CN115975036B - Pharmaceutical composition comprising stem cells and use thereof for treating cancer - Google Patents
Pharmaceutical composition comprising stem cells and use thereof for treating cancer Download PDFInfo
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Abstract
The present invention relates to a pharmaceutical composition comprising stem cells and the use thereof for the treatment of cancer. The monoclonal antibody for the breast cancer cells is specifically developed, has the characteristics of better combining the breast cancer cells and the effect of inhibiting the proliferation of the breast cancer cells, and has a better application prospect when being combined with the mesenchymal stem cells.
Description
Technical Field
The invention relates to the field of biology, in particular to a pharmaceutical composition containing stem cells and application of the pharmaceutical composition to treatment of cancers.
Background
The incidence and mortality rate of malignant tumors in China are still high, and the situation of coexistence of high-incidence cancer spectrums in developed countries and developing countries is faced. 1010 ten thousand new cancer cases and 620 ten thousand deaths are worldwide in 2000, and 2240 ten thousand cancer patients are present. Worldwide cancer morbidity and mortality have increased by about 22% over the past 10 years. World Health Organization (WHO) specialists predict that 80 million worldwide population in 2020, new cases of cancer will reach 2000 ten thousand, 1200 ten thousand die from cancer, and cancer will become one of the greatest public health problems worldwide; according to measurement, the economic loss caused by Chinese malignant tumor is up to 1400 billions of Yuanrenzen coins.
At present, the common tumor treatment methods such as chemotherapy, radiotherapy, surgical excision and the like have the defect of killing cancer cells and normal cells, so a new thought of 'fixed point clearance' or 'target treatment' for malignant tumors is provided for a long time, the biggest benefit of 'target treatment' for cancers is strong pertinence and remarkable effect, and the normal tissues are basically not damaged when the cancer cells are killed, so the method is considered to be the most promising research direction in future cancer treatment. Novel tumor targeting therapeutic methods including tumor antibody targeting drug therapy, gene therapy, virus therapy, cell vector targeting therapy and the like emerge. In particular, the antibody targeting drugs closely related to the research progress of functional genome and proteome are rapidly developed.
Due to the development of antibody technology, more than 10 kinds of antibodies have been reported worldwide, wherein more than 1000 kinds of genetically engineered antibodies and more than 200 kinds of humanized antibodies exist. Currently, more than 500 antibodies are internationally available for diagnosis and therapy, and 18 antibodies have been approved by the FDA to date, 8 of which are targeted antibodies for tumor therapy. Monoclonal antibodies are favored because they have the following technical advantages that are difficult to compare: namely, compared with the traditional biological medicine product, the method has the advantages of quicker development process, high target specificity of disease treatment and low side effect; the patient has good compliance in treatment, has the capability of carrying various medicines or toxins and the like to reach target targets, and has the capability of inducing immune response to diseases.
More and more experimental researches on antibody medicaments for treating tumors show that the antibody has a selective killing effect on tumor cells; animal experiments have higher curative effect or lower toxicity, and the animal experiments show specific distribution in vivo; has specific effect on tumor related molecular targets; has killing effect on drug-resistant tumor cells.
Antitumor mabs generally include class 2, one class is antitumor mabs, where the target to which these mabs are directed is typically a tumor-associated antigen or specific receptor on the surface of tumor cells; the other is an antitumor Mab conjugate, or Immunoconjugate (Immunoconjugate), consisting of a Mab and an antitumor moiety (radionuclide, toxin, and drug) 2, wherein the linker to the radionuclide is called a radioimmunoconjugate, the linker to the toxin is called an immunotoxin, and the linker to the drug is called a chemoimmunoconjugate. Such anti-tumor mabs, if capable of inhibiting proliferation of cancer stem cells themselves, can avoid the subsequent expense of extensive resources to develop conjugates.
Stem cells (stemcell) are a special class of cells that exist in the body and have self-renewing and multipotent differentiation potential. Stem cells can be classified into embryonic stem cells and adult stem cells. Embryonic Stem Cells (ESCs) are cells selected from the group of cells in an embryo or primordial germ cells by in vitro culture, and have developmental totipotency, theoretically capable of inducing differentiation into more than 200 cells in the body. Adult stem cells are undifferentiated cells that are present in already differentiated tissues and are capable of self-renewal and specialization into this type of tissue. For example, hematopoietic Stem Cells (HSCs), mesenchymal stem cells (mesenchymal stem cell, MSCs), neural stem cells, endothelial stem cells, skeletal muscle stem cells, and the like. ESCs are ethically limited by the need to destroy embryos by culturing, and have not been used clinically. Currently, stem cells that can be used in cancer therapy are HSCs and MSCs.
MSCs are a class of stem cells derived from mesoderm that have self-renewing and multipotent differentiation potential and are capable of differentiating into a variety of cells in vitro, such as bone cells, cartilage cells, and adipocytes. MSC studies began later than HSC studies, but developed very rapidly. The research shows that MSC can home to tumor cells directionally and survive and proliferate in tumor tissues, and after being subjected to gene modification in vitro, MSC can home to tumor stroma to kill tumor cells, and the characteristic makes MSC an excellent cell carrier for anti-tumor treatment.
The research shows that MSC can directly inhibit the growth of breast cancer cells. MSC is found to inhibit breast cancer cell growth and lung metastasis in a mouse breast cancer model, and the mechanism is that MSC induces apoptosis of tumor cells, so that the expression of apoptosis related factor apoptosis protease 3 in the tumor cells is increased. It has also been demonstrated that MSC can directly inhibit tumor cell growth by inhibiting the activity of phosphatidylinositol-3-kinase in target cells and Akt protein kinase in Akt signaling when contacted with tumor cells. In addition, MSC can secrete Wnt channel inhibitor Dickkopf-1 to inhibit proliferation of tumor cells. Research also shows that the expression of CD31 is detected to be reduced in the MSCs treatment group by using a molecular imaging technology after the MSCs are injected into tumor tissues of MDA-MB-231 human breast cancer in equal quantity, and the apoptosis index of tumors is obviously increased in the group, so that the MSCs can inhibit the growth of the tumors by inhibiting angiogenesis and promoting the apoptosis of tumor cells. The reinfusion of MSCs after tumorigenesis has been shown to inhibit tumor angiogenesis, and the timing of MSCs infusion may affect its effect on tumor angiogenesis. From the above studies, MSCs can be found to be useful in the treatment of cancer.
Although much research is currently directed to cancer treatment, the use of specific monoclonal antibodies in combination with MSCs has been rarely reported. If a functional monoclonal antibody of a specific targeted cancer stem cell can be obtained and combined with a stem cell treatment scheme, a new molecular targeted treatment scheme taking the cancer stem cell as a target is established, and an antibody candidate medicine and a treatment scheme are provided for treating cancer by the targeted cancer stem cell.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a monoclonal antibody specific to breast cancer cells and a medicament for treating breast cancer by using the antibody.
In one aspect of the present invention, there is provided a monoclonal antibody 4D21 specific for breast cancer cells, the light chain variable region sequence of which is as set forth in SEQ ID NO:1, and the heavy chain variable region sequence is shown as SEQ ID NO. 2.
Light chain variable region:
DIVITQRPALMAASPGEKVTITCVCYYVMKLPKQHWYQQKSGISPKPWIYVEYKMGVGVPARFSGSGSGTSYSLTITSMEAEDAATYYCGTWSHSMAKFGAGTKLELK
heavy chain variable region:
EVQLEESATELARPGASVKLSCKASGYIFSTLLCQWIKQRPGQGLEWIGLEENGQIKRICRMQYQGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGMEAKGSCWGLGTTLAVSS。
in particular embodiments, the antibody can specifically bind to breast cancer cells having affinity (e.g., by KD) of no more than 5.0X10 -6 M is not more than 1.0X10 -7 M is not more than 5.0X10 -7 M is not more than 1.0X10 -8 M is not more than 5.0X10 -8 M, or not more than 1.0X10) -9 M is not more than 5.0X10 -9 M. KD can be radiolabeled with Fab versions of antibodies of interest and their antigens, for example, by using known methodsAntigen binding assay (RIA).
Further, the antibody can also detect breast cancer, and the antibody is labeled with a detectable label.
Further, the detectable label is: a detectable molecule (also referred to as a detectable label) conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, the detectable label can be detected by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., CT scanning, MRI, ultrasound, fiber optic inspection, and laparoscopy). Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic bonds, radioisotopes, nucleic acids (e.g., DNA barcodes) and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). In one embodiment, "labeling an antibody" refers to the incorporation of another molecule into the antibody. For example, the tag is a detectable tag, e.g., incorporation of a radiolabeled amino acid or linkage of a biotin moiety to the polypeptide, which can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or enzyme activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
Pharmaceutical compositions, e.g., for treating or inhibiting proliferation of breast cancer cells, are also provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a monoclonal antibody disclosed herein and a pharmaceutically acceptable carrier.
Also provided is the use of a monoclonal antibody disclosed herein or a pharmaceutical composition comprising the antibody in the treatment, prevention or diagnosis of breast cancer in a subject.
The composition for administration may comprise a solution of the breast cancer specific antibody dissolved in a pharmaceutically acceptable carrier. Various aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally free of unwanted substances. These compositions may be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting agents and buffers, toxicity adjusting agents, and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of antibody in these formulations can vary widely and is selected primarily according to the particular mode of administration selected and the needs of the subject, depending on fluid volume, viscosity, body weight, etc.
Typical compositions for intravenous administration include from about 0.01 to about 30mg/kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of conjugate including antibody or antigen-binding fragment) per subject per day. The actual method of preparing the administrable composition is known or obvious to those skilled in the art. In some embodiments, the composition may be a liquid formulation comprising one or more antibodies, an antigen-binding fragment (e.g., an antibody or antigen-binding fragment that specifically binds breast cancer), in a concentration range of about 0.1mg/ml to about 20mg/ml, or about 0.5mg/ml to about 20mg/ml, or about 1mg/ml to about 20mg/ml, or about 0.1mg/ml to about 10mg/ml, or about 0.5mg/ml to about 10mg/ml, or about 1mg/ml to about 10mg/ml.
Furthermore, the invention also provides application of the breast cancer monoclonal antibody and the bone marrow mesenchymal stem cells in preparing a pharmaceutical composition for inhibiting proliferation of breast cancer cells.
The breast cancer cells are MCF-7 cells.
Advantageous effects
The monoclonal antibody for the breast cancer cells is specifically developed, has the characteristics of better combining the breast cancer cells and the effect of inhibiting the proliferation of the breast cancer cells, and has a better application prospect when being combined with the mesenchymal stem cells.
Drawings
FIG. 1 is a graph showing effect of monoclonal antibody on inhibiting MCF-7 growth
FIG. 2 is a graph showing the effect of monoclonal antibody on microsphere formation
FIG. 3 shows the tumor-inhibiting effect of monoclonal antibody alone or in combination with bone marrow mesenchymal stem cell nude mice
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 preparation of monoclonal antibodies specific for breast cancer cells
Immune cells: MCF-7 cells (cat# HTX1646, shenzhen Haodihua Biotechnology Co., ltd.).
Cell culture: MCF-7 was cultured with DMEM-F12 medium containing 10% fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin at 37℃with 5% CO 2 Culturing under saturated humidity, digesting with 0.25% trypsin-0.02% EDTA, and passaging. The experiment uses cells in logarithmic growth phase.
BALB/c mice, MCF-7 cell concentration was diluted to a single dose of 10 with PBS buffer 7 Equal volumes of Freund's complete adjuvant were added and the "water-in-oil" condition was achieved by repeated aspiration using a syringe. 3 BALB/c female mice with the age of 4-6 weeks are marked, and 10 is injected intraperitoneally 7 Primary immunization is carried out at a dosage; after two weeks, the same volume of Freund's incomplete adjuvant was repeatedly aspirated with a syringe to a "water-in-oil" state, and mice were immunized again in the same dose and in the same manner. The immunization method is the same as the first immunization and three immunization are performed every 2 weeks. Mice reaching the immunization titers were subjected to spleen booster immunization at 5 x10 doses 3 days prior to cell fusion 6 /only. Spleen cells from immunized mice were combined with NS-1, SP2/0 cells at a ratio of 10:1 fusion was performed under 50% peg 2000. HT medium was changed after 3d of selection with 18% HAT and after 6d was changed to 18% complete RPMI1640 medium.
The indirect ELISA method was used to screen positive hybridoma cells secreting antibodies, and in 96-well plates, 74 wells were grown with fused hybridoma cells, and 2 single colonies with higher A values (as shown in Table 1) were selected for cloning. After 3 subcloning, the positive detection rate of the clone reaches 100%, and the clones are 1F9 and 4D21 respectively.
TABLE 1 indirect ELISA detection of monoclonal antibody Positive Rate (blank control 0.016)
| Antibody numbering | 1F9 | 4D21 |
| OD value | 1.9986 | 1.9921 |
Preparation of 1F9, 4D21 monoclonal antibodies: BALB/c male mice over 6 weeks old were selected, and 0.5mL of liquid paraffin was injected into the peritoneal fluid of the mice 1 week before hybridoma cell inoculation. Collecting 1F9 and 4D21 hybridoma cells with good growth state, centrifuging to remove supernatant, washing with serum-free culture solution for 1 time, suspending in serum-free culture solution, and adjusting cell density to 2×10 6 Each mouse was injected intraperitoneally with 0.5mL of the cell suspension, individually. After 10d, the abdomen of the mice was seen to be obviously enlarged, the skin of the abdomen was disinfected, and ascites was extracted by puncturing the abdominal cavity with a syringe. Centrifuging at 2500r/min for 10min, removing upper layer oil, sucking pale yellow ascites, thawing in ice-water mixture, slowly adding saturated ammonium sulfate solution at a ratio of 1:1, and mixing until white precipitate appears. Centrifugation at 1000r/min for 10min, removal of supernatant, dissolution of the pellet with a volume of PBS (pH 7.45), transfer to a dialysis bag, dialysis against 20 volumes of binding buffer (provided in Protein ASefinose (TM) Kit) to remove high concentration ions, protein A affinity chromatography to obtain purified protein. The BCA protein quantification kit identified that the concentrations of 2 purified monoclonal antibodies 1F9, 4D21 were 0.521mg/mL and 0.642mg/mL, respectively. And subpackaging the purified antibody, and preserving at-20 ℃ for later use.
Histologic cross-reaction identification of monoclonal antibody specificity: the cell slide method is used for detecting histocytology cross reactivity, and the cross reactions of the 2 antibodies with human epithelial cells, MCF-7 cells, hela cells, CW-2 cells and LoVo cells are observed under a light microscope, so that the results show that the 2 antibodies react positively with the MCF-7 cells and react negatively with other normal cells or cancer cells, thus showing that the 2 antibodies have better specificity.
EXAMPLE 2 monoclonal antibody affinity identification
Affinity constants were determined by Friguent method: (1) coating: CB fluid dilution of MCF-7 cells to a concentration of 10 5 100 μl per well, overnight at 4deg.C. The plate was washed 1 time. (2) closing: 200. Mu.L ED blocking solution was left at 37℃for 2h. The plate was washed 5 times. (3) primary antibody: the initial concentration of the two antibodies of the invention is 50ng/mL, and the initial concentration of the antigen is 10 5 Per mL, then diluted in multiple ratios, 8 gradients were made and antigen-antibody mixture was left at 37℃for 1h. The reaction mixture was added thereto, and the mixture was left at 37℃for 1 hour in an amount of 100. Mu.L per well. The plate was washed 5 times. (4) secondary antibody: HRP-goat anti-mouse IgG, 100. Mu.L per well, was placed at 37℃for 1h. The plate was washed 5 times. (5) color development: adding 100 mu LTMB substrate color development liquid, and developing for 10min at 37 ℃ in a dark place; (6) terminate: adding 50 mu L of stop solution to stop the reaction; (7) colorimetric: OD value of each well was measured at 450nm/630nm dual wavelength. The calculation method comprises the following steps: (1) firstly, obtaining the average value of OD values of each antigen concentration gradient; (2) Calculating a B value, wherein b= (A0-Ai)/A0 (A0: OD at antigen concentration 0, ai: OD per concentration gradient); (3) obtaining 1-B, and calculating the value of B/(1-B); (4) And (3) using the antigen concentration i to make a scatter diagram of B/(1-B), and fitting a straight line to obtain a slope, wherein the slope is the affinity constant. The results are shown in Table 2 below.
TABLE 2 determination of the relative affinities of the antibodies of the respective strains
| Antibody numbering | 1F9 | 4D21 |
| Relative affinity (nM) | 9.83±0.23 | 6.27±0.17 |
From the results in Table 2, it can be seen that the 2 monoclonal antibodies obtained by the screening of the present invention have better affinity.
Example 3 identification of monoclonal antibody 4D21 light and heavy chain sequence and subtype identification
Extracting total RNA of hybridoma cells, synthesizing cDNA by reverse transcription, and amplifying by using the following primers:
the primer sequences are as follows: heavy chain: an upstream primer: 5'-TGAGGAGACGGTGACCGTGGTCCCTFGGCCCCAGAGGTGCAACTGCAGCAGTCAGG-3', the downstream primer is 5'-AGGTSMARCTGCAGSAGTCWGG-3' (S/M/R is a base-degenerate code). Light chain: an upstream primer: 5'-GATGTGAGCTCGTGATGACCCAGACTCC-3', downstream primer: 5'-GCGCCGTCTAGAATFAACACTCATTCCTGTTGAA-3'. Cloning the amplified product, performing gene sequencing, analyzing the variable region gene sequence, and respectively preparing and obtaining the light chain variable region sequence of the antibody, which is shown in SEQ ID NO:1, and the heavy chain variable region sequence is shown as SEQ ID NO. 2.
Light chain variable region:
DIVITQRPALMAASPGEKVTITCVCYYVMKLPKQHWYQQKSGISPKPWIYVEYKMGVGVPARFSGSGSGTSYSLTITSMEAEDAATYYCGTWSHSMAKFGAGTKLELK
heavy chain variable region:
EVQLEESATELARPGASVKLSCKASGYIFSTLLCQWIKQRPGQGLEWIGLEENGQIKRICRMQYQGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGMEAKGSCWGLGTTLAVSS。
meanwhile, the 4D21 monoclonal antibody is identified as an IgG antibody and as an IgG1 subtype by using a mouse monoclonal antibody subtype identification ELISA detection kit.
Example 4 experiments on inhibition of proliferation of breast cancer cells by 4D21 mab
Cell proliferation was detected by MTT assay: taking MCF-7 in logarithmic growth phase at 1×10 4 Inoculating 96-well plates, adding 200 μl of culture medium, standing at 37deg.C in 5% CO 2 Culturing under saturated humidity. After 24h cell adherence, serum-free culture solution is changed, and the cells are starved for 24h. Adding culture solution containing 4D21 monoclonal antibody, setting concentration gradient to make final concentration of the culture solution be 0, 1, 10, 20, 50, 100, 200 μg/mL, respectively, setting 3 repeated holes for each group, changing culture solution containing 4D21 every 3 days, and culturing for 9D. Culture was continued by adding 20. Mu.L of MTT (5 mg/mL) to each well 4 hours before the completion of the culture. The supernatant was discarded, 150. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, and after shaking for 10min, the optical density (D570 nm) of each well was measured by an ELISA reader, and the average of D570nm of a negative control group without monoclonal antibody was used as a control, and the above experiment was repeated 3 times. Cell proliferation inhibition rate calculation formula: proliferation inhibition (%) = (1 a experimental group D570 nm/control group D570 nm) ×100%. The results are shown in FIG. 1.
Inhibition of MCF-7 growth by 4D21 the inhibition rate of MCF-7 proliferation increases significantly with increasing mass concentration of 4D21. When the mass concentration of 4D21 reached 10. Mu.g/mL, the inhibition rate of MCF-7 proliferation was about 61.5% (FIG. 1).
Example 5 Effect of 4D21 monoclonal antibodies on microsphere formation
Preparing single cell suspension from MCF-7 in logarithmic phase, and collecting 1×10 4 The individual cells were incubated with a single antibody serum-free DMEM/F12 medium containing 20ng/mL EGF, 10ng/mL bFGF, 5% B27 and 5. Mu.g/mL insulin with or without a mass concentration of 10. Mu.g/mL at 37℃with 5% CO 2 Culturing under saturated humidity for 12d, changing liquid every 3 days, and observing microsphere formation and size. According to whether the monoclonal antibody is added into the culture solution, the culture solution is divided into a negative control group and a monoclonal antibody group, and a docetaxel positive control group (20 mug/mL) is additionally arranged. The number of 3 groups of microspheres was counted on day 1 of microsphere culture, respectively. Microsphere formation rate (MFE) =number of microspheres/number of single cells seeded. The results are shown in FIG. 2.
As can be seen from the results of fig. 2, MFE of the 4D21 mab group as well as the positive control group is significantly lower than the control group (P < o.01); the mab group MFE was only (0.63±0.10)% (fig. 2). And when the monoclonal antibody and the positive control group are cultured for 13 days, the microspheres formed by the monoclonal antibody and the positive control group are obviously smaller than those formed by the control group, which proves that the monoclonal antibody and the positive medicament can effectively inhibit the formation and the growth of the microspheres. More shows that the 4D21 monoclonal antibody has an inhibiting effect on the MCF-7 and has a more obvious inhibiting effect on microspheres formed by the MCF-7.
Example 6 Combined experiments with 4D21 mab alone or in combination with bone marrow mesenchymal stem cell nude mice
Human bone marrow mesenchymal stem cells (hBMSC), cat: PC-045h, wuhansai Otsi Biotechnology Co., ltd.
MCF-7 cells in logarithmic growth phase were washed 3 times with PBS, resuspended in PBS and adjusted to a concentration of 6X10 7 m1 was subcutaneously injected into the right inguinal region of each nude mouse with 0.2ml of the cell suspension, and the tumor formation was observed. Oncogenesis (tumor body long and diameter)>0.5 cm) and the treatment was started, the nude mice were randomly divided into 5 groups of 10A groups: group 4D21 mab (1 mg), group B: hBMSC cells (1X 10) 9 /ml,1m 1), group C: 4D21 mab (1 mg) +hBMSC cells (1 x 10) 9 Per ml,1m 1), group D (control): 0.9% NaC1 solution 1.5m1; group E (positive control group): 1mg docetaxel; all were injected tail vein. The treatment was repeated 1 time every 7d, the total treatment was 4 times, nude mice were sacrificed at 7d after the fourth administration, tumor mass was peeled off, and tumor mass was weighed. Tumor inhibition rate: { (control tumor mass-treatment tumor mass)/control tumor mass } x100%, the results are shown in FIG. 3.
From the results shown in fig. 3, the monoclonal antibodies, the positive control group and the monoclonal antibody combined hBMSC can effectively inhibit tumor growth, and particularly, compared with hBMSC, the effect of the 4D21 monoclonal antibody+hbmsc cells is obviously improved (P < o.01), and the tumor inhibition rate of the 4D21 monoclonal antibody+hbmsc cell group reaches (93.6±2.98)%. The tumor inhibition effect of the monoclonal antibody is better than that of a positive control group, and the monoclonal antibody has better application prospect.
It is to be understood that the invention 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 invention 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.
While the invention has been described and illustrated in detail as being sufficient to enable one skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses thereof will occur to those skilled in the art. Such modifications are intended to be included within the spirit of the invention and are to be limited only by the scope of the appended claims.
Claims (5)
1. A monoclonal antibody of breast cancer cells, the light chain variable region sequence of which is shown in SEQ ID NO:1, and the heavy chain variable region sequence is shown as SEQ ID NO. 2.
2. Use of a monoclonal antibody of breast cancer cells and human bone marrow mesenchymal stem cells in the preparation of a pharmaceutical composition for inhibiting proliferation of human breast cancer cells MCF-7; wherein, the light chain variable region sequence of the monoclonal antibody of the breast cancer cell is shown as SEQ ID NO:1, and the heavy chain variable region sequence is shown as SEQ ID NO. 2.
3. The use of claim 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
4. The use of claim 3, wherein the pharmaceutically acceptable carrier comprises a pH adjuster and a buffer.
5. The use according to claim 4, wherein the buffer is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride or sodium lactate.
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