A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

Abstract

Dendritic cells (DCs), as professional antigen-presenting cells (APCs), play a key role in the initiation and regulation of humoral and cellular immunity. DC vaccines loaded with different tumor-associated antigens (TAAs) have been widely used to study their therapeutic effects on cancer. A number of clinical trials have shown that DCs are safe as an antitumor vaccine and can activate certain anti-tumor immune responses; however, the overall clinical efficacy of DC vaccine is not satisfactory, so its efficacy needs to be enhanced. MUC1 is a TAA with great potential, and the immune checkpoint PD-L1 also has great potential for tumor treatment. Both of them are highly expressed on the surface of various tumors. In this study, we generated a novel therapeutic MUC1-Vax tumor vaccine based on the method of PD-L1-Vax vaccine we recently developed; this novel PD-L1-containing MUC1-Vax vaccine demonstrated an elevated persistent anti-PD-L1 antibody production and elicited a much stronger protective cytotoxic T lymphocyte (CTL) response in immunized mice. Furthermore, the MUC1-Vax vaccine exhibited a significant therapeutic anti-tumor effect, which significantly inhibited tumor growth by expressing a high MUC1⁺ and PD-L1⁺ level of LLC and Panc02 tumor cells, and prolonged the survival of cancer-bearing animals. Taken together, our study provides a new immunotherapy strategy for improving the cross-presentation ability of therapeutic vaccine, which may be applicable to pancreatic cancer, lung cancer and for targeting other types of solid tumors that highly express MUC1 and PD-L1.

Keywords: MUC1; PD-L1; dendritic cells; immunotherapy; tumor vaccine.

Figure 1

Expression and purification of recombinant protein MUC1-PDL1-IgG1 Fc (MUC1-Vax). (A) Plasmid-transfected E. coli lysed protein fractions were run on a 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. Lane M is the prestained protein molecular weight marker, lane 1 is the bacterial protein before induction, lane 2 is the bacterial protein after isopropyl β-D-1-thiogalactoside (IPTG) induction, and lane 3 is the purified MUC1-Vax protein, and lane 4 is the MUC1-Vax protein after dialysis. Recombinant protein MUC1-Vax (arrow) is indicated. (B) Western blot analysis of purified recombinant protein MUC1-Vax with anti-human His primary antibody. (C) Western blot analysis of purified recombinant protein MUC1-Vax with anti-human MUC1 antibody.

Figure 2

(A) C57BL/6 mice were subcutaneously inoculated with 2 × 10⁵ luciferase expressing LLC (LLC-MUC1-PDL1-Luc) or 3 × 10⁶ Panc02 (Panc02-MUC1-PDL1-Luc) cells by hypodermic injection (I.H.). The footpads were injected with 2 × 10⁶ BMDCs loaded with different proteins on the 7th and 14th days after tumor cell injection, respectively. Splenocytes of immunized mice were isolated 4 days after immunization and the activation of CD4⁺ T cells and CD8⁺ T cells (3 per group) and cytokine secretion were detected by flow cytometry. The tumor size, growth, and survival time of the remaining mice were observed (4–5 in each group). (B) Activation of CD4⁺ T cells, data are expressed as mean ± SD. (C) Secretion of IL-2 by CD4⁺ T cells, data are expressed as mean ± SD. (D) Secretion of IFN-γ by CD4⁺ T cells, data are presented as mean ± SD. (E) Secretion of IFN-γ by CD8⁺ T cells, data are presented as mean ± SD. * p < 0.05.

Figure 3

Whole blood, liver and kidney of mice were collected after 4 days of DC vaccine immunization, and serum was separated. (A) The serum levels of anti-PD-L1 IgG antibody in different groups were detected by ELISA, ** p < 0.01, **** p < 0.0001. (B) H&E staining analysis of liver and kidney sections. The original magnification of these pictures was ×20.

Figure 4

The tumor size of the tumor-bearing mice was measured with a vernier caliper every few days. tumor volume was monitored by the caliper is shown (A,C). The survival curves are shown (B,D). There are 4–5 mice in each group, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 5

Cell bioluminescence monitoring was performed in vivo on tumor mice inoculated with LLC-MUC1-PDL1-Luc every 6 days, as shown in figure (A) Bioluminescence in vivo of mice inoculated with LLC (n = 5), blank space means the mouse died; (B) In vivo bioluminescence of mice inoculated Panc02 (n = 3).