Department of Chemical Engineering and Materials Science , University of Minnesota-Twin Cities , 421 Washington Avenue SE , Minneapolis , Minnesota 55455 , United States.
Aleta Biotherapeutics , 27 Strathmore Road , Natick , Massachusetts 01760 , United States.
Biochemistry. 2019 Dec 3;58(48):4869-4881. doi: 10.1021/acs.biochem.9b00808. Epub 2019 Nov 21.
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.
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点。确定 CD19 与抗体结合的序列依赖性，能够增强对 CD19 靶向治疗药物的基础研究和更有针对性的开发。为了确定 CD19 上与抗体结合的表位，我们筛选了人类 CD19 细胞外结构域的综合单点饱和突变文库，以鉴定对 FMC63（嵌合抗原受体开发中使用的主要 CD19 抗体）以及 4G7-2E3 和 3B10 结合有害的突变，这些抗体已用于各种类型的 CD19 研究和开发。所有三种抗体在已发表的抗体 B43 表位附近都具有部分重叠但又不同的表位。FMC63 构象表位跨越空间相邻但遗传上相距较远的外显子 3 和 4 环。3B10 表位是一个线性肽序列，与 CD19 的结合亲和力为 440 pM。除了主要的表位作图目标外，突变耐受性数据还为进一步的 CD19 变体设计和分析提供了支持。一种设计的 CD19 变体，其所有 N-连接糖基化位点均被去除，在酵母展示环境中成功结合抗体，为无糖基化应用提供了一个先导。筛选耐热变体鉴定了突变，以进一步指导融合蛋白应用中 CD19 的稳定，并揭示了进化亲和力-稳定性的权衡。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。
