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.

The incidence of melanoma has steadily increased over the past decades [1], with approximately 325,000 new cases and 57,000 deaths worldwide due to melanoma described in 2020 [2]. At the time this phase I clinical study was initiated, dacarbazine (DTIC), was the only approved and available first-line treatment for patients with metastatic melanoma, with a median overall survival of 6–10 months [3‒5].

Since then, the treatment landscape for patients with metastatic melanoma has greatly evolved. Currently, the immune checkpoint inhibitors ipilimumab (an anti-CTLA-4 monoclonal antibody) [4, 6], nivolumab and pembrolizumab (both anti-PD-1 monoclonal antibodies), either as single agent or combination therapy [7‒11], next to targeted therapy with BRAF- and MEK-inhibitors in case of BRAFV600 mutated melanomas [12], have become standard-of-care treatment options. In addition, the combination of relatlimab (an anti-LAG-3 monoclonal antibody) plus nivolumab has recently become approved treatment [13], and the novel treatment strategy with adoptive cell therapy using tumor-infiltrating lymphocytes has demonstrated superior efficacy as second-line treatment in melanoma patients having failed prior anti-PD-1 treatment [14]. However, when this study was initiated in 2009, still little or no advance in the treatment field had been made and novel therapeutic options for patients with metastatic melanoma were being explored.

Cancer vaccines, such as DC, RNA and DNA vaccines, were considered appealing novel treatment modalities for solid tumors. As antigens are produced intracellularly by the patient’s own cells upon administration of a genetic vaccine, immunogenic epitopes can be presented in the context of both major histocompatibility complex (MHC) class I and class II molecules, resulting in systemic cellular immune responses [15]. Based on very promising preclinical experiments, both in small and large animal models [16], we set out to test a DNA vaccine-based treatment approach, consisting of naked DNA directly transferred to the patient’s skin with an intradermal application strategy. The DNA vaccine encodes a fusion protein of the non-toxic domain 1 of tetanus toxin fragment C and the immunodominant MART-1 epitope (ELAGIGILTV). In earlier preclinical experiments, a DNA vaccine could successfully be injected into the skin of mice and macaques by using an intradermal permanent make-up or tattoo device, resulting in gene expression in the skin and systemic induction of vaccine-specific CD4+ and CD8+ T cell responses [17, 18]. As animal furred skin is quite different from human skin, an ex vivo human skin model was used to optimize the settings for both the intradermal permanent make-up device and the amount of DNA vaccine as measured by gene expression [19].

Between February 2009 and January 2012, an open label, dose escalating phase Ia clinical trial in HLA-A*0201-positive metastatic melanoma patients was performed. Here, we report on the safety and on clinical and immunological outcomes of this study.

Patients

Patients with inoperable stage IIIC or IV, according to AJCC 7th edition, with disease progression under standard systemic treatment (DTIC, or temozolomide), were eligible for inclusion in this trial. Other inclusion criteria were: age ≥18 years, life expectancy ≥ 3 months, WHO performance status of 0–1, positive status for HLA-A*0201, expression of MART-1 and MHC class I by the tumor, normal organ function, and evaluable disease as measured by Response Evaluation Criteria in Solid Tumors (RECIST), v1.0 [20]. Exclusion criteria were a second malignancy, except for basal cell carcinomas and cervical carcinoma in situ, previous MART-1-specific immunotherapy, symptomatic brain metastases, severe cardiopulmonary comorbidities, pregnancy or lactation, immunosuppressive or anticoagulant medication, unwillingness to take adequate contraceptive measures.

Study Design and Objectives

This open label, dose-escalating, single centre, single arm, clinical phase Ia trial was conducted at the Netherlands Cancer Institute (NKI) and was approved by the Central Committee on Research Involving Human Subjects (NL.20284.000.08). The trial was conducted in accordance with the Declaration of Helsinki, the Medical Research Involving Human Subjects Act, and the ICH Harmonized Tripartite Guideline for Good Clinical Practice. All patients provided written informed consent before enrollment.

The primary objective of this trial was to evaluate the toxicity by dose escalation of naked DNA vaccination encoding a CD8+ T-cell epitope from the melanosomal antigen MART-1, genetically linked to the gene encoding domain 1 of tetanus toxin subunit C, called pDERMATT. In addition, the feasibility of a novel intradermal application strategy employing a permanent make-up or tattoo device was evaluated.

Secondary objectives were to investigate the efficacy of this naked DNA vaccine in inducing tumor-specific T cell immunity, as measured by accumulation of MART-1 specific T cells at the vaccination site in skin biopsies and staining of peripheral blood T cells. Other secondary endpoints were the objective response rate (ORR) according to RECIST v1.0, progression-free survival (PFS) and overall survival (OS).

A traditional 3 + 3 design was used for all dosage increases. The maximum tolerated dose (MTD) was defined as the dose level at which at most 1 of 6 patients experienced a dose limiting toxicity (DLT, see online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000537896 for the DLT criteria), with at least 2 patients experiencing a DLT at the next higher dose level. If the MTD is reached, a total of 6 patients would be treated at that level. If the MTD was not reached, a total of 6 patients would be treated at the highest dose level.

Vaccine Production

The DNA vaccine encoding the melanoma-associated epitope MART-1 and an immunostimulatory sequence (tetanus toxin fragment C), named pDERMATT (acronym for plasmid DNA Encoding Recombinant MART-1 And Tetanus Toxin fragment C), was developed and produced at the BioTherapeutics Unit situated in the hospital pharmacy of the Netherlands Cancer Institute.

pDERMATT is a minimal Escherichia coli-derived plasmid backbone (pVAX1) containing a pUC origin of replication, a Kanamycin resistance gene and a CMV early promoter that drives the gene of interest encoding the fusion protein of MART-1(26–35) E27L (ELAGIGILTV) MHC class I epitope fused to the carboxyl-terminus of tetanus toxin fragment C. For the manufacturing of the vaccine, a standard Good Manufacturing Practice production process was followed as described by Quaak et al. [21].

In preclinical models, the antigen expression and immunogenicity of the vaccine were dependent on the duration of tattooing, the DNA concentration as well as the repetition of tattooing, and dose increase by itself did not further influence the speed and size of the induced immune response, despite higher gene expression [18]. In order to optimize the DNA vaccination strategy, an ex vivo human skin model was used (data not shown). Based on these studies, the optimal dosing schedule for the vaccine was determined with the highest DNA concentration that could be manufactured (5 mg/mL), the optimal duration of tattooing per surface area (20 s/50 mm2) that did not lead to severe skin toxicity, and the optimal needle depth (1.5 mm) giving the highest epidermal gene expression.

pDERMATT was formulated as a lyophilized powder. The final product contains 2 mg pDNA and 40 mg sucrose as cryoprotector, and was dissolved in water for injection to 5 mg/mL pDNA before intradermal injection, resulting in an isotonic solution.

Study Procedures

Administration of pDERMATT [21] was performed using a novel intradermal application strategy employing a permanent make-up or tattoo device (Aella permanent make-up machine, provided by Medium-Tech GmbH, Berlin). Prior to vaccination, the skin was pretreated with an epilating cream to non-traumatically remove hair. The first 3 treated patients received 0.5 mg of vaccine over a skin surface of 4 cm2 (2 × 2 cm), as previous studies using an intramuscular DNA vaccine have shown that DNA amounts varying from 0.1 to 8 mg did not induce any detectable side effects [22‒26]. Administration of the vaccine on the upper part of a lower limb was performed on days 0, 3 and 6 followed by a boost on days 28, 31 and 34. If no DLT was observed in the first 3 patients, the dose escalation would be performed by increasing the skin surface area, instead of increasing DNA concentration or by injecting more vaccine into a fixed area of skin to prevent severe skin disruption. Subsequent dose levels would be 1 mg in 8 cm2 (2 × [2 × 2 cm]); then 2 mg in 16 cm2 (4 × 4 cm) and finally 4 mg in 32 cm2 (2 × [4 × 4 cm]). Intra-patient dose escalation was not permitted.

Safety, including local and systemic toxicity, was monitored according to the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 at the days of DNA vaccine injections, followed by weekly until week 8, and monthly thereafter by interviewing and physical examination. Patients underwent skin biopsies of the vaccination site at days 0 (prior to vaccination), 14 and 42 for immunologic monitoring. On day 42, an additional skin biopsy was taken of a non-vaccination site. Standard peripheral blood tests were performed on days 28 and 56. The first scheduled tumor response evaluation was performed around day 56 after vaccination using standard imaging techniques (CT-scan, MRI, X-ray or ultrasound), and a peripheral blood sample was taken to evaluate the presence of MART-1 specific T cells. See online Supplementary Figure 1 for a full overview of the study procedures.

Flow Cytometric Analysis of DNA Vaccine-Induced T Cell Response in Peripheral Blood

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood samples by Ficoll density-gradient centrifugation and cryopreserved until further experiments were conducted. For flow cytometric analysis, PBMCs harvested prior to, during and post vaccination were thawed and subsequently stained with a MART-1 specific HLA-A-*0201 multimer or an irrelevant HIV control multimer, and co-stained with APC-H7-conjugated CD8 antibody (BD), and ECD-conjugated anti-CD3 antibody (B Coulter) at 20°C for 15 min in FACS buffer (×1 PBS, 0.5% BSA and 0.02% sodium azide). Cells were washed three times in FACS buffer and analyzed by flow cytometry. Live cells were selected on SYTOTOX Blue exclusion (Invitrogen). Data acquisition was performed on a Cyan flow cytometer (Beckman Coulter) with FacsDiva software.

For the IFNγ-ELISPOT, PBMCs retrieved prior, during and post vaccination were thawed simultaneously. The PBMCs were stimulated with a MART-1 peptide at 2 × 105 cells per well. A CEF (HLA Class I Control) pool was used as positive control. After overnight incubation, IFNγ-producing spots were visualized using a standard staining protocol using an anti-IFN-γ detection antibody (U-CyTech, biosciences). Spots were quantified using the automated ELISPOT plate reader.

Flow Cytometric Analysis of DNA Vaccine-Induced T Cell Response at the Administration Site

The infiltration of antigen-specific T cells was measured in skin biopsies. For that, punch biopsies of the patients’ skin were taken prior to the first vaccination, and at 2 and 6 weeks after vaccination from the tattooed areas of interest. Furthermore, at week 6 an additional punch biopsy was taken from a non-vaccination site. Biopsies were transferred into well plates and cultured in complete culture media supplemented with interleukin-2 (IL-2) to allow T cell growth. After an average culture period of 4–6 weeks, T cells were stained with MART-1 specific HLA-A-*0201 multimer or an irrelevant HIV control multimer and co-stained with FITC-conjugated CD8 antibody (Pro Immune) and PE-Cy7-conjugated anti-CD3 antibody (B Coulter) at 20°C for 15 min in FACS buffer (1x PBS, 0.5% BSA and 0.02% sodium azide). Cells were washed 3 times in FACS buffer and analyzed by flow cytometry. Live cells were selected on SYTOTOX Blue exclusion (Invitrogen). Data acquisition was performed on a Cyan flow cytometer (Beckman Coulter) with FacsDiva software.

Statistical Considerations

No power calculation was performed as this was a phase I study. Statistical analysis was conducted in SPSS (version 22 for Windows; SPSS, Inc.). OS was calculated as the time from the date of the first tattoo until death, or last known follow-up. PFS was defined as the time from the date of the first tattoo until progressive disease (PD) per RECIST, v1.0, or last known follow-up. Kaplan-Meier estimates were used to determine OS and PFS.

Patients

Between January 2009 and January 2012, a total of 9 patients with metastatic melanoma were enrolled. Five out of 9 patients (56%) were male and the mean age at start of the study was 58 years (range 32–72). All patients had received prior systemic therapy and the majority (89%) had M1c disease. Furthermore, 3 patients had elevated LDH levels and one patient had asymptomatic central nervous system metastases. Baseline characteristics are summarized in Table 1.

Table 1.

Baseline characteristics

Sex, n (%) 
 Male 5 (56) 
 Female 4 (44) 
Age, years 
 Mean (range) 57.9 (32–72) 
WHO performance statusa, n (%) 
 0 7 (78) 
 1 2 (22) 
Brain metastases, n (%) 
 Symptomatic 
 Asymptomatic 1 (11) 
 None 8 (89) 
Baseline lactate dehydrogenase, n (%) 
 Normal 5 (56) 
 Elevatedb 3 (33) 
 Unknown 1 (11) 
M-staging of extent of metastases, n (%) 
 M1a 1 (11) 
 M1b 
 M1c 8 (89) 
Sex, n (%) 
 Male 5 (56) 
 Female 4 (44) 
Age, years 
 Mean (range) 57.9 (32–72) 
WHO performance statusa, n (%) 
 0 7 (78) 
 1 2 (22) 
Brain metastases, n (%) 
 Symptomatic 
 Asymptomatic 1 (11) 
 None 8 (89) 
Baseline lactate dehydrogenase, n (%) 
 Normal 5 (56) 
 Elevatedb 3 (33) 
 Unknown 1 (11) 
M-staging of extent of metastases, n (%) 
 M1a 1 (11) 
 M1b 
 M1c 8 (89) 

aThe World Health Organization (WHO) performance status of 0 indicates that the patient is asymptomatic and fully active; 1 that the patient is restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature; 2 ambulatory and capable of all self-care but unable to carry out any work activities.

bAn elevated level was considered to be a level above the upper limit of the normal range.

Study Conduct

The first 3 patients enrolled in the trial received 0.5 mg of the vaccine (cohort 1), the following 3 patients received 1 mg (cohort 2) and the last 3 patients received 2 mg (cohort 3). All patients received all 6 vaccinations as planned. Even though no DLT had been observed in the prior dose levels, no patients were enrolled in cohort level 4 (4 mg in 32 cm2), due to lacking clinical efficacy and an increased burden for patients, as a higher vaccination dose would require a larger skin surface for intradermal vaccine delivery.

Safety

No treatment-related deaths occurred during the course of this trial. The most common adverse events (AEs) of any grade, regardless of causality, were: superficial skin bleeding at site of injection (100%), local reaction at site of injection (100%), pain (78%), fatigue (56%), myalgia (56%), anorexia (56%) and flu-like symptoms (56%). The most common treatment-emergent AEs (TEAE) of any grade were: superficial skin bleeding at site of injection (100%), local reaction at site of injection (100%), pain (56%), fatigue (44%), myalgia (44%) and flu-like symptoms (44%) (see Table 2). Grade 3–4 TEAEs occurred in one (11%) patient (2 events). The most common grade 3–4 TEAE were fatigue (11%) and nausea (11%). One patient developed grade 4 dyspnoea due to disease progression with multiple pulmonary metastases and pleural effusion. Despite pleural fluid drainage, the patient died several weeks later from disease progression.

Table 2.

Adverse events of any cause

Adverse events of any causeTreatment emergent adverse events
all grades, n (%)grade 3 or 4, n (%)all grades, n (%)grade 3 or 4, n (%)
Gastrointestinal 
 Nausea 4 (44) 1 (11) 3 (33) 1 (11) 
 Vomiting 4 (44) 3 (33) 
 Obstipation 3 (33) 
 Diarrhea 2 (22) 1 (11) 
 Dehydration 1 (11) 
Generalized symptoms 
 Pain 7 (78) 5 (56) 
 Fatigue 5 (56) 2 (22) 4 (44) 1 (11) 
 Flu-like symptoms 5 (56) 4 (44) 
 Anorexia 5 (56) 1 (11) 3 (33) 
 Myalgia 5 (56) 4 (44) 
 Fever 3 (33) 3 (33) 
Respiratory disorder 
 Dyspnea 2 (22) 1 (11) 1 (11) 
Hematology 
 Decrease in hemoglobin 3 (33) 2 (22) 
 Decrease in lymphocyte count 2 (22) 2 (22) 
Skin disorder 
 Bleeding 9 (100) 9 (100) 
 Local toxicity 9 (100) 9 (100) 
 Rash 3 (33) 1 (11) 2 (22) 
Eye disorder 
 Conjunctivitis 1 (11) 1 (11) 
Increase in aminotransferase level 
 AST 2 (22) 1 (11) 
 ALT 1 (11) 
Increase in alkaline phosphatase 1 (11) 1 (11) 
Hypoalbuminemia 1 (11) 1 (11) 
Total number of adverse events 78 57 
Adverse events of any causeTreatment emergent adverse events
all grades, n (%)grade 3 or 4, n (%)all grades, n (%)grade 3 or 4, n (%)
Gastrointestinal 
 Nausea 4 (44) 1 (11) 3 (33) 1 (11) 
 Vomiting 4 (44) 3 (33) 
 Obstipation 3 (33) 
 Diarrhea 2 (22) 1 (11) 
 Dehydration 1 (11) 
Generalized symptoms 
 Pain 7 (78) 5 (56) 
 Fatigue 5 (56) 2 (22) 4 (44) 1 (11) 
 Flu-like symptoms 5 (56) 4 (44) 
 Anorexia 5 (56) 1 (11) 3 (33) 
 Myalgia 5 (56) 4 (44) 
 Fever 3 (33) 3 (33) 
Respiratory disorder 
 Dyspnea 2 (22) 1 (11) 1 (11) 
Hematology 
 Decrease in hemoglobin 3 (33) 2 (22) 
 Decrease in lymphocyte count 2 (22) 2 (22) 
Skin disorder 
 Bleeding 9 (100) 9 (100) 
 Local toxicity 9 (100) 9 (100) 
 Rash 3 (33) 1 (11) 2 (22) 
Eye disorder 
 Conjunctivitis 1 (11) 1 (11) 
Increase in aminotransferase level 
 AST 2 (22) 1 (11) 
 ALT 1 (11) 
Increase in alkaline phosphatase 1 (11) 1 (11) 
Hypoalbuminemia 1 (11) 1 (11) 
Total number of adverse events 78 57 

AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Clinical Activity

The median follow-up time was 5.9 months (range 2.1–107.9 months). At time of the first scheduled tumor response assessment, 8 out of 9 (89%) patients showed PD on CT-scan. One patient (11%) showed disease stabilization, lasting 353 days. The median OS for the entire cohort was 5.9 months (95% CI: 0.0–12.6), with a median PFS of 1.8 months (95% CI: 1.7–2.0). Three out of 8 patients (38%) who showed PD at time of first assessment received subsequent treatment lines. The remaining 5 patients died due to rapid PD shortly after participation in this trial (range 19–124 days).

Immunomonitoring Assessments

Antigen-specific T cell responses before and after vaccination were analyzed in peripheral blood and skin at the vaccine administration site. In the peripheral blood compartment, no increase in antigen-specific T cell response could be detected in any of the patients (data not shown).

Skin biopsies taken at day 0 (prior to vaccination), 14 and 42, were available from 5 patients (56%) at all time points, with 1 patient missing a pre-treatment biopsy, 1 patient missing a week 6 biopsy and two patients having insufficient quality of the week six biopsy. An antigen-specific MART-1 CD8+ T cell response could be observed after culturing the skin biopsy in 4 out of 5 evaluable patients (see Fig. 1 for a representative patient) after an average culture period of 4 weeks. This antigen-specific T cell response could not be observed in control skin biopsies (non-tattooed) of 2 patients, indicating that this influx of CD8+ T cell was vaccine induced. Since all biopsy samples were cultured for several weeks with high dose IL-2 to induce expansion before analysis, no conclusion on T cell frequency could be made.

Fig. 1.

Fluorescence-activated cell sorting of skin biopsies taken 6 weeks after vaccination of a representative patient. Biopsies were cultured in 6,000 IU IL-2 for 4 weeks. a shows a control of a non-vaccinated area. b shows a biopsy of a vaccinated area. Forty-one percent MHC tetramer MART-1 positive CD8+ T-cells can be found.

Fig. 1.

Fluorescence-activated cell sorting of skin biopsies taken 6 weeks after vaccination of a representative patient. Biopsies were cultured in 6,000 IU IL-2 for 4 weeks. a shows a control of a non-vaccinated area. b shows a biopsy of a vaccinated area. Forty-one percent MHC tetramer MART-1 positive CD8+ T-cells can be found.

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With immunohistochemistry, an increase in CD80 expression was seen in skin biopsies of all 5 patients. Furthermore, an increase in CD3 and T cell receptors was observed in 4 patients. A clear increase in lymphocytes in skin 6 weeks after tattoo from a vaccinated site was observed compared to pre-tattooed skin. No increase in lymphocytes was observed in a biopsy 6 weeks after tattoo from a non-vaccinated site (see Fig.). In 2 patients a slight decrease in CD1a was noted.

Fig. 2.

Immunohistochemistry of biopsied skin from a representative patient (a 71-year old male with lymph node and (sub)cutaneous melanoma metastases treated in dose cohort 3; 2.0 mg vaccine dose per tattoo). a Pre-tattooed skin. b 2 weeks after tattoo from vaccinated site. c 6 weeks after tattoo from non-vaccinated site. d 6 weeks after tattoo from vaccinated site.

Fig. 2.

Immunohistochemistry of biopsied skin from a representative patient (a 71-year old male with lymph node and (sub)cutaneous melanoma metastases treated in dose cohort 3; 2.0 mg vaccine dose per tattoo). a Pre-tattooed skin. b 2 weeks after tattoo from vaccinated site. c 6 weeks after tattoo from non-vaccinated site. d 6 weeks after tattoo from vaccinated site.

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This dose-escalating phase Ia study in patients with metastatic melanoma was the first trial clinically applying dermal DNA tattooing. Although the vaccine was deemed safe and well tolerated, no clinical responses were observed, with 1 patient reaching disease stabilization as best response. No patients were enrolled in the highest dose cohort. Although we cannot rule out that a higher dose might have induced clinical responses, a higher dose would also require an increased skin surface for intradermal vaccine delivery with a higher burden for patients. The absence of any clinical effects seen in the lower dose cohorts led to the decision not to enrol any subsequent patients in the highest dose cohort.

When investigating the immunological response, no increase in antigen-specific T cell response in the peripheral blood could be detected by ELISPOT assays or flow cytometry. Since responses were analyzed directly without any ex vivo re-stimulation, there is a possibility that T cell responses were present, but below the detection limit of the analysis methods applied. At the skin administration site, MART-1-specific T cell responses were detectable after culturing in 4 out of 5 evaluable patients. This interesting observation indicates that MART-1-specific T cell responses were induced, but that the T cells were either not systemically detectable anymore at the time of peripheral blood analysis as the majority of T cells possibly had homed to the skin, or that no systemic MART-1-specific T cell responses were induced, but tissue resident MART-1 specific T cells only proliferated locally in reaction to the tattoo vaccination.

The lack of clinical and systemic immunological responses in this trial could be a result of the poor immunogenicity of the vaccine. We have previously published the results of another DNA tattoo vaccination trial, using a genetic vaccine endoding a fusion protein of TTFC and the shuffled version of HPV16 E7 oncoprotein (TTFC-E7SH) [27]. Although this treatment was also well tolerated, no clinical responses at 3 months follow-up were seen. In addition, only low vaccine-induced immune responses were seen in 4/12 (33%) patients with usual type vulvar intraepithelial neoplasia (uVIN) [27].

In order to determine if optimization of the genetic design of the vaccine can improve clinical and immunological responses, we subsequently clinically evaluated novel vaccine formats with improved carrier-proteins in HPV type 16 induced premalignancies [28]. Using these novel vaccine formats, a phase I/II clinical trial was performed with 14 HPV16+ uVIN patients, whom were treated with a genetically enhanced DNA vaccine targeting E6 and E7, resulting in a clinical response (defined as >50% decrease in lesion size) in 43% of patients [29]. In this clinical study, 5/14 (38%) patients showed HPV-specific T-cell responses in peripheral blood, measured in ex vivo reactivity assays. These results indicate that genetic optimization of DNA vaccine format could potentially result in higher immunogenicity, although the clinical benefit of a genetic vaccine as monotherapy in metastasized patients remains challenging.

Although our trial showed no clinical benefit, the advances in the last decade in the treatment of melanoma with immune checkpoint inhibitors may also provide new possibilities for cancer vaccine development. In fact, the field of cancer vaccines is momentarily going through a renaissance period, especially with development of optimized DNA and RNA vaccines and neoantigen-specific vaccines, either alone or in combination with immune checkpoint inhibitors or adoptive cell therapy [30‒33]. For example, in a murine melanoma model combining an intradermal DNA vaccine with CTLA-4 and PD-1 blockade induced a strong antigen-specific immune response and resulted in a significant delay in tumor growth and prolonged survival [34]. Furthermore, in the currently recruiting KEYNOTE-D36 trial (NCT05309421), treatment naïve patients with metastatic or unresectable melanoma are treated with pembrolizumab in combination with EVX-01. EVX-01 is a personalized peptide-based therapeutic vaccine consisting of multiple peptides, representing a neoepitope only found in the patient’s tumor [35]. In the phase 1 EVX-01 trial, of the 5 evaluable patients, 1 patient had a complete response, while 2 other patients had a partial response [36]. In addition, combining a vaccine with immune checkpoint inhibition also seems feasible in the adjuvant setting. In the KEYNOTE-942 trial patients with radically resected high-risk cutaneous melanoma were randomized to receive pembrolizumab with or without mRNA-4157. mRNA-4157 is an mRNA-based individualized neoantigen therapy encoding for up to 34 neoantigens, tailored to an individual’s tumor and human leukocyte antigen. Recently published data show a prolonged recurrence-free survival in the combination group [37]. These recent developments could therefore also provide new opportunities to optimize clinical DNA (tattoo) vaccination in patients with advanced melanoma, but further research is required to optimize this treatment modality as either single-agent treatment or combination treatment.

We would like to thank Pia Kvistborg, Trees de Jong, Willeke van de Kasteele, and Sandra Adriaansz for their contributions to this trial.

This study protocol was reviewed and approved by the Central Committee on Research Involving Human Subjects (NL.20284.000.08). This study was conducted in accordance with the Declaration of Helsinki, the Medical Research Involving Human Subjects Act, and the ICH Harmonized Tripartite Guideline for Good Clinical Practice. Written informed consent was obtained from all participants prior to enrollment.

D.P. is currently employed at Galapagos NV. J.H. Beijnen is part-time employee and (in)direct stock holder of Modra Pharmaceuticals (small spin off company of the Netherlands Cancer Institute) and (partly) holds a patent on oral taxane formulations, which are clinically developed by Modra Pharmaceuticals. This is not related to the manuscript. J.H.B. has received grants from NEON therapeutics, BMS and Medimmune and is currently employee of Galapagos NV. J.H. has provided consultation, attended advisory boards, and/or provided lectures for BMS, CureVac, GSK, Imcyse, Iovance Bio, Instil Bio, Immunocore, Ipsen, Merck Serono, MSD, Molecular Partners, Novartis, Pfizer, Roche/Genentech, Sanofi, Scenic, Third Rock Ventures, has participated in the SAB of Achilles Tx, BioNTech US, Instil Bio, PokeAcell, T-Knife, Scenic and Neogene Therapeutics, and through this the NKI has received grant support from Amgen, Asher Bio, BioNTech, BMS, MSD, Novartis, Sastra Cell Therapy. All other authors have no competing interests to declare.

This study was not supported by any sponsor or funder.

All authors reviewed and approved the final manuscript. M.H.G.F., M.W.R. and J.S.W.B. contributed equally in the preparation and critical revision of the clinical data and the manuscript. M.H.G.F. analyzed the safety and clinical activity data. D.P. and F.V.-D. performed the immunomonitoring assessments. J.H. Beijnen., B.N., J.H.B. and J.B.A.G.H. were involved in the conceptualization and design of the study procedures and study protocol and made substantial contributions to the manuscript. J.B.A.G.H. was responsible for the decision to submit the manuscript and is the corresponding author of this manuscript.

Additional Information

Marnix H. Geukes Foppen, Maartje W. Rohaan, and Jessica S.W. Borgers contributed equally to this work.

Access to qualified academic scientific researchers may be granted. Data will be shared for proposals that are complete, for which the scientific request is valid and the data are available, consistent with safeguarding patient privacy and informed consent. Upon a scientifically sound request, data access can be obtained via the NKI’s scientific repository at repository@nki.nl, which will contact the corresponding author (J.B.A.G. Haanen). Data requests will be reviewed by the institutional review board of the Netherlands Cancer Institute (NKI) and will require the requesting researcher to sign a data access agreement with the NKI. Researchers must commit to transparency in publication.

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