Purpose: The combination of vaccines and chemotherapy holds promise for cancer therapy, but the effect of cytotoxic chemotherapy on vaccine-induced antitumor immunity is unknown. This study was conducted to assess the effects of systemic chemotherapy on ALVAC-CEA/B7.1–induced T-cell immunity in patients with metastatic colorectal cancer.

Experimental Design: Patients with metastatic colorectal cancer were treated with fluorouracil, leucovorin, and irinotecan and were also given ALVAC-CEA/B7.1 vaccine with or without tetanus toxoid adjuvant. Eligible patients were randomized to ALVAC followed by chemotherapy and booster vaccination (group 1), ALVAC and tetanus toxoid followed by chemotherapy (group 2), or chemotherapy alone followed by ALVAC in patients without disease progression (group 3). Humoral immune responses were measured by standard ELISA assay, and carcinoembryonic antigen (CEA)-specific T-cell responses were measured by IFN-γ enzyme-linked immunospot assay.

Results: One hundred eighteen patients were randomized to receive either ALVAC before and concomitantly with chemotherapy (n = 39), ALVAC with tetanus adjuvant before and concomitantly with chemotherapy (n = 40), or chemotherapy followed by ALVAC (n = 39). Serious adverse events were largely gastrointestinal (n = 30) and hematologic (n = 24). Overall, 42 patients (40.4%) showed objective clinical responses. All patients developed antibody responses against ALVAC, but increased anti-CEA antibody titers were detected in only three patients. Increases in CEA-specific T cells were detected in 50%, 37%, and 30% of patients in groups 1, 2, and 3, respectively. There were no differences in clinical or immune responses between the treatment groups.

Conclusion: The combination of ALVAC-CEA/B7.1 vaccine and systemic chemotherapy has an acceptable safety profile in patients with metastatic colorectal cancer. Systemic chemotherapy did not affect the generation of CEA-specific T-cell responses following vaccination.

Translational Relevance

The application of tumor vaccines has been promising because defined antigens are available, costimulation to improve T-cell activation is an established adjuvant strategy, and the toxicity of vaccination has been limited to date. Preliminary evaluation of one such vaccine, a nonreplicating canarypox virus (ALVAC) expressing the carcinoembryonic antigen (CEA) and T-cell costimulatory molecule, B7.1, shows promise in priming CEA-specific immunity but few objective clinical responses have been seen. Recent evidence suggests that certain chemotherapy agents might actually augment vaccine-induced T-cell responses through active tumor cell death and release of tumor-associated antigens. The effect of chemotherapy commonly used for colorectal cancer on induction of vaccine-induced immune responses, however, has not been reported. The authors sought to determine the feasibility of combining ALVAC-CEA/B7.1 vaccine with standard chemotherapy used in metastatic colorectal cancer patients and determine the effect on CEA-specific immune responses. The data suggest that chemotherapy does not inhibit vaccine-mediated immunity and provide further support for evaluating novel combinations of chemotherapy and tumor vaccines for colorectal and other cancers.

Globally, colorectal cancer accounts for more than 1 million new cases and 529,000 deaths annually (1). In the United States, colorectal cancer is the third most frequent cancer diagnosis and the second leading cause of cancer-related mortality (2). Interest in vaccines and immunotherapy for colorectal cancer has received increasing attention due to the identification of putative tumor-associated antigens, documentation of vaccine safety in phase I clinical trials, and evidence that vaccines can induce tumor antigen–specific immune responses in patients with advanced cancers (3, 4). The therapeutic effectiveness of such vaccines, however, has not been shown. An emerging paradigm for improving therapeutic responses of vaccines is to combine vaccination with cytotoxic chemotherapy, which may help promote protective immunity through release of targeted tumor antigens (5). The effect of specific chemotherapy combinations, however, on vaccine-induced immunity is not known.

The mainstay of colorectal cancer chemotherapy has been fluorouracil-based regimens, typically administered with leucovorin. Other cytotoxic agents with activity against colorectal cancer include irinotecan and oxaliplatin. Two randomized trials have shown improved survival with irinotecan in patients with metastatic colorectal cancer (6, 7) and further improvements in response rates were observed when irinotecan was combined with bolus and infusional fluorouracil therapy (8, 9). In contrast to cytotoxic chemotherapy, which directly targets malignant cells, vaccines act by stimulating the host immune response to generate effector cells or antibodies that target tumor-associated antigens expressed by tumor cells (10). CEA is an oncofetal antigen that when expressed within recombinant poxviruses has shown induction of HLA-restricted, CEA-specific T cells, suggesting that CEA can serve as a target for vaccine development (11, 12).

ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (3, 4, 13, 14). In a phase I clinical trial, an ALVAC virus expressing CEA showed an excellent safety profile and resulted in increased CEA-specific T-cell responses in selected patients; objective clinical responses, however, were not observed (15). Because full activation of T cells requires additional costimulatory signals, coexpression of weak self-antigens, such as CEA, with potent costimulatory molecules was used as a strategy to increase the potency of vaccination (16). The B7.1 (CD80) costimulatory molecule binds to CD28 on the surface of T cells and leads to cell proliferation and cytokine release (17, 18). Preclinical studies suggested that coexpression of B7.1 and CEA enhanced tumor-specific immunity and resulted in improved therapeutic responses (19). The inclusion of tetanus toxoid as an adjuvant to MHC class I–restricted peptide vaccines to provide nonspecific helper epitopes has also been shown to induce more robust CD8+ T-cell responses (20). To date, the addition of tetanus toxoid as an adjuvant to poxvirus vaccines has not been tested in the clinic.

A series of phase I clinical trials using an ALVAC virus expressing both CEA and B7.1 showed that vaccination was well tolerated and induced more robust CEA-specific immunity than previous trials with ALVAC expressing CEA alone (3, 4, 15). These trials also showed minor clinical responses that correlated with CEA-specific T-cell responses. The effect of chemotherapy on induction of CEA-specific immunity, however, has not been previously reported in a prospective manner. Thus, we conducted a phase II randomized, multi-institutional clinical trial to test the hypothesis that standard cytotoxic chemotherapy would not adversely affect the generation of CEA-specific T-cell responses induced by an ALVAC vaccine expressing CEA and B7.1.

Study objectives and patient eligibility. The primary objective of this trial was to evaluate the effects of ALVAC-CEA/B7.1 administered concurrently or sequentially with systemic chemotherapy on CEA-specific immunity in patients with metastatic colorectal carcinoma. A secondary objective was to evaluate safety and determine whether nonadsorbed tetanus toxoid could enhance vaccine-induced immunity.

Patients were eligible if they had a confirmed diagnosis of colorectal adenocarcinoma with documented metastatic disease. All patients were required to have an Eastern Cooperative Oncology Group performance status of 0 to 1 and life expectancy greater than 6 mo, to be ≥18 y of age, to be able to provide written informed consent, to be able to comply with all study procedures, and to have been fully recovered from surgery. Baseline laboratory values for study entry included hemoglobin ≥10 g/dL, granulocyte count ≥1,500/mm3, lymphocyte count ≥1,000/mm3, platelet count ≥100,000/mm3, serum creatinine <2.5 mg/dL, total bilirubin <1.5× the normal upper limits, and aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase <3× the normal upper limit, or <5× the normal upper limit if due to liver metastases. Patients had to be chemotherapy naive for treatment of metastatic disease.

Patients were excluded from the study if they were pregnant, had evidence of immunosuppression, required chronic steroids, or had significant comorbid medical problems, uncontrolled infections, autoimmune diseases, clinically active central nervous system disease, or the presence of a second primary tumor within the prior 5 y, except basal cell carcinoma or adequately treated in situ carcinoma of the cervix. Patients with known allergies to egg products, neomycin, or tetanus toxoid were also considered ineligible. Prior exposure to CEA-based immunotherapy was not allowed, and patients could not have received any other investigational product within 28 d of trial registration.

Vaccine preparation. ALVAC-CEA/B7.1 was prepared by recombinant engineering, as described elsewhere (3). Individual vials containing 1 × 107 CCID50/mL of freeze-dried vaccine were stored in a secured, monitored, alarmed refrigerator at 2°C to 8°C. On the day of vaccination, the vaccine was reconstituted with 1 mL of 0.4% NaCl diluent and shaken gently. A sterile syringe was used to inject 1 mL of solution s.c. in the deltoid region. For patients in group 2, 0.05 mL of tetanus toxoid (nonadsorbed) concentrate was diluted in 0.5 mL PBS, and 0.55 mL was either administered alone (priming) or admixed with the ALVAC-CEA/B7.1 vial (boosters).

Chemotherapy regimen. The chemotherapy regimen consisted of irinotecan, fluorouracil, and leucovorin. Initially, the fluorouracil was administered as a bolus, but the regimen was changed to a 46-h infusion after safety concerns were raised by the Food and Drug Administration regarding bolus fluorouracil in the three-drug regimen (21). Thus, the first 15 patients received IFL chemotherapy regimen (5), and the remaining 103 patients received the FOLFIRI chemotherapy regimen (6), which consisted of 180 mg/m2 irinotecan infused over 90 min; 400 mg/m2 leucovorin infused over 2 h, administered concomitantly (not mixed) with each irinotecan dose; 400 mg/m2 5-fluorouracil bolus injection, immediately following each leucovorin dose; and 2.4 g/m2 5-fluorouracil infused over 46 h, following the bolus administration. Dose reduction and/or treatment delays for hematologic and/or nonhematologic toxicities were allowed. All patients were treated with the intent of receiving a total of four 6-wk cycles. Chemotherapy was administered as shown in Fig. 1 for the IFL (Fig. 1A) and FOLFIRI (Fig. 1B) regimens.

Fig. 1.

Protocol schema. The first 15 patients received IFL chemotherapy (A) and the next 103 patients received FOLFIRI (B). Group 1 received three vaccinations (C) followed by vaccination on the first day of each chemotherapy cycle (V). Group 2 patients were treated in a similar manner but also received tetanus toxoid (T) at the indicated times. Group 3 had four cycles of chemotherapy and, if responding, received four vaccinations at the indicated time points. Bracketed areas were repeated for a total of four cycles.

Fig. 1.

Protocol schema. The first 15 patients received IFL chemotherapy (A) and the next 103 patients received FOLFIRI (B). Group 1 received three vaccinations (C) followed by vaccination on the first day of each chemotherapy cycle (V). Group 2 patients were treated in a similar manner but also received tetanus toxoid (T) at the indicated times. Group 3 had four cycles of chemotherapy and, if responding, received four vaccinations at the indicated time points. Bracketed areas were repeated for a total of four cycles.

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Study design and treatment. The clinical protocol was approved by the Institutional Review Board at each participating center, as well as by the NIH Office of Biotechnology Activities and each local institutional biosafety committee. Eligible patients were assigned sequential identification numbers and centrally randomized into one of three treatment groups. Group 1 patients received ALVAC-CEA/B7.1 vaccine (1 × 107 CCID50/mL/injection) by s.c. injection once weekly for 3 successive weeks before chemotherapy. The first chemotherapy cycle started 2 wk after the third vaccination, and patients received an additional booster vaccine on the first day of each of the four chemotherapy cycles for a total of seven vaccinations. The vaccine was administered 30 min before starting chemotherapy. Group 2 patients received the same treatment as group 1, except that a priming dose of nonadsorbed tetanus toxoid (5 Lf/injection) was given 2 wk before the first vaccination, and tetanus toxoid was administered concomitantly with each of the seven ALVAC vaccinations. Group 3 patients received four cycles of systemic chemotherapy; those patients free from disease progression went on to receive ALVAC-CEA/B7.1 (1 × 107 CCID50/mL/injection) vaccine 2 wk after completing chemotherapy by s.c. injection once weekly for 3 successive weeks and once again following a 2-wk rest period for a total of four vaccinations (Fig. 1).

Standard blood counts and serum chemistries were obtained within 28 d of enrollment, during the first week of vaccine treatment, and weekly during each cycle of chemotherapy. Peripheral blood mononuclear cells (PBMC) were collected for immune assays within 28 d of starting treatment and at weeks 1 and 3 of the first two cycles of chemotherapy for patients in groups 1 and 2 and on week 3 of chemotherapy cycle 2 for patients in group 3. An additional sample was collected at the end of the study period for all patients. Serum CEA and autoimmune markers (antinuclear antibody, rheumatoid factor, antimitochondrial antibody, and antimicrosomal antibody) were monitored. A chest X-ray and computed tomography scan of the chest, abdomen, and pelvis were required before study enrollment and after each cycle of chemotherapy. Clinical responses were evaluated using the Response Evaluation Criteria in Solid Tumors criteria.

Immunologic monitoring. Anti-ALVAC antibody titers were detected by standard ELISA assay. Sera from ALVAC-vaccinated donors were used as a positive control and reference control (with assigned ELISA units), and a pool of sera from healthy donors was used as a negative control. Plates were read at 450 nm after 10 min of color development and within 30 min of stopping the reaction using a SpectraMax 190 ELISA reader (Molecular Devices) and SoftMax Pro software.

T-cell responses were evaluated by IFN-γ enzyme-linked immunospot (ELISPOT) assay using Ficoll-separated and cryopreserved (Cryomed) PBMCs collected from patients before and at specific time points after vaccination, similar to methods described elsewhere (4). In HLA-A2+ patients, C1R.A2 cells were used as a source of antigen-presenting cells and were adjusted to 2 × 106/mL and irradiated with 10,000 rads using a Nordion γ-irradiator before pulsing with four separate peptide pools, including negative control (p72 ribosomal protein; 20 μg/mL), HLA-A2–restricted CEA peptides [CAP-1 (YLSGANLNL) and CAP-16D (YLSGADLNL; 7 μg/mL each)], CEA691 (IMIGVLVGV) and CEA694 (GVLVGVALI) peptides (7 μg/mL each), and positive control peptides (Flu285, CMVpp65, and EBV1508; 7 μg/mL each). Peptide-pulsed antigen-presenting cells were incubated for 2 to 3 h at 37°C in a 5% CO2 incubator, washed, and resuspended at 5 × 105/mL, and 100 μL were added to the ELISPOT wells for a final cell concentration of 5 × 104 per well. After overnight incubation, PBMCs were counted and the concentration was adjusted to 1 × 106/mL, and 100 μL were transferred to appropriate wells on the ELISPOT plate. The plates were treated as described previously (4). The number of individual spots per well was counted using an ELISPOT Image Analyzer and software (Cell Technology, Inc.). The precursor frequency was calculated as the number of spot-forming units from wells containing PBMC + antigen-presenting cell + peptide after subtraction of the background (PBMC + antigen-presenting cell alone) relative to the number of PBMC seeded per well.

Statistical methods. The primary end point of this trial was immune response to the vaccine. Safety was determined by recording adverse events by type, seriousness, intensity/grade (using National Cancer Institute Common Toxicity Criteria version 2.0), and relation to malignant disease, the vaccine, and chemotherapy. The number and percentage of patients in each group reporting each type of event were calculated. For neutropenia and diarrhea, which are the most common expected adverse events following chemotherapy, the number and percentage of patients experiencing events of at least grade 3 severity were calculated, along with the 95% exact confidence interval. Treatment arms were compared using a simple regression analysis.

For immune responses, the IFN-γ ELISPOT assay was used, which was similar to assays described by others (3, 4). An increase in the precursor frequency of CEA-specific T-cell responses at any time point of at least 2.4-fold greater than the baseline level was considered positive. Data were reported as a fold increase calculated as the mean T-cell precursor frequency at any time point divided by the mean T-cell precursor frequency at baseline. For each visit, the number and percentage of patients in each group who experienced a greater than 2.4-fold increase from baseline in T-cell reactivity to test peptides were calculated, as well as the number and percentage of patients in each group who experienced a 2.4-fold increase from baseline at any time during the study. For tetanus toxoid effects, a 4-fold increase in T-cell precursor frequency was selected as interesting for further study based on previous ALVAC trials, which established a SD of the log of the precursor frequency to be 0.74 and was based on an assumption that all patients receiving tetanus toxoid would experience a similar fold increase in immune response and the SD would remain the same (4). To document a 4-fold increase between groups using a one-sided t test with an α level of 0.05, a power of 90%, and a SD of 1.25, 15 evaluable HLA-A2+ patients per group were required.

Study population. One hundred eighteen patients were randomized to one of three treatment groups: 39 (33%) to group 1, 40 (34%) to group 2, and 39 (33%) to group 3. Patient characteristics are listed in Table 1. One patient in group 2 received only the priming dose of tetanus toxoid and withdrew from the trial before starting ALVAC-CEA/B7.1 therapy. In group 1, 3 (7.7%) patients received the vaccine but did not go on to chemotherapy, and 36 (92.3%) patients received both vaccine and chemotherapy. In group 2, 2 (5%) patients received only vaccine, and 37 (92.5%) patients received both vaccine and chemotherapy. In group 3, 15 (38.5%) patients had only chemotherapy, and 24 (61.5%) patients received both treatment regimens. Overall, 26 patients in each treatment group completed the trial as planned. The reasons for study discontinuation included death due to tumor progression (3), disease progression based on clinical judgment (8), or objective tumor measurements (19), patient noncompliance (1), withdrawn consent (4), or other nonspecified reasons (2).

Table 1.

Patient characteristics

CharacteristicsGroup 1 (ALVAC + chemotherapy), n (%)Group 2 (ALVAC + TT + chemotherapy), n (%)Group 3 (chemotherapy + ALVAC), n (%)
Sample size 39 (33.05) 40 (33.9) 39 (33.05) 
Mean age (y) 59.8 56.5 56.7 
Gender    
    Male 23 (59) 23 (57.5) 24 (61.5) 
    Female 16 (41) 17 (42.5) 15 (38.5) 
Ethnicity    
    Asian 0 (0) 3 (7.5) 2 (5.1) 
    Black 4 (10.3) 1 (2.5) 3 (7.7) 
    Caucasian 33 (84.6) 32 (80) 32 (82.1) 
    Hispanic 2 (5.1) 4 (10) 1 (2.6) 
    Other 0 (0) 0 (0) 1 (2.6) 
Mean CEA (ng/mL)    
    Screening 201.5 279.3 397.3 
    Final 44.0 76.2 12.0 
Performance status    
    0 21 (53.8) 26 (65) 28 (71.8) 
    1 18 (46.2) 14 (35) 11 (28.2) 
CharacteristicsGroup 1 (ALVAC + chemotherapy), n (%)Group 2 (ALVAC + TT + chemotherapy), n (%)Group 3 (chemotherapy + ALVAC), n (%)
Sample size 39 (33.05) 40 (33.9) 39 (33.05) 
Mean age (y) 59.8 56.5 56.7 
Gender    
    Male 23 (59) 23 (57.5) 24 (61.5) 
    Female 16 (41) 17 (42.5) 15 (38.5) 
Ethnicity    
    Asian 0 (0) 3 (7.5) 2 (5.1) 
    Black 4 (10.3) 1 (2.5) 3 (7.7) 
    Caucasian 33 (84.6) 32 (80) 32 (82.1) 
    Hispanic 2 (5.1) 4 (10) 1 (2.6) 
    Other 0 (0) 0 (0) 1 (2.6) 
Mean CEA (ng/mL)    
    Screening 201.5 279.3 397.3 
    Final 44.0 76.2 12.0 
Performance status    
    0 21 (53.8) 26 (65) 28 (71.8) 
    1 18 (46.2) 14 (35) 11 (28.2) 

Abbreviation: TT, tetanus toxoid.

Treatment characteristics and toxicity. In groups 1 and 2, 65.5% of the patients (51 of 79) received all seven vaccinations as planned. In group 3, 24 (61.5%) patients were free of disease progression after chemotherapy and went on to vaccination, with 2 patients receiving three immunizations and 22 patients receiving all four. The majority of patients in all three treatment groups received four cycles of chemotherapy (n = 79).

The adverse events were largely related to chemotherapy administration (Table 2). The most frequently observed grade 3 and 4 toxicities were related to hematologic and gastrointestinal events, including dehydration (n = 9), diarrhea (n = 12), nausea (n = 8), vomiting (n = 14), granulocytopenia (n = 18), and lymphopenia (n = 6). Fatigue was reported in 9 patients, and there were 12 reports of thromboembolic complications. There were no differences in grade 3 or 4 toxicities between the treatment groups (P = 0.74).

Table 2.

Adverse events

SystemAdverse eventWorst gradeGroup 1 (ALVAC + chemotherapy, n = 39), n (%)Group 2 (ALVAC + TT + chemotherapy, n = 40), n (%)Group 3 (chemotherapy + ALVAC, n = 39), n (%)
Constitutional Fatigue 4 (10.3) 4 (10) 1 (2.6) 
Gastrointestinal Dehydration 4 (10.3) 2 (5) 2 (5.1) 
 Diarrhea 3 (7.7) 7 (17.5) 2 (5.1) 
 Nausea 4 (10.3) 3 (7.5) 1 (2.6) 
 Vomiting 6 (15.4) 3 (7.5) 5 (12.8) 
Hematologic Granulocytes 5 (12.8) 3 (7.5) 10 (25.6) 
 Platelets 1 (2.6) 1 (2.5) 0 (0) 
 Hemoglobin 0 (0) 1 (2.5) 1 (2.6) 
 WBC 2 (5.1) 0 (0) 3 (7.7) 
 Lymphocytes 4 (10.3) 0 (0) 2 (5.1) 
Hepatic Bilirubin 0 (0) 1 (2.5) 1 (2.6) 
 ALT 0 (0) 1 (2.5) 0 (0) 
 ALP 0 (0) 3 (7.5) 1 (2.6) 
Renal Hypokalemia 0 (0) 1 (2.5) 1 (2.6) 
 Hyponatremia 0 (0) 1 (2.5) 0 (0) 
Vascular Thromboembolism 4 (10.3) 5 (12.5) 3 (7.7) 
SystemAdverse eventWorst gradeGroup 1 (ALVAC + chemotherapy, n = 39), n (%)Group 2 (ALVAC + TT + chemotherapy, n = 40), n (%)Group 3 (chemotherapy + ALVAC, n = 39), n (%)
Constitutional Fatigue 4 (10.3) 4 (10) 1 (2.6) 
Gastrointestinal Dehydration 4 (10.3) 2 (5) 2 (5.1) 
 Diarrhea 3 (7.7) 7 (17.5) 2 (5.1) 
 Nausea 4 (10.3) 3 (7.5) 1 (2.6) 
 Vomiting 6 (15.4) 3 (7.5) 5 (12.8) 
Hematologic Granulocytes 5 (12.8) 3 (7.5) 10 (25.6) 
 Platelets 1 (2.6) 1 (2.5) 0 (0) 
 Hemoglobin 0 (0) 1 (2.5) 1 (2.6) 
 WBC 2 (5.1) 0 (0) 3 (7.7) 
 Lymphocytes 4 (10.3) 0 (0) 2 (5.1) 
Hepatic Bilirubin 0 (0) 1 (2.5) 1 (2.6) 
 ALT 0 (0) 1 (2.5) 0 (0) 
 ALP 0 (0) 3 (7.5) 1 (2.6) 
Renal Hypokalemia 0 (0) 1 (2.5) 1 (2.6) 
 Hyponatremia 0 (0) 1 (2.5) 0 (0) 
Vascular Thromboembolism 4 (10.3) 5 (12.5) 3 (7.7) 

Abbreviations: ALT, alanine aminotransferase; ALP, alkaline phosphatase.

Tumor marker and clinical responses. Serum CEA levels were monitored at baseline and after each cycle of chemotherapy. At the screening visit, the mean CEA levels for groups 1, 2, and 3 were 201.5 (±119.9), 279.3 (±214.6), and 397.3 (±282.0), respectively. Following chemotherapy, there was a decrease in the mean serum CEA levels across all treatment groups to 18.3 (±8.0) in group 1 and 68.8 (±42.8) for group 2. The mean CEA level for group 3 decreased to 12.0 (±3.3).

Overall, 42 (40.4%) of 104 evaluable patients achieved an objective clinical response by Response Evaluation Criteria in Solid Tumors criteria (Table 3). Of the two patients who had a complete response, both were in group 3. Objective partial responses were seen in 15 (44.1%) patients in group 1, 10 (31.3%) patients in group 2, and 15 (39.5%) patients in group 3. Stable disease was observed in an additional 42 (37.5%) patients equally distributed across the treatment groups.

Table 3.

Best overall clinical responses

ResponseGroup 1 (ALVAC + chemotherapy, n = 34), n (%)Group 2 (ALVAC + TT + chemotherapy, n = 32), n (%)Group 3 (chemotherapy + ALVAC, n = 38), n (%)
Complete response 0 (0) 0 (0) 2 (5.3) 
Partial response 15 (44.1) 10 (31.3) 15 (39.5) 
Stable disease 14 (41.2) 15 (46.9) 13 (34.2) 
Progressive disease 5 (14.7) 5 (15.6) 5 (13.2) 
Not evaluable 0 (0) 2 (6.3) 3 (7.9) 
Total objective responses (complete + partial response) 15 (44.1) 10 (31.3) 17 (44.7) 
ResponseGroup 1 (ALVAC + chemotherapy, n = 34), n (%)Group 2 (ALVAC + TT + chemotherapy, n = 32), n (%)Group 3 (chemotherapy + ALVAC, n = 38), n (%)
Complete response 0 (0) 0 (0) 2 (5.3) 
Partial response 15 (44.1) 10 (31.3) 15 (39.5) 
Stable disease 14 (41.2) 15 (46.9) 13 (34.2) 
Progressive disease 5 (14.7) 5 (15.6) 5 (13.2) 
Not evaluable 0 (0) 2 (6.3) 3 (7.9) 
Total objective responses (complete + partial response) 15 (44.1) 10 (31.3) 17 (44.7) 

Immune responses. T-cell responses were assessed in HLA-A2+ patients who had completed at least two cycles of chemotherapy along with the accompanying vaccinations (groups 1 and 2) or had completed all four chemotherapy cycles and received at least one vaccination (group 3). Forty-two patients met the above criteria for T-cell analysis: 16 in group 1, 16 in group 2, and 10 in group 3. Humoral responses were done on samples from these patients and an additional 20 patients where samples were available.

All patients tested who received ALVAC-CEA/B7.1 developed an increase in IgG titers against ALVAC following vaccination (Fig. 2A). We did not detect significant changes in the anti-CEA antibody titers in the majority of patients following vaccination (Fig. 2B): three patients had slight increases and five patients showed a slight decrease. We did not directly measure antigen-antibody complexes in this study.

Fig. 2.

Antibody responses to ALVAC-CEA/B7.1 vaccine. Patient sera collected before (Pre) and after (Post) treatment were tested for anti-ALVAC titers (A) and anti-CEA titers (B) by standard ELISA assay. Bars, mean for each group.

Fig. 2.

Antibody responses to ALVAC-CEA/B7.1 vaccine. Patient sera collected before (Pre) and after (Post) treatment were tested for anti-ALVAC titers (A) and anti-CEA titers (B) by standard ELISA assay. Bars, mean for each group.

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The T-cell response was measured by direct ex vivo IFN-γ ELISPOT assay using HLA-A2–restricted CEA peptide pools with a 2.4-fold increase in precursor frequency defined as a “positive” response. The majority of patients in all three treatment groups showed T-cell reactivity against the recall antigens (Fig. 3). CEA-specific T cells were evaluated using a mixture of the HLA-A*0201 CAP-1 and modified CAP-1 peptides described by Schlom and colleagues (CAP-1 pool) or two other HLA-A2–restricted CEA peptides (CEA pool). Of the 16 group 1 patients eligible for T-cell analysis, 5 (31.3%) had a greater than 2.4-fold increase in CEA-specific T cells after vaccination and 8 (50%) had an increase in CAP-specific T-cell responses (Fig. 3A). An additional eight (50%) patients exhibited some reactivity against the CEA and CAP peptides but did not show a 2.4-fold increase. Among the 16 group 2 patients included in the analysis, 6 (37.5%) responded to the CEA peptides and 6 (37.5%) responded to the CAP peptides with a greater than 2.4-fold increase in T-cell response (Fig. 3B). An additional six patients reacted against CEA peptides, and five patients reacted against CAP peptides but did not increase by ≥2.4-fold. All patients in this cohort reacted to tetanus peptides at all time points measured (data not shown). The frequency of patients responding to CEA peptides and CAP peptides was similar for the groups 1 and 2. Of the 10 group 3 patients eligible for analysis, 2 (20%) exhibited a 2.4-fold or greater increase in T-cell response against the CEA peptides and 3 (30%) had an increase of greater than 2.4-fold against the CAP peptides (Fig. 3C). Another four (40%) patients had some increase in CEA-specific T-cell response but did not reach the 2.4-fold level.

Fig. 3.

Best T-cell response to ALVAC-CEA/B7.1 vaccine. HLA-A2+ patient PBMCs from treatment groups 1 (A), 2 (B), and 3 (C) were tested by direct ex vivo IFN-γ ELISPOT assay to standard recall peptides (CEF; Flu285, CMVpp65, and EBV1508), a pool of defined HLA-A2–restricted CEA peptides, CAP-1 and CAP-16D (CAP), or two novel CEA peptides, CEA691 and CEA694 (CEA). A p72 ribosomal protein antigen peptide was used as a negative control (data not shown). The positive T-cell response to CAP-1, CEA, and CEF peptides is shown as percentage (%) of patients.

Fig. 3.

Best T-cell response to ALVAC-CEA/B7.1 vaccine. HLA-A2+ patient PBMCs from treatment groups 1 (A), 2 (B), and 3 (C) were tested by direct ex vivo IFN-γ ELISPOT assay to standard recall peptides (CEF; Flu285, CMVpp65, and EBV1508), a pool of defined HLA-A2–restricted CEA peptides, CAP-1 and CAP-16D (CAP), or two novel CEA peptides, CEA691 and CEA694 (CEA). A p72 ribosomal protein antigen peptide was used as a negative control (data not shown). The positive T-cell response to CAP-1, CEA, and CEF peptides is shown as percentage (%) of patients.

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In this randomized pilot phase II clinical trial, we found that combination ALVAC-CEA/B7.1 vaccine/chemotherapy treatment was well tolerated and most patients completed the entire regimen. Toxicities were generally related to the expected chemotherapy side effects. The most common grade 3 and 4 side effects observed were gastrointestinal (n = 30), hematologic (n = 24), thromboembolic (n = 12), fatigue (n = 9), increase in hepatic enzymes (n = 7), and electrolyte imbalances (n = 3). There were no differences in the incidence of these events between the various treatment groups. Thus, combining ALVAC-CEA/B7.1 vaccine with systemic chemotherapy was feasible and there was no unexpected toxicity associated with the combination or sequencing of therapy.

Although the trial was not designed to detect clinical responses, we observed an overall objective response rate of 40.4% with two complete responses and an additional 37.5% of patients experienced stable disease. This compares quite favorably with the 39% objective response rate reported for FOLFIRI chemotherapy and 35% in the intention-to-treat analysis reported by Douillard et al. (8) and Saltz et al. (9). All patients were successfully immunized, as indicated by the appearance of high anti-ALVAC antibody titers (Fig. 2A). Despite the increased antiviral titers, we did not detect an appreciable increase in anti-CEA antibody titers following vaccination. Some patients had slightly elevated CEA-specific antibody titers before treatment and this is consistent with previous reports (22). The high levels of circulating CEA from metastatic tumor in our patient population may have induced a low level antibody response and it is possible that detection of anti-CEA antibody titers was hindered by antigen-antibody complex formation. The weak immunogenicity of CEA and the sensitivity of our assay may be other explanations for the lack of detectable anti-CEA titers.

Overall, 13 (31%) of the evaluable patients exhibited at least a 2.4-fold increase in CEA-specific T-cell precursors after vaccination. The mean fold increase in groups 1, 2, and 3 were 2.8, 2.4, and 2.6, respectively. Another 16 patients had increases but not above 2.4-fold, which we defined as cutoff for “positivity” in this study. In previous trials, a mean 2.4-fold increase in CEA-specific T-cell responses by ELISPOT was correlated with clinical outcomes and, hence, was used to define a positive immune response in this trial. This may have underestimated the true rate of positive responders because an additional 16 patients had elevated CEA-reactive T cells as defined by at least a 2-fold increase over baseline. In any event, the frequency of T-cell responses in this trial was similar to previously reported responses in phase I trials (3, 4). Thus, the administration of chemotherapy did not seem to significantly inhibit the induction of CEA-specific T-cell responses by ALVAC-CEA/B7.1 vaccination. Although there were more patients in group 1 with increased CEA-specific T-cell responses, the frequency was not statistically different from the other groups. The data also suggested that tetanus toxoid was not a useful adjuvant as we did not observe any significant difference in CEA-specific T-cell responses in the patients enrolled in group 2, although all patients reacted to tetanus. The rationale for including tetanus was based on the ability to induce nonspecific CD4+ T helper cell responses, which should help promote activation of CD8+ T-cell responses (23).

The clinical application of combination chemotherapy and vaccines will depend on a better understanding of the mechanisms by which such combinations augment therapeutic responses. Chemotherapy may augment antitumor immunity through a variety of mechanisms, including depletion of regulatory T cells, increased tumor cell apoptosis/necrosis, and subsequent antigen presentation, creating increased danger signals through tumor and tissue damage, reduced tumor burden, and hence tumor-induced immune suppression and through lymphodepletion-induced homeostatic repopulation (24). There is currently little data available on how individual cytotoxic chemotherapy agents influence immune responses and these agents may have pleiotropic actions, making it difficult to predict the outcome of any particular combination regimen. For example, 5-fluorouracil has been reported to promote apoptosis of activated peripheral blood T cells but may also serve to augment antitumor immunity through FasL-mediated killing by tumor-infiltrating T cells and increased antigen presentation (25, 26). The effects of irinotecan on immune responses is also poorly defined, with evidence for both increased and decreased CD4+ T-cell counts following exposure to the drug (27). The timing of vaccination in relation to systemic chemotherapy is another important variable to consider. For example, nonmyeloablative conditioning chemotherapy has been shown to induce significant clinical responses when administered before adoptively transferred T-cell therapy and improve host T-cell responses to vaccination after bone marrow transplantation (2831).

In conclusion, we have shown that vaccination with ALVAC-CEA/B7.1 vaccine is feasible in combination with fluorouracil, leucovorin, and irinotecan in patients with metastatic colorectal cancer. The regimen has an acceptable safety profile and induces antibody titers against the ALVAC vector and T-cell responses against CEA in some patients. Our data suggest that T-cell responses to the ALVAC-CEA/B7.1 vaccine were not impaired by the FOLFIRI chemotherapy combination. Future studies based on careful selection of agents to maximize therapeutic activity, inclusion of novel immune adjuvants, and optimizing the timing of administration will be necessary to better define the role of combination chemoimmunotherapy in patients with advanced colorectal cancer.

M. Yu, J. Caterini, M. DeBenedette, D. Salha, T. Vogel, and I. Elias are employed by Sanofi Pasteur.

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.

Note: This work has not been published or presented elsewhere.

1
Meyerhardt JA, Mayer RJ. Systemic therapy for colorectal cancer.
N Engl J Med
2005
;
352
:
476
–87.
2
Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005.
CA Cancer J Clin
2005
;
55
:
10
–30.
3
Horig H, Lee DS, Conkright W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule.
Cancer Immunol Immunother
2000
;
49
:
504
–14.
4
von Mehren M, Arlen P, Tsang KY, et al. Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas.
Clin Cancer Res
2000
;
6
:
2219
–28.
5
Hoos A, Parmiani G, Hege K, et al. A clinical development paradigm for cancer vaccines and related biologics.
J Immunother
2007
;
30
:
1
–15.
6
Cunningham D, Pyrhonen S, James RD, et al. Randomised trial of irinotecan plus supportive care versus supportive care alone after fluorouracil failure for patients with metastatic colorectal cancer.
Lancet
1998
;
352
:
1413
–8.
7
Rougier P, Van Cutsem E, Bajetta E, et al. Randomised trial of irinotecan versus fluorouracil by continuous infusion after fluorouracil failure in patients with metastatic colorectal cancer.
Lancet
1998
;
352
:
1407
–12.
8
Douillard JY, Cunningham D, Roth AD, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial.
Lancet
2000
;
355
:
1041
–7.
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
Horig H, Medina FA, Conkright WA, et al. Strategies for cancer therapy using carcinoembryonic antigen vaccines.
Expert Rev Mol Med
2000
;
2
:
1
–24.
11
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.
12
Zaremba S, Barzaga E, Zhu M, et al. Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
Cancer Res
1997
;
57
:
4570
–7.
13
Musey L, Ding Y, Elizaga M, et al. HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals.
J Immunol
2003
;
171
:
1094
–101.
14
Paoletti E. Applications of pox virus vectors to vaccination: an update.
Proc Natl Acad Sci U S A
1996
;
93
:
11349
–53.
15
Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen.
J Clin Oncol
1999
;
17
:
332
–7.
16
Liu B, Podack ER, Allison JP, et al. Generation of primary tumor-specific CTL in vitro to immunogenic and poorly immunogenic mouse tumors.
J Immunol
1996
;
156
:
1117
–25.
17
Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity.
Nat Rev Immunol
2004
;
4
:
336
–47.
18
Allison JP. CD28-B7 interactions in T-cell activation.
Curr Opin Immunol
1994
;
6
:
414
–9.
19
Akagi J, Nakagawa K, Egami H, et al. Induction of HLA-unrestricted and HLA-class-II-restricted cytotoxic T lymphocytes against MUC-1 from patients with colorectal carcinomas using recombinant MUC-1 vaccinia virus.
Cancer Immunol Immunother
1998
;
47
:
21
–31.
20
La Rosa C, Wang Z, Brewer JC, et al. Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice.
Blood
2002
;
100
:
3681
–9.
21
Rothenberg ML, Meropol NJ, Poplin EA, et al. Mortality associated with irinotecan plus bolus fluorouracil/leucovorin: summary findings of an independent panel.
J Clin Oncol
2001
;
19
:
3801
–7.
22
Marshall J. Carcinoembryonic antigen-based vaccines.
Semin Oncol
2003
;
30
:
30
–6.
23
Slingluff CL, Jr., Yamshchikov G, Neese P, et al. Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.
Clin Cancer Res
2001
;
7
:
3012
–24.
24
Gattinoni L, Powell DJ, Jr., Rosenberg SA, et al. Adoptive immunotherapy for cancer: building on success.
Nat Rev Immunol
2006
;
6
:
383
–93.
25
Cheng H, Liu Y, Liu S, et al. 5-Fluorouracil enhances apoptosis sensitivity of T lymphocytes mediated by CD3ε.
Cell Biochem Funct
2004
;
22
:
187
–95.
26
Yang S, Haluska FG. Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways.
J Immunol
2004
;
172
:
4599
–608.
27
Melichar B, Touskova M, Vesely P. Effect of irinotecan on the phenotype of peripheral blood leukocyte populations in patients with metastatic colorectal cancer.
Hepatogastroenterology
2002
;
49
:
967
–70.
28
Mackall CL, Hakim FT, Gress RE. Restoration of T-cell homeostasis after T-cell depletion.
Semin Immunol
1997
;
9
:
339
–46.
29
Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes.
Science
2002
;
298
:
850
–4.
30
Zoller M. Tumor vaccination after allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned tumor-bearing murine host.
J Immunol
2003
;
171
:
6941
–53.
31
Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.
J Clin Oncol
2005
;
23
:
2346
–57.