CN115028720B

CN115028720B - Exosomes and use of inhibitors to increase drug sensitivity in the treatment of cancer

Info

Publication number: CN115028720B
Application number: CN202210747574.XA
Authority: CN
Inventors: 洪子涵; 戴伟亮
Original assignee: Chengdu Future Health Technology Co ltd
Current assignee: Chengdu Fuzhi Future Technology Co ltd
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2023-09-05
Anticipated expiration: 2042-06-28
Also published as: CN115028720A

Abstract

The present application relates to the use of exosomes and inhibitors to increase drug sensitivity in the treatment of cancer. The application provides a monoclonal antibody specific to CK8, which can remarkably improve drug sensitivity of tumor cells. After the CK8 monoclonal antibody is used together with exosomes loaded with chemical drugs, the proliferation of tumors can be effectively inhibited, and the monoclonal antibody has good application prospects and application values.

Description

Exosomes and use of inhibitors to increase drug sensitivity in the treatment of cancer

Technical Field

The present application relates to the field of biology, more specifically to exosomes and the use of inhibitors to increase drug sensitivity in the treatment of cancer.

Background

According to the latest data of the world health organization, breast cancer has replaced lung cancer as the first tumor worldwide, 42 thousands of breast cancer patients are newly increased in China each year, and the incidence rate is increased by 3% to 4% per year in recent years. Advances in the comprehensive treatment of breast cancer play an important role, wherein antibody drugs are important therapeutic means.

Molecular targeted treatment of breast cancer, the most successful of the moxideclizumab (herceptin) drug for metastatic breast cancer that is overexpressed by HER-2. Developed by the company roche flag gene tek. FDA approval of HER-2 over-expression for treatment of metastatic breast cancer at 9 th 1998 has been a first-line drug. The medicine can save about 1/4 of refractory breast cancer patients and prolong lives, the sales of 2012 can reach 68.39 hundred million dollars worldwide, the 8 th medicine is ranked to be the most popular worldwide, and meanwhile, the superhost position of the Roche company for developing herceptin in developing antibody medicines for treating HER-2 breast cancer is laid. A clinical test shows that the trastuzumab combined with the first-line chemotherapeutic drug anthracycline, cyclophosphamide or taxol has better effect and higher survival rate compared with the single use of the same chemotherapeutic drug, so that the recurrence risk of patients is reduced by 46% -52% and the death risk is reduced by 1/3-53. This novel adjuvant therapy greatly improves the quality of life of the patient. FDA approval at 6/8 2012 for the treatment of HER-2 positive metastatic breast cancer was developed by the company lozenges gene taek, which is a monoclonal antibody for breast cancer applied in clinic following trastuzumab, which is the 1 st monoclonal antibody called HER dimerization inhibitor. A clinical trial with trastuzumab and pertuzumab was ongoing, with 808 HER-2 positive metastatic breast cancer patients randomly assigned, with control group receiving placebo, trastuzumab plus paclitaxel, and pertuzumab group receiving pertuzumab, trastuzumab plus paclitaxel, to evaluate progression free survival and overall survival. Compared with a control group, the average progression-free survival time of the pertuzumab group is prolonged from 12.4 months to l8.5 months, and the result proves that the combination of the pertuzumab and the trastuzumab and the paclitaxel can be used as the first-line treatment of HER-2 positive metastatic breast cancer, so that the progression-free survival time can be remarkably prolonged without increasing toxic and side effects on the heart. Studies have also shown that bevacizumab and pertuzumab in combination also have an effect superior to that of the single drug. HER-2 is taken as an emerging target of breast cancer, is favored by a plurality of scholars, is taken as a direction of developing breast cancer medicaments in a dispute, and is important to find a new target in another way.

Endocrine therapy is one of the means of systemic treatment of breast cancer. Some endocrine therapeutic agents inhibit and reduce estrogen synthesis and binding to breast cancer cells, thereby blocking tumor signaling pathways, resulting in cancer cell death, which plays an important role in reducing breast cancer recurrence and mortality risk, further prolonging survival of patients. However, these drugs are easily metabolized and do not exert their effect for a long period of time.

Cytokeratin (CK) is a largest family of intermediate filaments, which are predominantly distributed in epithelial cells and are integral components of the epithelial cytoskeleton. Normal, different types of epithelial cells exhibit specific CK markers, and studies have demonstrated that these cells retain this marker property upon tumorigenesis. Thus CK is one of the epithelial markers widely used in tumor research and pathological differential diagnosis. Current studies indicate that inhibition of CK8 activity can inhibit cancer cell activity and thus be used in cancer treatment.

In recent years, research on exosome delivery drugs has been increasing, and some small molecule chemical drugs, gene drugs, etc. have been successfully loaded into exosomes. Exosomes combine the advantages of cell and nanotechnology delivery of drugs, with exosomes being easier to store and safer than cell therapies. Such as exosome delivery of gene drugs, does not deposit at different sites in the body resulting in immune rejection. In addition, exosomes can be separated from body fluid of a patient and are modified to move back to the same patient, so that the possibility of immune response in clinic is greatly reduced; exosome delivery of drugs may improve the stability of the drug. For example, exosomes may protect nucleic acids from hydrolysis by ribozymes during transport. Meanwhile, exosomes can directly enter cell sap to avoid metabolic elimination, so that the circulation time of the medicine in the body is prolonged; the exosomes are nanoscale molecules and carry cell surface substances, so that the exosomes have strong capacity of penetrating various biological barriers; exosomes have a natural targeting ability based on donor cells. For example, the exosomes derived from tumor cells carry tumor specific antigens, proteins, RNA and other substances, and can play an anti-tumor immunity role.

At present, the treatment means for treating breast cancer by adopting exosome loaded medicines are not abundant, and alternative approaches are not enough, so that the method is worthy of further research.

Disclosure of Invention

The application overcomes the defects of the prior art and provides a medicine capable of effectively improving the drug sensitivity in the treatment of breast cancer.

In one aspect, a monoclonal antibody that specifically binds CK8 is provided, which antibody is capable of increasing the drug sensitivity of cancer cells.

Further, the CK8 monoclonal antibody of the application is a CK8-4D13 monoclonal antibody, and the light chain variable region sequence and the heavy chain variable region sequence of the monoclonal antibody are shown as follows:

the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of which is EVQLVESGGGLVQPGGSLRLSCAASGFSLSQYRQKWVRQAPGKGLEWVGISLWPALAGDCMISYFRFTISKDNSKNTLYLQMNSLRAEDTAVYYCARTFMRTSYIYTCCEWGQGTLVTVSS;

the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2, the amino acid sequence is AYQMTQSPSSVSASVGDRVTITCCGTVNKHKTRAWYQQKPGKAPKLLIYMKGYRACGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLRTTYYLGREDIVFGGGTKVEIK.

The amino acid sequence of CDR-H1 (in this specification CDR-H1 represents heavy chain CDR 1) is shown in QYRQK;

the amino acid sequence of CDR-H2 (in this specification CDR-H2 represents heavy chain CDR 2) is shown as ISLWPALAGDCMISYF;

the amino acid sequence of CDR-H3 (in this specification CDR-H3 represents heavy chain CDR 3) is shown as TFMRTSYIYTCCE;

the amino acid sequence of CDR-L1 (in this specification CDR-L1 represents light chain CDR 1) is shown as CGTVNKHKTRA;

the amino acid sequence of CDR-L2 (in this specification CDR-L2 represents light chain CDR 2) is shown as MKGYRAC;

the amino acid sequence of CDR-L3 (in this specification CDR-L3 represents light chain CDR 3) is shown as LRTTYYLGREDIV.

Furthermore, the application provides application of the CK8 monoclonal antibody CK8-4D13 in preparing a medicament for treating breast cancer, wherein the CK8 monoclonal antibody CK8-4D13 can improve the medicament sensitivity of drug-resistant tumor cells.

Furthermore, the application provides application of the CK8 monoclonal antibody CK8-4D13 and exosomes loaded with chemical anticancer drugs in preparation of drugs for treating breast cancer, wherein the CK8 monoclonal antibody CK8-4D13 can improve drug sensitivity of drug-resistant tumor cells.

Further, the exosomes are prepared by separating from the supernatant of the tumor cell culture fluid, the loading is prepared by mixing an equivalent amount of exosomes with the chemical anticancer drug, preparing the chemical anticancer drug loaded exosomes by an electroswitching method, the electric shock condition is that the voltage is 400V, the electric switching is carried out by a capacitor 125uF and a 4mm electroswitching cup, and then the separation and nonspecific binding of the chemical anticancer drug are removed by filtering with an inverted centrifugal ultrafiltration membrane.

Further, the anticancer drug of the present application is mitoxantrone, topotecan or daunorubicin.

Furthermore, the application provides application of the CK8 monoclonal antibody CK8-4D13 and the BCRP inhibitor in preparing medicaments for treating breast cancer, wherein the CK8 monoclonal antibody CK8-4D13 and the BCRP inhibitor can improve the medicament sensitivity of drug-resistant tumor cells.

Further, the medicament of the application further contains mitoxantrone, topotecan or daunorubicin.

Further, the BCRP inhibitor is KS176, and the structure of the BCRP inhibitor is shown in formula 1:

further, the medicament of the application also contains a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition may include one or more pharmaceutical excipients or carriers. The "pharmaceutical excipient or carrier" may be any suitable ingredient (e.g., suitable for use in a drug, for a dosage of a drug, for timing of drug release, for the disease state, or for the route of delivery) including, but not limited to, water (e.g., boiled water, distilled water, filtered water, pyrogen-free water, or water containing chloroform), sugar (e.g., sucrose, glucose, mannitol, sorbitol, xylitol, or syrups made therefrom), ethanol, glycerol, glycols (e.g., propylene glycol), acetone, ethers, DMSO, surfactants (e.g., ethanol, glycerol, glycols, acetone, ethers, DMSO). Anionic surfactants, cationic surfactants, zwitterionic surfactants, or nonionic surfactants (e.g., polysorbate)), oils (e.g., animal oils, vegetable oils (e.g., coconut oil or peanut oil) or mineral oils), oil derivatives (e.g., ethyl oleate, glycerol monostearate or hydrogenated glycerol), excipients, preservatives (e.g., cysteine, methionine, antioxidants (e.g., vitamins (e.g., a, E or C), selenium, retinyl palmitate, sodium citrate, citric acid, chloroform or parabens (e.g., methyl or propyl parabens), or combinations thereof.

In some embodiments, the pharmaceutical composition may be presented in a dosage form suitable for topical, subcutaneous, intrathecal, intraperitoneal, oral, parenteral, rectal, dermal, nasal routes. In other embodiments, the pharmaceutical composition may be present in a dosage form suitable for parenteral, mucosal, intravenous, subcutaneous, topical, intradermal, oral, sublingual, intranasal or intramuscular administration. The pharmaceutical composition may be, for example, a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel (including hydrogels), paste, ointment, cream, paste, pack, delivery device, suppository, enema, injection, implant, spray, aerosol or other suitable form.

In addition, the pharmaceutical compositions of the present application may also include other pharmaceutical agents, carriers, adjuvants, diluents and excipients. In certain embodiments, the carrier, excipient, or excipient may facilitate administration, delivery, and/or improve preservation of the composition. In other embodiments, the one or more carriers include, but are not limited to, saline solutions, such as physiological saline, ringer's solution, PBS (phosphate buffered saline), and general mixtures of various salts, including potassium salts and phosphate salts, with or without a sugar additive such as glucose. The carrier may include aqueous and nonaqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the body fluid of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. In other embodiments, the one or more excipients may include, but are not limited to, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances such as wetting agents, buffers or emulsifying agents may also be added to the compositions. Oral formulations may include commonly used excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

Advantageous effects

The application provides a monoclonal antibody specific to CK8, which can remarkably improve drug sensitivity of tumor cells. After the CK8 monoclonal antibody is used together with exosomes loaded with chemical drugs, the proliferation of tumors can be effectively inhibited, and the monoclonal antibody has good application prospects and application values.

Drawings

FIG. 1 SDS-PAGE map of recombinant protein CK8 expression

FIG. 2 is a graph showing subtype identification results of monoclonal antibodies

FIG. 3 shows a diagram of the results of specific identification of monoclonal antibodies

FIG. 4 is a graph showing the results of exosome-loaded drug-binding mAb therapy on tumor growth

Detailed Description

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

EXAMPLE 1 preparation of human CK8 protein

Based on the gene sequence of human CK8, the upstream and downstream primers were designed, wherein:

an upstream primer: 5' -CGGAATTCATGTCCATCAGGGT-3' (EcoRI cleavage site);

a downstream primer: 5' -GTCGACTCACTTGGGCAGGAC-3' (Sal/I cleavage site);

the human breast cancer cell line MCF7 was activated and cultured, RNA was extracted, cDNA was reverse transcribed, and PCR was performed using the above primers. The PCR cycle parameters were as follows: pre-denaturation at 94℃for 10min, denaturation at 94℃for 45s, annealing at 62℃for 45s, extension at 72℃for 2min, 30 cycles, and extension at 72℃for 10min. The PCR product is recovered by agarose gel electrophoresis and is connected with a T carrier, DH5a competent cells are transformed, positive clones are screened and identified, plasmids are extracted, restriction enzymes EcoRI and SalI are used for double digestion, a target gene fragment gel recovery kit is used for purification, recovery and quantification, the target gene fragment gel recovery kit is connected with an expression carrier pET-28a (+) subjected to double digestion by EcoRI and SalI by using T4DNA ligase, competent BL21 (DE 3) bacteria prepared by a calcium chloride conversion method are transformed, and recombinant bacterial colonies are picked for expansion culture through identification.

Individual colonies were picked overnight for resuscitation in LB containing kanamycin and then were plated at 1:100 inoculated with the same medium, aerated at 37℃until the A600 value was about 0.6, then added with human IPTG to a final concentration of 1mmol/L, induced at 30℃for 1h, 3h, 5h, and then subjected to cell disruption, 10ul of which was subjected to SDS-PAGE, resulting in the highest protein level induced to be expressed in 5h, as shown in FIG. 1. Centrifuging the bacterial liquid induced for 5 hours, and then purifying the protein by adopting a Ni column for later use.

As a result, as shown in FIG. 1, lane 2 shows that the band of interest at Mr58 000 is clearly stained after induction for 5 hours, lane 1 shows that the control strain is blank, and no band of interest is expressed, which indicates that the recombinant protein CK8 protein expressed in the study has a good expression level.

Example 2 preparation of human CK8 protein monoclonal antibody and Performance identification

100 μg of purified recombinant CK8 protein was mixed with 10 μg CpG, and after complete ultrasonic emulsification with an equivalent amount of Freund's incomplete adjuvant, the mixture was intramuscular injected into the buttocks of both sides of BALB/c mice, and 3 mice were immunized once on days 0, 14, 28. 7-10 d after the third immunization, taking a plurality of drops of mouse tail vein blood, separating serum, and determining the antibody titer to be 1 by an indirect immunofluorescence method (IFA): at 5000 and above, the mice with highest titers were selected and boosted once, and after 3d, the cell fusion assay was performed. Spleen lymphocyte suspensions were prepared by aseptically isolating the spleen of immunized mice: 10 number ratio with SP2/0 myeloma cells, low speed centrifugation to discard supernatant, slow addition of pre-warmed thawing promoter (50% PEG 4000) to the pellet, and volume fixation of HAT selection medium (20% fetal bovine serum) to 50 ml. Transferring into 96-well cell culture plate (100 μl/well), standing for 3 weeks, and selecting cell clone for transferring into 24-well plate for further culture. The positive clones secreting anti-CK 8 protein antibodies are screened by an indirect ELISA method by taking the supernatant of the cultured cells of a 24-well plate as a primary antibody and purified CK8 protein as a coating antigen. Positive clones obtained by screening are subjected to limited dilution by using an HA selection medium, then subcloning is continuously carried out for 3 times, and screening is carried out in the same way until the Kong Yangxing rate of all cells is 100%, so that hybridoma cell strains which stably secrete anti-CK 8 protein monoclonal antibodies are obtained, wherein one of the hybridoma cell strains is CK8-4D13. The obtained cell line was cultured in an expanded manner, and the culture supernatant was collected to obtain a cell line containing a large amount of monoclonal antibody CK8-4D13.

The titers of the monoclonal antibodies were determined by indirect ELISA. The ELISA plate (100. Mu.l/well) was coated with 10ml of purified recombinant CK8 protein, blocked at room temperature for 1h at 4℃overnight with 10% BSA, and after 1h incubation at 37℃with 1.0.05% PBST for 3 washes, 1:2000 dilution of HRP-labeled goat anti-mouse IgG.37℃for 1h followed by washing. The color developing agent ABTS is added, and the enzyme label instrument reads the absorbance (A) value of 405nm wavelength. Meanwhile, SP2/0 myeloma cell culture supernatant was used as a negative control. The highest dilution factor of the sample that produced a positive reaction was taken as its titer. The results are shown in Table 1.

TABLE 1 titers of monoclonal antibodies CK8-4D13

Monoclonal antibody	1：400	1:800	1:1600	1:3200	1:6400	Control
							CK8-4D13	1.697	1.152	0.934	0.628	0.303	0.056

As can be seen from the results in Table 1, the monoclonal antibody CK8-4D13 has a potency of more than 1:6400, and has a good effect.

Coating an ELISA plate with purified recombinant CK8 protein, and using 1: the 500-fold diluted CK8-4D13 monoclonal antibody is used as a primary antibody (murine), HRP-marked goat anti-mouse antibodies (IgG, igM, igA) and subclasses (IgG 1, igG2a, igG2b and IgG 3) are respectively used as secondary antibodies, human color developing agents are added after conventional incubation and plate washing, and an enzyme-labeling instrument reads absorbance (A) values at 405nm wavelength to identify mAbs and subclasses. The results are shown in FIG. 2.

As can be seen from the results of FIG. 2, the CK8-4D13 monoclonal antibody is IgG, specifically IgG2a.

Specificity identification: the enzyme-labeled plate was coated with 1. Mu.g of each of CK8 protein, MCF7 cell lysate, BSA, E.coli lysate, mouse fibroblast NIH3T3 lysate, and PBS wells were used as blank controls. After PBS washing, 2.5% skim milk was added and blocked at room temperature for 1h, after PBS washing, 1:10 dilutions of cell culture supernatant (monoclonal antibody, primary antibody), incubation at 37 ℃ for 1h, 3 washes with 0.05% pbst, 1:2000 dilution of HRP-labeled goat anti-mouse IgG (secondary antibody), incubation at 37℃for 1h, washing, adding a chromogenic agent ABTS, and reading absorbance (A) at 405nm by a microplate reader. The results are shown in FIG. 3.

As can be seen from the results of FIG. 3, the CK8-4D13 monoclonal antibody of the present application only binds to CK8 protein or MCF7 cell lysate expressing CK8 protein, but not to several other samples, and exhibits good specificity.

Binding ability identification: 1mg of the CK8-4D13 monoclonal antibody is taken and dispersed into 1mL of PBS buffer solution; the prepared antibody dispersion liquid is fixed on an aminopropyl sensor, the sensor fixed with the antibody is balanced in PBS buffer solution and then respectively acts with CK8 protein solutions with different concentrations, the spectrum phase difference caused by the combination of the CK8 protein on the antibody is recorded, after the combination is balanced, the sensor is placed in the PBS buffer solution, the spectrum phase difference caused by the dissociation of the protein from the antibody is recorded, and the affinity of the antibody to the protein is fitted according to the phase difference. Experimental results show that the prepared antibody has an affinity of 4.28X10 to CK8 protein ^-9 M, has excellent binding ability.

Meanwhile, the light chain amplification primer is adopted for amplification and sequencing, and the light chain variable region sequence of the CK8-4D13 monoclonal antibody is obtained as shown in SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO: 2.

EXAMPLE 3 evaluation of monoclonal antibody CK8-4D13 efficacy

The SRB method detects the sensitivity of cells to chemotherapeutics: mitoxantrone 800 μg/L was added to MCF-7/MX cells during culture to maintain resistance and stopped two weeks prior to the experiment. MCF-7/MX cells CK8-4D13 mab and/or 100. Mu.g/mL BCRP inhibitor KS176 (Bai Oret, cat# M01428) were added to the cell culture wells at a concentration of 100. Mu.g/mL, with the blank being medium only. After 24h of culture, the digested cells were inoculated into 96-well plates, 2000 cells/well, 3 multiplex wells, and the culture was continued for 24h until complete adherence, and after 48h of action with different concentrations of chemotherapeutics (mitoxantrone, topotecan and daunorubicin), the culture was terminated. Adding 50 μl of precooled 50% (mass/volume) trichloroacetic acid into each hole to fix cells, standing for 5min, and transferring into a refrigerator at 4deg.C for standing for 1 hr; taking out the culture plate, washing each hole with deionized water for 5 times to remove TCA, and drying in air; after complete drying, 100 μl of 0.4% SRB prepared with 1% acetic acid was added to each well, and after 30min of staining at room temperature, the liquid in each well was discarded; washing with 1% acetic acid for 5 times, removing unbound dye, and air drying; the wells were then dissolved in 150. Mu.l of 10mM Tris-lye pH 10.5 and shaken on a plate shaker for 5min, and the absorbance at 490nm was measured on a BioRad microplate reader. IC50 values were calculated using Prism software. The results are shown in Table 1.

Table 1 influence of groups of inhibitors on sensitivity of MCF-7/MX cells to chemotherapeutic agents

* Indicating significant P <0.05 difference compared to the non-dosed group.

As can be seen from Table 1, after the CK8 monoclonal antibody and the BCRP inhibitor are added to MCF-7/MX cells, the sensitivity to chemotherapeutics such as mitoxantrone, topotecan and daunorubicin is obviously improved, and the drug resistance phenotype is obviously reversed. Conclusion the combination of CK8 and BCRP inhibitor can obviously treat multi-drug resistance of MCF-7/MX, and has better treatment effect.

EXAMPLE 4 preparation of exosome-loaded drug and Combined CK8-4D13 mab treatment experiment

MCF-7 is selected as source cells of tumor exosomes, the culture medium is DMEM of 10% fetal bovine serum, and the DMEM is transferred into a cell incubator (constant temperature of 37 ℃ C., volume fraction, 5% CO) ₂ ) After 48h of medium culture, collecting tumor cell supernatant, subjecting the supernatant to gradient centrifugation (2,000Xg, centrifugation, 20min,10000 Xg, centrifugation for 30 min) to remove cell debris, and removing large vesicles by a 0.22 μm filter; filtering with 100000 Xg filtrate, centrifuging for 70min, discarding supernatant, and collecting 100 μThe bottom of the centrifuge tube is resuspended by LDPBS, the exosomes are recovered, and the exosomes are stored in a refrigerator at-80 ℃ for standby.

An equivalent amount of exosome was mixed with daunorubicin and the daunorubicin-loaded exosome was prepared by electrotransformation. The electric shock condition is that the voltage is 400V, the capacitor 125uF and the 4mm electric rotating cup conduct electric rotating. The free and nonspecifically bound daunorubicin is then removed by filtration through an inverted centrifugal ultrafiltration membrane.

Subcutaneous inguinal inoculation of mice 5X 10 ⁵ After MCF7/MX cells 7d, tumor bearing mice were randomly divided into 5 groups according to the random number table method: blank control group, daunorubicin-loaded exosome group, CK8-4D13 monoclonal antibody-combined daunorubicin-loaded exosome group, CK8-4D13 monoclonal antibody group and daunorubicin group; the treatment groups were given subcutaneously by tail vein injection (100. Mu.g/dose) according to the above groups, the blank was 100ul of physiological saline solution, and 50. Mu.g/dose of the monoclonal antibody was given in combination with daunorubicin-loaded exosomes, and after 2 hours, 50. Mu.g/dose was given to the exosomes. The other groups were dosed at 100 μg/dose. Dosing was performed once again after 7d interval, mice were sacrificed 14d after the first dosing, and tumor volume = short diameter was measured ² X long diameter/2. The results are shown in FIG. 4.

As can be seen from the results of fig. 4, after 14d of administration, the difference was statistically significant (P < 0.05) in each daunorubicin-containing treatment group compared to the blank group. The differences between the single mab-treated group and the blank control group were not statistically significant (P > 0.05), indicating that the inhibition of the single mab on cells was relatively small.

The tumor volume of the peripheral group of the CK8-4D13 monoclonal antibody combined daunorubicin-loaded exosome is only (73.5+/-7.8) mm ³ . Compared with the treatment of the exosome group with daunorubicin without adding the monoclonal antibody, the effect of the treatment is far better, which proves that the CK8-4D13 monoclonal antibody can obviously reduce the drug resistance of cancer cells, thereby improving the treatment effect.

It is to be understood that the application is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The application is capable of embodiments in addition to those described and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

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Claims

1. A monoclonal antibody CK8-4D13 against CK8, characterized in that the light chain variable region sequence and the heavy chain variable region sequence of the antibody are as follows:

the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2.

2. The use of the monoclonal antibody CK8-4D13 according to claim 1 for the preparation of a medicament for the treatment of breast cancer, wherein the monoclonal antibody CK8-4D13 of CK8 is capable of increasing the drug sensitivity of drug resistant breast cancer cells.

3. Use of CK8 monoclonal antibody CK8-4D13 in combination with exosomes loaded with a chemical anti-cancer drug, according to claim 1, for the preparation of a medicament for the treatment of breast cancer, wherein CK8 monoclonal antibody CK8-4D13 is capable of increasing the drug sensitivity of resistant breast cancer cells, said chemical anti-cancer drug being daunorubicin.

4. The use as claimed in claim 3, wherein the exosomes are prepared by separation from the supernatant of a breast cancer cell culture broth by mixing an equal amount of exosomes with a chemical anticancer drug, preparing a chemical anticancer drug-loaded exosomes by electroswitching under conditions of 400V voltage, 125uf capacitance, 4mm electroswitching, followed by filtration with an inverted centrifugal ultrafiltration membrane to remove free and non-specifically bound chemical anticancer drug which is daunorubicin.

5. The use of CK8 monoclonal antibody CK8-4D13 and BCRP inhibitor of claim 1 in the manufacture of a medicament for treating breast cancer wherein CK8 monoclonal antibody CK8-4D13 and BCRP inhibitor in combination increase the sensitivity of drug resistant breast cancer cells to the drug, the drug with increased sensitivity being mitoxantrone, topotecan or daunorubicin.