CD19细胞外域的精细表位作图促进了设计。,Biochemistry

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CD19细胞外域的精细表位作图促进了设计。
Biochemistry ( IF 2.9 ) Pub Date : 2019-11-21 , DOI: 10.1021/acs.biochem.9b00808
Justin R Klesmith ₁ , Lan Wu ₂ , Roy R Lobb ₂ , Paul D Rennert ₂ , Benjamin J Hackel ₁

Affiliation

B细胞表面蛋白CD19存在于整个细胞生命周期中，并在白血病中均匀表达，使其成为嵌合抗原受体工程免疫细胞治疗的靶标。鉴定CD19与抗体结合的序列依赖性使基础研究和CD19靶向治疗剂的开发更具针对性。为了鉴定CD19上的抗体结合表位，我们筛选了人CD19细胞外域的全面单点饱和突变文库，以鉴定有害于结合FMC63（嵌合抗原受体发育中使用的主要CD19抗体）以及4G7-的突变。 2E3和3B10已用于各种类型的CD19研发中。所有这三种抗体在已发表的抗体B43抗原决定簇附近均具有部分重叠的抗原决定簇。FMC63构象表位跨越外显子3和4的空间相邻但遗传距离远的环。3B10表位是线性肽序列，以440 pM的亲和力结合CD19。除了实现表位作图的主要目标外，突变耐受性数据还使其他CD19变体的设计和分析得以实现。在酵母展示环境中，已设计的CD19变体具有所有N-连接的糖基化位点，可成功去除结合的抗体，为无糖基化的应用提供了线索。筛选热稳定变异体可识别突变，以指导CD19进一步稳定用于融合蛋白应用，并揭示了进化亲和力-稳定性之间的权衡。这些对CD19序列-功能关系的基本见解，加深了我们对抗体介导的CD19靶向治疗剂的了解。

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Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design.

The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63—the dominant CD19 antibody used in chimeric antigen receptor development—as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity–stability trade-offs. These fundamental insights into CD19 sequence–function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.

更新日期：2019-11-21

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