Abciximab

点击上方的 行舟Drug ▲ 添加关注作者｜周永飞，杨敬鹏，常军亮，曹玉锋人源化单克隆抗体目前在肿瘤、自身免疫病、抗病毒等多个治疗领域发挥着越来越重要的作用。截止目前，FDA已有50多种人源化抗体药物获批上市，人源化抗体为人类疾病的治疗提供了广阔的思路。抗体人源化技术包括嵌合抗体技术、人源化抗体技术和全人源抗体技术，并随之衍生出单链抗体、双特异性抗体、纳米抗体等多种形式的小分子人源化单克隆抗体。本文对人源化单克隆抗体技术及人源化抗体的发展形势作一综述。最早的单克隆抗体（简称单抗）是1975年Milstein和Kohler发明的鼠源单抗［1］，虽然其具有广阔的应用前景，但由于恒定区可被人体免疫系统识别而产生免疫副反应，诱发人抗鼠抗体反应（human anti-mouse antibody reaction，HAMA）［2］，鼠源单抗的应用有诸多限制，目前主要应用于诊断领域。近年来，抗体技术的研究方向逐渐向人源方向发展［3］，迄今全球已上市的治疗性抗体药物有130多种，在抗肿瘤、抗病毒等多个领域发挥巨大作用［4］。抗体技术经40多年的发展，目前已有人鼠嵌合抗体、人源化抗体及全人源单抗等［5-6］，兔源及驼源抗体也逐渐应用于治疗领域，同时也出现了不同于原始抗体结构的抗体，如单链抗体、双特异性抗体、纳米抗体等，它们在传统抗体的结构上进行改进，使抗体的应用更具有靶向性、功能性和操作性，能够实现很多传统抗体所不能实现的功能。本文对近年来单抗人源化技术及其发展形势作一综述。1 单抗人源化技术1. 1 人鼠嵌合抗体（chimeric antibody）技术人鼠嵌合抗体是抗体人源化进程中最早研究的抗体，是一种基因工程表达的重组抗体［7］，通过将鼠源抗体可变区基因与人源抗体恒定区基因融合表达，使其人源序列占整个抗体的70%左右，大大降低了抗体的HAMA［8］。由于不同亚型的IgG恒定区与补体和Fc受体的作用能力不尽相同，筛选得到的抗体恒定区可产生抗体依赖的细胞介导的细胞毒作用补体依赖性的细胞毒作用（complement dependent cytotoxicity，CDC）等多种功能［9］，目前应用最多的为IgG形式的嵌合抗体。最早上市的人鼠嵌合抗体是Reopro，之后还有Sylant、Rituximab［10］、Infliximab等成功上市。由于人鼠嵌合抗体所保留的鼠源序列较多，在进行临床实验时仍会被人体的免疫系统所识别产生HAMA发生，同时，由于其免疫位点的改变，致使抗体的活性降低，在实际应用中往往需多次免疫才能达到预期效果［11］。1. 2 人源化抗体技术人源化抗体技术以人鼠嵌合抗体为基础继续进行改造，鼠源序列进一步减少，从而减少HAMA的发生［12］。抗体的可变区包含3个互补决定区（complementary determining region，CDR）和4个框架区（framework region，FR），人源化抗体通过抗体重构、框架区重构等技术来达到减少鼠源序列的目的。目前上市的人源化抗体有Trastuzumab、Bevacizumab、Ranibizumab、Aletuzumab等。1.2.1 抗体重构抗体重构也称为CDR移植，与嵌合抗体不同的是，抗体重构是将人源的FR序列克隆至抗体的相应部位，仅保留了鼠源的CDR序列。重构抗体的人源化程度可达90%以上，目前该方法是单抗人源化过程中最常用的方法［13］。由于重构抗体移植区域明显少于人鼠嵌合抗体，该抗体中异源序列的含量进一步减少，免疫原性显著降低，临床疗效良好。但抗体重构在实际应用中仍存在潜在的免疫原性，因此，CDR移植的发展方向是减少不必要的CDR的引入。1.2.2 特异性决定残基（specificity determining residue，SDR）移植随着研究的深入，人们发现CDR区并非所有的氨基酸序列均具有免疫原性，CDR区的某些特定的氨基酸残基可与抗原接触，从而识别特定抗原，但还有其他氨基酸序列未参与该过程，这些与抗原接触的氨基酸序列被称为SDR。在构建重组抗体的过程中，只需将SDR克隆至抗体的框架中，即可获得目标抗体，从而进一步降低鼠源序列［14］。1.2.3 框架区重构在CDR移植过程中，对FR进行的筛选选择与鼠源抗体序列最相似的人FR基因序列使该抗体具有更高的活性［15］。FR的修饰包括表面重塑与糖基化修饰两方面：表面重塑即将鼠源抗体FR表面氨基酸残基进行人源化，而这些氨基酸残基在免疫应答中并不能起到重要作用；糖基化修饰即改变原有的糖基化位点，从而使抗体的作用效力更高。框架区重构可得到亲和活性明显提升的抗体，是重构抗体中经常用到的手段［16］。1. 3 全人源抗体技术人源化单抗仍具有潜在的免疫原性，而且各种氨基酸的替换需要大量的实验，而全人源抗体的氨基酸序列均由人源抗体基因序列编码，理论上可达到100%的人源化［17］，见图1。目前应用最多的全人源抗体技术有转基因鼠技术和抗体库技术［18］。目前已上市的全人源抗体有Nivoluma、Pembrolizumab、Denosumab等。1.3.1 转基因鼠技术1994年，美国首先利用转基因小鼠生产人源化单抗。转基因小鼠技术将优化的编码人抗体DNA序列导入至免疫缺陷小鼠的基因组中［19］，由于缺乏鼠源性免疫球蛋白基因，在抗原免疫后，小鼠分泌的抗体均由人抗体的DNA序列编码，同时产生的抗体也不会被小鼠的免疫系统识别并产生其抗体［20-21］。由于该抗体是在体内产生，经历了正常的抗体装配和成熟，从而保证了产生的抗体具有很高的靶结合能力。已上市的Nivolumab、Denosumab、Panitumumab 等均是由转基因小鼠生产的。但比较显示，转基因动物产生的全人源单抗并没有野生型抗体产生的作用明显，另外，由于转基因小鼠免疫系统仍为小鼠本身的系统，不能产生和人体完全相同的抗体。1.3.2 抗体库技术抗体库技术首先需建立抗体库，目前有免疫抗体库和非免疫抗体库两种［22］。免疫抗体库的建立首先要通过疫苗注射等途径进行免疫，再从体内采集抗体，通过单细胞克隆技术等建立抗体库［23-24］。非免疫抗体库有半合成抗体库、全合成抗体库和天然抗体库3种：半合成抗体库是合成随机的CDR序列；全合成抗体库则是整个可变区完全由人工合成；天然抗体库的抗体基因来源于未免疫个体的淋巴细胞，由于未用特异性抗原免疫，可用于多种抗原的筛选，但所得抗体由于未经抗体亲和力成熟等过程，亲和力较低［25］。1.3.2.1 噬菌体展示技术（phage dispay）噬菌体展示技术将抗体库中可变区基因在噬菌体表面表达，经抗原抗体结合后，筛选出亲和力最高的抗体，从而用于制备人源化单抗产品［26］。由于该方法是在体外筛选，更易于自动化与高通量化。体外筛选还克服了免疫耐受性，能够筛选出识别高度保守靶点的蛋白。Necitumumab及Adalimumab等均是由噬菌体库筛选的［27］。但库容量的限制、宿主对氨基酸修饰的限制、细胞毒蛋白的限制等仍会影响目的蛋白质的表达。1.3.2.2 核糖体展示技术（ribosome display）与噬菌体展示技术不同的是，核糖体展示技术所产生的抗体不会存在于噬菌体表面，而是直接在非细胞培养系统中表达。该技术首先需利用PCR扩增目的基因DNA文库，再导入一系列转录元件进行体外转录，在无细胞翻译系统中进行翻译，使靶基因的翻译产物展示在核糖体表面，形成mRNA-蛋白质-核糖体三元复合体，再向翻译混合物中加入抗原进行筛选，以获得亲和力最高的蛋白质。核糖体展示技术作为一种无细胞展示系统，经20多年的发展，已成功应用于体外筛选特异性结合抗体［28］。与噬菌体展示技术相比，核糖体展示技术解决了噬菌体展示技术的抗体库容量问题，可表达包含毒性蛋白在内的所有蛋白质，实现了更高库容量的表达。1.3.2.3 mRNA 展示（mRNA display）技术mRNA展示技术的原理与核糖体展示技术基本相似，不同的是，mRNA展示系统翻译出来的蛋白是与mRNA通过接头连接在一起的，由于无核糖体的存在，更有利于筛选，而核糖体展示由于mRNA、多肽和核糖体三位一体，可能会影响接下来的筛选，丢失部分抗体，同时，由于其结合的不稳定性，很有可能导致筛选失败［29］，因此，mRNA展示技术是核糖体展示技术的改良与延伸。1.3.2.4 酵母展示技术（yeast display）酵母展示技术利用酵母作为载体，将外源基因通过融合表达的形式定位至酵母细胞表面，从而展现出最完整的蛋白结构以进行筛选。酵母作为一种真核生物，既能实现蛋白表达过程中的翻译后修饰过程，又能将蛋白的表位完全呈现出来，还可通过荧光素等标志物标记酵母细胞，继而利用流式细胞术进行实时和定量筛选。1.3.2.5 单细胞技术随着基因测序技术及流式细胞术的发展，近年来单细胞技术研究火热，在人源化抗体领域应用广泛。该技术可从建立好的抗体库内根据细胞表面标记分子筛选出单个细胞，细胞富集后利用RT-PCR直接获得抗体的重链和轻链基因［30］。免疫过疫苗的人外周血抗体库筛选出来的高特异性抗体可用来制备全人源抗体，免疫过疫苗的兔脾脏可用来筛选兔源单抗，经抗体人源化后即可应用于人体，Brolucizumab为全球首个获批的兔源单抗［31］。综上所述，抗体库技术彻底改变了单抗的制备方法，摆脱了对鼠源细胞的依赖，产生的抗体完全人源化，是当下研究的热点。2 新型抗体随着抗体技术的发展，抗体的结构也不仅局限于最原始的结构，逐渐发展出单链抗体、双特异性抗体、纳米抗体以及以DNA编码的单抗等。目前已有多种不同类型的人源化单抗上市。2. 1 单链抗体（single-chain Fv）单链抗体由于不含抗体恒定区，其相对分子质量要小于正常的抗体，同时也失去了与Fc 受体结合的功能，但也因此，其可塑性非常强，可与多种分子结合，从而介导不同的杀伤作用，如制备靶向作用的“生物炮弹”、抗体融合蛋白［32］、抗体-药物偶联物（antibody-drug conjugate，ADC）［33］等，同时，由于其相对分子质量小，产生HAMA的几率非常低［34］，已上市的单链抗体有Bren-tuximab vedotin 和Ado-trastuzumabemtansine等。单链抗体人源化的技术有多种，以噬菌体展示和核糖体展示技术最为常用，鼠源抗体经蛋白质测序后进行CDRs移植的方法也有文献报道［35］。但单链抗体也存在抗原性弱、效果不稳定的缺点，在实际应用中往往存在诸多限制。2. 2 双特异性抗体（bispecific antibody）双特异性抗体的可变区含有两种不同抗原的结合位点［36-37］，这两种结合位点可以是分别与效应细胞和肿瘤细胞结合的，从而激发出具有导向性的免疫反应［38-39］，也可以是靶向两种肿瘤位点的，以减少用药量以及临床审批的步骤，也会起到更强的免疫作用。目前双特异性抗体制备最常规的手段是基因工程方法，也可利用两种杂交瘤细胞融合、化学偶联法等方法制备［40］，现已成为抗体工程领域研究的热点，在肿瘤治疗及抗病毒领域应用广泛［41］。同时靶向CD19和CD3的Blinatumomab以及治疗血友病的Emicizumab均为该类抗体药物［42］。2. 3 纳米抗体（nanobody）纳米抗体是1993年最早从羊驼体内发现的抗体，其天然缺失轻链和CH1恒定区。由于其相对分子质量小，体内组织渗透性好，可很轻易地穿过血管到达靶标。天然的羊驼纳米抗体与人源抗体相比，其氨基酸残基差别并不大。目前已有利用噬菌体展示技术以及转基因小鼠技术制备全人源化纳米抗体的报道。针对von Willebrand因子的Caplacizumab-Yhdp作为全球首个获批的纳米抗体药物，为纳米单抗药物的研发提供了广阔的前景。由于目前上市的产品较少，未来纳米抗体的长期使用是否会产生免疫副反应仍需观察。2. 4 DNA 编码的单抗（DNA-encoded monoclonal antibody，dMAb）DNA编码的单抗是新兴起的一种全人源单抗技术。与当前常规治疗性抗体不同，其是在人体内产生，而并非在体外合成。与DNA疫苗类似，dMAb首先需构建出一种序列优化的质粒，用于编码一种单抗，借助CELLECTRA誖等公司的电穿孔技术将重组质粒转至人体内，使该单抗基因在人体中表达。dMAb可进行序列优化，从而使抗体在体内表达量显著提升，刺激强大的CD8+T细胞浸润，同时还能保持以往的抗原抗体结合能力。dMAb技术应用非常广泛，当前现有的人源化单抗均可由基因编码后在人体内表达，是一种抗体全人源化的全新手段。dMAb技术由于兴起较晚，目前还处于研发阶段，暂时无上市的单抗［43］。3 国内外抗体药物发展趋势目前国外人源化单抗的生产以创新为主，全球已有80多种单抗类药物获批上市，其中美国食品药品监督管理局已批准50多种。截至2019年5月，国内共批准单抗药品36个，其中国产14个，进口22个，与美国差距明显。随着多种原研单抗药物的专利到期，近年来国外生物类似药获批数量也逐渐增多，呈井喷式发展。截止目前，欧盟已有17种单抗类生物类似药获批上市，美国已有10种。最早获批上市的生物类似药是欧盟2013年批准的英夫利西单抗，2016年，美国安进公司（Amgen）阿达木单抗的生物类似药AMJEVITA在美国食品药品监督管理局获批上市，这也是阿达木单抗首个获批的抗体生物类似药。国内人源化单抗由于起步较晚，研发水平与发达国家存在较大差距，目前以进口药为主，但自研药以及生物类似药的研究也发展迅速。2015年2月，国家食品药品监督管理总局正式颁布《生物类似药研发与评价技术指导原则》（试行），规范了生物类似药的申报程序、注册类别和申报资料等相关注册要求，为生物类似药的研发提供了基本指导和依据。在政府的大力支持及行业和市场的合力驱动下，国内的生物类似药研究呈指数上升趋势，靶点主要集中在CD20、EFGR、VEGF、TNF和HER2，包括阿达木单抗、贝伐单抗、曲妥珠单抗、利妥昔单抗及英夫利昔单抗等在内的多种专利已过期或即将过期的原研单抗药物成为国内药企争先研发的热点。2019年2月，由上海复宏汉霖生物技术股份有限公司研发的首个国产利妥昔单抗生物类似药汉利康誖上市，意味着在生物类似药开发领域，我国生物医药产业已处在从“跟跑欧美”转向“与欧美并跑”的关键节点。4 展望近年来，在全球批准的新药中，人源单抗类药物所占比例越来越大，可见人源单抗具有很高的应用价值，为肿瘤、自身免疫性疾病等多种疾病的治疗提供了新的思路。虽然抗体人源化后理论上能降低HAMA，但目前尚无法完全消除，越来越多的报道显示，人源化抗体在应用过程中仍可产生HAMA［44］，因此，合理运用上述方法和技术创新，在原有的基础上减少抗体异源性，不断提高抗体的亲和力，开发出针对全新靶点的人源抗体，是抗体药物发展的必由之路，必将为新药开发开辟新的道路。另一方面，随着生物类似药的逐渐上市，未来以外企为主的竞争格局将逐渐被打破，鉴于生物类似药较原研药具有明显的价格优势，加上国家医保政策的强力支持，生物类似药将更好、更广泛地惠及患者，对医疗大健康产业持续性地产生积极影响。------------THE END------------文章信息源于公众号天咨医药，登载该文章目的为更广泛的传递行业信息，不代表赞同其观点或对其真实性负责。文章版权归原作者及原出处所有，文章内容仅供参考。本网拥有对此声明的最终解释权，若无意侵犯版权，请联系小编删除。学如逆水行舟，不进则退；心似平原走马，易放难收。行舟Drug每日更新 欢迎订阅+医药大数据|行业动态|政策解读

Glenn Mattes to step down as Chief Executive Officer and as a Member of the Board of Directors Dr. Harlan F. Weisman, Vice Chairman of the Board, appointed as Interim Chief Executive Officer FORT WORTH, Texas, Dec. 05, 2022 (GLOBE NEWSWIRE) -- TFF Pharmaceuticals, Inc. (NASDAQ: TFFP), a clinical-stage biopharmaceutical company focused on developing and commercializing innovative drug products based on its patented Thin Film Freezing technology platform, today announced that Glenn Mattes will step down as Chief Executive Officer and Member of the Board of Directors. Dr. Harlan F. Weisman, Vice Chairman of the Board, has been appointed Interim Chief Executive Officer, effective December 5, 2022. “On behalf of the entire Board of Directors and our shareholders, I would like to thank Glenn for his leadership and accomplishments,” said Dr. Aaron Fletcher, TFF Pharmaceuticals’ Chairman of the Board. “During Glenn’s tenure, TFF Pharmaceuticals completed its IPO financing, advanced two proprietary pipeline candidates into clinical testing, and began collaborations with many of the leading global biotechnology and pharmaceuticals companies. Under his leadership, Thin Film Freezing applications have expanded, and the platform is now recognized as a promising, next-generation technology in drug delivery. We thank Glenn for helping TFF Pharmaceuticals, and we wish him well in his future endeavors.” “I would like to thank the dedicated employees of TFF Pharmaceuticals for having the opportunity and privilege to work with all of you over the last five years on a truly unique and innovative technology that could have a great impact on many patients,” said Glenn Mattes. “I am confident that under Harlan’s leadership, this mission will continue, and I look forward to helping the Board in any way during this transition period for the Company.” “I am honored to serve as Interim Chief Executive Officer of TFF Pharmaceuticals, and I would like to thank our Board of Directors for this opportunity,” said Dr. Harlan F. Weisman. “Based upon the Company’s significant pipeline growth and impressive collaboration activity, I remain confident that we are only starting to realize the full therapeutic and commercial potential of Thin Film Freezing technology.” Harlan F. Weisman, M.D. has more than 30 years of experience as a senior healthcare executive responsible for the discovery, development, regulatory approval and launch of pharmaceutical, biopharmaceutical, medical device and diagnostic products. He is currently Managing Director of And-One Consulting, LLC, advising medical product companies, investment firms, and government and non-government healthcare organizations in formulating and implementing strategies for driving innovation in healthcare products and services. He is on advisory committees to the Johns Hopkins University Institute for Basic Biomedical Sciences and the Memorial Sloan Kettering Cancer Center Technology Development Fund. Dr. Weisman began his career in the pharmaceutical industry at Centocor, Inc., where he pioneered monoclonal antibodies as medical breakthroughs for wide therapeutic applications, including ReoPro®, REMICADE®, Simponi® and STELARA® which have combined annual sales of over $8 Billion. He was the head of R&D when Johnson & Johnson acquired Centocor in 1999. He then became President of J&J Pharmaceutical R&D, where he accelerated pipeline growth by advancing more than15 new molecular entities into late-stage development within 3 years, and gaining approvals of 10 drugs with over $15 Billion in annual sales. Dr. Weisman was promoted to Company Group Chairman, Pharmaceutical R&D, and had executive oversight of ALZA Corporation and J&J Pharmaceutical R&D, and led the acquisition and integration of 3-Dimensional Pharmaceuticals and later TransForm Pharmaceuticals. Dr. Weisman was Co-founder, Chairman and CEO of Flame Biosciences, a clinical stage company focused on the research, development, and commercialization of transformative therapies for cancer, from January 2020 to January 2022. Between December 2012 and December 2013, Dr. Weisman was Chairman and Chief Executive Officer of Coronado Biosciences, a biopharmaceutical company developing novel immunotherapies for autoimmune diseases and cancer. Dr. Weisman graduated from the University of Maryland with High Honors and the University of Maryland School of Medicine Cum Laude. After his residency in Internal Medicine at Mount Sinai Hospital in New York, he was a fellow in cardiovascular disease at Johns Hopkins Hospital, and was then appointed as Assistant Professor of Medicine at Johns Hopkins University School of Medicine, Consultant Cardiologist, Johns Hopkins Hospital, and Director of the Experimental Cardiac Pathology Laboratory. About TFF Pharmaceuticals’ Thin Film Freezing technology platformTFF Pharmaceuticals’ proprietary Thin Film Freezing (TFF) technology allows for the transformation of both existing compounds and new chemical entities into dry powder formulations exhibiting unique characteristics and benefits. The Thin Film Freezing process is a particle engineering process designed to generate dry powder particles with advantageous properties for inhalation, as well as parenteral, nasal, oral, topical and ocular routes of administration. The process can be used to engineer powders for direct delivery to the site of need, circumventing challenges of systemic administration and leading to improved bioavailability, faster onset of action, and improved safety and efficacy. The ability to deliver therapies directly to the target organ, such as the lung, allows TFF powders to be administered at lower doses compared to oral drugs, reducing unwanted toxicities and side effects. Laboratory data suggests the aerodynamic properties of the powders created by Thin Film Freezing can deliver as much as 75% of the dose to the deep lung. Thin Film Freezing does not introduce heat, shear stress, or other forces that can damage more complex therapeutic components, such as fragile biologics, and instead enables the reformulation of these materials into easily stored and temperature-stable dry powders, making therapeutics and vaccines more accessible for distribution worldwide. The advantages of Thin Film Freezing can be used to enhance traditional delivery or combined to enable next-generation pharmaceutical products. About TFF PharmaceuticalsTFF Pharmaceuticals, Inc. is a clinical-stage biopharmaceutical company engaging patented rapid freezing technology to develop and transform medicines into potent dry powder formulations for better efficacy, safety, and stability. The company’s versatile Thin Film Freezing (TFF) technology platform has broad applicability to convert most any drug, including vaccines, small and large molecules, and biologics, into an elegant dry powder highly advantageous for inhalation, with improved absorption so drugs can also be delivered to the eyes, nose and topically to the skin. TFF has two lead drug candidates in the clinic: Voriconazole Inhalation Powder and Tacrolimus Inhalation Powder and continues to expand its pipeline by collaborating with a broad array of pharmaceutical companies, academic institutions, and government partners to revolutionize healthcare around the globe. The TFF Platform is protected by 120+ patents issued or pending in the U.S. and internationally. To learn more about TFF Pharmaceuticals and its product candidates, visit the Company’s website at https://tffpharma.com. Safe Harbor This press release contains forward-looking statements regarding TFF Pharmaceuticals, Inc., including, the benefits of the Company’s TFF platform and the Company’s plans to add to its existing pipeline of product candidates. Those forward-looking statements involve known and unknown risks, uncertainties and other factors that could cause actual results to differ materially. Among those factors are: (i) the risk that the Company may not be able to successfully conclude clinical testing or obtain pre-market approval of any of its dry powder product candidates, (ii) no drug product incorporating the TFF platform has received FDA pre-market approval or otherwise been incorporated into a commercial drug product, (iii) the Company has no current agreements or understandings with any large pharmaceutical companies for the development of a drug product incorporating the TFF Platform, (iv) the risk that the Company will not be able to conclude a long-term commercial agreement with any third-party, and (vi) those other risks disclosed in the section “Risk Factors” included in the Company’s Quarterly Report on Form 10-Q filed with the SEC on November 14, 2022. TFF Pharmaceuticals cautions readers not to place undue reliance on any forward-looking statements. TFF Pharmaceuticals does not undertake, and specifically disclaims, any obligation to update or revise such statements to reflect new circumstances or unanticipated events as they occur, except as required by law. TFF Contacts: Corporate Contact:Bill Begien VP of Investor Relations TFF Pharmaceuticals, Inc.c (617) 842-2222bbegien@tffpharma.com Investor Relations Contact:Corey Davis, Ph.D.LifeSci Advisors(212) 915-2577cdavis@lifesciadvisors.com Media Contact:Raena Mina, Ph.D.LifeSci Communicationsrmina@lifescicomms.com