July 1, 2014 (Vol. 34, No. 13)
High Affinity and High Specificity through Application of RabMAb® Technology
G-protein coupled receptors (GPCRs) are seven-pass transmembrane proteins involved in numerous signaling pathways. Upon extracellular ligand binding and activation, members of this superfamily mediate processes such as neurotransmission, olfaction, and intercellular communication through cytoplasmic effectors.
Roughly 30% of currently marketed drugs exert their effects through GPCRs, and many others are druggable, untapped targets. Therapeutic development in the GPCR space and other arenas requires good tools, including antibodies. Potential uses of immunoreagents in GPCR drug pipelines include:
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Target discovery
Analysis of protein levels in affected tissues for identification of specifically expressed GPCRs -
Drug candidate generation and screening
Crystallization of GPCRs and structure-gathering for molecular docking and guided design of compounds
Pathway-neutral, ligand-free GPCR internalization assays for measuring activation upon compound application -
Lead discovery and evaluation
GPCR pathway delineation and pharmacodynamics experiments for comparative assessments and characterization of compounds -
Biomarker discovery and companion diagnostics
Generation of devices and assays for patient stratification and therapeutic selection based on GPCR expression levels
The Problem
High-quality antibodies to GPCRs are largely unavailable commercially. Those that are available often lack specificity and may not pick up low-abundance, endogenous proteins. As noted by Reubi et al., in Trends in Pharmaceutical Science, “…high affinity antibodies against GPCRs are extremely difficult to develop and…a majority of commercial receptor antibodies lack the sensitivity and the specificity required to detect receptors in native tissues, even though they may detect the high receptor levels seen in transfected cell lines.”1
The Solution
RabMAb® technology from Abcam enables the development of high-affinity, high-specificity antibodies to challenging targets such as GPCRs. The advantages of rabbit monoclonals stem from the uniqueness of the rabbit immune system. It has been widely observed that, compared to mice and other rodents, rabbits:
- Mount immune responses against a broader range of compounds
- Show less immune dominance toward carrier proteins
- Undergo more somatic gene conversion
- Possess longer and more heterogeneous CDR3 sequences
In addition, the rabbit spleen, the typical source of antibody-producing cells for generation of hybridomas, is larger and contains more cells than a mouse spleen. The combination of these properties results in a wider range of antibodies and therefore a better chance of finding the right experimental tools than with traditional technologies. Rabbit monoclonals also have on average 10–100x higher affinity than mouse monoclonals, and they are often better at detecting subtle differences such as slight sequence variations that distinguish members of a protein family.
Case Studies
The Abcam Custom Services team has worked with a number of investigators for the generation of rabbit monoclonal antibodies to GPCRs. The examples listed below give an overview of the performance of these antibodies in published research.
C-X-C chemokine receptor type 4 (CXCR4)
CXCR4 and its ligand CXCL12 influence cancer progression and metastasis through their roles in cell migration, proliferation, and angiogenesis, among other processes. CXCR4 is overexpressed in many tumor types, and its presence in affected tissues could be diagnostically and prognostically informative. Prior to the development of an anti-CXCR4 rabbit monoclonal (Figure 1), most immunostaining results pointed to a presumably artifactual nuclear localization of this receptor.
In comparing this rabbit monoclonal with previously characterized antibodies, side-by-side testing revealed more intense staining, lower background, and the expected plasma membrane distribution with the RabMAb primary. The new antibody therefore permits more extensive analysis of CXCR4 expression in fixed tumor samples.
µ Opioid Receptor (MOR)
MOR mediates the downstream effects of the widely used opioid analgesic morphine and related molecules, such as heroin, as well as the signaling pathways at the root of reward-based behaviors. Characterization of this receptor beyond immunohistochemical staining of frozen sections from rodents has been limited by the performance standards of available reagents.
To open up new experimental avenues via a variety of applications, a rabbit monoclonal antibody was developed using a peptide from the C-terminal region of MOR. This antibody works in a number of techniques, including Western blotting, immunoprecipitation (IP), immunohistochemistry, and immunocytochemistry (ICC) (Figure 2). This study identified localization of MOR in the pituitary, which had not been observable prior to this work, indicating a direct function there.
Performance of the anti-CXCL4 rabbit mAb UMB-2 in Western blotting (WB) and immunohistochemistry (IHC). UMB-2 specifically detects protein in wild-type (CXCR4+/+) but not in null (CXCR4–/–) mice by WB of brain homogenates (A) and IHC in FFPE brain tissue sections (B–F). E17 and P90 refer to embryonic and adult animals, respectively. Image used with permission from Fischer, T., et al., 2008, PLoS One 3: e4069.
Performance of the anti-MOR rabbit monoclonal antibody UMB-3 in WB, ICC, and IHC. (A) UMB-3 immunoprecipitates MOR from extract of wild-type (MOR+/+) but not null (MOR–/–) mice; the extra bands above and below the correct one (arrows) likely correspond to antibody eluted from the IP resin. The same antibody can be used for WB. (B) Immunoprecipitation of MOR by UMB-3 is blocked upon incubation with the immunizing peptide. In these panels, a polyclonal antibody from guinea pig (GP6) is used for WB detection of the IP. (C & D) Overexpressed MOR is present on HEK293 cell membranes and is internalized upon application of the opioid ligand etonitazene. (E & F) MOR is specifically detected in mouse brain sections by IHC. Image used with permission from Lupp, A., et al., 2011, Reg Pep 167: 9–13.
Figure 2
Somatostatin Receptor 2A (sst2A)
Somatostatin is an antiproliferative peptide involved in hormone secretion and other processes. Present in a number of human tumors, levels of its receptor sst2A have been shown to correlate with response to therapeutic somatostatin analogs in oncologic indications. Detection of sst2A, however, has been hampered by a lack of availability of good quality, specific antibodies.
A new antibody was raised to a C-terminal peptide of sst2A through the application of RabMAb technology. In both Western blotting and immunohistochemistry, the antibody specifically recognizes the protein of interest, and there is no reactivity in null animals. Direct comparisons in formalin-fixed, paraffin-embedded tissues reveal the increased signal relative to an available polyclonal. In addition, immunocytochemistry experiments produced novel data on the receptor localization relative to upstream and downstream peptide hormones.
Summary
RabMAb technology in combination with judicious screening has been successfully applied for making specific, sensitive antibodies to GPCRs. These rabbit monoclonals have the affinity required to detect native protein as well as a complete lack of cross-reactivity (according to criteria set by Michel et al. in Nayun-Schmiedeberg’s Archives of Pharmacology2), making them useful reagents for new investigations in GPCR research.
RabMAb technology is broadly applicable to other GPCRs and membrane proteins, a number of which are already available from Abcam (including the three presented here) alongside custom antibody generation services.
References
1Reubi, J.C., et al., 2013, Trends Pharm Sci 34: 676–688.
2Michel, M.C., et al., 2009, Nayun Schmied Arch Pharmacol 379: 385–388.
Amanda Hartman, Ph.D. ([email protected]), is RabMAb technology specialist at Abcam.
