CN115814069B

CN115814069B - Application of MIF gene knockout tumor cells in preparation of tumor vaccine

Info

Publication number: CN115814069B
Application number: CN202211348248.8A
Authority: CN
Inventors: 郑宇欢; 黄菁草; 徐娟
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-07-21
Anticipated expiration: 2042-10-31
Also published as: CN115814069A

Abstract

The invention relates to an application of MIF gene knockout tumor cells in preparation of tumor vaccines, and belongs to the field of tumor vaccines. The invention discovers for the first time that MIF plays an important role in inducing the immune escape process of Multiple Myeloma (MM), and NK cells, macrophages and T cells participate in the MIF-regulated MM immunosuppression and immune escape. MIF knockout MM cells do not induce tumor immunosuppressive microenvironments efficiently, and thus cannot escape immunity, so that tumors are not generated in the mouse transplanted tumor model. The mice after the MIF gene knockout MM cell injection stimulation can not only not generate MM, but also acquire MM immunity; normal MM cells are used in future for tumor transplantation, and MM does not occur. Therefore, the MIF gene knocked-out MM cell can be used as a tumor vaccine, effectively prevents MM, and has potential application prospect in blocking benign precancerous lesions from progressing to MM and reducing the occurrence rate of MM.

Description

Application of MIF gene knockout tumor cells in preparation of tumor vaccine

Technical Field

The invention belongs to the field of tumor vaccines, and particularly relates to application of MIF gene knockout tumor cells in preparation of tumor vaccines.

Background

Multiple Myeloma (MM) is a hematological malignancy that is well developed in the elderly, and has not been cured until now. MM is the second most common hematologic malignancy based on foreign epidemiological statistics. With the rapid progress of the aging of China, the incidence of MM in China is recently and continuously increased.

Macrophage migration inhibitory factor (macrophage migration inhibitory factor, abbreviated MIF) is a pro-inflammatory, pro-tumor cytokine with multiple biological functions. MIF can be synthesized and secreted by a variety of cells (e.g., monocytes, macrophages, etc.). In solid tumors, MIF is closely related to the growth, invasion, metastasis, etc. of various tumors. Mechanically, MIF can regulate the functions of cytotoxic T cells and NK cells and induce the production of immunosuppressive cells such as MDSCs, TAMs, etc. In 2018, piddock et al reported that MIF can promote bone marrow stromal cells to secrete IL-6 and IL-8 through c-MYC, thereby indirectly affecting the MM immune microenvironment.

Tumor vaccine is an important direction of tumor immunotherapy, and the basic concept is to introduce tumor antigen into a body in various forms to activate the body to perform anti-tumor immunity, so as to achieve the purposes of preventing, controlling or eliminating tumor. In the field of MM, attempts have been made to control MM using various forms of tumor vaccines, such as polypeptide vaccines, dendritic cell vaccines, and anti-idiotype antibody vaccines, with a certain clinical effect, but no substantial progress has been made. Tumor vaccines can be classified into prophylactic tumor vaccines and therapeutic tumor vaccines, and the major challenges facing therapeutic MM vaccine development are reduced MM cell immunogenicity and the immune-suppressive tumor microenvironment induced MM cells to evade immune recognition and clearance.

MM has a well-defined stage of benign precancerous lesions, known as "meaningless monoclonal gammaglobulinemia" (monoclonal gammopathy of undermined significance, MGUS). In the population over 50 years of age, about 3.2% of individuals suffer from MGUS, manifesting as abnormal serum monoclonal proteins and small numbers of bone marrow clonal plasma cells. MGUS patients have no associated symptoms or signs, but the proportion of MGUS patients who progress to malignant MMs is far higher than the general population, with about 1% of MGUS patients progressing to MMs each year. The molecular mechanism of MGUS to MM has not yet been fully resolved, nor has there been any intervention at present. The presence of MGUS provides a window period for early intervention of MM, or even MM prophylaxis. Therefore, development of a tumor vaccine having an MM preventive effect is of great importance in blocking the progress of MGUS into MM and reducing the occurrence rate of MM.

Disclosure of Invention

In order to solve the above problems in the prior art, an object of the present invention is to provide an application of MIF knockout tumor cells in preparing tumor vaccines, and another object of the present invention is to provide a tumor vaccine using MIF knockout tumor cells as an active ingredient.

The invention provides a tumor vaccine, which is a preparation prepared by taking MIF gene knocked-out tumor cells as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

Further, the tumor is multiple myeloma.

Further, the formulation is an injectable formulation.

The invention also provides application of the MIF gene knockout tumor cells in preparation of tumor vaccines.

Further, the tumor is multiple myeloma.

Further, the tumor vaccine is a prophylactic tumor vaccine or a therapeutic tumor vaccine.

Further, the tumor vaccine is a formulation that blocks the development of benign precancerous lesions, preferably monoclonal gammaglobulinemia of unknown significance, into tumors.

Further, the tumor vaccine is an agent for regulating NK cell immunity, regulating macrophage immunity and/or regulating T cell immunity.

The invention discovers for the first time:

1) Macrophage Migration Inhibitory Factor (MIF) plays an important role in inducing immune escape in Multiple Myeloma (MM). MM cells with MIF knockout (MIF-KO) do not produce tumors in the mouse transplantation tumor model because they are not able to effectively induce the generation of tumor immunosuppressive microenvironment and cannot escape.

2) MIF plays a regulatory role in the MM immunosuppression microenvironment, and NK cells, macrophages and T cells participate in MIF-regulated MM immunosuppression and immune escape.

3) The MM cells of MIF-KO are injected into the stimulated mice, so that the mice can not only not generate MM, but also acquire MM immunity; normal MM tumor transplantation is used again in the future, and MM does not occur. Thus, MM cells of MIF-KO can be used as tumor vaccine to effectively prevent MM.

4) As is well known to those skilled in the art, MM patients commonly have benign precancerous lesions known as Monoclonal Gammaglobulinemia (MGUS), which suffer from MM at a much higher rate than the general population, and can be considered as a high risk group for MM. If MGUS is effectively blocked from progressing to MM, MM prevalence will be greatly reduced. Therefore, the tumor vaccine with the MM preventing effect has potential application prospect in blocking MGUS from being progressed into MM and reducing the occurrence rate of MM.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1. Expression of MIF in multiple myeloma. A) MIF levels in bone marrow puncture supernatants of non-tumor patients or healthy volunteers (CTR-BM) and multiple myeloma patients (MM-BM). B) MIF expression levels in various cells, including bone marrow stromal cells from healthy volunteers (nmmscs), bone marrow stromal cells from multiple myeloma patients (MM-BMSCs), patient MM cells (pt.mm), and various human MM cell lines. C) Multiple myeloma expression MIF levels correlated with patient prognosis, with overall poor prognosis for MM patients with high MIF expression (left panel): in relapsed patients, patients with high MIF expression also had poor prognosis (right panel).

FIG. 2 MIF regulates multiple myeloma tumorigenesis. A) Mouse multiple myeloma 5T-MM model, immunized normal C57BL/Ka mice were taken, tail vein injected with wild type (wt) or MIF knockout (MIF-KO) mouse myeloma 5TGM1 cells, and the neoplastic condition was detected by in vivo bioluminescence experiments. B) Subcutaneous tumors were constructed using a subcutaneous injection method using a mouse multiple myeloma 5T-MM model, and tumor cells were present 1 week after MIF-KO tumor cell injection, but after 2 weeks the tumor cells disappeared, eventually failing to form tumors. C) In another mouse multiple myeloma model (mouse multiple myeloma cells P3X63Ag8U.1, injected as Balb/c mice in vivo), subcutaneous injection of MIF-KO at P3X63Ag8U.1 failed to form tumors, wt cell tumors. D) Mouse multiple myeloma cells 5TGM1 were injected subcutaneously into immunodeficient NSG mice and both MIF-KO cells and wt cells were able to normally form tumors. E) MIF was knocked out in C57BL/Ka mice such that there was no expression of MIF in the mouse bone marrow (myeloma site), and after knockout, mouse sequencing confirmed MIF knockout. F) Normal 5TGM1 cells were injected into wild type or MIF knockout mice via tail vein, which failed to form tumors, suggesting that MIF expression in bone marrow microenvironment also has an effect on MM tumor formation. G) Two viruses CK and MK (upper panel) were used in the mouse spontaneous plasma cell tumor model; after the two viruses infect the cells, the cells overexpress Myc and K-ras, and Western blot verification of MIF effect is knocked down (lower panel). H) Schematic of mouse spontaneous plasma cytoma model construction. I) In a mouse plasma cell primary tumor model, MK infects a cell building block, and the tumor formation efficiency is obviously reduced. J) Taking a mouse myeloma 5T-MM model, taking subcutaneous tumor tissues 5 days after tumor cell transplantation, and carrying out mass spectrometry cytometry detection, wherein myeloma cells are in a red frame, and NK cells are in a black frame. The results suggest that MIF-KO tumors have more NK cell infiltration in vivo and fewer myeloma cells survive. K) And adopting a mouse myeloma 5T-MM model, and carrying out flow cytometry detection on tumor tissues to confirm a mass spectrometry flow experiment result.

FIG. 3 MIF modulates multiple myeloma tumor immunity and its use as a multiple myeloma vaccine. A) Schematic diagram of experiments on multiple myeloma cell vaccine of MIF-KO, MM cells of MIF-KO are taken for subcutaneous injection into mice, and the mice cannot become tumor. Four weeks later, immunized mice were inoculated with normal MM tumor cells, control groups were inoculated with non-immunized mice, and after 3 weeks of inoculation, none of the immunized mice developed tumors. B) According to the previous experiment of panel a, the waiting time after the first immunization was prolonged to 3 months, and the immunized mice still did not become tumor. C) Injection into hindlimb bone of C57BL/Ka mice (Intra-bone marrow injection) 5TGM1 tumor cell schematic, showing that the hindlimb of the same mice was injected with MM by wt and MM by MIF-KO, respectively. D) The results of the foregoing experiment, panel C, were compared to bilateral hind limb injections of wt MM.

Detailed Description

The reagents and raw materials used in the following examples were commercially available, unless otherwise specified.

Example 1: preparation of MIF Gene knockout multiple myeloma cells

MIF gene knockout multiple myeloma cell 5TGM1 is constructed by using CRISPR/Cas9 technology.

The specific operation is as follows:

1. lentivirus packaging:

design and Synthesis of sgRNA: MIF knock-out small guide RNA (sgRNA) design was done on GPP Web Portal (The Genetic Perturbation Platform, https:// portals. Broadsetup. Org/GPP/public /). Was synthesized by the biotechnology company, genewiz (Jin Weizhi) in su.

The sequence information is as follows:

forward sequence CACCGTGTGCAGGCGATCGGACAGC (SEQ ID NO. 1);

the reverse sequence AAACGCTGTCCGATCGCCTGCACAC (SEQ ID NO. 2).

Sequencing after annealing the sgrnas to pHKO23 plasmid confirmed successful construction. pHKO23 sgRNA plasmid and lentiviral packaging plasmid psPAX2, pMD2.G were amplified by extraction from the plasmid. The three plasmids were transfected into HEK-293T cells by calcium chloride precipitation and the lentivirus-containing supernatant was collected. The collected virus supernatant was filtered through a 0.45 μm filter, and the virus was concentrated by precipitation using a lentiviral concentrate and stored at-80 ℃.

2. Lentivirus infection of 5TGM1 cells:

cell transfection was performed in 6-well plates, 2X10 per well ⁵ 2mL of complete medium, 100. Mu.L of concentrated virus and 2. Mu.L of Polybrene mother solution (8 mg/mL, final concentration 8. Mu.g/mL) were added to 5TGM1 cells. Placed in a incubator of 5% CO2 at 37 ℃. Cell exchange was performed 24h after lentivirus infection, 48h after infection, and puromycin (puromycin) was added to the medium at a ratio of 1:1000. And (3) replacing the liquid periodically until the cells are no longer dead and proliferate normally, and collecting the cell protein extract for protein level verification.

Monoclonal screening of MIF-KO cells:

sorting the 5TGM1 cells with the MIF expression reduced by a flow sorter, inoculating 1 cell per well to a 96-well plate for culture, gradually screening a stably-proliferated monoclonal cell strain, extracting protein and DNA, respectively confirming the MIF protein expression reduction of the cell strain again by WB, and carrying out Sanger sequencing on the DNA to verify that the sgRNA target sequence is subjected to DNA recombination.

Example 2: expression studies of MIF in multiple myeloma

1. Experimental method

Bone marrow specimens from multiple myeloma patients or healthy volunteers were derived from Cleveland medical center (Cleveland Clinic) in ohio. Multiple myeloma patients or healthy volunteers each have written consent for their bone marrow specimens for scientific research. The experimental results were passed by the ethical committee of the cleveland medical center.

(1) Bone marrow puncture specimen supernatant was tested for MIF expression levels in the samples using Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA). After the marrow puncture specimen is centrifuged at 800g in a centrifuge, supernatant is collected, insoluble precipitate is removed by centrifuging at 12000g in a low-temperature high-speed centrifuge at 4 ℃ for 15 minutes, the marrow supernatant is collected and stored at-80 ℃, the level of MIF secreted by sample cells is detected by using a human MIF specific ELISA kit, and P value is obtained by using Student's t detection. The results are shown in FIG. 1A.

(2) Mononuclear cells in the bone marrow sample are separated by Ficoll density gradient centrifugation, CD138 positive myeloma cells in the bone marrow of a patient are obtained by magnetic bead sorting through CD138 antibody labeling, and BMSC (bone marrow stroma cell) in the bone marrow of a patient with reference bone marrow and myeloma is CD138 negative cells obtained by magnetic bead sorting. The total RNA of the specimens and the multiple myeloma cell line is advanced by using a Trizol method, cDNA is obtained by reverse transcription, and the relative expression quantity of MIF mRNA of all samples is obtained by using SYBR Green to perform fluorescent quantitative PCR. Analysis of variance compares the differences between the groups and LSD-t test compares the groups. The results are shown in FIG. 1B.

(3) The published clinical information and gene expression data set (PMID: 16728703; related data set GEO ID: GSE 2658) of patients with multiple myeloma were analyzed by Kaplan-Meier survival analysis, and the P value was calculated by Mantel-Cox test. The results are shown in FIG. 1C.

2. Experimental results

The results are shown in FIG. 1. It can be seen that patients with multiple myeloma have high levels of MIF expression in the bone marrow (fig. 1A). MIF is a secreted protein, and high levels of MIF in bone marrow in patients with multiple myeloma are derived from both tumor cells and non-tumor cells in bone marrow (fig. 1B). Clinical data demonstrated a poor prognosis for MIF-high expressing multiple myeloma (fig. 1C).

Example 3: MIF-regulated multiple myeloma tumorigenesis study

1. Experimental method

(1) Mouse multiple myeloma 5T-MM model: the 5T-MM (5T murine myeloma model) model refers to a model in which C57BL/KaLwRijHsd (hereinafter referred to as C57 BL/Ka) mice-derived 5TGM1 multiple myeloma cell lines are cultured in vitro, and the cell lines are transplanted (intravenously or subcutaneously or intramedularly) into C57BL/Ka mice to form myeloma. The C57BL/Ka mouse is an inbred sub-line of the C57BL mouse, the aged C57BL/Ka mouse is prone to high-frequency monoclonal B cell diseases, and the 5TGM1 cell line is derived from the C57BL/Ka mouse with spontaneous multiple myeloma. The method comprises the steps of infecting 5TGM1 cells by using lentiviruses expressing luciferase (luciferase), expressing the luciferase in the 5TGM1 cells, transplanting the luciferase into a mouse body, injecting the luciferin (luciferan) into the mouse body by intraperitoneal injection (150 mg/kg), and detecting bioluminescence by using the luciferin as a substrate through a living animal imaging system to evaluate tumor burden in the mouse body.

In this experiment, 5TGM1 cell tails of wt (wild type) and MIF-KO (MIF gene knockout) were transplanted into wild type C57BL/Ka mice by intravenous injection, and three weeks later, by observation of a small animal in vivo imaging system, it was found that tumor cells of MIF-KO could not form tumors in wild type mice. The results are shown in FIG. 2A.

(2) 5TGM1 cells (cell number 1X 10) of wt or MIF-KO were injected subcutaneously in C57BL/Ka mice (n=5 per group) respectively, using a mouse multiple myeloma 5T-MM model ⁶ ) Mice were tested for subcutaneous tumor burden by in vivo imaging of the mice. The results are shown in FIG. 2B.

(3) Mouse multiple myeloma P3X-Balb/c model: P3X63Ag8U.1 is a murine myeloma cell line derived from Balb/c mice, which can be directly tumorigenic by inoculation into syngeneic Balb/c mice. P3X63Ag8U.1 cells (cell number 1X 10) of wt or MIF-KO were injected subcutaneously in Balb/c mice (n=5 each group) ⁶ ) The size of the mouse subcutaneous tumor was measured by vernier calipers. The results are shown in FIG. 2C.

(4) Severe immunodeficiency mouse NSG model: NSG mice are immunodeficient mice resulting from knockout of Prkdc gene and Il2rg gene, and are currently the most immunodeficient tool mice. The Prkdc (protein kinase, DNA-activated, catalytic polypeptide) gene mainly encodes the catalytic subunit of DNA-dependent protein kinase (DNA-PK), and is an important gene involved in double-strand DNA break repair, immunoglobulin and T cell receptor variable (V), diversity (D), and junction (J) segment recombination. Prkdc ^scid Mutations represent severe combined immunodeficiency, manifested by the loss of T, B cells, inability to mediate cellular and humoral immunity, and rejection of allografts and xenografts. Nonfunctional T cells and B cells, NK cells are low in activity, have no hemolytic complement activity, and are deficient in bone marrow development. The IL2rg gene codes for the gamma chain of the Intereukin-2 receptor (IL-2 Rgamma c, also known as CD 132) as cytokines IL2, IL-4, IL-7, IL-9, IL-15 with important immune functionsAnd the common receptor subunit of IL-21, the immune function of the mouse body after the gene knockout is seriously reduced, and cytokine signaling is blocked through various receptors, so that functional NK cell defects are caused. By forming tumors in the NSG mouse model, the effect of immune cells on tumor formation can be substantially excluded.

5TGM1 cells (cell number 1X 10) of wt or MIF-KO were injected subcutaneously in NSG mice (n=5 per group), respectively ⁶ ) Mice were tested for subcutaneous tumor burden by in vivo imaging of the mice. The results are shown in FIG. 2D.

(5) MIF-/-mouse strain: MIF gene was knocked out in C57BL/Ka mice using TALEN technology to achieve MIF gene frameshift mutation. Sequencing analysis of MIF knockout mutant mice revealed that the wild type DNA sequence (peak shape indicated by black arrow) and the mutant DNA sequence (peak shape indicated by red arrow) deleted 7 bases GGAGCTC, and that frame shift mutation occurred. The terminator TGA is introduced in advance after the frame shift mutation, the protein sequence is shortened, and the normal functional MIF protein can not be encoded.

Wt 5TGM1 cells (cell number 1X 10) were injected subcutaneously in C57BL/Ka mice (n=5 per group) of wt or MIF-KO, respectively ⁶ ) Mice were tested for subcutaneous tumor burden by in vivo imaging of the mice. The results are shown in FIGS. 2E-F.

(6) Mouse plasma cell primary tumor model: the model uses retroviral infection, in donor mouse T2B cells (IgM ⁺ B220 ⁺ CD38 ⁺ IgD ⁺ ) The human cMYC protein and KRAS (G12V) mutein were overexpressed, recipient mice were transplanted, and plasma cell primary tumor models were established in the recipient mice. The specific process is briefly described as follows: magnetic bead sorting IgM in spleen cells of donor mice using normal Balb/c mice as spleen cell donors ⁺ Cells were stimulated with LPS and IL4 to convert them to T2B cells. Retrovirus (control virus CK: expresses cMYC/KRAS12V and control shRNA; MIF knockdown virus MK: expresses shRNA expressing cMYC/KRAS12V and targeting MIF) infects T2B cells twice, and the above-mentioned infected cells are mixed with bone marrow cells derived from normal Balb/c mice, respectively. Tail vein injection of mixed cells, transplantation to corresponding group of recipient mice after lethal dose irradiationIn vivo, a mouse plasma cell primary tumor model is established. The specific flow is shown in fig. 2H. The packaged CK or MK virus was infected with the mouse NIH-3T3 cell line, and the overexpression of c-MYC and K-ras, and the effect of MK virus knocking down MIF expression were verified, and the results are shown in FIG. 2G. Survival of mice with CK and MK plasma cell primary tumor model was counted and the results are shown in FIG. 2I.

(7) 5TGM1 cells (cell number 5X 10) of wt or MIF-KO were injected subcutaneously in C57BL/Ka mice (n=5 per group) respectively using a mouse myeloma 5T-MM model ⁶ ) The subcutaneous tumor of the mouse is taken 5 days after the tumor is negative, and the subcutaneous tumor is ground and passes through a cell sieve with a pore diameter of 45 mu m to prepare single cell suspension. Samples were sent to a beijing norelsen source (Novegene) for mass cytometry detection, the results of which are shown in fig. 2J.

(8) Taking a mouse myeloma 5T-MM model, taking subcutaneous tumor to prepare single cell suspension after the mouse is neoplastic, and respectively marking a sample by using CD138 and NK1.1 streaming antibodies and detecting by using a streaming cytometer. The results are shown in FIG. 2K.

2. Experimental results

The results are shown in FIG. 2. It can be seen that in the mouse multiple myeloma 5T-MM model experiment, MIF-KO myeloma cells were unable to form tumors-in this experiment, tumor cells knocked out MIF using CRISPR-Cas9 system, but recipient mice expressed MIF normally (fig. 2A). In the subcutaneous tumor experiments in the above animal model, MIF-KO cells survived briefly after entering the body, but were eventually cleared by the body (FIG. 2B). A similar phenomenon was also seen in another murine myeloma model (P3X-Balb/C model: murine myeloma cells P3X63Ag8U.1, subcutaneously injected into Balb/C mice) (FIG. 2C). Both animal models were used to immunize normal mice. Subsequently, the present invention repeated experiments using severely immunodeficiency NSG mice, and found that myeloma cells of MIF-KO were capable of forming tumors in NSG mice (FIG. 2D). The phenomenon is described as follows: MIF affects the development of multiple myeloma by modulating an anti-myeloma immune response. In addition, MIF-/-mouse strains were constructed (FIG. 2E). Using MIF-/-mouse modeling, the present invention found that myeloma still failed to occur normally if the microenvironment was deficient in MIF (FIG. 2F). Finally, the present invention also demonstrates that lowering MIF expression results in a decrease in myeloma incidence using an spontaneous myeloma animal model whose principle is that cMYC protein and K-ras (G12V) mutein are overexpressed in plasma cells resulting in myeloma genesis, see fig. 2G,2 h. All the above data suggest that MIF low expression activates anti-myeloma tumor immunity. To further explore the mechanism, the present invention utilizes mass flow cytometry techniques. This technique detects cell surface markers from protein levels, thereby systematically determining the immune cell components of the sample. As shown in FIG. 2J, MIF-KO myeloma cells, after entering the mice, attracted more NK cells to infiltrate into the tumor mass, accompanied by a decrease in the number of myeloma cells. This finding was verified by flow cytometry (fig. 2K).

The experimental results show that MIF plays a regulating role in the MM immunosuppression microenvironment, and the MM immunosuppression and immune escape regulated by MIF are involved in NK cells and macrophages. The MIF-KO MM cell injection stimulated mice did not form multiple myeloma.

Example 4: MIF (MIF) for regulating tumor immunity of multiple myeloma and application of MIF as multiple myeloma vaccine

1. Experimental method

(1) Cell vaccine experiments using the mouse multiple myeloma 5T-MM model 5TGM1 cells of MIF-KO (cell number 1x 10) were subcutaneously injected in C57BL/Ka mice (n=5) ⁶ ) MIF-KO cells failed to form tumors subcutaneously in C57BL/Ka mice. After 4 weeks, 5C 57BL/Ka mice of the same week age were taken as a control group, and 5TGM1 cells (cell number 2X 10) were inoculated subcutaneously in the immunized group and the control group at the same time ⁶ ) In figure 3A, the mice were made tumor subcutaneously after 3 weeks, both control groups were made tumor subcutaneously, and none of the immunized mice were made tumor.

(2) Cell vaccine experiments using the mouse myeloma 5T-MM model 5TGM1 cells (cell number 1x 10) of MIF-KO were subcutaneously injected in C57BL/Ka mice (n=10) ⁶ ) MIF-KO cells failed to form tumors subcutaneously in C57BL/Ka mice. After 3 months, 10 mice of the same week-old C57BL/Ka were used as control group, and 5TGM1 cells (cell number 2X 10) were inoculated subcutaneously in the immunized group and the control group ⁶ ). As shown in fig. 3B, by in vivo imaging of small animals: the next day after inoculation, subcutaneous swelling in control and immune groupsThe tumor fluorescence intensity is equivalent, which indicates that the load of the two groups of inoculated tumor cells is the same; after 3 weeks, the fluorescence intensity of the subcutaneous tumor of the control group was enhanced, and the fluorescence of the subcutaneous tumor of the immunized mice was disappeared.

(3) Injecting into bone marrow cavity of mouse myeloma 5T-MM model, collecting 5TGM1 cells of wt or MIF-KO, after PBS resuspension, after mouse isoflurane induced anesthesia, maintaining anesthesia by mask, dehairing and skin preparation at injection site, sufficiently sterilizing povidone iodine solution, penetrating into bone marrow cavity of femur perpendicularly to femur patella surface of mouse by microinjector, injecting 10uL containing 2x10 ⁵ A suspension of individual tumor cells. 5TGM1 cells of wt or MIF-KO were injected into the femur on both sides of the same mouse, respectively. In figure 3C, live images of the small animals the next day after injection are shown, showing tumor cells in the bone marrow cavity.

(4) Intramedullary injections using the mouse myeloma 5T-MM model were used. Control group: injecting wt 5TGM1 cells into femur of the same mouse; immunization group: 5TGM1 cells of wt or MIF-KO were injected into the femur on both sides of the same mouse, respectively. The following day after injection, in vivo imaging of the small animals showed no difference in tumor cell load in the left and right femur bone marrow cavities of all mice in the group; two weeks after injection, the tumor burden in both femur of control mice increased compared to the second day after negative tumor, and the tumor burden in both femur of immunized mice decreased compared to the second day after negative tumor (fig. 3D).

2. Experimental results

The results are shown in FIG. 3. It can be seen that reinjecting wild type myeloma cells normally expressing MIF into mice vaccinated with 5TGM1 cells of MIF-KO, the mice were immunized against tumor cells and no longer tumorigenic (FIG. 3A). Tumor stimulation was still not tumorigenic 3 months after immunization (FIG. 3B), and it was deduced that 5TGM1 cells of MIF-KO activated long-term cellular immunity in mice, with involvement of memory T cells (memory T cells), that is, MIF was able to regulate anti-myeloma T cell immunity in addition to NK regulation. In addition, the invention adopts an intraosseous injection method, wild type or MIF-KO myeloma cells are inoculated on the bilateral hind limbs of the same mouse, and the mouse does not generate tumor.

The experimental results show that the MM cells of the MIF-KO are injected into the stimulated mice, so that the MM cannot occur, and the MM immunity can be obtained; normal MM tumor transplantation is used again in the future, and MM does not occur. MM cells of MIF-KO can be effective as tumor vaccine for preventing multiple myeloma.

In conclusion, the invention provides the application of MIF gene knockout tumor cells in preparing tumor vaccines. The invention discovers for the first time that MIF plays an important role in inducing the immune escape process of Multiple Myeloma (MM), and NK cells, macrophages and T cells participate in the MIF-regulated MM immunosuppression and immune escape. MIF knockout MM cells do not induce tumor immunosuppressive microenvironments efficiently, and thus cannot escape immunity, so that tumors are not generated in the mouse transplanted tumor model. The mice after the MIF gene knockout MM cell injection stimulation can not only not generate MM, but also acquire MM immunity; normal MM tumor transplantation is used again in the future, and MM does not occur. Therefore, the MIF gene knocked-out MM cell can be used as a tumor vaccine, effectively prevents MM, and has potential application prospect in blocking benign precancerous lesions from progressing to MM and reducing the occurrence rate of MM.

Claims

Use of mif knockout multiple myeloma tumor cells in the preparation of a multiple myeloma tumor vaccine.
2. Use according to claim 1, characterized in that: the tumor vaccine is a preventive tumor vaccine or a therapeutic tumor vaccine.
3. Use according to claim 1 or 2, characterized in that: the tumor vaccine is a formulation that blocks the development of benign precancerous lesions into tumors.
4. Use according to claim 3, characterized in that: the benign precancerous lesion is monoclonal gammaglobulinemia of unknown significance.
5. Use according to claim 1 or 2, characterized in that: the tumor vaccine is a preparation for regulating NK cell immunity, macrophage immunity and/or T cell immunity.