CN116063521A - 靶向bcma的抗体及其所构成的嵌合抗原受体 - Google Patents
靶向bcma的抗体及其所构成的嵌合抗原受体 Download PDFInfo
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Abstract
本发明属于生物医药领域,具体涉及一种靶向BCMA的抗体及其所构成的嵌合抗原受体。具体地,所述抗体包括轻链可变区和重链可变区,所述轻链可变区中的CDR依次如SEQ ID NO.:1‑3所示,所述重链可变区中的CDR依次如SEQ ID NO.:4‑6所示。
Description
技术领域
本发明属于生物医药领域,具体涉及一种靶向BCMA的抗体及其所构成的嵌合抗原受体。
背景技术
BCMA(CD269、TNFRSF17)是肿瘤坏死因子受体超家族的一员,于20世纪90年代初首次被发现。这种184个氨基酸的糖蛋白在B细胞成熟和分化为浆细胞中起主要作用。从结构上讲,BCMA由三个主要结构域组成:胞外段(氨基酸1-54,在8-21、24-37和28-41位置通过二硫键连接)、跨膜区(氨基酸55-77)和胞内段(氨基酸78-184)。BCMA在原初和记忆B细胞上几乎不存在,但在浆细胞分化期间选择性地诱导,在此期间其可以通过促进正常浆细胞和成浆细胞的存活而支持体液免疫。
相关研究表明BCMA是多发性骨髓瘤细胞系上最具选择性表达的受体,且其表达量随着疾病的发展而逐步递增,因此,BCMA是治疗多发性骨髓瘤(multiple myeloma,MM)的理想靶点。同时,BCMA还已经在来自患有霍奇金氏病(Hodgkin's disease)的患者的里斯二氏细胞(Reed-Sternberg cell,CD30+)上表达,目前已经通过敲落实验证明了BCMA有助于霍奇金氏病细胞系的增殖与存活。
利用免疫学治疗手段来攻克肿瘤,一直是免疫学在转化医学方面应用的重要方向。随着各种组学的发展,肿瘤细胞由于突变产生的免疫原性得到了广泛的认可,这为肿瘤免疫治疗奠定了理论的基础。自然杀伤(natural killer,缩写NK)细胞是非特异性免疫系统的重要组成部分,先天免疫系统反应的关键性介质细胞。NK细胞是一种广谱免疫细胞,具有快速发现和摧毁异常细胞(如癌症或病毒感染的细胞)的功能,而且不需要提前致敏或HLA配型,即可展示强大的溶解异常细胞的活性。使用免疫细胞(包括NK细胞)来治疗癌症是近年来的新趋势,经过CAR结构修饰后的NK细胞,能够高效的识别肿瘤细胞,并通过释放杀伤介质、诱导靶细胞凋亡等多种手段杀伤肿瘤细胞。这种新疗法有望为对传统手术、化疗和放疗无效的肿瘤提供了新的治愈希望。
发明内容
第一方面,本发明提供了一种抗体,所述抗体包括轻链可变区和重链可变区,所述轻链可变区中的CDR依次如SEQ ID NO.:1-3所示,所述重链可变区中的CDR依次如SEQ IDNO.:4-6所示。
优选地,所述轻链可变区的氨基酸序列如SEQ ID NO.:7所示。
优选地,所述重链可变区的氨基酸序列如SEQ ID NO.:8所示。
优选地,所述重链可变区和轻链可变区之间由linker连接。
优选地,所述linker包括柔性linker或刚性linker。
优选地,所述linker由G(Gly,甘氨酸)和/或S(Ser,丝氨酸)组成。
优选地,所述linker是(GGGGS)3,也即linker是GGGGSGGGGSGGGGS。
所述术语“CDR”指的是互补决定区或互补决定簇(complementarity-determiningregions,CDR)。它位于免疫球蛋白的超变区,超变区是抗体的抗原结合位,与抗原决定簇的结构互补。重链和轻链的可变区(variable region,V)分别称为VH和VL。VH和VL中各含有3个互补决定区,包括CDR1、CDR2和CDR3。VH和VL的3个CDR共同组成Ig的抗原结合部位(antigen-binding site),决定抗体的特异性,是抗体识别及结合抗原的部位。
另一方面,本发明提供了一种多肽复合物,所述多肽复合物中包括本发明所述抗体,所述多肽复合物还包括可检测标志物或药物。
优选地,所述抗体和可检测标志物、抗体和药物可以以任意方式连接,例如共价键、肽键连接等。
优选地,所述药物包括抗有丝分裂剂、抗肿瘤抗生素、烷化剂、细胞抑制剂、抗血管生成剂。
优选地,所述可检测标记物包括放射性标记物、化学发光标记物、荧光标记物、量子点、测温标记物或免疫聚合酶链式反应标记物。
优选地,所述荧光标记物包括5-荧光素、6-羧基荧光素、3'6-羧基荧光素、5(6)-羧基荧光素、6-六氯-荧光素、6-四氯荧光素、异硫氰酸荧光素、罗丹明、藻胆蛋白和R-藻红蛋白。
优选地,所述可检测标记物可以发出可检测信号。
优选地,所述可检测信号包括光学信号或电学信号。
另一方面,本发明提供了一种融合蛋白,所述融合蛋白是在本发明所述抗体的氨基端和/或羧基端连接结构域。
优选地,所述连接是通过肽键连接。
优选地,所述结构域包括以下任意一种或多种:
1)铰链区;
2)跨膜结构域;
3)信号转导结构域;
优选地,所述信号转导结构域包含共刺激结构域和/或初级信号传导结构域。
优选地,所述铰链区包括CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合。
优选地,所述铰链区的氨基酸序列如SEQ ID NO.:9所示。
优选地,所述跨膜结构域包括蛋白质的跨膜结构域,所述蛋白质包括:2B4基因表达的蛋白、T细胞受体的α、β或ζ链,CD28,CD3ε,CD45,CD4,CD5,CD8,CD9,CD16,CD22,CD33,CD37,CD64,CD80,CD86,CD123,CD134,CD137和CD154。
优选地,所述跨膜结构域的氨基酸序列如SEQ ID NO.:10所示。
优选地,所述共刺激结构域包括2B4、CD3ζ、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)的功能性信号传导结构域。
优选地,所述共刺激结构域的氨基酸序列如SEQ ID NO.:11所示。
优选地,所述初级信号传导结构域包括NKG2D初级信号、CD3-ζ、FcεRIγ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的一种蛋白或多种蛋白的任意组合的信号传导区。
优选地,所述初级信号传导结构域的氨基酸序列如SEQ ID NO:12所示。
优选地,所述融合蛋白是CAR(嵌合抗原受体)。
所述术语“CAR”即嵌合抗原受体,它赋予免疫细胞新的能力,以靶向特定的抗原蛋白。CAR通常包括三个主要部分:胞外域(抗原结合域和铰链区,所述抗原结合域通常为抗体,在本发明中优选为本发明所提供的抗体)、跨膜域和胞内域,胞内域可以包括信号转导结构域和共刺激结构域。
优选地,所述CAR的完整结构是轻链可变区-linker-重链可变区-铰链区-跨膜区-共刺激结构域-胞内信号转导结构域。
另一方面,本发明提供了一种分离的多核苷酸,所述多核苷酸编码前述抗体或融合蛋白。
另一方面,本发明提供了一种核酸构建体,所述核酸构建体上包含前述分离的多核苷酸。
优选地,所述核酸构建体包括质粒表达载体或病毒表达载体。
优选地,所述病毒表达载体包括慢病毒载体、腺病毒载体、腺相关病毒表达载体或其他类型病毒载体。
示例性的,所述病毒表达载体可以是pShuttle-CMV-lacZ,pShuttle-CMV-EGFP-C,pXC1,pBHGE3,pAAV-MCS,pAAV-RC,pHelper,pAAV-LacZ,pLK0.1-puro,pLK0.1-CMV-tGFP,pLKO.1-puro-CMV-tGFP,pLK0.1-CMV-Neo,pLK0.1-Neo,pLKO.1-Neo-CMV-tGFP,pLKO.1-puro-CMV-TagCFP等。
在一些实施方式中,所述载体上还可以携带基因工程中常用的调控元件,例如增强子、启动子、内部核糖体进入位点、其他表达控制元件(例如转录终止信号,或者多腺苷酸化信号和多聚U序列等)。
另一方面,本发明提供了一种细胞,所述细胞中包含或表达前述抗体、前述融合蛋白、前述分离的多核苷酸、前述核酸构建体中的一种或多种。
优选地,所述细胞包括原核细胞和真核细胞。
优选的,所述原核细胞包括细菌细胞,如农杆菌、大肠杆菌。
优选的,所述真核细胞包括动物细胞(例如人的细胞)、昆虫细胞、植物细胞、藻类细胞、真菌细胞(例如酵母细胞)。
优选地,所述动物细胞是人的免疫细胞。
优选地,所述免疫细胞包括T细胞、B细胞、K细胞和NK细胞中的一种或多种。
优选地,所述NK细胞包括自行制备的人源NK细胞或是商品化的NK细胞系。
优选地,所述NK细胞系包括NK-92、NKG、YT、NK-YS、HANK-1和NK L。
优选地,所述自行制备的人源NK细胞包括自体的或异体的NK细胞。
优选地,所述自行制备的人源NK细胞可以是由干细胞分化而来,或者是从单细胞中分离而来。
另一方面,本发明提供了一种药物组合物,所述药物组合物中包含前述抗体、前述融合蛋白、前述分离的多核苷酸、前述核酸构建体或前述细胞的一种或多种。
优选地,所述药物组合物中还可以包括其他治疗癌症的物质,具体例如:烷化剂(例如顺铂、卡铂、环磷酰胺、美法仑、苯丁酸氮芥、白消安和亚硝基脲);抗代谢物(例如抗叶酸药、氟嘧啶类药物、5-氟尿嘧啶和吉西他滨、替加氟、雷替曲塞、氨甲蝶呤、阿糖胞苷和羟基脲);抗肿瘤抗生素(例如蒽环类药物:阿霉素、博来霉素、阿霉素、道诺霉素、表柔比星、伊达比星、丝裂霉素-C、放线菌素和光神霉素);抗有丝分裂剂(例如长春花碱、长春碱、长春碱、长春地辛和长春瑞滨等长春花生物碱以及紫杉醇和紫杉醇等紫杉烷类);拓扑异构酶抑制剂(例如表鬼臼毒素:依托泊苷、替尼泊苷、拓扑替康、喜树碱);蛋白体抑制剂(例如硼替佐米Velcade);细胞抑制剂,例如抗雌激素(他莫昔芬、托瑞米芬、雷洛昔芬、屈洛昔芬和碘多芬),雌激素受体下调剂(例如氟维司汀),抗雄激素(例如比卡鲁胺、氟他米特、鲁米特、酸环丙孕酮);血管损伤剂(例如Combretastatin A4)。
优选地,所述药物组合物还包括药学上可接受的赋形剂和/或添加剂。
优选地,所述赋形剂包括但不限于缓冲体系、增稠剂、稳定剂、中和试剂、保湿剂。
优选地,所述添加剂包括但不限于充填剂、结合剂、增湿剂、助流剂、稳定剂、防腐剂、乳化剂。
优选地,所述药物组合物的给药途径为肠道给药或非肠道给药,如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔黏膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
优选地,所述药物组合物的给药剂型可以是液体剂型、固体剂型或半固体剂型。
另一方面,本发明提供了前述抗体、前述多肽复合物、前述融合蛋白、前述分离的多核苷酸、前述核酸构建体、前述细胞或前述药物组合物中任意一种或多种在制备药物中的应用。
优选地,所述药物是治疗BCMA相关癌症的药物。
优选地,所述药物是治疗BCMA异常表达(或高表达)的癌症的药物。
优选地,所述癌症包括实体瘤、非实体瘤;
优选地,所述实体瘤包括前列腺癌、膀胱癌、肾癌、肝癌、乳腺癌、卵巢癌、肺癌、肺腺癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、直肠癌、血管内皮瘤、睾丸癌、皮肤癌。
优选地,所述非实体瘤包括白血病、骨髓瘤、淋巴瘤等。
优选地,所述白血病可以按细胞分化成熟程度和自然病程可分为急性白血病(acuteleukemia,AL)和慢性白血病(chronicleukemia,CL)。优选地,所述AL(急性白血病)分为急性淋巴细胞白血病(acutelymphoblasticleukemia,ALL)和急性髓系白血病(acutemyeloidleukemia,AML)。优选地,所述CL(慢性白血病)可分为慢性髓系白血病(chronicmyeloidleukemia,CML)、慢性淋巴细胞白血病(chroniclymphocyticleukemia,CLL)及少见类型的白血病如毛细胞白血病、幼淋巴细胞白血病等。
优选地,所述骨髓瘤根据临床表现可以分为典型的骨髓瘤、冒烟型骨髓瘤、孤立性骨髓瘤、浆细胞白血病型骨髓瘤、不分泌型骨髓瘤、骨硬化型骨髓瘤等。
优选地,所述淋巴瘤包括霍奇金淋巴瘤或非霍奇金淋巴瘤。
另一方面,本发明提供过来一种治疗癌症的方法,所述方法包括向患者施用本发明前述抗体、前述多肽复合物、前述融合蛋白、前述分离的多核苷酸、前述核酸构建体、前述细胞或前述药物组合物中任意一种或多种。
另一方面,本发明提供了检测细胞是否表达BCMA的方法,所述方法包括将待测细胞与本发明所述抗体接触。
优选地,所述待测细胞获自受试者。
优选地,所述受试者患有或疑似患有癌症。
优选地,所述待测细胞来自组织、组织切片和体液中的一种或更多种。
另一方面,本发明提供了一种试剂盒或其在检测细胞是否表达BCMA中的应用,所述试剂盒中含有本发明所述抗体或前述多肽复合物。
另一方面,本发明提供了制备药物的方法,所述方法包括制备前述抗体、前述融合蛋白、前述分离的多核苷酸、前述核酸构建体、前述细胞或前述药物组合物中任意一种或多种的步骤。
附图说明
图1是筛选出的抗体的酶标仪的光学读数统计结果图。
图2是载体pTT5的结构图。
图3是本发明所提供的894抗体与细胞中的BCMA结合的能力检测结果图。
图4是使用本发明所提供的894抗体构建成CAR转导入NK细胞后的CAR阳性率统计结果图。
图5是本发明所提供的CAR-NK细胞的杀伤活性的检测结果图。
具体实施方式
实施例一、抗体筛选及验证
一、抗体库构建及抗体库质量检测
1、动物免疫
1)选择3只雌性、健康BALB/c小鼠,8~10周龄;
2)免疫前留少量血清,在检测效价时作为阴性对照;
3)将50μg抗原用PBS稀释至50μl,与等体积佐剂混合均匀,混合溶液共100μl;
4)采取小腿肌肉注射方法免疫1只小鼠,共免疫两次,分别在第0天和第21天进行;
5)初次免疫后第35天在小鼠尾巴取血,检测血清效价。
2、效价检测
1)包被:将检测原用pH9.6碳酸包被液稀释至5μg/ml,96孔酶标板每孔包被100μl,4℃,过夜;
2)封闭:用4%脱脂奶粉-PBS封闭,300μl/孔,37℃,2h;
3)一抗结合:弃孔内液体,用PBST洗三遍,加入小鼠抗血清,1:200、1:400、1:800、1:1600、1:3200、……1:1638400稀释,100μl/孔,37℃结合1h。分别设阴性血清和PBS对照孔,阴性血清1:1000稀释;
4)二抗结合:弃孔内液体,用PBST洗三遍,加入HRP-goat anti-mouse IgG(Fc),1:5000稀释,100μl/孔,37℃结合1h;
5)显色:弃孔内液体,用PBST洗五遍,加入TMB显色液,100μl/孔,避光显色;
6)终止:每孔加入50μl 2M H2SO4终止反应;
7)酶标仪读A450值。
3、构建抗体库
3.1分离RNA
1)破碎免疫小鼠脾脏,加入适量Trizol使细胞裂解充分;
2)裂解液13000rpm离心,弃去沉淀;
3)每1mL裂解液上清加入200μl氯仿,剧烈震荡30s,冰浴5min;
4)13000rpm离心10min,管内分为3层,小心吸出350-500μl上清。
5)加入等体积异丙醇混匀,-20℃静置30min,13000rpm离心10min。
6)弃去上清,沉淀用75%冰乙醇1ml清洗两次沉淀,将沉淀晾干,根据沉淀量用已经加入RRI的DEPC水复溶。
3.2PCR扩增基因
1)使用Oligo(dT)逆转录RNA得到cDNA;
2)使用小鼠抗体特征序列引物扩增轻重链基因;
3)重叠PCR得到ScFv片段。
3.3连接及转化
1)ScFv片段酶切
ScFv片段酶切反应体系如表1所示,37℃酶切过夜。
表1、ScFv片段酶切反应体系
试剂 | 体积 |
ScFv PCR产物 | 3μg |
酶1 | 3μl |
酶2 | 3μl |
10×buffer | 20μl |
<![CDATA[ddH<sub>2</sub>O]]> | Up to 200μl |
2)载体酶切
载体使用双酶切法,酶切体系如表2所示。
表2、载体酶切体系
试剂 | 体积 |
PNC | 10μg |
酶1 | 4μl |
酶2 | 4μl |
10×buffer | 20μl |
<![CDATA[ddH<sub>2</sub>O]]> | Up to 200μl |
配置好酶切体系后,将体系放于37℃水浴酶切2h,之后70℃水浴20min终止反应;
3)酶切后的片段、载体使用自产T4 DNA聚合酶连接,脱盐后用于电击转化;
4)从冰箱中取出分装好的感受态,每支加入10μl左右脱盐产物,轻轻吸打混合均匀;
5)取出1支混合液加入电击杯中,放入电击槽中,电击;
6)电击结束后立即取出电击杯,用2YT培养基洗出杯中菌液,转移至三角瓶中培养;
7)培养1h后,取样涂布平板测库容;
8)补加抗生素和葡萄糖,培养至OD600=1.0,取一部分菌液包装噬菌体,剩余培养至OD600>3.5,离心收菌;
3.4包装噬菌体
1)在上述OD600=1.0的菌液中,加入M13KO7浸染,混匀后放入37℃培养箱中静置30min;
2)静置后,37℃,180rpm缓慢摇晃30min;
3)侵染的菌液4℃,5000rpm离心10min,弃尽上清,用300mL2YT培养基(含有终浓度100μg/mL氨苄青霉素、50μg/mL卡那霉素)重悬沉淀,30℃,220rpm过夜;
4)300ml过夜菌9000rpm离心20min,量取200mL上清,加入1/4体积的PEG6000/NaCl,冰水浴沉淀4h;
5)冰浴后,4℃,9000rpm,离心20min,倒掉上清,根据沉淀的量加入3ml左右PBS溶解沉淀;
6)过滤除菌后,按体积加入一定量的50%的甘油,至甘油终浓度为17%;
7)混匀后,分装到EP管中,取部分进行噬菌体滴度测定和污染检测,其余-80℃冻存。
3.5测定噬菌体滴度
1)早上接种TG1单克隆于2YT无抗培养基中,37℃,220rpm培养至OD600≈1.0;
2)取噬菌体库梯度稀释到10-10,取100μl稀释液加入到200μL培养好的TG1中,37℃培养箱静置30min,全部涂布至氨苄抗性平板上,37℃过夜培养,第二天统计板上克隆数,计算滴度,TG1单克隆用于实施例2中的抗体筛选。
4、抗体库质量检测
4.1初级细菌文库污染检测
1)取冻存菌库,涂布200μl原液,并涂布10-2-10-6的梯度稀释液,30℃培养过夜;
2)平板菌落计数。烈性噬菌体的存在会在平板上产生溶解斑,或减少菌落,尤其是在用未稀释或低稀释原代细菌细胞库涂布的平板上。
4.2次级细菌文库污染检测
1)取冻存菌库,1:1000接种,37℃,220rpm培养至OD600>1,涂布200μl培养菌,30℃培养过夜;
2)烈性噬菌体的存在会在平板上产生溶解斑,或减少菌落。
4.3噬菌体文库污染检测
1)取噬菌体库,10μl侵染300μl OD600>0.8的TG1菌液,37℃静置30min。涂布200μl混合液,并涂布10-2-10-5梯度稀释液,30℃培养过夜;
2)常规感染噬菌体库,将噬菌体加入上述TG1培养基中,轻轻混合,37℃孵育30min;
3)涂在2YT-AG平板上,30℃培养过夜;
4)平皿菌落计数,烈性噬菌体的存在会在平板上产生溶解斑,或减少菌落,尤其是在用未稀释或低稀释原代细菌细胞库涂布的平板上。
二、抗体库筛选
1、实验材料
1)2YT培养基:Tryptone16 g,Yeast extract 10g,NaCl 5g,加去离子水定容到1L。2YT固体培养基含1.5%琼脂。
2YT+A(Amp)+G(glu)液体培养基:A:100μg/ml G:0.1%。
2YT+A(Amp)+G(glu)固体培养基:A:100μg/ml G:1.0%。
2)40%葡萄糖:40g葡萄糖加水定容至100ml(w/v),过滤除菌后分装,4℃保存。
3)氨苄青霉素:配制浓度(100mg/ml),0.5g AMP+5ml水,充分溶解,过滤除菌,分装1ml/管,-20℃保存。工作浓度(100μg/ml),1:1000稀释使用。
4)1×PBS缓冲液:NaCl 8.0g,KH2PO4 0.24g,Na2HPO4·12H2O 3.63g,KCl 0.2g,加去离子水定容到1L,盐酸调PH 7.4。
5)封闭液:2%M-PBS,1×PBS+2%Milk(w/v)充分溶解后,9000rpm离心2min取上清,现配现用。
6)包被液:0.1M NaHCO3,NaOH调PH9.6,过滤除菌,4℃保存。
7)洗涤液:0.1%PBST,0.3%PBST,0.5%PBST(T:Tween20,要配制成10%的母液使用)。
8)洗脱液:胰酶细胞消化液。
9)噬菌体沉淀液:PEG6000/NaCl(20%PEG/2.5M NaCl),称取40g PEG6000和29.22g NaCl,加入140ml水,在电磁炉上,开水浴至颗粒完全溶解,冷水降温到溶液澄清,定容至200ml,低磅灭菌。
2、实验方法
1)预试验:测定靶分子和对照分子的理化性质(PH值、浓度、溶液体系、分子量,SDS电泳)。
2)包被靶分子:用包被液(0.1M NaHCO3 PH9.6)将靶分子稀释至需要的合理浓度,每孔100μl,在湿盒中4℃静置过夜(如有对照分子,一起包被)。
3)封闭空白孔(扣除背景):用2%M-PBS封闭,300μl/孔,视情况封闭多少个孔。在湿盒中4℃静置过夜(2%M-PBS需9000rpm离心2min,取上清)。
4)划TG1平板:挑取TG1单克隆或取冻存菌于10ml 2YT液体培养基中,37℃,250rpm震荡培养至对数期,然后划无抗平板。
5)接TG1菌:挑取TG1单克隆于10ml 2YT液体培养基中,37℃,250rpm震荡培养至对数期(OD≈0.5)用于测滴度。
6)封闭靶分子:倒掉包被靶分子孔中的包被液,PBS洗板3次,每孔加入300μl封阻液(2%M-PBS),37℃静置1h。
7)扣除前背景:PBS洗板3次,将库(投入库的量约1.0×1013pfu)与1%M-PBS混合,然后100μl/孔,加入到封闭好的空白孔中,室温静置1h。
8)结合:PBS洗3次封闭好的步骤6)的包被孔,将扣除前背景的步骤7)的抗体库100μl/孔加入步骤6)的包被孔中结合,室温结合1h。
9)洗涤:0.1%、0.2%、0.3%或0.5%PBST洗板(每次液体要充满孔,枪头不能碰内壁),每次缓摇1min。若是需要较多克隆,进行8次洗板,若是一般淘选则进行9次洗板。PBST洗板多次过后再次用PBS洗板一次。
10)甘氨酸洗脱:100μl/孔,室温缓摇8min,吸出后用15μl/孔的Tris-HCl(pH 9.1)终止,得到洗脱产物,4℃保存。
11)测定滴度:测定洗脱产物噬菌体滴度。
12)接TG1单克隆菌:将TG1单克隆接种于20ml 2YT培养基37℃,250rpm震荡培养至对数期(OD=0.5)。
13)洗脱产物侵染:加入洗脱产物混匀,37℃静置30min。
14)补加抗性:补加Amp 4μl(终浓度20μg/ml),37℃,180rpm缓摇60min。
15)M13KO7侵染:向瓶中加入相应量的M13KO7(辅助噬菌体的数:菌体数≥10:1)37℃静置20min。
16)补加培养基:补加30ml 2YT培养基及6μl Amp(终浓度20μg/ml),180rpm缓摇60min。
17)收菌重悬:5000rpm离心10min收菌,菌体重悬在50ml 2YT+Amp(终浓度50μg/ml)+Kan(终浓度10μg/ml)中,30℃,220rpm过夜培养。
18)将过夜培养的培养物转入离心管中,4℃,12000rpm离心10min。上清转入另一离心管同样条件再离心。将上清的转入新离心管,加入1/4体积的PEG6000/NaCl,4℃沉淀4h。
19)4℃,12000rpm离心20min,倒掉上清液,瞬离一次,吸去残留上清。
20)重悬沉淀于0.5ml PBS中,4℃,12000rpm离心10min使残余细胞沉淀,上清转入另一新鲜离心管中,此为扩增产物。
21)测扩增产物滴度。
22)洗脱产物阳性率鉴定,鉴定反应体系及PCR程序如表3所示。
表3、洗脱产物阳性鉴定反应体系及PCR程序
23)第一轮淘选结束。根据第一轮淘选结果,通过适当降低靶分子包被的浓度,或者提高洗涤液中Tween20的浓度和洗涤次数及时间,降低靶分子与噬菌体库的结合时间和温度,提高淘选压力,进行第二,三轮淘选。以降低背景,进一步富集高亲和力的噬菌体。
注:直接包被法中扣除背景相对简单,一般只需要经噬菌体库及洗脱产物各一步扣除背景,有对照分子的应在包被有对照分子的ELISA平板内扣除对照背景。
三、单克隆噬菌体制备
1)在96孔培养板NEST(板1)A1-H11中加入2YT+A+G(终浓度Amp 100μg/ml,Glu0.1%),100μl/孔(将配好的2YT+A+G倒入无菌的一次性培养皿中,用排枪加入)。
2)用镊子夹住黄枪头从涂有2-E(3-E)洗脱产物的培养皿中挑取单克隆(蘸一下即可),然后在培养基中蘸一下使噬菌体接入。
3)将接好菌的培养板盖好盖子,用医用透明胶带先将盖子和板子封好,在放入拉口袋中,外用皮筋缠好,37℃,220rpm过夜培养。
4)准备新的96孔培养板NEST(板2),在A1-H11中加入2YT+A(终浓度Amp 100μg/ml),100μl/孔,(将配好的2YT+A倒入无菌的一次性培养皿中,用排枪加入)。
5)用排枪吸取20μl板1中的噬菌体,对应接入板2中。
6)将转接的培养板盖好盖子,用医用透明胶带先将盖子和板子封好,在放入拉口袋中,外用皮筋缠好,37℃,220rpm至OD=0.5左右。
7)加M13KO7:用2YT将M13KO7稀释至需要浓度,20μl/孔加入孔中,37℃静置30min后180rpm缓摇30min。
8)加2YT+A+K(终浓度Amp 50μg/ml,Kan 25μg/ml)100μl/孔(将配好的2YT+A+K倒入无菌的一次性培养皿中,用排枪加入)。
9)将培养板盖好盖子,用医用透明胶带先将盖子和板子封好,在放入拉口袋中,外用皮筋缠好,30℃,220rpm过夜培养。
10)取过夜培养板,加入4%M-PBS 80μl/孔,盖好盖子,用医用胶带封好,4000rpm,10min水平离心,(配平时要注意板子缺刻的位置)。此时phage体积是240μl/孔,用80μl/孔4%M-PBS稀释用于phage ELISA结合时使用。
四、单克隆与目的蛋白的Phage ELISA结合能力测定
1、步骤
1、包被:取ELISA平板用PH9.6包被液稀释靶蛋白,50μl/孔,在湿盒中37℃静置1h或4℃静置过夜。
2、封闭:甩出多余包被液,并倒置平板在干净纸巾上拍甩去除残液,PBS洗板3次。加入封阻液,300μl/孔,37℃在湿盒中静置1h。
3、一抗:甩出多余噬菌体溶液,并倒置平板在干净纸巾上拍甩去除残液,PBS洗板3次。用1%Milk-PBS 1:1000稀释HRP兔抗M13,加入50μl/孔,37℃在湿盒中静置1h。
4、显色:甩出多余溶液,并倒置平板在干净纸巾上拍甩去除残液,PBS洗板4次。TMB显色液,50μl/孔,2M H2SO4终止,450nm检测。
检测结果如附图1所示,附图展示了具有代表性阴性克隆和阳性克隆对应的酶标仪的光学读数。
实施例二、IgG重构及其抗体检测结果
1、获得抗体序列
将实施例1鉴定出的阳性单克隆进行抗体测序,经验证,893和894序列相同,是同一个抗体,具体序列是:
轻链CDR
CDR1:RSSQSIVHSNGNTYLE(Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly AsnThr Tyr Leu Glu,如SEQ ID NO.:1所示),
CDR2:KVSNRFS(Lys Val Ser Asn Arg Phe Ser,如SEQ ID NO.:2所示),
CDR3:FQGSHVPWT(Phe Gln Gly Ser His Val Pro Trp Thr,如SEQ ID NO.:3所示)
重链CDR
CDR1:GYTFTSYVMH(Gly Tyr Thr Phe Thr Ser Tyr Val Met His,如SEQ ID NO.:4示),
CDR2:YIIPYNDGTKYNEKFKG(Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys TyrAsn Glu Lys Phe Lys Gly,如SEQ ID NO.:5示),
CDR3:HGWDYAMDY(His Gly Trp Asp Tyr Ala Met Asp Tyr,如SEQ ID NO.:6示)
轻链的完整序列:
VVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLELKRT(如SEQ ID NO.:7所示)
重链的完整序列:
ELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDGTKYNEKFKGKATLTSDKSSSTVYMELSSLTSEDSAVYYCARHGWDYAMDYWGQGTSLTVSS(如SEQ ID NO.:8示)
2、构建抗体的表达质粒
使用载体pTT5(如附图2所示),采用PCR扩增抗体序列,利用酶切连接插入EcoRI和BamHI位点之间。
3、细胞转染法转染293细胞
1)细胞准备
293细胞(内部细胞冻存管编号:A07),调整密度为1×106/ml,125ml摇瓶内溶液体积补充DMEM+10%FBS至30ml,。
2)转染复合物配置
a、将30μg抗体表达质粒稀释至1.5ml KPM培养基(KPM终体积为摇瓶液体总量的10%),混匀;
b、将150μl T1噬菌体稀释至1.5ml KPM培养基,其中质粒:T1=1:5,混匀,室温孵育5min;
c、将T1噬菌体稀释液加入DNA稀释液,混匀,室温孵育30min,即得到转染复合物。
3)转染
a、将转染复合物加入细胞悬液,混匀;
b、37℃,CO2浓度5%的摇床内120rpm培养24h。
4)加入添加剂
培养24h后加入终浓度为0.5%的TN1溶液(Tryptone N1溶液,产品信息:Organotechni#19553,原溶液浓度10%)。
5)转染第6天,4400rpm离心10min收集上清。
4、蛋白A亲和层析纯化IgG抗体
1)样品(上清液)用0.45μm滤器过滤,留过滤液备用;
2)将1ml ProteinA色谱柱用五蒸水冲洗20个柱体积,再用PB冲洗20个柱体积,流速:1.0ml/min。
3)样品上样,流速:1.0ml/min,上样结束后用PB冲洗20个柱体积至基线。
4)0.1M柠檬酸-柠檬酸钠(PH=3.4)洗脱,收集洗脱液。(收集管中需事先加入约150μl的1M PH9.0 Tris-HCI缓冲液,防止抗体在过酸环境下失活)。
5)洗脱产物用2M PH=8.0Tris-HCI小心调PH值至5.0,静置10min,观察无沉淀后再调至6.0。
6)鉴定:12%SDS-PAGE检测,确认抗体存在。
5、IgG重构抗体检测
1)敏感性检测
利用细胞K562(BCMA阴性)和NCI-H929(BCMA阳性)检测本发明所述抗体(0μg、0.4μg、2μg、10μg)与细胞中的BCMA结合的能力,结果如图3所示,K562(左)没有表达BCMA,因此荧光强度低,NCI-H92表达内生性BCMA,被本发明所述抗体染色,峰右移。
实施例三、CAR-NK细胞的构建方法及体外功能评价
1、NK细胞制备
步骤1:从献血者获得PBMC:取全血20-40ml,缓慢的把全血加到15ml淋巴细胞分离液体,离心1200g 10min,轻柔获得上层血清,获取白膜层,尽量不接触ficoll层,加入40mlDPBS重悬细胞,离心400g 10min,去上清,再次加入40ml DPBS洗涤细胞,离心400g 10min,去上清,重悬于NK激活培养基(包括细胞因子II和细胞因子III)(依科赛NE000-N022)。
步骤2:获得上层血清置于56度30分钟,然后4度10分钟,1200g 10分钟,获得上清,为自体血清。
步骤3:从步骤1中获得PBMC后,使用10%灭活血清和NK激活培养基调整细胞浓度到2x106 cells/ml。
步骤4:72小时后,加入等量体积的含有10%自体血清的NK基础培养基(包含细胞因子III)
步骤5:48小时后,取细胞计数,使用NK基础培养基调整细胞密度至1.5x106cells/ml
步骤6:每隔48小时,重复步骤5,培养至14-16天收获NK细胞
2、CAR表达载体的构建
1)CAR结构各部分序列如下:
ScFv:
连接方式:轻链可变区(SEQ ID NO.:7)-linker-重链可变区(SEQ ID NO.:8)。
所述轻链可变区序列(如SEQ ID NO.:7所示):VVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLELKRT
所述linker是GGGGSGGGGSGGGGS
所述重链可变区序列(如SEQ ID NO.:8所示):ELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDGTKYNEKFKGKATLTSDKSSSTVYMELSSLTSEDSAVYYCARHGWDYAMDYWGQGTSLTVSS
也即,完整的ScFv序列如下:
VVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLELKRTGGGGSGGGGSGGGGSELVKPGASVKMSCKASGY
TFTSYVMHWVKQKPGQGLEWIGYIIPYNDGTKYNEKFKGKATLTSDKSSSTVYMELSSLTSEDSAVYYCARHGWDYA
MDYWGQGTSLTVSS
铰链区的氨基酸序列(如SEQ ID NO.:9所示):
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
跨膜区的氨基酸序列(如SEQ ID NO.:10所示):
IYIWAPLAGTCGVLLLSLVIT
共刺激结构域的氨基酸序列(如SEQ ID NO.:11所示):
LYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE
胞内信号转导结构域的氨基酸序列(如SEQ ID NO.:12所示):
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
综上,最优选地,本发明使用“轻链可变区-linker-重链可变区-铰链区-跨膜区- 共刺激结构域-胞内信号转导结构域”的核酸序列依次连接,然后表达得到的本发明所提供的CAR。
2)构建载体
此处构建,是由北京华大六合生物技术有限公司,合成基因片段,连接入表达载体pLenti-puro质粒,使用BamHI和AgeI酶切位点切开基因片段和表达质粒产生粘性末端,连入。
3、病毒包装
使用三代慢病毒包装系统共四质粒系统,包装三质粒pRSV-Rev,pMDLg/pRRE,pMD2.G,穿梭pLVX-puro,HEK293T细胞作为病毒生产细胞,混合比例pLenti-puro:pRSV-Rev:pMDLg/pRRE:pMD2.G 2:1:1:1,转染试剂使用脂质体mirus TransIT-293(mir2700),脂质体和DNA比例为3:1,转然后,三天收集上清,高速离心40000g 6小时。
4、病毒转导
将病毒按照感染浓度为10MOI直接加入NK细胞的培养基,同时加入polybrene 0.8μg/ml,孵育6小时候,去除含有病毒NK培养基,加入新鲜的NK培养基,继续培养48小时后,检测CAR表达。
5、CAR-NK细胞CAR表达效率检测
取感染过后的NK细胞0.5E6,PBS清洗两次后,检测BCMA CAR抗体(美天旎)1:100稀释,孵育60分钟后,PBS清洗两次,使用流式细胞仪检测阳性细胞数。确认CAR阳性率,894号抗体的阳性率为15.86%(附图4)。
6、细胞杀伤性实验
使用DELFIA EuTDA 试剂盒检测细胞毒性效果:H929细胞(骨髓瘤细胞)与BATDAreagent浓度2μl/ml预先混合孵育30min在37度,经过5次洗涤彻底洗脱细胞外残留reagent,按照比例H929:NK=1:1、1:2、1:4、1:8混合孵育2小时后,Nalm6细胞被杀伤后,reagent从H929中释放到细胞培养基中,由于不同杀伤效果,reagent释放的量不同,收集细胞上清液,包含reagent,混合ES溶液室温孵育15分钟,每3分钟,轻轻震动混匀。使用酶标仪TRF通道读取荧光数据(315nm激发640nm接收)。
计算公式为:%=(上清读数-自发光读数)/(细胞裂解最大读数-自发光读数),获得杀伤率。本发明所提供的894号抗体在1:1、2:1、4:1、8:1时的杀伤活性具体数值依次是:13.7%、15.4%、19.5%、30.2%(图5)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (10)
1.一种抗体,所述抗体包括轻链可变区和重链可变区,所述轻链可变区中的CDR依次如SEQ ID NO.:1-3所示,所述重链可变区中的CDR依次如SEQ ID NO.:4-6所示;
优选地,所述轻链可变区的氨基酸序列如SEQ ID NO.:7所示;
优选地,所述重链可变区的氨基酸序列如SEQ ID NO.:8所示;
优选地,所述重链可变区和轻链可变区之间由linker连接;
优选地,所述linker包括柔性linker或刚性linker;
优选地,所述linker由G和/或S组成;
优选地,所述linker是GGGGSGGGGSGGGGS。
2.一种多肽复合物,所述多肽复合物中包括权利要求1所述抗体,所述多肽复合物还包括可检测标志物或药物;
优选地,所述抗体与可检测标志物以任意方式连接;
优选地,所述抗体与药物以任意方式连接;
优选地,所述药物包括抗有丝分裂剂、烷化剂、细胞抑制剂、抗血管生成剂;
优选地,所述可检测标记物包括放射性标记物、化学发光标记物、荧光标记物、量子点、测温标记物或免疫聚合酶链式反应标记物;
优选地,所述荧光标记物包括5-荧光素、6-羧基荧光素、3'6-羧基荧光素、5(6)-羧基荧光素、6-六氯-荧光素、6-四氯荧光素、异硫氰酸荧光素、罗丹明、藻胆蛋白和R-藻红蛋白;
优选地,所述可检测标记物可以发出可检测信号;
优选地,所述可检测信号包括光学信号或电学信号。
3.一种融合蛋白,所述融合蛋白中含有权利要求1所述抗体和以下至少一种结构域:
1)铰链区;
2)跨膜结构域;
3)信号转导结构域;
优选地,所述信号转导结构域包含共刺激结构域和/或初级信号传导结构域;
优选地,所述铰链区包括CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合;
优选地,所述铰链区的氨基酸序列如SEQ ID NO.:9所示;
优选地,所述跨膜结构域包括蛋白质的跨膜结构域,所述蛋白质包括:2B4基因表达的蛋白、T细胞受体的α、β或ζ链,CD28,CD3ε,CD45,CD4,CD5,CD8,CD9,CD16,CD22,CD33,CD37,CD64,CD80,CD86,CD123,CD134,CD137和CD154;
优选地,所述跨膜结构域的氨基酸序列如SEQ ID NO.:10所示;
优选地,所述共刺激结构域包括2B4、CD3ζ、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1、ICOS和4-1BB的功能性信号传导结构域;
优选地,所述共刺激结构域的氨基酸序列如SEQ ID NO.:11所示;
优选地,所述初级信号传导结构域包括NKG2D初级信号、CD3-ζ、FcεRIγ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的一种蛋白或多种蛋白的任意组合的信号传导区;
优选地,所述初级信号传导结构域的氨基酸序列如SEQ ID NO:12所示;
优选地,所述融合蛋白是CAR;
优选地,所述CAR的完整结构是轻链可变区-linker-重链可变区-铰链区-跨膜区-共刺激结构域-胞内信号转导结构域。
4.一种分离的多核苷酸,所述多核苷酸编码权利要求1所述抗体或权利要求3所述融合蛋白。
5.一种核酸构建体,所述核酸构建体上包含权利要求4所述分离的多核苷酸;
优选地,所述核酸构建体包括质粒表达载体或病毒表达载体;
优选地,所述病毒表达载体包括慢病毒载体、腺病毒载体或腺相关病毒表达载体。
6.一种细胞,所述细胞中包含或表达权利要求1所述抗体、权利要求2所述多肽复合物、权利要求3所述融合蛋白、权利要求4所述的多核苷酸、权利要求5所述核酸构建体中的一种或多种;
优选地,所述细胞包括原核细胞和真核细胞;
优选的,所述原核细胞包括细菌细胞;优选地,所述细菌细胞包括农杆菌或大肠杆菌;
优选的,所述真核细胞包括动物细胞、昆虫细胞、植物细胞、藻类细胞或真菌细胞;
优选地,所述动物细胞是人的免疫细胞;
优选地,所述免疫细胞包括T细胞、B细胞、K细胞和NK细胞中的一种或多种;
优选地,所述NK细胞包括自行制备的人源NK细胞或是商品化的NK细胞系;
优选地,所述NK细胞系包括NK-92、NKG、YT、NK-YS、HANK-1和NK L;
优选地,所述自行制备的人源NK细胞包括自体的或异体的NK细胞;
优选地,所述自行制备的人源NK细胞是由干细胞分化而来,或者,是从单细胞中分离而来。
7.一种药物组合物,所述药物组合物中包含权利要求1所述抗体、权利要求2所述多肽复合物、权利要求3所述融合蛋白、权利要求4所述的多核苷酸、权利要求5所述核酸构建体或权利要求6所述细胞的一种或多种;
优选地,所述药物组合物中还包括其他治疗癌症的物质,所述物质包括烷化剂、抗代谢物、抗肿瘤抗生素、抗有丝分裂剂、拓扑异构酶抑制剂、蛋白体抑制剂、细胞抑制剂或血管损伤剂;
优选地,所述药物组合物还包括药学上可接受的赋形剂和/或添加剂;
优选地,所述赋形剂包括缓冲体系、增稠剂、稳定剂、中和试剂或保湿剂;
优选地,所述添加剂包括充填剂、结合剂、增湿剂、助流剂、稳定剂、防腐剂或乳化剂;
优选地,所述药物组合物的给药途径为肠道给药或非肠道给药;
优选地,所述药物组合物的给药剂型是液体剂型、固体剂型或半固体剂型。
8.权利要求1所述抗体、权利要求2所述多肽复合物、权利要求3所述融合蛋白、权利要求4所述的多核苷酸、权利要求5所述核酸构建体或权利要求6所述细胞或权利要求7所述药物组合物中任意一种在制备药物中的应用;
优选地,所述药物是治疗BCMA相关癌症的药物;
优选地,所述药物是治疗BCMA异常表达的癌症的药物;
优选地,所述癌症包括实体瘤、非实体瘤;
优选地,所述实体瘤包括前列腺癌、膀胱癌、肾癌、肝癌、乳腺癌、卵巢癌、肺癌、肺腺癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、直肠癌、血管内皮瘤、睾丸癌、皮肤癌;
优选地,所述非实体瘤包括白血病、骨髓瘤、淋巴瘤;
优选地,所述白血病可以按细胞分化成熟程度和自然病程可分为急性白血病和慢性白血病;优选地,所述急性白血病分为急性淋巴细胞白血病和急性髓系白血病;优选地,所述慢性白血病可分为慢性髓系白血病、慢性淋巴细胞白血病、毛细胞白血病、幼淋巴细胞白血病;
优选地,所述骨髓瘤包括典型的骨髓瘤、冒烟型骨髓瘤、孤立性骨髓瘤、浆细胞白血病型骨髓瘤、不分泌型骨髓瘤、骨硬化型骨髓瘤;
优选地,所述淋巴瘤包括霍奇金淋巴瘤或非霍奇金淋巴瘤。
9.一种方法,所述方法选自以下任意一种:
1)检测细胞是否表达BCMA的方法,所述方法包括将待测细胞与权利要求1所述抗体或权利要求2所述多肽复合物接触;
优选地,所述待测细胞获自受试者;
优选地,所述受试者患有或疑似患有癌症;
优选地,所述待测细胞来自组织、组织切片和体液中的一种或更多种;
2)制备药物的方法,所述方法包括制备权利要求1所述抗体、权利要求2所述多肽复合物、权利要求3所述融合蛋白、权利要求4所述的多核苷酸、权利要求5所述核酸构建体或权利要求6所述细胞或权利要求7所述药物组合物中任意一种或多种的步骤;
优选地,所述药物是治疗BCMA相关癌症的药物;
优选地,所述药物是治疗BCMA异常表达的癌症的药物。
10.一种试剂盒或其在制备检测细胞是否表达BCMA的产品中的应用,所述试剂盒中含有权利要求1所述抗体或权利要求2所述多肽复合物。
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