Introduction
Activating immunity against cancer in patients has been a difficult goal [
1] but randomized studies are now showing encouraging results in solid tumors [
2,
3], including prostate cancer [
4]. Prostate cancer immunotherapy is attractive at early biochemical detection of recurrence since rising prostate-specific antigen (PSA), even without radiologically measurable disease, identifies patients at risk who have very small volume disease [
5]. Vaccine targets, like Muc-1 [
6], PSA [
7,
8], prostatic acid phosphatase (PAP) [
9] or prostate-specific membrane antigen (PSMA) [
10‐
12], have been identified as promising targets [
13]. A randomized phase III trial showed that prostate-associated antigens can be effectively targeted by vaccination [
4]. The improved median survival of 4.1 months in late-stage disease was not mirrored by PSA changes [
4], an observation also made in other immunotherapy studies [
14]. Although Sipuleucel-T sets a treatment paradigm, producing a new patient-specific vaccine is a technical, financial and logistical challenge. Overall benefit remains small, indicating an unmet clinical need for better, ideally non-toxic, treatments to improve outcomes [
13].
Vaccination against cancer using exogenous peptide has been tested widely and may confer clinical effect in some settings [
15‐
17]. However, CD8
+ T-cell responses following vaccination using exogenous short peptides appear transient [
18] possibly due to the lack of T-cell help. Viral vector–based vaccines may overcome this problem and have shown promise in metastatic disease [
8,
19] with effects also on PSA doubling time (PSA-DT) at biochemical failure [
6]. However, viral vectors will either face pre-existing immunity or induce it on repeat injections. DNA vaccines avoid this problem and offer a novel delivery vehicle for the induction of peptide-specific responses.
We have designed DNA fusion-gene vaccines able to deliver tumor-derived peptides, together with microbial genes, to generate high levels of T-cell help [
20]. Our platform design includes a strongly immunogenic helper domain (DOM), derived from fragment C (FrC) of tetanus toxin, linked to a tumor-epitope sequence of choice [
20]. In pre-clinical models, DOM-epitope vaccines induce durable tolerance-breaking epitope-specific CD8
+ T-cell immunity, able to suppress a range of tumors [
20].
In mice expressing the HLA-A0201* transgene, the DOM-epitope vaccine design incorporating an epitope from PSMA (PSMA
27 VLAGGFFLL) [
20,
21] induced high levels of specific CD8
+ T cells able to kill tumor cells [
22]. We have now vaccinated patients with biochemically recurrent prostate cancer and, to optimize human translation, also evaluated delivery with electroporation (EP). EP has been reported to increase the potency of DNA vaccines by increasing antigen levels and stimulating local inflammation [
23], and its use is rapidly expanding in both infectious diseases and cancer vaccination. We found that this approach was safe, well tolerated and significantly increased antibody induction [
24].
We report here the effect of our DOM-epitope vaccine on T-cell immunity and clinical outcome. The vaccine reproducibly induces T-cell immunity to PSMA27 and significantly increases PSA-DT, and in spite of the small sample size, we identified a trend to increased time to next treatment compared to a control group of unvaccinated HLA-A2− patients. Taken together, these data support further randomized testing of the vaccine.
Discussion
In HLA-A2 transgenic mice, pDOM-PSMA
27 epitope vaccination stimulates strong peptide-specific CD8
+ T-cell responses [
22]. The PSMA
27 epitope is processed from PSMA, and induced T cells can kill human target cells, confirming PSMA
27 as a useful target for CD8
+ T-cell attack. The phase I/II study we present here takes these observations to the clinic. In HLA-A2
+ prostate cancer patients at biochemical failure, with low disease burden, vaccination significantly increased PSA-DT compared to pre-vaccination. We compared time to next treatment in vaccinated patients with a synchronous group of HLA-A2
− patients. The data suggest that pDOM-PSMA
27 vaccination could affect the natural history of prostate cancer and the suggestion that time to next treatment can be extended will need evaluation in a larger, randomized study. Whether HLA-A2 in its own right is an adverse prognostic factor has not been answered definitively, though there is a suggestion of link to prostate cancer incidence [
31], increased proportion of large tumors (T3b–T3c) and higher post-operative Gleason sums compared to the HLA-A2
− control group [
32]. An adverse effect of HLA-A2 on outcome would strengthen a clinical effect of vaccination.
The increase in PSA-DT became visible after >24 weeks after first vaccination, and in 14/30 patients, the increase was 200 % or greater. From a baseline of 12 months, PSA-DT increased to 17 months. While caution is needed in the absence of randomized controls [
33], a consistent story supporting an effect of vaccination at biochemical recurrence is emerging, where vaccination significantly increases PSA-DT [
6,
9,
34‐
36]. Within the limits of comparability between studies, it appears that our DNA vaccine, targeting a single PSMA epitope, is at least as effective as other more complex DNA- or peptide-based vaccines.
T cells against the DOM helper sequence expanded in almost all (29/30, 97 %) patients, demonstrating patients’ immunocompetence and the immunological performance of the vaccine. pDOM-PSMA
27 induced CD8
+ T-cell responses in 16/30 (55 %) of patients, using pre-defined assay criteria and a single round of in vitro culture. Comparison of immunogenicity between trials is hampered by widely varying assay systems used for immune monitoring, and additionally, only few studies are available that report this data in comparable clinical settings [
6,
9]. The dataset by McNeel et al. [
9] with a full-length DNA vaccine encoding PAP is most similar to our own, and in this study, 3/22 patients had measurable CD8
+ T-cell responses compared to 6/30 patients in our dataset ex vivo.
Incorporation of full-length antigen sequence into the DNA vaccine seems attractive since it would allow vaccination of all rather than to the 40 % of patients who carry HLA-A2 [
9]. However, there are cogent reasons for using a peptide-focused vaccine since the inductive power of the repositioned peptide is generally considerably higher than from full-length sequence [
37]. CD8
+ T cells specific for a single epitope are clearly capable of suppressing even an acute viral infection [
38]. Should escape from focused attack occur, a second vaccine against a different epitope could be used [
39], and we are exploring double attack in our current clinical trial against the WT-1 antigen [
40]. Although our vaccine design could readily incorporate tumor-derived MHC class II-binding epitopes, there is no clear evidence that these are required for the maintenance of cytotoxic T cells and there is a danger that regulatory T cells might be induced [
13,
41].
Viral vector–based vaccines have the problem of pre-existing or induced antiviral immunity. However, an MVA-MUC-1 vaccine induced an IFNγ
+ T-cell response to MUC-1 after short-term culture in 7/34 patients with prostate cancer [
6]. Pox viral delivery in metastatic disease also generated PSA peptide-specific CD8
+ T-cell responses in 13/29 patients following PSA-TRICOM vaccination [
42] and in 9/24 patients following MVA-Trovax vaccination [
43]. It appears that our approach has at least comparable immunogenicity. We would contend, however, that avoiding blocking immunity, likely to arise from MVA [
44], will be important for repeated vaccinations required to maintain attack on cancer. A concern at the outset of our study had been whether T-cell responses would be durable, as with some vaccines approaches CD8
+ T-cell responses can be lost rapidly and then not re-expand after repeated injection [
18]. Our data argue that with DNA vaccination this is not a problem with T-cell responses maintained to the end of the follow-up period.
To examine whether our DNA vaccine had sufficient potency to be scaled from mouse to human, we examined the delivery of our DNA vaccine using the Inovio Elgen100 device for the first time in the clinic. We had found pre-clinically [
22,
45] that EP increased antibody responses, with lesser increase in CD8
+ T-cell responses to our DNA fusion vaccine. In the clinic, this dichotomy is also evident with clear increases in antibody [
24] but only a trend for increase in both CD4
+ and CD8
+ T-cell responses with EP. After cross-over of 11/15 patients to EP boosting, there is a significant and durable increase to the end of the study but we can no longer assess the impact of the individual delivery modalities. It is intriguing to speculate why EP has an apparently smaller effect on T-cell responses compared to humoral responses. In the trial, this may simply be a reflection of very small patient numbers treated without electroporation, and a randomized dataset needs to evaluate the comparative question further. A possible explanation for both the murine and human data could be that unlike B-cell responses, where the increased muscular antigen expression after electroporation leads to higher humoral responses [
24], for T cells there may not be such a strict correlation with the quantity of antigen expressed by the muscle cells.
In summary, the pDOM-PSMA27 vaccine is safe, generates anti-PSMA responses in the majority of patients and is associated with an increase in PSA-DT. Use of EP was well tolerated and may increase T-cellular vaccine efficacy. These findings merit further testing in a randomized setting. Examining the vaccine-induced T cells for their ability to home to the tumor will be a critical component of further evaluation and may offer the tool to better identify a link between vaccine-induced immunity and clinical outcome.
Acknowledgments
This work was supported by the NIHR Southampton Experimental Cancer Medicine Centre, funded by Cancer Research UK and the Department of Health, UK. Clinical work was undertaken in the University Hospitals Southampton and at the Royal Marsden NHS Foundation Trust who received a proportion of its funding from the NHS Executive. The work was further supported by the Alan Willett Foundation, an unrestricted educational grant from Inovio Pharmaceuticals, the Institute of Cancer Research, the Bob Champion Cancer Trust and Cancer Research UK Section of Radiotherapy [CRUK] grant number C46/A2131. We acknowledge NHS funding to the NIHR Biomedical Research Centre and support by the Welcome Trust Clinical Research Facility, Southampton. Assay development and validation was in part supported by the Wallace Coulter Foundation.