Figure 2.

Sequences and characterization of Ab building blocks

(A) Sequences of CDRs that were diversified in the libraries, numbered according to the IMGT nomenclature. Dashes indicate gaps in the alignment. (B) Kinetic parameters and apparent affinities for Abs binding to immobilized antigens. All values were determined by BLI, except EC₅₀ values that were determined by titration in ELISA (indicated with (*)) or flow cytometry on CD3+ Jurkat cells (indicated with (#)). (C) Anti-EPH Ab specificity. The heat map represents the steady-state BLI signal for 100 nM anti-EPHA2 Fab or ELISA signal for 100 nM anti-EPHB2 VH-Fc (x-axis) binding to immobilized Fc-tagged EPH ECD or negative control Fc (y-axis), normalized to the highest signal for each antibody. (D) ELISA demonstrating binding of LUC and EPHA2 DATEs or d-DATEs at 50 nM. D-DATEs additionally contained the EPHB2 targeting arm and exhibited recognition of EPHB2 in addition to GLuc (Gaussia Luciferase) or EPHA2. Human and murine versions of EPHA2 and EPHB2 were used to assess species cross-reactivity. (E-F) Titration of GLuc-targeting DATE (LUC-1), (E) CD133-targeting DATE (133–1), or (F) EPCAM-targeting DATE (EC-1) binding to HCT116 cells that display CD133 and EPCAM, assessed by flow cytometry (n = 1, 2 technical replicates). (G) Titration of GLuc-targeting DATE (LUC-1) or EPHA2-targeting DATEs (A2-1, A2-2, A2-3, A2-4) binding to KP4 cells that display EPHA2, assessed by flow cytometry (representative of n = 2 independent experiments). (H) Titration of DATEs, containing mOKT3 or hOKT3 scFv, binding to Jurkat T cells that display human CD3, assessed by flow cytometry. The anti-CD3 scFv was fused to anti-GLuc Fab LUC-1 (n = 1, 3 technical replicates). All error bars represent standard deviation about the mean.