Immunogenicity of a Tripartite Cell Penetrating Peptide Containing a MUC1 Variable Number of Tandem Repeat (VNTR) and A T Helper Epitope

Abstract

Peptide-based vaccines for cancer have many advantages however, for optimization these immunogens should incorporate peptide epitopes that induce CD8, as well as CD4 responses, antibody and long term immunity. Cell penetrating peptides (CPP) with a capacity of cytosolic delivery have been used to deliver antigenic peptides and proteins to antigen presenting cells to induce cytotoxic T cell, helper T cell and humoral responses in mice. For this study, a tripartite CPP including a mucin 1 (MUC1) variable number of tandem repeat (VNTR) containing multiple T cell epitopes and tetanus toxoid universal T helper epitope peptide (tetCD4) was synthesised (AntpMAPMUC1tet) and immune responses investigated in mice. Mice vaccinated with AntpMAPMUC1tet + CpG show enhanced antigen-specific interferon-gamma (IFN-γ) and IL-4 T cell responses compared with AntpMAPMUC1tet vaccination alone and induced a Th1 response, characterised by a higher ratio of IgG2a antibody/IgG1 antibodies. Furthermore, vaccination generated long term MUC1-specific antibody and T cell responses and delayed growth of MUC1^+ve tumours in mice. This data demonstrates the efficient delivery of branched multiple antigen peptides incorporating CPP and that the addition of CpG augments immune responses.

Keywords: Mucin 1; TLR agonist; antigen delivery; immunogenicity; immunotherapy; membrane penetrating peptide; membrane translocating peptide; multiple antigen peptide; penetratin; vaccine.

Figure 1

(A) Structure of the AntpMAPMUC1tet immunogen. The HLA-A2 restricted CTL epitope and the H2-K^b epitope of the mucin 1 (MUC1) variable number of tandem repeat (VNTR) is denoted in bold type. (B) SDS-PAGE and western blot analysis of AntpMAPMUC1tet (lane 1), molecular weight markers (lane 2). Anti-MUC1 antibody, 1.3.14 was used for western blot analysis (lane 3). (C) Binding of anti-MUC1 antibodies to AntpMAPMUC1. AntpMAPMUC1tet was coated onto a 96-well microtitre plate and bound peptide detected with anti-MUC1 antibody, BC2 recognizing the DTR epitope (●) and 1.3.14 antibody recognizing the APPAH epitope (■) in the tripartite peptide, AntpMAPMUC1tet. (D)Tetanus toxoid CD4 T cell epitope (tetCD4) in AntpMAPMUC1tet is processed and presented by human MoDC to tetCD4-specific human T cell lines. MoDC were pulsed with equimolar concentrations (7.6 or 11.5 uM) of tetCD4, AntptetCD4, AntpMAPMUC1tet or Antp, OVA (non-specific control antigens) for 14 hr before the addition of responder cells for 15 h. Golgistop was added for a further 4 h before the cells were stained for CD4 and intracellular IFNγ. MoDC alone with or without OVA or Antp were used as negative controls for non-specific IFNγ production. Values represent IFNγ production in tetCD4-specific T cells ± SEM stimulated by pulsed MoDC from 3 different donors. * p < 0.05.

Figure 2

Uptake of AntpMAPMUC1tet peptide by dendritic cells (DC) in vitro. (A) DC were pulsed with AntpMAPMUC1tet peptide at either varying concentrations (5 to 200 µg/mL) for 60 min or (B) a constant dose of 100 µg/mL for set times between 5 and 60 min. Uptake was determined by flow cytometry as the percent surface staining subtracted from the percent intracellular staining (mean ± SEM, for 3 replicates). (C) DCs were incubated with 100 µg/mL Alexa-labelled AntpMAPMUC1tet (green) in chamber slides for 2 h, counterstained with Hoechst nuclear stain (blue), washed and visualized by confocal microscopy or (D) DC were pre-treated with 10 µg/mL cytochalasin D before AntpMAPMUC1tet pulsing.

Figure 3

AntpMAPMUC1tet targets APC in vivo. Popliteal lymph nodes were isolated from mice (n = 4) 18 h after i.d. immunisation with PBS or FITC-labelled AntpMAPMUC1tet. (A) Lymph node cells were stained with anti-CD3, CD19, CD11c and F4/80 antibodies. (B) Cells were labelled with CD11c, CD8 and DEC205 to separate DC subsets and analysed by flow cytometry. Each labelled population was analysed for FITC fluorescence intensity. Results are depicted as histograms representative of two separate experiments.

Figure 4

DC maturation in vivo by AntpMAPMUC1tet with and without CpG. C57BL/6 mice were injected i.d. with PBS, AntpMAPMUC1tet or AntpMAPMUC1tet + CpG. 18 h later popliteal lymph nodes were isolated and stained with the DC marker CD11c and maturation markers (CD40, CD80, CD86, and MHC class II) and assessed by flow cytometry. Representative histogram plots are shown (n = 3).

Figure 5

In vivo IFN-γ and IL4 response to AntpMAPMUC1tet and AntpMAPMUC1tet + CpG in C57BL/6. C57BL/6 mice were injected i.d. on day 0, 10 and 17 with PBS, 100 µg AntpMAPMUC1tet and AntpMAPMUC1tet + CpG. Number of IFN-γ secreting cells (A) and IL4-secreting cells (B) to MUC1K^b (SAPDTRPAP) and tetCD4 antigens were analysed by ELISpot assay. ConA (1 µg/mL) was used as an internal positive control (not shown). Results are shown as mean spot-forming units (SFU)/5 × 10⁵ cells ± SEM. Results are representative of three separate experiments. * p < 0.05, ** p < 0.001.

Figure 6

In vivo IFN-γ and IL4 response to AntpMAPMUC1tet and AntpMAPMUC1tet + CpG in HLA-A2 transgenic mice. HLA-A2 mice were injected i.d. on day 0, 10 and 17 with PBS, 100 µg AntpMAPMUC1tet and AntpMAPMUC1tet + CpG. Number of IFN-γ secreting cells (A) and IL4-secreting cells (B) to MUC1A2 (STAPPAHGV) and tetCD4 recall antigens were analysed by ELISpot assay. ConA (1 µg/mL) was used as an internal positive control (not shown). Results are shown as mean spot-forming units (SFU)/5 × 10⁵ cells ± SEM. Results are representative of three separate experiments. * p < 0.05, ** p < 0.001.

Figure 7

Cellular immune responses in AntpMAPMUC1tet immunised mice measured as in vivo CTL killing assays. (A) C57BL/6 or HLA-A2 mice were immunised i.d. with PBS, 100 µg AntpMAPMUC1tet or 100 µg AntpMAPMUC1tet + 50 µg CpG and the percent MUC1K^b or MUC1A2-specific lysis was determined eight days after immunisation calculated as: {[1 − (ratio CFSE^low/CFSE^high of PBS mice/ratio CFSE^low/CFSE^high of immunised mice)] × 100}. Representative histograms are shown. (B) Data is presented as mean % of killing ± SEM (n = 6). * p < 0.05, ** p < 0.001.

Figure 8

Tumour growth is delayed by immunization (A). C57BL/6 mice were immunised on days 0, 10 and 17 with PBS (●), 100 µg AntpMAPMUC1tet (■) or 100 µg AntpMAPMUC1tet + 50 µg CpG (▲) then inoculated subcutaneously 7 days after final immunisation with 2 × 10⁵ B16-MUC1 melanoma cells into the abdomen. Tumour growth was recorded. Data showing the product of individual perpendicular measurements (mm²) and days post tumour inoculation. Number of tumour-free mice (†) and number of surviving mice (★) is also shown. Kaplan–Meier survival curves for each immunised group are shown (B).

Figure 9

Long term in vivo IFN-γ and IL-4 responses to AntpMAPMUC1tet and AntpMAPMUC1tet + CpG. C57BL/6 mice were injected i.d on day 0, 10 and 17 with PBS, 100 µg AntpMAPMUC1tet and AntpMAPMUC1tet + CpG. Number of IFN-γ secreting cells (A) and IL4-secreting cells (B) to MUC1K^b (SAPDTRPAP) and tetCD4 antigens were analysed by ELISpot assay. ConA (1 µg/mL) was used as an internal positive control (not shown). Results are shown as mean spot-forming units (SFU)/5 × 10⁵ cells ± SEM. Results are representative of three separate experiments. * p < 0.05.

Figure 10

Long term cellular immune responses in AntpMAPMUC1tet immunised mice, measured as in vivo CTL killing assays. (A) C57BL/6 mice were immunised i.d. with PBS, 100 µg AntpMAPMUC1tet or 100 µg AntpMAPMUC1tet + 50 µg CpG and the percent MUC1K^b-specific lysis was determined eight days after immunisation calculated as: {[1 − (ratio CFSE^low/CFSE^high of PBS mice/ratio CFSE^low/CFSE^high of immunised mice)] × 100}. Representative histograms are shown. (B) Data is presented as mean % of killing ± SEM (n = 6). ** p <0 .001.

Figure 11

Short and long term MUC1-specific total IgG, IgG1 and IgG2a subclass antibody responses. C57BL/6 mice were injected on day 0, 10 and 17 i.d. with PBS, 100 µg AntpMAPMUC1tet or 100 µg AntpMAPMUC1tet + 50 µg CpG and 14 days after last immunisation mice (short term) (A) or 40 days after the last immunisation (long term) (B) were bled and total IgG, IgG1 and IgG2a antibody responses to Cp13-32 (MUC1 VNTR) were determined via ELISA. Data presented as mean titre ± SEM (n = 4). Statistical difference compared to naïve mice: * p < 0.05; ** p < 0.001.