Integral membrane proteins such as GPCRs and ion channels represent key molecules involved in cell signaling, cell homeostasis, and human disease, with one-third of all approved drugs targeting such proteins. Despite the clinical relevance of membrane protein targets, monoclonal antibodies (mAbs), especially those targeting conformational epitopes, have been difficult to develop.
As a result, fewer than a dozen mAbs have been described that target conformational epitopes on the 426 ion channels and 533 transporters in the human genome. Similarly, the vast majority of the 907 human GPCRs have no inhibitory or conformation-dependent mAbs available. While the number of FDA-approved therapeutic mAbs continues to grow, only two of these mAbs target multiple-spanning membrane proteins (CD20 and CD25) and none target the largest membrane protein families, GPCRs, ion channels, or transporters. The lack of monoclonal antibody reagents against this important class of proteins reflects the need for more effective mAb discovery approaches.
Antibodies that recognize conformation-dependent epitopes on membrane proteins are usually the most valuable type of antibody for therapeutics and diagnostics because they can bind to critical structures of the receptor that are required for function and can detect the protein as it exists on the cell surface. However, deriving such antibodies requires presenting membrane proteins to the immune system in their native conformation and orientation. Current immunization approaches using peptides, purified membrane proteins, membrane preparations, or whole cells have had limited success in eliciting conformational antibody responses against many membrane protein targets.
For example, widely used whole cell immunogens preserve native membrane protein topology, but often lack sufficient concentrations of the target protein due to low expression levels, protein toxicity, poor trafficking to the cell surface, or interference from other cellular proteins. New immunization approaches that offer high concentrations of complex membrane proteins in their native structure are essential for generating antibodies against clinically relevant membrane protein targets.
The Lipoparticle developed by Integral Molecular represents a useful technology for immunization and antibody discovery applications against membrane protein targets. Lipoparticles are virus-like particles (VLPs) that incorporate high concentrations of target membrane proteins in their native conformation. Lipoparticles are produced in mammalian cells by co-expressing a retroviral structural core polyprotein, Gag, along with a user-specified membrane protein which is correctly translated, folded, and post-translationally modified.
Gag core proteins self-assemble at the plasma membrane, where they bud off and capture target membrane proteins directly from the cell surface at concentrations of 50–200 pmol/mg, approximately 10–100 fold higher concentration than cells or membrane preparations. Immunization with Lipoparticles presents high concentrations of membrane proteins in their native topology to the immune system, making them well suited for antibody elicitation.
Generating Immune Responses
Lipoparticles have been used to generate reactive immune sera against all major classes of membrane proteins, including GPCRs, ion channels, transporters, oligomeric single-transmembrane proteins, and other multispanning membrane proteins. For example, Lipoparticles containing the GPCR CXCR4, the ion channel Hv1, the tetraspanin claudin-1, the transporter Glut1, and the single transmembrane (TM) oligomeric protein CD27 were each incorporated into immunogen-grade Lipoparticles and used to immunize mice.
In all cases, specific serum responses against each target were observed under native conditions by flow cytometry (Figure 1A) and under denaturing conditions by Western blot (Figure 1B). By comparison, traditional immunogens such as whole cells and membrane preparations yielded weaker responses. For example, transfected cells and membrane preparations containing the GPCR CXCR4 elicited weak immune responses to CXCR4 while the strongest immune responses (50% serum reactivity >1:500) were obtained with CXCR4 Lipoparticles (Figure 1C).