Targeting membrane proteins for antibody discovery using phage display

Abstract

A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b.

Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation

“…Various formats have been used to present complex membrane-dependent antigens for antibody discovery 35 . Of these, display on whole cells offers the most native-like conformation but also presents some of the most daunting challenges for biopanning given the high degree of background cell-surface "noise" that threatens to drown out target-specific binding events 36 . The GBid approach fostered success in this challenging endeavor by enabling the facile creation of a large library of 10 10 potentially unique transformants containing ~80% full-length Fab inserts, approaching the maximum phage display library size empirically achieved using "traditional" library creation approaches 37 .…”

“…In addition to the use of GBid, several other steps were taken to help ensure success in our quest to discover anti-hCD20 antibodies: (i) Use of an immune library enriched for hCD20-binders, as opposed to a naïve or completely synthetic library. Indeed, the discovery of binders to multi-pass membrane proteins from naïve libraries has reportedly proven difficult/impossible 36 . The choice of chickens as a source of immune diversity was particularly appealing given the large phylogenetic distance of chickens from humans, allowing the discovery of binders to potentially important conserved mammalian epitopes that would not be accessible in more conventional immune hosts such as mice.…”

An epitope‐directed antibody affinity maturation system utilizing mammalian cell survival as readout

“…The tendency is remarkable especially when we use membrane proteins as the target. To overcome these problems, a phage displaybased method using a native antigen has been developed, which utilizes a membrane protein antigen expressed on the mammalian cell surface (Jones et al, 2016). However, this method inherently exhibits nonspecific phage binding to other membrane proteins on the cell surface.…”

Detection of antibody-secreting cells specific for the cytomegalovirus and herpes simplex virus surface antigens

“…Ebola), but challenging for herpesviruses using highly complex entry machinery. Therefore, conventional screening strategies based on single recombinant proteins may be unsuitable to identify antibodies/B-cells specific for such antigens (de Alwis et al, 2012;Jones et al, 2016). Prior studies have shown that some dengue virus neutralizing antibodies target complex epitopes that exist in a correct conformational state only on the surface of the virion particle, but not on a recombinant soluble antigen (Smith et al, 2014).…”