以IgE/FcεRI信号通路为靶标治疗变态反应性疾病研究进展

变态反应性疾病已成为全球性的社会卫生问题,IgE与靶细胞表面高亲和力受体FcεRI结合是I型变态反应的关键步骤。针对IgE/FcεRI信号通路重要靶点进行药物设计是当前治疗变态反应性疾病药物研究的热点,主要集中在以IgE、IgE高亲和力受体FcεRI以及信号通路关键信号分子为靶点的抗体、多肽、疫苗、融合蛋白及小分子化合物等相关药物研究。本文综述了近年来以IgE/FcεRI信号通路为靶点的代表性药物在变态反应性疾病中的作用及其机制。     

Fc融合蛋白在药学领域的研究进展

Fc融合蛋白是指利用基因工程等技术将某种具有生物活性的功能蛋白分子与Fc片段融合而产生的新型重组蛋白,其不仅保留了功能蛋白分子的生物学活性,还具有一些抗体的性质,如通过结合相关Fc受体延长半衰期和引发抗体依赖细胞介导的细胞毒性效应等。对Fc融合蛋白及其在药学领域的研究进展进行了综述。     

融合蛋白技术在生物医药领域中的应用

在治疗性药物的研发中,效应分子与人免疫球蛋白IgG1 Fc片段或人血清白蛋白形成的融合蛋白疗效不变,但体内半衰期明显延长、药物耐受性增加、副作用减少.在疫苗研发中,抗原分子与毒素分子或Fc形成的融合蛋白是很好的新型疫苗,并能激发细胞免疫.     

融合蛋白技术在生物医药领域中的应用

在治疗性药物的研发中,效应分子与人免疫球蛋白IgG1 Fc片段或人血清白蛋白形成的融合蛋白疗效不变,但体内半衰期明显延长、药物耐受性增加、副作用减少.在疫苗研发中,抗原分子与毒素分子或Fc形成的融合蛋白是很好的新型疫苗,并能激发细胞免疫.     

治疗性抗体半衰期研究进展

对于治疗性抗体药物来说,半衰期是其重要的一个药代动力学（PK）参数,也是影响其临床应用可行性和实用性的重要因素。临床上常用的治疗性抗体药物主要以免疫球蛋白G(Ig G)为主,针对其半衰期改造的研究也在不断创新,目前延长Ig G半衰期的策略主要集中在可结晶片段（Fc）区域改造、等电点（p I）改造、糖基化修饰改造、抗原结合部位p H依赖性改造以及抗原结合钙离子依赖性改造等方面。近年来,驼源纳米抗体（Nb）逐渐成为治疗性抗体药物研究的热门领域,作为一种潜力巨大的小分子抗体,延长其半衰期具有重要意义,以蛋白融合、化学修饰、红细胞载体和多价Nb为主的策略被应用到Nb半衰期改造的研究中。通过对延长Ig G和Nb半衰期的一些现有策略和方法进行介绍,并简述半衰期改造后的应用现状,以期为更多其他的治疗性抗体半衰期改造的研究提供参考。     

生物制剂治疗强直性脊柱炎的近期疗效与安全性研究进展

目前,临床上治疗强直性脊柱炎(AS)的方法主要为使用肿瘤坏死因子-α(TNF-α)生物拮抗药治疗,且取得较好的疗效,但该类药物均有一定的不良反应. 近年来,国内外治疗AS的生物制剂主要有TNF-α单克隆抗体(英夫利昔单抗,infliximab)和重组人Ⅱ型TNF受体-抗体融合蛋白(rhTNFR:Fc),对它们的使用剂量、近期疗效、安全性进行评价.     

基于中国仓鼠卵巢细胞表达的Fc融合蛋白制备工艺的优化

目的 优化基于中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达的Fc融合蛋白制备工艺.方法 对于上游细胞培养工艺,先在摇瓶中分别对培养基配比和流加培养方案进行优化,再将优化工艺放大到生物反应器规模并确认其可行性.对于下游蛋白纯化工艺,分别对A蛋白亲和层析的洗脱条件和阴离子交换层析的平衡条件进行优化.结果 对于上游细胞培养工艺,以种子培养基∶基础培养基(CD OptiCHO)∶基础培养基(CDM4Mab)为2∶1∶1的配比配制培养基,并以3、6和9d补料的流加培养方案进行细胞培养,可获得理想的细胞密度和活率,且成本较低.对于下游蛋白纯化工艺,以pH3.3的洗脱液进行A蛋白亲和层析纯化时,Fc融合蛋白的回收率为94.7％,纯度为98.64％;以pH5.0～pH5.5的平衡液进行阴离子交换层析纯化时,Fc融合蛋白的回收率达到98％以上、纯度达到99％.结论 基于CHO细胞表达的Fc融合蛋白制备工艺的上游细胞培养和下游蛋白纯化工艺得到成功优化,这为此类抗体药物制备工艺的优化提供了思路.     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白优化治疗活动期类风湿关节炎的研究

目的： 通过对比重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白（rhTNFR：Fc）不同时间给药联合甲氨蝶呤（MTX）治疗活动性类风湿关节炎的临床疗效,探索重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白最佳治疗时间。方法： 2015年1月至2017年12月收治的活动性类风湿关节炎患者90例随机分为重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白上午治疗组和重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白下午治疗组,各45例。重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白上午治疗组给予基础药物（MTX）与上午（8：00~10：00）重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白治疗,重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白下午治疗组给予基础药物（MTX）与下午（16：00~18：00）重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白治疗,均治疗12周。比较治疗前后临床症状、实验室参数的变化情况及不良反应发生情况。结果： 治疗后2组患者的压痛关节数（TJC）、肿胀关节数（SJC）、血沉（ESR）、C反应蛋白（CRP）、基于28个关节的疾病活动指数（DAS28-ESR）、类风湿因子（RF）、抗环瓜氨酸肽抗体（ACPA）、血清肿瘤坏死因子α（TNF-α）、白介素6（IL-6）水平均较治疗前显著降低（P<0.05）,且重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白下午治疗组患者的TJC、SJC、ESR、DAS28-ESR、ACPA及血清IL-6水平显著低于重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白上午治疗组（P<0.05）;2组患者不良反应总体发生率相似（P>0.05）。结论： 重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白下午（16：00~18：00）给药联合甲氨蝶呤治疗活动性类风湿关节炎的疗效可能优于重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白上午（8：00~10：00）给药联合甲氨蝶呤,需进一步研究。     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白致药疹2例

2例患者（例1女,59岁;例2女,67岁）分别因中轴型脊柱关节炎和类风湿关节炎应用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白（rhTNFR：Fc）治疗。例1接受rhTNFR：Fc 50 mg皮下注射、1次/周。治疗3次后双侧上臂出现粟粒大小红色丘疹,伴瘙痒,自行停用rhTNFR：Fc后皮疹减轻;2周后再次使用rhTNFR...     

利用基因改造的鸡制备重组TNF受体／Fc融合蛋白

本研究构建了一种逆转录病毒载体,该载体含有编码人肿瘤坏死因子(TNF)受体2胞外域和人IgG1 Fc区融合蛋白(TNFR/ Fc)的基因表达框,期望成为治疗类风湿关节炎的有效药物。向发育中的鸡胚注射高滴度的病毒载体,孵化出的鸡能在血清     

A long-acting erythropoietin fused with noncytolytic human Fc for the treatment of anemia

The Fc fusion technology has been introduced to generate long-acting antagonistic drugs such as Enbrel, Orencia and Amevive. Here, Genexine created a novel noncytolytic hybrid Fc (hyFc) as a carrier of agonistic protein drugs using naturally existing IgD and IgG4 Fcs without any mutation in the hyFc region. The erythropoietin (EPO) fused with hyFc exhibited little binding activity to FcγR and C1q molecules that are main mediators for death of target cells. The EPO-hyFc showed higher in vitro and in vivo bioactivities than EPOIgG1 Fc and highly glycosylated EPO (Aranesp). Phase I clinical trial with EPO-hyFc is currently undergoing in Korea.     

Comparison of assay formats for drug-tolerant immunogenicity testing

Background: Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development. Results: Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(?) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive. Conclusions: Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.     

Clinical and pharmacological experience with etanercept

Tumour necrosis factor (TNF) is the dominant mediator of the cytokine cascade that causes inflammation and joint destruction in rheumatoid arthritis (RA). A new class of agent under investigation, the biological TNF inhibitors, inhibit the activity of TNF. Recombinant human TNF receptor p75 Fc fusion protein (TNFR:Fc; etanercept) blocks the activity of the cytokine TNF. Etanercept (Enbrel?) was recently approved by the Food and Drug Administration for the treatment of RA. Etanercept is a genetically engineered recombinant soluble TNF receptor fusion protein that binds to TNF body and inhibits its biologic action; it is the first biotechnology product approved for the treatment of RA. Remarkable progress has been achieved in the treatment of RA with the development of TNF antagonists. Recent clinical trials have improved our understanding of the major role played by the cytokine TNF in provoking joint inflammation in RA.     

Clinical and pharmacological experience with etanercept

Tumour necrosis factor (TNF) is the dominant mediator of the cytokine cascade that causes inflammation and joint destruction in rheumatoid arthritis (RA). A new class of agent under investigation, the biological TNF inhibitors, inhibit the activity of TNF. Recombinant human TNF receptor p75 Fc fusion protein (TNFR:Fc; etanercept) blocks the activity of the cytokine TNF. Etanercept (Enbrel) was recently approved by the Food and Drug Administration for the treatment of RA. Etanercept is a genetically engineered recombinant soluble TNF receptor fusion protein that binds to TNF body and inhibits its biologic action; it is the first biotechnology product approved for the treatment of RA. Remarkable progress has been achieved in the treatment of RA with the development of TNF antagonists. Recent clinical trials have improved our understanding of the major role played by the cytokine TNF in provoking joint inflammation in RA.     

重组Exendin-4-胰高血糖素样肽1/IgG4(Fc)融合蛋白的表达和活性研究

目的  构建表达长效胰高血糖素样肽1（glucagon-like peptide 1,GLP-1）受体激动剂重组Exendin-4-GLP-1/IgG4(Fc)融合蛋白的质粒载体,并研究该融合蛋白的活性。方法  将编码Exendin-4-GLP-1/IgG4(Fc)的重组基因插入表达载体pOptiVEC™-TOPO^®来构建重组质粒Exendin-4-GLP-1/IgG4(Fc)-pOptiVEC™-TOPO^®。将构建的重组质粒转染CHO/DG44细胞并收获表达产物后,分别用亲和层析法和免疫印迹法对表达产物进行纯化和检测。通过胰岛素释放实验确认重组融合蛋白对INS-1细胞胰岛素分泌的影响,同时在CD1小鼠中研究该融合蛋白对血糖的调节作用。结果  转染重组质粒的CHO/DG44细胞可成功表达Exendin-4-GLP-1/IgG4(Fc)。蛋白质印迹法检测显示,纯化的表达产物的相对分子质量（M_r）与预期相符（重组融合蛋白单体和二聚体的M_r分别约为35 000和70 000）。胰岛素释放实验表明,在葡萄糖浓度恒定的情况下INS-1细胞分泌的胰岛素量随重组融合蛋白浓度的升高而增加。CD1小鼠实验显示,重组融合蛋白对链脲佐菌素诱导的糖尿病小鼠的血糖具有调节作用,Exendin-4-GLP-1/IgG4(Fc)处理的糖尿病小鼠的血糖明显低于对照糖尿病小鼠（F=3194,P＜0.01）。结论  Exendin-4-GLP-1/IgG4(Fc)具有天然GLP-1的活性,可作为GLP-1受体激动剂用于2型糖尿病的治疗。     

Delivery of an erythropoietin-Fc fusion protein by inhalation in humans through an immunoglobulin transport pathway.

A novel drug delivery platform has been developed that utilizes a naturally occurring receptor known as the neonatal Fc receptor (FcRn). The receptor is specific for the Fc fragment of IgG and is expressed in epithelial cells where it functions to transport immunoglobulins across these cell barriers. It has been shown that FcRn is expressed in both the upper and central airways in non-human primates as well as in humans. Pulmonary delivery of an erythropoietin- Fc fusion molecule (EpoFc) was previously demonstrated in non-human primates using this FcRn pathway. We have now conducted a phase I clinical study to test whether the FcRn pathway functioned similarly in man using human erythropoietin (Epo) fused to the Fc portion of human IgG1. The design was a three leg, non-randomized study conducted in healthy male volunteers with rising doses (3, 10, and 30 microg/kg) of the fusion protein targeted to the central lung regions. Using a target range of 10-30% vital capacity and 15 breaths per minute, approximately 70% of the lung-deposited dose of aerosolized EpoFc was delivered safely and effectively to the central lung regions. We showed dose-dependent concentrations of the fusion protein in the serum and an increase in circulating reticulocytes was evident in the highest dose group, thus demonstrating that large therapeutic molecules can be delivered to humans via the lung, with retention of biological activity, using the FcRn-mediated transport pathway.     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白联合甲氨蝶呤治疗银屑病关节炎的临床疗效和安全性初步研究

目的评价重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc;商品名:益塞普)联合甲氨蝶呤治疗银屑病关节炎(PsA)患者的临床疗效以及安全性。方法本研究为对照开放性试验,共纳入16例活动期脊柱病型PsA患者,至少服用甲氨蝶呤10～15mg/周联合非甾体抗炎药3个月临床未缓解。试验组加用rhTNFR:Fc每周50mg皮下注射,对照组加用其他改善病情抗风湿药(DMARDs)(来氟米特3例,沙利度胺3例,柳氮磺吡啶2例)。主要评价指标为2,4,8,12周达到强直性脊柱炎(AS)评价标准(ASAS)20改善的比例以及银屑病皮损面积和严重性指数(PSI75),次要评价指标包括上述时间达到ASAS50改善的比例以及PSI50,ASAS评分系统各单项指标的变化。并同时记录2组药物不良反应。计量资料比较采用t检验或Wilcoxon秩和检验,计数资料比较采用χ2检验。结果第2周对照组所有指标较基线均无明显改善,而试验组均明显改善(P<0.05)。至12周时试验组及对照组达到ASAS20改善的患者数分别为7例(7/8)及2例(2/8),2组比较差异有统计学意义(P<0.05)。试验组ASAS50、PSI75、PSI50均明显优于对照组。2组均未见严重不良反应发生。结论 rhTNFR:Fc治疗PsA能明显改善脊柱关节症状,减轻皮损,并且具有良好的安全性和耐受性。     

类风湿关节炎患者应用重组人Ⅱ型肿瘤坏死因子α受体-抗体融合蛋白联合甲氨蝶呤治疗过程中并发淋巴瘤

1例81岁男性患者因类风湿关节炎病情加重,皮下注射重组人Ⅱ型肿瘤坏死因子α受体-抗体融合蛋白25 mg,2次/周;口服甲氨蝶呤10 mg,1次/周。治疗13个月后发现左下颌下无痛性肿物,切除肿物,病理确诊为腺淋巴瘤。术后停用重组人Ⅱ型肿瘤坏死因子α受体-抗体融合蛋白及甲氨蝶呤,随访8个月淋巴瘤无复发。