Genetically engineered bone marrow mesenchymal stem cell with homing characteristic and application of genetically engineered bone marrow mesenchymal stem cell

Abstract

The invention discloses a genetically engineered bone marrow mesenchymal stem cell with homing characteristic and application thereof, relates to the technical field of biomedicine, and is characterized in that effective treatment of glioma is realized through the genetically engineered bone marrow mesenchymal stem cell. MSCs are designed to secrete an anti-tumor immune cell factor IL-12, a CD47 nano antibody and nCD47-SLAMF7, the IL-12 can regulate and control the activity of T cells, and the nCD47-SLAMF7 can promote the phagocytosis of macrophages on in-situ tumors in a glioma microenvironment; engineered MSCs with different secretion characteristics are mixed and injected in situ to the glioma site, bypassing the blood brain barrier to provide high concentrations of bioactive proteins. The polypeptide can enhance M1 polarization infiltrating macrophages, stimulate macrophage-mediated tumor cell phagocytosis, activate antigen-presenting cells, promote effector CD8 + T cell infiltration and effectively control development of glioma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically, to a genetically engineered bone marrow mesenchymal stem cell with homing characteristics and application thereof. Background Art

[0002] Glioma is the most common primary tumor of the central nervous system. The special location and invasive growth characteristics of the tumor make the complete resection of glioma challenging. In addition, the existence of the blood-brain barrier and the interaction between neurons and tumors limit the effect of clinical radiotherapy and chemotherapy. Therefore, the average survival time of glioma patients is only 14 months. In recent years, immune checkpoint blockade (ICB) has made breakthroughs and has been successfully applied to the treatment of many types of tumors, but its therapeutic effect on glioma is not ideal. The resistance factors of glioma to immunotherapy include endogenous and exogenous elements. Endogenous factors involve the activation of the β-catenin signaling pathway, the high expression of PD-L1, and the high expression of immunosuppressive molecules such as IDO and PEG2. However, there are major technical challenges in changing the gene expression profile of glioma cells in vivo. On the other hand, exogenous factors include the infiltration of various immunosuppressive cells and the expression of related inhibitory molecules, among which macrophages account for more than half of the infiltrated immune cells and secrete immunosuppressive molecules such as IL-4, IL-10 and SIRRα. These molecules can effectively enable glioma cells to evade innate immune recognition, thereby limiting the effectiveness of adaptive immune responses. In addition, the trans-interaction of the CD47 / SIRPα axis and the cis-interaction of CD47 / SLAMF7 inhibit the phagocytosis and antigen presentation of tumor cells by phagocyte-like cells, thereby inhibiting the killing effect of innate immune cells on tumor cells and further inhibiting the direct recognition of adaptive immune cells. However, since CD47 is also expressed on blood cells and other normal cells, the effectiveness of anti-CD47 antibody therapy is limited.

[0003] Cell therapy plays an important role in anti-tumor immunity, including CAR-T cells, TCR-T cells, CAR-macrophages and CAR-NK cells, all of which have shown tumor-killing efficacy, but their application is limited by MHC restriction, tumor cell heterogeneity and immunosuppressive microenvironment. Mesenchymal stem cells (MSCs) are a type of cell with a low degree of differentiation, strong proliferation ability, high differentiation potential and the unique ability to "home" to tumors. MSCs are used in the treatment of preclinical GBM in various forms, including MSCs themselves, MSC-derived exosomes, oncolytic virus vectors, suicide protein expression vectors and immune activation factor vectors, but how to effectively regulate the differentiation of MSCs in GBM treatment and avoid damage to normal neural tissue outside the tumor area still needs to be explored. Summary of the invention

[0004] The purpose of the present invention is to provide a genetically engineered bone marrow mesenchymal stem cell with homing characteristics and its application, wherein the MSCs can effectively release IL-12 cytokines and nCD47-SLAMF7 fusion protein by activating Smad4 transcription factor in an environment rich in TGF-β. The nCD47-SLAMF7 fusion protein is composed of a signal peptide, a nano antibody that can block the function of CD47, and an extracellular region of SLAMF7 that can target tumor cells and mask their CD47 molecules, which can promote the uptake of tumor cells by SLAMF7-expressing macrophages and activate innate and cellular immunity in gliomas. In addition, the IL-12 released in the glioma microenvironment can effectively promote T cell proliferation and activation, and enhance the phagocytosis and antigen presentation ability of macrophages to tumor cells.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] In a first aspect of the present invention, a nCD47-SLAMF7 fusion protein is provided, wherein the amino acid sequence of the nCD47-SLAMF7 fusion protein is shown in SEQ ID NO.1.

[0007] In the second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the nCD47-SLAMF7 fusion protein.

[0008] The present invention is further configured as follows: the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2.

[0009] In a third aspect of the present invention, a nucleic acid sequence comprising a promoter sequence encoding a SMAD response element and encoding a nCD47-SLAMF7 fusion protein is provided.

[0010] In the fourth aspect of the present invention, a bone marrow mesenchymal stem cell that responds to TGF-β cytokine and expresses secretory nCD47-SLAMF7 fusion protein is provided. The bone marrow mesenchymal stem cell that expresses secretory nCD47-SLAMF7 fusion protein is obtained by transfecting the bone marrow mesenchymal stem cell with a lentivirus that expresses both a promoter sequence encoding a SMAD response element and a gene sequence encoding the nCD47-SLAMF7 fusion protein.

[0011] In a fifth aspect of the present invention, a method is provided comprising a promoter sequence encoding a SMAD response element and a nucleic acid sequence encoding an IL-12 cytokine.

[0012] In the sixth aspect of the present invention, a bone marrow mesenchymal stem cell that responds to TGF-β cytokine and then expresses secretory IL-12 cytokine is provided. The bone marrow mesenchymal stem cell that expresses secretory IL-12 cytokine is obtained by transfecting the bone marrow mesenchymal stem cell with a lentivirus that expresses both a promoter sequence encoding a SMAD response element and a gene sequence encoding the IL-12 cytokine.

[0013] The present invention is further configured as follows:

[0014] Obtaining MSCs that can respond to TGF-β cytokines and express secretory nCD47-SLAMF7 fusion protein or secretory IL-12: containing a promoter sequence encoding a SMAD response element and a nucleic acid sequence encoding a secretory nCD47-SLAMF7 fusion protein / IL-12, obtaining the nucleic acid molecule sequence by an in vitro de novo synthesis step, adding sequences complementary to the bases of a part of the sequence of a lentiviral vector plasmid at both ends of the molecule sequence, constructing the nucleic acid molecule sequence into a lentiviral vector plasmid by a DNA homologous recombinase, and then co-transfecting the HEK293 cell line with packaging plasmids such as PMD2G and PSPAX2, obtaining a lentivirus (carrying a puromycin resistance gene) containing the target nucleic acid molecule to transfect MSCs, and obtaining the desired MSCs cells;

[0015] The present invention is further configured as follows: MSCs expressing secretory nCD47-SLAMF7 fusion protein / IL-12 are obtained after being screened by puromycin.

[0016] In the seventh aspect of the present invention, a cell therapy composition is provided, comprising: a combined cell administration mode of MSCs that express secretory nCD47-SLAMF7 fusion protein or secrete IL-12 in response to TGF-β cytokine.

[0017] The present invention is further configured as follows: the promoter sequence of the SMAD response element is shown as SEQ ID NO.3, and the amino acid sequence of the IL-12 cytokine is shown as SEQ ID NO.4.

[0018] The present invention is further configured as follows: the MSCs expressing secretory nCD47-SLAMF7 fusion protein or the MSCs expressing secretory IL-12 cytokine further include an aqueous solution, and the aqueous solution is selected from water, physiological saline, glucose aqueous solution or cell culture medium.

[0019] The present invention is further configured as follows: the cell therapy composition can also be prepared into a cell therapy preparation, which comprises the cell therapy composition and also includes pharmaceutically acceptable excipients.

[0020] The above-mentioned pharmaceutically acceptable excipients are selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

[0021] The present invention is further configured as follows: the cell therapy preparation is selected from in situ injection.

[0022] In an eighth aspect of the present invention, a fixed ratio cell therapy composition is provided.

[0023] The fixed ratio cell therapy composition is a mixture of MSCs expressing secretory nCD47-SLAMF7 fusion protein and MSCs expressing secretory IL-12 cytokine, and the quantity ratio of the two is 1:1.

[0024] In the ninth aspect of the present invention, provided are the MSCs expressing secretory nCD47-SLAMF7 fusion protein or secretory IL-12 that can respond to TGF-β cytokine, the cell therapy composition, and the use of the fixed-ratio cell therapy composition in the preparation of a drug for resisting in situ glioma.

[0025] The above-mentioned gliomas in situ include astrocytic tumors, oligoglial tumors, ependymal tumors, astrocytic tumors and oligodendroglial tumors.

[0026] In summary, the present invention has the following beneficial effects:

[0027] 1. First, the present application found that MSCs have a strong TGF-β responsive homing ability, and in situ injected MSCs can actively migrate to the tumor lesion area;

[0028] 2. The present invention provides a secretory nCD47-SLAMF7 fusion protein that can respond to TGF-β cytokines and its application. MSCs expressing secretory nCD47-SLAMF7 fusion protein can release nCD47-SLAMF7 responsively under the stimulation of high concentrations of TGF-β in the tumor microenvironment, avoiding toxic effects on the nervous system in non-tumor areas. The fusion protein nCD47-SLAMF7 can inhibit the transmission of CD47 / SIRPα signals and promote the phagocytic effect of macrophages on glioma cells. At the same time, the SLAMF7 contained in the fusion protein can attract macrophages to search for tumor cells, further enhancing the targeted uptake of tumor cells by macrophages, thereby effectively initiating the innate immune response and activating cellular immunity through the antigen presentation effect.

[0029] 3. The present invention provides MSCs expressing secretory IL-12 that can respond to TGF-β cytokines and their applications. MSCs expressing secretory IL-12 can responsively release IL-12 under the stimulation of high concentrations of TGF-β in the tumor microenvironment, thereby avoiding toxic effects on the nervous system in non-tumor areas. The release of IL-12 in the tumor microenvironment can effectively maintain the killing function of effector T cells and promote the proliferation of effector T cells, effectively promoting the control of cellular immunity on in situ gliomas, thereby providing a new strategy for the treatment of in situ gliomas.

[0030] 4. The two methods for preparing and applying genetically engineered bone marrow mesenchymal stem cells with homing properties provided by the present invention have the advantages of simple operation, low cost, and avoiding damage to non-target areas, and have important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the lentiviral plasmid containing both the promoter sequence encoding the SMAD response element and the gene sequence encoding the nCD47-SLAMF7 fusion protein / IL-12 cytokine in Experimental Examples 1 and 2 of the present invention and the schematic diagram of the microenvironment response characteristics; ( Figure 1 Figure A is a schematic diagram of the construction structure of the lentiviral plasmid, and Figure B is a schematic diagram of MSCs transfected with the lentiviral plasmid responding to TGF-β signals in the microenvironment and releasing nCD47-SLAMF7 or IL-12;)

[0032] Figure 2 It is a graph showing the results of verifying that MSCs respond to TGF-β signals and release nCD47-SLAMF7 or IL-12 by confocal imaging in Experimental Examples 3 and 4 of the present invention; ( Figure 2 Figure A in the middle is a verification through confocal imaging that TGF-β signal stimulation promotes the expression of IL-12 cytokine in MSCs that have been transfected with lentiviral plasmids; Figure B is a verification through confocal imaging that TGF-β signal stimulation promotes the expression of nCD47-SLAMF7 fusion protein in MSCs that have been transfected with lentiviral plasmids;)

[0033] Figure 3 The secretion of IL-12 and nCD47-SLAMF7 in the supernatant derived from MSCs transfected with lentiviral plasmids was verified by SDS-PAGE experiment, Western blotting experiment and Elisa experiment in Experimental Example 5 of the present invention; ( Figure 3Figure A in the middle verifies the molecular weight of IL-12 and nCD47-SLAMF7 secreted by MSCs through 10% denatured SDS-PAGE gel; Figures B and D verify the responsiveness of MSCs transfected with lentiviral plasmids to TGF-β signals through Western blotting; Figures C and E confirm that MSCs transfected with lentiviral plasmids can release a large amount of target proteins under the stimulation of TGF-β signals through Elisa experiments.

[0034] Figure 4 The targeting properties of IL-12 and nCD47-SLAMF7 secreted from the supernatant of MSCs transfected with lentiviral plasmids on T cells and macrophages were verified in Experimental Examples 6 and 7 of the present invention;

[0035] Figure 5 The effect of IL-12 cytokine secreted from the supernatant of MSCs transfected with lentiviral plasmids on the proliferation and activation of effector T cells was verified in Experimental Example 8 of the present invention; Figure 5 Figure A shows the effect on the proliferation of effector T cells; Figure B shows the effect on the function of effector T cells.

[0036] Figure 6 The experimental example 9 of the present invention verifies the property of the nCD47-SLAMF7 fusion protein secreted in the supernatant of MSCs transfected with the lentiviral plasmid to promote the uptake of tumor cells by macrophages, and the effect of macrophages that take up tumor cells on effector T cells; ( Figure 6 Figure A is a schematic diagram of the principle of nCD47-SLAMF7 promoting macrophage uptake of tumor cells; Figure B is the verification through confocal imaging that nCD47-SLAMF7 can promote macrophage uptake of tumor cells; Figure C is the identification through flow cytometry that nCD47-SLAMF7 can promote macrophage uptake of tumor cells; Figure D is the verification through flow cytometry of the effect of macrophages that engulf tumor cells on the proliferation of effector T cells; Figure E is the verification through flow cytometry of the effect of macrophages that engulf tumor cells on the activation of effector T cells;)

[0037] Figure 7 The tumor microenvironment migration effect of MSCs expressing secretory nCD47-SLAMF7 fusion protein or IL-12 cytokine in vivo was verified in Experimental Example 10 of the present invention; ( Figure 7 Figure A is a schematic diagram of in situ injection of glioma cells and different MSCs cells; Figure B is a confocal microscope to verify the homing effect of MSCs expressing different target proteins at the tumor site;)

[0038] Figure 8The present invention's experimental example 11 verified the therapeutic effect of mixed injection of MSCs cells expressing secretory nCD47-SLAMF7 fusion protein or IL-12 cytokine on the in situ glioma model in vivo through animal experiments; ( Figure 8 Figure A is a schematic diagram of glioma model treatment and imaging; Figure B verifies the in vivo growth of glioma through small animal in vivo imaging; Figure C is a statistical analysis of the luciferase intensity produced by the glioma model of mice in different groups on day 0; Figure D is a statistical analysis of the luciferase intensity produced by the glioma model of mice in different groups on day 16; Figure E is a statistical analysis of the survival rate of mice with glioma;)

[0039] Fig. 9 This is an image of the efficiency of Cas9@CP-EX knocking out the ID1 gene in bone marrow mesenchymal stem cells in Experimental Example 11 of the present invention, which verifies the growth of gliomas in different groups and the homing characteristics of MSCs by immunofluorescence and laser confocal experiments;

[0040] Fig.10 The polarization state of the infiltrating macrophages and the infiltration of CD8 T cells in the glioma microenvironment were verified by immunofluorescence in Experimental Example 11 of the present invention;

[0041] Fig.11 The present invention provides a flowchart of two genetically engineered bone marrow mesenchymal stem cells with homing characteristics from preparation to action. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] Example 1: Construction of a lentiviral plasmid containing a SMAD response element gene sequence and a nCD47-SLAMF7 fusion protein gene sequence

[0044] This embodiment provides a method for screening a secretory nCD47-SLAMF7 fusion protein that responds to TGF-β. The specific scheme is as follows:

[0045] The amino acid sequence of the nCD47-SLAMF7 fusion protein is shown in SEQ ID NO.1.

[0046] SEQ ID NO.1:

[0047] MASPLTRFSLNLLLLGESIILGSGEADIVMTQSPATLSVTPGDRVSLSCRASQTISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGH GFPRTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGDLVKPGGSLKLSCAASGFTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNTLYLQIDS LKSEDTAIYFCARSLAGNADYWGQGTSVTVSSGGGSGGGGGGGSGGGGSGTLKKVAGALDGSVTFTLNITEIKVDYVVWTFNTFFLAMVKKDGVTSQSSNKERIVFPDGLYSMKLSQL KKNDSGAYRAEIYSTSSQASLIQEYVLHVYKHLSRPKVTIDRQSNKNGTCVINLTCSTDQDGENVTYSWKAVGQGDNQFHDGATLSIAWRSGEKDQALTCMARNPVSNSFSTPVFPQKLC EDAATDLTSLRG;

[0048] The SMAD response element promoter sequence and the nCD47-SLAMF7 fusion protein gene sequence were connected in series, and the corresponding nCD47-SLAMF7 base sequence is shown in SEQ ID NO.2:

[0049] SEQ ID NO.2:

[0050]

[0051] The SMAD response element gene sequence is shown in SEQ ID NO.3:

[0052] SEQ ID NO.3:

[0053] GGCCTAACTGGCCGGTACCGCTAGCCTCGATCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCG GGCCTAACTGGCCGGTACCGCTAGCCTCGATCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCG GGCCTAACTGGCCGGTACCGCTAGCCTCGATCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCG GGCCTAACTGGCCGGTACCGCTAGCCTCGATCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCCTCAGACCGTTAGACGCCAGGACGGGCTGTCAGGCTGGCGCCGCG;

[0054] The lentiviral vector plasmid pLV3-CMV-MCS-3×FLAG-Hyg (Cat. No. P55550, Wuhan Miaoling Biotechnology Co., Ltd.) was constructed by gene synthesis and homologous repair recombinase, which contains the puromycin resistance gene;

[0055] The lentiviral plasmid containing the SMAD response element and the nCD47-SLAMF7 fusion protein sequence was co-transfected with the packaging plasmids PSPAX2 and PMD2G into HEK293T cells using PEI reagent to obtain a lentiviral vector loaded with the target sequence plasmid;

[0056] The lentivirus was transfected into mouse bone marrow-derived mesenchymal stem cells (MSCs). After 48 hours, stem cell complete culture medium containing 0.8 μg / mL puromycin was added for screening. The screening period was 3 weeks.

[0057] The bone marrow mesenchymal stem cells prepared above were added with stem cell culture medium containing 10 ng / mL TGF-β and cultured for an additional 24 hours. The proportion of MSCs expressing secretory nCD47-SLAMF7 fusion protein was observed by flow cytometry in combination with flow cytometry antibodies that can target SLAMF7. Figure 1 As shown in Figures A and B.

[0058] Example 2. Construction of a lentiviral plasmid containing a SMAD response element gene sequence and an IL-12 cytokine gene sequence

[0059] This embodiment provides a method for screening bone marrow mesenchymal stem cells that express secretory IL-12 in response to TGF-β. The specific scheme is as follows:

[0060] The SMAD response element promoter sequence and the IL-12 gene sequence are connected in series, and the corresponding IL-12 base sequence is shown in SEQ ID NO.4:

[0061] SEQ ID NO.4:

[0062]

[0063] The lentiviral vector plasmid pLV3-CMV-MCS-3×FLAG-Hyg (Cat. No. P55550, Wuhan Miaoling Biotechnology Co., Ltd.) was constructed by gene synthesis and homologous repair recombinase, and the plasmid contained the puromycin resistance gene; the lentiviral plasmid containing the SMAD response element and the IL-12 sequence and the packaging plasmids PSPAX2 and PMD2G were co-transfected into HEK293T cells by PEI reagent to obtain the lentiviral vector loaded with the target sequence plasmid;

[0064] The lentivirus was transfected into mouse bone marrow-derived mesenchymal stem cells (MSCs). After 48 hours, stem cell complete culture medium containing 0.8 μg / mL puromycin was added for screening. The screening period was 3 weeks.

[0065] The bone marrow mesenchymal stem cells prepared above were added with stem cell culture medium containing 10 ng / mL TGF-β and cultured for an additional 24 hours. The proportion of MSCs expressing secretory IL-12 cytokines was observed by flow cytometry in combination with flow cytometry antibodies that can target IL-12. Figure 1 As shown in Figures A and B.

[0066] Example 3. MSCs that can respond to TGF-β signals express secretory nCD47-SLAMF7 fusion protein

[0067] The MSCs expressing EGFP signal were subjected to the lentiviral transfection and screening operation in Example 1. The lentiviral plasmid in this step additionally carried mcherry fluorescent protein for tracing the expression of nCD47-SLAMF7 fusion protein. The obtained bone marrow mesenchymal stem cells were cultured with stem cell culture medium containing 10 ng / mL TGF-β for an additional 24 hours, and the expression of the target protein by MSCs was observed using a confocal microscope. The experimental results are shown in FIG. Figure 3 shown.

[0068] from Figure 2 It can be seen from the results that after the preliminary screening method of Example 1, MSCs that release the target protein in response to TGF-β account for more than 95% of all MSCs, and no further screening is required.

[0069] Example 4. MSCs that can respond to TGF-β signals express secretory IL-12 factor

[0070] The MSCs expressing EGFP signal were subjected to the lentiviral transfection and screening operation in Example 1. The lentiviral plasmid in this step additionally carried mcherry fluorescent protein for tracing the expression of IL-12 factor; the obtained bone marrow mesenchymal stem cells were cultured with stem cell culture medium containing 10 ng / mL TGF-β for an additional 24 hours, and the expression of the target protein by MSCs was observed using a confocal microscope. The experimental results are shown in FIG. Figure 3 shown.

[0071] from Figure 2 It can be seen from the results that after the preliminary screening method of Example 1, MSCs that release the target protein in response to TGF-β account for more than 95% of all MSCs, and no further screening is required.

[0072] Example 5. MSCs can efficiently secrete target proteins into the supernatant under TGF-β signal stimulation

[0073] Take the MSCs secreting two different target proteins obtained in Example 1 and Example 2, and culture the two types of bone marrow mesenchymal stem cells with stem cell culture medium containing 10 ng / mL TGF-β for an additional 24 hours. Use a 10KD ultrafiltration concentration tube to concentrate the supernatant, and use SDS-PAGE experiment, Western blotting experiment and Elisa experiment to detect the relative molecular weight, structural translation accuracy and degree of extracellular secretion of nCD47-SLAMF7 fusion protein and IL-12 cytokine. The relevant experimental results are shown in the figure below. Figure 3 shown.

[0074] from Figure 3 As can be seen from the results of Figure A, the relative molecular weights of nCD47-SLAMF7 fusion protein and IL-12 are consistent with the theoretical design, proving the accuracy of the target protein length. Figure 3 Figure 3B and Figure 3D confirmed through Western blotting experiments that MSCs maintained the accuracy of translation of the target protein sequence; in addition, Figure 3 Figure C and Figure 3 The results in Figure E in the middle confirm that after MSCs respond to TGF-β, they can release a large amount of relevant target proteins into the supernatant, which is consistent with the experimental design principles in Examples 1 and 2.

[0075] Example 6. Targeting properties of nCD47-SLAMF7 fusion protein secreted by MSCs

[0076] CD8+T cells from the spleen of C57 mice were obtained using Miltenyi CD8 positive magnetic bead sorting kit; CD8+T cells and GL261 tumor cells were stained using biolegend's flow cytometry antibodies against CD47 (Cat. No. 127509) and IL-12R (Cat. No. 505217), and the expression of IL-12 receptor on the surface of T cells and CD47 on the surface of GL261 tumor cells was observed; CD8+T cells and GL261 cells were co-incubated with nCD47-SLAMF7 having mcherry fluorescent protein signal in Example 3, and then the targeting effect of nCD47-SLAMF7 on T cells and tumor cells was detected by flow cytometry, and the relevant results are shown in Figure 4.

[0077] from Figure 4 A picture and Figure 4 From the results in Figure B, it can be seen that saturation was reached when the ratio of exosomes and DMPE-PEG2000-DAEWVDVS co-incubation was 1:15, and incubation with a higher concentration of DMPE-PEG2000-DAEWVDVS could not promote the exosomes to carry a higher concentration of p-selectin targeting peptide.

[0078] Example 7. Targeting properties of IL-12 secreted by MSCs

[0079] The CD8+T cells of the spleen of C57 mice were obtained using the Miltenyi CD8 positive magnetic bead sorting kit; and the CD8+T cells and GL261 tumor cells were stained with flow cytometry antibodies against CD47 (Cat. No. 127509) and IL-12R (Cat. No. 505217) of biolegend to observe the expression of IL-12 receptor on the surface of T cells and CD47 on the surface of GL261 tumor cells; the CD8+T cells and GL261 cells were co-incubated with IL-12 having mcherry fluorescent protein signal in Example 4, and then the targeting effect of IL-12 on T cells and tumor cells was detected by flow cytometry. If the relevant results are as follows Figure 4 shown.

[0080] from Figure 4 A picture and Figure 4 From the results in Figure B, it can be seen that saturation was reached when the ratio of exosomes and DMPE-PEG2000-DAEWVDVS co-incubation was 1:15, and incubation with a higher concentration of DMPE-PEG2000-DAEWVDVS could not promote the exosomes to carry a higher concentration of p-selectin targeting peptide.

[0081] Example 8. Effect of IL-12 secreted by MSCs on antigen-specific OT-1 CD8+ T cells

[0082] The CD8+T cells of the spleen of OT-1 transgenic mice (Jackson Laboratory, Cat. No. 003831) were obtained using Miltenyi CD8 positive magnetic bead sorting kit, and OT-1T cells were stained with CFSE proliferation dye; the culture supernatant of ordinary MSCs and the culture supernatant containing secreted IL-12 were incubated with OT-1T cells respectively, and the OT-1T cells were stained with biolegend's membrane permeabilization solution and IFN-γ flow cytometry antibody (Cat. No. 505807) to observe the effect of MSCs supernatant containing IL-12 on T cell function. The relevant experimental results are shown in Figure 5 shown.

[0083] from Figure 5 The results show that the ordinary MSCs culture supernatant has a weak effect on the proliferation of OT-1T cells and the effect of inducing T cell activation. However, by co-incubating OT-1T cells with MSCs culture supernatant containing secretory IL-12, it can strongly promote the proliferation and activation of OT-1T cells within 24 hours and enhance the killing function of effector T cells.

[0084] Example 9. nCD47 fusion protein secreted by MSCs can promote phagocyte uptake of tumors and enhance antigen presentation

[0085] 1. Use 0.1 mg / mL PKH26 cell membrane dye to stain bone marrow-derived macrophages, and use 0.1 mg / mL PKH67 cell membrane dye to stain GL261-OVA cells (GL261 cell line expressing OVA protein); culture the two cells in a confocal imaging dish at a ratio of 1:1, and incubate the mixed cells with MSCs supernatant containing secretory nCD47-SLAMF7 for 24 hours; then fix the cells with 1% paraformaldehyde solution, stain the cell nuclei with 10 μg / mL DAPI solution, and then image the cells using a confocal microscope to observe the phenomenon of macrophages phagocytosing the GL261 tumor cell line; at the same time, use mixed cells incubated with different MSCs supernatants, and use flow cytometry to observe the phagocytosis of tumor cells by macrophages.

[0086] 2. The macrophages engulfing the GL261-OVA cells were incubated with the OT-1 cells stained with CFSE dye in Example 5 at a ratio of 1:5 for 24 hours, and then the OT-1T cells were stained with IFN-γ flow cytometry antibody (Cat. No. 505807). The proliferation and activation of OT-1T cells were observed using a flow cytometer. The results are as follows: Figure 6 shown.

[0087] Figure 6Figure A in the middle shows a schematic diagram of the mechanism of inducing macrophages to phagocytize tumor cells after adding nCD47-SLAMF7 fusion protein to the culture medium; Figure 6 The confocal imaging results in Figure B show that macrophages can efficiently phagocytose tumor cells in the culture medium containing nCD47-SLAMF7 fusion protein. Figure 6 The flow cytometry results in Figure C also confirmed that nCD47-SLAMF7 fusion protein has the effect of promoting macrophage phagocytosis of tumors compared with other MSCs culture supernatants. If culture supernatants containing IL-12 and nCD47-SLAMF7 fusion protein are added at the same time, the phagocytosis of tumor cells by macrophages can be promoted more efficiently. In addition, Figure 6 D Figure and Figure 6 The results in Figure E show that macrophages that have engulfed GL261-OVA can efficiently present OVA antigens on the surface of macrophages, effectively enhancing the proliferation and activation of antigen-specific T cells.

[0088] Example 10. In vivo tumor homing effect of secretory MSCs

[0089] To demonstrate the homing effect of MSCs themselves and the tumor homing effect of MSCs transfected with lentivirus, in this example, GL261 tumor cells and MSCs cells were simultaneously injected into C57 mice in situ, with the injection sites spaced about 500 μm apart, wherein GL261 expressed mcherry fluorescent protein (obtained by transfection screening using lentivirus plasmids containing the mcherry fluorescent protein sequence), MSCs themselves expressed GFP fluorescent protein (obtained by transfection screening using lentivirus plasmids containing the GFP fluorescent protein sequence), and astrocytes expressing green fluorescent protein were used as negative controls. One week after inoculation of the orthotopic tumor, the mice were subjected to cardiac perfusion, and the brain regions of the mice were obtained for frozen section imaging, and the relevant results are shown in the figure below. Figure 7 shown.

[0090] Figure 7 The schematic diagram in Figure A shows the flow chart of the experiment; Figure 7 The results in Figure B show that astrocytes cannot effectively migrate to the tumor area, while MSCs expressing secretory target proteins can efficiently infiltrate into the tumor area, confirming the strong tumor homing effect of MSCs in vivo.

[0091] Example 11. Effect of MSCs expressing secretory nCD47-SLAMF7 fusion protein or IL-12 factor that can respond to TGF-β signal on in vivo treatment of glioma in situ

[0092] Using stereotaxic equipment, GL261 cells expressing luciferase and mcherry were injected into the deep cortical region of the brain of C57 mice at a total of 2 million cells in PBS solution. On the 6th day, 5 million MSCs expressing GFP were injected near the tumor area in the same way, including 2.5 million MSCs that released nCD47-SLAMF7 fusion protein in response to TGF-β signal and 2.5 million MSCs that released IL-12 cytokine in response to TGF-β signal. On the 8th, 12th and 16th days, small animal live imaging was used to observe the growth of in situ gliomas in C57 mice in different treatment groups, and the survival rate was statistically analyzed. At the same time, the brains of mice in different treatment groups on the 16th day after tumor injection were taken for immunofluorescence operation to observe the growth of gliomas, the infiltration of MSCs in the tumor microenvironment, the infiltration of M2 macrophages in the tumor microenvironment, and the infiltration of CD8 T cells in the tumor microenvironment. The relevant results are as follows: Figure 8-10 shown.

[0093] Figure 8 The schematic diagram in Figure A shows the entire experimental process of MSCs in vivo treatment of glioma; Figure 8 As can be seen from the results of Figure B, the group injected with both MSCs that release nCD47-SLAMF7 fusion protein in response to TGF-β signal and MSCs that release IL-12 cytokine in response to TGF-β signal has the best effect in inhibiting the growth of in situ glioma. Figure 8 Figure C and Figure 8 Figure D shows the statistical results of the luminescence intensity of luciferase in situ gliomas in different groups of mice on days 0 and 16. Figure 8 Similar results to those in Figure B, and Figure 8 The results of Figure E also show that the group injected with MSCs that release nCD47-SLAMF7 fusion protein in response to TGF-β signal and MSCs that release IL-12 cytokine in response to TGF-β signal can most effectively prolong the survival rate of mice with orthotopic glioma model and has a certain probability of curing mice with orthotopic glioma model.

[0094] Fig. 9The immunofluorescence results of the whole mouse brain sections also showed that the development of in situ glioma in mice injected with MSCs that released nCD47-SLAMF7 fusion protein in response to TGF-β signal and MSCs that released IL-12 cytokine in response to TGF-β signal at the same time was the slowest, and MSCs had a strong co-localization with glioma; the development of in situ glioma in mice injected with MSCs that released nCD47-SLAMF7 fusion protein in response to TGF-β signal or MSCs that released IL-12 cytokine in response to TGF-β signal alone was slower than that in the PBS group, but the effect was not as good as that in the combined treatment group.

[0095] from Fig.10 The confocal results show that the proportion of M2 macrophages in the tumor microenvironment in the combined treatment group is the lowest (upper row of images, F4 / 80 antibody is green, purchased from biolegend, catalog number 123119; CD206 antibody is red, purchased from biolegend, catalog number 141712) and the number of effectively infiltrating CD8-positive T cells is the largest (lower row of images, CD8 antibody is red, purchased from biolegend, catalog number 126611), which proves the superiority of this combined treatment system in the treatment of in situ glioma models.

[0096] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A nCD47-SLAMF7 fusion protein, characterized in that: The amino acid sequence of the nCD47-SLAMF7 fusion protein is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the nCD47-SLAMF7 fusion protein according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A plasmid vector, characterized in that: The plasmid comprises a promoter sequence encoding a SMAD response element and the nucleotide sequence of the nucleic acid molecule of claim 2; The promoter sequence of the SMAD response element is shown in SEQ ID NO.

3.

5. A bone marrow mesenchymal stem cell that secretes nCD47-SLAMF7 fusion protein in response to TGF-β cytokine, characterized in that: The bone marrow mesenchymal stem cells secreting the nCD47-SLAMF7 fusion protein are obtained by transfecting the bone marrow mesenchymal stem cells with a plasmid expressing the nucleotide sequence comprising the promoter sequence encoding the SMAD response element and the nucleic acid molecule as claimed in claim 4.

6. A plasmid vector, characterized in that: The plasmid comprises a promoter sequence encoding a SMAD response element and a nucleic acid sequence encoding murine IL-12; The promoter sequence of the SMAD response element is shown in SEQ ID NO.3, and the amino acid sequence of the IL-12 cytokine is shown in SEQ ID NO.

4.

7. A bone marrow mesenchymal stem cell that secretes mouse-derived IL-12 cytokine in response to TGF-β cytokine, characterized in that: The bone marrow mesenchymal stem cells secreting the mouse-derived IL-12 cytokine are obtained by transfecting the bone marrow mesenchymal stem cells with a plasmid expressing the promoter sequence encoding the SMAD response element and the nucleic acid sequence encoding the mouse-derived IL-12 as described in claim 6.

8. A cell therapy composition, characterized in that: The cell therapy composition comprises the bone marrow mesenchymal stem cells secreting the nCD47-SLAMF7 fusion protein as described in claim 5 or the bone marrow mesenchymal stem cells secreting the mouse-derived IL-12 cytokine as described in claim 7.

9. The cell therapy composition according to claim 8, characterized in that: The cell composition also includes an aqueous solution, which is one of water, physiological saline, glucose aqueous solution or cell culture medium.

10. A fixed ratio cell therapy composition, characterized in that: The fixed-ratio cell therapy composition comprises the bone marrow mesenchymal stem cells expressing secretory nCD47-SLAMF7 fusion protein and the bone marrow mesenchymal stem cells expressing secretory IL-12 cytokine as described in claim 8, and the quantity ratio of the two is 1:

1.

11. The use of a cell therapy composition according to claim 8, characterized in that: The cell therapy composition can also be prepared into a cell therapy preparation, which comprises the cell therapy composition and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, an osmotic pressure regulator, a stabilizer, a suspending agent, a coating material, an anti-adhesive, an integrifier, a penetration enhancer, a pH regulator, a buffer, a surfactant, an absorbent, a diluent, a filter aid, and a sustained-release material.

12. The use of a cell therapy composition according to claim 11, characterized in that: The cell therapy preparation is preferably an in situ injection.

13. Use of any one of the bone marrow mesenchymal stem cells secreting nCD47-SLAMF7 fusion protein according to claim 5, the bone marrow mesenchymal stem cells secreting mouse-derived IL-12 cytokine according to claim 7, the cell therapy composition according to claim 8 or 9, and the fixed-ratio cell therapy composition according to claim 10 in the preparation of a drug for resisting in situ glioma.

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