History of GPCR and GPCR antibodies

The “side-chain” theory


In 1891, Paul joined the team of Robert Koch, to set a new institute dedicated to serum testing of diverse infections. Working on diphtheria toxin, Paul Ehrlich hypothesised that immune response to infectious agents occurs through the intermediate of cellular structures that he decided to name "side chains".


Building his theory on Emil Fisher work (interaction of the enzyme with its substrate), Paul pictured the interaction between antibody and antigen as a lock and a key structure. In 1897, Paul finally published and lectured his theory (1, 2) about the side chain.


Focusing on the “receptive substance”


Amongst the different “side-chains”, Raymond Alhquist chose to dig up on the pharmacological experiments of Langley on the ‘receptive substance’ (3) and rapidly suggested the existence of alpha and beta adrenotropic receptors. He called them alpha and beta, based on the abilities of these receptors to stimulate physiological processes in response to chemical entities close to epinephrine.


“The alpha adrenotropic receptor is associated with vasoconstriction and stimulation (…). The beta adrenotropic receptor is associated with most of the inhibitory functions and one excitatory function” (4).


In 1959, Robert was just graduated from the Bronx High School of Science. Curious about the concepts of Alhquist, he rapidly figured that the key to the understanding of the mechanisms involving receptors would be new technologies that didn’t rely on detection of a functional response. In early 70’s, the apparition of radioligand binding assays will prove him right. Robert Lefkowitz then developed a procedure to purify alpha (6) and beta (5) adrenergic receptors, demonstrating their existence.


The hypothetical “receptive substance” was so rebranded with a more concrete word. The term “cell receptor” was created.


From cyclic AMP to G protein


In 1957, Earl Wilbur discovered that the increased formation of phosphorylase observed in the presence of epinephrine and glucagon was mediated by a heat-stable factor. This factor was identified as a nucleotide called cyclic 3,5-adenosine monophosphate or cyclic AMP (cAMP) (7). A few years later, Earl Wilbur Sutherland found that epinephrine stimulates the cell to form cAMP by a specific enzyme, originally called adenyl cyclase, and then correctly named adenylyl cyclase or adenylate cyclase (8).


            At that time, we didn’t know if the enzyme was the receptor for epinephrine or not. But the work of Lefkowitz concluded that adrenergic receptor and adenylyl cyclase were two distinct macromolecules (9).


If this first puzzle has been solved, the second one was not long in coming.


Rodbell et al. started questioning the relationship between the receptor and the enzyme?


They demonstrated that guanosine triphosphate (GTP) plays an obligatory role in the activation of adenyl cyclase by glucagon (10). Gilman, working in the same lab, persevered and finally stumbled upon a 35kDa subunit of a guanine nucleotide binding protein. This heterotrimeric protein was name Gilman protein, or G protein (11).


Robert Lefkowitz then gained the paternity of the discovery of the related receptor, the G protein-coupled receptors or GPCRs.


A common seven-transmembrane or heptahelical structure


In 1984, Kobilka joined Lefkowitz when the latter was trying to determine the DNA sequence of the beta2-adrenergic receptor. Using recombinant bacteria to produce large quantity of DNA (Dixon et al. 1986), Kobilka discovered that all GPCRs possess seven domains that cross through the cell membrane.


This “snake” 7TM receptor changes its conformation in response to ligand. In fact, the hormone-GPCR complex interacts with the heterotrimeric G protein and opens the nucleotide-binding site so that GDP can leave and GTP can bind. Immediately, the α subunit dissociates from the βγ dimer (Gβγ) and binds the surface of adenylate cyclase in preference to Gβγ. The activation of adenylate cyclase triggers the production of cAMP that stimulates the phosphorylation of many proteins through protein kinase A (PKA). Activated PKA then conducts phosphorylation cascade to specific targets and alter their respective activity.


The challenges of generating G protein-coupled receptor (GPCR) antibodies


GPCRs are today the most important drug target for more than one third of all developed therapeutics due to their implication in many signaling pathways. The exact size of this cell-surface receptor superfamily is unknown, but 416 members and their respective ligands have already been identified and divided into three classes (12). Others are qualified as orphan receptors: these are seven-transmembrane receptors but for which no natural or synthetic ligands have been identified so far. Depending of the source, the total number of identified GPCRs could be between 800 to 1,000 entities.


Since serpentine receptors are identified in many living organisms (bacteria, virus, mammalians…), can be found mutated or over expressed in cancers, infections and inflammations (13, 14, 15), they revealed themselves as interesting biomarkers to target.


But the development of anti-GPCR agonist and antagonist antibodies remains challenging.


In fact, the conformation of the GPCR extracellular region is highly variable and the exposed area of the GPCR extracellular epitopes is limited. These bottlenecks imply the need to raise stereo-specific antibodies that recognize the native conformation of functional GPCR.



Our technique circumvents the need of difficult and costly transmembrane protein antigen preparation, and allows performing efficient cell-associated antigen-based screening, either by fluorescence-activated cell sorting analysis (FACS) or whole cell enzyme-linked immunosorbent assay (ELISA).


SYnAbs has already conducted several Pharma and Biotech projects, in the course of which we were able to demonstrate our know-how in the raising of unique stereo-specific monoclonal antibodies.