Phage display technology is a powerful approach for isolating human antibodies, where genetically modified phage carrying antibody fragments are used for panning and screening of antibody libraries (Figure 1A). Phage display enables screening of large and diverse antibody libraries (>1010 variants), allows for modifiable in vitro selection conditions for deriving antibodies with specific functionalities, and bypasses immune tolerance limitations, facilitating the selection of affinity reagents against even highly conserved protein targets.
However, phage panning with membrane protein antigens presents a unique set of challenges, including a requirement for high concentrations of target proteins that are not easily purified, a propensity for phage to stick nonspecifically to charged lipids and glycoproteins on cell membranes, and the need to retain native membrane protein structures during capture and panning procedures.
As a result of these limitations, phage display has largely focused on soluble proteins to date, with less than a dozen phage-derived mAbs in clinical studies targeting unique membrane proteins, nearly all of which are single transmembrane proteins rather than more complex multi-transmembrane proteins.
Integral Molecular is combining its Lipoparticle technology with optimized phage protocols to overcome the specific challenges associated with phage display for isolating antibodies against membrane protein targets (Figure 1B). Lipoparticles incorporate high concentrations of properly folded and oriented membrane proteins on their surface, such as specific GPCRs, ion channels, or transporters. Membrane proteins are incorporated on Lipoparticles at concentrations of 50–200 pmol/mg, 10 to 100 fold greater than cells or membrane preparations, providing a highly enriched target for phage clone selection.
Lipoparticles are particularly well suited for phage display as they are small (~150 nm), stable, and can be biotinylated, enabling efficient capture to commonly used streptavidin surfaces. Furthermore, Lipoparticles can be made without target receptors (Null Lipoparticles) or from heterologous cell types (human, murine, avian, etc.), enabling controlled negative selection steps and improved recovery of antigen-specific clones.
Conventional phage display protocols have now been optimized with Lipoparticle antigens, resulting in sensitive and specific selection of antibodies directed against membrane protein targets.
In a controlled panning experiment comparing a membrane protein antigen on Lipoparticles versus the same antigen in its purified form, similar numbers of reactive clones were recovered, demonstrating equivalent sensitivity between antigen types (Figure 1C).
Optimized phage panning with Lipoparticles provides enrichment of target-specific clones, with a round-dependent increase in reactivity and no significant reactivity against Null Lipoparticles (Figure 1D). Optimization of phage display with Lipoparticles is now enabling antibody campaigns against a wide variety of membrane protein classes, including GPCRs, ion channels, transporters, viral envelopes, and other complex multi-transmembrane protein targets.