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.