用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型
Summary
我们描述了人类外周血单核细胞(PBMC)–基于人体的异种移植小鼠模型,用于转化免疫肿瘤学研究。该协议可作为建立和描述类似I-O治疗评估模型的一般准则。
Abstract
近年来免疫肿瘤学(I-O)疗法的发现和发展是癌症治疗的一个里程碑。然而,治疗挑战依然存在。健壮且与疾病相关的动物模型是继续临床前研究和开发的重要资源,以解决一系列额外的免疫检查点。在这里,我们描述了人类外周血单核细胞(PBMC)-基于人性化异种移植模型。BGB-A317(Tislelizumab)是一种在晚期临床开发中研究性的人性化抗PD-1抗体,作为讨论平台设置、模型特征和药物疗效评估的例子。这些人性化的小鼠支持大多数经过测试的人类肿瘤的生长,从而允许在人类免疫和人类癌症的背景下评估I-O疗法。一旦建立,我们的模型是比较时间和成本效益,通常产生高度可重复的结果。我们建议本文中概述的协议可以作为建立小鼠模型的一般指南,该模型与人类PBMC和肿瘤重组为I-O研究。
Introduction
免疫肿瘤学(I-O)是一个迅速扩大的癌症治疗领域。研究人员最近开始意识到调节免疫系统作用对肿瘤的攻击作用。免疫检查点封锁已经证明了各种癌症类型的令人鼓舞的活动,包括黑色素瘤,肾细胞癌,头和颈部,肺,膀胱和前列腺癌1,2。与直接杀死癌细胞的目标疗法相反,I-O疗法能增强人体免疫系统攻击肿瘤3。
迄今为止,已经建立了许多相关的I-O动物模型。这些包括:1)小鼠肿瘤细胞系或肿瘤同源性小鼠;2)来自基因工程小鼠(GEM)或致癌物的自发性肿瘤;3) 功能性鼠免疫系统中与人类药物靶点敲的嵌合的GEM;4)用人类癌细胞或患者衍生异种移植物(PDXs)移植的具有重组人体免疫力的小鼠。每种模型都有明显的优势和局限性,这些优点和局限性已在其他地方广泛描述和审查。
复生人类免疫免疫缺陷小鼠作为转化I-O研究的临床相关方法,已日益受到重视。这通常是通过成人免疫细胞(例如,外周血单核细胞 (PMBC))5、6 或 2) 从脐带血或胎儿的造血干细胞 (HSC) 的移植来实现的肝脏7,8。这些人性化的小鼠可以支持人类肿瘤的生长,从而允许在人类免疫和人类癌症的背景下评估I-O疗法。尽管这些优点,但人性化小鼠在I-O研究中的应用通常受到一些顾虑的阻碍,例如模型开发时间长,成本相当高。
在这里,我们描述了一个基于人类PBMC的模型,可以广泛应用于翻译I-O研究。该模型在疗效研究中具有较好的时间和成本效益,具有很高的可重复性。它已被内部用于评估目前正在临床前和临床开发中的几种I-O疗法。BGB-A317(Tislelizumab),一种研究性的人性化抗PD-1抗体9,作为例子,讨论模型开发、表征以及抗肿瘤疗效分析的可能应用。
Protocol
在涉及人类参与者的研究中进行的所有程序都符合BeiGene和/或国家研究委员会的道德标准,以及1964年《赫尔辛基宣言》及其后来的修正或类似的道德标准。研究中的所有个人参与者都得到了知情同意。在涉及动物的研究中执行的所有程序都得到北金内部审查委员会的批准。该协议已针对人体化NOD/SCID小鼠的BGB-A317(Tislelizumab)的评估进行了专门调整。 1. 建立基于人PBMC的模型 </p…
Representative Results
按照此处介绍的程序,成功建立了基于 PBMC 的人性化异种移植模型。总之,在CP和DS治疗后,通过对嗜中性粒细胞和单细胞种群的流细胞测定分析,确定了NOD/SCID小鼠的CP骨髓消融效应(图1)。100毫克/千克CP加125毫克/千克DS被确定为最佳剂量,并在后来的研究中使用,因为该方案可导致嗜中性粒细胞和单核细胞的最大耗竭,而不会对小鼠造成严重毒性。其次,进行了人体PBMC和肿瘤移植。IHC?…
Discussion
近年来,我们对癌症发展和进展的认识有了显著的进步,重点是全面了解肿瘤细胞及其相关频闪。利用宿主免疫机制可以引起对癌细胞的更大影响,这是一种有前途的治疗策略。具有完整小鼠免疫系统的鼠模型,如合成和GEM模型,已被广泛用于研究检查点介导的免疫。使用这些模型的功效评估主要取决于代理抗小鼠靶抗体13,14。然而,人类和鼠的免疫系统之间的…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢实验室成员进行有益的讨论。这项工作得到了北京市科委生物医学和生命科学创新与培养研究项目的一部分支持。Z151100003915070(项目”新免疫肿瘤抗肿瘤药物BGB-A317的临床前研究”),也部分得到公司内部临床前研究资金的支持。
Materials
| PBMC separation /cell culture | |||
| Histopaque-1077 | Sigma | 10771 | Cell isolation |
| DMEM | Corning | 10-013-CVR | Cell culture |
| DPBS | Corning | 21-031-CVR | Cell culture |
| FBS | Corning | 35-076-CV | Cell culture |
| Penicillin-Streptomycin, Liquid | Gibco | 15140-163 | Cell culture |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200-114 | Cell culture |
| Matrigel | Corning | 356237 | CDX inoculation |
| FACS analysis | |||
| Deoxyribonuclease I from bovine pancreas | Sigma | DN25 | Sample preparation |
| Collagenase Type I | Sigma | C0130 | Sample preparation |
| Anti-mouse/human CD11b (M1/70) antibody | BioLegend | 101206 | FACS |
| Anti-mouse Ly-6C (HK1.4) antibody | BioLegend | 128008 | FACS |
| Anti-mouse Ly-6G (1A8) antibody | BioLegend | 127614 | FACS |
| Anti-human CD8 (OKT8) antibody | Sungene Biotech | H10082-11H | FACS |
| Anti-human CD279 (MIH4) antibody | eBioscience | 12-9969-42 | FACS |
| Anti-human CD3 (HIT3a) antibody | 4A Biotech | — | FACS |
| Guava easyCyte 8HT Benchtop Flow Cytometer | Millipore | 0500-4008 | FACS |
| Tumor/PDX implantation /dosing / measurement | |||
| Cyclophosphamide | J&K | Cat#419656, CAS#6055-19-2 | In vivo efficacy |
| Disulfiram | J&K | Cat#591123, CAS#97-77-8 | In vivo efficacy |
| Syringe | BD | 300841 | CDX inoculation |
| Hypodermic needles (14G) | Shanghai SA Mediciall & Plastic Instruments Co., Ltd. | 0.7*32 TW SB | PDX inoculation |
| Vernier Caliper (MarCal) | Mahr | 16ER | Tumor measurement |
| IVC individual ventilated cages | Lingyunboji Ltd. | IVC-128 | Animal facility |
| IHC | |||
| Leica ASP200 Vacuum tissue processor | Leica | ASP200 | IHC |
| Leica RM2235 Manual Rotary Microtome for Routine Sectioning | Leica | RM2235 | IHC |
| Leica EG1150 H Heated Paraffin Embedding Module | Leica | EG1150 H | IHC |
| Ariol-Clinical IHC and FISH Scanner | Leica | Ariol | IHC |
| Anti-human CD8 (EP334) antibody | ZSGB-Bio | ZA-0508 | IHC |
| Anti-human PD1 [NAT105] antibody | Abcam | ab52587 | IHC |
| Anti-human PD-L1 (E1L3N) antibody | Cell Signaling Technology | 13684S | IHC |
| Polink-2 plus Polymer HRP Detection System | ZSGB-Bio | PV-9001/9002 | IHC |
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Citer Cet Article
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