A monoclonal antibody and its use in the preparation of a medicament for treating tumors

Abstract

The application relates to the technical field of tumor treatment, in particular to a monoclonal antibody and application thereof in preparation of a tumor treatment drug. The monoclonal antibody comprises a monoclonal antibody B6 or a monoclonal antibody E6. The application uses early pregnancy factor monoclonal antibodies to be co-cultured with cancer cells in vitro, explores the influence of the early pregnancy factor monoclonal antibodies on the malignant biological behaviors of tumor cells, and finds that the two early pregnancy factor monoclonal antibodies B6 and E6 can significantly inhibit the proliferation, migration and clone formation of HeLa cells and Mcf-7 cells and promote the apoptosis of the cancer cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor treatment, in particular to a monoclonal antibody and application thereof in preparation of a tumor treatment drug. BACKGROUND

[0002] Early Pregnancy Factor (EPF) is a trace protein with immunosuppressive and growth regulatory effects, which was detected in the serum of pregnant mice by an Australian scholar 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 the expression of early pregnancy factor also exists in the serum of tumor patients, which not only promotes tumor progression as a growth factor, but also may simulate the maternal-fetal immune tolerance mechanism to help tumor cells evade immune clearance of the body.

[0003] Some scholars pointed out that EPF or EPF-like active substances in the serum of testicular cancer patients were positively expressed, while the healthy control group was negative, indicating that EPF has the potential value of a specific biomarker for early screening of testicular cancer. Some Chinese scholars measured the serum of 11 kinds of high-incidence tumor patients 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, but widely exists in various tumor patients and promotes the growth and proliferation of tumors, aggravates the patient's condition, and has a universal impact on the harm and influence of tumors. Therefore, it is necessary to use early pregnancy factor to prepare monoclonal antibodies for treating cancer. SUMMARY

[0004] In order to solve the above problems, the present application provides a monoclonal antibody and application thereof in preparation of a tumor treatment drug. The monoclonal antibodies B6 and E6 provided by the present application have inhibitory effects on the growth inhibition, migration and clone formation of human cervical cancer HeLa cells and human breast cancer Mcf-7 cells, and promote apoptosis, thereby providing a certain theoretical basis for treating cancer by using early pregnancy factor monoclonal antibodies.

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

[0006] The present application provides a monoclonal antibody, which comprises 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 as SEQ ID No. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 4.

[0009] The application further provides a medicine for treating tumors, comprising the monoclonal antibody in the above technical solution.

[0010] Preferably, the tumors include human breast cancer and / or human cervical cancer.

[0011] The application further provides the use of the monoclonal antibody in the above technical solution in the preparation of a medicine for treating tumors.

[0012] The application further provides the use of the monoclonal antibody in the above technical solution in the preparation of a medicine for inhibiting the growth of tumor cells.

[0013] The application further provides the use of the monoclonal antibody in the above technical solution in the preparation of a medicine for inhibiting the migration of tumor cells.

[0014] The application further provides the use of the monoclonal antibody in the above technical solution in the preparation of a medicine for inhibiting the cloning of tumor cells.

[0015] The application further provides the use of the monoclonal antibody in the above technical solution in the preparation of a medicine for promoting the apoptosis of tumor cells.

[0016] Preferably, the tumors include human breast cancer and / or human cervical cancer.

[0017] Beneficial effects:

[0018] The application uses early pregnancy factor monoclonal antibodies to co-culture with cancer cells in vitro to explore the influence of the early pregnancy factor monoclonal antibodies on the malignant biological behavior of tumor cells, and finds that the two early pregnancy factor monoclonal antibodies B6 and E6 can significantly inhibit the proliferation, migration, cloning formation of HeLa cells and Mcf-7 cells and promote the apoptosis of cancer cells. Compared with the previous scholars' research, the two monoclonal antibodies have better effects in inhibiting tumor proliferation and promoting apoptosis. The application further explores the cell cycle and finds that the B6 and E6 monoclonal antibodies can significantly block the G0 / G1 phase and G2 / M phase of DNA synthesis of cancer cells, which indicates that the B6 and E6 monoclonal antibodies promote the apoptosis of cancer cells and are closely related to the early and late stages of DNA synthesis, but the specific mechanism and path still need to be further explored. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

[0020] Figure 1Figure 1 SDS-PAGE analysis of rEPF expression. M: Protein Marker; 1: IPTG induced 15 μL; 2: IPTG induced 10 μL; 3: IPTG uninduced. Figure 2 SDS-PAGE analysis of srEPF expression. M: Protein Marker; 1: PET28a-EPF empty plasmid; 2: IPTG uninduced; 3: IPTG induced. Figure 3 SDS-PAGE analysis of srEPF purification and enzyme digestion. M: Protein Marker; 1: srEPF whole bacterial protein; 2: srEPF purified protein; 3: srEPF purified protein enzyme digestion; 4: EPF. Figure 4 SDS-PAGE analysis of srdEPF expression. M: Protein Marker; 1: uninduced control; 2-4: L-arabinose induced control; 5: lysis precipitate; 6: lysis supernatant. Figure 5 SDS-PAGE analysis of srdEPF purification. M: Protein Marker; 1: filtrate; 2: elution 1; 3: elution 2; 4: elution 3; 5: elution 4; 6: enzyme digestion product dEPF.

[0021] Figure 2 Figure 6 Indirect ELISA method for detecting hybridoma cell culture supernatant OD value.

[0022] Figure 3 Figure 7 SDS electrophoresis verification results of EPF mAb B6, E6 antibody purification. The light chain of the mAb is about 25 kD, and the heavy chain is about 50 kD. The obtained relatively pure monoclonal antibody can be used for subsequent experiments.

[0023] Figure 4 Figure 8 EPF mAb affinity curve.

[0024] Figure 5 Figure 9 EPF mAb subtype identification results.

[0025] Figure 6 Figure 10 WB specificity identification of mAb. A and B respectively show the reaction results of B6 antibody and E6 antibody with recombinant protein and enzyme-digested EPF. M: Protein marker. 1: srEPF. 2: EPF; C and D respectively show the reaction results of B6 antibody and E6 antibody with natural protein in tumor cell culture supernatant. M: Protein marker. 1: Human ovarian cancer cell A2780. 2: Human prostate cancer cell DU145. 3: Human hepatocellular carcinoma 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 7Prediction analysis results of the EPF mAb and EPF protein mutual combination; A is the AlphaFold predicted B6 single antibody structure, B is the AlphaFold predicted E6 single antibody structure, C is the AlphaFold predicted EPF protein structure, D is the AlphaFold predicted B6 and EPF complex formation score result, E is the AlphaFold predicted B6 and EPF complex visualization result, F is the AlphaFold predicted E6 and EPF complex visualization result, G is the stable binding mode of the formed complex, which presents a typical immunoglobulin fold structure;

[0027] Figure 8 B6 and E6 single antibody growth inhibition rates on HeLa cells and Mcf-7 cells;

[0028] Figure 9 B6 single antibody inhibits the migration of HeLa cells and Mcf-7 cells, A: B6 single antibody inhibits the migration distance of HeLa cells, B: B6 single antibody inhibits the migration distance of Mcf-7 cells, C: B6 single antibody inhibits the migration of HeLa cells and Mcf-7 cells;

[0029] Figure 10 E6 single antibody inhibits the migration of HeLa cells and Mcf-7 cells, A: E6 single antibody inhibits the migration distance of HeLa cells, B: E6 single antibody inhibits the migration distance of Mcf-7 cells, C: E6 single antibody inhibits the migration of HeLa cells and Mcf-7 cells;

[0030] Figure 11 B6 single antibody inhibits the colony formation of HeLa cells and Mcf-7 cells, A: B6 single antibody inhibits the colony formation number of HeLa cells, B: B6 single antibody inhibits the colony formation number of Mcf-7 cells, C: B6 single antibody inhibits the colony formation of HeLa cells and Mcf-7 cells;

[0031] Figure 12 E6 single antibody inhibits the colony formation of HeLa cells and Mcf-7 cells, A: E6 single antibody inhibits the colony formation number of HeLa cells, B: E6 single antibody inhibits the colony formation number of Mcf-7 cells, C: E6 single antibody inhibits the colony formation of HeLa cells and Mcf-7 cells;

[0032] Figure 13 B6 single antibody promotes the apoptosis of HeLa cells and Mcf-7 cells, A: B6 single antibody promotes the apoptosis of HeLa cells, B: B6 single antibody promotes the apoptosis of Mcf-7 cells, C: B6 single antibody promotes the apoptosis of HeLa cells and Mcf-7 cells;

[0033] Figure 14Apoptosis of HeLa cells and Mcf-7 cells promoted by E6 monoclonal antibody, A: Apoptosis of HeLa cells promoted by E6 monoclonal antibody, B: Apoptosis of Mcf-7 cells promoted by E6 monoclonal antibody, C: Apoptosis rates of HeLa cells and Mcf-7 cells to E6 monoclonal antibody;

[0034] Figure 15 Influences of B6 and E6 monoclonal antibodies on cell cycles of HeLa cells and Mcf-7 cells, A: Influences of B6 and E6 monoclonal antibodies on cell cycles of HeLa cells, B: Influences of B6 and E6 monoclonal antibodies on cell cycles of Mcf-7 cells; DETAILED DESCRIPTION

[0035] The present application 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, and the specificities are as follows:

[0036] SEQ ID No. 1:

[0037] MECSWILPFILSVTSGVYSLVQLQQSGAELARPGASVKLSCKASGYTFTNYWMQWVKQRPGQGLEWIGAIYPGDFDTRYTQKFKGKATLTADKSSNTAYMQLSSLASEDSAVYYCVRWGWGQGAYWGQGTTLTVSS;

[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 EEDAATYYCQHSRYLPWTFGGGTKLEIK.

[0044] The present application provides a medicament for treating tumor, comprising the monoclonal antibody according to the above technical solution. In the present application, the tumor preferably comprises human breast cancer and / or human cervical cancer. The present application does not have special limitation on the dosage form and preparation method of the medicament, which can be prepared according to the routine method by those skilled in the art.

[0045] The present application also provides the use of the monoclonal antibody according to the above technical solution in the preparation of a medicament for treating tumor. In the present application, the tumor preferably comprises human breast cancer and / or human cervical cancer. The present application does not have special limitation on the dosage form and preparation method of the medicament, which can be prepared according to the routine method by those skilled in the art.

[0046] The present application also provides the use of the monoclonal antibody according to the above technical solution in the preparation of a medicament for inhibiting the growth of tumor cells. In the present application, the tumor preferably comprises human breast cancer and / or human cervical cancer. The present application does not have special limitation on the dosage form and preparation method of the medicament, which can be prepared according to the routine method by those skilled in the art.

[0047] The present application also provides the use of the monoclonal antibody according to the above technical solution in the preparation of a medicament for inhibiting the migration of tumor cells. In the present application, the tumor preferably comprises human breast cancer and / or human cervical cancer. The present application does not have special limitation on the dosage form and preparation method of the medicament, which can be prepared according to the routine method by those skilled in the art.

[0048] The present application also provides the use of the monoclonal antibody according to the above technical solution in the preparation of a medicament for inhibiting the cloning of tumor cells. In the present application, the tumor preferably comprises human breast cancer and / or human cervical cancer. The present application does not have special limitation on the dosage form and preparation method of the medicament, which can be prepared according to the routine method by those skilled in the art.

[0049] The application also provides the use of the monoclonal antibody in the preparation of a drug for promoting tumor cell apoptosis. In the application, the tumor preferably includes human breast cancer and / or human cervical cancer. The application does not have special limitations on the dosage form and preparation method of the drug, and the skilled person in the art can prepare it according to the conventional method.

[0050] In order to further illustrate the application, the application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of the application.

[0051] Example 1

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

[0053] 1. Preparation of recombinant EPF antigen

[0054] Taking NCBI Reference Sequence: NM_174346.2 as a target, the gene is analyzed in the GenBank database to be conserved in animals such as cattle, sheep, and deer, and can encode early pregnancy factor (EPF) protein. The recombinant protein rEPF antigen with an immunosuppressive peptide segment (SEQ ID No. 6: FRDGDILGKYV) removed, srEPF with a SUMO fusion tag, and dimeric srdEPF with a fusion tag are prepared by using molecular biology techniques through the conventional processes of gene synthesis, vector construction, transformation and expression, identification and preservation, and protein purification. Figure 1 , which are used as subsequent immunogens and detection antigens. The EPF amino acid sequence is SEQ ID No. 5, and the srdEPF is formed by connecting two EPFs through a flexible linker (GGGGS) to increase the effective antigen epitopes and the probability of producing antibodies.

[0055] SEQ ID No. 5:

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

[0057] 2. Mouse immunization

[0058] Six- to eight-week-old female BALB / c mice were immunized with rEPF and srdEPF, respectively. A 100 μg dose per mouse was mixed with an equal volume of complete Freund's adjuvant, emulsified, and administered subcutaneously at multiple sites. Two weeks later, half the dose was mixed with an equal volume of incomplete Freund's adjuvant and used to immunize mice. Seven to ten days later, the mice were tail-chopping and bled. The srEPF was enzymatically digested and used as a test antigen for serum titer testing. When the serum titer reached 4000 or higher, the mice were ready for fusion. Three days prior to fusion, mice were immunized intraperitoneally without adjuvant at a dose of 100 μg per 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. HT medium was replaced for the first screening on the 7-9th day after fusion. The immunogens rEPF and srdEPF were used for the second positive screening by indirect ELISA. The srEPF and the enzyme-cut EPF 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 reacted with the recombinant proteins as shown below. 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, B6 had higher OD values ​​for the reaction with each recombinant protein. Among the monoclonal cell lines E6, H9, and D21 obtained using srdEPF as the immunogen, E6 had higher OD values ​​for the reaction with each recombinant protein, indicating that B6 and E6 had strong binding abilities with various EPF proteins. Therefore, in this example, B6 and E6 monoclonal antibodies were selected as key experimental antibodies.

[0061] Example 2

[0062] Preparation of anti-EPF monoclonal antibodies

[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 became noticeably swollen, the ascites was collected and centrifuged at 12,000 rpm / min to remove impurities. The ascites was then stored in a -80°C refrigerator.

[0065] 2. Antibody Purification

[0066] Ascites was purified using the caprylic acid-ammonium sulfate precipitation method. The ascites was removed and centrifuged at 12,000 rpm for 30 minutes to remove the upper fat. Acetate buffer was added and the pH was adjusted to 4.5. Caprylic acid 11 μL / mL was added dropwise while stirring. After 30 minutes, the mixture was allowed to stand for 2 hours. Centrifuged at 12,000 rpm for 30 minutes and the precipitate was discarded. 1 / 10 volume of PBS was added to the supernatant and the pH was adjusted to 7.2. Pre-prepared saturated ammonium sulfate was slowly added to the supernatant on a 4°C magnetic stirrer to a saturation of 45%, stirred for 30 minutes, and allowed to stand for 1 hour. Centrifuged at 12,000 rpm for 30 minutes 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 EPF mAb was diluted in series for indirect ELISA detection. The reaction curve was drawn and the affinity constant was 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. Taking EPF mAb B6 as an example, when the coating antigen concentration is 0.5μg / mL and 1.0μg / mL, [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 x 10 8 L / mol, 2.5 x 10 8 L / mol, and it is generally considered that Ka is between 10 7 ~ 10 12 L / mol, and when Ka is less than 10 7 L / mol, it is a low affinity antibody, so the six monoclonal antibodies prepared in this screening all belong to EPF mAb with good affinity.

[0074] 2. Antibody subtype detection

[0075] The six EPF monoclonal antibodies were identified by using mouse monoclonal antibody subtype kit (purchased from Luoyang Baoao Tong Experimental Material Center). The results are shown in Figure 5 , the OD 450nm value of monoclonal antibody is the highest when the subtype is IgG1, so the six monoclonal antibodies are all IgG1 subtype.

[0076] 3. Detection of antibody and recombinant protein and natural protein

[0077] The prokaryotic expressed recombinant protein srEPF, EPF and tumor cell culture supernatant were mixed with sample loading buffer to prepare samples, and after 15% SDS-PAGE electrophoresis, the proteins were transferred from the gel to 0.22 μM PVDF membrane by using wet transfer method at 200 mA for 1 h 30 min. 5% skim milk was blocked at room temperature for 2 h, and the prepared EPF B6, E6 monoclonal antibody was diluted 1:1000, and incubated at 4°C for 12 h. The membrane was washed with TBST for 3 times, each time for 10 min. The HRP labeled goat anti-mouse IgG was diluted 1:5000, and incubated at 37°C for 1 h. The membrane was washed with TBST for four times, and developed using M5 HiPer ECL Western HRP Substrate (ultra-sensitive ECL luminescent solution). The results are shown in Figure 6 , the prepared B6, E6 monoclonal antibodies can specifically bind to EPF recombinant protein and natural protein in tumor cell culture supernatant (human breast cancer cell MCF-7, human cervical cancer cell HeLa).

[0078] Example 4

[0079] Monoclonal antibody variable region amino acid sequence composition

[0080] After affinity, antibody titer, and reaction with recombinant antigen and natural antigen, the hybridoma cell strains B6 and E6 were selected, and the variable region was sequenced to reserve cell and gene resources for antibody production.

[0081] Cell culture and RNA extraction

[0082] Take the cells out of the liquid nitrogen tank, recover the cells, expand the culture, and collect the cultured cells; Cell RNA was extracted according to the Cell / Tissue Total RNA Isolation Kit V2 kit instructions. Reverse transcription was performed to obtain cDNA. Using universal primers for the variable region of IgG subtype mouse monoclonal antibodies and the above cDNA as a template, the variable regions of the light and heavy chains were PCR amplified. The amplified products were identified by DNA gel analysis and the PCR products were recovered by gel excision using the TIANGEN kit. The above light and heavy chain PCR products were ligated with the 5minTA / Blunt-Zero Cloning Kit (C601-02), transformed into Top10 competent cells, and colony PCR was performed using M13F(-47) / M13R(-48) primers. Positive clones were selected for sequencing.

[0083] Sequencing revealed that 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] Sequencing revealed that 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 DIVLTQSPASLAVSLGQRATISCRASKSVS TSAYSYMH WYQQKPGQPPKLLIY VASNLESGVPARFSGSGSGTDFTLNIHPLEEE DAATYYC QHSRYLPWTFGGGTKLEIK.

[0093] wherein the bold part is a 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 degree of mutual binding of monoclonal antibodies to EPF protein

[0096] In this example, the AlphaFold-based structure prediction strategy is used 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 three-dimensional structures of B6 and E6 monoclonal antibodies and EPF protein are predicted using the AlphaFold algorithm, and key prediction indicators including local residue confidence (pLDDT), interface prediction template modeling (ipTM), global prediction template modeling (pTM), and expected position error (EPE) are obtained, so as to systematically evaluate the configuration accuracy of the binding interface of the antibody and the target protein 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 three-dimensional structure prediction, and the higher the value, the more reliable the predicted position; ipTM (interface predicted template modeling score) interface prediction template modeling: quantitatively evaluates the local structure matching degree of the contact interface of the antibody and the EPF protein, and the higher the index, the better the prediction accuracy of the binding region; pTM (predicted template modeling score) global prediction template modeling: measures the similarity between the predicted overall protein structure and the real conformation, and provides a theoretical basis for subsequent functional verification; EPE (Expected Position Error) expected position error evaluation: represents the expected deviation between the predicted residue position and its actual position, and the smaller the error, the more accurate the prediction.

[0098] First, the AlphaFold is used to predict the three-dimensional structure of B6 monoclonal antibody (A), E6 monoclonal antibody (B) and EPF protein (C) respectively. Figure 7 Figure 7 Figure 7 ​​C) tertiary structure prediction was performed, and the reliability of the model was evaluated by the following parameters. The results show that: pLDDT: The pLDDT value of most of the interface residues of B6 mAb and EPF protein is > 90 Figure 7 D Blue area), the main chain (Cα atom) prediction error is less than close to the resolution accuracy of crystallography, indicating that the prediction confidence of this area is high and the structural stability is good. ipTM = 0.64: The interface topological 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 the local conformation such as side chain orientation still has optimization space. pTM: pTM = 0.72: indicates that the overall folding of the complex has high similarity with the native conformation (pTM > 0.5 is considered as a reliable model), reflecting that the overall structure topological accuracy of B6 mAb-EPF complex is high, supporting the subsequent functional research (such as mutation verification, molecular docking). In the EPE heat map, the prediction error of the green area is less than ( Figure 7 D Green area), indicating that the relative spatial positioning deviation of B6 mAb and EPF protein in the interface region is low, and the interface structure correlation is strong. Based on the above scoring results, the docking structure of B6 mAb and EPF protein was visualized and analyzed, and the results showed that the two formed a reasonable complex conformation, and the protein tertiary structure topology was complete. Among them, the heavy chain of B6 mAb is marked in green, the light chain is marked in yellow, and the EPF protein is marked in purple. (See Figure 7 E)

[0099] Similarly, the binding of E6 mAb and EPF protein was predicted and evaluated, and the results are as follows: pLDDT > 90: most of the regions have very high confidence Figure 7 F Blue area), indicating that the local structure prediction is stable. ipTM = 0.56: the interface matching degree is good, but there is still some optimization space. pTM = 0.67: the overall structure stability is high, and is suitable as the basis for subsequent functional research. EPE: the error of the binding region is small, and the predicted position is accurate (see Figure 7 F). The complex binding mode formed by the two is stable, showing a typical immunoglobulin folding structure. (See Figure 7 G), wherein the heavy chain of E6 mAb is marked in green, the light chain is marked in yellow, and the EPF protein is marked in purple.

[0100] The interaction between B6 mAb and EPF protein was predicted by AlphaFold, and the results showed that the two have good binding characteristics, and the scoring and energy analysis all support the stable binding, which can provide reliable theoretical basis for subsequent functional verification, mutation experiment and drug design.

[0101] Example 6

[0102] Effects 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 Cells were seeded in 96-well plates and cultured until adherent. The cells were divided into blank, control, and experimental groups. EPF-B6 and EPF-E6 monoclonal antibodies were added to the experimental groups at working concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL, respectively. After 24 hours of culture, the old culture medium was discarded and CCK8 working solution was added for 2 hours. The absorbance (OD) of each group was measured at a wavelength of 450 nm. The cell growth inhibition rate was calculated. Cell growth inhibition rate = [(Ac-As) / (Ac-Ab)] x 100% (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 groups 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 differences between the two groups were significant (P<0.01). Figure 8 Middle B).

[0105] The above results show 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 increased significantly, and there were significant differences in the effects of B6 and E6 on HeLa cells and Mcf-7 cells.

[0106] Example 7

[0107] Effects 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 seeded in a 6-well plate and cultured until the cell confluence reached approximately 90%. A scratch was then created in the center of the well using a sterilized 200 μL pipette tip. The cells were then randomly divided into a control group and an experimental group. The experimental groups were treated with EPF-B6 and EPF-E6 monoclonal antibodies at low (0.1 mg / mL), medium (0.4 mg / mL), and high (0.8 mg / mL) concentrations, respectively. Cell migration was observed under a microscope, photographed at designated locations, and the cell migration distance was measured to calculate the cell migration rate. Cell migration rate = (initial scratch width - designated 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, namely 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 it was co-cultured with HeLa cells at four experimental concentrations, the cell migration rates were (41.92±0.58)%, (19.42±0.63)%, (11.78±0.29)% and (6.88±0.67)% for 48 hours. Figure 9 A in middle, C in middle). When B6 was co-cultured with Mcf-7 cells at the control, low, medium, and high concentrations for 24 h, the cell migration rates were (32.32±0.55)%, (7.04±0.25)%, (6.54±0.50)%, and (6.46±0.32)%, respectively. When co-cultured with Mcf-7 cells 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. Figure 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 decreases significantly with the increase of B6 monoclonal antibody concentration. For Mcf-7 cells, the difference in cell migration rate among low, medium and high concentrations is small. The reason may be that at low concentrations, B6 monoclonal antibody significantly inhibits its migration effect and reaches the migration threshold.

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

[0112] E6 monoclonal antibody and Mcf-7 cells were co-cultured for 24 h at four experimental concentrations of control, low, medium, and high, and the cell migration rates were (31.90 ± 0.45) %, (24.02 ± 0.50) %, (17.88 ± 0.40) %, and (12.34 ± 0.23) %, respectively. The cell migration rates were (41.28 ± 0.49) %, (35.02 ± 0.41) %, (20.22 ± 0.26) %, and (18.34 ± 0.49) % after 48 h of co-culture

[0113] Example 8

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

[0115] HeLa cells and Mcf-7 cells in the logarithmic growth phase were inoculated into 6-well plates at 700 cells per well, and were 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 48 h of culture, the old culture medium was discarded and replaced with normal culture medium for continued culture for 10-14 days. When the number of cells in each colony was greater than 50 cells, the culture was terminated, and the cells were fixed and stained. The cell colony formation rate was calculated. Cell colony formation rate = (number of colonies formed / number of cells inoculated) x 100%.

[0116] The results showed that the colony formation rates of HeLa cells in the control group and at low, medium, and high concentrations of B6 monoclonal antibody were (78.76 ± 0.73) %, (68.81 ± 0.78) %, (37.33 ± 0.81) %, and (15.66 ± 0.50) %, respectively Figure 11The cloning formation rates of Mcf-7 cells were (75.71 ± 1.17) %, (65.14 ± 1.08) %, (38.57 ± 0.52) %, (15.29 ± 0.80) % (Medium A, 11C) respectively. Figure 11 Medium B, 11C).

[0117] The cloning formation rates of HeLa cells were (73.33 ± 0.50) %, (61.67 ± 0.64) %, (38.19 ± 0.58) %, (24.29 ± 0.57) % (Medium A, 12C) respectively. Figure 12 The cloning formation rates of Mcf-7 cells were (75.95 ± 0.95) %, (62.48 ± 0.68) %, (48.38 ± 0.44) %, (23.00 ± 0.89) % (Medium A, 12C) respectively. Figure 12 Medium B, 12C).

[0118] The above results show that B6 and E6 monoclonal antibodies both have a significant inhibitory effect on the cloning formation of HeLa cells and Mcf-7 cells, and the effect of B6 monoclonal antibody 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 taken and prepared into a single cell suspension and inoculated in a 6-well plate. After the cells were cultured to adhere to the wall, they were divided into a control group and an experimental group. The experimental group was 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 h of culture, the old culture medium was discarded. The cells were collected by a cell scraper, washed with pre-cooled PBS, resuspended, and 1 × 10 5 cells were collected and counted. The cells were treated with an Annexin v-FITC / PI double staining cell apoptosis kit. 5 uL of Annexin v-FITC and 10 uL of PI were added to 100 uL of Binding Baffer, and the cells were gently mixed and incubated at room temperature for 15 min in the dark. The apoptosis rate of the cells was detected by a cell flow cytometer.

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

[0123] The apoptosis rates of HeLa cells treated with E6 monoclonal antibody at four different concentrations (control, low, medium, and high) were 13.51%, 17.43%, 22.15%, and 48.95% (Medium A), respectively, and the apoptosis rates of Mcf-7 cells were 8.1%, 13.69%, 22.15%, and 51.73% (Medium B), respectively. Figure 14 Figure 14 The apoptosis rates of HeLa cells treated with B6 monoclonal antibody at four different concentrations (control, low, medium, and high) were 13.51%, 17.43%, 22.15%, and 48.95% (Medium A), respectively, and the apoptosis rates of Mcf-7 cells were 8.1%, 13.69%, 22.15%, and 51.73% (Medium B), respectively. Figure 13 Figure 14 The apoptosis rates of HeLa cells treated with B6 monoclonal antibody at four different concentrations (control, low, medium, and high) were 13.51%, 17.43%, 22.15%, and 48.95% (Medium A), respectively, and the apoptosis rates of Mcf-7 cells were 8.1%, 13.69%, 22.15%, and 51.73% (Medium B), respectively.

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

[0125] Example 10

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

[0127] HeLa cells and Mcf-7 cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 2x10 5 After 24 hours of culture, the cells were collected by cell scraping, washed twice with PBS, and then 1 mL of 70% ethanol was added for fixation at 4°C overnight. The cell cycle detection kit was used according to the instructions, and the cell cycle was detected by flow cytometry.

[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 was (62.14±0.99)%, (59.27±0.63)%, and (63.8±0.74)%, respectively, the S phase was (24.46±0.65)%, (18.20±0.48)%, and (19.04±0.27)%, respectively, and the G2 / M phase was (12.76±0.58)%, (21.49±0.39)%, and (16.32±0.52)%, respectively. Figure 15 ​​The proportion of G2 / M phase in the B6 monoclonal antibody group is significantly higher than that in the control group, and the proportion of G0 / G1 phase and G2 / M phase in the E6 monoclonal antibody group is significantly higher than that in the control group, which indicates that the cell cycle of HeLa cells is blocked in the G2 / M phase of DNA synthesis when the B6 monoclonal antibody acts on the HeLa cells, and the cell cycle of HeLa cells is blocked in the G0 / G1 phase and G2 / M phase of DNA synthesis when the E6 monoclonal antibody acts on the HeLa cells. Figure 15 The proportion of G0 / G1 phase and G2 / M phase in the B6 monoclonal antibody group is significantly higher than that in the control group, and the proportion of G0 / G1 phase and G2 / M phase in the E6 monoclonal antibody group is significantly higher than that in the control group, which indicates that the cell cycle of Mcf-7 cells is blocked in the G0 / G1 phase and G2 / M phase of DNA synthesis when the B6 monoclonal antibody and the E6 monoclonal antibody act on the Mcf-7 cells. The above results all indicate that the B6 monoclonal antibody and the E6 monoclonal antibody have a significant influence on the DNA replication cycle of HeLa cells and Mcf-7 cells.

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

[0130] The present application uses early pregnancy factor monoclonal antibodies to co-culture with cancer cells in vitro to explore the influence of the early pregnancy factor monoclonal antibodies on the malignant biological behavior of tumor cells, and finds that the two early pregnancy factor monoclonal antibodies B6 and E6 can significantly inhibit the proliferation, migration and clone formation of HeLa cells and Mcf-7 cells, and promote the apoptosis of cancer cells. Compared with the previous research of scholars, the two monoclonal antibodies have better effects in inhibiting tumor proliferation and promoting apoptosis. The present application further explores the cell cycle and finds that the B6 monoclonal antibody and the E6 monoclonal antibody can significantly block the G0 / G1 phase and the G2 / M phase of DNA synthesis of cancer cells, which indicates that the promotion of the B6 monoclonal antibody and the E6 monoclonal antibody on the apoptosis of cancer cells is closely related to the early stage and the late stage of DNA synthesis, but the specific mechanism and pathway still need to be further explored.

[0131] The monoclonal antibody of early pregnancy factor can accurately recognize and combine with the antigen on the surface of cancer cells, avoid damage to normal cells and reduce side effects in the treatment process. The experiment provides a new idea and target for the application of the monoclonal antibody of early pregnancy factor in clinical treatment of cancer, provides a theoretical basis for the development of new antibodies and antibody conjugated drugs, and has very broad application prospect and important significance.

[0132] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained without creativity on the basis of the present embodiments, which belong to the protection scope of the present application.

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: The monoclonal antibody according to claim 1.

3. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for treating tumors, wherein the tumor is human breast cancer and / or human cervical cancer.

4. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for inhibiting the growth of tumor cells, wherein the tumor is human breast cancer and / or human cervical cancer.

5. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for inhibiting tumor cell migration, wherein the tumor is human breast cancer and / or human cervical cancer.

6. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for inhibiting tumor cell colony formation, wherein the tumor is human breast cancer and / or human cervical cancer.

7. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for promoting apoptosis of tumor cells, wherein the tumor is human breast cancer and / or human cervical cancer.

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