This study was approved by the Dartmouth Committee for the Protection of Human Subjects and performed according to US FDA IND 9554. Approximately 4 weeks after resection of all detectable metastatic colorectal cancer, patients signed their informed consent. Eligibility criteria included Karnofsky performance status >60%, recall DTH to either Candida or tetanus and enough tumor cells to form the vaccine and do the immunoassays (>75 × 106 cells). Patients were excluded if they were receiving immunosuppressive drugs, had active autoimmune disease, HIV, hepatitis B or C. A leukopheresis was scheduled 1–2 weeks after the patient consented. A CT scan was performed just prior to pheresis to determine if residual measurable disease was present. Eligible patients were randomized in a blinded fashion into two groups: one had CD40L added to their DC culture, the other did not. The data was unblinded following completion of all immunoassays. Patients received their first DC vaccination 8 days after pheresis. The second and third vaccinations were given 3 weeks and 6 weeks after the first vaccination, respectively.
Preparation of Tumor Cells
Tumors were transferred from the Operating Room to Pathology, where under sterile conditions the margins of resection were evaluated and a sample was removed for histologic confirmation of adenocarcinoma. The tumor was then mechanically dissociated with a scalpel and filtered. The filtered cells were enumerated. Some were aliquotted for DTH testing, irradiated 10,000 cGy and cryopreserved at 2 million cells /mL in human AB serum (Gemini BioProducts, West Scramento, Ca) containing 10% DMSO (Sigma, St. Louis, MO). The remaining cells were combined with tumor chunks that did not go through the filter and were enzymatically digested using 4 mg/mL collagenase (Sigma) and 0.2 mg/mL DNAase (Sigma). These cells were then washed, irradiated 10,000 cGy, and subjected to 3 freeze thaw cycles in liquid nitrogen to form the tumor lysate.
Preparation of DC vaccines
Patients underwent leukopheresis using the Cobe Spectra Apheresis System (Lakewood, CO) in the Cellular Therapy Center of the Norris Cotton Cancer Center. The leukopheresis product was enriched for monocytes by counter flow centrifugation elutriation using a Beckman J6-MI elutriation centrifuge (Palo Alto, CA). Monocytes were cultured in Life Cell culture bags (Baxter Nexell Therapeutics, Deerfield, Ill) in AIM-V media (Life Technologies, Grand Island, NY) at an initial concentration of 2.5 × 106 cells/mL with 10 ng/mL rhuGM-CSF (Immunex, Seattle, WA) and 20 ng/mL rhuIL-4 (Schering Plough, Kennilworth, NJ) added on days 0, 3 and 6 of culture. On culture day 6 autologous tumor lysate was added at a ratio of one tumor cell equivalent (TCE) to one DC. Keyhole limpet hemocyanin (KLH) (Vacmune, Biosyn Corp, Carlsbad, CA) was added at 25 ug/mL. On culture day 7 recombinant human CD40L (Amgen, Thousand Oaks, CA) was added at 1 ug/mL to the cultures of those patients who randomized to this treatment. On culture day 8 DCs were harvested, viable cells were enumerated and administered if they met the following release criteria: viability > 70%, no organisms on gram stain, cultured samples with no growth and endotoxin levels of < 5 EU/kg. Five million DCs were injected in a volume of 0.5 mL into each of 2 inguinal lymph nodes under ultrasound guidance. The remainder of the DCs were cryopreserved in autologous serum/10% DMSO at 1 × 107 DCs/mL for the second and third vaccinations, which were performed 3 weeks and 6 weeks after the initial vaccination in the same nodal basin.
PBMCs were isolated from whole blood drawn from 3 different time points (prevaccine, one week after all vaccinations and 3 months after all vaccinations) using Ficoll-Paque Plus (GE Healthcare, Pittsburgh, PA). After separation, PBMCs were washed, then frozen in a 10% DMSO/FBS (Lonza, Portsmouth, NH) solution and stored at −140°C. PBMCs from all 3 time points were concurrently assayed.
Autologous DCs, cryopreserved after 6 days of culture, were thawed and washed in serum free AIMV (Gibco, Carlsbad, CA) medium, then re-suspended in AIMV supplemented with rhuGM-CSF at 500 IU/mL and rhuIL-4 at 20 ng/mL. DCs were incubated in 6 well Ultra Low Adhesion plates (Costar, Lowell, MA) at 37°C/5% CO2. After 24 hours of incubation, DCs were pulsed with antigens for 24 hours. The following antigens were used: KLH at 25 ug/mL and autologous tumor lysate at a ratio of 1 tumor cell equivalent (TCE) to 1 DC. DCs were then harvested and washed in serum free AIMV medium, then re-suspended in CM and used as stimulators. For peptide antigens DCs were first activated with rhuCD40L (1 ug/mL), then pulsed with peptides and B2microglobulin (Calbiochem, Gibbstown, NJ) at 3 ug/mL for 4hrs at 37°C. The HLA-A2 restricted peptides Ep-Cam p263–271 (GLKAGVIAV), CEA p571–579 (YLSGANLNL) and her-2/neu p654–662 (IISAVVGIL) (Peptide Technologies Corp. Gaithersburg, MD) were used at 20 ug/mL.
The ELISPOT assay was performed on 96 well Immobilon-P acrylic PVDF plates (Millipore, Billerica, MA) which were coated with 5 ug/mL mouse-anti human IFN-γ antibody, 1-DIK (Mabtech, Mariemont, OH) and incubated at 4°C overnight. After thawing, PBMCs were washed and transferred to tissue culture treated polystyrene flasks (Corning, Corning, NY) for a 10 minute adherence depletion at 37°C. Cells were harvested from flasks and then 3 × 105 cells were added to each well in triplicate. Antigen-pulsed DCs were added at 20,000 per well. PHA (Sigma) was used as a control at 20 ug/mL. After an 18 hour incubation, the plates were washed with PBS/0.05% Tween 20 (Sigma). The biotinylated detection antibody (IFNγ: Mab 7-B6-1 Biotin, Mabtech) was added at 1 ug/mL and incubated at 37°C for 2 hours. The plates were then washed, Avidin-Peroxidase-Complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) was added and plates were incubated at room temperature for 1 hour. AEC substrate (3-amino-9-ethylcarbazole, Sigma) was added for 4 minutes, and the plates were washed and analyzed using the KS ELISPOT Axioplan 2 Imaging system (Carl Zeiss, Hallbergmoos, Germany).
Restimulation ELISPOT Assay
PBMCs were thawed, washed and plated at 1 × 106/well in 24 well plates. DCs were antigen loaded as previously described and added to wells at 1 × 105/well. IL-12 (Peprotech, Rocky Hill, NJ) was added to each well at 1 ng/mL. On culture day 2, IL-2 (Peprotech) was added at 10 U/mL. Cells were incubated for an additional 5 days. On Day 6, the cells were harvested and plated onto a pre-coated IFN-γ ELISPOT plate (as described above) at a concentration of 3 × 104/well. Antigen-pulsed DCs were added to these cells and the ELISPOT plate was developed and analyzed as described above.
Dye Dilution Proliferation Assay (DDPA)
The DDPA was performed as previously described (15
). Briefly, PBMCs were thawed, washed, centrifuged and resuspended in Diluent C (Sigma) at a concentration of 2 × 107
/mL. An equal volume of Diluent C containing 4 uM PKH-67 (Sigma) was added. Cells were incubated for 3 minutes in the dark at room temperature. The PKH-67 was quenched by adding an equal volume of FBS for 1 minute. PBMCs were then washed, aliquotted at 1 × 106
cells/mL and stimulated with medium or DCs at a ratio of 1:10 DC to PBMC. After 7 days of culture at 37 C, 5% CO2 the cells were harvested, transferred to 12 × 75 mm tissue culture tubes (USA Scientific, Waltham, MA) and blocked with H-IgG (Sigma). Cells were stained with CD4 APC (Becton Dickson, Franklin Lakes, NJ) and CD8 PE (Becton Dickson), incubated on ice for 30 minutes and fixed with 1% paraformaldehyde. Cells were acquired on a Becton Dickson FacsCalibur and analyzed with Winlist and Modfit (Verity, Topsham, MA).
DTH tests were done by intradermal injection of 1 million mechanically dissociated, irradiated viable autologous tumor cells or 5 ug of KLH. Tests were read 2 days later and were considered positive if > 5 mm of induration was observed.
For the DDPA and ELISPOT assays, all samples were run in triplicate to calculate the mean percentage of proliferating cells and spots (± SD). A response for a particular patient at one time point was considered positive if the mean value obtained as a result of a specific antigenic stimulus (e.g. tumor lysate-pulsed DCs) was significantly greater (by t-test) than the mean value derived from the control stimulus (e.g. unpulsed DCs). Values reported are the response to the antigenic stimulus minus the response to the control stimulus.
The study was designed with a sample size of 24 patients to achieve a power of 80% to detect a difference in vaccine-induced immune responses in the DDPA assay between the CD40L and non-CD40L groups. The Kaplan-Meier product limit method was used to estimate relapse-free survival and overall survival. The log rank test was used to compare the survival of vaccine responders to non-responders.