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Phase I Study in Advanced Cancer Patients of a Diversified Prime-and-Boost Vaccination Protocol Using Recombinant Vaccinia Virus and Recombinant Nonreplicating Avipox Virus to Elicit Anti–Carcinoembryonic Antigen Immune Responses

From the Georgetown University Medical Center, Vincent T. Lombardi Cancer Center, Washington, DC; Laboratory of Tumor Immunology and Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD; and University of Virginia Health Sciences Center, Charlottesville, VA.

Address reprint requests to John L. Marshall, MD, Lombardi Cancer Center, 3800 Reservoir Rd NW, Washington, DC 20007; email marshallj{at}gunet.georgetown.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: This trial sought to determine, for the first time, the validity in human vaccinations of using two different recombinant vaccines in diversified prime-and-boost regimens to enhance T-cell responses to a tumor antigen.

PATIENTS AND METHODS: Eighteen patients with advanced tumors expressing carcinoembryonic antigen (CEA) were randomized to receive either recombinant vaccinia (rV)-CEA followed by three avipox-CEA vaccinations, or avipox-CEA (three times) followed by one rV-CEA vaccination. Subsequent vaccinations in both cohorts were with avipox-CEA. Immunologic monitoring was performed using a CEA peptide and the enzyme-linked immunospot assay for interferon gamma production.

RESULTS: rV-CEA followed by avipox-CEA was superior to the reverse order in the generation of CEA-specific T-cell responses. Further increases in CEA-specific T-cell precursors were seen when local granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose interleukin (IL)-2 were given with subsequent vaccinations. The treatment was extremely well tolerated. Limited clinical activity was seen using vaccines alone in this patient population. Antibody production against CEA was also observed in some of the treated patients.

CONCLUSION: rV-CEA was more effective in its role as a primer of the immune system; avipox-CEA could be given up to eight times with continued increases in CEA T-cell precursors. Future trials should use rV-CEA first followed by avipox-CEA. Vaccines specific to CEA are able to generate CEA-specific T-cell responses in patients without significant toxicity. T-cell responses using vaccines alone may be inadequate to generate significant anticancer objective responses in patients with advanced disease. Cytokines such as GM-CSF and IL-2 may play a key role in generating such responses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
CARCINOEMBRYONIC antigen (CEA) is a 180,000 molecular weight oncofetal glycoprotein expressed in the normal fetal colon. In adults, CEA has been found in lower levels in normal colonic mucosa and also in saliva, feces, serum, and colonic lavages.¹ CEA is overexpressed in virtually all colorectal adenocarcinomas and most adenocarcinomas of the pancreas, stomach, breast, and lung.^2-4 Many colorectal cancers and some carcinomas at other sites produce high levels of CEA that are measurable in sera.⁵ Because of this, CEA is one of the most widely used serologic markers of malignancy, especially in patients with colorectal cancer.

It has been proposed that CEA functions as an intercellular recognition and adhesion molecule.⁶ Increased CEA expression by a group of cells may promote metastasis through increased intercellular adhesions mediated by CEA. After metastasizing from a primary tumor, a group of adhesive cells may more easily survive to reach a distant organ and form a secondary tumor.

Using CEA as a target in immunologic-based therapies has two potential problems. First, given that CEA is a normal protein expressed in the body, it is likely that tolerance will exist to this protein. Secondly, if one were successful in generating such an immune response, the result could lead to autoimmune disease. On the other hand, if one were successful at this, the impact of such therapy would have tremendous clinical implications. Thus immunotherapy protocols are being designed to produce an immune response against CEA-bearing cancer cells by generating cytotoxic T lymphocytes (CTL) that lyse CEA-expressing cancer cells while sparing the normal CEA-expressing gut cells. This may be possible because CEA is expressed at higher levels in carcinoma cells versus normal colonic epithelial cells.⁷ A recombinant vaccinia virus containing the CEA gene (designated rV-CEA) has been developed.^8,9 This virus is capable of infecting professional antigen-presenting cells (APCs) and presenting CEA peptides to T lymphocytes in the context of HLA class I and II molecules, which in turn activate the corresponding CD8⁺ or CD4⁺ T cells.^8,10,11 The safety of rV-CEA has been documented in nonhuman primates.¹¹ In a phase I clinical trial, the safety of rV-CEA was demonstrated in humans; however, no significant antineoplastic effect was observed.^11-13 Possible reasons for the lack of clinical efficacy in these trials were (1) prior exposure to the vaccinia virus in all patients treated, which led to the development of antivaccinia immune responses on repeated dosings of the vaccine, (2) the advanced state of the tumors in patients, and (3) potentially compromised immune status of patients owing to prior chemotherapy regimens.

The phase I rV-CEA study demonstrated that CEA-specific T-cell responses could be generated in humans through administration of a vaccine.¹¹ This study also showed that CTL cell lines could be generated from peripheral-blood mononuclear cells (PBMCs) of rV-CEA–vaccinated patients in the presence of a CEA peptide, designated carcinoembryonic antigen peptide-1 (CAP-1). This 9-mer amino acid peptide (YLSGANLNL) has been shown to bind HLA-A2 class I molecules. Tumor cells expressing HLA-A2 molecules and CEA were lysed by CAP-1–specific CTL from HLA-A2–positive vaccinated patients, whereas non–HLA-A2–expressing cells were not lysed. This finding indicated that CTL-mediated lysis occurred in a major histocompatibility complex–restricted manner. Stable CTL lines derived by culture of PBMCs from rV-CEA–vaccinated patients with CAP-1 peptide and interleukin (IL)-2 have also been described.^14,15 Recently, a CAP-1 agonist epitope has been identified and designated (CAP-1-6D), which has been shown to activate T cells to even higher levels.^16,17

Another recombinant anti-CEA vaccine, avipox-CEA, has been developed.^9,18 The canarypox vector used in this trial has been termed ALVAC. Similar to rV-CEA, avipox-CEA contains the CEA gene in its genome but, unlike rV-CEA, cannot replicate in mammalian cells. Avipox viruses, such as ALVAC and fowlpox, infect mammalian cells, express their transgene product for 14 to 21 days before death of the cell, and then do not infect other cells. Therefore, systemic infections and the resulting influenza-like symptoms as seen with rV-CEA do not occur. Additionally, humans are unlikely to have had prior exposure to this virus. The safety of avipox-CEA has been documented in a phase I trial in patients with advanced carcinomas.¹⁹ A moderate but statistically significant increase in the number of CEA-specific CTL precursors was observed in seven of nine HLA-A2–positive patients treated with avipox-CEA; however, no true, objective anticancer effects were seen. Possible explanations for the low number of CTL precursors observed include decreased immune status and/or preexisting immune suppression related to the advanced state of disease in the patients studied.

Preclinical evidence has indicated that the combination of rV-CEA and avipox-CEA in diversified prime-and-boost protocols would in fact generate a more vigorous T-cell response than either vaccine alone.¹⁸ When rV-CEA was used to prime the immune system and avipox-CEA was used as a boost in the experimental model, CEA-specific T-cell responses were at least four times greater than those achieved with three vaccinations of avipox-CEA alone. Multiple boosts of avipox-CEA further potentiated these CEA-specific T-cell responses.¹⁸ This preclinical finding, combined with the results of the phase I trials using either rV-CEA or avipox-CEA alone, justified a phase I trial to validate this concept of diversified prime-and-boost vaccination protocol for the first time in patients with advanced carcinomas. Preclinical data also demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose IL-2 can potentiate the CEA-specific immune responses to rV-CEA vaccinations; little, if any, effect was seen when the cytokines were used alone.^20,21

In this study, we proposed to treat cancer patients with CEA-bearing tumors with rV-CEA (V) and avipox-CEA (A) to determine (1) the safety of the two agents in this population, (2) whether the sequence of administration (ie, VAAA v AAAV) has an effect on T-cell response, and (3) whether any objective responses could be achieved using vaccines alone in patients with metastatic disease. Although preclinical evidence supports the addition of cytokines to these vaccines,^20,21 our initial studies were performed with the vaccines alone to first document safety of the diversified prime-and-boost vaccine combination. An enzyme-linked immunospot (ELISPOT) assay was selected to monitor CEA-specific T-cell responses to a CEA 9-mer peptide. The ELISPOT assay used for interferon gamma (IFN-) production required only a 24-hour in vitro stimulation of PBMCs from patients, either pre- or postvaccination. An identical ELISPOT assay to an influenza (Flu) 9-mer peptide was used simultaneously as a control.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Eligibility
To be eligible for this trial, patients had to meet the following criteria: pathologic evidence for advanced, incurable (or high-risk) malignancy (patients with stage IV malignancy but without radiographic evidence of disease were eligible); serum CEA at least 10 ng/mL at some point in the past or tumor that stained positively for CEA by immunohistochemical techniques; age at least 18 years; anticipated survival of at least 6 months; ability to give informed consent; performance status of 0 or 1 (Eastern Cooperative Oncology Group); WBC count of at least 3,000/µL and platelet count of at least 100,000/µL; prothrombin time and partial thromboplastin time within normal ranges; normal serum creatinine level or creatinine clearance at least 60 mL/min; adequate immunologic function, defined by normal delayed-type hypersensitivity, normal CD4:CD8 ratio (> 1) or normal immunoelectrophoresis; human immunodeficiency virus seronegativity; no other diagnoses of altered immune function; no prior radiation to more than 50% of all nodal groups; and no concurrent use of corticosteroids. Contraindications to enrollment included history of another malignancy in the past 2 years, prior radiation to the pelvis, recent major surgery, pregnancy or lactation, serious intercurrent illness, and clinically evident brain metastasis. Patients who received avipox-CEA in a previous clinical trial were able to participate in this trial, provided that they still met the eligibility criteria. Three such patients were enrolled; these patients were enrolled to explore the role of a delay in vaccinations on the immune system T-cell response and were evaluated separately from the other patients in this trial. These patients are clearly identified in the Results section, and results from these patients were not included in the evaluations to define the optimal prime-and-boost protocol.

Treatment
Twelve HLA-A2–positive patients were initially selected for this study because of requirements of the T-cell assay used for monitoring purposes (see Immunologic Monitoring Methods: ELISPOT and Antibody Assays). It was unknown whether priming the immune system with a more potent immunogen (rV-CEA) would produce a greater T-cell response compared with boosting the immune system with this immunogen. Therefore, patients were randomly assigned to one of two study cohorts. The first cohort received one vaccination of rV-CEA followed by three vaccinations of avipox-CEA (designated VAAA). The second cohort received vaccinations in the reverse order (AAAV). Three HLA-A2–negative patients were randomized to each cohort to expand the clinical experience in HLA-A2–negative patients and further document safety. All vaccinations were administered 4 weeks apart, with each 28-day period constituting a cycle. Cycles of 28 days were considered optimal, because longer intervals would be unacceptable for cancer patients and shorter intervals might lessen the immune response. rV-CEA (1.0 x 10⁷ pfu) was administered intradermally into the deltoids. Avipox-CEA (2.5 x 10⁷ plaque-forming units [pfu]) was administered subcutaneously in two equally divided doses (for volume purposes), using the Biojector 2000 (Bioject Inc., Portland, OR) needle-free system, into the arm, thighs, or buttocks (the injection site was rotated). Dose levels for both vaccines were documented in previous clinical trials.^11,19 Patients in both cohorts were monitored before each injection and 4 weeks after the final injection by physical examination, measurement of performance status, complete blood cell counts, blood chemistry profile, urinalysis, and measurement of CEA level; PBMCs for T-cell immunologic monitoring and serum for measuring antibody production against CEA were also taken at these time intervals. Tumor responses were evaluated after every two treatment cycles.

Investigational Use of GM-CSF and IL-2 as Vaccine Adjuvants
Preclinical studies with vaccines have shown that local administration of GM-CSF at the vaccination site^21-23 or systemic administration of low-dose IL-2 after rV-CEA vaccination²⁰ enhanced CEA-specific CTL responses when compared with the use of rV-CEA alone. After undergoing four cycles of vaccine treatment (VAAA or AAAV), patients who had no evidence of progressive disease could elect to continue to receive vaccinations with avipox-CEA with GM-CSF (Leukine; Immunex Corporation, Seattle, WA) for two cycles. GM-CSF was prepared by reconstituting GM-CSF in lyophilized powder form with sterile water to a concentration of 500 µg/mL for injection per United States Pharmacopeia. GM-CSF 100 µg was patient-administered as close to the most recent vaccination sites (ie, both arms) as possible, beginning on the day of avipox-CEA vaccination and for three consecutive days thereafter. If patients continued to show no further disease progression after two cycles, patients could elect to receive avipox-CEA with GM-CSF and IL-2 (Proleukin; Chiron, Emeryville, CA). IL-2 was prepared by reconstituting lyophilized IL-2 to a final concentration of 18.0 x 10⁶ IU/m². The solution was stored at 4°C and administered at room temperature within 48 hours of reconstitution. IL-2 was patient-administered on days 7 through 11 of each cycle at a dosage of 6.0 x 10⁶ IU/m². Both the GM-CSF and IL-2 were provided to us by the Cancer Therapy Evaluation Program of the National Cancer Institute, Bethesda, MD.

Vaccine Preparation
rV-CEA is a live vaccinia virus prepared from the Wyeth New York Board of Health strain of vaccine. It has been genetically engineered using a plasmid vector to carry a copy of the human CEA gene in the viral 30K gene (Hind III M fragment). The vaccine was manufactured by Therion Biologics Corporation (Cambridge, MA). Virus for vaccination was grown in the CV-1 monkey kidney cell line. The vaccine was stored in vials of 1.0 x 10⁹ pfu/mL. Vials were kept at -70°C until the day of administration and thawed before use at room temperature, at which point 10 µL (1.0 x 10⁷ pfu) of the vaccine was administered intradermally into the deltoids. Any remaining units of vaccine were stored at 4°C for no more than 4 days. Vaccine preparation was performed in a sterile hood.

Avipox-CEA is a recombinant canarypox virus (ALVAC) that contains the entire human CEA gene. The vaccine was manufactured by Pasteur-Mérieux Serums et Vaccins (Marcy, France)/Virogenetics (Troy, NY). The canarypox strain from which ALVAC was derived was first isolated at the Rentschler Bakteriologisches Institute (Lauphein, Württemberg, Germany), where it was attenuated by serial passage in chick embryo fibroblasts. The recombinant virus was grown and generated on chick embryo fibroblasts from pathogen-free flocks qualified for vaccine production. The vaccine was stored in vials of 2.5 x 10⁷ pfu/0.2 mL. Vaccine vials were kept at -70°C until the day of administration. They were then thawed at room temperature or in a 37°C water bath. The sample in the vial was diluted with sterile saline to a total volume of 500 µL and then divided into two 250-µL syringes for the Bioject system. Dilutions were performed in a sterile hood.

Immunologic Monitoring Methods: ELISPOT and Antibody Assays
Normal HLA-A2 donor PBMCs were obtained from the Clinical Center blood bank of the National Institutes of Health. Normal and patient PBMC samples were stored in liquid nitrogen at a concentration of 1 x 10⁷ cells/mL. Cells were thawed and cultured overnight in RPMI-1640 complete medium (Life Technologies, Inc, Gaithersburg, MD) at 37°C at 5% CO₂ before performing the ELISPOT assay. A modification of the ELISPOT assay, measuring IFN- production, was used to determine the T-cell CTL precursor frequency specific for the CAP-1-6D peptide^16,17 in both pre- and postvaccination PBMCs. Briefly, 96-well millimeter high-affinity plates (Millipore Corporation, Bedford, MA) were coated with 100 µL/well of capture monoclonal antibody against IFN- at a concentration of 10 µg/mL for 12 hours at room temperature. Plates were blocked for 30 minutes with RPMI 1640 plus 10% human antibody serum. A total of 2 x 10⁵ PBMCs were added to each well. CAP-1-6D–pulsed C1R-A2 cells were added into each well as APC at an effector:APC ratio of 1:3. Unpulsed C1R-A2 cells were used as a negative control. HLA-A2 binding Flu matrix peptide 59-66 was used as a positive peptide control.²⁴ Cells were incubated for 24 hours and lysed with phosphate buffered saline (PBS)-Tween (0.05%). Biotinylated anti–IFN- diluted to 2 µg/mL in PBS-Tween containing 1% bovine serum albumin (BSA) was added and incubated overnight in 5% CO₂ at 37°C. Plates were washed three times and developed with avidin alkaline phosphatase (GIBCO/BRL, Grand Island, NY) for 2 hours. After washing the plates three times, each well was examined for positive dots. The number of dots in each well was counted by two separate investigators in a blinded manner, and the frequency of responding cells was determined for a total of 1.2 x 10⁶ effector cells plated.

Western Blot Analysis
Purified preparations of native CEA, recombinant CEA, and BSA (1 µg of each) were electrophoresed on denaturing sodium dodecyl sulfate/4% to 20% gradient Trisglycine polyacrylamide gels (Novex, San Diego, CA) and electroblotted using Trisglycine transfer buffer (Novex) to nitrocellulose membranes (0.45-µm pore size; Novex) for 2 hours at 4°C. A See-Blue stain marker (Novex) was included as a molecular weight standard on all membranes. The membranes were incubated overnight at 4°C in PBS containing 5% BSA to prevent nonspecific protein binding. This and each additional incubation and washing were performed on a shaking apparatus. The membranes were then washed four times (10 min/wash) with PBS containing 0.025% Tween-20 (Bio Rad Laboratories, Hercules, CA). Patient serum was diluted in PBS containing 1% BSA and 5% normal goat serum (Life Technologies) and incubated with the membrane for 5 hours at room temperature. Pooled normal human sera (Gemini Bio Products, Calabasas, CA), the HuCol-1 anti-CEA antibody (1 µg/mL), and human immunoglobulin G (IgG; 1 µg/mL) (Jackson ImmunoResearch, West Grove, PA) were used as controls. Each membrane received 15 mL of appropriately diluted patient sera or controls. Membranes were then washed sequentially under stringent conditions (10 min/wash) with PBS containing 0.3%, 0.1%, 0.05%, and 0.025% Tween-20. The wash with 0.025% Tween-20 was performed twice. Goat antihuman IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was diluted 1:20,000 in PBS containing 1% BSA and was incubated with the membranes for 1 hour at room temperature. The membranes were washed four times (10 min/wash) at room temperature and then kept overnight at 4°C with PBS containing 0.025% Tween-20. The membranes were first treated with the electrochemiluminescence detection reagent (Amersham Life Sciences, Arlington Heights, IL) and then exposed to Kodak Biomax MR film (Eastman Kodak, Rochester, NY) and developed.

Statistical Methods
Eighteen patients (12 HLA class I A2⁺ and six HLA class I A2^-) were randomized to either the VAAA or AAAV study cohort. Once accrued to the study, each patient was randomly assigned to either cohort with the stipulation that no more than nine patients could comprise each cohort. A statistical comparison of the two study cohorts (VAAA and AAAV) was performed by assuming that the distribution of ln(change in CEA-specific T-cell precursor frequency) was approximately normal and that it was reasonable to analyze the postvaccination precursor frequency minus the prevaccination frequency for each sample as representing the change in precursor frequency for each patient. For example, the change in CEA-specific T-cell precursor frequency for patient no. 4 would be postvaccination ln(1/87,500) - prevaccination ln(1/200,000). This analysis revealed the percentage increase or decrease in precursor frequency for each patient pre- versus postvaccination. The two study cohorts were compared by calculating the mean of the percentage increase or decrease in precursor frequency for each cohort.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eighteen patients were enrolled in this phase I study. Patient characteristics are listed in Tables 1 and 2. Of 18 patients, nine were randomized to receive VAAA (cohort 1). The remaining patients were randomized to receive AAAV (cohort 2). Six of the patients in each cohort were HLA-A2–positive for immunologic monitoring purposes. Three HLA-A2–negative patients were randomized to each cohort to expand the safety profile in HLA-A2–negative patients. Patient no. 17 (AAAV, HLA-A2–positive) was removed from the study after two vaccinations because of disease progression and was not replaced because sufficient toxicity data were obtained from the other patients in the AAAV cohort. Patients no. 3 (VAAA) and 9 (AAAV), both HLA-A2–negative, were also removed from study because of disease progression. All other patients received four monthly doses of the vaccines according to their cohort schedule. Nine patients with no evidence of disease progression at the completion of the initial four cycles of vaccinations elected to continue receiving avipox-CEA with GM-CSF. Seven of these patients elected to add IL-2 to the treatments after two cycles of avipox-CEA with GM-CSF.

Clinical Response
No objective antitumor responses were observed in any patients treated. Two of the patients (nos. 6 and 7) from these two cohorts remain on study currently and are being treated with monthly cycles 16 and 17, respectively. However, because both patients are HLA-A2–negative, we cannot determine their T-cell response. Patient no. 6 has metastatic colon cancer to his liver and was vaccinated after having a complete radiographic response to fluorouracil in October 1997. He remains without evidence of disease with a serum CEA level of 0.7 more than 21 months after his initial vaccination. Patient no. 7 had metastatic colon cancer to his liver, which was resected, and has now been on the vaccine study for more than 20 months. His CEA level has increased to 54.4, but he still has no radiographic evidence of disease, except for a positive positron-emission tomography scan in his abdominal nodes more than 20 months after initiation of vaccination. Patient no. 15 has pseudomyxoma peritonei with metastatic disease in her lungs and abdomen, including measurable disease in her abdominal wall. After having stable disease for 6 months, she had a minor reduction in the size of her abdominal nodes and a decrease in her CEA level when IL-2 was added to her regimen. Her carcinoma progressed in her lungs only and she was taken off study after 10 cycles on therapy. Patients no. 1, 2, and 19 were on study for a long period, but each had no evidence of disease at the beginning and were found to have experienced disease progression on therapy after 8, 10, and 10 months, respectively.

Immunologic Responses
T-cell assays using the HLA-A2–binding CEA agonist peptide (CAP-1-6D) and Flu matrix peptides were used to investigate T-cell responses in patients positive for the HLA-A2 allele. Ficoll-purified PBMCs from each of these patients were purified and viably frozen at approximately 1 x 10⁷ cells/mL. PBMCs were obtained prevaccination and 4 weeks after each vaccination cycle for each patient. The ELISPOT assays, using the CEA and Flu peptides and PBMCs from each patient prevaccination and 4 weeks after each vaccination cycle, were performed simultaneously and coded. PBMCs were assayed after only 24 hours in culture in the presence of peptide to rule out effects of in vitro selection of T-cell populations. Results were expressed as precursor frequency of IFN-–secreting cells in response to the given peptide; a higher number of precursors is expressed by a smaller number in the denominator of the precursor frequency. As seen in Table 3, responses to the Flu matrix 9-mer peptide were quite similar before and after the vaccinations. These data, and the use of an aliquot (from frozen vials) of PBMCs from a normal donor and the Flu peptide, also served as an internal control for the ELISPOT assay.

View larger version (15K): [in this window] [in a new window]   Fig 1. Changes in CEA-specific T-cell precursor frequencies before the first vaccination (pre) and after 4 vaccinations (post) from patients treated with VAAA (A) and AAAV (B). The ELISPOT assay using the CEA peptide and IFN- production was used.

Antibody Assay Results
All patients treated on this trial were analyzed to determine whether they produced antibodies directed against CEA. Four patients have shown such a result: results from two of these patients are depicted in Figs 2 and 3. Serum from patient no. 21 (Fig 2) showed no reactivity in Western blot analysis to either native CEA or recombinant CEA, using prevaccination serum. Postvaccination serum from this patient showed IgG reactivity to both CEA preparations in Western blot. No reactivity was seen to the control antigen BSA using either pre- or postvaccination serum. Interestingly, patient no. 15 had preexisting antibodies to native CEA (Fig 3). However, after four vaccinations, this patient was found to have increased IgG antibody against native CEA as well as reactivity against recombinant CEA. No reactivity was seen to the control antigen BSA using either pre- or postvaccination serum (Fig 3).

View larger version (44K): [in this window] [in a new window]   Fig 2. Induction of CEA-specific IgG responses after vaccination. CEA-specific IgG in serum (1:100 dilution) from patient no. 21 pre- (left) and postvaccination (right) versus native CEA (nCEA), recombinant CEA (rCEA), and BSA (control).

View larger version (40K): [in this window] [in a new window]   Fig 3. Induction of CEA-specific IgG responses after vaccination. CEA-specific IgG in serum (1:40 dilution) from patient no. 15 pre- and postvaccination. Each panel contains 3 lanes that include purified nCEA, rCEA, and BSA (control). (right) Results with normal human IgG (HuIgG).

View larger version (14K): [in this window] [in a new window]   Fig 4. The impact of GM-CSF on T-cell responses. Patients no. 15 (A), 14 (B), and 5 (C) received rV-CEA (V) followed by 3 vaccinations with avipox-CEA (A), all without cytokine. At the fifth vaccination (VAAAA), all patients received avipox-CEA with recombinant GM-CSF. Depicted are T-cell responses to the 9-mer Flu peptide () and to the 9-mer CEA peptide () using the ELISPOT assay.

View larger version (13K): [in this window] [in a new window]   Fig 5. The impact on CEA-specific T-cell responses of multiple vaccinations of avipox-CEA escalated with the addition of GM-CSF and IL-2. Patients no. 21 (A) and 2 (B and C) received the AAAV vaccination regimen without the addition of cytokine. (A and B) No enhancement in T-cell responses for Flu () or CEA () after 4 vaccinations. Vaccinations no. 5 and 6 with avipox-CEA (A) also included GM-CSF. Vaccinations no. 7 through 9 were with avipox-CEA and GM-CSF followed by low-dose IL-2. Vaccination no. 10 consisted of avipox-CEA with GM-CSF. (C) Expanded scale of (B) showing results postvaccination 5 through 10 for patient no. 2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
This phase I study demonstrates for the first time the safety in humans of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus. This study also compared, for the first time, two diversified vaccination schedules (VAAA v AAAV) by monitoring the level of CEA-specific T-cell precursors in HLA-A2–positive patients. On the basis of a statistical analysis of the two vaccination schedules, VAAA was concluded to be the more effective dose schedule. This result, however, must be evaluated considering variations in the patient population comprising each study group. Variations in tumor burden (four of six patients in the AAAV cohort had evidence of metastatic disease compared with three of six patients in the VAAA cohort), tumor size, primary tumor site, lowered immune status, and/or immune suppression may have been confounding variables in this comparison of the two dose schedules, although no such variations were found (Table 2). We thus conclude that VAAA remains the preferable dosage schedule over AAAV because VAAA produced positive CEA-specific T-cell responses in all six patients assayed, whereas AAAV showed responses in only two of five patients. Furthermore, the VAAA cohort showed increased average T-cell responses ( 328%) compared with the AAAV cohort (approximately 80% increase). These studies validate, for the first time, a diversified prime-and-boost vaccination protocol in patients.

This study also began to investigate the effects of local GM-CSF and low-dose IL-2 when administered after vaccination with avipox-CEA. It seems that both of these cytokines were effective in increasing the frequency of CEA-specific T-cell precursors in all six HLA-A2–positive patients assayed. However, it cannot be determined at this time whether the increase in CEA T-cell responses is due to either the addition of cytokines, additional vaccinations, or both. Patients are currently being accrued to the second stage of this study, in which the safety and efficacy of GM-CSF and IL-2 during the initial four cycles of vaccinations are being investigated. Patients no. 2 and 21 (both in the AAAV cohort), who did not respond immunologically to the initial four cycles of vaccinations, showed marked responses after GM-CSF was added to the vaccinations. The planned phase I/II study investigating the safety and efficacy of GM-CSF and IL-2 will more conclusively test the benefit of including low-dose IL-2 in these treatments.

The use of this diversified prime-and-boost vaccination protocol is not limited to the 50% of the population that is positive for HLA-A2. Although immunologic monitoring was conducted for patients who were HLA-A2–positive for proof of concept, these vaccines can potentially elicit T-cell responses in patients of any other HLA type, because CEA peptides have already been identified that elicit cytolytic T-cell responses in vitro for HLA-A24, HLA-A3, and other alleles.^10,24-27 Efforts to expand the number of monitoring tools are ongoing. Antibody responses were also observed in some patients on this study; this, of course, could give an additional measure of CEA-specific immune responses in HLA-A2–negative patients, as well as suggest stimulation of the humoral arm of the immune system by these vaccines. Nonetheless, the ELISPOT assay proved to be quite effective in measuring CEA-specific T-cell immune responses, and there is the suggestion that clinical responses may mirror immune responses in some patients (patient no. 15 and others are now in the stage II portion of the trial).

Despite measurable CEA-specific T-cell responses in patients enrolled in this study, no objective anticancer effects were observed. The reason remains unclear at this time but may be related to the hypothesis that some patients with advanced cancer are unable to respond to immunologic therapy because of lowered immune status and/or preexisting immune suppression. The lowered immune status of cancer patients has been demonstrated through a decrease in the chain of the T-cell receptor and by a shift from a type 1 T-cell response to a type 2 T-cell response. The presence of putative immune inhibitors, such as transforming growth factor beta or IL-10, may be responsible for the immune suppression observed in cancer patients.^28-30 Another explanation for the lack of clinical efficacy is that the number or affinity of T cells generated by the vaccines, given no preexisting immune suppression or lowered immune status, may not have been sufficient to elicit a measurable reduction in tumor size and/or progression. More potent vaccine strategies, such as the incorporation of a triad of costimulatory molecules²⁷ into the vectors used here and the insertion of the CEA enhancer agonist epitope^16,17 into these vectors, are examples of such planned innovations. Finally, the size and/or high interstitial pressures of tumor masses may have prevented T cells from penetrating the tumor(s). Subsequent studies will try and define the role of tumor burden on the ability of patients to generate an immune response.

We have reported here for the first time the use of a diversified prime-and-boost vaccination protocol using two different recombinant vectors in humans and have validated that the VAAA dose schedule is preferable to AAAV for use in future studies. Moreover, these studies have demonstrated, for the first time, that avipox-CEA can be given up to eight times with continued increases in CEA T-cell responses. These studies thus form the rational basis for the use of diversified prime-and-boost vaccine strategies in less advanced disease settings.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted December 7, 1999; accepted June 26, 2000.

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