Wilms’ tumour antigen 1 Immunity via DNA fusion gene vaccination in haematological malignancies by intramuscular injection followed by intramuscular electroporation: a Phase II non-randomised clinical trial (WIN)

BackgroundIn the UK almost 7000 people are diagnosed with leukaemia each year, but despite continuing advances in diagnosis and treatment with new drugs, such as the tyrosine kinase inhibitors, the majority of these patients will eventually die from their disease. Until quite recently, the only treatment to offer the possibility of long-term disease-free survival was allogeneic stem cell transplantation. However, this carries a substantial risk of mortality and is available to only a minority of patients.ObjectivesThe aim of the study was to test the hypothesis that molecular and clinical responses, induced by T lymphocytes (T cells), can be predicted by increases in the number of CD8+ (cluster of differentiation 8-positive) T cells specific for the vaccine-encoded T-cell epitopes. This project also aimed to build on the established programme of deoxyribonucleic acid (DNA) fusion-gene vaccination delivered by intramuscular injection, exploiting a unique experience with electroporation, to induce durable immune responses with the aim of controlling disease by precision attack of the tumour by CD8+ T cells.MethodA non-randomised, open-label, single-dose-level Phase II clinical trial in two patient groups [chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML)] on stable doses of imatinib. Human leucocyte antigen A2-positive (HLA A2+) patients were vaccinated with two DNA vaccines: (1) p.DOM–WT1-37 (epitope sequence: VLDFAPPGA); and (2) p.DOM–WT1-126 (epitope sequence: RMFPNAPYL). The HLA A2-negative patients formed an unvaccinated control group. The sample size for the HLA A2+ group was originally determined following Simon’s optimal Phase II trial design (Simon R. Optimal two-stage designs for phase II clinical trials.Control Clin Trials1989;10:1–10). This was changed to A’Hern’s single-stage design during the course of the trial (A’Hern RP. Sample size tables for single-stage phase II designs.Stat Med2001;20:859–66), which was endorsed by the trial’s independent oversight committees.ResultsThe study included 12 patients with CML who were vaccinated and nine patients with CML who were unvaccinated as the control group. Both the vaccines and the electroporation were safe, with no new or unexpected toxicities. The evaluation adverse events of special interest (heart, bone marrow, renal) did not reveal safety concerns. TwoBCR–ABL(breakpoint cluster region–Abelson murine leukaemia viral oncogene homolog 1) responses were observed, both of which were defined as a major response, with one in each group. Two Wilms’ tumour antigen 1 (WT1) molecular responses were observed in the vaccinated group and one was observed in the control group. At an immunological level, the vaccine performed as expected.ConclusionsThe study met its primary decision-making target with one major molecular response inBCR–ABLtranscript levels. Overall, the data showed, in this clinical setting, the immunogenicity and safety of the vaccine.LimitationsThe study did not complete recruitment and there were multiple hurdles that contributed to this failure. This is disappointing given the robust induction immune responses againstWT1T-cell responses in 7 out of 10 evaluable patients.Future workEvaluation of the p.DOM–WT1 vaccines in AML remains attractive clinically, but it is unlikely to be feasible at this time. Combination of the DNA vaccine approach with strategies to expand T-cell responses with immunomodulatory antibodies is in development.Funding detailsThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council (MRC) and National Institute for Health Research (NIHR) partnership, and Bloodwise.

Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors

Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88 NF-kB, while the inhibition of transgene expression depends on TRIF IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88 NF-kB signaling.

Genetic (DNA) Vaccines

With the development of various branches of medicine and biology the classical ideas about means to prevent infectious diseases have changed. Nowadays in different countries of the world, investigations are carried out intensively in the sphere of genetic vaccines. Distinctive feature of DNA-vaccination is long lasted expression in eukaryotic cell cytoplasm of nucleic acids encoding synthesis of immunogenic proteins. Genetic vaccines induce both humoral and cellular responses accompanied by production of large pool of immunological memory cells. A number of questions regarding features of gene-engineered construction and transfer of DNA-vaccines into the cells of macroorganism, structure of DNA-vaccines and mechanisms of immune response generation are considered in the review. Attention is paid on the safety of gene vaccination and ways to improve its efficiency.

Antirabies DNA Immunization

Cited are literary data related to the development of DNA vaccines against rabies virus. Research results regarding gene vaccination of different models of laboratory animals and different ways of vaccine introduction are presented. Possibility to potentiate immunogenicity of DNA vaccines using adjuvants and cytokines is considered. Ways of improving of polynucleotide vaccines are discussed.