CN110494543A - 通过声学的亲和细胞提取 - Google Patents

通过声学的亲和细胞提取 Download PDF

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CN110494543A
CN110494543A CN201780070614.9A CN201780070614A CN110494543A CN 110494543 A CN110494543 A CN 110494543A CN 201780070614 A CN201780070614 A CN 201780070614A CN 110494543 A CN110494543 A CN 110494543A
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N·罗季奥诺娃
B·罗斯-约翰斯鲁德
B·利普肯斯
R·吉尔曼申
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Abstract

将施加有功能化材料的珠暴露于声场以捕获珠或使珠穿过。珠可包括或不含铁磁材料。珠对于宿主可以是生物相容的或生物降解的。珠的尺寸可以在一定范围内变化，和/或是异质的或同质的。珠的组成可包括高、中或低声学对比材料。功能化材料的化学成分可与现有方法相容。

Description

通过声学的亲和细胞提取
背景技术
生物材料的分离已经在各种情况中应用。例如，用于将蛋白质与其他生物材料分离的分离技术用于许多分析方法。
发明内容
生物材料的分离可以通过分布在流体室中的功能化材料来实现，所述功能化材料结合特定的靶材料，例如重组蛋白质和单克隆抗体或细胞。功能化材料(例如用亲和蛋白质包被的微载体)被声驻波的波节和波腹捕获。在该方法中，功能化材料被捕获而没有接触(例如，使用机械通道、导管、镊子等)。
附图说明
参考附图，下面更详细地描述附图，其中：
图1是在磁场中使用顺磁珠的分离方法的示意图；
图2是在声场中使用声学珠的分离方法的示意图；
图3是具有珠的CD3+T细胞复合体的图像；
图4是没有CD3-T细胞的珠的图像，以证明选择的特异性；
图5是以带有链霉抗生物素蛋白和生物素缀合物可得的异质(heterogenous)珠的图像；
图6是同质(homogenous)琼脂糖珠的图像；
图7是用于加工珠的微型声学系统的照片；
图8是分离结果的照片；
图9是可以与珠一起使用的亲和技术的图解；
图10、11和12是彼此形成复合体的链霉抗生物素蛋白缀合珠和生物素缀合珠的显微照片；
图13是鉴定有红细胞、树突细胞和T细胞的细胞悬浮液的显微照片；
图14是细胞的明视场图像的显微照片；
图15是与细胞结合的抗CD4抗体的绿色荧光的明视场图像的显微照片；
图16是细胞的明视场图像的显微照片；
图17是与细胞结合的抗CD4抗体的品红色荧光的明视场图像的显微照片；
图18是一系列显微照片，示出了在具有过量珠和过量细胞的环境中珠-细胞复合体的实例；和
图19是用于亲和结合的活化化学成分(chemistry)实例的图解。
具体实施方式
生物材料(例如蛋白质或细胞)的亲和分离是通过使用与表面(例如微珠)共价结合的配体与蛋白质或细胞相互作用由此使得蛋白质或细胞与微珠上的配体结合来实现的。
配体是与生物分子形成复合体的物质。通过蛋白质-配体结合，配体通常是通过结合靶蛋白质上的位点产生信号的分子，结合通常导致靶蛋白质的构象的改变。配体可以是与蛋白质材料结合的小分子、离子或蛋白质。配体和结合配偶体之间的关系是电荷、疏水性和分子结构的函数。结合通过分子间力(例如离子键、氢键和范德华力)发生。对接的缔合实际上通过解离是可逆的。配体和靶分子之间可测量的不可逆共价键在生物系统中是典型的。
能够与受体结合、改变受体功能并引发生理反应的配体被称为受体的激动剂。与受体结合的激动剂可以根据可以引发多少生理反应以及根据产生生理反应所需的激动剂浓度来表征。高亲和配体结合意味着相对低浓度的配体足以最大限度地占据配体-结合位点并引发生理反应。Ki水平越低，越有可能在待处理抗原和接受抗原之间存在化学反应。低亲和结合(高Ki水平)意味着在结合位点被最大占据并且实现对配体的最大生理反应之前需要相对高浓度的配体。二价配体由作为配体的两个连接的分子组成，并用于科学研究以检测受体定时器并研究其性质。
T细胞受体或TCR是在T细胞或T淋巴细胞表面上发现的分子，其负责识别作为与主要组织相容性复合体(MHC)分子结合的肽的抗原片段。TCR和抗原肽之间的结合具有相对低的亲和并且是退化的(degenerative)。
参考图1，顺磁珠(例如以名称Dynabeads出售的铁或铁磁珠)已用于实现亲和提取。用功能化材料包被的磁珠与复合体混合物中的生物靶结合，以允许使用磁场将靶材料从复合体混合物中分离出来。珠带有以高特异性亲和结合各种靶的分子。将珠注入复合体混合物中并孵育以结合靶。通过磁铁将珠与附着在珠上的靶一起提取。
微尺寸的珠，例如Dynabeads，是可得的，其尺寸约为4.5μm。可以使用纳米尺寸的珠，例如Myltenyi，其尺寸约为50nm。可以使用的一些亲和分子包括抗体、适体、寡核苷酸和受体等。亲和结合的靶可以包括生物分子、细胞、外来体、药物等。
参考图2，示出了具有高声学对比和亲和化学成分的珠。就用于亲和结合的具有功能化材料包被或组合物而言，这些声学珠可以以与磁珠完全相同的方式使用。将声学珠设计成利用声场从复合体混合物或流体中提取。声学珠可以直接用于在使用磁珠的细胞制备、生物化学、诊断、传感器等中开发的所有应用中。
声学珠可以使用与磁珠所使用的相同的表面和亲和化学成分。声学珠相对于磁珠的这种易于替代具有许多优点，包括简化对应用的批准，以及简化应用。
声学珠可以制成生物相容的。这种珠可以以不同的尺寸生产，这允许在尺寸区分声场中基于尺寸的连续分离，例如可以提供角度场分级分离技术。声学珠可以与封闭的基于声学的系统相结合，从而为治疗细胞制备提供连续的端到端循环。该功能提供了磁珠提取的替代方案，同时保留了目前存在的亲和化学成分的使用，其可以直接转移到声学珠。声学珠可以是分离操作中的可消耗产品。
在一个实例中，使用公开的Memorial Sloan Kettering Cancer Center(MSKCC)方案进行概念验证试验，以从患者的血液中提取CD3+T细胞。在试验中，使用顺磁珠，并用声场代替磁场。从患者血液中提取CD3+T细胞的方法是制备CAR(嵌合抗原受体)T细胞的组成部分。目前的方法基于商业可得的CD3 Dynabeads。在试验中，努力使方案差异最小化，包括在培养液而不是血液中进行实验。由于来自培养液的CAR T细胞制备工作的几个步骤，认为差异减小。溶剂密度增加，使T细胞“声学不可见”或不易受到声场影响。Dynabeads的小尺寸可以提供与细胞类似的声学对比，从而使分离公差(tolerance)更小。该试验使用JurkatCD3+和CD3- T细胞系作为模型。CD3-细胞用作非特异性捕获的对照。
现在参考图3和4，显示了试验结果的图像。将细胞悬浮液与CD3 Dynabeads(其结合CD3+细胞)一起孵育。使混合物穿过声学系统，所述声学系统捕获磁珠(有或没有细胞)。收集的细胞在培养中成功生长。图3和图4中的图像是通过明视场图像与荧光图像的重叠获得的。珠是黑色并带有微红的自发荧光。活细胞呈荧光红色。珠直径为4.5微米。图3显示了具有珠的CD3+ T细胞复合体，其证明了该技术的效率。图4显示没有提取CD3- T细胞，这证明了该技术的特异性和选择性。
现在参考图5和6，显示了使用声学珠的试验结果。在该试验中，使用琼脂糖珠作为声学珠。这些珠可以从几个制造商获得现成的，并且不是顺磁的或者几乎没有至不含铁或铁磁含量。一些琼脂糖珠具有简化抗体附着的表面修饰。它们也由生物相容材料组成，这对治疗解决方案很重要。图5显示ABTBeads，其是相对便宜的、异质的(20-150μm)、现成的珠，带有链霉抗生物素蛋白和生物素缀合物的珠是可得的。图6显示了CellMosaic琼脂糖珠，其倾向于相对昂贵、同质的(20-40μm)，并且可以按订单配置任何修饰。
声学珠可以被捕获在声场中，例如多维声驻波。参考图7，显示了为声学应用开发的微型声学系统，其用于捕获声学珠。较小尺寸的系统有助于减少对较大量昂贵试剂的需求并允许处理小体积样品。
参考图8，显示了CellMosaic琼脂糖珠在声学系统中逃逸(左栏)和捕获(右)。声学系统的捕获效率可以达到90％+。
参考图9，示出了活化声学珠的灵活方法。在该方法中，使用链霉抗生物素蛋白-生物素复合体将抗体附着在琼脂糖珠上。该复合体被广泛应用于生物化学，而且非常稳定。具有缀合的链霉抗生物素蛋白的琼脂糖珠以抗体-生物素缀合物可商购获得。
评估了链霉抗生物素蛋白珠和生物素珠的功能。参考图10-12，链霉抗生物素蛋白缀合的珠和生物素缀合的珠在混合时如预期的那样显示彼此形成复合体。
可能需要从悬浮液中获得CD4+和CD8+(分别为“辅助”和“杀伤”T细胞)的单独分离，并以期望的比例将它们混合以实现有效治疗。为此，可以提供对CD4和CD8受体有亲和力的声学珠。用小鼠脾脏制备的细胞悬浮液进行试验以获得实例。参考图13，提供了红细胞、树突细胞和T细胞的鉴定。使用Invitrogen耗尽试剂盒分别从4只脾脏中分离了约2000万(20M)和1800万(18M)的CD4+和CD8+ T细胞。两种细胞系均可生长，并且CD4和CD8-T细胞均为约8.2-8.6μm。
在该试验中，免疫学验证了CD4+和CD8+分离的细胞。参考图14和15，获得了CD4受体存在的验证。Alexa488抗CD4抗体用于估计从小鼠脾脏纯化后分离的CD4-T细胞的量。图14示出了明视场图像，其中小圆圈是焦平面中的细胞。图15显示了与细胞结合的抗CD4抗体的荧光。图16示出了明视场图像，其中小圆圈是焦平面中的细胞。图17显示了与细胞结合的抗CD4抗体的荧光。图15和17中分别是不同颜色的绿色和品红色，可以允许对结果进行多重分析，例如CD4/CD8比率。
现在参考图18，显示了试验结果，其中链霉抗生物素蛋白缀合的琼脂糖珠与生物素缀合的抗CD3抗体和CD3+Jurkat T细胞一起使用。清楚地说明了珠和细胞的亲和组合。可以在声场中分离珠以从混合物中提取细胞。
使用声学系统中的声学亲和珠已经显示概念验证和性能验证。所公开的方法和系统允许使用现成的试剂和目前可得的声学系统。亲和性可以靶向任何类型的所需T细胞或标志物，包括CD3+、CD4+、CD8+。声学珠可以具有高、中或低对比因子，其可以影响珠如何响应声场，例如被推向声学波节或波腹，或者穿过场。
珠可以由各种材料和组合组成，这允许开发具有声学性能和生物相容性的最佳化学成分。可处理珠用于分离、分选或在分离方法中有用的任何其他功能。当与调谐声学系统一起使用时，专门设计的声学珠的性能可以匹配或超过顺磁珠的性能。
现有的化学成分可以与声学珠一起使用，并结合尺寸和结构同质性的规格来实现声学和分离性能的期望结果。珠可以由复合构造组成，以提高声学效率。声学系统提供了管理小尺寸的灵活性，通过加热管理，以及使用流体学来获得单独使用顺磁珠不可能获得的结果。声学珠的生物相容性和/或生物降解性以及简化的处理允许与CAR T细胞制备的现有硬件相整合。亲和声学珠可用于许多环境，包括模型环境，例如掺有靶细胞和鼠脾脏提取物的动物血液。因此，声学珠可以与现有系统协作使用，并且可以针对目标应用而设计和制造。珠可以提供有声学活性或中性的芯，并且珠本身可以为了高、中或低声学对比而配置。珠的尺寸可以为了组合分离和亲和而配置，例如某种尺寸的珠可以包括靶向某种生物材料的功能化材料，而另一种尺寸的珠可以被功能化以靶向另一种生物材料，每种生物材料可以被同时并连续地在封闭或流动的系统中分离。珠可以设计成在窄的或相对宽的范围内具有同质的尺寸分布。可以使用各种亲和化学成分，包括链霉抗生物素蛋白-生物素复合体和免疫球蛋白或适体。珠可以针对易于制造和/或针对保质期而设计。珠可以与批准的化学成分一起使用，使得它们可以容易地整合到使用批准的化学成分的已知系统中。
参考图19，显示了活化化学成分的实例的图示。所示的活化化学成分适用于本文所述的声学亲和珠。
上面讨论的方法、系统和设备是实例。可以适当地省略、替换各种配置，或添加各种过程或组件。例如，在替代配置中，可以以与所描述的顺序不同的顺序进行所述方法，并且可以添加、省略或组合各种步骤。而且，关于某些配置描述的特征可以与各种其他配置组合。可以以类似的方式组合配置的不同方面和元件。此外，技术发展以及因此的许多元件是实例，并不限制本公开或权利要求的范围。
在说明书中给出了具体细节以提供对实例配置(包括实施)的透彻理解。然而，可以在没有这些具体细节的情况下实践配置。例如，已经示出了熟知的方法、结构和技术，而没有不必要的细节以避免模糊配置。该描述仅提供实例配置，并且不限制权利要求的范围、适用性或配置。相反，前面对配置的描述提供了用于实现所述技术的描述。在不脱离本公开的精神或范围的情况下，可以对元件的功能和安排进行各种改变。
而且，可以将配置描述为描绘成流程图或框图的方法。尽管每个操作可以将操作描述为顺序方法，但是许多操作可以并行或同时进行。另外，可以重新排列操作的顺序。方法可能具有附图中未包括的附加阶段或功能。
已经描述了若干实例配置，可以使用各种修改、替代构造和等价物而不脱离本公开的精神。例如，上述元件可以是更大系统的组件，其中其他结构或方法可以优先于本发明的应用或以其他方式修改本发明的应用。此外，可以在考虑上述元件之前、期间或之后进行许多操作。因此，以上描述不限制权利要求的范围。

Claims (3)

1.用于分离方法的材料，包括：
不含铁磁材料的珠；和
在珠上的功能化材料，用于吸引靶生物材料。
2.一种分离材料的系统，包括：
用于产生声场的装置；
多个珠，其包括与每个珠结合的生物材料，所述珠在被呈递给声场时被捕获或穿过声场。
3.一种分离材料的方法，包括：
将功能化材料应用于多个珠；
将珠暴露于对功能化材料具有亲和性的生物材料，以使生物材料与珠结合；和
将珠暴露于声场以捕获珠或使珠穿过。
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