Name: generation of car t cells for adoptive therapy in the context of glioblastoma standard of care
Uploaded: 2015-02-16T14:00:00
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?...

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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SO...

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。

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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Name of Material</td>
			<td style="width:172px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:449px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCL-Eco Retrovirus Packaging Vector</td>
			<td>Imgenex</td>
			<td>10045P</td>
			<td>Helper vector for generating CAR retrovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Retronectin</td>
			<td>ClonTech/Takara</td>
			<td>T100B</td>
			<td>Facilitates retroviral transduction of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>HEK293 culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Life technologies</td>
			<td>11058-021</td>
			<td>Transfection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM L-Glutamine&#160;</td>
			<td>Life technologies</td>
			<td>25030-081</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Sodium Pyruvate</td>
			<td>Life technologies</td>
			<td>11360-070</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Life technologies</td>
			<td>11140-050</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">55 mM 2-Mercaptoethanol&#160;</td>
			<td>Life technologies</td>
			<td>21985-023</td>
			<td>Reducing agent to remove free radicals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin (50 mg/mL)&#160;</td>
			<td>Life technologies</td>
			<td>15750-060</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GemCell U.S. Origin Fetal Bovine Serum</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Provides growth factors and nutrients for in vitro cell growth&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA), Fraction V&#8212;Standard&#160;Grade&#160;</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pharm Lyse (10X concentrate)&#160;</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm BioCoat Culture Dishes with Poly-D-Lysine&#160;</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temozolomide</td>
			<td>Best Pharmatech</td>
			<td>N/A</td>
			<td>Lyophilized powder prepared on the day of administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL&#160;</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（...

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 ...

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...