A pharmacologically active monoclonal antibody against the human melanocortin-4 receptor: effectiveness after peripheral and central administration

Abstract

The hypothalamic melanocortin-4 receptor (MC4R) is a constituent of an important pathway regulating food intake and energy expenditure. We produced a monoclonal antibody (mAb) directed against the N-terminal domain of the MC4R and evaluated its potential as a possible therapeutic agent. This mAb (1E8a) showed specific binding to the MC4R in human embryonic kidney 293 cells expressing the human MC4R and blocked the activity of the MC4R under basal conditions and after stimulation with alpha-melanocyte-stimulating hormone (alpha-MSH). The inverse agonist action of Agouti-related protein was significantly enhanced in the presence of mAb 1E8a. After a single intracerebroventricular injection into the third ventricle, mAb 1E8a (1 microg) increased 24-h food intake in rats. After 7 days of continuous intracerebroventricular administration, mAb 1E8a increased food intake, body weight, and fat pad weight and induced hyperglycemia. Because the complete mAb was ineffective after intravenous injection, we produced single-chain variable fragments (scFvs) derived from mAb 1E8a. In pharmacokinetic studies it was demonstrated that these scFvs crossed the blood-brain barrier and reached the hypothalamus. Consequently, the scFv 1E8a increased significantly food intake and body weight in rats after intravenous administration (300 mug/kg). The pharmacological profile of mAb 1E8a and the fact that its scFv was active after peripheral administration suggest that derivatives of anti-MC4R mAbs may be useful in the treatment of patients with anorexia or cachexia.

Fig. 1

Immunocytochemistry of HEK-293 cells transfected with hMC4R or hMC3R. Nuclear labeling (blue) is merged with immune labeling (red). a and b, specific membrane labeling was seen in HEK-293 cells expressing hMC4R when incubated with the mAb 1E8a (a) but not in HEK-293 cells expressing hMC3R (b). c, no labeling was observed with mAb 2G2 in hMC4R expressing cells. Magnification: 400×. d, immunoprecipitation of hMC4R and rMC4R. Lanes 1 and 2 show the results obtained with hMC4R and hMC3R, respectively, with mAb 1E8a, and lanes 5 and 6 show the corresponding results with rMC4R and rMC3R, respectively. Lanes 3 and 4 show control experiments with mAb 2G2. Purified mAb 1E8a precipitated both hMC4R (lane 1) and rMC4R (lane 5) but not hMC3R or rMC3R (lanes 2 and 6), whereas mAb 2G2 was inactive (lanes 3 and 4). e, inhibition of hMC4R immunoprecipitation by mAb 1E8a using increasing concentration of the NT peptide. Results are presented as percentage of inhibition of the signal obtained without NT peptide in function of the [NT]. Mean ± S.D. are calculated from three independent experiments. n = 5 measurements/experiment.

Fig. 2

Intracellular cAMP formation in HEK-293 cells transfected with hMC4R. a, concentration-response curves obtained with purified mAb 2G2. The mAb 2G2 had no effect on basal cAMP production. b, concentration-response curves obtained with α-MSH in the presence or absence of 100 nM of mAb 2G2 (▴) or PBS ( ). The presence of mAb 2G2 had no effect on the concentration-response curve of α-MSH. c, concentration-response curves obtained with purified mAb 1E8a. The concentration-dependent decrease in the intracellular cAMP content suggests an inverse agonist effect of mAb 1E8a. d, concentration-response curves obtained with α-MSH in the presence or absence of 100 nM mAb 1E8a (♦) or PBS ( ). The reduced maximum efficacy of α-MSH in the presence of mAb 1E8a suggests that this mAb acts as a noncompetitive antagonist. Data are presented as means ± S.D. calculated from three independent experiments. e, concentration-response curves obtained with AgRP in the presence of 100 nM mAb 1E8a (♦) or PBS ( ). The leftward shift of the AgRP response curve in the presence of mAb 1E8a suggests that this mAb acts in synergy with AgRP. Data are presented as means ± S.D. calculated from three independent experiments. ***, p < 0.001, F test.

Fig. 3

Time course of agonist-induced internalization of MC4R. HEK-293 cells stably expressing hMC4R were exposed to 200 nM α-MSH coupled to rhodamine red for various lengths of time. a, confocal images show the distribution of MC4R (red) at the selected time points (t = 0, 10, 30, and 45 min) after agonist stimulation in the presence (+) or absence (−) of mAb 1E8a. b, quantitative analysis of time course of agonist-induced MC4R internalization. Digitized fluorescence intensity on the cell surface membrane and total cellular fluorescence intensity were quantified as described under Materials and Methods. The graphic represents the MC4R internalization expressed as OD as a function of time in the presence (+) or absence (−) of mAb 1E8a. The presence of mAb 1E8a did not interfere with the internalization of the hMC4R.

Fig. 4

a, 24-h food intake in rats that received an intracerebroventricular injection of 1 μg of mAb 1E8a, BSA, or mAb 2G2. The injection of mAb 1E8a induced a significant increase in food intake. Data are presented as mean ± S.E.M. ##, p < 0.01 two-way ANOVA with repeated measures; *, p < 0.05; **, p < 0.01; ***, p < 0.001 Bonferroni post hoc test. b, body weight change of rats 24 h after intracerebroventricular injections. mAb 1E8a induced an increase in body weight compared with rats that received either mAb 2G2 or BSA. $, p < 0.05; $$, p < 0.01, Student’s t test. c, Seven-day food intake in rats, which received a continuous intracerebroventricular infusion of either 1 μg/day of mAb 1E8a or mAb 2G2. The infusion of mAb 1E8a induced a significant increase in food intake. Data are presented as mean ± S.E.M. ##, p < 0.01 two-way ANOVA with repeated measures. *, p < 0.05; **, p < 0.01, Bonferroni post hoc test. d, body weight change of rats during 7 days of continuous intracerebroventricular infusion. mAb 1E8a induced an increase in body weight compared with rats that received mAb 2G2. #, p < 0.05 two-way ANOVA with repeated measures. e and f, fasting glycemia (e) and fat pads weight (f) corrected for body weight (BW) of rats that received a continuous intracerebroventricular infusion of mAb 1E8 or mAb 2G2. $, p < 0.05, Student’s t test.

Fig. 5

Nucleotidic and amino acid sequences of scFv 1E8a (top) and scFv 2G2 (bottom). The hypervariable loops are underlined.

Fig. 6

Purification of the scFv 1E8a by Ni-NTA chromatography. a, Western blot probed with an anti-His₆ Ab that revealed the presence of the His₆-tagged scFv 1E8a. b, SDS-polyacrylamide gel electrophoresis stained with Coomassie blue, PE, flow-through (FT), wash fraction (W), and the elution of the purified scFv protein (see Materials and Methods).

Fig. 7

Intracellular cAMP production in HEK-293 cells transfected with hMC4R. a, concentration-response curve obtained with purified scFv 2G2. The scFv 2G2 had no effect on basal cAMP production. b, concentration-response curves obtained with α-MSH in the presence or absence of 100 nM scFv 2G2 ( ) or PBS ( ). The presence of the scFv 2G2 had no effect on the concentration-response curve of α-MSH. c, concentration-response curve obtained with purified scFv 1E8a. The concentration-dependent decrease in cAMP formation suggests an inverse agonist effect of the scFv 1E8a. d, concentration-response curves obtained with α-MSH in the presence or absence of 10 nM scFv 1E8a (⋄), 100 nM scFv 1E8a (♦), or PBS (▪). The reduced maximum efficacy of α-MSH in the presence of scFv 1E8a suggests that this mAb acts as a noncompetitive antagonist. Data are presented as means ± S.D. calculated from three independent experiments. **, p < 0.01; ***, p < 0.001, F test.

Fig. 8

a–d, the uptake of I-scFv by cortex (a), cerebellum (b), hypothalamus (c), and hippocampus (d) in comparison with whole brain as a function of Expt. e, comparison of I-scFv and I-Alb uptake by whole brain.

Fig. 9

a, 24-h food intake in rats that received an intracerebroventricular injection of 1 μg of scFv 1E8a or scFv 2G2. The injection of scFv 1E8a induced a significant increase in food intake. Data are presented as mean ± S.E.M. #, p < 0.05, two-way ANOVA with repeated measures and Bonferroni post hoc test; **, p < 0.01. b, body weight change in rats 24 h after injection. The injection of scFv 1E8a induced a smaller loss of body weight compared with rats that received scFv 2G2. $, p < 0.05, Student’s t test. c, 24-h food in-take in rats that received an intravenous injection of 300 μg/kg of scFv 1E8a or scFv 2G2. The injection of scFv 1E8a induced a significant increase in food intake. Data are presented as means ± S.E.M. ##, p < 0.01, repeated measures two-way ANOVA with Bonferroni post hoc test; *, p < 0.05; **, p < 0.01. d, body weight change in rats 24 h after injection. The scFv 1E8a significantly prevented the body weight loss seen in rats that received scFv 2G2. $$, p < 0.01, Student’s t test.