作者要感谢卡尔博士6月（宾夕法尼亚大学），帮助和提供帮助的SB系统的人工APC的克隆＃4和Perry博士哈克特（明尼苏达大学）。 格兰特支持：癌症中心的核心格兰特（CA16672）; RO1（CA124782，CA120956，CA141303），R33（CA116127），P01（CA148600）; SPORE（CA136411）;Ĵ区伟业基金，宝来惠康基金;吉尔森Longenbaugh基金;癌症预防研究所得克萨斯州CLL全球研究基金会，国防部的Noelan L. Bibler房地产哈里T Mangurian，小，基金白血病免疫治疗研究所的个性化癌症治疗白血病和淋巴瘤协会淋巴瘤研究基金会; MDACC的姊妹机构网络基金，米勒基金会;先生香草西门子;先生和夫人乔H.秤;托马斯·斯科特先生，美国国家癌症研究基金会;儿科癌症研究基金会;生产AssistancE细胞疗法（PACT），威廉·劳伦斯和布兰奇·休斯儿童的基金会。

<html> 0日或之前 1。从PB和UCB分离单个核细胞（MNC） <ol><li>稀释与等量的PB和UCB与PBS-EDTA四卷。 </li><li>慢慢层稀释血液（25毫升）到聚蔗糖（12毫升）在50毫升离心管中（s）和在400×g离心30 - 40分钟（无制动器）的离心机。 </li><li>收集并转移到新鲜的50毫升离心管中使用移液管的单核细胞组分（接口）。 </li><li>调出音量用PBS-EDTA至50毫升，450×g离心10分钟，离心。 </li><li>吸液的上清液，轻轻地重悬细胞沉淀（s）在50毫升的完全培养基（CCM）和在400×g离心10分钟，离心。 </li><li>轻轻地重新悬浮细胞沉淀池在CCM，并进行细胞计数采用台盼蓝排除方法（Cellometer，PBMC程序）。 </li><li> MNC现在可以用于电穿孔（核转染）或冷冻保存，以备将来使用。 </li></ol>题“&gt; 2。下游，用于电穿孔的T细胞在第0天<ol><li>如果使用冷冻保存的跨国公司，快速解冻足够的细胞进行全面电（2×10 8，增加了大约20％，占细胞在离心过程中的损失和孵育2小时），在37°C水浴。如果使用的是新鲜分离的MNC继续执行步骤2.3。 </li><li>轻轻重新悬浮并转移细胞的一个适当大小的离心管中（）中含有预先温热的完整的苯酚 - 免的RPMI培养基（PF-RPMI），在200×g离心10分钟（无制动器），吸出物上清液和离心机。 </li><li>重新悬浮在PF-RPMI的MNC，进行细胞计数（Cellometer）和转移细胞的一个适当大小的细胞培养容器（s）在10 6细胞/ ml的浓度。 </li><li>孵育在加湿37°C / 5％CO 2的培养箱中培养2小时±30分钟。 </li><li>无菌离心管中（），在200×g离心5分钟（没有刹车）的自旋转移的MNC，吸出上清液，并轻轻ŕE-暂停，并结合细胞沉淀（S），PF-RPMI。 </li><li>进行小区的计数（Cellometer）和计算所需的细胞悬浮液（2×10 8 MNC）的体积。 </li><li>计算出的量的转移到一个无菌的50毫升离心管中，在200×g离心10分钟（无制动器）的自旋。 </li><li>吸取上清液，无残留的媒体依然存在，轻轻重悬敲击侧管。 </li></ol> 3。 0天电（核转），MNC（满量程使用10比色皿） <ol><li>预孵育的无菌的12孔板，用10口井，含有4毫升的温暖的PF-RPMI中加湿37°C / 5％CO 2培养箱中。 </li><li>准备和预暖龙沙Nucleofector解决人类T细胞试剂盒（再按照生产商的说明， <a href="http://www.lonza.com" target="_blank">www.lonza.com</a> ）在生物安全柜（BSC）的环境温度。 </li><li>准备的Nucleofector解决方案/ DNA预混插件克加入100μl的补充Nucleofector溶液，15微克的转座子（超螺旋的DNA质粒指定为CD19RCD28/pSBSO）和5微克的转座（超螺旋的DNA质粒指定为的pCMV-SB11）每反应/反应杯。 </li><li>分散的细胞沉淀（从步骤2.8）通过轻轻拍打离心管的一侧，并重新悬浮于核转染溶液/ DNA主混合物（最终细胞浓度：2×10 7个 cells/100微升）。 </li><li>小心转移100μl的细胞悬液（3.4）10（10）龙沙核转染试管，小心避免气泡产生。 </li><li>一次点击的比色皿，并electroporate使用程序U-014（未受刺激的T细胞）。 </li><li>的比色皿，在12孔板（步骤3.1）传送给BSC。 </li><li>收获的电穿孔的细胞从每个比色皿中，使用一个Amaxa公司精尖的移液管，加入〜500微升预先温热的培养基中从相应的井（12孔板的Ste制备p 3.1），并返回板加湿37°C / 5％CO 2的培养箱中培养2小时±30分钟。 </li><li>经过2个小时的孵化，收获和所有井的细胞转移到无菌的离心管中。 </li><li>通过离心洗涤细胞，在140×g离心8分钟，环境温度下，无制动和吸液并丢弃上清液使得无残留介质覆盖细胞沉淀。 </li><li>分散细胞沉淀通过轻轻拍打离心管的一侧，并轻轻重新悬浮在CCM中，以实现单细胞悬浮液。 </li><li>进行细胞计数，并调节细胞浓度至10 6细胞/ ml在CCM。 </li><li>细胞悬浮液转移到细胞培养烧瓶中（s）和在孵化器中过夜。 </li><li>工艺步骤2.1到3.8的重复为控制：EGFP转染的细胞（5×10 6个细胞/反应杯用5μgAmaxa公司控制EGFP的超螺旋质粒，pmaxGFP）。 </li></ol> 1天1 日和随后的刺激周期 4。第1天的CAR表达的流式细胞仪分析<ol><li>收获的电穿孔的细胞，并进行细胞计数采用台盼蓝排斥法（血球）。 </li><li>着色漆与CD3，CD4，CD8，和人IgG的Fcγ（作为CAR的表达的测定）的特异性的抗体的细胞（1〜2×10 6）。 </li><li>收购细胞FACS刀魂和使用FCS Express软件，计算CAR表达的数据进行分析。 <ol><li>计算CAR +细胞，在培养由下式： （编号：活细胞总数）×（％CAR +细胞）=号的CAR +细胞 </li></ol></li></ol> 5。 AAPC（克隆＃4）的制备第1天。 AAPC（克隆＃4）来源于合作快递所需的T细胞共刺激分子的K562细胞（亲本获得了美国典型培养物保藏） <ol><li>解冻的一份冻结的100 Gy的照射人工AP​​C在37°水浴。 </li><li>通过离心将细胞洗涤两次，在400×g离心10分钟在CCM中，计数使用Cellometer（台盼蓝排斥）。 </li><li>计算可行的人工APC所需的刺激： （CAR +细胞数）×2 =号的照射人工APC </li></ol> 6。的人工APC介导的刺激CAR + T细胞1日开始的第一和后续刺激周期<ol><li>将电穿孔的细胞（表达，CAR）和γ照射人工APC（克隆＃4）混合比为1:2（CAR +细胞：可行人工APC）在CCM中在无菌容器中。 <ol><li> 注意的人工APC的比调整为基于流式细胞术在电穿孔后的天表达CAR。 </li></ol></li><li>加入IL-21（30纳克/毫升）的细胞悬浮液。 </li><li>等分试样在T-75cm 2的烧瓶中（s）和/或Vue生命文化袋在10 6细胞/毫升的浓度，并返回到incubat或。 </li></ol> 3，5天 7。续文化CAR + T细胞<ol><li>执行半介质改变，补充，IL-21，和保持T细胞，在10 6细胞/毫升的浓度。 </li></ol> 7天 8。首先的人工APC介导的刺激周期的结束<ol><li>收获细胞，计算和染色CD3，CD4，CD8，并与Fcγ（CAR），并继续执行步骤10.1。 </li></ol> 9。枯竭的CD56 +细胞（通常在7至14天后电） <ol><li>执行CD56耗尽使用顺磁性珠，如果CD56 + CD3 阴性淋巴细胞≥10％。 </li></ol>刺激循环＃2，＃3，＃4对应于8→14天，15→21天，与天为22→28 10。递归AAPC传播性T细胞在临床上有足够数量的增加<ol><li>重复stimulatioN处理（4次），描述在步骤4.3-8.1。 </li><li>加入IL-2（50U/ml）的文化开始的7天，14和21，和然后在每个媒体的变化（每周3次，上周一至周三，周五上课）。 </li><li>冷冻保存（归档）多余的T细胞。 <ol><li>使用控制率冰柜冷冻的T细胞。 </li></ol></li></ol>第28天 11。最后的人工APC介导的刺激周期结束：收获T细胞<ol><li>冻存T细胞释放测试和输液。 </li></ol>

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没有利益冲突的声明。

转座子和转座系统，如从piggyBac转12,19和SB 18,20-22，非病毒性的基因治疗的方法，是一个替代病毒临床级CAR + T细胞介导的转导。其潜在的人类基因治疗1,6,23的基因转移系统的基础上被选择了作为对SB。我们开发的SB换位（引入一个CAR）和递归添加γ照射人工AP​​C（检索转基因的T细胞的稳定表达一个CAR）作为符合TAA-特异性T细胞在制造平台技术的双重技术与cGMP的I / II期临床试验（ 图4）。 γ照射人工APC上共培养28天（4 7天刺激周期）后，我们通常能够生成适合人类应用至少〜10 10转基因的T细胞。根据需要，可以进行额外的刺激周期吨Ø产生大量的转基因T细胞。此外，如果需要更少CAR + T细胞，电穿孔和传播的方法可以被缩减雇用较少的比色皿和弘扬只是一个在开始时，每个子集的数值扩展的T细胞增殖对人工APC随后的几轮刺激周期。大部分的电穿孔，并传播的T细胞输注稳定表达​​的CAR收获。的产物，CD4 +和CD8 + T细胞表达我们的第二代车包括内存/天真的表型的细胞，并表现出三个特点重新定向的特异性。首先，转基因的T细胞的具体裂解CD19 +的目标，第二，响应于CD19 +刺激细胞产生IFN-γ，和，第三，增殖响应于CD19 +饲养细胞，所有在CAR-依赖性13,18。我们的方法结合吃了转基因CAR通过电子转移的非病毒DNA质粒的SB系统，可以进行静态主要来自PB和UCB的T细胞。我们和其他转基因K562细胞作为AAPC有效地传播的临床足够数量的T细胞输注24,25。人工APC和组织文化环境（ 例如增加了IL-21）已被修改，以产生患者和供体来源的CD19-特异性T细胞输注造血干细胞移植后（ 表1）13,18。我们可以生产汽车+ T细胞PB简单地通过静脉穿刺，避免了由单采获得MNC从PB的成本，不适和不便。注入同种异基因脐血移植后T细胞的能力获得大量的CAR + T细胞，少量的跨国公司特别有吸引力。体积小，不愿透露姓名的新生儿捐助防止再访问这个人在以后的时间点，只有数量有限的收获MNC可作为起始原料制造T细胞与造血，以避免干扰。制造过程中的进一步前进到目前正在进行中包括一个高吞吐量加上一个完全封闭的WAVE生物反应器，以尽量减少处理的电穿孔的移动设备。骨料，SB和人工APC呼吁平台以产生CD19-特异性CAR + T细胞，可以适于以产生大量的转基因的T细胞可识别细胞表面的替代TAAS符合cGMP。

<table border="1"><tbody><tr><td> 试剂 </td><td> 供应商 </td><td> 目录编号 </td><td></td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> DNA质粒表达GFP（pmaxGFP） </td><td>龙沙</td><td> VPA-1002（套机） </td><td></td></tr><tr><td> PBS </td><td>西格玛</td><td> D8537 </td><td></td></tr><tr><td> CliniMACS PBS-EDTA </td><td>美天旎生物技术公司</td><td> 70026 </td><td></td></tr><tr><td> HYQ RPMI-1640 </td><td> Thermo Scientific的</td><td> SH30096.01 </td><td></td></tr><tr><td>苯酚免费RPMI-1640 </td><td> Thermo Scientific的</td><td> SH30605.01 </td><td></td></tr><tr><td> Hyclone公司的胎牛血清</td><td> Thermo Scientific的</td><td> SH3007003HI </td><td></td></tr><tr><td> GlutaMAX（100x）的</td><td> Gibco公司</td><td> 3505-061 </td><td></td></tr><tr><td>用Ficoll-Paque </td><td> GE Healthcare公司</td><td> 17-1440-02 </td><td></td></tr><tr><td>重组人IL-21的</td><td> PeproTech公司</td><td> AF-200-21 </td><td></td></tr><tr><td>重组人IL-2（Proleukin） </td><td>诺华公司</td><td> NDC 65483-116-07 </td><td></td></tr><tr><td>人类T细胞试剂盒（Nucleofector解决方案） </td><td>龙沙</td><td> VPA-1002 </td><td></td></tr><tr><td> CliniMACS CD56试剂</td><td>美天旎</td><td> 70206 </td><td></td></tr><tr><td>为人类CD3 FITC缀合的小鼠单克隆抗体特异性</td><td> BD Biosciences公司</td><td> 349201 </td><td></td></tr><tr><td> APC-共轭与小鼠单克隆抗体特异于人CD4 </td><td> BD Biosciences公司</td><td> 340443 </td><td></td></tr><tr><td> PerCPCy5.5-共轭与小鼠单克隆抗体特异于人CD8 </td><td> BD Biosciences公司</td><td> 341051 </td><td></td></tr><tr><td> PE共轭到F（&#39;）2片段山羊抗体特异于人的Fcγ </td><td> Invitrogen公司</td><td> H10104 </td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> S2002 </td><td></td></tr><tr><td></td><td></td><td></td><td> 耗材 </td></tr><tr><td> 12孔板</td><td>康宁</td><td> 3513 </td><td></td></tr><tr><td> T-75厘米2烧瓶</td><td>康宁</td><td> 430641 </td><td></td></tr><tr><td>培养袋</td><td> VUE生活</td><td> 290C，750C </td><td></td></tr><tr><td> 50 mL离心管</td><td> BD Biosciences公司</td><td> 352098 </td><td></td></tr><tr><td></td><td></td><td></td><td><s仲&gt;设备</td></tr><tr><td> Amaxa公司Nuceleofector II </td><td>龙沙</td><td> AAB-1001 </td><td></td></tr><tr><td> Cellometer </td><td> Nexcelom生命科学</td><td> Cellometer视觉</td><td></td></tr><tr><td> SORVALL传奇RT离心机</td><td> SORVALL </td><td> 75004377 </td><td></td></tr><tr><td> BD流式细胞仪刀魂</td><td> BD Biosciences公司</td><td> 342975 </td><td></td></tr><tr><td>控制率冰柜</td><td>刨床</td><td> Kryo 750 </td><td></td></tr><tr><td>照射器</td><td> CIS国际生物</td><td> IBL-437 C＃09433 </td><td></td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td colspan="4"> 表达的分子（内源性和引进） </td></tr><tr><td> 细胞系 </td> &lt;TD&gt; 通用名 </td><td> 电池类型 </td><td> 表达的分子 </td></tr><tr><td> CJKT64.86.41BBL.GFP-IL15.CD19 </td><td>克隆4 </td><td> K562 </td><td> CD137L，CD64，CD86，CD19，GFP表达与膜结合型IL-15 </td></tr></tbody></table> 的完全培养基（CCM）的 （存储在4℃下长达30天） HYQ RPMI-1640/Phenol-free RPMI-1640 10％FBS的 1％GlutaMAX 流式细胞仪缓冲液 （保存在4°C至30日） PBS 2％FBS的 0.1％的叠氮化钠</td></tr></tbody></table>

clinical application of sleeping beauty and artificial antigen presenting cells to genetically modify t cells from peripheral and umbilical cord blood

临床级T细胞的效力，可以提高结合基因治疗，免疫治疗造出一种生物制品与潜在的优越的（ⅰ）识别的肿瘤相关抗原（TAAS），（ⅱ）持续输注后，（ⅲ）迁移到肿瘤部位，及（iv）能够回收的效应功能，在肿瘤微环境的潜力。大多数T细胞工程为人类应用遗传操作方法使用逆转录病毒和慢病毒的稳定表达CAR 1-3。这种方法虽然符合现行良好生产规范（GMP），可以是昂贵的，因为它依赖于临床级的重组病毒数量有限的生产设施的制造和释放。由于DNA物种可以产生临床级在约1/10的成本尿激酶原，电转移的非病毒质粒是一个有吸引力的替代转导蚂蚁GMP级的病毒。为了提高效率的整合，我们人类应用4-8适应睡美人 （SB）转座子和转。我们的SB系统使用两个DNA包含编码感兴趣的基因（ 例如， 第二代CD19-特异性CAR转基因，CD19RCD28指定）和一个转座（ 例如，SB11）的转基因插入到TA二核苷酸重复9-11转座子的质粒。要生成临床足够数量的转基因的T细胞中，我们使用K562-衍生的人工抗原呈递细胞（人工APC）（克隆＃4）修饰以表达一个TAA（ 例如 CD19）以及T细胞共刺激分子CD86和CD137L，一个膜结合版本的白细胞介素（IL）-15（肽融合改良的IgG4 Fc区域）和CD64（FC-γ受体1）为加载的单克隆抗体（单抗）12。在这份报告中，我们将演示程序，可以进行compliaNCE与cGMP产生特定的CAR + CD19-T细胞适合人类应用。这是通过两个DNA质粒，一个SB的座子（CD19RCD28）和稳定的整合体通过检索一个SB转座（SB11），然后每7天增加（刺激周期），γ-照射人工AP​​C同步电转移（克隆＃4）的存在下，可溶性重组人IL-2和IL-21的13。通常为4个周期（28天连续培养）进行稳定表达CAR的T细胞，产生临床上有吸引力的数字。可以应用这种方法来制造的临床级CD19-特异性T细胞衍生的T细胞从外周血（PB）或脐带血（UCB）。此外，这种方法可以被利用来产生不同类型的肿瘤的T细胞配对的特殊性，介绍了车载表达的TAA，通过CAR的认可，在人工APC。

我们报告说，电转移DNA质粒和传播的T细胞对γ照射人工AP​​C可以用来产生临床吸引力来自PB和UCB人类应用T细胞的数目。这些转基因T细胞表达的的TAA CD19，独立的主要组织相容性复合体介绍了车载承认。将SB-衍生的DNA质粒，以表达为（i）转座子，2 第二代CAR（CD19RCD28）信号通过CD28和CD3-ε14，和（ⅱ）的转座，SB11 15，已被先前描述的13，16,17 。在目前的研究中所用的质粒魏斯曼临床生物制造设备（麦迪逊，威斯康星州）的商业化生产。人工APC（克隆＃4），K562细胞（亲本系得自美国典型培养物保藏中心），共表达所需的T细胞共刺激分子（分别介绍了在3 90％的分子在细胞表面上的人工APC）来自如先前描述的12。在这里，我们表明，CD19-特异性T细胞可以产生单个核细胞（MNC）来自PB或UCB，使用SB换位介绍车AAPC此外，数值扩大T细胞的CAR-依赖性（ 图1 ，4）13,18。 10的比色皿（2×10 7 MNC /比色皿），电穿孔为每个收件人使用15微克的DNA质粒（CD19RCD28/pSBSO）编码转座子（CAR）和5μg的DNA质粒（质粒pCMV-SB11）编码转座（SB11）。如果跨国公司限制或缩减实验室工作的比色皿的数量可以减少。电穿孔的天被定义为“第0天”刺激周期＃1。作为流式细胞仪和培养条件的控制，自​​体T细胞是模拟电穿孔的（没有DNA质粒）和数值扩大γ照射人工AP​​C（克隆＃4）已预先装入的OKT3交叉链接CD3维持T细胞上扩散。我们routinely评估电转效率和可行性的T细胞在电穿孔后的一天（ 图2B）。控制DNA质粒（指定pmaxGFP），和CAR在这一初始时间点的表达EGFP反映的集成和游离质粒的蛋白表达。通常情况下，一天电击后，我们测量EGFP表达〜60％〜40％（ 图2A）和CAR的表达与T细胞活力在40％至50％之间。可溶性重组人IL-2和IL-21的存在下，γ照射人工AP​​C递推添置检索T细胞稳定表达CAR（CD19RCD28）。 CD3 阴性 CD56 + NK细胞被耗尽从培养物中，使用特定的CD56-顺磁性珠，如果这些NK细胞的百分比是≥10％，特别是如果T细胞上表达的百分比CAR是低。这种枯竭防止干扰的能力AAPC迅速的NK细胞过度生长，维持增殖的n的CAR + T细胞。有时，在过去两年刺激周期进行CAR + T细胞，NK细胞耗尽，但引入了损失所需的细胞共同表达CD56 + T细胞一些汽车。 T细胞生长在一个封闭的系统使用Vue公司过去14天的生活文化袋功能。转基因和传播性T细胞的一个子集通常被冷冻保存，作为未来的分析和存档材料的来源，是在第14天或第21天（结束刺激周期＃2和＃3）共同培养AAPC解冻如果随后无法预料的问题发生在制造过程中。 T细胞通常采收约28天的文化（ 图3），经常表达&gt; 90％的汽车和80％可行的（ 图2C，D）。之前我们已经表明，四个星期后共同培养AAPC的平均折膨胀的CD3 + T细胞是19,800±11,313与CA<html> R +表达为90％±7.5 13。这些T细胞的冷冻保存和接受的过程和释放测试，通知上所生产的产品的安全性和疗效的潜力。版本进行测试，符合临床实验室改进修正案（CLIA）来生成一个证书之前注入到收件人的临床试验分析。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50070/50070fig1highres.jpg" src="/files/ftp_upload/50070/50070fig1.jpg" /> 图1。步骤概述CAR + T细胞从PB和UCB electroporate和传播的过程。 <a href="https://www.jove.com/files/ftp_upload/50070/50070fig1large.jpg" target="_blank">点击此处查看大图</a> 。 upload/50070/50070fig2highres.jpg“SRC =”/ files/ftp_upload/50070/50070fig2.jpg“/&gt; 图2。从PB的转基因的T细胞的表征（A）的EGFP的表达在第0天的第一刺激周期，以评估的基因转移的效率。表达CD19特定CAR（CD19RCD28），流式细胞仪检测细胞CD3 +，CD8 +，CD4 + T细胞（B）约24小时后，电穿孔和（C）后28日内，共同培养AAPC。 UCB来源的T细胞相似的表达CAR。 （D）反应动力学，CAR表达。 <a href="https://www.jove.com/files/ftp_upload/50070/50070fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="4.5in" fo:src="/files/ftp_upload/50070/50070fig3highres.jpg" src="/files/ftp_upload/50070/50070fig3.jpg" /> 图3。传播PB衍生车+ T细胞。评估数字扩大的CD3 +和CAR + T细胞来自PB通过反复共培养在重组人可溶性IL-2和IL-21存在下，γ照射人工APC。向上的箭头表示增加的γ射线辐照人工APC标记的每一个刺激周期的开始。来自UCB-CAR + T细胞表现出类似的数字扩展。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50070/50070fig4highres.jpg" src="/files/ftp_upload/50070/50070fig4.jpg" /> 图4。概略的SB和人工APC系统的制造过程中使用基因修改和传播CAR +衍生的T细胞从PB和UCB。CD19-特异性CAR + T细胞所产生的电转移SB-衍生的超螺旋DNA的质粒和随后的共培养K562衍生AAPC（克隆＃4）的存在ØF重组人可溶性IL-2和IL-21。 <a href="https://www.jove.com/files/ftp_upload/50070/50070fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <table border="1"><tbody><tr><td width="5"> T细胞源 </td><td width="9"> 转座子* </td><td width="9"> 转座子 </td><td width="26"> T细胞输注 </td><td width="1"> IRB＃ </td><td width="1"> NIH-OBA＃ </td><td width="1"> IND＃ </td></tr><tr><td>自体（病人派生）** </td><td> CD19RCD28 </td><td> SB11 </td><td>自体造血干细胞移植后</td><td> 2007-0635 </td><td> 0804-922 </td><td> 14193 </td></tr><tr><td>异体（供体来源） </td><td> CD19RCD28 </td><td> SB11 </td><td>异基因造血干细胞移植后</td><td> 2009-0525 </td><td> 0910-1003 </td><tD&gt; 14577 </td></tr><tr><td>异体（供体来源） </td><td> CD19RCD28 </td><td> SB11 </td><td>异基因脐带血移植后</td><td> 2010-0835 </td><td> 1001年至1022年</td><td> 14739 </td></tr></tbody></table> *表1中。临床试验在MDACC美国食品药品管理局的主持下，注入CD19-特异性CAR + T细胞上传播AAPC。通过强制表达的SB转座子编码的第二代车，指定CD19RCD28，CD28和CD3-ε信号通过，而具体的T细胞CD19。参考5 **试用。

Watch this Scientific Journal Video about Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood at JoVE

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T细胞表达CD19-特异性嵌合抗原受体（CAR）被注入我们的第一个人类基因治疗试验中研究治疗B细胞恶性肿瘤。我们描述了使用基因改造的T细胞<em&gt;睡美人</em（SB）系统地介绍具体的CD19-CAR和选择性传播的名牌CD19 +人工抗原提呈细胞。

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

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JoVE Journal

Immunology and Infection

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

18.2K 次观看.  U.T. MD Anderson Cancer Center. T细胞表达CD19-特异性嵌合抗原受体（CAR）被注入我们的第一个人类基因治疗试验中研究治疗B细胞恶性肿瘤。我们描述了使用基因改造的T细胞睡美人

视频: Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

科研

免疫与感染

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

人脐血巨分化与血小板形成的 CD34+单元格

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...