Combined Vaccination with NY-ESO-1 Protein, Poly-ICLC, and Montanide Improves Humoral and Cellular Immune Responses in Patients with High-Risk Melanoma

Abstract

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1-specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

Conflict of interest statement

Conflict of interest: NB is on the SAB of Neon Therapeutics, Tempest, and Check Point Sciences, and a consultant for Genentech. NB also receives research support from Merck. R.S. works as clinical scientist at Genentech, Inc. No potential conflicts of interest were disclosed by other authors.

©2019 American Association for Cancer Research.

Figures

Figure 1:

Evolution of antibody titers against NY-ESO-1 by ELISA. A. Titer comparisons for NY-ESO-1 at screening and first follow-up (FU1) time points. B. Titer comparisons for NY-ESO-1 at different time points over the vaccination period. Arm 1 (n=3): 100 μg NY-ESO-1 protein emulsified in 1.1 mL montanide ISÄ−51 VG + 0.35 mg Poly-ICLC, Arm 2 (n=3): 100 μg NY-ESO-1 protein emulsified in 1.1mL montanide ISÄ−51 VG + 0.70 mg Poly-ICLC, Arm 3 (n=3): 100 μg NY-ESO-1 protein emulsified in 1.1mL montanide ISÄ−51 VG + 1.40 mg Poly-ICLC, Arm A (n=12): 100 μg NY-ESO-1 protein emulsified in 1.40 mg Poly-ICLC without montanide ISÄ−51 VG, Arm B (n=10): 100 μg NY-ESO-1 protein emulsified in 1.1 mL montanide ISÄ−51 VG + 1.40 mg Poly-ICLC. C. Seroreactivity to specific regions of the NY-ESO-1 protein were mapped using 20 mer overlapping peptides covering the entire protein.

Figure 2:

CD4+ and CD8+ T-cell responses from selected patients throughout vaccination with NY-ESO-1. A. Representative dot plot showing CD4+ and CD8+ T cell responses (patient 34, Arm B). CD4+ and CD8+ T cells were isolated from PBMCs of patients and then stimulated in vitro with NY-ESO-1 overlapping peptides. NY-ESO-1-specific T cell responses were evaluated by ICS at day 14 (CD8+ T; bottom panel) and day 21 (CD4+ T; upper panel). B. Number of NY-ESO-1-specific T cell responders based on the production of IFN-γ by CD4+ and CD8+ T cells, during treatment course. C. Quantification of CD4+ and CD8+ IFN- γ+ production by intracellular cytokine staining at different time points over the vaccination treatment. Arm A (blue), n=12 and Arm B (red), n=9. The time points represent responses measured at screening (SCRN), and through different cycles. The CnDx designation refers to cycle (C) and day (D) of blood draw.

Figure 3:

Recognition of processed NY-ESO-1 protein by vaccine-induced NY-ESO-1-specific T cells. A. Avidity of vaccine-induced NY-ESO-1-specific CD4+ T cells, after vaccination (FU) is shown. PBMCs from patients in arm A and arm B (2 from each group) were cultured in the presence of NY-ESO-1 OLPs for 12 days. On that day, cells were re-stimulated with serial dilutions of NY-ESO-1-specific peptides (1 μM-1 nM) (P5, 7, and/or 17) and GolgiPlug as well as GolgiSTOP were added after 1h of peptide stimulation. Then, intracellular cytokine staining was performed to assess flow cytometry. Percentage of CD4+ IFN-γ+ cells were determined at different peptide concentrations; peptides: P5 (AA81–100), P7 (AA119–143), and P17 (AA161–180). N=2 (arm A; patient 25 responded against P5 and 7; patient 33 responded against P5 and 17), n=2 (arm B; patient 27 responded against P5 and 7; patient 35 responded against P5 and 17). B. Recognition of NY-ESO-1 peptides (OLPs) presented by MoDCs. MoDCs from an arm B patient (patient 32) were pulsed with NY-ESO-1 OLPs for 4 h and then co-cultured with PBMCs isolated from screening and FU time points, or C. PBMCs from the same patient were expanded for 10 days in the presence of NY-ESO-1 OLPs. At day 10, cells were harvested and re-stimulated with MoDCs pulsed with OLPs (1 μM) and GolgiPlug as well as GolgiSTOP were added after 1h of peptide stimulation. Intracellular cytokine staining was performed to assess flow cytometry.

Figure 4:

Analysis of immune cell infiltration at the injection site and location of inflammatory cells by immunohistochemistry (IHC). Scale bar equals to 200 μm. A. IHC analysis of immune cell infiltration was performed for each arm and different markers for specific immune cell populations were used, including CD3, CD4, CD8, CD20, and CD11c. 0: No expression of the marker of interest, 1: single cells or small clusters (