Combination therapy of umbilical cord mesenchymal stem cells and monoclonal antibodies for cancer treatment

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of umbilical cord mesenchymal stem cells and monoclonal antibodies in the combined treatment of cancer. The monoclonal antibody is specifically directed against the human PAFAH1B3 protein, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:2. The PAFAH1B3 protein monoclonal antibody of the present invention can significantly inhibit the proliferation of pancreatic cancer cells. The combined use of umbilical cord mesenchymal stem cells and the PAFAH1B3 protein monoclonal antibody has an even better inhibitory effect on the proliferation of pancreatic cancer cells. The monoclonal antibody can be used to prepare anticancer drugs, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of umbilical cord mesenchymal stem cells and monoclonal antibodies in the combined treatment of cancer. Background Art

[0002] Pancreatic cancer, a tumor that originates in the pancreatic ductal epithelium, is one of the most lethal malignancies in humans. Its pathogenesis remains unclear, and the majority of patients develop between the ages of 40 and 65. Currently, pancreatic cancer treatments primarily include chemotherapy, radiotherapy, and targeted therapies. However, these therapies have a high recurrence rate and significant side effects. Therefore, there is a need to develop safe biological agents that can inhibit the growth of pancreatic cancer cells while also providing targeted therapeutic options.

[0003] Mesenchymal stem cells, including those derived from bone marrow, umbilical cord, and adipose tissue, possess a certain degree of self-renewal capacity and can proliferate in large numbers, but they can also undergo aging and apoptosis. Under specific culture conditions, mesenchymal stem cells can differentiate into different cell types. Human umbilical cord stem cells, isolated from human umbilical cord tissue, possess strong differentiation potential and, under specific induction conditions, can differentiate into various tissue cells, including nerves, muscles, liver, and myocardium. Currently, they are widely used in tissue engineering and regenerative medicine.

[0004] The uncontrolled proliferation, invasion, and migration of pancreatic cancer cells are the root cause of death in pancreatic cancer patients. Monoclonal antibodies are a very important approach to pancreatic cancer treatment. By leveraging the body's autoimmune mechanisms, pancreatic cancer treatment can improve patient acceptability and tolerance, further reducing disease recurrence. Related studies have shown that the PAFAH1B3 gene is closely associated with the proliferation, invasion, and migration of pancreatic cancer cells. Therefore, there is an urgent need to develop safe biologics targeting the PAFAH1B3 protein or related molecules upstream and downstream of PAFAH1B3 to improve cancer treatment efficacy and provide patients with new medication options. Summary of the Invention

[0005] In order to fill the deficiencies of the prior art, the first purpose of the present invention is to provide an umbilical cord mesenchymal stem cell and monoclonal antibody composition that can effectively inhibit the proliferation of pancreatic cancer cells and provide a new approach for the treatment of pancreatic cancer.

[0006] The second purpose of the present invention is to provide an application of umbilical cord mesenchymal stem cells and monoclonal antibodies in the combined treatment of cancer.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] Umbilical cord mesenchymal stem cells and a monoclonal antibody composition, wherein the monoclonal antibody is a monoclonal antibody specifically targeting human PAFAH1B3 protein, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2.

[0009] Furthermore, the subtype of the monoclonal antibody is IgG1.

[0010] Furthermore, the umbilical cord mesenchymal stem cells are P2-P5 generation umbilical cord mesenchymal stem cells.

[0011] Furthermore, the method for preparing P2-P5 generation umbilical cord mesenchymal stem cells comprises the following steps:

[0012] (1) Wharton's jelly was removed from the umbilical cord, and the Wharton's jelly was minced into tissue blocks. The tissue blocks were mixed with culture medium and cultured in a cell culture incubator at 37°C and 5% CO2.

[0013] (2) When umbilical cord mesenchymal stem cells appear around the tissue block, the medium is completely replaced and recorded as P0;

[0014] (3) When the cell confluence reaches 80-85%, the umbilical cord tissue pieces and culture medium are discarded, 0.25% trypsin is added for digestion, the digestion solution is removed by centrifugation, and the culture medium is added for resuspending. The cells are then subcultured to obtain P2-P5 umbilical cord mesenchymal stem cells.

[0015] Furthermore, the subculture ratio in step (3) is 1:2.

[0016] The second object of the present invention is achieved by adopting the following technical solution:

[0017] Application of the above-mentioned umbilical cord mesenchymal stem cells and monoclonal antibody composition in combined treatment of cancer.

[0018] Furthermore, the cancer is pancreatic cancer.

[0019] Compared with existing technologies, the present invention's primary beneficial effects lie in the fact that the PAFAH1B3 protein monoclonal antibody specifically binds to the human PAFAH1B3 protein and significantly inhibits the proliferation of pancreatic cancer cells that highly express the protein. Combining the PAFAH1B3 protein monoclonal antibody with umbilical cord mesenchymal stem cells effectively reduces the proliferation and invasion of pancreatic cancer cells, and thus has promising application prospects for the preparation of anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the morphology of P3 umbilical cord mesenchymal stem cells;

[0021] Figure 2 This is a diagram showing the specific identification results of the PAFAH1B3 protein monoclonal antibody;

[0022] Figure 3 is the inhibition rate of PAFAH1B3 protein monoclonal antibody on pancreatic cancer cell proliferation;

[0023] Figure 4 The effects of PAFAH1B3 protein monoclonal antibody and umbilical cord mesenchymal stem cells on the proliferation of pancreatic cancer;

[0024] Figure 5 The effects of PAFAH1B3 protein monoclonal antibody and umbilical cord mesenchymal stem cells on pancreatic cancer tumor size;

[0025] Figure 6 The effects of PAFAH1B3 protein monoclonal antibody and umbilical cord mesenchymal stem cells on pancreatic cancer tumor weight. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents and instruments used, unless otherwise specified, are conventional products obtained through commonly purchased channels.

[0027] Example 1

[0028] Preparation of umbilical cord mesenchymal stem cells:

[0029] (1) Take the umbilical cord of a healthy full-term newborn and wash it with PBS in a clean bench to remove residual blood. Remove the Wharton's jelly from the umbilical cord, cut the Wharton's jelly into tissue blocks, wash it three times with normal saline, and culture the tissue blocks in DMEM medium supplemented with 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) in a cell culture incubator at 37°C and 5% CO2. Change half of the medium every 2 days.

[0030] (2) When umbilical cord mesenchymal stem cells appear around the tissue block, the medium is completely replaced and recorded as P0;

[0031] (3) When the cell confluence reaches 80%, the umbilical cord tissue pieces and culture medium are discarded, 0.25% trypsin is added for digestion, the digestion liquid is removed by centrifugation, and DMEM medium is added for resuspending and subculture to obtain P3 generation umbilical cord mesenchymal stem cells (such as Figure 1 shown).

[0032] Example 2

[0033] Preparation of monoclonal antibodies against PAFAH1B3 protein:

[0034] S1. Immunization of experimental animals: 6 healthy female mice of about 11 weeks old with good growth status were selected for immunization, and 1 healthy female mouse that was not immunized was used as a negative control. Recombinant human PAFAH1B3 protein (immunogen) was injected into the peritoneal cavity of the mice at a dose of 50 μg / mouse. The injection method was subcutaneous multi-point injection. Immunization was performed once every two weeks for a total of 3 times. The recombinant human HPAFAH1B3 protein was mixed with Freund's complete adjuvant for the first immunization, and with Freund's incomplete adjuvant for the 2nd-3rd immunization. The titer of mouse antibodies was determined by indirect ELISA, and the mice with the highest antibody titer were selected for subsequent related experiments.

[0035] S2. Hybridoma Cell Fusion: Prepare a myeloma cell suspension from well-growing myeloma cells in the logarithmic phase. Sacrifice the female mouse with the highest antibody titer from step S1 by cervical dislocation, remove the spleen, and prepare a splenocyte suspension. Fusion of myeloma cells and splenocytes at a ratio of 10:1 in the presence of 50% polyethylene glycol. Five days after cell fusion, replace the culture medium with HAT selective medium. Seven days later, collect the cell culture supernatant and screen for positive hybridoma cells using indirect ELISA.

[0036] S3. Subcloning and screening: Subclone the positive hybridoma cells by limiting dilution method, pick the monoclonal stable cell line with the highest positive value, expand the culture and freeze it.

[0037] S4. Preparation of mouse ascites: Six healthy female mice, about 11 weeks old and in good condition, were intraperitoneally injected with 0.5 mL of liquid paraffin. One week later, the hybridoma cells obtained in step S3 were injected into the mice at an inoculum size of 5 × 10 5 The mouse abdomen was observed. When the mouse abdomen was noticeably bulging, ascites was collected. The collected ascites was centrifuged at 4000 rpm for 30 minutes. The supernatant was diluted with buffered saline. Silica powder was added to the ascites, and the mixture was incubated at room temperature for half an hour with shaking. The mixture was then centrifuged for 30 minutes to obtain the supernatant containing the PAFAH1B3 antibody.

[0038] S5. Affinity chromatography of PAFAH1B3 antibody supernatant: equilibrium - loading - washing - elution. Equilibration: Equilibrate the affinity chromatography column with buffer. Loading: Load the anti-PAFAH1B3 monoclonal antibody supernatant onto the affinity chromatography column. Washing: After loading, wash twice with buffer B1 and buffer B2 until the UV absorbance value stabilizes, then stop washing. Elution: After washing, elute the affinity chromatography fluid with buffer B3. When the UV absorbance value reaches 100 mAU, collect the protein solution for later use. Determine the protein concentration using the BCA method, adjust the antibody concentration to 5 mg / mL with PBS buffer, aliquot the purified antibody, lyophilize it, and store it at -20°C.

[0039] S6. Analysis of monoclonal antibody sequences: The PAFAH1B3 protein monoclonal antibody was identified by sequencing. The amino acid sequence of the heavy chain variable region of the monoclonal antibody was shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody was shown in SEQ ID NO: 2.

[0040] Heavy chain variable region (SEQ ID NO: 1):

[0041] EVQLVESGAELAQPGASVKVSCAATGYIFSAYDIHWVKQAPGHGLELIDINNP DNGDLGWKEIDTGFTILTSDTSKNTAYMQLSRSESEDTAVYYEARTYFRFAYLAWL AYVTVQGTLVT;

[0042] Light chain variable region (SEQ ID NO: 2):

[0043] AYQMTQSPSSVPKPGTVTVTISCRVSQSLYGMSANWQKWYLISPPQRGRTSPK DNGNRFARFSGRTINGDKLTIFFTLMEAEDVRYEDLFFYYCFFGSHVPYTFGLGTKL ELK.

[0044] Test Example 1

[0045] Monoclonal antibody subtype detection:

[0046] According to the instructions of the monoclonal antibody typing multifunctional detection kit, identify the subtype of the PAFAH1B3 protein monoclonal antibody. The operation is as follows: take 100 ng of PAFAH1B3 antigen and add it to the ELISA plate, and coat it overnight at 4°C. Add PBST buffer, incubate at 37°C for 3 hours, and then block the ELISA plate. Dilute the PAFAH1B3 protein monoclonal antibody prepared in Example 2 to a concentration of 1 μg / mL, add 100 μL / well to the ELISA plate, and incubate at 37°C for 1 hour. Add 100 μL of 1 μg / mL 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, mix gently, cover with a sealing film, and incubate at 37°C for 1 hour. Wash the plate with PBST buffer to remove free matter, then develop with TMB colorimetric solution for 10 minutes in the dark. Terminate the reaction by adding 100 μL of sulfuric acid to each well. Measure the OD value at 450 nm. The well with the darkest color or the highest OD value corresponds to the PAFAH1B3 protein monoclonal antibody isotype.

[0047] Table 1 Subtype detection results

[0048]

[0049] From the results in Table 1, it can be seen that the monoclonal antibody against PAFAH1B3 protein is of IgG1 subtype.

[0050] Test Example 2

[0051] Specificity identification of PAFAH1B3 monoclonal antibodies:

[0052] PAFAH1B3 recombinant protein, BSA protein, and SDS-containing loading buffer (2×) were mixed in a 1:1 volume ratio. Human pancreatic cancer cell lines SW1990 were lysed and subjected to SDS-PAGE. After electrophoresis, blocking buffer was added for 2 hours. PAFAH1B3 monoclonal antibody was used as the primary antibody and incubated overnight at 4°C to allow the membrane protein to react. The cells were then rinsed three times with PBST buffer and once with PBS to remove unbound antibody and other impurities. Alkaline phosphatase-conjugated anti-mouse secondary antibody was added and incubated for 1 hour at room temperature with gentle shaking. The cells were then rinsed three times with PBST buffer and once with PBS. The cells were then scanned using an infrared imaging system to determine if the monoclonal antibody specifically recognized the PAFAH1B3 protein.

[0053] The results are as follows Figure 2As shown in the figure, lanes 1, 2, and 3 correspond to human pancreatic cancer cell SW1990 lysate, PAFAH1B3 recombinant protein, and BSA protein samples, respectively. The results show that both the human pancreatic cancer cell SW1990 lysate and the recombinant PAFAH1B3 protein form specific bands with the monoclonal antibody PAFAH1B3, while the BSA protein in lane 3 does not. This indicates that the monoclonal antibody PAFAH1B3 specifically binds to PAFAH1B3 in cancer cells and to the recombinant PAFAH1B3 protein, but has no significant binding affinity to BSA, demonstrating that the PAFAH1B3 monoclonal antibody has good specificity.

[0054] Test Example 3

[0055] Functional verification of PAFAH1B3 protein monoclonal antibody:

[0056] A negative control group, PAFAH1B3 protein monoclonal antibody groups at 100 μg / mL, 200 μg / mL, and 300 μg / mL concentrations, and a 200 μg / mL cetuximab positive control group were set up, with 6 replicates for each concentration. Human pancreatic cancer cells SW1990 in the logarithmic growth phase were obtained, digested with trypsin, and the cell suspension concentration was adjusted to 1×10 6 Cells were plated at 400 μL per well and incubated in DMEM medium containing 10% FBS, 1% penicillin, and streptomycin sulfate. After cell attachment, the above drugs were added. After 48 hours of culture, the culture medium was discarded and 100 μL of DMEM medium containing MTT was added to each well for an additional 4 hours. The culture medium was discarded and 100 μL of DMSO was added to each well. After shaking, the absorbance of each well at 490 nm was measured using a microplate reader. The cell growth inhibition rate was calculated as (1 - absorbance of experimental group / absorbance of control group) × 100%. The effects of PAFAH1B3 protein monoclonal antibodies on pancreatic cancer cell proliferation were analyzed based on absorbance and inhibition rate, and compared with the positive control group, cetuximab.

[0057] The results are as follows Figure 3 As shown in the results, PAFAH1B3 protein monoclonal antibody significantly inhibited the proliferation of human pancreatic cancer cell line SW1990. The inhibitory effect was greatest at a concentration of 200 μg / mL, making 200 μg / mL the optimal concentration. At a concentration of 200 μg / mL, the cell growth inhibition rate reached (92.12±1.67)%, significantly higher than the positive control group (91.26±1.83)% with cetuximab.

[0058] Test Example 4

[0059] Effects of monoclonal antibodies and umbilical cord mesenchymal stem cells on the proliferation of pancreatic cancer:

[0060] Frozen human pancreatic cancer cells SW1990 were quickly thawed in a 37°C water bath and cultured in DMEM medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL). Human pancreatic cancer cells SW1990 in the logarithmic growth phase were used for subsequent experiments. The cells were digested with trypsin and the cell suspension density was adjusted to 1×10 6 100 μL of cell suspension was inoculated into 96-well plates for culture. Six parallel controls were set up in each group to reduce errors. After 24 h of culture, 0.9% saline (negative control group), 1×10 umbilical cord mesenchymal stem cells, and 1×10 5 cells / mL (mesenchymal stem cell group), 200 μg / mL PAFAH1B3 monoclonal antibody (monoclonal antibody group), 1×10 umbilical cord mesenchymal stem cells 5 The researchers used a microplate reader to measure the absorbance of cells at 24, 48, and 72 hours after the infusion of umbilical cord mesenchymal stem cells (UCMSCs), monoclonal antibodies, and the combination of UCMSCs and monoclonal antibodies, along with cetuximab, on the proliferation of pancreatic cancer cells SW1990.

[0061] The results are as follows Figure 4 As shown in the figure, at 72 hours, the OD value of the negative control group was 1.51, the OD value of the positive control group was 0.14, and the OD value of the combination group was 0.06. The results show that the combination of umbilical cord mesenchymal stem cells and PAFAH1B3 protein monoclonal antibody can significantly inhibit the proliferation of human pancreatic cancer cells SW1990, and the inhibition of pancreatic cancer cell proliferation is slightly stronger than that of the positive control group. At 72 hours, the OD value of the umbilical cord mesenchymal stem cell group was 0.38, and the OD value of the monoclonal antibody group was 0.12. Although the umbilical cord mesenchymal stem cell group did not inhibit the proliferation of human pancreatic cancer cells SW1990 as significantly as the monoclonal antibody group, it still had a good inhibitory effect compared to the negative control group. Overall, the combination of umbilical cord mesenchymal stem cells and monoclonal antibodies has a good synergistic effect and can significantly inhibit the proliferation of human pancreatic cancer cells SW1990.

[0062] Test Example 5

[0063] Effects of umbilical cord mesenchymal stem cells and monoclonal antibodies on tumor size and weight:

[0064] Human pancreatic cancer cells in the logarithmic growth phase were prepared into a cell suspension and 1×10 cells of the cell suspension were inoculated into female nude mice of about 6 weeks old. 5 / mouse, a subcutaneous pancreatic cancer tumor model was established in female nude mice. The mice with successful tumor transplantation were divided into 5 groups, 8 mice in each group. They were injected with 0.9% saline (negative control group), 2mg / kg monoclonal antibody (monoclonal antibody group), 1×10 6 / kg umbilical cord mesenchymal stem cells (mesenchymal stem cell group), 2mg / kg monoclonal antibody and 1×10 6 The mice were inoculated with umbilical cord mesenchymal stem cells (100mg / kg) and cetuximab (2mg / kg) (positive control group). The growth of tumors in different groups was observed by inoculating different drugs. The nude mice were weighed and the tumor size was measured using a vernier card. The long diameter L and short diameter W of the tumor were calculated using the formula V = 0.5LW. 2 Estimated tumor volume (mm 3 ), data processing was performed to plot a tumor growth curve. After 32 days, the mice were sacrificed, the skin was peeled, the tumors were separated, and the weight and volume of the tumors were measured to comprehensively investigate and evaluate the therapeutic effect.

[0065] The results are as follows Figure 5 As shown in the figure, at 32 days, compared with the negative control group, the monoclonal antibody group, umbilical cord mesenchymal stem cell group, combined group, and positive control group were able to inhibit the size of the tumor. The combined group was able to significantly inhibit the size of the tumor, and its inhibitory ability was slightly stronger than that of the positive control group. Although the effect of the umbilical cord mesenchymal stem cell group was not as significant as that of the monoclonal antibody group, it also had a good inhibitory effect compared with the negative control group. The experimental results are shown in the figure. Figure 6 As shown, the tumor weights of the combination group and the negative control group were 0.31±0.23g and 0.82±0.15g, respectively. The combination group significantly inhibited tumor weight, showing the strongest ability to suppress tumor weight, slightly stronger than the positive control group. In summary, the combination of monoclonal antibodies and umbilical cord mesenchymal stem cells has a good synergistic effect, inhibiting tumor volume and weight.

[0066] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A composition of umbilical cord mesenchymal stem cells and monoclonal antibodies, characterized in that: The monoclonal antibody is a monoclonal antibody specific for human PAFAH1B3 protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

2.

2. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 1, wherein: The subtype of the monoclonal antibody is IgG1.

3. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 1, wherein: The umbilical cord mesenchymal stem cells are P2-P5 generation umbilical cord mesenchymal stem cells.

4. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 3, wherein: The method for preparing P2-P5 umbilical cord mesenchymal stem cells comprises the following steps: (1) Wharton's jelly was removed from the umbilical cord, and the Wharton's jelly was minced into tissue blocks. The tissue blocks were mixed with culture medium and cultured in a cell culture incubator at 37°C and 5% CO2. (2) When umbilical cord mesenchymal stem cells appear around the tissue block, the medium is completely replaced and recorded as P0; (3) When the cell confluence reaches 80-85%, the umbilical cord tissue pieces and culture medium are discarded, 0.25% trypsin is added for digestion, the digestion solution is removed by centrifugation, and the culture medium is added for resuspending. The cells are then subcultured to obtain P2-P5 umbilical cord mesenchymal stem cells.

5. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 4, wherein: The subculture ratio in step (3) is 1:

2.

6. Use of the umbilical cord mesenchymal stem cell and monoclonal antibody composition according to any one of claims 1 to 5 in the preparation of a drug for treating pancreatic cancer.

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