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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 293T cells | ATCC | CRL-11268 | |
| BSA | Sigma Aldrich | A7030-10G | |
| CD3/CD28 beads | Gibco (ThermoFisher) | 11161D | |
| D-Luciferin, Potassium Salt | Goldbio | LUCK-1G | |
| DMEM | Gibco (ThermoFisher) | 11965092 | |
| DNA in vitro transfection reagent (polyjet) | SignaGen Laboratories | SL100688 | |
| EDTA | Sigma Aldrich | E9884-100G | |
| FBS | Gibco (ThermoFisher) | 10437028 | |
| Gag/pol plasmid | Addgene | 14887 | |
| GFP plasmid | Addgene | 11150-DNA.cg | |
| Penicilin-Streptomycin | Gibco (ThermoFisher) | 15140122 | |
| Recombinant human IL-2 | R&D Systems | 202-IL-010/CF | |
| Retronectin | Takara | T100B | |
| Trypsin | Gibco (ThermoFisher) | 25-300-120 | |
| VSV-G plasmid | Addgene | 8454 |
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