Use of CCL3 in immunotherapy of multiple myeloma

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of CCL3 in immunotherapy of multiple myeloma. On one hand, the invention provides application of a preparation for detecting CCL3 expression in preparation of a product for analyzing drug-resistant recurrence risk after multiple myeloma immunotherapy and a product for analyzing severity of an immunosuppressive microenvironment of multiple myeloma; on the other hand, the invention provides application of the IMNP in preparation of a product for immunotherapy of multiple myeloma; in addition, the invention also provides an application of the IMNP combined PD-L1 antibody in preparation of an immunotherapy product for multiple myeloma. According to the invention, a new observation and analysis means is provided for immunotherapy of multiple myeloma, and the immunotherapy effect change in the immunotherapy process of multiple myeloma can be better monitored and evaluated; the IMNP improves the myeloma immunosuppressive microenvironment by efficiently removing CCL3 molecules, reduces the risk of drug-resistant relapse in the process of multiple myeloma immunotherapy, and improves the effect of MM immunotherapy by combining the IMNP with a PD-L1 antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of CCL3 in immunotherapy of multiple myeloma. Background Art

[0002] Multiple myeloma (MM) is the second largest tumor in the blood system. It mainly invades the bone marrow and can also form infiltration foci outside the bone marrow, such as in the liver, spleen, kidney, lymph nodes, skin, lung, thyroid, etc. The latter is a manifestation of rapid progression of the disease, indicating a poor prognosis. The emergence of new drugs for the treatment of multiple myeloma (such as proteasome inhibitors, immunomodulators, CD38 monoclonal antibodies, etc.) and the application of standard clinical treatment models including induction therapy, consolidation therapy and maintenance therapy have significantly improved the progression-free survival (PFS) and overall survival (OS) of MM patients. However, MM is still incurable and recurrence of the disease is inevitable. The key reason is that the bone marrow immunosuppressive microenvironment supports and protects MM minimal residual disease and induces its drug resistance, making it difficult to completely eliminate minimal residual disease. A large amount of clinical data shows that the degree of elimination of minimal residual disease is closely related to the PFS and OS benefits of MM patients. Effectively regulating the bone marrow immunosuppressive microenvironment to completely eliminate minimal residual disease and to prolong the patient's PFS as much as possible in order to cure MM has important clinical value.

[0003] Inflammatory factors are an important component of the MM bone marrow immunosuppressive microenvironment. The progression of MM is often accompanied by a significant increase in the levels of a large number of inflammatory factors. Chemokine ligand 3 (CCL3) is an important class of inflammatory chemokines, mainly derived from MM cells and the bone marrow microenvironment (such as osteoclasts). In a large number of studies in the past, the study of CCL3 mainly included the characterization of its chemotactic ability, and extended to its role in autoimmune inflammatory processes and hematological cancers (such as multiple myeloma). The dual role of CCL3 has been revealed, that is, as an effective immune activator and promoter of inflammatory disease processes, but no studies have revealed the role of CCL3 in the formation of the immunosuppressive microenvironment in myeloma.

[0004] A large number of studies have shown that the bone marrow microenvironment of MM patients mainly manifests an immunosuppressive phenotype that supports tumor growth (i.e., an immunosuppressive microenvironment), such as abnormal dendritic cell function leading to antigen presentation defects, an increase in the number of M2 macrophages, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) with immunosuppressive effects, a decrease in the number and dysfunction of cytotoxic T cells with MM killing effects, and a significant increase in immunosuppressive soluble cytokines such as TGF-β, IL-10, and IL-8. Studies have shown that the state of the bone marrow microenvironment in MM patients is related to the degree of clearance of minimal residual disease, and regulating the bone marrow microenvironment is conducive to the complete clearance of minimal residual disease. Therapeutic drugs (such as immunomodulators and CD38 monoclonal antibodies) or treatment methods (such as autologous hematopoietic stem cell transplantation) with certain bone marrow microenvironment regulation effects have achieved excellent therapeutic effects in the treatment of MM. However, MM patients still cannot avoid drug resistance and relapse after the above treatment, indicating that the existing means of reshaping the bone marrow immunosuppressive microenvironment for the treatment of MM still cannot fully meet clinical needs. In addition, the PD-L1 antibody, which has achieved excellent efficacy in some solid tumors such as breast cancer and melanoma, has unsatisfactory efficacy in MM. Therefore, new means of regulating the immunosuppressive microenvironment of myeloma need to be developed urgently. Summary of the invention

[0005] In view of the above problems, the present invention provides the use of CCL3 in the immunotherapy of multiple myeloma, mainly to solve the problems that new detection methods for the severity of the bone marrow immunosuppressive microenvironment need to be supplemented and improved, and the regulation methods of the bone marrow immunosuppressive microenvironment need to be developed urgently.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] The first aspect provides the use of a preparation for detecting CCL3 expression in the preparation of a product for detecting and analyzing the risk of drug-resistant relapse after immunotherapy for multiple myeloma; wherein, high expression of CCL3 indicates a high risk of drug-resistant relapse, and the CCL3 expression level is positively correlated with the risk of drug-resistant relapse after immunotherapy for multiple myeloma. In the immunotherapy of multiple myeloma, when CCL3 expression is high, it indicates that the immunosuppression in the bone marrow microenvironment is more severe, which can lead to drug-resistant relapse after immunotherapy for myeloma. The risk of drug-resistant relapse after immunotherapy for multiple myeloma can be evaluated and analyzed before and after immunotherapy, for example, the expected immunotherapy effect can be evaluated based on the expression of CCL3 before immunotherapy, and the immunotherapy effect can also be evaluated based on the expression of CCL3 after immunotherapy. Furthermore, when the risk of drug-resistant relapse is high, it indicates that the immunotherapy effect is not good.

[0008] The level of CCL3 expression can be distinguished based on general clinical knowledge, such as the expression of CCL3 in the corresponding parts or cells, and the CCL3 expression level as a reference for treatment. The detection of CCL3 expression can be carried out by conventional methods, such as IHC, WB, ELISA, etc. The "product" mentioned in the text can at least be interpreted as detection reagents, kits, detection equipment, etc., and its specific form is not specifically limited, the same as below.

[0009] The second aspect provides the use of a preparation for detecting CCL3 expression in the preparation of a product for detecting and analyzing the severity of the immunosuppressive microenvironment of multiple myeloma; wherein the CCL3 expression level is positively correlated with the severity of immunosuppression. The severity of the immunosuppressive microenvironment of myeloma can be known by the expression of CCL3. When CCL3 expression is high, it indicates that the microenvironment immunosuppression of myeloma is more serious, that is, when CCL3 expression is high, the microenvironment immunosuppression of myeloma is serious, which provides guidance for clinical diagnosis and treatment. In addition to clinical applications, it can also be used in scientific research projects. When verifying the therapeutic effects of different drugs on myeloma, the suppression of the immune response can be understood by monitoring the expression of CCL3, so as to clarify whether the research object drug can play an effective therapeutic role in myeloma. The specific means of detecting the expression of CCL3 can adopt existing technologies without special limitation.

[0010] The third aspect provides a method for preparing IMNPs and the IMNPs prepared by the method.

[0011] The preparation method of IMNP comprises the following steps: mixing the macrophage membrane obtained by co-incubating IFN-γ with macrophages and nanocarriers or nanoliposomes to prepare IMNP.

[0012] The purpose of co-incubating macrophages with IFN-γ is to optimize the stimulation of macrophages with IFN-γ, and then use the macrophage membrane to coat the nanocarrier or fuse it on the surface of nanoliposomes to obtain IMNP.

[0013] As for the preparation of IMNP, any one or more of the conditions in the preparation process can be selected:

[0014] Reaction condition 1: when preparing membrane-coated nanocarriers: inject acetone containing polyester materials into water, and then remove the acetone to obtain nanocarriers, wherein the polyester materials can be at least any one of PLGA (polylactic acid-co-glycolic acid), PLA (polylactic acid), and PCL (polycaprolactone), but are not limited thereto, and the acetone can be removed by evaporation; when preparing membrane fusion liposomes: after the lipid material film is formed, add the membrane material during hydration, mix well, and then perform ultrasound, and extrude to form membrane fusion nanoliposomes, and the nanoliposomes can be prepared by existing technology;

[0015] Reaction condition 2: when the cell membrane is selected from any of conventional RAW264.7 macrophage membranes, neutrophil membranes, hybrid membranes of different types of biological membranes, or cell membranes of gene-edited cells (not limited thereto), they should all be equivalently within the scope of the claims of the present invention;

[0016] Reaction condition three: macrophage membrane and nanocarriers were mixed and then sonicated in an ice bath to obtain IMNPs;

[0017] Reaction condition 4: For the materials used to prepare IMNP, they can also be limited according to the situation:

[0018] One of them is that the stimulation concentration of IFN-γ is generally 10±0.5ng / ml, and of course, those that fluctuate outside this range but have similar effects to the present invention should still be within the scope of the present invention;

[0019] Second of them, each material can be directly ultrasonically reacted in pure water.

[0020] Nanocarriers take PLGA nanoparticles as an example: IMNPs are prepared using RAW264.7 macrophages and polyester material PLGA. PLGA nanoparticles can be directly prepared using existing materials or existing preparation methods. 500 microliters of acetone dissolved with 5 mg of PLGA polymer is quickly injected into 1 ml of deionized water. After the acetone is evaporated by a vacuum pump, PLGA nanoparticles can be obtained. After adding 1 mg of IFN-γ stimulated RAW264.7 macrophage membrane and mixing evenly, ice bath ultrasound (power of 200 watts) is used for 5 minutes to obtain IMNPs; large-scale preparation can be adjusted according to actual conditions.

[0021] The IMNPs prepared by the preparation method of IMNPs involved in any of the above situations have good biocompatibility. The particle size of IMNPs is generally about 100 nm, the potential is about -20 mv, and they are regular spherical under an electron microscope, but are not limited to the above characteristics. Any IMNPs that can be consistent with the IMNPs prepared by any of the above methods or have the same coating structure according to conventional material identification methods should be within the scope of the present invention.

[0022] The fourth aspect provides the use of IMNP in the preparation of an adsorption preparation of CCL3; wherein the CCL3 is present in the immunosuppressive microenvironment of multiple myeloma. IMNP can effectively remove CCL3 in the immunosuppressive microenvironment of myeloma. Of course, the removal mentioned in this article does not mean complete removal. It is a relative expression, the purpose of which is to reduce the content of CCL3 in the immunosuppressive microenvironment of multiple myeloma, and to improve the immunosuppressive microenvironment of multiple myeloma and reduce the inhibitory effect of the immune response. This type of adsorption preparation has both medical uses and can be used as an experimental reagent. For example, in an animal model or a cell model, the amount of CCL3 can be controlled by the adsorption preparation.

[0023] The fifth aspect provides the use of nanomedicines with a scavenging effect of CCL3 in the preparation of products for immunotherapy of multiple myeloma; wherein the nanomedicine with a scavenging effect of CCL3 refers to any of the aforementioned IMNPs or nanomedicines formed by any of the membranes of RAW264.7 macrophages, neutrophil membranes, hybrid membranes of different types of biological membranes, and cell membranes of gene-edited cells coated with nanocarriers or fused on the surface of nanoliposomes. In some specific examples, nanomedicines with a scavenging effect of CCL3 are used to prepare products that inhibit the recurrence of drug resistance after immunotherapy of multiple myeloma. The drug resistance recurrence involved can specifically be the recurrence of drug resistance after immunotherapy of multiple myeloma PD-L1 antibodies, or nanomedicines with a scavenging effect of CCL3 are used to prepare products that resist drug resistance in immunotherapy of multiple myeloma to resist drug resistance.

[0024] Among them, the effect of IMNP is at least manifested in one of the following: the IMNP is used to improve the immunosuppressive microenvironment of the bone marrow, and / or the IMNP is used to inhibit the osteoclast induction effect of CCL3, and / or the IMNP is used to inhibit the proliferation and migration of myeloma cells, and / or reduce the risk of drug resistance recurrence after immunotherapy of multiple myeloma, and / or improve the immunotherapy effect of myeloma PD-L1 antibody; as long as one of them is met, it is within the scope of the present invention, but it is not limited to the above four performances. Through any of the above regulatory effects, it is shown that IMNP is beneficial to the immunotherapy of multiple myeloma, and can reduce the situation of MM drug resistance recurrence to a certain extent, improve the effect of immunotherapy, and have a significant gain effect. "At least one of the following" can be understood as at least one of the listed ones, but it is not required to be one of the listed ones.

[0025] In terms of improving the immunosuppressive microenvironment of the bone marrow: the purpose is to reduce the immune cells that support the growth of myeloma cells in the immunosuppressive microenvironment of myeloma, increase the immune cells with myeloma cell-killing effect, and thereby eliminate minimal residual disease and reduce its drug resistance, so as to prolong the PFS and OS of MM patients. Nanomedicines with the scavenging effect of CCL3 can be used in the preparation of drugs that inhibit the resistance of multiple myeloma immunotherapy. Nanomedicines with the scavenging effect of CCL3 are used to inhibit the resistance of multiple myeloma PD-L1 antibody immunotherapy.

[0026] As for inhibiting the osteoclast induction effect of CCL3 and the proliferation and migration of myeloma cells: the purpose is to inhibit osteoclast differentiation, inhibit the proliferation and migration of myeloma cells, and then inhibit the progression of myeloma, and can also improve the immunosuppressive microenvironment of myeloma. Of course, it is not required that CCL3 can inhibit all the aforementioned mechanisms at the same time in actual application to be within the scope of the present invention. As long as any one of them is met, it should be regarded as within the scope of the present invention. For example, inhibiting myeloma cell proliferation and inhibiting myeloma cell migration are two mechanisms. If one of them is met and is beneficial to the treatment of multiple myeloma, it should be within the scope of the present invention.

[0027] The sixth aspect provides the use of IMNP combined with PD-L1 antibody in the preparation of immunotherapy products for multiple myeloma. IMNP combined with PD-L1 antibody can play a significant role in the treatment of multiple myeloma, and the effect of IMNP combined with PD-L1 antibody is significantly different from that of IMNP group and PD-L1 antibody group, which reflects that IMNP and PD-L1 antibody have a synergistic effect in the treatment of multiple myeloma, which can more effectively avoid the occurrence of drug resistance recurrence.

[0028] Among them, immunotherapy products for multiple myeloma are used to kill tumor cells in the bone marrow, and / or immunotherapy products for myeloma are used to inhibit extramedullary infiltration foci of myeloma in the spleen. Killing tumor cells in the bone marrow: Its main effect is to kill myeloma tumor cells and also reduce their migration. Inhibiting extramedullary infiltration foci of myeloma in the spleen: That is, IMNP combined with PD-L1 antibodies can effectively inhibit the formation of infiltration foci of multiple myeloma outside the bone marrow (extramedullary infiltration or extramedullary invasion: such as metastasis of multiple myeloma to the spleen), specifically inhibiting the formation of infiltration foci of multiple myeloma in the spleen, thereby alleviating the condition of patients with rapidly progressive or advanced multiple myeloma and prolonging their survival.

[0029] The present disclosure provides a new observation and analysis method for immunotherapy of multiple myeloma, which can better monitor and evaluate the changes in the immunotherapy effect during the immunotherapy of multiple myeloma. The present invention also provides a nano drug constructed by IFN-γ-stimulated optimized macrophage membrane-coated nanoparticles, which has a stronger CCL3 adsorption effect than ordinary macrophage membrane-coated nanoparticles, improves the immunosuppressive microenvironment of myeloma by efficiently removing CCL3 molecules, reduces the risk of drug resistance recurrence during the immunotherapy of multiple myeloma, and improves the effect of MM immunotherapy by combining IMNP with PD-L1 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the flow chart of this research project.

[0031] Figure 2 The expression of CCL3 is correlated with the overall prognosis and immunosuppression of multiple myeloma. The expression level of CCL3 in myeloma patients and healthy people (A); the analysis of CCL3 expression level and the prognosis of myeloma patients (B); the correlation analysis of CCL3 level and myeloma immunosuppressive molecules (C); the correlation analysis of CCL3 level and different types of stromal cells in myeloma (D).

[0032] Figure 3 Optimization, preparation and characterization of MNPs. The effects of different concentrations of IFN-γ on the expression levels of CRR1 and CCR5 on the macrophage membrane were analyzed by flow cytometry (A-D) and Western (E&F); the distribution of CCR1 and CCR5 on IMNPs (G&H); the particle size (I), potential (J), electron microscopy (K) and stability (L) of IMNPs.

[0033] Figure 4 The figures show the scavenging effect of IMNP on CCL3 and its effect on the proliferation and migration of myeloma cells. Evaluation of the scavenging effect of IMNP and its control group on CCL3 (A); Evaluation of the scavenging effect of IMNP on CCL3 in different media (B); Evaluation of the scavenging effect of fixed IMNP concentration on different concentrations of CCL3 (C); Evaluation of the scavenging effect of different concentrations of IMNP on fixed concentration of CCL3 (D); Time-dependent effect of IMNP on CCL3 scavenging (E); IMNP scavenging effect on CCL3 derived from 5TGM1 myeloma cell line (F) and the effect on 5TGM1 cell proliferation (G&H) and migration (I).

[0034] Figure 5IMNP inhibits CCL3-induced osteoclast differentiation. Flow cytometry (A&B) and TRAP staining (C) were used to evaluate the effect of IMNP on CCL3-induced osteoclast differentiation; Elisa was used to detect the expression of APRIL, the effector molecule of CCL3 (D).

[0035] Figure 6 Qualitative (A) and quantitative analysis (B&C) of tumor burden in 5TGM1-Luc model mice under different treatments (PBS group, RNP group, MNP group, IMNP group, PD-L1 antibody group, and IMNP combined with PD-L1 antibody group).

[0036] Figure 7 The survival curves (A), peripheral blood globulin levels (B), bone marrow CCL3 levels (C&G upper row), peripheral blood myeloma cell proportion (D), bone marrow myeloma cell proportion (E&G lower row) and spleen myeloma cell proportion (F) of 5TGM1-Luc model mice under different treatments (PBS group, RNP group, MNP group, IMNP group, PD-L1 antibody group and IMNP combined with PD-L1 antibody group).

[0037] Figure 8 Figure 3 Bone MDSCs and CD8 in spleen and bone marrow of 5TGM1-Luc model mice under different treatments (PBS group, RNP group, MNP group, IMNP group, PD-L1 antibody group, and IMNP combined with PD-L1 antibody group). + T cells, GranB + CD8 + T cells, CD4 + The ratio of T cells to Tregs cells.

[0038] In the figure: * indicates that the p value is less than 0.05, ** indicates that the p value is less than 0.01, *** indicates that the p value is less than 0.001, and **** indicates that the p value is less than 0.0001. The smaller the p value, the more significant the difference. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with specific research projects.

[0040] 1. Design and preparation of IMNPs

[0041] The preparation of IMNPs is as follows: S1, inject acetone containing PLGA into water; S2, evaporate and remove acetone to obtain PLGA nanoparticles; S3, add 10ng / ml IFN-γ stimulated RAW264.7 macrophage membrane and mix, then obtain IMNPs by ice bath ultrasound, the ratio of macrophage membrane to PLGA nanoparticle input is 1:2. The cell membrane content is 2.5mg, and the PLGA content is 5mg, and both are placed in pure water for ultrasound reaction.

[0042] The macrophage membrane may also be any one of a conventional RAW264.7 macrophage membrane, a neutrophil membrane, a hybrid membrane of different types of biological membranes, and a cell membrane of a gene-edited cell.

[0043] 2. CCL3 expression is associated with the immunosuppressive microenvironment of myeloma

[0044] By analyzing the data from public databases (two datasets: GSE47552 and MMRF), we found that the expression of CCL3 in myeloma patients was significantly higher than that in healthy people ( Figure 2 Middle A), high expression of CCL3 indicates poor overall prognosis in myeloma patients ( Figure 2 In middle B), the expression of CCL3 in myeloma patients is positively correlated with the expression levels of multiple myeloma immunosuppressive molecules such as GEM, HACVR2, and CTLA4 ( Figure 2 C), positively correlated with the distribution of immunosuppressive cells (such as Treg and M2 macrophages), and negatively correlated with the distribution of cells related to immune activation, such as osteoblasts and Th1 cells ( Figure 2 (D) This suggests that CCL3 is associated with the severity of the myeloma immunosuppressive microenvironment and is expected to be an effective therapeutic target for reversing the myeloma immunosuppressive microenvironment.

[0045] 3. RAW264.7 cell membrane optimized by IFN-γ stimulation

[0046] IFN-γ stimulated macrophage membranes were preferred for preparation of IMNPs and characterization. Figure 3 Chinese AD) and Western ( Figure 3 The results of E&F suggest that 10 ng / ml IFN-γ is the best choice for increasing the expression of CCL3's natural receptors CCR1 and CCR5 on the macrophage membrane. The preparation of IMNPs using macrophage membranes stimulated with 10 ng / ml IFN-γ can well preserve CCR1 and CCR5 on the surface of macrophage membranes. Figure 3 GH), IMNP particle size is about 100nm ( Figure 3 I), potential -20mv ( Figure 3 Middle J), ​​regular spherical shape under electron microscope ( Figure 3The particle size did not change significantly after being stored at 4°C for one week, indicating that IMNPs have good stability ( Figure 3 Middle L).

[0047] IV. Scavenging effect of IMNP on CCL3

[0048] After the nanomedicine was mixed with CCL3 for a period of time, the supernatant was ultracentrifuged and the residual CCL3 was measured by enzyme-linked immunosorbent assay to evaluate the scavenging effect of IMNP on CCL3. The results showed that IMNP had better CCL3 adsorption capacity than MNP (conventional macrophage membrane-coated nanoparticles), RNP (red blood cell membrane-coated nanoparticles) and NP (nanoparticles without cell membrane coating) ( Figure 4 (A), which may be related to the fact that the levels of CCL3 receptors such as CCR1 and CCR5 on the surface of IMNP are significantly higher than those of MNP, which can more efficiently adsorb and remove CCL3. The adsorption effect of IMNP on CCL3 is different in different media ( Figure 4 Middle B), IMNP scavenged CCL3 in a concentration-dependent manner ( Figure 4 C&D) and time-dependent methods ( Figure 4 Middle E), IMNP also has a certain clearance effect on CCL3 derived from 5TGM1 myeloma cell line ( Figure 4 F), IMNP treatment can inhibit the effect of CCL3 on the proliferation of 5TGM1 cells ( Figure 4 G&H) and migration ( Figure 4 The promoting effect of I).

[0049] 5. IMNPs can inhibit the osteoclast-inducing effect of CCL3 and suppress APRIL expression

[0050] CLL3 and IMNP were mixed or not mixed and then ultracentrifuged to obtain the supernatant for inducing osteoclast differentiation. Flow cytometry ( Figure 5 A&B) and TRAP staining ( Figure 5 The results in middle C) showed that IMNP could inhibit the osteoclast-inducing effect of CCL3. Elisa results further showed that the expression of APRIL, an effector molecule of CCL3, decreased in the IMNP-treated group ( Figure 5 (middle D).

[0051] 6. IMNP combined with PD-L1 significantly enhances the immunotherapy effect against myeloma

[0052] Myeloma model mice bearing 5TGM1-Luc were established. After different treatments (PBS group, RNP group, MNP group, IMNP group, PD-L1 antibody group, and IMNP combined with PD-L1 antibody group), the tumor burden of the model mice was regularly monitored by small animal in vivo imaging. Figure 6A) and semi-quantitative ( Figure 6 The results in Figure 2 (B&C) showed that IMNP combined with PD-L1 antibody treatment can significantly reduce the tumor burden of myeloma model mice.

[0053] The combined treatment of IMNP and PD-L1 antibody significantly prolonged the survival curve of model mice ( Figure 7 A), significantly reducing the peripheral blood globulin level of model mice ( Figure 7 B), CCL3 levels in bone marrow ( Figure 7 C& Figure 7 Middle G upper row), peripheral blood ( Figure 7 Middle D), bone marrow tissue ( Figure 7 China E& Figure 7 Middle G lower row) and spleen tissue ( Figure 7 The flow cytometry and immunohistochemistry results in the middle (F) showed that the combined treatment group could significantly reduce the myeloma cell load in peripheral blood, bone marrow and spleen compared with other treatment groups, and the IMNP combined with PD-L1 antibody group had a significant difference in effect compared with other groups. + The proportion of T cells ( Figure 8 ), and pharmacodynamic results ( Figure 6 & Figure 7 ) to remain consistent.

[0054] It is clear to those skilled in the art that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention, and existing technical means can be used for the parts not detailed in the text. They all fall within the protection scope of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.

Claims

1. Application of a preparation for detecting CCL3 expression in the preparation of a product for analyzing the risk of drug resistance recurrence after immunotherapy for multiple myeloma; wherein: High expression of CCL3 indicates a high risk of drug-resistant recurrence.

2. The use of a preparation for detecting CCL3 expression in the preparation of a product for analyzing the severity of the immunosuppressive microenvironment in multiple myeloma; wherein: The expression level of CCL3 was positively correlated with the severity of immunosuppression.

3. A method for preparing IMNPs, characterized in that: The method comprises the following steps: mixing the macrophage membrane obtained by co-incubating IFN-γ with macrophages with nanocarriers or nanoliposomes to prepare IMNP; wherein the macrophage membrane is coated with the nanocarrier, or the macrophage membrane is fused with the surface of the nanoliposome.

4. The method for preparing IMNP according to claim 3, characterized in that: When preparing a membrane-coated nanocarrier: injecting acetone containing a polyester material into water, and then removing the acetone to obtain a nanocarrier, wherein the polyester material is at least any one of PLGA, PLA, and PCL; or, when preparing a membrane-fused liposome: after a lipid material film is formed, adding a membrane material during hydration, mixing, ultrasonicating, and extruding to form a membrane-fused nanoliposome; The macrophage membrane is the cell membrane of RAW264.7 cell line.

5. The method for preparing IMNP according to claim 3, characterized in that: The stimulating concentration of IFN-γ was 10±0.5 ng / ml.

6. The IMNPs prepared by the method for preparing IMNPs according to any one of claims 3 to 5.

7. Use of the IMNP in claim 6 in preparing an adsorption preparation of CCL3; wherein, The CCL3 is present in the immunosuppressive microenvironment of multiple myeloma.

8. Application of nanomedicine with CCL3 scavenging effect in the preparation of immunotherapy products for multiple myeloma; wherein, The nanomedicine with the CCL3 scavenging effect is at least the IMNP in claim 6 or a nanomedicine formed by coating a nanocarrier or fusing on the surface of a nanoliposome with any of the membranes of RAW264.7 macrophages, neutrophil membranes, hybrid membranes of different types of biological membranes, and cell membranes of gene-edited cells; The effect of IMNP is at least one of the following: the IMNP is used to improve the immunosuppressive microenvironment of the bone marrow, and / or the IMNP is used to inhibit the osteoclast induction effect of CCL3, and / or the IMNP is used to inhibit the proliferation and migration of myeloma cells, and / or reduce the risk of drug resistance and recurrence after immunotherapy for multiple myeloma, and / or improve the immunotherapy effect of myeloma PD-L1 antibody.

9. Use of the IMNP in claim 6 in combination with a PD-L1 antibody in the preparation of an immunotherapy product for multiple myeloma.

10. The use according to claim 9; wherein: Immunotherapy products for multiple myeloma are used to kill tumor cells in the bone marrow, and / or immunotherapy products for myeloma are used to inhibit extramedullary infiltration of myeloma in the spleen.

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