Phase I trial of multidrug resistance-associated protein 3-derived peptide in patients with hepatocellular carcinoma

Multidrug resistance-associated protein 3 (MRP3) is a carrier-type transport protein belonging to the ABC transporters. In this study, we investigated the safety and immunogenicity of a MRP3-derived peptide (MRP3 765 ) as a vaccine and characterized the MRP3-specific T cell responses induced. Twelve hepatocellular carcinoma (HCC) patients treated with hepatic arterial infusion chemotherapy (HAIC) were enrolled. The MRP3-derived peptide was emulsified in incomplete Freund’s adjuvant and administered via subcutaneous immunization three times weekly. No serious adverse drug reactions to the peptide vaccine were observed, and the vaccination was well tolerated. The vaccination induced MRP3-specific immunity in 72.7% of the patients. In a phenotypic analysis, the largest post-vaccinated increase in MRP3-specific T cells was due to an increase in cells with the effector memory phenotype. Among the 12 patients, one patient showed a partial response, nine showed a stable disease, and two showed a progressive disease. The median overall survival time was 14.0 months. In conclusion, the safety, effects of immune boosting, and possible prolongation of overall survival by the MRP3-derived peptide demonstrate the potential of the peptide to provide clinical benefit in HCC patients.


INTRODUCTION
Hepatocellular carcinoma (HCC), the sixth most frequent type of cancer worldwide, is an important public health concern because its incidence has continued to increase in Western and Asian countries [1; 2].Advanced HCC is treated with sorafenib as the standard treatment.This is the sole systemic agent available and can prolong survival for 2.3-2.8months [3; 4].Hepatic arterial infusion chemotherapy (HAIC) has been used as an alternative therapy to sorafenib in Japan and other Asian countries [5; 6].However, when no curative treatment is administered at the time of the diagnosis of advanced HCC, the median survival of patients is currently less than 1 year [7].Thus, there is a continuing need for novel treatment strategies to improve the outcomes of advanced HCC patients.Some recently developed immunotherapies have turned out to be promising strategies for several advanced cancers.As with other cancers, immunotherapy is expected to provide new treatments for HCC.
Multidrug resistance-associated protein 3 (MRP3) is a carrier-type transport protein belonging to the ABC transporters that transport substances against a concentration gradient in an ATP energy-dependent manner [8].It is expressed at high levels in the Mizukoshi  small and large intestines, pancreas, placenta, and adrenal cortex [9], and recent studies have reported that its expression is enhanced in various cancer cells [10; 11; 12].We previously demonstrated that the MRP3 expression level in HCC tissue was significantly higher than in non-cancerous tissue and that MRP3-specific cytotoxic T cells (CTLs), which showed cytotoxicity against HCC cells overexpressing MRP3, could be induced regardless of liver function, blood α-fetoprotein (AFP) level, and the stage of HCC [13].These results suggest that MRP3 may be useful as a target antigen in HCC immunotherapy.
Clinical trials using MRP3-derived peptides have been performed in cancer patients, and the results have shown that the peptide vaccine is well tolerated and effective for patients with glioblastoma and prostate cancer [14; 15].However, the safety and efficacy of MRP3-derived peptide vaccines in HCC patients remain unclear.
The results of recent studies have also suggested that combination therapies of a peptide vaccine and conventional chemotherapy can enhance the anti-tumor effects of peptides [16; 17].In this study, the safety and immunological effects of a MRP3-derived peptide used as a potential vaccine in HCC patients treated with HAIC were investigated.

Patients
Twelve HLA-A24-positive HCC patients were enrolled (Table 1).The inclusion criteria were a histological or radiological diagnosis of primary HCC according to the American Association for the Study of Liver Diseases (AASLD) guidelines for the management of HCC [18], a Karnofsky score of ≥70%, ≥20 years of age, informed consent, and the following normal baseline hematological parameters: hemoglobin ≥8.5 g/dL, white blood cell count ≥2,000/µL, platelet count ≥50,000/µL, creatinine disorders, including splenectomy and radiation to the spleen; corticosteroid or anti-histamine therapy; currently lactating or pregnant; history of organ transplantation; or difficulty in follow up.

Treatment protocol
The present study was designed as a phase I trial.Primary endpoints were the evaluation of the safety and immunological effects of the MRP3 765 peptide vaccine.The secondary endpoint was the anti-tumor response.After starting treatment, HCC progression was evaluated with dynamic CT or MRI every 2-3 months.
After diagnosis, all patients were treated with HAIC using pegylated (PEG) interferon (IFN)-α-2b/5-fluorouracil (FU) + cisplatin at Kanazawa University Hospital between April 2011 and December 2012.The patients received a continuous hepatic arterial infusion of FU (5-FU, Kyowa Hakko, Tokyo, Japan) at a dose of 300 mg/m 2 /day for 5 days in the first and second weeks (for 120 h) using an infusion pump (Baxter Infusor SV1, Tokyo, Japan), as described previously [19].The maximum amount of FU infused over 5 days was 2,500 mg.PEG IFN α-2b (Schering-Plough, Osaka, Japan) at a dose of 1.0 µg/kg was administered s.c. on days 1, 8, 15, and 22.A dose of 20 mg/m 2 Cisplatin (Nippon Kayaku, Tokyo, Japan) was given by a hepatic arterial infusion over 1.  Adverse events/toxicities were categorized and graded using the Common Terminology Criteria of Adverse Events (CTCAE; ver.3.0).Criteria for discontinuation included unacceptable toxicity and disease progression, defined as progressive disease (PD) according to the RECIST criteria.
All patients provided written informed consent for their participation in the study in accordance with the Helsinki declaration.This study (trial registration: UMIN000005678) was approved by the regional ethics committee (Medical Ethics Committee, Kanazawa University, No. 1018).

Peptides and preparation of PBMCs
The HCC patients were immunized with the MRP3 765 peptide, which was synthesized Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.

IFN-γ ELISPOT assay
IFN-γ ELISPOT assays were performed, as reported previously [23].The number of spots in control wells was fewer than 10 in all ELISPOT assays.Responses to the MRP3-derived peptides were considered positive if more than the mean + 2 SD specific spots were detected in healthy normal donors and the number of spots detected in the presence of an antigen was at least two-fold that detected in its absence.

Flow cytometric analysis
Peptide MRP3 765 -specific tetramer was used for the detection of peptide Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.vaccine-induced CTLs.Peptide HIVenv 584 -specific tetramer was used as a negative control of tetramer assay.All tetramers were purchased from Medical Biological Laboratories Co., Ltd.(Nagoya, Japan).PBMCs were stained with anti-CD8-APC (Becton Dickinson, Tokyo, Japan), anti-CCR7-FITC (eBioscience, Tokyo, Japan), anti-CD45RA-PerCP-Cy5.5 antibodies (eBioscience, Tokyo, Japan), and with tetramer-PE for 30 min at room temperature.Cells were washed, fixed with 0.5% paraformaldehyde/PBS, and analyzed using a Becton Dickinson FACSAria II system.In the tetramer assays with the negative control tetramer, we did not observe more than 0.03% tetramer-positive cells in any assay.Based on the results of the negative control, responses to MRP 765 -specific tetramer were considered positive if more than 0.03% tetramer positive cells were detected.At least 1,000,000 PBMCs were acquired for each tetramer assay.For the detection of Tregs and MDSCs, the following anti-human monoclonal antibodies were used: anti-CD4 (Becton Dickinson), anti-CD14 (Becton Dickinson), anti-CD25 (Becton Dickinson), anti-CD127 (Becton Dickinson), and anti-HLA-DR (Becton Dickinson).

Statistical analysis
Data are expressed as means ± SD.Paired t-tests were used for statistical analyses of the frequency of MRP3 765 peptide-specific T cells, Tregs, and MDSCs before and after HAIC with the MRP3 765 peptide vaccine.We used the Kaplan-Meier method to estimate the distribution of progression-free survival (PFS) and overall survival (OS) rates.PFS was calculated from the first day of HAIC to the date of radiographic disease progression using the RECIST criteria.OS was calculated from the first day of HAIC to the date of death from any cause.A per-protocol statistical analysis was performed using the SPSS software (SPSS, Inc., Chicago, IL, USA).A p value < 0.05 was considered to indicate statistical significance.Mizukoshi

Patient profiles
Clinical profiles of the patients are shown in Table 1.All patients but one (patient C2) received 3-6 vaccinations and were evaluated with immunological analyses.The etiology of liver diseases consisted of HCV, HBV, and other for 7, 0, and 5 patients, respectively.Ten patients had liver cirrhosis, proven by liver biopsy.The TNM stage was classified according to the Liver Cancer Study Group of Japan (LCSGJ): 4, 3, and 5 patients had stages II, III, and IV, respectively.

Toxicity of the MRP3-derived peptide vaccine
Adverse events/toxicities were categorized and graded using the CTCAE.As shown in Table 2, three (25.0%)patients showed grade 1 fever.One (8.3%), three (25.0%),two (16.7%), and one (8.3%)patient developed dizziness, ascites, gastric ulcer, and arthralgia, respectively.Blood examinations revealed reductions in neutrophil or platelet counts in each patient.These adverse events were considered to be related to HAIC, as reported previously [5; 24].No severe adverse event related to the MRP3 765 peptide vaccine was observed.Mizukoshi

CTL response
CTL responses before and 2 weeks after final vaccination were evaluated using an IFN-γ ELISPOT assay.Comparisons of the frequency of each peptide-specific CTL before and 2 weeks after vaccination in all patients except one (patient C2) are shown in Figure 1A.The frequencies of MRP3 765 peptide (peptide 1)-specific CTLs before vaccination were 0-2 per 3×10 5 PBMCs.The frequencies of HIVenv 584 (peptide 2)-and CMVpp65 328 peptide (peptide 3)-specific CTLs before vaccination were 0-2.5 and 1-230 per 3×10 5 PBMCs, respectively.When T cell responses against a single peptide with >2.9 specific spots (i.e., > mean + 2 SDs of the frequency of MRP3 765 -specific T cells in healthy controls) and a two-fold increase were defined as significant, a significant increase in the MRP3 765 peptide was observed in 8 of 11 (72.7%)patients (patients A1, A3, B3, C1, C3, C4, C5, and C6).In

Detection of Tregs and MDSCs
In addition to the analysis of MRP3 765 peptide-specific CD8 + T cells, we examined the frequency of Tregs and MDSCs in peripheral blood to identify the effects of HAIC with the peptide vaccine on immune suppressor cells.The population of Tregs was detected as CD4 + CD25 + CD127 -/low cells, as reported previously (Fig. 2A) [26].The population of MDSCs was detected as CD14 + HLA-DR -/low cells, as reported previously (Fig. 2B) [26].
The frequencies of MRP3 765 peptide-specific CD8 + T cells (Fig. 2C), Tregs (Fig. 2D) and of MDSCs (Fig. 2E) in peripheral blood before and after HAIC with peptide vaccine are shown for each patient.

Kinetics of peptide-specific CTLs after vaccination
To evaluate the kinetics of MRP3 765 peptide-specific CTLs after vaccination, the Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.frequency of CTLs 24 weeks after vaccination was examined in six patients (A3, B3, C1, C4, C5, and C6) whose PBMCs were available for ELISPOT assays.The frequency of MRP3 765 peptide-specific CTLs, evaluated using an IFN-γ ELISPOT assay 24 weeks after vaccination was 0-3 cells per 3×10 5 PBMCs, and no patient maintained a level of >10 cells per 3×10 5 PBMCs (Fig. 3A).With the exception of three patients (patients A3, B3, and C6), the frequencies of CMV-and HIV-derived peptide-specific CTLs did not decrease during the study course,

Clinical outcomes
A summary of the immune and tumor responses in all patients after vaccination with the MRP3 765 peptide is shown in Table 3.According to the RECIST criteria, among the 12 patients, one patient showed a partial response (PR), nine showed a stable disease (SD), and two showed a progressive disease (PD).The overall survival time for each patient is presented in Table 3.At the time of writing, all patients have shown disease progression with death, and the median overall survival time was 14.0 months (95% CI = 9.6-18.5 months)(Fig.3B), which is longer than those in previous studies including patients treated with HAIC without peptide vaccination (median OS = 12.0-12.6months) [24; 27].
The representative clinical course of the patient with PR is shown in Figure 4.The patient with PR (Patient B3) had a large HCC in the right lobe of the liver and was vaccinated with the MRP3 765 peptide weekly, for a total of six times.After vaccination, elevated serum AFP and DCP values normalized within 100 days (Fig. 4A), and the size of the HCC (Fig. 4B), evaluated by CT, was getting smaller.During the vaccination, ALT was elevated to the abnormal limit, but it normalized after the final vaccination (Fig. 4A).

DISCUSSION
In previous studies, we performed a simultaneous and comparative analysis of immune responses to 27 different CTL epitopes derived from 14 TAAs using PBMCs from HCC patients to identify suitable TAA-derived epitopes for HCC immunotherapy [28].We found that MRP3-derived peptides were frequently recognized by T cells and were capable of generating peptide-specific CTLs in HCC patients, suggesting that these peptides were immunogenic.
In this phase I study, immunotherapy using the MRP3 765 peptide in a formulation with the Montanide ISA-51 adjuvant was shown to be safe and immunogenic.The maximum toxicity observed was grade 3, according to the common terminology criteria, and it was actually considered to be due to the HAIC.Adverse events observed frequently were fever, ascites, and gastric ulcer, and these are also known common adverse events of HAIC [5; 24].IFN-γ ELISPOT assays revealed that vaccination with the MRP3 765 peptide induced MRP3-specific immunity in 8 of 11 (72.7%)vaccinated patients.In previous studies of peptide vaccines for HCC, AFP, hTERT, and glypican-3 have been targeted as tumor-associated antigens for the treatment of advanced HCC [29; 30; 31; 32; 33].In these studies, peptide-specific T cells were reported to be induced in 0-80% of the Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.patients vaccinated.Thus, the induction rate of TAA-derived peptide-specific T cell responses in MRP3 765 peptide vaccination was similar to those of AFP-or glypican-3-derived peptides, suggesting that the MRP3 765 peptide was immunogenic.
In phenotypic analyses of CTLs induced by the MRP3 765 peptide vaccine, the post-vaccination increase in MRP3 765 peptide-specific CTLs was due to increases in cells with the CD45RA -/CCR7 -(effector memory) and CD45RA + /CCR7 -(effector) phenotypes.A similar phenotypic analysis was reported by Butterfield et al. [34], who identified AFP-specific CTLs induced by AFP-derived peptide-pulsed DCs that were both naïve and of the central memory phenotype and failed to complete differentiation into effector and effector memory T cells.Differences between the present study and the previous one involved the administration method of the peptide vaccine and the use of combined therapy.In the previous study, they used dendritic cells pulsed with multiple peptides and with no additional treatment.All the patients in this study simultaneously received HAIC with the peptide vaccination.Recent studies reported that anti-tumor chemotherapy produced its anti-tumor effects by not only direct cytotoxic effects against tumor cells but also by the elimination or inactivation of cells with suppressive effects on tumor immunity, such as Tregs and MDSCs [35; 36].In fact, the frequency of Tregs decreased after the treatment, which was consistent with the previously reported Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.responses of Tregs to chemotherapy against colon cancer using cyclophosphamide and against non-small cell lung cancer using paclitaxel [37; 38].This may indicate more favorable conditions for the induction of peptide-specific T cells with effector memory or effector phenotypes.
Although peptide-specific CTLs and the effector memory phenotype could be induced in this study, the anti-tumor effects for HCC were limited, which is consistent with previous studies of peptide vaccines for HCC [31; 33].One reason might be the slow speed and weakness of the anti-tumor effects induced by the peptide vaccine.In this study, the induction rate of MRP3 765 peptide-specific T cells and their frequencies during the first course of vaccination were low.Another reason may be the short life of MRP3 765 peptide-specific T cells.The frequency of MRP3 765 peptide-specific T cells decreased after 6 months in most patients, suggesting 3-6 injections of the MRP3 765 peptide was not sufficient to induce long-lived T cells.Furthermore, the advanced HCC stages of the patients or continued HAIC may affect the short life of MRP3 765 peptide-specific T cells.
The frequency of MDSCs increased significantly after the treatment in some of the patients.This phenomenon is considered to be unfavorable to anti-tumor immunity.In significant increase (more than 10%) of MDSCs (Table 3).In addition, all of the patients without a significant increase of MDSCs after vaccination showed PR or SD as a best study response.Although it is difficult to clearly state the relationship between the increase of MDSCs and the anti-tumor effect of vaccination because of small number of patients in this study, these results suggest that the small number of MDSCs in PBMCs might be favorable conditions for the induction of peptide-specific CTLs and contribute to the anti-tumor effect of peptide vaccination.
Regarding the effects of MRP3 765 peptide on the prognosis of HCC patients, obviously this is limited because our study was designed as a phase I study and did not involve a large population.However, the results of the present study suggest that the overall survival times of the patients treated with HAIC with the peptide vaccine was longer than those in previous studies including patients treated with HAIC with no peptide vaccination.To confirm these results, larger, later-stage clinical trials are necessary.

HCC.
In conclusion, the feasibility of a MRP3-derived peptide vaccine has been demonstrated based on its safety, its effect on immune boosting, and even the possible prolongation of overall survival time.The potential to eradicate advanced HCC using a combination therapy of a peptide vaccine and chemotherapy should be further evaluated in additional studies.showed that the frequencies of CD45RA -/CCR7 + (central memory), CD45RA -/CCR7 - (effector memory), and CD45RA + /CCR7 -(effector) T cells were 0.4%, 74.4%, and 24.8% of MRP3 765 CD8 + tetramer + T cells, respectively.In contrast, phenotypic analysis of total CD8 + T cells after vaccination showed that the frequencies of CD45RA -/CCR7 + Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.
et al.Phase I Trial of MRP3-Derived peptide for HCC.

Furthermore, we characterized
the phenotype of MRP3-specific T cells induced by the peptide and monitored the MRP3-specific immune response, the numbers of regulatory T Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC. cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and the anti-tumor effects on HCC after vaccination.Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.

≤1. 5
mg/dL, and Child-Pugh class A or B. Regarding tumor stage, the following patients were included: patients who had (1) severe vascular invasion (i.e., vascular invasion in the main trunk to the secondary branches of the portal vein or invasion in the right, middle, or left hepatic vein) and (2) multiple intrahepatic lesions (i.e., five or more nodules in the left and/or right lobes, as confirmed by radiology).Exclusion criteria included severe cardiac, renal, pulmonary, hematological, or other systemic diseases associated with a discontinuation risk.Such diseases included human immunodeficiency virus (HIV) infection; prior history of other malignancies; history of surgery, chemotherapy, or radiation therapy within 4 weeks of the trial; immunological Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.
5 h on Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.

days 1 and 8
prior to the administration of FU and after appropriate hydration and antiemetic medication.A treatment cycle comprised 4 weeks of drug administration, including the administration of IFN, and a subsequent 2-week rest period.Patients also received 0.03-3.0mg MRP3-derived peptide (MRP3 765 ) vaccine on days 1, 8, and 15 during HAIC.The peptide was administered emulsified with incomplete Freund's adjuvant (Montanide ISA-51 VG; SEPPIC, Paris, France) by subcutaneous immunization.Vaccination was also performed during the second course of HAIC.
et al.Phase I Trial of MRP3-Derived peptide for HCC.
et al.Phase I Trial of MRP3-Derived peptide for HCC.
contrast, no increase in the frequency of HIVenv 584 peptide-specific CTLs was observed in any patient.The frequency of CMVpp65 328 peptide-specific CTLs increased in 3 of 11 (27.2%)patients (patients A2, B3, and C6).Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.
765 peptide-specific CTLs before and after vaccination are shown in Figure 4C.The frequency of MRP3 765 peptide-specific CTLs increased after vaccination.However, the frequencies of HIVenv peptide-specific CTLs were unchanged during vaccination, and those of CMVpp65 peptide-specific CTLs fluctuated.Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.
fact, among the 8 patients who showed MRP3 765 peptide-specific CTLs in PBMCs after 1 or 2 course vaccination, 5 patients (patients B3, C1, C3, C4 and C5) did not show a Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.
Specifically, HAIC with more than 6 times injection of MRP3-derived peptide or the combination with the inhibitors of MDSCs such as low dose gemcitabine, cisplatin or 5-FU should be tried as a next clinical trial.Furthermore, previous studies have demonstrated that Sorafenib decreases the number of MDSCs [39; 40], and therefore the vaccination of MRP3-derived peptide with Sorafenib might be effective for patients with Mizukoshi et al.Phase I Trial of MRP3-Derived peptide for HCC.

Figure 2 .
Figure 2. Frequencies of Tregs and MDSCs in peripheral blood of HCC patients before

Figure 4 .
Figure 4. Representative clinical course of the patient with PR. (A) Time course of serum