Intradermal Naked DNA Vaccination by DNA Tattooing for Mounting Tumor-Specific Immunity in Stage IV Melanoma Patients: A Phase I Clinical Trial.

INTRODUCTION: Naked DNA vaccination could be a powerful and safe strategy to mount antigen-specific cellular immunity. We designed a phase I clinical trial to investigate the toxicity of naked DNA vaccines encoding CD8+ T-cell epitope from tumor-associated antigen MART-1 in patients with advanced melanoma. METHODS: This dose escalating phase Ia clinical trial investigates the toxicity and immunological response upon naked DNA vaccines encoding a CD8+ T-cell epitope from the tumor-associated antigen MART-1, genetically linked to the gene encoding domain 1 of subunit-tetanus toxin fragment C in patients with advanced melanoma (inoperable stage IIIC-IV, AJCC 7th edition). The vaccine was administrated via intradermal application using a permanent make-up or tattoo device. Safety was monitored according to CTCAE v.3.0 and skin biopsies and blood samples were obtained for immunologic monitoring. RESULTS: Nine pretreated, HLA-A*0201-positive patients with advanced melanoma expressing MART-1 and MHC class I, with a good performance status, and adequate organ function, were included. With a median follow-up of 5.9 months, DNA vaccination was safe, without treatment-related deaths. Common treatment-emergent adverse events of any grade were dermatologic reactions at the vaccination site (100%) and pain (56%). One patient experienced grade 4 toxicity, most likely related to tumor progression. One patient (11%) achieved stable disease, lasting 353 days. Immune analysis showed no increase in vaccine-induced T cell response in peripheral blood of 5 patients, but did show a MART-1 specific CD8+ T cell response at the tattoo administration site. The maximum dose administered was 2 mg due to lack of clinical activity. CONCLUSION: We showed that the developed DNA vaccine, applied using a novel intradermal application strategy, can be administered safely. Further research with improved vaccine formats is required to show possible clinical benefit of DNA vaccination.

Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma.

BACKGROUND: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma. METHODS: In this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti-cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight). Infusion of at least 5×109 TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival. RESULTS: A total of 168 patients (86% with disease refractory to anti-programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P<0.001); 49% (95% CI, 38 to 60) and 21% (95% CI, 13 to 32) of the patients, respectively, had an objective response. Median overall survival was 25.8 months (95% CI, 18.2 to not reached) in the TIL group and 18.9 months (95% CI, 13.8 to 32.6) in the ipilimumab group. Treatment-related adverse events of grade 3 or higher occurred in all patients who received TILs and in 57% of those who received ipilimumab; in the TIL group, these events were mainly chemotherapy-related myelosuppression. CONCLUSIONS: In patients with advanced melanoma, progression-free survival was significantly longer among those who received TIL therapy than among those who received ipilimumab. (Funded by the Dutch Cancer Society and others; ClinicalTrials.gov number, NCT02278887.).

Signatures of recent activation identify a circulating T cell compartment containing tumor-specific antigen receptors with high avidity.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

Autotaxin impedes anti-tumor immunity by suppressing chemotaxis and tumor infiltration of CD8+ T cells.

Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Differential effects of PD-1 and CTLA-4 blockade on the melanoma-reactive CD8 T cell response.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

HPV-16 E6/E7 DNA tattoo vaccination using genetically optimized vaccines elicit clinical and immunological responses in patients with usual vulvar intraepithelial neoplasia (uVIN): a phase I/II clinical trial.

BACKGROUND: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis. METHOD: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays. RESULTS: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response. CONCLUSIONS: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit. TRIAL REGISTRATION NUMBER: NTR4607.

Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up.

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses. PURPOSE: Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial. EXPERIMENTAL: Ten patients were treated with TIL therapy. Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed. Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products. RESULTS: Five out of 10 patients, who were all anti-PD-1 naïve at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years. Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed. For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion. CONCLUSION: The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity. In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial.

Transgenerational transfer of gene-modified T cells.

Tumor immunotherapy using gene-modified T cells has already met with considerable success in the treatment of metastatic melanoma and B cell lymphoma. With improving patient prognoses, new questions arise. In particular, the long-term consequences of treatment among individuals of childbearing age could now be considered. Former patients can carry a cohort of transgenic memory T cells long after treatment has ceased and the effector T cell population has contracted. When patients become parents well after treatment is completed, expectant mothers may still pass transgenic T cells to their unborn children. Consequences should be more measurable if the mother also breastfeeds the baby. Maternal T cells may shape immune responses in the child, can tolerize the child to maternal antigens, and might cause either beneficial or adverse effects in the offspring. The hypothesis put forth is that transgenic T cells transferred from mother to child during and after pregnancy might have consequences that have not been adequately considered to date. Depending on the targeted antigen and the MHC eventually required to present it, such transfer may be beneficial, uneventful or even damaging. Such potential consequences are addressed in this paper. The transgenic T cells might form a pocket of memory T cells in secondary lymphoid organs of the child, expand upon antigen stimulation, and react. However, simple measures might be devised to avoid any reason for concern. These considerations provide ample incentive to probe transgenerational transfer of transgenic T cells.

Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRPα axis and a target for cancer immunotherapy.

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells2-5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.

Small-scale GMP production of plasmid DNA using a simplified and fully disposable production method.

In the past years, the demand for small batches of clinical grade plasmid DNA has been growing. For that purpose, we designed and qualified a scaled-down Good Manufacturing Practices (GMP) production method, able to produce small batches (1-4 mg) of plasmid. The developed method does not require any complex production equipment and utilizes only disposable production materials, which makes it easy to implement and simplifies line-clearance. We have successfully used this method to produce several small batches of two different plasmids. The produced plasmids, both formulated in an Electroporation Buffer, are mixed and filled into small, single-use, aliquots. Quality control confirmed the robustness of the developed method and a stability study showed that the final formulation is stable for at least two years. The final patient formulation will be subsequently used in a phase I/II clinical trial in which retina cells of patients with Age Related Macular Degeneration, are transfected. The presented production method can be generically used for other plasmid constructs and final formulation designs.

Adoptive transfer of tumor-infiltrating lymphocytes in melanoma: a viable treatment option.

The treatment of metastatic melanoma patients with autologous tumor-infiltrating lymphocytes (TIL) shows robust, reproducible, clinical responses in clinical trials executed in several specialized centers over the world. Even in the era of targeted therapy and immune checkpoint inhibition, TIL therapy can be an additional and clinically relevant treatment line. This review provides an overview of the clinical experiences with TIL therapy thus far, including lymphodepleting regimens, the use of interleukin-2 (IL-2) and the associated toxicity. Characteristics of the TIL products and the antigen recognition pattern will be discussed, as well as the current and upcoming production strategies, including the selective expansion of specific fractions from the cell product. In addition, the future potential of TIL therapy in melanoma and other tumor types will be covered.

Treatment With Tumor-infiltrating Lymphocytes in Advanced Melanoma: Evaluation of Early Clinical Implementation of an Advanced Therapy Medicinal Product.

Tumor-infiltrating lymphocytes (TIL)-therapy in advanced melanoma is an advanced therapy medicinal product (ATMP) which, despite promising results, has not been implemented widely. In a European setting, TIL-therapy has been in use since 2011 and is currently being evaluated in a randomized controlled trial. As clinical implementation of ATMPs is challenging, this study aims to evaluate early application of TIL-therapy, through the application of a constructive technology assessment (CTA). First the literature on ATMP barriers and facilitators in clinical translation was summarized. Subsequently, application of TIL-therapy was evaluated through semistructured interviews with 26 stakeholders according to 6 CTA domains: clinical, economic, patient-related, organizational, technical, and future. In addition, treatment costs were estimated. A number of barriers to clinical translation were identified in the literature, including: inadequate financial support, lack of regulatory knowledge, risks in using live tissues, and the complex path to market approval. Innovative reimbursement procedures could particularly facilitate translation. The CTA survey of TIL-therapy acknowledged these barriers, and revealed the following facilitators: the expected effectiveness resulting in institutional support for an internal pilot, the results of which led to the inclusion of TIL-therapy in a national coverage with evidence development program, the availability of an in-house pharmacist, quality assurance expertise and a TIL-skilled technician. Institutional and national implementation of TIL-therapy remains complex. The promising clinical effectiveness is expected to facilitate the adoption of TIL-therapy, especially when validated through a randomized controlled trial. Innovative and conditional reimbursement procedures, together with the organization of knowledge transfer, could support and improve clinical translation of TIL and ATMPs.

HPV16 E7 DNA tattooing: safety, immunogenicity, and clinical response in patients with HPV-positive vulvar intraepithelial neoplasia.

BACKGROUND: Usual type vulvar intraepithelial neoplasia (uVIN) is caused by HPV, predominantly type 16. Several forms of HPV immunotherapy have been studied, however, clinical results could be improved. A novel intradermal administration route, termed DNA tattooing, is superior in animal models, and was tested for the first time in humans with a HPV16 E7 DNA vaccine (TTFC-E7SH). METHODS: The trial was designed to test safety, immunogenicity, and clinical response of TTFC-E7SH in twelve HPV16+ uVIN patients. Patients received six vaccinations via DNA tattooing. The first six patients received 0.2 mg TTFC-E7SH and the next six 2 mg TTFC-E7SH. Vaccine-specific T-cell immunity was evaluated by IFNγ-ELISPOT and multiparametric flow cytometry. RESULTS: Only grade I-II adverse events were observed upon TTFC-E7SH vaccination. The ELISPOT analysis showed in 4/12 patients a response to the peptide pool containing shuffled E7 peptides. Multiparametric flow cytometry showed low CD4+ and/or CD8+ T-cell responses as measured by increased expression of PD-1 (4/12 in both), CTLA-4 (2/12 and 3/12), CD107a (5/12 and 4/12), or the production of IFNγ (2/12 and 1/12), IL-2 (3/12 and 4/12), TNFα (2/12 and 1/12), and MIP1ß (3/12 and 6/12). At 3 months follow-up, no clinical response was observed in any of the twelve vaccinated patients. CONCLUSION: DNA tattoo vaccination was shown to be safe. A low vaccine-induced immune response and no clinical response were observed in uVIN patients after TTFC-E7SH DNA tattoo vaccination. Therefore, a new phase I/II trial with an improved DNA vaccine format is currently in development for patients with uVIN.

Long-term Survival and Clinical Benefit from Adoptive T-cell Transfer in Stage IV Melanoma Patients Is Determined by a Four-Parameter Tumor Immune Signature.

The presence of tumor-infiltrating immune cells is associated with longer survival and a better response to immunotherapy in early-stage melanoma, but a comprehensive study of the in situ immune microenvironment in stage IV melanoma has not been performed. We investigated the combined influence of a series of immune factors on survival and response to adoptive cell transfer (ACT) in stage IV melanoma patients. Metastases of 73 stage IV melanoma patients, 17 of which were treated with ACT, were studied with respect to the number and functional phenotype of lymphocytes and myeloid cells as well as for expression of galectins-1, -3, and -9. Single factors associated with better survival were identified using Kaplan-Meier curves and multivariate Cox regression analyses, and those factors were used for interaction analyses. The results were validated using The Cancer Genome Atlas database. We identified four parameters that were associated with a better survival: CD8+ T cells, galectin-9+ dendritic cells (DC)/DC-like macrophages, a high M1/M2 macrophage ratio, and the expression of galectin-3 by tumor cells. The presence of at least three of these parameters formed an independent positive prognostic factor for long-term survival. Patients displaying this four-parameter signature were found exclusively among patients responding to ACT and were the ones with sustained clinical benefit. Cancer Immunol Res; 5(2); 170-9. ©2017 AACR.

Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor.

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.

Manufacture of gene-modified human T-cells with a memory stem/central memory phenotype.

Advances in genetic engineering have made it possible to generate human T-cell products that carry desired functionalities, such as the ability to recognize cancer cells. The currently used strategies for the generation of gene-modified T-cell products lead to highly differentiated cells within the infusion product, and on the basis of data obtained in preclinical models, this is likely to impact the efficacy of these products. We set out to develop a good manufacturing practice (GMP) protocol that yields T-cell receptor (TCR) gene-modified T-cells with more favorable properties for clinical application. Here, we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for retroviral transduction, followed by expansion in the presence of IL-7 and IL-15, production of a well-defined clinical-scale TCR gene-modified T-cell product could be achieved. A major fraction of the T-cells generated in this fashion were shown to coexpress CD62L and CD45RA, and express CD27 and CD28, indicating a central memory or memory stemlike phenotype. Furthermore, these cells produced IFNγ, TNFα, and IL-2 and displayed cytolytic activity against target cells expressing the relevant antigen. The T-cell products manufactured by this robust and validated GMP production process are now undergoing testing in a phase I/IIa clinical trial in HLA-A*02:01 MART-1-positive advanced stage melanoma patients. To our knowledge, this is the first clinical trial protocol in which the combination of IL-7 and IL-15 has been applied for the generation of gene-modified T-cell products.

Intradermal vaccination by DNA tattooing.

DNA vaccination is an attractive vaccination method. First, the production of plasmid DNA as a vaccine is considerably more cheap and simple than the production of recombinant protein. Second, the expression cassette of DNA vaccines can readily be modified, making DNA vaccines highly flexible. Finally, in animal models, DNA vaccination is able to induce potent cellular immune responses. Over the past decade, the focus in the DNA vaccination field has in large part moved from intramuscular immunization towards dermal administration. As a natural "porte d'entrée" for pathogens, the skin is rich in antigen-presenting cells, which are required for generating an efficient antigen-specific immune response. This chapter describes a DNA vaccination protocol that utilizes a simple tattooing device for the dermal delivery of plasmid DNA. This technique, called DNA tattooing, is capable of generating high frequencies of antigen-reactive T cells in mice and macaques.

Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7.

To allow vaccination irrespective of HLA type, DNA vaccines encoding full-length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full-length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic "gene-shuffled" variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full-length and gene-shuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full-length and the gene-shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein-70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T-cell responses. The E7-specific CD8(+) T cells induced by TTFC fusion vaccines were functional in terms of IFN-γ production, formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene-shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full-length and gene-shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV-positive carcinomas in future studies.

DNA vaccination in oncology: current status, opportunities and perspectives.

After almost 20 years of research, DNA vaccination is still a relatively young technique in the vaccine-toolbox. DNA vaccines can easily be modified by conventional cloning techniques, are relatively easy to produce and might be particularly useful for therapeutic vaccination against intracellular pathogens and cancer. After the early pre-clinical successes, DNA vaccination moved into the clinic and numerous trials have been performed thus far. In the oncology field, these trials aimed for the induction of cellular immunity directed against tumor specific antigens. Although DNA vaccines proved to be well tolerated, and elicited some immune activation in patients, robust immune activation followed by clinical responses has not been observed yet. Nevertheless, several promising strategies are currently under development to increase the performance of the current generation DNA vaccines. Future research has to demonstrate whether these strategies are able to give DNA vaccination a defined position in cancer treatment.