PO.CH01.06 · 化学
开发单B细胞平台以加速抗体发现与筛选
Developing a single B cell platform to accelerate antibody discovery and screening
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摘要 Abstract
中文摘要
快速高效地分离抗原特异性B细胞是抗体发现过程中的一个关键瓶颈，尤其对于复杂的、维持小而特异表位的或低免疫原性的抗原而言。为解决这一挑战，我们开发了一个新一代单B细胞发现平台，重点是同时利用荧光激活细胞分选（FACS）和CellCelector（CC）来最大限度地回收低群体、高价值的B细胞。通过同步进行初步筛选，该平台能够最大化其互补优势：FACS通过精确的多参数门控对大量淋巴细胞群进行深度采样，而CC能够确保对更脆弱的B细胞亚群进行灵活的、图像引导的回收。这种双模式工作流程提高了单细胞检索效率，减少了采样偏差，并改善了下游重组抗体表达的成功率。为了展示平台性能，我们采用了一个靶向DM1的案例研究，DM1是一种在ADC开发中广泛使用的美登素类载荷。使用荧光DM1偶联物富集并筛选抗原特异性记忆B细胞。FACS显示成功率为73%，在设定门控参数后，选择了96个具有rIgG+/hIgG-DM1+/rIgM-结合的抗体。在49个独特CDR3抗体中，45个对DM1具有特异性，并首先通过ELISA筛选。亲和力高于纳摩尔水平的抗体通过生物膜层干涉技术（BLI）和ELISA进一步确认，凸显了19个针对DM1的潜在候选物。结合表征显示19个抗体表现出高于纳摩尔范围的亲和力，证实了对DM1部分的稳健识别。这些结果凸显了结合平行的FACS和CellCelector工作流程以捕获多样化、高亲和力抗体库的能力，即使针对复杂的小分子载荷也是如此。总体而言，该平台提供了一条可扩展的、高置信度的抗体发现途径，支持ADC载荷靶向、机制研究以及新一代生物治疗药物的开发。
查看英文原文 English abstract
Rapid and efficient isolation of antigen-specific B cells is a critical bottleneck during antibody discovery, especially for antigens that are complex, maintain small and specific epitopes, or have a low immunogenicity. To solve this challenge, we developed a next-generation single B cell discovery platform focusing around utilizing both fluorescence-activated cell sorting(FACS) and CellCelector(CC) to maximize the recovery of low-population, high-value B cells. By performing an initial screen concurrently, this platform is able to maximize its complementary strengths: FACS with the deep sampling of large lymphocyte populations with precise multi-parametric gating, while CC can ensure a flexible, image-guided recovery of more fragile B cell subsets. This dual-mode workflow increases single-cell retrieval efficiency, reduces sampling bias, and improves downstream recombinant antibody expression success rates. To demonstrate platform performance, we employed a case study targeting DM1, a maytansinoid payload widely used in ADC development. Antigen-specific memory B cells were enriched and screened using fluorescent DM1 conjugates. FACS revealed a success rate of 73%, and after setting the gating parameters, 96 Abs with rIgGwith rIgG+/hIgG-DM1+/rIgM- binding was selected. Out of the 49 unique CDR3 Abs, 45 were specific to DM1 and first screened by ELISA. Antibodies with an affinity above nanomolar levels were further confirmed by biolayer interferometry (BLI) and ELISA, highlighting 19 potential candidates against DM1. Binding characterization revealed that 19 antibodies exhibited affinities above the nanomolar range, confirming robust recognition of the DM1 moiety. These results highlight the power of combining parallel FACS and CellCelector workflows to capture diverse, high-affinity antibody repertoires, even against complex small-molecule payloads. Overall, the platform provides a scalable, high-confidence route for antibody discovery supporting ADC payload targeting, mechanistic studies, and the development of next-generation biotherapeutics.
利益披露 Disclosure
S. Chiang, None..
W. Ren, None..
A. Ouyang, None..
Y. Sun, None..
X. Li, None.