NAFLD is a metabolic disease and positively associated with hypercholesterolemia that is increasingly acknowledged as the key factor of excessive lipid uptake and lipotoxicity in liver resulting in pathologic insult.3, 41 It is projected to develop into cirrhosis with increasing risk of hepatocellular carcinoma and may be the main inducement of liver transplantation.42 Despite its prevalence and seriousness, NAFLD still lacks effective therapeutics.38 ANGPTL3 is a key lipid metabolism regulator leading to high levels of plasma TG, TC and LDL-C.10 In this study, we investigated whether inhibiting ANGPTL3 is an effective approach for NAFLD treatment. Herein, we have developed a VHH-Fc fusion protein against ANGPTL3, which significantly ameliorated hepatic lipid accumulation and liver injury in HF/HCCD-induced NAFLD mice through reducing serum lipid levels of TG, TC and LDL-C.
Although NAFLD was availably treated by reducing blood lipids and blood glucose, anti-fibrosis and anti-inflammatory in animal models, it still affects approximately 25% of the population worldwide.39 Until now, exclusive Saroglitazar was the approved drug for the treatment of NAFLD, which is a PPAR-α/γ agonist.7 Unfortunately, the PPAR-α/γ agonist has been reported to increase about 2-4% of body weight and to have adverse reactions including peripheral oedema precipitated in probably 5% of treated patients and fatal heart failure occurred in approximately 11% of treated patients.40 The traditional lipids-reducing agents statins were not applicable to NAFLD treatment as for its intolerance or side effects.41 Thus, a safe and efficient targets for NAFLD is urgently needed. ANGPTL3, as a depressor of LPL and EL, was recently revealed elevated expression levels in liver of NAFLD patients and contributed to increased circulating ANGPTL3. 16 Interestingly, neither the homozygous loss-of-function of ANGPTL3 nor the inhibitors of ANGPTL3 all did generate adverse effects with preeminent safety and tolerability, but leaded to very low TG, VLDL, LDL and non-esterified fatty acids (NEFAs).42, 43 Our results suggested ANGPTL3 inhibitor showed splendid reduction of TG (44.2%), TC (36.6%) and LDL-C (54.4%). More importantly, its multiple administrations significantly reduced 18.3% of liver weight, ameliorated liver coloration and morphology, improved hepatic lipid accumulation and protected HF/HCCD-related liver injury. All those results indicated blocking ANGPTL3 was a novel therapeutic approaches for NAFLD with satisfactory efficacy and safety.
The existing ANGPTL3 inhibiting tactics includes the monoclonal antibody (evinacumab), ASO (IONIS-ANGPTL3-LRx) and the CRISPR/Cas9 gene knockout technology, whereas neither of them is employed for NAFLD therapy. Evinacumab and the ASO-based drugs are expensive and the CRISPR/Cas9 gene knockout technology haven’t been approved for in clinical use.19–21 Nanobodies, which are promising therapeutic agents, display higher binding ability, better penetration and superior stability. Recently, caplacizumab, which is a bivalent VHH for thrombotic thrombocytopenic purpura treatment, has been regarded as the first nanobody approved by the FDA.35, 44, 45 The development and characterization of VHHs that specifically block ANGPTL3 have not been reported so far. Hence, we developed a nanobody as the ANGPTL3 inhibitor by establishing a naïve phage-display VHHs library about 5×109 after immunizing alpaca with hANGPTL3 (S17-K170). We obtained 11 specific VHH candidates against hANGPTL3 (S17-K170) with different CDRs sequences by indirect ELISA from the library.
The VHHs have prominently short circulation half-life due to its small size of approximately 15kDa and is restricted to disease therapy. Multiple methods have been applied to benefit its half-life period in vivo, such as VHHs can be conjugated with human serum albumin, fused to IgG-Fc and PEGylated.46–48 Meanwhile, previous studies revealed that bivalent nanobody had better affinity than monovalent modality. In particular, the bivalent VHHs fused with Fc showed above 10 times higher affinity and stronger therapeutic effects than monovalent in vitro and in vivo, which also emerged longer half-life of about 15 days when compared to that of monovalent (~30 min) and tandem bivalent VHHs (~60 min).48, 49 In this study, the 11 candidate VHHs were fused to human IgG1 Fc domain to construct the bivalent VHHs-Fc fusion proteins and then successfully expressed in ExpiCHO cells, followed by its purification by protein A affinity chromatography. The pharmacodynamics results of single administration suggests that the VHHs-Fc fusion protein has long half-life in vivo as the durable lipid-reducing effect of 12 days.
The full length and the cleaved CCD of ANGPTL3 proteins all can regulate lipid metabolism by inhibiting the enzyme activity of LPL and EL.8 Although C27-Fc had ~ 12 times higher affinity to the cleaved CCD of hANGPTL3 than C44-Fc, its affinity against full length of human and mouse ANGPTL3 proteins remained unsatisfactory. Thus, we ultimately selected C44-Fc for further study its therapeutic effects on NAFLD due to its high affinity and LPL de-repression of both the human full length and CCD of ANGPTL3 along with crossing reaction to mouse ANGPTL3s. The affinities against human and mouse ANGPTL3 proteins of C44-Fc were about 1.7 ~ 3.2 times higher than evinacumab. But in terms of IC50 of rescuing ANGPTL3s-mediated suppression of LPL activity and the lipid-reducing effect as well as relieving function in NAFLD, C44-Fc performed no significant difference with evinacumab. This is probably because of the different ANGPTL3-binding epitopes between C44-Fc and evinacumab.
In addition to its high affinity, the C44-Fc had many merits in the discovery and development process as a therapeutic antibody compared to evinacumab. The productivity of Camelidae antibody C44-Fc in ExpiCHO cells reached ~500 mg/L that was twice as much as evinacumab and isotype control human IgG1. Besides, previous research reported nanobodies had excellent thermostability and chemostability,28, 50 likewise, we detected the thermal stability and the forced degradation of C44-Fc. The results notarized nanobody (C44-Fc) had a better thermostability (Tm1 59.7°C) than evinacumab (Tm1 57.3°C) and had no obvious degradation or aggregation at low pH, high pH, high temperature (40°C for 28 days) and freeze-thaw. All these results indicated that C44-Fc could decrease the expenditure of production, storage and transportation in the future development.
Consistent with those results, we had developed a VHH-Fc fusion protein as the ANGPTL3 inhibitor that showed remarkable lipid-lowering and therapeutical effect on HF/HCCD-induced NAFLD mice, which indicated the C44-Fc fusion protein was likely to be a prospective curative agent for hypercholesterolemic and NAFLD patients. It also might be a latent treatment in other metabolic disorders such as cardiovascular diseases. Nonetheless, further research such as humanization, pharmacodynamics study in cynomolgus monkeys can be performed to develop it into a therapeutic drug.