Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer

Between 2007 and 2012, 42 patients with unresectable or recurrent pancreatic cancer histologically confirmed as invasive ductal carcinoma by endoscopic ultrasound-guided fine-needle aspiration were treated at the Department of Digestive Surgery and Surgical Oncology (Department of Surgery II) of the Yamaguchi University Graduate School of Medicine. This therapy was not a clinical trial, but a medical treatment approved as advanced health care by the Japanese Ministry of Health, Labor and Welfare, and provided for all patients who could pay the cost for this therapy and who met the basic criteria as described below. We retrospectively summarized safety and efficacy of this therapy. The study protocol was also approved by the Institutional Review Board for Human Use at the Yamaguchi University School of Medicine. Written informed consent was obtained from all patients.

Eligibility criteria were as follows: age of ≥20 years; life expectancy ≥3 months; and adequate hepatic, renal, and bone marrow function (serum creatinine level, <2.0 mg/dl; bilirubin level, <3.0 g/dl; platelet count, ≥75,000/ml; total white blood cell count ≥3,000/ml and ≤15,000/ml). All patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–2 at the time of initial consultation.

Patients were treated with GEM (1000 mg/m²) for 3 weeks (on days 1, 8, and 15) followed by 1 week of rest, while MUC1-DCs suspended in 2 ml saline were injected intradermally in the inguinal region as maximum available cell products, and MUC1-CTLs suspended in 100 ml saline were given intravenously as maximum available cell products on day 18 every 4 weeks (Figure 1a). This AIT was repeated until progressive disease (PD) was recognized.

Adverse events were evaluated according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE)[28]. Computed tomography (CT) scan or magnetic resonance imaging (MRI) examination was made before the treatment. Tumors were staged with the UICC classification system.

CT or MRI was made after the first 3 transfers and was repeated every 4 to 6 weeks after the treatment. Patients were assigned a response category according to the Response Evaluation Criteria in Solid Tumors (RECIST) Committee[29].

Peripheral blood mononuclear cells (PBMCs) were harvested with the COBE Spectra Apheresis System (COBE BCT, Inc., Lakewood, CO, USA). PBMCs from 3000 ml of blood were enriched by density gradient centrifugation with Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). The PBMCs were incubated for 45 min in a 5% CO₂ atmosphere at 37°C in serum-free AIM-V medium (Gibco, Paisley, Scotland). Plastic-adherent cells were used for the generation of DCs, while non-adherent cells were used for the generation of CTLs.

MUC1-CTLs were induced as previously described[15]. Briefly, non-adherent cells were cultured in AIM-V with the MUC1-expressing human pancreatic cancer cell line YPK-1 (HLA-A2402) inactivated with 0.2 mg/ml mitomycin C (Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan). The effector-to-stimulator cell ratio was 1,000:1. On days 3, 5, and 7, recombinant human interleukin 2 (IL-2) (Shionogi Pharmaceutical Co., Tokyo, Japan) was added to the cultures at a final concentration of 10 units/ml (U/ml). The plates were incubated in a 5% CO₂ atmosphere at 37°C. On day 10, MUC1-CTLs were washed 3 times with saline, suspended in 100 ml saline and administered intravenously (Figure 1b).

Cytotoxicity assays of MUC1-CTLs induced from a healthy volunteer with the HLA-A 24/26 were performed as previously described (Additional file1: Figure S1a)[15]. Briefly, target cells (1 × 10⁶/ml) were labeled for 60 min at 37˚C with 100 μCi/ml radioactive sodium chromate (⁵¹Cr) (Amersham Japan, Tokyo, Japan). The cells were then washed 4 times in RPMI-1640 medium (Sigma-Aldrich). Labeled cells were resuspended in culture medium (1 × 10⁵/ml). Effector cells consisting of induced MUC1-CTLs were suspended at 0.5, 1.0 or 2.0 × 10⁶/ml. Effector cell suspension (0.1 ml) was added to a microplate (Falcon Plastics, Cockeysville, MD) with 0.1 ml target cells, to yield an effector to target cell ratio of 5:1, 10:1 or 20:1. All experiments were performed in triplicate. Plates were incubated for 4 h at 37˚C in a CO₂ incubator. The amount of ⁵¹Cr released into each well was determined with a γ counter (Auto Well Gamma System ARC-202, Aloka, Tokyo, Japan). The percentage of cytotoxicity was calculated as follows:

To measure the spontaneous ⁵¹Cr release of target cells in the absence of effector cells, target cells were mixed with 0.1 ml culture medium. To obtain maximal a ⁵¹Cr release, target cells were treated with 0.1 ml 0.1 N hydrochloric acid[15].

Antibody inhibition assay of cytotoxicity of induced MUC1-CTLs induced from a healthy volunteer with the HLA-A 24/26 was described previously (Additional file1: Figure S1b)[15]. Briefly, anti-CD3, -CD4, -CD8 and anti-class I mAbs (each diluted at 1:50) were used for blocking assays and were purchased from Dako Corp., Carpinteria, CA. The MUC1-expressing pancreatic cancer cell line YPK-1 was used as the target cell. Effector cells consisting of induced MUC1-CTLs were incubated with mAb at the indicated concentrations for 45 min at 37˚C, washed 3 times in RPMI-1640 medium and suspended at 2 × 10⁶/ml. Target cells were labeled with ⁵¹Cr as described above, washed 4 times and resuspended in culture medium (1 × 10⁵/ml). Effector cell suspension (0.1 ml) was added with 0.1 ml target cells to yield a 20:1 effector to target cell ratio for cytotoxicity assays as described above. For anti-MUC1 mAb blocking, target cells were preincubated for 1 h at 37˚C with anti-MUC1 mAb MY.1E12 (diluted at 1:200), kindly provided by Dr Tatsuo Irimura, Department of Cancer Biology and Molecular Immunology, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan. Cytotoxicity assays were performed as described above[15].

Adherent cells were cultured in AIM-V medium containing 800 U/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (Osteogenetics GmbH, Wurzburg, Germany) and 500 U/ml IL-4 (Osteogenetics GmbH). On days 3 and 6, GM-CSF and IL-4 were added to the cultures at a final concentration of 400 U/ml and 250 U/ml respectively. On day 6, immature DCs (imDCs) were cultured in AIM-V medium containing 1000 U/ml tumor necrosis factor-α (TNF-α) (R&D Systems, Minneapolis, MN, USA). On day 10, floating and loosely adherent cells were collected as mature DCs (mDCs). The mDCs were washed once, suspended in AIM-V medium, and adjusted to a final cell density of 2 × 10⁶ cells/ml. Subsequently, 400 μl of the cell suspension was mixed with 10 μg of MUC1-mRNA and electroporated in a 4-mm cuvette by using a BTX 830 square-wave electroporator (Harvard Apparatus, Holliston, MA, USA). Electroporation settings were adjusted to a single pulse, 400 V, 500 μs. Subsequently, MUC1-DCs were washed 3 times with saline, suspended in 2 ml saline and injected intradermally in the inguinal region (Figure 1b).

EGFP mRNA electroporated DCs were checked for EGFP expression 18 h after transfection by an EPICS Flow Cytometer. Gating was performed on cells exhibiting a large forward scatter (FSC) and side scatter (SSC) profile in order to allow exclusion of contaminating autologous lymphocytes. Gated DCs were then evaluated for EGFP expression. Non-transfected DCs were used as a control.

Induced DC subsets were analyzed with mAbs against surface antigens. All mAbs were purchased from Coulter (Hialeah, FL, USA). FITC-conjugated anti-CD80 (B7-1), -CD83 (HB-15), -CD14 (B1), -HLA-ABC and –HLA-DR (I2) were used. PE-conjugated anti-CD86 (B7-2) and -CD40 were also used according to the manufacturer's instructions. Samples were analyzed with an EPICS Flow Cytometer (Coulter Electronics, Inc., Hialeah, FL, USA) at a fluorescence excitation wavelength of 488 nm at 200–500 mW. For each sample, 5,000 DCs were analyzed.

PBMCs (obtained before the treatment and one month after 3 transfers) were enriched by density gradient centrifugation with Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). Cells were aliquoted for MDSC and Treg analysis. Cells were incubated with energy-coupled dye-phycoerythrin-Texas Red (ECD)- conjugated anti-human CD4 (T4), FITC-conjugated anti-human CD25 (Beckman Coulter), VioBlue-conjugated anti-human CD11b (M1/70.15.11.5) (Miltenyi Biotech, Bergisch Gladbach, Germany), FITC-conjugated anti-human CD33 (HIM3-4) (eBioscience, San Diego, CA, USA) or the corresponding isotype control Abs (Beckman Coulter) for 30 min at 4°C. For Treg intra-nuclear Foxp3 analysis, after treatment with rat serum and permeabilization buffer for 15 min at 4°C, cells were incubated with rat PE-labeled human Foxp3 Ab (PCH101) (eBioscience, San Diego, CA, USA) or the appropriate isotype control (Beckman Coulter) for 30 min at 4°C, then washed, re-suspended in 1% paraformaldehyde (PFA) in Dulbecco’s phosphate-buffered saline (D-PBS) (Nissui pharmaceutical, Tokyo, Japan), and stored at 4°C in the dark until flow cytometric analysis. Two-color flow cytometry was performed with an EPICS flow cytometer (Coulter Electronics, Inc., Hialeah, FL). Treg analysis was performed using at least 30,000 cells that were gated in the region of the lymphocyte population, whereas for MDSC analysis, all cells including the region of mononuclear cells and the polymorphonuclear leukocyte population were analyzed. Lymphocytes were gated in FSC and SSC profiles. Tregs were identified as CD4 + CD25+ Foxp3+ and calculated as a percentage of CD4+ lymphocytes. MDSCs were identified as CD11b + CD33+[30] and calculated as a percentage of total PBMC.

Frozen PBMCs, obtained before the treatment, after first treatment and after third treatment, were thawed prior to use and rested overnight in 10 U/ml benzonase nuclease (Novagen) at 37°C 5% CO₂ in a humidified incubator, and then used in the next step. PBMCs (responder cells) were cultured with the MUC1-mRNA electroporated PBMCs (stimulator cells) at the responder cells to stimulator cells (R/S ratio) of 1:1, and then were measured for interferon-γ (IFN-γ) responses using ex vivo ELISPOT assay. Nitrocellulose bottomed 96-well Multiscreen plates (Millipore, UK Ltd) were coated with anti-human-IFN-γ mAb (Mabtech, UK) overnight at 4°C. PBMCs were plated in 100 μl final volume and plates were incubated for 18–20 h in a 5% CO₂ atmosphere at 37°C. Assays were performed in triplicate and the results were averaged. Plates were washed and developed. Number of spots on the plate was counted by Eliphoto Scan (Minerva Tech, Tokyo, Japan). MUC1 specific spots of IFN-γ were counted and calculated as described below.

A; PBMCs co-cultured with PBMCs electroporated with MUC1 mRNA

B; PBMCs co-cultured with PBMCs electroporated without mRNA

Indium oxine (¹¹¹In-Oxine) labeled DC study was performed in one patient with stable disease (SD). DCs were labeled according to the protocols supplied by the manufacturer (Nihon Medi-Physics, Hyogo, Japan). Mature-DCs were resuspended in platelet-poor autologous plasma (CFP1) and incubated for 15 min at room temperature with radioactive ¹¹¹In-Oxine (1 mCi) (Nyconmed Amersham Inc., Buckinghamshire, UK). After two washes to eliminate the unbound isotope, the cells were resuspended in a total volume of 1.5 ml of CFP1. Radiolabelling of the DCs and culture supernatant was evaluated with a gamma camera, after which DCs were intradermally inoculated 10 cm from inguinal lymph nodes. Scintigraphic images of the depot were acquired with a gamma camera 0, 2, 24, and 48 h after injection.

Patient characteristics and clinical outcomes are summarized in Table 1. Of 42 patients receiving AIT with MUC1-DCs and MUC1-CTLs plus GEM, 1 patient with recurrence had complete response (CR) (2.4%), 3 patients with stage III (n = 1) and stage IV (n = 2) had partial response (PR) (7.1%), 22 patients with stage III (n = 11), stage IV (n = 7) and recurrence (n = 4) had SD (52.4%), and 16 patients with stage III (n = 2), stage IV (n = 10) and recurrence (n = 4) had PD (38.1%). The disease control rate was 61.9%.Images from gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced (Gd-EOB-DTPA) MRI and CT scans of a patient with CR are shown in Figure 2. He had liver metastasis after curative surgery (Figure 2a and b). After 3 transfers, liver metastasis disappeared completely (Figure 2c and d). In contrast, the other 6 patients who had liver metastasis before this therapy had PD and the median survival time (MST) was 6.3 months (data not shown).

The 1-year survival rate was 51.1%, and the MST was 13.9 months in all patients (Figure 3a). Liver metastasis during therapy appeared in only 5 of 35 patients without liver metastasis before treatment. The 1-year survival rate and MST in 35 patients who received more than 1 × 10⁷ MUC1-DCs per injection were significantly better than for 7 patients who received less than 1 × 10⁷cells (60.3% vs. 0%, 16.1 months vs. 6.2 months, p = 0.0036) (Figure 3b). Administration times and total number of MUC1-CTLs and MUC1-DCs were 6.2 ± 0.8 times, 4.5 ± 0.7 × 10⁹ cells and 12.9 ± 1.8 × 10⁷ cells in the high dose group, and 3.1 ± 0.2 times, 1.5 ± 0.2 × 10⁹ cells and 1.9 ± 0.5 × 10⁷ cells in the low dose group. The 1-year survival rate and MST in 36 patients who received more than 3 × 10⁸ MUC1-CTLs per injection were significantly better than for 6 patients who received less than 3 × 10⁸ cells (56.6% vs. 16.7%, 15.1 months vs. 5.2 months, p = 0.0060) (Figure 3c). Administration times and total number of MUC1-CTLs and MUC1-DCs were 6.3 ± 0.8 times, 4.6 ± 0.7 × 10⁹ cells and 12.4 ± 1.8 × 10⁷ cells in the high dose group, and 2.3 ± 0.3 times, 0.5 ± 0.1 × 10⁹ cells and 2.4 ± 0.5 × 10⁷ cells in the low dose group. The 1-year survival rate and MST in 30 patients who received both more than 1 × 10⁷MUC1-DCs per injection and 3 × 10⁸ MUC1-CTLs per injection were significantly better than for the other 12 patients (66.7% vs. 10.4%, 16.5 months vs. 5.7 months, p = 0.0002) (Figure 3d). Administration times and total number of MUC1-CTLs and MUC1-DCs were 6.8 ± 0.9 times, 5.0 ± 0.8 × 10⁹ cells and 14.4 ± 2.0 × 10⁷ cells in the high dose group, and 2.9 ± 0.3 times, 1.3 ± 0.3 × 10⁹ cells and 2.5 ± 0.5 × 10⁷ cells in the low dose group. There was no significant bias of patient characteristics between the high dose group and low dose group (Table 2).

A comparative analysis revealed that there were no significant differences between the clinically good (CR/PR/SD) and poor responders (PD) for age, sex, disease stage and neutrophil/lymphocyte ratio, except for pretreatment liver metastasis (p = 0.0081) and the number of DCs (p = 0.0015). The number of CTLs per injection was trend to higher in the clinically good responders (p = 0.0537) (Table 3).

The major grade 3 and 4 adverse events are summarized in Table 4. The most common grade 3or 4 hematologic adverse event was neutrophils (31%). Grade 3 or 4 anorexia (14.3%) was the most nonhematologic adverse event. No AIT-related adverse events such as rash, fever, chill, and injection site reaction were observed. There was no clinical or radiological evidence of autoimmune reaction in any of the patients.

Induced MUC1-CTLs (HLA-A24/26) showed strong cytotoxicity against pancreatic cancer cell lines (YPK-1; HLA-A24, and YPK-3; HLA-A02) which expressed MUC1 antigen on the cell surface in an HLA- unrestricted manner. However, cytotoxicity against the esophageal cancer cell lines (YES-1 and -2), which did not express MUC1, was low (Additional file1: Figure S1a).

Anti-CD3 mAb or anti-CD8 mAb inhibited CTL cytotoxicity against YPK-1 cells. Anti-MUC1 mAb also inhibited cytotoxicity in these cells. Anti-class I mAb showed no inhibition of CTL cytotoxicity (E:T = 20:1; anti-CD3, 66.5%; -CD4, 26.9%; -CD8, 76.9%; anti-class I, 11.8% and anti-MUC1, 64.1%) (Additional file1: Figure S1b)[15]. These results showed that cytotoxicity of induced MUC1-CTLs was MUC1 specific and HLA-unrestricted.

To confirm the translation efficiency from introduced mRNA to protein, the mDCs were electroporated with EGFP mRNA. The expression coefficient of EGFP was more than 95% (Figure 4a). This result indicates that DCs can translate the introduced mRNAs to encoded protein.

A comparison of surface marker expression between imDCs and mDCs was shown (Figure 4c). Twenty nine patients were evaluable. The expression of each antigen in imDCs was found in 17.5 ± 2.9% (CD80), 67.1 ± 3.1% (CD86), 2.8 ± 0.7% (CD83), 92.6 ± 1.7% (CD40), 9.5 ± 2.3% (CD14), 98.6 ± 0.3% (HLA-ABC) and 92.8 ± 1.8% (HLA-DR). The expression of each antigen in mDCs was found in 92.1 ± 1.5% (CD80), 96.5 ± 0.7% (CD86), 75.6 ± 3.3% (CD83), 98.9 ± 0.5% (CD40), 4.6 ± 3.4% (CD14), 99.4 ± 0.2% (HLA-ABC) and 96.6 ± 1.1% (HLA-DR). The percentage of CD80+, CD83+, and CD86+ DCs was extremely increased in mDCs as compared with imDCs (P < 0.0001). A high expression level of CD40 was also observed in mDCs (P = 0.0004). HLA-class I and HLA-class II expression levels were identical between imDCs and mDCs.

We assessed negative immune factors focusing on CD11b + CD33+ cells (n = 14) and CD4+ CD25+ Foxp3+ cells (n = 18) in PBMCs before and one month after 3 transfer. Before treatment there was no difference in the percentage of CD11b + CD33+ cells between patients with CR, PR, and SD (15.1 ± 2.2, n = 11) and patients with PD (17.8 ± 3.9, n = 3). After treatment, the percentage of CD11b + CD33+ cells in patients with CR, PR and SD (10.2 ± 1.6) was significantly lower than patients with PD (21.9 ± 4.0) (p = 0.0430) (Figure 5b). Before treatment there was no difference in the percentage of CD4+ CD25+ Foxp3+ cells between patients with CR, PR and SD (0.68 ± 0.20, n = 14) and patients with PD (1.03 ± 0.62, n = 4). After treatment, the percentage of CD4+ CD25+ Foxp3+ cells in patients with CR, PR and SD (0.57 ± 0.18) was significantly lower than patients with PD (1.33 ± 0.41) (p = 0.0495) (Figure 5f).

IFN-γ ELISPOT assay was performed to evaluate the specific responses to MUC1 of immune effector cells from 6 patients. The number of MUC1 specific spots was 6.8 ± 2.3, 13.1 ± 5.8 and 23.2 ± 13.9, prior to treatment, after first treatment and after third treatment, respectively. More IFN-γ spots were observed in post-treatment compared with pretreatment PBMC samples. Although IFN-γ activity was increased in this assay, it was still unclear which cells had high activity such as CTL or NK cells (Figure 6).

Scintigraphic images obtained from one patient 2 h and 48 h after DC administration demonstrated that ¹¹¹In-oxine labeled DCs accumulated at the injection site and regional lymph nodes 2 h after injection. Forty-eight hours after injection, the accumulation extended into distant lymph nodes, but still remained in the injection site and regional lymph node (Figure 2e and f).