Pharmacological characterisation of a monoclonal antibody raised against a thermo-stabilised conformation of the turkey β₁-adrenoceptor in CHO-K1 cells.

M Soave1, A Brown2, J Woolard1, SJ Hill1. 1University of Nottingham, Nottingham, UK, 2Heptares Therapeutics Ltd., Welwyn Garden City, UK

The β₁-adrenoceptor (β₁-AR) is an important regulator of cardiac function (1). It has been shown that autoimmune antibodies directed against the second extracellular loop (ECL2) of the β₁-AR play a role in the pathogenesis of idiopathic dilated cardiomyopathy (1). Recently, a series of murine monoclonal antibodies were raised against the purified thermo-stabilised turkey β₁AR protein that was originally used to solve its crystal structure (β₁-m23 StaR) (2). One of these monoclonal antibodies (mAb3) was shown to bind to ECL2 of purified β₁-AR StaR (2). Here, we have characterised the pharmacological properties of mAb3 in Chinese Hamster Ovary cells (CHO-K1) expressing the turkey β₁-AR.

CHO-K1 cells stably expressing either a truncated turkey β₁AR (tβtrunc; (3)), or a variant containing thermo-stabilising mutations (tβ6-m23; (3)) were studied using [3H]-CGP 12177 radioligand binding, [3H]-cAMP accumulation and CRE-mediated SPAP production (as described in (4)). Confocal imaging of mAb3 binding (method adapted from (5)) to fixed cells was performed on a Zeiss LSM 710 microscope, using a polyclonal goat anti-mouse-Rhodamine secondary antibody.

MAb3 was able to inhibit the specific binding of [3H]-CGP 12177 to CHO-K1 cells expressing tβtrunc or tβ6-m23 β₁-ARs (Log IC₅₀ -7.91 ± 0.08; -7.83 ± 0.12, n=7, respectively). Confocal imaging confirmed that mAb3 bound specifically to turkey β₁-ARs expressed on the surface of each cell type. No binding of mAb3 was detected in un-transfected CHO-K1 cells. MAb3 was able to inhibit isoprenaline-induced [3H]-cAMP accumulation and CRE-mediated SPAP production in CHO tβ6-m23 cells, whilst having no significant effect on the [3H]-cAMP accumulation response mediated by the partial agonist CGP 12177 (Table 1). MAb3 alone was unable to elicit any agonist response at either receptor.

Table 1 - Log EC₅₀ values and % maximal stimulation of [3H]-cAMP accumulation and CRE-mediated SPAP production compared to 10μM isoprenaline in CHO tβ6-m23 CRE-SPAP cells in response to isoprenaline or CGP 12177 in combination with differing concentrations of mAb3. Data are mean ± s.e.m. from five separate experiments. *p<0.05; one-way ANOVA followed by Bonferroni’s post-hoc test comparing Log EC₅₀ and % iso max of mAb3 treatment to either isoprenaline or CGP 12177 alone.

In summary, this study demonstrates the ability for an antibody directed against ECL2 to bind selectively to the turkey β₁-AR and inhibit isoprenaline-induced cAMP and CRE-SPAP responses.

(1) – Deubner N. et al., (2010), Eur J Heart Fail, 12, 753-762

(2) – Hutchings C.J. et al., (2014), mAbs, 6, 1-16

(3) – Baker J.G. et al., (2011), Naunyn Schmiedebergs Arch Pharmacol, 384, 71-91

(4) – Baker J.G. (2010), PLoS One, 5, e15487

(5) – Self T.J. et al., (2005), Brit J Pharmacol, 146, 612-624