PD-L1和OX40靶点肿瘤免疫疗法研究进展

恶性肿瘤作为危害人类健康最严重的疾病之一,其诱因较多、早期无明显症状且易发生转移.随着分子生物学技术的发展和对肿瘤病理机制深入研究,肿瘤免疫疗法逐渐成为人类研究的重点.其中免疫检查点PD-L1是肿瘤免疫治疗经典靶点,它可以与PD-1结合通过激活信号通路抑制T细胞活性,促进肿瘤生长.2016年,第一个PD-L1抑制剂阿特...     

OX40、OX40L分子在乳腺癌中的表达及其临床意义

目的研究OX40、OX40L分子在乳腺癌发生、发展中的作用。方法运用免疫组化S-P法检测OX40、OX40L分子在58例乳腺癌中的表达,并分析它们与乳腺癌临床病理指标的关系。结果OX40、OX40L分子在乳腺癌组织的阳性表达均显著高于正常乳腺上皮(均为P<0.01),且与临床分期、肿瘤瘤块大小均显著相关(均为P<0.01),与淋巴结转移无关(P>0.05)。结论OX40、OX40L分子在乳腺癌中的发生、发展中起着不同程度的作用,其检测可对肿瘤恶性程度判定、预后判断和进一步治疗提供依据。     

OX40和OX40L在胃癌及癌前病变中的表达分析

目的研究Oxford 40（OX40）和OX40配体（OX40L）在胃癌及癌前病变中的表达及其意义。方法收集复旦大学附属中山医院内镜中心胃部内镜黏膜下剥离术标本43例,分为慢性非萎缩性胃炎、胃黏膜上皮轻中度不典型增生、胃黏膜上皮重度不典型增生或胃癌3组,采用EnVision二步法检测各种病变中OX40、OX40L的实质细胞表达,并分析其与病变临床病理特征的关系。结果 OX40的实质细胞表达在胃黏膜上皮重度不典型增生中较胃癌、胃黏膜上皮轻中度不典型增生、慢性非萎缩性胃炎中高（P〈0.05）。OX40L的实质细胞表达在胃黏膜上皮轻中度不典型增生、胃黏膜上皮重度不典型增生或胃癌中较慢性非萎缩性胃炎中高（P〈0.05）。结论 OX40、OX40L参与机体抗不典型增生和抗肿瘤免疫反应,其表达的变化可能与胃癌的发生、发展有关。     

OX40-OX40L相互作用对C57BL/6小鼠淋巴细胞活性的影响

目的探讨OX40-OX40L相互作用对C57BL/6小鼠脾淋巴细胞增殖及细胞因子表达的影响。方法应用MTT法检测小鼠淋巴细胞增殖;ELISA法检测淋巴细胞表达细胞因子IL-4、IL-2及INF-γ表达。结果与对照组比较,Anti-OX40特异性刺激OX40-OX40L轴后,小鼠淋巴细胞增殖增强,分泌IL-2和INF-γ含量明显增加,48h细胞增殖最佳;IL-4含量无明显变化。应用特异性anti-OX40L抗体阻断OX40-OX40L轴后,淋巴细胞增殖及分泌细胞因子IL-2和INF-γ含量明显受到抑制。结论 OX40-OX40L相互作用能够调控C57BL/6小鼠脾淋巴细胞增殖及促炎性细胞因子表达。     

人可溶性OX40L蛋白ELISA检测试剂盒的研制及其在自身免疫性疾病诊断中的应用

目的研制特异性检测人可溶性OX40L蛋白（sOX40L）的ELISA试剂盒,探讨其在自身免疫性疾病临床诊断中的应用。方法利用两株识别不同抗原表位的鼠抗人OX40L单克隆抗体1G1和4C12分别作为包被抗体和检测抗体,采用双抗体酶联免疫夹心法研制特异性检测人sOX40L的ELISA方法,并对其稳定性、精确性和特异性进行分析。结果①建立的检测人sOX40L酶联免疫试剂盒在抗原浓度为3.91～250ng/ml范围内有良好的线性关系,并具有良好的稳定性、较高的准确性和特异性。②利用该ELISA方法检测到sOX40L蛋白在肾病综合征和Graves’病患者血清中表达水平均显著高于健康对照组。结论成功建立了检测人sOX40L蛋白的ELISA试剂盒,应用该方法检测到sOX40L在多种自身免疫病患者血清中异常高表达。该试剂盒在OX40L分子基础研究以及自身免疫病的临床诊断方面具有潜在的应用价值。     

NFATc1与OX40-OX40L在ApoE-/-小鼠动脉粥样硬化中的表达及意义

目的 探讨活化T细胞核因子c1(NFATc1)与OX40-OX40配体(OX40L)在载脂蛋白E基因敲除(ApoE-/-)小鼠动脉粥样硬化(AS)中的表达及意义.方法 选取16只ApoE-/-小鼠作为手术组,应用颈动脉硅胶圈植入法快速制作斑块模型;另选16只C57BL/6J小鼠作为对照组.免疫组化检测颈动脉粥样斑块中NFATc1表达,RT-PCR和流式细胞术分别检测淋巴细胞NFATc1、OX40及OX40L mRNA和蛋白表达.结果 NFATc1在手术组颈动脉斑块中显著表达,而在对照组中不表达;手术组NFATc1、OX40、OX40L mRNA和蛋白表达均明显高于对照组(P＜0.01);ApoE-/-小鼠NFATc1与OX40、OX40L表达均呈正相关(r分别为0.938、0.964,P＜0.01).结论 NFATc1与OX40-OX40L在AS发生发展中发挥着重要作用,NFATc1可能是受OX40-OX40L调控的下游分子.     

替罗非班对急性冠状动脉综合征患者经皮冠状动脉介入术后血浆可溶性OX40L的影响

目的:探讨替罗非班对急性冠状动脉综合征(ACS)患者经皮冠状动脉介入术(PCI)后血浆可溶性OX40L(sOX40L)水平的影响。方法:66例ACS患者设为ACS组,并根据治疗方法不同分为支架组和替罗非班组,支架组给予常规PCI介入治疗,替罗非班组给予PCI联合替罗非班治疗。另选择同期在我院疑似冠心病并经冠状动脉造影检查确认冠脉无狭窄者30例为对照组。测定术前1天、术后即刻及术后24h、48h、10d血浆sOX40L和超敏C反应蛋白(hs-CRP)水平。结果:术前ACS组血浆sOX40L和hs-CRP水平显著高于对照组(P<0.05);在ACS不同的临床类型中,心肌梗死(MI组)sOX40L和hs-CRP水平显著高于不稳定型心绞痛(UA组)(P<0.05);在不同的冠脉病变支数中,sOX40L和hs-CRP含量:三支病变组>双支病变组>单支病变组,各组间差异有统计学意义(P<0.05);术后即刻及术后24h、48h、10d替罗非班组sOX40L水平显著低于支架组(P<0.05);替罗非班治疗后无严重并发症发生。结论:ACS患者血浆sOX40L水平显著上升,且与冠状动脉粥样硬化病变的严重程度相关;替罗非班可降低ACS患者PCI后血浆sOX40L水平。     

紫草素对特应性皮炎小鼠TSLP/OX40L通路及Th1/Th2平衡的影响#br# #br#

摘要：目的　探究紫草素对特应性皮炎（AD）小鼠胸腺基质淋巴细胞生成素（TSLP）/OX40配体（OX40L）通路及辅助性T细胞（Th）1/Th2平衡的影响。方法　采用局部外涂2,4-二硝基氟苯（DNFB）法制备AD小鼠模型。采用随机数字表法将模型小鼠分为模型组,紫草素低（20 mg/kg）、中（30 mg/kg）、高（40 mg/kg）剂量组和阳性对照组（泼尼松龙,10 mg/kg）,每组15只;另取15只作为正常对照组。各给药组大鼠按10 mL/kg灌胃相应药物,模型组和正常对照组灌胃等体积溶剂,每日1次,连续15 d。分别于给药后第5、10和15天观察小鼠搔抓行为;于给药后第7、15天评价小鼠皮损状况;末次给药结束后,HE染色观察小鼠皮损组织病理学变化;流式细胞术检测小鼠外周血中Th1/Th2比例;酶联免疫吸附试验（ELISA）检测小鼠血清中TSLP、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-4和干扰素（IFN）-γ水平;Western blot法检测小鼠皮损组织TSLP和OX40L蛋白表达水平。结果　与正常对照组相比,模型组小鼠搔抓行为增加,皮肤炎性损伤加重;皮损组织中表皮和真皮区域均有密集的炎性细胞浸润,表皮增厚;外周血Th2细胞、TSLP、TNF-α和IL-4水平以及皮损组织TSLP和OX40L蛋白表达水平显著升高（P＜0.05）,外周血Th1细胞、Th1/Th2比例和IFN-γ水平显著下降（P＜0.05）。与模型组相比,紫草素低、中、高剂量组小鼠第5、10、15天的搔抓行为减少,第7、15天皮肤炎性损伤依次减轻（P＜0.05）;小鼠表皮和真皮区域炎性细胞浸润和表皮增厚依次减轻,外周血Th2细胞、TSLP、TNF-α和IL-4水平以及皮损组织TSLP和OX40L蛋白表达水平依次降低（P＜0.05）,Th1细胞、Th1/Th2比例和IFN-γ水平依次升高（P＜0.05）。结论　紫草素可能通过抑制TSLP/OX40L通路维持AD小鼠外周血中Th1/Th2平衡,改善皮肤炎性损伤。     

肾移植急性排斥受者外周血OX40 mRNA和Th1的变化及意义

目的 探讨外周血单个核细胞 OX40 mRNA和外周血 CD3+CD8- T辅助细胞（Th）1、Th2检测用于评估肾移植术后急性排斥的应用价值。方法 收集 2016年 1月—2017年 1月天津市第一中心医院器官移植中心行肾移植手术的 40例肾移植受者及 20名健康志愿者纳入研究,肾移植受者分为急性排斥组 20例和肾功能稳定组 20例。应用TaqMan实时荧光定量聚合酶链式反应（RT-PCR)定量检测 3组外周血单个核细胞（PBMCs）中 OX40 mRNA水平,应用流式细胞术检测外周血 CD3+CD8-T淋巴细胞中 IFN-γ（Th1）和 IL-4（Th2）表达率。ROC曲线评价 OX40和 Th1对预 测急性排斥的诊断价值。结果 肾移植术后急性排斥组 PBMCs 中 OX40 mRNA水平明显高于肾功能稳定组以及健康对照组（P＜0.01）,肾功能稳定组 PBMCs 中 OX40 mRNA水平与健康对照组差异无统计学意义（P＞0.05）。急性排斥组 Th1 表达率明显高于肾功能稳定组和正常对照组（P＜0.01）,3 组 Th2 表达率差异无统计学意义（P＞0.05）。OX40 mRNA 诊断急性排斥反应的 ROC 曲线下面积（AUC）为 0.903（95%CI：0.825~0.980）;Th1 的 AUC 为 0.731（95%CI：0.587~0.874）;两者联合诊断的 AUC 为 0.930（95%CI：0.859~1.001）。结论 监测肾移植受者外周血 OX40mRNA及 Th1细胞可以预测急性排斥反应。     

Cancer combinatorial immunotherapy using anti-OX40 agonist and anti-PD-L1 antagonist: a patent evaluation of US2018256711A1

Introduction: OX40 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of OX40 and consequently improving the immune response in the various types of cancer. Authors of patent US2018256711A1 propose a method to eradicate cancer that utilizes anti-OX40 agonist antibody in combination with anti-PD-L1 antagonist antibody.

Areas covered: Patent US2018256711A1 describes a method of cancer combinatorial treatment consisting of the utilization of a pharmaceutical cocktail containing anti-OX40 and an anti-PD-L1 antibody.

Expert opinion: The results of the clinical trials only support trials regarding the tolerability of combinatorial therapy, even when the objectives of determining the safety and pharmacokinetics of the treatment are proposed.     

OX40Ig修饰大鼠脂肪间充质干细胞的实验研究

目的构建含OX40融合蛋白(OX40Ig)基因修饰的大鼠脂肪间充质干细胞(ADSCs)即ADSCs^OX40Ig,探讨其在体外对淋巴细胞的免疫调节作用。方法构建pcDNA3.1(+)/OX40Ig融合基因表达载体,采用核转染技术转染大鼠ADSCs,Western blot检测ADSCs中OX40Ig表达水平。ADSCs与大鼠异基因淋巴细胞共培养后分为对照组(反应细胞+刺激细胞)、ADSCs组(反应细胞+刺激细胞+ADSCs)及ADSCs^OX40Ig组(反应细胞+刺激细胞+ADSCs^OX40Ig),MTT法检测淋巴细胞增殖抑制率,逆转录聚合酶链反应(RT-PCR)检测淋巴细胞内干扰素(IFN)-γ、白细胞介素(IL)-10及转化生长因子(TGF)-βmRNA表达。结果经测序鉴定验证,pcDNA3.1(+)/OX40Ig质粒构建成功,转染ADSCs后OX40Ig蛋白呈高表达;与ADSCs组比较,ADSCs^OX40Ig显著增强异基因淋巴细胞增殖抑制率(P<0.05);对照组、ADSCs组及ADSCs<...     

Therapeutic peptidomimetic strategies for autoimmune diseases: costimulation blockade*

Abstract: Cognate interactions between immune effector cells and antigen‐presenting cells (APCs) govern immune responses. Specific signals occur between the T‐cell receptor peptide and APCs and nonspecific signals between pairs of costimulatory molecules. Costimulation signals are required for full T‐cell activation and are assumed to regulate T‐cell responses as well as other aspects of the immune system. As new discoveries are made, it is becoming clear how important these costimulation interactions are for immune responses. Costimulation requirements for T‐cell regulation have been extensively studied as a way to control many autoimmune diseases and downregulate inflammatory reactions. The CD28:B7 and the CD40:CD40L families of molecules are considered to be critical costimulatory molecules and have been studied extensively. Blocking the interaction between these molecules results in a state of immune unresponsiveness termed ‘anergy’. Several different strategies for blockade of these interactions are explored including monoclonal antibodies (mAbs), Fab fragments, chimeric, and/or fusion proteins. We developed novel, immune‐specific approaches that interfere with these interactions. Using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis mediated by central nervous system (CNS)‐specific T‐cells, we developed a multi‐targeted approach that utilizes peptides for blockade of costimulatory molecules. We designed blocking peptide mimics that retain the functional binding area of the parent protein while reducing the overall size and are thus capable of blocking signal transduction. In this paper, we review the role of costimulatory molecules in autoimmune diseases, two of the most well‐studied costimulatory pathways (CD28/CTLA‐4:B7 and CD40:CD40L), and the advantages of peptidomimetic approaches. We present data showing the ability of peptide mimics of costimulatory molecules to suppress autoimmune disease and propose a mechanism for disease suppression.     

Therapeutic strategies against protein misfolding in neurodegenerative diseases

Background: Neurodegenerative diseases are a group of chronic and progressive disorders of the nervous system. A hallmark event in these diseases is the misfolding and accumulation in the brain of protein aggregates. Objective: In this article, we describe the knowledge of the mechanism of protein misfolding and aggregation, its role in neurodegeneration and the diverse therapeutic targets for intervention. We also critically review various strategies under development to discover drugs attacking this process. Conclusion: In spite of the substantial progress on understanding the critical role of protein misfolding and aggregation, drugs effective against this process are still years away from approval.     

不同剂量瑞舒伐他汀对急性冠脉综合征患者血清sOX40L、MMP-9的影响

目的　探讨不同剂量瑞舒伐他汀对急性冠脉综合征（ACS）患者血清可溶性OX40配体（sOX40L）、基质金属蛋白酶9（MMP-9）的影响。方法　选择ACS患者60例,空白对照组20例。随机将ACS患者分为20 mg瑞舒伐他汀治疗组（20mg组）30例和10 mg瑞舒伐他汀治疗组（10mg组）30例,比较治疗前后各组间血清sOX40L、MMP-9的水平变化。结果　瑞舒伐他汀治疗2周后,两治疗组患者血清sOX40L、MMP-9水平均较治疗前明显降低,差异有统计学意义（P<0.01）。其中20mg组较10mg组降低更为明显,差异有统计学意义（P<0.05）。 结论　ACS患者早期使用较大剂量瑞舒伐他汀可明显减少冠状动脉粥样斑块基质成分的降解和炎症反应,从而起到稳定动脉粥样硬化斑块,改善ACS患者的预后的作用。     

Research Article: Effective and Specific Inhibition of the CD40–CD154 Costimulatory Interaction by a Naphthalenesulphonic Acid Derivative

Costimulatory interactions are important regulators of T-cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small-molecule inhibitors of the CD40–CD154 costimulatory protein–protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration-dependently inhibit the CD154(CD40L)-induced cellular responses in human THP-1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40–CD154 interaction with sub-micromolar activity, but it also has considerably more than 100-fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF-R1–TNF-α, BAFF-R(CD268)–BAFF(CD257/BLys), OX40(CD134)–OX40L(CD252), RANK(CD265)–RANKL(CD254/TRANCE), or 4-1BB(CD137)–4-1BBL. There is now sufficient structure-activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction.     

P40家族成员及其在自身免疫性疾病中的作用

作为P40家族中研究最为广泛的细胞因子,白细胞介素-12(Interleukin-12)其最显著的特点是有助于Th1细胞分化,参与由Th1细胞所介导的免疫性疾病。而白细胞介素-23(Interleukin-23)与Th17细胞增殖有关,最新发现Th17介导的免疫反应在实验性变态反应性脑脊髓炎(EAE)和多发性硬化症(MS)疾病方面发挥重要作用,并且其分化途径的提出对传统的Th1/Th2细胞免疫也带来了新的挑战[1]。该家族的另外两个成员P40单体和P40同源二聚体也参与免疫调控,实验证明P40分子不仅在细胞内感染及炎症过程中起着重要的调节作用,而且与银屑病、多发性硬化症、Crohn’s病等多种自身免疫性临床疾病的发病密切相关。     

Naloxone can improve the anti-tumor immunity by reducing the CD4CD25Foxp3 regulatory T cells in BALB/c mice

New strategies that stimulate cell-mediated immunity (CMI) against tumors and inhibit regulatory T cells are needed to improve the outcome of cancer immunotherapy. The aim of this study was to enhance the anti-tumor immunity of gp96 vaccine through naloxone administration. Therefore, we used BALB/c mouse model of fibrosarcoma tumor and analyzed the tumor size, splenocyte proliferation, spleen and tumor-infiltrated lymphocytes. Tumor and spleen CD4⁺CD25⁺Foxp3⁺ regulatory T lymphocytes, cytotoxic activity of the splenocytes, IFN-γ and IL-4 secretion were assessed to describe the anti-tumor immune response. Our findings showed that co-administration of gp96 and naloxone has resulted in a significant reduction in CD4⁺CD25⁺Foxp3⁺ regulatory T cells in the spleen. The results indicated that on days 27 and 32 the tumors in the gp96 + Nal group grew significantly slower. Moreover, co-administration of gp96 and naloxone significantly increased the intra-tumor CD8⁺ T cells and cytotoxic activity. In addition the results indicated a significant increase in the proliferation of splenocytes and IFN-γ production. Our results demonstrate that naloxone is an effective immunoadjuvant in cancer immunotherapy.     

Therapeutic strategies by modulating oxygen stress in cancer and inflammation

Oxygen is the essential molecule for all aerobic organisms, and plays predominant role in ATP generation, namely, oxidative phosphorylation. During this process, reactive oxygen species (ROS) including superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) are produced as by-products, while it seems indispensable for signal transduction pathways that regulate cell growth and reduction-oxidation (redox) status. However, during times of environmental stress ROS levels may increase dramatically, resulting in significant damage to cell structure and functions. This cumulated situation of ROS is known as oxidative stress, which may, however, be utilized for eradicating cancer cells.It is well known that oxidative stress, namely over-production of ROS, involves in the initiation and progression of many diseases and disorders, including cardiovascular diseases, inflammation, ischemia-reperfusion (I/R) injury, viral pathogenesis, drug-induced tissue injury, hypertension, formation of drug resistant mutant, etc. Thus, it is reasonable to counter balance of ROS and to treat such ROS-related diseases by inhibiting ROS production. Such therapeutic strategies are described in this article, that includes polymeric superoxide dismutase (SOD) (e.g., pyran copolymer-SOD), xanthine oxidase (XO) inhibitor as we developed water soluble form of 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP), heme oxygenase-1 (HO-1) inducers (e.g., hemin and its polymeric form), and other antioxidants or radical scavengers (e.g., canolol).On the contrary, because of its highly cytotoxic nature, ROS can also be used to kill cancer cells if one can modulate its generation selectively in cancer. To achieve this goal, a unique therapeutic strategy was developed named as “oxidation therapy”, by delivering cytotoxic ROS directly to the solid tumor, or alternatively inhibiting the antioxidative enzyme system, such as HO-1 in tumor. This anticancer strategy was examined by use of O₂⁻ or H₂O₂-generating enzymes (i.e., XO and d-amino acid oxidase [DAO] respectively), and by discovering the inhibitor of HO-1 (i.e., zinc protoporphyrin [ZnPP] and its polymeric derivatives). Further for the objective of tumor targeting and thus reducing side effects, polymer conjugates or micellar drugs were prepared by use of poly(ethylene glycol) (PEG) or styrene maleic acid copolymer (SMA), which utilize EPR (enhanced permeability and retention) effect for tumor-selective delivery. These macromolecular drugs further showed superior pharmacokinetics including much longer in vivo half-life, particularly tumor targeted accumulation, and thus remarkable antitumor effects.The present review concerns primarily our own works, in the direction of “Controlling oxidative stress: Therapeutic and delivery strategy” of this volume.     

Acid-switchable nanoparticles induce self-adaptive aggregation for enhancing antitumor immunity of natural killer cells

Deficiency of natural killer (NK) cells shows a significant impact on tumor progression and failure of immunotherapy. It is highly desirable to boost NK cell immunity by upregulating active receptors and relieving the immunosuppressive tumor microenvironment. Unfortunately, mobilization of NK cells is hampered by poor accumulation and short retention of drugs in tumors, thus declining antitumor efficiency. Herein, we develop an acid-switchable nanoparticle with self-adaptive aggregation property for co-delivering galunisertib and interleukin 15 (IL-15). The nanoparticles induce morphology switch by a decomposition-metal coordination cascade reaction, which provides a new methodology to trigger aggregation. It shows self-adaptive size-enlargement upon acidity, thus improving drug retention in tumor to over 120 h. The diameter of agglomerates is increased and drug release is effectively promoted following reduced pH values. The nanoparticles activate both NK cell and CD8⁺ T cell immunity in vivo. It significantly suppresses CT26 tumor in immune-deficient BALB/c mice, and the efficiency is further improved in immunocompetent mice, indicating that the nanoparticles can not only boost innate NK cell immunity but also adaptive T cell immunity. The approach reported here provides an innovative strategy to improve drug retention in tumors, which will enhance cancer immunotherapy by boosting NK cells.