Phage display screening and generation of antibodies

Supported lipid bilayers were prepared on Proplate Microtiter Plates. FLS assays were performed according to our usual methods (Walrant et al., 2015). Briefly, assay mix comprising 6 mg/ml Xenopus egg extracts, 2 mM DTT, 5 µM actin, energy regenerating system (Energy Mix; final concentration 2 mM ATP, 15 mM creatine phosphate, and 2 mM MgCl₂) in XB was added to the supported bilayers and incubated for 15 min at room temperature (Walrant et al., 2015). For the mature FLS condition, 2 µl phalloidin (Thermo Fisher Scientific, P3457) from a 10 mg/ml methanol stock was added and incubated for a further 5 min followed by three washes with XB and fixation with 4% formaldehyde for 75 min. For the washed FLS condition, after 15 min of FLS growth, three washes with XB were performed then the FLS stabilized by addition of 2 µl phalloidin. After a 5 min incubation, assays were washed three times with XB and fixed with 4% formaldehyde in XB for 75 min. For the early FLS condition, the assay mix was removed after 3 min and washed three times with XB. 2 µl phalloidin was added, and the mix was incubated for 5 min, then fixed with 4% formaldehyde in XB for 75 min. Fixed FLS were kept at 4°C for up to 3 d. Phenotypic selections were performed using the CAT2.0 human scFv phage display library (Lloyd et al., 2009; Vaughan et al., 1996). Three rounds of selection were performed against each of three different filopodia structures: the mature, the washed, and the early time point. Wells were washed with PBS and then blocked for 1 h with PBS-Marvel milk powder (3% wt/vol). 10⁹–10¹² phages were blocked for 1 h at room temperature in PBS-Marvel milk powder (3% wt/vol) and then transferred to wells coated with lipid bilayers and purified FLS proteins to deplete nonspecific and known binders. The blocked phages were subsequently incubated with the appropriate FLS condition for 1 h at room temperature and any unbound phage removed by a series of wash cycles using PBS-Tween (0.1% vol/vol) and PBS. Bound phage particles were eluted by addition of 5 µg/ml trypsin, infected into Escherichia coli TG1 bacteria and rescued for the next round of selection (Vaughan et al., 1996).

Following three rounds of phage display selection, individual scFv were prepared as phage supernatants, and the round 2 and 3 outputs screened by phage ELISA for binding to the corresponding filopodia structures used for selections (Osbourn et al., 1996; Vaughan et al., 1996). Nunc Maxisorp (Immobiliser) plates (Thermo Fisher Scientific, 44–2404-21) coated with lipid bilayers and a mix of purified TOCA-1, fascin, Arp2/3 complex, N-WASP, Ena, and VASP as described in (Dobramysl et al., 2019 Preprint) were used for negative selection. Specific clones, defined as those which gave more than threefold signal over background, were sequenced, expressed in TG1 E. coli, and purified via the C-terminal His tag by immobilized nickel chelate chromatography (Bannister et al., 2006; Lloyd et al., 2009).

Antibodies were converted from scFv to whole immunoglobulin G1 triple mutant (IgG1-TM, IgG1 Fc sequence incorporating mutations L234F, L235E, and P331S) antibody format essentially as described by Persic et al. (1997) with the following modifications. An OriP fragment was included in the expression vectors to facilitate use with CHO-transient cells and to allow episomal replication. The variable heavy domain was cloned into a vector containing the human heavy chain constant domains and regulatory elements to express whole IgG1-TM heavy chain in mammalian cells. Similarly, the variable light domain was cloned into a vector for the expression of the human light chain (lambda) constant domains and regulatory elements to express whole IgG light chain in mammalian cells. The plasmids were cotransfected into CHO-transient mammalian cells (Daramola et al., 2014) and IgG proteins purified from cell culture medium using Protein A chromatography. The purified IgG were analyzed for aggregation and degradation purity using size exclusion chromatography–-HPLC and by SDS-PAGE.