Stabilizing GPCRs in Their Therapeutically Relevant Conformation to Discover Therapeutic Antibodies
The discovery of therapeutic antibodies to G-protein coupled receptors (GPCRs), one of the most attractive drug target classes for targeted therapy, remains challenging. The vast majority of GPCR antibodies in (pre-)clinical development are antagonists, blocking GPCR signaling (Hutchings 2020). To date, only two GPCR antibodies are FDA-approved: mogamulizumab inhibiting CC-chemokine receptor 4 function for the treatment of T cell lymphoma, and erenumab blocking calcitonin gene-related peptide receptor for migraine (Liu et al. 2021).
The Confo® technology platform is based on ConfoBodies®, camelid single domain antibody fragments (VHHs) which form a complex with the desired conformational state of the GPCR (Rasmussen et al. 2011a and b; Staus et al. 2014 and 2016). In small molecule drug discovery, ConfoBodies are used for active state-directed drug screening and structure-guided elaboration of small molecules (Pardon et al. 2018). We were interested to test whether the same principle could be applied to antibody discovery.
We will show the unique potential of ConfoBody stabilized GPCR conformations as critical reagents to facilitate de novo discovery of VHHs with desired conformation specificity to human GPCRs. Following camelid immunization with a clinically validated GPCR stabilized with an active state-specific ConfoBody, a sequence diverse panel of extracellular binding, nM potency agonistic VHH antibodies was identified. Of note, the entire discovery procedure was run independently from purified protein. We will present in vitro and in vivo data with monovalent and Fc formatted VHH, confirming full agonist pharmacology of the antibodies, and showing a novel ‘ligand mimicking’ mode of agonism, independent from receptor cross-linking. We will show an agonistic VHH enabled high resolution cryo-EM GPCR active state structure.
In addition to the above example, we have meanwhile successfully deployed the same principle on two additional GPCRs, further demonstrating the potential of the Confo® technology for driving therapeutic antibody discovery.
Hutchings C. A review of antibody-based therapeutics targeting G protein-coupled receptors: an update. Expert Opin Biol Ther (2020) 20:925-935.
Liu Q, Garg P, Hasdemir B, Wang L, Tuscano E, Sever E, Keane E, Lujan Hernandez AG, Yuan TZ, Kwan E, Lai J, Szot G, Paruthiyil S, Axelrod F, Sato AK. Functional GLP-1R antibodies identified from a synthetic GPCR-focused library demonstrate potent blood glucose control. MAbs (2021) 13: e1893425.
Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK. Structure of a nanobody-stabilized active state of the b2 adrenoceptor. Nature (2011a) 469: 175-80.
Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature (2011b) 477:549-55.
Staus, DP, Wingler, LM, Strachan, RT, Rasmussen, SG, Pardon, E, Ahn, S, Steyaert, J, Kobilka, BK, and Lefkowitz, RJ. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol Pharmacol (2014), 85, 472–481.
Staus, DP, Strachan, RT, Manglik, A, Pani, B, Kahsai, AW, Kim, TH, Wingler, LM, Ahn, S, Chatterjee, A, Masoudi, A, Kruse, AC, Pardon, E, Steyaert, J, Weis, WI, Prosser, RS, Kobilka, BK, Costa, T, and Lefkowitz, RJ. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature (2016), 535, 448–452.
Pardon E, Betti C, Laeremans T, Chevillard F, Guillemyn K, Kolb P, Ballet S, Steyaert J. Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors. Angewandte Chemie (2018) 57:5292-5295.