Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design
Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design
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DOI：
10.1021/acs.biochem.9b00808
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发表时间：
2019-12-03
期刊：
影响因子：
2.9
通讯作者：
Hackel, Benjamin J.
中科院分区：
文献类型：
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作者：
Klesmith, Justin R.;Wu, Lan;Hackel, Benjamin J.
B细胞表面蛋白CD19存在于细胞的整个生命周期，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点。识别CD19与抗体结合的序列依赖性有助于基础研究和更有针对性的CD19靶向治疗药物的开发。为了确定CD19上的抗体结合表位，我们筛选了一个完整的人CD19胞外区单位点饱和突变文库，以鉴定对结合FMC63以及4G7-2E3和3B10有害的突变。FMC63是嵌合抗原受体发育中使用的主要CD19抗体，4G7-2E3和3B10已用于各种类型的CD19研究和开发。所有这三种抗体在已公布的抗体B43表位附近都有部分重叠但不同的表位。FMC63构象表位位于外显子3和4上，在空间上相邻，但在遗传上距离很远。3B10表位是一个线性多肽序列，与CD19以440 pm的亲和力结合。除了他们绘制表位图的主要目标外，突变耐受性数据还支持了其他CD19变体的设计和分析。设计的CD19变异体在酵母展示环境中成功地去除了所有N-连接的糖基化位点结合抗体，这为糖基化应用提供了先导。对热稳定变异体的筛选发现了突变，以指导融合蛋白应用中CD19的进一步稳定，并揭示了进化亲和力和稳定性之间的权衡。这些对CD19序列-功能关系的基本见解增强了我们对抗体介导的CD19靶向治疗的理解。
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.