Simultaneously blocking ANGPTL3 and IL-1β for the treatment of atherosclerosis through lipid-lowering and anti-inflammation

Objective

Blood lipid levels play a critical role in the progression of atherosclerosis. However, even with adequate lipid reduction, significant residual cardiovascular risk remains. Therefore, it is necessary to seek novel therapeutic strategies for atherosclerosis that can not only lower lipid levels but also inhibit inflammation simultaneously.

Methods

The fusion protein FD03-IL-1Ra was designed by linking the Angiopoietin-like 3 (ANGPTL3) nanobody and human interleukin-1 receptor antagonist (IL-1Ra) sequences to a mutated human immunoglobulin gamma 1 (IgG1) Fc. This construct was transfected into HEK293 cells for expression. The purity and thermal stability of the fusion protein were assessed using SDS-PAGE, SEC-HPLC, and differential scanning calorimetry. Binding affinities of the fusion protein to ANGPTL3 and IL-1 receptor were measured using Biacore T200. The biological activity of the fusion protein was validated through in vitro experiments. The therapeutic efficacy of the fusion protein was evaluated in an ApoE-/- mouse model of atherosclerosis, including serum lipid level determination, histological analysis of aorta and aortic sinus sections, and detection of inflammatory and oxidative stress markers. ImageJ software was utilized for quantitative image analysis. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test.

Results

The FD03-IL-1Ra fusion protein was successfully expressed, with no polymer formation detected, and it demonstrated good thermal and conformational stability. High affinity for both murine and human ANGPTL3 was exhibited by FD03-IL-1Ra, and it was able to antagonize hANGPTL3's inhibition of LPL activity. FD03-IL-1Ra also showed high affinity for both murine and human IL-1R, inhibiting IL-6 expression in A549 cells induced by IL-1β stimulation, as well as suppressing IL-1β-induced activity inhibition in A375.S2 cells. Our study revealed that the fusion protein effectively lowered serum lipid levels and alleviated inflammatory responses in mice. Furthermore, the fusion protein enhanced plaque stability by increasing collagen content within atherosclerotic plaques.

Conclusions

These findings highlighted the potential of bifunctional interleukin-1 receptor antagonist and ANGPTL3 antibody fusion proteins for ameliorating the progression of atherosclerosis, presenting a promising novel therapeutic approach targeting both inflammation and lipid levels.