CN113274502B - Compositions for specific type three-negative breast cancer immunotherapy - Google Patents
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- CN113274502B CN113274502B CN202110487230.5A CN202110487230A CN113274502B CN 113274502 B CN113274502 B CN 113274502B CN 202110487230 A CN202110487230 A CN 202110487230A CN 113274502 B CN113274502 B CN 113274502B
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Abstract
本发明公开了用于特定型三阴乳腺癌免疫治疗的组合物。发明人鉴定了异常的B7‑H3糖基化,并表明在TNBC肿瘤中,NXT基序上B7‑H3的N‑糖基化与其蛋白稳定性和免疫抑制有关。岩藻糖基转移酶FUT8催化B7‑H3发生N‑聚糖核心岩藻糖基化以维持其高表达。FUT8基因敲除可挽救TNBC细胞糖基化的B7‑H3介导的免疫抑制功能。FUT8过表达介导的B7‑H3糖基化异常在TNBC患者中具有重要的生理意义和临床意义。值得注意的是,核心岩藻糖基化抑制剂2F‑Fuc和抗PDL1联合使用可增强对B7‑H3阳性TNBC肿瘤的治疗效果。这些发现表明,靶向FUT8‑B7‑H3轴可能是改善TNBC患者抗肿瘤免疫反应的一种有前途的策略。The invention discloses a composition for immunotherapy of specific type triple-negative breast cancer. The inventors identified aberrant B7‑H3 glycosylation and showed that N‑glycosylation of B7‑H3 at the NXT motif is associated with its protein stability and immunosuppression in TNBC tumors. Fucosyltransferase FUT8 catalyzes the N-glycan core fucosylation of B7‑H3 to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7‑H3-mediated immunosuppressive function in TNBC cells. Abnormal B7‑H3 glycosylation mediated by FUT8 overexpression has important physiological and clinical significance in TNBC patients. Notably, the combination of the core fucosylation inhibitor 2F‑Fuc and anti-PDL1 enhanced the therapeutic effect against B7‑H3-positive TNBC tumors. These findings suggest that targeting the FUT8‑B7‑H3 axis may be a promising strategy to improve antitumor immune responses in TNBC patients.
Description
技术领域technical field
本发明涉及乳腺癌的治疗,特别涉及用于提高具有B7-H3蛋白N-糖基化修饰异常的三阴乳腺癌免疫治疗疗效的组合物及其应用。The invention relates to the treatment of breast cancer, in particular to a composition for improving the curative effect of immunotherapy on triple-negative breast cancer with abnormal N-glycosylation modification of B7-H3 protein and its application.
背景技术Background technique
三阴乳腺癌(Triple Negative Breast Cancer, TNBC)指缺乏ER、PR和人类表皮生长因子受体2 (HER-2)蛋白表达的一种乳腺癌亚型。临床上,TNBC是一种侵袭性亚型,占所有确诊乳腺癌病例的15%至20%,且在较年轻的女性和非洲裔或非洲裔美国妇女中更为普遍。TNBC以浸润性导管癌为主,其特点是分化差、增殖能力强、肿瘤体积大。与其他乳腺癌亚型转移到骨和软组织相比,TNBC易转移扩散到肺和大脑。此外, TNBC的5年生存率约为70%,较其他亚型80%比率低。TNBCs可以细分为7个子类。这些亚类包括基底样BL1和BL2、间充质细胞样M、间充质干细胞样MSL、腔内雄激素受体表达型LAR和免疫调节型IM。TNBC不同亚型间具有异质性,在肿瘤之间存在着形态学、突变表型和信号转导谱的差异。Triple negative breast cancer (Triple Negative Breast Cancer, TNBC) refers to a subtype of breast cancer that lacks ER, PR and human epidermal growth factor receptor 2 (HER-2) protein expression. Clinically, TNBC is an aggressive subtype that accounts for 15% to 20% of all diagnosed breast cancer cases and is more prevalent in younger women and African-American or African-American women. TNBC is mainly invasive ductal carcinoma, which is characterized by poor differentiation, strong proliferative ability, and large tumor volume. TNBC tends to metastasize to the lung and brain compared to other breast cancer subtypes that metastasize to bone and soft tissue. In addition, the 5-year survival rate of TNBC is about 70%, which is lower than the 80% rate of other subtypes. TNBCs can be subdivided into seven subcategories. These subtypes include basal-like BL1 and BL2, mesenchymal-like M, mesenchymal stem-like MSL, luminal androgen receptor-expressing LAR, and immunomodulatory IM. There is heterogeneity among different subtypes of TNBC, and there are differences in morphology, mutation phenotype and signal transduction profile between tumors.
TNBC缺乏特异性靶点,以蒽环类药物和紫杉醇为主的化疗仍是早期和晚期TNBC患者的主要治疗方案,有效治疗此类恶性侵袭性乳腺癌将是一个巨大的挑战。近年的临床试验结果证明,新辅助化疗方案中增加铂和钌类药物可提高化疗敏感的TNBC患者的手术治疗效果。尽管进行了全面和积极的治疗,仍有超过50%的TNBC患者出现复发,其中超过37%的患者在5年内死亡。这可能是由于复发患者中存在尚未明确的多药耐药分子机制,削弱了化疗药对恶性肿瘤的治疗疗效。因此,寻求新的有效治疗TNBC的方法成为乳腺癌研究的主要热点之一。TNBC lacks specific targets. Chemotherapy based on anthracyclines and paclitaxel is still the main treatment option for patients with early and advanced TNBC. Effective treatment of this type of malignant invasive breast cancer will be a huge challenge. The results of clinical trials in recent years have proved that adding platinum and ruthenium drugs to the neoadjuvant chemotherapy regimen can improve the surgical treatment effect of chemotherapy-sensitive TNBC patients. Despite comprehensive and aggressive treatment, more than 50% of TNBC patients relapse, and more than 37% of them die within 5 years. This may be due to the unidentified molecular mechanism of multidrug resistance in relapsed patients, which weakens the therapeutic effect of chemotherapy drugs on malignant tumors. Therefore, seeking new and effective methods for treating TNBC has become one of the main hot spots in breast cancer research.
三阴乳腺癌中仅有小部分患者对现有免疫检查点抑制剂治疗有效,治疗反应率远不及其他肿瘤好,因此急需要探索新的有效的免疫治疗方案。Only a small number of patients with triple-negative breast cancer are effective to the existing immune checkpoint inhibitors, and the treatment response rate is far lower than that of other tumors. Therefore, it is urgent to explore new and effective immunotherapy options.
B7-H3又称CD276或B7RP-2,作为肿瘤特异性相关抗原,在非免疫方面也发挥着重要作用。B7-H3在调节肿瘤细胞的糖酵解、迁移、增殖和化疗耐药中发挥重要作用。虽然前期有证据证明B7-H3可以促进肿瘤免疫应答,但越来越多的证据提示B7-H3在肿瘤中扮演着负性调节的角色。Roth 等对823位前列腺癌根治术后的患者进行队列分析,发现B7-H3在前列腺上皮内瘤的患者中高表达,但正常前列腺的组织中B7-H3的表达水平较低,其染色强度与肿瘤转移、复发及肿瘤特异性死亡成正相关, 此类患者中B7-H3的表达水平与免疫抑制效率的相关性更强。这些数据提示发明人B7-H3在前列腺癌免疫应答中发挥着抑制的作用。同样在肾癌、子宫内膜癌、乳腺癌、结肠癌、卵巢癌、胰腺癌、及非小细胞性肺癌患者中B7-H3的表达也可以作为不良预后的指标。在神经母细胞瘤和神经胶质瘤中,肿瘤细胞表面的4Ig-B7-H3与NK细胞表面抑制性受体结合后抑制NK细胞调节的细胞杀伤能力。Chen等的最新的研究中发现,巨噬细胞与肺癌细胞共培养后可诱导巨噬细胞B7-H3的表达,B7-H3相关的肿瘤特异性巨噬细胞可强烈抑制T细胞调节的免疫应答反应。此外,B7-H3通过上调IL-10和下调IL-12等细胞因子的分泌,使得肿瘤发生免疫逃逸进而促进肿瘤的发生发展。目前B7-H3在抗肿瘤中发挥的作用尚存争议,明确其具体功能及调控的分子机制将为肿瘤免疫治疗提供新的思路。B7-H3是一种高度糖基化的蛋白质。然而,调节癌细胞中糖基化B7-H3表达的分子机制以及糖基化B7-H3影响免疫反应的分子机制仍然不清楚。B7-H3, also known as CD276 or B7RP-2, as a tumor-specific associated antigen, also plays an important role in non-immune aspects. B7-H3 plays an important role in regulating glycolysis, migration, proliferation and chemotherapy resistance of tumor cells. Although previous evidence has proved that B7-H3 can promote tumor immune response, more and more evidence suggests that B7-H3 plays a negative regulatory role in tumors. Roth et al. conducted a cohort analysis of 823 patients after radical prostatectomy and found that B7-H3 was highly expressed in patients with prostate intraepithelial neoplasia, but the expression level of B7-H3 in normal prostate tissues was low, and its staining intensity was related to that of tumors. Metastasis, recurrence, and tumor-specific death were positively correlated, and the expression level of B7-H3 in such patients was more correlated with the efficiency of immunosuppression. These data suggest to the inventors that B7-H3 plays a suppressive role in the prostate cancer immune response. Similarly, the expression of B7-H3 in patients with renal cancer, endometrial cancer, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, and non-small cell lung cancer can also be used as an indicator of poor prognosis. In neuroblastoma and glioma, 4Ig-B7-H3 on the surface of tumor cells binds to inhibitory receptors on the surface of NK cells and inhibits NK cell-mediated cell killing. The latest research by Chen et al. found that the co-culture of macrophages and lung cancer cells can induce the expression of macrophage B7-H3, and B7-H3-related tumor-specific macrophages can strongly inhibit the immune response regulated by T cells . In addition, B7-H3 promotes the occurrence and development of tumors by up-regulating the secretion of IL-10 and down-regulating the secretion of IL-12 and other cytokines, making tumors immune escape. At present, the role of B7-H3 in anti-tumor is still controversial, and clarifying its specific function and molecular mechanism of regulation will provide new ideas for tumor immunotherapy. B7-H3 is a highly glycosylated protein. However, the molecular mechanisms that regulate the expression of glycosylated B7-H3 in cancer cells and how glycosylated B7-H3 affects immune responses remain unclear.
岩藻糖基化,特别是核心岩藻糖基化,是N-糖链中最常见的癌变之一。α-1,6-岩藻糖基转移酶(α-1,6-岩藻糖基转移酶,FUT-8)是目前已知的唯一一种在N-糖链核心产生α-1,6-岩藻糖基化结构的酶。据报道,FUT8在乳腺癌、肺癌、前列腺癌、肝细胞癌、结直肠癌和黑色素瘤等多种癌症中表达上调,表明FUT8与肿瘤生物学特性和患者预后有关。FUT8在肿瘤中的具体作用依然不够明确,现有技术未有通过调控FUT-8来治疗肿瘤。鉴于其只是一种糖基转移酶,并不会影响蛋白固有的性质,一般认为难以作为肿瘤的治疗靶点。Fucosylation, especially core fucosylation, is one of the most common cancers in N-glycans. α-1,6-fucosyltransferase (α-1,6-fucosyltransferase, FUT-8) is the only one known to produce α-1 at the core of the N-glycan chain, 6- Enzymes for fucosylation structures. It has been reported that FUT8 is upregulated in various cancers such as breast cancer, lung cancer, prostate cancer, hepatocellular carcinoma, colorectal cancer, and melanoma, suggesting that FUT8 is related to tumor biological characteristics and patient prognosis. The specific role of FUT8 in tumors is still not clear enough, and the prior art has not treated tumors by regulating FUT-8. Since it is only a glycosyltransferase and does not affect the inherent properties of the protein, it is generally considered difficult to be a therapeutic target for tumors.
发明内容Contents of the invention
本发明的目的在于克服现有的技术的至少一种不足,提供一种可以显著提高三阴乳腺癌治疗疗效的技术。The purpose of the present invention is to overcome at least one deficiency of the existing technology and provide a technology that can significantly improve the therapeutic effect of triple-negative breast cancer.
大多数三阴性乳腺癌(TNBC)患者对抗PD1/PDL1免疫治疗无反应,提示有必要探索免疫检查点靶点。B7-H3是一种高度糖基化的蛋白质。然而,B7-H3糖基化调节的机制以及糖基是否参与免疫抑制尚不清楚。发明人鉴定了异常的B7-H3糖基化,并表明在TNBC肿瘤中,NXT基序上B7-H3的N-糖基化与其蛋白稳定性和免疫抑制有关。岩藻糖基转移酶FUT8催化B7-H3发生 N-聚糖核心岩藻糖基化以维持其高表达。FUT8基因敲除可挽救TNBC细胞糖基化的B7-H3介导的免疫抑制功能。FUT8过表达介导的B7-H3糖基化异常在TNBC患者中具有重要的生理意义和临床意义。值得注意的是,核心岩藻糖基化抑制剂2F-Fuc和抗PDL1联合使用可增强对B7-H3阳性TNBC肿瘤的治疗效果。这些发现表明,靶向FUT8-B7-H3轴可能是改善TNBC患者抗肿瘤免疫反应的一种有前途的策略。Most patients with triple-negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, suggesting the need to explore immune checkpoint targets. B7-H3 is a highly glycosylated protein. However, the mechanism by which B7-H3 glycosylation is regulated and whether glycosyls are involved in immunosuppression remains unclear. The inventors identified aberrant B7-H3 glycosylation and showed that N-glycosylation of B7-H3 at the NXT motif is associated with its protein stability and immunosuppression in TNBC tumors. Fucosyltransferase FUT8 catalyzes the N-glycan core fucosylation of B7-H3 to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7-H3-mediated immunosuppressive function in TNBC cells. Abnormal B7-H3 glycosylation mediated by FUT8 overexpression has important physiological and clinical implications in TNBC patients. Notably, the combination of
本发明所采取的技术方案是:The technical scheme that the present invention takes is:
本发明的第一个方面,提供:A first aspect of the present invention provides:
B7-H3蛋白核心岩藻糖基化修饰干预剂在制备三阴乳腺癌免疫治疗增效剂中的应用。Application of B7-H3 protein core fucosylation modification intervention agent in the preparation of triple-negative breast cancer immunotherapy potentiator.
在一些实例中,所述三阴乳腺癌为糖基化B7-H3阳性的三阴乳腺癌。In some examples, the triple negative breast cancer is glycosylated B7-H3 positive triple negative breast cancer.
在一些实例中,所述糖基化为N-聚糖核心岩藻糖基化。In some examples, the glycosylation is N-glycan core fucosylation.
在一些实例中,所述核心岩藻糖基化修饰干预剂选自糖基转移酶FUT8表达抑制剂、糖基转移酶FUT8活性抑制剂、核心岩藻糖类似物中的至少一种。In some examples, the core fucosylation modification intervention agent is selected from at least one of glycosyltransferase FUT8 expression inhibitors, glycosyltransferase FUT8 activity inhibitors, and core fucose analogs.
在一些实例中,所述核心岩藻糖类似物选自2-氟-L-岩藻糖、6-炔基岩藻糖中的至少一种。In some examples, the core fucose analogue is selected from at least one of 2-fluoro-L-fucose and 6-alkynyl fucose.
在一些实例中,所述糖基转移酶FUT8表达抑制剂为FUT8的siRNA或sgRNA。In some examples, the glycosyltransferase FUT8 expression inhibitor is siRNA or sgRNA of FUT8.
在一些实例中,所述siRNA的序列为siFUT8#1: 5’-CUGCAGUGUGGUGGGUGUCTT-3’或siFUT8 #2: 5’-AGGUCUGUCGAGUUGCUUATT-3’; 所述sgRNA的序列为 sgRNA2: 5’-CACCGACAGCCAAGGGTAAATATGG-3’ 或 sgRNA7 5’-CACCGTGAAGCAGTAGACCACATGA-3’。In some examples, the sequence of the siRNA is siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT-3' or siFUT8#2: 5'-AGGUCUGUCGAGUUGCUUATT-3'; the sequence of the sgRNA is sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' or sgRNA7 5'-CACCGTGAAGCAGTAGACCACATGA-3'.
在一些实例中,所述免疫治疗为anti-PDL1免疫治疗。In some examples, the immunotherapy is anti-PDL1 immunotherapy.
本发明的第二个方面,提供:A second aspect of the present invention provides:
组合物在制备治疗三阴乳腺癌制剂中的应用,所述三阴乳腺癌为糖基化B7-H3阳性的三阴乳腺癌,所述组合物包括:Application of the composition in the preparation of preparations for treating triple negative breast cancer, the triple negative breast cancer is glycosylated B7-H3 positive triple negative breast cancer, the composition comprising:
至少一种B7-H3蛋白核心岩藻糖基化修饰干预剂;以及At least one B7-H3 protein core fucosylation modification intervening agent; and
至少一种免疫治疗制剂。at least one immunotherapeutic agent.
在一些实例中,所述三阴乳腺癌为糖基化B7-H3阳性的三阴乳腺癌。In some examples, the triple negative breast cancer is glycosylated B7-H3 positive triple negative breast cancer.
在一些实例中,所述糖基化为N-聚糖核心岩藻糖基化。In some examples, the glycosylation is N-glycan core fucosylation.
在一些实例中,所述核心岩藻糖基化修饰干预剂选自糖基转移酶FUT8表达抑制剂、糖基转移酶FUT8活性抑制剂、核心岩藻糖类似物。In some examples, the core fucosylation modification intervention agent is selected from a glycosyltransferase FUT8 expression inhibitor, a glycosyltransferase FUT8 activity inhibitor, and core fucose analogs.
在一些实例中,所述核心岩藻糖类似物选自2-氟-L-岩藻糖、6-炔基岩藻糖中的至少一种。In some examples, the core fucose analogue is selected from at least one of 2-fluoro-L-fucose and 6-alkynyl fucose.
在一些实例中,所述糖基转移酶FUT8表达抑制剂为FUT8的siRNA或sgRNA。In some examples, the glycosyltransferase FUT8 expression inhibitor is siRNA or sgRNA of FUT8.
在一些实例中,所述siRNA的序列为siFUT8#1: 5’-CUGCAGUGUGGUGGGUGUCTT-3’或siFUT8 #2: 5’-AGGUCUGUCGAGUUGCUUATT-3’;所述sgRNA的序列为 sgRNA2: 5’-CACCGACAGCCAAGGGTAAATATGG-3’ 或 sgRNA7 5’-CACCGTGAAGCAGTAGACCACATGA-3’。In some examples, the sequence of the siRNA is siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT-3' or siFUT8#2: 5'-AGGUCUGUCGAGUUGCUUATT-3'; the sequence of the sgRNA is sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' or sgRNA7 5'-CACCGTGAAGCAGTAGACCACATGA-3'.
在一些实例中,所述免疫治疗制剂选自为anti-PDL1免疫治疗制剂。In some examples, the immunotherapeutic formulation is selected from anti-PDL1 immunotherapeutic formulations.
本发明的有益效果是:The beneficial effects of the present invention are:
本发明的一些实例,提供了一种有前途的改善TNBC患者抗肿瘤免疫反应策略,打破了现有三阴乳腺癌缺乏有效治疗策略的不足,为延长三阴乳腺癌患者的生存时间提供了一种新的策略。Some examples of the present invention provide a promising strategy for improving the anti-tumor immune response of TNBC patients, break through the lack of effective treatment strategies for existing triple-negative breast cancer, and provide a way to prolong the survival time of patients with triple-negative breast cancer new strategy.
附图说明Description of drawings
图1:三阴乳腺癌组织样品中B7-H3蛋白表达情况;(A、B) B7-H3蛋白在原发性乳腺癌患者样本中的表达。蛋白质印迹分析具有代表性的乳腺癌患者样本中的糖基化B7-H3表达。免疫组化分析乳腺癌患者样本癌与癌旁中的B7-H3表达。(C) 免疫组化分析数据中B7-H3表达与患者总生存关系的曲线。Figure 1: B7-H3 protein expression in triple-negative breast cancer tissue samples; (A, B) B7-H3 protein expression in primary breast cancer patient samples. Western blot analysis of glycosylated B7-H3 expression in representative breast cancer patient samples. Immunohistochemical analysis of B7-H3 expression in cancer and paracancerous samples of breast cancer patients. (C) Curve of the relationship between B7-H3 expression and overall survival of patients in immunohistochemical analysis data.
图2:生物信息学分析B7-H3 mRNA在乳腺癌中的表达情况;(A) BC GenExMiner 网站分析TCGA数据集中乳腺癌的B7-H3表达与患者总生存曲线。根据Hu’S SSP和SCMGENE的两种算法对患者进行分层分析。(B)Kaplan-Meier网站分析TCGA数据集中乳腺癌的B7-H3表达与无复发生存(RFS)和无病生存(DMFS)曲线。通过学生t检验具有统计学意义。所有误差线均表示为3个独立实验的平均值± SD。Figure 2: Bioinformatics analysis of B7-H3 mRNA expression in breast cancer; (A) BC GenExMiner website analysis of B7-H3 expression and overall survival curve of breast cancer in TCGA dataset. Patients were stratified according to two algorithms of Hu’S SSP and SCMGENE. (B) Kaplan-Meier website analysis of B7-H3 expression and recurrence-free survival (RFS) and disease-free survival (DMFS) curves of breast cancer in the TCGA dataset. Statistically significant by Student's t-test. All error bars are expressed as mean ± SD of 3 independent experiments.
图3:三阴乳腺癌细胞系中B7-H3的糖基化修饰类型分析;(A) B7-H3蛋白的糖基化修饰类型。用肽-N-糖苷酶F(PNGase F),糖苷内切酶(Endo H)和双糖O-聚糖酶(O-glycanase)处理细胞裂解物,并通过蛋白质印迹分析进行分析。 (B)用N-连接或O-连接的糖基化抑制剂处理细胞,并通过蛋白质印迹对B7-H3表达进行分析。实心圆,糖基化B7-H3;星形,非糖基化B7-H3。 (C)衣霉素处理细胞,使用流式细胞仪分析细胞表面B7-H3蛋白表达。。Figure 3: Analysis of glycosylation modification types of B7-H3 in triple-negative breast cancer cell lines; (A) Glycosylation modification types of B7-H3 protein. Cell lysates were treated with peptide-N-glycosidase F (PNGase F), endoglycosidase (Endo H) and disaccharide O-glycanase (O-glycanase) and analyzed by Western blot analysis. (B) Cells were treated with N-linked or O-linked glycosylation inhibitors and B7-H3 expression was analyzed by western blot. Solid circles, glycosylated B7-H3; stars, non-glycosylated B7-H3. (C) Cells were treated with tunicamycin, and the expression of B7-H3 protein on the cell surface was analyzed by flow cytometry. .
图4:构建糖基化和非糖基化形式的B7-H3三阴乳腺癌细胞株;(A) N-糖基化模体结构、人源和鼠源B7-H3氨基酸序列示意图。(B、D)本研究中使用的B7-H3 NQ突变体的示意图。数字表示B7-H3上的氨基酸位置 (C、E)通过蛋白质印迹分析检测敲除,糖基化和非糖基化的B7-H3细胞裂解物。Figure 4: Construction of glycosylated and non-glycosylated B7-H3 triple-negative breast cancer cell lines; (A) N-glycosylation motif structure, human and mouse B7-H3 amino acid sequences. (B, D) Schematic representation of the B7-H3 NQ mutant used in this study. Numbers indicate amino acid positions on B7-H3 (C, E) Knockout, glycosylated and aglycosylated B7-H3 cell lysates were examined by Western blot analysis.
图5:糖基化修饰增强B7-H3蛋白的稳定性;(A、B、C,D)指定时间间隔用20 mM环己酰亚胺(CHX)处理MDA-MB-231细胞(A)、HCC1806细胞(B)和HEK293T细胞(C)、表达B7-H3-Flag的MDA-MB-231细胞(D),并通过蛋白质印迹分析检测B7-H3蛋白。通过ImageJ定量糖基化或非糖基化B7-H3的蛋白水平,并用GAPDH标准化 (E)指定的间隔用20mM放线菌酮(CHX)处理细胞,用或不用MG132(100mM)处理6小时,并通过蛋白质印迹分析进行分析 (F) 抑制糖基化B7-H3增强泛素化修饰。B7-H3-8NQ转染的HEK293T细胞用或不用MG132处理后,进行B7-H3免疫沉淀(IP)和蛋白质印迹分析。(G)使用流式细胞仪对细胞表面B7-H3蛋白进行分析。Figure 5: Glycosylation modification enhances the stability of B7-H3 protein; (A, B, C, D) MDA-MB-231 cells were treated with 20 mM cycloheximide (CHX) at specified time intervals (A), HCC1806 cells (B) and HEK293T cells (C), MDA-MB-231 cells (D) expressing B7-H3-Flag, and B7-H3 protein detected by Western blot analysis. Protein levels of glycosylated or non-glycosylated B7-H3 were quantified by ImageJ and normalized with GAPDH. (E) Cells were treated with 20 mM cycloheximide (CHX) with or without MG132 (100 mM) for 6 hr at indicated intervals, and analyzed by Western blot analysis (F) Inhibition of glycosylation B7-H3 enhances ubiquitination modification. B7-H3-8NQ transfected HEK293T cells were treated with or without MG132, followed by B7-H3 immunoprecipitation (IP) and Western blot analysis. (G) Analysis of cell surface B7-H3 protein using flow cytometry.
图6:糖基化B7-H3抑制了T细胞介导的三阴乳腺癌细胞死亡。 (A、B、C)敲除,糖基化和非糖基化的B7-H3三阴乳腺癌细胞与或不与活化的T细胞共培养,在6小时检测T细胞杀伤肿瘤细胞的百分比。Figure 6: Glycosylated B7-H3 inhibits T cell-mediated triple-negative breast cancer cell death. (A, B, C) Knockout, glycosylated and non-glycosylated B7-H3 triple-negative breast cancer cells were co-cultured with or without activated T cells, and the percentage of T cells killing tumor cells was detected at 6 hours.
图7:糖基化B7-H3在体外抑制T细胞增殖。(A、B)在敲除,糖基化和非糖基化的B7-H3三阴乳腺癌细胞存在下,用抗CD3和抗CD28刺激的CD4+ T细胞和CD8+ T细胞的增殖(A)和活性(B)。Figure 7: Glycosylated B7-H3 inhibits T cell proliferation in vitro. (A, B) Proliferation of CD4 + T cells and CD8 + T cells stimulated with anti-CD3 and anti-CD28 in the presence of knockout, glycosylated and non-glycosylated B7-H3 triple-negative breast cancer cells (A ) and activity (B).
图8:体外糖基化B7-H3对三阴乳腺癌细胞的增殖和侵袭能力无影响。(A)糖基化(B7-H3-WT)和非糖基化(B7-H3-8NQ)的B7-H3三阴乳腺癌细胞增殖测定,通过CellTiterGlo进行分析,每个条件重复三次。(B) 糖基化(B7-H3-WT)和非糖基化(B7-H3-8NQ)的B7-H3三阴乳腺癌细胞的平板克隆形成测定。 (C) 糖基化(B7-H3-WT)和非糖基化(B7-H3-8NQ)的B7-H3三阴乳腺癌细胞进行transwell实验,通过计数26小时后迁移到跨孔插入物基底侧的细胞数来量化迁移细胞。Figure 8: In vitro glycosylation of B7-H3 has no effect on the proliferation and invasion of triple-negative breast cancer cells. (A) Proliferation assay of glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8NQ) B7-H3 TNBC cells analyzed by CellTiterGlo in triplicate for each condition. (B) Plate colony formation assay of glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8NQ) B7-H3 triple-negative breast cancer cells. (C) Glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8NQ) B7-H3 triple-negative breast cancer cells migrated to the transwell insert substrate after 26 hours by counting in a transwell assay Side by side cell number to quantify migrated cells.
图9:糖基化的B7-H3抑制移植瘤中免疫细胞的浸润和活性。(A) 过表达了糖基化(B7-H3-WT)和非糖基化(B7-H3-4NQ)的鼠源B7-H3敲除三阴乳腺癌细胞株4T1,异体接种至Balb/c小鼠,在指定的时间点测量肿瘤体积,测量肿瘤重量(n=6)。 (B) 从4T1肿瘤中分离TIL,检测其中的CD4+CD8+ T细胞和NK细胞群比例,在CD8+ T细胞中的GzmB和IFN阳性T细胞的频率。Figure 9: Glycosylated B7-H3 inhibits the infiltration and activity of immune cells in xenografts. (A) The murine B7-H3 knockout triple-negative breast cancer cell line 4T1 overexpressing glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ) was allogeneically inoculated into Balb/c For mice, tumor volumes were measured at indicated time points, and tumor weights were measured (n = 6). (B) TILs were isolated from 4T1 tumors, and the proportion of CD4 + CD8 + T cells and NK cell populations, and the frequency of GzmB and IFN positive T cells among CD8 + T cells were detected.
图10:糖基化的B7-H3不影响移植肿瘤在Balb/c SCID小鼠中的生长。(A)过表达了糖基化(B7-H3-WT)和非糖基化(B7-H3-4NQ)的鼠源B7-H3敲除三阴乳腺癌细胞株4T1,异体接种至Balb/c SCID小鼠并观察肿瘤生长。在指定的时间点测量肿瘤体积,测量肿瘤重量(n=11)。 (B)过表达了糖基化(B7-H3-WT)和非糖基化(B7-H3-4NQ)的人源B7-H3敲除三阴乳腺癌细胞株MDA-MB-231,异体接种至Balb/c SCID小鼠并观察肿瘤生长。在指定的时间点测量肿瘤体积,测量肿瘤重量(n=7)。Figure 10: Glycosylated B7-H3 does not affect the growth of transplanted tumors in Balb/c SCID mice. (A) Mouse-derived B7-H3 knockout triple-negative breast cancer cell line 4T1 overexpressing glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ), allogeneic inoculation into Balb/c SCID mice and observe tumor growth. Tumor volume was measured at indicated time points, and tumor weight was measured (n = 11). (B) Human B7-H3 knockout triple-negative breast cancer cell line MDA-MB-231 overexpressing glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ), allogeneic inoculation to Balb/c SCID mice and observe tumor growth. Tumor volume was measured at indicated time points, and tumor weight was measured (n=7).
图11:FUT8催化B7-H3发生糖基化修饰。(A)在MDA-MB-231和HCC1806细胞系中敲除FUT8并通过蛋白质印迹分析检测进行B7-H3蛋白。 (B) 实时荧光定量PCR显示敲低FUT8对B7-H3 mRNA表达无影响(C)用阴性对照或FUT8 sgRNA敲除的MDA-MB-231-WT/8NQ细胞的全细胞裂解物进行LcH亲和层析,通过蛋白质印迹方法分析FUT8敲低对B7-H3表达的影响。(D)用阴性对照或Fut8基因的gRNA单独转导的MDA-MB-231-WT/8NQ细胞进行流式细胞术分析,检测核心岩藻糖(Lens culinaris凝集素[LCA])和B7-H3在细胞表面的表达。Figure 11: FUT8 catalyzes the glycosylation modification of B7-H3. (A) FUT8 was knocked out in MDA-MB-231 and HCC1806 cell lines and B7-H3 protein was detected by western blot analysis. (B) Real-time fluorescent quantitative PCR showed that knockdown of FUT8 had no effect on B7-H3 mRNA expression (C) LcH affinity was performed with negative control or whole cell lysates of FUT8 sgRNA knockout MDA-MB-231-WT/8NQ cells Chromatography, the effect of FUT8 knockdown on B7-H3 expression was analyzed by Western blot. (D) Flow cytometric analysis of MDA-MB-231-WT/8NQ cells transduced with negative control or Fut8 gene gRNA alone to detect core fucose (Lens culinaris lectin [LCA]) and B7-H3 expression on the cell surface.
图12:沉默FUT8恢复糖基化B7-H3对免疫的抑制作用。(A)siRNA FUT8的蛋白质印迹分析(B)在过表达了糖基化(B7-H3-WT)和非糖基化(B7-H3-4NQ)的人源B7-H3敲除三阴乳腺癌细胞株MDA-MB-231中敲低FUT8,然后与或不与活化的T细胞共培养,共培养6小时后通过流式检测T细胞介导的肿瘤细胞杀伤的百分比。(C)流式细胞术分析在FUT8沉默糖基化B7-H3的三阴乳腺癌细胞存在下, 用抗CD3和抗CD28刺激的CD4+T细胞增殖情况。(D、E、F、G)在FUT8沉默糖基化B7-H3的三阴乳腺癌细胞存在下,用抗CD3和抗CD28刺激的CD4+T细胞和CD8+T细胞表达IL-2和IFNγ指标比例。Figure 12: Silencing of FUT8 restores the immunosuppressive effect of glycosylated B7-H3. (A) Western blot analysis of siRNA FUT8 (B) overexpression of glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-4NQ) human B7-H3 knockout triple-negative breast cancer FUT8 was knocked down in the cell line MDA-MB-231, and then co-cultured with or without activated T cells. After 6 hours of co-culture, the percentage of T cell-mediated tumor cell killing was detected by flow cytometry. (C) Flow cytometry analysis of the proliferation of CD4+ T cells stimulated with anti-CD3 and anti-CD28 in the presence of FUT8-silenced glycosylated B7-H3 triple-negative breast cancer cells. (D, E, F, G) CD4+ T cells and CD8+ T cells stimulated with anti-CD3 and anti-CD28 express IL-2 and IFNγ in the presence of FUT8-silenced glycosylated B7-H3 triple-negative breast cancer cells Indicator ratio.
图13:阻断核心岩藻糖基化下调B7-H3表达并增强T细胞毒性。(A、C)用DMSO或200μM或300μM 2F-Fuc处理MDA-MB-231细胞(A)和4T1细胞(C),用流式细胞术分析细胞表面B7-H3表达。(B)用DMSO或400μM2F-Fuc预处理的糖基化和非糖基化的B7-H3三阴乳腺癌细胞,然后与或不与活化的T细胞共培养,共培养6小时后通过流式检测T细胞介导的肿瘤细胞杀伤的百分比。Figure 13: Blockade of core fucosylation downregulates B7-H3 expression and enhances T cell toxicity. (A, C) MDA-MB-231 cells (A) and 4T1 cells (C) were treated with DMSO or 200 μM or 300
图14:阻断B7-H3核心岩藻糖基化增强PD-L1抗体抗肿瘤免疫敏感性。(A) 治疗组(对照,抗PD-L1,2F-Fuc,抗PD-L1和2F-Fuc)的示意图。(B)异体移植4T1-B7-H3KO-B7-H3-WT肿瘤并按治疗方案进行处理,观察的Balb/c小鼠的肿瘤生长情况,在指定的时间点测量肿瘤体积及最终瘤重(n=5。)(C)免疫组化检测不同处理组肿瘤B7-H3的表达。(D)CD8+T细胞,CD4+T细胞和NK细胞中IFNγ阳性细胞的频率(E)Tunel染色检测不同处理组肿瘤的凋亡情况。Figure 14: Blocking B7-H3 core fucosylation enhances the anti-tumor immune sensitivity of PD-L1 antibody. (A) Schematic representation of treatment groups (control, anti-PD-L1, 2F-Fuc, anti-PD-L1 and 2F-Fuc). (B) 4T1-B7-H3KO-B7-H3-WT tumor was xenografted and treated according to the treatment plan, the tumor growth of Balb/c mice was observed, and the tumor volume and final tumor weight were measured at the designated time points (n =5.) (C) Immunohistochemical detection of B7-H3 expression in tumors of different treatment groups. (D) Frequency of IFNγ-positive cells in CD8+T cells, CD4+T cells and NK cells (E) Tunel staining to detect tumor apoptosis in different treatment groups.
具体实施方式detailed description
下面结合实验,进一步说明本发明的技术方案。The technical solution of the present invention will be further described below in combination with experiments.
鉴定三阴乳腺癌中B7-H3的糖基化类型及位点Identification of glycosylation types and sites of B7-H3 in triple-negative breast cancer
1.1 B7-H3主要以糖基化形式高表达于大部分乳腺癌组织中1.1 B7-H3 is mainly highly expressed in the form of glycosylation in most breast cancer tissues
为明确B7-H3蛋白在人乳腺癌肿瘤组织中的表达情况,发明人随机选取15对乳腺癌与匹配的癌旁组织蛋白进行WB检测分析,WB结果提示B7-H3蛋白主要表达在90-110KD(黑色圆圈指示处)分子量范围内,然而B7-H3的相对分子量为45-66KD,根据前期文献报道B7-H3存在糖基化修饰,发明人猜想肿瘤组织中B7-H3可能以糖基化形式存在;另外对比于正常乳腺组织,发明人发现在肿瘤组织中B7-H3蛋白的表达较高,这说明在乳腺癌组织中高表达糖基化形式B7-H3 (图1A);为进一步明确是否B7-H3蛋白高表达与三阴乳腺的不良预后相关,发明人对来自中山大学肿瘤防治中心的1999-2005年病理科的三阴乳腺癌样本进行切片和免疫组化染色,并统计每例样品中正常组织和肿瘤组织的染色评分。免疫组化依据评分依据中位数进行生存分析,组化结果提示肿瘤组织中B7-H3显著高正常组织(图1B),患者生存分析发现B7-H3高表达组患者生存时间比B7-H3低表达组更短,结果具有统计学差异(P=0.033,图1C)。In order to clarify the expression of B7-H3 protein in human breast cancer tumor tissue, the inventor randomly selected 15 pairs of breast cancer and matched paracancerous tissue proteins for WB detection and analysis. The WB results indicated that B7-H3 protein was mainly expressed at 90-110KD (indicated by the black circle) is within the molecular weight range, but the relative molecular weight of B7-H3 is 45-66KD. According to previous literature reports, there is glycosylation modification of B7-H3. The inventor guessed that B7-H3 in tumor tissue may be in the form of glycosylation In addition, compared with normal breast tissue, the inventors found that the expression of B7-H3 protein in tumor tissue was higher, which indicated that the glycosylated form of B7-H3 was highly expressed in breast cancer tissue (Figure 1A); in order to further clarify whether B7 The high expression of -H3 protein is related to the poor prognosis of triple-negative breast cancer. The inventors sectioned and immunohistochemically stained triple-negative breast cancer samples from the Pathology Department of Sun Yat-Sen University Cancer Prevention and Control Center from 1999 to 2005, and counted the number of samples in each sample. Staining scoring of normal and tumor tissues. Survival analysis was performed based on the median score of immunohistochemistry. Histochemical results showed that B7-H3 in tumor tissue was significantly higher than that in normal tissue (Figure 1B). Survival analysis of patients found that the survival time of patients with high expression of B7-H3 was shorter than that of patients with B7-H3 The expression group was shorter and the results were statistically different (P=0.033, Figure 1C).
仅在三阴乳腺癌中B7-H3 mRNA水的高表达与不良预后相关High expression of B7-H3 mRNA water only in triple-negative breast cancer is associated with poor prognosis
为观察B7-H3 mRNA水平的差异与三阴乳腺癌预后的相关性,发明人使用bc-GenExMiner网站分析TCGA数据库中B7-H3 mRNA高低与患者总生存期(OS)的关系,采用SCMGENE与 Hu's SSP两种算法分析比较B7-H3 mRNA表达与乳腺癌各分子亚型预后的相关关系。结果提示仅在Basal-like型的三阴乳腺癌中上述两种计算方法均有统计学差异,B7-H3 mRNA高表达的患者,其总生存期较短(OS)(P=0.0116和P=0.0046, 图2A)。同时发明人也使用KM-Plot网站进一步分析TCGA数据库中B7-H3 mRNA的表达差异与患者无复发生存(RFS)和无远处转移生存期(DMFS)的相关性,发现仅在Basal-like型患者中B7-H3 mRNA表达与RFS 和DMFS呈负相关,且具有统计学意义(P=0.0043,P=0.047),而其他乳腺癌亚型没有此特征(图2B)。In order to observe the correlation between the difference in B7-H3 mRNA level and the prognosis of triple-negative breast cancer, the inventors used the bc-GenExMiner website to analyze the relationship between the level of B7-H3 mRNA in the TCGA database and the overall survival (OS) of patients, using SCMGENE and Hu's Two SSP algorithms were used to analyze and compare the correlation between B7-H3 mRNA expression and the prognosis of each molecular subtype of breast cancer. The results indicated that only in Basal-like triple-negative breast cancer, there were statistical differences between the above two calculation methods. Patients with high expression of B7-H3 mRNA had shorter overall survival (OS) (P=0.0116 and P=0.0116, respectively). 0.0046, Figure 2A). At the same time, the inventor also used the KM-Plot website to further analyze the correlation between the expression difference of B7-H3 mRNA in the TCGA database and the recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) of patients, and found that only in Basal-like type B7-H3 mRNA expression was negatively correlated with RFS and DMFS in patients with statistical significance (P=0.0043, P=0.047), while other breast cancer subtypes did not have this feature (Fig. 2B).
在三阴乳腺癌中B7-H3主要发生N-糖基化修饰B7-H3 mainly undergoes N-glycosylation modification in triple-negative breast cancer
2015年Min-Huey Chen等通过糖基化质谱的方式鉴定出口腔癌细胞中的B7-H3主要发生N-糖基化修饰并鉴定了8个N-糖基化位点.为进一步确定三阴乳腺癌中B7-H3是否存在其他糖基化修饰形式,发明人选取MDA-MB-231和HCC1806三阴乳腺癌细胞株,用去除所有N-糖糖链结构的肽-N-糖苷酶F(PNGase F)、去除高甘露糖和部分低聚糖的糖苷内切酶(Endo H)、去除所有O连接的双糖O-聚糖酶(O-glycanase)分别处理两株细胞株蛋白,结果显示当使用PNGase F时B7-H3由110KD(黑色圆圈)显著下降至70KD(黑色星号),而Endo H和O-glycanase无显著变化,这提示B7-H3主要发生N-糖基化修饰(图3A)。使用N-连接糖基化抑制剂Tunicamycin(TM),和O-连接糖基化抑制剂Thiamet G及PUGNAc处理MDA-MB-231和Hcc1806三阴乳腺癌细胞株,发明人发现N-糖基化抑制剂TM能够显著抑制糖基化B7-H3的表达使其分子量下降至70KD(图3B)。另外发明人用不同浓度TM处理MDA-MB-231和HCC1806细胞,流式检测处理 24小时的时候细胞膜表面B7-H3蛋白的表达情况,流式结果提示N-连接糖基化抑制剂处理后细胞膜表面的B7-H3明显减少,并与TM的浓度和处理时间相关(图3C)。In 2015, Min-Huey Chen et al. identified by glycosylation mass spectrometry that B7-H3 in oral cancer cells mainly undergoes N-glycosylation modification and identified 8 N-glycosylation sites. Whether there are other glycosylation modification forms of B7-H3 in breast cancer, the inventors selected MDA-MB-231 and HCC1806 triple-negative breast cancer cell lines, and used peptide-N-glycosidase F( PNGase F), endoglycosidase (Endo H) to remove high mannose and partial oligosaccharides, and O-glycanase (O-glycanase) to remove all O-linked disaccharides were used to treat the proteins of the two cell lines, and the results showed that When PNGase F was used, B7-H3 decreased significantly from 110KD (black circle) to 70KD (black asterisk), while Endo H and O-glycanase had no significant changes, which suggested that B7-H3 mainly undergoes N-glycosylation modification (Fig. 3A). The inventors found that N-glycosylation Inhibitor TM can significantly inhibit the expression of glycosylated B7-H3 and reduce its molecular weight to 70KD (Fig. 3B). In addition, the inventors treated MDA-MB-231 and HCC1806 cells with different concentrations of TM, and detected the expression of B7-H3 protein on the cell membrane surface by flow cytometry after 24 hours of treatment. B7-H3 on the surface was significantly reduced and correlated with the concentration of TM and treatment time (Fig. 3C).
构建糖基化B7-H3的三阴乳腺癌细胞模型Construction of triple-negative breast cancer cell model with glycosylated B7-H3
N-糖基化修饰主要发生在一段保守的氨基酸序列Asn-X-Thr/Ser (X ≠ P)中,细胞内合成的聚糖链通过相关转移酶连接至肽链天冬酰胺的酰胺氮上(图4A),依据该模体结构对人源(8个位点:N91,N104,N189,N215,N309,N322,N407,N433)和鼠源(4个位点:N91,N104,N189,N215)的基因序列进行突变改造,将保守序列中的天冬酰胺突变为谷氨酰胺(图4B,4D),成功构建得到人源糖基化B7-H3-WT和突变B7-H3-8NQ质粒,及鼠源糖基化B7-H3-WT和突变B7-H3-4NQ质粒;同时在N端连接Flag标签序列,构建于含有启动子CMV的载体上以促进其在细胞中的表达。通过CRISPR-Cas9基因敲除技术靶向敲除人源三阴乳腺癌MDA-MB-231和HCC1806细胞和鼠源三阴乳腺癌4T1细胞株中的B7-H3,筛选出单克隆B7-H3-KnockOut细胞株,同时在敲除细胞株的基础上回复表达糖基化形式和非糖基化形式质粒,构建出糖基化形式和非糖基化形式B7-H3的人源(MDA-MB-231-B7-H3KO-WT和MDA-MB-231-B7-H3KO-8NQ, HCC1806-B7-H3KO-WT和HCC1806-B7-H3KO-8NQ)与鼠源三阴乳腺癌细胞模型(4T1-B7-H3KO-WT和4T1-B7-H3KO-4NQ);WB实验结果显示人源糖基化B7-H3分子量大约110kd,突变后约70KD,而鼠源的分别为55KD和40KD(图4C,4E)。N-glycosylation modification mainly occurs in a conserved amino acid sequence Asn-X-Thr/Ser (X ≠ P), and the glycan chain synthesized in the cell is connected to the amide nitrogen of the peptide chain asparagine through a related transferase (Fig. 4A), according to the motif structure, human (8 sites: N91, N104, N189, N215, N309, N322, N407, N433) and mouse sources (4 sites: N91, N104, N189, The gene sequence of N215) was mutated, and the asparagine in the conserved sequence was mutated into glutamine (Figure 4B, 4D), and the human glycosylated B7-H3-WT and mutant B7-H3-8NQ plasmids were successfully constructed , and mouse-derived glycosylated B7-H3-WT and mutant B7-H3-4NQ plasmids; at the same time, the Flag tag sequence was connected to the N-terminus, and constructed on a vector containing the promoter CMV to promote its expression in cells. Targeted knockout of B7-H3 in human triple-negative breast cancer MDA-MB-231 and HCC1806 cells and mouse triple-negative breast cancer 4T1 cell line by CRISPR-Cas9 gene knockout technology, screening out monoclonal B7-H3- KnockOut cell line, on the basis of the knockout cell line, restore the expression of glycosylated form and non-glycosylated form of the plasmid, and construct the human source of glycosylated form and non-glycosylated form of B7-H3 (MDA-MB- 231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ, HCC1806-B7-H3KO-WT and HCC1806-B7-H3KO-8NQ) and mouse triple negative breast cancer cell model (4T1-B7- H3KO-WT and 4T1-B7-H3KO-4NQ); WB experiment results showed that the molecular weight of human glycosylated B7-H3 was about 110kD, and about 70KD after mutation, while that of mouse was 55KD and 40KD respectively (Figure 4C, 4E).
蛋白发生糖基化其稳定性增强,在细胞膜上的表达增加Glycosylation of the protein increases its stability and increases its expression on the cell membrane
为明确N-糖基化修饰是否影响B7-H3蛋白的稳定性,用蛋白合成抑制剂环己酰亚胺(CHX)处理MDA-MB-231、HCC1806和HEK293T细胞株,收集不同处理时间点细胞进行WB检测,结果显示非糖基化的B7-H3(黑色星号)降解较糖基化蛋白快(图5A-C)。在MDA-MB-231-B7-H3KO细胞中过表达带Flag标签的糖基化与非糖基化B7-H3,对比其降解速率也能得出一致的结论(图5D)。使用MG132阻断蛋白酶体途径和自噬溶酶体途径,同时予以CHX抑制蛋白合成, MG132处理组的非糖基化B7-H3并未随着处理时间的延长而出现明显下降,这提示非糖基化形式B7-H3主要通过蛋白酶体途径发生降解(图5E)。为探索非糖基化B7-H3是否能发生泛素化修饰,发明人在HEK 293T细胞中外转B7-H3-8NQ及野生型泛素(Ub),当两个质粒共同转染时B7-H3-8NQ可以发生泛素化修饰,且在MG132阻断降解后其泛素化水平增高(图5F)。检测上述构建细胞模型中B7-H3在细胞膜上的表达情况,仅当B7-H3发生糖基化时该蛋白在细胞膜上的表达才明显增高,这提示糖基化修饰不仅可以增B7-H3蛋白的稳定性还可以促进它在细胞膜上表达(图5G)。In order to clarify whether N-glycosylation modification affects the stability of B7-H3 protein, MDA-MB-231, HCC1806 and HEK293T cell lines were treated with protein synthesis inhibitor cycloheximide (CHX), and cells were collected at different treatment time points WB detection was performed, and the results showed that non-glycosylated B7-H3 (black asterisk) was degraded faster than glycosylated protein (Fig. 5A-C). In MDA-MB-231-B7-H3KO cells, glycosylated and non-glycosylated B7-H3 with Flag tag was overexpressed, and a consistent conclusion could be drawn by comparing the degradation rate (Fig. 5D). Using MG132 to block the proteasome pathway and autophagy-lysosome pathway, and at the same time giving CHX to inhibit protein synthesis, the non-glycosylated B7-H3 in the MG132 treatment group did not decrease significantly with the prolongation of treatment time, which suggested that non-glycosylated B7-H3 The kylated form B7-H3 was mainly degraded by the proteasomal pathway (Fig. 5E). In order to explore whether non-glycosylated B7-H3 can undergo ubiquitination modification, the inventors transfected B7-H3-8NQ and wild-type ubiquitin (Ub) in HEK 293T cells. When the two plasmids were co-transfected, B7-H3 -8NQ can undergo ubiquitination modification, and its ubiquitination level increases after MG132 blocks degradation (Fig. 5F). Detecting the expression of B7-H3 on the cell membrane in the cell model constructed above, the expression of the protein on the cell membrane is significantly increased only when B7-H3 is glycosylated, which suggests that glycosylation modification can not only increase the expression of B7-H3 protein The stability of can also promote its expression on the cell membrane (Fig. 5G).
、糖基化B7-H3介导三阴乳腺癌发生免疫逃逸、Glycosylated B7-H3 mediates immune escape in triple-negative breast cancer
2.1 体外T淋巴细胞对糖基化B7-H3三阴乳腺癌细胞的杀伤能力显著降低2.1 The killing ability of T lymphocytes to glycosylated B7-H3 triple-negative breast cancer cells in vitro was significantly reduced
为探索糖基化与非糖基化B7-H3对肿瘤免疫差异,发明人用CD28和CD3抗体在体外非特异性的激活健康献血者的T淋巴细胞三至六天,将构建成功的B7-H3-WT和B7-H3-8NQ过表达的MDA-MB-231与HCC1806三阴乳腺癌细胞株与效应T细胞按15:1共培养6-12小时;收集细胞流式细胞分析仪检测凋亡指标Caspase-3阳性的肿瘤细胞比例。流式细胞术结果显示T细胞诱导糖基化B7-H3高表达的三阴乳腺癌细胞的凋亡能力显著减低(图6A,图6C)。MDA-MB-231细胞株培养结束后,吸取部分上清用乳酸脱氢酶试剂盒检测T细胞杀伤肿瘤的效率;乳酸脱氢酶检测结果发现T细胞对糖基化B7-H3细胞的杀伤比率明显下降,但对非糖基化B7-H3的杀伤能力强(图6B)。In order to explore the difference between glycosylated and non-glycosylated B7-H3 on tumor immunity, the inventors used CD28 and CD3 antibodies to non-specifically activate T lymphocytes of healthy blood donors in vitro for three to six days, and successfully constructed B7-H3 -WT and B7-H3-8NQ overexpressed MDA-MB-231 and HCC1806 triple-negative breast cancer cell line and effector T cells were co-cultured at a ratio of 15:1 for 6-12 hours; cells were collected to detect apoptosis indicators by flow cytometry The percentage of tumor cells positive for Caspase-3. The results of flow cytometry showed that the apoptosis ability of T cells inducing triple-negative breast cancer cells with high expression of glycosylated B7-H3 was significantly reduced ( FIG. 6A , FIG. 6C ). After the MDA-MB-231 cell line culture was completed, a part of the supernatant was drawn to detect the tumor killing efficiency of T cells with a lactate dehydrogenase kit; the results of lactate dehydrogenase detection showed the killing ratio of T cells to glycosylated B7-H3 cells Significantly decreased, but the killing ability to non-glycosylated B7-H3 was strong (Fig. 6B).
体外糖基化B7-H3三阴乳腺癌细胞抑制T淋巴细胞的增殖Glycosylated B7-H3 triple-negative breast cancer cells inhibit the proliferation of T lymphocytes in vitro
为进一步确定糖基化B7-H3与非糖基化对T细胞功能的影响,用活细胞荧光标记染料羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)染色T细胞,上述构建成功的三阴乳腺癌细胞株经80Gy剂量照射后,与染色标记的T细胞共同种入已经包被了CD28和CD3抗体的孔板中,共培养4-7天后用流式细胞术检测T细胞的增殖情况。实验结果表明,糖基化B7-H3细胞株可以显著抑制CD4+和CD8+T细胞的增殖而非糖基化B7-H3-8NQ对T细胞增殖抑制作用不明显,这提示糖基化B7-H3具有抑制T细胞的增殖能力和活性(图7A,7B)。In order to further determine the effect of glycosylated B7-H3 and non-glycosylated on T cell function, T cells were stained with living cell fluorescent labeling dye hydroxyfluorescein diacetate succinimide lipid (CFSE), and the above-mentioned successful construction After the triple-negative breast cancer cell lines were irradiated at a dose of 80Gy, they were planted together with stained and labeled T cells into well plates coated with CD28 and CD3 antibodies, and the proliferation of T cells was detected by flow cytometry after co-cultivation for 4-7 days Condition. The experimental results show that glycosylated B7-H3 cell lines can significantly inhibit the proliferation of CD4 + and CD8 + T cells, but glycosylated B7-H3-8NQ has no obvious inhibitory effect on T cell proliferation, which suggests that glycosylated B7- H3 has the ability to inhibit the proliferation and activity of T cells (Figure 7A, 7B).
体外B7-H3蛋白发生糖基化修饰对三阴乳腺癌细胞的增殖迁移无显著影响Glycosylation modification of B7-H3 protein in vitro has no significant effect on the proliferation and migration of triple-negative breast cancer cells
接下来,为了明确糖基化和非糖基化B7-H3除肿瘤免疫以外的其他对肿瘤的影响,发明人利用体外MTT实验和克隆形成实验研究糖基化和非糖基化B7-H3对肿瘤细胞增殖和克隆形成有无差异,连续7天观察记录细胞生长情况,生长曲线提示糖基化与非糖基化B7-H3三阴乳腺癌细胞株的增殖能力无显著无差异;克隆形成观察到相似现象,糖基化与非糖基化B7-H3细胞的克隆形成能力无区别(图8A,8B)。将上述构建成功肿瘤细胞株放置Transwell小室中培养16-24小时观察不同肿瘤细胞穿透小室通透膜的数量,用以评估肿瘤细胞的迁移能力,实验结果证明基化与非糖基化B7-H3对三阴乳腺癌细胞的迁移能力的影响无差别(图8C)。Next, in order to clarify the effects of glycosylated and non-glycosylated B7-H3 on tumors other than tumor immunity, the inventors used in vitro MTT experiments and clone formation experiments to study the effects of glycosylated and non-glycosylated B7-H3 on tumors. Whether there is any difference in tumor cell proliferation and clone formation, observe and record the cell growth for 7 consecutive days, the growth curve shows that there is no significant difference in the proliferation ability of glycosylated and non-glycosylated B7-H3 triple-negative breast cancer cell lines; clone formation observation Seeing a similar phenomenon, there was no difference in the clonogenic ability of glycosylated and non-glycosylated B7-H3 cells (Fig. 8A, 8B). The successfully constructed tumor cell lines were placed in the Transwell chamber and cultured for 16-24 hours to observe the number of different tumor cells penetrating the permeable membrane of the chamber to evaluate the migration ability of the tumor cells. The experimental results proved that sylated and non-glycosylated B7- There was no difference in the effect of H3 on the migration ability of triple-negative breast cancer cells ( FIG. 8C ).
糖基化B7-H3显著促进免疫正常小鼠移植瘤生长并抑制肿瘤中T淋巴细胞的浸润和活性Glycosylated B7-H3 significantly promotes the growth of xenograft tumors in normal immune mice and inhibits the infiltration and activity of T lymphocytes in tumors
将4T1-B7-H3KO-WT, 4T1-B7-H3KO-4NQ两组细胞株在免疫正常Balb/c小鼠腹部乳房脂肪垫接种,构建糖基化与非糖基化B7-H3的三阴乳腺癌动物模型;定期测量记录小鼠肿瘤体积,实验结束后取出小鼠肿瘤称重;实验结果发现糖基化B7-H3能明显促进小鼠移植瘤生长,统计学分析发现小鼠肿瘤体积和瘤重较4T1-B7-H3KO-4NQ组别显著增加(图9A)。解离小鼠肿瘤进行流式,分析小鼠肿瘤中浸润免疫细胞;对比 4T1-B7-H3KO-WT组与4T1-B7-H3KO-4NQ流式结果,发现糖基化B7-H3组其浸润的CD4+T细胞CD8+T细胞和NK细胞数量明显减少,而且浸润的CD8+T细胞中的活性指标IFN-γ及Granzyme的含量也明显减低(图9B).以上证据提示糖基化B7-H3可以通过抑制肿瘤中免疫细胞的浸润和活性来抑制肿瘤免疫,从而促进肿瘤的发生发展.Two groups of cell lines 4T1-B7-H3KO-WT and 4T1-B7-H3KO-4NQ were inoculated in the abdominal mammary fat pad of immune normal Balb/c mice to construct triple negative mammary glands with glycosylated and non-glycosylated B7-H3 Cancer animal model; regularly measure and record the tumor volume of the mice, and take out the tumors of the mice after the experiment and weigh them; the results of the experiment found that glycosylated B7-H3 can significantly promote the growth of transplanted tumors in mice, and statistical analysis found that the tumor volume and tumor volume of mice The weight was significantly increased compared with the 4T1-B7-H3KO-4NQ group (Fig. 9A). Dissociate mouse tumors for flow cytometry, and analyze the infiltrating immune cells in the mouse tumors; compare the results of flow cytometry between the 4T1-B7-H3KO-WT group and the 4T1-B7-H3KO-4NQ group, and found that the infiltrating immune cells in the glycosylated B7-H3 group The number of CD4 + T cells, CD8 + T cells and NK cells was significantly reduced, and the content of activity indicators IFN-γ and Granzyme in the infiltrating CD8 + T cells was also significantly reduced (Figure 9B). The above evidences suggest that glycosylated B7-H3 It can inhibit tumor immunity by inhibiting the infiltration and activity of immune cells in tumors, thereby promoting the occurrence and development of tumors.
2.5 糖基化B7-H3对免疫缺陷小鼠移植瘤生长无显著影响2.5 Glycosylated B7-H3 has no significant effect on the growth of transplanted tumors in immunodeficient mice
为进一步验证糖基化B7-H3主要是通过对免疫系统的调节来发挥促进肿瘤的作用,发明人将4T1-B7-H3KO-WT, 4T1-B7-H3KO-4NQ两株细胞株分别接种于免疫缺陷的Balb/c Nude裸鼠腹部双侧乳房脂肪垫,定期测量记录小鼠肿瘤体积,实验结束后取出小鼠肿瘤称重;小鼠肿瘤生长曲线提示糖基化B7-H3在免疫缺陷小鼠中对肿瘤的生长无影响,对小鼠肿瘤瘤重进行统计分析发现4T1-B7-H3KO-WT组与 4T1-B7-H3KO-4NQ组无差异(图10A)。另外发明人还将人源的MDA-MB-231-B7-H3KO-WT和MDA-MB-231-B7-H3KO-8NQ细胞株也接种于免疫缺陷的Balb/c Nude裸鼠腹部双侧乳房脂肪垫,定期测量记录。实验结果与上述鼠源细胞株结论一致,人源糖基化和非糖基化B7-H3细胞株的成瘤能力和对肿瘤体积和瘤重的影响无明显差别(图10B)。以上结果发明人进一步得三阴乳腺癌动物模型中糖基化B7-H3主要通过免疫系统影响肿瘤生长。In order to further verify that glycosylated B7-H3 mainly plays a role in promoting tumors through the regulation of the immune system, the inventors inoculated two cell lines, 4T1-B7-H3KO-WT and 4T1-B7-H3KO-4NQ, in the immune system, respectively. The bilateral mammary fat pads in the abdomen of deficient Balb/c Nude nude mice were regularly measured and recorded for the tumor volume of the mice. After the experiment, the tumors of the mice were taken out and weighed. The medium had no effect on the growth of the tumor, and the statistical analysis of the tumor weight of the mice found that there was no difference between the 4T1-B7-H3KO-WT group and the 4T1-B7-H3KO-4NQ group ( FIG. 10A ). In addition, the inventors also inoculated the human MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ cell lines into the abdominal bilateral breast fat of immunodeficient Balb/c Nude nude mice Pad, periodic measurement records. The experimental results were consistent with the conclusions of the mouse-derived cell lines above, and there was no significant difference between the human-derived glycosylated and non-glycosylated B7-H3 cell lines in their tumorigenic ability and their effects on tumor volume and tumor weight (Fig. 10B). From the above results, the inventors further found that in animal models of triple-negative breast cancer, glycosylated B7-H3 mainly affects tumor growth through the immune system.
、B7-H3发生糖基化修饰的分子机制, Molecular mechanism of glycosylation modification of B7-H3
为探索FUT8与B7-H3之间的关系,发明人选取不同的sgRNA导向敲除MDA-MB-231和HCC1806细胞株中的FUT8,并检测B7-H3的表达情况,实验结果发现当敲除FUT8时糖基化的B7-H3蛋白显著减少但B7-H3的mRNA无显著变化,这提示FUT8可以通过调节B7-H3糖基化修饰来影响B7-H3的表达,但对B7-H3的mRNA无影响(图11A,11B)。为进一步明确B7-H3能否发生核心岩藻糖修饰,发明人利用能与核心岩藻糖特异性结合的凝集素LCH(LCA)抗体,进行凝集素富集实验(主要用来反应FUT8调节的核心岩藻糖蛋白的表达情况),结果显示将FUT8敲除之后核心岩藻糖型的B7-H3显著减少,且FUT8降低时对总的糖基化B7-H3也有一定影响(图11C)。用流式方式检测FUT8敲除后MDA-MB-231细胞膜上B7-H3的表达情况,结果发现FUT8可以显著减少细胞膜表面核心岩藻蛋白的表达(图11D),且B7-H3在膜上的表达也明显减少,这提示抑制FUT8后核心岩藻糖的B7-H3在细胞膜上的表达也减少(图11D)。In order to explore the relationship between FUT8 and B7-H3, the inventors selected different sgRNAs to guide the knockout of FUT8 in MDA-MB-231 and HCC1806 cell lines, and detected the expression of B7-H3. The experimental results found that when knocking out FUT8 The glycosylated B7-H3 protein was significantly reduced but the mRNA of B7-H3 was not significantly changed, which suggested that FUT8 could affect the expression of B7-H3 by regulating the glycosylation modification of B7-H3, but had no effect on the mRNA of B7-H3. Effect (Fig. 11A, 11B). In order to further clarify whether B7-H3 can undergo core fucose modification, the inventors used the lectin LCH (LCA) antibody, which can specifically bind to core fucose, to conduct lectin enrichment experiments (mainly used to reflect the FUT8-regulated The expression of core fucoglycoprotein), the results showed that the core fucosylated B7-H3 was significantly reduced after FUT8 was knocked out, and the reduction of FUT8 also had a certain impact on the total glycosylated B7-H3 (Figure 11C). The expression of B7-H3 on the MDA-MB-231 cell membrane was detected by flow cytometry, and it was found that FUT8 can significantly reduce the expression of core fucoidin on the cell membrane surface (Figure 11D), and the expression of B7-H3 on the membrane The expression was also significantly reduced, suggesting that the expression of B7-H3 of core fucose on the cell membrane was also reduced after inhibition of FUT8 ( FIG. 11D ).
通过B7-H3负向调控T细胞的杀伤能力和活性Negative regulation of killing ability and activity of T cells by B7-H3
前期实验证明高表达糖基化的B7-H3三阴乳腺癌细胞能够明显抑制T淋巴细胞对其的杀伤且显著抑制T淋巴细胞的增殖。发明人使用RNA干扰技术在MDA-MB-231-B7-H3KO-WT和MDA-MB-231-B7-H3KO-8NQ细胞株基础上敲低FUT8(siRNA分别为siFUT8#1: 5’-CUGCAGUGUGGUGGGUGUCTT-3’(SEQ ID NO.:1); siFUT8 #2: 5’-AGGUCUGUCGAGUUGCUUATT-3’(SEQ ID NO.:2); sgRNA2: 5’-CACCGACAGCCAAGGGTAAATATGG-3’(SEQ ID NO.:3) 或sgRNA7 5’-CACCGTGAAGCAGTAGACCACATGA-3’(SEQ ID NO.:4)),48小时后将处理的肿瘤细胞与T淋巴细胞共培养,用流式的方式检测细胞凋亡指标Caspase-3的阳性比例,流式结果提示敲低FUT8后可以显著解除糖基化B7-H3对T淋巴细胞杀伤肿瘤细胞的抑制作用,但不改变T淋巴细胞对非糖基化B7-H3细胞的杀伤能力(图12A,12B)。另外发明人RNA干扰技术敲低MDA-MB-231-B7-H3KO-WT细胞株中的FUT8,将处理的肿瘤细胞经80Gy剂量照射后,与用活细胞荧光标记染料羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)染色的T淋巴细胞共培养4-7天,流式的方式检测T淋巴细胞的增殖和活性指标IL-2和IFN-γ,从流式分析结果发明人发现,当敲低糖基化B7-H3细胞株中的FUT8可以明显增强CD4+T细胞的增殖(图12C),并且增强CD4+T细胞和CD8+T中IL-2和IFN-γ表达的比例(图12D,12E,12F,12G)。以上实验结果提示敲低FUT8后增强了T细胞对糖基化B7-H3细胞株的杀伤,并且解除了糖基化B7-H3对T细胞活性的抑制作用。Previous experiments have proved that B7-H3 triple-negative breast cancer cells with high expression of glycosylation can significantly inhibit the killing of T lymphocytes and significantly inhibit the proliferation of T lymphocytes. The inventors used RNA interference technology to knock down FUT8 on the basis of MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ cell lines (siRNAs are siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT- 3' (SEQ ID NO.: 1); siFUT8 #2: 5'-AGGUCUGUCGAGUUGCUUATT-3' (SEQ ID NO.: 2); sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' (SEQ ID NO.: 3) or sgRNA7 5'-CACCGTGAAGCAGTAGACCACATGA-3' (SEQ ID NO.: 4)), after 48 hours, the treated tumor cells were co-cultured with T lymphocytes, and the positive ratio of the apoptosis index Caspase-3 was detected by flow cytometry. The formula results suggested that knocking down FUT8 could significantly release the inhibitory effect of glycosylated B7-H3 on T lymphocytes killing tumor cells, but did not change the killing ability of T lymphocytes on non-glycosylated B7-H3 cells (Fig. 12A, 12B ). In addition, the inventor knocked down FUT8 in the MDA-MB-231-B7-H3KO-WT cell line by RNA interference technology, irradiated the treated tumor cells with a dose of 80Gy, and combined with the living cell fluorescent labeling dye hydroxyfluorescein diacetate Succinimide lipid (CFSE) stained T lymphocytes were co-cultured for 4-7 days, and the proliferation and activity indicators IL-2 and IFN-γ of T lymphocytes were detected by flow cytometry. From the results of flow cytometry analysis, the inventors found that, Knockdown of FUT8 in the glycosylated B7-H3 cell line can significantly enhance the proliferation of CD4 + T cells (Fig. 12C), and enhance the ratio of IL-2 and IFN-γ expression in CD4 + T cells and CD8 + T cells (Fig. 12D, 12E, 12F, 12G). The above experimental results suggested that knocking down FUT8 enhanced the killing of T cells against glycosylated B7-H3 cell lines, and relieved the inhibitory effect of glycosylated B7-H3 on T cell activity.
、靶向干预FUT8降低B7-H3表达以增强三阴乳腺癌对免疫治疗的敏感性、 Targeted intervention of FUT8 reduces the expression of B7-H3 to enhance the sensitivity of triple-negative breast cancer to immunotherapy
4.1 抑制FUT8通过降低B7-H3增强体外T细胞对三阴乳腺癌细胞的杀伤能力4.1 Inhibition of FUT8 enhances the killing ability of T cells against triple-negative breast cancer cells in vitro by reducing B7-H3
2-Fluoro-L-Fucose(2F-Fuc)是一类能岩藻糖基化抑制剂,2F-Fuc进入细胞中与岩藻糖基化原料GDP-Fuc竞争GDP,形成GDP-2F-Fuc并抑制天然GDP-Fuc的合成,导致岩藻糖结构的减少。为探索2F-Fuc是否可以减少糖基化B7-H3的表达,发明人使用不同浓度2F-Fuc处理MDA-MB-231-B7-H3KO-WT、MDA-MB-231- B7-H3KO-8NQ和4T1-B7-H3KO-WT细胞株,四天后用流式方法检测细胞膜表面的表面B7-H3蛋白的表达,流式结果提示加入2F-Fuc处理人源和鼠源的乳腺癌细胞株后细胞膜表面的B7-H3均显著减少(图13A,13C),这提示2F-Fuc可以减少糖基化B7-H3在细胞膜上的表达。为明确2F-Fuc降低糖基化B7-H3表达是否能够恢复T细胞对肿瘤细胞的杀伤能力,予以2F-Fuc预处理MDA-MB-231-B7-H3KO-WT和MDA-MB-231-B7-H3KO-8NQ细胞株,四天后与T淋巴细胞共培养,并用流式检测细胞凋亡指标Caspase-3阳性比率;结果发现用2F-Fuc预处理糖基化B7-H3细胞株对比于未处理组其T细胞的杀伤能力显著增强,而2F-Fuc对T淋巴细胞杀伤非糖基化肿瘤细胞的能力没有显著影响(图13B),这说明2F-Fuc主要通过降低糖基化B7-H3的表达来增强T淋巴细胞对肿瘤细胞的杀伤。2-Fluoro-L-Fucose (2F-Fuc) is a class of fucosylation inhibitors. 2F-Fuc enters cells and competes with fucosylation raw material GDP-Fuc for GDP, forming GDP-2F-Fuc and Inhibits the synthesis of native GDP-Fuc, resulting in the reduction of fucose structures. To explore whether 2F-Fuc can reduce the expression of glycosylated B7-H3, the inventors used different concentrations of 2F-Fuc to treat MDA-MB-231-B7-H3KO-WT, MDA-MB-231-B7-H3KO-8NQ and 4T1-B7-H3KO-WT cell line, after four days, the expression of B7-H3 protein on the surface of the cell membrane was detected by flow cytometry. B7-H3 of all the cells were significantly reduced (Fig. 13A, 13C), suggesting that 2F-Fuc can reduce the expression of glycosylated B7-H3 on the cell membrane. In order to clarify whether 2F-Fuc can reduce the expression of glycosylated B7-H3 and restore the ability of T cells to kill tumor cells, MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7 were pretreated with 2F-Fuc -H3KO-8NQ cell line was co-cultured with T lymphocytes four days later, and the positive ratio of cell apoptosis index Caspase-3 was detected by flow cytometry; the results showed that the glycosylated B7-H3 cell line pretreated with 2F-Fuc was compared with the untreated cell line The killing ability of T cells in the group was significantly enhanced, while 2F-Fuc had no significant effect on the ability of T lymphocytes to kill non-glycosylated tumor cells (Fig. Expression to enhance the killing of tumor cells by T lymphocytes.
靶向干预FUT8联合PD-L1单抗能显著抑制小鼠移植瘤生长和增强T淋巴细胞活性Targeted intervention of FUT8 combined with PD-L1 monoclonal antibody can significantly inhibit the growth of transplanted tumors and enhance the activity of T lymphocytes in mice
上述结果可知2F-Fuc可以通过降低糖基化B7-H3的表达来增强免疫杀伤作用。予以2F-Fuc和DMSO分别预处理4T1-B7-H3-KO-WT细胞株7天后,将两种不同处理的4T1-B7-H3-KO-WT细胞株接种于两组免疫正常的Balb/c小鼠腹部乳房脂肪垫;接种一周后将上述两组小鼠各自再均分成两组,分别予以PBS+Isotype,PBS+Anti-PD-L1, 2F-Fuc +Isotype和2F-Fuc +Anti-The above results show that 2F-Fuc can enhance the immune killing effect by reducing the expression of glycosylated B7-H3. After 4T1-B7-H3-KO-WT cell lines were pretreated with 2F-Fuc and DMSO for 7 days, two differently treated 4T1-B7-H3-KO-WT cell lines were inoculated into two groups of Balb/c cells with normal immunity. Abdominal mammary fat pad of mice; One week after inoculation, the above two groups of mice were divided into two groups, and were given PBS+Isotype, PBS+Anti-PD-L1, 2F-Fuc+Isotype and 2F-Fuc+Anti-
PD-L1处理小鼠(图14A);细胞株经2F-Fuc处理所接种的小鼠,在接种后进行2F-Fuc灌胃处理,每周三次;Anti-PD-L1和Isotype每间隔3天腹腔注射治疗,共计三次。定期观察小鼠肿瘤体积并记录绘制生长曲线,实验结束后将小鼠肿瘤解离进行流式检测不同组别中浸润的免疫细胞数量及活性。根据生长曲线发明人发现对照组与单药处理组的肿瘤大小无明显区别,但2F-Fuc和Anti-PD-L1联合使用时可以显著减小小鼠肿瘤体积和瘤重(图14B),免疫组化分析发现2F-Fuc处理后肿瘤组织中B7H3表达显著减少(图14C),流式分析发现2F-Fuc和Anti-PD-L1联合可以明显增加CD4+T细胞、CD8+T细胞和NK细胞的活性指标IFN-γ的表达(图14D),Tunel染色分析显示2F-Fuc和Anti-PD-L1联合可以增强肿瘤的凋亡比例。由此发明人可以得出2F-Fuc和Anti-PD-L1联合可以通过增强肿瘤中免疫细胞的功能,达到抗肿瘤的效果。Mice were treated with PD-L1 (Figure 14A); the inoculated mice were treated with 2F-Fuc by the cell line, and 2F-Fuc was administered to the mice after inoculation, three times a week; the interval between Anti-PD-L1 and Isotype was 3 days Intraperitoneal injection treatment, a total of three times. The tumor volume of the mice was regularly observed and the growth curve was recorded and drawn. After the experiment, the tumors of the mice were dissociated and the number and activity of infiltrating immune cells in different groups were detected by flow cytometry. According to the growth curve, the inventors found that there was no significant difference in the tumor size between the control group and the single-drug treatment group, but the combined use of 2F-Fuc and Anti-PD-L1 could significantly reduce the tumor volume and tumor weight of the mice (Figure 14B). Histochemical analysis found that the expression of B7H3 in tumor tissue was significantly reduced after 2F-Fuc treatment (Figure 14C), and flow cytometry analysis found that the combination of 2F-Fuc and Anti-PD-L1 could significantly increase CD4 + T cells, CD8 + T cells and NK cells The expression of the activity index IFN-γ (Figure 14D), Tunel staining analysis showed that the combination of 2F-Fuc and Anti-PD-L1 can enhance the apoptotic ratio of the tumor. From this, the inventors can conclude that the combination of 2F-Fuc and Anti-PD-L1 can achieve anti-tumor effects by enhancing the function of immune cells in tumors.
结论:in conclusion:
在三阴乳腺癌中B7-H3蛋白主要发生N-糖基化修饰,糖基化修饰使其稳定性增强、在膜表面的表达增高;糖基化B7-H3的三阴乳腺癌细胞对T细胞的杀伤不耐受,并能抑制T细胞的增殖、浸润和活性,从而介导了三阴乳腺癌发生免疫逃逸。岩藻糖转移酶FUT8正向调节B7-H3的核心岩藻糖基化,并影响其免疫抑制作用。B7-H3及FUT8表达增高均与三阴乳腺癌患者的不良预后相关,且在三阴乳腺癌组织中B7-H3与FUT8呈正相关。靶向干预FUT8通过降低B7-H3糖基化水平可提高三阴乳腺癌对PD-L1抗体治疗的敏感性。In triple-negative breast cancer, B7-H3 protein mainly undergoes N-glycosylation modification, which enhances its stability and increases the expression on the membrane surface; glycosylated B7-H3 triple-negative breast cancer cells have Cell killing intolerance, and can inhibit the proliferation, infiltration and activity of T cells, thus mediating the immune escape of triple-negative breast cancer. The fucosyltransferase FUT8 positively regulates the core fucosylation of B7-H3 and affects its immunosuppressive effect. Increased expressions of B7-H3 and FUT8 are associated with poor prognosis in patients with triple-negative breast cancer, and B7-H3 is positively correlated with FUT8 in triple-negative breast cancer tissues. Targeted intervention of FUT8 can improve the sensitivity of triple-negative breast cancer to PD-L1 antibody therapy by reducing the level of B7-H3 glycosylation.
以上是对本发明所作的进一步详细说明,不可视为对本发明的具体实施的局限。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的简单推演或替换,都在本发明的保护范围之内。The above is a further detailed description of the present invention, and should not be regarded as a limitation to the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.
<110> 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所)<110> Sun Yat-sen University Cancer Center (Sun Yat-sen University Affiliated Cancer Hospital, Sun Yat-sen University Cancer Institute)
<120> 用于特定型三阴乳腺癌免疫治疗的组合物<120> Composition for immunotherapy of specific type triple-negative breast cancer
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Claims (1)
- Application of a B7-H3 protein core fucosylation modification intervention agent in preparation of a triple-negative breast cancer immunotherapy synergist, wherein the triple-negative breast cancer is glycosylated B7-H3 positive triple-negative breast cancer, the glycosylation is N-glycan core fucosylation, the core fucosylation modification intervention agent is siRNA of FUT8, the sequence of the siRNA is siFUT8#1: 5.
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CN108949984A (en) * | 2018-07-25 | 2018-12-07 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所) | Application of the gene DESI2 in three negative breast cancer diagnosis, prognosis evaluation and treatment |
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