Ligustrazine hydrogel and application thereof in improving ability of chimeric antigen receptor T cell immunotherapy in targeted killing of triple negative breast cancer

Abstract

The invention discloses ligustrazine hydrogel and application of the ligustrazine hydrogel in improving the ability of chimeric antigen receptor T cell immunotherapy for targeted killing of triple negative breast cancer. The ligustrazine hydrogel is formed by mixing ligustrazine with endogenous hydrogen peroxide reactive hydrogel composed of polyethylene glycol dimethacrylate and ferrous chloride. After the ligustrazine hydrogel disclosed by the invention is fixed in a tumor, TMP can continuously promote angiogenesis and expand tumor blood vessels to remodel the tumor blood vessels by activating expression of vascular endothelial growth factors (VEGF) and nitric oxide synthetase / nitric oxide (eNOS / NO) axes in vascular endothelial cells, so that the tumor hypoxia condition is relieved; cAR-T tumor infiltration, survival and effector functions are improved, so that tumor growth is inhibited. The invention provides a simple and effective method for the first time, namely, the treatment effect of CAR-T cells on solid tumors is enhanced by remodeling tumor blood vessels through the ligustrazine hydrogel.

Description

Technical Field

[0001] The present invention belongs to the research field of Chinese medicine extracts, hydrogels, and CAR-T cell immunotherapy for triple-negative breast cancer, and specifically relates to ligustrazine hydrogel and its application in enhancing the ability of chimeric antigen receptor T cell immunotherapy to target and kill triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) lacks expression of progesterone receptor, estrogen receptor and human epidermal growth factor receptor 2 (HER2) and is the most aggressive subtype of breast cancer. It is reported that 45-70% of TNBC patients show overexpression of epidermal growth factor receptor (EGFR, also known as HER1), which has been used as a target for the invention of new therapies to expand our arsenal against TNBC. Unlike small molecule EGFR inhibitors (such as erlotinib, icotinib, gefitinib, etc.), HER1-CAR-T cell engineering represents a promising way to treat HER1-positive TNBC tumors because they have great potential to induce sustained or even complete anti-tumor effects. However, similar to other CAR-T cell therapies for the treatment of corresponding solid tumors, the efficacy of HER1-CAR-T cells in the treatment of TNBC tumors is still far from satisfactory due to the complex tumor microenvironment (TME) and many other factors. For example, tumor hypoxia is a common feature of solid tumors, caused by irregular blood vessels and elevated metabolism, which has been shown to inhibit the viability and function of CAR-T cells. Recent progress has shown that the simultaneous delivery of CAR-T cells with stimulatory cytokines or TME remodeling agents can effectively enhance the effects of these CAR-T cells on corresponding solid tumors by eliminating tumor immunosuppression. Therefore, the development of effective TME remodeling strategies is of great practical significance for improving the therapeutic effect of HER1-CAR-T cells.

[0003] Over the past few decades, efforts have been made to find effective TME remodeling strategies, both in the clinic and in preclinical exploration. Since abnormalities in tumor vasculature are closely related to tumor hypoxia, tumor acidity, tumor immunosuppression, etc., normalizing tumor vasculature through different mechanisms has attracted tremendous research interest. Therefore, it has been shown to have the versatility to directly inhibit tumor growth and benefit other cancer treatment modalities by promoting tumor accumulation and intratumoral diffusion of different cancer therapeutics, such as small molecule chemotherapeutics and nanotherapeutics. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a ligustrazine hydrogel for improving the ability of CAR-T cell immunotherapy to target and kill TNBC, which can promote angiogenesis and dilate tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia, improving CAR-T tumor infiltration, survival and effector function, and thus inhibiting tumor growth.

[0005] The present invention also provides the application of the ligustrazine hydrogel in enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer.

[0006] Technical solution: To achieve the above-mentioned purpose of the invention, the ligustrazine hydrogel described in the present invention is used to enhance the ability of CAR-T cell immunotherapy to target and kill TNBC, characterized in that the ligustrazine hydrogel is formed by mixing ligustrazine with an endogenous hydrogen peroxide-reactive hydrogel composed of polyethylene glycol dimethacrylate and ferrous chloride.

[0007] The ligustrazine hydrogel is prepared by mixing polyethylene glycol dimethacrylate, ferrous chloride, hydrogen peroxide, and water-soluble ligustrazine, and then incubating the mixture at room temperature until a gel is formed.

[0008] The ligustrazine hydrogel of the present invention is used in promoting angiogenesis and dilating tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia.

[0009] The application of the ligustrazine hydrogel described in the present invention in improving the tumor infiltration, survival and effector function of CAR-T.

[0010] Among them, the ligustrazine hydrogel activates the expression of vascular endothelial growth factor (VEGF) and the nitric oxide synthase / nitric oxide (eNOS / NO) axis in vascular endothelial cells, continuously promotes angiogenesis and dilates tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia and improving CAR-T tumor infiltration, survival and effector function.

[0011] The ligustrazine hydrogel of the present invention is used in the preparation of a drug for promoting angiogenesis and dilating tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia.

[0012] The ligustrazine hydrogel of the present invention is used in the preparation of drugs for improving CAR-T tumor infiltration, survival and effector function.

[0013] The ligustrazine hydrogel of the present invention is used in enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer.

[0014] Among them, ligustrazine hydrogel combined with CAR-T therapy can significantly inhibit triple-negative breast cancer.

[0015] The application of the ligustrazine hydrogel of the present invention in the preparation of a drug for enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer.

[0016] The application of the ligustrazine hydrogel described in the present invention in improving the ability of CAR-T cell immunotherapy to target and kill TNBC.

[0017] The application of the ligustrazine hydrogel of the present invention in the preparation of drugs or reagents for improving the targeted killing ability of TNBC in CAR-T cell immunotherapy.

[0018] The pharmaceutical composition for enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer of the present invention comprises the ligustrazine hydrogel as an active ingredient and a pharmaceutically acceptable excipient.

[0019] Wherein, the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

[0020] Furthermore, the ligustrazine hydrogel described in the present invention is used in combination with HER1-CAR-T in the preparation of drugs for the treatment of triple-negative breast cancer.

[0021] The present invention proposes for the first time the use of a hydrogel prepared from the Chinese medicinal extract ligustrazine in remodeling tumor blood vessels to enhance the treatment of solid tumors by CAR-T cells.

[0022] The present invention mixes water-soluble ligustrazine (TMP) with an endogenous hydrogen peroxide (H2O2) reactive hydrogel composed of polyethylene glycol dimethacrylate (PEGDMA) and ferrous chloride (FeCl2). The obtained TMP-loaded PEGDMA hydrogel (TMP@PEGgel) can be fixed in the tumor by activating VEGF expression and eNOS / NO axis of vascular endothelial cells, effectively remodeling tumor blood vessels, thereby continuously alleviating tumor hypoxia. In addition, this TMP@PEGgel treatment has been shown to enhance the infiltration of intravenously injected HER1-CAR-T cells, thereby promoting their survival and effector function in MDA-MB-468 and MDA-MB-231TNBC tumors. It was further found that TMP@PEGgel assisted HER1-CAR-T cell therapy can synergistically inhibit the growth of TNBC tumors. Based on the above research conclusions, remodeling tumor blood vessels with TMP@PEGgel treatment can enhance the therapeutic effect of HER1-CAR-T cell therapy on TNBC tumors.

[0023] The present invention proposes an injectable hydrogel composed of polyethylene glycol dimethacrylate (PEGDMA) and ferrous chloride (FeCl2) that reacts to endogenous hydrogen peroxide (H2O2), encapsulates the Chinese herbal extract ligustrazine (TMP), and fixes it in the tumor. After TMP is fixed in the tumor, it can activate the expression of vascular endothelial growth factor (VEGF) and the nitric oxide synthase / nitric oxide (eNOS / NO) axis in vascular endothelial cells, continuously promote angiogenesis and expand tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia, improving CAR-T tumor infiltration, survival and effector function, and inhibiting tumor growth. The present invention proposes for the first time a simple and effective method, that is, to enhance the therapeutic effect of CAR-T cells on solid tumors by reshaping tumor blood vessels with ligustrazine hydrogel.

[0024] Due to abnormal blood vessels in the tumor, the efficiency of immune cells such as CAR-T entering the tumor is low. At the same time, even after entering, the harsh tumor microenvironment such as hypoxia also inhibits the immune function of CAR-T. The present invention uses hydrogel to release TMP for a long time, improves tumor blood vessels and tumor microenvironment for a long time, and enhances the anti-tumor ability of CAR-T. The present invention uses ligustrazine hydrogel for the first time to enhance CAR-T targeted killing and inhibit triple-negative breast cancer. Compared with the injection of ligustrazine (TMP) alone, ligustrazine hydrogel (TMP@PEGel) can dilate tumor blood vessels for a longer time and improve the hypoxic microenvironment of the tumor. Compared with the injection of CAR-T alone, TMP@PEGel combines with CAR-T, and the proportion of CAR-T infiltration in the tumor is higher, the survival time is longer, the cytokine release is higher, and the tumor inhibition ability is better. The present invention first discovered that TMP@PEGel enhances CAR-T, and TMP@PEGel synergizes with CAR-T to achieve significant anti-tumor effects. In addition, directly using TMP to coordinate CAR-T anti-tumor treatment will result in direct drug administration into the tumor, which will be metabolized too quickly and cannot maintain the efficacy for a long time. At the same time, due to intravenous administration, too little drug enters the tumor, and if the amount is increased, it will have side effects on the whole body.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0026] The ligustrazine hydrogel (TMP@PEGgel) prepared by the present invention is the first to discover that TMP@PEGgel can effectively reshape tumor blood vessels by activating VEGF expression and eNOS / NO axis of tumor vascular endothelial cells, thereby achieving sustained relief of tumor hypoxia.

[0027] The present invention has found through experimental research that after TMP@PEGgel is injected into the tumor, it can enhance the infiltration of intravenously injected HER1-CAR-T cells, thereby promoting their survival and effector function in MDA-MB-468 and MDA-MB-231TNBC tumors. It is further found that TMP@PEGgel synergistically with HER1-CAR-T cell therapy can synergistically inhibit the growth of TNBC tumors; therefore, the ligustrazine hydrogel of the present invention can be used in the preparation of drugs that enhance CAR-T targeted killing and inhibit triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The construction of HER1-CAR-T cells; (ab) schematic diagrams show the third-generation Con-CAR (a) and HER1-CAR (b) structures. (cd) Real-time PCR (c) and Western blotting were used to detect the CD3ζ expression levels of Con-CAR-T cells and HER1-CAR-T cells.

[0029] Figure 2 TMP activates VEGF expression and eNOS / NO axis in vascular endothelial cells; (ab) Cell viability of HUVEC and HAEC cells cultured with different concentrations of TMP. (cd) Western blot analysis (c) and corresponding semi-quantitative analysis (d) of VEGF expression levels in HUVEC and HAEC cells treated with TMP. (ef) Western blot analysis (e) and corresponding semi-quantitative analysis (f) of p-eNOS and eNOS expression levels in HUVEC and HAEC cells treated with TMP. (g) NO production levels in HUVEC and HAEC cells treated with TMP. (h) Schematic diagram of TMP activation of VEGF expression and eNOS / NO axis. (**p<0.01, or*p<0.05).

[0030] Figure 3 Preparation of TMP@PEGgel; (a) Photographs of mixed solutions of PEGDMA and H2O2 with (right) and without (left) FeCl2 solution. (b) Representative scanning microscopy images of the prepared PEGgel. (c) UV-Vis-NIR spectrum of TMP. (d) Calibration curve and regression equation of TMP. (e) Cumulative release curve of TMP from TMP@PEGgel hydrogel.

[0031] Figure 4Figure 1. TMP@PEGgel-mediated tumor vascular remodeling and tumor hypoxia relief; (a) Schematic diagram of the experimental plan. (b) Fluorescence imaging of tumor sections collected from mice injected with rhodamine B 3 or 10 days after PEGgel and without PEGgel. (cd) NO production levels in MDA-MB-468 tumors (c) and MDA-MB-231 tumors (d) with different treatments as indicated. (e) Confocal microscopy imaging of blood vessels on MDA-MB-468 tumor sections using anti-CD31 and corresponding secondary antibodies. (fg) Semi-quantitative statistical analysis of total blood vessel density (f) and effective blood vessel area (g) based on the images shown in e. (h) Confocal microscopy imaging of blood vessels on MDA-MB-231 tumor sections using anti-CD31 and corresponding secondary antibodies. (ij) Semi-quantitative statistical analysis of total blood vessel density (i) and effective blood vessel area (j) based on the images shown in h. For simplicity, the color codes in Figures e and j are used to represent the corresponding treatments. (kn) Confocal microscopy images of different tumor sections with pinodazole-specific (kl) and HIF-1-specific staining (mn) after different treatments as indicated (***p<0.001, **p<0.01, or *p<0.05).

[0032] Figure 5 TMP@PEGgel promotes the infiltration, survival, and effector function of HER1-CAR-T cells in TNBC tumors; (a) Schematic diagram of the experimental timeline. (bc) Confocal microscopy imaging of RFP-positive HER1-CAR-T cells in MDA-MB-468 (b) and MDA-MB-231 tumors (c), with different treatments as shown. (de) Flow cytometric analysis of the percentage of RFP-positive HER1-CAR-T cells in MDA-MB-468 (d) and MDA-MB-231 tumors (e), with different treatments as shown. (fk) Intratumoral secretion levels of IL-2 (fg), TNF-α (hi), and IFN-γ (jk) in MDA-MB-468 and MDA-MB-231 tumors, as shown. (***p<0.0 01, **p<0.01, or *p<0.05).

[0033] Figure 6 TMP@PEGgel enhances HER1-CAR-T cell therapy for TNBC tumors; (a) Schematic diagram of the experimental timeline. (b, c) Average and individual tumor growth curves of MDA-MB-468 (b) and MDA-MB-231 (c) tumors in mice with different treatments are shown. (n = 5) Color codes are used to represent the corresponding treatments. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0035] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0036] The biological materials, reagents and kits used in the present invention can all be obtained through conventional commercial purchases, and the experimental techniques used are all conventional operations in the art or are performed with reference to the instructions of the corresponding products.

[0037] The sources or models of the biological materials used in the present invention are as follows:

[0038] TNBC cell line MDA-MB-231

[0039] TNBC cell line MDA-MB-468

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

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

[0042] Table 1 Sources of biological and chemical reagents

[0043]

[0044]

[0045] Example 1

[0046] Preparation and characterization experiments of PEGgel:

[0047] The substances in the system were mixed according to the final concentration. Specifically, PEGDMA (10wt%) was mixed with FeCl2 (1.6mM), H2O2 (0.5mM) and TMP (400ug / mL) using a vortex mixer for 10 seconds, and then incubated at room temperature until the gel formed TMP@PEGgel (about 1min). At the same time, the hydrogel PEGgel prepared without adding TMP was used as a control.

[0048] The experimental results show that ( Figure 3 In a and b), PEGDMA monomers can form hydrogels within one minute after adding H2O2 and FeCl2. Both substances can undergo Fenton reaction to generate highly active hydroxyl groups (-OH), which are widely used polymerization initiators, such as Figure 3a and b, without FeCl2, are in liquid state and cannot form gel, but with FeCl2, they are in gel state. The microstructure of the prepared hydrogels was observed by scanning electron microscopy (SEM, Zeiss Gemini 500). Figure 3 b, the obtained PEGgel showed a microscopic porous network structure, and the structure of TMP@PEGgel was similar to that of PEGgel. Further, the release of TMP was evaluated in PBS at 37°C, and the released TMP was analyzed using a UV-visible spectrophotometer (PerkinElmer). By recording the absorbance of TMP at 294nm, it was shown that the TMP-loaded PEGgel (TMP@PEGgel) was able to achieve sustained release of TMP ( Figure 3 (ce).

[0049] Example 2

[0050] Cell experiments:

[0051] PBMCs cells were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd. PBMCs were inoculated and cultured in AIM VTM complete medium supplemented with anti-CD3 / CD28 beads (2.5 μL / mL) and stimulated with 5% carbon dioxide at 37°C. After 2 weeks, T cells were induced for 1 day with lentivirus containing CD8 signal, CD8 Hinge, CD28, 4-1BB, CD3ζ and HER1-specific single-chain fragment variable (scFv). After that, CAR-T cells were collected after 3 days of culture with AIM VTM complete medium containing anti-CD3 / CD28 beads according to the standard method. Then, the experimental results showed ( Figure 1 (ad) The CD3ζ expression levels on these obtained con-CAR-T and HER1-CAR-T cells were evaluated by real-time PCR and Western blotting. Overexpression of CD3ζ confirmed that CAR-T cells were successfully constructed, and the constructed con-CAR-T and HER1-CAR-T were used for subsequent experiments.

[0052] To evaluate the cytotoxicity of TMP on endothelial cells, HUVEC and HAEC (1×10 4 The cells were cultured with fresh medium supplemented with different concentrations of TMP at 37°C for another 24 hours, and then the cell viability was measured using the cell counting kit-8 (CCK-8) method. The steps were to add 10 μL of CCK-8 solution and 90 μL of complete medium to each well, and continue to culture in the incubator for 1 hour, and then use a microplate reader to detect the absorbance value at 450 nm. The experimental results showed that ( Figure 2TMP has no obvious cytotoxicity to human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) at a concentration of 0-500 nM, proving its safety

[0053] To evaluate the efficacy of TMP treatment in activating vascular endothelial growth factor expression and eNOS / NO axis, HUVEC and HAEC were seeded in 6-well plates and cultured for 12 hours, and then cultured with TMP (500nM) at 37°C for another 24 hours. The expression levels of VEGF, p-eNOS, eNOS and β-Actin in these cells were measured by Western blotting. The basic steps are to add the collected protein samples to the SDS-PAGE gel wells, electrophoresed, and transferred to the membrane. The PVDF membrane was blocked with 5% skim milk powder. The PVDF membrane was incubated with primary antibodies for VEGF, p-eNOS, eNOS and β-Actin, and then incubated with secondary antibodies. The developer was added, and the expression of VEGF, p-eNOS, eNOS and β-Actin proteins was detected by the biomolecular imaging analysis system. The experimental results show ( Figure 2 (cf) TMP treatment (500 nM, 24 h) resulted in a significant increase in the expression of VEGF and p-eNOS in HUVEC and HAEC cells, but did not significantly impair the expression of total eNOS. The effect of TMP treatment on NO production was measured using a NO detection kit. The experimental results showed that ( Figure 2 TMP treatment was able to promote the release of NO from HUVEC and HAEC cells.

[0054] In vivo animal experiments:

[0055] Female Balb / c nude mice (4 weeks old) were purchased from the Model Animal Research Center of Nanjing University. The Medical Ethics Committee of Nanjing Normal University approved the animal experiments in this study. HTB-132 TM ) or MDA-MB-231( HTB-26 TM )) cells (5×10 6 ) were injected subcutaneously into the mammary fat pad of each mouse to generate a tumor model of the TNBC cell line.

[0056] In the in vivo sustained release study, rhodamine B@PEGgel (prepared using the method of Example 1, with ligustrazine replaced by rhodamine B) and free rhodamine B solution (ensuring rhodamine B = 1 mg / kg) were injected into TNBC tumors. At 15 minutes, 3 days, and 10 days after the corresponding injections, the tumors of the treated mice were removed, frozen, sectioned, counterstained with DAPI, and observed under a confocal microscope to record the fluorescence of rhodamine B. The experimental results showed that ( Figure 4 In Figures ab), obvious Rhodamine B fluorescence was observed in the tumor sections of mice with PEGgel-immobilized Rhodamine B at 3 and 10 days after injection (pi). In contrast, only obvious Rhodamine B fluorescence was observed in the tumor sections of mice at 3 days after injection.

[0057] In order to conduct relevant research on the evaluation of NO improving tumor hypoxia in vivo, after the mouse tumor was implanted for 2 weeks, TMP@PEGgel (TMP = 1 mg / kg), PEGgel and TMP (TMP = 1 mg / kg) were injected accordingly, where PEGgel and TMP@PEGgel were injected in equal volumes, and an equal volume of PBS was injected as a control. Three days and ten days after the injection, TNBC tumor mice were sacrificed to prepare tissue lysate and tumor slices. The effect of TMP treatment on tumor NO production was measured using a NO detection kit. The experimental results showed that ( Figure 4 cd), compared with intratumoral injection of PBS and PEGgel in mice, treatment with TMP@PEGgel (TMP = 1 mg / kg) significantly increased NO production after 3 and 10 days, while treatment of tumor-bearing mice with free TMP only resulted in increased NO production in the tumor at 3 days. The experimental results showed ( Figure 4 en), TMP and TMP@PEGgel treatment for 3 days effectively increased tumor vascular intensity as demonstrated by immunofluorescence detection of CD31. The percentage of effective blood vessels was also increased by measuring the intact margin of blood vessels after 3 days. However, only TMP@PEGgel treatment increased intratumoral vascular density and percentage of effective blood vessels for 10 days. Further, pimonidazole staining was performed, and another batch of mice treated in the same way were injected intraperitoneally with pimonidazole 90 minutes before euthanasia. Tumors were then collected for hypoxia staining using the hypoxyprobe-1plus kit. After TMP@PEGgel treatment, the pimonidazole-specific fluorescence signal was greatly suppressed on tumor sections collected from the two TNBC tumor mice by microscopic observation. Meanwhile, treatment with free TMP only resulted in suppression of pimonidazole-specific fluorescence on tumor sections collected after 3 days, while the interference of other treatments on pimonidazole-specific fluorescence on tumor sections collected at two time intervals was negligible. In addition, tumor sections from mice treated with free TMP and TMP@PEGgel showed significantly reduced expression of hypoxia-inducible factor (HIF)-1α by standard immunofluorescence staining, and the evolution trend was similar to the results of the aforementioned pimonidazole staining method.

[0058] To evaluate the in vivo tumor infiltration, survival, and effector function of HER1-CAR-T cells, 30 mice bearing TNBC tumors were randomly divided into 6 groups and received the following treatments: 1) PBS, 2) Con-CAR-T injection, 3) HER1-CAR-T injection, 4) PEGgel fixation + HER1-CAR-T injection, 5) TMP injection + HER1-CAR-T injection, and 6) TMP@PEGgel fixation + HER1-CAR-T injection, in which 5 × 10 CAR-T cells were injected into the two groups. 6 , TMP@PEGgel and TMP (TMP = 1 mg / kg), PEGgel and PBS were kept at the same volume as TMP@PEGgel. Then the mice were intravenously injected with 5×10 6 Con-CAR-T cells and HER1-CAR-T cells, both of which had intrinsic expression of RFP within 1 day. Tumors were frozen or homogenized 3 and 10 days after TMP@PEGgel injection, and RFP fluorescence was recorded by confocal microscopy and flow cytometry to record tumor infiltration and survival of the two CAR-T cells, respectively. The experimental results showed ( Figure 5 In the tumor sections of the TMP@PEGgel combined with HER1-CAR-T cell injection group, obvious RFP fluorescence signals were detected on days 3 and 10. TMP treatment only led to a significant increase in the tumor infiltration of HER1-CAR-T cells on day 3, while other treatments had little effect on the tumor infiltration ability of HER1-CAR-T. In addition, the tumors were wet weighed and homogenized using the corresponding ELISA kits to measure the secretion of IL-2, TNF-α, and IFN-γ. The experimental results showed that ( Figure 5 fk), within 10 days, TMP@PEGgel promoted the secretion of cytokines by HER1-CAR-T cells infiltrating into TNBC tumors. Meanwhile, injection of TMP only promoted a significant increase in the intratumoral secretion of IL-2, TNF-α, and IFN-γ by HER1-CAR-T cells within 3 days, and this effect could not last for 10 days. In sharp contrast, other treatments had less effect on the secretion of these pro-inflammatory cytokines.

[0059] In terms of in vivo combined therapy, 6 groups of TNBC cells MDA-MB-468 ( HTB-132 TM ) tumor mice (about 100mm 3) received the same intratumoral injection of TMP@PEGgel, PEGgel, and TMP on day 0 (TMP@PEGgel and TMP (TMP = 1 mg / kg, PEGgel and TMP@PEGgel maintained the same volume), but received three injections of Con-CAR-T cells or HER1-CAR-T cells (5 × 10 6 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 × length × width × width. The weight of each mouse was monitored weekly with a digital balance. The experimental results showed that ( Figure 6 In Figures ac), TMP@PEGgel combined with HER1-CAR-T injection showed the highest efficacy in inhibiting the growth of MDA-MB-468 tumors, with one tumor completely disappearing, while the other four tumors showed no significant growth within 63 days after tumor treatment. In sharp contrast, the other treatments only slightly delayed tumor growth, and all of these mice died within 56 days of the corresponding treatment. In addition, this excellent effect of TMP@PEGgel-assisted HER1-CAR-T treatment was also shown in inhibiting the growth of TNBC cells MDA-MB-231 ( HTB-26 TM ) was also confirmed in tumor growth.

[0060] In summary, the present invention combines ligustrazine hydrogel with CAR-T to inhibit triple-negative breast cancer by targeting and killing. Compared with the injection of ligustrazine (TMP) alone, ligustrazine hydrogel (TMP@PEGel) can dilate tumor blood vessels for a longer period of time and improve the hypoxic microenvironment of the tumor. Compared with the injection of CAR-T alone, TMP@PEGel combined with CAR-T has a higher proportion of CAR-T infiltration in the tumor, a longer survival time, a higher amount of cytokine release, and better tumor inhibition ability. The present invention utilizes a specific TMP@PEGel to enhance CAR-T, and TMP@PEGel synergizes with CAR-T to achieve significant anti-tumor effects.

Claims

1. A ligustrazine hydrogel for improving the ability of CAR-T cell immunotherapy to target and kill TNBC, characterized in that: The ligustrazine hydrogel is formed by mixing ligustrazine with an endogenous hydrogen peroxide reactive hydrogel composed of polyethylene glycol dimethacrylate and ferrous chloride.

2. The ligustrazine hydrogel for improving the ability of CAR-T cell immunotherapy to target and kill TNBC according to claim 1, characterized in that: The ligustrazine hydrogel is preferably prepared by mixing polyethylene glycol dimethacrylate, ferrous chloride, hydrogen peroxide, and water-soluble ligustrazine, and then incubating at room temperature until a gel is formed.

3. A use of the ligustrazine hydrogel according to claim 1 in promoting angiogenesis and dilating tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia.

4. A use of the ligustrazine hydrogel according to claim 1 in improving CAR-T tumor infiltration, survival and effector function.

5. The use according to claim 3 or 4, characterized in that: The ligustrazine hydrogel activates the expression of vascular endothelial growth factor (VEGF) and the nitric oxide synthase / nitric oxide (eNOS / NO) axis in vascular endothelial cells, continuously promotes angiogenesis and dilates tumor blood vessels to reshape tumor blood vessels, thereby alleviating tumor hypoxia and improving CAR-T tumor infiltration, survival and effector function.

6. An application of the ligustrazine hydrogel according to claim 1 in enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer.

7. The use according to claim 6, characterized in that: Ligustrazine hydrogel combined with CAR-T therapy can significantly inhibit triple-negative breast cancer.

8. Use of the ligustrazine hydrogel according to claim 1 in the preparation of a drug for enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer.

9. A pharmaceutical composition for enhancing CAR-T targeted killing and inhibiting triple-negative breast cancer, characterized in that: It comprises the ligustrazine hydrogel as claimed in claim 1 as an active ingredient and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

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