CN111116750B

CN111116750B - Monoclonal antibody of specific targeting bile duct cancer stem cell and application thereof

Info

Publication number: CN111116750B
Application number: CN202010027979.7A
Authority: CN
Inventors: 彭菲; 顾超
Original assignee: Heyuan Biotechnology Tianjin Co ltd
Current assignee: Heyuan Biotechnology Tianjin Co ltd
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2020-09-11
Anticipated expiration: 2040-01-10
Also published as: CN111116750A

Abstract

The invention discloses a monoclonal antibody targeting bile duct cancer stem cells, and a preparation method and application thereof. The cholangiocarcinoma stem cell antibody comprises a heavy chain variable region and a light chain variable region. The invention also provides a method for separating the cholangiocarcinoma stem cells and a method for preparing the corresponding monoclonal antibody, wherein the antibody can better combine the cholangiocarcinoma stem cells and inhibit the growth of the corresponding stem cells, so that the antibody can be applied to medicines for treating tumors and separation of the corresponding stem cells.

Description

Monoclonal antibody of specific targeting bile duct cancer stem cell and application thereof

Technical Field

The invention relates to the field of biological pharmacy, in particular to a monoclonal antibody capable of targeting bile duct cancer stem cells and application thereof.

Background

Tumor Stem cells (CSCs), also known as Cancer Stem cells, or Cancer Stem cells, refer to cells in a tumor that have the ability to self-renew and can give rise to heterogeneous tumor cells. It is now clear that tumor stem cells play an important role in tumor progression and development of therapy resistance, the most prominent being in hematological cancers such as leukemia. Therefore, researchers have proposed that tumor stem cells play an important role in the processes of tumor development, development and metastasis, but what is the tumor stem cells is not accurately defined at present. Tumor stem cells and tumor tissue cells are different. These cells differentiate themselves slowly but are resistant to many physicochemical insults (e.g., radiation and chemotherapy). Chemotherapy and radiotherapy also promote proliferation of tumor stem cells. Tumor stem cells are considered to be the source of recurrence of tumor therapy because even if most tumor cells are killed, these stem cells can differentiate into tumor cells by definition as long as the tumor stem cells remain. This theory provides new direction and visual angle for our recognization of the origin and nature of tumors, as well as clinical tumor treatment.

Cholangiocarcinoma occupies the first place among biliary system malignancies, with cholangiocarcinoma of the hepatic gate being the most common. Because early symptoms are atypical, the misdiagnosis rate of early diagnosis of the hepatic portal cholangiocarcinoma is high, the surgical resection rate is low, the prognosis is poor, and the 5-year survival rates of intrahepatic cholangiocarcinoma, distal extrahepatic cholangiocarcinoma and hepatic portal tumor after surgery are 22% -44%, 27% -37% and 11% -41%, respectively. Under the condition of great progress of surgical techniques at present, the recurrence rate of bile duct cancer is still high even through active operations. Therefore, adjuvant chemotherapy combined with surgery is of great importance.

Sell, et al 1989, proposed the presence of biliary cancer stem cells in a chemically-induced liver cancer rat model. Subsequently, several researchers demonstrated that disorders in the mechanism of self-renewal of hepatic stem/progenitor cells were an early event in cholangiocellular carcinogenesis. The multidrug resistance of tumors is the main cause of difficult radical cure of tumors, failure of chemotherapy and tumor recurrence. Tumor stem cells have been shown to be chemoresistant and radioresistant, as well as specific for promoting angiogenesis. The tumor stem cells can express an ATP-binding cassette transporter (ABC), so that the tumor stem cells can pump out anti-tumor drugs and toxic substances; the active DNA repair capacity and anti-apoptotic capacity of tumor stem cells like normal stem cells also contribute to tumor survival in chemotherapy and radiotherapy, and therefore, a treatment regimen combining targeted therapy against tumor stem cells with traditional anti-cancer therapies can effectively eliminate tumor cells and control cancer recurrence.

Since the first reported human Acute Myeloid Leukemia (AML) stem cells with the phenotype of CD34+/CD38 by Lapidot et al in 1994, more and more solid tumor stem cells were discovered, the tumor stem cell population is different, some cancer cells with characteristic surface markers have stem cell activity, the molecular markers can be used singly or in combination to identify and separate the tumor stem cells, and the cholangiocarcinoma stem cells also have characteristic surface markers. CD133 positive cells have higher proliferative capacity and in vivo tumorigenic capacity, and therefore CD133 is an important marker for biliary tract cancer stem cells, and in addition, CD44 and EpCAM are also important markers for biliary tract cancer.

At present, targeted separation treatment aiming at tumor stem cells is expected to make up for the defects of the existing clinical treatment means and improve the prognosis of patients with cholangiocarcinoma. At present, no research report of high-activity targeted human bile duct cancer stem cell treatment is seen.

Disclosure of Invention

The invention aims to overcome the defect that the specific antibody of the bile duct cancer stem cell is lacked at present, and provides the bile duct cancer stem cell with high affinity and strong specificity, and a preparation method and application thereof.

The invention also provides a method for separating and preparing the bile duct cancer stem cells.

Further, the invention provides a method for preparing the bile duct cancer stem cell monoclonal antibody.

The invention also provides a bile duct cancer stem cell monoclonal antibody which is a 4H5 antibody. Specifically, the sequence is shown as follows:

4H5 light chain sequence:

DIQLTQSPSGVSASVGRRVTITCTSCAQGSIPDGWYQQKPGKAPKLLIYVSRDSTVGVPSRFSGSGQGTEFTATISSLQPSDFATYYCDANADGGYQSFGPTTKVDIK(SEQ ID NO:1)；

4H5 heavy chain sequence:

QVQLQQSGPGLVGPSQTLSLTCATSGDTVSCSGQTPSWIRQSPSRGLEWLGVSAGGGASSTSGVQLAS IRMTINADTSKNQVSLHQNSVTKEDTAVYYCARQTCYSSCGPWGQGTLVTVSS(SEQ ID NO:2)；

further, the antibodies of the invention are full-length antibodies, and the full-length proteins of the antibodies are conventional in the art and comprise a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region form a full-length protein of a fully human antibody. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3 or IgG 4.

The invention also provides a nucleic acid encoding the antibody described above.

The preparation method of the antibody is a conventional preparation method in the field. The preparation method preferably comprises the following steps: isolated from an expression transformant which recombinantly expresses the protein or obtained by artificially synthesizing a protein sequence. The following method is preferably obtained by separating the protein from an expression transformant which recombinantly expresses the protein: cloning the nucleic acid molecule which codes the protein and has point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant, and obtaining the protein by separation and purification through culturing the obtained recombinant expression transformant.

The invention provides an application of the monoclonal antibody in treating tumors.

The invention provides an application of the monoclonal antibody in treating bile duct cancer.

The invention also provides a pharmaceutical composition, which comprises the monoclonal antibody and a corresponding pharmaceutical carrier.

The invention provides an application of the pharmaceutical composition in treating bile duct cancer.

The invention also provides a method for detecting the bile duct cancer stem cells, which comprises the following steps: the monoclonal antibody is contacted with a sample to be detected in vitro, and the combination of the protein and the sample to be detected is detected.

The invention provides application of the antibody in preparation of a kit for detecting cholangiocarcinoma.

The means of detection of such binding is conventional in the art, and is preferably FACS detection.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the cholangiocarcinoma stem cell antibody has good affinity with cholangiocarcinoma stem cells, and can inhibit the proliferation of the stem cells after the antibody is combined with the stem cells. Therefore, the antibody drug prepared by the antibody can effectively kill bile duct cancer tumor stem cells, is used for treating tumors, and has a good application prospect.

Drawings

FIG. 1 is a graph showing the inhibition rate of cell proliferation.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 isolated preparation of biliary cancer Stem cells

CCLP-1 human cholangiocarcinoma cells, purchased from Biotech, Inc., Ginie, Guangzhou.

(1) Culturing cancer cells by preparing serum-free culture medium with DMEM/F12+ EGF (20ng/mL) + bFGF (20ng/mL) + 4% B27, collecting bile duct cancer CCLP-1 cells in logarithmic growth phase, adding appropriate amount of pancreatin for digestion, mechanically blowing to form single cell suspension, counting by cell counting plate, adding serum-free culture medium, and adjusting cell density to 5 × 10³L, seeded in ultra-low adhesion 6-well plates at 2mL per well (i.e., 10 per well)⁴And each cell) is observed by using an inverted microscope every day, the change of the cell is photographed under the condition of 400 times, 500L of serum-free culture medium is added every 2-3 days, after 10 days, the clone cell balls are collected, blown out and placed in an ultra-low adhesion 6-pore plate for continuous culture and amplification.

(2) Isolated culture of bile duct cancer stem cells

Collecting the clone cell balls cultured without serum in the step (1), centrifuging for 1000r/min × 5min, pouring out the supernatant, adding 5mL of buffer solution, centrifuging again after blowing, pouring out the supernatant, adding 1mL of pancreatin, blowing into single cell suspension, adding complete culture medium to stop digestion, centrifuging again, pouring out the supernatant, adding 3mL of buffer solution, centrifuging again after uniformly blowing, pouring out the supernatant, adding 100 microliters of buffer solution, uniformly blowing, adding 10 microliters of PE-Anti CD133 and 5 microliters of Anti-EpCAM-FITC, incubating for 20min on ice, loading on a machine for flow cell sorting, and sorting out CD133⁺EpCAM^highStem cell-like cells. After cell counting 10⁴Inoculating the cells on an ultra-low adhesion 6-well plate, adding 2mL serum-free culture medium into each well, placing at 37 deg.C and 5% CO₂Culturing under 100% humidity condition, adding 500 microliter serum-free culture medium every 2-3 days, collecting clone cell balls after 10 days, blowing off the clone cell balls, and placing the clone cell balls into an ultra-low adhesion 6-pore plate for relayContinuously culturing and amplifying for 2d, and collecting cells for later use.

Example 2 preparation of cholangiocarcinoma Stem cells-SP cells

Preparing bile duct cancer stem cells into single cell suspension, and adding Hoechst33342 dye (the final mass concentration is 5m 1); verapamil (final concentration 50g/m1) was added to the control group. Incubating for 90min at 37 ℃, discarding the supernatant, washing with precooled PBS for 3 times, adding PI to a final mass concentration of 2ml, detecting and sorting side population cells (sP) in the PI by a flow cytometer, and obtaining the bile duct cancer stem cell-SP.

Example 3 preparation of monoclonal antibody hybridoma cells targeting biliary cancer stem cells

The prepared bile duct cancer stem cells are injected to the abdominal subcutaneous part of BALB/c (nu/nu) mice (total 6 mice), and each mouse is injected with 1 × 10⁶One stem cell, 1 time every 2 weeks, was continuously immunized for 12 months.

When the serum titer of the immunized mice reaches 1:50000, 1 x10 is taken⁸Spleen cells of immunized mice and 2 × 10⁷SP2/O cells in logarithmic growth phase were fused and seeded in methylcellulose dishes containing HAT for 12d, and isolated, larger cell colonies, i.e., hybridoma cells, were picked and seeded in 96-well plates for continued culture. After culturing, hybridoma clone grows in 3426 holes, so that a large-capacity monoclonal antibody library containing 3426 clones is established.

EXAMPLE 4 screening of hybridoma mAbs

Inoculating a 96-well plate to the bile duct cancer stem cell, culturing for 30h by using an SFM culture medium, detecting the reaction of 3426 hybridoma supernatants in a monoclonal antibody library and the bile duct cancer stem cell by using a fixed cell immunofluorescence method, wherein the hybridoma monoclonal antibody showing a fluorescence positive reaction comprises 301 strains, and screening the 301 strains of positive monoclonal antibodies by using a living cell immunofluorescence method to obtain 97 strains of positive hybridoma monoclonal antibodies specifically combined with the bile duct cancer stem cell membrane. The 97 monoclonal antibodies are detected by the cholangiocarcinoma stem cell-SP and the non-cholangiocarcinoma stem cell-SP which are sorted by a flow cytometer, and the result shows that 21 monoclonal antibodies only have positive reaction with the cholangiocarcinoma stem cell-SP, which indicates that the hybridoma monoclonal antibodies have specific monoclonal antibodies for resisting the cholangiocarcinoma stem cell. Table 1 shows the results of immunofluorescence staining of the most positive 3 monoclonal antibodies.

Example 5 identification of hybridoma monoclonal antibodies

CD133 is a marker of biliary cancer stem cells. In order to further identify the identification condition of the specific marker of the 3 monoclonal antibodies with better positive, a polychrome fluorescence method is adopted to detect the co-expression condition of the hybridoma monoclonal antibodies in the bile duct cancer stem cell-SP and the non-bile duct cancer stem cell-SP with CD133, CD44 and EpCAM respectively. The results show that the 3 hybridoma monoclonal antibodies and the CD133 can simultaneously recognize the cholangiocarcinoma stem cell-SP and show double-color immunofluorescence co-staining of red fluorescence (hybridoma monoclonal antibody) and green fluorescence (anti-CD 133 monoclonal antibody) (table 2), and the 3 hybridoma monoclonal antibodies can specifically recognize the cholangiocarcinoma stem cell.

TABLE 2 immunofluorescence staining for detection of hybridoma monoclonal antibodies, CD133 binding to cholangiocarcinoma stem cells-SP (. 100)

Example 6 hybridoma monoclonal antibody type identification

Monoclonal antibody subclass identification kit purchased by the homogeneous biotechnology (shanghai) ltd, cat no: QY282, monoclonal antibody type identification. A second antibody of a mouse antibody common site is adopted to coat a microporous plate, the microporous plate is combined with the added mouse antibody in culture supernatant, then antibodies of various types and subclasses of mice marked by HRP are added for respective reaction, finally, a TMB substrate system is used for developing color and is stopped by dilute sulfuric acid, and then the class or the subclass of the monoclonal antibody to be detected is judged by detecting absorbance through a microplate reader. As shown in Table 3, all of the 3 hybridoma monoclonal antibodies were IgM-type monoclonal antibodies, and no complex-type clone was observed.

TABLE 3 monoclonal antibody type test results

McAb	Heavy chain	Light chain
			4H5	IgM	-
7H1	IgM	-
			11H8	IgM	-

Example 7 CCK-8 method for examining the Effect of monoclonal antibodies on the proliferation of cholangiocarcinoma Stem cells

The bile duct cancer stem cell in the logarithmic growth phase is expressed as 1 × 10³Inoculating the cells into a 96-well plate, culturing for 24h, removing culture solution, adding hybridoma supernatant of 3 antibodies, culturing for 3D, changing the culture solution for 1 time, continuously culturing for 3D, removing the supernatant, adding 0 microliters of CCK-8 into each well, and measuring an optical density (D) value, wherein SP2/O cell culture supernatant is used as a negative control in the experiment, the inhibition rate of cell proliferation is calculated according to the following formula, namely the inhibition rate (%) (control group D-experimental group D)/control group D × 100% and the result is shown in figure 1, 3 hybridoma monoclonal antibodies can inhibit the proliferation of the bile duct cancer stem cells, the inhibition rate is 63% -89%, and the 3 hybridoma monoclonal antibodies are functional monoclonal antibodies which have specific recognition and can inhibit the proliferation of the bile duct cancer stem cells.

EXAMPLE 84 determination of the amino acid sequence of the light and heavy chain variable regions of the H5 monoclonal antibody

Total RNA isolation by centrifugation the 4H5 hybridoma cells obtained in example 4 were harvested 2-9 × 10⁷The number of the main components is one,add 1mL Trizol mix well and transfer to a 1.5mL centrifuge tube and let stand at room temperature for 5 minutes. 0.2mL of chloroform was added, the mixture was shaken for 15 seconds, allowed to stand for 10 minutes, centrifuged at 12000g at 4 ℃ for 5 minutes, and the supernatant was transferred to a new 1.5mL centrifuge tube. 0.5mL of isopropanol was added, the solution in the tube was gently mixed, left to stand at room temperature for 10 minutes, centrifuged at 12000g for 15 minutes at 4 ℃ and the supernatant was discarded. Adding 1mL of 75% (v/v) ethanol, gently washing the precipitate, centrifuging at 4 ℃ and 12000g for 5 minutes, removing the supernatant, drying the precipitate in the air, and adding DEPC-treated water to dissolve (water bath at 55 ℃ promotes dissolution for 10 minutes) to obtain the total RNA.

Reverse transcription and PCR: mu.g of total RNA was taken and prepared into a 20. mu.L system, and after reverse transcriptase was added, the reaction was carried out at 42 ℃ for 60 minutes and at 7 ℃ for 10 minutes to terminate the reaction. Preparing a 50 mu L PCR system comprising 1 mu L cDNA, 25pmol of each primer, 1 mu L DNA polymerase and a matched buffer system, 250 mu mol dNTPs; setting PCR program, pre-denaturation at 95 deg.c for 3 min, denaturation at 95 deg.c for 30 sec, annealing at 55 deg.c for 30 sec, extension at 72 deg.c for 35 sec, and extension at 72 deg.c for 5min after 35 cycles to obtain PCR product. Wherein the kit used for reverse transcription is PrimeScript RT Master Mix purchased from Takara under the cat # RR 036; the kit used for PCR included Q5 super fidelity enzyme, purchased from NEB under cat number M0492.

Cloning and sequencing: 5 mu.L of PCR product is taken to carry out agarose Gel electrophoresis detection, and a positive detection sample is purified by using a column recovery kit, wherein the recovery kit is Gel & PCR Clean-up and is purchased from MACHEREY-NAGEL with the commodity number of 740609. Carrying out a ligation reaction: 50ng of sample, 50ng of T carrier, 0.5 mu L of ligase, 1 mu L of buffer solution and 10 mu L of reaction system react at 16 ℃ for half an hour to obtain a ligation product. Wherein the linked kit is T4DNA ligase purchased from NEB under the accession number M0402; mu.L of the ligation product was added to 100. mu.L of competent cells (Ecos 101 component cells, from Yeastern, cat # FYE607) and ice-washed for 5 minutes, then heat-shocked in a water bath at 42 ℃ for 1 minute, then placed back on ice for 1 minute, then 650. mu.L of antibiotic-free SOC medium was added and thawed on a shaker at 37 ℃ for 30 minutes at 200 RPM. 200. mu.L of the suspension was spread on LB solid medium containing antibiotics and incubated overnight at 37 ℃. The next day, a 30. mu. LPCR system was configured using primers M13F and M13R on a T-carrier, colony PCR was performed, colonies were dipped with a pipette tip and aspirated into the PCR reaction system, and 0.5. mu.L of the colonies were pipetted onto another LB solid culture dish containing 100nM ampicillin to preserve the strains. After the PCR reaction, 5. mu.L of the sample was removed and subjected to agarose gel electrophoresis, and the positive sample was subjected to sequencing and analysis (Shanghai Biotech sequencing). The sequencing result shows that the amino acid sequence of the heavy chain variable region of the 4H5 monoclonal antibody is SEQ ID No.2, and the amino acid sequence of the light chain variable region is SEQ ID No. 1.

Example 9 flow cytometry to assess the binding Capacity of antibodies to Stem cells

The stem cells isolated in example 1 in the logarithmic growth phase were collected, washed 2 times with PBS and then resuspended in FACS buffer (PBS solution containing 2% fetal bovine serum). Adjust density at 2x10⁵The individual cells/well were plated in 96-well U-plates, centrifuged at 300g for 5 minutes, the supernatant decanted, the 4H5 antibody solution was added as a gradient and incubated on ice for 40 minutes. Washing cells for 2 times, adding 100 mu L/hole phycoerythrin fluorescence labeled goat anti-mouse secondary antibody, incubating for 40 minutes at 4 ℃ in the dark, washing cells for 3 times, adding 100 mu L of FACS buffer solution for resuspension in each hole, blowing the cells evenly, and detecting on a machine. The fluorescence intensity of each well of cells was measured using a flow cytometer model II Canto, BD. Data processing using Graphpad prism software gave an EC50 concentration value of 102.7pM for 4H5 antibody-bound cells. In addition, the binding constant of the 4H5 antibody detected by a common Kd detection method (ELASA method) is 230.4pM, and the antibody has good binding property.

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 and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or 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.

As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

While the invention has been described and illustrated in detail as being sufficient to enable those 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 as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

Sequence listing

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<120> monoclonal antibody of specific targeting bile duct cancer stem cell and application thereof

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Claims

1. The separated monoclonal antibody targeting the bile duct cancer stem cell comprises a heavy chain variable region and a light chain variable region, and is characterized in that the amino acid sequence of the heavy chain variable region is shown in a sequence table SEQ ID No.2, and the amino acid sequence of the light chain variable region is shown in a sequence table SEQ ID No. 1.

2. A nucleic acid encoding the monoclonal antibody of claim 1.

3. A recombinant expression vector comprising the nucleic acid of claim 2.

4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.

5. A pharmaceutical composition comprising the monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier.

6. The use of the monoclonal antibody of claim 1 for the preparation of an antitumor medicament, characterized in that: the tumor is bile duct cancer.

7. Use of the monoclonal antibody of claim 1 for the preparation of a kit for the detection of cholangiocarcinoma.