Sodium alginate hydrogel for expressing catalase chimeric antigen receptor NK cells and application of sodium alginate hydrogel

Abstract

The invention discloses sodium alginate hydrogel of a chimeric antigen receptor NK cell for expressing catalase and application of the sodium alginate hydrogel. The hydrogel is formed by mixing endogenous calcium ion reactive hydrogel composed of HER1-CAR-CAT-NK and sodium alginate. After the hydrogel disclosed by the invention is injected into an operation excision part, HER1-CAR-CAT-NK cells are decomposed into oxygen by catalyzing main active oxygen (hydrogen peroxide) in a tumor, so that the tolerance of the cells to high-level oxidative stress and hypoxia in the tumor is improved, and the hydrogel shows obviously enhanced durability and effect functions in triple negative breast cancer, and therefore, the hydrogel can be used for preparing a medicine for treating the triple negative breast cancer. The local cell therapy not only can inhibit the growth of local primary residual tumors, but also can stimulate the whole-body anti-tumor activity and inhibit the growth of distant tumors. According to the present invention, the tumor oxidative stress and the anoxic microenvironment are remodeled through the ALG of HER1-CAR-CAT-NK so as to inhibit the postoperative recurrence of TNBC;

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of research on catalase, sodium alginate hydrogel, CAR-NK cell inhibiting postoperative recurrence of triple-negative breast cancer, and specifically relates to a sodium alginate hydrogel of chimeric antigen receptor NK cell expressing catalase and application thereof. BACKGROUND

[0002] Breast cancer is a common cancer with high morbidity and mortality in women, and triple-negative breast cancer (TNBC) is the most aggressive subtype. In clinical practice, surgical resection is still the preferred treatment strategy for TNBC patients, and is reasonably combined with different adjuvant therapies (such as chemotherapy, radiotherapy and targeted therapy) to achieve better therapeutic effect. However, due to the limited efficacy and obvious side effects of these adjuvant therapies, TNBC patients still have serious metastasis in brain, lung, bone and other parts within five years after the first round of treatment, and the overall recurrence rate is very high. Therefore, it is now an urgent task to develop alternative strategies to improve the safety and efficiency of TNBC treatment.

[0003] Chimeric antigen receptor NK (CAR-NK) cell therapy can simultaneously rely on the inherent tumor cell recognition ability of NK cells and engineered CARs to distinguish malignant cells from healthy cells. Compared with CAR-T cell therapy, the safety of CAR-NK cell therapy is significantly improved, and therefore it is considered to be one of the most promising immunotherapies for treating TNBC and other cancers. However, similar to other types of cell therapy, the current CAR-NK cell therapy also has limited effect on the treatment of solid tumors, because the abnormal tumor microenvironment (TME) of solid tumors causes a series of biological obstacles, limiting the intratumoral infiltration and survival of CAR-NK cells. Hydrogen peroxide (H2O2) is a major reactive oxygen species in most solid tumors, which induces pro-cancerous or anti-cancerous processes by participating in the regulation of apoptosis, cell cycle progression and proliferation of cancer cells, and the formation of tumor immune suppression. The scientific community has been exploring how to convert hydroxyl radicals from hydroxyl radicals through Fenton reaction to induce direct cancer cell death; or decompose hydroxyl radicals into oxygen through catalase or similar nanocatalysts to enhance the effect of other oxygen-consuming cancer treatments. To this end, the tumor recognition ability and effector function of CAR-NK cells are improved to enhance their efficacy on glioblastoma and other solid tumors. However, although TMEs can severely weaken the tumor eradication ability of CAR-NK cells through different mechanisms, few studies have reported that CAR-NK cell therapy is enhanced by directly reprogramming resistance to TMEs (such as hypoxia, oxidative stress, acidity). SUMMARY

[0004] Invention purposes: In view of the problems existing in the prior art, the present application provides a sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells for inhibiting the recurrence of triple-negative breast cancer after surgery, which realizes the preparation of catalase-expressing chimeric antigen receptor NK cells and sodium alginate hydrogel for the first time. The catalase can promote the decomposition of hydrogen peroxide into oxygen, improve the tolerance of chimeric antigen receptor NK cells to high-level oxidative stress and hypoxia in tumors, enhance the function of chimeric antigen receptor NK cells, and thus improve the application of chimeric antigen receptor NK cells in inhibiting the recurrence and metastasis of triple-negative breast cancer after surgery.

[0005] The present application also provides the application and composition of the sodium alginate hydrogel of the catalase-expressing chimeric antigen receptor NK cells in inhibiting the recurrence of triple-negative breast cancer after surgery.

[0006] Technical scheme: In order to achieve the above-mentioned purposes, the present application provides a sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells, which is characterized by being composed of catalase-expressing chimeric antigen receptor NK cells and sodium alginate.

[0007] The sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells is mixed with catalase-expressing chimeric antigen receptor NK cells, sodium alginate and calcium chloride, and then incubated at room temperature until the gel is formed.

[0008] The chimeric antigen receptor NK cells (CAR-NK) include catalase-expressing chimeric antigen receptor NK cells (HER1-CAR-CAT-NK).

[0009] The sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells of the present application is used to improve the tolerance of CAR-NK cells to high-level oxidative stress and hypoxia in tumors.

[0010] The sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells of the present application is used to improve the tumor persistence and effector function of CAR-NK cells.

[0011] The sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells promotes the decomposition of hydrogen peroxide into oxygen in tumors, improves the tolerance of CAR-NK cells to high-level oxidative stress and hypoxia in tumors, and thus improves the tumor persistence and effector function of CAR-NK cells.

[0012] The sodium alginate hydrogel of catalase-expressing chimeric antigen receptor NK cells of the present application is used to prepare a drug or reagent for inhibiting the recurrence of triple-negative breast cancer after surgery.

[0013] The application of the sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase in inhibiting the recurrence and metastasis of triple-negative breast cancer after surgery in the preparation of a drug or reagent for inhibiting the recurrence of triple-negative breast cancer after surgery.

[0014] The pharmaceutical composition for inhibiting the recurrence of triple-negative breast cancer after surgery of the application comprises the sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase as an active ingredient and a pharmaceutically acceptable adjuvant.

[0015] The pharmaceutical composition is an injection.

[0016] The application of the sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase in inhibiting the recurrence of triple-negative breast cancer after surgery in the preparation of a drug or reagent for inhibiting the recurrence of triple-negative breast cancer after surgery in remodeling the high level oxidative stress and hypoxic microenvironment of tumors to enhance the chimeric antigen receptor NK cell in the postoperative treatment of triple-negative breast cancer.

[0017] The application proves that the HER1-CAR-CAT-NK cell expressing CAT has significantly improved tolerance to high-concentration H2O2 and hypoxia, and can continuously alleviate the hypoxia of tumors after being locally fixed by the injectable alginate hydrogel. Therefore, this local HER1-CAR-CAT-NK cell therapy can effectively inhibit the growth of postoperative TNBC by promoting the continuous existence and effector function of the cells in the residual tumor mass. This local HER1-CAR-CAT-NK cell therapy can gradually migrate to distant metastatic tumors, and thus can also inhibit the growth of distant tumors in a patient xenograft (PDX) mouse model. This study proves that simultaneously reprogramming the tumor microenvironment rich in H2O2 and hypoxia is a simple and effective method for promoting CAR-NK cell therapy to target TNBC tumors, and is expected to realize clinical transformation in the future.

[0018] The application proposes to genetically engineer the HER1-CAR-NK cell to express catalase, and to gradually release the HER1-CAR-CAT-NK cell by wrapping it with a sodium alginate hydrogel. After being fixed in the residual tumor after surgical resection, catalase-mediated decomposition of endogenous H2O2 and alleviation of tumor hypoxia can enhance the therapeutic efficacy of the cell on solid tumors. This further means that CAR-NK cells and other living cell therapies can be genetically engineered to express tumor microenvironment regulating molecules or other functional molecules, thereby enhancing the therapeutic efficacy on solid tumors. In addition, this local administration of CAR-NK cells is expected to effectively inhibit the recurrence of local and distant metastatic tumors, while reducing the safety risk and cost, and is expected to realize clinical transformation in the future.

[0019] The present application first proposes a simple and effective method, that is, inhibiting the postoperative recurrence of triple-negative breast cancer by sodium alginate hydrogel of chimeric antigen receptor NK cells expressing catalase.

[0020] Surgical resection is the main treatment for triple-negative breast cancer (TNBC) patients, but the postoperative local recurrence and metastatic recurrence rates are high. Therefore, it is of great significance to develop effective adjuvant strategies to inhibit the high recurrence rate of postoperative tumors in TNBC patients. Chimeric antigen receptor engineered natural killer (CAR-NK) cell therapy is a very promising strategy, which can specifically recognize and eliminate TNBC tumor cells, and is safer. Therefore, it is very urgent and important to find an effective strategy to enhance the immunotherapy of CAR-NK cells on TNBC tumors. Hydrogen peroxide (H2O2) is a major reactive oxygen species in most solid tumors, which can trigger pro-cancer or anti-cancer processes and form tumor immunosuppression. In order to eliminate the negative effects of H2O2 and hypoxia in the tumor microenvironment on CAR-NK cell survival and effector function, HER1-CAR-NK cells were genetically engineered with catalase (CAT), which can decompose endogenous H2O2 into oxygen, thereby reducing tumor hypoxia. Therefore, the tolerance of HER1-CAR-CAT-NK cells expressing CAT to high concentrations of H2O2 and hypoxia is significantly improved. In the current clinic, CAR-NK cells usually need to be injected intravenously multiple times to achieve effective treatment of solid tumors, because the tumor accumulation effect of CAR-NK cells is limited, and even if it is safer than other treatment methods, it will also bring unnecessary side effects to patients. Therefore, the present application uses an injectable alginate hydrogel to encapsulate HER1-CAR-CAT-NK cells and locally inject them into the resection cavity. The HER1-CAR-CAT-NK cells gradually released from this in situ formed ALG depot can enter the adjacent tumor mass, thereby effectively eliminating H2O2 and reducing hypoxia, and promoting the persistence and effector function of HER1-CAR-CAT-NK cells in the residual tumor mass, improving the inhibition of postoperative residual TNBC tumors. In addition, they can also gradually migrate to distant metastatic tumors by entering the blood circulation, thereby inhibiting the growth of distant tumors in patient xenograft (PDX) mouse models.

[0021] The tumor microenvironment is harsh, including abnormal blood vessels, acidity, hypoxia, ROS, immunosuppression, etc. At present, many drugs, enzymes, etc. can improve the tumor microenvironment, but if the immune cells overexpress, there may be problems: 1. The survival rate of immune cells decreases. 2. The original function of immune cells decreases. 3. Or these substances cannot express the original function of improving the tumor microenvironment in immune cells. The present application firstly assembles CAT in CAR-NK cells, and the above problems do not exist. Meanwhile, NK cells are different from T cells in that NK cells are more sensitive to H2O2, so CAR-NK cells loaded with CAT are more meaningful than CAR-T cells loaded with CAT in resisting the hydrogen peroxide microenvironment of tumors. In fact, if CAR-T cells are directly loaded with CAT, it cannot further enhance the high level of oxidative stress and hypoxia in tumors, enhance the function of chimeric antigen receptor cells, and cannot further enhance the application of chimeric antigen receptor cells to the inhibition of postoperative triple-negative breast cancer recurrence and metastasis, and its effect is obviously not as good as CAR-NK cells loaded with CAT.

[0022] The CAT element in the HER1-CAR-CAT-NK cell constructed in the present application can assist in decomposing hydrogen peroxide in the tumor to generate oxygen, improve the tolerance of HER1-CAR-CAT-NK cells to high ROS and hypoxic environment of the tumor, and further improve the inhibition ability of the tumor. The CAT element in the CAR-NK cell in the present application can not change the original function of the CAR-NK, and the CAT element can also decompose hydrogen peroxide into oxygen, further improving the killing ability of the CAR-NK to the solid tumor. The present application further prepares sodium alginate hydrogel to wrap the HER1-CAR-CAT-NK cells, and the long-acting sustained-release HER1-CAR-CAT-NK cells can work for a long time after a single injection.

[0023] The HER1-CAR-CAT-NK cells in the present application have a significantly higher tolerance to hydrogen peroxide and hypoxia than the HER1-CAR-NK cells, and the inhibition ability of the HER1-CAR-CAT-NK cells to the solid tumor is also significantly higher than that of the HER1-CAR-NK cells. Based on the improvement of the HER1-CAR-CAT-NK by the CAT, the ALG synergizes to further improve the CAR-NK to improve the tumor treatment effect. The ALG provides support for the HER1-CAR-CAT-NK structure in the tumor, provides growth support for the HER1-CAR-CAT-NK cells, so that the HER1-CAR-CAT-NK cells can proliferate around the tumor, and the long-acting sustained-release makes the cells injected once grow into the tumor for a long time to inhibit the tumor function.

[0024] Advantages: Compared with the prior art, the present application has the following advantages:

[0025] The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase prepared by the application first found that the CAT in the HER1-CAR-CAT-NK cell can decompose H2O2 in the tumor into oxygen, thereby continuously relieving the high H2O2 and hypoxia phenomenon of the tumor.

[0026] The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase prepared by the application can release the HER1-CAR-CAT-NK cell from the in-situ formed ALG depot, which can enter the adjacent tumor mass, thereby effectively eliminating H2O2 and relieving hypoxia, and promoting the continuous existence and effector function of the HER1-CAR-CAT-NK cell in the residual tumor mass, improving the inhibition of postoperative residual TNBC tumor; in addition, they can also gradually migrate to distant metastatic tumors by entering the blood circulation, thereby inhibiting the growth of distant tumors in a patient-derived xenograft (PDX) mouse model; therefore, the sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase prepared by the application is applied in the drug for inhibiting the recurrence of triple-negative breast cancer after operation.

[0027] The sodium alginate hydrogel prepared by the application, in which the sodium alginate is combined with the chimeric antigen receptor NK cell expressing catalase, is applied in the drug for inhibiting the recurrence of triple-negative breast cancer after operation, and has a remarkable synergistic effect. The ALG hydrogel in the application is non-toxic and harmless to the HER1-CAR-CAT-NK cell, does not affect the growth of the HER1-CAR-CAT-NK cell therein, and has good permeability to absorb nutrients, support and nutrients for the HER1-CAR-CAT-NK cell; at the same time, the ALG hydrogel can maintain the survival of the HER1-CAR-CAT-NK cell in the tumor for a long time and release the HER1-CAR-CAT-NK cell. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1Figure 1. Characterization of HER1-CAR-CAT-NK cells; (A) Schematic diagram of the structure of con-CAR, HER1-CAR and CAT. (B-F) The results of flow cytometry (B-C), real-time quantitative PCR (D-E) and Western blotting (F) for detecting the expression levels of CD3ζ and CAT in NK cells, con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells, as indicated by color code. (G) Photos of H2O2 solution producing oxygen bubbles in culture with different NK cells, as indicated by color code. (H-I) Relative cell viability (H) and percentage of viable cells (I) of different NK cells, as indicated by color code. (J) Percentage of viable cells of different NK cells cultured with different concentrations of H2O2 for 24 hours. (K) Concentration-dependent H2O2 production mediated by glucose oxidase. (L) Percentage of viable cells of viable NK cells cultured with glucose oxidase for 24 hours. (M) Relative cell viability of con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells cultured under normoxia, hypoxia and hypoxia plus H2O2 conditions for 24 hours.

[0029] Figure 2 Figure 2. Resistance of HER1-CAR-CAR-NK cells to H2O2-rich and hypoxia conditions; (A-B) Levels of granzyme B (A) and perforin (B) secreted by different CAR-NK cells in co-culture with parental and brain metastatic MDA-MB-231 cells. (C-D) Cytolysis ability of different CAR-NK cells against parental MDA-MB-231 cells (C) and brain metastatic MDA-MB-231 cells (D) under different culture conditions, as indicated by color code. (E) Schematic diagram of Transwell migration assay. (F-G) Optical microscope observation (F) and corresponding semi-quantitative analysis (G): migration rate of parental and brain metastatic MDA-MB-231 cells after co-culture with different CAR-NK cells under different conditions, as indicated by color code. (H) Schematic diagram of Transwell invasion assay. (I-J) Optical microscope observation (I) and corresponding semi-quantitative analysis (J): invasion rate of parental and brain metastatic MDA-MB-231 cells after co-culture with different CAR-NK cells under different conditions.

[0030] Figure 3Safe local delivery of HER1-CAR-CAT-NK cells by ALG hydrogel; (A) Photographs of gelation behavior of ALG solutions at different concentrations in the presence of CaCl2. (B) Rheological properties of gels formed by ALG solutions at different concentrations in CaCl2solution. (C) Coomassie blue staining of ALG hydrogel lysates cultured in standard medium and PBS for 1 h. (D-E) Optical microscope images (D) and relative cell viability (E) of con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells cultured for 7 days with ALG hydrogel. (F) Number of different CAR-NK cells released from ALG hydrogel during culture. (G-I) Secretion levels of granzyme B (G) and perforin (H) and specific cytolytic capacity (I) of CAR-NK cells with and without ALG encapsulation co-cultured with parental and brain metastatic MDA-MB-231 cells, as indicated by color code. (J-L) Secretion levels of granzyme B (J) and perforin (K) and specific cytolytic capacity (L) of HER1-CAR-CAT-NK cells co-cultured with parental and brain metastatic MDA-MB-231 cells under H2O2condition and hypoxia condition.

[0031] Figure 4 ALG hydrogel potentiates tumor infiltration and enhanced effector functions of HER1-CAR-CAT-NK cells; (A) Experimental procedure. (B-F) Infiltration frequency of RFP-expressing CAR-NK cells in parental and brain metastatic MDA-MB-231 tumors was analyzed by confocal microscopy (B), flow cytometry (C-D) and real-time quantitative PCR (E-F) at day 17 and day 21, as indicated by color code. (G) Representative pimonidazole fluorescence imaging of tumor sections collected from parental and brain metastatic MDA-MB-231 tumor mice by confocal microscopy at day 17 and day 21, as indicated by color code. (H-K) Secretion levels of granzyme B (J-K) and perforin (L-M) in parental and brain metastatic MDA-MB-231 tumor mice intratumorally after treatment at day 17 and day 21, as indicated by color code.

[0032] Figure 5ALG assisted HER1-CAR-CAT-NK cell treatment can inhibit the postoperative recurrence of TNBC; (A) Experimental scheme of parental MDA-MB-231 tumor mice. (B-D) Average tumor growth curve (B), survival curve (C) and body weight (D) of parental MDA-MB-231 tumor mice under different treatment methods, as indicated by color code. (E) Experimental scheme of brain metastasis MDA-MB-231 tumor mice. (F-I) Average tumor growth curve (F), survival curve (G), body weight (H) and brain section of brain metastasis MDA-MB-231 tumor mice stained by H&E (I).

[0033] Figure 6 ALG assisted HER1-CAR-CAT-NK cell treatment can inhibit the distant metastasis of TNBC PDX tumor; (A) Experimental scheme. (B-F) Average tumor growth curve (B-C) and tumor weight (D-E) of primary treatment and distant untreated tumor, and body weight (F) of TNBC PDX tumor mice under different treatment methods. (G) Proportion of RFP positive CAR-NK cells in the blood of TNBC PDX tumor mice under different treatment methods (indicated by color bar), and analyzed by real-time quantitative PCR on day 21. (H) Immunofluorescence imaging of RFP positive CAR-NK cells in distant untreated TNBC PDX tumor sections of mice under different treatment methods (indicated by color bar), and analyzed on day 21. (I-J) Intratumoral secretion levels of granzyme B (I) and perforin (J) in distant TNBC PDX tumors of mice under different treatment, indicated by color code, and analyzed on day 21. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with the drawings and examples.

[0035] The materials, reagents and the like used in the examples can be obtained from commercial channels unless otherwise specified.

[0036] The biological materials, reagents and kits used in the application can be obtained by conventional commercial purchase, and the experimental techniques used are conventional operations in the art or are operated according to the instructions of the corresponding goods.

[0037] The biological material sources or models used in the application are as follows:

[0038] TNBC cell MDA-MB-231( HTB-26 TM );

[0039] 293T / 17 cells( CRL-11268 TM );

[0040] PBMCs cells were purchased from Meixun (Shanghai) Biotechnology Co., Ltd.

[0041] The sources of biological and chemical reagents used in the present application are shown in Table 1:

[0042] Table 1 Sources of biological and chemical reagents

[0043]

[0044]

[0045] Example 1

[0046] Cell experiment:

[0047] MDA-MB-468 cells, 293T / 17 cells, MDA-MB-231 were purchased from American Type Culture Collection (ATCC). The method for constructing brain metastatic MDA-MB-231 cells was according to the literature: Weiguang Liu, Proc Natl Acad Sci US A. 2022 May 31; 119(22): e2200230119. All cell lines were cultured in DMEM medium containing 10% FBS, and maintained at 37℃ in a humidified incubator containing 5% CO2.

[0048] NK cells were obtained from peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation. Then, the NK cells were cultured in NK cell culture medium supplemented with 10% human serum for two weeks.

[0049] HER1-CAR-CAT-NK cells were constructed by transducing primary NK cells with lentiviral vectors containing HER1-specific third-generation CAR and Catalase elements. HER1-CAR-NK cells and con-CAR-NK cells were constructed by transducing primary NK cells with lentiviral vectors containing HER1-specific third-generation CARs and non-targeted third-generation CARs, respectively, and the expression of CD3ζ and CAT was detected by Western blotting, real-time PCR and flow cytometry to confirm whether con-CAR-NK, HER1-CAR-NK, HER1-CAR-CAT-NK were successfully constructed. HER1-CAR-NK cells were constructed by transfecting NK cells obtained from human peripheral blood mononuclear cells (PBMCs) with third-generation CAR expression vectors fused with HER1-specific single-chain variable sequence Figure 1A). HER1-CAR-CAT-NK cell construction is to transfect the isolated NK cells with the above HER1-CAR expression vector and catalase (CAT) expression vector at the same time. At the same time, the non-specific CAR-NK cell (con-CAR-NK) construction is to transfect the NK cells with the empty third-generation CAR vector.

[0050] The specific construction of con-CAR-NK cells, HER1-CAR-NK cells, and HER1-CAR-CAT-NK cells is as follows: first, the third-generation CAR plasmid containing CD8 signal, CD8 hinge, CD28, 4-1BB and CD3ζ sequences (Yan Liu et al. Cell Prolif. 2020 Aug; 53(8): e12858.) is fused with sequences encoding HER1-specific single-chain fragment variable (scFv) (Yan Liu et al. Small. 2024 Jan 11: e2307521.) to construct a HER1-CAR vector. The non-specific third-generation CAR plasmid is used as con-CAR (con-CAR-NK) (Yan Liu et al. Cell Prolif. 2020 Aug; 53(8): e12858.). Figure 1 A). Subsequently, NK cells obtained from human peripheral blood mononuclear cells (PBMC) are transduced with lentivirus expressing HER1-CAR to generate HER1-CAR-NK cells, and NK cells are transduced with lentivirus containing con-CAR expression plasmid to generate Con-CAR-NK cells. Specifically: CAR (HER1-CAR / con-CAR), pMD2.G, psPAX2 lentivirus plasmid is mixed with lipo2000 at a ratio of 4:1:3 to transfect 293T / 17 cells, and fresh complete medium is replaced 8 hours after transfection. Three days later, the culture solution is collected, filtered with a 0.45 μm filter, the virus is concentrated by ultracentrifugation, and high-titer lentivirus solution is collected. Add the lentivirus to the NK cells to obtain the relevant con-CAR-NK and HER1-CAR-NK cells. HER1-CAR-NK cells are transduced with lentivirus expressing CAT to obtain HER1-CAR-CAT-NK cells. Specifically: CAT (purchased from sinobiological) expressing lentivirus plasmid, pMD2.G, psPAX2 are mixed with lipo2000 at a ratio of 4:1:3 to transfect 293T / 17 cells, and fresh complete medium is replaced 8 hours after transfection. Three days later, the culture solution is collected, filtered with a 0.45 μm filter, the virus is concentrated by ultracentrifugation, and high-titer lentivirus solution is collected. Add the lentivirus to the HER1-CAR-NK cells to obtain the HER1-CAR-CAT-NK cells.

[0051] The expression of CD3ζ and CAT at the transcriptional and protein levels was detected by flow cytometry, real-time quantitative PCR and Western blotting, respectively, to confirm the successful construction of these con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells. Figure 1 B-F).

[0052] To study the CAT enzyme activity of HER1-CAR-CAT-NK cells, 3x10 3 cells (NK cells, con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells) were suspended in PBS containing H2O2 (1 mM) for 30 minutes, and then imaged using a digital camera. The results showed that only HER1-CAR-CAT-NK cells cultured in H2O2 (1 mM) solution rapidly produced gaseous oxygen, which further confirmed that CAT had enzyme activity in HER1-CAR-CAT-NK cells Figure 1 G) MTT method: 5x10 4 cells (NK cells, con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells) were planted in 96-well plates, 3 replicates, 20-30 μL of 5 mg / mL MTT solution was added, and the plates were incubated in a 37°C, 5% CO2 cell incubator. At the end of the incubation, the culture medium was removed by centrifugation, 150 μL of DMSO was added, and the plates were shaken on a shaker at room temperature for 10-30 min. The OD490 absorbance value was detected on a microplate reader to detect cell viability. Iodinated propidium dye staining method: log phase NK cells, con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells were collected, centrifuged at 1000 rpm for 5 min, and the culture medium was discarded. The cells were washed twice with 1x PBS at 1000 rpm for 5 min each time. PI dye 5 μL was added, and the plates were incubated at 4°C in the dark on a shaker for 20 min. The PI fluorescence was analyzed by flow cytometry.

[0053] MTT method and iodinated propidium dye staining showed that the expression of HER1-CAR and CAT did not affect the viability of these NK cells Figure 1 H-I) PI staining detection showed that H2O2 culture (5-15 μmol / L, 24 hours) caused significant death of NK cells, con-CAR-NK cells and HER1-CAR-NK cells, and the mode of death was positively correlated with the increase in H2O2 concentration. In contrast, the interference of H2O2 culture on the viability of HER1-CAR-CAT-NK cells was negligible Figure 1J). Furthermore, since glucose oxidase catalyzes the oxidation of glucose to D-gluconic acid-δ-lactone and H₂O₂, these NK cells also exhibited a similar trend after incubation with glucose oxidase. Figure 1 These results collectively demonstrate that HER1-CAR-CAT-NK cells exhibit exceptional tolerance to physiological concentrations of H2O2 exposure.

[0054] To investigate the tolerance of NK cells and three types of CAR-NK cells to H2O2 or hypoxia, they were seeded in 96-well plates (5 × 10⁶ cells per well). 4 Cells were cultured in aerobic or hypoxic (<2% O2) conditions for 24 hours with H2O2 (5-15 μmol / L) or glucose oxidase (0.1-0.3 mU / mL). Cell viability or live cells were then assessed using the MTT assay or propidium iodide staining method. Results showed that, according to the standard MTT assay, hypoxic culture (<2% O2, 24 hours) significantly reduced the cell viability of con-CAR-NK cells, HER1-CAR-NK cells, and HER1-CAR-CAT-NK cells compared to normoxic culture. Figure 1 Adding H2O2 (5 μM) further reduced the survival rate of con-CAR-NK and HER1-CAR-NK cells under hypoxic culture. However, due to the introduction of catalase, the addition of H2O2 rescued HER1-CAR-CAT-NK cells from hypoxic culture-induced cell death. These results collectively demonstrate that HER1-CAR-CAT-NK cells exhibit exceptional tolerance to both H2O2-rich and hypoxic environments because CAT can decompose H2O2 into oxygen.

[0055] To investigate the effects of H2O2 or hypoxia on CAR-NK cell-specific lysis, cells were pre-seeded in 96-well plates (1 × 10⁶ cells per well). 4MDA-MB-231 cells and brain-transplanted MDA-MB-231 cells were co-cultured with con-CAR-NK cells, HER1-CAR-NK cells, or HER1-CAR-CAT-NK cells at different feeding ratios under normoxic or hypoxic conditions for 24 hours (ELISA ratio 3:1 for all cells; LDH ratios 1:1, 3:1, and 5:1 for all cells). Subsequently, the secretion levels of granzyme B and perforin in the supernatants were measured by ELISA, and the concentration of LDH in the supernatants was measured by the LDH cytotoxicity assay. The ELISA results showed that when HER1-CAR-NK cells or HER1-CAR-CAT-NK cells were co-cultured with parental or brain-transplanted MDA-MB-231 cells, the secreted amounts of granzyme B and perforin were comparable and significantly higher than those when con-CAR-NK cells were co-cultured with the aforementioned target cells. Furthermore, H2O2 and hypoxia significantly inhibited the secretion of granzyme B and perforin by HER1-CAR-NK cells when co-cultured with parental cells and brain-transferred MDA-MB-231 cells, but only slightly affected the secretion of granzyme B and perforin by HER1-CAR-CAT-NK cells. Figure 2 AB). Meanwhile, LDH assays revealed that HER1-CAR-CAT-NK cells exhibited superior cytotoxicity against both parental and brain-transferred MDA-MB-231 cells under normoxic, H2O2-rich, and hypoxic conditions. Figure 2 However, HER1-CAR-NK cells can only effectively eliminate these target cells under normoxic conditions, which means that this HER1-CAR-CAT-NK cell is a promising candidate cell for eliminating hypoxic and H2O2-rich solid tumors.

[0056] To investigate the inhibitory effects of H2O2 or hypoxia on the migration ability of CAR-NK cells to target cancer cells, MDA-MB-231 cells and brain-transferred MDA-MB-231 cells (5 × 10⁶ cells per chamber) were used. 4 Cells were pre-seeded in the upper layer of 8μm pores with different ratios of con-CAR-NK cells, HER1-CAR-NK cells, or HER1-CAR-CAT-NK cells (all 1:1) and co-cultured for 24 hours in the presence or absence of H2O2 (5μM) and under normoxic or hypoxic conditions. Cells adhering to the lower cavities were then fixed with paraformaldehyde (4%), stained with crystal violet (0.1%), and observed under an optical microscope. Figure 2 E). Cell migration assays revealed that HER1-CAR-NK cells or HER1-CAR-CAT-NK cells significantly inhibited the migration of parental or brain-transferred MDA-MB-231 cells from the upper end of the ventricle to its lower end. Figure 2F-G). And under H2O2-rich and hypoxic conditions, the migration ability of HER1-CAR-NK cells was significantly inhibited. In sharp contrast, the ability of HER1-CAR-CAT-NK cells to inhibit the migration of parental and brain metastatic MDA-MB-231 cells was little disturbed by H2O2-rich and hypoxia.

[0057] To investigate the inhibitory effect of H2O2 or hypoxia on the ability of CAR-NK cells to inhibit the invasion of target cancer cells, MDA-MB-231 cells and brain metastatic MDA-MB-231 cells were pre-plated in the upper chamber of a chamber coated with a layer of Matrigel (about 2 mm) (pore size 8 μιη). Then, they were co-cultured with con-CAR-NK cells, HER1-CAR-NK cells or HER1-CAR-CAT-NK cells (1 x 10 5 ) in the presence or absence of H2O2 (5 μΜ) and under normoxic or hypoxic conditions for 24 hours. Then, the MDA-MB-231 cells and brain metastatic MDA-MB-231 cells adhered to the lower chamber were fixed, stained and imaged as described above. It was found by the cell invasion experiment that HER1-CAR-CAT-NK cells could effectively inhibit the invasion of parental and brain metastatic MDA-MB-231 cells under different culture conditions Figure 2 H-J). However, HER1-CAR-NK cells could only effectively inhibit the invasion of these target cells under normoxic conditions. These results collectively indicate that HER1-CAR-CAT-NK cells have a stronger ability to inhibit cell invasion under H2O2-rich and hypoxic culture conditions.

[0058] This example effectively demonstrates that HER1-CAR-CAT-NK cells have a stronger ability to kill tumor cells and inhibit metastasis than HER1-CAR-NK cells under H2O2-rich and hypoxic culture conditions.

[0059] Example 2

[0060] Preparation and characterization of ALG hydrogel

[0061] To investigate the gelation ability of ALG (sodium alginate), ALG (sodium alginate) solutions of different concentrations from 0 mg / mL to 15 mg / mL were treated with a calcium chloride solution containing Ca 2+ ([Ca 2+ ] = 1 mM). Rheological experiments were performed on an Anton Paar rheometer to determine the elastic modulus (G') of the ALG hydrogel formed at 37°C. The experimental results demonstrated that commercial ALG solutions with a concentration higher than 5 mg / mL were treated with a Ca 2+ solution (H2O, [Ca2+ After treatment with 1mM, it will rapidly gel ( ). Figure 3 A). Rheological analysis revealed that, after Ca... 2+ The increase in storage modulus (G') of solution-treated ALG solutions (5, 10, and 15 mg / mL) was time- and concentration-dependent. Figure 3 B) indicates that the ALG solution in Ca 2+ It exhibits rapid gelation properties in the presence of these substances.

[0062] To investigate the adsorption capacity of ALG hydrogel for nutrients, an ALG (sodium alginate) solution (10 mg / mL) was used with a solution containing Ca... 2+ ALG hydrogels were formed by treating the cells with a 1 mM solution, and then immersed in complete cell culture medium containing FBS or PBS for 1 hour. The adsorbed proteins in the ALG hydrogels were then detected by polyacrylamide gel electrophoresis and Coomassie brilliant blue staining. The results showed that the ALG hydrogels immersed in complete cell culture medium for 1 hour could adsorb serum proteins (…). Figure 3 C), which indicates that it can exchange nutrients with the surrounding environment after being loaded with CAR-NK cells.

[0063] To investigate the effect of ALG hydrogel coating on the proliferation behavior of CAR-NK cells, con-CAR-NK cells, HER1-CAR-NK cells, and HER1-CAR-CAT-NK cells (1×10⁻⁶) were used. 5 CAR-NK cells were coated with 50 μL of ALG solution (10 mg / mL) and CaCl2 solution (final concentration 0.5 mM) for 10 min to form hydrogels in 24-well plates. After gelation, excess calcium chloride solution was aspirated, and the cells were washed with PBS. The daily growth of CAR-NK cells was observed and recorded under an optical microscope. Optical microscopy revealed that con-CAR-NK cells, HER1-CAR-NK cells, and HER1-CAR-CAT-NK cells, encapsulated in ALG hydrogel and cultured in conditioned medium, exhibited similar proliferation behavior, characterized by a gradual increase in cell cluster size over 7 days. Figure 3 D) indicates that the hydrogel is harmless to HER1-CAR-CAT-NK cells and can support cell growth.

[0064] To investigate the release of CAR-NK cells from ALG hydrogel, 5 × 10⁻⁶ cells were used. 6CAR-NK cells were mixed with 50 μL of ALG (10 mg / mL) solution in a 12-well plate to form droplets, and then treated with a final concentration of 0.5 mM CaCL2solution for 10 min to solidify the CAR-NK ALG hydrogel. After removing the CaCL2solution, the CAR-NK ALG hydrogel was washed twice with PBS and then cultured with complete medium. The survival rate of CAR-NK cells encapsulated in the ALG hydrogel was determined by CCK-8 assay. The number of cells released from the ALG hydrogel into the culture medium was calculated by cell counting every day. Cell proliferation assay showed that con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells exhibited the same proliferation trend Figure 3 E). In addition, under normal culture conditions, con-CAR-NK cells, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells encapsulated in ALG can gradually release and proliferate Figure 3 F), which shows that, on the basis of the foregoing, the ALG hydrogel is not only non-toxic and harmless to CAR-NK cells, but also can provide good growth support. At the same time, it can gradually release CAR-NK cells as a cell bank, so that they can subsequently act on tumor cells. The HER1-CAR-NK cells or HER1-CAR-CAT-NK cells released from the corresponding ALG hydrogel were collected after 7 days for subsequent experiments. If the ALG hydrogel is not used, only CAR-NK cells are used, and the CAR-NK cells will be used up and there will be no remaining CAR-NK cells. After combining with the ALG hydrogel, the CAR-NK cells can grow in the hydrogel, gradually release, and have a long-term effect after a single injection.

[0065] To investigate the killing effect of CAR-NK cells with or without ALG hydrogel coating on target cells, MDA-MB-231 cells and brain metastatic MDA-MB-231 cells (1 x 10 4 cells per well) were pre-seeded in a 96-well plate and co-cultured with HER1-CAR-NK cells, HER1-CAR-CAT-NK cells, or HER1-CAR-NK cells or HER1-CAR-CAT-NK cells released from the corresponding ALG hydrogel at a ratio of 1:1 effector cells to target cells for 24 hours. The secretion levels of granzyme B and perforin or the LDH concentration in each supernatant were determined by ELISA detection or LDH cytotoxicity detection. ELISA detection found that, under normoxic conditions, HER1-CAR-NK cells and HER1-CAR-CAT-NK cells released from the ALG hydrogel and HER1-CAR-NK cells and HER1-CAR-CAT-NK cells without ALG hydrogel coating secreted granzyme B and perforin at levels comparable to those of the parent or brain metastatic MDA-MB-231 cellsFigure 3 Furthermore, LDH assays revealed that, under normoxic conditions, the presence or absence of ALG coating had minimal impact on the specific cytolytic efficacy of HER1-CAR-NK and HER1-CAR-CAT-NK cells against parental or brain-transferred MDA-MB-231 cells. Figure 3 I).

[0066] To investigate the killing effect of CAR-NK cells coated with or uncoated with ALG hydrogel on target cells under H2O2 or hypoxic conditions, MDA-MB-231 cells and brain-transferred MDA-MB-231 cells (1×10⁶ cells per well) were used. 4 (Number of cells) were pre-seeded in 96-well plates with HER1-CAR-NK cells, HER1-CAR-CAT-NK cells, or HER1-CAR-NK cells released from the corresponding ALG hydrogel, or HER1-CAR-CAT-NK cells at a 1:1 effector-to-target cell ratio, and co-cultured for 24 hours under H2O2 or hypoxic conditions. The secretion levels of granzyme B and perforin, or the LDH concentration in each supernatant, were determined by ELISA or LDH cytotoxicity assay. ELISA showed that this ALG encapsulation negligibly weakened the resistance of HER1-CAR-CAT-NK cells to H2O2 enrichment and hypoxic conditions. This was reflected in the fact that, regardless of whether HER1-CAR-CAT-NK cells were ALG encapsulated, the secretion of granzyme B and perforin, as well as their ability to specifically lyse target cells, remained unaffected under H2O2 enrichment and hypoxic conditions when co-cultured with parental or brain-transferred MDA-MB-231 cells. Figure 4 JL).

[0067] The HER1-CAR-NK cells released from the ALG in this embodiment, the HER1-CAR-CAT-NK cells coated with ALG, and the cells not coated with ALG were compared. The killing ability of the HER1-CAR-CAT-NK cells released from the ALG coating under hydrogen peroxide and hypoxic environment was unchanged, indicating that the CAR-NK cells coated with ALG did not change the killing ability of the tumor, i.e., did not change the hydrogen peroxide and hypoxic tolerance of the HER1-CAR-CAT-NK cells and the killing effect on the tumor under such conditions. At the same time, this embodiment proves that the ALG hydrogel can soak nutrients for the growth of HER1-CAR-CAT-NK cells, and the ALG is non-toxic and harmless to the HER1-CAR-CAT-NK cells, supporting the growth of the HER1-CAR-CAT-NK cells therein. And the HER1-CAR-CAT-NK cells still retain their function on the tumor after being released from the ALG hydrogel. Therefore, the ALG hydrogel of the HER1-CAR-CAT-NK has the potential to serve as a cell bank for CAR-NK.

[0068] In addition, this embodiment constructs a sodium alginate hydrogel expressing catalase chimeric antigen receptor NK cells at the optimal ratio, including the concentration of sodium alginate (the hardness of the gel structure is appropriate), and the concentration of calcium chloride (can quickly promote gelation and does not affect the cells therein).

[0069] Example 3

[0070] In vivo animal experiments:

[0071] 4-week-old female NSG mice were purchased from the Animal Model Research Center of Nanjing University. All animal experiments were approved by the Medical Ethics Committee of Nanjing Normal University. MDA-MB-231 cells and brain metastatic MDA-MB-231 cells (5×10 6 were injected into the right mammary fat pad of each mouse to establish an orthotopic TNBC tumor model. 5×10 6 and 2×10 6 isolated TNBC tumor cells were injected into the right and left mammary fat pads of each mouse to generate a bilateral PDX tumor model.

[0072] To evaluate the in vivo tumor infiltration, survival, and effector function of HER1-CAR-CAT-NK cells in the presence and absence of ALG hydrogel, mice carrying TNBC tumors were randomly divided into four groups, and part of the tumors were removed on day 15 (about 50% remained). Then on day 15, HER1-CAR-CAT-NK cells (5×10 6 ) containing ALG solution (10 mg / ml) (with calcium ions in the tumor in vivo without addition); and ordinary con-CAR-NK cells (5×106 ), HER1-CAR-NK cells (5 x 10 6 ), HER1-CAR-CAT-NK cells (5 x 10 6 ) were injected into the resection cavity. The tumor infiltration and survival of CAR-NK cells were evaluated by detecting the expression level of RFP, which was intrinsically expressed by CAR-NK cells, in tumors collected on day 17 and day 21 by confocal microscopy, real-time quantitative PCR and flow cytometry. The tumor hypoxia level was determined by detecting pimonidazole by immunofluorescence. In addition, the secretion level of granzyme B and perforin was determined by enzyme-linked immunosorbent assay. By recording RFP fluorescence under confocal microscope, it was found that the ALG hydrogel-encapsulated HER1-CAR-CAT-NK cells showed stronger tumor infiltration effect at both time intervals Figure 4 A,B). However, the tumor masses of mice receiving ordinary con-CAR-NK cells, HER1-CAR-NK cells or HER1-CAR-CAT-NK cells treatment only showed effective tumor infiltration of corresponding CAR-NK cells on day 17, even though the effect of ALG hydrogel-encapsulated con-CAR-NK cells, HER1-CAR-NK was still poor. Similar trends were also observed by recording intratumoral RFP signals by flow cytometry and real-time quantitative PCR Figure 4 C-F). These results showed that ALG hydrogel could significantly promote the tumor infiltration of HER1-CAR-CAT-NK cells, which had significant differences with other groups. Immunofluorescence staining with commercial pimonidazole as a hypoxia detection probe showed that ALG-assisted HER1-CAR-CAT-NK cell treatment significantly reduced tumor hypoxia in residual parental and brain metastatic MDA-MB-231 tumors collected on day 17 and day 21 Figure 4 G). By ELISA test, it was found that the HER1-CAR-CAT-NK cell treatment group caused the secretion of granzyme B and perforin to be about 9.8 times higher than the ordinary con-CAR-NK cell treatment group on day 17, regardless of whether it was coated with ALG hydrogel Figure 5 H-K). And only the ALG-coated HER1-CAR-CAT-NK cell treatment group still maintained a high level of granzyme B and perforin secretion on day 21. Compared with the con-CAR-NK cell treatment group, the HER1-CAR-NK cell treatment group only produced moderate levels of granzyme B and perforin on day 17 and day 21. These results collectively indicated that ALG-encapsulated HER1-CAR-CAT-NK cell treatment had stronger target cell killing power.

[0073] To evaluate the ability of ALG hydrogel in conjunction with HER1-CAR-CAT-NK cell therapy in inhibiting post-surgical recurrence, four groups of TNBC tumor partial resection mice (with about 10% of tumor mass remaining) received the above-mentioned treatments (con-CAR-NK cells, HER1-CAR-NK cells, HER1-CAR-CAT-NK cells, and ALG hydrogel-encapsulated HER1-CAR-CAT-NK). The length and width of each tumor were recorded weekly with a digital caliper, and the tumor volume was calculated according to the formula: volume = 1 / 2 x length x width x width. A digital balance was used to monitor the body weight of each mouse. In addition, on the day of mouse death, the brains of orthotopic brain metastatic MDA-MB-231 tumor mice treated with various treatments were collected and sectioned for H&E staining. By recording the tumor size, it was found that the ALG hydrogel-encapsulated HER1-CAR-CAT-NK cell treatment had the highest efficacy, completely eradicating the residual tumor mass within 112 days, and no post-surgical local recurrence was observed Figure 5 A-C). The other HER1-CAR-NK cell and HER1-CAR-CAT-NK cell treatments alone could only slightly delay the growth of the residual tumor mass, with median survival times of 76 days and 97 days, respectively, slightly longer than the 68-day median survival of con-CAR-NK cell-treated mice. In addition, these treatments only caused a slight decrease in body weight in the first week after treatment, which should be attributed to the surgical resection Figure 5 D). These results show that this ALG-assisted HER1-CAR-CAT-NK cell treatment can effectively eliminate HER1-expressing TNBC tumors without significant side effects, even with ALG hydrogel-encapsulated con-CAR-NK cells, HER1-CAR-NK cells, which are still significantly weaker than ALG hydrogel-encapsulated HER1-CAR-CAT-NK.

[0074] Similarly, 4 groups of mice carrying brain metastatic MDA-MB-231 tumors received the same treatments as above, and it was observed that ALG-assisted HER1-CAR-CAT-NK cell treatment completely inhibited the growth of residual tumors, and no post-surgical local recurrence was observed within 119 days Figure 5 E-G). In contrast, HER1-CAR-NK cell treatment alone and HER1-CAR-CAT-NK cell treatment alone could only moderately delay the growth of the residual tumor mass, with median survival times of 90 days and 104 days, respectively, longer than the 75-day median survival of con-CAR-NK cell-treated mice. In addition, the body weight of these treatment mice fluctuated slightly throughout the monitoring period Figure 5 H), further demonstrating the advantages of ALG-assisted HER1-CAR-CAT-NK synergistic therapy.

[0075] No obvious metastatic foci were found in the brain sections of cured mice collected at day 119 by hematoxylin-eosin (H&E) staining Figure 6 I) In sharp contrast, brain sections of the dead mice treated with con-CAR-NK cells, HER1-CAR-NK cells, and HER1-CAR-CAT-NK cells alone showed obvious metastatic foci. These results collectively indicate that ALG-assisted HER1-CAR-CAT-NK cell therapy can effectively suppress local and distant recurrence of highly metastatic TNBC tumors after surgery, which can be due to its superior tumor-killing ability in H2O2 and hypoxia-rich microenvironments.

[0076] To evaluate the ability of ALG-assisted HER1-CAR-CAT-NK cell therapy in suppressing local and distant tumor recurrence after surgery, the primary large tumor of each tumor-bearing bilateral PDX mouse was first partially resected, and then received the same treatments described above. The tumor volume and body weight of each mouse were recorded as described above. In addition, the distribution of CAR-NK was evaluated using real-time quantitative PCR by detecting the expression level of RFP, which is intrinsically expressed in CAR-NK, in blood collected at day 21 after various treatments. Confocal microscopy was used to evaluate the distant tumor infiltration and survival of CAR-NK by detecting the expression level of RFP in distant tumors collected at day 21 after various treatments. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of granzyme B and perforin in distant tumors collected at day 21 after various treatments. It was found that ALG-assisted HER1-CAR-CAT-NK cell therapy not only completely suppressed the growth of the primary residual tumor mass, but also was the most effective in suppressing the growth of distant untreated tumors Figure 6 A-E) In sharp contrast, compared with con-CAR-NK cell therapy, HER1-CAR-NK cell and HER1-CAR-CAT-NK cell therapy alone could only moderately suppress the growth of the primary residual tumor mass and distant tumors. In addition, these treatment methods did not produce obvious side effects, as only slight weight loss was observed in the first week after surgical treatment Figure 6 F) By recording the RFP signal of CAR-NK cells using real-time quantitative PCR, it was found that the blood samples collected from mice at day 21 after ALG-assisted HER1-CAR-CAT-NK cell therapy had the highest RFP level compared to those from mice treated with other methods Figure 6 G) In addition, it was found that tumor sections from mice treated with ALG-assisted HER1-CAR-CAT-NK cells showed the highest level of RFP fluorescence compared to those from mice treated with other methods by confocal microscopy Figure 6H). Therefore, by corresponding ELISA detection, it was found that the secretion levels of granzyme B and perforin in the distant tumor after ALG assisted HER1-CAR-CAT-NK cell treatment were higher than those of other treatment methods ​ I-J). In summary, these results show that this ALG assisted HER1-CAR-CAT-NK cell can exist in the resection cavity for a long time, gradually enter the peripheral blood circulation and accumulate in the distant tumor, thereby effectively synergistically inhibiting the growth of the distant tumor.

[0077] The short-time effect of HER1-CAR-CAT-NK cells in the embodiments of the present application is significantly stronger than that of con-CAR-NK cells and HER1-CAR-NK, which embodies the importance of CAT chimerization, and the ALG assisted HER1-CAR-CAT-NK further has a synergistic effect in long-term treatment.

Claims

1. A sodium alginate hydrogel expressing chimeric antigen receptor NK cells for catalase, characterized by, The endogenous calcium ion reactive hydrogel is formed by mixing the chimeric antigen receptor NK cell expressing catalase and sodium alginate.

2. The sodium alginate hydrogel expressing catalase chimeric antigen receptor NK cells according to claim 1, characterized in that, The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase is mixed by mixing the chimeric antigen receptor NK cell expressing catalase, sodium alginate and calcium chloride, and then incubated at room temperature until the gel is formed.

3. The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase of claim 1 is used to improve the tolerance of CAR-NK cells to high levels of oxidative stress and hypoxia in tumors.

4. The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase of claim 1 is used to improve the tumor persistence and effector function of CAR-NK cells.

5. Use according to claim 3 or 4, characterized in that, The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase improves the tolerance of CAR-NK cells to high levels of oxidative stress and hypoxia in tumors by catalyzing the decomposition of hydrogen peroxide in tumors into oxygen, thereby improving the tumor persistence and effector function of CAR-NK cells.

6. The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase of claim 1 is used to prepare a drug or reagent for inhibiting the postoperative recurrence of triple-negative breast cancer.

7. Use according to claim 6, characterized in that, The sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase improves the inhibition of postoperative recurrence and metastasis of triple-negative breast cancer by chimeric antigen receptor NK cells, and is used to prepare a drug or reagent for inhibiting the postoperative recurrence of triple-negative breast cancer.

8. A pharmaceutical composition for inhibiting postoperative recurrence of triple-negative breast cancer, characterized by, It contains the sodium alginate hydrogel of the chimeric antigen receptor NK cell expressing catalase as claimed in claim 1 as an active ingredient and a pharmaceutically acceptable adjuvant.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition is preferably an injection.

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