Tn-MUC1 DC Vaccination of Rhesus Macaques and a Phase I/II Trial in Patients with Nonmetastatic Castrate-Resistant Prostate Cancer

Abstract

MUC1 is a glycoprotein expressed on the apical surface of ductal epithelial cells. Malignant transformation results in loss of polarization and overexpression of hypoglycosylated MUC1 carrying truncated carbohydrates known as T or Tn tumor antigens. Tumor MUC1 bearing Tn carbohydrates (Tn-MUC1) represent a potential target for immunotherapy. We evaluated the Tn-MUC1 glycopeptide in a human phase I/II clinical trial for safety that followed a preclinical study of different glycosylation forms of MUC1 in rhesus macaques, whose MUC1 is highly homologous to human MUC1. Either unglycosylated rhesus macaque MUC1 peptide (rmMUC1) or Tn-rmMUC1 glycopeptide was mixed with an adjuvant or loaded on autologous dendritic cells (DC), and responses were compared. Unglycosylated rmMUC1 peptide induced negligible humoral or cellular responses compared with the Tn-rmMUC1 glycopeptide. Tn-rmMUC1 loaded on DCs induced the highest anti-rmMUC1 T-cell responses and no clinical toxicity. In the phase I/II clinical study, 17 patients with nonmetastatic castrate-resistant prostate cancer (nmCRPC) were tested with a Tn-MUC1 glycopeptide-DC vaccine. Patients were treated with multiple intradermal and intranodal doses of autologous DCs, which were loaded with the Tn-MUC1 glycopeptide (and KLH as a positive control for immune reactivity). PSA doubling time (PSADT) improved significantly in 11 of 16 evaluable patients (P = 0.037). Immune response analyses detected significant Tn-MUC1-specific CD4⁺ and/or CD8⁺ T-cell intracellular cytokine responses in 5 out of 7 patients evaluated. In conclusion, vaccination with Tn-MUC1-loaded DCs in nmCRPC patients appears to be safe, able to induce significant T-cell responses, and have biological activity as measured by the increase in PSADT following vaccination. Cancer Immunol Res; 4(10); 881-92. ©2016 AACR.

Figure 1

Groups of five rhesus macaques were immunized at baseline, week 3 and week 8 with either the unglycosylated rmMUC1 (A) or the Tn-rm-MUC1 (B) peptides emulsified with GLA adjuvant. Blood samples were taken from each animal at various time points for the analysis of humoral and cellular immune responses. IgG titers, as determined by ELISA (gray bars), and frequency of INFγ-positive PBMCs as determined by ELISPOT (black bars) are presented. Assays were performed in triplicate.

Figure 2

Groups of five rhesus macaques were immunized at baseline, week 3 and week 8 with monocyte-derived dendritic cells loaded with either the unglycosylated rmMUC1 (A) or the Tn-rm-MUC1 (B) peptides. Blood samples were taken from each animal at various time points for the analysis of humoral and cellular immune responses. IgG titers, as determined by ELISA (gray bars), and frequency of INFγ⁺ PBMCs as determined by ELISPOT (black bars) are presented. Assays were performed in triplicate.

Figure 3

Box and whiskers plot of PSADT calculated for each patient at pre-vaccination and post-vaccination period comprising the first 200 days following the first immunization. Data represent the mean and range for the 16 patients. The mean PSADT during the on-study vaccination period is significantly increased compared to the pre-vaccination period (p=0.037, unpaired Student t-test).

Figure 4

Total frequency of Tn-MUC1 or KLH-specific memory CD4⁺ and CD8⁺ T cells at different time points following vaccination. Patients’ PBMCs were stimulated in vitro with Tn-MUC1-DCs or KLH-DCs and the total frequency of functional subsets (either positive for IL2, TNFα, INFγ or CD107a) of CD4⁺ or CD8⁺ T cells was determined. Samples from each time point were assayed in monoplicate. Total functionality was calculated by summing all functional subsets and normalized to 10⁶ memory CD45RA⁻ CD4⁺ or CD45RA⁻ CD8⁺ T cells.

Figure 5

Frequency of regulatory T cells following vaccination. Patients’ PBMCs obtained at various time points were analyzed for the presence of CD4⁺ T cells expressing any combination of FoxP3, CD25 and Ki67. Data from weeks 2, 4, 6 and 8 were included in the <2 month group whereas data from weeks 24, 26, 52 and 54 were included in the >6 month group. Response profiles were generated using Boolean analysis of 3 functional response gates, resulting in 7 separate functional T cell subsets. The frequency of each CD4⁺ T cell subset at baseline, <2 months and > 6 months is presented in bar graphs. Each dot in the bar graphs represents data from one PBMC sample (see bar chart legend for color code). The bar graph provides fine detail on frequencies within each of the individual 7 categories; medians are shown. Assays were performed in triplicate. Pie charts represent the distribution of regulatory and non-regulatory subsets within the CD4⁺ T-cell pool at the three periods. Each color in the pie charts represents one of the CD4⁺ T-cell subsets (see pie slice color code presented below the bar graph). P values identify whether the distribution of subsets analyzed differed between periods. Subsets which were statistically different compared to the baseline time point are identified (#: p < 0.05, Wilcoxon test).