Targeting PCSK9, through an innovative cVLP-based vaccine, enhanced the therapeutic activity of a cVLP-HER2 vaccine in a preclinical model of HER2-positive mammary carcinoma

Abstract

Background: HER2-targeted therapies have revolutionized the treatment of HER2-positive breast cancer patients, leading to significant improvements in tumor response rates and survival. However, resistance and incomplete response remain considerable challenges. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a novel therapeutic strategy for the management of dyslipidemia by enhancing the clearance of low-density lipoprotein cholesterol receptors, however recent evidence also shows links between PCSK9 and cancer cells. We present an innovative immunization approach combining capsid virus-like particle (cVLP)-based vaccines against HER2 and PCSK9.

Methods: The therapeutic activity of the combined vaccine was evaluated in female mice challenged with HER2-positive mammary carcinoma cells. Controls included untreated mice and mice treated with cVLP-PCSK9 and cVLP-HER2 as standalone therapies. Antibodies elicited by vaccinations were detected through ELISA immunoassay. The functional activity of the antibodies was tested in 3D-soft agar assay on human HER2 + + + trastuzumab sensitive and resistant cells.

Results: Mice vaccinated with cVLP-HER2 + cVLP-PCSK9 displayed tumor regression from the 40th day after cell challenge in 100% of mice remaining tumor-free even 4 months later. In contrast, 83% of mice treated with cVLP-HER2 vaccine alone experienced an initial tumor regression, followed by tumor relapse in 60% of subjects. Untreated mice and mice treated with the cVLP-PCSK9 vaccine alone developed progressive tumors within 1-2 months after cell injection. The combined vaccine approach elicited strong anti-human HER2 antibody responses (reaching 1-2 mg/ml range) comprising multiple immunoglobulins isotypes. cVLP-PCSK9 vaccine elicited anti-PCSK9 antibody responses, resulting in a marked reduction in PCSK9 serum levels. Although the anti-PCSK9 response was reduced when co-administered with cVLP-HER2, it remained significant. Moreover, both cVLP-HER2 + cVLP-PCSK9 and cVLP-HER2 alone induced anti-HER2 antibodies able to inhibit the 3D growth of human HER2 + + + BT-474 and trastuzumab-resistant BT-474 C5 cells. Strikingly, antibodies elicited by the combined vaccination were more effective than those elicited by the cVLP-HER2 vaccine alone in the inhibition of trastuzumab-resistant C5 cells.

Conclusions: The results indicate that cVLP-PCSK9 vaccination shows adjuvant activity when combined with cVLP-HER2 vaccine, enhancing its therapeutic efficacy against HER2-positive breast cancer and holding promise in overcoming the challenges posed by resistance and incomplete responses to HER2-targeted therapy.

Keywords: Breast cancer; Cancer progression; HER2; PCSK9; Therapeutic cancer vaccines; Therapy resistance; Virus-like particle (VLP)-based vaccine.

Fig. 1

Therapeutic efficacy of cVLP-PCSK9 and cVLP-HER2 vaccines as standalone or combined approach. A Experimental design; B Kaplan–Meier tumor free survival plot, n = 6; *p < 0.05; ***p < 0.01; long-rank test; C Tumor growth curves, each point represents the mean (and SEM) of all mice in each group, n = 6; D Single mice growth curves of each treated group

Fig. 2

Anti-HER2 polyclonal antibody response elicited by cVLP-HER2 vaccine. A Anti-HER2 antibody titers measured by ELISA on human HER2^ECD, n = 6. Each point represents the mean (and SEM) of mouse groups shown in Fig. 1; B HER2 specific IgM and IgG isotypes elicited by cVLP-HER2 vaccinations, n = 2–4. Each point represents the mean (and SEM). Histogram reports the mean (and SEM) peak value of each isotype after the last vaccination

Fig. 3

Anti-PCSK9 responses induced by cVLP-PCSK9 vaccine. A Mean (and SEM) of anti-PCSK9 antibody titers measured by ELISA represented as logarithm of Optical Density (OD) value multiplied by dilution factor of the serum sample, n = 6. Vehicle vs. cVLP-PCSK9, at least p < 0.01, from day 56; Vehicle vs. cVLP-HER2 + cVLP-PCSK9, p < 0.05, from day 98; cVLP-HER2 vs. cVLP-PCSK9, at least p < 0.05, from day 56; cVLP-PCSK9 vs. cVLP-HER2 + cVLP-PCSK9, at least p < 0.05, from day 98; cVLP-HER2 vs. cVLP-HER2 + cVLP-PCSK9, p < 0.05, day 98. All statistics were carried out by Welch t-test. B Percentage of the increase of anti-PCSK9 total antibodies after the last vaccination. Histogram reports mean (and SEM) of each group. *p < 0.05 vs Vehicle, + p < 0.05 vs cVLP-HER2, #p < 0.05 at least vs cVLP-HER2 + cVLP-PCSK9 by Welch’s t-test; C PCSK9 protein levels in mouse serum (µg/mL), each point represents the mean (and SEM) for each experimental group, n = 5–6. D Percentage of the increase of anti-PCSK9 protein inhibition after the last vaccination. Histogram reports mean (and SEM) of each group. + p < 0.05 vs cVLP-HER2, #p < 0.05 at least vs cVLP-HER2 + cVLP-PCSK9 by Welch’s t-test

Fig. 4

Inhibition of human breast cancer cell 3D agar colony growth by antibodies elicited by vaccinations. Upper panel, colony inhibition assay on BT-474 (HER2 +  +  + , trastuzumab sensitive) and BT-474 C5 (HER2 +  +  + , trastuzumab resistant), n = 2. Each bar represents the mean (and SEM) number of colonies larger than 90 µm as counted in two independent cultures with the aid of a micrometer. *p < 0.05 vs untreated, °p < 0.05 vs cVLP-PCSK9, + p < 0.05 vs cVLP-HER2, by t student’s test, unpaired. Lower panel, representative pictures of live agar colonies were acquired with an inverted microscope (dark-field, 25X)