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Clinical Trials |
1 Cancer Research Cooperative Laboratory at the National Taiwan University Hospital, Division of Cancer Research, National Health Research Institutes, 2 Department of Internal Medicine and 3 Laboratory Medicine, National Taiwan University Hospital; and 4 Graduate Institute of Biomedical Technology, Taipei Medical Universty, Taipei, Taiwan, Republic of China
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Six patients with the HLA-A*2402 genotype and 4 patients with the HLA-A*0201 genotype were enrolled. A single CEA peptide (YLSGANLNL) or two CEA peptides (QYSWFVNGTF and TYACFVSNL) were used for patients with the HLA-A*0201 or HLA-A*2402 genotype, respectively. Autologous DCs were generated by culturing adherent mononuclear cells with interleukin 4 and granulocyte macrophage colony-stimulating factor for 6 days. Maturation of DCs was then induced with tumor necrosis factor 
for 40 h. Mature DCs were pulsed with appropriate CEA peptides for 2 h. After washing, 1 million peptide-pulsed DCs were injected into one inguinal lymph node under sonographic guidance. Each patient received four injections.
Results: No grade II/III toxicity or autoimmunity was observed. An increase in the number of CEA-specific T cells after DC vaccination could be detected in 7 of 10 (70%) patients. Two (20%) patients had stable disease for at least 12 weeks. One of these 2 patients experienced a transient decrease in CEA levels during the treatment period and also had the most significant T-cell response against the immunizing CEA peptides.
Conclusions: These results suggest that our vaccination procedure can generate or boost specific T-cell responses and may provide clinical benefit in certain cancer patients.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dendritic cells (DCs) are the most potent type of antigen presenting cells in the human body, and are involved in the regulation of both innate and adoptive immune responses (4, 5, 6) . Vaccination with DCs loaded with tumor-specific antigens or TAAs has been shown to induce protective immune responses in several animal models (7 , 8) . The results of several clinical trials that have investigated the use of tumor antigen-pulsed DC vaccines for the treatment of various advanced malignancies have been reported (9 , 10) . One important issue in applying DC-based cancer immunotherapy is the choice of the appropriate TAA. For colorectal cancer, several different approaches have been developed for boosting the immune response against carcinoembryonic antigen (CEA; Ref. 11 ). Elevated expression of CEA is observed in most colorectal carcinomas. Although CEA expression is also observed in normal colon epithelial cells, the expression levels tend to be relatively low (12) . Immune responses to CEA have been reported in many cancer patients (13 , 14) , indicating that the tolerance to CEA is incomplete. Thus, enhancing the immune response of a patient against CEA may trigger a stronger immune attack against tumor cells that express high levels of CEA. DCs present antigens to T cells in an HLA-restricted manner. Three CTL epitopes on CEA have been identified: one epitope is presented by HLA-A*0201 (15 , 16) and two are presented by HLA-A*2402 (17 , 18) . It has been demonstrated that CTLs specific for these epitopes can be generated from peripheral blood mononuclear cells (PBMCs) of normal donors and that these CTLs are capable of killing tumor cells expressing CEA and the appropriate HLA molecules (19, 20, 21) .
Colorectal cancer ranks third on the cancer mortality list in Taiwan. Therefore, we conducted a pilot clinical trial of DC-based immunotherapy to treat metastatic colorectal carcinomas that have failed standard chemotherapy. In our study, autologous DCs were pulsed with peptides representing known CTL epitopes on CEA. Phase I trials using CEA peptides in DC-based immunotherapy against malignancies have been reported by other investigators (22 , 23) . However, our current study significantly differs from these studies in at least three aspects. First, in our study, synthetic CEA peptides that can bind to either HLA-A*0201 or HLA-A*2402, which are both common genotypes in Taiwan, were used in the appropriate patients. Previous clinical trials have primarily used the HLA-A*0201-restricted peptide because of the predominance of HLA-A*0201 expression in Caucasian populations (22) . Secondly, peptide-loaded and tumor necrosis factor (TNF)-matured DCs, rather than immature DCs, were used for vaccination. It has been shown that mature DCs are more potent stimulators of T cells and more resistant to immune suppressive factors released by tumor cells. Furthermore, they can maintain their activated phenotypes after withdrawal of cytokines (10) . Therefore, immunization with mature DCs pulsed with antigenic peptides is likely to induce a stronger immune response. Finally, the DC vaccine was introduced into patients by intranodal injection instead of i.v. or intradermal injection. Recent reports have suggested that intradermal injection of DCs results in more efficient migration to lymph nodes than s.c. or i.v. injections (24 , 25) . However, the number of DCs that can migrate to lymph nodes after intradermal injection is still very limited. The direct intranodal injection of DC method used in our study should greatly increase the number of peptide-pulsed DCs in lymph nodes and should, therefore, result in a stronger immune response. Because of these important differences, our study can provide valuable information that was not available in previous trials. In this study, we have demonstrated that intranodal vaccination with DCs pulsed with CEA peptides is feasible and safe for late-stage colorectal cancer patients. Moreover, such treatment can generate CEA-specific T-cell responses in most cancer patients. Additional clinical studies are required to better evaluate the efficacy of similar treatment approaches.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3,500/mm3, neutrophil 1,500/mm3, lymphocyte 1,000/mm3, platelet 100,000/mm3, bilirubin 
1.5 times normal range, and creatinine 2.0 times the upper limit of normal. Patient performance status ranged from 0–1 on the ECOG scale. Patients who had central nervous system metastasis, autoimmune disease, or active acute/chronic infection, and patients who received chemotherapy, steroid, or biological treatment within 6 weeks before enrollment were excluded from this study. Patient characteristics are shown in Table 1
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(Strathmann). On day 8, floating and loosely adherent cells were collected as mature DCs
Vaccine Preparation and the Vaccination Protocol.
Mature DCs (1.5 x 106) were suspended in 1 ml of AIM-V medium and pulsed with appropriate CEA peptides (50 µg/ml each) for 2 h according to the HLA-typing of the patient. One peptide with the sequence YLSGANLNL was used for patients with the HLA-A*0201 genotype (15
, 16)
. Two peptides with the sequences QYSWFVNGTF (peptide-1) and TYACFVSNL (peptide-2) were used for patients with the HLA-A*2402 genotype (17
, 18)
. All of the peptides were synthesized by AnaSpec Inc. (San Jose, CA) under GMP conditions. After extensive washing, 1 x 106 DCs were suspended in 0.3 ml PBS containing 1% autologous plasma and injected into one inguinal lymph node under sonographic guidance. Patients were vaccinated once a week for 3 weeks followed by a single booster 2 weeks later. Whole blood (30 ml) was collected from patients 2 weeks after the first and the last injections. PBMCs were purified and cryopreserved for evaluation of the immune responses against the CEA peptides.
Flow Cytometry Analysis.
Cells to be analyzed for the expression of surface markers were stained with different fluorescence-labeled monoclonal antibodies (mAbs) and then analyzed using a flow cytometer (EPICS XL-MCL; Beckman Coulter). The mAbs used in this study were: FITC-anti-HLA-DR, phycoerythrin-anti-CD86, FITC-anti-CD80, phycoerythrin-anti-CD83, phycoerythrin-anti-CD14 (Immunotech, Marseille Cedex, France), and FITC-anti-CD40 (Serotec, Oxford, United Kingdom). Isotype-matched control mAbs were obtained from Immunotech and Caltag (Burlingame, CA).
IFN-
Enzyme-Linked Immunospot (ELISPOT) Assay.
The ELISPOT assay was performed as described previously (2
, 26 , 27)
. Briefly, PBMCs (2 x 105/well x 5 wells) obtained before, during, and after vaccination were cultured individually with 50 µg/ml of the immunizing CEA peptide (one peptide for HLA-A*0201 and two separate peptides for HLA-A*2402 patients) or a control peptide in 96-well filtration plates (Multiscreen-IP; Millipore, Bedford, MA) precoated with an anti-IFN- mAb (Mabtech AB, Nacka, Sweden). After a 24-h incubation, the wells were extensively washed and then stained with a biotin-labeled anti-IFN- mAb (Mabtech) followed by avidin-conjugated alkaline phosphatase. The alkaline phosphatase substrate was then added to allow development of a blue color, and the number of blue spots (representing the numbers of IFN- secreting cells) was counted. Counting was done in a blinded manner to prevent undesired bias. The HLA-A*0201-restricted CEA peptide was used as a negative control for samples from HLA-A*2402 patients and vice versa. Phorbol 12-myristate-13-acetate plus ionomycin (Sigma, St. Louis, MO) was used as a positive control (27)
. The number of spots in the negative control normally ranges from 0 to 26, and the number of spots in the positive control normally ranges from 124 to 195. The average number of spots in the negative control group was subtracted from that of the test group. The final number of CEA-specific spots was adjusted with a normalizing factor obtained from the positive control group containing ionomycin and phorbol 12-myristate-13-acetate, based on the assumption that the total number of cells that could be nonspecifically stimulated to produce IFN- by phorbol 12-myristate-13-acetate plus ionomycin in PBMCs remains the same before and after DC vaccination.
Intracellular IFN- Flow Cytometry Analysis.
The intracellular cytokine flow cytometry analysis was performed as described previously (28)
. Briefly, PBMCs from different time points were cultured with the immunizing CEA or control peptides for 18 h. The GolgiStop reagent (BD PharMingen) was added during the last 4 h of culture to block protein secretion. Cells were harvested, washed, and stained with a phycoerythrin-labeled anti-CD8 mAb (Immunotech). After washing, cells were fixed, permeabilized with Cytofix/Cytoperm (BD PharMingen), and stained with a FITC-labeled anti-IFN- mAb (BD PharMingen). Cells (25,000 cells) were subjected to flow cytometry analysis to determine the percentage of CD8+, IFN--producing cells. The HLA-A*0201-restricted CEA peptide was used as a negative control for samples from HLA-A*2402 patients and vice versa. The percentage of the control group was always <0.05 and was subtracted from that of the test group.
Toxicity and Clinical Evaluation.
Toxicity grading was done and recorded according to National Cancer Institute Common Toxicity Criteria Scale, version 2.0. The clinical responses were defined as follows: (a) stable disease, <25% change in size with no new lesions developing for 6 weeks; and (b) disease progression, the appearance of new lesions or >25% increase in the area of existing lesions.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7% of input PBMCs. DCs obtained on days 6 and 8 (after incubation with TNF- for 40 h) were stained for several surface markers to monitor their characteristics. Day 6 DCs displayed a typical immature DC staining profile: HLA-DR+, CD86+, CD80dim, CD14dim, and CD83dim. Day 8 DCs generally displayed a more mature DC phenotype, HLA-DR+, CD86+, CD80+, CD14–, CD40+, and CD83+ (Table 2
; Fig. 1
). However, substantial variations did exist among different patients, which may reflect their different previous chemotherapy protocols. Due to the limited patient number, the influence of such variations on the generation of immune responses or clinical responses is not entirely clear at present.
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Evaluation of Immune Responses against CEA.
The secondary end point of this study was to evaluate the immune response against CEA before and after DC vaccination. Cryopreserved PBMCs obtained before the study (day 0), 1 week after the second injection (day 22), and 2 weeks after the fourth injection (day 50) were thawed on the same day for analysis. Additional PBMCs were obtained on day 120 from patients 5 and 7, who both had stable disease. PBMCs were stimulated with the immunizing CEA peptide(s) or a control peptide, and the number of peptide-specific T cells was determined by IFN- ELISPOT assay and intracellular IFN- flow cytometry analysis. Minor to moderate increases in the number of CEA peptide-specific, IFN--producing T cells were observed in all 4 of the HLA-A*0201 patients and in 3 of the 6 HLA-A*2402 patients by ELISPOT assay (Fig. 2)
. These 3 HLA-A*2402 patients responded to both HLA-A*2402-restricted CEA peptides used in the DC vaccine. Using intracellular IFN- flow cytometry analysis, a substantial increase in the percentage of CEA-specific T cells was detected in patients 4 and 6, whereas a minor increase was observed in patients 1 and 2 (Fig. 3
; Table 3
). Although 7 patients had an enhanced CEA-specific T-cell response with the ELISPOT assay, only patients 1 and 6 also experienced an increased T-cell response as detected by intracellular cytokine flow cytometry analysis. The source of the discrepancy between these results is not known. However, it has been reported that the sensitivity of ELISPOT assay is higher than intracellular cytokine flow cytometry analysis (29)
. In addition, because of the limitation of our clinical blood samples, we can only use 25,000 cells from each setting for flow cytometry analysis. Thus, the results of our intracellular cytokine analysis are less informative. Nevertheless, these results suggest that our vaccination procedure can boost or generate specific T-cell responses in some cancer patients.
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. There was a transient decrease (from 496 to 189.2 ng/ml) in the CEA level in patient 5 during the treatment period. Interestingly, this patient, who was HLA-A*2402, also had the most significant T-cell responses against both CEA peptides as determined by ELISPOT. Unfortunately, concurrent T-cell responses analyzed by intracellular cytokine flow cytometry were not available for this patient. Although patients 1 and 6 had an increased T-cell response that was detected by both methods, they had progressive disease when evaluated 6 weeks after the first vaccination. Stabilization of CEA level in patients 4 and 10 was observed during the treatment period (up to day 50). These 2 patients also displayed an increase in T cells against the immunizing CEA peptides with intracellular cytokine flow cytometry or ELISPOT analysis. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The secondary end point of this study was to evaluate the immune response against the CEA peptide before and after DC vaccination. We observed a substantial increase in the number of CEA-specific T cells by ELISPOT or intracellular cytokine flow cytometry in most patients (70%) after DC vaccinations, which indicates that our vaccination procedure may generate or boost a CEA-specific T-cell response in certain patients. Although not a primary goal of this trial, we followed the clinical outcomes of these patients. Two of the 10 (20%) patients experienced disease stabilization for at least 12 weeks, which is comparable with or slightly better than the outcome of other similar studies (20
, 21)
. However, most patients had progressive disease, which was expected because they tended to have a heavy tumor burdens at study entry, which was reflected by high CEA levels (day 0; Table 3
).
Our study design differed from previous DC vaccination studies in three important ways. First, both HLA-A*0201 and HLA-A*2402-restricted CEA peptides were used. This allowed us to include a greater proportion of Taiwanese patients. Enhanced CEA-specific T-cell responses were observed in both HLA-A*0201 and HLA-A*2402 patients. In the 3 HLA-A*2402 patients who had increased CEA-specific T-cell responses, both HLA-A*2402-restricted CEA peptides included in the DC vaccine were recognized. One HLA-A*0201 patient and 1 HLA-A*2402 patient had disease stabilization. The sample number is too small to draw any firm conclusions, but these results suggest that our DC vaccination protocol may be applicable to both HLA-A*0201 and HLA-A*2402 patients.
Secondly, TNF--matured DCs rather than immature DCs were used for our vaccine preparation. It has been shown that mature DCs are more potent stimulators of T cells, are more resistant to immune-suppressive factors released by tumor cells, and can maintain an activated phenotype after cytokine withdrawal. Therefore, immunization with mature DCs pulsed with antigenic peptides is expected to induce a stronger immune response. The expression of both CD80 and CD83 on DCs was enhanced after stimulation with TNF-. Recent studies by other investigators have suggested that the use of both TNF- and IFN- can impart DCs with an improved capacity for promoting T-helper 1 and CTL responses (30)
. Therefore, we will also adapt to this approach in future vaccination regimens.
Thirdly, the peptide-pulsed DCs were introduced into patients by intranodal injection instead of i.v. or intradermal injection. The direct intranodal injection of DCs in our study was expected to increase the number of peptide-pulsed DCs in lymph nodes and, therefore, elicit a better immune response. However, it is not known whether injected DCs localize to the correct compartment in the lymph node or whether the disturbance of lymph nodes by liquid injection interferes with their function. An alternative approach is to s.c. inject the DC vaccine together with an inflammation-inducible agent nearby lymph nodes. The resulting inflammatory microenvironment at the injection site might additionally modulate the coinjected DCs and promote their migration to the lymph nodes through the pathway of DCs exposed to invading pathogens.
Taken together, results from this pilot clinical study are very encouraging. We have proposed a Phase II study to better evaluate and improve the efficacy of such DC-based immunotherapy. The valuable information obtained from this study and our next phase II study should help us in the design and execution of future clinical trials of DC-based cancer immunotherapy.
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FOOTNOTES |
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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.
Requests for reprints: Chuan-Cheng Wang, Department of Internal Medicine, Chung Shan Medical University Hospital, No. 110, Section 1, Chien-Kuo North Road, 402 Taichung, Taiwan. Phone: 886-4-24739595, extension 4721; Fax: 886-4-24728905; E-mail: cshy666{at}csh.org.tw
Received 10/20/03; revised 11/29/03; accepted 12/ 3/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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