Selective Targeting and Potent Control of Tumor Growth Using an EphA2/CD3-Bispecific Single-Chain Antibody Construct

Abstract

The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells.

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

“…19 Meanwhile, instead of increasing the killing potency of T cells, higher E:T ratios appeared to only accelerate the T cell killing process. 19,23,49–52 Consistently, the data we analyzed also suggested that the higher E:T ratios only accelerate the T cell killing process (τ), but do not affect the intrinsic potency value (EC 50 ). Our model used two exponential functions to correlate both effector cell concentrations (k τ1 ) and target-to-effector cell ratios (k τ2 ) to τ.…”

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

“…The gene encoding the HER2-binding affibody molecule was recovered from a vector containing a (Z HER2:342 ) 2 construct previously described by Orlova et al [30]. The vector was first restricted with endonucleases AccI and NotI, followed by ligation of a gene fragment (containing an SfiI restriction site) encoding the (GGGGS) 3 linker. The gene encoding the EGFR-binding affibody molecule was recovered from a vector containing a Z EGFR:1907 construct previously described by Friedman et al [31].…”

“…The gene encoding the EGFR-binding affibody molecule was recovered from a vector containing a Z EGFR:1907 construct previously described by Friedman et al [31]. A gene fragment (containing an SfiI restriction site) encoding the (GGGGS) 3 linker was introduced to the vector by amplifying the Z EGFR:1907 fragment with specific primers introducing the linker, followed by PstI/AccI and SfiI/AccI restriction and ligation. A second affibody gene construct was introduced head-to-tail, giving a vector encoding the (Z EGFR:1907 ) 2 with a N-terminal (GGGGS) 3 linker fragment.…”

“…A gene fragment (containing an SfiI restriction site) encoding the (GGGGS) 3 linker was introduced to the vector by amplifying the Z EGFR:1907 fragment with specific primers introducing the linker, followed by PstI/AccI and SfiI/AccI restriction and ligation. A second affibody gene construct was introduced head-to-tail, giving a vector encoding the (Z EGFR:1907 ) 2 with a N-terminal (GGGGS) 3 linker fragment. The two vectors encoding the dimeric HER2-and EGFR-binding entities were subsequently PstI/SfiI-restricted and the fragment encoding the HER2-binding affibody linker moiety was ligated into the (Z EGFR:1907 ) 2 -containing vector.…”

“…The bound protein was eluted with imidazole buffer (20 mM sodium phosphate, 0.5 M NaCl and 500 mM imidazole, pH 7.4) and then applied to a PD-10 column (GE Healthcare) equilibrated with PBS (pH 7.4), and eluted with PBS. The proteins were denoted (Z HER2:342 ) 2 -(G 4 S) 3 [(Gly 4 -Ser) 3 ]-(Z EGFR:1907 ) 2 and (Z HER2:342 ) 2 -(G 4 S) 3 -(Z EGFR:1907 ) 2 -Cys. Protein concentrations were estimated from A 280 using the appropriate absorption coefficient for each protein.…”