody promotes apoptosis of HeLa cells. B: E6 monoclonal antibody promotes apoptosis of Mcf-7 cells. C: The apoptosis rate of HeLa cells and Mcf-7 cells induced by E6 monoclonal antibody.

[0034] Fig.15 Effects of B6 and E6 monoclonal antibodies on the cell cycle of HeLa cells and Mcf-7 cells. A: Effects of B6 and E6 monoclonal antibodies on the cell cycle of HeLa cells. B: Effects of B6 and E6 monoclonal antibodies on the cell cycle of Mcf-7 cells. DETAILED DESCRIPTION

[0035] The present invention provides a monoclonal antibody, including monoclonal antibody B6 or monoclonal antibody E6; the amino acid sequence of the heavy chain variable region of the monoclonal antibody B6 is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody E6 is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.4, which are specifically as follows:

[0036] SEQ ID No.1:

[0037] MECSWILPFILSVTSGVYSLVQLQQSGAELARPGASVKLSCKASGYTFTN YWMQWVKQRPGQGLEWIGAIYPGDFDTRYTQKFKGKATLTADKSSNTAYM QLSSLASEDSAVYYCVRWGWGQGAYWGQGTTLTVSS;

[0038] SEQ ID No.2:

[0039] MSVPTQVLGLLLLWLTGARCDIQMTQSSASQSASVGETVTITCRASENIY SYLAWFQQRQGKSPQLLIYNAETLAEGVPSRFSGSGSGTQFSLKINSLQPEDF GTYYCQHHYGSPWTFGGGTKLEIK;

[0040] SEQ ID No.3:

[0041] MGWSCIILFLVATATGVHSQVQLQQPGAALVKPGAPVKLSCKASGYTFT KYWMNWMKQRPGRGLEWIGRIDPSDSETHYNQNFRDKATLTVDKSSSTAYI QLSSLTSEDSAVYYCTRSGNYAGAMDYWGQGTSVTVSS;

[0042] SEQ ID No.4:

[0043] METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVS TSAYSYMHWYQQKPGQPPKLLIYVASNLESGVPARFSGSGSGTDFTLNIHPLE EEDAATYYCQHSRYLPWTTFGGGTKLEIK.

[0044] The present invention provides a drug for treating tumors, comprising the monoclonal antibody described in the above technical solution. In the present invention, the tumor preferably comprises human breast cancer and / or human cervical cancer. The present invention does not specifically limit the dosage form and preparation method of the drug, and those skilled in the art can follow the routine.

[0045] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for treating tumors. In the present invention, the tumor preferably includes human breast cancer and / or human cervical cancer. The present invention has no special restrictions on the dosage form and preparation method of the drug, and those skilled in the art can follow the routine.

[0046] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for inhibiting tumor cell growth. In the present invention, the tumor preferably includes human breast cancer and / or human cervical cancer. The present invention has no special restrictions on the dosage form and preparation method of the drug, and those skilled in the art can follow the routine.

[0047] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for inhibiting tumor cell migration. In the present invention, the tumor preferably includes human breast cancer and / or human cervical cancer. The present invention has no special restrictions on the dosage form and preparation method of the drug, and those skilled in the art can follow the routine.

[0048] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for inhibiting tumor cell cloning. In the present invention, the tumor preferably includes human breast cancer and / or human cervical cancer. The present invention does not specifically limit the dosage form and preparation method of the drug, and those skilled in the art can follow the routine.

[0049] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a drug for promoting tumor cell apoptosis. In the present invention, the tumor preferably includes human breast cancer and / or human cervical cancer. The present invention has no special restrictions on the dosage form and preparation method of the drug, and those skilled in the art can follow the routine.

[0050] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Preparation of hybridoma cell line secreting anti-EPF monoclonal antibody

[0053] 1. Preparation of recombinant EPF antigen

[0054] NCBI Reference Sequence: NM_174346.2 was used as the target. The gene was conserved in cattle, sheep, deer and other animals according to the GenBank database analysis, and could encode early pregnancy factor (EPF) protein. The recombinant protein rEPF antigen with the immunosuppressive peptide (SEQID No.6: FRDGDILGKYV) removed, srEPF with SUMO fusion tag and dimer srdEPF with fusion tag were prepared by molecular biology technology through conventional processes such as gene synthesis, vector construction, transformation and expression, identification and seed preservation, and protein purification. The expression products were shown in SDS-PAGE electrophoresis. Figure 1 , used as the immunogen and detection antigen for subsequent experiments. The amino acid sequence of the EPF is as shown in SEQ ID No. 5, and the srdEPF is formed by connecting two EPFs via a flexible linker (GGGGS) to increase its effective antigen epitopes and the probability of producing antibodies.

[0055] SEQ ID No.5:

[0056] MAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAV GSGSKGKGGEIQPVSVKVGDKVLLPEYGGTKVVLDDKDYFL FRDGDILGKY V D, the underlined part is the immunosuppressive peptide segment.

[0057] 2. Mouse Immunization

[0058] Female balb / c mice aged 6-8 weeks were immunized with rEPF and srdEPF respectively; the dose of 100 μg / mouse was mixed with an equal volume of Freund's complete adjuvant, emulsified completely, and multiple immunizations were performed subcutaneously in the peritoneum; two weeks later, half the dose was mixed with an equal volume of incomplete Freund's adjuvant, and mice were immunized in the same manner; 7-10 days later, the tails of mice were cut and blood was collected, and srEPF was digested and used as a detection antigen to detect serum titer. When the serum titer reached more than 4000, fusion was prepared; 3 days before fusion, mice were immunized by intraperitoneal injection without adjuvant, and the immunization dose was 100 μg / mouse.

[0059] 3. Hybridoma cell fusion and screening

[0060] The spleen of the immunized mouse was taken, and the mouse B lymphocytes were collected after grinding, dispersion and centrifugation. The cells were fused with PEG1450 at a ratio of 7:1 (B lymphocytes: SP2 / 0), placed in a 96-well cell culture plate using HAT medium, and cultured in a carbon dioxide incubator. On the 7th to 9th day after fusion, the HT medium was replaced for the first screening, and the immunogens rEPF and srdEPF were used for the second positive screening by indirect ELISA. Then, srEPF and EPF after enzyme digestion were used for the third screening by indirect ELISA. The hybridomas with high OD values ​​were subcloned by limited dilution cloning until all were positive. The cell lines were expanded and frozen. The OD values ​​of the culture supernatants of the relevant monoclonal cell lines and the reactions with the recombinant proteins are shown in the following table. Figure 2 .Depend on Figure 2 It can be seen that among the monoclonal cell lines B6, F6, and F8 obtained using rEPF as the immunogen, the OD value of the reaction between B6 and each recombinant protein is relatively high, and among the monoclonal cell lines E6, H9, and D21 obtained using srdEPF as the immunogen, the OD value of the reaction between E6 and each recombinant protein is relatively high, indicating that B6 and E6 have strong binding abilities with various EPF proteins. Therefore, in this example, B6 and E6 monoclonal antibodies are selected as key experimental antibodies.

[0061] Example 2

[0062] Preparation of anti-EPF monoclonal antibody

[0063] 1. Ascites Preparation

[0064] Balb / c female mice aged about 10-12 weeks were selected, and each mouse was intraperitoneally injected with 500 μL of liquid paraffin. One week later, 1×10 6 After 5 days, the mice were continuously observed. When their abdomens were obviously swollen, the ascites was collected and centrifuged at 12000 rpm / min to remove impurities and then stored in a -80°C refrigerator.

[0065] 2. Antibody Purification

[0066] The ascites was purified by the caprylic acid-ammonium sulfate precipitation method. The ascites was removed and centrifuged at 12000rpm for 30min to remove the upper fat. Acetate buffer was added and the pH was adjusted to 4.5. Caprylic acid 11μL / mL was added dropwise while stirring. After 30min, it was allowed to stand for 2 hours. Centrifuged at 12000rpm for 30min and the precipitate was discarded. 1 / 10 volume of PBS was added to the supernatant and the pH was adjusted to 7.2. Slowly add the pre-prepared saturated ammonium sulfate to 45% on a 4℃ magnetic stirrer, stir for 30min, and let stand for 1 hour. Centrifuge at 12000rpm for 30min to retain the white precipitate, which was resuspended in PBS to obtain the purified EPF mAb.

[0067] Example 3

[0068] Monoclonal antibody identification

[0069] 1. Antibody Affinity Determination

[0070] Identification of affinity constant (Ka): The microplate was coated with EPF at mass concentrations of 0.5 and 1 mg / L, and the EPF mAb was diluted in multiples for indirect ELISA detection. The reaction curve was drawn and calculated according to the formula

[0071]

[0072] Where n = [Ag]t / [Ag′]t; [Ag]t and [Ag′]t represent the molar concentrations of the two antigen coatings; [Ab]t and [Ab′]t represent the maximum OD at two different coating concentrations. 450nm The antibody molar concentration corresponding to half the value.

[0073] The affinity of the 6 monoclonal antibodies screened was determined by indirect ELISA, and the OD 450nm The affinity curve was drawn by comparing the logarithmic value of the antibody concentration ( Figure 4 ), calculate OD 450nm The affinity constant is calculated according to the formula when the concentration of the binding antibody reaches 50%. Taking EPF mAb B6 as an example, when the coating antigen concentration is 0.5μg / mL and 1.0μg / mL, the formulas for [Ab]t and [Ab′]t are 1.35×10 -10 mol / L and 9.22×10 -11 mol / L, calculated Ka = 1.4 × 10 9 L / mol. Similarly, the affinity constants of F8, F6, E6, H9, and D21 were 1.2×10 9 L / mol, 8.7×10 8 L / mol, 2.8×10 7L / mol, 4.2×10 8 L / mol, 2.5×10 8 L / mol, and it is generally believed that Ka is between 10 7 ~10 12 L / mol, it is a high affinity antibody, and when Ka is less than 10 7 L / mol, it is a low-affinity antibody, so the 6 monoclonal antibodies obtained in this screening are all EPF mAbs with good affinity.

[0074] 2. Antibody subtype detection

[0075] The subtypes of six EPF monoclonal antibodies were identified using a mouse monoclonal antibody subtype kit (purchased from Luoyang Bio-Tong Experimental Materials Center). Figure 5 The results show that the OD of monoclonal antibodies is IgG1. 450nm The value was the highest, so it was determined that the six monoclonal antibodies were all of IgG1 subtype.

[0076] 3. Detection of antibodies, recombinant proteins and native proteins

[0077] The prokaryotically expressed recombinant proteins srEPF, EPF and tumor cell culture supernatant were mixed with loading buffer to prepare samples. After 15% SDS-PAGE electrophoresis, the proteins were transferred from the gel to a 0.22μM PVDF membrane using a wet transfer method with a reaction condition of 200mA and 1h30min. Blocked with 5% skim milk at room temperature for 2h, the prepared EPF B6 and E6 monoclonal antibodies were diluted 1:1000 and incubated at 4°C for 12h. The membrane was washed 3 times with TBST, each time for 10min. HRP-labeled goat anti-mouse IgG was diluted 1:5000 and incubated at 37°C for 1h. The membrane was washed four times with TBST and developed using M5 HiPer ECL Western HRP Substrate (ultra-sensitive ECL luminescent liquid). The results are shown in the figure. Figure 6 The results showed that the prepared B6 and E6 monoclonal antibodies could specifically bind to EPF recombinant protein and EPF natural protein in tumor cell culture supernatant (human breast cancer cell MCF-7, human cervical cancer cell HeLa).

[0078] Example 4

[0079] Amino acid sequence composition of monoclonal antibody variable region

[0080] Based on affinity, antibody titer, and reactivity with recombinant and natural antigens, hybridoma cell lines B6 and E6 were selected, and variable region sequencing was performed to retain cell and gene resources for antibody production.

[0081] Cell culture and RNA extraction

[0082] Take the cells out of the liquid nitrogen tank, resuscitate and expand the culture, and collect the cultured cells; Cell / Tissue Total RNA Isolation KitV2 kit instructions, extract cell RNA. Reverse transcription to obtain cDNA. Using the universal primers for the variable region of the IgG subtype mouse monoclonal antibody, the above cDNA was used as a template to perform PCR amplification of the variable region of the light chain and heavy chain, and the amplified products were identified by DNA gel, and the PCR products were recovered by gel cutting using the kit of TIANGEN. The above light chain and heavy chain PCR products were connected with 5minTA / Blunt-Zero Cloning Kit (C601-02), transformed into Top10 competent cells, and colony PCR was performed with M13F(-47) / M13R(-48) primers, and positive clones were selected for sequencing.

[0083] After sequencing, the sequence of the heavy chain variable region (VH) of the B6 monoclonal antibody is shown in SEQ ID No. 1, and the sequence of the light chain variable region (VL) is shown in SEQ ID No. 2;

[0084] B6 heavy chain amino acid sequence (136aa)

[0085]

[0086] B6 light chain amino acid sequence (127aa)

[0087]

[0088] After sequencing, the sequence of the heavy chain variable region (VH) of the E6 monoclonal antibody is shown in SEQ ID No. 3, and the sequence of the light chain variable region (VL) is shown in SEQ ID No. 4;

[0089] E6 heavy chain amino acid sequence (138aa)

[0090] MGWSCIILFLVATATGVHS QVQLQQPGAALVKPGAPVKLSCKASGYTF T KYWMNWMKQRPGRGLEWIG RIDPSDSETHYNQNFRDKATLTVDKSSSTAYI QLSSLTSEDSAVYYCTR SGNYAGAMDYWGQGTSVTVSS;

[0091] E6 light chain amino acid sequence (131aa)

[0092] METDTLLLWVLLLWVPGSTG DIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMH WYQQKPGQPPKLLIY VASNLESGVPARFSGSGSGTDFTLNIHPLEEE DAATYYC QHSRYLPWTFGGGTKLEIK.

[0093] The bold part is Leader sequence-, the underlined part is FR1, the bold italic part is CDR1, the italic part is FR2, the bold underlined part is CDR2, the double underlined part is FR3, the wavy line part is CDR3, and the rest is FR4.

[0094] Example 5

[0095] Analysis of the binding degree between monoclonal antibodies and EPF protein

[0096] This example uses a structure prediction strategy based on AlphaFold to perform computational biology analysis on the interaction between B6 and E6 monoclonal antibodies and EPF protein. AlphaFold is based on protein sequence information and integrates local and global features to achieve high-precision prediction of protein three-dimensional structure.

[0097] In this example, the AlphaFold algorithm was used to predict the three-dimensional structures of B6 and E6 monoclonal antibodies and EPF protein, respectively, and key prediction indicators including local residue confidence (pLDDT), interface prediction template modeling (ipTM), global prediction template modeling (pTM) and expected position error (EPE) were obtained, thereby systematically evaluating the configuration accuracy of the antibody-target protein binding interface in the complex and the reliability of the overall structure. These parameters include: pLDDT (predicted Local Distance Difference Test) local residue confidence score: reflects the confidence of each amino acid residue in the three-dimensional structure prediction. The higher the value, the more reliable the predicted position; ipTM (interface predicted template modeling score) interface prediction template modeling: quantitatively evaluates the local structural matching degree of the contact interface between the antibody and EPF protein. The higher the index, the better the prediction accuracy of the binding area; pTM (predicted template modeling score) global prediction template modeling: measures the similarity between the predicted overall protein structure and the actual conformation, providing a theoretical basis for subsequent functional verification; EPE (Expected Position Error) expected position error evaluation: indicates the expected deviation between the predicted residue position and its actual position. The smaller the error, the more accurate the prediction.

[0098] First, AlphaFold was used to analyze the B6 monoclonal antibody ( Figure 7 A), E6 monoclonal antibody ( Figure 7 Middle B) and EPF protein ( Figure 7The tertiary structure was predicted by C) and the reliability of the model was evaluated by the following parameters. The results showed that: pLDDT: The pLDDT value of most of the binding interface residues of B6 monoclonal antibody and EPF protein was >90 ( Figure 7 The blue area in D), the prediction error of the main chain (Cα atom) is less than It is close to the crystallographic resolution accuracy, indicating that the prediction confidence in this area is high and the structural stability is good. ipTM = 0.64: ​​The interface topology matching degree is above the medium level (ipTM>0.5 is considered reliable), indicating that the spatial arrangement of the interface residues is reasonable, but local conformations such as side chain orientations still have room for optimization. pTM: pTM = 0.72: It shows that the overall folding of the complex has a high similarity to the natural conformation (pTM>0.5 is considered a credible model), reflecting that the overall structural topology accuracy of the B6 monoclonal antibody-EPF complex is high, supporting subsequent functional studies (such as mutation verification, molecular docking). In the EPE heat map, the prediction errors in the green area are all less than ( Figure 7 The green area in D indicates that the relative spatial positioning deviation between B6 monoclonal antibody and EPF protein in the interface area is low, and the interface structure is highly correlated. Based on the above scoring results, the docking structure of B6 monoclonal antibody and EPF protein was visualized and analyzed. The results showed that the two formed a reasonable complex conformation, and the protein tertiary structure was topologically complete. Among them, the heavy chain of B6 monoclonal antibody is marked in green, the light chain is marked in yellow, and the EPF protein is marked in purple. (See Figure 7 Middle E)

[0099] Similarly, the binding of E6 monoclonal antibody to EPF protein was predicted and evaluated, and the results were as follows: pLDDT>90: Most regions have extremely high confidence ( Figure 7 The blue area in F indicates that the local structure prediction is stable. ipTM = 0.56: The interface matching is good, but there is still room for optimization. pTM = 0.67: The overall structure is relatively stable and is suitable as a basis for subsequent functional studies. EPE: The error in the binding area is small and the predicted position is accurate (see Figure 7 F). The complex formed by the two has a stable binding mode and presents a typical immunoglobulin folding structure. (See Figure 7 (G), in which the heavy chain of E6 monoclonal antibody is marked in green, the light chain is marked in yellow, and the EPF protein is marked in purple.

[0100] AlphaFold was used to predict the interaction between B6 monoclonal antibody and EPF protein. The results showed that the two had good binding characteristics, and various scores and energy analyses supported their stable binding. The prediction results can provide a reliable theoretical basis for subsequent functional verification, mutation experiments and drug design.

[0101] Example 6

[0102] Effect of EPF monoclonal antibody on cancer cell proliferation

[0103] Human cervical cancer HeLa cells and human breast cancer Mcf-7 cells in the logarithmic growth phase were taken and 5×10 3 Inoculate in 96-well plates and culture until cells adhere to the wall. Divide into blank group, control group, and experimental group. EPF-B6 and EPF-E6 monoclonal antibodies were added to the experimental group, and the working concentrations were 0.1mg / mL, 0.2mg / mL, 0.4mg / mL, 0.6mg / mL, and 0.8mg / mL. After culturing for 24 hours, the old culture medium was discarded and CCK8 working solution was added for incubation for 2 hours. The absorbance (OD) value of each group was measured at a wavelength of 450nm. Calculate the cell growth inhibition rate. Cell growth inhibition rate = [(Ac-As) / (Ac-Ab)] x100% (As: absorbance of the experimental group, Ac: absorbance of the control group, Ab: absorbance of the blank group).

[0104] The results showed that the growth inhibition rates of B6 monoclonal antibody at 0.2 mg / mL on HeLa cells and Mcf-7 cells were (21.84±3.58)% and (31.08±2.26)%, respectively; at 0.8 mg / mL, the growth inhibition rates of B6 monoclonal antibody at 0.8 mg / mL on HeLa cells and Mcf-7 cells were (77.88±3.64)% and (70.45±1.92)%, respectively. The difference between the two was significant (P<0.01)( Figure 8 (A). The growth inhibition rates of HeLa cells by E6 monoclonal antibody at 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL were (21.17±1.35)%, (42.92±0.98)%, (60.08±1.51)%, and (74.99±1.03)%. The growth inhibition rates of Mcf-7 cells at the above experimental concentrations were (17.13±0.57)%, (32.35±0.51)%, (48.50±1.18)%, and (63.15±0.07)%, respectively. The difference between the two was significant (P<0.01)( Figure 8 (middle B).

[0105] The above results indicate that both EPF-B6 and EPF-E6 monoclonal antibodies can significantly inhibit the growth of HeLa cells and Mcf-7 cells. With the increase of concentration, the cell growth inhibition rate significantly increases, and there are significant differences in the effects of B6 and E6 on HeLa cells and Mcf-7 cells.

[0106] Example 7

[0107] Effect of EPF monoclonal antibody on cancer cell migration

[0108] Human cervical cancer HeLa cells and human breast cancer Mcf-7 cells in the logarithmic growth phase were taken and 2×10 5 Cells were inoculated in a 6-well plate and cultured until the cell confluence was about 90%. A sterilized 200uL pipette tip was used to artificially create scratches in the center of the well. The cells were randomly divided into a control group and an experimental group. The experimental groups were added with low concentrations (0.1mg / mL), medium concentrations (0.4mg / mL), and high concentrations (0.8mg / mL) of EPF-B6 and EPF-E6 monoclonal antibodies, respectively. The cell migration state was observed under a microscope, fixed-point photos were taken, the cell migration distance was measured, and the cell migration rate was calculated. Cell migration rate = (initial scratch width - fixed-point scratch width) / initial scratch width × 100%.

[0109] The results showed that when B6 monoclonal antibody was co-cultured with HeLa cells at four experimental concentrations, control group, low, medium and high, the cell migration rates were (28.12±0.28)%, (13.72±0.13)%, (6.70±0.81)%, and (4.10±0.31)% for 24 hours; when co-cultured with HeLa cells for 48 hours, the cell migration rates were (41.92±0.58)%, (19.42±0.63)%, (11.78±0.29)%, and (6.88±0.67)% ( Fig. 9 When B6 and Mcf-7 cells were co-cultured for 24 h at the control group, low, medium, and high concentrations, the cell migration rates were (32.32±0.55)%, (7.04±0.25)%, (6.54±0.50)%, and (6.46±0.32)%, respectively. When B6 and Mcf-7 cells were co-cultured for 48 h, the cell migration rates were (41.10±0.21)%, (15.28±0.45)%, (11.66±0.18)%, and (9.74±0.42)%, respectively. Fig. 9 9 in B, 9 in C).

[0110] The above results show that B6 monoclonal antibody can significantly inhibit the migration of HeLa cells and Mcf-7 cells, and for HeLa cells, the cell migration rate significantly decreases with the increase of B6 monoclonal antibody concentration. For Mcf-7 cells, the cell migration rate is slightly different when compared with the three concentrations of low, medium and high. The reason may be that at low concentrations, B6 monoclonal antibody significantly inhibits its migration effect and reaches the migration threshold.

[0111] When E6 monoclonal antibody was co-cultured with HeLa cells at four experimental concentrations, control group, low, medium, and high, for 24 hours, the cell migration rates were (27.76±0.47)%, (20.42±0.16)%, (13.56±0.82)%, and (6.74±0.80)%. When co-cultured for 48 hours, the cell migration rates were (36.58±0.36)%, (30.18±0.36)%, (22.20±0.82)%, and (15.44±0.58)%. Fig.10 Figure 10 (A), Figure 10 (C). When E6 monoclonal antibody was co-cultured with Mcf-7 cells at four experimental concentrations, control group, low, medium, and high, for 24 hours, the cell migration rates were (31.90±0.45)%, (24.02±0.50)%, (17.88±0.40)%, and (12.34±0.23)%. When co-cultured for 48 hours, the cell migration rates were (41.28±0.49), (35.02±0.41), (20.22±0.26), and (18.34±0.49)%. Fig.10 10 (in B, 10 (in C).

[0112] When E6 monoclonal antibody acted on HeLa cells and Mcf-7 cells, there was a significant difference in the inhibitory effect on migration compared with the control group. The above results show that both B6 and E6 monoclonal antibodies can significantly inhibit the migration of HeLa cells and Mcf-7 cells, showing concentration and time dependence.

[0113] Example 8

[0114] Effect of EPF monoclonal antibody on cancer cell clone formation

[0115] HeLa cells and Mcf-7 cells in the logarithmic growth phase were inoculated in 6-well plates at 700 cells per well and divided into a control group and an experimental group. The experimental groups were added with low concentration (0.1 mg / mL), medium concentration (0.4 mg / mL), and high concentration (0.8 mg / mL) of EPF-B6 and EPF-E6 monoclonal antibodies, respectively. After 48 hours of culture, the old culture medium was discarded and replaced with ordinary culture medium for 10-14 days. When the number of cells formed in each clone was greater than 50 cells, the culture was terminated and fixed and stained. The cell clone formation rate was calculated. Cell clone formation rate = (number of clones formed / number of cells inoculated) × 100%.

[0116] The results showed that the clone formation rates of B6 monoclonal antibody on HeLa cells at four different experimental concentrations, namely, (78.76±0.73)%, (68.81±0.78)%, (37.33±0.81)%, (15.66±0.50)% ( Fig.11The clone formation rates of Mcf-7 cells were (75.71±1.17)%, (65.14±1.08)%, (38.57±0.52)%, and (15.29±0.80)% respectively. Fig.11 (B in 11, C in 11).

[0117] The clone formation rates of E6 monoclonal antibody on HeLa cells at four different experimental concentrations, namely, control group, low, medium, and high concentrations, were (73.33±0.50)%, (61.67±0.64)%, (38.19±0.58)%, and (24.29±0.57)%, respectively. Fig.12 The clone formation rates of Mcf-7 cells were (75.95±0.95)%, (62.48±0.68)%, (48.38±0.44)%, (23.00±0.89)% ( Fig.12 12 in B, 12 in C).

[0118] The above results indicate that both B6 and E6 monoclonal antibodies have a significant inhibitory effect on the clone formation of HeLa cells and Mcf-7 cells in a concentration-dependent manner, and the inhibitory effect of B6 monoclonal antibody on the clone formation of the two cells is better than that of E6 monoclonal antibody.

[0119] Example 9

[0120] Effect of EPF monoclonal antibody on apoptosis of cancer cells

[0121] HeLa cells and Mcf-7 cells in the logarithmic growth phase were prepared into single cell suspensions and inoculated into 6-well plates. The cells were cultured until they adhered to the wall and divided into control and experimental groups. The experimental groups were added with low concentration (0.1 mg / mL), medium concentration (0.4 mg / mL), and high concentration (0.8 mg / mL) of EPF-B6 and EPF-E6 monoclonal antibodies, respectively. After culturing for 48 hours, the old culture medium was discarded. The cells were collected with a cell scraper, washed with pre-cooled PBS and resuspended. 1×10 5 Cells were treated with Annexinv-FITC / PI double staining cell apoptosis kit. 5uL Annexinv-FITC1 and 10uLPI were added to 100uLBinding Baffer, the cells were gently mixed, incubated at room temperature in the dark for 15min, and the cell apoptosis rate was detected by cell flow cytometry.

[0122] The results showed that the apoptosis rates of HeLa cells in the control group, low, medium and high concentrations of B6 monoclonal antibody were 13.51%, 15.48%, 46.9% and 57.76% respectively. Fig.13 A), the apoptosis rates of Mcf-7 cells were 8.1%, 17.51%, 32.97%, and 56.47% respectively ( Fig.13 (middle B).

[0123] The apoptosis rates of HeLa cells in the control group, low, medium and high concentrations of E6 monoclonal antibody were 13.51%, 17.43%, 22.15% and 48.95% respectively. Fig.14 A), the apoptosis rates of Mcf-7 cells were 8.1%, 13.69%, 22.15%, and 51.73% respectively ( Fig.14 B). The apoptosis rate of the two cell lines was significantly different when the B6 monoclonal antibody was in the middle concentration (P<0.01) ( Fig.13 C), the apoptosis rates of the two cell lines were significantly different when the E6 monoclonal antibody was at low and high concentrations (P<0.01) ( Fig.14 Middle C).

[0124] The above results show that both B6 and E6 monoclonal antibodies can significantly promote the apoptosis of HeLa cells and Mcf-7 cells in a concentration-dependent manner, and there are differences in the apoptosis effects of B6 and E6 monoclonal antibodies on different cancer cells.

[0125] Example 10

[0126] Effects of EPF monoclonal antibody on cancer cell cycle

[0127] HeLa cells and Mcf-7 cells in the logarithmic growth phase were cultured at 2×10 5 The cells were inoculated in 6-well plates and cultured until the cells adhered to the wall. 0.4 mg / mL B6 and E6 monoclonal antibodies were added to the experimental group cells respectively. After culturing for 24 h, the cells were collected with a cell scraper, washed twice with PBS, added with 1 mL of 70% ethanol, and fixed at 4°C overnight. The cell cycle was detected by flow cytometry after using a cell cycle detection kit according to the instructions.

[0128] The results showed that the G0 / G1 phase of HeLa cells in the control group, B6 monoclonal antibody group, and E6 monoclonal antibody group were (62.14±0.99)%, (59.27±0.63)%, and (63.8±0.74)%, respectively; the S phase were (24.46±0.65)%, (18.20±0.48)%, and (19.04±0.27)%, respectively; the G2 / M phase were (12.76±0.58)%, (21.49±0.39)%, and (16.32±0.52)%, respectively. Fig.15Middle A). Compared with the control group, the proportion of G2 / M phase in the B6 monoclonal antibody group increased significantly, and the proportion of G0 / G1 phase and G2 / M phase in the E6 monoclonal antibody group increased significantly, indicating that the cell cycle of HeLa cells was blocked in the G2 / M phase of DNA synthesis when B6 monoclonal antibody acted on HeLa cells, and the cell cycle of HeLa cells was blocked in the G0 / G1 phase and G2 / M phase of DNA synthesis when E6 monoclonal antibody acted on HeLa cells. Compared with the control group, B6 monoclonal antibody group, and E6 monoclonal antibody group, the G0 / G1 phase was (47.47±0.68)%, (53.30±0.44)%, and (57.67±0.29)%, respectively; the S phase was (43.84±0.21)%, (27.18±0.44)%, and (23.39±0.47)%, respectively; the G2 / M phase was (8.52±0.51)%, (18.60±0.05)%, and (13.22±0.27)%, respectively. Fig.15 (B). Compared with the control group, the proportion of G0 / G1 and G2 / M phases in the B6 monoclonal antibody group increased significantly, and the proportion of G0 / G1 and G2 / M phases in the E6 monoclonal antibody group increased significantly, indicating that the cell cycle was blocked in the G0 / G1 and G2 / M phases of DNA synthesis when B6 and E6 monoclonal antibodies acted on Mcf-7 cells. The above results all indicate that B6 and E6 monoclonal antibodies have a significant effect on the DNA replication cycle of HeLa cells and Mcf-7 cells.

[0129] Cancer has become one of the major public health issues worldwide. In recent years, a large number of patients die from cancer every year worldwide. As the main killer threatening human survival, cancer seriously affects human health and life. With the deepening of molecular biology research and the progress of precision tumor treatment, molecular targeted drugs, as a new type of drug in recent years, play an increasingly important role in the treatment of cancer. Monoclonal antibodies are widely used in clinical research due to their strong specificity, high affinity and low toxicity.

[0130] The present invention utilizes early pregnancy factor monoclonal antibodies to co-culture with cancer cells in vitro to explore their effects on the malignant biological behaviors of tumor cells, and finds that both early pregnancy factor monoclonal antibodies B6 and E6 can significantly inhibit the proliferation, migration, cloning of HeLa cells and Mcf-7 cells and promote apoptosis of cancer cells, and compared with previous studies by scholars, these two monoclonal antibodies are more effective in inhibiting tumor proliferation and promoting apoptosis. The present invention further explores the cell cycle and finds that B6 and E6 monoclonal antibodies can significantly block the G0 / G1 phase and G2 / M phase of DNA synthesis in cancer cells, which indicates that B6 and E6 monoclonal antibodies promote apoptosis of cancer cells and are closely related to the early and late stages of DNA synthesis, but their specific mechanisms and pathways still need to be further explored.

[0131] The monoclonal antibody of early pregnancy factor can accurately identify and bind to the antigen on the surface of cancer cells, avoiding damage to normal cells and reducing side effects during treatment. This experiment provides new ideas and targets for the application of monoclonal antibody of early pregnancy factor in clinical treatment of cancer, and provides a theoretical basis for the development of new antibodies and antibody-drug conjugates, which has a very broad application prospect and important significance.

[0132] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that including monoclonal antibody B6 or monoclonal antibody E6; The amino acid sequence of the heavy chain variable region of the monoclonal antibody B6 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2; The amino acid sequence of the heavy chain variable region of the monoclonal antibody E6 is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

2. A drug for treating tumors, characterized in that: Comprising the monoclonal antibody according to claim 1.

3. The drug according to claim 2, characterized in that The tumors include human breast cancer and / or human cervical cancer.

4. Use of the monoclonal antibody according to claim 1 in the preparation of drugs for treating tumors.

5. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for inhibiting tumor cell growth.

6. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for inhibiting tumor cell migration.

7. Use of the monoclonal antibody according to claim 1 in the preparation of drugs for inhibiting tumor cloning.

8. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for promoting tumor cell apoptosis.

9. The use according to any one of claims 4 to 8, characterized in that: The tumors include human breast cancer and / or human cervical cancer.

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