Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer

Abstract

Background: Dendritic cells (DCs) enhance the quality of anti-tumor immune response in patients with cancer. Thus, we posit that DC-based immunotherapy, in conjunction with toll-like receptor (TLR)-3 agonist poly-ICLC, is a promising approach for harnessing immunity against metastatic or locally advanced unresectable pancreatic cancer (PC).

Methods: We generated autologous DCs from the peripheral blood of HLA-A2+ patients with PC. DCs were pulsed with three distinct A2-restricted peptides: 1) human telomerase reverse transcriptase (hTERT, TERT572Y), 2) carcinoembryonic antigen (CEA; Cap1-6D), and 3) survivin (SRV.A2). Patients received four intradermal injections of 1 × 107 peptide-pulsed DC vaccines every 2 weeks (Day 0, 14, 28, and 42). Concurrently, patients received intramuscular administration of Poly-ICLC at 30 μg/Kg on vaccination days (i.e., day 0, 14, 28, and 42), as well as on days 3, 17, 21, 31, 37, and 45. Our key objective was to assess safety and feasibility. The effect of DC vaccination on immune response was measured at each DC injection time point by enumerating the phenotype and function of patient T cells.

Results: Twelve patients underwent apheresis: nine patients with metastatic disease, and three patients with locally advanced unresectable disease. Vaccines were successfully manufactured from all individuals. We found that this treatment was well-tolerated, with the most common symptoms being fatigue and/or self-limiting flu-like symptoms. Among the eight patients who underwent imaging on day 56, four patients experienced stable disease while four patients had disease progression. The median overall survival was 7.7 months. One patient survived for 28 months post leukapheresis. MHC class I -tetramer analysis before and after vaccination revealed effective generation of antigen-specific T cells in three patients with stable disease.

Conclusion: Vaccination with peptide-pulsed DCs in combination with poly-ICLC is safe and induces a measurable tumor specific T cell population in patients with advanced PC.

Trial registration: NCT01410968 ; Name of registry: clinicaltrials.gov; Date of registration: 08/04/2011).

Figures

Fig. 1

Phenotypic characterization of dendritic cells. a Schematic diagram showing the different time points for vaccination and analysis. b Dendritic cells (DCs) were prepared from each patient (see Methods). Before treatment administration the DCs were characterized using the flurochrome-conjugated antibodies for cell surface expression of CD11c, CD86, HLA-DR, and CD14. The data was acquired using BD Accuri flow cytometer and analyzed using FlowJo. The numerical values adjacent to the histogram represent the mean fluorescence intensity (MFI)

Fig. 2

Overall and Progression-free survival. Kaplan-Meier curves show OS and PFS vs. time since leukapheresis in months

Fig. 3

Characterization of the post-vaccination lymphocyte profile. PBMC was obtained from patients post vaccination (on days 14, 28, 42, 56). Immune cells were stained using the multiple fluorochrome-conjugated antibodies to determine the lymphocyte subsets. The data was acquired using BD FACS Aria and analyzed using FlowJo software. The percentages of cellular subsets are plotted against different time points. Each patient’s data is represented by points at each time point (blue = stable disease; black = progression) connected over time. The green circle represents the data from long survivor patient #13. The red circles represent the overall mean value at each time point

Fig. 4

Quantitative and qualitative characterization of antigen specific T cell response post-vaccination. The durability of the antigen specific T cell response after peptide pulsed DC vaccination was determined by enumerating the difference in antigen specific T cells using the PBMCs prepared from peripheral blood drawn prior to vaccination and d56 post-vaccination. a The tetramer staining for the Cap1, Tert, and SRV shows the T cells reactive to these epitopes in the PBMCs for various patients at different time points. Thin red lines indicate patients with stable disease; thin black lines patients with progression. The line obtained from the data of long survivor patient has patient ID #13 marked next to it. Thick black line indicates fitted regression model. The data was acquired using BD FACS Aria and analyzed using FlowJo software. b ELISPOT assay was performed as detailed in the methods. The antigen specific re-stimulation with the tumor peptide epitope leading to secretion of the effector cytokine IFNγ was determined by quantifying the differences in ELISPOT’s between PBMC from pre vaccination (d0) vs. post-vaccination (d56) samples. Overnight stimulation of the pre-vaccination and post-vaccination PBMCs with the three tumor epitope peptides and CEF peptide pool was done. The CEF peptide stimulation served as positive control for the assay. Subtracting the spots seen in the unstimulated well normalized the data, and IFNγ spots were plotted at baseline vs. at day 56. Patient data is indicated by unique symbols on the plot, with red for stable disease and black for progression. Patient with long survival is indicated with black circle around his/her symbol in all of the plots. An x = y line is included to demonstrate changes from baseline (points above the line indicate increases; points below the line indicate decreases). c The supernatant collected after overnight re-stimulation of the pre-vaccination and post-vaccination PBMCs with the tumor epitope peptides was used to determine the IFNγ levels (pg/ml) using ELISA. The differences in pre-vaccination and post-vaccination levels were plotted. The PBMCs were also stimulated in parallel with the CEF peptide pool that served as positive control for the assay. Patient data is indicated by unique symbols on the plot, with red for stable disease and black for progression. Patient with long survival is indicated with black circle around his/her symbol in all of the plots. An x = y line is included to demonstrate changes from baseline (points above the line indicate increases; points below the line indicate decreases)