Application of ANXA1 protein or antagonist thereof in preparation of medicine for treating multiple myeloma

Abstract

The invention provides an application of an ANXA1 protein or an antagonist thereof in preparation of a medicine for treating multiple myeloma. After the multiple myeloma BCMA immunotherapy, when BCMA negative recurrence occurs, in multiple myeloma cells, a BCMA receptor signal channel is reduced, ANXA1 protein expression quantity is increased, proliferation of the multiple myeloma cells is promoted, and CAR-T cell immunocompetence and immune persistence are inhibited at the same time. The antagonist of the ANXA1 protein reduces the expression quantity of the ANXA1 protein, inhibits the proliferation of multiple myeloma cells, and recovers the immunocompetence and immune persistence of CAR-T cells at the same time. According to the application, the biological function of the ANXA1 in the MM cells is defined, the action mechanism of the ANXA1 antagonist for treating the BCMA negative MM cells and improving the activity of the CAR-T cells is proved, a valuable theoretical basis is provided for improvement of clinical myeloma CAR-T cell treatment, and finally the disease-free lifetime of a patient after BCMA-targeted immunotherapy is prolonged.

Description

Technical Field

[0001] This application belongs to the field of combined targeted therapy and immunotherapy, and relates to the use of ANXA1 protein or its antagonist in the preparation of a medicament for treating multiple myeloma. Background Art

[0002] B cell maturation antigen (BCMA) is a membrane receptor protein necessary for the maturation and growth of normal plasma cells. It activates the NF-κb signaling pathway by binding to the natural ligands APRIL (a proliferation-inducing ligand) and BAFF (an activator), promoting cell proliferation (see reference / patent: O'Connor, B.P. et al. BCMA is essential for the survival of long-lived bone marrow plasma cells. The Journal of experimental medicine 199, 91-98, doi: 10.1084 / jem.20031330 (2004)). Multiple myeloma (MM) cells are generally considered to highly express BCMA specifically. Currently, in the treatment of relapsed / refractory multiple myeloma (RRMM), antigen chimeric receptor T cell (CAR-T) technology targeting BCMA has achieved great success as a revolutionary gene-editing T cell immunotherapy (see reference / patent: Brudno, J.N. et al. T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma. Journal of clinical oncology 36, 2267-2280, doi: 10.1200 / JCO.2018.77.8084 (2018)).The inventor team and domestic collaborating units, through exploratory trials and long-term follow-up of the targeted BCMA CAR-T cell therapy, found that approximately 15% of RRMM patients could achieve disease-free survival of more than 5 years, but more than half of the patients would still relapse and progress within 2 years after treatment (see literature / patent: Xu, J., et al. Exploratory trial of abiepitopic CAR T-targeting B cell maturation antigen in relapsed / refractory multiple myeloma. Proceedings of the National Academy of Sciences of the United States of America 116, 9543-9551, doi: 10.1073 / pnas.1819745116 (2019). Xu, J., et al. Long-term remission and survival in patients with relapsed or refractory multiple myeloma after treatment with LCAR-B38M CAR T cells: 5-year follow-up of the LEGEND-2 trial. Journal of Hematology & Oncology. 2024 Apr 24; 17(1): 23.). The inventors found through the analysis of tumor cells from patients who relapsed after targeted BCMA immunotherapy that there was a phenomenon of loss of BCMA target antigen on the surface of relapsed myeloma cells, which prevented CAR-T cells from recognizing them and led to immune escape (see literature / patent: Shuangshuang Yang, Jie Xu , et al. Neutrophil activation and clonal CAR-T re-expansion underpinning cytokine release syndrome during ciltacabtagene autoleucel therapy in multiple myeloma. Nature Communications. 2024 Jan 8; 15(1): 360.).

[0003] To reduce the risk of BCMA-negative recurrence, on the basis of the BCMA single target, bispecific CAR-T cell immunotherapy prepared by adding a second target has been exploratory applied to relapsed and refractory multiple myeloma, such as anti-GPRC5D / BCMA CAR-T, anti-CD19 / BCMA CAR-T, etc. For example, US Patent US20220267438A1 provides multispecific antibodies that bind BCMA, GPRC5D, and CD3 and multispecific antigen-binding fragments thereof, and also provides related polynucleotides capable of encoding the provided multispecific antibodies or multispecific antigen-binding fragments, cells expressing the provided multispecific antibodies or multispecific antigen-binding fragments, and related vectors and detectable-labeled multispecific antibodies or multispecific antigen-binding fragments. In addition, this application also provides methods for generating and using the provided multispecific antibodies and multispecific antigen-binding fragments. Patent WO2024170001A1 provides CAR-T cells targeting BCMA and CD19. Specifically, a parallel expression structure of a chimeric antigen receptor (CAR) for B cell maturation antigen (BCMA) and CD19 antigen molecules is provided, and the CD28 co-stimulatory domain motif is optimized; this patent also provides the use of the chimeric antigen receptor in adoptive T cell therapy for B cell-related disorders.

[0004] In summary, the technical problems of the prior art are as follows: The existing multi-target CAR-T cell immunotherapy technology has been exploratory applied to relapsed and refractory multiple myeloma. However, their efficacy is comparable to that of BCMA single-target products, and the phenomenon of recurrence still exists. How to formulate a combination drug therapy plan combined with CAR-T cell therapy to overcome BCMA-negative recurrence has become a difficulty in the field of CAR-T cell therapy for multiple myeloma and is also the core problem to be solved by the present technical invention. Summary of the Invention

[0005] Based on the above-mentioned disadvantages existing in the prior art, the purpose of this application is to provide the use of ANXA1 protein or its antagonist in the preparation of a drug for treating multiple myeloma.

[0006] ANXA1 protein is a calcium-regulated phospholipid-dependent membrane-binding protein. Under normal circumstances, it is highly expressed in multiple cells such as the respiratory system, kidneys, brain tissue, neutrophils, and macrophages. Under ischemia-reperfusion, oxidative stress, and the action of glucocorticoids, ANXA1 can be significantly upregulated. It specifically interacts with the N-formyl peptide receptor (FPR) and participates in biological processes such as inflammation, phagocytosis, proliferation, differentiation, and apoptosis. During these processes, ANXA1 can inhibit the NF-κb signaling pathway and the production of pro-inflammatory factors, exerting an anti-inflammatory effect and immunosuppressive function. Interestingly, ANXA1 is also closely related to the occurrence, invasion, and metastasis of certain tumors, such as solid tumors like gastric cancer, breast cancer, and glioma (see literature / patent: Guo, C., Liu, S. & Sun, M. Z. Potential role of Anxa1 in cancer. Future oncology 9, 1773-1793, doi:10.2217 / fon.13.114(2013).).

[0007] Previous work by the inventors through research at multiple levels such as primary specimens after multiple myeloma-targeted BCMACAR-T treatment, database source data, and in vitro experiments on cell lines has shown that ANXA1 protein is abnormally highly expressed in BCMA-negative MM cells and can promote the proliferation of MM cells. However, the specific role of ANXA1 in MM has rarely been reported. Currently, only one study has shown that knocking down the ANXA1 transcription level in vitro can achieve an anti-MM effect similar to that of bortezomib (bortezomib can delay the growth of tumors including multiple myeloma). The method of combining bortezomib with ANXA1 knockdown treatment has significantly higher cytotoxicity to tumors than bortezomib alone, suggesting that ANXA1 may play an important role in the occurrence and development of MM (reference / patent: Jia, C., Kong, D., Guo, Y., Li, L. & Quan, L. Enhanced antitumor effect of combination of annexin A1 knockdown and bortezomib treatment in multiple myeloma in vitro and in vivo. Biochemical and biophysical research communications 505, 720-725, doi:10.1016 / j.bbrc.2018.09.140(2018).).

[0008] Based on the above purposes and existing research content, the present application provides the following technical solutions:

[0009] One of the technical solutions of the present invention provides the use of ANXA1 protein or its antagonist in the preparation of a drug for treating multiple myeloma.

[0010] Further, the multiple myeloma refers to: relapsed and refractory multiple myeloma with BCMA-negative recurrence after BCMA immunotherapy; the BCMA immunotherapy refers to cell therapy with BCMA CAR-T cells.

[0011] Further, this application confirms that: after BCMA-targeted CAR-T cell therapy for multiple myeloma, in MM cells with BCMA-negative recurrence, when the BCMA receptor signaling pathway is downregulated, the function of ANXA1 protein produces a compensatory effect, the expression level of ANXA1 protein is upregulated, promoting the proliferation of multiple myeloma cells, and at the same time inhibiting the immune activity and persistence of CAR-T cells. The compensatory effect means that after the original BCMA-mediated cell proliferation signal is downregulated, in order to maintain proliferation and growth, the cell will positively regulate and increase the expression of ANXA1. Increasing ANXA1 will activate the AMPK signaling pathway of the cell to stimulate cell proliferation, thereby producing a compensatory effect, and cell proliferation becomes stronger, which can also be called abnormal overexpression.

[0012] Further, this application clarifies through in vitro and in vivo studies that the ANXA1 antagonist can inhibit the proliferation of BCMA-negative myeloma and restore the cytotoxicity of CAR-T cells to BCMA-positive myeloma.

[0013] Further, the drug for treating multiple myeloma includes an active ingredient and excipients.

[0014] Further, the active ingredient is: an ANXA1 antagonist, and the addition amount of the active ingredient in the drug is 20 - 50 mg / mL of liquid drug or 20 - 50 mg / g of solid drug; the excipients are pharmaceutically acceptable carriers for improving the processing performance of the active ingredient.

[0015] Further, the pharmaceutically acceptable carrier includes at least any one of a filler, a diluent, a lubricant, a binder, or a disintegrant.

[0016] The filler / diluent is used to increase the weight / volume of the tablet, improve the processing properties and performance of the product, such as powder fluidity, compressibility, granulation performance, uniformity, content uniformity, disintegration, dissolution, tablet integrity, friability, physicochemical stability performance; the filler / diluent is preferably any one or a mixture of lactose, mannitol, microcrystalline cellulose, calcium dihydrogen phosphate, cellulose lactose, or microcrystalline cellulose colloidal silica co-processed product.

[0017] The lubricant is used for: ① improving the electrostatic distribution on the ionic surface; ② improving the roughness of the ionic surface and reducing the frictional force; ③ improving the selective adsorption of gas and weakening the van der Waals force between particles. The lubricant is preferably any one or a mixture of stearic acid, magnesium stearate, calcium stearate, sodium stearyl fumarate, hydrogenated vegetable oil or glyceryl behenate.

[0018] The binder is an adjuvant that imparts appropriate viscosity to non-viscous or insufficiently viscous materials by virtue of its inherent viscosity. It not only helps in the formation and fluidity of granules during the manufacturing process but also contributes to the integrity of tablets during tablet pressing. The binder is preferably any one or a mixture of pregelatinized starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, copovidone, microcrystalline cellulose, ethyl cellulose or methyl cellulose.

[0019] The disintegrant is a substance used to eliminate the binding force generated by the binder or high compression, so that the tablet can be quickly broken into fine particles in gastrointestinal fluid, enabling the active ingredient to be quickly dissolved and absorbed to exert its effect. The disintegrant is preferably any one or a mixture of sodium starch glycolate, calcium silicate, croscarmellose sodium or low-substituted hydroxypropyl cellulose.

[0020] Furthermore, the active ingredient needs to be mixed with at least one pharmaceutically acceptable carrier to prepare a drug; the dosage form of the drug is any one of oral preparations, injection preparations, topical skin preparations or inhalation preparations for the respiratory system.

[0021] The second technical solution of the present invention provides a combined treatment drug for multiple myeloma. The combined treatment drug includes an ANXA1 protein antagonist and a BCMA-targeted CAR-T cell. The ANXA1 protein antagonist has a synergistic effect with the BCMA-targeted CAR-T cell, and the ANXA1 protein antagonist is used to enhance the therapeutic effect of the BCMA-targeted CAR-T cell.

[0022] Compared with the prior art, the present application has at least the following advantages / beneficial effects:

[0023] (1) The present application clarifies the biological function of ANXA1 in MM cells, confirms the mechanism of action of the ANXA1 antagonist in treating BCMA-negative MM cells and improving the activity of CAR-T cells, provides a valuable theoretical basis for the improvement of clinical myeloma CAR-T cell therapy, and ultimately prolongs the disease-free survival period of patients after BCMA-targeted immunotherapy.

[0024] (2) This application shows that ANXA1 is a potential target for alleviating BCMA-negative myeloma. This discovery is based on patient evidence, through clinical research samples and cell analysis, and verified in animal models, confirming the clinical significance of the ANXA1 targeting strategy, that is, the ANXA1 antagonist WRW4 for optimizing BCMA CAR-T therapy. At the same time, more RRMM patients are encouraged to adopt high-resolution technologies to accurately and rapidly identify antigen-deficient tumors after targeted immunotherapy.

[0025] (3) This application first innovatively explores the relationship between ANXA1 and the activity of BCMA-negative MM cells. Currently, the role of ANXA1 in MM has been rarely reported, and it is also unknown how MM cells that do not rely on the BCMA receptor survive in the long term. However, the inventor's previous work has suggested that BCMA-negative MM cells may rely on ANXA1 for survival. Therefore, whether ANXA1 is a signature oncoprotein of BCMA-negative MM and whether it can be a drug target are not only the research focus of this application but also the innovation points. Secondly, the technical solution provided by this application "kills two birds with one stone" and provides a new method for optimizing the therapeutic effect of CAR-T cells. Achieving a cure for MM remains a huge challenge. From the current clinical trial results worldwide, most RRMM patients will still relapse and progress within 2-3 years after CAR-T treatment. In addition to improving the structure of CAR-T cells themselves, the "CAR-T + X regimen" is also a worthy exploration path. This technical solution first proposes a combined regimen targeting the ANXA1 protein, which not only focuses on attacking BCMA-negative MM cells but also analyzes whether downregulating ANXA1 can relieve a negative factor in the myeloma microenvironment and provide better conditions for the long-term survival of CAR-T cells. This innovative concept of "killing two birds with one stone" will enrich the means of CAR-T cell therapy. Brief Description of the Drawings

[0026] The present invention will be further described in detail below in conjunction with the drawings of the specification.

[0027] Figure 1 The antagonist WRW4 that inhibits ANXA1 signal transduction can reduce this inhibitory effect on CAR-T activity;

[0028] Figure 2 The ANXA1 antagonist WRW4 interferes with the decrease in PD-1 levels caused by rhANXA1 treatment;

[0029] Figure 3 Western blot analysis confirmed that as ANXA1 activity was blocked by the ANXA1 antagonist WRW4, CAR-T activity was significantly enhanced;

[0030] Figure 4When the ANXA1 antagonist WRW4 inhibits ANXA1 signal transduction, the levels of the oxidative stress markers CM-H2DCFDA and MitoSOX both decrease;

[0031] Figure 5 For the in vitro treatment of BCMA-negative myeloma using a single-drug strategy, after treatment with the ANXA1 antagonist WRW4, the proliferative ability of 8226 lacking BCMA is significantly weakened;

[0032] Figure 6 When WRW4 is used in combination with BCMA-targeted CAR-T cells, the CAR-T cells can effectively kill BCMA-positive myeloma, while in BCMA-knockout tumors, the effectiveness of CAR-T is weakened;

[0033] Figure 7 For the changes in tumor volume over time after drug treatment in different groups in a myeloma mouse model, after treatment with WRW4, its growth ability is significantly lower than that of myeloma not treated with WRW4;

[0034] Figure 8 When WRW4 is introduced under CAR-T treatment conditions, the proliferative ability of BCMA-deficient myeloma is significantly reduced;

[0035] Figure 9 Tumor biopsy immunofluorescence detection confirms that there is CAR-T cell infiltration in the vector group, while there is no infiltration in the BCMA-negative myeloma group;

[0036] Figure 10 This application provides a schematic diagram of the use of the ANXA1 antagonist WRW4 for the treatment of BCMA-negative recurrence after BCMA immunotherapy for multiple myeloma. Detailed implementation mode

[0037] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0038] There are no special restrictions on the sources of all raw materials of the present invention, and those purchased on the market or prepared according to the conventional methods well-known to those skilled in the art are all acceptable. The techniques involved in the present invention, such as cell culture, Western blot analysis, flow cytometry, tumor biopsy immunofluorescence detection, etc., are all conventional operations well-known to those skilled in the art, and the specific methods are not elaborated in detail in this application.

[0039] WRW4 is a specific formyl peptide receptor-like 1 (FPRL1) antagonist, purchased from SELLECK Biotechnology Co., Ltd. in the United States.

[0040] The MM cell line 8226 was purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0041] Example 1

[0042] ANXA1 is considered to have an immunosuppressive effect on inflammation. In this example, it is hypothesized that high ANXA1 expression will have a negative impact on lymphocyte function. Therefore, anti-BCMACAR-T cells, the antagonist WRW4, and MM cells 8226 (including the Vector group without BCMA knockout and the sg-BCMA-6 group with BCMA knockout) (sg-BCMA-6: the targeting sequence used to knockout BCMA in MM cells 8226. Among them, the forward sequence of sg-BCMA-6 is TNFRSF17(BCMA)-gRNA6-F-5’-GAGAGGCCTCGAGTACACGG-3’, and the reverse sequence of sg-BCMA-6 is TNFRSF17(BCMA)-gRNA6-R-5’-CCGTGTACTCGAGGCCTC TC-3’) were co-cultured. The total volume of each group was 1 mL. The specific experimental group variable settings are as follows:

[0043] Control group: 2×10 5 cells / mL MM cells 8226 + 1 μM DMSO (Vector group);

[0044] Experimental groups:

[0045] (1) 1×10 5 cells / mL MM cells 8226 + 1×10 5 cells / mL targeted BCMACAR-T cells (Vector group);

[0046] (2) 1×10 5 cells / mL MM cells 8226 sg-BCMA-6 + 1×10 5 cells / mL targeted BCMA CAR-T cells (BCMA knockout group);

[0047] (3) 1×10 5 cells / mL MM cells 8226 sg-BCMA-6 + 1×10 5 cells / mL targeted BCMA CAR-T cells + 1 μM antagonist WRW4 (BCMA knockout + drug treatment group);

[0048] Flow cytometry analysis was performed on several experimental groups, and the experimental results are as Figure 1As shown, compared with the control group, the mean fluorescence intensity (MFI) of the experimental group with targeted BCMA CAR-T cells increased significantly (compare column 2 with column 1); this example also verified that when CAR-T and 8226 cells were incubated, in the case of downregulation of tumor BCMA, the activity of CD69 + (a cell activation index) activated CAR-T cells was weakened (compare column 3 with column 2); in addition, by adding the antagonist WRW4 that inhibits ANXA1 signal transduction, this inhibitory effect on CAR-T can be alleviated (compare column 4 with column 3).

[0049] Example 2

[0050] In this example, recombinant human ANXA1 (rhANXA1, used to overexpress ANXA1 and upregulate ANXA1) was further used in the co-culture of CAR-T and 8226 cells. The total volume of each group was 1 mL. The specific experimental group variable settings were as follows:

[0051] Control group: 1×10 5 cells / mL MM cell 8226 + 1×10 5 cells / mL targeted BCMA CAR-T cells;

[0052] Experimental group:

[0053] (1) 1×10 5 cells / mL MM cell 8226 + 1×10 5 cells / mL targeted BCMA CAR-T cells + 100 ng / mL rhANXA1;

[0054] (2) 1×10 5 cells / mL MM cell 8226 + 1×10 5 cells / mL targeted BCMA CAR-T cells + 100 ng / mL rhANXA1 + 1 μM antagonist WRW4;

[0055] Flow cytometry analysis was performed on several experimental groups. The results were as Figure 2 shown. Under rhANXA1 treatment, the level of ANXA1 was upregulated, and the expression of PD-1 (programmed death receptor 1 CD279, an important immunosuppressive molecule) on CAR-T cells increased significantly ( Figure 2 ); however, when WRW4 interfered with rhANXA1, the level of ANXA1 was downregulated (WRW4 is an antagonist of ANXA1 and can competitively bind to the receptor FPR2 of ANXA1, so it can interfere with both endogenous ANXA1 and exogenous rANXA1), and then a decrease in the level of PD-1 was observed ( Figure 2 ).

[0056] Then, Western blot analysis was performed on the CAR-T cells sorted from the co-culture systems of the above several experimental groups. Calnexin protein was used as an internal reference in the experiment to represent the cell loading level. The results are as Figure 3 shown, which confirmed that: with the blockade of ANXA1 activity, the level of PD-1 decreased, and it also demonstrated a similar change in the level of the TIGIT protein (the full name is T cell immune receptor with Ig and ITIM domains, also known as WUCAM, Vstm3, VSIG9, which belongs to cell surface proteins with immunosuppressive functions) and PD-1.

[0057] Meanwhile, in this example, the above several experimental groups were purified by flow cytometry for quantitative analysis of the mitochondrial oxidative stress marker CM-H2DCFDA (a chloromethyl derivative of H2DCFDA, which enters cells by passive diffusion and is mainly used to detect reactive oxygen species ROS in cells). The results are as Figure 4 shown, indicating that: after treatment with rhANXA1, the level of CM-H2DCFDA in CAR-T cells increased significantly; importantly, when the ANXA1 antagonist WRW4 inhibited ANXA1 signaling, both the levels of CM-H2DCFDA and MitoSOX (referring to the level of superoxide in intracellular mitochondria, MitoSOX is a cationic fluorescent probe that can penetrate the cell membrane and enter mitochondria, and produces strong red fluorescence after reacting with superoxide ions, thus can be measured by methods such as fluorescence microscopy, flow cytometry or spectrophotometer; it is a marker of the level of cellular oxidative stress, and the higher it is, the worse the CAR-T activity) decreased.

[0058] Example 3 In vitro treatment of BCMA-negative myeloma experiment

[0059] The above data indicate that in BCMA-negative defective myeloma, the high expression of ANXA1 may lead to CAR-T dysfunction and weaken the tumor killing ability of CAR-T against BCMA-positive MM. The ANXA1 antagonist can inhibit the proliferation of BCMA-negative myeloma and restore the cytotoxicity of CAR-T cells against BCMA-positive myeloma.

[0060] In view of the above results, in this example, a single-drug strategy and a combination regimen were used to treat BCMA-negative myeloma in vivo and in vitro. The total volume of each group was 1 mL. The specific experimental group variable settings are as follows:

[0061] Control group (Control): 1×10 5 cells / mL MM cells 8226 + 1 μM DMSO (Vector group);

[0062] Experimental group:

[0063] (1) 1×10 5 cells / mL MM cell line 8226 + 1×10 5 cells / mL BCMA-targeted CAR-T cells (Vector group);

[0064] (2) 1×10 5 cells / mL MM cell line 8226 sg-BCMA-6 + 1×10 5 cells / mL BCMA-targeted CAR-T cells (BCMA knockout group);

[0065] (3) 1×10 5 cells / mL MM cell line 8226 sg-BCMA-6 + 1×10 5 cells / mL BCMA-targeted CAR-T cells + 1 μM antagonist WRW4 (BCMA knockout + drug treatment group);

[0066] After Annexin V treatment of the above experimental groups, flow cytometry was used to detect the fluorescence intensity, cell proliferation and apoptosis as Figures 5 - 6 shown: The results showed that compared with the vector group, after treatment with WRW4, the proliferation ability of 8226 cells lacking BCMA was significantly weakened ( Figure 5 ). When WRW4 was used in combination with BCMA-targeted CAR-T cells, flow cytometry detected apoptosis of 8226 cells (BCMA + Annexin V + ). As expected, CAR-T cells could effectively kill BCMA-positive myeloma (columns 1, 2), while in BCMA-knockout tumors, the effectiveness of CAR-T was weakened (columns 3, 4 compared with columns 1, 2)( Figure 6 ). However, after combined treatment with CAR-T and WRW4, this inhibited activity was significantly restored ( Figure 6 ).

[0067] Example 4 In Vivo Treatment of BCMA-Negative Myeloma in a Myeloma Mouse Model

[0068] Construction of a myeloma mouse model:

[0069] On day -10 (10 days before the start of the experiment), each mouse (NSG mouse, purchased from Shanghai Model Organisms Center, Inc.) was injected with 6×10 6Myeloma cells were cultured for 10 days, and then the mice were divided into two groups: (1) One group started from day 0 and was injected with the WRW4 inhibitor (2.5 mg / kg) or an equal volume of PBS buffer twice a week, and the tumor volume was measured once a week; (2) Another group, on the basis of the operation in group (1), was also intravenously injected with 2×10 6 CAR-T cells (experimental group) or UTD (non-CAR-T cell control group) for treatment.

[0070] The tumor volume was plotted as a curve, as shown in Figure 7 and Figure 8 shown:

[0071] In the myeloma mouse model, the tumor volume of BCMA knockout increased rapidly, while after treatment with WRW4 alone, its growth ability was significantly lower than that of myeloma without WRW4 treatment ( Figure 7 ).

[0072] Subsequently, the effect of the WRW4+CAR-T combination regimen on the inhibition of BCMA-negative myeloma was evaluated in in vivo experiments ( Figure 8 ). The results showed that although treated with CAR-T, the myeloma lacking BCMA still grew rapidly, which was contrary to the vector group effectively eradicated by CAR-T cells ( Figure 8 ). Notably, after introducing WRW4 under the condition of CAR-T treatment, the proliferation ability of BCMA-negative myeloma was significantly reduced ( Figure 8 ). Immunofluorescence detection of tumor biopsy confirmed that there was CAR-T cell infiltration in the vector group, while there was no infiltration in the BCMA-negative myeloma group ( Figure 9 ).

[0073] In summary, as shown in Figure 10 , the examples of this application provide evidence that the lack of BCMA antigen and impaired CAR-T cells in MM jointly lead to the recurrence of myeloma in anti-BCMA CAR-T therapy. Combining with the mouse experiment, within the first month after the infusion of BCMA-targeted CAR-T cells, the ANXA1 antagonist was infused twice a week by intravenous injection. At the same time, for efficacy evaluation, in the examples, first, the myeloma disease was evaluated, referring to the IMWG standard, and then one month after CAR-T treatment, the residual BCMA-negative cells were detected by high-throughput transcriptome sequencing. The ANXA1 antagonist WRW4 can effectively inhibit the proliferation of BCMA-negative myeloma that escapes CAR-T surveillance.

[0074] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. Obviously, those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. Application of ANXA1 protein in the preparation of drugs for treating multiple myeloma.

2. Application of ANXA1 protein antagonists in the preparation of drugs for treating multiple myeloma.

3. Use of the ANXA1 protein according to claim 1 in preparing a drug for treating multiple myeloma or use of the ANXA1 protein antagonist according to claim 2 in preparing a drug for treating multiple myeloma, characterized in that: The multiple myeloma refers to: relapsed refractory multiple myeloma with BCMA-negative relapse after BCMA immunotherapy.

4. The use according to claim 3, characterized in that: The BCMA immunotherapy refers to cell therapy using targeted BCMA CAR-T cells.

5. The use according to claim 3, characterized in that: The medicine for treating multiple myeloma comprises active ingredients and excipients, wherein the active ingredient is an ANXA1 antagonist.

6. The use according to claim 5, characterized in that: The ANXA1 antagonist is WRW4.

7. The use according to claim 5, characterized in that: The auxiliary material is a pharmaceutically acceptable carrier selected from at least any one of a filler, a diluent, a lubricant, a binder or a disintegrant.

8. The use according to any one of claims 5 to 7, characterized in that: The drug is any one of an oral preparation, an injection preparation, a topical preparation for skin application or an inhalation preparation for the respiratory system.

9. A combined therapeutic drug for multiple myeloma, characterized in that: The combined therapeutic drug includes an ANXA1 protein antagonist and targets BCMA CAR-T cells.

10. A combined therapeutic drug for multiple myeloma according to claim 9, characterized in that: The ANXA1 protein antagonist has a synergistic effect with targeted BCMACAR-T cells, and the ANXA1 protein antagonist is used to enhance the therapeutic effect of targeted BCMACAR-T cells.

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