GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性
Summary
在此, 我们提出了一种在大肠杆菌中产生双特异性抗体 GPC3-S-Fab 的协议.纯化 GPC3-S-Fab 对 GPC3 阳性肝癌细胞具有较强的细胞毒性作用。
Abstract
该协议描述了双特异性抗体 (bsAb) 的构建和功能研究, GPC3-S-Fab。bsAbs 可以通过两个不同的手臂识别两个不同的表位。bsAbs 已积极研究他们的能力, 直接招募免疫细胞杀死肿瘤细胞。目前, 大多数 bsAbs 是以重组蛋白的形式生产的, 它们要么是含有 fc 的 bsAbs, 要么是没有 fc 区域的较小的 bsAb 衍生物。本研究通过将 anti-GPC3 抗体 GC33 与 anti-CD16 单域抗体相结合, 设计了一种基于抗体片段 (GPC3-S-Fab) 的双特异性抗体。GPC3-S-Fab 可在大肠杆菌中表达, 并经两种亲和 chromatographies 纯化。纯化的 GPC3-S-Fab 可以通过招募自然杀伤细胞来明确地结合和杀死 GPC3 阳性肝癌细胞, 这表明 GPC3-S-Fab 在肝癌治疗中的潜在应用。
Introduction
单克隆抗体现在广泛用于癌症治疗1。由于抗体的柔韧性, 各种基于抗体的格式已经得到了积极的探索。与单克隆抗体相比, bsAbs 有两种不同的抗原结合模块, 能够同时识别两个不同靶点, 有效地触发免疫效应细胞的招募, 以靶向和杀死肿瘤细胞2。
当前的重组 bsAb 格式通常可以分配给两个类: fc 包含 bsAbs 和 bsAbs, 而没有 fc 区域。与大多数哺乳动物细胞中所含 fc 格式相比, 没有 fc 区域的 bsAbs 具有较小尺寸的优点, 更容易在微生物表达系统中产生, 并且能更有效地穿透肿瘤组织。3。
没有 Fc 区域的 bsAbs 通常是通过链接单个绑定基团 (如单链变量片段 (scFvs) 或3) 来形成的。如果没有稳定的域, 基于抗体碎片的 bsAbs 通常会损害热稳定性、低溶解度或增加聚集4、5的潜力。相比之下, 以 bsAbs 为基础的 heterodimerization, 由于 CH1 和 CL 在原生的晶圆4,6, 更稳定。
由重链抗体 (VHHs, 也称为单域抗体) 的可变域是天然重链抗体的活性抗原结合片段7。VHHs 具有亲和力高、常规 IgGs8特异性强、免疫原化低、细菌表达率高的特点9。与抗体片段相比, VHHs 具有较高的热稳定性10。与基团相比, VHHs 的尺寸较小, 因为缺乏 CH1 和 CL。因此, 通过将 bsAb 与单域抗体、VHH 相连接获得的 S 型, 设计并研究了其抗肿瘤作用11、12。
本文介绍了 hGC3313与 anti-CD16a VHH14相结合的 GPC3-S-Fab 结构。周质在大肠杆菌(大肠杆菌)中的表达能有效地产生 GPC3-S-Fab。GPC3-S-Fab 的功能性研究表明 GPC3-S-Fab 是肝癌治疗的一个有前途的策略。因此, GPC3-S-Fab 在替代技术上的优势和适用于以前研究的参考, 包括易于生产和纯化, 以及更稳定的 bsAbs。
哺乳动物表达系统和原核表达系统已被用来表达各种形式的 BsAbs。与哺乳动物表达系统相比,大肠杆菌蛋白表达系统具有产量高、成本低、省力、遗传操作方便、转化效率高15的优点。对于 bsAbs 在大肠杆菌中的表达, 有两种基本策略: 细胞质中的表达和细胞质与外细胞膜之间 periplasm 的表达15。与细胞质的还原环境相比, periplasm 是一种比较氧化的环境, 促进了蛋白质16的正确折叠和共表达。正确的折叠对 bsAbs 的溶解度、稳定性和功能生成起着关键的作用。因此, 在 s–政局的 N 端添加了一个信号序列, 直接分泌到大肠杆菌的 periplasm17。为了确保正确的折叠、溶解度、热稳定性和构象稳定性, 减少抗体的复杂性和大小经常被使用16。S-晶圆的格式包括一个工厂和一个 VHH, 这是表达很好的细菌系统可能由于简单的结构和小规模。
GPC3 是在这种 GPC3-S-Fab 双特异性抗体格式中选择的。Glypican-3 (GPC3) 是肝素硫酸盐 (HS) 蛋白多糖家族的成员, 通过 glycosylphosphatidylinositol (GPI)18锚定在细胞表面。GPC3 是抗原70% 肝癌 (HCC) 病例, 占大多数肝癌19,20,21,22。由于 GPC3 在正常组织中很少表达, GPC3 被建议为肝癌的潜在靶向。对 GPC3 产生了多个鼠标抗体。然而, 只有 GC33 显示有限的抗肿瘤活动22, 它没有表现出临床疗效的病人。在这项研究中, GPC3-S-Fab 被证明能够招募 NK 细胞杀死 GPC3 肿瘤细胞14。
为了招募 NK 细胞, 使用了 anti-CD16 VHH。CD16a 是一种低亲和 IgG 受体, 主要表现为自然杀伤细胞、巨噬素、单核和 T 细胞的某些亚型。它涉及抗体依赖性细胞细胞毒性 (ADCC) 的 NK 细胞23。人 NK 细胞可分为两类, CD56-CD16+ 和 CD56+CD16。与 CD56+CD16− nk 细胞相比, CD56-CD16+ nk 细胞可以释放出更高水平的穿孔和颗粒酶 B, 从而呈现出强烈的细胞毒性24。枯否细胞 (KCs), 表达 CD16a, 是肝脏中的巨细胞。枯否细胞在25肝癌的抑癌中起着重要作用。因此, bsAbs 靶向 CD16a 可能是一个更有希望的策略比接触 T 细胞对抗肝癌。
Protocol
Representative Results
Discussion
在这项研究中, 我们提出了一个新的格式 bsAbs, GPC3-S-Fab, 它可以招募 NK 细胞靶向 GPC3 阳性肿瘤细胞。s-工厂是基于自然的晶圆格式, 添加一个 anti-CD16 VHH11,12。与含 Fc 区的 bsAbs 相比, GPC3-S-Fab 可以很容易地在细菌 periplasm 中产生。
利用该协议中描述的表达式和纯化策略, 得到了大量的可溶性和功能 GPC3-S-Fab。为便于周质表达, 政局在 N 终…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了广东省 (2016A050503028) 研究 & 开发计划的资助。
Materials
| Shaking incubator | Thermo Fisher | MAXQ 4000 | |
| Shaking incubator | Zhicheng | ZWYR-D2402 | |
| Centrifuge | Cence | GL-10MD | |
| Centrifuge | Beckman coulter | Avanti j-26S XPI | |
| Centrifuge | eppendorf | 5810R | |
| Ultraviolet spectrophotometer | Thermo Fisher | Nanodrop | |
| Analytical polyacrylamide gel electrophoresis apparatus | Mini-PROTEAN® Tetra | Bio-rad | |
| Trans-blot apparatus | Criterion | Bio-rad | |
| Imaging system | Bio-rad | chemidoc tm XRS+ | |
| Fast Protein Liquid chromatogram | GE Healthcare | AKTA avant | |
| GF column | GE Healthcare | 28-9909-44 Superdex 200 Increase 10/300 GL | |
| Flow Cytometer | Beckman coulter | FC500 | |
| Centrifuge | eppendorf | 5702R | |
| Envision plate reader | TECAN | Infinite F50 | |
| Anti His-tag | eBioscience | 14-6657-82 | |
| anti-Flag-tag | Sigma | F1804 | |
| anti-human(H&L)-488 | A11013 | Invitrogen | |
| Anti-mouse IgG HRP-linked antibody | Cell Signaling | 7076S | |
| Ni-NTA-Agarose | Tribioscience | TBS9202-100 | |
| IgG-CH1 affinity resin | Thermo Fisher | 194320005 | |
| Ficoll-Plaque Plus | GE Healthcare | 17-1440-03 | |
| NK cell enrichment kit | Stemcell | 19055 | |
| Magnet | Stemcell | 18000 | |
| CCK8 kit | Dojindo | CK04 | |
| DMEM | Gibco | C11995500CP | |
| RPMI-1640 | Gibco | C11875500CP | |
| Fetal Bovine Serum (FBS) | Sigma | F2442 | |
| Trypsin | Gibco | 15050-057 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | Cell culture |
| Standard marker | Sigma Aldrich | MWGF200 | Gel filtration |
| Isopropyl-b-D-thio-galactopyranoside (IPTG) | VWR chemicals | VWRC0487-100G | |
| Dialysis tubing | Sigma Aldrich | D0655-100FT | |
| Knanamycine | VWR | VWRC0408-100G | |
| Ampicillin | VWR | VWRC0339-100G | |
| Tryptone | Thermo Fisher | LP0042B | |
| Yeast Extract | Thermo Fisher | LP0021B | |
| NaCl | Sangon Biotech | A100241 | |
| Trizma base | Sigma Aldrich | T6791-1KG | |
| EDTA | Sigma Aldrich | V900106 | |
| Glycine | aladdin | A110752-500g | |
| KCl | aladdin | P112134-500g | |
| MgCl | Sigma Aldrich | V900020 | |
| Agar | Sangon Biotech | A505255-0250 | |
| Potassium Phosphate, Monobasic Anhydrous (KH2PO4) | VWR | 7778-77-0 | |
| Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4) | VWR | 7558-79-4 | |
| 2-Mercaptoethanol | VWR | 60-24-2 | |
| Phenylmethyl Sulfonyl Fluoride (PMSF) | VWR | 329-98-6 | |
| Lysozyme | Sigma Aldrich | L6876-25G | |
| Coomassie Brilliant Blue R250 | VWR | VWRC0472-50G | |
| Bromophenol blue | Sangon Biotech | A500922-25G | |
| Bovine Serum Albumin (BSA) | VWR | VWRC0332-100G | |
| Glycerol | Sigma Aldrich | V900122 | |
| 100mm x 20mm plastic dish | Corning | 430167 | |
| 25cm2 flask | Corning | 430639 | |
| 96 well cell culture cluster | Corning | 3599 | |
| Sucrose | Sangon Biotech | A610498-0005 | |
| CHO | the Type Culture Collection of the Chinese Academy of Sciences | GNHa 3 | |
| MHCC-97H | the Type Culture Collection of the Chinese Academy of Sciences | SCSP-528 | |
| HepG2 | the Type Culture Collection of the Chinese Academy of Sciences | TCHu 72 | |
| Huh7 | the Type Culture Collection of the Chinese Academy of Sciences | TCHu182 | |
| Hep3B | the Type Culture Collection of the Chinese Academy of Sciences | TCHu106 | |
| NK92 | ATCC | CRL2408 |
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