Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β1-adrenergic (β1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β1AR. Immunization with the β1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β1AR to which the mAbs bind. The anti-β1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β1AR agonism

A monoclonal antibody raised against a thermo-stabilised β1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of 1- adrenoceptors expressed in stable cell lines

Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β1-adrenoceptor (β1AR-m23 StaR), on β1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ1-AR, but not its human homologue. Specific binding of mAb3 to tβ1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey 1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β1-AR conformational states and oligomeric complexes

Human Metapneumovirus Fusion Protein Vaccines That Are Immunogenic and Protective in Cotton Rats

Human metapneumovirus (hMPV) is a recently described paramyxovirus that is a major cause of upper and lower respiratory infection in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We previously reported the development of the cotton rat model of hMPV infection and pathogenesis (J. V. Williams et al., J. Virol. 79:10944-10951, 2005). We report here the immunogenicity of an hMPV fusion (F) protein in this model. We constructed DNA plasmids that exhibited high levels of expression of hMPV F in mammalian cells (DNA-F). These constructs were used to develop a novel strategy to produce highly pure, soluble hMPV F protein lacking the transmembrane domain (FΔTM). We then immunized cotton rats at 0 and 14 days with either control vector, DNA-F alone, DNA-F followed by FΔTM protein, or FΔTM alone. All groups were challenged intranasally at 28 days with live hMPV. All three groups that received some form of hMPV F immunization mounted neutralizing antibody responses and exhibited partial protection against virus shedding in the lungs compared to controls. The FΔTM-immunized animals showed the greatest degree of protection (>1,500-fold reduction in lung virus titer). All three immunized groups showed a modest reduction of nasal virus shedding. Neither evidence of a Th2-type response nor increased lung pathology were present in the immunized animals. We conclude that sequence-optimized hMPV F protein protects against hMPV infection when delivered as either a DNA or a protein vaccine in cotton rats