Engineering and characterization of humanized CD19-targeted scFvs. (A) The murine anti-CD19 scFv FMC63 (black) was subjected to CDR grafting and in silico assessment, resulting in the generation of 2 humanized variants, huCD19R(4D5) (blue) and huCD19R(VH4vκ1) (red). (B) CD19-targeted scFvs (red with yellow CDRs) were displayed on the surface of yeast cells as N-terminal fusions to a flexible polypeptide linker (black) and agglutinin 2 (Aga2p; light blue). Fusions were anchored to the yeast cell wall via disulfide linkage to Aga1p (dark blue). scFv expression is detected by antibody labeling of a C-terminal c-Myc epitope (green) and antigen binding is detected using fluorescently tagged CD19 extracellular domain (purple). (C) Successful expression of FMC63 or humanized variants scFvs on the yeast surface was detected by c-Myc epitope tag labeling. One representative trial is shown. (D) The binding affinity of the indicated scFvs was tested using flow cytometry with titration of biotinylated recombinant CD19 extracellular domain and fit to a 1:1 equilibrium binding model. Data are presented as mean ± standard deviation of 3 trials. Titration curve fits are calculated based on the average estimated K_d for each clone. Concentrations on the x-axis are in nanomolar (nM). (E) The thermal stabilities of the indicated scFvs were determined by heating yeast displaying the indicated scFvs followed by labeling with either 50 nM biotinylated recombinant CD19 extracellular domain (FMC63) or conformation-specific binder biotinylated protein L (humanized variants). The fluorescence proportion of foldedness was determined by flow cytometry. Data are fit to a 2-state unfolding model. Thermal stabilities are presented as mean ± standard deviation of 3 to 4 trials.