Evolocumab

All animal experiments were approved by the Animal Use and Care Committee of Beijing Chest Hospital (2021-003). Eight-week-old syngeneic female C57BL/6J mice, purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd., were inoculated subcutaneously with Lewis lung carcinoma (LLC) cells (1.0 × 10⁶ per mouse). Five micrograms of anti-PCSK9 monoclonal antibody (evolocumab, Amgen, CA, USA) was intratumorally injected on days 5, 7, 9, and 11 in the PCSK9i group and the doublet group. Three micrograms of anti-CD137 agonist (a rat anti-mouse CD137 agonist, 1D8, US patent #7,754,209 B2, SEQ ID Nos. 101 and 103, mIgG2a isotype) was intratumorally injected on days 9, 11, and 13 in the aCD137 group and the doublet group. Tumor size was measured using a caliper and calculated using the formula—volume = (length)(width)²/2—every 2 days afterward. Body weights were also monitored every 2 days. The endpoint was defined as the time in which a progressively growing tumor reached 15 mm in its longest dimension or 2,000 mm³ in diameter. Mice were also euthanized when they experienced open skin lesions, were dead, or failed to thrive.

Alirocumab (Sanofi), Evolocumab (Amgen), or non-specific human IgG2 control antibody (BP0301; Bio X Cell) was diluted in 0.9% isotonic saline solution and i.p. applied at a final concentration of 1 µg/g bodyweight. In vitro, blocking of LRP1 function was achieved by using 15 µg/ml mouse anti-11E2 antibody [35 (link)] in comparison to equal amounts of a non-specific mouse IgG control (12-371; Sigma-Aldrich). Human Aβ was immunoprecipitated and detected by mouse anti-IC16 [45 (link)] and the α-chain/full length of LRP1 recognized by mouse anti-11E2 antibody [14 (link)]. Rabbit anti-1704 antibody was used to identify the LRP1 β-chain [46 (link)]. Rabbit anti-LDL receptor antibody 3/43 was kindly provided by Joachim Herz (UT Southwestern, USA). Rabbit anti-PCSK9 (ab125251), rabbit anti-Apolipoprotein E (ApoE) (ab150032), chicken anti-rabbit (ab6831), chicken anti-mouse (ab6706), HRP-conjugated donkey anti-chicken (ab16349), and Alexa488-conjugated goat anti-mouse antibody (ab150117) were purchased from Abcam. Rabbit anti-β-actin (A2066) and HRP-conjugated goat anti-rabbit antibody (A5278) were purchased from Sigma-Aldrich. HRP-conjugated donkey anti-mouse antibody (715–035-151) was purchased from Dianova.

Oral lipid-lowering drugs were purchased as follows: atorvastatin from Nipro (Osaka, Japan), rosuvastatin from Tokyo Chemical Industry (Tokyo, Japan), probucol from Alfresa Pharma Corp. (Osaka, Japan), and ezetimibe from Nihon Pharmaceutical Industry (Tokyo, Japan). Atorvastatin (2 mg/kg), rosuvastatin (1 mg/kg), probucol (45 mg/kg) or ezetimibe (0.5 mg/kg) was included in a piece of banana that was consumed by the LDLR KO monkeys once a day for 4 weeks. An anti-PCSK9 antibody, evolocumab, was purchased from Amgen (Thousand Oaks, CA, USA). One subcutaneous injection of evolocumab (6 mg/kg) was performed in LDLR KO monkeys. Blood samples were collected from the monkeys before and 2 and 4 weeks after the administration of statins, probucol and ezetimibe, and before and 1 and 2 weeks after the administration of evolocumab. Plasma total cholesterol concentration was measured by the LabAssay Cholesterol kit (Fujifilm Wako), and plasma LDL and HDL concentrations were outsourced to Oriental Yeast.

The interaction between PCSK9 and LDLR in the presence of heparin was analyzed using a PCSK9-(biotinylated)-LDLR-binding assay kit (BPS Bioscience), according to the manufacturer’s protocol. Briefly, microtiter plate wells coated with LDLR extracellular domain were incubated with biotinylated PCSK9 in the presence of heparin (5 or 50 U/ml), 5 nManti-PCSK9 (BPS Bioscience #71207), evolocumab (Amgen), or mAb 5E11. Following washing, wells were incubated with HRP-labeled streptavidin and binding of PCSK9 to the LDLR extracellular domain was assessed by addition of HRP substrate and evaluation of signal using a chemiluminescence microplate reader.