基于唾液酸酶和岩藻糖转移酶的抗体—细胞偶联物构建

目的 通过基于唾液酸酶和岩藻糖转移酶的糖链工程将曲妥珠单抗高密度偶联到人外周血白血病T细胞Jurkat细胞,并评价偶联细胞对HER2+靶细胞的识别活性。方法 首先通过凝集素染色和流式细胞术探究去唾液酸化对细胞上α-1,3FucT催化的岩藻糖编辑效率的影响。然后制备含有叠氮标签的曲妥珠单抗,与GDP-炔基岩藻糖通过生物正交反应构建GDP-岩藻糖-抗体偶联物。再利用α-1,3FucT将GDP-岩藻糖-抗体偶联物转移至唾液酸酶处理过的细胞表面,构建抗体-细胞偶联物,并通过流式细胞术和共聚焦成像验证。最后通过荧光成像观察抗体-细胞偶联物对靶细胞SK-BR-3细胞的识别。结果 细胞表面糖链的末端α-2,6唾液酸切除后能够显著提升α-1,3FucT催化的岩藻糖编辑的效率,成功制备GDP-岩藻糖-抗体偶联物并转移至细胞表面得到曲妥珠单抗-Jurkat偶联物,去唾液酸化显著增加了Jurkat细胞偶联曲妥珠单抗的容量,构建的曲妥珠单抗-Jurkat偶联物具备HER2+细胞靶向性。结论 细胞糖链工程可实现细胞偶联分子容量的调节,应用于抗体对免疫细胞的功能化,也为构建海洋糖类活性化合物偶联细胞提供了新的思路...     

PI3K/Akt信号通路活化在曲妥珠单抗耐药机制意义的研究

目的探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)信号转导通路激活是曲妥珠单抗耐药的重要靶点之一,为乳腺癌曲妥珠单抗耐药的靶向治疗提供理论基础。方法对人乳腺癌细胞株BT474连续处理建立了耐曲妥珠单抗的耐药亚株BT-HerR,FISH法对耐药细胞株BT-HerR做Her-2表型分析,MTT法检测曲妥珠单抗对BT474和BT-HerR细胞的体外增殖抑制情况,流式细胞仪检测曲妥珠单抗干预后细胞的凋亡变化,PI3K/Akt抑制剂LY294002干预细胞后Western blot检测p-Akt表达。结果耐药细胞株BT-HerR Her-2基因表达为强阳性;曲妥珠单抗干预细胞72 h后,细胞的体外增殖受到抑制且随着浓度的升高而增强;经曲妥珠单抗处理后比较BT474与BT-HerR细胞凋亡率,差异具有显著性(P<0.01);曲妥珠单抗仅能抑制BT474的Akt蛋白磷酸化,LY294002则能同时抑制BT474的BT-HerR Akt蛋白磷酸化。结论曲妥珠单抗耐药细胞Akt蛋白磷酸化活化,PI3K/Akt抑制剂LY294002能明显抑制曲妥珠单抗耐药细胞Akt蛋白磷酸化,PI3K/Akt信号转导通路与曲妥珠单抗耐药存在明确相关性。     

治疗慢性淋巴细胞白血病的新药:奥比妥珠单抗

2013年11月1日美国食品和药物管理局批准奥比妥珠单抗(又名GA101)联合苯丁酸氮芥用于初治的慢性淋巴细胞白血病患者。奥比妥珠单抗是一种经过Fc段修饰的人源糖基化的Ⅱ型抗CD20单克隆抗体,其抗体依赖细胞介导的细胞毒作用及直接细胞毒作用强于利妥昔单抗,补体依赖细胞毒作用弱于利妥昔单抗,药物活性和疗效较高。奥比妥珠单抗具有较好的耐受性,常见的不良事件是输液反应、中性粒细胞减少、血小板减少、贫血、发热等。此外,在治疗CD20阳性非霍奇金淋巴瘤的一系列临床试验中,奥比妥珠单抗也表现出了令人欣喜的结果。     

抗体-药物偶联药物的研究进展

抗体-药物偶联物（ADC）也称为免疫偶联物,属于靶向抗肿瘤药物,由药物、抗体以及偶联抗体和药物的连接物所组成。当抗体和肿瘤细胞表面的抗原特异性结合时,抗体-药物偶联物可将药物成功释放到肿瘤细胞特定部位。本文主要概述抗体-药物偶联物的作用机理、分类以及相关药物的临床进展并重点介绍最新上市的药物曲妥珠单抗-美登木素I（T-DM1,Kadcyla）。     

表没食子儿茶素没食子酸酯联合曲妥珠单抗对HER2过表达乳腺癌细胞增殖的影响及其机制

目的 研究表没食子儿茶素没食子酸酯(EGCG)联合曲妥珠单抗对人表皮生长因子受体2(HER2)过表达乳腺癌细胞增殖的影响及其作用机制.方法 表达纯化曲妥珠单抗;用CCK-8细胞增殖检测试剂盒(CCK8)检测不同浓度EGCG、曲妥珠单抗及两药联用对HER2过表达乳腺癌细胞BT474、SK-BR-3的增殖抑制作用;用Wes...     

抗乳腺癌药曲妥珠单抗-DM1

由罗氏集团子公司Genentech研制的抗癌药曲妥珠单抗-DM1（T-DM1）是将ImmunoGen公司的高活性抗有丝分裂产品DM1与罗氏公司的治疗性人源化抗HER2抗体曲妥珠单抗（trastuzumab,Herceptin）连接而形成的一种肿瘤激活前药（TAP）免疫结合物,即细胞毒药物-HER2抗体偶联物,也称“武装抗体”。     

伊尼妥单抗联合吡咯替尼治疗原发耐药HER2阳性晚期乳腺癌1例

约10%～30%的乳腺癌患者存在人表皮生长因子受体2(Human epidermal growth factor receptor-2,HER-2)过表达,后者与较差的预后相关。多种抗HER2药物的出现改变了这一现状,其中,曲妥珠单抗仍是一线标准治疗,而曲妥珠单抗治疗失败后的二线及以上治疗靶向药物选择成为新的困难与挑战。伊尼妥单抗是一种优化细胞毒性作用(Antibody-dependent cell-medicated cytotoxicity, ADCC)效应的新型单抗药物,通过改造抗体的Fc段,显示出优于曲妥珠单抗的疾病控制时间及良好的安全性。基础研究显示,小分子酪氨酸激酶抑制剂吡咯替尼在阻断细胞内的HER2 ATP位点的同时,进一步提高了单抗药物的ADCC效应,两药联合的治疗模式具有潜在的临床获益。本文报道了1例曲妥珠单抗原发耐药的HER-2阳性晚期乳腺癌患者,二线治疗失败后应用长春瑞滨联合伊尼妥单抗及吡咯替尼三线治疗,获得了超过19个月的无进展生存期(Progression-free survival, PFS)。     

HER2阳性的进展期胃癌目前和未来的靶向治疗

目前对胃癌中人表皮生长因子受体(2HER2)的预测价值存有争议。当前的治疗指南已经把检测胃癌中HER2状态作为标准化操作。最近,在治疗HER2阳性进展期胃癌中,曲妥珠单抗已经成为一线靶向治疗用药,而原发与继发性药物抵抗则成为主要问题,需要新的治疗策略来克服这种抵抗。许多HER2阳性进展期胃癌患者在接受曲妥珠单抗治疗后都出现疾病进展,必须接受二线方案治疗。新的靶向药物,诸如酪氨酸激酶抑制剂(TKI)拉帕替尼、哺乳动物雷帕霉素靶蛋白(mTOR)通路抑制剂依维莫司、热休克蛋白90(HSP90)抑制剂AUY922、HER二聚化抑制剂帕妥珠单抗以及抗体-药物偶联物曲妥珠单抗-emtansine(T-DM1),在以曲妥珠单抗为基础的一线治疗失败时可以成为二线治疗用药。     

抗体偶联药物在乳腺癌治疗中的研究进展

抗体偶联药物(ADC)是一类治疗乳腺癌的新型靶向药物,由连接子将化疗药物同单克隆抗体(单抗抗偶联而成,以单抗作为载体将化疗药物靶向运输至目标肿瘤细胞内发挥抗肿瘤作用。根据ADC作用于不同靶点的抗原,分为靶向人表皮生长因子受体2(HER2)、人滋养细胞表面抗原2 (Trop-2)及其他分子等类型。目前,全球范围内已有3种ADC获批治疗乳腺癌,除用于HER2阳性乳腺癌的曲妥珠单抗-美坦新偶联物(T-DM1)、曲妥珠单抗-德鲁替康(T-DXd)外,还有可使三阴性乳腺癌(TNBC)获益的戈沙妥珠单抗(SG)。ADC在HER2阳性乳腺癌治疗中疗效显著,同时在治疗晚期TNBC和部分HER2低表达的乳腺癌中也取得了重要进展,为不同乳腺癌分子分型的人群提供了更多选择。     

抗体偶联药物研究进展

抗体-药物偶联物(Antibody-drug conjugates,ADCs)由“弹头”药物(细胞毒药物)、抗体以及偶联抗体和药物的偶联链3部分组成,其作为一种新型药物,结合了抗体的靶向特异性和小分子药物的细胞毒作用,已成为当今肿瘤研究领域的热点。根据其细胞毒药物来源可分为陆地与海洋两大类,本文总结了近年来这两大类抗体偶联药物的研究进展,并从靶点的特异性、抗体的亲和力、高效的细胞毒分子及合适的偶联链等方面对ADCs的发展关键进行了简要评述。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.