Name: manufacturing chimeric antigen receptor (car) t cells for adoptive immunotherapy
Uploaded: 2019-12-17T12:00:00
Description: Watch this Scientific Journal Video about Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy at JoVE.com

这项工作部分得到了诺华制药公司通过与宾夕法尼亚大学（迈克尔·米隆）的研究联盟以及圣巴尔德里克基金会学者奖（萨巴·加塞米）提供的资金的支持。

所有动物研究都得到宾夕法尼亚大学动物护理和使用委员会的批准。
1. T细胞激活、转导和扩张
<ol><li>与抗CD3/CD28磁珠（例如，dynabet）混合，在6孔细胞培养皿中，以每T细胞3个珠的比例激活新鲜或冷冻保存的初级人类T细胞。X-VIVO 15培养基中的培养T细胞辅以5%正常人AB血清、2mM L-谷氨酰胺、20 mM HEPES和IL2（100单位/mL）。在扩张过程中将T细胞的浓度保持在106T细胞/mL。培?...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+for+acute+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.</a> The New England Journal of Medicine. 368 (16), 1509-1518 (2013).</li><li>Kalos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cells+with+Chimeric+Antigen+Receptors+Have+Potent+Antitumor+Effects+and+Can+Establish+Memory+in+Patients+with+Advanced+Leukemia.">T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia.</a> Science Translational Medicine. 3 (95), 95ra73 (2011).</li><li>Kochenderfer, J. N., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+B-cell+cancer+with+T+cells+expressing+anti-CD19+chimeric+antigen+receptors.">Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.</a> Nature Reviews. Clinical Oncology. 10 (5), 267-276 (2013).</li><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+for+sustained+remissions+in+leukemia.">Chimeric antigen receptor T cells for sustained remissions in leukemia.</a> The New England Journal of Medicine. 371 (16), 1507-1517 (2014).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+in+chronic+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.</a> The New England Journal of Medicine. 365 (8), 725-733 (2011).</li><li>Porter, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+persist+and+induce+sustained+remissions+in+relapsed+refractory+chronic+lymphocytic+leukemia.">Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.</a> Science Translational Medicine. 7 (303), 303ra139 (2015).</li><li>Maude, S. L., Teachey, D. T., Porter, D. L., Grupp, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+chimeric+antigen+receptor+T-cell+therapy+for+acute+lymphoblastic+leukemia.">CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.</a> Blood. 125 (26), 4017-4023 (2015).</li><li>Kochenderfer, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoma+Remissions+Caused+by+Anti-CD19+Chimeric+Antigen+Receptor+T+Cells+Are+Associated+With+High+Serum+Interleukin-15+Levels.">Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels.</a> Journal of Clinical Oncology. 35 (16), 1803-1813 (2017).</li><li>Bollard, C. M., Rooney, C. M., Heslop, H. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptively+transferred+effector+cells+derived+from+naive+rather+than+central+memory+CD8++T+cells+mediate+superior+antitumor+immunity.">Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (41), 17469-17474 (2009).</li><li>Klebanoff, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+memory+self/tumor-reactive+CD8++T+cells+confer+superior+antitumor+immunity+compared+with+effector+memory+T+cells.">Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (27), 9571-9576 (2005).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+human+central+memory-derived+effector+CD8++T+cells+in+immunodeficient+mice.">Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice.</a> Blood. 117 (6), 1888-1898 (2011).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+naive+and+central+memory+derived+CD8++effector+cell+engraftment+fitness+and+function+following+adoptive+transfer.">Comparison of naive and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer.</a> Oncoimmunology. 5 (1), e1072671 (2016).</li><li>Fraietta, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+response+and+resistance+to+CD19+chimeric+antigen+receptor+(CAR)+T+cell+therapy+of+chronic+lymphocytic+leukemia.">Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.</a> Nature Medicine. 24 (5), 563-571 (2018).</li><li>Ghassemi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Ex+Vivo+Culture+Improves+the+Antileukemic+Activity+of+Chimeric+Antigen+Receptor+(CAR)+T+Cells.">Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells.</a> Cancer Immunology Research. 6 (9), 1100-1109 (2018).</li><li>Milone, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+receptors+containing+CD137+signal+transduction+domains+mediate+enhanced+survival+of+T+cells+and+increased+antileukemic+efficacy+in+vivo.">Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 17 (8), 1453-1464 (2009).</li><li>Cieri, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+and+IL-15+instruct+the+generation+of+human+memory+stem+T+cells+from+naive+precursors.">IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.</a> Blood. 121 (4), 573-584 (2013).</li><li>Cui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7-Induced+Glycerol+Transport+and+TAG+Synthesis+Promotes+Memory+CD8+T+Cell+Longevity.">IL-7-Induced Glycerol Transport and TAG Synthesis Promotes Memory CD8 T Cell Longevity.</a> Cell. 161 (4), 750-761 (2015).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closely+related+T-memory+stem+cells+correlate+with+in+vivo+expansion+of+CAR.CD19-T+cells+and+are+preserved+by+IL-7+and+IL-15.">Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15.</a> Blood. 123 (24), 3750-3759 (2014).</li><li>Singh, N., Perazzelli, J., Grupp, S. A., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+memory+phenotypes+drive+T+cell+proliferation+in+patients+with+pediatric+malignancies.">Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies.</a> Science Translational Medicine. 8 (320), 320ra323 (2016).</li></ol>

在这里，我们描述了测量CAR T细胞的功能和功效的方法，这些细胞在整个外体培养过程中以不同的间隔收获。我们的方法提供全面的检测分析，旨在评估增殖能力以及体外效应器功能。我们描述了如何通过CAR刺激测量CAR T细胞活性，并使用在对数扩张阶段的第3天和第9天收获的CAR T细胞进行详细白血病异种移植模型。
在比较在外体扩张期间在不同时间点采集的CAR T细胞的功效存?...

奇美抗原受体（CARs）提供了一种有希望的方法，可以针对不同的肿瘤抗原重定向T细胞。CARs 是结合抗原靶点的合成受体。虽然其精确组成是可变的，但 CA 通常包含 3 个不同的域。细胞外域直接结合到靶抗原，通常由通过细胞外铰链与CAR相连的单链抗体片段组成。第二个域，通常来自T细胞受体（TCR）复合物的CD3*链，促进CAR参与后的T细胞激活。包括第三个成本领域，以增强T细胞功能，移植，代谢和持久性。CAR T细胞治疗在各种造血恶性肿瘤中的成功，包括B细胞急性淋巴细胞白血病（ALL）、慢性淋巴细胞白血病（CLL）和多发性骨髓瘤，凸显了这种方法1、2、3、4、5、6的治疗前景。最近美国食品和药物管理局 （FDA） 批准两种 CD19 特异性 CAR T 细胞疗法，针对儿科和年轻成人的 tisagenlecleucel 和用于扩散大 B 细胞淋巴瘤的 axicabagene 胆珠，强化了 CAR T 细胞治疗的转化价值。
基于CAR T的方法包括从外周血分离T细胞、活化、基因改造和外体扩张。分化是调节CAR T细胞疗效的重要参数。因此，在外体培养过程中限制T细胞分化可增强注入产物的移植、扩张和坚持能力，在采用转移2、7、8、9之后提供长期免疫监测。T细胞由几个不同的子集组成，包括：幼稚的T细胞（Tn）、中央记忆（Tcm）、效应器记忆（Tem）、效应器分化（T）和干细胞记忆（Tscm）。效应者分化T细胞具有强效细胞解细胞能力;然而，他们是短暂的和移植差10，11，12。相反，具有较少分化表型的T细胞，包括幼稚的T细胞和Tcm在采用细胞转移13、14、15、16、17、18之后表现出优异的增生和增殖能力。预制造产品中收集的T细胞的组成可能因患者而异，并与CAR T细胞的治疗潜力相关。在起始阿普西斯产品中，具有天真型免疫表型的T细胞比例与移植和临床反应高度相关19。
培养持续时间是影响CAR T细胞分化的重要参数，为采用转移做准备。我们最近开发了一种方法，使用缩写培养范式20生成高质量的CAR T细胞。利用我们的方法，我们表明，有限的培养导致CAR T细胞具有优越的效应功能和持久性后，在白血病的异种移植模型的收养转移。在这里，我们介绍可靠生成CART19细胞的方法（用于表达CD33和4-1BB信令域的抗CD19 scFv的自体T细胞），并包括分析的详细说明，在采用转移之前提供对CAR T生物活性和有效性的洞察。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti CD3/CD28 dynabeads</td>
			<td>Thermo Fisher</td>
			<td>40203D</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD8</td>
			<td>BD Biosciences</td>
			<td>555369</td>
			<td>RRID:AB_398595</td>
		</tr>
		<tr>
			<td>APC-H7 Mouse anti-Human CD8 Antibody</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>RRID:AB_1645481</td>
		</tr>
		<tr>
			<td>BD FACS Lysing Solution 10X Concentrate</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Trucount Absolute Counting Tubes</td>
			<td>BD Biosciences</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-human CD4 Antibody</td>
			<td>BioLegend</td>
			<td>317444</td>
			<td>RRID:AB_2561866</td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-human CD3 Antibody</td>
			<td>BioLegend</td>
			<td>317322</td>
			<td>RRID:AB_2561911</td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>Life Technolohgies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads,</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-Human CD197 (CCR7) Antibody</td>
			<td>BD Pharmingen</td>
			<td>561271</td>
			<td>RRID:AB_10561679</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD4</td>
			<td>BD Biosciences</td>
			<td>555346</td>
			<td>RRID:AB_395751</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human AB serum</td>
			<td>Valley Biomedical</td>
			<td>HP1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2 IS, premium grade</td>
			<td>Miltenyi</td>
			<td>130-097-744</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>28030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintillation counter, MicroBeta trilux</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Violet</td>
			<td>Molecular Probes</td>
			<td>L34964</td>
			<td></td>
		</tr>
		<tr>
			<td>Multisizer Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na251CrO4</td>
			<td>Perkin Elmer</td>
			<td>NEZ030S001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue anti-human CD14 Antibody</td>
			<td>BioLegend</td>
			<td>325616</td>
			<td>RRID:AB_830689</td>
		</tr>
		<tr>
			<td>Pacific Blue anti-human CD19 Antibody</td>
			<td>BioLegend</td>
			<td>302223</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD45RO Antibody</td>
			<td>BD Biosciences</td>
			<td>555493</td>
			<td>RRID:AB_395884</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-human CD95 (Fas) Antibody</td>
			<td>BioLegend</td>
			<td>305610</td>
			<td>RRID:AB_493652</td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-human CD27 Antibody</td>
			<td>Beckman Coulter</td>
			<td>A54823</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red-free medium</td>
			<td>Gibco</td>
			<td>10373-017</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure SDS Solution, 10%</td>
			<td>Invitrogen</td>
			<td>15553027</td>
			<td></td>
		</tr>
		<tr>
			<td>Via-Probe</td>
			<td>BD Biosciences</td>
			<td>555815</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Gibco</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin - K+ Salt</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td></td>
		</tr>
	</tbody>
</table>

manufacturing chimeric antigen receptor (car) t cells for adoptive immunotherapy

采用免疫疗法对治疗癌症和传染病有希望。我们描述了一种用嵌合抗原受体（CAR）转导原发人类T细胞并扩展其后代前体的简单方法。我们包括检测，以测量CAR表达以及分化，增殖能力和细胞解细胞活性。我们描述检测，以测量在CAR T细胞中产生效应细胞因子和炎症细胞因子分泌物。该方法为采用免疫治疗的临床前模型培养CAR T细胞提供了可靠、全面的方法。

使用上述方法，我们刺激和扩展T细胞3或9天（图1A，B）。我们还通过测量细胞表面表达的独特糖蛋白的丰度，分析了它们的分化特征，如图1C中概述的浇注策略所示。在外体培养过程中，我们显示出一个逐渐向效应器分化的转变（图1D）。我们评估了CAR T细胞对抗原的反应作用和增殖能力。我?...

Watch this Scientific Journal Video about Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy at JoVE.com

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

我们描述了一种可靠地生成嵌合抗原受体（CAR）T细胞的方法，并测试其体外和体内的分化和功能。

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 ...

脱利-at-at功能能力，在输液后，具有促进、增殖和cit-el-uric活性，是有效采用免疫疗法的关键组成部分。例如，在实体肿瘤中，CAR T细胞可以穿透实体肿瘤，并接受最佳的抗原刺激;然而，它们的整体感染和增殖在人体内受到阻碍。这种方法的主要好处是，它可以保留 CAR T 细胞的质量或 CAR T 细胞的分化状态，而无需产生最终分化或耗尽的 T 细胞，这将降低增殖能力或输液后的有效功能差。这种技术可能很困难，因为T细胞对细胞浓度或培养的血管的表面面积非常敏感。以及他们的代谢需求。从本质上讲，T细胞需要在整个过程中保持快乐，否则它们不会生长。要开始这个程序，请设置6个井文化菜肴。通过将新鲜或低温保存的原人类T细胞与抗CD3 CD28磁珠混合，在培养皿的井中，每T细胞三个珠子的比例激活它们。培养X-Vivo 15培养的T细胞，辅以正常人AB血清、L-谷氨酰胺、血尿和IL2。在膨胀过程中，以每毫升一百万T细胞的浓度激活T细胞。在37摄氏度下培养它们，用20%的氧气、5%的二氧化碳和95%的湿度来培养它们。经过一夜的刺激，在激活的T细胞中加入扁病毒超自然。计算达到三到五之间多重感染所需的超级钠的体积。使用相同的条件继续培养细胞。第三天，收集低温保存细胞的代表性等分。在低温保存之前，通过轻轻移液和磁分离去除磁珠。准备含有 PBS 的冻结介质，其 0.5% DMSO。并储存在四摄氏度，直到准备使用。接下来，将T细胞在300倍G下离心5分钟。丢弃上一杯，加入五毫升的PBS。以 300 次 G 再次离心细胞五分钟，然后丢弃 PBS。我们把小气暂停在一毫升的冷低温保鲜介质中。将T细胞冷冻在冷冻容器中。并在零下80摄氏度下储存48小时。在此之后，将冷冻细胞转移到液氮中。在五毫升PBS中清洗其余T细胞一次，以消除任何残留载体。在300次G下离心5分钟。然后，以每毫升50万细胞的浓度，将PBS和T细胞培养中细胞的佩利暂停。将 T 细胞分成两个培养物，设计为 45 和 9。使用计数珠子和对人 CD4 和 CD8 的单克隆抗体，通过流式细胞法计算 T 细胞。以及一种活力染料。每隔一天重新喂养培养物，以保持每毫升50万个细胞的浓度。第五天，计数和冷冻保存第五天文化，如前面描述。第七天，用PBS清洗50万个细胞。并在100微升荧光激活细胞分选缓冲液中重新挂起它们。然后，通过免疫染色检测 CAR 表面蛋白表达，通过流式细胞学进行荧光结合抗 CAR 19 白痴型检测 CAR 表面蛋白表达。第九天计数和冷冻保存第九天文化如前所述。在这项研究中，测量了在整个X-Vivo培养文化中以不同间隔采集的 CAR T细胞的功能和功效。使用本视频中描述的方法，T细胞被刺激和扩大三到九天。此处所示的浇注策略所示的细胞分化轮廓通过测量细胞表面表达的明显糖蛋白的丰度进行分析。在 X-Vivo 培养过程中，逐渐向效应器分化转变。然后评估 CAR T细胞对抗原反应的效应函数和增殖能力。扩展较少的细胞在功能上优于在较长持续时间内广泛培养的细胞。在人类异种移植小鼠模型中，比较了不同时间段收获的 CAR T 细胞的所有效力。九天细胞表现出一种依赖剂量的抗卢克反应，完全反应值300万;低剂量50万的疗效丧失。然而，当天三个细胞显示持续肿瘤控制在高和低剂量。此反应与小鼠外周血液中 CAR-T 19 的绝对计数相关。总体而言，这些结果提供了证据，证明 CAR T 细胞的收获更早，优于后来收获的细胞。按照这个程序，可以进行额外的功能检测，如海马测定，以测量其代谢特性，如氧气消耗或螺旋能力。也可以进行几轮再刺激，以深入了解其分化和 CAR T 细胞的持久性。以这项工作为基础，我们和其他研究人员正在从事研究，以进一步缩写文化过程。它也可以扩展到其他癌症抗原的核心方法设计中。使用扁病毒时，必须始终小心。但是，即使扁病毒被设计为复制无能。

Research

JoVE Journal

Cancer Research

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

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

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

A Spheroid Killing Assay by CAR T Cells

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

用于向癌细胞输送 sirna 的外质体的制备

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

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

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 ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

使用 CRISPR/Cas9 在 CAR-T 电池中敲除 GM-CSF

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from SÃƒÆ’Ã‚Â©zary Syndrome Patients for Transcriptomic Profiling

从Sézary综合征患者中分离人外周血单核细胞和CD4 + T细胞以进行转录组学分析

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

使用[18F]四氟硼酸盐正电子发射断层扫描/计算机断层扫描对嵌合抗原受体T细胞进行动态成像

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

使用急性淋巴细胞白血病患者来源的异种移植小鼠模型评估嵌合抗原受体T细胞相关毒性

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...