The challenges behind G protein-coupled receptor (GPCR) antibodies

Bernd Gerhartz, Vice President of Technology Development, outlines what we’re doing to overcome the difficulties associated with GPCR antibody production. 

​​G protein-coupled receptors play a role in many diseases, including cancer, infection, and inflammation1. As such, these cell-surface signal transmission receptor proteins are important targets for therapeutics and diagnostics and there is a considerable demand to produce antibodies against them.

Bernd Gerhartz, Vice President of Technology Development here at Abcam explains why they are so in demand, “GPCRs are a cornerstone in cell signaling. They are the transmission between a signal from the external environment of the cell and translating that into a downstream signaling cascade leading to a cellular response. Pharmaceutically, GPCRs are extremely important, the biggest class of pharmaceutical drugs are against GPCRs. There is a lot of ongoing research on GPCRs and their use as drug targets.”

GPCRs and other multispan transmembrane proteins exist in a lipophillic environment, so they are not soluble in the usual conditions.​​

However, because they are difficult to prepare and isolate in solution, and they have small exposed area of extracellular epitope,1 GPCRs are notoriously difficult targets to generate antibodies against, “Usually, when we carry out antibody generation, we express a protein in a soluble form, and this is used as the immunogen to inject into the host animal. With GPCRs and other membrane proteins, it is very difficult to express them recombinantly and isolate them in a soluble form to produce a native folded antigen. Usually, due to the nature of a soluble protein it will keep its native fold in a hydrophilic environment. GPCRs and other multispan transmembrane proteins, however, exist in a lipophillic environment, so they are not soluble in the usual conditions.”

We are starting to look at ways that we can address some of the issues to improve antibody production against GPCRs in future.

“Maintaining the structure of the GPCR is important when you are generating the antibody, especially if you want to use applications like flow cytometry where the antibody will only recognize the extracellular region of the GPCR protein. For any application that requires the native structure of a GPCR, it is extremely difficult to generate specific, high-affinity antibodies.”

Despite the difficulty in producing specific, high-affinity antibodies to GPCRs for such applications, we are starting to look at ways that we can address some of the issues to improve antibody production against GPCRs in future, as Bernd explains, “There is no one fix-all solution, but what we are doing is generating overexpression cell lines, and instead of immunizing with a single protein, we immunize with a complete cell. The overexpressing cell will contain many more GPCRs on the cell surface, and you stand a better chance of generating high-affinity antibodies for your GPCRs."

“The cell lines we use for this have an extremely high expression of the GPCR. There is a high level of cross-reactivity between different GPCRs due to the similarity in their protein sequences, so we need to validate the antibodies we produce by testing them for cross-reactivity with other GPCRs.”​

GPCR antibodies

The production of RabMAb® rabbit monoclonal anti-GPCR antibodies at Abcam is beginning to take off with rabbit monoclonal antibodies to nearly 100 GPCRs on our catalog. Our new recombinant RabMAb® rabbit monoclonal against CXCR2 (ab225732) was designed specifically for immunohistochemistry using a synthetic peptide immunogen.

CXCR2 is an important GPCR in immuno-oncology, an area of great interest right now for cancer research.​​

CXCR2 and one of its many ligands CXCL8 (IL-8) are important regulators in cancer development and the CXC family of chemokines and their GPCRs including CXCR2 have been shown to have great potential as prognostic and predictive biomarkers within diagnostic tests2 . Bernd explains that “CXCR2 is an important GPCR in immuno-oncology, an area of great interest right now for cancer research. This signaling pathway stimulates angiogenesis and MMP activation during tumor progression. It is also involved in immune system suppression, allowing the tumor to survive in the host undetected by the immune response."

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Bernd Gehartz

Photo: ​​Bernd Gerhartz, Vice President of Technology Development​

Our RabMAb® platform is particularly suited to the development of antibodies against GPCRs, “RabMAb antibodies are very potent, and this is very important for GPCRs that are not highly expressed on the cell. For IHC, it is especially important that we have a very potent antibody that recognizes a GPCR like CXCR2. We also have many RabMAb antibodies for GPCR ligands and their downstream signaling molecules.”

At Abcam, we currently have many antibodies against GPCRs in our catalog, but we’re working hard to make even more. Further developing the methods described here by Bernd will enable us to produce even more high-affinity antibodies for GPCRs suitable for use in a wide range of applications. Each GPCR is unique and requires careful antibody production and validation, but we are committed to delivering these targets to our customers. Over time more and more GPCRs appearing on our catalog because we know just how important these proteins are for the future of cancer research.

The  CXCR2 RabMAb® rabbit monoclonal antibody (ab225732) is also available in a PBS-only (BSA and azide-free) version (ab245982), which is ideal for conjugation with different labels, like metal-conjugation for Imaging Mass CytometryTM.


References

1) Jo, M., & Jung, S. T. (2016). Engineering therapeutic antibodies targeting G-protein – coupled receptors. Experimental & Molecular Medicine48(2), e207-9.

2) Heras, S. C., Martínez-balibrea, E., Heras, S. C., & Martínez-balibrea, E. (2018). CXC family of chemokines as prognostic or predictive biomarkers and possible drug targets in colorectal cancer, 24(42), 4738–4749. 


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