CN119874900B - A monoclonal antibody and its application in preparing drugs for treating tumors - Google Patents

Abstract

The invention relates to the technical field of tumor treatment, in particular to a monoclonal antibody and application thereof in preparing a medicine for treating tumors. The monoclonal antibody comprises monoclonal antibody B6 or monoclonal antibody E6. The invention researches the influence of the early pregnancy factor monoclonal antibody on the malignant biological behavior of tumor cells by utilizing the in-vitro co-culture of the early pregnancy factor monoclonal antibody and the cancer cells, and discovers that the B6 and E6 two early pregnancy factor monoclonal antibodies can obviously inhibit proliferation, migration, clone formation and promote apoptosis of HeLa cells and Mcf-7 cells.

Description

Monoclonal antibody and application thereof in preparation of tumor treatment drugs

Technical Field

The invention relates to the technical field of tumor treatment, in particular to a monoclonal antibody and application thereof in preparing a medicine for treating tumors.

Background

Early pregnancy factor (EarlyPregnancyFactor, EPF) is a trace protein with immunosuppression and growth regulation effects detected by australian scholars in 1974 in pregnant mouse serum, and can protect embryos 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. Research shows that the early pregnancy factor is expressed in the serum of tumor patient, and the early pregnancy factor can be used as growth factor to promote tumor progress and simulate maternal-fetal immune tolerance mechanism to help tumor cell avoid immune elimination.

The scholars indicate that EPF or EPF-like active substances in the serum of testicular cancer patients are positively expressed, and the healthy control groups are negative, which proves that the EPF has potential value as a specific biomarker for early screening of testicular cancer. The serum of 11 tumor patients with higher incidence rate is measured by students in China, and EPF exists in high incidence rate cancers such as ovarian cancer, cervical cancer, melanoma, colorectal cancer, lymphoma, liver cancer and the like. This suggests that early pregnancy factors are not present alone in a tumor, are widely present in many tumor patients and promote the growth and proliferation of tumors, exacerbate the disease condition of the patient, and have a general detrimental effect on tumors. There is therefore a need for the use of early pregnancy factors for the preparation of monoclonal antibodies for the treatment of cancer.

Disclosure of Invention

In order to solve the problems, the invention provides a monoclonal antibody and application thereof in preparing medicines for treating tumors, and the monoclonal antibodies B6 and E6 provided by the invention discuss that the monoclonal antibodies have inhibiting effects on growth inhibition, migration and clone formation of human cervical cancer HeLa cells and human breast cancer Mcf-7 cells, promote apoptosis and provide a certain theoretical basis for treating cancers by using early pregnancy factor monoclonal antibodies.

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

the invention provides a monoclonal antibody, comprising a monoclonal antibody B6 or a monoclonal antibody E6;

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

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.

The invention also provides a medicine for treating tumors, which comprises the monoclonal antibody.

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

The invention also provides application of the monoclonal antibody in preparing a medicine for treating tumor.

The invention also provides application of the monoclonal antibody in preparation of a medicament for inhibiting tumor cell growth.

The invention also provides application of the monoclonal antibody in preparation of medicines for inhibiting tumor cell migration.

The invention also provides application of the monoclonal antibody in preparing a tumor cell cloning inhibiting medicament.

The invention also provides application of the monoclonal antibody in preparation of medicines for promoting apoptosis of tumor cells.

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

The beneficial effects are that:

The invention utilizes the in vitro co-culture of the early pregnancy factor monoclonal antibody and the cancer cells to explore the influence of the early pregnancy factor monoclonal antibody on the malignant biological behaviors of the cancer cells, discovers that the B6 and E6 monoclonal antibodies can obviously inhibit the proliferation, migration, clone formation and promote the apoptosis of HeLa cells and Mcf-7 cells, and compared with the research of early scholars, the two monoclonal antibodies have better effects on inhibiting the proliferation and promoting the apoptosis of the tumors, the invention further explores the cell cycle, discovers that the B6 and E6 monoclonal antibodies can obviously block the G0/G1 phase and the G2/M phase of the DNA synthesis of the cancer cells, and shows that the B6 and E6 monoclonal antibodies promote the apoptosis of the cancer cells to be closely related to the early stage and the later stage of the DNA synthesis, but the specific mechanism and the passage of the monoclonal antibodies still need to be further explored.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 shows recombinant EPF SDS-PAGE analysis, A.SDS-PAGE detection rEPF expression. M: protein Marker, 1: IPTG induction 15. Mu.L, 2: IPTG induction 10. Mu.L, 3: IPTG non-induction. B.SDS-PAGE detection srEPF expression. M: protein Marker, 1: PET28a-EPF empty plasmid, 2: IPTG non-induction, 3: IPTG induction. C.SDS-PAGE detection srEPF purification and digestion. M: protein Marker, 1: srEPF holoprotein, 2: srEPF purified protein, 3: srEPF purified protein digestion, 4: EPF.D.SDS-PAGE detection srdEPF expression. M: protein Marker, 1: non-induction control, 2-4:L-arabinose induction control, 5: cleavage precipitation, 6: cleavage supernatant E.SDS-PAGE detection srdEPF purification. M: protein Marker, 1: filtrate, 2: elution 4: 6; enzyme digestion product of 4: dEPF);

FIG. 2 shows the detection of OD values of hybridoma cell culture supernatants by an indirect ELISA method;

FIG. 3 is a diagram showing the result of SDS electrophoresis verification of EPF monoclonal antibodies B6 and E6, wherein the light chain of the monoclonal antibody is about 25kD, and the heavy chain of the monoclonal antibody is about 50kD, so that a purer monoclonal antibody can be obtained and can be used for subsequent experiments.

FIG. 4 is an EPF mAb affinity curve;

FIG. 5 shows EPF mAb subtype identification results;

FIG. 6 shows the specific identification of monoclonal antibody WB, A, B shows the reaction results of B6 antibody, E6 antibody, recombinant protein and digested EPF, M protein marker.1 srEPF.2 EPF, C, D shows the reaction results of B6 antibody, E6 antibody and natural protein in tumor cell culture supernatant, 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 Ishiwaka-H-12.6 human cervical cancer cell HeLa;

FIG. 7 shows the results of a prediction analysis of the interaction of EPF mAb and EPF protein, wherein A is AlphaFold the predicted B6 monoclonal antibody structure, B is AlphaFold the predicted E6 monoclonal antibody structure, C is AlphaFold the predicted EPF protein structure, D is AlphaFold the predicted B6 and EPF complex scoring result, E is AlphaFold the predicted B6 and EPF complex visualization result, F is AlphaFold the predicted E6 and EPF complex visualization result, G is the stable binding mode of the complex formed, and typical immunoglobulin folding structure is presented;

FIG. 8 shows the growth inhibition of HeLa cells and Mcf-7 cells by B6, E6 mab;

FIG. 9 shows migration inhibition of HeLa cells and Mcf-7 cells by B6 mAb, migration inhibition of HeLa cells by B6 mAb, migration inhibition of Mcf-7 cells by B6 mAb, migration inhibition of HeLa cells, mcf-7 cells by B6 mAb;

FIG. 10 shows migration inhibition of HeLa cells and Mcf-7 cells by E6 mAb, migration inhibition of HeLa cells by E6 mAb, migration inhibition of Mcf-7 cells by E6 mAb, migration inhibition of HeLa cells, mcf-7 cells by E6 mAb;

FIG. 11 shows the inhibition of clone formation of HeLa cells and Mcf-7 cells by B6 mAb, the inhibition of clone formation number of HeLa cells by B6 mAb, the inhibition of clone formation number of Mcf-7 cells by B6 mAb, and the inhibition rate of clone formation of HeLa cells and Mcf-7 cells by B6 mAb;

FIG. 12 shows the inhibition of clone formation of HeLa cells and Mcf-7 cells by E6 mAb, the inhibition of clone formation number of HeLa cells by E6 mAb, the inhibition of clone formation number of Mcf-7 cells by E6 mAb, and the inhibition of clone formation rate of HeLa cells and Mcf-7 cells by E6 mAb;

FIG. 13 shows that B6 mab promotes apoptosis of HeLa cells and Mcf-7 cells, A B6 mab promotes apoptosis of HeLa cells, B6 mab promotes apoptosis of Mcf-7 cells, and C B6 mab has apoptosis rate on HeLa cells and Mcf-7 cells;

FIG. 14 shows that E6 mab promotes apoptosis of HeLa cells and Mcf-7 cells, A that E6 mab promotes apoptosis of HeLa cells, B that E6 mab promotes apoptosis of Mcf-7 cells, C that E6 mab has apoptosis rate on HeLa cells, mcf-7 cells;

FIG. 15 is a graph showing the effect of B6, E6 mab on HeLa cells and the Mcf-7 cell cycle, A: the effect of B6, E6 mab on HeLa cell cycle, B: the effect of B6, E6 mab on Mcf-7 cell cycle;

Detailed Description

The invention provides a monoclonal antibody, which comprises a monoclonal antibody B6 or a monoclonal antibody E6, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody B6 is shown as SEQ ID No.1, the amino acid sequence of a light chain variable region is shown as SEQ ID No.2, the amino acid sequence of a heavy chain variable region of the monoclonal antibody E6 is shown as SEQ ID No.3, and the amino acid sequence of a light chain variable region is shown as SEQ ID No.4, and the specific steps are as follows:

SEQ ID No.1:

MECSWILPFILSVTSGVYSLVQLQQSGAELARPGASVKLSCKASGYTFTN YWMQWVKQRPGQGLEWIGAIYPGDFDTRYTQKFKGKATLTADKSSNTAYM QLSSLASEDSAVYYCVRWGWGQGAYWGQGTTLTVSS;

SEQ ID No.2:

MSVPTQVLGLLLLWLTGARCDIQMTQSSASQSASVGETVTITCRASENIY SYLAWFQQRQGKSPQLLIYNAETLAEGVPSRFSGSGSGTQFSLKINSLQPEDF GTYYCQHHYGSPWTFGGGTKLEIK;

SEQ ID No.3:

MGWSCIILFLVATATGVHSQVQLQQPGAALVKPGAPVKLSCKASGYTFT KYWMNWMKQRPGRGLEWIGRIDPSDSETHYNQNFRDKATLTVDKSSSTAYI QLSSLTSEDSAVYYCTRSGNYAGAMDYWGQGTSVTVSS;

SEQ ID No.4:

METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVS TSAYSYMHWYQQKPGQPPKLLIYVASNLESGVPARFSGSGSGTDFTLNIHPLE EEDAATYYCQHSRYLPWTFGGGTKLEIK.

The invention provides a medicine for treating tumors, which comprises the monoclonal antibody. In the present invention, the tumor preferably comprises human breast cancer and/or human cervical cancer. The invention is not particularly limited in the dosage form, the preparation method and the like of the medicament, and can be used by a person skilled in the art according to the conventional method.

The invention also provides application of the monoclonal antibody in preparing a medicine for treating tumor. In the present invention, the tumor preferably comprises human breast cancer and/or human cervical cancer. The invention is not particularly limited in the dosage form, the preparation method and the like of the medicament, and can be used by a person skilled in the art according to the conventional method.

The invention also provides application of the monoclonal antibody in preparation of a medicament for inhibiting tumor cell growth. In the present invention, the tumor preferably comprises human breast cancer and/or human cervical cancer. The invention is not particularly limited in the dosage form, the preparation method and the like of the medicament, and can be used by a person skilled in the art according to the conventional method.

The invention also provides application of the monoclonal antibody in preparation of medicines for inhibiting tumor cell migration. In the present invention, the tumor preferably comprises human breast cancer and/or human cervical cancer. The invention is not particularly limited in the dosage form, the preparation method and the like of the medicament, and can be used by a person skilled in the art according to the conventional method.

The invention also provides application of the monoclonal antibody in preparing a tumor cell cloning inhibiting medicament. In the present invention, the tumor preferably comprises human breast cancer and/or human cervical cancer. The invention is not particularly limited in the dosage form, the preparation method and the like of the medicament, and can be used by a person skilled in the art according to the conventional method.

The invention also provides application of the monoclonal antibody in preparation of medicines for promoting apoptosis of tumor cells. In the present invention, the tumor preferably comprises human breast cancer and/or human cervical cancer. The invention is not particularly limited in the dosage form, the preparation method and the like of the medicament, and can be used by a person skilled in the art according to the conventional method.

The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.

Example 1

Preparation of hybridoma cell lines secreting anti-EPF monoclonal antibodies

1. Preparation of recombinant EPF antigen

NM_174346.2 is taken as a target, the gene is analyzed by GenBank database to be conserved in animals such as cattle, sheep, deer and the like, early Pregnancy Factor (EPF) proteins can be encoded, and recombinant protein rEPF antigen with immune suppression peptide segment removed (SEQ ID No.6: FRDGDILGKYV), srEPF with SUMO fusion promotion tag and dimer srdEPF with fusion promotion tag are prepared by conventional processes such as gene synthesis, vector construction, transformation and expression, identification and seed preservation, protein purification and the like by utilizing molecular biology technology, and the expressed product SDS-PAGE electrophoresis is shown as figure 1, and is used as immunogen and antigen for detection in subsequent experiments. The EPF amino acid sequence is shown as SEQ ID No.5, and srdEPF is formed by connecting two EPFs through a flexible linker (GGGGS) so as to increase the effective epitope and increase the probability of antibody production.

SEQ ID No.5:

MAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAV GSGSKGKGGEIQPVSVKVGDKVLLPEYGGTKVVLDDKDYFLFRDGDILGKY VD, The underlined part is the immunosuppressive peptide fragment.

2. Immunization of mice

Female balb/c mice of 6-8 weeks old are immunized by rEPF and srdEPF respectively, mixed with equal volume of Freund's complete adjuvant according to the dosage of 100 mug/dose, emulsified completely, subcutaneously multipoint immunized in abdominal cavity, half dosage after two weeks mixed with equal volume of incomplete Freund's adjuvant, the mice are immunized equally, the mice are collected by tail cutting for 7-10 days, srEPF is cut by enzyme and then used as detection antigen to detect serum titer. Serum titer reaches over 4000, and the mice are subjected to intraperitoneal injection immunization 3 days before fusion, and the immunization dose is 100 mug/mouse without adjuvant.

3. Hybridoma cell fusion and screening

Taking spleen of immunized mice, grinding, dispersing and centrifuging, collecting B lymphocytes of the mice, performing cell fusion by using PEG1450 according to the proportion of 7:1 (B lymphocytes: SP 2/0), placing the cells in a 96-well cell culture plate by using a HAT culture medium, and placing the cells in a carbon dioxide incubator for culture. The HT culture medium is changed for the first screening on the 7 th to 9 th days after fusion, the immunogens rEPF and srdEPF are utilized for the second screening positive by an indirect ELISA method, the srEPF and the EPF after enzyme digestion are utilized for the third screening by an indirect ELISA method, and the hybridoma with high OD value is combined for subcloning to be positive. And (5) performing expansion culture and freezing storage on the cell strain. The OD values of the reaction of the culture supernatants of the related monoclonal cell lines with the recombinant proteins are shown in FIG. 2. As can be seen from fig. 2, in the monoclonal cell lines B6, F6 and F8 obtained by using rEPF as the immunogen, the OD value of the reaction between B6 and each recombinant protein was high, and in the monoclonal cell lines E6, H9 and D21 obtained by using srdEPF as the immunogen, the OD value of the reaction between E6 and each recombinant protein was high, which means that the binding capacity between B6 and E6 and each EPF protein was high, so in this example, the monoclonal antibodies B6 and E6 were selected as the key experimental antibodies.

Example 2

Preparation of anti-EPF monoclonal antibodies

1. Ascites preparation

Balb/C female mice with the age of about 10-12 weeks are selected, each mouse is injected with 500 mu L of liquid paraffin in an intraperitoneal mode, 1X 10 ⁶ hybridoma cells prepared in example 1 are injected in the intraperitoneal mode after one week, after 5 days, the mice are continuously observed, after the abdomen of the mice is obviously swelled, ascites are collected, centrifugation is carried out at 12000rpm/min, impurities are removed, and the mice are stored in a refrigerator at-80 ℃.

2. Antibody purification

And purifying the ascites by adopting an octanoic acid-ammonium sulfate precipitation method. Taking out the ascites, centrifuging at 12000rpm for 30min, and removing upper fat. Acetic acid buffer was added to adjust the pH to 4.5. The octanoic acid 11. Mu.L/mL was added dropwise with stirring, and after 30 minutes, the mixture was allowed to stand for 2 hours. High speed centrifugation at 12000rpm for 30min, the pellet was discarded. 1/10 volume of PBS was added to the supernatant, and the pH was adjusted to 7.2. Slowly adding pre-prepared saturated ammonium sulfate to make the saturation degree of the saturated ammonium sulfate 45% on a magnetic stirrer at the temperature of 4 ℃, stirring for 30min, and standing for 1 hour. And (3) centrifuging at a high speed of 12000rpm for 30min to leave a white precipitate, and re-suspending by PBS to obtain the purified EPF mAb.

Example 3

Monoclonal antibody identification

1. Antibody affinity assay

Affinity constant (Ka) identification, namely, EPF coating the micro-pore plate with mass concentration of 0.5 and 1mg/L respectively, performing indirect ELISA detection by dilution of EPF mAb, drawing a reaction curve, and calculating according to a formula

Wherein n= [ Ag ] t/[ Ag ' ] t, [ Ag ] t and [ Ag ' ] t represent the molar concentration of the two antigen coatings, and [ Ab ] t and [ Ab ' ] t represent the molar concentration of the corresponding antibody when the reaction reaches half of the highest OD _450nm value under two different coating concentrations.

Affinity determination was performed on the 6 monoclonal antibodies screened by indirect ELISA to obtain an affinity curve of OD _450nm values and the logarithmic value of antibody concentration (FIG. 4), the concentration of bound antibody was calculated when OD _450nm values reached 50%, and affinity constants were calculated according to the formula. Taking EPF mAb B6 as an example, when the concentration of the coating antigen is 0.5 mug/mL and 1.0 mug/mL, the [ Ab ] t and the [ Ab' ] t are respectively 1.35X10 ^-10 mol/L and 9.22X10 ^-11 mol/L according to the formula, ka=1.4X10 ⁹ L/mol is calculated, and similarly, the affinity constants of F8, F6, E6, H9 and D21 are respectively 1.2×10⁹L/mol、8.7×10⁸L/mol、2.8×10⁷L/mol、4.2×10⁸L/mol、2.5×10⁸L/mol, and are generally considered to be high affinity antibodies when Ka is 10 ⁷～10¹² L/mol, and when Ka is less than 10 ⁷ L/mol, the monoclonal antibodies of 6 strains prepared by the screening all belong to EPF mAbs with better affinities.

2. Antibody subtype detection

Six strains of EPF mab were subtype identified using the mouse mab subtype kit (purchased from the loyang baioton experimental materials center). As shown in FIG. 5, the OD _450nm was highest when the subtypes were IgG1, and thus, it was determined that the six monoclonal antibodies were all of the IgG1 subtype.

3. Detection of antibodies and recombinant proteins and native proteins

The prokaryotic expression recombinant protein srEPF, EPF and tumor cell culture supernatant are mixed with a loading buffer solution for sample preparation, and after 15% SDS-PAGE electrophoresis, the protein is transferred from the gel to a 0.22 mu M PVDF membrane by adopting a wet transfer mode under the reaction condition of 200mA and 1h for 30 min. Sealing 5% skim milk for 2h at normal temperature, diluting with prepared EPF B6 and E6 monoclonal antibodies by 1:1000 times, and incubating for 12h at 4 ℃. The TBST film was washed 3 times for 10min each. HRP-labeled goat anti-mouse IgG was diluted 1:5000-fold and incubated at 37℃for 1h. TBST was washed four times and developed using M5 HIPER ECL WESTERN HRP Substrate (hypersensitive ECL luminophore). As shown in FIG. 6, the prepared B6 and E6 monoclonal antibodies can specifically bind to EPF recombinant proteins and EPF natural proteins in tumor cell culture supernatants (human breast cancer cells MCF-7 and human cervical cancer cells HeLa).

Example 4

Amino acid sequence constitution of variable region of monoclonal antibody

Through affinity, antibody titer, reaction with recombinant antigen and natural antigen, hybridoma cell strains B6 and E6 are selected, variable region sequencing is carried out, and cell and gene resources are reserved for producing the antibody.

Cell culture and RNA extraction

Taking out cells from liquid nitrogen tank, resuscitating and culturing, collecting cultured cells, and mixing according to the following stepsCell/Tissue Total RNA Isolation KitV kit instructions for extracting cellular RNA. Reverse transcription to obtain cDNA. The variable region universal primer of the IgG subtype mouse monoclonal antibody is used, the cDNA is used as a template to carry out PCR amplification on the variable regions of the light chain and the heavy chain, the amplified products are identified by DNA gel, and the PCR products are recovered by gel cutting through a kit of TIANGEN company. The light chain and heavy chain PCR products were ligated with 5minTA/Blunt-Zero Cloning Kit (C601-02), top10 competent was transformed, colony PCR was performed with M13F (-47)/M13R (-48) primers, and positive clones were selected for sequencing.

Sequencing and detecting, wherein the sequence of a heavy chain variable region (VH) of the B6 monoclonal antibody is shown as SEQ ID No.1, and the sequence of a light chain variable region (VL) is shown as SEQ ID No. 2;

B6 heavy chain amino acid sequence (136 aa)

B6 light chain amino acid sequence (127 aa)

Sequencing and detecting, wherein the sequence of a heavy chain variable region (VH) of the E6 monoclonal antibody is shown as SEQ ID No.3, and the sequence of a light chain variable region (VL) is shown as SEQ ID No. 4;

e6 heavy chain amino acid sequence (138 aa)

MGWSCIILFLVATATGVHSQVQLQQPGAALVKPGAPVKLSCKASGYTF TKYWMNWMKQRPGRGLEWIGRIDPSDSETHYNQNFRDKATLTVDKSSSTAYI QLSSLTSEDSAVYYCTRSGNYAGAMDYWGQGTSVTVSS;

E6 light chain amino acid sequence (131 aa)

METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVS TSAYSYMHWYQQKPGQPPKLLIYVASNLESGVPARFSGSGSGTDFTLNIHPLEEE DAATYYCQHSRYLPWTFGGGTKLEIK.

Wherein the bold portion is a Leader sequence-, the underlined portion is FR1, the bold italic portion is CDR1, the italic portion is FR2, the bold underlined portion is CDR2, the double underlined portion is FR3, the wavy line portion is CDR3, and the rest is FR4.

Example 5

Analysis of the degree of interaction between monoclonal antibodies and EPF proteins

In this example, a AlphaFold-based structure prediction strategy was used to calculate the biological analysis of the interactions between B6, E6 monoclonal antibodies and EPF proteins. AlphaFold integrates local and global features based on protein sequence information, thereby realizing high-precision prediction of the three-dimensional structure of the protein.

In this embodiment, the three-dimensional structures of the B6 and E6 monoclonal antibodies and EPF proteins are predicted by AlphaFold algorithm, and key prediction indexes including local residue confidence (pLDDT), interface prediction template modeling (ipTM), global prediction template modeling (pTM) and Expected Position Error (EPE) are obtained, so that the configuration accuracy of the antibody-target protein binding interface in the complex and the reliability of the overall structure are systematically evaluated. The parameters comprise pLDDT (PREDICTED LOCAL DISTANCE DIFFERENCE TEST) local residue confidence scores, ipTM (INTERFACE PREDICTED TEMPLATE) interface prediction template modeling, quantitative evaluation of local structure matching degree of an antibody and EPF protein contact interface, higher index shows that the combination region prediction precision is better, pTM (PREDICTED TEMPLATE) global prediction template modeling, measurement of similarity between a predicted whole protein structure and a real conformation, theoretical basis for subsequent functional verification, EPE (ExpectedPosition Error) expected position error assessment, and prediction accuracy.

First, three-level structure prediction was performed on B6 mab (a in fig. 7), E6 mab (B in fig. 7), and EPF protein (C in fig. 7) using AlphaFold, respectively, and reliability of the model was evaluated by the following parameters. The results show that pLDDT B6 mab has a pLDDT value >90 (D blue region in FIG. 7) with most of the binding interface residues with EPF protein, and the prediction error of the backbone (C.alpha.atom) is less thanNear the accuracy of crystallographic resolution, this region is shown to have high confidence in prediction and good structural stability. ipTM =0.64 the degree of matching of the interface topology is at a level above medium (ipTM >0.5 is considered reliable), which means that the spatial arrangement of the interface residues is reasonable, but the local conformations such as side chain orientation still have space to be optimized. pTM: ptm=0.72: shows that the overall folding of the complex has a high similarity to the native conformation (pTM >0.5 is considered as a trusted model), reflecting a high topological accuracy of the overall structure of the B6 mab-EPF complex, supporting subsequent functional studies (e.g. mutation validation, molecular docking). In the EPE heatmap, the prediction error of the green region is smaller than that of the EPE heatmap(D green area in FIG. 7), it shows that the relative spatial positioning deviation of the B6 monoclonal antibody and the EPF protein in the interface area is lower, and the interface structure association is stronger. Based on the scoring result, the docking structure of the B6 monoclonal antibody and the EPF protein is visually analyzed, and the result shows that the docking structure and the EPF protein form a reasonable complex conformation, and the tertiary structure of the protein is topologically complete. Wherein, the heavy chain of B6 monoclonal antibody is marked green, the light chain is marked yellow, and EPF protein is marked purple. (see E in FIG. 7)

Similarly, the binding of E6 mab to EPF protein was predicted and evaluated as follows: pLDDT >90 with very high confidence in most regions (F blue region in FIG. 7), indicating stable local structure prediction. ipTM =0.56. Interface matching degree is good, but there is still a certain optimization space. ptm=0.67 overall structural stability is higher, suitable as the basis of subsequent functional study. EPE-the error in the binding region is small and the predicted position is accurate (see F in FIG. 7). The complex formed by the two has stable combination mode and presents a typical immunoglobulin folding structure. (see G in fig. 7), wherein the heavy chain of E6 mab is indicated in green, the light chain in yellow, and EPF protein in purple.

The interaction of B6 monoclonal antibody and EPF protein is predicted by AlphaFold, and the result shows that the B6 monoclonal antibody and the EPF protein have good combination characteristics, and each score and energy analysis support stable combination, and the prediction result can provide reliable theoretical basis for subsequent functional verification, mutation experiments and drug design.

Example 6

Effect of EPF monoclonal antibodies on proliferation of cancer cells

Taking human cervical cancer HeLa cells and human breast cancer Mcf-7 cells in logarithmic phase, inoculating 5×10 ³ cells per well into 96-well plate, and culturing until the cells adhere to wall. The groups were divided into blank group, control group and experimental group. EPF-B6 and EPF-E6 monoclonal antibodies are added into the experimental group, the working concentrations are 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL and 0.8mg/mL, after 24h of culture, the old culture medium is discarded, and CCK8 working solution is added for 2h of incubation. Absorbance (OD) values were measured for each group at a wavelength of 450 nm. Cell growth inhibition was calculated. Cell growth inhibition = [ Ac-As ]/(Ac-Ab) ] x100% (As: absorbance of experimental group, ac: absorbance of control group, ab: absorbance of blank group).

The results showed that the growth inhibition rates of B6 mab on HeLa cells and Mcf-7 cells at 0.2mg/mL were (21.84.+ -. 3.58)%, and (31.08.+ -. 2.26)%, respectively, and the growth inhibition rates of B6 mab on HeLa cells and Mcf-7 cells at 0.8mg/mL were (77.88.+ -. 3.64)%, and (70.45.+ -. 1.92)%, respectively, and the difference was significant (P < 0.01) (FIG. A). E6 mAb was found to have a growth inhibition ratio of (21.17.+ -. 1.35)%, of (42.92.+ -. 0.98)%, of (60.08.+ -. 1.51)%, of (74.99.+ -. 1.03)% to HeLa cells at 0.2mg/mL,0.4mg/mL,0.6mg/mL, and 0.8 mg/mL. 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) (B in fig. 8).

The results show that the EPF-B6 and EPF-E6 monoclonal antibodies can obviously inhibit the growth of HeLa cells and Mcf-7 cells, the cell growth inhibition rate is obviously increased along with the increase of the concentration, and the action effects of B6 and E6 on the HeLa cells and the Mcf-7 cells are obviously different.

Example 7

Effect of EPF monoclonal antibodies on cancer cell migration

Taking human cervical cancer HeLa cells and human breast cancer Mcf-7 cells in logarithmic growth phase, inoculating 2X 10 ⁵ cells per well into a 6-well plate, culturing until the cell fusion degree is about 90%, manually manufacturing scratches at the center of the well by using a sterilized 200uL gun head, and randomly dividing the cells into a control group and an experimental group. The experimental groups are respectively added with EPF-B6 and EPF-E6 monoclonal antibodies with low concentration (0.1 mg/mL), medium concentration (0.4 mg/mL) and high concentration (0.8 mg/mL), the cell migration state is observed under a lens, the fixed point is photographed, the cell migration distance is measured, and the cell mobility is calculated. Cell mobility= (initial scratch width-fixed point scratch width)/initial scratch width x 100%.

The results showed that B6 mab was co-cultured with HeLa cells for 24h at the control, low, medium and high concentrations of four experiments, with cell mobilities of (28.12±0.28)%, of (13.72±0.13)%, of (6.70±0.81)%, of (4.10±0.31)%, and of (41.92±0.58)%, of (19.42±0.63)%, of (11.78±0.29)%, of (6.88±0.67)% (a in fig. 9, of C) for 48 h. B6 and Mcf-7 cells were co-cultured at four experimental concentrations of control, low, medium and high for 24 hours, and the cell mobilities were (32.32.+ -. 0.55)%, (7.04.+ -. 0.25)%, (6.54.+ -. 0.50)%, and (6.46.+ -. 0.32)%, respectively, and were (41.10.+ -. 0.21)%, (15.28.+ -. 0.45)%, and (11.66.+ -. 0.18)%, and (9.74.+ -. 0.42)%, respectively, for 48 hours, and were co-cultured with Mcf-7 cells, respectively (B, 9, C in FIG. 9).

The results show that the B6 monoclonal antibody can obviously inhibit migration of HeLa cells and Mcf-7 cells, the cell mobility of the HeLa cells is obviously reduced along with the increase of the concentration of the B6 monoclonal antibody, and the difference of the cell mobility of the Mcf-7 cells is smaller compared with the difference of the cell mobility of the low, medium and high concentrations, probably because the B6 monoclonal antibody extremely obviously inhibits the migration effect of the B6 monoclonal antibody to reach the migration threshold value at the low concentration.

E6 mAb and HeLa cells were co-cultured at four experimental concentrations of control, low, medium and high for 24 hours, the cell mobilities were (27.76.+ -. 0.47), (20.42.+ -. 0.16), (13.56.+ -. 0.82), (6.74.+ -. 0.80)%, and (36.58.+ -. 0.36), (30.18.+ -. 0.36), (22.20.+ -. 0.82)%, and (15.44.+ -. 0.58)% (A, 10 in FIG. 10). E6 mAb and Mcf-7 cells were co-cultured at four experimental concentrations of control, low, medium and high for 24 hours, and the cell mobilities were (31.90.+ -. 0.45)%, (24.02.+ -. 0.50)%, (17.88.+ -. 0.40)%, and (12.34.+ -. 0.23)%, respectively, and were co-cultured for 48 hours, and were (41.28.+ -. 0.49), and (35.02.+ -. 0.41)%, and (20.22.+ -. 0.26)%, and (18.34.+ -. 0.49)% (B in FIG. 10, C).

When the E6 monoclonal antibody acts on HeLa cells and Mcf-7 cells, the migration inhibition effect is obviously different from that of a control group. The results show that both B6 and E6 monoclonal antibodies can obviously inhibit migration of HeLa cells and Mcf-7 cells, and show concentration and time dependence.

Example 8

Effect of EPF monoclonal antibodies on the formation of cancer cell clones

HeLa cells and Mcf-7 cells in logarithmic growth phase are taken, 700 cells per hole are inoculated into a 6-hole plate and are divided into a control group and an experimental group, the experimental group is respectively added with EPF-B6 and EPF-E6 monoclonal antibodies with low concentration (0.1 mg/mL), medium concentration (0.4 mg/mL) and high concentration (0.8 mg/mL), after 48 hours of culture, old culture medium is discarded, the culture medium is replaced by common culture medium to continue to be cultured for 10-14 days, and when the number of cells forming each clone is more than 50, the culture is stopped, and the fixed dyeing is carried out. The cell clone formation rate was calculated. Cell clone formation rate= (clone formation number/number of inoculated cells) ×100%.

The results showed that the cloning efficiency of B6 mab was (78.76 ±0.73)%, (68.81 ±0.78)%, (37.33 ±0.81)%, and (15.66±0.50)%, respectively, for HeLa cells at four different experimental concentrations of control, low, medium, and high (fig. 11 a, 11C). Cloning efficiency for Mcf-7 cells was (75.71.+ -. 1.17)%, (65.14.+ -. 1.08)%, respectively (38.57.+ -. 0.52)%, and (15.29.+ -. 0.80)% (C in FIGS. 11B and 11).

E6 mAb was cloned at four different concentrations of control, low, medium and high, respectively (73.33.+ -. 0.50)%, and (61.67.+ -. 0.64)%, and (38.19.+ -. 0.58)%, and (24.29.+ -. 0.57)% (C in FIG. 12A, 12), and (75.95.+ -. 0.95)%, and (62.48.+ -. 0.68)%, and (48.38.+ -. 0.44)%, and (23.00.+ -. 0.89)%, respectively, for Mcf-7 cells.

The results show that the B6 and the E6 monoclonal antibodies have remarkable inhibition effects on clone formation of HeLa cells and Mcf-7 cells, show concentration dependence, and have better inhibition effects on clone formation of the B6 monoclonal antibodies on the two cells than the E6 monoclonal antibodies.

Example 9

Effect of EPF monoclonal antibodies on apoptosis of cancer cells

HeLa cells and Mcf-7 cells in logarithmic growth phase are taken, prepared into single cell suspension, inoculated into a 6-hole plate, cultured until the cells adhere to the wall, and divided into a control group and an experimental group, wherein the experimental group is respectively added with EPF-B6 and EPF-E6 monoclonal antibodies with low concentration (0.1 mg/mL), medium concentration (0.4 mg/mL) and high concentration (0.8 mg/mL), and the old culture medium is discarded after 48 hours of culture. Cells were collected with a cell scraper, washed with pre-chilled PBS and resuspended, and 1X 10 ⁵ cells were harvested and counted and treated with Annexinv-FITC/PI double-stained apoptosis kit. 100uLBinding Baffer is added with 5uLAnnexinv-FITC1 and 10uLPI, the cells are gently mixed, incubated for 15min at room temperature and in a dark place, and the apoptosis rate is detected by a cell flow meter.

The results showed that the apoptosis rates of B6 mab were 13.51%, 15.48%, 46.9%, 57.76% for HeLa cells (a in fig. 13) and 8.1%, 17.51%, 32.97%, 56.47% for Mcf-7 cells, respectively, at four different experimental concentrations, low, medium, and high, respectively (B in fig. 13).

E6 mab was 13.51%, 17.43%, 22.15%, 48.95% apoptosis rate on HeLa cells (FIG. 14A) and 8.1%, 13.69%, 22.15%, 51.73% apoptosis rate on Mcf-7 cells (FIG. 14B) respectively, at four different experimental concentrations of control, low, medium and high. B6 mab showed a significant difference in apoptosis rate of two cells at medium concentration (P < 0.01) (C in fig. 13), and E6 mab showed a significant difference in apoptosis rate of two cells at low and high concentration (P < 0.01) (C in fig. 14).

The results show that both the B6 monoclonal antibody and the E6 monoclonal antibody can obviously promote apoptosis of HeLa cells and Mcf-7 cells, show concentration dependence, and have different apoptosis effects of the B6 monoclonal antibody and the E6 monoclonal antibody on different cancer cells.

Example 10

Effect of EPF monoclonal antibodies on cancer cell cycle

HeLa cells and Mcf-7 cells in logarithmic growth phase are taken and inoculated into a 6-hole plate according to 2X 10 ⁵ cells per hole, the cells are cultured until the cells are attached to the wall, the experimental group cells are respectively added with 0.4mg/mL of B6 monoclonal antibody and E6 monoclonal antibody, after 24 hours of culture, the cells are collected by a cell scraper, after PBS is used for cleaning twice, 1mL of 70% ethanol is added, the cells are fixed at 4 ℃ overnight, and the cell cycle is detected by a cell flow meter after the cell cycle detection kit is operated according to the description.

The results showed that the HeLa cell control group, the B6 mab group and the E6 mab group were (62.14.+ -. 0.99)%, (59.27.+ -. 0.63)%, and (63.8.+ -. 0.74)%, respectively, the S phase was (24.46.+ -. 0.65)%, and (18.20.+ -. 0.48)%, and (19.04.+ -. 0.27)%, and the G2/M phase was (12.76.+ -. 0.58)%, and (21.49.+ -. 0.39)%, and (16.32.+ -. 0.52)%, respectively, in the G0/G1 phase. Compared with the control group, the B6 monoclonal antibody group has significantly increased G2/M phase ratio, and the E6 monoclonal antibody group has significantly increased G0/G1 phase and G2/M phase ratio, which indicates that the cell cycle of the B6 monoclonal antibody is blocked in the G2/M phase and the cell cycle of the E6 monoclonal antibody is blocked in the G0/G1 phase and the G2/M phase of the DNA synthesis when acting on HeLa cells. The Mcf-7 cell control group, the B6 mab group and the E6 mab group were (47.47.+ -. 0.68)%, (53.30.+ -. 0.44)%, and (57.67.+ -. 0.29)%, respectively, the S phase was (43.84.+ -. 0.21)%, and (27.18.+ -. 0.44)%, and (23.39.+ -. 0.47)%, respectively, and the G2/M phase was (8.52.+ -. 0.51)%, and (18.60.+ -. 0.05)%, and (13.22.+ -. 0.27)%, respectively, in the G0/G1 phase (B in FIG. 15). Compared with the control group, the B6 monoclonal antibody group has significantly increased G0/G1 phase and G2/M phase, and the E6 monoclonal antibody group has significantly increased G0/G1 phase and G2/M phase, which indicates that the cell cycle is blocked in the G0/G1 phase and G2/M phase of DNA synthesis when the B6 and E6 monoclonal antibodies act on Mcf-7 cells. The above results all demonstrate that B6, E6 mab has a significant effect on DNA replication cycle in HeLa cells and Mcf-7 cells.

Cancer has become one of the major public health problems worldwide. In recent years, a large number of patients die each year worldwide from cancer, which is a major killer threatening human survival, severely affecting human health and life. With the progress of molecular biology research and tumor accurate treatment, molecular targeted drugs play an increasingly important role in the progress of cancer treatment as a brand new drug in recent years. Monoclonal antibodies are widely used in clinical studies due to their high specificity, high affinity, and low toxic effects.

The invention utilizes the in vitro co-culture of the early pregnancy factor monoclonal antibody and the cancer cells to explore the influence of the early pregnancy factor monoclonal antibody on the malignant biological behaviors of the cancer cells, discovers that the B6 and E6 monoclonal antibodies can obviously inhibit the proliferation, migration, clone formation and promote the apoptosis of HeLa cells and Mcf-7 cells, and compared with the research of early scholars, the two monoclonal antibodies have better effects on inhibiting the proliferation and promoting the apoptosis of the tumors, the invention further explores the cell cycle, discovers that the B6 and E6 monoclonal antibodies can obviously block the G0/G1 phase and the G2/M phase of the DNA synthesis of the cancer cells, and shows that the B6 and E6 monoclonal antibodies promote the apoptosis of the cancer cells to be closely related to the early stage and the later stage of the DNA synthesis, but the specific mechanism and the passage of the monoclonal antibodies still need to be further explored.

The early pregnancy factor monoclonal antibody can accurately identify and bind to 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 thought and target point for applying the early pregnancy factor monoclonal antibody to clinical treatment of cancers, provides a theoretical basis for development of novel antibodies and antibody coupling medicaments, and has very broad application prospect and important significance.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (7)

1. A monoclonal antibody comprising monoclonal antibody B6 or monoclonal antibody E6;

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

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.

2. A medicament for treating tumors comprising the monoclonal antibody of claim 1.

3. Use of the monoclonal antibody of claim 1 for the preparation of a medicament for the treatment of a tumor, said tumor being human breast cancer and/or human cervical cancer.

4. Use of the monoclonal antibody of claim 1 for the preparation of a medicament 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 of claim 1 for the preparation of a medicament for inhibiting tumor cell migration, wherein the tumor is human breast cancer and/or human cervical cancer.

6. Use of the monoclonal antibody of claim 1 for the preparation of a medicament for inhibiting the formation of clones of tumor cells, said tumor being human breast cancer and/or human cervical cancer.

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