CN116554357A - 一种双特异性嵌合抗原受体（car）及其在制备抗肿瘤药物中的应用 - Google Patents
一种双特异性嵌合抗原受体（car）及其在制备抗肿瘤药物中的应用 Download PDFInfo
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- CN116554357A CN116554357A CN202310567676.8A CN202310567676A CN116554357A CN 116554357 A CN116554357 A CN 116554357A CN 202310567676 A CN202310567676 A CN 202310567676A CN 116554357 A CN116554357 A CN 116554357A
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Abstract
本发明提供了一种双特异性嵌合抗原受体(CAR)及其在制备抗肿瘤药物中的应用，其中选择ROR1和PD‑L1为双重靶标，构建嵌合抗原受体，可提高肿瘤特异性，防止对正常细胞的误损伤，降低毒副作用；所述ROR1和PD‑L1抗原结合域具有全新的CDR区，能够与目标抗原高亲和力结合；嵌合抗原受体结构中设有4‑1BB和CD28双共刺激因子，可提高免疫细胞的激活程度，改善抗肿瘤效果；嵌合抗原受体免疫细胞与化疗药物联合使用，可进一步提高抗肿瘤效果，促进免疫因子和趋化因子分泌，延长动物生存期。
Description
技术领域：
本发明属于生物技术研发领域，具体提供了一种双特异性嵌合抗原受体(CAR)及其在制备抗肿瘤药物中的应用。
背景技术：
在过去十年中，细胞免疫疗法已成为抗击癌症的新的强大治疗支柱，通过使用经过基因修饰以表达嵌合抗原受体(CAR)的T细胞，在患有晚期血液恶性肿瘤的患者中取得了惊人的成功(参见Westin J.R.et al,Andreadis C.Efficacy and safety of CD19-directed CAR-T cell therapies in patients with relapsed/refractory aggressiveB-cell lymphomas:Observations from the JULIET,ZUMA-1,and TRANSCENDtrials.Am.J.Hematol.2021；96:1295–1312)。鉴于难治性淋巴瘤/白血病患者的高反应率和相当大比例的长期完全缓解，FDA和EMA批准了几种用于治疗急性淋巴细胞白血病(acutelymphoblastic leukemia，ALL)的CAR-T细胞产品，适应症包括弥漫性大B细胞淋巴瘤(diffuse large B cell lymphoma，DLBCL)、滤泡性淋巴瘤(follicular lymphoma，FL)和多发性骨髓瘤(multiple myeloma，MM)等等。
然而，大部分患有实体瘤的患者迄今并未表现出对CAR-T细胞疗法的持久缓解，在实体瘤患者中，经常会观察到原发性CAR-T细胞衰竭，并且在CAR-T细胞输注后缺乏初始反应和疾病仍不断进展。实体瘤患者的CAR-T细胞疗法的临床疗效相对滞后，尽管进行了大量的临床前和临床研究，但针对实体瘤的CAR-T细胞产品迄今未能获得FDA的批准，其主要原因在于，一方面实体瘤中的CAR-T细胞疗法面临与血液恶性肿瘤类似的障碍，例如抗原关闭和CAR-T细胞功能障碍；另一方面，实体瘤对CAR-T细胞提出了独特的挑战，例如缺乏合适的靶抗原和存在肿瘤保护性免疫抑制微环境。针对在实体肿瘤治疗中存在的问题，研究人员提出了如下的解决思路：
第一，目标抗原的上调，与淋巴瘤/白血病中的CD19或BCMA不同，它们在肿瘤细胞上均匀表达并且仅由可有可无的细胞类型(例如正常B细胞)共表达，实体瘤中潜在的CAR靶抗原的表达更加异质并且通常不限于肿瘤细胞或可有可无的细胞类型。例如，HER2在癌和胶质母细胞瘤中表现出可变表达，但也在肺上皮细胞中发现，这在接受HER2特异性CAR-T细胞治疗结肠癌转移至肺和肝的患者中引起了致命的肺毒性(参见Liu X.et al，Drivingbetter and safer HER2-specific CARs for cancer therapy.Oncotarget.2017；8:62730–62741)。值得注意的是，该CAR的结合部分基于广泛用于治疗HER2阳性乳腺癌的抗体曲妥珠单抗，并且不会引起这些严重的副作用，这表明CAR-T细胞对实体瘤中某些抗原具有特异性的效力和潜在危险，因此选择适当的目标抗原治关重要。
第二，改善CAR-T细胞功能，在实体瘤中，CAR-T细胞的功能受到CAR-T细胞增殖不足和功能失调的CAR-T细胞积累的阻碍，这些细胞显示出T细胞耗竭的迹象。检查点阻断为常规的解决思路，CAR-T细胞和检查点阻断抗体的组合已进入血液恶性肿瘤的临床评估阶段，通过打破检查点介导的耐药性来促进实体瘤CAR-T细胞治疗的大部分报告数据都处于临床前阶段。研究发现，PD-1阻断对于CD28共刺激的CAR-T细胞特别重要，因为与使用4-1BB共刺激的CAR-T细胞相比，PD-1在遇到抗原后上调更多。另一种处理方法则通过表观遗传手段进行，Weber等人引入了CAR-T细胞瞬时静息的概念，以允许通过重塑与T细胞耗竭相关的表观遗传特征来恢复功能，通过使用达沙替尼(一种可逆地关闭CAR信号传导的市售激酶抑制剂)，在T细胞衰竭开始之前或获得详尽表型后对CAR-T细胞施加短暂休息，CAR-T细胞扩增过程中达沙替尼的存在可减少强直性CAR信号转导(参见Weber E.W.et al.Transientrest restores functionality in exhausted CAR-T cells through epigeneticremodeling.Science.2021；372:eaba1786)。
第三，强化免疫细胞激活刺激，CAR-T细胞共刺激的加入标志着CAR-T细胞疗法迄今为止最大的突破之一，有研究证明了通过共表达4-1BB和CD28共刺激CAR-T细胞(28z-41BB CAR-T)实现了优异的抗肿瘤活性。通过CD28和4-1BB的联合刺激最终导致T细胞持久性增加并减轻T细胞耗竭，在药理学上，28z-41BB CAR-T细胞的设计可以通过将CD28共刺激的CAR-T细胞与激动性4-1BB抗体相结合来辅助抗4-1BB疗法改善了实体瘤小鼠模型中HER2特异性CAR-T细胞的细胞因子分泌、增殖和抗肿瘤活性(参见Mardiana S.etal.AMultifunctional Role for Adjuvant Anti-4-1BB Therapy in Augmenting AntitumorResponse by Chimeric Antigen Receptor T Cells.Cancer Res.2017；77:1296–1309)。
第四，与化学抗肿瘤药物联用，化疗一直是多种恶性肿瘤的标准治疗方法，患者通常会接受多轮化疗方案以控制肿瘤生长。在临床上，化疗药物环磷酰胺和氟达拉滨通常在CAR-T细胞治疗之前使用，以消除淋巴细胞并改善CAR-T细胞植入。有研究表明，化疗可以消耗免疫抑制细胞，促进炎性肿瘤微环境形成，破坏肿瘤基质，并改善CAR-T细胞向肿瘤的募集。
综上所述，设计和制备新型的CAR-T细胞治疗技术成为抗肿瘤研究的重点，本发明提供了一种CAR-T细胞，包括靶向ROR1和PD-L1双特异性抗原结构域，使用CD28和4-1BB作为双共刺激因子，所述CAR-T细胞还可与化疗药物联用，以便提高抗肿瘤活性，促进趋化因子分泌，调整免疫因子表达，延长动物生存周期，提供了新的抗肿瘤药物研发思路。
发明内容
解决上述技术问题，本发明的第一方面提供了一种双特异性嵌合抗原受体，所述嵌合抗原受体包括信号肽、抗ROR1结构域、抗PD-L1结构域、铰链区、跨膜区、共刺激因子和胞内区；所述抗ROR1结构域包括氨基酸序列如SEQ ID NO.1所示的重链可变区和氨基酸序列如SEQ ID NO.2所示的轻链可变区；所述抗PD-L1结构域包括氨基酸序列如SEQ ID NO.3所示的重链可变区和氨基酸序列如SEQ ID NO.4所示的轻链可变区
受体酪氨酸激酶样孤儿受体1(receptor tyrosine kinase-like orphanreceptor 1,ROR1)是近年来发现的新型肿瘤靶点，它在许多人类肿瘤(例如乳腺癌、卵巢癌、肺癌)中的表达升高，并与癌症的发生、进展和转移有关，但是在正常的成人器官和组织中ROR1低水平表达，包括大脑、肝脏、膀胱、肾脏、卵巢、胰腺、骨髓、脂肪组织、淋巴和内分泌系统，以及胃肠道和呼吸道等。
ROR1配体Wnt5a是一种在癌组织中高表达的细胞因子。在三阴性乳腺癌(TNBC)细胞系MDA-MB-231中，Wnt5a与ROR1结合以发出信号并激活PI3K/AKT/CREB通路，该通路控制BCL2、CCND1和CCNB1等基因，这些基因参与癌细胞的存活和增殖(参见Zhang S,et al.ROR1is expressed in human breast cancer and associated with enhanced tumor-cellgrowth.PLoS One.(2012)7:e31127)。Zhang等研究证实，与正常卵巢样本相比，人类卵巢癌活组织检查中ROR1 mRNA的高表达水平，并表明ROR1作为TAA和此类肿瘤的有前途的治疗靶点(参见.Zhang H,et al.ROR1 expression correlated with poor clinical outcomein human ovarian cancer.Sci Rep.(2015)4:5811)。Zheng等发现，与邻近的非肿瘤组织细胞相比，肺腺癌细胞也表达升高水平的ROR1，ROR1高表达与恶性细胞的属性显着相关，并且与较差的总体存活率相关(参见ZhengY,et al.ROR1 is a novel prognosticbiomarker in patients with lung adenocarcinoma.Sci Rep.(2016)6:36447)。在这种癌症中，ROR1下调促凋亡分子(Bak、Caspase-3、Caspase-7)并上调抗凋亡分子(Bcl-2和Bcl-XL)和参与细胞周期的蛋白质，例如CDK4和细胞周期蛋白E1。
目前已有研究人员构建靶向ROR1的CAR-T细胞，所述CAR-T细胞多是基于靶向ROR1的单克隆抗体而开发，相关的ROR1的单克隆抗体包括2A2、R12、XBR1-402和UC-961等等，例如Hudecek等人设计了包含源自2A2和R12mAb的scFvs的antiROR1 CAR-T，评估了所述CAR-T细胞对ROR1+肿瘤细胞的效应活性，并证明了铰链长度在增强CAR-T细胞的细胞毒性作用方面的相关性(参见Hudecek M,et al.The B-cell tumor–associated antigen ROR1can betargeted with T cells modified to express a ROR1-specific chimeric antigenreceptor.Blood.(2010)116:4532–41)。CN114929751A公开了一种ROR1特异性嵌合抗原受体及其治疗应用，所述CAR包括信号肽、ROR1抗原结合结构域、铰链、跨膜结构域、共刺激结构域和细胞内信号结构域，上述CAR修饰的免疫细胞适合用于治疗恶性肿瘤，如癌症、慢性淋巴细胞白血病(CLL)和急性淋巴细胞白血病(ALL)。CN111741757A也公开了包含ROR1抗原结合结构域的嵌合抗原受体，可抑制白血病和淋巴瘤细胞增殖。
免疫检查点抑制剂PD-1/PD-L1，可以抑制T细胞的活化、增殖和存活，是肿瘤免疫逃逸的重要途径，基于该信号通路已经开发出了多种抗肿瘤抗体药物，并且PD-1/PD-L1也被广泛用于CAR-T细胞的开发中。Jiang等构建了靶向c-Met/PD-L1的双特异CAR-T细胞，与单价c-Met CAR-T细胞或PD-L1 CAR-T细胞相比，用这种双特性CAR修饰的T细胞对c-Met和PD-L1阳性肝细胞癌细胞表现出更好的抗肿瘤活性(参见Wei Jiang，et al.Bispecific c-Met/PD-L1CAR-T Cells Have Enhanced Therapeutic Effects on HepatocellularCarcinoma,Front Oncol,.2021,10(11)546586)。
本发明基于ROR1和PD-L1靶点，开发出了靶向ROR1和PD-L1的双特异性CAR-T细胞，并且增加了4-1BB和CD28双共刺激因子，以便同时提高抗肿瘤的靶向性和有效性，改善抗肿瘤效果。
进一步的，所述共刺激因子选自4-1BB和CD28。
进一步的，所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.5所示。
进一步的，所述双特异性嵌合抗原受体的核苷酸序列如SEQ ID NO.6所示。
本发明的第二方面提供了一种嵌合抗原受体免疫细胞，所述免疫细胞表达所述的双特异性嵌合抗原受体。
进一步的，所述免疫细胞选自T细胞、NK细胞和巨噬细胞。
进一步的，所述免疫细胞选自T细胞。
现有技术中，已经采用嵌合抗原受体修饰不同免疫细胞，对其进行基因工程改造，进而获得相应的抗肿瘤细胞，所述免疫细胞包括但不限于T细胞、NK细胞和巨噬细胞等等。其中，T细胞是研究最早、应用最为广泛的免疫细胞，并且接种目前全球已经有5款CAR-T细胞产品获批用于治疗肿瘤。
本发明的第三方面提供了一种药物组合物，包括所述的嵌合抗原受体免疫细胞和化疗药物，所述化疗药物选自环磷酰胺、吉西他滨、奥沙利铂、甲氨蝶呤、多西紫杉醇和阿霉素中的一种或多种。
进一步的，所述化疗药物为奥沙利铂。
已有研究结果表明，多种化疗药物，如烷化剂(如环磷酰胺)、铂类化合物(如顺铂、卡铂、奥沙利铂)、抗代谢药(如甲氨蝶呤)、蒽环类药物、DNA甲基转移酶抑制剂和纺锤体毒物(如紫杉烷类药物)等等，可与CAR-T细胞联用，提高抗肿瘤效果。这些药物可引起深度免疫抑制，增强抗肿瘤免疫力，减轻肿瘤诱导的免疫抑制，增强细胞毒性免疫细胞的抗肿瘤活性以及改善免疫细胞向肿瘤部位的运输。因此，化疗的这些积极作用可以克服CAR-T细胞疗法治疗实体癌中的障碍，本发明中将所述化疗药物与CAR-T细胞组合，从而增强抗肿瘤疗效。
本发明的第四方面提供了所述的双特异性嵌合抗原受体和/或所述的嵌合抗原受体免疫细胞和/或所述的药物组合物在制备抗肿瘤药物中的应用。
有益效果
本发明提供了一种双特异性嵌合抗原受体、药物组合物及其在制备抗肿瘤药物中的应用，具有以下优势：
(1)选择ROR1和PD-L1为双重靶标，构建嵌合抗原受体，可提高肿瘤特异性，防止对正常细胞的误损伤，降低毒副作用；
(2)所述ROR1和PD-L1抗原结合域具有全新的CDR区，能够与目标抗原高亲和力结合；
(3)嵌合抗原受体结构中设有4-1BB和CD28双共刺激因子，可提高免疫细胞的激活程度，改善抗肿瘤效果；
(4)嵌合抗原受体免疫细胞与化疗药物联合使用，可进一步提高抗肿瘤效果，促进免疫因子和趋化因子分泌。
附图说明
图1：嵌合抗原受体结构示意图；
图2：CAR-T细胞抑制肿瘤细胞生长；
图3：CAR-T细胞联合不同化疗药物抑制肿瘤细胞生长；
图4：动物生存期；
图5：动物血清IFN-γ表达水平；
图6：动物血清CCL-5表达水平。
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂生物材料、检测试剂盒，如无特殊说明，均可从商业途径获得。
实施例1嵌合抗原受体设计
本发明提供的嵌合抗原受体为双特异性嵌合抗原受体，如图1所示，依次包括信号肽、抗ROR1结构域、抗PD-L1结构域、铰链区、跨膜区、共刺激因子和胞内区，其中所述抗ROR1结构域包括氨基酸序列如SEQ ID NO.1所示的重链可变区和氨基酸序列如SEQ ID NO.2所示的轻链可变区；所述抗PD-L1结构域包括氨基酸序列如SEQ ID NO.3所示的重链可变区和氨基酸序列如SEQ ID NO.4所示的轻链可变区，所示抗原结合域可与目标抗原高亲和力结合，双靶点的设计可以更大程度的降低脱靶效应。
有研究表明，铰链区的长度对于嵌合抗原受体免疫细胞的抗肿瘤活性具有较大影响，通常铰链区的长度延长抗肿瘤活性增加，因此本发明所述嵌合抗原受体的铰链区选用长度较大的CD8铰链区。为提高免疫细胞的激活程度，本发明所述嵌合抗原受体中选用了4-1BB和CD28作为双共刺激因子，可提高T细胞激活程度，发挥强力抗肿瘤作用。使用柔性连接子将各个元件连接，所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.5所示，核苷酸序列如SEQ ID NO.6所示。
实施例2CAR-T细胞制备
通过PCR法在编码嵌合抗原受体的核苷酸序列两端引入XhoⅠ/Eco RⅠ酶切位点，将所述核苷酸片段克隆导入逆转录病毒载体中，然后将所述逆转录病毒载体与辅助质粒共同转染293T细胞中，于37℃、5％CO2条件下培养过夜。在转染72h后收集上清，2000×g、4℃离心10min，获得逆转录病毒上清液，得到携带所述嵌合抗原受体基因的逆转录病毒RT-CAR。
使用淋巴细胞分离液从健康志愿者外周血中分离获得单个核细胞，使用免疫磁珠法筛选CD8+T细胞。将所述T细胞接种于含10％FBS的DMEM培养基中，同时培养基中加入终浓度为200U/mL IL-2，培养24h后，按病毒与细胞50:1的比例加入所述逆转录病毒载体RT-CAR，培养4-6天后收获CAR-T细胞。
实施例3CAR-T细胞体外抗肿瘤实验
3.1CAR-T细胞抑制肿瘤细胞生长
ROR1在多种肿瘤细胞中均有表达，包括但不限于白血病、淋巴瘤、乳腺癌、卵巢癌、肺腺癌、肝癌等，本实施例中以乳腺癌MCF-7为研究对象，考察所述CAR-T细胞的抗肿瘤作用。
复苏并培养MCF-7细胞至对数生长期，调节细胞密度，按每孔1×105个细胞接种于96孔板中，37℃、5％CO2条件下培养过夜，按照CAR-T细胞与肿瘤细胞1:1,5:1和10:1的比例加入所述CAR-T细胞，每组设立3个复孔，同时设立空白对照孔和实验对照孔(不加入CAR-T细胞)，培养48h。每孔加入10μL CCK-8溶液，在37℃，5％CO2条件下培养0.5-1h，然后10min内用酶标仪测定在OD450nm处的吸光度，计算肿瘤抑制率，所述肿瘤抑制率＝(实验对照孔OD值-CAR-T细胞孔OD值)/(实验对照孔OD值-空白对照孔OD值)×100％。
结果如图2所示，加入CAR-T细胞可显著抑制肿瘤细胞生长，且随CAR-T比例的增大，肿瘤抑制率增加，但5:1和10:1组的肿瘤抑制率类似，未见显著差异，说明使用5:1比例已经能够有效杀死肿瘤细胞。
3.2CAR-T细胞与化疗药物联用
已有研究结果表明，多种化疗药物，如烷化剂(如环磷酰胺)、铂类化合物(如顺铂、卡铂、奥沙利铂)、抗代谢药(如甲氨蝶呤)、蒽环类药物、DNA甲基转移酶抑制剂和纺锤体毒物(如紫杉烷类药物)等等，可与CAR-T细胞联用，提高抗肿瘤效果，本发明中尝试选择可与所述CAR-T细胞产生协同抗肿瘤效果的化疗药物，构成相应药物组合物，以便改善疗效。
本节中，按5:1比例将CAR-T细胞与MCF-7细胞共培养后，分别加入终浓度为100μg/mL的环磷酰胺、吉西他滨、奥沙利铂、甲氨蝶呤、多西紫杉醇和阿霉素，培养48h后采用CCK-8法测量并计算肿瘤抑制率，具体方法同3.1节。结果如图3所示，CAR-T细胞与化疗药物联用后，抗肿瘤效果出现了不同程度的改善，其中与奥沙利铂和阿霉素的抗肿瘤能力最强，环磷酰胺、吉西他滨、甲氨蝶呤、多西紫杉醇等效果次之。
实施例4CAR-T细胞体内抗肿瘤实验
4.1动物模型制备与给药
收集处于对数生长期的MCF-7细胞，用无菌PBS洗涤3次，调整细胞密度；取4-6周龄BALB/c裸鼠，用注射器在小鼠右侧腋附近皮下注射1×107个肿瘤细胞，每天观察肿瘤生长情况，当小鼠皮下肿瘤体积达到80-100mm3时进行后续实验。
将模型小鼠随机分为3组，每组10只，分别为：CAR-T组，每周尾静脉注射1×107个CAR-T细胞；联合组，每周尾静脉注射1×107个CAR-T细胞和100mg/kg体重的奥沙利铂；实验对照组，每周尾静脉注射等体积生理盐水。上述各组共治疗4周。
4.2动物生存周期观测
每天观察动物的生存状况，并进行记录，实验完成后给出记录数据，使用GraphPadPrism 8软件绘制生存曲线，如图4所示，使用CAR-T治疗可延长动物生存时间，且使用CAR-T与奥沙利铂联合治疗，可大幅度延长生存时间，说明这种疗法对于乳腺癌治疗更加有效。
4.3血清因子表达水平检测
治疗28周后，眼眶取血，3000g离心15min，去上层血清，使用ELISA试剂盒(购自R&D公司)检测血清中的IFN-γ和CCL-5浓度，具体方法按照试剂盒说明书进行。
IFN-γ是重要的免疫调节因子，可介导多种免疫反应，抑制肿瘤细胞生长。如图5所示，经过CAR-T细胞治疗后，动物体内IFN-γ水平升高，而联合奥沙利铂后，这种似乎趋势得到了强化，但为表现出显著差异。
CCL-5体内重要的趋化因子，可促进T细胞运输，将其募集于肿瘤组织中，从而发挥强烈的抗肿瘤作用。如图6所示，单独使用CAR-T细胞，对于CCL5的表达水平影响不大，而联合奥沙利铂后，CCL5表达水平幅度提高，说明奥沙利铂可能主要通过提高趋化因子表达水平，而促进CAR-T的肿瘤杀伤作用。
Claims (10)
1.一种双特异性嵌合抗原受体，其特征在于，所述嵌合抗原受体包括信号肽、抗ROR1结构域、抗PD-L1结构域、铰链区、跨膜区、共刺激因子和胞内区；所述抗ROR1结构域包括氨基酸序列如SEQ ID NO.1所示的重链可变区和氨基酸序列如SEQ ID NO.2所示的轻链可变区；所述抗PD-L1结构域包括氨基酸序列如SEQ ID NO.3所示的重链可变区和氨基酸序列如SEQID NO.4所示的轻链可变区。
2.如权利要求1所述双特异性嵌合抗原受体，其特征在于，所述共刺激因子选自4-1BB和CD28。
3.如权利要求2所述双特异性嵌合抗原受体，其特征在于，所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.5所示。
4.如权利要求2所述双特异性嵌合抗原受体，其特征在于，所述双特异性嵌合抗原受体的核苷酸序列如SEQ ID NO.6所示。
5.一种嵌合抗原受体免疫细胞，其特征在于，所述免疫细胞表达权利要求1-4任一项所述的双特异性嵌合抗原受体。
6.如权利要求5所述的嵌合抗原受体免疫细胞，其特征在于，所述免疫细胞选自T细胞、NK细胞和巨噬细胞。
7.如权利要求6所述的嵌合抗原受体免疫细，其特征在于，所述免疫细胞选自T细胞。
8.一种药物组合物，包括权利要求5-7所述的嵌合抗原受体免疫细胞和化疗药物，所述化疗药物选自环磷酰胺、吉西他滨、奥沙利铂、甲氨蝶呤、多西紫杉醇和阿霉素中的一种或多种。
9.如权利要求8所述的药物组合物，所述化疗药物为奥沙利铂。
10.如权利要求1-4任一项所述的双特异性嵌合抗原受体和/或权利要求5-7所述的嵌合抗原受体免疫细胞和/或权利要求8-9任一项所述的药物组合物在制备抗肿瘤药物中的应用。
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