Identification of a novel allosteric GLP-1R antagonist HTL26119 using structure- based drug design.

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.

Corrigendum: Crystal structure of the GLP-1 receptor bound to a peptide agonist.

This corrects the article DOI: 10.1038/nature22800.

Crystal structure of the GLP-1 receptor bound to a peptide agonist.

Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.

Extra-helical binding site of a glucagon receptor antagonist.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro.

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.

Preclinical development of hybrid cell vaccines for multiple myeloma.

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We propose that hybrid cells, formed by fusing professional antigen-presenting cells with malignant plasma cells, would induce immune responses capable of mediating tumour regression. The human B-lymphoblastoid cell line, HMy2, was fused in vitro with CD138+ bead-separated myeloma plasma cells from five patients with MM. The hybrid cell lines generated in these studies grew stably in tissue culture, and maintained their phenotypic and functional characteristics, providing self-renewing cell lines with potential for therapeutic vaccination. The hybrid cells stimulated allogeneic and autologous T-cell proliferative responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, and directly activated both CD4+ and CD8+ T-cell responses. The enhanced T-cell stimulation correlated with expression of CD80 on the hybrid cells, and was inhibited by CTLA4-Ig fusion protein. The hybrid cell lines expressed several tumour-associated antigens known to be expressed in myeloma. These data show that self-replicating cell lines with enhanced immunostimulatory properties and potential for therapeutic vaccination can be generated by in vitro fusion of ex vivo myeloma cells and B-lymphoblastoid cell lines.