GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice

Abstract

Background: Patients with cancer undergoing chemotherapy experience cachexia with anorexia, body weight loss, and the depletion of skeletal muscles and adipose tissues. Effective treatment strategies for chemotherapy-induced cachexia are scarce. The growth differentiation factor 15 (GDF15)/GDNF family receptor alpha-like (GFRAL)/rearranged during transfection (RET) axis is a critical signalling pathway in chemotherapy-induced cachexia. In this study, we developed a fully human GFRAL antagonist antibody and investigated whether it inhibits the GDF15/GFRAL/RET axis, thereby alleviating chemotherapy-induced cachexia in tumour-bearing mice.

Methods: Anti-GFRAL antibodies were selected via biopanning, using a human combinatorial antibody phage library. The potent GFRAL antagonist antibody A11 was selected via a reporter cell assay and its inhibitory activity of GDF15-induced signalling was evaluated using western blotting. To investigate the in vivo function of A11, a tumour-bearing mouse model was established by inoculating 8-week-old male C57BL/6 mice with B16F10 cells (n = 10-16 mice per group). A11 was administered subcutaneously (10 mg/kg) 1 day before intraperitoneal treatment with cisplatin (10 mg/kg). Animals were assessed for changes in food intake, body weight, and tumour volume. Plasma and key metabolic tissues such as skeletal muscles and adipose tissues were collected for protein and mRNA expression analysis.

Results: A11 reduced serum response element-luciferase reporter activity up to 74% (P < 0.005) in a dose-dependent manner and blocked RET phosphorylation up to 87% (P = 0.0593), AKT phosphorylation up to 28% (P = 0.0593) and extracellular signal regulatory kinase phosphorylation up to 75% (P = 0.0636). A11 inhibited the action of cisplatin-induced GDF15 on the brainstem and decreased GFRAL-positive neuron population expressing c-Fos in the area postrema and nucleus of the solitary tract by 62% in vivo (P < 0.05). In a melanoma mouse model treated with cisplatin, A11 recovered anorexia by 21% (P < 0.05) and tumour-free body weight loss by 13% (P < 0.05). A11 significantly improved the cisplatin-induced loss of skeletal muscles (quadriceps: 21%, gastrocnemius: 9%, soleus: 13%, P < 0.05) and adipose tissues (epididymal white adipose tissue: 37%, inguinal white adipose tissue: 51%, P < 0.05).

Conclusions: Our study suggests that GFRAL antagonist antibody may alleviate chemotherapy-induced cachexia, providing a novel therapeutic approach for patients with cancer experiencing chemotherapy-induced cachexia.

Keywords: Cancer cachexia; Chemotherapy; Cisplatin; GDF15; GFRAL; GFRAL antagonist antibody.

Figure 1

Anti‐GFRAL antibodies were selected by solution‐phase biopanning. (A) Strategy for anti‐GFRAL antibody selection using a human combinatorial antibody phage library. Black dotted lines represent GFRAL‐binding phage pool. (B) Polyclonal phage ELISA for phage pools from each round of biopanning. (C) Heavy‐chain complementarity‐determining region 3 (H‐CDR3) amino acid sequences of the anti‐GFRAL antibody clones. (D) Binding of the anti‐GFRAL antibodies to both human and mouse GFRAL extracellular domains verified by ELISA. (E) Luciferase reporter assay of HEK293 cells transfected with human GFRAL, human RET, and SRE‐luciferase genes for the selection of the most potent GFRAL antagonist antibody with inhibitory activity against GDF15‐induced luminescent signal (n = 5). Data are presented as the mean ± standard error of the mean (SEM), analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn's test. Statistical differences in post hoc testing are indicated as ns = non‐significant, ***P < 0.005.

Figure 2

The antibody A11 binds to GFRAL and inhibits the signal transduction of the GDF15/GFRAL/RET axis. (A) SPR sensorgram showing the binding kinetics of the antibody A11 to the recombinant human GFRAL extracellular domain. Black box indicates concentration of antibody A11 (1.89–31.25 nM). (B) Flow cytometry analysis of the control antibody and antibody A11 binding in wild‐type (WT) and human GFRAL gene‐transfected HEK293 cells. Black box indicates concentration of antibody A11 (0–50 nM). (C) Representative immunostaining images of commercial anti‐GFRAL antibody (left) and antibody A11 (right) binding in WT and human GFRAL gene‐transfected HEK293 cells. The white scale bar represents 30 μm. (D) Western blot analysis showing the inhibitory effect of the antibody A11 on the phosphorylation of RET, AKT, and ERK in human GFRAL/RET gene‐transfected HEK293 cells. The gel is representative of three independent experiments.

Figure 3

The antibody A11 attenuates cisplatin‐induced cachexia in vivo Normal: control mice with PBS injections, Cis/Con: mice with cisplatin and control antibody injections, Cis/A11: mice with cisplatin and the antibody A11 injections (n = 7 mice per group). (A) Cumulative food intake per mouse on days 2–5. (B) Body weight change compared with the weight before drug administration followed up to day 5. (C) Weights of isolated skeletal muscles (quadriceps, gastrocnemius, and soleus). (D) Weights of isolated adipose tissues (eWAT and iWAT). (E) Plasma levels of GDF15 on day 5, determined by ELISA. Data are presented as the mean ± SEM, analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn's test. Statistical differences in post hoc testing are indicated as ns = non‐significant, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.

Figure 4

The antibody A11 suppresses c‐Fos accumulation on the hindbrain of cisplatin‐treated mice Normal: control mice with PBS injections, Cis/Con: mice with cisplatin and control antibody injections, Cis/A11: mice with cisplatin and the antibody A11 injections (n = 5 mice per group). (A) Representative sections of the area postrema (AP) and the nucleus of the solitary tract (NTS) showing c‐Fos expression in GFRAL‐expressing neurons 4 h after cisplatin treatment on day 4. White arrows represent neurons stained with both GFRAL and c‐Fos, indicating activation by cisplatin‐induced GDF15. The white scale bars represent 150 μm and the yellow scale bar represents 20 μm. The CC represents central canal. (B) Quantification of cisplatin‐induced c‐Fos expression in the AP/NTS 4 h after cisplatin injection. Each point represents one mouse. Data from each mouse represent quantification from 6 alternative sections. (C) Number of GFRAL‐positive neurons in the AP/NTS that co‐express c‐Fos 4 h after cisplatin injection. Each point represents one mouse. Data from each mouse represent quantification from 6 alternative sections. Data are presented as the mean ± SEM, analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn's test. Statistical differences in post hoc testing are indicated as ns = non‐significant, *P < 0.05, **P < 0.01.

Figure 5

The antibody A11 ameliorates cisplatin‐induced cachexia in a mouse model of melanoma Normal: control mice without tumour, TB/Con: tumour‐bearing mice with control antibody injections, TB/A11: tumour‐bearing mice with the antibody A11 injections, TB/Cis/Con: tumour‐bearing mice with cisplatin and control antibody injections, TB/Cis/A11: tumour‐bearing mice with cisplatin and the antibody A11 injections (n = 10–16 mice per group). (A) Cumulative food intake per mouse on days 13–18. (B) Body weight change compared with the weight before drug administration followed up to day 17. (C) Weights of isolated skeletal muscles (quadriceps, gastrocnemius, and soleus). (D) Weights of isolated adipose tissues (eWAT and iWAT). (E) Plasma GDF15 levels on day 18, determined by ELISA. Data are presented as the mean ± SEM, analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn's test. Statistical differences in post hoc testing are indicated as ns = non‐significant, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.