Name: generation of car t cells for adoptive therapy in the context of glioblastoma standard of care
Uploaded: 2015-02-16T14:00:00.000+00:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

这个协议是基于其中10只小鼠用10 7 CAR t各自细胞进行了处理的实验设计。这意味着10 8 CAR T细胞，将需要;屈服应为5×10 7 -1×10 8被高估到帐户中生存能力的损失。以下协议进行缩放，以产生大约200×10 6个细胞。然后将细胞静脉内施用给雌性C57BL / 6小鼠用9天建立同基因的EGFRvIII阳性颅内肿瘤，从现有KR158B星形细胞瘤或B16黑色素瘤细胞系的开发。与之同时CAR T细胞的生成，荷瘤小鼠服用临床相应剂量的lymphodepletive TMZ（60毫克/公斤）和WBI（16.5戈瑞）的。 小鼠维护和无病原体条件在杜克大学医学中心（DUMC）下饲养。所有的动物实验是按照批准杜克大学机构的协议进行人的动物护理和使用委员会（IACUC）。 1.政府替莫唑胺对荷瘤小鼠天0到4: <ol><li>计算要注射的小鼠的总数和由0.5乘以此确定需要（ 例如，30只小鼠X0.5毫升=15毫升TMZ）TMZ溶液的体积和加入1.5毫升该体积占溢出（16.5毫升TMZ ）。 </li><li> 3毫克乘以这个总量/毫升，以确定冻干TMZ所需要的重量（ 例如，16.5×3 = 49.5毫克TMZ）。称量到50毫升锥形此。 注意:TMZ是通过吸入，食入，并与眼睛，皮肤接触和黏膜毒性。咨询与减少暴露风险的建议，机构的职业安全和/或环境健康和保健部门。 </li><li>由0.15乘以总体积来确定的二甲基亚砜的体积（DMSO）中所需的（ 例如，16.5 X0.15 =2.475毫升DMSO）中。过滤消毒的DMSO此体积（加上额外的2 ml至占溢出）通过0.2微米的过滤器进入无菌管中。添加2.475毫升无菌DMSO中至TMZ粉末。 </li><li>放置TMZ / DMSO溶液中的水的烧杯中在热板上在65℃下10-15分钟。一旦TMZ完全溶解，该溶液应成为颜色浅黄色;如果溶液变成粉红色，则TMZ降低，和一个新的解决方案应该用新鲜很多TMZ的制备。 </li><li>计算的无菌盐水中所需的总体积（0.85×16.5毫升=1​​4.025毫升盐水）乘以0.85的适当体积。加入15毫升无菌盐水以50ml锥形。而TMZ被溶解在DMSO中，热盐水至65℃。 </li><li>涡流的TMZ / DMSO溶液，以确保该TMZ粉末完全溶解，并慢慢加入温热生理盐水到TMZ / DMSO溶液。 </li><li>配制后立即完全加载15×1毫升结核菌素syringES与施用TMZ解决4天建立荷瘤小鼠。 </li><li>通过重复4天1这个过程总共5 TMZ施用。 </li></ol>荷瘤小鼠的2全脑照射天2至4: <ol><li>功率上的X射线照射，并允许它预热到全电压。确保适当的过滤器是到位，并输入正确的电压和电流设置（ 图2A）。 注:该辐照装置的剂量应建立预先确定合适的电压设定和网格布局（ 图2A，B）。 </li><li>计算的时间是必要的，以导致5.5戈瑞X射线照射的长度。例如，如果X射线被递送以2格雷/分钟的速度，然后2.75分钟是必要提供一个总的5.5戈瑞。输入适当的持续时间。 注: 表2鼠标WBI一个剂量第二临床等效于人类。在高级别胶质瘤中的背景下，60戈瑞分馏WBI（2戈瑞馏分，5天/周，连续6周）是护理的临床标准，并且在这里所用的鼠等价物是16.5戈瑞（5.5戈瑞×3）。 </li><li>通过加入2ml氯胺酮和1ml甲苯噻嗪以17毫升盐水，使得最终溶液为10毫克/毫升氯胺酮和1mg / ml的甲苯噻嗪制备氯胺酮/赛拉嗪用于全身麻醉的新鲜溶液。 </li><li>称重小鼠并管理10微升氯胺酮/赛拉嗪的腹膜内每克体重，使得动物接受氯胺酮的剂量为100毫克/公斤和10毫克/千克甲苯噻嗪。小鼠应充分镇静，并在氯胺酮/甲苯​​噻嗪管理2分钟明显呼吸。 </li><li>保持镇静动物眼用水分，轻轻擦在每只眼睛的少量人工泪液软膏。 </li><li>放置镇静的小鼠上，使得磁头被定位在接收中的最高的X射线的区域的网格张力（ 图2B，C）。从颈部与适当向下屏障体尺寸铅管阻断全身的X射线递送（ 图2D）。 </li><li>放置位置的小鼠的X射线束，使得激光表示的X射线束的焦点是在坐标（0,0）上的网格布局下。开始透视交付。 </li><li>当透视交付完成后，取出在一个温暖的加热垫的辐射和地方的动物。不要用有意识的动物容纳镇静剂动物直到动物重新获得足够的意识，保持胸骨recumbence。一旦动物能保持胸骨recumbence，将有意识的动物，早在其相应的笼子里。 </li><li>重复上天3和4，此过程总共三个分次剂量的辐射。 </li></ol> 3.转染 -1天: <ol><li>通过加入50ml的胎牛血清（FBS）的500毫升的Dulbecco并购准备D10媒体odified Eagle培养基（DMEM）中。 </li><li>通过加入5.5毫升L-谷氨酰胺，5.5毫升丙酮酸钠，5.5毫升非必需氨基酸，和5.5毫升青霉素/链霉素（青霉素/链霉素）至500ml的RPMI-1640培养基制备的T细胞培养基（TCM）的。接着，添加550微升2-巯基乙醇和550微升庆大霉素。最后，添加50毫升FBS。 </li><li>收获在体外培养的HEK293T细胞，并把以7.5×10 6个细胞/ D10在媒体ml的浓度。 </li><li>板10毫升D10媒体和加入1毫升HEK293T细胞悬浮液中的每一个16×10cm的聚D-赖氨酸（PDL）包被的板。 </li><li>孵育过夜，在37℃，5％的CO 2。 </li></ol>第0天: <ol start="6"><li>转染细胞替换之前用10毫升新鲜的D10至少30分钟的媒体。 </li><li>确定载体质粒所需的转染的量，PCL-生态矢量，和脂质体转染试剂通过板的总数乘以+ 1（16 + 1 = 17）14.1微克载体质粒（14.1×17 = 239.7微克），9.9微克的pCL-生态载体（9.9×17 = 168.3微克），和60微升脂质体转染试剂（60×17 = 1020微升）。所有试剂应该在室温下制备溶液之前。 </li><li>标签两管A和B管A，增加239.7微克载体质粒和168.3微克PCL-生态向量（1.5×17）毫升降低血清改进的Eagle培养基（RS-MEM）。在管B，加入1020微升脂质体转染试剂（1.5×17）毫升RS-MEM。 </li><li>孵育A和B分别为5分钟，在室温下。 </li><li>混合A和B一起轻轻（涡旋1-2秒或颠倒数次）并孵育20分钟，在室温下，以形成脂质/ DNA复合物。 </li><li>添加脂质/ DNA复合物滴至HEK293T细胞，10 cm培养皿。 </li><li>孵育在37℃，6-8小时或过夜（不超过24小时）。 </li><li>后6-8小时温育后，用12ml的新鲜TCM进行病毒生产更换介质。这镀金媒体将用于在第2天的病毒上清用于T细胞转导。 </li></ol> 4.脾切除和T细胞的制备第0天: <ol><li>倾10毫升医药到50ml锥形并置于冰上脾集合。 </li><li>通过CO 2窒息和次级断头牺牲动物的适当数目:将动物关在笼子里接收的CO 2在10-30％的笼体积/分钟的流速，每美国兽医医学协会准则（不超过5个动物可以是同时牺牲），直到呼吸终止，并在此后两分钟。从CO 2室中移除动物和斩首。 注:6-12周龄雌性C57BL / 6小鼠会产生约4.5-5×10 7脾健脾之一。这里，4个脾脏将收获约200×10 6个细胞。 </li><li>莱使得它的右侧朝上的鼠标和喷用70％的乙醇。随着镊子，抢薄下方左侧胸腔皮肤褶皱切一的切口略微用剪刀。剥开皮，小心抢薄用钳子腹膜折，切一小腔。 </li><li>脾是一个小的，长形，暗红色的器官，类似于一个扁平豆;微妙抢与镊子和消费税由切掉周围的结缔组织脾脏。将切除脾脏到含有冰10毫升中医的锥形。 </li><li>倒脾脏超过70微米的网状细胞过滤并通过用5毫升注射器的内部的钝端糖化以产生单细胞悬浮液分解。最多两个脾脏应该每个滤网进行分类。 </li><li>使用小体积中医仔细清洗下分解来收集任何剩余的脾细胞的过滤器。池中的所有分类脾脏成单细胞悬液。把最终体积50毫升中医一洗了二维自旋，在300×g离心10分钟。 </li><li>通过加入5ml 10×裂解缓冲液至45毫升无菌水制备的1×裂解缓冲液中的溶液。为了消除红血细胞，重悬沉淀在5ml每脾1×裂解缓冲液，在50毫升的锥形，轻轻吹打上下充分混合。如果超过5脾，用250毫升离心管。放置锥形或离心管在37℃水浴中5分钟。 </li><li>除去从水浴裂解反应，并添加医药以1:1的比例用裂解缓冲液中以中和反应。通过在300 XG旋转10分钟洗净。 </li><li>吸去上清液并充分重悬沉淀在中医（2毫升/脾，8毫升总计4脾脏）通过上下抽吸。 </li><li>通过加入10微升细胞悬浮液以190微升台盼蓝（1:20稀释）计数细胞。乘以20×10 4×总体积（8毫升）中的4网格正方形1得到的数，以获得总的细胞数目（ 例如，125细胞×20×10 4个</s达&gt; X8毫升= 200×10 6脾细胞）。 </li><li>稀释细胞以2×10 6个细胞/ ml，在中医的浓度，补充有2微克/ ml的伴刀豆球蛋白A（ConA刺激）和50IU / ml重组人白介素-2（的rhIL-2）。因此，对于200×10 6个脾细胞中，添加92毫升媒体8毫升细胞，200微克的ConA，和5000 IU的rhIL-2。 </li><li>加入2毫升细胞的24孔组织培养处理板各孔中，以使得4×10 6个细胞每孔（ 例如100毫升的细胞将需要大约4板）。 </li><li>孵育过夜，在37℃，5％的CO 2。 </li></ol> 5.转导第1天: <ol><li> 。预算非组织培养的数处理所需的转导24孔板乘以脾脏2收获的次数（需要为4脾脏8片）。 </li><li>接着，计算出的重组人纤连蛋白片段（RHFF）溶液至所需体积乘以需要外套板（总数井+ 3）×0.5 ML（8板×24井= 192 + 3 = 195）×0.5毫升=97.5毫升。 </li><li>通过将总体积25微克（97.5×25 = 2437.5微克）乘以制备含有RHFF以25微克/ ml的浓度97.5毫升磷酸盐缓冲盐水（PBS）。加入这一数额RHFF的97.5毫升的PBS。 </li><li>涂层的非组织培养处理的24孔板中加入每孔0.5毫升PBS / RHFF溶液。 </li><li>孵育过夜，在4℃。 </li></ol>第2天: <ol start="6"><li>倾倒的PBS / RHFF溶液从非组织培养处理过的24孔板中。 </li><li>加入1毫升/孔的2％牛血清白蛋白（BSA）的PBS，并在室温下孵育30分钟。 </li><li>紧紧颠覆板拆下BSA。加入2毫升的PBS洗涤。 </li><li>通过将媒体从每个HEK293T PDL板放入250毫升离心管中收集的病毒上清和自旋10分钟，在500×g。 </li><li>仔细转让苏病毒pernatant成新鲜250毫升离心管中，作为确保不打扰可能形成细胞沉淀。 </li><li>添加新鲜TCM到病毒上清，使得最终体积比所需RHFF包被的孔的量​​3毫升更多。例如，对于192 RHFF包被的孔，使病毒上清的量，以192 +3毫升=195毫升。 </li><li>在每口井的3倍 - 轻轻吹打上下2从孵化器，并重新悬浮培养脾细胞。转移到250毫升的锥形。如果使用多道移液器，使用转移将有助于加快这一步骤之前无菌水库。算如前所述和旋转，在300×g离心10分钟。 </li><li>添加的rhIL-2病毒上清以50IU / ml的浓度。例如，添加50×195 = 9,750 IU的rhIL-2195毫升病毒上清液。 </li><li>重悬在病毒上清的脾细胞以1×10 6个细胞/ ml的</li><li>加入1毫升/孔的脾细胞悬浮液，以RHFF包覆的24孔板。 </li><li>根据以下设置自旋90分钟:770×g离心，加速度= 4，制动/减速= 0时，32℃。 </li><li>准备用50 IU / ml的白细胞介素-2中医的解决方案。例如，通过加入万IU的rhIL-2制备200中医溶液。离心后加入1 ml的rhIL-2 /中的每个孔中。 </li><li>培养过夜，在37℃在5％CO 2。 </li></ol> 6. CAR T细胞培养和收获天3和4: <ol><li>如果T细胞达到&gt; 80％汇合，细胞可能会被分割（这通常发生在第3天或4天）。分裂的细胞，轻轻吸管上下各孔中的2-3倍，并移动1毫升从每个孔到一个新的24孔组织培养处理板的新井。然后，用50IU / ml的IL-2加入1毫升新鲜TCM到每个孔，使得最终体积为2毫升所有孔中。 </li><li>如果细胞不达到&gt; 80％汇合，通过缓慢地从每个顶部吹打关闭1毫升介质以及执行半媒体变化。避免ðisturbing细胞定居在底部，同时消除媒体。加入1毫升含50国际单位/毫升的rhIL-2的新鲜TCM。 </li></ol>第5天: <ol start="3"><li>重悬CAR T细胞轻轻吹打向上并在每个下降3倍井，并转移到250毫升的离心管中。旋转细胞在300×g离心10分钟。 </li><li>完全吸出上清，而不会干扰颗粒。用PBS洗一次，计数细胞如前所述，和洗涤用PBS第二时间。一个典型的汽车的T细胞产率是每孔约1×10 6个细胞（8板×24孔= 192×10 6个CAR T细胞）。 </li><li>重悬的细胞在PBS中的5×10 7 / ml的用于注射1×10 7中的200微克的体积的浓度（ 例如，对于192×10 6个汽车的T细胞，重悬洗涤沉淀在3.84毫升PBS）中。 </li></ol> 7. CAR T细胞当局荷瘤小鼠第5天: <ol><li> TRANSPO室温细胞在冰上动物设施用于静脉注射。负载500-1000微升到胰岛素注射器27日 - 一个31 G针，以确保所有气泡被排出。 </li><li>抢鼠标尾部的基础，并将在管限制器。 </li><li>拉拉紧与静脉朝上的尾部，并且通过插入其平行于所述尾静脉滑行针在皮肤下大约1-2毫米到静脉。慢慢地排出200微升进入静脉。如果针正确地插入静脉，体积会容易流入;否则就会出现阻力和针将需要从尾部取出并正确地重新插入。 </li></ol>

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The authors have no conflicts of interests to declare.

这里所描述的治疗时间表旨在模拟护理临床标准，并充分利用其影响汽车继治疗。 CAR T细胞的剂量，TMZ治疗方案，和放射治疗施用可以被修改，以提高在体内的T细胞活性，淋巴细胞缺失，以及肿瘤杀伤。 TMZ治疗方案可以增加以得到主机myeloablation和过继转移的细胞30的膨胀增加。此外，TMZ的lymphodepletive效果可以通过低剂量被概括（4 - 6 Gy）进行单级分的全身照射（TBI），从而绕过SOC的杀肿瘤性质。这里采用的辐射方案模拟60戈瑞的生物等效剂量;然而，级分和分数剂量数目可以被调谐以模仿其他临床剂量（ 见表2）。重要的是，我们的SOC的模型利用生物等效剂量的WBI的，而不是外部束辐射灶性递送到肿瘤和马rgins，如常被用在临床上，并因此可能导致在较低剂量的辐射递送至肿瘤和毒性对健康小鼠脑的风险增加。尽管有这些限制，但是，WBI是一个公认的技术在临床前设置模拟临床放疗。 CAR T细胞剂量可以增加或减少，以观察其对肿瘤杀伤和总生存率11剂量效应。此外，处理和递送途径的时间能够被修改，以看见疗效差异。例如，汽车的T细胞可以被递送颅内，以确保活性在肿瘤部位。 CAR阳性T细胞的产率可以根据转导的效率而变化。确保成功转导的一种方法是通过进行一个绿色荧光蛋白（GFP） - 控制整个过程。要做到这一点，1 HEK293T10厘米PDL板可分配的GFP转染的T细胞的单井和转导到卵石n表示GFP在3-5天。在转导效率的一个关键因素是对T细胞活化和增殖在加入病毒上清的程度。我们注意到，我们的T细胞活化，这是通过加入刀豆素A的培养基中来实现方法的一个重要的差异，而用在临床上，通过CD3和CD28激动剂单克隆抗体来实现。虽然后者是常规使用的，用于产生病人的逆转录病毒转导的CAR T细胞和也已成功地在鼠的T细胞的转导，我们先前确定的ConA刺激导致更好和更一致的转导37。我们还观察到，总体CAR T细胞产量可基于当步骤5.18进行上会有所不同。如果遇到很差CAR T细胞的产量，我们建议的步骤5.17，而不是等待5-6小时培养后立即执行步骤5.18。尽管这可能会影响转导的效率，我们没有观察到显著DIFferences在我们的研究CAR表面表达。 以下转导，CAR的T细胞可以被量化并通过流式细胞仪表型;我们染色了EGFRvIII的汽车的T细胞与2色面板由一链霉抗藻红蛋白的（​​SA / PE）缀合biotynlated EGFRvIII的衍生多聚体和抗CD3。可替代地，中亚也可以染色，使用山羊抗人的F（ab&#39;）表达这一直2 -生物素初级和SA / PE二级抗体先前所述36。我们经常观察中小鼠脾细胞11％55-70 CAR的表达。之前我们已经表明，交付的T细胞与这款车表达谱可以很容易地交通大脑和治疗肿瘤。 TMZ-或TBI诱导淋巴细胞减少增强过继转移的细胞的克隆扩增，增加汽车阳性细胞的频率和它们的整体的抗肿瘤反应。 CAR T细胞可以通过染色的外周血与一个被跟踪体内ppropriate四聚体;这对于理解CAR T细胞的存活时间和主机调理对CAR T细胞功能和持久性的影响的有用的技术。替代地，如果没有四聚体是可用于抗原受体，荧光报道可以包含在CAR载体构建体，或CAR T细胞可以标记在体外用羧基琥珀酰亚胺酯（CFSE）给药之前用于体内跟踪。 根据不同的肿瘤模型中，免疫治疗平台，和SOC方案中，施用一起过继疗法或疫苗的临床的SOC可导致毒性。 TMZ是导致非特异性细胞杀伤的DNA烷基化剂，和加强剂量可导致体重减轻和发病率在小鼠宿主。高剂量和WBI延长分数可能会导致脑积水。另外，从强疫苗和/或过继转移的T细胞应答的抗炎作用可导致发病因有毒细胞因子风暴。同样重要的是要注意的全身麻醉对病态动物的影响。如果TMZ和WBI导致显著毒性，然后氯胺酮/赛拉嗪的剂量可降低20-25％，以减少死亡的危险，因为动物只需要镇静和固定10分钟用于递送的小的辐射剂量（2- 8戈瑞）。 主机空调是所有免疫的平台，包括过继转移的关键。由于免疫试验是SOC的范围内经常进行评估，免疫疗法的临床前评估应此设置中完成复述的临床情况。我们在座借力lymphodepletive和SOC的使用CAR T细胞的杀瘤效应与过继治疗的一个例子。该方案是，可以适用于其他免疫治疗的平台，以评估相结合的SOC的影响的有力工具。

胶质母细胞瘤（GBM）是最常见的原发性恶性脑肿瘤，并且总是致命的。手术切除加上护理化疗和放疗的非特异性标准未能完全消除恶性细胞，导致少于15个月的患者一预后不佳这种疾病1。与此相反，免疫治疗提供了一种精确的方法用于特异性靶向肿瘤细胞，并因此具有作为一个非常有效的治疗平台抵押品毒性2-4的风险降低的潜力。 T细胞工程体外表达嵌合抗原受体（车）提供了一个通用的策略肿瘤免疫治疗。通过融合的抗体的胞外可变区与一种或多种细胞内的T细胞信号传导分子（多个），以代替全长主要组织相容性复合体的生成的CARs体（MHC）-restricted T细胞受体5。的抗体样抗原recogniti此模式上允许反应活性的抗原特异性T细胞识别并在没有MHC的响应肿瘤抗原，并且可以适合于几乎无限抗原剧目。 设计对多种肿瘤抗原的CAR T细胞表现出临床前功效和卓越的承诺在门诊6-9。具体而言，在GBM的上下文中，一辆汽车的T细胞平台靶向表皮生长因子受体变体III（EGFRvIII的），肿瘤特异性突变在细胞表面上表达的10，被证明延长生存胶质瘤的小鼠11。尽管他们的通用性，然而，汽车过继疗法的临床效益尚未完全实现，在部分肿瘤相关的免疫抑制和免疫逃避12-16以及在建立和维持抗原特异性T细胞在体内的挑战所致。借力护理（SOC）的标准与免疫治疗可以潜在地克服这几条升仿制品，从而在两者的临床前和临床的设置增强的功效。 SOC进行后切除GBM由大剂量替莫唑胺（TMZ），一个DNA烷化剂17，和全脑照射（WBI）1。这些治疗方法都是通过推测肿瘤MHC表达18-20上调及抗原的死肿瘤细胞17,19,21,22脱落，以协同与肿瘤疫苗。实际上，在加入的TMZ 20,23或18,24的WBI导致在临床前设置免疫为主的治疗增强抗肿瘤功效。此外，像许多非特异性的细胞毒性化学治疗剂，TMZ是已知的导致全身性淋巴细胞25,26，其可以被利用作为宿主调节过继疗法平台27-29的一种手段。 TMZ介导的淋巴细胞缺失已显示增强抗原特异性T细胞的频率和功能，导致一个ADOP的增加的效力略去治疗平台对颅内肿瘤的30。在CAR疗法的上下文中，淋巴细胞缺失用作宿主调节的通过减少内源性抑制性T细胞31的数目，并通过减少竞争因子33诱导的稳态增殖32，从而提高抗肿瘤活性11,34的装置。定GBM SOC与免疫平台之间的协同关系，评价新颖代管疗法和疫苗平台在SOC的上下文是用于绘制关于功效有意义的结论是至关重要的。 在这个协议中，我们概述了鼠EGFRvIII的特异性CAR T细胞一起TMZ和WBI的生成和静脉内给药于小鼠的EGFRvIII阳性颅内肿瘤（参见图1进行治疗的时间表）的方法。简言之，将汽车的T细胞是由逆转录病毒转导所作体外 。人胚胎肾（HEK）293T细胞用DNA /脂质复合物（包含CAR载体和PCL-生态质粒），以产生病毒，然后将其用于转导被收获并在平行培养的活化鼠脾细胞进行转染。在汽车发生的过程中，轴承的EGFRvIII阳性颅内肿瘤的鼠宿主施用分馏全脑的X射线照射和全身TMZ治疗的剂量相当于临床SOC。 CAR的T细胞，然后静脉内递送至lymphodepleted主机。 下面的过程是在七个不同的阶段中描述:（1）管理的替莫唑胺对荷瘤小鼠，（2）全脑荷瘤小鼠的辐射;（3）转染，（4）脾切除和T细胞的制备，（5 ）转导，（6）CAR T细胞培养和收获，以及荷瘤小鼠（7）CAR T细胞管理。这些阶段包括跨越6-7天，并同时执行多个步骤。

<table><tbody><tr><td>pCL-Eco Retrovirus Packaging Vector</td><td>Imgenex</td><td>10045P</td><td>Helper vector for generating CAR retrovirus</td></tr><tr><td>Concanavalin A</td><td>Sigma Aldrich</td><td>C2010</td><td>Non-specific mitogen to induce T cell proliferation and viral transduction</td></tr><tr><td>Retronectin</td><td>ClonTech/Takara</td><td>T100B</td><td>Facilitates retroviral transduction of T cells</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668-019</td><td>Transfection reagent</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life technologies</td><td>11995-065</td><td>HEK293 culture media</td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875-093</td><td>T cell culture media</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Life technologies</td><td>11058-021</td><td>Transfection media</td></tr><tr><td>200 mM L-Glutamine</td><td>Life technologies</td><td>25030-081</td><td>T cell culture media supplement</td></tr><tr><td>100 mM Sodium Pyruvate</td><td>Life technologies</td><td>11360-070</td><td>T cell culture media supplement</td></tr><tr><td>100X MEM Non-Essential Amino Acids Solution</td><td>Life technologies</td><td>11140-050</td><td>T cell culture media supplement</td></tr><tr><td>55 mM 2-Mercaptoethanol</td><td>Life technologies</td><td>21985-023</td><td>Reducing agent to remove free radicals</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life technologies</td><td>15140-122</td><td>T cell culture media supplement</td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>Life technologies</td><td>15750-060</td><td>T cell culture media supplement</td></tr><tr><td>GemCell U.S. Origin Fetal Bovine Serum</td><td>Gemini Bio Products</td><td>100-500</td><td>Provides growth factors and nutrients for &lt;em&gt;in vitro&lt;/em&gt; cell growth</td></tr><tr><td>Bovine Serum Albumin (BSA), Fraction V&amp;ndash;Standard Grade</td><td>Gemini Bio Products</td><td>700-100P</td><td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td></tr><tr><td>Pharm Lyse (10x concentrate)</td><td>BD Biosciences</td><td>555899</td><td>Lyses red blood cells during splenocyte processing</td></tr><tr><td>70 &amp;mu;m Sterile Cell Strainers</td><td>Corning</td><td>352350</td><td>Filters away large tissue particles during splenocyte processing</td></tr><tr><td>100 mm BioCoat Culture Dishes with Poly-D-Lysine</td><td>Corning</td><td>356469</td><td>Promotes HEK293 cell adhesion to maximize proliferation after transfection</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Temozolomide</td><td>Best Pharmatech</td><td>N/A</td><td>Lyophilized powder prepared on the day of administration</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Life Sciences</td><td>D2650</td><td>Necessary for complete dissolution of temozolomide</td></tr><tr><td>Saline</td><td>Hospira</td><td>IM 0132 (5/04)</td><td>Solvent for temozolomide and ketamine/xylazine</td></tr><tr><td>Ketathesia HCl</td><td>Henry Schein Animal Health</td><td>11695-0701-1</td><td>Ketamine solution</td></tr><tr><td>AnaSed</td><td>Lloyd Inc</td><td>N/A</td><td>Xylazine sterile solution 100 mg/ml</td></tr></tbody></table>

generation of car t cells for adoptive therapy in the context of glioblastoma standard of care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

通过转导与EGFRvIII的CAR逆转录病毒载体11中产生充足的T细胞。该载体，MSGV1，从SFGtcLuc_ITE4向量35，其中包含了鼠干细胞病毒（MSCV）长末端重复序列，扩展的gag区和包络剪接位显影（剪接供体，SD和剪接受体，SA），以及病毒包装信号（ψ）。含有人抗EGFRvIII的单链可变片段（scFv）139，在串联与鼠CD8TM的EGFRvIII的CAR，CD28，4-1BB，和CD3ζ胞内区域，被克隆到反转录病毒载体下游的NcoI位点（ 图3） 。 以下转导，CAR的T细胞可以被量化并通过流式细胞术表型。 EGFRvIII的CAR的T细胞可以用2色面板由一链霉抗藻红蛋白的（​​SA / PE）被可视化共轭biotynlated EGFRvIII的衍生多聚体和抗CD3 FITC。用文化和传导普罗特ocols这里描述，我们经常观察中小鼠脾细胞（ 图4A）11 55-70％CAR的表达。可替代地，中亚也可以染色，使用山羊抗人的F（ab&#39;）表达这一直2 -生物素初级和SA / PE二级抗体先前所述36。 CAR的T细胞可通过加入其他的荧光标记的抗体，以两色轿厢面板进一步表型。例如，染色的CD8和CD4表明转导的CARs的70％是CD8 + T细胞，而20％是CD4 + T细胞（ 图4B）。 CAR T细胞与此表达谱已示出能够容易地通信到大脑和治疗颅内肿瘤。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td dir="LTR"> 体重 </td><td dir="LTR"> 音量 </td></tr><tr><td dir="LTR"> 25克</td><td dir="LTR"> 0.50毫升</td></tr><tr><td diR ="LTR"&gt;24克</td><td dir="LTR"> 0.48毫升</td></tr><tr><td dir="LTR"> 23克</td><td dir="LTR"> 0.46毫升</td></tr><tr><td dir="LTR"> 22克</td><td dir="LTR"> 0.44毫升</td></tr><tr><td dir="LTR"> 21克</td><td dir="LTR"> 0.42毫升</td></tr><tr><td dir="LTR"> 20克</td><td dir="LTR"> 0.40毫升</td></tr><tr><td dir="LTR"> 19克</td><td dir="LTR"> 0.38毫升</td></tr><tr><td dir="LTR"> 18克</td><td dir="LTR"> 0.36毫升</td></tr><tr><td dir="LTR"> 17克</td><td dir="LTR"> 0.34毫升</td></tr><tr><td dir="LTR"> 16克</td><td dir="LTR"> 0.32毫升</td></tr><tr><td dir="LTR"> 15克</td><td dir="LTR"> 0.30毫升</td></tr><tr><td dir="LTR"> 14克</td><td dir="LTR"> 0.28毫升</td></tr><tr><td dir="LTR"> 13克</td><td dir="LTR"> 0.26毫升</td></tr><tr><td dir="LTR"> 12克</td><td dir="LTR"> 0.24毫升</td></tr><tr><td dir="LTR"> 11克</td><td dir="LTR"> 0.22毫升</td></tr></tbody&gt; </table> 表1:基于动物重量替莫唑胺剂量替莫唑胺剂量基础上计算的60毫克/公斤的总剂量。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td dir="LTR"> ð </td><td dir="LTR"> ð </td><td dir="LTR"> ＃分数 </td><td dir="LTR"> BED </td><td dir="LTR"> SOC </td></tr><tr><td dir="LTR"> 16.5 </td><td dir="LTR"> 5.5 </td><td dir="LTR"> 3 </td><td dir="LTR"> 62 </td><td dir="LTR"> GBM </td></tr><tr><td dir="LTR"> 14 </td><td dir="LTR"> 7 </td><td dir="LTR"> 2 </td><td dir="LTR"> 63 </td><td dir="LTR"> GBM </td></tr><tr><td dir="LTR"> 20 </td><td dir="LTR"> 4 </td><td dir="LTR">五</td><td dir="LTR"> 60 </td><td dir="LTR"> GBM </td></tr><tr><td dir="LTR"> 12 </td><td dir="LTR"> 6 </td><td dir="LTR"> 2 </td><td dir="LTR"> 48 </tD&gt; <td dir="LTR"> LGA </td></tr><tr><td dir="LTR"> 16 </td><td dir="LTR"> 4 </td><td dir="LTR"> 4 </td><td dir="LTR"> 48 </td><td dir="LTR"> LGA </td></tr><tr><td dir="LTR"> 21 </td><td dir="LTR"> 3 </td><td dir="LTR"> 7 </td><td dir="LTR"> 52.5 </td><td dir="LTR"> LGA </td></tr><tr><td dir="LTR"> 12 </td><td dir="LTR"> 3 </td><td dir="LTR"> 4 </td><td dir="LTR">三十</td><td dir="LTR">会见</td></tr><tr><td dir="LTR"> 16 </td><td dir="LTR"> 2 </td><td dir="LTR"> 8 </td><td dir="LTR"> 32 </td><td dir="LTR">会见</td></tr></tbody></table> 表2:临床相关的生物等效的辐射剂量的辐射剂量根据床计算值= D [1+ D /（α/β）]，其中D =总剂量，D =分馏剂量，α/β= 2）。的总剂量施用WBI与鼠宿主示于第计递送Ë分数剂量以及由那些剂量馏分建模的人用剂量。对于模型的目的，也显示了这床是标准治疗的恶性肿瘤。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52397/52397fig1highres.jpg" width="600" /> 图1:时间线治疗荷瘤小鼠和护理化疗并发体外 CAR代标准和全脑照射肿瘤植入（A）后第4天开始。在这段时间间隔，产生CAR T细胞和体外培养（B）和主机调理的全部课程后，交付使用。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52397/52397fig2highres.jpg" width="600" /> 图2:布局和设置的辐射传递。X射线是根据320千伏和10毫安剂量递送，以根据所期望的剂量（A）的选择的持续时间变化。的X射线辐射的焦点字段是一个窄2.5厘米区域。镇静动物根据电网（B），使得他们的头在于最高的X射线强度的区域设置;（C）表示从X射线束的一端的视图。大约有4只动物，可根据该网格布局（B，D）是辐射下，在透视政府的一门课程。铅管是用来屏蔽体，留下暴露WBI（D）只有他们的头。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/52397/52397fig3highres.jpg" width="600" /> 图3:改性SFGtcLuc_ITE4逆转录病毒载体，MSGV1，用于转导CAR T细胞和产生的。含有人抗EGFRvIII的单链可变片段（scFv）139，在串联与鼠CD8TM，CD28，4-1BB，和CD3ζ胞内区域EGFRvIII的CAR插入，通过鼠干细胞病毒（MSCV）长末端重复序列侧翼序列（LTR ）。上游CAR刀片的是扩展gag区和包络剪接位点（剪接供体，SD和剪接受体，SA），和病毒包装信号（ψ）。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52397/52397fig4highres.jpg" width="600" /> 图4:EGFRvIII的汽车是表达的T细胞的表面上 48小时以下转导，T细胞进行染色用于使用LEEKKGNYVVTDHC-K（生物素）-NH 2 /链霉亲和PE组成的多聚体的EGFRvIII的CARs的表面表达。来自同一供体未转导的T细胞被染色也为阴性对照。数据表明CAR的T细胞（y-轴）阳性:STA的数量进不去由PE缀合的EGFRvIII的多聚体（x轴），门上的CD3 +细胞为T细胞标记物，其中，表达被发现是60.9％（A）中 。染色的CD4-PerCP-经Cy5.5和CD8-APC CAR T细胞表现出21.7％和70.9％，分别为（B）的表达谱。

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

21.3K 次观看.  Duke University. The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care. 

视频: Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

同源小鼠 B 细胞淋巴瘤模型对 CD19 轿车 T 细胞的临床前评价

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

科研

癌症研究

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

颅内插管植入小鼠连续局部区域嵌合抗原受体（CAR）T细胞输注

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

医学

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

免疫与感染

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

符合GMP的基因修饰T细胞生成程序

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

生物工程

在同基因模型中生成小鼠 CAR-T 细胞的简化方案

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

作者聚焦:增强同基因肿瘤模型中的 CAR-T 细胞功能 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

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此视频中的章节

同源小鼠 B 细胞淋巴瘤模型对 CD19 轿车 T 细胞的临床前评价

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

颅内插管植入小鼠连续局部区域嵌合抗原受体（CAR）T细胞输注

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

符合GMP的基因修饰T细胞生成程序

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

小鼠嵌合抗原受体（CAR）-T细胞的高效生成