CN116769724B

CN116769724B - Mesenchymal stem cells carrying killing switch and application thereof in tumor treatment

Info

Publication number: CN116769724B
Application number: CN202311033606.0A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Zaiyiu Beijing Biotechnology Co ltd
Current assignee: Zaiyiu Beijing Biotechnology Co ltd
Priority date: 2023-08-17
Filing date: 2023-08-17
Publication date: 2023-10-27
Anticipated expiration: 2043-08-17
Also published as: CN116769724A

Abstract

The application provides a mesenchymal stem cell with a killing switch and application thereof in tumor treatment, wherein the mesenchymal stem cell is introduced with a suicide gene, the suicide gene is cytosine deaminase, and the cytosine deaminase is subjected to sequence transformation and has stronger catalytic activity; the tumor immunosuppressant comprises the mesenchymal stem cells, 5-FC and monoclonal antibodies, wherein the monoclonal antibodies are targeted CD44 antibodies, targeted CD133 antibodies and/or targeted OLIG2 antibodies, so that the tumor stem cells can be effectively killed, drug-resistant cells are prevented from appearing, and the tumor treatment effect is improved.

Description

Mesenchymal stem cells carrying killing switch and application thereof in tumor treatment

Technical Field

The application belongs to the field of biotechnology research and development, and particularly provides a mesenchymal stem cell carrying a killer switch and application thereof in tumor treatment.

Background

Tumors have become the largest "killer" threatening human health, and so far researchers have developed a variety of means for treating malignant tumors, with chemotherapy being the most common and most widely used therapy. Chemotherapy drugs kill most cells in tumors, but long term use can give rise to tumor resistance, probably because tumor cells receiving chemotherapy release extracellular vesicles, resulting in the establishment of drug-resistant cell subsets, and systemic administration can adversely affect normal cells due to poor targeting of the chemical, thus causing unpleasant toxic side effects. There is therefore an increasing interest in developing new strategies that can more effectively target prodrugs to tumor cells to increase efficacy and reduce toxicity.

Gene-directed enzyme prodrug therapy (GDEPT) is one of the most important and successful methods of prodrug delivery, showing great application prospects in cancer treatment. GDEPT uses transgenically encoded enzymes to convert the prodrug into an active therapeutic metabolite. GDEPT is typically composed of three parts: inactive drug (prodrug), gene encoding an enzyme that converts the inactive prodrug into an active drug, and vector. The therapy generally comprises the following three steps: first, cloning the coding gene into a vector and delivering to tumor cells with or without the vector; in the second step, the gene is transcribed into mRNA and subsequently translated into enzymes within the tumor cell; in a third step, the prodrug is administered systemically and taken up by the same cell, and then the prodrug can be converted to a cytotoxic drug by intracellular enzymes. Since gene expression may be controlled by a tumor cell specific promoter, the enzyme and its associated enzymatic reactions can be directed precisely against tumor cells, leaving other cells unaffected even if phagocytosed to the gene and prodrug (see Both GW. Recent progress in gene-directed enzyme prodrug therapy: an emerging cancer treatment, current Opin Mol Ther 2009;11 (4): 421-32). The prodrug is preferentially converted to the drug and the toxic drug is only produced in tumor cells with minimal contact with healthy cells, so that the therapeutic index of the prodrug can be much higher than that of conventional cancer chemotherapeutics.

The ideal GDEPT enzyme should be specifically expressed in tumor cells or expressed in relatively high proportions and should have high catalytic activity so that the tumor cells can convert the prodrug even at low substrate concentrations. The ideal prodrug of GDEPT should be non-toxic or minimally toxic prior to activation by the enzyme, but highly toxic after activation by the enzyme. Furthermore, the prodrug should be efficiently taken up by tumor cells, have a high affinity for the transduced enzyme, and a low affinity for the unrelated endogenous enzyme. In order to effectively kill tumor cells, the cytotoxic metabolites of the prodrugs should have a long half-life. During the last two decades, many enzyme/prodrug systems have been studied, the most widely used of which are herpes simplex virus thymidine kinase (herpes simplex virus thymidine kinase, HSV-TK) and Ganciclovir (GCV), cytosine deaminase (cytosine deaminase, CD) and 5-fluorocytosine (5-FC), cytochrome P450 and cyclophosphamide/ifosfamide (CPA/IFA)), as well as nitroreductase and CB1954.

Among the many GDEPT enzymes, cytosine deaminase is a well studied biological enzyme, and several studies have compared the efficacy of CD/5-FC with HSV-Tk/GCV systems, showing that CD/5-FC is significantly better than HSV-TK/GCV, the superior effect of CD/5-FC may be attributed to its larger bystander effect, the bystander effect of GCV is primarily dependent on gap junctions, while the effect of 5-FU is mediated by passive diffusion. Cytosine deaminase is present only in bacteria and fungi, and catalyzes the deamination of cytosine to uracil, an important member of the pyrimidine nucleotide rescue pathway. GDEPT therapy using CD has almost entirely focused on one prodrug, 5-FC.5-FU has been widely used in cancer chemotherapy, but high doses are required for anticancer activity and patients cannot tolerate these high doses. Compared to 5-FU, 5-FC is less toxic. Thus, the CD/5-FC system reduces systemic toxicity that may be caused by 5-FU alone. Because CD is capable of converting 5-FC to 5-FU, toxicity of 5-FU is only directed against CD-expressing tumor cells (see Kurozumi K, et al Apoptosis induction with 5-fluorodeoxynine/cytosine deaminase gene therapy for human malignant glioma cells mediated by adenovirus. J neuroonol. 2004;66 (1-2): 117-27).

CD/5-FC therapy has been studied in a variety of in vitro and in vivo animal models of cancer. Studies have shown that MDA-MB-231 breast Cancer cells transfected with E.coli CD are 1000-fold more sensitive to 5-FC than control cells, and that complete cytotoxicity can be induced in co-cultures with uninfected cells by only 10% cell transfection, and that intratumoral injection of adenovirus-encoded CD and systemic 5-FC can control MDA-MB 231 breast Cancer xenografts in nude mice and intracranial human glioma xenografts in Severe Combined Immunodeficiency (SCID) mice (see Li Z, et al Enzyme/prodrug Gene therapy approach for breast Cancer using a recombinant adenovirus expressing Escherichia coli cytosine deaminase. Cancer Gene Ther. 1997;4 (2): 113-7). Similar findings also appear in CD/5-FC treatment of colon and Prostate cancers (see O' Keefe DS, et al, prostate-specific suicide gene therapy using the Prostate-specific membrane antigen promoter and, advanced. Prostate. 2000;45 (2): 149-57). Currently the CD/5-FC system has been started for clinical trials, one ongoing pilot feasibility study was directed to the treatment of recurrent high-grade gliomas (NCT 01172964), while the other two patients were recruited to solid tumors (NCT 01562626) and malignant brain tumors (NCT 01470794).

One of the challenges of GDEPT therapy is the development of efficient gene delivery systems to optimize enzyme gene expression and increase the efficacy of GDEPT, a variety of delivery systems have been explored to target GDEPT systems to tumors, including viral vectors, liposomes, nanoparticles, mesenchymal stem cells, naked DNA, and the like. Initially, the focus was on viral vectors which are easy to prepare, efficient to transfect, easy to genetically engineer, but the safety issues associated with viral vectors have prompted efforts to develop non-viral vectors. Mesenchymal stem cells (Mesenchymal stem cell, MSC) are multipotent stem cells with tumor homing capacity, thus becoming part of the tumor microenvironment, this particular capacity of mesenchymal stem cells inspires us to develop a therapeutic approach based on gene-directed enzyme/prodrug therapies. MSC have been used to deliver the GDEPT gene, which is expressed as CD to convert 5-FC to 5-FU in vitro using MSC or Neural stem cells, and this system has been shown to have the effect of inhibiting tumor growth and prolonging survival time of tumor mice (see Aboody KS, et al, neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical scaffolds. Sci Transl Med. 2013;5 (184): 184ra 59). In addition, MSC-mediated prodrug gene therapy has been effective in inhibiting the growth of human colon, melanoma, and prostate cancers in nude mice.

Notably, mesenchymal stem cells currently have limitations as gene vectors. First, targeting tumors has limited efficiency, hampering the efficiency of gene delivery to tumors, and to overcome this limitation, methods to enhance tumor tropism of MSCs have been proposed and tested, including altering tumor microenvironment and modifying MSC surface molecules to obtain better adhesion and tumor infiltration (see klop AH, et al Tumor irradiation increases the recruitment of circulating mesenchymal stem cells into the tumor microenvironment. Cancer res 2007;67 (24): 11687-95). The second limitation stems from the relationship between mesenchymal stem cells and tumor growth. It remains controversial whether MSCs promote tumor growth, and suicide genes are the most desirable payloads for mesenchymal stem cells for safety reasons, as they can destroy the cell carrier after delivery.

In order to overcome the defects of the existing GDEPT therapy, the application provides a mesenchymal stem cell with a killing switch, which carries a cytosine deaminase gene, can convert 5-FC into 5-FU in vivo to play an anti-tumor role, and can be used with a targeted CD133 antibody to prevent the occurrence of tumor drug resistance, further improve the anti-tumor activity and prolong the life cycle of animals.

Disclosure of Invention

In a first aspect, the application provides a mesenchymal stem cell carrying a killing switch, wherein the mesenchymal stem cell is introduced with a suicide gene, the suicide gene is cytosine deaminase, and the amino acid sequence of the suicide gene is shown as SEQ ID NO. 2.

The application utilizes the tumor cell homing ability of the mesenchymal stem cells, uses the mesenchymal stem cells as an immunotherapy carrier to transport suicide genes, and can avoid systemic adverse reactions and effectively kill tumor cells by being matched with prodrug 5-FC, thereby providing a tumor treatment scheme with high safety.

Further, the nucleotide sequence of the suicide gene is shown as SEQ ID NO. 3.

Further, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the mesenchymal stem cells are CD73+, CD90+, CD34-, and CD 45-cells.

In a second aspect, the application provides a tumor immunosuppressant, which comprises the mesenchymal stem cells, 5-FC and a monoclonal antibody, wherein the monoclonal antibody is a targeted CD44 antibody, a targeted CD133 antibody and/or a targeted OLIG2 antibody.

Furthermore, the monoclonal antibody is a targeting CD133 antibody, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5.

The development of tumor resistance is associated with a variety of mechanisms of action, including immune checkpoint shutdown, gene mutation, key enzyme inactivation, etc., where cancer stem cells play a key role in this process. The concept of tumor stem cells was first proposed by Park et al in 1971 to consider tumors as diseases driven by a self-renewing subpopulation of CSCs that have the ability to differentiate into different cell populations that make up the tumor entity, and even though some treatments could completely regress the tumor entity, there may remain sufficient tumor stem cells to cause recurrence of the tumor, suggesting that treatment with only tumor stem cells more specifically might result in a more durable therapeutic effect, even curing metastatic tumors. Currently, tumor stem cells have been identified and isolated from tumors of the hematopoietic system, breast, lung, prostate, colon, brain, head and neck, and pancreas.

Tumor stem cells are not unscrupulous, and often express some tumor markers on their surface, such as CD44, CD133, CD117, OLIG2, etc., where CD44 and CD133 are the most common tumor stem cell markers, CD117 is also observed in a variety of tumor stem cells such as ovarian cancer, glioma, liver cancer, etc., oligodendrocyte transcription factor 2 (OLIG 2) is a central nervous system limiting transcription factor, plays a key role in glial progenitor proliferation and CNS development, OLIG2 is also widely expressed in gliomas, and plays an important role in gliomagenesis and tumor phenotype plasticity. Studies have shown that CD133 expression levels on tumor cell surfaces are increased following development of 5-FU resistance (see Hee Yi, hee-Jung Cho, et al, effect of 5-FU and MTX on the Expression of Drug-resistance Related Cancer Stem Cell Markers in Non-small Cell Lung Cancer Cells, korea J Physiol Pharmacol. 2012 Feb; 16 (1): 11-16.)

According to the application, the mesenchymal stem cells subjected to genetic engineering modification are used in combination with the antibodies targeting different tumor stem cell surface markers, so that not only can the proliferation of the tumor stem cells be inhibited, but also the occurrence of drug resistance can be prevented, thereby improving the anti-tumor effect, and the research shows that the effect of combining with the anti-CD 133 antibody is optimal.

The third aspect of the application provides an application of the mesenchymal stem cells or the tumor immunosuppressant in preparing an anti-tumor medicament.

Further, the tumor is glioma, colorectal cancer or ovarian cancer.

Advantageous effects

The application provides a mesenchymal stem cell carrying a killing switch and application thereof in tumor treatment, and the mesenchymal stem cell has the following advantages:

(1) The suicide gene cytosine deaminase is introduced into the mesenchymal stem cells, so that the medicine administration treatment in vivo can be realized, and systemic toxic and side effects are prevented;

(2) The cytosine deaminase gene is modified, so that the cytosine deaminase has stronger deamination effect and the enzyme activity is enhanced;

(3) The genetically modified mesenchymal stem cells are combined with monoclonal antibodies, especially with antibodies targeting CD133, so that the growth of the tumor stem cells can be inhibited, and the occurrence of tumor recurrence and drug resistance can be prevented;

(4) The stem cell treatment method provided by the application can effectively inhibit the growth of tumors in animals, regulate the expression of immune factors and improve the treatment effectiveness.

Drawings

Fig. 1: genetically modified mesenchymal stem cells MSC-CD inhibit proliferation of a variety of tumors;

fig. 2: MSC-CD cells and antibodies used in combination to inhibit proliferation of drug-resistant tumor cells;

fig. 3: tumor volume changes in animal models of different treatment groups;

fig. 4: immune factor expression levels in serum of model animals.

Description of the embodiments

The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the application and are not intended to limit the application in any way. All techniques implemented based on the above description of the application should be within the scope of the application as claimed.

The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.

Example 1 sequence optimization of cytosine deaminase

A variety of cytosine deaminase enzymes have been reported in the prior art to be useful in tumor therapy, including E.coli, yeast derived cytosine deaminase enzymes, and the application is based on the research of E.coli cytosine deaminase enzymes, wherein the amino acid sequence of the cytosine deaminase enzyme is searched and selected in NCBI database, the Genebank number of which is WP_219385354.1, and the amino acid sequence of which is shown as SEQ ID NO. 1.

In early studies, researchers tried to modify the structure of cytosine deaminase sequences by various site-directed mutagenesis, sequence truncation, and the corresponding mutations were designated CD-M01, CD-M02, CD-M03, CD-M04, and CD-M05, respectively. Synthesizing the mutant gene through total genes, designing primers to carry out PCR amplification by utilizing homologous recombination, and respectively introducing subclones into corresponding sites of a vector pET32a to obtain recombinant plasmids; recombinant E.coli BL21 (DE 3)/pET 32a-CD was obtained by heat shock transformation of E.coli BL21 (DE 3). Culturing at 37deg.C for 12 hr/min, centrifuging to collect thallus, removing supernatant medium, adding fermentation medium, culturing at 37deg.C for 8 hr/min, adding lactose to final concentration of 2.0g/L, cooling to 30deg.C, inducing expression for 24 hr, centrifuging to collect thallus. The mutant enzyme activity was determined by adding 10g/L wet cell and 20mM substrate 5-FC, the reaction medium was phosphate buffer pH 8.0, the total system was 1mL, and incubated at 37℃for 90 minutes. The reaction was then quenched with 10% trichloroacetic acid and the mixture was centrifuged at 4 ℃ to collect the supernatant. The presence of 5-FC and 5-FU was detected by absorbance at 290nm and 255nm, respectively, and the relative enzyme activities were calculated. As a result, as shown in Table 1, the enzyme activities of the respective mutations were changed to different extents, wherein the mutant CD-M03 was the strongest in activity, far higher than the wild type, and the mutant was selected for subsequent experiments, and the amino acid sequence thereof was shown as SEQ ID NO.2, and the nucleotide sequence thereof was shown as SEQ ID NO. 3.

TABLE 1 mutant relative enzyme Activity

	Relative enzyme Activity
		Wild type CD	100%
CD-M01	165%
		CD-M02	85%
CD-M03	260%
		CD-M04	186%
CD-M05	90%

EXAMPLE 2 modification of mesenchymal Stem cells Using cytosine deaminase Gene

2.1 Lentiviral vector preparation

Designing corresponding primers, amplifying a CD-M03 gene sequence by a PCR technology, introducing the gene sequence into a lentiviral vector plasmid vector pCDH-EF1 alpha-copGGFP-T2A-Puro by an enzyme digestion ligation reaction, and mixing the pCDH-EF1 alpha-copGGFP-T2A-Puro plasmid vector with virus packaging plasmids pSPAX2 and pMD2G according to a ratio of 4:3:2 and incubated with PEI (available from Polyscience, USA) and Opti-MEM medium to transfect 293T cells, see PEI transfection reagent instructions for specific transfection procedures. The DMEM complete medium is changed 24 hours after transfection, the supernatant is collected 48 hours and 72 hours after liquid change, and after centrifugation for 15 minutes at 4 ℃ and 3000g, the supernatant is filtered by a 0.45 mu m filter membrane, split-packed into EP tubes and transferred to-80 ℃ for preservation.

2.2 mesenchymal Stem cell preparation

Separating and preparing the mesenchymal stem cells from umbilical cord tissues in this section, washing the umbilical cord with 0.9% physiological saline to remove impurities, putting the washed umbilical cord into a new culture dish, cutting off the ligature parts at the two ends, cutting off the small umbilical cord along the spiral trend of the venous blood vessel, and removing the arterial blood vessel and the venous blood vessel in the umbilical cord; cutting the residual umbilical cord into a plurality of segments 2-3 cm long small segments, and washing 3 times by using sterile physiological saline; tearing tissue from Huatong with tissue forceps, and cutting into pieces 1-8 mm ³ Is a small block of (2); inoculating the tissue into cell culture dish, adding 4mL complete culture medium, and adding 5% CO at 37deg.C ₂ And culturing under saturated humidity until the mesenchymal stem cells climb out of the tissue. After the cells grow out, the cells are digested by pancreatin and collected by centrifugation, and detected by a flow cytometer, and the cells are CD73+, CD90+, CD34-, and CD 45-cells, so that the follow-up requirements are met.

2.3 mesenchymal Stem cell Gene modification

Mesenchymal stem cells obtained in section 2.2 were subcultured, and after the cells had reached the logarithmic growth phase, lentiviruses were transfected with an infection coefficient of moi=5, and a virus transfection reagent polybeen (purchased from Sigma) was added. 37 ℃ and 5% CO ₂ And culturing under saturated humidity conditions, after 48h of transfection, centrifuging to collect cells, and identifying by a flow cytometer, wherein the cells can express the cytosine deaminase,transfection efficiency was up to 75% and the cells were designated MSC-CD.

EXAMPLE 3 cytosolic deaminase Gene-modified mesenchymal Stem cells inhibit tumor cell growth

The inhibition of tumor by cytosine deaminase gene modified mesenchymal stem cells was examined using glioma cell line U251, colon cancer cell line HCT15 and ovarian cancer cell line SKOV-3 (purchased from Shanghai cell institute) as subjects.

Inoculating the tumor cells into 96-well plate, 5000 cells per well, 37 deg.C, 5% CO ₂ And after 4 hours of incubation under saturated humidity, MSC-CD cells were added at a ratio of 5:1, with 500. Mu.g/mL of 5-FC added, and with control wells of culture wells with only 500. Mu.g/mL of 5-FC added. After 48h of culture, the cell viability was checked by CCK8 method, which comprises the following steps: the culture medium was discarded, the residual liquid was aspirated, 100. Mu.L of serum-free medium and 10. Mu.L of CCK8 were added per well under a dark condition, the 96-well plate was placed in a 37℃incubator for 1 hour under dark conditions, the absorbance OD450nm was measured using an enzyme-labeled instrument, and the measurement was repeated 3 times to obtain an average value. The cell viability was calculated as = (experimental OD value-blank OD value)/(control OD value-blank OD value) ×100% by the formula.

As shown in FIG. 1, the MSC-CD cells provided by the application have obvious inhibition effects on glioma cells, colon cancer cells and ovarian cancer cells, have the strongest inhibition effect on HCT15 cells, and have relatively poor SKOV-3 cells after U251 cells.

EXAMPLE 4 use of cytosine deaminase Gene-modified mesenchymal Stem cells in combination with antibodies

5-FU is a thymidylate synthase inhibitor which is converted into 5-fluorouracil deoxynucleotide (5F-dUMP) in cells, and inhibits deoxythymidylate synthase, preventing methylation of deoxyuridylate (dUMP) into deoxythymidylate (dTMP), thereby affecting DNA synthesis. As broad-spectrum antitumor drugs, 5-FU has obvious inhibition effect on esophageal cancer, gastric cancer, intestinal cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, ovarian cancer, chorionic epithelial cancer, bladder cancer, head and neck tumor, glioma and the like, but drug resistance is easy to generate in the use process, and researches show that the drug resistance is related to tumor stem cells, and MSC-CD cells are combined with an antitumor antibody in the section so as to effectively kill the tumor stem cells and prevent the occurrence of drug resistance.

In this section, a glioma cell line U251 is used as a basis, a 5-FU drug-resistant cell line is cultivated by a stepwise adaptation method, and the drug-resistant tumor cells can grow in 200 mug/mL of 5-FU after domestication. In this example, MSC-CD was examined in combination with a monoclonal antibody, and monoclonal antibodies targeting CD44, CD133, CD117, OLIG2, which were previously prepared by the inventors, were selected as candidate drugs for inhibition of drug-resistant cells, respectively. Inoculating the drug-resistant tumor cells into 96-well plates, 5000 cells per well, 37 ℃ and 5% CO ₂ And after culturing for 4 hours under saturated humidity, adding MSC-CD cells according to the proportion of 5:1, simultaneously adding 500 mug/mL of 5-FC, and then randomly dividing into 5 groups, wherein each group is provided with 3 compound holes, and the compound holes are respectively as follows: anti CD44 group, adding targeted CD44 monoclonal antibody, and the final concentration is 100 mug/mL; anti CD133 group, adding targeted CD133 monoclonal antibody, and the final concentration is 100 mug/mL; anti CD117 group, adding targeted CD117 monoclonal antibody, wherein the final concentration is 100 mug/mL; anti OLIG2 group, adding targeted OLIG2 monoclonal antibody, and the final concentration is 100 mug/mL; control group, add equal volume of medium. Cell viability was measured after 48h incubation using CCK8 procedure, see example 3 for specific procedures.

As shown in fig. 2, the effect of the single use of MSC-CD cells on drug-resistant tumor cells is very small, and after the single use of MSC-CD cells and monoclonal antibodies are combined, the anti-tumor effect is enhanced, but the monoclonal antibodies targeting CD117 do not seem to have obvious synergistic effect, and the combined effect of the monoclonal antibodies targeting CD133 and OLIG2 is better, so that the monoclonal antibodies are optimal combined therapeutic agents because CD133 is a surface marker commonly existing in various tumor stem cells and has higher adaptability. Through sequence determination, the amino acid sequence of the heavy chain variable region of the target CD133 monoclonal antibody provided by the application is shown as SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5.

EXAMPLE 5 in vivo inhibition of tumor cell growth by cytosine deaminase Gene-modified mesenchymal Stem cells

5.1 preparation of tumor animal models and treatment with drug administration

In this section, glioma cell line U251 was used to prepare a mouse tumor model, 1×10 was taken ⁶ The individual cells were inoculated subcutaneously in the armpit of the right forelimb of nude mice, the tumor formation was observed daily and recorded, and the tumor volume was grown to 100mm ³ In the above cases, successful molding was indicated. Taking 30 mice successfully molded, randomly dividing the mice into 3 groups, and respectively: MSC-CD group, tail vein injection 1X 10 ⁶ MSC-CD cells injected daily with 500 mg/kg of 5-FC; MSC-CD+CD133 group, tail vein injection 1×10 ⁶ MSC-CD cells were injected with 500 mg/Kg of 5-FC per day, and 50mg/Kg of CD 133-targeting monoclonal antibody every 3 days; in the control group, an equal volume of physiological saline was injected daily. The various drugs were administered for 30 days, and the therapeutic effect was observed.

5.2 cell therapy can inhibit tumor growth

Tumor size was observed every 2 days, and the long diameter (L) and short diameter (W) of the tumor were recorded, and the tumor volume (V) was calculated as follows: v tumor= (long diameter l×short diameter W/2). The results are shown in FIG. 3, where MSC-CD cells are able to delay tumor growth, but tumor inhibition begins to decrease at the end of the administration period, especially in the last week, and tumor volume begins to increase rapidly, which may be associated with the development of 5-FU resistance, whereas MSC-CD cells in combination with anti-CD 133 antibodies do not, indicating that this combination therapy is effective in killing tumor stem cells and inhibiting the development of resistance.

5.3 cell therapy modulating immune factor expression

The expression level of tumor immune factors in serum was examined in this section to examine the effect of the stem cell therapy on the immune environment. After 15 days of treatment, the blood of the mice is taken and placed in an anticoagulant tube for standing at room temperature for 1h, and the blood is centrifuged at 4 ℃ and 2000rmp for 20min to obtain the serum of the mice. The serum was assayed for TNF- α and IFN- γ content using ELISA kits (ex Abcam, USA) and the specific procedures were followed according to the kit instructions.

The results are shown in FIG. 4, where the levels of TNF- α and IFN- γ are both increased after treatment with MSC-CD cells, and where IFN- γ is more pronounced, especially in the combination of MSC-CD cells with anti-CD 133 antibodies, the IFN- γ expression levels are greatly increased, indicating that this approach is beneficial for mobilizing immune responses in vivo against tumor growth.

The present application is not limited to the above-mentioned embodiments, but is not limited to the above-mentioned embodiments, and any person skilled in the art can make some changes or modifications to the equivalent embodiments without departing from the scope of the present application.

Claims

1. A tumor immunosuppressant, characterized in that: the monoclonal antibody is a targeting CD133 antibody, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5; the mesenchymal stem cells are mesenchymal stem cells carrying a killing switch, and are introduced with suicide genes, wherein the suicide genes are cytosine deaminase, and the amino acid sequence of the suicide genes is shown as SEQ ID NO. 2.

2. The use of a tumor immunosuppressant as claimed in claim 1 for the preparation of an antitumor drug, wherein: the tumor is glioma, colorectal cancer or ovarian cancer.