Name: a gpc3-targeting bispecific antibody, gpc3-s-fab, with potent cytotoxicity
Uploaded: 2018-07-12T13:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity at JoVE.com

这项工作得到了广东省 (2016A050503028) 研究 & 开发计划的资助。

所有的程序, 包括人的血液收集被孙中山大学伦理委员会批准。
1. GPC3-S-Fab 设计策略
<ol><li>设计 GPC3-S-Fab 通过链接一个 anti-GPC3 (人性化的 GC3313) 与 anti-CD16 VHH14 (图 1)。</li>
	<li>将 VH-CH1-CD16-VHH 和 pET26b 和 pET21a 载体合成并克隆为先前报告的12。</li>
</ol>2. 变革和文化
<ol><li>将含有 VH-CH1-CD16 (图1A) 和 100 pET26b 的 pET21a 的 100 ng 添加到100µL 的主管BL21 (DE3) 细胞中。拌匀, 在冰上孵育30分钟, 在42摄氏度的水浴中孵化 45-九十年代, 转移, 并在冰上孵化 3-5 分钟。</li>
	<li>将500µL 溶源性汤 (磅) 培养基放入含有 BL21 (DE3) 细胞的管中, 然后在37摄氏度处孵化, 150 rpm 45-60 分钟100µL 的 BL21 (DE3) 细胞在含有50µg/毫升卡那霉素和100µg/毫升氨苄西林的 LB 琼脂板上的分布, 然后孵育37°c 12-16 小时。<ol>
<li>准备溶源性肉汤 (磅) 培养基: 1% 磅/卷胰蛋白胨, 0.5% 磅/卷酵母萃取物, 1% 磅/卷氯化钠。</li>
	</ol>
</li>
	<li>接种细胞从一个单一的殖民地成5毫升的超级汤 (锑) 培养基含有50µg/毫升卡那霉素和100µg/毫升氨苄西林, 和文化在37摄氏度, 220 rpm, 一夜之间。然后, 将1毫升的培养物接种到100毫升的锑培养基中, 其中含有50µg卡那霉素和100µg/毫升氨苄西林, 培养在37摄氏度, 220 rpm 为 4 h。然后, 将10毫升的培养物转移到1升的锑培养基中, 含卡那霉素50 µg/毫升,氨苄西林100 µg/毫升, 培养37摄氏度, 220 rpm。<ol>
<li>准备超级肉汤培养基: 3.2% 胰蛋白胨/卷, 2% 个/卷酵母萃取物, 0.5% 个/卷氯化钠。</li>
	</ol>
</li>
	<li>当外径600达到 0.6-0.8, 加入异丙基 d-硫 galactopyranoside (IPTG) 到最终浓度为0.2 毫米。文化在16°c, 180 转每分钟为 36-48 h 为周质表示。</li>
</ol>3. GPC3-S-Fab 周质纯化
<ol><li>周质分数准备<ol>
<li>收集细胞通过离心在 4000 x g, 4 °c 30 分钟, 丢弃培养基, 并权衡细胞。</li>
		<li>并用重悬在4毫升的冰冷蔗糖溶液 (20 毫米的三盐酸, pH 7.5; 25% (重量/卷) 蔗糖和1毫米 EDTA) 每克细胞, 并在冰上孵育15分钟。</li>
		<li>离心机在 8500 x g (桌上部离心机, 固定的角度转子), 4 °c 为20分钟. 去掉上清 (蔗糖分数) 并将其保存在冰上。</li>
		<li>并用重悬在冰冷的5毫米氯化镁2和1毫米的 PMSF (每克细胞4毫升) 混合的颗粒。添加40µL 的溶菌酶的股票 (15 毫克/毫升) 每克, 并在冰上孵化30分钟。</li>
		<li>离心机在 8500 x g (桌上部离心机, 固定角度转子), 4 °c 为20分钟. 转移上清 (周质分数), 并保存在冰上。</li>
		<li>结合蔗糖分数和周质分数, 离心机在 3万 x g, 4 °c 30 分钟. 取出上清, 并将其保存在冰上的50毫升锥形管中。</li>
	</ol>
</li>
	<li>镍 NTA 亲和纯化<ol>
<li>并用重悬1毫升的镍 NTA 琼脂糖通过混合彻底达到均匀悬浮, 除去1毫升的镍 NTA 琼脂糖到一个新鲜的15毫升圆锥管, 并增加10柱容积 (CV) 平衡缓冲 (25 毫米的三 HCl, pH 7.5; 1 米氯化钠) 到平衡。</li>
		<li>离心机在 400 x g 的5分钟, 并仔细清除上清。然后, 将镍 NTA 琼脂糖加入含有蔗糖分数和周质分数的50毫升管中。岩石在4°c 为 2 h。</li>
		<li>离心机的混合物在 400 x g, 4 °c 为5分钟. 小心地将上清液移至另一条新鲜冰冷的管子作为未绑定的分数。将镍 NTA 琼脂糖转移到重力柱上。</li>
		<li>添加10个洗涤缓冲器 (25 毫米的三盐酸, pH 7.5; 1 米的氯化钠; 20 毫米, 30 毫米或40毫米咪唑) 到列, 并收集洗脱作为洗涤分数。 注意: 这些解决方案应添加到增加咪唑浓度的顺序。</li>
		<li>添加3个洗脱缓冲液 (25 毫米的三盐酸, pH 7.5; 300 毫米氯化钠; 100 毫米, 200 毫米, 300 毫米或400毫米咪唑) 到专栏并且收集洗脱作为洗脱分数 1, 2, 3 和4。 注意: 这些解决方案应添加到增加咪唑浓度的顺序。</li>
		<li>透析洗脱2升的 PBS (137 毫米氯化钠, 2.7 毫米的氯化钾, 10 毫米的 Na2HPO4, 2 毫米的第 2 PO4,pH 7.4) 使用透析油管 (12.4 kDa) 在4°c 为 2 h. 检查是否存在 GPC3-S-Fab 由 12% SDS 页, 然后通过考马斯蓝染色。</li>
	</ol>
</li>
	<li>IgG-CH1 亲和纯化<ol>
<li>将1毫升的 IgG-CH1 亲和树脂转化为50毫升圆锥管, 首先用 PBS 清洗珠子。然后, 在步骤3.2.6 后添加包含 GPC3-S-Fab 的透析解决方案。岩石在4°c 为 2 h。</li>
		<li>离心机在 400 x g 为5分钟, 4 °c。小心地将上清液移到另一条新鲜冰冷的管子上, 将树脂转移到重力柱上。</li>
		<li>将洗涤缓冲器 (PBS) 的10个 CV 添加到柱上, 并将洗脱作为洗涤分数。</li>
		<li>通过添加1米的三 pH 值 9.0 (每个洗脱分数的每毫升0.1 毫升) 准备收集管。洗脱 3 CV 洗脱缓冲器 (0.1 米甘氨酸盐酸盐, pH 2.7), 并收集洗脱分数;重复3次。</li>
		<li>用透析油管 (12.4 kDa) 在4摄氏度洗脱2升的 PBS 进行透析. 重复两次。</li>
		<li>用 ultramicrospectrophotometer 测定蛋白质浓度 (摩尔消光系数: 91105. 1 A280 = 0.69 毫克/升)。</li>
	</ol>
</li>
</ol>4. SDS 页和西方印迹分析
<ol><li>将10µL 的样品放入 5x SDS 加载缓冲器中, 混合均匀。将蛋白质混合物煮沸100摄氏度, 5 分钟。<ol>
<li>准备 5x SDS 装载缓冲器: 250 毫米的三盐酸, pH 值 6.8, 10% 量/卷 SDS, 0.5% 溴酚/卷蓝色, 50% 卷/卷甘油, 5% 卷/卷 beta 巯基乙醇。</li>
	</ol>
</li>
	<li>准备一个 SDS 页凝胶与加载凝胶 (5%) 和分离凝胶 (12%), 如前所述26,27,28。</li>
	<li>加载10µL 的煮蛋白质混合物和蛋白质标记, 并运行 SDS 页。</li>
	<li>用考马斯亮蓝溶液晃动20分钟, 然后执行脱色。</li>
	<li>对于西方印迹, 转移到 PVDF 膜后, 用 TBST (20 毫米的三 HCl、150毫米氯化钠、0.1% 卷/卷补 20) + 5% 小时脱脂牛奶在室温下, 在震动时将膜堵塞。</li>
	<li>用原抗体 (反他或反旗) 孵育膜, 稀释 1:2, 000 TBST + 5% 脱脂奶1小时。</li>
	<li>在室温下用 TBST 洗5分钟, 重复3次。</li>
	<li>在二级抗体中孵化膜 (与酶联的抗小鼠 Fc 抗体) 稀释 1:2, 000 TBST + 5% 脱脂牛奶1小时。</li>
	<li>在室温下用 TBST 洗5分钟, 重复3次。</li>
	<li>每片添加1毫升 HPR 基片, 开发并拍摄图像。</li>
</ol>5. 凝胶过滤分析
<ol><li>使用以下栏: 列容积为24毫升;PBS pH 值7.4 的平衡缓冲器;室温下0.5 毫升/分的流动速率;样品体积为200µL。</li>
	<li>使用以下蛋白质体积和浓度: 500 µL 1.2 毫克/毫升 GPC3-S-Fab。离心机在 2万 x g 30 分钟, 并采取200µL 加载。</li>
	<li>对于蛋白质标记的体积和浓度, 采取100µL 细胞色素 C (2 毫克/毫升, 12.4 kDa), 140 µL 的酸酐酶 (3 毫克/毫升, 29 kDa), 140 µL 的白蛋白 (10 毫克/毫升, 66 kDa) 和200µL 酒精脱氢 (5 毫克/毫升, 150 kDa) 成一管。离心机在 2万 x g 30 分钟, 并采取200µL 加载。</li>
	<li>每次运行前, 以0.5 毫升/分钟的流速清洗至少2的蒸馏水。</li>
	<li>平衡至少有 2 CV 的平衡缓冲器 (PBS, pH 值 7.4), 流速为0.5 毫升/分钟。</li>
	<li>自动注入0.5 毫升/分钟的样品来装载样品。</li>
	<li>洗脱与平衡缓冲器在0.5 毫升/分钟, 并检测在280和 215 nm。</li>
</ol>6. 流式细胞仪分析
<ol><li>文化 HepG2 (GPC3 阳性), Hep3B (GPC3 阳性), Huh7 (GPC3 阳性), MHCC-97H (GPC3 阴性) 细胞在100毫米 x 20 毫米的塑料菜肴含有 DMEM 培养基10% 胎牛血清 (血清), 青霉素 (100 µg/毫升) 和链霉素 (50 µg/毫升) 在37°c,5% CO2。</li>
	<li>培养的 CHO (GPC3 阴性) 细胞和 CHO/GPC3 细胞窝藏全长人类 GPC3 cDNA (GPC3 阳性) 在100毫米 x 20 毫米塑料菜肴含有 RPMI 1640 培养基10% 血清, 青霉素 (100 µg/毫升), 和链霉素 (50 µg/毫升) 在37摄氏度, 5% CO2。</li>
	<li>在 25cm2瓶含有 RPMI 1640 培养基10% 血清, 青霉素 (100 µg/毫升), 和链霉素 (50 µg/毫升) 在37摄氏度, 5% CO2的 NK92/CD16 中, 培养全长人 CD16 cDNA (CD16 阳性)。</li>
	<li>当细胞 70-90% 汇合时, 丢弃培养基, 用2毫升的 PBS 冲洗细胞。添加1毫升0.25% 胰蛋白酶和孵育37°c 2-3 分钟 (或直到细胞开始从表面分离)。添加1毫升的完全生长培养基, 以中和胰蛋白酶和重新暂停细胞通过轻轻吹打上下。</li>
	<li>用 Neubauer hemocytometer 计数细胞数, 用台盼蓝检测细胞活力。</li>
	<li>为每个细胞线收集 3 x 106细胞。添加1毫升 PBS + 0.2% BSA, 以重新悬浮细胞。离心机在 200 x g, 4 °c 为5分钟. 重复两次。</li>
	<li>添加0.3 毫升 PBS + 0.2% BSA, 以重新悬浮细胞, 并转移100µL (约 100万) 到每5毫升聚苯乙烯圆底管。 注意: 请确保从这里到管道的所有步骤都应在冰上保持冷。当抗体与细胞表面抗原结合时, 复合体将开始内部化。通过保持寒冷, 这个过程会大大减慢。</li>
	<li>添加10µL 的主要抗体 (GPC3-S-Fab 或人类 IgG1, 最终浓度5µg/毫升) 每管, 并在冰上孵化1小时。</li>
	<li>添加1毫升 PBS + 0.2% BSA 清洗, 离心机在 200 x g, 4 °c 为5分钟. 重复三次。</li>
	<li>在100µL 的 PBS + 0.2% BSA 中重新暂停, 然后添加0.5 µL 的二级抗体 (抗人 IgG (H + L)-488, 最后浓度10µg/毫升) 和在冰上孵育1小时. 注: 从这一步, 请保持细胞不受光线的影响。</li>
	<li>添加1毫升 PBS + 0.2% BSA 清洗, 离心机在 200 x g, 4 °c 为5分钟. 重复三次。</li>
	<li>根据标准协议29, 用流式细胞仪对单元进行分析。用488纳米激光获取细胞激发。</li>
</ol>7. 细胞毒性化验
<ol><li>准备肿瘤细胞。<ol>
<li>文化 HepG2、Hep3B、Huh7、MHCC-97H 和 CHO 细胞系, 如步骤 6.1-6.5 所述。</li>
		<li>稀释细胞到 0.5 x 105/毫升, 和板材100µL 细胞 (每井5000个细胞) 在96井板材。文化 6-8 h 允许细胞附加。</li>
	</ol>
</li>
	<li>制备人外周血单个核细胞 (PBMCs)。<ol>
<li>用50毫升锥形管中的 PBS, 稀释新鲜的准备好的血液。例如, 用10毫升的 PBS 稀释10毫升血液。</li>
		<li>将15毫升淋巴细胞分离培养基转化为新鲜的50毫升圆锥管。慢慢将稀释后的血液转移到淋巴细胞分离培养基上。 注意: 保持管子垂直。不要破坏淋巴细胞分离培养基的表面。</li>
		<li>离心机在 400 x g 为35分钟在室温下与刹车。</li>
		<li>慢慢移除上血浆层, 并采取在血浆淋巴细胞分离培养基界面中含有 PBMCs 的白血层。</li>
		<li>稀释 PBMCs 的双卷 PBS + 2% 的血清。离心机在 400 x g 5 分钟。</li>
		<li>用 PBS + 2% 的血清冲洗细胞两次。离心机在 400 x g 的5分钟. 计算步骤6.5 中所述的单元格数。</li>
	</ol>
</li>
	<li>准备 NK 细胞。<ol>
<li>准备 PBMCs 悬浮在 5 x 107细胞/毫升的 PBS + 2% 的血清, 并把它们放在一个5毫升聚苯乙烯圆底管。</li>
		<li>添加人类 NK 细胞浓缩鸡尾酒在50µL/毫升细胞。混合好, 室温孵育10分钟。</li>
		<li>从 NK 细胞浓缩试剂盒中并用重悬磁性颗粒, 混合均匀, 确保吹打向上和向下一致悬浮5倍以上。将悬浮磁性粒子转移到100µL/毫升到细胞悬浮。混合好, 室温孵育5分钟。</li>
		<li>通过增加 PBS + 2% 的血清, 使细胞悬浮量达到2.5 毫升的总体积。通过轻轻吹打 2-3 倍, 将管中的细胞混合在一起。把管子 (没有盖子) 放进磁铁里。让磁性珠子在室温下安定下来2.5 分钟。</li>
		<li>卸下磁铁。在一个连续的运动中, 将管子倒置, 将富细胞悬浮液倒入新管中。</li>
		<li>按步骤6.5 中的说明计算单元格数。</li>
	</ol>
</li>
	<li>建立细胞毒性反应。<ol>
<li>使用相应的培养基将 NK 细胞稀释为 5 x 105细胞/毫升。将 NK 细胞悬浮液中的100µL 添加到96井板上镀的肿瘤细胞中。</li>
		<li>添加10µL 的 GPC3-S-Fab 在不同浓度, 最高剂量的最终浓度为30µg/毫升, 3 倍稀释, 9 点 (三复制)。</li>
		<li>孵育在37°c, 5% CO2为 72 h。</li>
		<li>经过72小时的孵化, 去除培养基, 并添加100µL 的 DMEM 培养基含有10µL 的 CCK8 试剂, 每个井在96井板, 然后孵化在37摄氏度。</li>
		<li>添加 CCK8 试剂后, 在1、2、3和 4 h 上读出 450 nm 的板材。</li>
		<li>分析数据。计算存活率为 (OD450 GPC3-S-Fab + 效应 + 肿瘤OD450培养基)/(OD450肿瘤-OD450培养基) x 100% 和 (OD450 GPC3-S-Fab + 肿瘤-OD450培养基)/(OD450肿瘤-OD450中等) x 100%。效应细胞为 NK 细胞。</li>
	</ol>
</li>
</ol>

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作者声明没有利益冲突。

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...

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。

<table><tbody><tr><td>Shaking incubator</td><td>Thermo Fisher</td><td>MAXQ 4000</td><td></td></tr><tr><td>Shaking incubator</td><td>Zhicheng</td><td>ZWYR-D2402</td><td></td></tr><tr><td>Centrifuge</td><td>Cence</td><td>GL-10MD</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman coulter</td><td>Avanti j-26S XPI</td><td></td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>5810R</td><td></td></tr><tr><td>Ultraviolet spectrophotometer</td><td>Thermo Fisher</td><td>Nanodrop</td><td></td></tr><tr><td>Analytical polyacrylamide gel electrophoresis apparatus</td><td>Mini-PROTEAN&amp;reg; Tetra</td><td>Bio-rad</td><td></td></tr><tr><td>Trans-blot apparatus</td><td>Criterion</td><td>Bio-rad</td><td></td></tr><tr><td>Imaging system</td><td>Bio-rad</td><td>chemidoc tm XRS+</td><td></td></tr><tr><td>Fast Protein Liquid chromatogram</td><td>GE Healthcare</td><td>AKTA avant</td><td></td></tr><tr><td>GF column</td><td>GE Healthcare</td><td>28-9909-44 Superdex 200 Increase 10/300 GL</td><td></td></tr><tr><td>Flow Cytometer</td><td>Beckman coulter</td><td>FC500</td><td></td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>5702R</td><td></td></tr><tr><td>Envision plate reader</td><td>TECAN</td><td>Infinite F50</td><td></td></tr><tr><td>Anti His-tag</td><td>eBioscience</td><td>14-6657-82</td><td></td></tr><tr><td>anti-Flag-tag</td><td>Sigma</td><td>F1804</td><td></td></tr><tr><td>anti-human(H&amp;amp;L)-488</td><td>A11013</td><td>Invitrogen</td><td></td></tr><tr><td>Anti-mouse IgG HRP-linked antibody</td><td>Cell Signaling</td><td>7076S</td><td></td></tr><tr><td>Ni-NTA-Agarose</td><td>Tribioscience</td><td>TBS9202-100</td><td></td></tr><tr><td>IgG-CH1 affinity resin</td><td>Thermo Fisher</td><td>194320005</td><td></td></tr><tr><td>Ficoll-Plaque Plus</td><td>GE Healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>NK cell enrichment kit</td><td>Stemcell</td><td>19055</td><td></td></tr><tr><td>Magnet</td><td>Stemcell</td><td>18000</td><td></td></tr><tr><td>CCK8 kit</td><td>Dojindo</td><td>CK04</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>C11995500CP</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>C11875500CP</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15050-057</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Cell culture</td></tr><tr><td>Standard marker</td><td>Sigma Aldrich</td><td>MWGF200</td><td>Gel filtration</td></tr><tr><td>Isopropyl-b-D-thio-galactopyranoside (IPTG)</td><td>VWR chemicals</td><td>VWRC0487-100G</td><td></td></tr><tr><td>Dialysis tubing</td><td>Sigma Aldrich</td><td>D0655-100FT</td><td></td></tr><tr><td>Knanamycine</td><td>VWR</td><td>VWRC0408-100G</td><td></td></tr><tr><td>Ampicillin</td><td>VWR</td><td>VWRC0339-100G</td><td></td></tr><tr><td>Tryptone</td><td>Thermo Fisher</td><td>LP0042B</td><td></td></tr><tr><td>Yeast Extract</td><td>Thermo Fisher</td><td>LP0021B</td><td></td></tr><tr><td>NaCl</td><td>Sangon Biotech</td><td>A100241</td><td></td></tr><tr><td>Trizma base</td><td>Sigma Aldrich</td><td>T6791-1KG</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>V900106</td><td></td></tr><tr><td>Glycine</td><td>aladdin</td><td>A110752-500g</td><td></td></tr><tr><td>KCl</td><td>aladdin</td><td>P112134-500g</td><td></td></tr><tr><td>MgCl</td><td>Sigma Aldrich</td><td>V900020</td><td></td></tr><tr><td>Agar</td><td>Sangon Biotech</td><td>A505255-0250</td><td></td></tr><tr><td>Potassium Phosphate, Monobasic Anhydrous (KH2PO4)</td><td>VWR</td><td>7778-77-0</td><td></td></tr><tr><td>Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4)</td><td>VWR</td><td>7558-79-4</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>VWR</td><td>60-24-2</td><td></td></tr><tr><td>Phenylmethyl Sulfonyl Fluoride (PMSF)</td><td>VWR</td><td>329-98-6</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma Aldrich</td><td>L6876-25G</td><td></td></tr><tr><td>Coomassie Brilliant Blue R250</td><td>VWR</td><td>VWRC0472-50G</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sangon Biotech</td><td>A500922-25G</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>VWR</td><td>VWRC0332-100G</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>V900122</td><td></td></tr><tr><td>100mm x 20mm plastic dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>25cm2 flask</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>96 well cell culture cluster</td><td>Corning</td><td>3599</td><td></td></tr><tr><td>Sucrose</td><td>Sangon Biotech</td><td>A610498-0005</td><td></td></tr><tr><td>CHO</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>GNHa 3</td><td></td></tr><tr><td>MHCC-97H</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>SCSP-528</td><td></td></tr><tr><td>HepG2</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>TCHu 72</td><td></td></tr><tr><td>Huh7</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>TCHu182</td><td></td></tr><tr><td>Hep3B</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>TCHu106</td><td></td></tr><tr><td>NK92</td><td>ATCC</td><td>CRL2408</td><td></td></tr></tbody></table>

a gpc3-targeting bispecific antibody, gpc3-s-fab, with potent cytotoxicity

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。

GPC3-S-Fab 净化
GPC3-S-Fab 从大肠杆菌中纯化, 经两步亲和纯化, 先用镍 NTA 琼脂糖, 其次 IgG-CH1 亲和纯化。两步亲和纯化后, GPC3-S-Fab 被纯化为同质性, 两链接近 1:1 (图 2A)。VH-CH1-CD16 VHH 和 cl 多肽的存在可以通过它们独特的 C 终端标记来识别, 反他的大约 25 kDa, 以及 VH-CH1-VHH 大约 38 kDa 的反旗 (...

Watch this Scientific Journal Video about A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity at JoVE.com

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

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。

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

a-gpc3-targeting-bispecific-antibody-gpc3-s-fab-with-potent

Research

JoVE Journal

Cancer Research

8.5K 次观看.  Sun Yat-Sen University. 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。

视频: A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

靶性抗原特异性治疗抗体的肿瘤及组织分布分析

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

科研

癌症研究

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

生物化学

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

生物工程

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

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as valency, spatial arrangement, interdomain distance, and Fc mutations affect the anti-tumor efficacy of these therapies, commonly by influencing the homing of T cells to tumors, which remains a major challenge. Here, we describe a method to transduce activated human T cells with luciferase, allowing in vivo tracking of T cells during T-BsAb therapy studies. The ability of T-BsAbs to redirect T cells to tumors can be quantitatively evaluated at multiple time points during treatment, allowing researchers to correlate the anti-tumor efficacy of T-BsAbs and other interventions with the persistence of T cells in tumors. This method alleviates the need to sacrifice animals during treatment to histologically assess T cell infiltration and can be repeated at multiple time points to determine the kinetics of T cell trafficking during and after treatment.

Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies.

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 ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

免疫与感染

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

通过抗原肽和Fc-III三氧化二甲物的双功能结合阻断靶向抗体

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

人诱导多能干细胞衍生的嵌合抗原受体表达自然杀伤细胞的研究进展

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...