CN108047333A - 以cd33为靶点的特异性抗体、car-nk细胞及其制备和应用 - Google Patents
以cd33为靶点的特异性抗体、car-nk细胞及其制备和应用 Download PDFInfo
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- CN108047333A CN108047333A CN201810036254.7A CN201810036254A CN108047333A CN 108047333 A CN108047333 A CN 108047333A CN 201810036254 A CN201810036254 A CN 201810036254A CN 108047333 A CN108047333 A CN 108047333A
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Abstract
本发明提供一种抗CD33抗体或其抗原结合片段，其中所述抗体包含重链可变区(VH)和/或轻链可变区(VL)，其中所述重链可变区包含重链VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包含轻链VLCDR1、VLCDR2、VLCDR3。本发明提供以CD33为靶点的抗体或其抗原结合片段，并以此所构建的CD33 CAR‑NK细胞为单克隆细胞株培养并扩增所得，其性状稳定，可用于大规模生产制备。CD33 CAR‑NK细胞以CD33分子为靶抗原，能够特异性地杀死或杀伤髓系白血病细胞，可作为髓系白血病类疾病的治疗药物，用于CD33分子高表达的髓系白血病的治疗。
Description
技术领域
本发明涉及生物医药领域，具体涉及以CD33为靶点的特异性抗体，以CD33为靶点的CAR-NK细胞及其制备和应用。
背景技术
嵌合抗原受体(chimeric antigen receptor，缩写CAR)修饰的免疫细胞使用遗传工程手段修饰免疫细胞使其表达外源性抗肿瘤基因。CAR基因主要包括细胞外识别域和细胞内信号转导结构域：前者用于识别肿瘤表面特异性分子和后者用于启动识别肿瘤表面分子后的免疫细胞应答，发挥细胞毒作用，其主要以T-细胞为载体。
CAR-T，全称是Chimeric AntigenReceptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法。嵌合抗原受体T细胞(CAR-T细胞)是将能识别某种肿瘤抗原的抗体的抗原结合部与CD3-ζ链或4-1BB等胞内元件在体外偶联为一个嵌合基因，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。患者的T细胞被“重编码”后，生成大量肿瘤特异性的CAR-T细胞。CD33是髓系细胞分化抗原，分子量为67KD，主要分布在髓系血细胞，特别是在分化的早期阶段。其胞内区含有免疫酪氨酸抑制基序(ITIM)，所以它可能具有通过募集信号分子来调节细胞的生长与分化等功能。CD33在90％以上的急性髓系白血病患者都有表达。在造血干细胞表面没有表达,在成熟的粒细胞和其他组织也没有表达,因此CD33成为髓系白血病治疗的良好靶点。
目前经过大量的研究证明CD33可作为治疗髓系白血病的靶点用于CAR的构建。然而制备CAR-T的T细胞只能来自患者自身，不能异体回输，导致其不仅生产成本高，而且不能大批量生产和制备，使其应用受到了极大的限制。
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)的特异功能，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新趋势，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
有鉴于此，本发明提供了一种以CD33为靶点的抗CD33抗体或其抗原结合片段，以及能够表达该抗体或抗原结合片段的CAR-NK细胞及其制备和应用，该细胞性状稳定，可大批量生产和制备，可进行异体回输。
本发明一方面提供一种以CD33为靶点的抗CD33抗体或其抗原结合片段，其中所述抗体包含重链可变区(VH)和/或轻链可变区(VL)，其中所述重链可变区包含重链VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包含轻链VLCDR1、VLCDR2、VLCDR3，其中所述VHCDR1的氨基酸序列如SEQ ID NO：1所示的序列或其同源序列；所述VHCDR2的氨基酸序列如SEQ ID NO：2所示的序列或其同源序列；所述VHCDR3的氨基酸序列如SEQ ID NO：3所示的序列或其同源序列；所述VLCDR1的氨基酸序列如SEQ ID NO：4所示的序列或其同源序列；所述VLCDR2的氨基酸序列如SEQ ID NO：5所示的序列或其同源序列；所述VLCDR3的氨基酸序列如SEQ IDNO：6所示的序列或其同源序列。
示例性地，所述VH链的氨基酸序列如SEQ ID NO：7所示的序列或其同源序列。
示例性地，所述VL链的氨基酸序列如SEQ ID NO：8所示的序列或其同源序列。
示例性地，所述同源序列与原序列的同源性为60％以上，例如，约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上等。
示例性地，所述VH链和VL链直接连接或通过连接肽连接。
在本发明提供的一个具体实施方式中，以CD33为靶点的抗CD33抗体或其抗原结合片段包括VH链和VL链。
在本发明一个具体实施方式中，所述VH链和VL链通过连接肽连接。
在本发明一个具体实施方式中，所述抗CD33抗体或其抗原结合片段的氨基酸序列如SEQ ID NO：9所示的序列或其同源序列。
在本发明提供的一个具体实施方式中，所述抗CD33抗体或其抗原结合片段与CD33的亲和力达到1.697×10-7M以上。
本发明另一方面提供一核苷酸序列，其能够表达上述抗CD33抗体或其抗原结合片段。
在本发明提供的一个具体实施方式中，所述的核苷酸序列包含SEQ ID NO：10所示的序列或其简并序列，和/或SEQ ID NO：11所示的序列或其简并序列。
在本发明提供的一个具体实施方式中，所述的核苷酸序列如SEQ ID NO：12所示的序列或其简并序列。
示例性地，所述简并序列与原序列的同源性为60％以上，例如，约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上等。
本发明另一方面提供上述抗CD33抗体或其抗原结合片段的制备方法，其包括：建立噬菌体抗体库并从中筛选能与CD33结合的抗体或抗体片段。其具体过程包括：
①制备M13KO7辅助噬菌体；
②利用M13KO7辅助噬菌体建立噬菌体抗体库；
③利用CD33抗原从步骤②中的抗体库中筛选单链噬菌体DNA；以及任选地，
④将步骤③中获得的单链噬菌体DNA进行表达，并纯化。
本发明另一方面提供一种Anti CD33CAR-NK细胞，该细胞能够表达嵌合抗原受体，所述嵌合抗原受体包含上述抗CD33抗体或其抗原结合片段。
在本发明的一个具体实施方式中，所述嵌合抗原受体还包含跨膜结构域和/或共刺激信号传导区。
示例性地，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域。
优选地，所述的抗CD33抗体或其抗原结合片段能够特异性结合肿瘤特异性抗原CD33，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。
示例性地，所述嵌合抗原受体是结构为SCFV-CD8-4-1BB-CD3ζ的融合蛋白，该融合蛋白能够特异性识别CD33分子。
在本发明的一个具体实施方式中，所述SCFV-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：13所示或其同源序列等。
示例性地，所述SCFV-CD8-4-1BB-CD3ζ融合蛋白的核苷酸的序列如SEQ ID NO：14所示或其简并序列等。
在本发明的一个具体实施方式中，所述Anti CD33 CAR-NK细胞能够有效地杀伤或杀死K562细胞等。
本发明另一方面提供上述Anti CD33 CAR-NK细胞的制备方法，其包括如下步骤：
(1)合成和扩增SCFV-CD8-4-1BB-CD3ζ融合蛋白基因，将所述SCFV-CD8-4-1BB-CD3ζ融合蛋白基因克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染293T细胞，包装和制备慢病毒；
(3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到CD33 CAR-NK细胞。
本发明还提供上述抗CD33抗体或其抗原结合片段，和/或上述核苷酸序列表达的抗CD33抗体或其抗原结合片段，和/或上述Anti CD33 CAR-NK细胞在制备治疗和/或预防高表达CD33分子的肿瘤及相关疾病的药物中的应用。
示例性地，Anti CD33 CAR-NK细胞在制备治疗高表达CD33分子的髓系白血病的药物中的应用，尤其在制备治疗高表达CD33分子的急性髓系白血病的药物中的应用。
本发明还提供了一种药物组合物，其包括上述抗CD33抗体或其抗原结合片段，和/或上述核苷酸序列表达的抗CD33抗体或其抗原结合片段，和/或上述Anti CD33 CAR-NK细胞，以及任选地，药学上可接受的辅料。
本发明至少具有如下优势之一：
本发明提供的以CD33为靶点的抗CD33抗体或其抗原结合片段，并以此所构建的Anti CD33CAR-NK细胞为单克隆细胞株培养并扩增所得，其性状稳定，可用于大规模生产制备。Anti CD33CAR-NK细胞以CD33分子为靶抗原，能够特异性地杀死或杀伤髓系白血病细胞，可作为髓系白血病类疾病的治疗药物，用于CD33分子高表达的髓系白血病的治疗。
附图说明
图1所示为本发明实施例提供的pRRSLIN-ScFV(anti CD33)-CD8TM-4-1BB-CD3ζ慢病毒转染载体的结构示意图。
图2所示为本发明实施例提供的流式细胞仪检测CD33CAR-NK细胞阳性率的结果图；图2a为NK-92对照组，图2b为CD33NK-92实验组。
图3所示为本发明实施例提供的CD33CAR-NK细胞杀伤K562细胞的实验结果图。
具体实施方式
除非另有定义，本发明中使用的所有技术和科学术语具有与本发明所述技术领域的普通技术人员通常理解的相同含义。
具体而言，本发明所述的编码SCFV-CD8-4-1BB-CD3ζ融合蛋白的核苷酸的序列是任何能够编码该融合蛋白的任何DNA序列，优选地，该序列为SEQ ID NO：14或其互补序列。另一方面，本发明所述的编码SCFV-CD8-4-1BB-CD3ζ融合蛋白的核苷酸的序列可为在严紧条件下与由SEQ ID NO：14的核苷酸序列进行杂交、且编码该融合蛋白的多核苷酸或其互补序列；
本文所述的“严紧条件”，可以为低严紧条件、中严紧条件、高严紧条件中的任一种，优选为高严紧条件。示例性地，“低严紧条件”可为30℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“中严紧条件”可为40℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“高严紧条件”可为50℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件。本领域技术人员应当理解温度越高越能得到高同源性的多核苷酸。另外，本领域技术人员可以选择影响杂交的严紧度的温度、探针浓度、探针长度、离子强度、时间、盐浓度等多个因素形成的综合结果来实现相应的严紧度。
除此之外可杂交的多核苷酸还可以为，通过FASTA、BLAST等同源性检索软件用系统设定的默认参数进行计算时，与编码序列号6的多核苷酸具有约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上同一性的多核苷酸。
核苷酸序列的同一性，可以使用Karlin及Altschul的算法规则BLAST(Proc.Natl.Acad.Sci.USA 87：2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873,1993)来确定。基于BLAST算法规则的程序BLASTN、BLASTX已被开发(Altschul SF,et al:JMol Biol 215:403,1990)。使用BLASTN分析碱基序列时，如使参数为score＝100、wordlength＝12；此外使用BLASTX分析氨基酸序列时，如使参数为score＝50、wordlength＝3；使用BLAST和Gapped BLAST程序时，采用各程序的系统可设定默认参数值。
本发明中，术语“抗体”指的是与抗原特异性结合的免疫球蛋白分子。抗体可为源于自然源或源于重组源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。抗体通常为免疫球蛋白分子的四聚物。本发明中的抗体可以以多种形式存在，包括例如，多克隆抗体、单克隆抗体、Fv、Fab和F(ab)2，以及单链抗体和人源化抗体等(Harlow等，1999,In:Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY；Harlow等，1989,In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York；Houston等，1988,Proc.Natl.Acad.Sci.USA 85:5879-5883；Bird等，1988,Science 242:423-426)。
术语“抗体片段”指的是完整抗体的一部分，并指的是完整抗体的抗原决定可变区。抗体片段的例子包括但不限于Fab、Fab'、F(ab')2和Fv片段，由抗体片段形成的线性抗体、scFv抗体和多特异性抗体。
除非另有规定，“编码核苷酸”包括为彼此简并版本并编码相同的氨基酸序列的所有核苷酸序列。编码蛋白质的核苷酸序列可包括内含子。
术语“慢病毒”指的是逆转录病毒科的属，其能够有效感染非周期性和有丝分裂后的细胞；它们可传递显著量的遗传信息进入宿主细胞的DNA，以便它们是基因传递载体的最有效的方法之一。
术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。很多载体在本领域中是已知的，包括但不限于线性多核苷酸、与离子或两性分子化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语也应被解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，诸如例如聚赖氨酸化合物、脂质体等等。病毒载体的例子包括但不限于，腺病毒载体、腺伴随病毒载体、逆转录病毒载体等等。
术语“癌症”被定义为以畸变细胞的快速和失控生长为特征的疾病。癌症细胞可局部蔓延或通过血流和淋巴系统蔓延至身体的其他部分。各种癌症的例子包括但不限于乳腺癌、前列腺癌、卵巢癌、子宫颈癌、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、淋巴瘤、白血病、肺癌等等。
如本文所使用的，“包含”与“包括”、“含有”或“特征在于”同义，并且是包括在内的或开放性的，并且不排除另外的未陈述的元件或方法步骤。术语“包含”在本文中的任何表述，特别是在描述本发明的方法、用途或产品时，应理解为包括基本上由所述组分或元件或步骤组成和由所述组分或元件或步骤组成的那些产品、方法和用途。本文示例性描述的本发明适当地可以在不存在本文未具体公开的任何一种或多种元件、一种或多种限制的情况下进行实践。
本文已采用的术语和表述用作描述性而不是限制性术语，并且在此种术语和表述的使用中不预期排除所示和所述特征或其部分的任何等价物，但应认识到各种修饰在请求保护的本发明的范围内是可能的。因此，应当理解尽管本发明已通过优选实施方案和任选特征具体公开，但本领域技术人员可以采用本文公开的概念的修饰和变化，并且此类修饰和变化被视为在如由附加权利要求定义的本发明的范围内。
本文中出现的英文名称不区分大小写；CD33CAR-NK、CD33-CAR NK表示的含义相同；CD33 CAR-NK与Anti CD33-CAR NK表示相同的含义，均表示抗CD33分子的CAR-NK细胞；NK-92、NK92均表示NK92细胞。
本发明所述的“NK”为人体正常NK细胞或NKT细胞或NK细胞系，其包括NK-92细胞，YT细胞，NKL细胞，HANK-1细胞，NK-YS细胞，KHYG-1细胞，SNK-6细胞和IMC-1细胞等。本发明的具体实施例中以NK-92细胞为例进行说明。
为更清楚地说明本发明，现结合如下实施例进行详细说明，但这些实施例仅仅是对本发明的示例性描述，并不能解释为对本申请的限制。
实施例1CD33人源抗体的筛选
2×YT液体培养基：16g蛋白胨，10g酵母提取物，5g氯化钠，800mL水，用氢氧化钠调pH至7.0，加水定容至1000mL，121℃灭菌20min。
2×YT-G：2×YT培养基中加入2％的葡萄糖。
2×YT-AK：2×YT培养基中加入1001g/mL的氨苄青霉素和50 1g/mL的卡那霉素。
一、准备M13KO7辅助噬菌体
1.在37℃条件下，用不同稀释浓度的辅助噬菌体M13K07感染对数生长期的TG1细菌细胞30分钟，之后将其涂布在琼脂平板上。
2.将TG1菌斑克隆挑到3mL 2YT培养基中进行培养。在37℃下培养2小时。
3.将步骤2中的培养物转移到1L 2YT培养基中，加入卡那霉素至50μg/m，37℃培养16小时。
4.离心(10min at 5000g)去除细菌细胞，在上清中加入噬菌体沉淀剂收集噬菌体。
5.计算噬菌体滴度，之后将其稀释到1×1013pfu/mL并保存在-20度冰箱中，备用。
二、准备噬菌体抗体库
1.取噬菌体库甘油菌接种到500ml培养基中，在37℃、250rpm条件下至OD值为0.8-0.9。
2.将步骤一制备的M13KO7加入上述步骤1中，其终浓度为5×109pfu/mL，37℃静置30min，之后在200rpm转速下，恒温摇床培养30min。
3.离心(2200g，15min)步骤2中的培养液，收集细菌细胞，用2YT-AK培养基重悬菌泥，在30℃条件下300rmp转速下过夜培养。
4.离心(7000g，15min，4℃)步骤3中的培养液，去除细菌细胞，将上清收集到1L瓶中。
5.向步骤4中的上清中加入噬菌体沉淀剂，收集噬菌体沉淀。
6.离心(7000g，15min)，去除上清，收集噬菌体沉淀，将噬菌体沉淀加入8ml PBS。
7.重新离心(12,000g，10min)去除细菌碎片，上清中保留噬菌体，备用。(请确认：该部分没有裂解细胞步骤)
三、抗体筛选
1.将CD33抗原以5μg/mL的浓度，在4℃条件下包被孔板2h，之后洗涤三次。利用封闭液在4℃条件下封闭过夜。
2.倒掉封闭液并洗涤，将噬菌体(步骤二中所制备)与100μg/mL的人IgG蛋白重悬在封闭液中(含2％牛奶)并加入孔板中室温孵育1h，之后洗涤。
3.通过胰蛋白酶消化结合在孔板上噬菌体，用于后续第二轮筛选。
4.按上述1-3中的流程重复三轮筛选。
5.通过沉淀获得单链噬菌体DNA，即单链抗体基因序列，并测序鉴定。
实施例2：CD33抗体的表达与鉴定
将实施例1获得的单链噬菌体DNA克隆到pCDNA3.4载体上，构建pCDNA3.4-ScFV(anti-CD33)表达载体，将该载体瞬转至CHO细胞进行表达，通过镍株纯化该单链抗体序列用于后续分析。
用SPR方法鉴定筛选到的单链抗体与CD33蛋白的亲和力，从中选择与CD33蛋白的亲和力最高的单链抗体，并将其命名为单链抗体3-3。单链抗体3-3与CD33分子亲和力如表1所示。
表1：单链抗体3-3与CD33分子亲和力
| sequence | Ka(1/Ms) | Kd(1/s) | KD(M) |
| 3-3 | 1.65E+4 | 0.028 | 1.697E-7 |
其中，单链抗体3-3的VH链的氨基酸序列如SEQ ID NO.7所示，其核苷酸序列如SEQID NO.10所示；单链抗体3-3的VL链的氨基酸序列如SEQ ID NO.8所示，其核苷酸序列如SEQID NO.11所示；单链抗体3-3的氨基酸序列如SEQ ID NO.9所示，其核苷酸序列如SEQ IDNO.12所示。
实施例3慢病毒表达载体的制备
基因合成SCFV(anti CD33)-CD8-4-1BB-CD3ζ融合基因序列(其氨基酸序列如SEQID NO：13所示，基因序列如SEQ ID NO：14所示)。通过酶切，将其转化连接到PRRSLIN载体中，基因上游为EP-1α启动子。载体转化Stbl3大肠杆菌菌株，氨苄青霉素筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，获得PRRLSIN-SCFV(anti CD33)-CD8-4-1BB-CD3ζ慢病毒转染载体(如图1所示)。
实施例4慢病毒的制备
(1)转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。
(2)准备溶液A和溶液B
溶液A：6.25ml 2×HEPES buffer缓冲液(5个大皿一起包装的量，效果最好)。
溶液B：分别加入以下质粒的混合物：112.5μg PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ(target plasmid)；39.5μg pMD2.G(VSV-G envelop)；73μg pCMVR8.74(gag，pol，tat，rev)；625μl 2M钙离子溶液。溶液B总体积：6.25ml。
充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液B，静置5-15分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。(不要旋转培养皿)放置于培养箱中培养16-18小时。更换新鲜培养基，继续培养，分别在48小时和72小时后收集含病毒的上清液。500g，25℃离心10分钟。PES膜(0.45μm)过滤。以70％乙醇消毒贝克曼库尔特Ultra-clear SW28centrifuge tubes，并置于紫外灯下消毒30分钟。将已过滤的含慢病毒的上清液转移至离心管中。在离心管底部小心铺上一层20％蔗糖(每8ml上清液加1ml蔗糖)。以PBS平衡离心管，25000rpm(82，700g)，4℃离心2小时。小心取出离心管，倒掉上清液，倒置离心管去掉残余液体。加入100μl PBS，密封离心管，在4℃放置2小时，每20分钟轻轻涡旋一次，500g离心1分钟(25℃)，收集病毒上清。冰上冷却后，置于-80℃保存。
实施例5 CD33 CAR-NK-92细胞的制备
将NK-92细胞密度调整至2-3×105/ml，按体积比(V/V)病毒载体：细胞培养基＝1:5-10的比例添加病毒载体(实施例4所制备的)，同时添加聚凝胺8μg/ml。4h之后，补加等量的新鲜的完全培养基将细胞密度调整至1×105/ml继续培养。次日，将所有的细胞离心，加入新鲜的培养基，继续培养。每隔1-2天进行补液，使维持细胞密度在2-3×105/ml。72h后进行CAR抗体染色，同时流式分选CD33CAR NK-92阳性细胞并扩大培养。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。
利用流式检测CAR NK-92细胞阳性率，流式检测结果如图2a和图2b所示。图2a和图2b中，所采用的抗体为APC荧光标记，在横坐标上进行表示，NK92细胞若成功表达CAR分子，该信号值将显著升高。从图2a和图2b中可以看出，APC荧光标记的信号值显著升高，表明NK-92细胞成功表达出CAR分子，CAR-NK92阳性率为99.81％。
实施例6 CD33 CAR-NK细胞对体外肿瘤杀伤效果的评估
利用CCK-8方法检测CD33CAR-NK细胞对过表达CD33分子的K562细胞的杀伤效果。实验操作方法如下：
(1)在24孔板中配制1ml K562细胞悬液(2X10^4个/孔)。将培养板在培养箱预培养12h。
(2)弃去24孔板的培养上清，每孔加入1ml效应细胞，效应细胞与靶细胞数目的比例为5:1。培养基对照孔只加1ml培养基，每个实验置三个复孔。效应细胞与靶细胞共孵育4小时。
(3)每孔加入100ul CCK-8溶液，将培养板在培养箱内孵育2h。
(4)用酶标仪测定在450nm处的吸光度。
(5)杀伤率＝(As-Ab)/(Ac-Ab)X100％；
As：试验孔(含有肿瘤细胞的培养基、CCK-8、CAR-NK)；
Ac：对照孔(含有肿瘤细胞的培养基、CCK-8)；
Ab：空白对照(不含细胞和CAR-NK的培养基、CCK-8)；
CD33CAR-NK细胞体外肿瘤杀伤效果评估的实验结果如图3所示。如图3所示实验结果显示，与NK92对照组比较，本发明所制备的CD33CAR NK-92细胞能够显著杀伤过表达CD33分子的K562靶细胞株，但对普通K562细胞杀伤不明显。
本发明的CD33CAR NK-92是将CD33CAR分子侵染NK92细胞株并经过流式筛选获得单一克隆细胞，将性状稳定杀伤活性高的CAR NK92单克隆细胞株培养并扩增获得。该细胞可以用于大规模生产制备，可以用于不同病人且不会产生GVHR排斥。CD33CAR NK-92细胞与CAR-T细胞相比，无需分离病人外周血单核细胞(PBMC)，无需特异活化T细胞和制备CAR-T细胞(该过程需要病人等待10天以上时间)，不需要个性定制且能用于多个病人，缩短了时间，CD33CAR-NK92细胞可以大批量制备培养，病人即来即用；另一方面，常规制备的CAR-T细胞，由于是分离病人的T细胞经病毒感染而制备的，T细胞不是同一个单克隆来源，而分选出的CAR-NK92细胞来源于同一个单克隆，性状和活性均一且稳定，便于大规模生产和质控；同时与NK92细胞相比，由于进行了CAR载体导入，其杀伤活性特异，肿瘤治疗效果更加明显。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
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<213> 人工序列(Artificial Sequence)
<220>
<223> VLCDR3
<400> 6
Gln Gln Arg Thr Ala Trp Pro Tyr Thr
1 5
<210> 7
<211> 115
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Anti CD33 VH
<400> 7
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Arg Thr Gly Ala Arg Thr Ile Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Lys Asp Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 8
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Anti CD33 VL
<400> 8
Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45
Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Thr Ala Trp Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 9
<211> 238
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Anti CD33
<400> 9
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Arg Thr Gly Ala Arg Thr Ile Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Lys Asp Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
145 150 155 160
Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
165 170 175
Leu Leu Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
180 185 190
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
195 200 205
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Thr Ala Trp
210 215 220
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
225 230 235
<210> 10
<211> 345
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Anti CD33 VH
<400> 10
gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtgagcacc atccgcaccg gcgcccgcac catctacgcc 180
gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgccaa gaccaaggac 300
cccttcgact actggggcca gggcaccctg gtgaccgtga gcagc 345
<210> 11
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Anti CD33 VL
<400> 11
accgacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgaccgcgtg 60
accatcacct gccgcgccag ccagagcatc agcagctacc tgaactggta ccagcagaag 120
cccggcaagg cccccaagct gctgtacagc gccagcaccc tgcagagcgg cgtgcccagc 180
cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag cgcaccgcct ggccctacac cttcggccag 300
ggcaccaagg tggagatcaa gcgc 324
<210> 12
<211> 714
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Anti CD33
<400> 12
gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtgagcacc atccgcaccg gcgcccgcac catctacgcc 180
gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgccaa gaccaaggac 300
cccttcgact actggggcca gggcaccctg gtgaccgtga gcagcggcgg cggcggcagc 360
ggcggcggcg gcagcggcgg cggcggcagc accgacatcc agatgaccca gagccccagc 420
agcctgagcg ccagcgtggg cgaccgcgtg accatcacct gccgcgccag ccagagcatc 480
agcagctacc tgaactggta ccagcagaag cccggcaagg cccccaagct gctgtacagc 540
gccagcaccc tgcagagcgg cgtgcccagc cgcttcagcg gcagcggcag cggcaccgac 600
ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag 660
cgcaccgcct ggccctacac cttcggccag ggcaccaagg tggagatcaa gcgc 714
<210> 13
<211> 461
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> ScFV(anti CD33)- CD8TM-4-1BB-CD3ζ
<400> 13
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Arg Thr Gly Ala Arg Thr Ile Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Lys Asp Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
145 150 155 160
Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
165 170 175
Leu Leu Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
180 185 190
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
195 200 205
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Thr Ala Trp
210 215 220
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Thr
225 230 235 240
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
245 250 255
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
260 265 270
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
275 280 285
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
290 295 300
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
305 310 315 320
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
325 330 335
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
340 345 350
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
355 360 365
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
370 375 380
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
385 390 395 400
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
405 410 415
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
420 425 430
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
435 440 445
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210> 14
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ScFV(anti CD33)- CD8TM-4-1BB-CD3ζ
<400> 14
gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60
agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtgagcacc atccgcaccg gcgcccgcac catctacgcc 180
gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgccaa gaccaaggac 300
cccttcgact actggggcca gggcaccctg gtgaccgtga gcagcggcgg cggcggcagc 360
ggcggcggcg gcagcggcgg cggcggcagc accgacatcc agatgaccca gagccccagc 420
agcctgagcg ccagcgtggg cgaccgcgtg accatcacct gccgcgccag ccagagcatc 480
agcagctacc tgaactggta ccagcagaag cccggcaagg cccccaagct gctgtacagc 540
gccagcaccc tgcagagcgg cgtgcccagc cgcttcagcg gcagcggcag cggcaccgac 600
ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag 660
cgcaccgcct ggccctacac cttcggccag ggcaccaagg tggagatcaa gcgcaccacg 720
acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 780
cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 840
gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 900
ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 960
ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1020
gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1080
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1140
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1200
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1260
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1320
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1380
cgctaa 1386
Claims (18)
1.抗CD33抗体或其抗原结合片段，其中所述抗体包含重链可变区(VH)和/或轻链可变区(VL)，其中所述重链可变区包含重链VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包含轻链VLCDR1、VLCDR2、VLCDR3，其中所述VHCDR1的氨基酸序列如SEQ ID NO：1所示的序列或其同源序列；所述VHCDR2的氨基酸序列如SEQ ID NO：2所示的序列或其同源序列；所述VHCDR3的氨基酸序列如SEQ ID NO：3所示的序列或其同源序列；所述VLCDR1的氨基酸序列如SEQ IDNO：4所示的序列或其同源序列；所述VLCDR2的氨基酸序列如SEQ ID NO：5所示的序列或其同源序列；所述VLCDR3的氨基酸序列如SEQ ID NO：6所示的序列或其同源序列。
2.如权利要求1所述的抗CD33抗体或其抗原结合片段，所述VH链的氨基酸序列如SEQID NO：7所示的序列或其同源序列；以及任选地，所述VL链的氨基酸序列如SEQ ID NO：8所示的序列或其同源序列。
3.如权利要求1或2所述的抗CD33抗体或其抗原结合片段，所述同源序列与原序列的同源性为60％以上，优选地，所述同源性为90％以上。
4.如权利要求1或2所述的抗CD33抗体或其抗原结合片段，所述VH链和VL链直接连接或通过连接肽连接，优选地，所述抗CD33抗体或其抗原结合片段的氨基酸序列如SEQ ID NO：9所示的序列或其同源序列。
5.如权利要求1或2所述的抗CD33抗体或其抗原结合片段，所述抗CD33抗体或其抗原结合片段与CD33的亲和力达到1.697×10-7M以上。
6.表达权利要求1-5中任一项所述的抗CD33抗体或其抗原结合片段的核苷酸序列。
7.如权利要求6所述的核苷酸序列,其中所述的核苷酸序列包含SEQ ID NO：10所示的序列或其简并序列，和/或SEQ ID NO：11所示的序列或其简并序列，优选地，所述核苷酸序列如SEQ ID NO：12所示的序列或其简并序列。
8.如权利要求7所述的核苷酸序列，所述简并序列与原序列的同源性为60％以上，优选地，所述同源性为90％以上。
9.权利要求1-5中任一项所述的抗CD33抗体或其抗原结合片段或权利要求6-8中任一项所述的核苷酸所表达的抗CD33抗体或其抗原结合片段的制备方法，其包括：在宿主细胞中表达权利要求6-8中任一项所述的核苷酸。
10.Anti CD33 CAR-NK细胞，其能够表达嵌合抗原受体，所述嵌合抗原受体包含权利要求1-5中任一项所述的抗CD33抗体或其抗原结合片段，和/或权利要求6-8中任一项所表达的抗CD33抗体或其抗原结合片段，和/或权利要求9所制备的抗CD33抗体或其抗原结合片段。
11.如权利要求10所述的Anti CD33 CAR-NK细胞，所述嵌合抗原受体还包含跨膜结构域和/或共刺激信号传导区；优选地，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域；优选地，所述抗CD33抗体或其抗原结合片段能够特异性结合肿瘤特异性抗原CD33，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。
12.如权利要求11所述的Anti CD33 CAR-NK细胞，所述嵌合抗原受体是结构为scFV-CD8-4-1BB-CD3ζ的融合蛋白，其中所述的scFV能够特异性结合CD33分子，优选地，所述SCFV-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：13或其同源序列。
13.如权利要求12所述的Anti CD33 CAR-NK细胞，所述SCFV-CD8-4-1BB-CD3ζ融合蛋白的编码核苷酸的序列如SEQ ID NO：14所示或其简并序列。
14.如权利要求10-13中任一项所述的Anti CD33 CAR-NK细胞，其能够有效地杀伤和/或杀死K562细胞。
15.权利要求10-14中任一项所述Anti CD33 CAR-NK细胞的制备方法，其包括如下步骤：
(1)合成和扩增编码权利要求12或13所述的scFV-CD8-4-1BB-CD3ζ融合蛋白的核苷酸，并将所述核苷酸克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染细胞，包装和制备慢病毒；
(3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到CAR-NK细胞。
16.一种药物组合物，其包含根据权利要求1-5中任一项所述的抗CD33抗体或其抗原结合片段，和/或权利要求6-8中任一项所述的核苷酸序列所编码的抗CD33抗体或其抗原结合片段，和/或权利要求9所制备的抗CD33抗体或其抗原结合片段，和/或权利要求10-14中任一项所述的Anti CD33 CAR-NK细胞，和/或权利要求15所制备的Anti CD33 CAR-NK细胞。
17.如权利要求16所述的药物组合物，其还包括药学可接受的辅料。
18.权利要求1-5中任一项所述的抗CD33抗体或其抗原结合片段，和/或权利要求6-8中任一项所述的核苷酸序列所编码的抗CD33抗体或其抗原结合片段，和/或权利要求9所制备的抗CD33抗体或其抗原结合片段，和/或权利要求10-14中任一项所述的Anti CD33 CAR-NK细胞，和/或权利要求15所制备的Anti CD33 CAR-NK细胞在制备治疗和/或预防高表达CD33的肿瘤及相关疾病的药物中的用途；优选的，所述高表达CD33的肿瘤为髓系白血病。
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| CN113271965B (zh) * | 2019-01-11 | 2023-11-03 | 湖南远泰生物技术有限公司 | Cd37抗体和cd37-car-t细胞 |
| US12005081B2 (en) | 2019-04-30 | 2024-06-11 | Senti Biosciences, Inc. | Chimeric receptors and methods of use thereof |
| CN112851814A (zh) * | 2020-03-17 | 2021-05-28 | 西安宇繁生物科技有限责任公司 | 一种靶向bcma的全人源单链抗体及其制备方法与应用 |
| CN112851814B (zh) * | 2020-03-17 | 2023-07-07 | 西安宇繁生物科技有限责任公司 | 一种靶向bcma的全人源单链抗体及其制备方法与应用 |
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