Antibody Strategies for Membrane Protein Targets
Membrane-embedded proteins, including G protein-coupled receptors (GPCRs) and ion channels, are currently the most important class of therapeutic targets, and antibodies directed against them are highly sought for therapeutic, diagnostic, and research purposes. Despite substantial interest in these targets, high-quality antibodies against membrane proteins have been challenging to generate using conventional means.
Deriving antibodies against membrane proteins
Given the importance of membrane protein targets, private and academic laboratories have tried numerous approaches to raise antibodies against them (Table 1). The most common way of generating these antibodies is immunization with whole cells over-expressing the target protein. This allows membrane protein targets to be displayed in their native conformation without mechanical or detergent disruption. Typically, stable murine cells expressing a human membrane protein are used to immunize mice. Using this approach, a number of antibodies against highly expressing membrane proteins have been developed. Transiently-transfected cells, membrane preparations, and cells selected for the highest expression levels have been used to improve this approach, with varying degrees of success. However, for many important membrane protein targets the ability of cell-based immunogens to elicit high-quality antibodies has been limited by low membrane-protein expression, the abundance of non-specific proteins, and target protein toxicity during cell line selection.
|Immunogen||Native Conformation||Immunogen Concentration||Immunogen Purity|
|Reconstituted Protein in Vesicles||+/-||+++||+++|
|Table 1: Comparison of different immunogens used for the production of antibodies against complex membrane protein targets. (Source: Integral Molecular)|
Purified, reconstituted membrane proteins do not always maintain their native structure, but, when feasible, have resulted in highly significant monoclonal antibodies. For example, crystallography of the ?2-adrenergic receptor (?2-AR) was enabled by a conformational antibody developed against the purified GPCR.1 The ?2-AR protein was detergent solubilized, reconstituted into phospholipid vesicles, and then used as an immunogen in mice. Extensive screening resulted in the isolation of a conformation-dependent antibody that stabilized a critical flexible region of the receptor, enabling its crystallization. Similarly, another conformation-dependent antibody played a crucial role in one of the first crystal structures of an ion channel, the KcsA potassium channel.4 The limitation of this approach is the requirement for purified, solubilized, and reconstituted membrane protein in its structurally intact form, which is a difficult requirement for many membrane proteins.
Integral Molecular has taken a different approach to the challenge of presenting native membrane protein immunogens by developing the Lipoparticle.3 Lipoparticles are virus-like particles that incorporate high concentrations of target membrane proteins in their native conformation. Lipoparticles are produced from mammalian cells by co-expressing the retroviral structural core polyprotein, Gag, along with a desired membrane protein. Gag core proteins self-assemble at the plasma membrane, where they bud off and capture target membrane proteins (Figure 1). Lipoparticles are approximately 150 nm in diameter, so are readily suspended in aqueous solutions that can be used for inoculation. Because membrane proteins within Lipoparticles are derived directly from the cell surface without mechanical disruption or detergents, the native structure and orientation of the membrane proteins are retained. Unlike traditional sources of membrane proteins (Figure 2), Lipoparticles do not contain cytoplasmic proteins or inverted membrane proteins that can result in an unfocused immune response. Typical concentrations of specific membrane proteins in Lipoparticles are 50-200 pmol/mg, approximately 10 to 100 fold more concentrated than in cells or membrane preparations. As a result, Lipoparticles represent a high quality immunogen.
About the Authors
Soma S.R. Banik’s most current research both at Integral Molecular and elsewhere has focused on viral drug targets. Benjamin J. Doranz has been studying viruses and membrane proteins for the past 20 years.
1. Day PW, Rasmussen SG, Parnot C, Fung JJ, Masood A, Kobilka TS, Yao XJ, Choi HJ, Weis WI, Rohrer DK, Kobilka BK. A monoclonal antibody for G protein-coupled receptor crystallography. Nat Methods 2007; 4:927-9.
2. Timmerman P, Puijk WC, Meloen RH. Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology. J Mol Recognit 2007; 20:283-99.
3. Willis S, Davidoff C, Schilling J, Wanless A, Doranz BJ, Rucker J. Virus-like particles as quantitative probes of membrane protein interactions. Biochemistry 2008; 47:6988-90.
4. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature 2001; 414:43-8.
This article was published in Drug Discovery & Development magazine: Vol. 12, No. 9, October, 2009, pp. 14-17.