Purpose: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen–derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells.

Experimental Design: HLA-A2–positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-γ intracellular cytokine assays were done to evaluate CTL reactivity.

Results: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1–specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1–specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen–specific CD8+ cells decreased by an average 14%.

Conclusions: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1–specific T cells were observed in 47% of patients after vaccination.

Despite advances in occult blood screening (1), colonoscopy (2), surgery (3), radiotherapy, and adjuvant (46) chemotherapy, colorectal cancer is still the second leading cause for cancer-related mortality in industrialized countries in men and the third leading cause in women. Palliative chemotherapy regimens have been improved by adding topoisomerase-I inhibitors and platin-based cytotoxic drugs. Currently, the majority of patients with newly diagnosed metastatic colorectal cancer are treated with irinotecan or oxaliplatin together with high-dose 5-fluorouracil (5-FU) and leucovorin (7, 8). However, even with therapy, patients with Unio Internationale Contra Cancrum stage IV disease reach an average overall survival time of 15 to 20 months (9, 10).

In this setting, immunotherapy offers a potential complementary approach to chemotherapy, especially in the setting of minimal residual disease and limited prior chemotherapy that may adversely affect immune responsiveness. Because vaccination strategies result in slower times to response than cytotoxic chemotherapy, high tumor burdens may interfere with vaccination strategies by limiting survival time and also by hampering the immune system and decreasing specific T-cell functions as reported in hematologic malignancies (11). However, vaccination approaches require the choice of multiple factors such as epitopes, antigen source (peptide, peptide pool, protein, vector, lysed tumor cells, and whole tumor cells), antigen presentation (dendritic cells, transfected tumor cells, and CD40-activated B cells), adjuvants, route of administration (s.c., i.m., i.v., and intradermal), and vaccination schedule.

This study was designed to answer two critical questions in the field of tumor immunity. Because many patients with metastatic colorectal cancer are currently treated with irinotecan/high-dose 5-FU/leucovorin chemotherapy, the first aim of the study was to determine whether concurrent immunization at the time of irinotecan/high-dose 5-FU/leucovorin chemotherapy resulted in a measurable immune response. With this approach, we were able to immunize patients who had had limited prior chemotherapy and excellent performance status. We monitored the effect of chemotherapy on the cellular immune system with viral recall antigens. As an immunotherapeutic target, we selected CAP-1, which is the immunodominant MHC class I HLA-A2–restricted nonamer epitope of the carcinoembryonic antigen (CEA), a 180 kDa protein which is expressed by over 90% of colorectal tumors (12). CTL lines specific for CAP-1 have been generated from patients immunized with a recombinant CEA vaccine and are able to kill CEA-expressing tumor cells in vitro (13). CAP-1 has been successfully used before in CEA-positive cancer patients to generate immune responses (14).

The second aim of the study was to directly compare the immunologic efficacies of granulocyte macrophage colony-stimulating factor (GM-CSF), bacterial DNA sequences with nonmethylated CpG motifs (CpG-ODN or dSLIM molecules), and dendritic cells in a randomized fashion. Recent studies are controversial on the relative efficacy of GM-CSF, CpG-containing dSLIM molecules, interleukin-2 (IL-2), and dendritic cells as vaccine adjuvants (1520). Additionally, we determined feasibility and toxicities of this combination regimen.

Patients. This was an open-label prospective randomized clinical phase I/II trial of HLA-A2–positive patients with primary metastatic colorectal cancer. The protocol was approved by the institutional review committee and by the German Drug Administration. All patients signed informed consent. Inclusion criteria required an age of between 18 and 75 years, positive HLA-A2 status, elevated serum CEA (>5μg/L) and/or CEA-positive tumor, untreated metastatic disease, chemotherapy-free interval after adjuvant treatment of at least 6 months, Karnofsky index >70%, life expectancy of at least 3 months, sufficient bone marrow and liver function, HIV and hepatitis B and C negativity, absence of central nervous system metastases, no immunosuppressant medication, and negative pregnancy test. Between December 1999 and May 2001, a total of 70 consecutive patients with primary metastatic colorectal cancer were screened according to the inclusion criteria. Fifty-three patients were excluded, of whom 21 were HLA-A2 negative, 15 had been pretreated for their metastatic disease, 1 was older than 75 years, 2 had a normal serum CEA, 3 patients refused informed consent, 2 suffered from renal impairment, 3 had rapid progressive tumors and needed immediate chemotherapy, 2 had no measurable lesions, 3 had a low Karnofsky score, and 1 had a change of histology (non-adeno). Seventeen patients were eligible and enrolled in the study. They had a median age of 60 years (range: 32-75 years); 8 were male and 9 were female; 13 had been diagnosed with colon cancer and 4 with rectal cancer. The mean serum CEA level was 266 ± 453 μg/L.

Study design. All patients received a complete clinical examination including computed tomography scans of chest and abdomen/pelvis, electrocardiogram, echocardiography, pulmonary function test, as well a laboratory workup. At baseline and after three cycles of chemoimmunotherapy, patients underwent a leukapheresis to obtain peripheral blood mononuclear cells for diagnostic and therapeutic purposes. Toxicities were continuously documented and graded according to the WHO criteria. Patients were randomized to receive CAP-1, GM-CSF and IL-2 or CAP-1, dSLIM and IL-2 or CAP-1, and IL-2 alone. In addition, patients were randomized to be vaccinated with or without CAP-1–pulsed autologous dendritic cells as the primary vaccine. Two vaccinations were given 2 and 1 week before the first cycle of chemotherapy. Alternating with the three cycles, two vaccinations were given in the chemotherapy-free interval with a 1-week pause. After the third cycle, patients were vaccinated weekly until progressive disease. The study design is illustrated in Fig. 1.

Fig. 1.

Patients were first randomized (circled R) to receive CAP-1 and IL-2 with different adjuvants (dSLIM, GM-CSF, or none). Subsequently, they were randomized to receive their first vaccination with or without pulsed autologous dendritic cells. Vaccinations (V) and chemotherapy (ChT) were given in an alternating schedule, starting with two vaccinations.

Fig. 1.

Patients were first randomized (circled R) to receive CAP-1 and IL-2 with different adjuvants (dSLIM, GM-CSF, or none). Subsequently, they were randomized to receive their first vaccination with or without pulsed autologous dendritic cells. Vaccinations (V) and chemotherapy (ChT) were given in an alternating schedule, starting with two vaccinations.

Close modal

Chemotherapy. Patients were treated with 80 mg/m2 irinotecan, 2,000 mg/m2 high-dose 5-FU, and 500 mg/m2 leucovorin for three cycles (six weekly administrations) as described before (21, 22), if progressive disease did not occur during therapy.

Vaccine production and administration. Double stem-loop immunomodulators (dSLIM-30L1), which are covalently closed dumbbell-shaped DNA molecules containing unmethylated CpG motifs, were produced from 5′-CCTAGGGGTTACCACCTTCATTGGAAAACGTTCTTCGGGGCGTTCTTAGGTGGTAACC-3′ oligodeoxynucleotides (ODN) under conditions resembling good manufacturing practice as described (20, 23). CAP-1 peptide (YLSGANLNL) was synthesized under good manufacturing practice–like conditions as well. Clinical grade molgramostim (GM-CSF) was purchased from Novartis Pharma GmbH (Nürnberg, Germany). Clinical grade aldesleukin (IL-2) was purchased from Chiron GmbH (Marburg, Germany). Vaccine preparations were mixed under sterile conditions and frozen at −80°C. Shortly before administration, vaccines were thawed at room temperature and given s.c. at altering sites of the upper arm. Depending on the randomization, the first vaccine consisted of an average of 1 × 107 CAP-1–pulsed dendritic cells or 50 μg CAP-1 with 250 μg dSLIM, or 50 μg CAP-1 with 50 μg GM-CSF, or 50 μg CAP-1 alone. The second and all following vaccinations contained 50 μg CAP-1 together with 1 × 106 IU IL-2 and either 250 μg dSLIM, 50 μg GM-CSF, or no adjuvant.

Generation and maturation of dendritic cells. CD14-positive cells were isolated from leukapheresis material by immunomagnetic enrichment technique using the CliniMACS device (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the guidelines of the manufacturer. Purified CD14 cells (purity grade >97%) were cultured in serum-free CellGenix medium (CellGenix, Freiburg, Germany) containing Glutamax I (Life Technologies, Inc., Karlsruhe, Germany) in the presence of 800 units/mL GM-CSF and 500 units/mL IL-4 (R&D Systems GmbH, Wiesbaden, Germany). On days 3 and 6, 50% fresh medium with cytokines was added. Cells were separated by Ficoll centrifugation on day 6 to purge them from dead cells. On day 7, cells were transferred to medium containing Glutamax I, 800 units/mL GM-CSF, 500 units/mL IL-4, 1 μg/mL prostaglandin E2 (Sigma-Aldrich, Deisenhofen, Germany), 20 ng/mL tumor necrosis factor (Sigma-Aldrich), 1,000 units/mL IL-6 (R&D Systems), and 10 μg/mL anti-CD40 antibody (BD Biosciences, Hamburg, Germany). Mature dendritic cells were harvested, washed, and characterized by flow cytometry on day 10. Dendritic cells were incubated with 50 μg CAP-1 peptide in 500 μL 0.9% NaCl for 2 hours at 37°C. Afterwards, dendritic cells were washed and resuspended in 1 mL of 0.9% NaCl solution for s. c. administration.

Clinical assessment of response. Patients were restaged after each cycle of chemoimmunotherapy and evaluated for response after complete treatment by computed tomography of involved sites and extensive laboratory workup. Patients were monitored monthly for serum CEA levels to assess the clinical efficacy of immunotherapy after completion of chemotherapy and while undergoing weekly vaccinations. A complete response (CR) was defined as the disappearance of all tumor signs, a partial response (PR) as the regression of all lesions by at least 30% in diameter, and progressive disease as an increase of at lease 20% in diameter or occurrence of new lesions. Stable disease was if neither PR nor progressive disease criteria were met.

Immune assessment. State-of-the-art immune assessment (24) including MHC class I tetramer analysis and intracellular cytokine assay was done on all samples. Assays were applied according to Dana-Farber Cancer Institute Immune Assessment Laboratory standard operating procedures, which were developed by thorough research and testing. Daily quality assessment and quality control was applied to all involved reagents and machines. Peripheral blood mononuclear cells from leukapheresis were isolated by Ficoll centrifugation and frozen in liquid nitrogen before performance of assays. In case of progressive disease, 50 mL of blood were drawn from the patient after the last treatment instead of leukapheresis. Cells were phenotypically evaluated by flow cytometry for monocyte, T-cell, and B-cell markers (CD14, CD3, CD4, CD8, CD14, CD19, CD20, CD45, CD45RA, and CD45R0). Peptides for in vitro use were purchased from New England Peptides (Gardner, MA).

Flow cytometry. Uncompensated digital acquisition was done on a five-color flow cytometer (Beckman-Coulter FC500, Miami, FL). Data were compensated and analyzed with FlowJo for Macintosh software version 4.3 (Treestar, Ashland, OR).

MHC class I tetramer analysis. Biotinylated HLA-A2 monomers were synthesized in association with different peptides and β2-microglobulin as previously described (25). Monomers were multimerized with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR). Peripheral blood mononuclear cells, 2 × 106 to 4 × 106, were stained with 2 μg tetramer and anti-CD27 FITC, anti-CD45RA ECD, anti-CD8 PC7, anti-CD4 PC5, anti-CD14 PC5, and anti-CD19 PC5 (all antibodies from Beckman-Coulter). For quantitation of vaccine-specific CD8+ T cells, CAP-1 tetramers were used. Human T-cell lymphotrophic virus type I TAX (LLFGYPVYV) tetramers served as negative control. To evaluate recall antigen–specific memory CTL, peripheral blood mononuclear cells were stained with cytomegalovirus (CMV) pp65 (NLVPMVATV) and EBV BMLF-1 (GLCTLVAML) tetramers. Cells were gated on lymphocyte population, CD4/CD14/CD19-negative cells (“bin gate,” as described before; ref. 24), and CD8. The limit of detection for CAP-1–specific CTL was 0.02% as defined by staining peripheral blood mononuclear cells from 12 HLA-A2–positive healthy volunteers.

IFN-γ intracellular cytokine assay. Thawed peripheral blood mononuclear cells were incubated at 37°C and 5% CO for 1 hour with either 10 μg/mL CAP-1 peptide, 10 μg/mL HIV RT-POL (ILKEPVHGV) as negative control, or 2 μg/mL staphylococcal enterotoxin B as positive control. To block secretion of cytokines, 10 μg/mL brefeldin A (Sigma, St. Louis, MO) was added. Cells were incubated for 5 hours at 37°C and 5% CO2. Afterwards, cells were stained with CAP-1 tetramers as described above for 20 minutes at room temperature and later fixed and permeabilized. To detect intracellular cytokines, cells were stained with anti–IFN-γ FITC (BD Biosciences), anti-CD69 ECD, anti-CD4/14/19, and anti-CD8 PC7 (Beckman-Coulter) for 20 minutes at room temperature and analyzed by flow cytometry.

In vitro peptide stimulation of T cells. To expand CAP-1–specific T cells, thawed peripheral blood mononuclear cells were incubated with 1 μg/mL of CAP-1 peptide in a 96-well plate (2 × 106 cells/mL) in the presence of 20 IU/mL of IL-2 (Chiron Corp., Emeryville, CA) at 37°C and 5% CO2 for 8 days. IL-2 was refreshed on day 4 (20 IU/mL). Cells were further phenotypically and functionally analyzed by tetramer staining and intracellular cytokine assays as described above.

Generation of a CAP-1–specific T-cell line. CD40-activated B cells generated from peripheral blood mononuclear cells of patients and healthy donors were used to stimulate autologous CD8+ T cells (26). CTLs were cultured in T-cell medium in 24-well plates in the presence of 10 ng/mL IL-7 (Endogen, Inc., Woburn, MA) on day 0 and 20 IU/mL IL-2 on days 1 and 4. Restimulation of T cells was done with irradiated, 1 μg/mL CAP-1–pulsed CD40-activated B cells every week. Cells were repeatedly tested with CAP-1 tetramers for specific CTL. Functional analysis was done with the Europium release assay as previously described (27).

Statistics. All statistical tests were done with the statistics software SPSS V11.0 for Windows (SPSS, Inc., Chicago, IL). A Student's t test was used to calculate differences between pre- and post-therapeutic CAP-1–specific CD8+ cell frequencies. The same test was applied to changes in recall antigen CD8+ cell frequencies before and after one and three cycles of chemotherapy, respectively. Calculations of time to progression and overall survival were done with a Kaplan-Meier analysis. Differences in survival between treatment groups were calculated with a log-rank test. Probabilities (P values) smaller than 0.05 (below 5%) were considered as statistically significant.

Patient treatment

Seventeen patients (13 colon and 4 rectal cancers) were enrolled in this trial. Twelve patients received the planned three cycles of chemoimmunotherapy, two patients received only one cycle (six administrations of irinotecan/5-FU/leucovorin), and three patients discontinued before the first cycle had been finished because of progressive disease. Two of the 17 patients were randomized to receive CAP-1, GM-CSF, and IL-2; six received CAP-1, dSLIM, and IL-2; and five patients received CAP-1 and IL-2 only. CAP-1–pulsed dendritic cells were administered during the first vaccination to four patients, three of whom received CAP-1, dSLIM, and IL-2 in the consecutive vaccinations and one was randomized to continue with CAP-1, GM-CSF, and IL-2. The study was discontinued after 17 patients, as it became apparent that neither of the adjuvants provided superiority in eliciting a high CAP-1–specific immune response and enough data had been acquired to analyze the other objectives of the study.

Toxicity

Chemotherapy was generally well tolerated. There were no treatment delays due to blood count, nausea/vomiting, or pain. WHO grade 3 diarrhea occurred in four cases, which did not require hospitalization. Vaccinations caused mild local reactions (swelling, induration, or erythema) at the sites of injection in six cases. One patient reported transient chills and a slight increase in temperature (38°C) that resolved within a couple of hours.

Clinical response

Clinical response to combined modalities. Five of 17 patients (29%) achieved a CR (one patient with curative liver metastasis resection and two patients with metastatic disease of the peritoneum), one (6%) a PR, and five (29%) patients had stable disease combining for a general response rate of 35% (CR + PR). Six (36%) patients showed progressive disease, five during the first cycle and one at the end of the third.

Of the five patients who were treated without any adjuvants, one achieved a CR, one had stable disease, and three patients progressed (response rate 1/5; 20%). Of four patients who had received CAP-1–pulsed dendritic cells, one achieved a CR, one had stable disease, and two patients progressed. The group of 13 patients without dendritic cell–based vaccines showed four CR, one PR, four stable disease, and four progressive disease. In the GM-CSF group with three patients, one PR and two progressive disease occurred. dSLIM vaccines (n = 9) showed four CR, four stable disease, and only one progressive disease (response rate 4/9; 44%) response rate. Comparisons of dSLIM-receiving patients (n = 9; response rate 44.4%) with patients who did not receive dSLIM (n = 8; response rate 25%) indicated a favorable response to dSLIM treatment although the numbers were too small to perform statistical analysis.

After a median observation time of 29 months (range: 22-34 months), 11 of 17 patients had died due to their disease and 5 patients had relapsed. One patient remained free of disease. He had a stage IV cancer with elevated serum CEA and underwent curative resection of two liver metastases before start of therapy. The median time to progression of relapsed/progressive patients was 8 months (range: 1-15 months) and the median survival 17 months (range: 2-34 months). A log-rank test did not reveal any statistically significant differences in time to progression or survival between the groups dendritic cell/nondendritic cell and adjuvant/no adjuvant (data not shown). Table 1 summarizes patient characteristics and clinical results.

Table 1.

Characteristics and clinical results of all enrolled patients

Patient IDSexAge (y)Primary tumorSites of metastasesSerum CEA (μg/L)Primary vaccineBoostNo. cycles of three plannedClinical responseFFTF (mo)Survival (mo)
P01 48 Rectum Liver, lung, bone, adrenal gland 12 CAP-1 CAP-1 + IL-2 0.3 PD 
P02 66 Colon Liver 23 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 PR 15 34 
P03 44 Colon Liver 602 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 SD 21 
P04 70 Colon Liver 142 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 SD 12 32 
P05 69 Colon Liver, lung, mediastinum 196 DC-CAP-1 CAP-1 + GM-CSF + IL-2 0.5 PD 
P06 32 Colon Liver, spleen, ovaries, pelvis, peritoneum 11 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 CR 12 26 
P07 60 Colon Liver 792 CAP-1 CAP-1 + IL-2 3.0 SD 11 15 
P08 57 Rectum Lung 16 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 SD 17 
P09 44 Rectum Liver 19 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 CR 12 28 
P10 64 Colon Liver, lung 881 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 SD 10 28 
P11 38 Colon Liver 21 DC-CAP-1 CAP-1 + dSLIM + IL-2 1.0 PD 10 
P12 72 Colon Liver, spleen, lung, mediastinum 1690 CAP-1 CAP-1 + IL-2 0.5 PD 
P13 43 Rectum Liver, bone, pararectal, paraaortal lymph nodes 21 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 PD 13 
P14 61 Colon Liver 23 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 CR 11 
P15 75 Colon Liver 44 CAP-1 CAP-1 + IL-2 0.5 PD 
P16 62 Colon Peritoneum 22 CAP-1 CAP-1 + IL-2 3.0 CR 13 21 
P17 55 Colon Liver (primary resection) <5* CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 CR 24 24 
Patient IDSexAge (y)Primary tumorSites of metastasesSerum CEA (μg/L)Primary vaccineBoostNo. cycles of three plannedClinical responseFFTF (mo)Survival (mo)
P01 48 Rectum Liver, lung, bone, adrenal gland 12 CAP-1 CAP-1 + IL-2 0.3 PD 
P02 66 Colon Liver 23 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 PR 15 34 
P03 44 Colon Liver 602 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 SD 21 
P04 70 Colon Liver 142 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 SD 12 32 
P05 69 Colon Liver, lung, mediastinum 196 DC-CAP-1 CAP-1 + GM-CSF + IL-2 0.5 PD 
P06 32 Colon Liver, spleen, ovaries, pelvis, peritoneum 11 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 CR 12 26 
P07 60 Colon Liver 792 CAP-1 CAP-1 + IL-2 3.0 SD 11 15 
P08 57 Rectum Lung 16 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 SD 17 
P09 44 Rectum Liver 19 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 CR 12 28 
P10 64 Colon Liver, lung 881 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 SD 10 28 
P11 38 Colon Liver 21 DC-CAP-1 CAP-1 + dSLIM + IL-2 1.0 PD 10 
P12 72 Colon Liver, spleen, lung, mediastinum 1690 CAP-1 CAP-1 + IL-2 0.5 PD 
P13 43 Rectum Liver, bone, pararectal, paraaortal lymph nodes 21 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 PD 13 
P14 61 Colon Liver 23 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 CR 11 
P15 75 Colon Liver 44 CAP-1 CAP-1 + IL-2 0.5 PD 
P16 62 Colon Peritoneum 22 CAP-1 CAP-1 + IL-2 3.0 CR 13 21 
P17 55 Colon Liver (primary resection) <5* CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 CR 24 24 

Abbreviations: DC, dendritic cells; SD, stable disease; PD, progressive disease; FFTF, freedom from treatment failure.

*

P17 had a CEA-positive tumor.

Clinical response to immunotherapy. During the three cycles, it was not possible to discriminate clinical effects of treatment modalities because vaccinations and chemotherapy were given simultaneously. After completion of chemotherapy, vaccinations were the only treatment for the patients. Thus, clinical response to immunotherapy could be evaluated. Of 12 patients who completed three cycles, all but one showed increasing serum CEA levels within 1 month after chemotherapy was discontinued. The patient with decreasing serum CEA levels had undergone a curative resection of liver metastases. He had a measurable increase of CAP-1–specific CTL. Figure 2 shows the course of serum CEA in all evaluable patients.

Fig. 2.

Normalized CEA levels of eight patients who received weekly booster vaccinations after three cycles of immunochemotherapy. All but one patient showed increasing levels after discontinuation of cytostatic drugs (dotted line).

Fig. 2.

Normalized CEA levels of eight patients who received weekly booster vaccinations after three cycles of immunochemotherapy. All but one patient showed increasing levels after discontinuation of cytostatic drugs (dotted line).

Close modal

Immunologic response

Peptide/MHC class I tetramers. Peptide/MHC class I tetramers were used to detect CAP-1–specific CD8+ T cells, which were analyzed either freshly thawed or after a weeklong in vitro CAP-1 stimulation to enhance sensitivity. At baseline, CAP-1–specific CD8+ cells were observed in unstimulated samples of 8 patients (47%; CD8+ specific frequency: 0.045 ± 0.035%) and in 11 patients (65%) after in vitro stimulation (0.110 ± 0.085%). An immunologic response, defined as an increase of specific CD8+ cells after three cycles (or fewer in progressive patients), was found in unstimulated samples of 4 patients (24%; 0.045 ± 0.024% to 0.171 ± 0.077%; P > 0.05). After an in vitro stimulation with CAP-1 peptide, 8 patients (48%) showed a significant increase of specific CD8+ from pre- to post-vaccination (0.058 ± 0.031% to 0.204 ± 0.139%; P = 0.04). Responders included four patients who had received CAP-1/dSLIM/IL-2 as adjuvants, one of the CAP-1/GM-CSF/IL-2 arm, and three patients who received CAP-1 and IL-2 only (all progressive disease within first cycle). Two of eight patients had received dendritic cell in their first vaccine. Figure 3 shows examples of two patients responding to vaccination therapy with an increase of CAP-1–specific CD8+ cells.

Fig. 3.

Example of two patients (PAT14 and PAT17) responding to three cycles of immunochemotherapy with an increase of CAP-1–specific CTLs (CD8+). PAT14 showed a significant CAP-1 response at baseline (0.045% CD8+). As a control tetramer, TAX HLTV-1 was used (bottom).

Fig. 3.

Example of two patients (PAT14 and PAT17) responding to three cycles of immunochemotherapy with an increase of CAP-1–specific CTLs (CD8+). PAT14 showed a significant CAP-1 response at baseline (0.045% CD8+). As a control tetramer, TAX HLTV-1 was used (bottom).

Close modal

IFN-γ intracellular cytokine assay. Samples were processed freshly thawed and after a 1-week CAP-1 peptide in vitro stimulation to increase sensitivity of this assay. At baseline, T cells of two patients (12%; CD8+ specific frequency: 0.27 ± 0.03%) showed an increased IFN-γ secretion to CAP-1 peptide and in three patients (18%; CD8+ frequency: 0.39 ± 0.19) after in vitro stimulation. Immunologic response to CAP-1 from prevaccination to postvaccination was detected in two unstimulated patient samples (12%; from 0.00 to 0.03 ± 0.003; P > 0.05) and in four stimulated patient samples (24%; from 0.00 ± 0.00 to 0.27 ± 0.15; P = 0.05). Among responders were two patients vaccinated with CAP-1/dSLIM/IL-2, one with CAP-1/GM-CSF/IL-2 (three cycles completed) and one with CAP-1/IL-2 (not completed). One patient had received dendritic cell in the first vaccine. The results from tetramer analysis and IFN-γ intracellular cytokine assay showed no correlation. Table 2 summarizes the immunologic results.

Table 2.

Immunologic results of all enrolled patients

Patient IDPrimary vaccineBoostNo. cycles of three plannedTet CD8+ CAP-1 frq preTet CD8+ CAP-1 frq postTetramer increase pre/postICC CD8+ CAP-1 frq preICC CD8+ CAP-1 frq postICC increase pre/post
P01 CAP-1 CAP-1 + IL-2 0.3 0.08 0.13 0.65 0.00 − 
P02 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 0.00 0.01 − 0.00 0.41 
P03 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.01 0.02 − 0.00 0.04 
P04 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.07 0.04 − 0.00 0.00 − 
P05 DC-CAP-1 CAP-1 + GM-CSF + IL-2 0.5 0.14 0.00 − 0.00 0.00 − 
P06 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 0.00 0.00 − 0.01 0.22 
P07 CAP-1 CAP-1 + IL-2 3.0 0.09 0.04 − 0.21 0.20 − 
P08 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.09 0.04 − 0.30 0.02 − 
P09 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.04 0.01 − 0.00 0.00 − 
P10 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 0.01 0.15 0.00 0.00 − 
P11 DC-CAP-1 CAP-1 + dSLIM + IL-2 1.0 0.05 0.15 0.05 0.00 − 
P12 CAP-1 CAP-1 + IL-2 0.5 0.02 0.09 0.00 0.00 − 
P13 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 0.01 0.06 0.00 0.00 − 
P14 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.09 0.31 0.02 0.01 − 
P15 CAP-1 CAP-1 + IL-2 0.5 0.05 0.07 0.00 0.39 
P16 CAP-1 CAP-1 + IL-2 3.0 0.00 0.00 − 0.05 0.02 − 
P17 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.01 0.13 0.00 0.00 − 
Patient IDPrimary vaccineBoostNo. cycles of three plannedTet CD8+ CAP-1 frq preTet CD8+ CAP-1 frq postTetramer increase pre/postICC CD8+ CAP-1 frq preICC CD8+ CAP-1 frq postICC increase pre/post
P01 CAP-1 CAP-1 + IL-2 0.3 0.08 0.13 0.65 0.00 − 
P02 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 0.00 0.01 − 0.00 0.41 
P03 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.01 0.02 − 0.00 0.04 
P04 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.07 0.04 − 0.00 0.00 − 
P05 DC-CAP-1 CAP-1 + GM-CSF + IL-2 0.5 0.14 0.00 − 0.00 0.00 − 
P06 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 0.00 0.00 − 0.01 0.22 
P07 CAP-1 CAP-1 + IL-2 3.0 0.09 0.04 − 0.21 0.20 − 
P08 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.09 0.04 − 0.30 0.02 − 
P09 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.04 0.01 − 0.00 0.00 − 
P10 DC-CAP-1 CAP-1 + dSLIM + IL-2 3.0 0.01 0.15 0.00 0.00 − 
P11 DC-CAP-1 CAP-1 + dSLIM + IL-2 1.0 0.05 0.15 0.05 0.00 − 
P12 CAP-1 CAP-1 + IL-2 0.5 0.02 0.09 0.00 0.00 − 
P13 CAP-1 + GM-CSF CAP-1 + GM-CSF + IL-2 3.0 0.01 0.06 0.00 0.00 − 
P14 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.09 0.31 0.02 0.01 − 
P15 CAP-1 CAP-1 + IL-2 0.5 0.05 0.07 0.00 0.39 
P16 CAP-1 CAP-1 + IL-2 3.0 0.00 0.00 − 0.05 0.02 − 
P17 CAP-1 + dSLIM CAP-1 + dSLIM + IL-2 3.0 0.01 0.13 0.00 0.00 − 

NOTE: Frequencies (%) of CAP-1–specific CTL (CD8+ CAP-1 frq) were obtained from tetramer staining at baseline (Tet pre) and after the last therapy cycle (Tet post) as well as from intracellular cytokine assays (ICC pre/post) in prestimulated samples. Background CD8+ frequencies were subtracted from CAP-1 CD8+ frequencies. A response was defined as more than 0.02% tetramer-positive CAP-1–specific CD8+ CTLs.

Impact of chemotherapy on T cells. The cytotoxic effects of three cycles of irinotecan/high-dose 5-FU/leucovorin on the cellular immunity were evaluated by CD3+/CD4+ and CD3+/CD8+ frequencies as well as the responses to viral recall antigens before and after completion of treatment. In addition, peripheral blood mononuclear cells of patients who had received only one cycle due to progressive disease were evaluated. Twelve patients completed all three cycles. Their average CD4+ and CD8+ cell frequencies were not affected by chemotherapy (at baseline: 45 ± 14% and 26 ± 13%; after completion of therapy: 43 ± 14% and 26 ± 11%). Five patients who underwent only one or fewer cycles of chemotherapy showed similar average frequencies (CD4+ and CD8+ at baseline: 41 ± 18% and 21 ± 5%; after vaccination: 43 ± 19% and 23 ± 16%). CMV pp65– and EBV BMLF-1–specific CD8+ cell frequencies were assessed in all patients at baseline and after therapy. All but one patient had detectable CD8+ responses to the recall antigens. The average recall antigen CD8+ frequency at baseline was 0.64 ± 0.70%, which was similar to that of 12 healthy donors (0.61 ± 0.89%). Patients who were treated with three cycles (n = 12) showed an average decrease of recall antigen CD8+ frequency from 0.65 ± 0.68% to 0.47 ± 0.51%, which was statistically significant (P = 0.017). This is an average decrease of recall antigen–specific CD8+ cells by 14% if baseline values were set to 100%. In contrast, five patients with a therapy of one cycle (6 weeks) or fewer had an increase in recall antigen CD8+ from 0.62 ± 0.73% to 1.81 ± 2.14%. This was compared with an average increase of 310%, if baseline frequencies were set to 100%. Due to the small numbers, these results were not statistically significant. The recall antigen tetramer results of four patients from each group are depicted in Fig. 4.

Fig. 4.

Flow cytometric CMV or EBV peptide tetramer analysis in 4 of 11 patients who completed three cycles (two left columns) and 4 of 5 patients who received only one or fewer cycles (two right columns). Patients with three cycles showed a significant decrease of CMV/EBV-specific CD8+ frequencies after therapy, whereas an increase was observed in patients with one or fewer cycles.

Fig. 4.

Flow cytometric CMV or EBV peptide tetramer analysis in 4 of 11 patients who completed three cycles (two left columns) and 4 of 5 patients who received only one or fewer cycles (two right columns). Patients with three cycles showed a significant decrease of CMV/EBV-specific CD8+ frequencies after therapy, whereas an increase was observed in patients with one or fewer cycles.

Close modal

Generation of a CAP-1–specific T-cell line. A CAP-1–specific T-cell line (>1% specificity) could not be generated after multiple stimulations with CD40-activated B cells in seven different donors (patients and healthy controls). The maximum frequency that could be achieved was 0.5% specific CD8+ cells in three of six patients. However, when CAP-1 was substituted by the heteroclitic peptide CAP1-6D, peptide stimulations gave rise to a specific T-cell line in one healthy donor (Fig. 5).

Fig. 5.

Tetramer analysis of a CAP-1–specific CTL line (top) generated from peripheral blood mononuclear cells by multiple stimulation with the heteroclitic peptide CAP1-6D, which was able to lyse CAP-1– and CAP1-6D–pulsed T2 cells in a Europium release cytotoxicity assay (bottom).

Fig. 5.

Tetramer analysis of a CAP-1–specific CTL line (top) generated from peripheral blood mononuclear cells by multiple stimulation with the heteroclitic peptide CAP1-6D, which was able to lyse CAP-1– and CAP1-6D–pulsed T2 cells in a Europium release cytotoxicity assay (bottom).

Close modal

The presented combination of vaccination-based immunotherapy and irinotecan/high-dose 5-FU/leucovorin chemotherapy is a feasible combination regimen for primary metastatic colorectal cancer patients. The side effects were well tolerated and the treatment could be administered as planned.

The clinical overall response rate to the combination regimen was 35%. Only one patient showed a continuously decreasing CEA level after completion of chemotherapy, indicating that the vaccination strategies did not have a clinical effect after the third cycle in the rest of the patients. However, it cannot be ruled out that the vaccinations had a transient effect during the first 5 months of combination therapy.

It is known that immune responses to viral vaccines during chemotherapy are lower than expected in healthy people (28). Therefore, it was an important aim of this trial to determine whether the irinotecan/high-dose 5-FU/leucovorin chemotherapy regimen had an adverse effect on the memory CTL repertoire. Our data clearly show that three cycles of irinotecan/high-dose 5-FU/leucovorin reduce the relative number of EBV- or CMV-specific CTL by an average 14% without having an effect on the absolute CD8+ cell count. It is likely that chemotherapy eliminates antigen-specific memory CTLs, which do not recover as quickly as other CD8+ subsets. One intriguing observation is that 0.5 to 1 cycle of irinotecan/high-dose 5-FU/leucovorin increased recall antigen–specific CTL frequencies, although this was not statistically significant due to the small patient group. We speculate that a short administration of irinotecan/high-dose 5-FU/leucovorin chemotherapy shuts off regulatory immunologic mechanisms such as CD4+/CD25+ T cells and thereby increases antigen-specific responses. This is supported by recent observations in mice, which were treated with chemotherapy-modulated vaccinations (29). Cyclophosphamide and doxorubicin could enhance a tumor-specific CTL response by abrogating the suppression of CTL by CD4+/CD25+ T cells.

Our tetramer results indicate that CEA is not a neoantigen for most of the patients. Nagorsen et al. (30) reported that one third of HLA-A2–positive patients with colorectal cancer show T-cell responses to the CAP-1 peptide by IFNγ ELIspot. Although these cells can be detected by highly sensitive methods, they remain ineffective for tumor control. Almost 50% of our patients showed an immunologic response to vaccination (increase of CAP-1–specific CTL). IFNγ secretion was observed in T cells of 24% of the patients after in vitro stimulation, which is lower than the result of the tetramer analysis. This could be due to nonfunctional T cells or to the lower sensitivity of the IFNγ intracellular cytokine assay, which has been reported before (24, 31).

We did not find any striking differences in immunologic response between adjuvants (CAP-1/GM-CSF/IL-2 versus CAP-1/dSLIM/IL-2 versus CAP-1/IL-2) or between the use of the primary vaccine with or without dendritic cells. Therefore, we discontinued the trial after 17 enrolled patients. Although the immunologic response of our trial looks convincing (50% patients with increase of CAP-1 tetramer–positive CTL), the clinical efficacy does not. Unfortunately, this has been observed in numerous tumor vaccination studies (14, 29, 32). In our and other phase I/II studies, we are trying to immunize an already “infected” host, who is overwhelmed by an exponential growth of billions of destructive tumor cells. This has not worked with vaccines against already existing infectious diseases. To break the T-cell ignorance towards the tumor, it is necessary to elucidate questions about optimal vaccine preparation, timing and schedule, dosage, and adjuvants. We speculate that we will not experience consistent clinical responses in tumor patients if we do not reach tumor antigen–specific CTL frequencies comparable to those against virus antigens. It may be necessary to increase dosage of peptide and/or adjuvants. Whereas the role of GM-CSF as an adjuvant is still controversial (15, 16, 18), DNA molecules with unmethylated CpG motifs (CpG-ODN or dSLIM) are promising candidates in preclinical immunologic studies (17, 19, 20, 33, 34). This is one of the first studies to administer CpG-ODN/dSLIM as an adjuvant in cancer patients. The phosphorodiester-based dSLIM was well tolerated and did not cause any systemic side effects at a dosage of 250 μg.

CAP-1 has been used in other vaccination trials before. In a phase I study, Morse et al. (14) vaccinated 21 HLA-A2– and CEA-positive cancer patients with CAP-1–pulsed dendritic cells. Patients were treated in three dose escalation groups (1 × 107, 3 × 107, and 1 × 108 dendritic cells, i.v.) weekly or biweekly with a maximum of four immunizations. No major toxicities could be observed. Two patients showed clinical responses (one minor and one stable disease); all other had progressive disease. Immunologic response was measured by skin delayed-type hypersensitivity measurements. Two patients had new delayed-type hypersensitivity responses to CEA. Five patients showed a CEA delayed-type hypersensitivity before treatment, confirming our results that CEA is not a neoantigen in many cancer patients.

After we had started our trial, CAP1-6D, a heteroclitic peptide of CAP-1, was synthesized and reported to enhance the sensitization of CTL by 100 to 1,000 times (35, 36). Fong et al. (37) isolated peripheral dendritic cells after systemic administration of Flt3 ligand, pulsed the cells with CAP1-6D, and administered the vaccine to 12 colon and non–small-cell lung cancer patients with abnormal serum CEA. Tumor regression could be documented in two patients, and one patient experienced a mixed response. Seven patients developed CAP1-6D–specific CTL as evaluated by tetramer analysis. The patients achieved specific CTL frequencies of over 1% in contrast to our patients who showed weaker responses. This is probably due to the higher potency of CAP1-6D.

In conclusion, the presented chemoimmunotherapy is a feasible and safe combination therapy that shows clinical and immunologic efficacy. Neither of the adjuvants provided a superior CTL immune response although the numbers were too small for statistical analysis. In addition, the use of peptide-pulsed dendritic cells in the primary vaccine did not enhance immunity to CAP-1 compared with peptide/adjuvants alone. Irinotecan/high-dose 5-FU/leucovorin chemotherapy only slightly affected antigen-specific CTLs. In addition, our data suggest that a limited administration of chemotherapy could enhance specific CTL responses and increase the efficacy of future vaccination strategies.

Grant support: Aventis Pharmaceuticals AG, Germany. S. Ansén was supported by the Dr. Mildred Scheel Stiftung der Deutschen Krebshilfe.

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.

We thank Nicole Severing for her great support on processing leukapheresis material and HLA typing, Wanyong Zeng, M.D., for technical support, and Thomas Zander, M.D., for fruitful discussions.

1
Mandel JS, Church TR, Bond JH, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer.
N Engl J Med
2000
;
343
:
1603
–7.
2
Lieberman DA, Weiss DG. One-time screening for colorectal cancer with combined fecal occult-blood testing and examination of the distal colon.
N Engl J Med
2001
;
345
:
555
–60.
3
Hayashi N, Egami H, Kai M, Kurusu Y, Takano S, Ogawa M. No-touch isolation technique reduces intraoperative shedding of tumor cells into the portal vein during resection of colorectal cancer.
Surgery
1999
;
125
:
369
–74.
4
Moertel CG, Fleming TR, Macdonald JS, et al. Fluorouracil plus levamisole as effective adjuvant therapy after resection of stage III colon carcinoma: a final report.
Ann Intern Med
1995
;
122
:
321
–6.
5
Porschen R, Bermann A, Loffler T, et al. Fluorouracil plus leucovorin as effective adjuvant chemotherapy in curatively resected stage III colon cancer: results of the trial adjCCA-01.
J Clin Oncol
2001
;
19
:
1787
–94.
6
Andre T, Boni C, Mounedji-Boudiaf L, et al. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer.
N Engl J Med
2004
;
350
:
2343
–51.
7
Kuebler JP, de Gramont A. Recent experience with oxaliplatin or irinotecan combined with 5-fluorouracil and leucovorin in the treatment of colorectal cancer.
Semin Oncol
2003
;
30
:
40
–6.
8
Goldberg RM, Sargent DJ, Morton RF, et al. A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer.
J Clin Oncol
2004
;
22
:
23
–30.
9
Saltz LB, Cox JV, Blanke C, et al. Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group.
N Engl J Med
2000
;
343
:
905
–14.
10
Goldberg RM, Sargent DJ, Morton RF, et al. A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer.
J Clin Oncol
2004
;
22
:
23
–30.
11
Maecker B, Anderson KS, Bergwelt-Baildon MS, et al. Viral antigen-specific CD8+ T-cell responses are impaired in multiple myeloma.
Br J Haematol
2003
;
121
:
842
–8.
12
Wagener C, Petzold P, Kohler W, Totovic V. Binding of five monoclonal anti-CEA antibodies with different epitope specificities to various carcinoma tissues.
Int J Cancer
1984
;
33
:
469
–75.
13
Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J. Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine.
J Natl Cancer Inst
1995
;
87
:
982
–90.
14
Morse MA, Deng Y, Coleman D, et al. A Phase I study of active immunotherapy with carcinoembryonic antigen peptide (CAP-1)-pulsed, autologous human cultured dendritic cells in patients with metastatic malignancies expressing carcinoembryonic antigen.
Clin Cancer Res
1999
;
5
:
1331
–8.
15
von Mehren M, Arlen P, Gulley J, et al. The influence of granulocyte macrophage colony-stimulating factor and prior chemotherapy on the immunologic response to a vaccine (ALVAC-CEA B7.1) in patients with metastatic carcinoma.
Clin Cancer Res
2001
;
7
:
1181
–91.
16
Samanci A, Yi Q, Fagerberg J, et al. Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen.
Cancer Immunol Immunother
1998
;
47
:
131
–42.
17
Baral RN, Saha A, Chatterjee SK, et al. Immunostimulatory CpG oligonucleotides enhance the immune response of anti-idiotype vaccine that mimics carcinoembryonic antigen.
Cancer Immunol Immunother
2003
;
52
:
317
–27.
18
Ramanathan RK, Potter DM, Belani CP, et al. Randomized trial of influenza vaccine with granulocyte-macrophage colony-stimulating factor or placebo in cancer patients.
J Clin Oncol
2002
;
20
:
4313
–8.
19
Stern BV, Boehm BO, Tary-Lehmann M. Vaccination with tumor peptide in CpG adjuvant protects via IFN-γ-dependent CD4 cell immunity.
J Immunol
2002
;
168
:
6099
–105.
20
Kochling J, Konig-Merediz SA, Stripecke R, et al. Protection of mice against Philadelphia chromosome-positive acute lymphoblastic leukemia by cell-based vaccination using nonviral, minimalistic expression vectors and immunomodulatory oligonucleotides.
Clin Cancer Res
2003
;
9
:
3142
–9.
21
Vanhoefer U, Harstrick A, Achterrath W, Cao S, Seeber S, Rustum YM. Irinotecan in the treatment of colorectal cancer: clinical overview.
J Clin Oncol
2001
;
19
:
1501
–18.
22
Mitry E, Douillard JY, Van CE, et al. Predictive factors of survival in patients with advanced colorectal cancer: an individual data analysis of 602 patients included in irinotecan phase III trials.
Ann Oncol
2004
;
15
:
1013
–7.
23
Wittig B, Marten A, Dorbic T, et al. Therapeutic vaccination against metastatic carcinoma by expression-modulated and immunomodified autologous tumor cells: a first clinical phase I/II trial.
Hum Gene Ther
2001
;
12
:
267
–78.
24
Keilholz U, Weber J, Finke JH, et al. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.
J Immunother
2002
;
25
:
97
–138.
25
Altman JD, Moss PA, Goulder PJ, et al. Phenotypic analysis of antigen-specific T lymphocytes.
Science
1996
;
274
:
94
–6.
26
Bergwelt-Baildon MS, Vonderheide RH, Maecker B, et al. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.
Blood
2002
;
99
:
3319
–25.
27
Blomberg K, Granberg C, Hemmila I, Lovgren T. Europium-labelled target cells in an assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence.
J Immunol Methods
1986
;
86
:
225
–9.
28
Ring A, Marx G, Steer C, Harper P. Influenza vaccination and chemotherapy: a shot in the dark?
Support Care Cancer
2002
;
10
:
462
–5.
29
Belardelli F, Ferrantini M, Parmiani G, Schlom J, Garaci E. International meeting on cancer vaccines: how can we enhance efficacy of therapeutic vaccines?
Cancer Res
2004
;
64
:
6827
–30.
30
Nagorsen D, Keilholz U, Rivoltini L, et al. Natural T-cell response against MHC class I epitopes of epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer.
Cancer Res
2000
;
60
:
4850
–4.
31
Whiteside TL, Zhao Y, Tsukishiro T, Elder EM, Gooding W, Baar J. Enzyme-linked immunospot, cytokine flow cytometry, and tetramers in the detection of T-cell responses to a dendritic cell-based multipeptide vaccine in patients with melanoma.
Clin Cancer Res
2003
;
9
:
641
–9.
32
Foon KA, John WJ, Chakraborty M, et al. Clinical and immune responses in advanced colorectal cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen.
Clin Cancer Res
1997
;
3
:
1267
–76.
33
Davila E, Kennedy R, Celis E. Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG-oligodeoxynucleotide adjuvant, and CTLA-4 blockade.
Cancer Res
2003
;
63
:
3281
–8.
34
Sandler AD, Chihara H, Kobayashi G, et al. CpG oligonucleotides enhance the tumor antigen-specific immune response of a granulocyte macrophage colony-stimulating factor-based vaccine strategy in neuroblastoma.
Cancer Res
2003
;
63
:
394
–9.
35
Tsang KY, Zhu M, Nieroda CA, et al. Phenotypic stability of a cytotoxic T-cell line directed against an immunodominant epitope of human carcinoembryonic antigen.
Clin Cancer Res
1997
;
3
:
2439
–49.
36
Zaremba S, Barzaga E, Zhu M, Soares N, Tsang KY, Schlom J. Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
Cancer Res
1997
;
57
:
4570
–7.
37
Fong L, Hou Y, Rivas A, et al. Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy.
Proc Natl Acad Sci U S A
2001
;
98
:
8809
–14.