Effects of immunisation against PCSK9 in mice bearing melanoma

Abstract

Introduction: Inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) is an established modality for the treatment of hypercholesterolaemia. However, the impact of PCSK9 inhibition in other situations such as cancer remains largely unknown. The current study was conducted to study the effects of PCSK9 inhibition on cancer endpoints in mice bearing melanoma.

Material and methods: To generate antiPCSK9 antibody in vivo, a nanoliposomal antiPCSK9 vaccine adsorbed to 0.4% Alum adjuvant was subcutaneously injected in C57BL/6 mice four times with bi-weekly intervals. Two weeks after the last immunisation, mice were subcutaneously inoculated with B16F0 melanoma cells. After a tumour mass was palpable (approximately 10 mm³), the mice were randomly divided into four groups and subjected to different treatment protocols: (1) PBS (untreated control), (2) vaccine group, (3) the combination of vaccine and a single dose of liposomal doxorubicin (Doxil^®), and (4) liposomal doxorubicin (positive control) group. To determine therapeutic efficacy, mouse body weight, tumour size, and survival were monitored every three days for 36 days.

Results: The nanoliposomal antiPCSK9 vaccine was found to efficiently induce specific antibodies against PCSK9 in C57BL/6 mice, thereby reducing plasma levels and function of PCSK9. Tumour volumes in the vaccinated group were not significantly different from those in the liposomal doxorubicin, combination, and control groups. The time to reach endpoint (TTE) values of the vaccine (28 ±5 days), combination (30 ±6 days), liposomal doxorubicin (34 ±2 days), and control (31 ±2 days) groups were not significantly different, either. Furthermore, the tumour growth delay (TGD) values of the vaccine (-11.5 ±15.4%), liposomal doxorubicin (7.75 ±6.5%), combination (-6 ±20.77%), and control (0 ±7.5) groups were not significantly different. Finally, there was no significant difference between the median survival time and lifespan of the vaccinated versus other tested groups.

Conclusions: The nanoliposomal PCSK9 vaccine did not adversely affect the growth of melanoma tumour nor the survival of tumour-bearing mice.

Keywords: PCSK9; cancer; immunisation; liposome; melanoma; nanoparticle; vaccine.

Figure 1

Schematic view of linking between peptide and DSPE-PEG-Maleimide

Figure 2

A summary of study design

Figure 3

Assessment of conjugation between DSPE-PEG-Mal and the IFPT peptide at the time zero and 48 h after starting of reaction. Lipid (DSPEPEG- Mal) is dissolved in the mobile phase and ascends to the top of the TLC plate (spots in the top of the left and middle lines), but peptide is bound to the silica and remains in the spotting point (the middle line). After 48, lipid bound to peptide and stayed in the point of spotting, and therefore the lipid spot on the top of the reaction mixture line disappeared, indicating the conjugation of the IFPT peptide and DSPE-PEG-Mal linker

Figure 4

HPLC chromatogram of DSPE-PEG-IFPT micelles and reference standard IFPT peptide. The retention time of reference standard was observed at 2.2 min, and it was found to be the same with free peptide present in the micelle sample

Figure 5

AntiPCSK9 vaccine efficacy. A – AntiPCSK9 antibody titres (ODmax/2) generated upon 4 immunisations in a bi-weekly interval (signed by arrows) have been evaluated upon 12 weeks post prime immunisation. B – Plasma levels of PCSK9 in the vaccine and control group were 17 ±5 ng/ml and 58 ±9 ng/ml, respectively. C – Direct detection of antibodies bound to plasma PCSK9 in plasma samples from vaccinated and control mice. Increased OD₄₅₀ is indicative for vaccine-generated anti-PCSK9 antibodies, which directly target PCSK9. D – In vitro PCSK9/LDLR binding assay. Plasma sample of vaccine group could decrease PCSK9 binding to LDLR by 50%, when compared with plasma sample of control group. Values are expressed as means ± SD (n = 3 replicates of the pooled samples of 10 mice per group). Significance compared to control values was analysed by unpaired 2-tailed Student’s t-test

Figure 6

A – The weight monitoring curve exhibits point by point alterations of the body weight during 36 days in the control, vaccine, and Doxil group. B – The integrated areas under the body weight curve (AUC_{body weight}) over 36 days demonstrate overall weight changes. The body weight loss was not significantly different between the vaccine, Doxil, and the control group. Animal body weight was measured every 3 days. Data are presented as the mean ± SD (n = 8). P < 0.05 was considered as the level of statistical significance

Figure 7

The tumour growth curve (A) shows increase of tumour size during 36 days in the control, vaccine, and Doxil group. Measuring the time to reach endpoint (TTE) (B) and tumour growth delay (TGD) (C) showed no significant difference between the studied groups. Tumour volume (mm³) was measured every 3 days. Data are presented as the mean ± SD (n = 8). Results with p < 0.05 were considered as statistically significant

Figure 8

Kaplan-Meier curves exhibit the survival rate of the control, vaccine, and Doxil group. There was no significant difference between the compared groups. Data are presented as the mean ± SD (n = 8). P < 0.05 was considered as the level of statistical significance