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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N., Arbabi-Ghahroudi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+single-domain+antibodies+in+bacterial+systems.">Expression of single-domain antibodies in bacterial systems.</a> Methods in Molecular Biology. 911, 257-275 (2012).</li><li>Arbabi-Ghahroudi, M., Tanha, J., MacKenzie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotic+expression+of+antibodies.">Prokaryotic expression of antibodies.</a> Cancer and Metastasis Reviews. 24 (4), 501-519 (2005).</li><li>Spiess, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies+with+natural+architecture+produced+by+co-culture+of+bacteria+expressing+two+distinct+half-antibodies.">Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies.</a> Nature Biotechnology. 31 (8), 753-758 (2013).</li><li>Filmus, J., Selleck, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypicans:+proteoglycans+with+a+surprise.">Glypicans: proteoglycans with a surprise.</a> Journal of Clinical Investigation. 108 (4), 497-501 (2001).</li><li>Baumhoer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican+3+expression+in+human+nonneoplastic,+preneoplastic,+and+neoplastic+tissues:+a+tissue+microarray+analysis+of+4,387+tissue+samples.">Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples.</a> American Journal of Clinical Pathology. 129 (6), 899-906 (2008).</li><li>Llovet, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+signature+to+discriminate+dysplastic+nodules+from+early+hepatocellular+carcinoma+in+HCV+cirrhosis.">A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis.</a> Gastroenterology. 131 (6), 1758-1767 (2006).</li><li>Zhu, Z. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+glypican-3+expression+differentiates+the+majority+of+hepatocellular+carcinomas+from+benign+hepatic+disorders.">Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders.</a> Gut. 48 (4), 558-564 (2001).</li><li>Hsu, H. C., Cheng, W., Lai, P. 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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&#174; 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&#38;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><a name="RANGE!A50">Coomassie Brilliant Blue R250</a></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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

科研

癌症研究

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

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 ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

自动差分核染色测定，准确测定三元细胞毒性

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an autoinjector apparatus, trained operators can achieve more efficient and consistent transplantation results compared to those obtained using a manual injector. Here, we cover topics in a chronological fashion: from the crossing of Drosophila lines, to the induction and dissection of the primary tumor, transplantation of the primary tumor into a new adult host and continued generational transplantation of the tumor for extended studies. As a demonstration, here we use Notch intracellular domain (NICD) overexpression induced salivary gland imaginal ring tumors for generational transplantation. These tumors can first be reliably induced in a transition-zone microenvironment within larval salivary gland imaginal rings, then allografted and cultured in vivo to study continued tumor growth, evolution, and metastasis. This allotransplantation method can be useful in potential drug screening programs, as well as for studying tumor-host interactions.

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol provides detailed guidance for the initial and continued generational allotransplantation of Drosophila tumors into the abdomen of adult hosts for studying various aspects of neoplasia. Using an autoinjector apparatus, researchers can achieve improved efficiency and tumor yields compared to those achieved by traditional, manual methods.

使用可编程自动纳升注射器在 黑色果蝇 中植入肿瘤异体

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an ...

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), relying on collateral flow to the liver through the gastroduodenal artery (GDA). In the current manuscript, the technical conduct of robotic DP-CAR is outlined. The greater curve of the stomach is mobilized with care to avoid sacrificing the gastroepiploic vessels. The stomach and liver are retracted cephalad to facilitate dissection of the porta hepatis. The hepatic artery (HA) is dissected and encircled with a vessel loop. The gastroduodenal artery (GDA) is carefully preserved. The common HA is clamped and triphasic flow in the proper HA via the GDA is confirmed using intra-operative ultrasound. A retropancreatic tunnel is made over the superior mesenteric vein (SMV). The pancreas is divided with an endovascular stapler at the neck. The inferior mesenteric vein (IMV) and splenic vein are ligated. The HA is stapled proximal to the GDA. The entire specimen is retracted laterally with further dissection cephalad to expose the superior mesenteric artery (SMA). The SMA is then traced back to the aorta. The dissection continues cephalad along the aorta with the bipolar energy device used to divide the crural fibers and celiac nerve plexus. The specimen is mobilized from the patient's right to left until the origin of the celiac axis is identified and oriented towards the left. The trunk is circumferentially dissected and stapled. Additional dissection with hook cautery and the bipolar energy device fully mobilizes the pancreatic tail and spleen. The specimen is removed from the left lower quadrant extraction site and one drain is left in the resection bed. A final intra-operative ultrasound of the proper HA confirms pulsatile, triphasic flow in the artery and liver parenchyma. The stomach is inspected for evidence of ischemia. Robotic DP-CAR is safe, feasible and when used in conjunction with multi-modality therapy, offers potential for long-term survival in selected patients.

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection DP-CAR for Pancreatic Cancer: Surgical Planning and Technique

We present our operative approach to robot assisted distal pancreatectomy, splenectomy, and celiac axis resection (DP-CAR), demonstrating that the procedure is safe and feasible with proper planning, patient selection, and surgeon experience.

机器人协助切除腹腔轴切除术 （DP-CAR） 胰腺癌:手术规划和技术

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), ...

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

摘要

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此视频中的章节

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

靶性抗原特异性治疗抗体的肿瘤及组织分布分析

自动差分核染色测定，准确测定三元细胞毒性

监测三D培养中的癌细胞入侵和T细胞毒性

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

使用可编程自动纳升注射器在黑色果蝇中植入肿瘤异体

机器人协助切除腹腔轴切除术（DP-CAR）胰腺癌:手术规划和技术

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

摘要

探索更多视频

此视频中的章节

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

靶性抗原特异性治疗抗体的肿瘤及组织分布分析

自动差分核染色测定，准确测定三元细胞毒性

监测三D培养中的癌细胞入侵和T细胞毒性

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

使用可编程自动纳升注射器在 黑色果蝇 中植入肿瘤异体

机器人协助切除腹腔轴切除术 （DP-CAR） 胰腺癌:手术规划和技术

使用可编程自动纳升注射器在黑色果蝇中植入肿瘤异体

机器人协助切除腹腔轴切除术（DP-CAR）胰腺癌:手术规划和技术