A pharmaceutical composition for improving the activity of gamma delta-t cells against triple-negative breast cancer and application thereof

Abstract

The present application relates to a kind of drug composition for improving the activity of γδ-T cell to triple-negative breast cancer and its application, belong to tumor treatment technical field.The present application is handled γδ-T cell after low-dose taxane drug, it is found that γδ-T cell can keep good activity, can enhance anti-tumor function.Compared with γδ-T cell without prior treatment, whether taxane drug and γδ-T cell are combined to handle triple-negative breast cancer cell, or using taxane drug pre-treatment γδ-T cell and its cell conditioned medium handle triple-negative breast cancer cell, can reduce triple-negative breast cancer cell activity and promote tumor cell death in different degrees.This shows that low-dose taxane drug can effectively reshape γδ-T cell function, improve its killing effect on triple-negative breast cancer.Therefore, taxane drug can be used to prepare γδ-T cell anti-tumor enhancer.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, and in particular to a pharmaceutical composition that enhances the activity of γδ-T cells against triple-negative breast cancer and its application. Background Technology

[0002] Triple-negative breast cancer (TNC) is a subtype of breast cancer that is negative for both estrogen receptor (ER) and progesterone receptor (PR) and does not exhibit HER2 overexpression or gene amplification. It is characterized by high invasiveness, a high risk of recurrence and metastasis, and limited treatment options. Current treatment options for TNC primarily include surgery, radiotherapy, chemotherapy, immunotherapy, and their combination therapies. Among these, taxanes, such as paclitaxel and docetaxel, are commonly used chemotherapy drugs in the clinical treatment of TNC. They can inhibit tumor cell proliferation and induce tumor cell death by stabilizing microtubules and interfering with mitosis.

[0003] γδ-T cells are a type of T cell with innate-like immune characteristics. They can recognize and kill tumor cells through non-MHC-restricted mechanisms and secrete a variety of cytokines and anti-tumor-related molecules. Due to their strong tumor recognition ability and low risk of graft-versus-host disease, γδ-T cells have potential applications in tumor cell therapy.

[0004] Existing technologies include regimens combining taxanes with immune checkpoint inhibitors for the treatment of triple-negative breast cancer, regimens using γδ-T cells for immunotherapy of solid tumors, and research on the anti-tumor effects of γδ-T cell-derived exosomes or secreted products. However, these regimens primarily focus on the direct chemotherapeutic effects of taxanes, immune checkpoint therapy, or the intrinsic anti-tumor effects of γδ-T cells, respectively, and have not yet provided specific technical solutions for regulating γδ-T cell function with low-dose taxanes and enhancing their anti-triple-negative breast cancer effects.

[0005] (1) Triple-negative breast cancer lacks clear targets for endocrine therapy and HER2-targeted therapy. Current treatment options are still mainly chemotherapy, and the efficacy and safety still need to be improved. (2) Taxanes may cause adverse reactions such as peripheral neurotoxicity and bone marrow suppression at conventional doses, and some tumor cells may gradually develop drug resistance. (3) When γδ-T cells are used alone to treat solid tumors, their anti-tumor activity, persistence, and adaptation to the tumor microenvironment are still insufficient; (4) Existing taxane drugs for the treatment of TNBC have problems such as toxic side effects, drug resistance and limited efficacy; γδ-T cells alone have problems such as insufficient anti-tumor activity and limited persistence when treating solid tumors; existing combination regimens focus on the direct effect of taxanes on tumor cells, and lack technical solutions to reshape the function and secretion profile of γδ-T cells using low-dose taxanes. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical composition and its application for enhancing the activity of γδ-T cells against triple-negative breast cancer.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of taxane drugs in the preparation of γδ-T cell antitumor enhancers.

[0008] This invention, through treatment of γδ-T cells with low doses (0.1-100 nM) of taxanes, found that γδ-T cells maintained good activity and enhanced anti-tumor related functions. Compared with untreated γδ-T cells, both combined treatment of triple-negative breast cancer cells with taxanes and γδ-T cells, and treatment of triple-negative breast cancer cells with taxane-pretreated γδ-T cells and their conditioned medium, reduced triple-negative breast cancer cell activity and promoted tumor cell death to varying degrees. This indicates that low-dose taxanes can effectively remodel γδ-T cell function and enhance their killing effect on triple-negative breast cancer. Therefore, taxanes can be used to prepare γδ-T cell anti-tumor enhancers.

[0009] Secondly, this invention provides the application of taxane drugs in the preparation of agents that enhance the activity of γδ-T cells against drug-resistant tumors. Preferably, the drug resistance is resistance to taxanes.

[0010] This invention demonstrates through experiments that the combined use of γδ-T cells and taxanes can enhance the killing effect on taxane-resistant triple-negative breast cancer cells, and can reduce the cell activity of organoids derived from triple-negative breast cancer patients, inhibit organoid growth, or promote tumor cell death. Therefore, taxanes can be used to prepare enhancers of γδ-T cells against drug-resistant tumors, especially showing potential application value against taxane-resistant triple-negative breast cancer.

[0011] Thirdly, the present invention provides the use of taxane drugs in the preparation of formulations that enhance the release or expression levels of ANXA6 and / or PPP2R1A proteins in γδ-T cells.

[0012] This invention experimentally demonstrates that treatment of γδ-T cells with low-dose taxanes can increase the expression levels of ANXA6 and / or PPP2R1A proteins in γδ-T cells and promote the release of ANXA6 and / or PPP2R1A into the cell culture supernatant or conditioned medium. Therefore, taxanes can be used to prepare formulations that regulate the expression or release of ANXA6 and / or PPP2R1A in γδ-T cells.

[0013] Fourthly, this invention provides the application of ANXA6 protein in the preparation of drugs for anti-tumor or drug-resistant tumors.

[0014] Fifthly, the present invention provides the application of PPP2R1A protein in the preparation of drugs for anti-tumor or drug-resistant tumors.

[0015] In a sixth aspect, the present invention provides the use of ANXA6 protein and / or PPP2R1A protein in the preparation of drugs that enhance the antitumor activity of γδ-T cells or in drugs that enhance the anti-drug-resistant tumor activity of γδ-T cells.

[0016] This invention demonstrates through experiments that exogenous supplementation with ANXA6 and / or PPP2R1A can reduce the activity of triple-negative breast cancer cells or taxane-resistant triple-negative breast cancer cells, increase the proportion of tumor cell apoptosis or death, and enhance the antitumor effect of γδ-T cell conditioned medium treated with low-dose taxanes. Therefore, ANXA6 and / or PPP2R1A proteins can be used for antitumor therapy and to enhance the antitumor activity of γδ-T cells.

[0017] In a preferred embodiment, the tumor is triple-negative breast cancer; the taxane drug is selected from paclitaxel, docetaxel, or cabazitaxel.

[0018] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a taxane drug and γδ-T cells.

[0019] Experiments have shown that, due to the synergistic anti-tumor effect of taxanes and γδ-T cells, this composition can effectively inhibit the growth of triple-negative breast cancer and overcome drug resistance.

[0020] Eighthly, the present invention provides a method for preparing a γδ-T cell conditioned medium, the method comprising the following steps: (1) Inoculate γδ-T cells into culture medium and culture them; (2) After adding taxane drugs to the culture system in step (1), continue culturing for 22-50 hours; the final concentration of the taxane drugs is 0.1-100 nM; (3) Retain the cells in the culture system of step (2), remove the culture medium containing taxane drugs, and wash the γδ-T cells; (4) After adding the γδ-T cells from step (3) to fresh culture medium and culturing for 22-50 hours, centrifuge and collect the culture supernatant to obtain the γδ-T cell conditioned culture medium.

[0021] For example, the culture time after treatment with taxane drugs in step (2) can be 22, 24, 30, 36, 42, 48 or 50 hours; preferably, the culture time is 24 hours.

[0022] For example, the final concentration of the taxane drug in step (2) can be 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, or 100 nM. Preferably, the concentration of the taxane drug is 10 nM.

[0023] For example, in step (4), the culture time in the fresh culture medium can be 22, 24, 30, 36, 42, 48 or 50 hours; preferably, the culture time is 48 hours.

[0024] In a ninth aspect, the present invention provides the use of the conditioned medium prepared by the method described above in the preparation of drugs for antitumor or anti-drug-resistant tumors.

[0025] Experiments have shown that conditioned medium for γδ-T cells treated with taxanes can significantly inhibit the proliferation of triple-negative breast cancer cells and induce their apoptosis, thereby exerting anti-tumor effects and overcoming drug resistance.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a novel use of taxane drugs combined with γδ-T cells for the treatment of triple-negative breast cancer, which is different from the traditional treatment method that relies solely on taxane drugs to directly kill tumor cells; (2) This invention utilizes low-dose taxane drugs to enhance the antitumor activity of γδ-T cells, which can improve the anti-triple-negative breast cancer effect under lower drug dosage conditions and help reduce the toxic side effects related to high-dose chemotherapy drugs; (3) This invention is not only applicable to ordinary triple-negative breast cancer cells, but also to taxane-resistant triple-negative breast cancer cells, patient-derived organoids and animal models, and has good prospects for translational application; (4) The present invention found that low doses of taxane drugs can regulate the secretion of anti-tumor related factors by γδ-T cells, including ANXA6 and / or PPP2R1A, providing a mechanistic basis for the combined treatment of triple-negative breast cancer with taxane drugs and γδ-T cells. (5) The present invention can be used to prepare pharmaceutical compositions, cell therapy products, combination therapy preparations or kits for treating triple-negative breast cancer, and has high clinical application value. Attached Figure Description

[0027] Figure 1This diagram illustrates the significant differential effects of taxanes on triple-negative breast cancer cells and γδ-T cells (A: Cell viability of MDA-MB-231 triple-negative breast cancer cells after treatment with paclitaxel (PTX) or docetaxel (DTX); B: Cell viability of MDA-MB-468 triple-negative breast cancer cells after treatment with PTX or DTX; C: Cell viability of γδ-T cells after treatment with PTX or DTX; D: Live cell count of γδ-T cells after treatment with PTX or DTX; E: Cell counts of triple-negative breast cancer cells and γδ-T cells after treatment with PTX or DTX; F: Apoptosis of triple-negative breast cancer cells and γδ-T cells after treatment with PTX or DTX). Figure 2 The diagram illustrates the killing effect of taxanes combined with γδ-T cells on triple-negative breast cancer cells (A: The combined killing effect of taxanes and γδ-T cells on triple-negative breast cancer cells (Example 2, first method); B: The killing effect of taxanes on triple-negative breast cancer cells after pretreatment of γδ-T cells (Example 2, second method)). Figure 3 Schematic diagram illustrating the effect of taxane-induced γδ-T cells in releasing anti-tumor-related molecules and exerting anti-triple-negative breast cancer activity (A: Indirect killing of triple-negative breast cancer cells by γδ-T cells pretreated with taxanes (Example 2, second method); B: Killing of triple-negative breast cancer cells by conditioned medium of γδ-T cells treated with taxanes (Example 2, third method); C: Effect of taxanes on apoptosis of γδ-T cells; D: Effect of taxanes on damage and lysis of γδ-T cells). Figure 4 A schematic diagram illustrating the successful establishment and validation of a paclitaxel-resistant triple-negative breast cancer cell model. Figure 5 Schematic diagram illustrating the effect of low-dose taxanes combined with γδ-T cells on anti-taxane-resistant triple-negative breast cancer; Figure 6 A schematic diagram of an organoid model derived from patients with drug-resistant triple-negative breast cancer, using low-dose taxanes combined with γδ-T cells (AB: origin and construction of drug-resistant triple-negative breast cancer organoids; CD: combined effect of γδ-T cells and taxanes in drug-resistant organoid models). Figure 7Schematic diagram illustrating how taxane-induced ANXA6 and PPP2R1A release promotes γδ-T cell-mediated killing of triple-negative breast cancer cells (A: Effect of taxanes on proteins secreted by γδ-T cells; B: ELISA detection of ANXA6 and PPP2R1A levels in conditioned medium; C: Killing effect of ANXA6 and PPP2R1A on triple-negative breast cancer cells; D: Effect of ANXA6 and PPP2R1A on apoptosis of triple-negative breast cancer cells). Figure 8 A schematic diagram illustrating the functional validation of ANXA6 and PPP2R1A as mediators of taxane-enhanced γδ-T cell-mediated killing of triple-negative breast cancer (AB: WB and ELISA results showing siRNA knockdown efficiency; C: Killing effect of γδ-T cells in conditioned medium after siRNA knockdown and killing effect of conditioned medium after addition of recombinant protein). Figure 9 This diagram illustrates the synergistic antitumor effect of low-dose taxanes combined with γδ-T cells in vivo (AC: effects of combined use of γδ-T cells and taxanes on changes in tumor volume, weight, proliferation level, and γδ-T cell infiltration in triple-negative breast cancer tumors; DE: safety assessment of combined treatment with γδ-T cells and taxanes). Figure 10 To investigate the effect of low-dose taxanes combined with γδ-T cells on the inhibition of taxane-resistant triple-negative breast cancer in vivo (A, C: Effects of combined use of γδ-T cells and taxanes on tumor volume, tumor proliferation level, and γδ-T cell infiltration in drug-resistant triple-negative breast cancer; B: Safety assessment of combined treatment with γδ-T cells and taxanes in drug-resistant triple-negative breast cancer). Figure 11 This is a schematic diagram illustrating the molecular mechanism by which taxane drugs enhance the antitumor efficacy of γδ-T cells.

[0028] ‌ Detailed Implementation

[0029] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] Example 1: Investigating the effects of taxane drugs on γδ-T cells 1. Experimental Method: Peripheral blood was drawn from healthy donors and diluted 1:1 with PBS. The diluted blood was then slowly added to the supernatant of the lymphocyte separation medium. After density gradient centrifugation, the white membrane layer cells were collected to obtain peripheral blood mononuclear cells (PBMCs). The PBMCs were then washed with PBS, and the supernatant was removed by centrifugation to obtain the PBMC cell pellet for subsequent culture.

[0031] The isolated PBMCs were seeded in RPMI 1640 complete medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and IL-2, with zoledronic acid added to stimulate γδ-T cell expansion. Fresh medium containing IL-2 was added periodically during culture to maintain cell growth. Around day 9, the expanded cells were collected for subsequent phenotypic identification and functional experiments.

[0032] The expanded γδ-T cells were divided into a control group and a taxane-treated group. The taxane-treated groups were treated with either paclitaxel or docetaxel at final concentrations of 0.1-100 nM for 24 hours.

[0033] Triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 were treated with taxanes, respectively.

[0034] After treatment, the number of viable cells was detected by trypan blue staining and an automated cell counter; apoptosis was detected by Annexin V / PI flow cytometry.

[0035] 2. Experimental Results: Flow cytometry analysis of CD3 and TCR Vδ2 confirmed that the expanded cells were mainly CD3⁺TCR Vδ2⁺ γδ-T cells.

[0036] The results are as follows Figure 1 The results showed that the cell viability of triple-negative breast cancer cells decreased with increasing concentrations of paclitaxel (PTX) or docetaxel (DTX). Figure 1 A, B); while γδ-T cells maintained good activity under low-dose (0.1-10 nM) paclitaxel or docetaxel treatment. Figure 1 C and D) show a trend of enhancing anti-tumor related functions; taxanes have significantly different effects on apoptosis of triple-negative breast cancer cells and γδ-T cells. At the same concentration, PTX or DTX significantly increased the apoptosis rate of triple-negative breast cancer cells compared to γδ-T cells. Figure 1 (E, F), thus it can be seen that taxanes have significantly different effects on triple-negative breast cancer cells and γδ-T cells.

[0037] Example 2: The killing effect of taxane-based drug pretreatment on γδ-T cells on triple-negative breast cancer cells 1. Experimental Method: Triple-negative breast cancer cells MDA-MB-231 and / or MDA-MB-468 were seeded in culture plates. After the cells adhered and reached a suitable growth state, they were treated as follows, wherein the taxane drug was paclitaxel or docetaxel at concentrations of 0, 10, and 100 nM: The first approach involves co-acting taxanes and γδ-T cells on triple-negative breast cancer cells. Specifically, taxanes and γδ-T cells are co-added to the culture system of triple-negative breast cancer cells at an effector-to-target ratio of 10:1, allowing them to act together on the tumor cells. After co-culturing for 48 hours, changes in luciferase signal in triple-negative breast cancer cells are detected to evaluate the relative survival rate and killing rate of tumor cells. Decreased luciferase signal indicates a reduction in surviving tumor cells and may reflect increased tumor cell death or lysis.

[0038] The second method involves pretreating γδ-T cells with taxanes before co-culturing them with triple-negative breast cancer cells. Specifically, γδ-T cells are pretreated with taxanes in vitro for 24 hours. After treatment, the cells are collected by centrifugation and washed 2-3 times with PBS or fresh culture medium to remove or reduce residual free taxanes. The pretreated γδ-T cells are then co-cultured with triple-negative breast cancer cells at an effector-to-target ratio (5:1 / 10:1) for 48 hours. Changes in luciferase signal in the tumor cells are detected to evaluate the relative survival and killing rate of tumor cells. A decrease in luciferase signal indicates a reduction in surviving tumor cells and may reflect increased tumor cell death or lysis.

[0039] The third method involves using conditioned medium released from γδ-T cells treated with taxanes to act on triple-negative breast cancer cells. Specifically, (1) γδ-T cells are seeded in a culture medium and cultured; (2) a final concentration of 10 or 100 nM taxanes is added to the culture system in step (1) and cultured for another 24 hours; (3) the culture medium containing taxanes is removed, and the γδ-T cells are washed with PBS or fresh culture medium to remove or reduce the residue of free taxanes; (4) fresh culture medium is added to the washed γδ-T cells and cultured for another 48 hours. After centrifugation to remove cells and cell debris, the culture supernatant is collected to obtain the γδ-T cell conditioned medium. The conditioned medium is then added to the triple-negative breast cancer cell culture system and co-cultured for 48 hours. The changes in luciferase signal of tumor cells are then detected to evaluate the relative survival rate and killing rate of tumor cells. A decrease in luciferase signal indicates a reduction in surviving tumor cells and may reflect an increase in tumor cell death or lysis.

[0040] To rule out the possibility that the antitumor activity in the conditioned medium originated from the release of contents due to γδ-T cell death or lysis, the culture supernatant of γδ-T cells from each group was collected, and the cell damage was detected using an LDH release assay. If the LDH levels in the paclitaxel-pretreated or docetaxel-pretreated γδ-T cell groups were not significantly higher than those in the control γδ-T cell group, it indicates that low-dose taxane pretreatment did not lead to significant cell lysis, and the antitumor activity in the supernatant is more likely derived from active secretion or extracellular release mechanisms.

[0041] When evaluating tumor cell killing activity using luciferase activity, the luciferase signal value of the control group tumor cells was used as 100%, and the relative luciferase activity or relative survival rate of each experimental group was calculated. Relative survival rate (%) = (Luciferase signal value of experimental group / Luciferase signal value of control group) × 100%; Killing rate (%) = 100% − relative survival rate (%). A decreased luciferase signal indicates a reduced number of surviving tumor cells.

[0042] 2. Experimental Results: The results are as follows Figure 2-3 The results showed that taxanes synergistically interacted with γδ-T cells (first mode) compared to untreated γδ-T cells. Figure 2 A) and γδ-T cells pretreated with low doses (10, 100 nM) of taxanes (second method) Figure 2 B), the third method ( Figure 3 Both methods have a stronger killing effect on triple-negative breast cancer cells. Whether triple-negative breast cancer cells are pretreated with taxanes and then treated with γδ-T cells in conditioned medium (the second method), or triple-negative breast cancer cells are pretreated with taxanes and then treated with γδ-T cells in conditioned medium (the third method), both methods can reduce the activity of triple-negative breast cancer cells and promote tumor cell death to varying degrees.

[0043] The above results indicate that pretreatment with low-dose taxanes can significantly enhance the γδ-T cell-mediated anti-triple-negative breast cancer activity. This enhancement can be manifested both in the killing process of γδ-T cells in direct contact with tumor cells and at least partially through the release of soluble antitumor-related components by γδ-T cells.

[0044] Example 3: Effect of taxane drugs combined with γδ-T cells on drug-resistant triple-negative breast cancer cells 1. Experimental Method: Triple-negative breast cancer cells MDA-MB-231-LUC and MDA-MB-468-LUC, expressing luciferase, were selected to establish paclitaxel-resistant triple-negative breast cancer cell models using either low-concentration continuous induction or stepwise concentration-increase induction. Specifically, MDA-MB-231-LUC cells were continuously cultured in a culture system containing 5 nM paclitaxel to obtain MDA-MB-231-LUC-R paclitaxel-resistant cells; MDA-MB-468-LUC cells were continuously cultured in a culture system containing 2 nM paclitaxel to obtain MDA-MB-468-LUC-R paclitaxel-resistant cells. The culture medium was changed regularly during the culture process, and appropriate drug selection pressure was maintained until the cells could stably proliferate at the corresponding paclitaxel concentration.

[0045] The sensitivity of parental triple-negative breast cancer cells and paclitaxel-resistant triple-negative breast cancer cells to paclitaxel was detected using the CCK-8 assay, and their IC50 values ​​were calculated to evaluate the success of establishing the paclitaxel-resistant cell model. Furthermore, a clonogenic assay was used to detect the long-term proliferative capacity of parental and resistant cells under paclitaxel treatment conditions to verify the growth advantage of resistant cells under paclitaxel stress. Figure 4 ).

[0046] Drug-resistant triple-negative breast cancer cells MDA-MB-231-LUC-R and MDA-MB-468-LUC-R were co-cultured with γδ-T cells for 48 hours. Changes in luciferase signal in the tumor cells were detected to evaluate the relative survival and killing rate of tumor cells. Decreased luciferase signal indicated a reduction in surviving tumor cells and could reflect increased tumor cell death or lysis.

[0047] Tumor tissue from patients with triple-negative breast cancer was collected, mechanically minced, and / or enzymatically digested to obtain tumor cells or tumor cell masses, which were then seeded into basement membrane extract for three-dimensional culture. The tumors were cultured in triple-negative breast cancer organoid culture medium, with 10 μM Y-27632 added for 2–3 days during the early stage of organoid formation to promote the formation and growth of patient-derived triple-negative breast cancer organoids. After stable organoid formation, a low-dose paclitaxel treatment or maintenance was used to establish a patient-derived organoid model of triple-negative breast cancer that is relatively resistant to taxane drugs. Figure 6 AB).

[0048] Ordinary γδ-T cells, paclitaxel-pretreated γδ-T cells, or corresponding γδ-T cell conditioned media were co-cultured or jointly treated with organoids derived from triple-negative breast cancer patients. The effector-to-target ratio of γδ-T cells to organoid tumor cells was set to 10:1, and the co-culture time was set to 48 hours. After treatment, organoid activity, morphological changes, volume changes, or cell viability were measured to evaluate the antitumor effect of paclitaxel-pretreated γδ-T cells or their conditioned media on organoids derived from triple-negative breast cancer patients and their taxane-dependent tolerance model.

[0049] 2. Experimental Results: The results are as follows Figure 5 and Figure 6 As shown, the combined use of γδ-T cells and taxanes can enhance the killing effect on taxane-resistant triple-negative breast cancer cells. Figure 5 ), and can reduce the cell activity of organoids derived from triple-negative breast cancer patients, inhibit organoid growth, or promote tumor cell death ( Figure 6 CD).

[0050] The above results indicate that the present invention is applicable not only to conventional triple-negative breast cancer cell models, but also to taxane-resistant triple-negative breast cancer models and patient-derived organoid models, thus having potential application value in improving the treatment effect of taxane-resistant triple-negative breast cancer.

[0051] Example 4: Validation of the combined antitumor effect of ANXA6 and / or PPP2R1A 1. Experimental Method: (1) Collect conditioned medium or culture supernatant of γδ-T cells treated with low dose taxanes, and use proteomics and ELISA to detect the content or release level of ANXA6 and / or PPP2R1A in the conditioned medium or culture supernatant to screen for antitumor-related molecules released by γδ-T cells induced by low dose taxanes.

[0052] (2) Knockdown of ANXA6 and / or PPP2R1A expression in γδ-T cells using siRNA. The knockdown effect was verified by detecting the protein expression levels of ANXA6 and / or PPP2R1A in γδ-T cells using Western blot and by detecting the release levels of ANXA6 and / or PPP2R1A in the conditioned medium of γδ-T cells using ELISA. Subsequently, the conditioned medium of γδ-T cells with knocked-down ANXA6 and / or PPP2R1A was collected and used to treat triple-negative breast cancer cells expressing luciferase or taxane-resistant triple-negative breast cancer cells. After a certain treatment time, luciferase substrate was added, and changes in luciferase signal in tumor cells were detected to evaluate the killing effect of the γδ-T cell conditioned medium on tumor cells. Decreased luciferase signal indicated a reduction in surviving tumor cells and an increase in cell lysis or death.

[0053] (3) Human recombinant ANXA6 protein and / or human recombinant PPP2R1A protein were exogenously added to the culture system of triple-negative breast cancer cells or taxane-resistant triple-negative breast cancer cells. The final concentrations of recombinant ANXA6 protein were set to 0, 1, 5, and 10 μg / mL; the final concentrations of recombinant PPP2R1A protein were set to 0, 5, 10, and 20 μg / mL. For combined supplementation experiments, recombinant ANXA6 protein and recombinant PPP2R1A protein were added simultaneously. After a certain treatment time, the changes in the luminescence signal of tumor cells were detected by luciferase activity assay to evaluate the killing effect of exogenously supplemented ANXA6 and / or PPP2R1A on triple-negative breast cancer cells and taxane-resistant triple-negative breast cancer cells.

[0054] (4) In the replenishment experiment, ANXA6 and / or PPP2R1A expression in γδ-T cells was first knocked down using siRNA, and the conditioned medium of the knocked-down γδ-T cells was collected. Subsequently, recombinant human ANXA6 protein and / or recombinant human PPP2R1A protein were exogenously supplemented into the conditioned medium to reach a preset final concentration. The conditioned medium supplemented with recombinant proteins was then used to treat triple-negative breast cancer cells expressing luciferase or taxane-resistant triple-negative breast cancer cells. After a certain period of treatment, the changes in luciferase signal in tumor cells were detected to evaluate whether exogenous replenishment of ANXA6 and / or PPP2R1A could restore or partially restore the antitumor killing effect of the knocked-down γδ-T cell conditioned medium.

[0055] The amino acid sequences of the ANXA6 and PPP2R1A proteins involved in this embodiment are derived from the UniProtKB / Swiss-Prot database, and the species is human (Homo sapiens).

[0056] The canonical isoform sequence of human Annexin A6 (ANXA6), UniProtKB database accession number P08133, isotype identifier P08133-1, sequence length 673 amino acids, database search date July 14, 2026. The amino acid sequence of human ANXA6 protein is shown in SEQ ID NO:1: (SEQ ID NO:1); The canonical isoform sequence of the 65 kDa regulatory subunit Aα isoform (PPP2R1A) of human serine / threonine protein phosphatase 2A is available in the UniProtKB database under accession number P30153, isoform identifier P30153-1, and is 589 amino acids in length. The database search date was July 14, 2026. The amino acid sequence of the human PPP2R1A protein is shown in SEQ ID NO:2. (SEQ ID NO:2).

[0057] 2. Experimental Results: (1) γδ-T cells treated with low-dose taxanes can release antitumor-related molecules ( Figure 7 A), the levels of ANXA6 and / or PPP2R1A in the conditioned medium or culture supernatant detected by ELISA were significantly increased ( Figure 7 B).

[0058] (2) Compared with the control γδ-T cell conditioned medium, the γδ-T cell conditioned medium obtained after knocking down ANXA6 and / or PPP2R1A showed a decreased antitumor effect on triple-negative breast cancer cells, indicating that ANXA6 and / or PPP2R1A are involved in the anti-triple-negative breast cancer effect mediated by γδ-T cells treated with low-dose taxanes. Figure 8 AC).

[0059] (3) Exogenous supplementation with ANXA6 and / or PPP2R1A can reduce the activity of triple-negative breast cancer cells, increase the proportion of tumor cell death, and enhance or partially restore the antitumor effect of γδ-T cell conditioned medium treated with low-dose taxanes. Figure 7 CD and Figure 8 C).

[0060] The above results indicate that ANXA6 and / or PPP2R1A are involved in the anti-triple-negative breast cancer activity mediated by γδ-T cells after treatment with low-dose taxanes, and can promote apoptosis or death of triple-negative breast cancer cells.

[0061] Example 5: Effects of taxanes combined with γδ-T cells in a triple-negative breast cancer animal model 1. Experimental Method: (1) A triple-negative breast cancer tumor-bearing animal model can be established by inoculating triple-negative breast cancer cells (MDA-MB-231 cells). After the tumor grows to a suitable size, the animals are divided into a control group, a taxane-only treatment group, a γδ-T cell-only treatment group, and a taxane-plus-γδ-T cell treatment group. After administration, tumor volume, tumor weight, animal body weight, and histopathological changes are monitored regularly, and indicators related to proliferation or immune cell infiltration in the tumor tissue can be further detected. In the taxane-plus-γδ-T cell treatment group, taxanes and γδ-T cells are administered simultaneously.

[0062] (2) A taxane-resistant triple-negative breast cancer tumor-bearing animal model can be established by inoculating taxane-resistant triple-negative breast cancer cell lines (MDA-MB-468-LUC-R cells). After the tumors grow to a suitable size, they are treated according to the above grouping method, and the tumor volume, tumor weight, animal body weight, histopathological changes and related anti-tumor indicators are detected.

[0063] 2. Experimental Results The results are as follows Figure 9 AC results showed that, compared with the control group, the taxane-only treatment group, or the γδ-T cell-only treatment group, the taxane-plus-γδ-T cell treatment group significantly inhibited the growth of triple-negative breast cancer tumors. Figure 10As shown, further, in a taxane-resistant triple-negative breast cancer animal model, taxane-based drugs combined with γδ-T cell treatment can still inhibit tumor growth, indicating that the present invention can be used to treat triple-negative breast cancer and can be used to improve the treatment effect of taxane-resistant triple-negative breast cancer.

[0064] Animal weight and histopathological examination of major organs showed that, under the administration conditions, the combination of taxanes and γδ-T cell treatment did not result in significantly increased acute toxicity or severe tissue damage, indicating that this combination regimen has good short-term safety and tolerability. Figure 9 DE).

[0065] Example 6 This embodiment 6 provides a method for preparing a γδ-T cell conditioned medium, the method comprising the following steps: (1) Inoculate γδ-T cells into culture medium and culture them; (2) Add taxane drugs to the culture system in step (1) to a final concentration of 10 nM and continue culturing for 24 hours; (3) Remove the culture medium containing taxanes and wash γδ-T cells with PBS or fresh culture medium to remove or reduce free taxane residues; (4) After adding fresh culture medium to the washed γδ-T cells and culturing for 48 hours, centrifuge to remove cells and cell debris, and collect the culture supernatant to obtain the γδ-T cell conditioned culture medium.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of taxane drugs in the preparation of γδ-T cell antitumor enhancers.

2. Application of taxane drugs in the preparation of agents that enhance the activity of γδ-T cells against drug-resistant tumors.

3. Application of taxane drugs in the preparation of formulations that enhance the release or expression levels of ANXA6 protein and / or PPP2R1A protein in γδ-T cells.

4. Application of ANXA6 protein in the preparation of drugs for anti-tumor or drug-resistant tumors.

5. Application of PPP2R1A protein in the preparation of drugs for anti-tumor or drug-resistant tumors.

6. The use of ANXA6 protein and / or PPP2R1A protein in the preparation of drugs that enhance the antitumor activity of γδ-T cells or in drugs that enhance the anti-drug-resistant tumor activity of γδ-T cells.

7. The application according to any one of claims 1-6, characterized in that, The tumor is triple-negative breast cancer; and / or, the taxane drug is selected from paclitaxel, docetaxel, or cabazitaxel.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises taxanes and γδ-T cells.

9. A method for preparing a γδ-T cell conditioned culture medium, characterized in that, The method includes the following steps: (1) Inoculate γδ-T cells into culture medium and culture them; (2) After adding taxane drugs to the culture system in step (1), continue culturing for 22-50 hours; the final concentration of the taxane drugs is 0.1-100 nM; (3) Retain the cells in the culture system of step (2), remove the culture medium containing taxane drugs, and wash the γδ-T cells; (4) After adding the γδ-T cells from step (3) to fresh culture medium and culturing for 22-50 hours, centrifuge and collect the culture supernatant to obtain the γδ-T cell conditioned culture medium.

10. The use of the conditioned medium prepared by the method described in claim 9 in the preparation of drugs for antitumor or anti-drug-resistant tumors.

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