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Cancer Research

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

Published: February 10, 2023
doi:
10.3791/64535
, , , , , , , ,
1T Cell Engineering Laboratory,Mayo Clinic, Rochester, 2Division of Hematology,Mayo Clinic, Rochester, 3Mayo Clinic Graduate School of Biomedical Sciences,Mayo Clinic, Rochester, 4Department of Molecular Medicine,Mayo Clinic, Rochester, 5Regenerative Sciences PhD Program,Mayo Clinic, Rochester, 6Department of Immunology,Mayo Clinic, Rochester

Summary

在这里，我们描述了一种方案，其中使用急性淋巴细胞白血病患者来源的异种移植模型作为评估和监测CD19靶向嵌合抗原受体T细胞相关毒性的策略。

Abstract

嵌合抗原受体T（CART）细胞疗法已成为治疗多种CD19+ 恶性肿瘤的有力工具，这导致FDA最近批准了几种CD19靶向CART（CART19）细胞疗法。然而，CART细胞疗法与一组独特的毒性有关，这些毒性具有自己的发病率和死亡率。这包括细胞因子释放综合征（CRS）和神经炎症（NI）。临床前小鼠模型的使用在CART技术的研究和开发中至关重要，用于评估CART功效和CART毒性。测试这种过继细胞免疫疗法的可用临床前模型包括同系、异种移植、转基因和人源化小鼠模型。没有单一的模型可以无缝地反映人体免疫系统，每个模型都有优点和缺点。本文旨在描述一种患者来源的异种移植模型，该模型使用急性淋巴细胞白血病患者的白血病原始细胞作为评估CART19相关毒性，CRS和NI的策略。该模型已被证明可以概括CART19相关的毒性以及临床上看到的治疗效果。

Introduction

嵌合抗原受体T（CART）细胞疗法彻底改变了癌症免疫治疗领域。它已被证明可成功治疗复发/难治性急性淋巴细胞白血病 （ALL）、大 B 细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤和多发性骨髓瘤 1,2,3,4,5,6,7，最近获得了 FDA 的批准。尽管在临床试验中取得了初步成功，但CART细胞疗法的毒性通常很严重，偶尔会致命。CART细胞治疗后最常见的毒性包括CRS和NI的发展，也称为免疫效应细胞相关神经毒性综合征（ICANS）8,9。CRS是由于CART细胞在体内的过度活化和大量扩增引起的，导致随后分泌多种炎性细胞因子，包括干扰素-γ、肿瘤坏死因子-α、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-6（IL-6）。这会导致低血压、高烧、毛细血管渗漏综合征、呼吸衰竭、多器官衰竭，在某些情况下会导致死亡10,11。CRS在CART19细胞疗法后50-100%的病例中发展11,12,13。ICANS是与CART细胞治疗相关的另一种独特的不良事件，其特征是全身性脑水肿，意识模糊，反应迟钝，失语，运动无力，偶尔癫痫发作9,14。任何级别的ICANS发生在多达70%的患者中，据报道3-4级发生在20-30%的患者中5,10,15,16。总体而言，CRS和ICANS很常见，可能是致命的。

CART细胞治疗后ICANS的管理具有挑战性。大多数ICANS患者也会出现CRS17，通常可以用IL-6受体拮抗剂托珠单抗或类固醇18治疗。之前的一份报告显示，托珠单抗的早期干预降低了严重CRS的发生率，但不影响ICANS19的发病率或严重程度。目前，ICANS没有有效的治疗方法或预防剂，研究预防策略至关重要20。

骨髓细胞和相关细胞因子/趋化因子被认为是CRS和ICANS21发展的主要驱动力。虽然CRS与细胞因子的极端升高和T细胞扩增直接相关，但ICANS的病理生理学在很大程度上是未知的22,23。因此，当务之急是建立一个小鼠模型，概括CART细胞治疗后的这些毒性，以研究其机制并制定预防策略。

目前有多种临床前动物模型用于研究、优化和验证 CART 细胞的功效，以及监测其相关毒性。这些包括同系、异种移植、免疫功能正常的转基因、人源化转基因和患者来源的异种移植小鼠，以及灵长类动物模型。然而，这些模型中的每一个都有缺点，有些不能反映CART细胞24,25的真正功效或安全问题。因此，必须仔细选择适合研究预期目标的最佳模型。

本文旨在描述用于评估CART细胞相关毒性，CRS和NI的方法，使用ALL患者来源的异种移植（PDX）体内模型（图1）。具体来说，在这里描述的方法中，按照先前描述的方案使用作者实验室中产生的CART19细胞。简而言之，通过密度梯度技术从健康供体外周血单核细胞（PBMC）中分离人T细胞，在第0天用CD3 / CD28磁珠刺激，并在第1天用由CD19靶向单链可变片段组成的CARs等地转入4-1BB和CD3ζ信号结构域。然后将这些 CART 细胞扩增，在第 6 天去珠，并在第 8 天冷冻保存 26,27,28,29,30。如前所述，小鼠接受淋巴细胞消耗治疗，然后施用患者来源的白血病原始细胞（ALL）28。首先，通过下颌下采血验证肿瘤植入。在建立适当的肿瘤负荷后，将CART19细胞施用于小鼠。然后，每天对小鼠称重以评估健康状况。进行小动物磁共振成像（MRI）以评估NI，同时进行尾部出血以评估T细胞扩增和细胞因子/趋化因子的产生。强烈建议将下面描述的技术用作在PDX模型中研究CART细胞相关毒性的模型。

Protocol

本协议遵循妙佑医疗国际机构审查委员会 （IRB）、机构动物护理和使用委员会 （IACUC A00001767） 和机构生物安全委员会 （IBC， Bios00000006.04） 的指南。 注意：用于小鼠的所有材料必须是无菌的。 1.向NSG小鼠注射白消安 获得雄性，8-12周龄，免疫功能低下，NOD-SCID IL2rγnull（NSG）小鼠，并在注射前称重。注意：为了统计显着性，建议每…

Representative Results

该协议的目的是使用来自ALL患者肿瘤细胞的PDX小鼠模型评估CART细胞相关毒性（图1）。首先，NSG小鼠接受腹膜注射白消安（30mg / kg），目的是免疫抑制它们并促进CART细胞植入28。第二天，他们收到了来自所有患者的~5×106 个PBMC（IV）。通过尾部出血测定 监测 小鼠的植入~13周。收集并裂解红细胞，然后进行流式细胞术制备以评估CD19 + ?…

Discussion

在本报告中，描述了一种使用 ALL PDX 模型评估 CART 细胞相关毒性的方法。更具体地说，该模型试图模仿两种危及生命的毒性，CRS和NI，患者在输注CART细胞后经常经历。它概括了在临床上观察到的CART毒性的许多特征：体重减轻，运动功能障碍，神经炎症，炎性细胞因子和趋化因子产生，以及不同效应细胞浸润到中枢神经系统8,20,28。<…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院（R37CA266344,1K99CA273304）、国防部（CA201127）、妙佑医疗国际K2R管道（S.S.K.）、妙佑医疗国际个体化医学中心（S.S.K.）和Predolin基金会（R.L.S.）的部分支持。此外，我们要感谢妙佑医疗国际核磁共振核心设施的工作人员。 图 1 是在 BioRender.com 中创建的

Materials

 APC Anti-Human CD19 Biolegend 302211
Alcohol Prep Pad Wecol 6818
Analyze 14.0 software AnalyzeDirect Inc. N/A https://analyzedirect.com/analyze14/
Artificial tears (Mineral oil and petrolatum) Akorn 17478-062-35 Topical ophtalmic gel to prevent eye dryness
BD FACS Lysing Solution  BD 349202 Red blood cells lysing buffer
BD Micro-Fin IV insulin syringes BD 329461
Brillian Violet 421 Anti-Human CD45 Biolegend 304032
Bruker Avance II 7 Tesla  Bruker Biospin N/A MRI machine
Busulfan (NSC-750) Selleckchem S1692
CountBright absolute counting beads Invitrogen C36950
CytoFLEX System B4-R2-V2 Beckman Coulter C10343 flow cytometer
Dulbecco's Phosphate-Buffered Saline Gibco 14190-144 
ERT Control/Gating Module  SA Instruments Model 1030 Small Animal Monitoring Respiratory and Gating System
Fetal bovine serum Millipore Sigma F8067
Hemocytometer Bright-Line Z359629-1EA
Human AB Serum; Male Donors; type AB; US Corning 35-060-CI
Isoflurane (Liquid) Sigma-Aldrich 792632
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation Invitrogen L34966
Microvette 500 Lithium heparin Sarstedt 20.1345.100 Blood collection tube
MILLIPLEX Huma/Cytokine/Chemokine Magnetic Beads Panel Millipore Sigma HCYTMAG-60K-PX38 Immunology Multiplex Assay to identify cytokines and chemokines
Omniscan Ge Healthcare Inc. 0407-0690-10 Gadolinium-based constrast agent
Pd Anti-Mouse CD45 Biolegend 103106
Penicillin-Streptomycin-Glutamine (100x), Liquid Gibco 10378-016
Round Bottom Polysterene Test tube Corning 352008
Sodium Azide, 5% (w/v) Ricca Chemical 7144.8-16
Stainless Steel Surgical Blade Bard-Parker 371215
X-VIVO 15 Serum-free Hematopoietic Cell Medium Lonza 04-418Q
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CAR-TAcute Lymphoblastic LeukemiaPatient-derived XenograftToxicity AssessmentMotor WeaknessWeight LossCytokine AnalysisMRI ImagingBreathing MonitoringParaVision Software

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Manriquez Roman, C., Sakemura, R. L., Kimball, B. L., Jin, F., Khadka, R. H., Adada, M. M., Siegler, E. L., Johnson, A. J., Kenderian, S. S. Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model. J. Vis. Exp. (192), e64535, doi:10.3791/64535 (2023).
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