A monoclonal antibody and cytokine composition and its application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a monoclonal antibody and a cytokine composition and its application. The light chain variable region sequence of the ZSCAN4 protein monoclonal antibody provided by the present invention is shown in SEQ ID NO.4, and the heavy chain variable region sequence is shown in SEQ ID NO.5. The cytokine is a cytokine secreted by bone marrow mesenchymal stem cells. In the monoclonal antibody and cytokine composition provided by the present invention, the ZSCAN4 protein monoclonal antibody can specifically bind to the human ZSCAN4 protein, and has the effect of reducing the vitality of colon cancer cells. After using it together with the cytokine, it can effectively reduce the number of colon cancer cell clones in a short time to treat colon cancer. The monoclonal antibody and cytokine composition provided by the present invention has a better inhibitory effect on colon cancer cells than cetuximab, and has a good prospect for medical application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a monoclonal antibody and cytokine composition and application thereof. Background Art

[0002] ZSCAN4 (zinc finger and scanning domain-containing protein 4) is a transcription factor that encodes an N-terminal SCAN domain connected to four zinc finger domains. Consistent with its structure, ZSCAN4 has been shown to both bind DNA and interact with other proteins. ZSCAN4 was originally discovered in mice as an embryonic gene, a nuclear protein expressed only at the 2-cell stage of preimplantation development in vivo (8-cell stage in humans), and inactivated after embryogenesis.

[0003] Studies have shown that ZSCAN4 plays a role in human cancer. Cancer has many similarities with embryonic stem cells, such as immortality and expression characteristics of pluripotent genes, and the ability of ZSCAN4 to activate early embryonic genes matches the role played by reactivated pluripotent factors in cancer. First, by using a reporter gene under the ZSCAN4 promoter, it was demonstrated that ZSCAN4 is enriched in tumor spheres and co-expressed with cancer stem cell (CSC) markers. ZSCAN4 depletion leads to a reduced ability to form spheres in vitro and severely affects the growth of xenograft tumors in mice. Secondly, in cells with high ZSCAN4 expression, the expression of CSC markers such as BMI1, CD44, OCT3 / 4, SOX2, NANOG, and KLF4 was significantly upregulated, while knocking down ZSCAN4 led to a significant downregulation of these factors.

[0004] CSCs are a type of slow-cycling, highly tumorigenic cells that are very similar to normal tissue stem cells and are considered to be the cause of tumor heterogeneity, treatment resistance, and cancer recurrence. CSCs have been found in a variety of cancers, including brain cancer, colon cancer, lung cancer, prostate cancer, head and neck cancer, melanoma, and ovarian cancer. Therefore, it is possible to treat many cancers by targeted destruction of CSCs within the tumor microenvironment. Therapies targeting multiple CSC targets and genotoxic therapies may improve patient prognosis. Therefore, we need to discover CSC targets and fill the gap in CSC-directed therapies.

[0005] Colon cancer is a common malignant digestive system tumor, and its incidence and mortality have been on the rise worldwide in recent years. Therefore, the treatment of colon cancer is very important in medical research. The main treatments for colon cancer include surgery, radiotherapy, chemotherapy, biological therapy, and traditional Chinese medicine. However, the side effects caused by surgery and radiotherapy and chemotherapy cannot be ignored, and traditional Chinese medicine has the disadvantages of insufficient therapeutic effect and slow effect. At present, the relevant research on finding effective anti-colon cancer drugs with few side effects has become a very important and promising topic at home and abroad. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a monoclonal antibody and cytokine composition that can target cancer cells overexpressing ZSCAN4 protein and inhibit cancer cell proliferation.

[0007] The second purpose of the present invention is to provide an application of a monoclonal antibody and cytokine composition.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A monoclonal antibody and cytokine composition, wherein the monoclonal antibody is a ZSCAN4 protein monoclonal antibody, the light chain variable region sequence of which is shown in SEQ ID NO.4, and the heavy chain variable region sequence of which is shown in SEQ ID NO.5.

[0010] Preferably, the light chain variable region of the monoclonal antibody comprises:

[0011] CDR1: GMSVEW; CDR2: NTSGGRTDY; CDR3: YGNFYLELVAMAEGRF.

[0012] Furthermore, the heavy chain variable region of the monoclonal antibody comprises:

[0013] CDR1: RSQKSN; CDR2: GSPKNYANN; CDR3: GWRTSRALYNPNTWVTS.

[0014] Preferably, the subtype of the monoclonal antibody is IgG1.

[0015] Preferably, the cytokine is a cytokine secreted by bone marrow mesenchymal stem cells.

[0016] Preferably, the preparation method of the cytokine is:

[0017] (1) The bone marrow mesenchymal stem cells obtained by subculture were inoculated into the culture medium and incubated at 37°C and 5% CO 2 The cells were cultured under normal oxygen concentration until the cell confluence reached 60%, and then placed in 37°C, 5% CO 2 and 5% O 2 Culture in the environment, restore normal oxygen concentration after 4 to 8 hours, and continue to culture until the cell confluence reaches 90%;

[0018] (2) collecting the cell culture supernatant in step (1), performing ultrafiltration and dialysis, and collecting the retentate;

[0019] (3) Add a lyophilization protectant to the retentate of step (2) and freeze-dry.

[0020] Preferably, the culture medium in step (1) is DMEM / F12 culture medium containing 10% FBS.

[0021] Preferably, the ultrafiltration dialysis operation in step (2) is: performing ultrafiltration dialysis through a 50KD ultrafiltration membrane to obtain a dialysate; then passing the dialysate through a 100D ultrafiltration membrane to collect the intercepted liquid.

[0022] Preferably, the freeze-drying protective agent in step (3) is trehalose, and its mass content in the retentate is 2%.

[0023] The second object of the present invention is achieved by adopting the following technical solution:

[0024] The monoclonal antibody and cytokine composition is used in preparing medicine for treating cancer.

[0025] Preferably, the cancer is colon cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] In the monoclonal antibody and cytokine composition provided by the present invention, the ZSCAN4 protein monoclonal antibody can specifically bind to the human ZSCAN4 protein, and has the effect of reducing the vitality of colon cancer cells. When used together with cytokines, it can effectively reduce the number of colon cancer cell clones in a short period of time to treat colon cancer. The monoclonal antibody and cytokine composition provided by the present invention has a better inhibitory effect on colon cancer cells than cetuximab, and has a good prospect for medical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the structure of the recombinant vector for producing the ZSCAN4 protein of the present invention;

[0029] Figure 2 is the mouse serum titer;

[0030] Figure 3 This is a diagram showing the specificity identification results of the monoclonal antibody against the ZSCAN4 protein of the present invention;

[0031] Figure 4 The inhibition of colon cancer cell proliferation by ZSCAN4 protein monoclonal antibody;

[0032] Figure 5 The numbers of colon cancer cell clones formed in different experimental groups. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0034] Table 1 Sources of cells and reagents

[0035]

[0036] Example 1

[0037] Construction of HEK293A cell line stably expressing human ZSCAN4 recombinant protein:

[0038] To construct a ZSCAN4 overexpression lentiviral vector, primers were designed based on the human ZSCAN4 gene (SEQ ID NO.1):

[0039] Upstream primer: 5′-TTCCGGTGAATTCAGTCTACAACAAGCCCA-3′ (SEQ ID NO. 2)

[0040] Downstream primer: 5′-GGCGGGATCCTTCATTTATTCAACAGAAAT-3′ (SEQ ID NO. 3)

[0041] The target fragment of human ZSCAN4 was obtained by PCR and inserted into the EcoRI and BamHI sites of the vector pLVX-IRES-ZsGreen1 (the structure of the insertion site is shown in Figure 1 As shown). According to the instructions of the Lipofectamine 3000 kit, pLVX-IRES-ZsGreen1, psPAX and pMD2.G plasmids were co-transfected into HEK-293T cells to produce lentivirus. HEK293A cells were transfected with these lentiviruses to obtain HEK293A cells HEK293A-ZSCAN4 that stably expressed human ZSCAN4 recombinant protein. The human ZSCAN4 recombinant protein was harvested after culturing in a medium (DMEM + 10% FBS) containing 0.2 μg / mL puromycin for 7 days and purified by ion exchange chromatography.

[0042] SEQ ID NO.1

[0043]

[0044] Example 2

[0045] Preparation of ZSCAN4 protein monoclonal antibody:

[0046] The ZSCAN4 protein prepared in Example 1 was used as an immunization antigen, and Balb / c female mice were immunized by multiple subcutaneous injections in the abdomen, with a total of 10 mice injected. The antigen was diluted to 2 mg / mL, Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for subsequent immunization. The entire immunization process was performed three times, with 200 μg injected each time. Starting one week after the second immunization, serum titer was tested one week after each immunization. Blood was collected from the mouse eye frame, and after standing at room temperature for 2 hours, serum was collected by centrifugation. The mouse serum antibody titer was detected by indirect ELISA, and the mouse with the highest titer was selected for hybridoma cell fusion. The ELISA method is operated as follows: Take 100 μL of 10 μg / mL ZSCAN4 recombinant protein to coat the plate, and coat it overnight at 4°C. The plates were blocked with 50 mg / L skim milk powder at 37°C for 1 h. The plates were washed with PBST and then 100 μL / well of mouse serum diluted with PBST was added (dilution gradient 1:1000, 1:2000, 1:4000, 1:6000, 1:8000, 1:10000, 1:15000, 1:20000, 1:25000, 1:30000, 1:35000, 1:40000, 1:45000). Unimmunized mouse serum was used as a negative control. The plates were incubated at 37°C for 1 h. After washing, 100 μL of HRP-labeled goat anti-mouse antibody was added to each well. The plates were incubated at 37°C for 40 min, then washed with PBST, developed with TMB for 15 min, terminated the reaction with 2 mol / L sulfuric acid, and placed in an enzyme reader to read the OD450 nm value.

[0047] The results are as follows Figure 2 As shown, the antibody titer of the serum of mouse No. 4 is about 40,000, which has the best effect. Mouse No. 4 was killed by cervical dislocation and soaked in 75% alcohol for 5 minutes. Carefully cut the abdomen of the mouse with forceps and scissors, lift the spleen and separate the spleen, and transfer it to a cell culture dish with incomplete culture medium. A sterile syringe punctures and grinds the spleen to release spleen cells. The ground cell suspension is filtered through a 70μm filter, then centrifuged, resuspended in HAT medium, counted, and evenly distributed into a 96-well plate. Splenocytes and SP2 / 0 cells are mixed in a ratio of 10:1, fused with PEG, and then the effect of PEG is terminated with incomplete culture medium. The fused cells are resuspended in HAT medium and plated in a 96-well plate and placed at 37°C and 5% CO 2After 10 days of HAT screening culture, the supernatant was taken for ELISA detection, and the mother clones with high OD value and good growth status were selected for subcloning, and the subcloning was repeated 3 times.

[0048] The hybridoma cells were subjected to antibody variable region gene sequencing, and the sequence of the light chain variable region of the monoclonal antibody obtained by sequencing was shown in SEQ ID NO.4, and the sequence of the heavy chain variable region was shown in SEQ ID NO.5.

[0049] SEQ ID NO.4

[0050] EVMVAESGLAGVKPGASLKLSMVASAFNYS GMSVEW WVRTTMEK NTSGGRTDY RFTISRETVKNSLTINMSSLKSEDTAMYYCVR YGNFYLELVAMAEGRF WGQLNSCYGNS

[0051] SEQ ID NO.5

[0052] AVIQESALTTNCGETASITC QSqR WVQEKMERLFTAVLG GSPKNYANN GVPLRFSGSLIGDKAGVNITGAQTEDEAIWYC GWRTSRALYNPNTWVTS FTKASGH

[0053] 1×10 7 The subcloned cells were intraperitoneally injected into Balb / c mice. On the 13th day, the mouse ascites was collected and filtered with a 0.2μm filter. The ascites was purified by ion exchange chromatography and then added to a concentration column. It was centrifuged at 5000rpm / min for 15min and PBS was added for replacement to obtain the concentrated monoclonal antibody. The concentration of the antibody was adjusted to 5mg / mL for later use.

[0054] Preparation of cytokines:

[0055] Bone marrow mesenchymal stem cells at P3 were inoculated into DMEM / F12 medium containing 10% FBS and incubated at 37°C and 5% CO 2 The cells were cultured under 37°C and 5% CO. 2 , 5% O 2The cells were treated with hypoxia for 6 hours under the condition of 40 °C, and then continued to be cultured in a carbon dioxide incubator with normal oxygen concentration. When the cells grew to 90% confluence, the supernatant was aspirated for collecting cytokines, centrifuged at 3000 r / min for 20 minutes to remove cell debris, and the supernatant was collected. The supernatant was added to Millipore's Amicon-Ultra-15 ultrafiltration tube for ultrafiltration and concentration, and ultrafiltration and dialysis were performed through a 50KD ultrafiltration membrane to obtain the dialysate; the dialysate was then passed through a 100D ultrafiltration membrane to collect the intercepted liquid, which was the bone marrow mesenchymal stem cell cytokine concentrate. 2% trehalose was added and freeze-dried, and stored at -80 °C for use.

[0056] Test Example 1

[0057] Specificity identification of monoclonal antibodies against ZSCAN4 protein:

[0058] ZSCAN4 recombinant protein, BSA protein, and PD-1 protein were mixed with loading buffer and denatured. Proteins were separated by SDS-PAGE electrophoresis, and the proteins in the gel were transferred to a nitrocellulose membrane using a semi-dry gel transfer apparatus. ZSCAN4 protein monoclonal antibody was added for incubation to react with the protein on the membrane, and the membrane was washed with TBST to remove unbound antibodies and other impurities. Alkaline phosphatase-labeled anti-mouse secondary antibody was added to further amplify the signal. Color development and observation of the results were performed to determine whether the monoclonal antibody has the ability to specifically recognize ZSCAN4 protein.

[0059] The results are as follows Figure 3 As shown, the ZSCAN4 protein monoclonal antibody can only specifically bind to the ZSCAN4 protein, but has no obvious binding characteristics with the other proteins, indicating that the ZSCAN4 protein monoclonal antibody prepared by the present invention has good specificity.

[0060] Test Example 2

[0061] Affinity determination of ZSCAN4 protein monoclonal antibody:

[0062] Antibody affinity determination: The obtained ZSCAN4 recombinant protein was coated overnight at 4°C, with coating concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, respectively. At the same time, blank control wells (no antigen, coating solution added) were set up. After blocking and washing, 100 μL of ZSCAN4 protein monoclonal antibody diluted in multiple ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800 was added to each well, and the plate was placed at 37°C for 1 hour after shaking. After washing, 100 μL of HRP-labeled goat anti-mouse antibody (1:3000 dilution) was added to each well, incubated at 37°C for 40 minutes, washed with PBST, and developed with TMB color system. The optical density of each well was measured at 450nm with an enzyme reader. The reciprocal of the average antibody concentration when the different concentrations of antigen reached half of the maximum optical density in the ELISA method represented the relative affinity of the monoclonal antibody. The results are shown in Table 2.

[0063] Table 2 Affinity and titer determination results of ZSCAN4 protein monoclonal antibodies

[0064]

[0065] It can be seen from Table 2 that the monoclonal antibody of the present invention has good affinity.

[0066] Test Example 3

[0067] ZSCAN4 protein monoclonal antibody subtype detection:

[0068] According to the instructions of the SBA monoclonal antibody typing multifunctional detection kit, the subtype detection of ZSCAN4 protein monoclonal antibody was performed as follows: First, add ZSCAN4 protein antigen to each well of the ELISA plate and coat it overnight at 4°C to ensure that the antigen is fully adsorbed on the plate. Incubate with PBST buffer at 37°C for 2 hours to block the ELISA plate. Take 100 μL / well of the ZSCAN4 protein monoclonal antibody to be detected and add it to the ELISA plate, incubate at 37°C for 1 hour to achieve specific binding of the antibody and antigen. Add 100 μL of 500-fold diluted HRP-labeled goat anti-mouse IgA-HRP, IgM-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, κ-HRP, and λ-HRP secondary antibodies to different wells, incubate at 37°C for 1 hour to specifically bind to the primary antibody. The plate was washed with PBST buffer to remove unbound substances, and then TMB colorimetric solution was used for colorimetric reaction under light-protected conditions. After 10 minutes, 2M sulfuric acid was added to terminate the reaction. The OD value was detected at a wavelength of 450 nm, and the subtype of the ZSCAN4 protein monoclonal antibody was determined based on the absorbance value.

[0069] Table 3 Subtype detection results

[0070]

[0071] From the results in Table 3, it can be seen that the monoclonal antibody against ZSCAN4 protein is of IgG1 subtype.

[0072] Test Example 4

[0073] Functional verification of ZSCAN4 protein monoclonal antibody:

[0074] Human colon cancer HT-29 cells in logarithmic growth phase were collected and plated at 3×10 4 100 μg / mL of DMEM medium containing MTT was added to each well and incubated for 4 hours. The culture medium was discarded and 150 μL of DMSO was added to each well. After oscillation for 10 minutes, the absorbance of each well at a wavelength of 490 nm was measured with an enzyme-linked immunosorbent assay to calculate the cell viability. Cell viability = average absorbance value / absorbance value of the blank control group × 100%. 100 μg / mL of cetuximab was used as a positive control.

[0075] The results are as follows Figure 4As shown, the ZSCAN4 protein monoclonal antibody of the present invention has the effect of reducing the viability of human colon cancer HT-29 cells, thereby affecting cell proliferation. When the concentration of the ZSCAN4 protein monoclonal antibody is 200 μg / mL, the effect of reducing cell viability is better than that of the positive control cetuximab, and when the concentration of the ZSCAN4 protein monoclonal antibody is increased to 400 μg / mL, the effect of reducing cell viability is not significantly improved compared with the treatment of 200 μg / mL of the ZSCAN4 protein monoclonal antibody, so 200 μg / mL of the ZSCAN4 protein monoclonal antibody is the optimal concentration.

[0076] Test Example 5

[0077] Efficacy of monoclonal antibodies combined with cytokines:

[0078] Human colon cancer HT-29 cells in the logarithmic growth phase were taken and inoculated in 6-well culture plates at 500 cells / well. After culturing with HT-29 cell-specific culture medium for 12 hours, 200 μg / mL ZSCAN4 protein monoclonal antibody (experimental group 1), 100 μg / mL bone marrow mesenchymal stem cell factor (experimental group 2), 200 μg / mL ZSCAN4 protein monoclonal antibody and 100 μg / mL bone marrow mesenchymal stem cell factor (experimental group 3) were added, and a negative control group and a 200 μg / mL cetuximab positive control group were set up. The culture medium was changed every 3 days and the cell state was observed. On the 10th day, the number of cells in most single clones was greater than 50, and the culture was terminated. After discarding the culture medium, the cells were washed twice with PBS, 1 mL of 4% paraformaldehyde was added, and fixed at room temperature for 30 minutes, then stained with 0.1% crystal violet solution for 10 minutes, and the staining solution was discarded and washed 3 times with double distilled water, and the number of cell clones was observed and counted.

[0079] The results are as follows Figure 5 As shown, the number of clones formed after 10 days of treatment with ZSCAN4 protein monoclonal antibody, bone marrow mesenchymal stem cell factor, and the combination of ZSCAN4 monoclonal antibody and stem cell factor was significantly reduced. The combination of ZSCAN4 monoclonal antibody and stem cell factor had the most obvious inhibitory effect on HT-29 cell clones, exceeding the positive control cetuximab.

[0080] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A monoclonal antibody and cytokine composition, characterized in that: The monoclonal antibody is a ZSCAN4 protein monoclonal antibody, the light chain variable region sequence is shown in SEQ ID NO.4, and the heavy chain variable region sequence is shown in SEQ ID NO.5; the subtype of the monoclonal antibody is IgG1; The preparation method of the cytokine is: (1) The bone marrow mesenchymal stem cells obtained by subculture were inoculated into the culture medium, cultured at 37°C and 5% CO2 normal oxygen concentration until the cell confluence reached 60%, and then cultured in an environment of 37°C, 5% CO2 and 5% O2. After 4 to 8 hours, the normal oxygen concentration was restored and the culture was continued until the cell confluence reached 90%; (2) collecting the cell culture supernatant in step (1), performing ultrafiltration and dialysis, and collecting the retentate; (3) adding a lyophilization protective agent to the retentate of step (2) and lyophilizing; The ultrafiltration dialysis operation described in step (2) is: ultrafiltration dialysis is performed through a 50KD ultrafiltration membrane to obtain a dialysate; the dialysate is then passed through a 100D ultrafiltration membrane to collect the intercepted liquid.

2. The monoclonal antibody and cytokine composition according to claim 1, characterized in that: The culture medium in step (1) is DMEM / F12 culture medium containing 10% FBS.

3. The monoclonal antibody and cytokine composition according to claim 1, characterized in that: The freeze-drying protective agent in step (3) is trehalose, and its mass content in the retentate is 2%.

4. The use of the monoclonal antibody and cytokine composition according to any one of claims 1 to 3, characterized in that: Application in preparing medicine for treating colon cancer.

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