Developing antibodies to membrane spanning targets such as ion channels and GPCR family members
G-protein-coupled receptors (GPCRs) and ion channels are instrumental players in many signaling transduction pathways, making them the largest class of targets for therapeutics and drug discovery. There are over 350 non-olfactory GPCRs that are the receptors for a very diverse range of ligands, from small ions (e.g., calcium, neurotransmitters, metabolites), to peptides and glycoprotein hormones. Because GPCRs play a role in many diseases, including cancer, infection, and inflammation, there is considerable demand to produce antibodies against these cell-surface signal transmission receptor proteins.
The challenges associated with GPCR antibody development involve GPCR’s unique characteristic of multiple membrane spanning regions. They have only a small, exposed hydrophilic area on cellular membranes making them difficult for antibodies to target. In addition to the small exposed epitope regions, developing GPCR antigens for antibody development is also a challenge. Developing peptides against the exposed extracellular or intracellular regions can be useful for antibody development; however, many of these antibodies developed against linear peptides may not bind to the native protein because GPCRs and other membrane proteins exist in a lipophilic environment. In addition, they are not soluble under usual conditions, making GPCR antigens very difficult to express recombinantly and isolate in a soluble form for traditional in-vitro assays.
To overcome the challenge of developing antibodies for applications like flow cytometry or any application that requires the native structure of a GPCR, maintaining the structure of the GPCR is crucial. This is why we start with generating over-expressing cell lines, which will contain many more GPCRs on the cell surface. In addition, instead of immunizing with a single protein, we immunize with a complete cell which increases the chances of generating antibodies for GPCRs.
Rabbit monoclonal antibodies are particularly suited to detect GPCRs due to their high affinity and specificity. Even with the use of over-expressing cell lines to ameliorate the challenges associated with isolating GPCR antigens and their naturally lower expression levels, GPCRs may still have lower expression levels compared to other antigens. Therefore, more potent antibodies are required for developing anti-GPCR antibodies that perform well in applications such as IHC. The screening strategy used for recombinant rabbit monoclonal antibodies favors the selection of ideal antibodies capable of binding to GPCRs as plasma cell isolation screens for fully matured B-cells with high affinity, specificity, and potency.