2024, Vol. 40中国科学院微生物研究所、中国微生物学会主办
文章信息
- 顾光磊, 方敏
- GU Guanglei, FANG Min
- 单克隆抗体的研究进展及上市药物分析
- Advances of monoclonal antibodies and analysis of marketed antibody drugs
- 生物工程学报, 2024, 40(5): 1431-1447
- Chinese Journal of Biotechnology, 2024, 40(5): 1431-1447
- 10.13345/j.cjb.230779
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文章历史
- Received: November 13, 2023
- Accepted: January 13, 2024
- Published: January 26, 2024
引用本文
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1975年，生物学家利用细胞融合技术，将可在体外培养并一直增殖的小鼠骨髓瘤细胞与经过所需抗原刺激过的纯系小鼠B淋巴细胞融合，产生了以杂交瘤细胞方式制备单克隆抗体的技术[1]。自1986年全球首个鼠源单抗药物上市以来[2]，经过30多年的发展，单抗药物已经成为生物制药中最热门的领域之一，治疗方向覆盖肿瘤、心血管疾病、自身免疫病、神经性疾病和抗感染等领域。抗体药物作为一种有独特功能的靶向性生物治疗药物，以其高安全性、高特异性和高有效性等特点，成为近年来治疗性药物研发的热点。其中全人源单抗相较于其他抗体形式，具有最高的理论安全性，将成为治疗性抗体未来的发展趋势，快速高效地构建高特异性、高亲和力及高安全性的全人源抗体显得尤为重要。目前，国内抗体药物的市场还远没有得到充分发掘，抗体药物在我国的医药研发投入及市场占比远低于欧美发达国家。
1 单克隆抗体的类型 1.1 鼠源单抗最早期的单克隆抗体均为鼠源单抗(mouse-derived antibodies)，来源于小鼠具有分泌特异性抗体能力的B淋巴细胞融合可无限增殖的骨髓瘤细胞而产生的杂交瘤细胞。首个抗移植后免疫排斥反应的鼠源单抗muromonab-CD3 (OKT3)在1986年获得美国食品药品监督管理局(Food and Drug Administration, FDA)批准使用[3]。鼠源单抗在理论及实践上的应用成为解决医学、生物学等重大问题的重要手段之一。然而，鼠源单抗在临床应用中显示出较多问题，如容易产生人抗鼠抗体(human anti-mouse antibodies, HAMA)反应而被当作人体异源蛋白被清除[4]；在应用于人体时活性低，且其Fc片段不能有效结合人类Fc受体，无法发挥抗体依赖性细胞毒性作用以及有效激活人体的补体系统[5]；在人体内稳定性差[6-7]，甚至有些会引起严重的过敏反应等[8]。因此，人们开始致力于人源化抗体的研究。
1.2 人鼠嵌合单抗第二代抗体是人鼠嵌合单抗(human-mouse chimeric antibodies)，是通过基因工程改造的重组抗体类型[9]，通过将鼠源抗体的可变区片段与人源抗体恒定区片段的基因进行融合表达，使人源基因序列占整个抗体蛋白的大部分，降低了单抗的HAMA反应。但由于人鼠嵌合单抗中保留的鼠源序列还占有一定比例，在临床中仍可能会发生HAMA反应，且因为其抗体结构的改变，抗体的生物活性有所降低，影响了人鼠嵌合单抗在临床应用中的效果[10-11]。
1.3 人源化单抗第三代抗体是人源化抗体(humanized antibody)，是以人鼠嵌合抗体为基础继续改造，将鼠源抗体轻链和重链的高变区插入人源抗体框架区，进一步减少抗体中鼠源序列的比例，使人源序列达到95%，以此减少HAMA的产生[12]。人源化单抗技术包括骨架区重构、抗体重构等。抗体重构也被称为互补决定区(complementarity-determining regions, CDR)移植，是将人源抗体框架区克隆至抗体相应部位，单抗仅保留鼠源抗体CDR，进一步降低了抗体蛋白中的鼠源序列比例，免疫原性相较于前两代单抗显著降低，但其抗原亲和力较鼠源单抗明显降低[13]。骨架区重构包括糖基化修饰和抗体表面重塑，糖基化修饰为改变抗体原有的糖基化位点，提高抗体活力，表面重塑则将鼠源框架区氨基酸残基进行人源化修改，达到降低鼠源序列的目的[14]。
1.4 全人源单抗第四代抗体是全人源单克隆抗体(human monoclonal antibody)，抗体蛋白的氨基酸序列均由人源基因编码[15]，进入人体内后发生超敏或排斥反应的可能性最低。目前制备全人源单克隆抗体的技术包括转基因鼠技术、抗体库技术以及单个B细胞技术等。转基因鼠技术将优化的人源抗体DNA序列导入到免疫缺陷鼠的基因组中[16]，其分泌的抗体均由人源抗体的基因编码。抗体库技术需要建立特定抗体库，目前有免疫抗体库和非免疫抗体库这2种[17-18]。单个B细胞技术需要先筛选出抗原特异性的单个B细胞，再利用逆转录PCR和巢式PCR直接获得抗体轻重链基因[19-21]。
2 制备全人源单克隆抗体的技术 2.1 转基因小鼠技术转基因动物为抗体药物的开发提供了一个可靠的平台，转基因动物具有无需人源化、生产抗体多样性强、抗体在体内进行亲和力成熟和克隆选择等优点。但是，在转基因小鼠中产生与人类相似的基因库需要人类抗体基因V、D和J片段在转基因小鼠中的高表达并进行重排[22]。为了克服这些问题，不同的策略已成功地用于生成表达人类抗体库的动物[23-24]。1989年，科学家们克隆了第一个人类重链结构，包含2个人类重链可变区基因，用于与人类重链连接簇(joining region of heavy chain, JH)相连的多样性片段(diversity region, D)和μ恒定区[25]，将这个25 kb的结构体作为微粒质粒显微注射到小鼠受精卵中，允许其随机插入到小鼠的基因组中，在这些转基因小鼠中，约4%的B淋巴细胞表达了人抗体μ链。1992年，Taylor等[26]克隆了人类抗体κ轻链结构，其中包含人类抗体κ可变区(Vκ)、κ连接簇(Jκ)和κ恒定区(Cκ)。转基因小鼠技术将优化过的编码人抗体的基因序列插入至免疫缺陷小鼠的基因组中[16]，由于小鼠缺乏自身免疫球蛋白基因，在经过特定抗原免疫后，小鼠分泌产生的抗体均由插入的人源抗体基因序列编码，同时产生的人源抗体也不会被小鼠缺陷的免疫系统识别并清除[22]。由于抗体是在小鼠体内产生的，经历了正常的克隆选择和亲和力成熟过程，保证了产生的抗体具备正常的抗原结合亲和力和特异性。但有研究表明[26]，转基因小鼠产生的全人源抗体并没有母本抗体的作用明显。另外，由于生产抗体的转基因小鼠免疫系统与人类免疫系统的差异，抗体在克隆选择和亲和力成熟等方面与人体有差异，无法产生和人体完全一致的抗体。
2.2 噬菌体展示技术George P Smith和Gregory P Winter教授获得了2018年诺贝尔化学奖，以奖励他们致力于“多肽和抗体的噬菌体呈现技术”。前者开发了噬菌体展示技术，它可以用于新蛋白质的发现[27]，后者将噬菌体展示技术应用于抗体的发现和分离[28]。噬菌体展示技术也被用于CDR定点突变和抗体的亲和力成熟。从噬菌体展示库中鉴定单克隆抗体始于抗体库的构建。来自人外周血单个核细胞的mRNA被逆转录为cDNA，然后使用特异性抗体引物扩增不同的轻重链可变区基因[29-30]，将其连接到噬菌体展示载体上。噬菌体展示技术通过将所选抗体库中抗体的可变区基因表达在噬菌体表面，筛选出与特定抗原结合亲和力高的抗体，进而用其制备全人源单克隆抗体[31]。基于这些技术，第一个全人源治疗性抗体——阿达木单抗(HUMIRA®)，一种人抗肿瘤坏死因子α (anti-tumor necrosis factor α, TNF-α)抗体[32]，在2002年被美国FDA批准用于类风湿性关节炎的治疗。该方法在体外筛选，更易于提高通量与实现自动化生产，但抗体库容量的限制、细胞毒蛋白的限制和宿主对抗体蛋白氨基酸修饰的限制等仍会影响抗体的表达和筛选。此外，类似的展示技术还包括mRNA展示技术、核糖体展示技术及酵母展示技术等。
2.3 单个B细胞技术随着高通量测序及流式细胞术的普及和发展，单细胞技术在生产人源化抗体方面应用广泛，该技术可从患者或接种疫苗人群的外周血中根据抗原和细胞表面标记物筛选出单个B细胞，再利用逆转录PCR和巢式PCR直接获得抗体轻重链基因。单个B细胞技术的优点是只需少量细胞就可以快速高效地筛选出潜在的抗体轻重链天然配对的单克隆抗体，在新发、突发传染病等紧急事件中，可以快速获得针对病原微生物的高亲和力抗体。使用显微操作[33-34]、激光捕获[35]和荧光细胞分选[34, 36]，可以从外周血单个核细胞或淋巴组织中分离出抗原特异性的单个B细胞。抗原包被的磁珠[37]和荧光标记的抗原[38-39]常被用于筛选抗原特异性B细胞，这一过程被称为抗原诱饵。
在过去的10年中，通过单个B细胞技术获得单克隆抗体的研究越来越深入。虽然单个B细胞技术具有不可替代的优势，但该技术仍存在一些尚未解决的问题。例如，抗原标记、抗原分类(如单体、二聚体乃至四聚体)和引物组的设计都是成功筛选单克隆抗体的重要因素。以单个B细胞技术结合新一代测序技术开发新的诊断和临床治疗用的单克隆抗体是未来的发展趋势，研究人员将高通量测序与单个B细胞技术结合，成功筛选出针对新冠病毒(SARS-CoV-2)的中和性单克隆抗体[40]。
3 抗体片段及抗体类似物 3.1 单链抗体和抗原结合区片段(scFv和Fab)单链抗体(single-chain antibodies, scFv)和抗原结合区片段(fragment of antigen binding, Fab)是目前应用较多的重组抗体片段形式[41]。scFv由抗体轻重链可变区片段组合而成，缺失抗体恒定区，其分子量远小于常规IgG抗体，约25 kDa。scFv失去与细胞表面Fc受体结合的能力，可塑性强，可结合多种分子而介导不同的杀伤作用，如抗体与药物偶联(antibody-drug conjugate, ADC)[42]以及带有靶向作用的抗体融合蛋白[43]等。scFv分子量较小，产生HAMA的可能性很低[44]。但是，scFv也存在一些限制，如特性不稳定、半衰期短等。Fab的分子量大小是scFv的2倍，约50 kDa，由完整的轻链和重链的可变区以及一个恒定区结构域组成，并通过二硫键连接[45-47]。
目前各类scFv和/或Fab的衍生抗体种类较多，如用2个或3个短肽接头连接单体scFv组成多价scFv[48]，通过二硫键连接2个Fab片段生产F (ab′) 2片段[49]，由Fab和scFv通过短肽接头形成Fab-scFv融合抗体[46]，以及可以同时靶向肿瘤抗原和免疫效应细胞从而杀伤肿瘤细胞的双特异性抗体等[50]。目前上市的单链抗体药物有用于治疗眼科病变的单链抗体BEOVU® (Brolucizumab-dbll)以及用于治疗白血病的单链双特异性抗体BLINCYTO® (Blinatumomab)等。
3.2 单结构域抗体(single-domain antibodies)只有重链结构的重链抗体(heavy-chain antibodies, HCAbs)最初在重链疾病中被发现[51]，它们缺乏抗体轻链和部分重链结构域，无法有效识别抗原，而后来在骆驼科物种的血清中发现了具备完全功能的无轻链抗体[52]和鲨鱼等某些软骨鱼类的重链抗体[53-54]。这些独特的HCAbs能够仅通过一个单一的重链结构域识别靶标，即可变的重链结构域，简称为VHH。该结构域也被称为纳米抗体(nanobody)或单结构域抗体，代表了具有抗原结合功能的最小分子量抗体片段，大小仅为12−15 kDa，其在组织中具有高渗透性，在高温或变性条件等恶劣环境中具有高稳定性，并且容易在细菌中生产。HCAbs可以识别在热和恶劣化学条件下形成的不寻常的抗原表位，拓展了新抗体制备的技术方法[55-58]。然而，HCAbs在低分子量分析物的鉴定中的应用存在局限性，如霉菌毒素、农药或抗生素，这可能是由于HCAbs缺乏传统抗体及其他重组抗体中存在的轻链结构[59-61]。
3.3 抗体类似物(antibody mimics)抗体类似物包括模拟肽、核酸适配体、分子印迹聚合物和非免疫球蛋白支架等[62-65]。抗体类似物完全在体外生产，抗原识别位点理论上可以识别任何想要的靶标。与抗体相比，抗体类似物的优点包括在恶劣环境下的高稳定性、易于化学修饰、生产重复和变性后易于再生等。然而，抗体类似物对某些类型分子的亲和力不高，特别是那些含有疏水基团及带负电的分子。另外，抗体类似物的交叉反应性也限制了该类型分子的一些应用。
4 核酸抗体由于临床级的重组蛋白表达纯化复杂、生产周期长、储存及运输条件苛刻等，导致单克隆抗体药物的成本高昂。核酸抗体是将含有抗体信息的基因(DNA或mRNA)导入细胞中，通过细胞编码生产相应抗体以发挥保护或预防作用。相较于传统抗体，核酸抗体具有无需复杂的蛋白体外表达纯化过程、序列设计简单、生产成本低等优点，越来越受到各国研究人员的重视[66-68]。
4.1 DNA抗体DNA抗体是利用载体将含有抗体信息的DNA导入宿主细胞，经过转录、翻译等过程后表达相应的抗体以发挥功能。目前，DNA抗体主要的递送载体包括：病毒载体，常用的有腺病毒(adenovirus, AdV)载体和腺相关病毒(adeno-associated virus, AAV)载体等；质粒DNA (plasmid DNA, pDNA)等非病毒载体。AAV载体是最常用的病毒载体，在递送DNA方面起到了重要的作用，但其存在将外源基因整合到宿主染色体上的潜在风险[69-71]。pDNA很难入核，且只有在有丝分裂过程中才被转录[72]，这限制了抗体基因的表达。此外，外源性DNA的引入可能激活细胞质中的cGAS-STING信号通路，诱导Ⅰ型干扰素(interferon-Ⅰ, IFN-Ⅰ)的产生，从而触发针对pDNA的免疫应答[73-75]。
4.2 mRNA抗体mRNA抗体是将编码抗体轻重链的DNA体外转录为mRNA，再通过载体将其导入细胞质，翻译合成抗体蛋白，进而发挥功能。脂质纳米颗粒(lipid nanoparticle, LNP)是近年来最常用的递送载体[76]，此外基因枪、电穿孔、鱼精蛋白、阳离子纳米乳及阳离子聚合物脂质体在递送mRNA方面也较为常用[72]。选择合适的递送方式可以有效避免mRNA降解、提高递送效率及安全性。真核生物mRNA的转录后修饰十分复杂，体外转录的mRNA缺乏相应的修饰，导致其稳定性和蛋白翻译效率较低，通过加帽、加尾以及加入修饰碱基(如假尿苷及5-甲基胞苷等)等均可提高mRNA的稳定性及翻译效率[77-78]。另外，外源性mRNA可以被核内体中的Toll样受体(Toll-like receptors, TLRs)以及细胞质中的视黄酸诱导基因蛋白Ⅰ (retinoic acid-inducible gene Ⅰ, RIG-Ⅰ)和黑色素瘤分化相关基因5 (melanoma differentiation-associated gene 5, MDA5)等受体所感知，激活先天免疫反应，限制抗体蛋白的表达[79]。目前，mRNA抗体在应对病毒感染及治疗肿瘤方面的研究正在加紧进行，其中针对基孔肯雅病毒的mRNA-1944已完成Ⅰ期临床试验[80]，表明了mRNA抗体的安全性及达到适当循环抗体水平的潜力。
5 单克隆抗体药物上市情况自1997年靶向CD20以治疗癌症的Rituximab获批上市以来，截至2023年10月1日，美国FDA已批准163种单克隆抗体药物，适应证涵盖癌症、血液病、免疫病、神经系统疾病及病原体感染等多个领域。随着基因技术的发展及现代医学的深入，单克隆抗体药物的研发愈发火热，新获批的单抗药物逐年增多，针对的靶点及适应证也愈发广泛。下面对获批上市的163种单抗药物的抗体类型、适应证、研发地区等进行综合分析。
5.1 抗体类型获批上市的163种单克隆抗体药物可分为人鼠嵌合抗体、人源化抗体、全人源抗体以及抗体偶联药物和双特异性抗体5个类型(图 1A)，其中人源化抗体有64种，是占比最多的抗体类型，占全部上市单抗药物的39%；其次是55种全人源抗体，占全部药物的34%；人鼠嵌合抗体有19种，占全部药物的12%；另外，抗体偶联药物和双特异性抗体分别有15种和10种，占比9%和6% (表 1)。人鼠嵌合抗体是人们为了降低鼠源单抗可能造成的HAMA以提高单克隆抗体安全性的早期尝试，前期的单克隆抗体药物为该类型抗体，但其安全性始终存在问题。因此，鼠源抗体和人鼠嵌合抗体的人源化对于提高前期研发药物的安全性至关重要，人源化抗体目前占较高比例。艾伯维公司的HUMIRA® (Adalimumab)是第一个获批上市的全人源单克隆抗体药物[81]。随着生物技术的发展，具有更高安全性的全人源抗体的构建及筛选愈发方便，大多数新批准上市的抗体药物均为此类型[82]，全人源抗体将逐渐占据抗体药物市场的主导地位。另外，抗体偶联药物和双特异性抗体的安全性及疗效不断提高，这2种类型将会在抗体药物市场占据更大比例。例如第一三共株式会社研发的偶联曲妥珠单抗和拓扑异构酶抑制剂的抗体偶联药物ENHERTU® (Fam-trastuzumab deruxtecan-nxki，代号为DS-8201)，在治疗HER2+乳腺癌方面疗效显著，对HER2低表达乳腺癌患者也有较好的疗效，并且对曲妥珠单抗(T-DM1)耐药后的肿瘤也有效，成为新一代治疗乳腺癌的“神药”。其他新型抗体形式如单链抗体、单结构域抗体等的研发也在继续深入，相信未来将会有更多的新型抗体药物获批上市。
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| 图 1 单克隆抗体药物上市情况 Fig. 1 Monoclonal antibody drugs on the market. A：批准上市的163种单抗药物分为5种类型. B：单抗药物主要用于治疗癌症、免疫病等. C：单抗药物热门靶点主要有白细胞介素、肿瘤坏死因子、CD20和HER2等. D：批准上市的单抗药物研发主要分布于欧美发达地区. E：以基因泰克为首的10个企业占据全部单抗药物的50% A: The 163 monoclonal antibody drugs approved for marketing are divided into five groups. B: Monoclonal antibody drugs are mainly used for the treatment of cancer, immune diseases, etc. C: Popular targets of monoclonal antibody drugs mainly include interleukin, tumor necrosis factor, CD20, HER2, etc. D: The research and development of monoclonal antibody drugs approved for marketing are mainly distributed in developed regions in Europe and America. E: Ten companies led by Genentech account for 50% of all monoclonal antibody drugs. |
| |
| Commercial name | Active ingredients | Target | Indications | R&D company |
| ADCETRIS | Brentuximab vedotin | CD30 | Hodgkin lymphoma, anaplastic large cell lymphoma | Seattle genetics |
| BESPONSA | Inotuzumab ozogamicin | CD22 | Relapsed or refractory acute lymphoblastic leukemia in adults | Wyeth Pharmaceuticals |
| BLENREP | Belantamab mafodotin-blmf | BCMA | Multiple myeloma | GSK |
| ELAHERE | Mirvetuximab soravtansine-gynx | FRα | Epithelial ovarian cancer, fallopian tube cancer, and primary peritoneal cancer | ImmunoGen |
| ENHERTU | Fam-trastuzumab deruxtecan-nxki | HER2 | Metastatic breast cancer, unresectable or metastatic non-small cell lung cancer | Daiichi Sankyo |
| KADCYLA | Ado-trastuzumab emtansine | HER2 | Advanced (metastatic) breast cancer | Genentech |
| MYLOTARG | Gemtuzumab ozogamicin | CD33 | Acute myeloid leukemia | Wyeth Pharmaceuticals |
| MYOSCINT | Imciromab pentetate | Myosin | Detecting cardiac hypertrophy | Sentoko |
| PADCEV | Enfortumabvedotin-ejfv | Nectin-4 | Refractory bladder cancer | Astellas |
| POLIVY | Polatuzumab vedotin-piiq | CD79b | Relapsed or refractory diffuse large B-cell lymphoma | Genentech |
| PROSTASCINT | Capromab pendetide | PSMA | Detect prostate cancer | Settogen |
| TIVDAK | Tisotumab vedotin-tftv | TF | Cervical cancer | Higgin |
| TRODELVY | Sacituzumab govitecan-hziy | Trop-2 | Metastatic triple-negative breast cancer in adults | Immunomedics |
| ZEVALIN | Ibritumomab tiuxetan | CD20 | Non-hodgkin lymphoma | Spectrum Pharmaceuticals |
| ZYNLONTA | Loncastuximab tesirine-lpyl | CD19 | Certain types of relapsed or refractory large B-cell lymphoma | ADC Pharmaceuticals |
| BLINCYTO | Blinatumomab | CD19 and CD3 | Acute lymphoblastic leukemia | Amgen |
| COLUMVI | Glofitamab-gxbm | CD20 and CD3 | Relapsed or refractory diffuse large B-cell lymphoma | Genentech |
| ELREXFIO | Elranatamab | BCMA and CD3 | Relapsed or refractory multiple myeloma in adults | Pfizer |
| EPKINLY | Epcoritamab-bysp | CD20 and CD3 | Relapsed or refractory diffuse large B-cell lymphoma | Genmab |
| HEMLIBRA | Emicizumab | FIXa and FX | Hemophilia A | Genentech |
| LUNSUMIO | Mosunetuzumab-axgb | CD20 and CD3 | Relapsed or refractory follicular lymphoma in adults | Genentech |
| RYBREVANT | Amivantamab-vmjw | EGFR and cMET | Non-small cell lung cancer | Janssen Biotech |
| TALVEY | Talquetamab-tgvs | GPRC5D and CD3 | Relapsed or refractory multiple myeloma in adults | Janssen Biotech |
| TECVAYLI | Teclistamab-cqyv | BCMA and CD3 | Relapsed or refractory multiple myeloma in adults | Janssen Biotech |
| VABYSMO | Faricimab-svoa | VEGF-A and Ang-2 | Neovascular (wet) age-related macular degeneration, diabetic macular edema | Genentech |
| Data from the United States Food and Drug Administration: Drugs@FDA: FDA-Approved Drugs, as of 10/1/2023. | ||||
目前获批上市的单克隆抗体药物涵盖了癌症、免疫病、血液病等众多领域，随着新一代生物技术的发展，越来越多的疾病领域出现了单克隆抗体药物的身影(图 1B)。针对癌症的单克隆抗体药物最早获批上市，其始终是单抗药物研发的热点，在163种药物中有52种是抗癌药物，占据全部单抗药物近1/3，主要治疗多发性骨髓瘤、乳腺癌、结直肠癌及非小细胞肺癌等恶性肿瘤；免疫病是单抗药物研发的第二大领域，目前已批准上市了34种治疗药物，约占全部单抗药物的1/5，其主要适应证为多发性硬化症、类风湿性关节炎、克罗恩病及强直性脊柱炎等；血液病领域批准上市的单克隆抗体也占据较大比例(28种，17%)，主要针对治疗淋巴细胞白血病及血友病等疾病；皮肤病领域单抗药物有9种，占比6%，主要治疗斑块状银屑病及特应性皮炎等；神经系统疾病领域的单抗药物有8种，占比5%，主要治疗偏头痛及阿尔茨海默病等；直接靶向病原体以治疗病原体感染的单抗药物有8种，占比5%，包括艰难梭菌、炭疽杆菌、埃博拉病毒、艾滋病病毒及呼吸道合胞病毒的感染；眼科疾病领域有7种单抗药物，主要治疗黄斑变性、黄斑水肿等；治疗代谢性疾病的单抗药物有6种，主要治疗高胆固醇血症、糖尿病等；针对呼吸系统疾病的单抗药物有5种，均用于治疗哮喘。
在获批上市的163个单抗药物中存在多个热门靶点，如白细胞介素类、TNF-α及CD20等(图 1C)。目前，有21个单抗药物针对白细胞介素类(intercytokines, IL-5、IL-6、IL-17和IL-23等)，18个针对肿瘤坏死因子(TNF-α)，14个针对CD20，12个针对人表皮生长因子受体2 (human epidermal growth factor receptor 2, HER2)，12个针对血管内皮生长因子(vascular endothelial growth factor, VEGF)，9个针对程序性死亡受/配体(programmed death receptors/ligands, PD-1/PD-L1)，4个针对CD19，4个针对降钙素基因相关肽(calcitonin gene-related peptides, CGRP)，4个针对表皮生长因子受体(epidermal growth factor receptors, EGFR)，3个针对补体(C5)。
5.3 研发地区及企业绝大多数被批准上市的单抗药物的研发都集中在欧美发达国家，其中美洲(主要是美国)拥有105个单抗药物，占研发总量60%的单抗药物，欧洲拥有39个单抗药物，占全部单抗药物的24%，而亚洲仅拥有19个单抗药物，占比12% (图 1D)。虽然163个单抗药物由60多家公司研发，但有10家公司占据了50%的药物数量(图 1E)，其中以基因泰克为首21个，安进12个，强生8个，诺华7个，辉瑞、阿斯利康、再生元各6个，礼来、赛特瑞恩、森托科各5个。亚洲获批上市的19个单抗药物中有9个来自韩国，分属于赛特瑞恩及三星Bioepis；日本6个，分别由协和麒麟、第一三共、卫材、武田制药及安斯泰来研发；以色列梯瓦公司研发2个；印度太阳制药研发1个；由我国百奥泰研发的Tocilizumab生物仿制药TOFIDENCE®于2023年9月29日被美国FDA批准上市，TOFIDENCE®是靶向IL-6的人源化单克隆抗体，被批准用于治疗类风湿性关节炎、多关节幼年特发性关节炎及系统性幼年特发性关节炎。
5.4 生物仿制药生物仿制药是与已批准的生物原研药高度相似的一种生物药[83]，单克隆抗体药物中同样存在众多获批上市的生物仿制药，在获批上市的单抗药物中有8个原研药已有生物仿制药(表 2)。艾伯维公司研发的HUMIRA® (Adalimumab)是世界上最畅销的单抗之一，靶向TNF-α，在2002年首次被批准用于治疗类风湿性关节炎，并后续获批用于治疗幼年特发性关节炎、银屑病关节炎、强直性脊柱炎、克罗恩病、溃疡性结肠炎、斑块状银屑病、化脓性汗腺炎及葡萄膜炎，在获批上市的单抗药物中有9个是它的生物仿制药，包括IDACIO® (费森尤斯·卡比)、YUFLYMA® (赛特瑞恩)、HYRIMOZ® (山德士)、CYLTEZO® (勃林格殷格翰)、ABRILADA® (辉瑞)、YUSIMRY® (Coherus)、AMJEVITA® (安进)、HADLIMA® (三星Bioepis)以及HULIO® (迈兰)。森托科研发的REMICADE® (Infliximab)同样靶向TNF-α，被批准用于治疗克罗恩病、溃疡性结肠炎、类风湿性关节炎等，有4个生物仿制药获批上市，包括RENFLEXIS® (三星Bioepis)、AVSOLA® (安进)、INFLECTRA® (赛特瑞恩)及IXIFI® (辉瑞)。
| Target | Commercial name | Active ingredients | R&D company |
| TNF-α | HUMIRA | Adalimumab | AbbVie |
| IDACIO | Adalimumab-aacf | Fresenius Kabi | |
| YUFLYMA | Adalimumab-aaty | Setrian | |
| HYRIMOZ | Adalimumab-adaz | Sanders | |
| CYLTEZO | Adalimumab-adbm | Boehringer Ingelheim | |
| ABRILADA | Adalimumab-afzb | Pfizer | |
| YUSIMRY | Adalimumab-aqvh | Coherus | |
| AMJEVITA | Adalimumab-atto | Amgen | |
| HADLIMA | Adalimumab-bwwd | Samsung Bioepis | |
| HULIO | Adalimumab-fkjp | Mylan Pharmaceuticals | |
| TNF-α | REMICADE | Infliximab | Sentoko |
| RENFLEXIS | Infliximab-abda | Samsung Bioepis | |
| AVSOLA | Infliximab-axxq | Amgen | |
| INFLECTRA | Infliximab-dyyb | Setrian | |
| IXIFI | Infliximab-qbtx | Pfizer | |
| HER2 | HERCEPTIN | Trastuzumab | Genentech |
| KANJINTI | Trastuzumab-anns | Amgen | |
| OGIVRI | Trastuzumab-dkst | Mylan | |
| ONTRUZANT | Trastuzumab-dttb | Samsung Bioepis | |
| HERZUMA | Trastuzumab-pkrb | Setrian | |
| TRAZIMERA | Trastuzumab-qyyp | Pfizer | |
| VEGF | AVASTIN | Bevacizumab | Genentech |
| VEGZELMA | Bevacizumab-adcd | Setrian | |
| MVASI | Bevacizumab-awwb | Amgen | |
| ZIRABEV | Bevacizumab-bvzr | Pfizer | |
| ALYMSYS | Bevacizumab-maly | Amneal | |
| VEGF-A | LUCENTIS | Ranibizumab | Genentech |
| SUSVIMO | Ranibizumab | Genentech | |
| CIMERLI | Ranibizumab-eqrn | Coherus | |
| BYOOVIZ | Ranibizumab-nuna | Samsung Bioepis | |
| CD20 | RITUXAN | Rituximab | Genentech |
| TRUXIMA | Rituximab-abbs | Setrian | |
| RIABNI | Rituximab-arrx | Amgen | |
| RUXIENCE | Rituximab-pvvr | Pfizer | |
| IL-6 | ACTEMRA | Tocilizumab | Genentech |
| TOFIDENCE | Tocilizumab-bavi | Biotech | |
| α4 integrin | TYSABRI | Natalizumab | Biogen Idec |
| TYRUKO | Natalizumab-sztn | Sanders | |
| Original drugs shaded gray; Data from the United States Food and Drug Administration: Drugs@FDA: FDA-Approved Drugs, as of 10/1/2023. | |||
基因泰克公司的HERCEPTIN® (Trastuzumab)靶向HER2，用于治疗乳腺癌及胃或胃食管交界腺癌，有5个生物仿制药获批上市，包括KANJINTI® (安进)、OGIVRI® (迈兰)、ONTRUZANT® (三星Bioepis)、HERZUMA® (赛特瑞恩)以及TRAZIMERA® (辉瑞)。基因泰克公司的AVASTIN® (Bevacizumab)靶向VEGF，用于治疗转移性结直肠癌、非鳞状非小细胞肺癌、复发性胶质母细胞瘤和转移性肾细胞癌等，有4个生物仿制药获批上市：VEGZELMA® (赛特瑞恩)、MVASI® (安进)、ZIRABEV® (辉瑞)及ALYMSYS® (Amneal)。基因泰克的LUCENTIS®/SUSVIMO® (Ranibizumab)靶向VEGF-A治疗黄斑变性及黄斑水肿等疾病，有2个生物仿制药，包括CIMERLI® (Coherus)和BYOOVIZ® (三星Bioepis)。同样是基因泰克公司研发的RITUXAN® (Rituximab)靶向CD20，用于治疗非霍奇金淋巴瘤、慢性淋巴细胞白血病及寻常型天疱疹等疾病，有3个生物仿制药获批上市，包括TRUXIMA® (赛特瑞恩)、RIABNI® (安进)和RUXIENCE® (辉瑞)。基因泰克研发的ACTEMRA® (Tocilizumab)靶向IL-6，用于治疗类风湿关节炎、巨细胞动脉炎及新冠病毒感染，是首个批准用于治疗COVID-19的单克隆抗体。其唯一一个获批上市的生物仿制药是由我国研发的TOFIDENCE® (百奥泰)，该仿制药也是我国第一个获美国FDA批准上市的单抗药物。百健艾迪研发的TYSABRI® (Natalizumab)靶向α4整合素，用以治疗多发性硬化症及克罗恩病，有1个生物仿制药TYRUKO® (山德士)获批上市。
6 展望随着高通量测序、生物信息学等技术的发展，获得高安全性、高有效性、高稳定性的抗体日益便利，抗体药物在全球药物市场中占有的比例将不断扩大。安全高效的单克隆抗体在癌症、免疫病、血液病和病原体感染等疾病的诊断或治疗中发挥重要的作用[82]。目前，仍有很多疑难杂症是以现在的医学技术难以医治，单克隆抗体药物可能是解决这些疑难杂症极具潜力的途径之一。全人源单克隆抗体以其独特的安全性成为当前单抗药物研发的趋势，将逐渐取代其他类型的抗体，抗体偶联药物以及新形式的抗体如双特异性抗体也将会有进一步的发展[84]。通过病毒载体、DNA或RNA转移所选抗体基因序列在体内表达单抗是一种新的抗体生产形式，该方式可以避免抗体大规模生产的高成本以及蛋白表征等问题[85-87]。
单抗药物的安全性一直是一个令人担忧的问题，有报道表明一些单抗(如Infliximab、Adalimumab、Certolizumab和Golimumab等)与治疗期间淋巴瘤和其他恶性肿瘤的风险增加之间存在关联[88-91]。此外，在治疗剂量下，单抗诱导的致畸性的生物学机制已被证实[92]。选择合适的靶点是生产高安全性、高有效性抗体的首要条件，一个新且有效靶点的发现很可能意味着某一类疾病的攻克。热门靶点的深入研究以及新靶点的鉴定都应成为我国单克隆抗体药物研发的重点，在新兴生物技术的推动下逐步追赶欧美发达国家的步伐。
亚洲地区在抗体药物市场上占据的份额较少，仅韩国及日本有多个单抗药物获批上市。以色列、印度及我国仅有1−2个获批上市，且大多数单抗药物均为生物仿制药，相对缺少创新性。由于原研药的高成本及高利润，通常单抗药物的价格十分高昂，这也促进了生物仿制药的研发[82]。为了应对生物仿制药的竞争，开发原研药的公司会尝试为现有的单克隆抗体增加新的临床应用，或是改进生产工艺以降低生产成本，但有些生物仿制药的性能优于原研药，甚至会比其对应的原研药更早获批上市。生物仿制药会降低单抗药物的使用成本，让更多的患者获得单抗药物治疗，但在一定程度上会阻碍原研药的研发，因为其损害了初研公司的利益。
目前，我国仅有一个生物仿制药获美国FDA批准上市，国内对抗体的研究还远落后于欧美发达国家，仍需要投入大量的财力及人力来促进抗体技术的发展。可喜的是我国对单抗药物的研发越来越重视，在实验室水平上生产了大量各类型的单抗。但由于抗体的商业生产及临床检验等受到资金限制，我国单抗药物的转化率仍处于较低水平，亟需大量资金的注入，促使有效单抗候选药物能尽快服务于社会并进一步推动国内单抗药物的研发。
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