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Tutorials : Jan 15, 2011 ( )
Antibodies Against Membrane Protein Targets
Lipoparticle Technology Facilitates Discovery in This Challenging Protein Class!--h2>
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).
The addition of chemical moieties to proteins represents a strategy utilized in many phases of mAb discovery to capture and visualize target proteins. For example, the biotinylation of proteins enables their attachment to avidin surfaces with high affinity while the addition of fluorophores allows for protein localization, quantification, and sorting. These biochemical modifications have been applied to Lipoparticles to enable membrane proteins to be immobilized on solid supports and visualized without disrupting the conformation of incorporated receptor targets during mAb characterization.
For example, biotinylated Lipoparticles attached to neutravidin microplates have been used to screen antibodies and hybridoma supernatants by ELISA for highly reactive monoclonal antibodies (Figure 2A). Biotinylated Lipoparticles immobilized onto avidin wells and beads have also been used to enrich phage libraries for reactive antibodies (Figure 2B). Finally, biotinylated Lipoparticles have been immobilized onto streptavidin-coated biosensor chips to enable detailed kinetic measurements of mAb binding to membrane protein targets (Figure 2C).
Lipoparticles that have been biochemically modified to contain fluorophores enable advanced visualization, quantification, and sorting of target proteins by microscopy, microplate fluorescent readers, and flow cytometry (Figure 2D). For example, fluorescent Lipoparticles have been used to facilitate antibody discovery strategies such as yeast display where yeast cells express a library of antibodies and those with desired specificity are sorted by their interactions with fluorescent Lipoparticles.
Lipoparticles represent a novel approach to discovering antibodies against previously intractable and clinically relevant membrane protein targets. When used as an immunogen, the high concentration of structurally intact membrane proteins helps elicit a focused and robust immune response. Modifications to Lipoparticles, including biotinylation and fluorescence, can facilitate a variety of screening strategies for isolating and characterizing monoclonal antibodies. These tools have the potential for helping develop key diagnostic and therapeutic reagents against this challenging class of proteins.
Soma S. R. Banik, Ph.D. ([email protected]), and Eli Berdougo, Ph.D., are involved in scientific communication, and Benjamin J. Doranz, Ph.D., is president and CSO at Integral Molecular.
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