Vaccination Of Malignant Lymphoma Patients With WT1 Peptide-Pulsed Dendritic Cells Induces Immunological and Clinical Responses: A Pilot Study
Authors: Ota Shuichi , Ogasawara Masahiro 
Published: October 2019

Abstract

Background
With the advent of monoclonal antibodies such as rituximab, the duration of remission and overall survival in B-cell non-Hodgkin’s lymphoma has improved significantly. However, new therapeutic approaches are needed for patients with refractory disease. Active immunizations using dendritic cells (DCs) loaded with ideotype proteins or tumor lysates have shown safety with limited efficacy. Wilms’ tumor 1 (WT1) gene is frequently overexpressed in lymphoma and is considered to be one of the ideal tumor antigens for cancer immunotherapy. In the present pilot study, we treated lymphoma patients with WT1 peptide-pulsed DC and examined safety, clinical and immunological responses to the vaccination.

Methods
5 HLA-A*24:02(A24)+patients (4 males, 1 female; aged 51-90 years; 2 with diffuse large B cell lymphoma, 1 with mantle cell lymphoma, 1 with follicular lymphoma and 1 with peripheral T cell lymphoma) with relapsed or refractory disease were enrolled in the present study. Autologous DCs were generated from patients' peripheral blood monocytes which were separated by an adherence technique from mononuclear cells collected by apheresis. Monocytes were cultured with GM-CSF and IL-4 followed by maturation with OK432 and PGE2. HLA-A24-restricted, modified 9-mer WT1 peptide-loaded mature DCs were administered intradermally every 2 weeks 5-7 times in combination with OK432. Induction of vaccine-induced T cell responses was monitored by a HLA-tetramer assay and a flow cytometry analysis. Cytotoxicity against WT1 peptide pulsed target cells was tested in a LDH release assay.

Results
The treatment was well tolerated and none of the patients experienced more than grade 2 adverse events during the treatment period. The most common adverse events were mild, transient erythema at injection sites and low grade fever. Of 5 patients, 1 had CR, 1 had PR, 2 had SD and 1 had PD following one course of the treatment. Survival of patients achieving CR, PR or SD (responder) after one course of DC vaccination was longer than those who did not respond to the treatment. Duration of survival in responders was 56, 32, 24 and 19 months after initiation of therapy. Three responders are still alive in remission. Increase in positivity of WT1-specific CD8+ T cells was observed in responders after one course of treatment; the percentage of HLA-A24 restricted WT1 tetramer positive cells was 0.12±0.12% before vaccination, it increased to 6.82±10.2% after the first course. WT1 tetramer positivity in one of the responding patients who received 3 courses of vaccination increased from 0.06% to 24.5% following one course of vaccination. High level of positivity persisted thereafter (14.6% and 14.6% after second and third course, respectively). Cytotoxic T cells (CTLs) generated by in vitro stimulation with WT1 peptide showed cytotoxic activity in an LDH release assay; percent specific lysis against WT1 peptide-pulsed HLA-A24+ target was 64.8% at effector/target ratio 30 to1. CTL did not lyse irrelevant HIV peptide-pulsed HLA-A24+ target and WT1 peptide-pulsed HLA-A24- target, demonstrating antigen specificity and HLA restriction. Absolute number of lymphocytes, CD3+ T cells, CD4/CD8 ratio and NK cells increased by 1.10±0.38, 2.03±1.78, 1.27±0.36, 1.31±0.74 fold, respectively, following the first course of DC vaccination in responders. On the other hand, absolute number of CD4+CD25+Foxp3+ regulatory T cells (Treg) decreased by 50.7%. Lin-CD33+HLA-DR- myeloid-derived suppressor cells (MDSC) decreased from 17.8%±10.1 to 9.0±2.5% (38.3% reduction). Similarly, monocytic MDSC (CD14+HLA-DRlow/-) and granurocytic MDSC (CD15+CD33+) decreased by 34.4% and 34.0%, respectively, indicating that DC vaccination may contribute to the reversal of immunosuppression by Treg and MDSC.

Conclusions
The present study demonstrated that a vaccination with WT1 peptide-pulsed DC was well tolerated and no serious adverse event was observed. DC vaccination elicits both innate and acquired cellular immune responses correlated with clinical effects. These results suggest WT1 peptide-pulsed DC vaccination might be a promising novel strategy for the treatment of malignant lymphoma patients with relapsed or refractory disease.

Disclosures:
No relevant conflicts of interest to declare.