p53-Reactive T Cells Are Associated with Clinical Benefit in Patients with Platinum-Resistant Epithelial Ovarian Cancer After Treatment with a p53 Vaccine and Gemcitabine Chemotherapy

More than 70% of patients with ovarian cancer present with advanced disease (stage III/IV) (1) and undergo aggressive surgical staging and cytoreduction, followed by systemic chemotherapy. Around 60%–80% of patients initially respond to platinum-based chemotherapy (cisplatin/carboplatin) in combination with paclitaxel (2, 3). However, the vast majority later relapse and eventually develop chemoresistant disease. Hence, new treatments are being actively pursued. The discovery that some cytotoxic agents can interfere with immunosuppressive pathways has led to novel immunotherapy agents being combined with conventional chemotherapy.

Standard-of-care options for platinum-resistant ovarian cancer include single-agent gemcitabine chemotherapy. Intracellular metabolism of gemcitabine produces the active diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleosides, which inhibit DNA synthesis. Similar to other agents used in the setting of platinum-resistant disease, gemcitabine has relatively low efficacy in extending survival (4). In patients with platinum- and paclitaxel-resistant ovarian cancer, the response rate to single-agent gemcitabine ranges from 13% to 17% with PFS of 3–5 months (5–7). However, gemcitabine may induce beneficial effects that could help overcome immune suppression and enhance responses to immunotherapy. Although gemcitabine therapy often induces neutropenia, it has shown positive immunomodulatory effects in pancreatic cancer patients, including increased frequency of peripheral CD3⁺CD45RO⁺ memory T cells (8), decrease of Tregs (9, 10), reduced MDSCs (10), and increased monocytes and dendritic cells (11). Proposed mechanisms for immunomodulation in cancer patients include inhibition of STAT3 phosphorylation in myeloid cells (10) and preventing the conversion of effector T cells to Tregs (12). In addition, reduced TGFβ and arginase-1 in cancer patients' plasma has been reported after gemcitabine therapy (10).

There is clinical evidence that gemcitabine may enhance responses to a variety of immunotherapies, including antigen-pulsed DC (dendritic cells) (13), peptides (14–16), and mAbs (17). However, most of these studies were conducted in pancreatic cancer and there is little published data on this approach in ovarian cancer patients. Dijkraaf and colleagues compared combinations of synthetic long p53 peptides, IFNα, and gemcitabine to treat patients with platinum-resistant ovarian cancer. Gemcitabine significantly reduced the frequency of MDSCs and all patients vaccinated with the p53 peptide vaccine demonstrated strong vaccine-induced p53-specific T-cell responses (18). Therefore, it seemed logical to combine gemcitabine chemotherapy with our p53-based vaccine.

Wild-type p53 protein maintains normal cell division and mutations in this gene are present in the majority of solid tumors (19). p53 gene mutations result in the accumulation of high levels of oncogenic p53 protein within tumor cells. In contrast, the concentration of normal p53 in healthy cells is low, making p53 an attractive target for immunotherapy of a wide range of malignancies. Because p53 is an autoantigen widely expressed throughout development, immunologic tolerance could limit the effectiveness of p53-directed immunotherapy. Wild-type p53 is not presented on the surface of parenchymal cells in healthy adults (20); however, humans can mount anti-p53 immune responses when p53 is presented as an antigen. The detection of endogenous anti-p53 reactive T cells in cancer patients suggests a lack of p53-specific tolerance at the T-cell level (21). Several groups have generated human CTL against wild-type p53 peptides in vitro (22, 23). Therefore, p53 immunotherapy aims to exploit the incomplete tolerance and stimulate immune-mediated killing of p53-overexpressing tumor cells. If dominant mutations in p53 occurred, which generated tumor specific antigens, this would provide ideal targets for a cancer vaccine. Unfortunately, the sites of p53 mutations are highly variable among tumor types and patients, and do not correspond to immunogenic T-cell epitopes (24). Therefore, p53-based vaccines commonly utilize the wild-type sequence.

Clinical trials targeting p53 by administration of synthetic peptides have yielded promising results. Administration of the canarypox virus ALVAC to patients with unresectable colorectal cancer demonstrated that 2 of 5 patients receiving the highest vaccine dose developed p53-specific responses (25). Ad-p53–pulsed dendritic cells have been evaluated in in cancer patients with advanced malignancies (26). The majority of patients generated potent p53-specific immunity and evidence of clinical benefit was demonstrated. No evidence of autoimmunity or serious adverse events were reported. Leffers and colleagues reported that a p53-synthetic long-peptide vaccine was well-tolerated and induced p53-specific T-cell responses in patients with recurrent ovarian cancer (27). When the vaccine was combined with cyclophosphamide, a reduction in Tregs was observed and 2 of 10 patients showed stable disease (28). Another study utilizing combinations of p53 peptides, montanide, cytokines, and DCs in ovarian cancer patients also reported the induction of p53-specific immune responses (29).

We have developed a strategy using the genetically engineered version of the MVA virus (Modified Vaccinia Ankara) to immunize patients with the wild-type p53 antigen (p53MVA). Using a viral vector to deliver full-length p53 has the potential to generate sustained antigen expression and the presentation of numerous antigenic determinants on different HLA molecules. MVA has a demonstrated safety record, being used in numerous clinical trials with only mild side-effects. Despite its inability to propagate in most mammalian cells, MVA still efficiently expresses viral and exogenous recombinant genes making it a potent antigen delivery platform. Furthermore, due to the inactivation of immune evasion genes, MVA vectors demonstrate useful adjuvant properties (30). MVA vectors are taken up by antigen-presenting cells such as DCs, allowing cross presentation of transgene-encoded antigens and priming of specific T-cell responses (30).

In a first-in-human, single-agent trial, a dose of 5.6 × 10⁸ pfu of p53MVA was well tolerated and immunogenic, but no clinical responses were apparent (31). Therefore, we designed a study that combined the vaccine with the immunomodulatory chemotherapy agent gemcitabine. The primary study objectives were to assess the immunogenicity and tolerability of the combined treatment. The secondary objectives were to assess any changes in the frequency of immunosuppressive cells in the periphery and clinical responses to therapy. Here we report the findings of this study in regard to safety, clinical response, and immunologic endpoints.

Participants were women with recurrent epithelial ovarian, peritoneal, or fallopian tube cancer with evidence of tumoral p53 overexpression by IHC (≥10% of cells within the tumor staining positive) or p53 mutational analysis. The Institutional Review Board approved the conduct of the study (ClinicalTrials.gov Identifier NCT02275039), which was in accordance with the Declaration of Helsinki and Good Clinical Practice. Prior to treatment, all patients signed the informed consent. Patients with platinum-resistant disease, that is, those showing recurrence or progression within 0–6 months following platinum-based chemotherapy were eligible. Patients with borderline platinum sensitivity, that is, those with documented disease recurrence or progression within 6–12 months following platinum-based therapy were also eligible. Other eligibility criteria included: ≤ 2 chemotherapy lines for recurrent disease, 18 years of age or older, Karnofsky Performance Status of 80–100, and a life expectancy of greater than 3 months. Adequate bone marrow, renal, and liver functions were required. Prior exposure to gemcitabine was not allowed. Patients with brain metastasis, or those receiving any additional investigational agents, radiotherapy, or systemic corticosteroids were excluded. In addition, patients with immunodeficiency (including HIV and organ graft related), a history of autoimmune disease, severe environmental allergies, or myopericarditis were ineligible.

This was a single institution, dose de-escalating phase I study of p53MVA vaccination in combination with gemcitabine, in women with recurrent ovarian cancer and tumoral p53 overexpression. The primary endpoints were safety and the frequency of circulating p53-responsive T cells. Secondary endpoints were PFS and the frequency of circulating Tregs and MDSCs. As systemic corticosteroids were prohibited from this study, pretreatment with dexamethasone was not given prior to chemotherapy. A three vaccination schedule was employed, as previously evaluated in our single-agent study. Gemcitabine was given prior to each p53MVA vaccination to reduce immunosuppression and enhance the vaccine response. Gemcitabine was administered at a dose of 1,000 mg/m² as a 30-minute intravenous (IV) infusion according to a modified standard-of-care schedule, on days 1 and 8 every 21 days. The p53MVA vaccine was administered at a dose of 5.6 × 10⁸ pfu p53MVA, intramuscularly on day 15 of the first 3 cycles of gemcitabine. The safety and immunogenicity of this p53MVA dose was established by our previous single-agent, phase I study conducted in patients with gastrointestinal malignancies (31). All subjects were monitored for one hour in the clinic after each immunization for temperature changes and local reactions at the injection site. All subjects were contacted 24 and 48 hours after each immunization to record any vaccine-related complications. In the case of drug-related adverse events, dose de-escalation was employed, with reduced doses of 800 mg/m² gemcitabine and 1.0 × 10⁸ pfu of p53MVA permitted. Only one dose reduction was allowed for both study agents. After completing p53MVA vaccination, patients continued single-agent gemcitabine on days 1 and 8, every 21 days until confirmed disease progression or unacceptable toxicity. See Fig. 1 for the complete treatment schedule.

p53MVA is a Modified Vaccinia Ankara vector expressing the full-length, wild-type human p53 gene. The p53MVA vaccine product was manufactured at the Center for Biomedicine and Genetics at City of Hope using GMP-grade materials and the final formulation was diluted in PBS and 7.5% lactose. The vaccine was frozen in high-dose and low-dose vials, and was thawed and administered as described previously (31).

Dose-limiting toxicity (DLT) and adverse events (AE) were graded by the NCI Common Terminology Criteria of Adverse Events (CTCAE) version 4.3. This was a de-escalating phase I study. The MTD was defined as less than 2 patients out of 6 with a DLT, and required at least 6 patients to be treated at that dose. DLT was assessed during the first 2 cycles of treatment. To be evaluable for DLT, patients must have received at least 3 out of 4 doses of gemcitabine and 2 doses of p53MVA, or must have experienced a DLT. Patients who required a dose reduction for gemcitabine or p53MVA were evaluable, provided they received the number of doses specified above. Unevaluable patients were replaced to allow the target accrual of 6–12 evaluable patients to be reached. DLTs were defined as any of the following events during the first 2 cycles of treatment and attributable to study procedures: any ≥ grade 3 hematologic toxicity that did not improve to baseline within 2 weeks, any ≥ grade 3 nonhematologic toxicity, any ≥ grade 2 autoimmune, allergic reactions (including bronchospasm or generalized urticaria), and any ≥ grade 2 myocarditis thought to be related to p53MVA. All patients underwent baseline radiologic evaluation (CT or PET/CT). Restaging scans were obtained every 12 weeks until progressive toxicity, intolerable toxicity, or patient's request to discontinue treatment. Clinical response was determined by irRECIST (immune-related criteria).

A library of overlapping p53 peptides were synthesized in the laboratory as described previously (32) and frozen in 100% DMSO at a concentration of 1 mg/mL. A human CMV pp65 peptide pool (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD) was used as a positive control. Peripheral blood samples were collected from patients by venipuncture in ACD tubes and processed within an hour. PBMCs were purified by density-gradient separation using Ficoll-Paque Plus (GE Healthcare). PBMCs were cultured in serum-free conditions in X-VIVO 20 medium (Lonza) and tested for reactivity to p53 peptides as described previously (31). In vitro stimulation was carried out for 24 hours in the presence of the following peptides: p53 (10 μg/mL), CMV pp65 (1 μg/mL), or a negative control (DMSO alone). After 24 hours, CD4⁺ and CD8⁺ T cells were analyzed for the expression of the activation marker CD137 by flow cytometry. The CD137 assay has been validated for both CD8⁺ and CD4⁺ responses, and there is evidence that it detects a broader repertoire of antigen-specific T cells than cytokines such as IFNγ (33). Cells from each in vitro stimulation were co-stained with anti-CD4, anti-CD8, and anti CD137-PE or a PE-labeled isotype control. CD137 positivity was assessed relative to the isotype control PE signal. Flow cytometric analyses were carried out using a Gallios (Beckman Coulter) flow cytometer. All data was analyzed with Kaluza software. Patients showing an increase in p53-responsive T cells above baseline levels, that reached a minimum of 0.5% of the CD4⁺ or CD8⁺ gate, were defined as T-cell responders.

Flow cytometry analysis of cell surface molecules on PBMCs was conducted using antibodies from Becton Dickinson or eBioscience. Cells were washed and stained with antibody for 30 minutes at room temperature, in the dark, in the presence of 1% FBS. For Treg intranuclear FOXP3 analysis, permeabilization was performed using the eBioscience anti-FOXP3 staining set, according to the manufacturer's instructions. Tregs were identified as CD3⁺CD4⁺CD25⁺CD127^low/−FOXP3⁺ and expressed as a percentage of CD4⁺ cells. MDSCs were identified as HLADR⁻LIN1^low/−CD33⁺CD11b⁺ and expressed as a percentage of the live PBMC population. T-cell phenotyping included analysis of the frequency of PD-1⁺ T cells within the CD4⁺ and CD8⁺ compartments. Flow cytometry gating was carried out as described previously (31). Blood samples from age and sex matched healthy donor controls were obtained through the City of Hope Michael Amini Transfusion Medicine Center.

We evaluated baseline % p53-reactive T cells, MDSCs and Tregs along with their nadir, peak, maximum increase (or decrease), and defined a % p53-reactive CD4 and CD8 vaccine response as an increase from baseline greater than 0.5%. This 0.5% increase corresponded to defining CD4 responders as patients whose peak CD4 counts were above the median for the study patients. Statistical comparison of PFS based on single factors was evaluated by the log-rank test with the median PFS estimates and Kaplan–Meier method. Multivariate Cox regression was used to adjust for age, stage, and prior lines of therapy, along with step-wise forward model selection, and were summarized with a HR, the 95% confidence interval, and used Wald statistics. Values ≤ 0.05 were considered significant. Correlation structure of the variables was evaluated with Pearson correlation coefficient. Comparison of the frequency of Tregs, MDSCs, and PD-1⁺ T cells between patients and healthy donors were carried out by Wilcoxon two-sided exact test, as were differences in baseline MDSCs, Tregs, or T-cell subsets between stage III and stage IV patients or between relevant variables and vaccine responders and nonresponders (e.g., MDSCs and Tregs). Statistical calculations were carried out using R version 3.4.2 and SAS version 9.4.

Participants were recruited from ovarian cancer patients attending Medical Oncology clinics at the City of Hope National Medical Center between December 2014 and November 2016. Twelve patients began study treatment, but 1 patient came off study prior to p53MVA vaccination due to gemcitabine-related adverse events. The remaining 11 patients (median age 59, range 41–76) were evaluated for toxicity of p53MVA/gemcitabine, clinical outcome, and immunologic response. The characteristics of all patients treated with the combination are summarized in Table 1. Ten of the vaccinated patients were platinum resistant; this included one patient who relapsed at 6 months and was considered platinum resistant. The prior lines of previous therapy for recurrent disease ranged from 0 to 2. One patient ended participation after one cycle of treatment due to rapid progression of disease. One patient withdrew after two cycles of therapy and 2 patients after only one cycle, due to gemcitabine toxicity.

Table 1 details the adverse events that were related to therapy. No DLTs were seen on this study. Injection site reaction (ISR) was the most commonly reported side-effect of p53MVA vaccination. The most common adverse events attributed to gemcitabine were neutropenia and skin rash. p53MVA immunization in combination with gemcitabine was well tolerated in the majority of patients, but 3 of 11 patients came off study within 2 months due to gemcitabine-attributed adverse events. Gemcitabine without steroid pretreatment was intolerable in some patients. This included 1 patient with allergic skin reaction, one with grade 2 pneumonitis, and 1 patient with a grade 2 CNS adverse event (reversible posterior leukoencephalopathy syndrome) that resolved rapidly after cessation of therapy and a short course of steroids (Table 2)

In a total of 11 patients receiving a minimum of one treatment cycle, no complete responses were observed. Three patients demonstrated stable disease (SD) by irRECIST criteria. A partial response (PR) was seen in 1 patient on the second posttherapy CT scan (64% reduction according to irRECIST criteria). This patient (patient 4), showed a dramatic CA-125 decline from 2,637 to 150 over a 6-month period. The PFS in treated patients ranged from 0.95 to 9.2 months (median 3 months). Protocol prespecified follow-up of patients was insufficient for an overall survival assessment.

p53-specific T-cell responses were evaluated by quantification of CD137⁺ T cells after stimulation with p53 peptides. The CD137 marker has been validated for measuring both CD8⁺ and CD4⁺responses (33–35), with the suggestion that this assay may detect a broader repertoire of antigen-specific T cells than measurement of IFNγ (33). Representative flow cytometry plots from patient 3 are shown in Figs. 2A and 3A. The fluorescence from the negative control stimulation was used to set the negative gates. Figure 2B shows the longitudinal frequency of p53-reactive T cells before and after vaccination in the CD4⁺ and CD8⁺ compartments in all 11 patients. Only one postvaccine blood draw was available from patients 2, 5, and 10 due to early withdrawal. In two responders, the expansion of p53-reactive T cells was greatest after the first vaccination; however, in other responders, the peak responses occurred after two or three rounds of p53MVA therapy.

The frequency of Tregs and MDSCs detected in the periphery did not differ significantly from healthy control samples, and did not decline significantly after one cycle of therapy when analyzed as a group (Fig. 4A). A decline in Tregs and MDSCs after therapy was detected in 7 of 11 and 6 of 11 patients, respectively (Supplementary Fig. S1 and S2), although in some cases, these changes were small and transient. The frequency of PD-1⁺ T cells at baseline was higher in patients compared with healthy controls, with this reaching statistical significance in the CD8⁺ compartment (Fig. 4B).

An objective measure of immunologic response was applied to the data set. “T-cell responders” were defined as patients showing an increase in p53-responsive T cells after therapy ≥ 0.5% of the CD4⁺ or CD8⁺ population. According to this criteria, 5 of 11 patients showed a CD4⁺ response and 6 of 11 showed a CD8⁺ T-cell response. p53-responders showed longer PFS than non-p53 responders (Figs. 2C and 3C), with this association reaching statistical significance. Of note, the one patient who was a CD8⁺ responder, but not a CD4⁺ responder had poor PFS. CD4⁺ peak frequency, CD4⁺ peak frequency above median (equivalent to a CD4⁺ responder in this patient population), also were associated with better PFS (see Supplementary Table S1). Maximum increase in p53-reactive CD4⁺ and CD8⁺ T cells, and peak p53-reactive CD8⁺ T cells also had a marginally significant, positive impact on PFS.

Patients with stage IV disease showed inferior PFS, and baseline MDSCs had a marginally negative impact on PFS (see Supplementary Table S1). Interestingly, stage IV patients had a lower expansion of p53-reactive CD4⁺ T cells (mean of 0.21 vs. 0.78, P < 0.05), and higher MDSC frequencies (mean of 0.72 vs. 0.28, P < 0.05) than stage III patients (see Supplementary Table S2 for correlations). Combined with small sample size, this correlational structure limits the interpretation of multivariate analysis. As a result, while adjusting for age, stage, and number of prior lines of therapy (0 vs. 1 or 2) we did not see any of the variables (including adjusted variables) achieve statistical significance (see Supplementary Table S1). Consequently, the forward stepwise regression final model included only CD4 response. CD8 response was less predictive and we cannot conclude that it is an important prognostic feature independent from CD4 response. There was no significant difference in baseline, peak, and lowest frequency of Tregs and MDSCs between immunologic responders and nonresponders (Supplementary Fig. S1B and S2B). The difference in p53 vaccine response or PFS between patients that did or did not show a fall in Tregs or MDSC was not statistically significant (Supplementary Fig. S1C and S2C).

In this report, we describe the phase I clinical trial of a p53MVA vaccine in combination with gemcitabine chemotherapy in platinum-resistant ovarian cancer. We observed expected toxicities related to gemcitabine in the absence of steroid premedication. p53MVA toxicity appeared limited to injection site reactions. Clinical efficacy was not a primary endpoint for this study but we observed durable disease control in 4 of 11 patients. One partial response yields a response rate of 9%, which is inferior to published reports of single-agent gemcitabine. However, 4 of 11 patients are alive at the time of this submission, two showing overall survival exceeding two years. The effect of p53MVA and gemcitabine on survival is difficult to assess, however, as many patients received additional chemotherapy agents after leaving the study and protocol-defined follow-up was limited to 12 months. Therefore, PFS was taken as the primary indicator of clinical activity. The median PFS from all 11 patients was 3 months (range 0.95–9.2 months).

An expansion of p53-reactive CD4⁺ and CD8⁺ T cells after vaccination was seen in 5 of 11 patients, approximately half the participants. Patients were defined as “immunologic responders” if they showed a rise in p53-reactive T cells above baseline and reached a minimum of 0.5% of the positive CD4⁺ or CD8⁺ T-cell gate (as defined by cytometry). Patients not reaching this level were defined as “immunologic nonresponders.” Some responses were low and in 3 of 11 patients, only one posttherapy blood draw was available. However, despite these limitations, a statistically significant relationship between immunologic and clinical response was apparent. The continuous measures of peak values and change, as well as dichotomizing patients based on the peak CD4⁺ percentage supported this conclusion. Patients with an anti-p53 T-cell response, that is, those defined as “immunologic responders” showed significantly longer PFS than “immunologic nonresponders.” This is similar to other immunotherapy clinical trials demonstrating improved clinical outcome associated with immunologic response, in a range of tumor types (36–39).

One of the primary aims of combining the vaccine with gemcitabine chemotherapy was to reduce the immunosuppression from Tregs and MDSCs and enhance the immunologic and clinical response. Surprisingly, both the peripheral Treg and MDSC frequencies detected were in the same range as healthy donor samples (Fig. 4A). Peripheral blood measurements may not reflect the frequency of Tregs and MDSC within the tumor microenvironment. Studies in tumor-bearing mice report variable frequencies of spleen, lung, tumor, lymph node, and peripheral blood MDSCs. Furthermore, there is no consensus on which compartment depletion of MDSCs is most important for enhancing antitumor immune responses (40).

During the treatment phase, 7 of 11 and 6 of 11 of our patients showed a decline in Tregs and MDSCs, respectively (Supplementary Figs. S1A and S2A). There was no statistically significant effect of baseline, lowest, or peak Tregs or MDSCs between p53 “responders” and “nonresponders” (Supplementary Fig. S1B and S2B). The anti-p53 vaccine response did not show a significant relationship with decline in Tregs or MDSCs (Supplementary Figs. S1C and S2C). Patients showing a decline in Tregs had a higher mean PFS than patients with no detectable Treg decline, but this did not reach statistical significance (Supplementary Fig. S1C). Factors such as disease stage, chemotherapy dose, administration schedule, time and location of blood sampling may contribute to variable MDSC levels reported by independent studies (40). A study in pancreatic cancer patients showed that blood MDSCs were highly variable pre- and post-gemcitabine treatment. However, when patients with higher baseline MDSCs showed a fall in MDSC, this was associated with greater immunologic response (41). In our study, individual patient's MDSC decline was not associated with improved response; however, all our patients had baseline MDSCs within the range for healthy donors. Perhaps, below a certain level, MDSC decline has minimal impact on antitumor immunity. However, our study did show that baseline MDSCs were marginally associated with worse outcome, were more prevalent in stage 4 patients, and were marginally associated with CD4 and CD8 changes after vaccination.

Murine studies suggest that MDSC frequencies vary depending on the interval between chemotherapy administration and blood sampling. MDSC levels may initially increase after chemotherapy, with a fall in MDSC not detectable until several weeks later (42). Other studies show an initial fall after chemotherapy, followed by a rebound of MDSCs in the spleen and tumor (43). A study of pancreatic cancer patients showed that after a resting period, peripheral MDSCs, Tregs, and plasma TGFβ1 reverted to levels before gemcitabine (10). It is possible that Treg and MDSC frequencies in our patients were dynamic during treatment, and our blood drawing schedule was not optimal to detect gemcitabine-induced declines. In addition, we did not measure factors such as arginase-1 or TGFβ1 in the plasma, which could have improved the detection of immunomodulation in our responders.

It is unclear why approximately half the patients responded immunologically to p53 vaccination and half did not. The p53MVA/gemcitabine combination was not well tolerated in some patients, resulting in early withdrawal of 3 patients. This could be due to the fact that steroids were not permitted on this study. Dexamethasone is commonly used as premedication prior to gemcitabine at baseline, or at the onset of mild allergic reactions to chemotherapy. The fact that patients on this trial did not receive steroid premedication could account for the higher than expected rate of allergic reactions to gemcitabine that caused early withdrawal. If these patients had been able to complete combination treatment, this may have resulted in better immunologic and clinical responses. However, 4 of the patients that did complete combination therapy did not respond clinically, with progressive disease detected on the first posttreatment scan.

None of the patients on this study had received more than two lines of platinum-based chemotherapy for recurrent disease. Considering that multiple lines of chemotherapy are common in this patient population, our study participants would not be considered heavily pretreated. However, even though the inclusion criteria allowed a platinum-free interval of ≤ 12 months, the majority of patients (9/11) had platinum-resistant disease. Of the two patients with borderline platinum sensitivity, one patient had low-grade serous carcinoma who had a rising CA 125 on carboplatin and taxol, but did not meet the GOG criteria for biochemical progression. This patient was labeled as borderline platinum-sensitive, as radiographic progression was documented at 11 months after completing platinum therapy. This likely reflected the natural course of disease rather than borderline sensitivity to platinum agents.

Immune-based therapies that have induced durable responses in melanoma and lung cancer patients have shown limited success in ovarian cancer patients, probably because of the ability of ovarian malignancies to block the development of antitumor immune responses (44). Analysis of ten clinical trials with 2,285 ovarian patients showed disappointing responses to various immunotherapy agents. Meta-analysis of six studies showed that there was no significant difference in overall survival and recurrence-free survival between patients treated with immunotherapy compared with controls (45). To our knowledge, there is only one other published report of p53 immunotherapy combined with gemcitabine in platinum-resistant ovarian cancer patients. In a three arm trial, Dijkgraaf and colleagues treated 6 patients with a combination of p53 peptides, gemcitabine, and IFNα. This combination achieved disease control in 3 of 6 of these patients (two partial responses and one stable disease) after therapy (18).

Expansion of p53-reactive T cells after p53MVA/gemcitabine therapy was associated with a small but statistically significant extension of PFS. This is encouraging, especially in a patient population with such poor prognosis. If this p53 response could be induced in all vaccinated patients, and the magnitude and duration of the response expanded further, greater clinical benefit may be achievable. This might be possible if the vaccine was coupled to an immunomodulatory agent with greater stimulatory activity than gemcitabine. Strategies which have shown potential in ovarian cancer, and hence could be combined with the p53MVA vaccine, include TGFβ and IDO blockade. Anti-TGFβ has shown potential in ovarian murine models (46). Elevated IDO expression is associated with poor prognosis in ovarian cancer patients (47), making the IDO inhibitor, epacadostat, another candidate. The mesothelin cancer vaccine in combination with epacadostat is being evaluated in platinum-resistant ovarian cancer (48). Another potential agent is Ontak, a Treg depletion agent, is currently being investigated with a DC vaccine in ovarian cancer patients (48).

Other immunomodulatory agents that could be combined with the p53MVA vaccine include antibodies that block PD-1/PD-L1 and CTLA-4. Hodi and colleagues reported that anti-CTLA-4 combined with a tumor cell vaccine stimulated a four-year remission in an ovarian cancer patient (49). These agents are being intensely evaluated in numerous tumor types. However, ovarian cancer response rates to single-agent checkpoint inhibition have been low. Combinations of these agents may be more effective (48). Our immunophenotyping data revealed that in contrast to Treg and MDSC levels, the frequency of PD-1⁺CD8⁺ T cells were higher in our trial participants compared with healthy controls (Fig. 4B). This is in agreement with our immunophenotyping data from patients with gastrointestinal malignancies (31) and raises the possibility of combining PD-1/PD-L1 blockade with the p53MVA vaccine in this patient population. We recently reported the outcome of a triple-negative breast cancer patient treated with p53MVA and the anti-PD-1 antibody, pembrolizumab. The patient experienced a complete remission, accompanied by a p53-specific T-cell response (50). Therefore, we are evaluating the potential of this combination of agents in patients with platinum-resistant ovarian cancer.

No potential conflicts of interest were disclosed.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conception and design: N.R. Hardwick, P. Frankel, R. Morgan, J.D.I. Ellenhorn, D.J. Diamond, M. Cristea

Development of methodology: N.R. Hardwick, L. Leong, R. Morgan, D.J. Diamond, M. Cristea

Acquisition of data (acquired and managed patients, provided facilities, etc.): N.R. Hardwick, W. Tsai, F. Kos, L. Leong, R. Tinsley, M. Eng, S. Wilczynski, M. Cristea

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N.R. Hardwick, P. Frankel, C. Ruel, F. Kos, R. Morgan, V. Chung, R. Tinsley, M. Cristea

Writing, review, and/or revision of the manuscript: N.R. Hardwick, P. Frankel, L. Leong, R. Morgan, R. Tinsley, M. Eng, D.J. Diamond, M. Cristea

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Tsai, T. Kaltcheva, R. Morgan, R. Tinsley, M. Eng, J.D.I. Ellenhorn, M. Cristea

Study supervision: D.J. Diamond, M. Cristea

Other (clinical research nurse coordinator-supervised pt care/protocol compliance per protocol): J. Kilpatrick

This work was supported by the City of Hope Chairs Discretionary Fund and utilized City of Hope Cancer Center funded Core Facilities (award number P30CA033572). The authors thank Dr. Bernard Moss (NIAID) for providing MVA 1974/NIH clone 1. They also thank the following City of Hope staff and departments: the Investigational Drug Service, the Bio-Specimen coordinators, the Center for Biomedicine and Genetics, and the Office of IND Development and Regulatory Affairs. Research reported in this publication included work performed in the Analytical Cytometry, Biostatistics, and Pathology Core facilities.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.