Name: initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting
Uploaded: 2018-03-08T12:30:00.000+00:00
Duration: 11 min 58 s
Description: Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

这项工作由癌症研究协会 (加拿大) 和 CIMS 资助。作者感谢 Samia 博士 Mohand 和吉恩-弗朗索瓦 Beaudoin 的帮助。天使洛佩兹博士 (南澳大利亚大学) 为单克隆 A14。

所描述的体内动物实验是根据一项经批准的议定书和中心 Hospitalier Universitaire de 舍布鲁克伦理学委员会动物实验的道德准则进行的。
1. 抗体肽共轭
注: ChAcNLS 可在任何商业肽制造商或大学附属的多肽综合服务平台上合成。ChAcNLS 的合成可以在参考34中找到。对于程序1和2使用单克隆 7G3, 这是特定的白血病抗原 IL-3Rα 39。
<ol><li>在一个1.7 毫升的离心管, 准备10毫克/毫升 (1 毫克总抗体) 溶液的7G3 在磷酸盐缓冲盐水 (PBS), pH 7.6。</li>
	<li>将磺-当局在二甲基亚砜 (亚砜) 中溶解, 浓度为5-10 毫米。涡流解决方案, 以达到完全溶解的最佳效果。</li>
	<li>添加到包含7G3 溶解磺-当局溶液的管 (通常在 5-20 ul 之间取决于 sulfo-SMCC-to-7G3 所需的摩尔比)。建议对10、25和50到1的 sulfo-SMCC-to-7G3 比率进行测试。室温孵育1小时。</li>
	<li>
在 PBS、pH 值7.0 中使用超滤装置和缓冲交换纯化和浓缩 maleimide-衍生物7G3。离心机在 8000 x g 左右, 约5分钟丢弃, 并填充 PBS, pH 值7.0 和离心机。
	<ol>
<li>执行这7-8 次以完全交换缓冲区。将过滤装置倒在干净的离心管中, 以恢复浓缩7G3 蛋白。离心机为2分钟, 在 1000 x g. 回收量应约0.3 毫升</li>
	</ol>
</li>
	<li>在含有 maleimide 活化7G3 的管中, 增加2倍摩尔的 ChAcNLS 相对于上一步所用的 sulfo-SMCC-to-7G3 比, 并在4摄氏度一夜之间孵化。例如, 如果使用了10到1的 sulfo-SMCC-to-7G3 比率, 则使用20到1的 ChAcNLS-to-7G3 比率。总成交量不应显著改变。 注意: 虽然 ChAcNLS 在共轭过程中不会引起降水, 但这是可能发生的其他肽。在反应过程中观察管内沉淀的迹象, 这很可能是由于多肽是疏水引起的。在这些情况下, 它可能值得重新研究的兴趣肽, 以便设计变化, 以提供增加的亲水性。</li>
	<li>使用超滤装置将 7G3 ChAcNLS 浓缩到所需浓度和 PBS pH 值7.4 的缓冲交换 (见步骤 1.4)。总蛋白回收率应为原起始材料的≥75%。</li>
	<li>使用12% 聚丙烯酰胺凝胶 (提供足够的分离 ChAcNLS 共轭重和轻链从 nonconjugated 链), 执行 SDS 页评估构建的 7G3 ChAcNLS 候选者。混合10微克的 7G3-ChAcNLS 在页面加载缓冲区包含 2 ul β-巯基乙醇和负载入井。</li>
	<li>设置适当的电压 (120 V 为1小时12% 凝胶) 和运行电泳。当考马斯染料前端到达凝胶底部时, 停止电泳。 注意: 不要让考马斯染料前面的凝胶。</li>
	<li>从电泳装置中取出凝胶, 用含有10% 伏/v 的冰乙酸和20% 伏/钒甲醇的脱色溶液冲洗。</li>
	<li>采取凝胶的相片和用标尺和测量距离 (cm) 迁移为标准和7G3 共轭物。计算保留系数 (Rf) 值, 这是迁移距离除以凝胶长度 (从何处将蛋白质加载到考马斯迁移前端)。将分子量 (兆瓦) 与每个蛋白质标准中的射频值进行记录, 并推断7G3 共轭物的大小。</li>
	<li>用1768.5 克/摩尔 (兆瓦 ChAcNLS) 除以7G3 共轭和未修改7G3 之间的差异, 计算每种抗体的肽数量。</li>
	<li>采取考马斯染色凝胶的数字图像, 进行密度分析。在这一点上, 选择一个潜在的候选者可以根据 AC 与最大数量的 ChAcNLS 分子, 不包含重要的聚集物种。</li>
</ol>2. 细胞内聚集评价的共焦显微术
注意: 重要的是要测试的细胞选择性的肽修饰抗体之前, 开发配方与有效载荷的利益。由于达也被证明具有非特异性细胞穿透的倾向, 它值得首先分析细胞内积累和细胞选择性, 然后采取昂贵的有效载荷的开发步骤。因此, 程序1还应修改同种特定无关的控制抗体。对于该协议的其余部分, 我们将使用 10 ChAcNLS 分子每抗体修改 ACs。图 2中描述了包括过程2和3的关键步骤的示意图。
注意: 该协议的这一步骤涉及对多聚甲醛的处理和操作。处理时请遵照制造商的指示操作。
<ol><li>治疗靶 TF-1a 细胞与200毫微米的 7G3-ChAcNLS 包括改良控制抗体。孵育 5 x 106抗原阳性细胞与共轭物为 1 h 在37°c。</li>
	<li>1小时后, 取出上清, 用1毫升3x 的冰冷 PBS 冲洗。添加新鲜的媒体和孵化一个额外的小时在37摄氏度。</li>
	<li>
在增加的小时以后, 去除上清和洗涤3x 在1毫升冰冷 PBS。添加0.5 毫升的 PBS, 含0.25% 胰蛋白酶和乙二胺四乙酸 (EDTA), 37 摄氏度, 3 至30分钟。 注: 在初步试验中, 建议不要胰蛋白酶处理细胞, 以确定交流细胞内积聚的差异。这个协议促进了胰蛋白酶的使用, 我们展示了如何改善不同交流候选者细胞内积累效率的评估。<ol>
<li>通过目视检查所有要分离的单元格, 测试不同的孵化时间。此外, 孵化细胞整除数与生存能力染色解决方案和执行流细胞分析, 如前所述的 40. </li>
		<li>中和胰蛋白酶与1.5 毫升细胞培养 RPMI 1640 media/10%fetal 牛血清。离心细胞在 500 x g 5 分钟, 去除上清, 并且洗涤3x 在0.5 毫升冰冷 PBS。</li>
	</ol>
</li>
	<li>固定细胞在0.5 毫升 pbs 包含1% 多聚甲醛和1% 蔗糖在冰上30分钟洗涤细胞3x 在0.5 毫升冰冷 pbs 并且离心机在 250 x g 在5毫升 pbs 中0.5 个 Permeabilize 细胞 0.15% ml 在冰上包含分钟的海卫 X-100。在冰冷的 PBS 中洗涤细胞并重复离心。</li>
	<li>
悬浮细胞在0.1 毫升 PBS 包含2微克/毫升 (或制造商推荐浓度) 的抗鼠 (同种特异) Fc 次级多克隆抗体共轭到 AlexaFluor 647 在室温下在黑暗中的1小时。
	<ol>
<li>离心机在 250 x g 5 分钟和洗涤细胞3x 在0.5 毫升冰冷 PBS。在0.5 毫升 PBS 中悬浮细胞。 暂停: 单元格可以存储在冰箱中。 注意: 必须选择一个二级抗体, 识别出正确的同种的单克隆的兴趣。AF647 是由于其远红外辐射而选择的, 不干扰碘碘 (PI) 染色的细胞核。</li>
	</ol>
</li>
	<li>添加 PI 到10微克/毫升浓度的容器与细胞。接下来, 将 1 x 105单元格安装到玻璃幻灯片上, 使用安装介质并盖上玻璃盖玻片。</li>
	<li>检查细胞与计划的60X 油浸泡目标 NA 1.42 在反向激光扫描共聚焦显微镜。检测 PI 荧光使用 488 nm 氩激光和光谱扫描棱镜设置为 600-650 nm。为 AF647 荧光使用633毫微米氦氖激光和光谱扫描棱镜设置为 650-700 毫微米。依次收集 PI 和 AF647 的荧光辐射。 注意: 在单元格的评估和比较中使用相同的设置。但是, 与非 trypsinized 单元格相比, 您必须调整 trypsinized 单元格的设置。</li>
	<li>使用显微镜软件获取图像。使用 1024 x 1024 像素的串行水平光学剖面收集图像, 并通过整个细胞厚度在0.5 微米间隔内进行2X 线平均。在最大荧光强度下, 以四连续切片显示图像为堆叠 z 投影。</li>
	<li>用显微镜软件分析细胞。记录共轭物的细胞分布格局。具体来说, 评估细胞质内的细胞内荧光是否被分组, 靠近细胞表面或弥漫和均匀。还要评估每个细胞的相对荧光强度。</li>
</ol>3. 核素交流结构和细胞分馏法评价64铜胞内分娩效率
注意: 程序3和4涉及放射性的处理和操纵。在执行这些步骤之前, 研究人员应该已经批准了他们的家庭机构的辐射安全局批准的安全培训和协议。
注: 对于程序3和 4, 我们使用单克隆 A14, 这是特定的浸润性膀胱癌抗原 IL-5Rα41。此外, 由于64Cu 的短半衰期, 所有实验都是时间敏感的。一般而言, 最好不要等待过去1周来执行体外实验, 而不超过72小时用于体内研究。
<ol><li>在1.7 毫升管悬浮 22 '-(7-(2-(25-dioxopyrrolidin-1-基) 氧)-2-乙醛基)-14, 7-triazonane-14-己) diacetic 酸 (不-NHS) 在亚砜到浓度10毫米。</li>
	<li>反应50倍摩尔过量的不-nhs 与 A14 (2 毫克起始材料) 在0.1 米碳酸氢钠在 pH 8.6 为 1 h 在室温下的容积≤0.5 毫升 (最后的非 NHS 卷不应超过 2% v/v 的总体积)。</li>
	<li>净化和缓冲交换 NOTA-A14 使用一个超滤装置在 PBS, pH 值 7.6 (见步骤 1.4)。</li>
	<li>对于后续的 ChAcNLS 连接, 请执行步骤 1.2-1.6。</li>
	<li>反应 NOTA-A14-ChAcNLS (250 微克批次) 与 100 megabecquerel (MBq) 64CuCl2在 0.1 M 碳酸氢钠, pH 5.5 为 1 h 在37°c ≤0.1 毫升。64铜是在 CIMS 42上的 TR PET 回旋加速器上生产的。</li>
	<li>将64Cu-A14-ChAcNLS 集中在 PBS pH 值7.4 中, 使用超滤装置达到所需浓度 (见步骤 1.4)。</li>
	<li>
用色谱条和 0.1 M, pH 值5.5 柠檬酸钠 (ITLC 淋洗) 作为溶剂, 确定64Cu-A14-ChAcNLS 的 radiolabeling 效率 (ITLC)。将反应溶液的 0.5 ul 整除应用于 ITLC 条。执行 autoradiograph, 获取数字图像。
	<ol>
<li>执行密度测量以获得绑定和自由64Cu 的比例。如果可用64Cu 内容为 > 5%, 请返回上一步并执行其他净化操作。</li>
		<li>此外, 使用考马斯染色进行 SDS 页来评估聚合。执行第二个 SDS 页, 然后 autoradiograph 的凝胶, 以确定是否有核素的聚合物种。</li>
	</ol>
</li>
	<li>
亲和结合质量检查点: 孵育64Cu-A14 共轭在增加浓度 (0-100 nM) 以重复与 1 x 106目标细胞每毫升。要估计非特异性绑定, 请将64Cu-A14 与细胞的重复样本混合在100倍摩尔超过未标记 A14 的情况下。
	<ol>
<li>在冰上孵化1小时后, 在伽玛计数器中冲洗细胞和计数总束缚抗体样本和总非特异性束缚抗体样本。图特定绑定 (总绑定抗体-非特异性绑定抗体) 反对无活性自由64Cu-A14 (nM) 在化验中使用。通过使用图形软件进行非线性回归确定离解常数 (Kd)。</li>
	</ol>
</li>
	<li>对于64cu 细胞积累研究: 以 100 nM 的64铜标记的 A14 共轭物治疗细胞 1 h, 6 h, 24 h 在37°c, 如前所述33。包括其他参数, 如在未修改的 A14 存在的情况下进行治疗, 以阻止 IL-5Rα地点和4摄氏度, 以阻断受体介导的内部化。</li>
	<li>在定义的时间点, 如前述步骤 2.3-2.3.2, 添加0.5 毫升的 PBS, 含有0.25% 胰蛋白酶和 EDTA 在37°c, 其次是中和和洗涤。</li>
	<li>在 1% NP-40, 10 毫米三 pH 7.5, 10 毫米氯化钠, 3 毫米氯化镁2缓冲器的等离子膜裂解缓冲液中孵化细胞为10分钟. 离心机在 90 x g 的等离子膜裂解细胞为5分钟。</li>
	<li>取出上清液并放置在新鲜的管子中。这代表细胞质分数。在冰冷的 PBS 中洗涤原子核 3x, 并将洗涤液加入胞浆部分。</li>
	<li>确保含有核和细胞质馏分的瓶子密封。然后将它们放在校准为64Cu 的伽玛计数器中, 以便将原始计数转换为 MBq。</li>
	<li>
通过对 Lamin、限制性核蛋白和 Rab7, 对全细胞裂解液、核和细胞质馏分进行了丰富的细胞质蛋白分析, 确定核分离的质量。
	<ol>
<li>将 5 x 106单元格与 500 ul 的无线电免疫沉淀检测 (马国贤) 缓冲器和在3.12、1% 和 2% NP-40 的步骤4% 中的等离子膜裂解缓冲液治疗。此外, 在 500 ul 马国贤缓冲液中分离出的细胞核, 以获得分离的核蛋白。</li>
		<li>用4x 的冷丙酮样品体积, 沉淀分离的核、细胞质和全细胞蛋白, 60 分钟-20 摄氏度。离心机在 1.3万 x g 的10分钟和醒酒上清, 小心不要去除蛋白颗粒。添加 100 ul 的 PBS, 以溶解蛋白质。</li>
		<li>将10微克的蛋白质装入10% 页凝胶的每个井中。按照 1.7-1.9 的描述进行电泳。执行以前在 refeference 43中描述的西方印迹传输。</li>
		<li>找出最符合 40 kDa 的阶梯标记。用这个标记把膜切成两半。用 Lamin A/C 特异抗体探测含有较高兆瓦蛋白的印迹。用7特异性抗体探测膜与下兆瓦蛋白。西方污点是一个众所周知的技术, 并没有在本议定书中描述。</li>
	</ol>
</li>
</ol>4. 肿瘤靶向的 PET 成像评价
注: 在小鼠体内植入肿瘤细胞以产生异位移植的技术是众所周知的, 大多数实验室都有为其肿瘤系统量身定做的内部协议。因此, 议定书没有涵盖这一点。异种移植模型将非肥胖糖尿病/重症合并 immunodeficient (点头/免疫缺陷) 小鼠, IL-5Rα-positive 浸润性膀胱肿瘤 HT-1376 和 HT-B9。IL-5Rα-positive 浸润性膀胱肿瘤细胞包括 > 66% 的总细胞在异种移植。相比之下, 仅有11% 的 IL-5Rα-positive HT-B9 细胞包含在发育中的移植性41中。因此, 该模型提供了一个很好的例子, 以评估交流肿瘤靶向两个肿瘤, 代表可预见的病人肿瘤异质性。
<ol><li>在含有≥ 4 (点头/免疫缺陷) 小鼠的组中, 向尾静脉注入20-30 微克 (6-9 MBq) 64Cu-A14-ChAcNLS、 64Cu-A14-NLS 和64Cu-A14。</li>
	<li>在同一天放置一个圆柱形幻影 (24.8 毫升), 其中包含 5 MBq 的64铜, 将每秒的放射性计数转换为每克组织的注射剂量 (%ID/克)。</li>
	<li>等待48小时, 然后麻醉老鼠放置在一个感应腔内, 1.5 升/分钟的氧流与2% 异氟醚。在植入 PET 扫描仪前, 将无菌眼膏应用于鼠眼。</li>
	<li>将鼠标迅速转移到一个宠物扫描仪表中的头俯卧位置与鼻锥的异氟醚。定位呼吸和直肠探头, 并监测温度和呼吸率, 以确保在实验中保持生理条件, 如前所述的44。</li>
	<li>使用常规采样模式设置和 250-650 凯文的能量窗口开始获取 PET 数据。通常, 每只鼠标45分钟的静态扫描足以为高质量的 PET 图像的重建和生产提供足够的重合事件。</li>
	<li>在64铜交流扫描之后, 从扫描仪中取出鼠标并将其放回笼子中。允许鼠标在返回动物设施之前充分恢复。</li>
	<li>利用三维最大似然期望最大化算法的20次迭代获得重建图像。</li>
	<li>
使用酰胺软件对肿瘤和视觉上应评的器官进行兴趣区 (ROI) 分析。
	<ol>
<li>绘制 ROIs 获取定量%ID/g 数据手动使用3D 手绘工具设置, 并仔细描绘肿瘤或相邻大腿肌肉。ROI 中每个像素的计数将从上一步骤4.2 转换为%ID/g。</li>
	</ol>
</li>
	<li>比较64Cu-A14、 64Cu-A14-ChAcNLS、 64Cu-A14-NLS 图像以进行肿瘤对比, 以及定量 ROI%ID/g 和肿瘤到背景比率。</li>
</ol>

作者没有什么可透露的

系统地提供抗癌药物的主要目标是增加肿瘤部位的积聚, 并在癌细胞中吸收, 并减少健康组织中有害的副作用。交流靶向肿瘤细胞提供分子有效载荷是一种非常有前景的治疗和检测肿瘤的方法。然而, endosome 和下游溶酶体退化所引起的疗效仍然是一个重要的挑战。虽然该协议利用 ChAcNLS 肽作为下一代 ACs 的一个例子, 但所描述的程序可以适应任何被开发的多肽修饰交流, 目的是增加细胞内交付的负载?...

抗体共轭 (ACs) 是生物成熟成为一个转化类的有效药物改善癌症治疗和检测肿瘤。ACs 由与分子有效载荷 (如放射性同位素、小分子和生物毒素) 共轭的单克隆抗体组成, 能够将这些有效载荷传递给具有精致靶抗原亲和性和特异性的癌细胞。因此 ACs 有可能显著减少非特异性毒性, 并增加肿瘤部位的有效载荷活动。治疗, ACs 运输细胞毒性小分子 (通常称为抗体-药物共轭) 已被批准处理乳腺癌和霍奇金淋巴瘤谁已经失败常规治疗1,2. 此外, 运输放射性同位素 (通常称为 radioimmunoconjugates) 的 ACs 也在开发中。通过交流传输放射性同位素进行成像, 确认前列腺癌转移3。随着更多的治疗 acs 提交批准4, 乐观是高的未来 acs, 以改善癌症护理5。
然而, 在运送化疗或放射性同位素时, ACs 很难有效地在靶细胞内积聚这些有效载荷。在许多情况下, 这方面显著地有助于 ACs 无法提供持久无疾病生存或高对比度肿瘤成像6,7。一般而言, 一旦 ACs 结合其目标抗原, 它们就会通过称为受体介导的吞的过程内部化。然后, ACs 被包裹在内涵体内并被贩运到溶酶体中, 用于降解和有效载荷释放 8. 细胞内的贩运过程给 ACs 带来了挑战, 以达到高有效载荷特异性和抗靶向癌细胞的功效。例如, 许多抗原, 如 Her2 (治疗交流曲妥珠单抗-emtansine 靶) 可以在前 30分钟9 中回收多达85% 的绑定抗体。此外, 一旦降解发生, 释放的化疗和放射性同位素可以通过增加表达和/或活动的膜相关传输蛋白10,11积极出口。溶酶体的降解也阻碍了新的生物有效载荷的传递, 如治疗酵素和寡核苷酸, 可以被停用12,13。从本质上讲, 癌细胞是高效的废除细胞内积累的有效载荷由 ACs 提供。
该协议描述了如何实现 ACs-耦合到病毒衍生肽的概念, 特别是为了逃避 endosome 诱捕和本地化到细胞核。这种复杂的操作宿主细胞系统, 这并不奇怪, 病毒衍生蛋白和肽作为潜在的生物的发展早已根深蒂固的治疗研究14。为了有效地进入宿主细胞, 在数以百万年的时间里, 病毒进化来获得一种特殊的蛋白质集合, 能够利用正常的生理哺乳动物细胞系统。对于通过受体介导的吞内化的病毒, 他们也面临着逃避贩运到溶酶体的挑战, 那里的蛋白酶局部浓度的冲击可能会对生存有问题。一种具有良好特征的病毒衍生肽, 用于药物的传递, 以逃避 endosome 诱捕是人体免疫机能丧失病毒 transactivator 转录 (达) 蛋白15。达能通过传感低 pH 值来逃避 endosome 诱捕, 在这种情况下, 蛋白质构象的变化发生, 使达能够插入和扰乱细胞膜膜16。这就产生了能进入细胞质的达-负载共轭物。与此协议相关的第二个病毒操作元素是用于将治疗基因和药物传递给核的方法17。病毒已经进化到成功地操纵宿主细胞机械, 通过核孔复合体通过核膜的进展。蜂窝大分子包含 (或结合含有的蛋白质) 核定位信号 (NLSs), 以绑定到核转运蛋白 (例如karyopherins α和β), 通过 NPC 提供必要的运动。病毒已经开发出含有 NLS 序列的蛋白质, 使它们能够利用宿主细胞传输蛋白来传送到细胞核18。
许多 ACs 以前已经功能性与达-和 NLS 衍生肽, 并测试他们的能力积累在癌细胞和靶向肿瘤19,20,21,22 ,23,24,25,26,27,28,29,30 (表 1)。提供细胞毒性有效载荷的研究表明, 通过病毒衍生肽进行修饰的 acs 能够显著增加细胞积累、细胞毒性和肿瘤死亡, 超过未修改的 ACs 22,26。这个新的交流类的一个共同特点是他们的建设。通常情况下, 多肽含有一个末端半胱氨酸, 提供游离巯基。抗体首先反应的是 noncleavable 双交联剂, 其中包含N-琥珀 (NHS) 和 maleimide 组在相反的一端。NHS 酯在单克隆抗体上与主要胺反应形成酰胺键。与自由 maleimide 组的反应, 然后反应与巯基组对肽形成一个硫酯键, 从而连接肽和单克隆。虽然同型交联剂已使用 28, 但 heterobifunctional 交联剂更常用于构建病毒衍生肽-ACs22,23,26, 31,32。本议定书特别使用交联 sulfosuccinimidyl 4-(n-马来) 环己烷-1-羧酸酯 (磺当局), 以便于使用, 因为它被用于经批准的抗体-药物共轭曲妥珠单抗-emtansine, 在许多病毒衍生肽-ACs 8,22,23,26,31,32。月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 是初步确定共轭效率和半定量的多肽每单克隆的主要方法。共焦显微术使用荧光标记的二次抗体特定于单克隆, 通常是初步评价病毒衍生肽修饰 ACs 细胞内分布特性的方法。迄今为止, 放射性同位素是由病毒衍生的肽修饰 ACs 提供的主要有效载荷。放射性同位素是有利的, 因为细胞中的放射性可以很容易地通过伽玛计数来量化。此外, 被翻译成老鼠的人类癌症模型的 ACs 为研究人员提供了评估肿瘤靶向性的能力, 例如单光子发射计算机断层扫描和正电子发射层析成像 (PET)。23,32,33. 一般而言, 研究人员主要使用的构造和验证测试方法为在初始开发阶段对病毒衍生肽进行了改进的 ACs 提供了很好的评估, 以便有效地进入和交付靶细胞内的有效载荷和靶向肿瘤。
达-和 NLS 修饰的 ACs 已经照亮了进一步改善肿瘤细胞和肿瘤内有效载荷的关键领域。关于 NLS 修改的 ACs, 细胞内积累的效率可以是适度的23,31,34。细胞内积聚效率低下是由持续的细胞膜诱捕引起的。在体内肿瘤靶向也可以减少与达-和 NLS 修改 ACs。有效序列的达和 NLS 含有几个正电荷残留物。当附加到抗体时, 总阳离子电荷可以显著增加35。因此, 经和 NLS 改良的 ACs 增加了对健康组织的吸收, 并增加了快速的血液清除量。
我们的小组开发了一个复合化合物组成的胆酸与 NLS (ChAcNLS;图 1)。ChAcNLS 修饰的 acs 能够增加所提供的放射性同位素的胞内积累和改善肿瘤靶向性, 与 NLS 修改和传统 ACs 33,34。胆酸的机制是由选择 nonenveloped 病毒的能力所激发的, 不能依靠膜融合来利用胆酸通过神经酰胺的形成来触发 endosome 逃逸。例如, 猪肠道病毒新兵胆酸激活 sphingomyelinase, 催化鞘磷脂水解成神经酰胺 36, 37, 38. 这破坏细胞膜膜, 并允许病毒逃逸。因此, 胆酸是另一个病毒衍生成分, 补充 NLS。
随着这一领域的向前推进和未来的进步发生在负载交付由 ACs 修改与病毒衍生肽, 这是一个适当的时间来提供视觉示范其生物化学和功能特性的初步发展。在这里, 我们描述我们的协议, 初步评估病毒衍生肽修饰 ACs 的有效而简单的确定细胞内积累和肿瘤靶向在早期发展。我们使用商业上可用的抗体7G3 和 A14 作为示例模型系统。步骤1描述了使用 SDS 页作为一种方法, 允许对构造的 ACs 进行 "转/不走" 决策。程序2描述了一种使用 trypsinization 的方法, 用于改善交流细胞内分布和积累的可视化。步骤3描述了一种改进细胞内分馏的方法, 以准确确定核定位。在这个过程中, 我们利用负载64Cu (t1/2 = 12.7 h), 因为它易受细胞外流, 是正电子发射器10。因此, 程序4描述了在体内肿瘤靶向特征的 PET 成像, 以可视化肿瘤摄取相对于背景 (即nontarget 健康组织), 并确定该例交流是否可以具体和有效地靶向肿瘤。这些方法足以为研究人员开发的 ACs 修改与病毒衍生肽, 以确定候选者进一步提高。

<table><tbody><tr><td>Sulfo-SMCC</td><td>Thermo Scientific</td><td>22122</td><td>There are many homo- and hetero-bifunctional maleimide crosslinkers to choose from.</td></tr><tr><td>Amicon Ultra-0.5 mL Centrifugal Filters</td><td>EMD Millipore</td><td>UFC505096</td><td>There are pack sizes of 8, 24, and 96. Choose according to your needs.</td></tr><tr><td>Precision Plus Protein Kaleidoscope Standards</td><td>BioRad</td><td>1610375EDU</td><td>Mulicolor recombinant proteins from 10 - 250 kDa.</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Scientific</td><td>25200056</td><td>100 or 500 mL volumes to choose from.</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td><td>Thermo Scientific</td><td>A-21235</td><td>1 - 10 &amp;mu;g/mL recommended</td></tr><tr><td>NOTA-NHS</td><td>CheMatech</td><td>C100</td><td></td></tr><tr><td>Lamin A/C antibody (N-18)</td><td>Santa Cruz Biotechnology</td><td>sc-6215</td><td></td></tr><tr><td>Rab7 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-376362</td><td></td></tr><tr><td>A14 mAb</td><td>BD Biosciences</td><td>555902</td><td></td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Thermo Scientific</td><td>NP0007</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M3148-25ML</td><td></td></tr><tr><td>TF-1a cells</td><td>ATCC</td><td>ATCC CRL-2003</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>ATCC</td><td>ATCC 30-2001</td><td></td></tr><tr><td>RIPA lysis and extraction buffer</td><td>Thermo Scientific</td><td>89900</td><td></td></tr><tr><td>AMIDE medical imaging software</td><td>available at amide.sourceforge.net</td><td></td><td>Completely free download</td></tr><tr><td>FluoView FV1000 Confocal Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Fluoview Software</td><td>Olympus</td><td></td><td>www.olympus-lifescience.com</td></tr><tr><td>ITLC strips</td><td>Biodex</td><td>150-771</td><td></td></tr></tbody></table>

initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting

用病毒衍生肽修饰的抗体共轭物 (acs) 是一种潜在强大的肿瘤细胞传递剂, 用于癌症治疗和影像学的分子有效载荷, 原因是细胞积累超过了目前的 ACs。在早期交流体外开发过程中, 荧光技术和 radioimmunoassays 对确定细胞内定位、积累效率和靶细胞特异性都有足够的作用。目前, 对于制备细胞以评价交流细胞内聚集和定位的标准化方法尚无共识。对使用病毒衍生肽进行修饰的 ACs 的初步测试是至关重要的, 特别是如果已经建立了几个候选者。通过荧光法测定细胞内的细胞积累, 可影响其表面的背景信号, 使堆积物的解释复杂化。对于 radioimmunoassays, 通常被处理的细胞被分割, 并测量不同细胞间的放射性。然而, 细胞裂解从细胞到细胞不同, 通常细胞核和细胞质隔间没有充分的隔离。这可能会产生关于负载传递属性的误导性数据。核素病毒衍生肽修饰 ACs 在肿瘤荷载小鼠中的静脉注射和核素显像是确定肿瘤靶向和有效载荷传递特性的有力方法, 在体内阶段发展.然而, 这是一个相对较新的进展, 很少有团体以这种方式评估病毒衍生的肽修饰 ACs。我们描述治疗细胞的处理, 以更准确地评估病毒衍生的肽修饰交流积累时, 使用共焦显微镜和 radioimmunoassays。具体地说, 一种 trypsinizing 单元格移除单元格表面绑定 ACs 的方法。我们还提供了一种改善细胞分馏的方法。最后, 本协议提供了一个体内方法, 使用正电子发射断层扫描 (PET) 评估肿瘤小鼠的初始肿瘤靶向性。我们使用放射性同位素64Cu (t1/2 = 12.7 h) 作为本协议中的一个示例负载。

在程序1中, 用磺当局作为交联剂 ChAcNLS 改性7G3 的结构是非常可靠的。通常, 当加载到12% 凝胶和分析的 SDS 页, 这导致可区分逐步增加兆瓦比例增加 sulfo-SMCC-to-7G3 比率使用, 并允许重和轻链单独评估 ChAcNLS共轭 (图 3)。7G3 反应在 10, 20, 25 和50到 1 sulfo-SMCC-to-7G3 比率跟随射频测量和兆瓦推断结果在 3, 7, 10 和 20 ChAcNLS 分子每7G3。通过密度测定, 7G3 3-10 ChAcNLS, 单体的百?...

Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

病毒衍生肽与抗体结合 (ACs) 是一种获得动力的方法, 由于可能提供的分子负载增加肿瘤细胞积累。利用常用的方法来评估肽共轭、AC 和有效载荷的胞内积累和肿瘤靶向, 这项协议帮助研究人员在关键的初步发展阶段。

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

initial-evaluation-antibody-conjugates-modified-with-viral-derived

Research

JoVE Journal

Bioengineering

7.6K 次观看.  Université de Sherbrooke. 病毒衍生肽与抗体结合 (ACs) 是一种获得动力的方法, 由于可能提供的分子负载增加肿瘤细胞积累。利用常用的方法来评估肽共轭、AC 和有效载荷的胞内积累和肿瘤靶向, 这项协议帮助研究人员在关键的初步发展阶段。

视频: Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

科研

癌症研究

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 ...

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 ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

用快速 Bioorthogonal 反应生成抗体-药物共轭物的非规范氨基酸的遗传编码

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...

化学

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

使用磁性荧光珠对 PDL1-vaxx 抗体阻断进行流式分析

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide (&#34;promiscuous&#34; T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

作者聚焦：PDL1-vaxx 肽疫苗诱导的抗体阻断 PD-1/PD-L1 相互作用的基于磁荧光珠的双报告基因流分析 

Checkpoint inhibitors are important targets in developing therapies for the battle against cancer. This report introduces a novel PDL1 peptide-based cancer vaccine, PDL1-Vaxx, which induces neutralizing polyclonal antibody production that blocks PD-1/PDL1 complex formation. This work also details the development and testing of a fluorescent bead-based assay for analyzing this activity.

基于磁性荧光珠的PDL1-Vaxx肽疫苗诱导的PD-1/PD-L1相互作用抗体阻断的双报告流分析

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...

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 ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

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靶性抗原特异性治疗抗体的肿瘤及组织分布分析

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用快速 Bioorthogonal 反应生成抗体-药物共轭物的非规范氨基酸的遗传编码

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

基于磁性荧光珠的PDL1-Vaxx肽疫苗诱导的PD-1/PD-L1相互作用抗体阻断的双报告流分析

基于磁性荧光珠的PDL1-Vaxx肽疫苗诱导的PD-1/PD-L1相互作用抗体阻断的双报告流分析

基于磁性荧光珠的PDL1-Vaxx肽疫苗诱导的PD-1/PD-L1相互作用抗体阻断的双报告流分析

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

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用