Name: tracking bispecific antibody-induced t cell trafficking using luciferase-transduced human t cells
Uploaded: 2023-05-12T12:05:00
Description: Watch this Scientific Journal Video about Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells at JoVE.com

作者要感谢Vladimir Ponomarev博士分享本文代表性结果部分描述的实验中使用的荧光素酶构建体。

以下程序已由纪念斯隆凯特琳的机构动物护理和使用委员会评估和批准。
1. 用荧光素酶转染293T细胞并收获病毒上清液
<ol><li>293T细胞培养<ol><li>通过将以下内容添加到一升DMEM中来制备培养基：110 mL热灭活胎牛血清（FBS），11 mL青霉素链霉素。</li>
		<li>解冻5 x 106 293T细胞，并将其转移到T175烧瓶中，培养基如上所述制备。</li>
		<li>每 10 天以 1：10 的比例拆分...

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD2+or+HER2+targeting+T+cell+engaging+bispecific+antibodies+to+treat+osteosarcoma.">GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma.</a> Journal of Hematology &amp; Oncology. 13 (1), 172 (2020).</li><li>Wu, Z., Guo, H. F., Xu, H., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+tetravalent+anti-GPA33/anti-CD3+bispecific+antibody+for+colorectal+cancers.">Development of a tetravalent anti-GPA33/anti-CD3 bispecific antibody for colorectal cancers.</a> Molecular Cancer Therapeutics. 17 (10), 2164-2175 (2018).</li><li>Lin, T. Y., Park, J. A., Long, A., Guo, H. F., Cheung, N. 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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=T+cell+engaging+bispecific+antibodies+targeting+CD33+IgV+and+IgC+domains+for+the+treatment+of+acute+myeloid+leukemia.">T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia.</a> Journal for Immunotherapy of Cancer. 9 (5), e002509 (2021).</li><li>Li, H., Er Saw, P., Song, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+strategies+for+next-generation+bispecific+antibody-based+antitumor+therapeutics.">Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics.</a> Cellular and Molecular Immunology. 17 (5), 451-461 (2020).</li><li>Rajabzadeh, A., Hamidieh, A. A., Rahbarizadeh, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinoculation+and+retronectin+highly+enhance+the+gene+transduction+efficiency+of+Mucin-1-specific+chimeric+antigen+receptor+(CAR)+in+human+primary+T+cells.">Spinoculation and retronectin highly enhance the gene transduction efficiency of Mucin-1-specific chimeric antigen receptor (CAR) in human primary T cells.</a> BMC Molecular and Cell Biology. 22 (1), 57 (2021).</li><li>Kleeman, B., 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=A+guide+to+choosing+fluorescent+protein+combinations+for+flow+cytometric+analysis+based+on+spectral+overlap.">A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.</a> Cytometry Part A. 93 (5), 556-562 (2018).</li><li>Park, J. A., Santich, B. H., Xu, H., Lum, L. G., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+ex+vivo+armed+T+cells+using+recombinant+bispecific+antibodies+for+adoptive+immunotherapy+with+reduced+cytokine+release.">Potent ex vivo armed T cells using recombinant bispecific antibodies for adoptive immunotherapy with reduced cytokine release.</a> Journal for Immunotherapy of Cancer. 9 (5), e002222 (2021).</li><li>Park, J. A., Wang, L., Cheung, N. 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虽然T-BsAb blinatumomab已被批准用于CD19阳性血液系统恶性肿瘤，但T-BsAbs在实体瘤中的成功实施已被证明要困难得多。Catumaxomab是一种针对上皮细胞粘附分子（EPCAM）的T-BsAb，被批准用于治疗卵巢癌患者的恶性腹水，但该药物的生产随后因商业原因而停止19。没有其他T-BsAb被批准用于实体瘤，这强调了与这种治疗相关的挑战。细胞因子释放综合征（CRS）引起的毒性是一个常见问题，尽?...

T 细胞结合双特异性抗体 （T-BsAb） 是一种工程抗体，用于通过一个结合臂结合 T 细胞和通过另一个结合臂结合肿瘤抗原，为多克隆 T 细胞提供人工特异性。该技术已成功应用于血液系统癌症（靶向CD19的blinatumomab1），并且许多T-BsAbs也处于各种实体瘤的临床前和临床开发中2。T-BsAbs以主要组织相容性复合体（MHC）非依赖性方式接合多克隆T细胞，因此即使是下调人白细胞抗原（HLA）的肿瘤也容易受到这种类型的治疗3，4。T-BsAbs已经以数十种不同的形式开发，在T细胞和肿瘤结合臂的化合价和空间排列，域间距离以及包含Fc结构域方面存在差异，这会影响半衰期并且可以诱导效应功能（如果存在5）。我们实验室先前的工作表明，这些因素显着影响T-BsAbs的抗肿瘤功效，效力差异高达1，000倍6。通过这项工作，我们将IgG-[L]-scFv格式确定为T-BsAb的理想平台（有关T-BsAb格式的更多详细信息，请参阅代表性结果部分），并将该平台应用于包括GD2（神经母细胞瘤），HER2（乳腺癌和骨肉瘤），GPA33（结直肠癌），STEAP1（尤因肉瘤），CD19（B细胞恶性肿瘤）和CD33（B细胞恶性肿瘤）在内的靶点7，8，9，10，11，12，13.
在实体瘤中成功实施T-BsAb疗法的主要挑战之一是克服免疫抑制肿瘤微环境（TME）以驱动T细胞向肿瘤的运输14。上述影响T-BsAb功效的因素对T-BsAb有效诱导T细胞归巢到肿瘤的能力有显著影响，但这种影响很难在 体内 系统中实时评估。本手稿详细介绍了在T-BsAbs临床前研究中使用荧光素酶转导的T细胞来评估治疗期间实验免疫功能低下小鼠模型中T细胞转运到各种组织的应用。该方法的总体目标是提供一种评估肿瘤和其他组织中T细胞浸润的方法，以及实时洞察T细胞归巢动力学和持久性，而无需在治疗期间牺牲动物。对于越来越多的专注于细胞免疫疗法的研究人员来说，在临床前动物 模型中追踪体内 T细胞的能力至关重要。我们的目标是提供我们用于跟踪荧光素酶转导T细胞的方法的彻底，详细的描述，以使其他研究人员能够轻松复制该技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3/CD28 beads</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11161D</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Goldbio</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA in vitro transfection reagent (polyjet)</td>
			<td>SignaGen Laboratories</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco (ThermoFisher)</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gag/pol plasmid</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP plasmid</td>
			<td>Addgene</td>
			<td>11150-DNA.cg</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Retronectin</td>
			<td>Takara</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G plasmid</td>
			<td>Addgene</td>
			<td>8454</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking bispecific antibody-induced t cell trafficking using luciferase-transduced human t cells

T细胞结合双特异性抗体（T-BsAbs）处于实体瘤临床前开发和临床试验的不同阶段。化合价、空间排列、域间距离和Fc突变等因素影响这些疗法的抗肿瘤疗效，通常是通过影响T细胞归巢到肿瘤，这仍然是一个主要挑战。在这里，我们描述了一种用荧光素酶转导活化的人T细胞的方法，允许在T-BsAb治疗研究期间对T细胞进行 体内 跟踪。T-BsAbs将T细胞重定向到肿瘤的能力可以在治疗期间的多个时间点进行定量评估，使研究人员能够将T-BsAbs和其他干预措施的抗肿瘤功效与T细胞在肿瘤中的持久性相关联。该方法减轻了在治疗期间牺牲动物以组织学评估T细胞浸润的需要，并且可以在多个时间点重复以确定治疗期间和治疗后T细胞运输的动力学。

如步骤4.3所述，小鼠可以在成像过程中以不同的位置定向，以评估不同组织中T细胞的存在。仰卧位可以评估肺部的T细胞，这在注射后的早期时间点很常见。皮下异种移植物朝上的侧位用于最好地评估 T 细胞向肿瘤的运输。雌性 C.Cg-Rag2tm1Fwa Il2rgtm1Sug / JicTac小鼠用于本手稿中描述的所有实验。图1显示了来自实验的图像，其中携带GD2阳性异种移植物的小?...

Watch this Scientific Journal Video about Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells at JoVE.com

Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

在这里，我们描述了一种用荧光素酶转导人T细胞的方法，以促进双特异性抗体诱导的T细胞向肿瘤的 体内跟踪， 以评估T细胞结合双特异性抗体的抗肿瘤功效和机制。

使用荧光素酶转导的人T细胞追踪双特异性抗体诱导的T细胞运输

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as ...

该方法允许用户轻松跟踪体内T细胞，以评估归巢动力学并监测T细胞持久性。该技术的主要优点是可以在同一只动物的多个时间点重复，而无需牺牲流式细胞术或免疫组织化学。这种技术也可以修改用于跟踪其他类型的免疫细胞。要培养293T细胞，通过加入110毫升热灭活胎牛血清和11毫升青霉素链霉素来制备1升DMEM。解冻5次10至第六个293T细胞，并将它们转移到含有DMEM的T175烧瓶中。每三天将细胞分成 1 到 10 个，持续 6 天，直到细胞汇合超过 90%。对于转染，使用移液器将1.5毫升培养基转移到两个50毫升的管中。向一个试管中加入 10 微克 VSVG 质粒、20 微克 gag pol 和 20 微克点击甲虫红 TDtomato 或 CBRTDR 荧光素酶质粒并混合。将100微升DNA体外转染试剂加入另一管中并混合。将两个试管在室温下孵育五分钟。将含有DNA的体外转染试剂的培养基滴液转移到含有DNA质粒的管中，并用移液管轻轻混合。在室温下孵育20分钟。在孵育期间，通过加入5至10毫升0.05%胰蛋白酶来分离293T细胞。细胞分离后，加入10毫升培养基，以800 G离心五分钟。将细胞重悬于1.5毫升培养基中。将含有DNA体外转染试剂和DNA质粒的培养基加入293T细胞中，并在37摄氏度下孵育30分钟。孵育后，将悬浮液转移到T175烧瓶中。加入 18 毫升培养基，在 37 摄氏度下孵育过夜。第二天，用玻璃移液管小心地吸出培养基，不要分离细胞，并用18毫升新鲜培养基代替。同样，在 37 摄氏度下孵育过夜。第二天，使用放在冰上的移液管除去病毒上清液，并以800G离心5分钟以沉淀细胞和碎片。使用0.22微米过滤器过滤病毒上清液，并立即将其放在冰上。对于人T细胞的体外活化，将10毫升含有0.1%牛血清白蛋白或BSA和2毫摩尔EDTA的PBS加入无菌15毫升管中。然后要清洗珠子，将珠子添加到管中并将管子放在磁性架中。两分钟后，从管中取出PBS。制备DMEM后，加入洗涤珠和白细胞介素-2或IL-2至每毫升30国际单位的最终浓度。使用血细胞仪和台盼蓝染色剂计数 T 细胞。计数后，将所需数量的T细胞添加到培养基中，并轻轻倒置试管进行混合。在24孔组织培养板的每个孔中接种含有200万个T细胞，磁珠和IL-2的2.5毫升培养基。在37摄氏度的加湿5%二氧化碳中培养。每天检查20倍物镜下的T细胞，持续三天，以确定何时用荧光素酶转导。接下来，要制备逆转录凝连蛋白板，将两毫升每毫升20微克的逆转录菌素在PBS中加入未处理的六孔板的孔中，并在室温下孵育两个小时。在不划伤板底部的情况下吸出逆转录菌素，并在六孔板中每孔加入三毫升 2% BSA 的 PBS。在室温下孵育30分钟。接下来，对于棘刺，吸出BSA并用三毫升PBS洗涤孔一次。继续或更换新的PBS，并将板盖在4摄氏度下过夜。第二天，每孔加入两毫升CBRTDR荧光素酶病毒上清液，并在32摄氏度下以1， 240 G离心90分钟。对于转导，计数 T 细胞后，吸出病毒上清液并在每毫升含有 30 国际单位的 DMEM 中，每孔接种 200 万个 T 细胞。检查T细胞两天。当细胞几乎汇合时，使用移液管轻轻地将它们从板底部洗掉，并将每个孔转移到单独的T75烧瓶中。加入9毫升培养基，每个烧瓶总共15毫升体积。第二天，加入15毫升培养基，并通过流式细胞术确认转导成功。麻醉小鼠后，使用26号针头，在100微升培养基中逆眶注射2000万个T细胞。皮下施用1， 000单位重组IL-2以支持体内T细胞存活。然后眶后或腹膜内给予双特异性抗体。对于体内成像，在麻醉小鼠中用26号针头通过眶后注射施用100微升三毫克D-荧光素。要捕获图像，请将鼠标放在成像仪的光密室中，使承载异种移植物的侧面朝上。使用采集控制面板，选择发光、照片和叠加。将曝光时间设置为自动，将像素合并设置为中等，将光圈设置为1并获取图像。在第一张图像之后，设置与第一个图像自动计算的曝光时间相匹配，以便可以直接比较后续图像。用鼠标仰卧拍摄另一组图像以评估肺部T细胞的存在。武装T细胞比非武装T细胞更快地贩运到肿瘤，提前两天离开肺部。通过生物发光测量T细胞随时间浸润到肿瘤中的定量，并表示为肿瘤轮廓上积分的总群数或每像素的辐射度。注射荧光素酶转导的T细胞后28天可以监测T细胞的运输，从而可以在整个治疗过程中跟踪T细胞的归巢和持久性。显示 T 细胞转运到 DKO 小鼠中 HER2 阳性人乳腺癌患者来源的异种移植物中。与对照双特异性抗体相比，使用未沉默的FC处理HER2双特异性抗体对肿瘤生长没有影响。相比之下，单独使用含有N297A突变或与K322A突变组合的HER2双特异性抗体治疗能够完全阻止肿瘤生长。对于具有沉默突变的组，与对照双特异性抗体和未沉默的HER2双特异性抗体相比，T细胞发光信号在注射后第3天达到更高的峰值，并持续处于更高的水平。携带神经母细胞瘤患者来源的异种移植物的小鼠表现出Ly6C阳性巨噬细胞的消耗显着增加了T细胞归向肿瘤，导致肿瘤生长减少并延长生存期。这种效果在骨肉瘤患者来源的异种移植物中更为明显。在靶向肿瘤抗原STEAP1的双特异性抗体中，IGGL SCFV格式在第一剂T细胞后第六天导致T细胞浸润显着增加，并在治疗期间持续存在。体外细胞毒性测定无法预测此类观察结果。在体内跟踪T细胞后，用户仍然可以进行免疫组织化学，以更具体地确定T细胞在肿瘤或其他正常组织中的位置。

Research

JoVE Journal

Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

科研

癌症研究

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

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 ...

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, ...

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 ...

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

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

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 ...

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 ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

细胞毒性细胞因子诱导的杀伤性T细胞的分离和扩张，用于癌症治疗

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...