Search

Background: Advanced non-small cell lung cancer (NSCLC) has remained challenging to treat effectively. This study aimed to investigate the clinical effects and safety of immunotherapy with dendritic cells and cytokine-induced killer cells (DC-CIK) administered with chemotherapy (CT) in this malignancy., Methods: We have developed a new clinical trial design termed as the prospective patient's preference-based study (PPPS). Consecutive patients (n = 135) with advanced NSCLC were treated with DC-CIK administered with CT or mono-therapy (CT or DC-CIK alone)., Results: For all the patients, the median PFS was 5.7 months and the median OS was 17.5 months. The 1-year PFS and OS rates were 29.4% and 58.2%, respectively. The 1-year PFS and OS rates for DC-CIK plus CT were significantly higher than that in the group of patients who received DC-CIK alone and CT alone (P < 0.05). The number of adoptively infused DC-CIK cells was associated with clinical efficacy. After adjusting for competing risk factors, DC-CIK combined with CT and infused number of CIKs remained independent predictors of PFS and OS. Phenotypic analysis of peripheral blood mononuclear cells showed that CD8+CD28+, and CD8+CD28- T cells, changed significantly in all groups (P < 0.01). The CD3+ T cells increased in the chemotherapy plus immunotherapy and the immunotherapy alone group (P < 0.01), while CD3-CD16+CD56 T cells decreased in the chemotherapy plus immunotherapy and the immunotherapy alone group (P < 0.01)., Conclusions: DC-CIK combined with chemotherapy administration resulted in numerically superior PFS and OS compared with monotherapy in advanced NSCLC.

Despite advances in research and treatment modalities, colorectal cancer still accounts for around half a million deaths yearly worldwide. Traditional and even newer pharmaceutical therapeutic regimens are limited in terms of tolerance, efficacy and cross-resistance. Additional non-cross resistant therapies with non-overlapping toxicities are needed to improve the outcome for patients with colorectal cancer. Cancer vaccines, designed to activate immune effectors (T-cells and antibodies) to prevent recurrence or treat advanced cancers, have now demonstrated clinical benefit in prostate cancer and lymphoma. Because immune effector infiltration into colon tumours is associated with improved clinical outcome, vaccines intended to activate immune responses against colon cancer have generated significant interest. This review discusses data supportive of the immune responsiveness of colorectal cancer, as well as the current status of colon cancer vaccines under development including those based on whole tumour cells or lysates, peptide or protein antigens, anti-idiotype antibodies, viral vectors, and dendritic cells. We also discuss challenges to colon cancer vaccine development, such as tumour associated mechanisms for immune evasion, and how future strategies may address these challenges.

Treatment with myeloablative chemotherapy and autologous peripheral blood stem cell (PBSC) transplantation followed by vaccination with autologous dendritic cells (DCs) treated with tumor antigens is a promising therapeutic strategy for several types of cancer. Obtaining sufficient numbers of both PBSCs and DCs is central to this approach. Previously, it has been shown that administration of Flt-3-Ligand (FL) combined with either G-CSF or GM-CSF mobilizes large numbers of PBSCs in patients with cancer. In the current study, we sought to determine whether these same cytokines could simultaneously mobilize DCs into the PBSC leukapheresis collection. DCs were analysed in PBSC leukapheresis samples obtained from five patients with high-risk breast cancer who received G-CSF alone as priming prior to leukapheresis, four patients who received FL+G-CSF and five patients who received FL+GM-CSF. DCs were defined as cells with a lin(dim/-) HLA-DR+ CD11c+ phenotype. The proportions of DCs in the FL+G-CSF and FL+GM-CSF samples were significantly higher than in pre-mobilization peripheral blood and G-CSF leukapheresis samples. The mean yield of DCs/kg in the FL+GM-CSF samples was also significantly higher than the mean yield of DCs in the G-CSF samples. The FL+G-CSF and FL+GM-CSF mobilized DCs were immature by morphologic and phenotypic criteria but stimulated allogeneic T-cells at levels similar to DCs generated in culture from PBMCs. Overnight culture?of the immature DCs obtained from patients receiving either FL+G-CSF or FL+GM-CSF in TNF-alpha?resulted in the generation of mature DCs. In summary, administration of FL in combination with GM-CSF and G-CSF to patients with breast cancer can mobilize large numbers of immature DCs into PBSC leukapheresis collections., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

Objective: To demonstrate the feasibility of inducing tumor antigen-specific immune responses in patients with metastatic cancer using total tumor RNA-loaded dendritic cells (DCs)., Summary Background Data: The authors have shown that DCs transfected with mRNA encoding defined tumor antigens induce tumor antigen-specific T-cell responses in vitro and in vivo. There may be significant advantages to inducing immune responses against the entire repertoire of antigens expressed by a patient's autologous tumor., Methods: RNA was extracted from a metastatic colon cancer and used to load autologous DCs. The DCs were coincubated with autologous T cells and the cytolytic activity of the T cells was assessed by the ability to lyse the autologous tumor cells. RNA was then extracted from a metastatic lung cancer and used to load autologous DCs, followed by four injections of the DC vaccine given every 4 weeks. Tumor antigen-specific cytotoxic T lymphocyte activity was then evaluated by testing peripheral blood mononuclear cells for their ability to lyse an antigen-expressing target., Results: DCs transfected with the total RNA content of autologous tumor cells stimulated antigen-specific T-cell responses that are capable of recognizing and lysing autologous, primary tumor cells in vitro. Tumor-specific immune responses were induced in a patient with a carcinoembryonic antigen-expressing adenocarcinoma after immunization with autologous DCs transfected with total tumor RNA., Conclusions: DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of tumor antigens of surgically resected malignancies.

Purpose: To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer., Patients and Methods: Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 microg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured., Results: After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 +/- 1.0 x 10(3)/mm(3) to 11.2 +/- 3.8 x 10(3)/mm(3) (mean +/- SD, P: =. 0001). The percentage of CD11c(+)CD14(-) DCs in PBMCs increased from 2.4% +/- 1.8% to 8.8% +/- 4.7% (P: =.004). Delayed-type hypersensitivity (DTH) responses to recall antigens (CANDIDA:, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P: =.06, P: =.03, and P: =.08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not., Conclusion: Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.

Dendritic cells (DCs) may arise from multiple lineages and progress through a series of intermediate stages until fully mature, at which time they are capable of optimal antigen presentation and T-cell activation. High cell surface expression of CD83 is presumed to correlate with full maturation of DCs, and a number of agents have been shown to increase CD83 expression on DCs. We hypothesized that interleukin 12 (IL-12) expression would be a more accurate marker of functionally mature DCs capable of activating antigen-specific T cells. We used combinations of signaling through CD40, using CD40 ligand trimer (CD40L), and interferon gamma to demonstrate that CD83 expression is necessary but not sufficient for optimal production of IL-12 by DCs. Phenotypically mature DCs could be induced to produce high levels of IL-12 p70 only when provided 2 simultaneous stimulatory signals. By intracellular cytokine detection, we determined that only a subset of cells that express high levels of CD80 and CD83 generate large amounts of IL-12. DCs matured with both signals are superior to DCs stimulated with the individual agents in activating antigen-specific T cell in vitro. These findings have important implications regarding the identification, characterization, and clinical application of functionally mature DCs.

Dendritic cells play a central role in the presentation of antigen to naïve T-cells and the induction of primary immune responses. Preclinical studies have established that dendritic cells loaded with antigens ex vivo induce potent antitumor and antiviral immune responses in vitro and in vivo. This has lead to a proliferation of clinical trials testing their effectiveness in humans, particularly with advanced malignancies. The few reported studies suggest that clinically relevant immune responses may be induced against some types of malignancies. Many questions regarding the best type of dendritic cell, degree of maturity, choice of antigen, route and schedule of administration, targeting to lymphoid tissue and use of additional adjuvants will need to be answered in preclinical and clinical studies as the field of dendritic cell-based immunotherapy progresses.

Immunization with ex vivo-generated, tumor antigen-loaded dendritic cells (DC) has been proposed as a strategy for reducing relapses following high-dose chemotherapy, but the ideal time and method for obtaining DC progenitors are unknown. We determined the percentage yield, phenotype, and function of DC generated over 7 days in GM-CSF and IL-4-supplemented, serum-free medium from PBMC obtained from breast cancer and lymphoma patients at the time of their initial presentation for transplant, cytokine or chemotherapy plus cytokine-mobilized leukapheresis, and following granulocyte recovery from high-dose chemotherapy. The median yield of large dendritic-like cells as a percentage of the starting number of PBMC was similar for all the mobilization strategies (11.6%-13.8%) studied and at all time points (9.9%-12.7%), except the yield was lower from the pretherapy, unmobilized peripheral blood (6.3%). The phenotype of the generated cells was similar for the various mobilization procedures, and there were no differences in allostimulatory function of the DC from any of the groups. We conclude that functional DC may be generated equally well from mobilized PBPC and PBPC obtained after high-dose chemotherapy.

Clinical trials of active immunotherapy strategies against viral infections and malignancies are increasingly using dendritic cells (DC) generated in vitro from peripheral blood mononuclear cells (PBMC) in media supplemented with granulocyte macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4). Although GM-CSF appears necessary, it is not well established whether other cytokines have advantages or could replace IL-4 in clinical preparations. IL-13 is a Th2-derived cytokine that shares a variety of biologic functions with IL-4, such as inhibition of monocyte differentiation and upregulation of major histocompatibility complex (MHC) molecules on cell surfaces. In the present study, the authors compared IL-4 and IL-13 in their abilities to induce DC differentiation and found that adherent PBMC cultured in GM-CSF and IL-13 displayed features characteristic of DC generated in media containing IL-4. They formed cellular clumps and had extensive dendrites. Fluorescence-activated cell sorter analysis showed that they expressed a high level of MHC class II and the costimulatory molecule CD86, and did not express the lineage markers CD3, CD14, CD16, or CD20. They were also equally potent stimulators of allogeneic lymphocytes in the mixed lymphocyte reaction. Moreover, the authors demonstrated that they were capable of inducing antigen-specific CD8+ cytotoxic T cells efficiently.

The application of dendritic cells (DC) to the active immunotherapy of cancer currently relies on the generation of potent DC capable of presenting tumor antigens such as carcinoembryonic antigen (CEA). It is unknown whether the T cells of patients with advanced malignancies can be reliably stimulated against tumor antigens by their autologous DC. In this study, starting with the peripheral blood mononuclear cells (PBMC) of patients with metastatic malignancies expressing CEA, autologous DCs were generated in vitro in serum-free media supplemented with GM-CSF and IL-4. The DCs from HLA A2 positive patients were loaded with the CEA peptide CAP-1 and the DCs from HLA A2 negative patients were depleted of bystander lymphocytes and loaded with mRNA encoding CEA. The DC preparations were tested to determine their phenotype and were used to stimulate autologous PBMC twice, separated by 10-14 days. The stimulated cells were then tested for their ability to lyse CEA-expressing target cells. We successfully generated an adequate number of DC for a clinical trial from all patients. The harvested DC preparations contained 49% DC and 87% DC if depleted of bystander lymphocytes. Phenotypic analysis showed the typical pattern of CD11c+ CD40+ CD86+ HLA-DR+ CD80(low) CD83(low) CD14(low). All preparations but one were able to stimulate CEA-specific cytotoxic T-lymphocyte (CTL) activity, suggesting that the majority of patients are not anergic to CEA and possess functional DC. The CTL activity was similar for the CEA peptide and CEA RNA-loaded DC.

Dendritic cells (DCs), antigen-presenting cells capable of priming naive T cells to specific antigens in an HLA-restricted fashion, have been demonstrated to induce protective T cell-mediated immunity in tumor-bearing animals. We performed this study to test the safety, feasibility, and clinical response of immunizations with in vitro-generated DCs, loaded with an HLA-A2-restricted peptide fragment of the tumor antigen carcinoembryonic antigen (CEA), for the treatment of patients with advanced CEA-expressing malignancies. Cell preparations enriched for autologous DCs were generated from the patients' plastic adherent peripheral blood mononuclear cells in serum-free media supplemented with granulocyte macrophage colony-stimulating factor and interleukin-4. Within the cell preparation, 66% of the cells expressed the phenotype typical for DCs (CD86high, HLA-DRhigh, and CD14low). The DCs were loaded with the CEA peptide CAP-1 and cryopreserved. Groups of three to six patients received four weekly or biweekly i.v. infusions of the CAP-1-loaded DC in escalating dose levels of 1 x 10(7), 3 x 10(7), and 1 x 10(8) cells/dose. A subset of the patients in the last group also received intradermal injections of 1 x 10(6) DCs. There were no toxicities directly referable to the treatments. One patient had a minor response, and one had stable disease. Skin punch biopsy at DC injection sites demonstrated pleomorphic infiltrates in the three patients evaluated. We conclude that it is feasible and safe to generate and administer large numbers of previously cryopreserved DCs loaded with CAP-1 peptide to patients with advanced malignancies.

Present clinical studies of active immunotherapy for malignancies using dendritic cells (DCs) require elucidation of the sites where DCs localize after injection. We evaluated the pattern of distribution of in vitro-generated, antigen-loaded, human DCs labeled with indium-111 oxyquinoline after i.v., s.c., and intradermal injection. Whereas the DCs injected i.v. localized in the lungs and then redistributed to the liver, spleen, and bone marrow, they were not detected in lymph nodes or tumors. A small percentage of DCs injected intradermally migrated rapidly to the regional lymphatics in some individuals. No lymph node activity was detected after s.c. injection. Our results demonstrate that DC distribution to sites of lymphoid tissue is dramatically affected by the mode of administration.

Dendritic cells (DCs), matured by CD40-ligand (CD40L), undergo marked changes in their ability to process and present antigen, resulting in augmented lymphocyte stimulatory activity. We demonstrate that the form of the tumor antigen (peptide or genetic material) used to load the DCs dictates the required sequence of antigen loading and maturation for antitumor immunotherapy. Optimal stimulation of carcinoembryonic antigen (CEA)-specific CTLs by peptide-loaded DCs occurs when DCs from cancer patients are matured with CD40L before exposure to CEA peptide, whereas optimal stimulation by RNA-transfected DCs occurs when the DCs are loaded with CEA RNA before maturation with CD40L.

Thymic dendritic cells (DCs) appear to have distinct biologic and functional properties compared with DCs in other tissues. Currently, little is known about human thymic DCs because they have been difficult to isolate and culture in vitro. Here, we report that human thymic stroma can support the development of primitive human hemopoietic stem cells into mature DCs without cytokine or serum supplementation. Coculture of CD34+CD38-lineage (lin)- and CD34+CD38+lin- umbilical cord blood cells with thymic stromal monolayers induced 43 +/- 17-fold and 32 +/- 16-fold expansions, respectively, of umbilical cord blood progenitors and also generated large numbers of cells with the morphologic, phenotypic, and functional characteristics of mature DCs. These cells expressed class I and class II MHC, CD1a, CD2, CD4, CD11c, CD40, CD45, CD80, CD83, and CD86 and were potent stimulators of allogeneic T cell activation. Primitive hemopoietic progenitors also developed into mature DCs in a novel tissue culture system of thymic nodules wherein thymic epithelial cells and fibroblasts were grown in nodular aggregates in vitro. These results demonstrate that human thymic stroma efficiently supports the development of CD34+CD38-lin- cord blood cells into mature DCs. In addition, the culture conditions described in this report are useful systems for studying the ontogeny of human DCs in thymic microenvironments.

Dendritic cells (DC) generated from the peripheral blood mononuclear cells of healthy individuals or from cancer patients transfected with carcinoembryonic antigen (CEA) mRNA stimulate a potent CD8+ cytotoxic T lymphocyte (CTL) response in vitro. DCs are effectively sensitized with RNA in the absence of reagents commonly used to facilitate mammalian cell transfection. RNA encoding a chimeric CEA/LAMP-1 lysosomal targeting signal enhances the induction of CEA-specific CD4+ T cells, providing a strategy to induce T-help that may be necessary to generate and/or maintain an optimal CD8+ CTL response in vivo. CEA RNA-transfected DCs also serve as effective targets in cytotoxicity assays, thus providing a general method for inducing, as well as measuring, CEA-specific CTL responses across a broad spectrum of HLA haplotypes.

While the promise of harnessing the immune system for a therapeutic effect has remained largely unfulfilled for many years, the discovery of the central role of dendritic cells in stimulating antigen-specific immune responses has prompted new enthusiasm for immunotherapy of malignancies. Elucidation of the pathways of dendritic cell development and trafficking, acquisition and processing of antigen, and stimulation of T cells has suggested methods for generating and antigen-loading dendritic cells for use in immunotherapy protocols. Animal models have demonstrated that dendritic cells can stimulate protective antitumor responses in vivo. Phase I clinical trials have been initiated to address the safety and feasibility of immunizations with dendritic cells in humans with various malignancies.

Using a murine metastasis model, we have previously shown that antigen-presenting cells (APC) loaded with unfractionated peptides derived from poorly immunogenic, highly metastatic tumor cells represent a potent form of tumor vaccine. The antimetastatic effect of peptide pulsed APC could be further enhanced by pretreating the cells with antisense oligonucleotides directed against the TAP-2 gene to increase the density of specific peptide-major histocompatibility complex (MHC) class I complexes and thereby improve the APC function of the treated cells (Nair SK et al., J Immunol 1996;156:1772). In this study, we investigated whether similar strategies can be used to enhance the potency of human dendritic cells (DC) to present antigen. We show that human DC pulsed with peptides encoding known cytotoxic T-lymphocyte (CTL) epitopes stimulate both memory and primary CTL responses in vitro after two cycles of stimulation with the peptide-pulsed DC. Two approaches were used to increase the density of specific peptide-MHC complexes on the surface of DC. One approach was to inhibit transporter associated with antigen presentation (TAP) function using TAP antisense oligonucleotides. The second approach was to inhibit the endogenous generation of the peptide epitopes by pretreating the DC with a proteasome inhibitor. Treatment of DC with either TAP antisense oligonucleotides or with a proteasome inhibitor resulted in a dramatic enhancement of primary CTL induction.

Objective: The purpose of the study was to characterize the requirements in terms of precursors, developmental pathways, and media for the generation of large numbers of mature dendritic cells (DC) under conditions acceptable for use in adjuvant, active immunotherapy strategies for surgically treated malignancies., Summary Background Data: Although limited previously by the small numbers accessible, DC-based immunotherapies for malignancy have become more realistic with the development of methods for efficiently generating larger numbers of DC from peripheral blood mononuclear cells (PBMC) in vitro, but these methods rely on clinically unacceptable culture conditions (such as inclusion of fetal bovine serum), necessitating the development of methods for generating functionally equivalent DC in serum-free conditions., Methods: Plastic-adherent PBMC (from healthy donors and patients with cancer) were incubated for 7 days with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) with and without tumor necrosis factor-alpha (TNF-alpha) in fetal bovine serum-containing and serum-free media and were analyzed by Wright's stain for morphology, flow cytometry for phenotype, and mixed lymphocyte reaction for allostimulatory function., Results: Growth in either serum-containing or serum-free media supplemented with GM-CSF and IL-4 yielded a similarly heterogeneous population of cells, 6% to 10% of which had the morphology (large cells with thin projections), immunophenotype (including CD83+), and function of mature DC. Tumor necrosis factor-alpha significantly augmented the number of these mature DC, whereas preculture depletion of CD14+ PBMC virtually eliminated them., Conclusions: Generation of mature DC in the authors' serum-free clinically applicable conditions is similar to serum-containing conditions and requires CD14+ precursors, differentiation through a CD14-CD83- immature stage under the influence of GM-CSF and IL-4, and maturation into a CD83+ DC under the influence of TNF-alpha.

Editorial. Focuses on the function of dendritic cells in the induction of primary immune response for cancer. Activation of T cells essential for cellular immune response; Infected cells capacity to stimulate responses after infection of dendritic cells; Development of dendritic cell-based vaccines; Modification of dendritic cells.