作者感谢罗伯特·舒斯特（Robert Schuster）在数据验证方面的帮助。

mAbScale 的高级工作流程 如图 2 所示。每个步骤都有更复杂的内部决策分支、循环和组合函数。描述计算过程的详细算法工作流程见 补充图 1。应用程序输出以电子表格格式保存在用户选择的文件夹中。输出文件由多个单独的工作表组成，这些工作表可以归类为用户输入、分子量计算和平均同位素质量数推导的参考（补充表中提供了示例输出）。用户输入的工作表包括用户输入的蛋白质氨基酸序列和其他信息、平均元素质量和聚糖质量，用于计算元素组成和不同分子量。分子量计算表包括各种形式的化学组成、有和没有糖基化和化学修饰的还原质量数，以及有和没有糖基化和化学修饰的完整质量数。如果用户在用户输入页面中输入两种不同的 HC 和/或两种不同的 LC，则将自动生成含有半抗体质量的纸，因为半抗体是需要相对于所需异二聚体进行鉴定和定量的主要杂质。mAbScale 的源代码可通过以下存储库访问:https://github.com/kkhatri99/mAbScale。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65298/65298fig02.jpg"> 图 2:使用该应用计算元素组成和质量所涉及的步骤概述。 颜色编码可用于链接到 补充图1中描述的工艺流程。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65298/65298fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
1. 打开 mAbscale 应用程序
<ol><li>双击可执行文件的图标打开软件应用程序。</li>
</ol>2. 序列输入
<ol><li>在标有 1 的相应文本框中输入重链和轻链序列，不带任何空格。<ol><li>对于 bsAbs，在标记为 2 的第二组文本框中添加其他重链或轻链。对于具有相同重链和轻链的mAb，留 2 个空白。</li>
		<li>选中 N-Terminal Cyclization 和/或 C-Terminal Clipping 复选框（如果这些重链端子变体适用）。</li>
		<li>将任何化学修饰（包括ADC分子的接头和有效载荷）添加到"重链化学模组"和/或"轻链化学模组"文本框中。<ol><li>将修饰指定为元素组成，例如 CaCl2。修饰将被添加到相应的蛋白质亚基或链中。 注:化学成分也可以通过在元素组成前面加上-符号来从亚基或链中减去。例如，-H2O 将从亚基组成和质量中减去水分子。</li>
		</ol></li>
	</ol></li>
</ol>3. 指定二硫键的数量
<ol><li>在标记为"二硫 化物总数"的文本框中指定蛋白质分子中二硫键的数量。</li>
	<li>在未还原的 HC 二硫 化物文本框中输入未还原的 HC 二硫化物的数量，在未还原的 LC 二硫化物文本框中输入未还原的 LC 二硫 化物的数量，具体取决于还原程度（完全还原与部分还原）。 注:mAb亚基的简化质量数分析涉及二硫键连接的重链和轻链的还原/分离。</li>
	<li>如果 mAb 轻链上存在糖基化，请选中 Light Chain is Glycosylated 复选框。</li>
</ol>4. 设置输出文件夹并运行应用程序
<ol><li>单击" 浏览 "按钮为"输出文件夹"文本框选择 输出文件夹 。</li>
	<li>在 Excel 文件（无分机） 文本框中输入不带文件扩展名的输出文件名（自动另存为 .xlsx）。</li>
	<li>单击 "提交 "按钮开始应用程序。输出文件可以在指定的文件夹中找到。 注:元素质量和聚糖列表可以通过分别编辑带分隔符的文本文件 Element_Mass.csv （补充编码文件 1）和 聚糖.csv （补充编码文件 2）来定制。这些文件必须与 mAbScale.exe （补充编码文件 3）可执行文件放在同一文件夹中，应用程序才能执行。应用程序将在执行一次后自动关闭。如果需要第二次计算，用户将不得不再次启动应用。</li>
</ol>

基于 Python 的生物治疗药物实体精确质量计算工具

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该软件在 Apache 2.0 许可下发布。葛兰素史克研究与发展有限公司版权所有 （2022）。保留所有权利。根据 Apache 许可证 2.0 版（以下简称"许可证"）获得许可;除非遵守许可，否则您不得使用此文件。您可以在 http://www.apache.org/licenses/LICENSE-2.0 获取许可证的副本。除非适用法律要求或书面同意，否则根据许可分发的软件按"原样"分发，不附带任何明示或暗示的保证或条件。请参阅许可证，了解许可证下管理权限和限制的特定语言。L.C.是葛兰素史克（GSK）的员工。T.H. 和 K.K. 作为 GSK 的员工开发了该软件，现在分别是 Merck 和 Moderna 的合作伙伴。

mAbScale提供了一个直观的用户界面，可以灵活地改变质量数和元素计算的构建模块。用户应对目标分子有基本的了解，以便使用该应用程序，得出正确的质量数，并解释结果。例如，由于完整或减少质量数的行数众多，完整或减少的质量输出表可能会让人不知所措，因为默认的聚糖数据库包含88种N-连接聚糖，这些聚糖通常存在于治疗性抗体的Fc部分，并且该应用程序计算了数据库18</s...

在过去的二十年里，生物治疗药物已经发展成为现代制药工业的中流砥柱。SARS-CoV2 大流行和其他危及生命的疾病进一步增加了对更快、更广泛地开发生物制药分子的需求 1,2,3。
生物治疗药物分子量与其他分析检测相结合，对于分子的鉴定至关重要。完整和减少的亚基质量数用于整个发现和开发生命周期，作为旨在保持质量的控制策略的一部分，如QTPP（质量目标产品简介）4中所述。
生物制药行业的分析开发在很大程度上依赖于质量测量，以使用肽图分析或多属性方法（MAM）监测进行完整质量分析和深度表征。利用现代质谱 （MS） 平台的这些技术的核心是能够提供高分辨率的精确质量 （HR/AM） 测量。大多数HR/AM仪器的质量精度在0.5-5 ppm范围内，与质量范围成正比。准确测量完整大分子质量数的能力能够快速、可靠地鉴定大分子治疗药物。由于使用大分子（>10 kDa）的典型实验条件无法获得同位素分辨率，因此必须计算平均质量数以进行比较和鉴定5,6。
典型的完整或亚基蛋白质质谱图代表了整体蛋白型图谱，其中包含翻译后修饰 （PTM） 产生的各种分子形式的复合信息以及任何一级结构差异，例如片段或序列变体。这些测量相对容易和高通量的特性使它们对表征和作为过程监测控制具有吸引力7,8。这些实验的数据分析通常要求用户定义分子形式（PTM或其他分子形式的范围）的搜索空间。对于糖基化蛋白，这种搜索空间很大程度上是由糖型异质性驱动的。多个 PTM、二硫键构型以及沿一级结构的其他变化的组合使得计算所有可能的分子形式成为一项繁琐的任务。因此，手动计算可能的分子形式是一个耗时、耗费资源的过程，极有可能出现人为错误。
在这里，我们提出了一种质量计算工具，该工具是考虑到生物治疗分子的最重要特征而开发的，例如mAb、bsAb、ADC等。该工具允许轻松合并搜索空间变量，以实现质量和元素组成的一致计算。该工具的模块化特性将使其能够进一步发展并应用于其他模式的质量计算和质量匹配。
GUI模块允许用户指定质量计算的输入，如图 1所示;具体来说，用户输入轻抗体链和重抗体链的单字母氨基酸序列。重链 N 端环化和 C 端赖氨酸剪切的常见修饰作为复选框包括在内。此外，可以通过相应的 Chem Mod 文本框从这些蛋白质链中添加/减去化学式/元素组成。这允许用户灵活地添加元素组成，包括多个翻译后修饰或ADC中的小分子有效载荷。由于大多数治疗性单克隆抗体被设计为去除轻链中的糖基化位点，因此轻链中的糖基化是可选的，可以使用 GUI 上的复选框进行指定。
抗体完整质量数分析的典型变化是亚基质量数分析，其中轻链通过减少链间二硫键从重链中分离出来。根据所用还原剂的强度，链内二硫键可能被裂解，也可能不被切割。用户可以根据IgG亚型或半胱氨酸偶联ADC9灵活地输入二硫键的总数。
该应用程序以自下而上的方式计算质量，其中首先计算单个重链和轻链的元素组成。接下来，通过调整计算出的元素组成来解释重链（HC）N端环化Lys-clipping。然后将任何指定的化学修饰应用于重链和/或轻链。根据分析类型和用户指定的二硫键模式，调整两条多肽链的氢数量。糖基化的HC和轻链（LC）（可选）质量数是根据用户的输入计算的。最后，将多个HC和LC质量数合并，并自动更新二硫键数以计算完整质量数。
对于较大的分子（如完整的蛋白质），当使用具有典型分辨能力的质谱仪时，由于加性质量缺陷，无法测量单同位素质量数。取而代之的是，测量或报告标称或平均质量 5,10,11,12,13。平均元素质量可以根据用于策划质量的来源而变化14,15。虽然元素质量的差异可能很小，但对于大分子分子量计算来说，它们加起来可以产生显著的值。软件应用程序中默认使用的平均元素质量如附表1所示。对于生物制药研发（R&D）领域等受监管的环境，保持一致的分子量非常重要，因为质量的变化可能意味着在监管备案期间分子实体发生变化。为了实现元素质量使用的一致性，软件工具中包含了元素质量字典，作为逗号分隔值 （csv） 文本文件:Element_Mass.csv（补充编码文件 1）。同样，包括mAb上常见的聚糖组合物的精选列表:聚糖.csv（补充编码文件2）。这两个文件都保存在与可执行应用程序相同的文件夹位置，用户可以修改以使用特定的元素质量数列表或聚糖库。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65298/65298fig01.jpg"> 图 1:mAbScale 应用程序的 GUI 界面。 GUI模块允许用户指定质量计算的输入。用户输入轻抗体链和重抗体链的单字母氨基酸序列。重链 N 端环化和 C 端赖氨酸剪切的常见修饰作为复选框包含在内。化学式/元素组成可以通过相应的 Chem Mod 文本框进行添加/减去。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65298/65298fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

<table><tbody><tr><td>Acquity UPLC system&amp;nbsp;</td><td>Waters Corp., Milford, MA</td><td>N/A</td><td>Modular system</td></tr><tr><td>Antibody-drug conjugate (ADC)</td><td>GlaxoSmithKline</td><td>N/A</td><td>Proprietory molecule</td></tr><tr><td>BEH 200 SEC column&amp;nbsp;</td><td>Waters Corp., Milford, MA</td><td>176003904</td><td></td></tr><tr><td>Bispecific mAb</td><td>GlaxoSmithKline</td><td>N/A</td><td>Proprietory molecule</td></tr><tr><td>Byos</td><td>Protein Metrics, Cupertino, CA</td><td>https://proteinmetrics.com/byos/&lt;br/&gt; Version 4.5</td><td></td></tr><tr><td>GPMAW</td><td>GPMAW</td><td>http://www.gpmaw.com/</td><td></td></tr><tr><td>LC-MS grade water&amp;nbsp;</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>W6-1</td><td></td></tr><tr><td>mAb standard&amp;nbsp;</td><td>Waters Corp., Milford, MA</td><td>186009125</td><td>Waters Humanized mAb Mass Check Standard</td></tr><tr><td>mAbScale</td><td>GlaxoSmithKline</td><td>Apache License, Version 2.0&amp;nbsp;</td><td></td></tr><tr><td>Xevo G2 Q-TOF mass spectrometer</td><td>Waters Corp., Milford, MA</td><td>N/A</td><td>Modular system</td></tr></tbody></table>

an open-source framework for mass calculation of antibody-based therapeutic molecules

生物治疗肿块是验证身份和结构完整性的一种手段。完整蛋白质或蛋白质亚基的质谱 （MS） 为生物制药开发的不同阶段提供了一种简单的分析工具。当MS的实验质量数在理论质量数的预定义质量数误差范围内时，蛋白质的身份得到确认。虽然有几种计算工具可用于计算蛋白质和肽分子量，但它们要么不是为直接应用于生物治疗实体而设计的，要么由于付费许可证而存在访问限制，要么需要将蛋白质序列上传到主机服务器。 
我们开发了一种模块化质量数计算程序，可以轻松测定治疗性糖蛋白（包括单克隆抗体 （mAb）、双特异性抗体 （bsAb） 和抗体-药物偶联物 （ADC））的平均或单同位素质量数和元素组成。这个基于 Python 的计算框架的模块化特性将允许该平台在未来扩展到其他模式，例如疫苗、融合蛋白和寡核苷酸，并且该框架也可用于自上而下的质谱数据查询。通过创建具有图形用户界面 （GUI） 的开源独立桌面应用程序，我们希望克服在无法将专有信息上传到基于 Web 的工具的环境中使用的限制。本文介绍了mAbScale工具在不同基于抗体的治疗方式中的算法和应用。

选择多种单克隆抗体来代表不同类型的单克隆抗体。选择市售的mAb标准品来表示在Fc区域具有相同重链、相同轻链和一个N-连接糖基化位点的常规mAb。此外，还选择了具有额外轻链 N-连接糖基化的单克隆抗体、双特异性单克隆抗体和抗体-药物偶联物 （ADC） 单克隆抗体，以扩大应用范围。 表1总结了这些示例mAb的化学成分、计算质量数、测量质量和质量数误差。mAbScale报告的蛋白质化学...

Watch this Scientific Journal Video about Advancing Biotherapeutic Mass Calculation by Introducing mAbScale, a Python-Based Desktop Application at JoVE.com

作者聚焦:通过推出基于 Python 的桌面应用程序 mAbScale 推进生物治疗药物质量计算

本文介绍了如何使用软件应用程序mAbScale来计算基于单克隆抗体的蛋白质疗法的质量数。

用于基于抗体的治疗分子质量数计算的开源框架

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides ...

对大蛋白质分子进行精确质量数计算的挑战是与测量相关的质量缺陷。因此，只能测量平均质量。微小的元素质量差异可能会累积，导致大的辅助治疗分子（如单克隆抗体）出现显着差异。我们的工具明确解决了基于抗体的疗法（如 MAP、双特异性和抗体药物偶联物）在一致质量数计算方面的挑战。由于涉及专有信息，商业生物制药开发领域的研究人员不得不依赖手动计算或不依赖于与外部服务器通信的工具。现有工具要么需要许可证，要么没有考虑到生物制药开发的特定需求。MapScale为用户和开发人员提供了用户友好的界面和适应性强的开源框架。它支持维护多个版本的元素质量和修饰列表，并允许列表共享以保持计算的一致性。可以使用内部服务器或基于云的部署在企业级别共享代码，并具有受控访问以保护专有信息。

an-open-source-framework-for-mass-calculation-antibody-based

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules

1.4K 次观看.  GlaxoSmithKline. 对大蛋白质分子进行精确质量数计算的挑战是与测量相关的质量缺陷。因此，只能测量平均质量。微小的元素质量差异可能会累积，导致大的辅助治疗分子（如单克隆抗体）出现显着差异。我们的工具明确解决了基于抗体的疗法（如 MAP、双特异性和抗体药物偶联物）在一致质量数计算方面的挑战。由于涉及专有信息，商业生物制药开发领域的研究人员不得不依赖手动计...

视频: 作者聚焦:通过推出基于 Python 的桌面应用程序 mAbScale 推进生物治疗药物质量计算

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

科研

生物化学

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

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

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

免疫与感染

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

通过质量光度法快速测定抗体抗原亲和力

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

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

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

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 ...

生物工程

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

细胞法检测酶替代疗法中细胞吸收的抗体

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

生物学

Synthetic Antigen Controls for Immunohistochemistry

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 &#176;C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities.
This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.