Development of Escherichia coli cell surface display for selection of single domain antibodies from immune libraries

Resumen

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work, we demonstrate that the native ß-domains of EhaA autotransporter and Intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin ß-domains to display individual sdAbs and sdAb libraries obtained after immunization of camelids with different proteins of biomedical interest, i.e. the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC), human fibrinogen (Fib) and A431 cells displaying human epidermal growth factor receptor (EGFR). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the ß-domain of Intimin showed higher antigen-binding capacity. The sdAb libraries against TirMEHEC and Fib cloned in both E. coli display platforms were screened for antigen binding clones by MACS using purified biotinylated antigen. High affinity binders were selected by both display systems, although more efficiently with the intimin ß- domain. The specificity of the selected clones against their respective antigen was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. In addition, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions. Further, we used the ß-domain of intimin to display a sdAb library against human EGFR and developed a method for the direct selection of this sdAb library on cells. Bacteria displaying the anti-EGFR sdAb immune library were subjected to consecutive rounds of selection on EGFRnegative cells (i.e. murine fibroblast cell line, NIH-3T3 2.2) and EGFR-positive cells HER14 (i.e. NIH-3T3 2.2 transfected cells expressing human EGFR) to enrich for EGFR-specific binding clones. EGFR-specific clones that bound HER14 cells and not NIH-3T3 2.2 cells were identified and their specific binding to EGFR confirmed by flow cytometry analysis using biotinylated EGFR-Fc fusion. In addition, we used E. coli display to characterize the affinity and binding of these sdAbs to EGFR-Fc in the presence or absence of an excess of EGF, a natural ligand of EGFR. Our data demonstrates that E. coli display allows the selection of sdAbs against relevant tumor-associated antigens from libraries generated by cell immunization and performance of direct selection on tumor cells without the need for purified antigens.