Anna-Karin Roos

A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer.

Prostate-specific antigen (PSA) is a serine protease secreted at low levels by normal luminal epithelial cells of the prostate and in significantly higher levels by prostate cancer cells. Therefore, PSA is a potential target for various immunotherapeutical approaches against prostate cancer. DNA vaccination has been investigated as immunotherapy for infectious diseases in patients and for specific treatment of cancer in certain animal models. In animal studies, we have demonstrated that vaccination with plasmid vector pVAX/PSA results in PSA-specific cellular response and protection against tumour challenge. The purpose of the trial was to evaluate the safety, feasibility and biological efficacy of pVAX/PSA vaccine in the clinic. A phase I trial of pVAX/PSA, together with cytokine granulocyte/macrophage-colony stimulating factor (GM-CSF) (Molgramostim) and IL-2 (Aldesleukin) as vaccine adjuvants, was carried out in patients with hormone-refractory prostate cancer. To evaluate the biologically active dose, the vaccine was administered during five cycles in doses of 100, 300 and 900 μg, with three patients in each cohort. Eight patients were evaluable. A PSA-specific cellular immune response, measured by IFN-γ production against recombinant PSA protein, and a rise in anti-PSA IgG were detected in two of three patients after vaccination in the highest dose cohort. A decrease in the slope of PSA was observed in the two patients exhibiting IFN-γ production to PSA. No adverse effects (WHO grade >2) were observed in any dose cohort. We demonstrate that DNA vaccination with a PSA-coding plasmid vector, given with GM-CSF and IL-2 to patients with prostate cancer, is safe and in doses of 900 μg the vaccine can induce cellular and humoral immune responses against PSA protein.

Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment.

Background Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. Methodology/Principal Findings This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. Conclusions/Significance This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.

Optimization of Skin Electroporation in Mice to Increase Tolerability of DNA Vaccine Delivery to Patients

Electroporation has, during the last years, proven to be a very successful delivery method for DNA vaccines and has now reached clinical evaluation. Although intramuscular electroporation is practical in animal models, intradermal electroporation might be more suitable for clinical administration. Skin is the most accessible organ of the body and has professional antigen-presenting cells in large amounts; thus, skin is an ideal target for DNA vaccine delivery. Moreover, intradermal electroporation has clear clinical benefits such as improved safety and tolerability. This article describes improvements for effective and tolerable DNA delivery to skin. The time of pulse delivery has been shortened by 90% and even pulse programs of 240-ms total duration generate robust immune responses. We show that a single vaccination using an optimized gene delivery generates (i) high and consistent protein expression in vivo, (ii) cytotoxic antigen-specific T cells expressing both IFNgamma and CD107a (lysosomal-associated membrane protein 1). Furthermore, application of a topical anesthetic cream prior to vaccination does not affect the number or function of the antigen-specific T cells induced. This suggests that local anesthesia can be used to further decrease the sensation of pulse delivery in patients.

Plasmid DNA vaccination using skin electroporation promotes poly-functional CD4 T-cell responses

Plasmid DNA vaccination using skin electroporation (EP) is a promising method able to elicit robust humoral and CD8+ T‐cell immune responses while limiting invasiveness of delivery. However, there is still only limited data available on the induction of CD4+ T‐cell immunity using this method. Here, we compare the ability of homologous prime/boost DNA vaccinations by skin EP and intramuscular (i.m.) injection to elicit immune responses by cytokine enzyme‐linked immunosorbent spot (ELISPOT) assay, as well as study the complexity of CD4+ T‐cell responses to the human immunodeficiency virus antigen Gag, using multiparamater flow cytometry. We find that DNA vaccinations by skin EP and i.m. injection are capable of eliciting both single‐ and poly‐functional vaccine‐specific CD4+ T cells. However, although DNA delivered by skin EP was administered at a five‐fold lower dose it elicited significant increases in the magnitude of multiple‐cytokine producers compared with i.m. immunization suggesting that the skin EP could provide greater poly‐functional T‐cell help, a feature associated with successful immune defense against infectious agents.

DNA Vaccination for Prostate Cancer

DNA-based cancer vaccines have been used successfully in mice to induce cytotoxic T lymphocytes (CTLs) specific for prostate antigens. Translation of a prostate-specific antigen (PSA) DNA vaccine into a phase I clinical trial demonstrated that PSA-specific immune responses could be induced but at a significantly lower level compared with those in mice. To enhance the efficacy of DNA vaccination against prostate cancer, we have explored and optimized intradermal electroporation as an effective way of delivering a PSA DNA vaccine. The results demonstrated that intradermal DNA vaccination using low amounts of DNA, followed by two sets of electrical pulses of different length and voltage, effectively induced PSA-specific T cells. Here we describe in detail how to perform intradermal DNA electroporation to induce high gene expression in skin and, more important, how to induce and analyze PSA-specific T cell responses.

A modified epitope identified for generation and monitoring of PSA-specific T cells in patients on early phases of PSA-based immunotherapeutic protocols

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8(+) T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53-61, was able to activate high levels of PSA-specific CD8(+) T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53-61 was also the only peptide that induced human T cells to produce IFNgamma after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNgamma producing CD8(+) T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.