A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease

Figures

• 

  Fig. 1. Schematic presentation of the whole experiment. The whole experimental procedure includes immunization with ANGPTL3, B-cells separation, VHH library construction, phage-display to select ANGPTL3-specific VHH, sanger sequencing, VHH and VHH-Fc fusion proteins expression, affinity determination, in vitro LPL assay and pharmacodynamic evaluation in hypercholesterolemic and NAFLD mice model

• 

  Fig. 2. The affinity test of anti-hANGPTL3-CCD VHHs and epitope binning assay. a Identification of ANGPTL3 specific VHHs from 48 clones specifically binding with hANGPTL3 after three rounds bio-panning. b The SDS-PAGE analysis of the C27-His6, C44-His6 and C46-His6. M: Maker; lane 1: C27-His6; lane 2: C44-His6; lane 3: C46-His6. c–e The affinity of C27-His6, C44-His6 and C46-His6 binding to hANGPTL3 (S17-P220)-His6. f The assay for hANGPTL3-binding epitopes of the C44-His6 was performed against evinacumab by the dual antibody sandwich ELISA. The horizontal and vertical axes represent the concentrations of the C44-His6 and the OD450 value

• 

  Fig. 3. The affinity test of C44-Fc with human and mouse ANGPTL3 proteins. a Diagram of the C44 fused with human IgG1 Fc fragments. b The SDS-PAGE analysis of the C44-Fc. M: Maker; lane 1: SDS-PAGE analysis of C44-Fc in reduced gel; lane 2: SDS-PAGE analysis of C44-Fc in non-reduced gel. c, g and k The affinity of C44-His6, C44-Fc and evinacumab binding to hANGPTL3 (S17-E460)-His10. d, h and l The affinity of C44-His6, C44-Fc and evinacumab binding to mANGPTL3 (S17-T206)-His6. e, i and m The affinity of C44-His6, C44-Fc and evinacumab binding to mANGPTL3 (S17-T455)-His10. f and j The affinity of C44-Fc and evinacumab binding to hANGPTL3 (S17-P220)-His6

• 

  Fig. 4. The biophysical stability evaluation of C44-Fc in vitro. a SEC-HPLC profile of C44-Fc and evinacumab. b Time course analysis of C44-Fc expression levels in ExpiCHO cells. The culture supernatants of ExpiCHO cells transiently transfected with C44-Fc, evinacumab and control Ab expression plasmid were collected on days 5–12. c and d SEC-HPLC analysis of C44-Fc and evinacumab at low or high pH. C44-Fc and evinacumab were preserved at pH 5.0 or pH 9.0 and 40 ℃ for 72 h. e SEC-HPLC analysis of C44-Fc and evinacumab at 40 ℃ for 28 days. f The freeze–thaw stability analysis of C44-Fc and evinacumab by SEC-HPLC

• 

  Fig. 5. C44-Fc suppresses ANGPTL3-induced blockade of LPL. The vertical axis indicates the incremental concentrations of four kinds of ANGPTL3 proteins or antibody including of C44-Fc, evinacumab and control Ab (n = 3). The vertical axis indicates the relative fluorescence units (RFU) at 482/515 nm (excitation/emission)

• 

  Fig. 6. C44-Fc reduces serum TG, TC and LDL-C levels in hypercholesterolemic mice. C57BL/6 mice were induced by HF/HCCD-fed for 4 weeks to establish hypercholesterolemic mice model and then treated with isotype control Ab (25 mg/kg), C44-Fc (10 mg/kg or 25 mg/kg) and evinacumab (25 mg/kg). Serum TG (a), TC (c) and LDL-C (e) levels were tested enzymatically (n = 6–8). The TG (b), TC (d) and LDL-C (f) reduction levels of C44-Fc or evinacumab-treated group from control Ab-treated group were also measured. g Postheparin plasma LPL activities of HF/HCCD-fed mice treated with control Ab, C44-Fc and evinacumab (n = 6). h Lipid tolerance test were performed 4 days after control Ab, C44-Fc and evinacumab treatment (n = 6). †, § and * respectively represent the Student’s t-test of 10 mg/kg C44-Fc, 25 mg/kg C44-Fc and 25 mg/kg evinacumab treatment compared with control Ab

• 

  Fig. 7. C44-Fc administrations reduce serum lipid levels for 6 weeks. a Schematic illustration of experimental design to assess the therapeutic effects of C44-Fc in NAFLD mice after multiple administrations. NAFLD model in C57BL/6 mice were induced by HF/HCCD-fed for 8 weeks and then treated with 25 mg/kg of isotype control Ab, 25 mg/kg of C44-Fc, or 25 mg/kg of evinacumab weekly. Serum TG (b), TC (c) and LDL-C (d) levels were weekly tested 4 days after antibody administrations (n = 6). Plasma lipoproteins of cholesterol (e) were separated by HPLC and measured in each fraction. § and * respectively represent the Student’s t-test of 25 mg/kg C44-Fc and 25 mg/kg evinacumab treatment compared with control Ab

• 

  Fig. 8. C44-Fc treatment relieves hepatic steatosis induced by HF/HCCD feeding. Body weight (a), representative liver morphology (b) and liver weight (c) of mice were presented (n = 6). d The TG contents of livers were measured (n = 5) by commercial kits. e Representative H&E staining of liver tissues. f Representative images and quantification (g) of oil red O staining are presented (n = 3). The levels of Serum ALT (h) and AST (i) were measured at the terminus of the research (n = 6). § and * respectively represent the Student’s t-test of 25 mg/kg of C44-Fc and 25 mg/kg of evinacumab treatment compared with control Ab