Affinity BIO have developed the Retained Display (ReD) system, whereby a scFv library based upon a human scaffold is displayed on the cell wall of permeablised E. coli cells. Cells displaying the library of scFv antibodies are screened by incubating with fluorophore-conjugated target protein, followed by FACS to enrich for clones that bind to the target. Multiple rounds of scFv display, target binding and FACS permits rapid screening of the library. Individual clones are then analysed by flow cytometry in a multi-well format, to identify optimal binders. 

We have used the ReD system to isolate novel single chain antibodies that recognise the human CD3 heterodimer, a component of the T cell receptor complex. Each TCR contains two CD3 heterodimers, one gamma-epsilon and one delta-epsilon; therefore, two ReD library screens were conducted in parallel, one targeting each heterodimer. CD3 target proteins were synthesised as IgG-Fc fusions; therefore binding reactions included differentially-labelled IgG to facilitate counter screening. Dual colour FACS was used to preferentially enrich for clones that recognise the CD3 moiety, rather than Fc. Following multiple rounds of FACS selection and enrichment, individual clones were tested for binding to both CD3 gamma-epsilon and CD3 delta-epsilon, to isolate antibodies that recognise epitopes common to both heterodimers.  

Using this approach, we have demonstrated the capacity of the ReD system to rapidly identify novel antibodies, while customising the screening process to focus on specific regions of the target protein. The novel anti-CD3 antibodies identified are undergoing further characterisation for use in the development of future immunotherapeutics.