Patients enrolled in this trial were self-referred to either the Shukokia Clinic or the Tokyo Clinic and Research Institute. All patients had recurrent or stage IV malignancies having failed prior standard surgical and/or adjuvant therapy. Additional enrollment criteria included the presence of tumors that measured 3 cm or less and a minimum lapse of three months from prior therapy to protocol enrollment. Patients gave written consent to the procedures and protocols after detailed explanation and discussion. The consent forms and procedures were approved by the Institutional Review Boards of the respective institutions.

Figure 1 illustrates an overview of the treatment protocol. Prior to treatment, the extent of disease and the location of metastases were established by PET-CT (Positron Emission Tomography—Computed Tomography). The patients underwent leukapheresis to obtain monocytes for differentiation into iDC and a monocyte depleted T-cell enriched population for preparation of activated T-cells (AT). After preparation of these reagents, a portion of the iDC was combined with lymphocyte conditioned media, a multi-cytokine based adjuvant (LCM), and KLH. This cell mixture was equally divided based on the number of sites to be injected, the volume of each aliquot was adjusted to ∼2 mL with PBS, and the individual lesions were injected under CT guidance. AT were infused the following day. Approximately 3 days later, the injected tumor sites received Intensity-Modulated Radiotherapy (IMRT) in divided doses. Two to three days following the last dose of radiation, the tumor sites were again injected with iDC suspended in LCM (without KLH) and AT infused the next day. Blood samples were obtained prior to protocol initiation and periodically thereafter to monitor serum levels of tumor markers and the development of anti-KLH antibodies. The PET-CT exams were repeated 6 weeks after the last treatment and periodically thereafter. The treatment cycle was repeated for several patients who developed lesions at new sites.

Leukaphereses were performed on a COBE Spectra blood separator (Gambro KK, Tokyo, Japan) using the program for collection of the mononuclear cell population (MNC) (version 7.1). MNC were further purified by density gradient centrifugation, the cells washed, and a portion used to isolate monocytes for differentiation into iDC and to prepare AT. Remaining MNC were cryopreserved in AIMV media (Invitrogen Gibco, Tokyo, Japan) containing 10% DMSO and stored in vapor phase of liquid nitrogen. To prepare iDC and AT from cryopreserved MNC, the MNC were thawed in a 37 °C water bath and washed twice in AIMV medium. iDC and AT were prepared as described below.

iDC were differentiated from monocytes by the loosely adherent method as previously described [40]. In brief, PBMC (∼6 × 10⁸) were suspended in 20 mL AIMV media and distributed to four T75 cm² polystyrene flasks containing 10 mL of AIMV media. After two hours at 37 °C, non-adherent cells were removed, transferred to conical tubes, and reserved for AT preparation (see below). Fifteen milliliters of DC growth medium consisting of AIMV medium supplemented with 800 IU/mL GM-CSF and 500 U/mL IL-4 (CellGenix, Germany) was added to each flask containing adherent cells. Flasks were incubated at 37 °C with 5% CO₂ and supplemented with an equal volume of growth media on day 3. The iDCs were harvested on day 7 and either prepared for injection or cryopreserved in AIMV medium containing 20% autologous serum and 10% DMSO.

Approximately 6–9 × 10⁸ non-adherent cells obtained from the monocyte isolation described above were suspended in 20 mL of AIMV medium containing 10% autologous serum and supplemented with 1000 IU/mL IL-2 (Proleukin, Novartis, Emeryville, CA) and distributed into 4 flasks pre-treated with 10 mL of PBS containing 5 mcg/mL anti-CD3 (Orthoclone OKT3 injection, Janssen Pharmaceutical KK, Japan). Flasks were incubated for 7 days at 37 °C with 5% CO₂ and supplemented with an equal volume of media at day 3 or 4. Three hours prior to harvesting, 1 mcg/mL ionomycin (Sigma, St. Louis, MO) was added to the medium. The cells were harvested, washed three times, and portions prepared either for infusion or cryopreservation as described above.

Preparation of products of activated lymphocytes that were used as an adjuvant in these protocols and the cytokine/chemokine content of the adjuvant are detailed elsewhere [41]. In brief, lymphocytes were suspended in 50 mL XVIVO 10 medium (Cambrex, Walkersville, MD) containing human T-expander CD3/CD28 Dynabeads (Invitrogen Dynal AS, Oslo, Norway) at 1 cell to 1 bead ratio. The combination was incubated at 37 °C in 5% CO₂ for 2 days. Cytokine-rich supernatants were harvested by centrifugation at 300 × g for 7 min and stored at 4 °C for later use. Sterility and endotoxin testing were carried out as described below.

Seven days prior to cell harvest, presence of microbial contaminants was tested by incubation of cultured cells on agar at 37 °C with subsequent inspection for bacterial growth. Endotoxin levels (<0.5 EU/mL) were determined using a commercially available chromogenic endotoxin assay kit according to the manufacturer's instruction (Toxicolor system LS-50M, Seikagaku Corp., Tokyo, Japan). Only those cultures with a negative test result were administered.

A standard flow cytometry labeling protocol was used to determine cell surface marker expression on iDCs using fluorochrome-conjugated monoclonal antibodies to CD11c, CD14, CD40, CD80, CD83, CD86 and HLA-DR (BD Pharmingen, Japan). AT were evaluated for expression of CD3, CD4, CD8, CD11c, CD14, CD19, CD25, CD45, CD56, CD154, and HLA-DR following culture. Minimally 5,000 events were acquired on a BD FACSCalibur (BD Biosciences, Japan) and data were analyzed using Cell Quest analysis software. The phenotype of the injected iDC and infused AT is summarized in Table 1.

iDC and AT were thawed in a 37 °C water bath approximately 1 hour prior to planned injection. One milliliter of AIMV media was added to each thawed vial, suspensions were incubated for 2 min at 22 ± 3 °C (room temperature, RT) and cells were transferred into 50 mL of media and centrifuged at 300 × g for 7 min to remove DMSO. Cells were resuspended in fresh media, counted, and sterility and endotoxin testing samples removed. Remaining iDC were suspended in PBS containing 10% LCM. Based on the number of sites to be injected, the iDC were distributed into two or more microtubes and placed on ice for transport to the clinic where they were injection into the patient's metastatic lesions. AT were suspended in 100 mL of normal saline and infused IV over a period of 30–40 min.

Intensity Modulated Radiotherapy was delivered to each site injected with iDC. The appropriate prescribed total dose was determined to be such that the biologically effective dose was 72 Gy according to the linear quadratic model. The fraction size was optimized such that the estimated radiation induced late toxicity of surrounding normal tissue would be equal to or less than grade 2 based on the NCI-CTC version 2.0 scaling system.

Patients were monitored for adverse reactions to therapy. Indices checked include vital signs (temperature, blood pressure, pulse, respiration), blood chemistry and hematology, diaphoresis, arthralgia, and pain and/or swelling at the injection site. Other signs and symptoms that might be associated with radiation or the injection of cells by insertion of a needle through various organs in order to reach metastatic lesions that are present in body cavities (i.e., peri-aortic lymph nodes) were monitored.

PET-CT imaging was used to assess response to treatment following RECIST criteria. The first exam was conducted ∼6 weeks after the end of the last treatment cycle with periodic follow-up CT or PET-CT exams. Complete response (CR) was defined as the resolution of the treated site and no new lesions at distant sites at follow-up with radiographic exams. Patients who had an initial complete response at the treated sites at the 6-week CT exam but developed radiographic evidence of disease on subsequent evaluation were designated as having recurrent disease (RD) even though they experienced a disease-free interval. Finally, progressive disease (PD) was defined as no appreciable diminution in the size of the treated lesion and/or the appearance of new lesions at first follow-up PET-CT exam.

Serum antibodies specific for KLH and mesothelin were detected by sandwich ELISA. Test wells were coated with 1.0 mcg/mL KLH (Calbiochem, San Diego, CA) in 1 × PBS or 20 mcg/mL mesothelin lysate (Novus Biologicals) in 0.1 M carbonate buffer. For KLH ELISA, control wells were treated with 1 × PBS only. Plates were incubated overnight at 4 °C, washed with PBST (PBS + 0.05% Tween 20), and blocked with 200 μL of 5% non-fat dried milk/PBST for 2 h at RT. After additional washing, 100 μL of serum diluted in blocking buffer was added in triplicate, plates were incubated at RT for 1 h, washed, and incubated for another hour with 100 μL of peroxidase-conjugated goat anti-human IgG (KPL, Gaithersburg, MD) diluted to 1:30,000 for KLH or 1:1,000 for mesothelin. After washing, assays were developed with 100 μL/well of SureBlue TMB Peroxidase Substrate (KPL) for 30 min at RT and terminated by addition of 1 N HCl. Optical density was measured at 450 nm using a VersaMax ELISA reader (Molecular Devices, Sunnyvale, CA). OD_450nm of specific anti-KLH antibodies was determined by subtracting the mean OD for wells coated with PBS alone (background) from mean OD obtained in wells coated with KLH. For anti-mesothelin ELISA, data was reported as percent increase in OD_450nm units by comparing the reactions of sera collected before and after treatment.

PBMC collected from a breast cancer patient at enrollment and during treatment were stimulated with 10 mcg/mL freeze-thaw cell lysates of the breast cancer cell line, MCF7. Cells were cultured at 10⁶ cells/mL in 96-well trays for 48 h. Cell culture supernatants were harvested and analyzed for cytokine content by Luminex 100 multiple cytokine detection assay following the manufacturer's instruction (Luminex 100, Austin, TX).

Patient demographics, diagnosis, and an overview of past treatment information are shown in Table 2. The distribution of cancers treated on this protocol reflects the incidence of the different cancers in Japan. In addition, the distribution of patient age is typical for patients with the different cancers who had received and failed prior therapy. The majority of patients were treated with surgery at some time during the course of their disease and all patients received adjuvant therapy consisting of either radiation or chemotherapy, or both.

A summary of the extent of patients' diseases, number of metastatic lesions observed radiographically, doses of iDC and AT, IMRT dosages and periodicity of delivery, treatment-related toxicity and outcome is provided in Table 3. There is considerable variation amongst the cohort with respect to the scope of existing disease at the time of enrollment, as evidenced by the number of sites identified by PET-CT. The listed sites of recurrence represent the reappearance of cancer at or near the location of primary disease as well as multiple metastases at various anatomic locations. For each patient, the total number of iDC injected was dependent on the number recovered from the cultures. The cells were equally divided amongst their identified sites of metastasis and local recurrence. The number of AT infused varied according to the number of cells recovered from the cultures. Each of the metastatic sites was treated with fractionated doses of IMRT followed by re-injection of iDC at these sites and infusion of AT. Importantly, there were no complications related to the intratumoral injection of iDC and no toxicity related to intravenous infusion of AT. Radiation associated toxicities were observed in seven patients and do not appear to be related to tumor type, location of tumor, radiation dose, or number of fractions delivered.

Therapeutic response was evaluated by PET-CT or CT at 6 weeks after the last treatment and periodically thereafter. The patients on this study are listed in Table 3 ordered by days of disease-free survival. At the 6-week evaluation, five of 26 patients had progressive disease while the remaining patients exhibited complete response at the treatment site. Of these 21 patients, eight were found on subsequent follow up to have recurrent disease at sites distant from that of the original treatment. The remaining thirteen patients had complete responses over the duration of their evaluation period. The estimated restricted mean disease-free time in the 21 patients who had initial response is 345 days and the estimated median is 377 days.

Examples of radiographic evidence of response to treatment are illustrated in Figure 2. Panel A shows the pre- and post-treatment CT of a breast cancer patient with metastatic disease confined to the supraclavicular region, probably lymph nodes. Another breast cancer patient had a complete response when treated for metastatic breast cancer involving the mediastinum and the chest wall (Figure 2B). The radiographic images of a patient with stomach cancer (Figure 2C) illustrate multiple metastatic lesions in the supraclavicular region, mediastinum, and periaortic lymph nodes at enrollment that completely responded to treatment. Figure 2D illustrates resolution of metastatic tumors at sites distant from the areas receiving iDC injection and IMRT in a patient with cervical cancer.

In addition to radiographic studies, serum tumor markers were used to monitor treatment response. Representative results are shown in Figure 3. NCC-ST-439 levels declined after treatment in serum samples obtained from a patient with metastatic breast cancer (Figure 3A) while serum CEA levels fell to normal after treatment and remained at that level in the follow-up period for a patient with metastatic gastric carcinoma (Figure 3B).

The results of tumor marker studies in patients where markers of disease are generally accepted to be informative are summarized in Table 4. The values shown in this table were obtained in serum samples drawn at initial evaluation (Pre), at the first or second evaluation after completion of iDC injections and AT infusions (Post-1), and at the latest post-treatment evaluation (Post-2). Serum tumor marker levels decreased in post-treatment samples of all patients tested, returning to normal in seven of the 11 patients indicating that tumor load was significantly decreased with the treatment process.

KLH was co-administered with the initial iDC injections as a marker to investigate the ability of iDC to acquire antigen from their environment and generate an immune response. Twenty-three of the 26 patients were immunized with 1mg of KLH combined with the first iDC and LCM preparation that was subsequently apportioned according to the number of sites to be injected. Serum or plasma samples obtained prior to treatment and at periodic intervals thereafter were tested for antibodies to KLH. Anti-KLH antibodies were detected in 16 of the 23 vaccinated patients (data not shown). The kinetics of antibody response in four representative patients is illustrated in Figure 4.

Interestingly, the frequency of KLH immunity was higher in the complete response group (81.82%) than in the combined recurrent and progressive disease groups (58.33%). To determine if KLH responses were correlated with response to therapy, Kaplan-Meier analysis of disease-free survival of the two groups were compared (Figure 5). The data suggest that the patients who responded to KLH immunization had a better overall survival. However, the difference was not statistically significant (p = 0.31) when analyzed using the exact log-rank test from StatXact.

We have begun to investigate immune responses to antigens that may be overexpressed by tumors of different histological origin. Specifically, we questioned whether treatment induced a humoral response to mesothelin, a protein that has been shown to be overexpressed in pancreatic, gastric, ovarian, and lung carcinoma. Our preliminary results are presented in Figure 6 where post-treatment sera from three lung cancer patients showed an increase in anti-mesothelin antibody titer above the titer detected in sera obtained before therapy. Sera from two patients with ovarian and pancreatic cancer showed no changes.

Using an alternate approach, we exposed PBMC from a patient with breast cancer to lysates from the breast cancer cell line, MCF-7, and measured cytokine production as a criteria of antigen recognition and response. As shown in Table 5, there was a significant increase in the amount of IL-10, IL-1β, IL-6 and TNFα produced by cells obtained 25 days post-treatment compared to cells procured at enrollment and after therapy. Interestingly, levels of IL-2, IFNγ, and IL-17 did not increase upon exposure to lysates. We speculate but have no proof that the response observed is due to recognition of antigenic determinants shared by the MCF-7 cell line and the patient's tumor.