Functional phage display of two murine α/β T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance

Abstract

Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the present study was to explore phage display of soluble T-cell receptors (TCRs). A library platform that supports engineering and selection of improved TCRs to be used as detection reagents for specific antigen presentation will be very useful. In such applications, high, equal and clone independent display levels are a prerequisite for 'fair' selection. Therefore, we explored how different pIII fusion formats and modes affected the display levels of two murine alpha/beta TCRs. Both are derived from T-cell clones associated with the MOPC315 myeloma model. The results show that the design of the pIII fusion particle significantly affects the subsequent display levels. Furthermore, successful display may be obtained both in phagemid and phage versions. Importantly, improvement of poor display can be achieved by over-expressing the periplasmic chaperone FkpA.

Reliable Titration of Filamentous Bacteriophages Independent of pIII Fusion Moiety and Genome Size by using Trypsin to Restore Wild-Type pIII Phenotype

“…The phagemids used have been described in a previous study (4). All are ampicillin-resistant (amp R ), encode T-cell receptor (TCR) or antibody domains fused to pIII, and range in size from 4.9 kb to 6.6 kb.…”

“…Regular M13K07 encodes wt pIII and therefore directs display of varying, but lower, numbers of pIII fusion proteins. Titration based on infection efficiency was done by spot titration (7), and ELISA was done as described in Reference 4. Acetylated trypsin was from Sigma-Aldrich (St. Louis, MO, USA) and a trypsin/EDTA premix was obtained from BioWhittaker (Lonza Group Ltd., Visp, Switzerland).…”

“…Acetylated trypsin was from Sigma-Aldrich (St. Louis, MO, USA) and a trypsin/EDTA premix was obtained from BioWhittaker (Lonza Group Ltd., Visp, Switzerland). SDS PAGE and Western blot analysis was performed as described previously (4). …”

“…(A) A fixed amount of VCSM13- or a scTCR-encoding phagemid (pFKPDN-scTCR V7A10B2, described in Reference 4), rescued by M13K07 (K) or Hyperphage (H), was digested with 0, 50, or 500 g acetylated trypsin, or by a trypsin/EDTA premix corresponding to 125 g trypsin. Digestion was done for 1 h at room temperature before the samples were titrated.…”

“…The experiment was performed in triplicate and the mean sd is shown. Key to graph: scTCR A, pFKPDN-scTCR V4B2A1; scTCR B, the same clone as shown in Figure 1A; both are described in Reference 4. (B) Phagemids that display different pIII fusions (scTCR, cFab, or Fab) were rescued by M13K07 or Hyperphage followed by titration with or without 5 min trypsin digestion (using the trypsin/EDTA premix).…”