水木视界vol.3丨基于冷冻电镜和次世代测序技术的抗体发现

2022-01-29 13:48 水木未来

在分析对疫苗或感染的免疫反应时，其中一个限制性步骤是对单克隆抗体的分离和定性。

转载自GEN News:

"Hybrid Tech Uses Electron Microscopy and Nextgen Sequencing to Speed Antibody Discovery"

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斯克里普斯研究所的科学家们设计了一种具有大幅缩短现代疫苗开发流程潜力的方法，并展示了他们如何应用高分辨率的冷冻电镜（cryo-EM）数据来快速在原子水平上表征由疫苗或感染引起且与选定的病毒靶点相结合的抗体。该方法利用一种 "从结构到序列"的计算机算法，可以将冷冻电镜确定的单克隆抗体（mAb）结构与产生该结构的DNA序列联系起来。

Scientists at Scripps Research have devised a method that may be able to shortcut one of the big steps in modern vaccine development. The researchers showed that they could apply high-resolution, low-temperature electron microscopy (cryo-EM) data to rapidly characterize antibodies – elicited by a vaccine or infection – that bind to a desired target on a virus at an atomic level. The method utilises a “structure-to-sequence” computer algorithm that can relate monoclonal antibody (mAb) structure determined by cryo-EM, to the DNA sequence that would produce that structure.

该研究的通讯作者、斯克里普斯研究所的结构和计算生物学系教授Andrew Ward博士提到："COVID-19的大流行突出了对高效快速的疫苗和抗病毒技术的需求。我们乐观地认为，这种新方法将通过极大缩短抗体的发现流程来帮助我们满足这一需求。"

“The COVID-19 pandemic has highlighted the need for robust and rapid vaccine and antiviral technologies,” said study senior author Andrew Ward, PhD, a professor in the Department of Integrative Structural and Computational Biology at Scripps Research. “We are optimistic that our new approach will help fill that need by greatly streamlining antibody discovery.”

Andrew Ward和他的同事们在《Science Advances》上的 "从结构到序列，基于冷冻电镜的抗体发现"一文中描述了他们的技术。(DOI: 10.1126/sciadv.abk2039)

Ward and colleagues described their technology in Science Advances, in a paper titled “From structure to sequence: Antibody discovery using cryoEM.”

文章作者们指出，单克隆抗体的分离和定性是对疫苗或感染的免疫反应分析中的一大痛点。"显著降低传统抗体分离方法效率的步骤之一是筛选mAb库，以确定具有所需表位特异性的克隆......对感染或疫苗接种的免疫反应进行综合分析是极其费力且昂贵的。" 正如该研究的共同第一作者Aleksandar Antanasijevic博士所提到的："一般来说，识别对病毒有用的抗体需要对产生抗体的B细胞进行费力的分类和测试，以找到正确的抗体。这一过程往往需要数月时间。"

A key rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies, the authors noted. “One of the rate-limiting steps with traditional methods for antibody isolation is screening mAb libraries to identify the clones with desired epitope specificity … Comprehensive analyses of immune responses to infection or vaccination are laborious and expensive.” As study co-first author, Aleksandar Antanasijevic, PhD, pointed out, “Traditionally, identifying antibodies that are useful against a virus involves the laborious sorting and testing of antibody-producing B cells to find the right ones—a process that takes months.”

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单克隆抗体的释放

Release of monoclonal antibodies, illustration.

[Nanoclustering/Science Photo Library/Getty Images]

研究人员的部分成果归功于最近冷冻电镜的改进，这种技术使用电子束来标记目标并成像，其结果准确度远高于普通光学显微镜。例如，在8月发表于《Nature Communications》上的一项研究中，研究人员使用高分辨率冷冻电镜快速而精确地绘制了猕猴的抗体与HIV包膜蛋白的合成版本结合的位置，这些合成版本正在被开发并用于潜在的HIV疫苗。他们写道："最近，我们开发了一种方法（cryoEMPEM），即使用冷冻电镜（cryoEM）在免疫血清的水平上表征由疫苗接种或感染引起的多克隆抗体（pAb）反应。

The researchers’ feat was enabled in part by recent improvements in cryo-EM, a technology that uses a beam of electrons to illuminate and image targets far below the scale of ordinary light microscopy. In a study published in Nature Communications in August, for example, the researchers used high-resolution cryo-EM to rapidly and precisely map where antibodies in rhesus macaque monkeys bind to synthetic versions of the HIV envelope protein that are being developed for potential HIV vaccines. “Recently, we developed an approach that uses cryo–electron microscopy (cryoEM) for characterization of polyclonal antibody (pAb) responses elicited by vaccination or infection (cryoEMPEM) on the level of immune sera,” they wrote.

在研究中，研究者们将这一研究思路向前推进了一步。他们采用了一种 "从结构到序列"的计算机算法，该算法可以将冷冻电镜确定的抗体结构与产生该结构的DNA序列联系起来。他们写道："在这项研究中，我们通过引入一种从结构观察中识别功能性抗体序列的方法，扩大了冷冻电镜数据的适用性。"

For their new study, the investigators took this line of research a step further. They employed a “structure-to-sequence” computer algorithm that can relate antibody structure determined by cryo-EM, to the DNA sequence that would produce that structure. “In this study, we expanded the applicability of cryoEMPEM data by introducing a method for identification of functional antibody sequences from structural observations,” they wrote.

为了实现这一点，研究小组建立了一个含有猕猴的所有抗体编码的DNA序列数据库，该数据库可以通过快速对动物淋巴结中产生抗体的B细胞的遗传物质进行批量测序而获得。将该算法应用于冷冻电镜数据和抗体序列库后，科学家们便能将冷冻电镜图像中的选定抗体与序列数据库中定义的独特抗体进行高准确度匹配。

To enable this, the team assembled a library of all the antibody-encoding DNA sequences from the rhesus macaque monkeys, which could obtained by quickly bulk sequencing the genetic material from antibody-producing B-cells from the animals’ lymph nodes. Applying the algorithm to the cryo-EM data and the antibody sequence library, the scientists could reliably match an antibody of interest in their cryo-EM images to a unique antibody defined in the sequence database.

研究小组表示，他们可以通过使用序列数据制作一个单克隆抗体的副本来确认结果的准确性，并通过冷冻电镜验证该抗体与最初成像的抗体以相同的方式结合。"......我们开发了一种直接从冷冻电镜图像中确定mAb序列的方法，"该团队说。"这种由电子显微镜（EM）和次世代测序（NGS）组成的复合方法使多克隆Fabs（Fv）可变区域的序列分配成为可能，包括互补性决定区域（CDRs）......这种方法为基于单个B细胞分选、杂交瘤和噬菌体展示技术的传统mAb发现方法提供了一种替代方法。"

The researchers showed that they could confirm the accuracy of the result by making copies of a monoclonal antibody using the sequence data, and verifying with cryo-EM that the antibody bound in an identical way to the antibody that was originally imaged. “ .. we developed a method to determine mAb sequences directly from cryoEMPEM maps,” the team stated. “This hybrid approach, consisting of electron microscopy (EM) and next-generation sequencing (NGS), enabled sequence assignment of variable regions of polyclonal Fabs (Fv) including the complementarity-determining regions (CDRs) … This approach provides an alternative to traditional mAb discovery methods based on single B cell sorting, hybridoma, and phage display technologies.”

斯克里普斯研究所科学家和研究的共同第一作者Charles Bowman博士说。"有了这种新方法，我们只需10天左右便能从病人的血样中采集到所有由感染及免疫引发的目标抗体"

“With this new method we can go from blood sample collection from infected or immunized patients to identifying all the elicited antibodies of interest in about ten days,” stated staff scientist and study co-first author Charles Bowman, PhD.

文章作者们还指出，在文中概念的验证过程中，他们使用了对某种特定抗原具有特异性的淋巴结B细胞。然而，他们推测这种方法也适用于从其他来源获得的B细胞，如外周血、脾脏、骨髓或浆细胞，并且无需为抗原结合进行预选。对此，他们表示："通过使用冷冻电镜直接对血清抗体进行成像的方法，我们有了丰度、亲和力和克隆性的指标。

The authors also noted that their reported proof of concept study used lymph node B cells with specificity for a particular antigen. However, they anticipate that their approach will work with B cells obtained from other sources, such as peripheral blood, spleen, bone marrow, or plasma cells, and without the presorting for antigen binding. “By directly imaging the serum antibodies using cryoEM, we have a proxy for abundance, affinity, and clonality,” they commented.

科学家们现在正在完善他们的技术，以优化其效率和可用性，以期将其应用于以下几个领域：快速评估人类对实验性HIV疫苗的抗体反应；为自身免疫性疾病开发抗体阻断疗法；以及发现可以治疗性地击中细胞上其他蛋白质靶点的抗体。

The scientists are now refining their technique to optimize its speed and usability, and are applying it to several areas: to rapidly evaluate human antibody responses to experimental HIV vaccines; to develop antibody-blocking treatments for autoimmune diseases; and to discover antibodies that could therapeutically hit other protein targets on cells.

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斯克里普斯研究所开发的一种新方法：利用冷冻电镜技术快速识别用于疫苗开发的抗体。在这个例子中，该算法从数据库中筛选了大约100,000到1,000,000个可能的抗体序列，以确定与他们的冷冻电镜图像（透明的灰色表面）中观察到的抗体最匹配的序列（左边）。[斯克里普斯研究所]

A new method developed at Scripps Research uses cryoEM technology to more quickly identify antibodies for use in vaccine development. In this example, the algorithm screened between approximately 100,000 to 1,000,000 possible antibody sequences from the database to identify the sequence (left) that best matches the antibody observed in their cryoEM images (transparent gray surface). [Scripps Research]

研究者们预测：在未来，冷冻电镜技术和"从结构到序列"的算法改进将允许我们更快速地识别抗体：只需依靠高分辨率的冷冻电镜结构图像，而不需要对B细胞进行DNA测序。

They expect that future improvements in cryo-EM technology and structure-to-sequence algorithms will allow the even more rapid identification of antibodies using high-resolution structural images alone, with no need for DNA sequencing of B cells.

Antanasijevic说："这种从结构到序列的算法在免疫学和其他领域有很大的潜力。"我们设想有一天能够用它来研究蛋白质之间的相互作用，例如，发现某个蛋白质的结合配体"。正如作者所总结的那样，"......我们的方法将为发现毫安抗体和分析感染和疫苗接种的抗体反应打开新的大门。目前的COVID-19大流行便突出了对这种高效且快速的技术的需求"。

“This structure-to-sequence approach has a lot of potential in immunology and beyond,” Antanasijevic said. “We envision being able to use it someday to study protein-to-protein interactions generally, for example, to discover a given protein’s binding partners.” As the authors concluded, “ … our approach will open up new doors for both the discovery of mAbs and analyzing antibody responses to infection and vaccination. The ongoing COVID-19 pandemic has highlighted the need for such robust and rapid technologies.”

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