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Keywords:

  • immunotherapy;
  • immunomodulation;
  • cytokines;
  • anergy;
  • dendritic cells;
  • neoplasms;
  • bone marrow;
  • treatment outcome;
  • interleukin 2;
  • granulocyte-macrophage–colony stimulating factor

Abstract

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

BACKGROUND

Advances in immunotherapy for the treatment of patients with malignant disease have led to increasingly successful use of these methods in the clinical setting. This review presents findings from recent studies that have explored improved methods for the presentation of tumor-associated antigens and for the restoration of tumor specific immune responses using cytokine therapy.

METHODS

A review of human clinical trial research on immune cytokines from 1995 (MEDLINE) to the present was conducted. Particular attention was focused on articles that reported results from Phase II or later clinical studies in patients with malignant disease.

RESULTS

The defects in cellular immunity commonly seen in patients with malignancies often are expressed as tumor specific anergy. Reversing patient tolerance to tumor antigens may be accomplished by treatment with immunoregulatory cytokines, such as Flt-3 and granulocyte-macrophage–colony stimulating factor, that mature and activate dendritic cells. Published clinical studies indicate that granulocyte-macrophage–colony stimulating factor stimulates antigen-presenting cells and has promising antitumor activity as an adjunct or as stand-alone therapy for patients with malignant disease, including leukemia, melanoma, breast carcinoma, prostate carcinoma, and renal cell carcinoma.

CONCLUSIONS

Immune-modulating cytokines may be used alone or in combination with other treatments to help restore immune function, improve response to tumor-associated antigens, and reduce the toxic effects of standard antitumor therapies. The evolving understanding of how dendritic cells regulate immune responses and promising results from published studies of immune-enhancing cytokines in the treatment of patients with malignant disease support the conduct of randomized clinical trials to confirm the clinical benefit of these immunotherapeutic strategies. Cancer 2003;97:1797–809. © 2003 American Cancer Society.

DOI 10.1002/cncr.11247

Observations of tumor regressions after bacterial infection led William B. Coley of New York City to explore the use of bacterial extracts as a nonspecific treatment for patients with malignant disease in the early part of the 20th century. In the ensuing years, interest in immunotherapeutic methods for the treatment of patients with malignant disease waned after the demonstration of clinical responses to chemotherapy and after concerns arose regarding the biologic basis of immunotherapies. More recently, the commercial development of numerous hematopoietic cytokines and antibodies targeted to different hematopoietic cell subsets, along with a better understanding of immune regulation, has encouraged researchers to reapproach immunotherapy for patients with malignant disease.

Recently completed and current clinical trials support the theory that self-tolerance to tumor antigens represents an immunologic balance that may be tipped toward antitumor responses. Investigators are evaluating whether immunotherapies can restore and enhance antitumor effector mechanisms that have been lost due to the suppressive effects of the malignancy itself or due to the cytotoxic effects of prior therapies. They also wanted to test whether cytokine immunotherapies, used alone or in combination with chemotherapy, may induce clinically meaningful responses or may improve survival in the adjuvant setting.

Despite a promising outlook, the application of immunotherapy in the treatment of patients with malignant disease is beset with a number of unique challenges. The diminished ability of the immune system to respond to tumor cells may be due to acquired defects in the effector pathways, to immunosuppressive factors, or to tumor cell escape mechanisms.1 Most tumors fail to elicit specific autologous immune responses, because 1) the patient has developed tolerance to tumor-associated antigens (TAAs),2 2) the tumor has developed in an immunopriveleged site,3 or 3) the tumor has suppressed the activity of responding cytotoxic T-lymphocytes (CTLs) through the expression of apoptotic ligands, e.g., Fas-Fas ligand.4 In addition, many tumors can secrete immunosuppressive factors, such as interleukin-10 (IL-10) and transforming growth factor-β.5, 6

Research in the area of tumor vaccines has posed yet another set of challenges. Immunization strategies that employ commonly used mammalian vectors, such as adenoviruses and vaccinia viruses, run the risk of poor responses due to preexisting immunity to these carrier molecules in much of the adult population.7 Even when nonmammalian systems are adopted, issues related to the immunodominance displayed by some of the unique carrier antigens may overwhelm the patient's response to the more self-like tumor antigens.8 Furthermore, although efforts to develop antitumor vaccines have met with some success in clinical trials, there remains some concern that the presentation of self-antigens in tandem with tumor antigens will induce autoimmunity.9 Although a good deal of work continues in the area of tumor vaccine development and vaccine delivery methods, cytokine therapies likely will be an important adjunct to tumor vaccines by helping to break immunologic tolerance and enhancing cytotoxic effects.

The goal for cytokine therapy as a component of immunotherapy for patients with malignant disease is to restore or enhance immune status by replenishing needed immune factors or by enhancing pathways that can diminish tolerance or anergy. In this review, the current state of cytokine therapy is discussed as a means to enhance immune effector pathways and to reverse immunosuppressive mechanisms.

Defective Cellular Immunity

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

A considerable body of data indicates that patients with malignant disease have a deficiency in cellular immunity independent of the immunosuppressive effects of chemotherapy and radiotherapy. Diminished response to tumor antigens has been noted in a host of malignancies, including ovarian carcinoma,10 renal cell carcinoma (RCC),11 melanoma,5, 12 acute myelogenous leukemia (AML),13 and lymphoma.14 Immunosuppression of the host can be expressed as antigen specific tolerance15 or as a generalized immunodeficiency and has been attributed to a wide variety of tumor-induced, failed immune processes.16 Studies into the responsible immune mechanism have implicated molecular and cellular functions that lead either to the induction of tolerance or to immune system evasion.1, 17

Patients with malignant disease often are tolerant to de novo tumors a result of the lack of TAA specific T-cells.2 A number of conceptual frameworks have been used to explain this anergic state. One such theory posits that many TAAs also have been identified as developmental or embryonic antigens; thus, central tolerance to these self-antigens may occur early in development.18 Similarly, intrathymic deletion of T-cells responsive to TAAs that also are expressed at low levels in lymphoid organs may occur when antigen is presented in the absence of generalized inflammatory signals.19 Anergy to newly developed tumors also can occur when interference in antigen presentation occurs through low-efficiency major histocompatibility complex (MHC) presentation to effector cells or from a lack of costimulatory signals.20 Each of these mechanisms leads to the impairment of the host response to early tumor antigens at a time when the tumor burden is still low and prior to the development of iatrogenic or tumor-induced, generalized, immunosuppressive states.

Impaired T-cell responses have been reported in patients with colorectal carcinoma, pancreatic carcinoma, and a variety of hematologic malignancies. Recent studies of nonresponsive T-cells have implicated the down-regulation of costimulatory molecules, including the T-cell receptor (TCR) ζ-chain, although this remains controversial.11 In Hodgkin disease, defective TCR ζ-chain expression can be reversed in vitro by stimulation with CD3 and CD28 cross linking, returning T-cells to normal function and levels of interleukin 2 (IL-2) secretion to normal.21 In patients with ovarian carcinoma, signal transduction at the TCR surface is reduced in patients with ζ-chain specific expression that is inhibited by a 14-kD soluble factor derived from the patients' ascites.10 This down-regulation of ζ-chain expression has been associated with prolonged exposure to tumor necrosis factor (TNF)22 and with apoptotic CD3 positive cells from patients with advanced melanoma.23 Important costimulatory roles also are played by other molecules, such as CTLA-4 and CD28, which have been investigated for their role in the suppression of T-cell activation.24

Abnormal Patterns of Immune Cytokines

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

In patients with malignant disease, alterations in the patterns of cytokine production that lead to decreased cellular immunity are evidenced by the down-regulation of many cytokines, such as TNF, interferons (IFNs), and IL-2. Woo et al. reported that soluble factors in supernatants from renal tumor explants could inhibit the production of IL-2 and IFN-γ by peripheral blood lymphocytes and could suppress T-cell proliferation.25 In another study, T-cells from patients with gastric carcinoma showed simultaneous decreases in cytokine expression and ζ-chain expression and increases in caspase-3 and T-cell apoptosis.26 Rayman et al. found evidence of T-cell infiltration of tumors but no antitumor response: Analysis of the tumor cell explants demonstrated that the reduction of IL-2 and IL-2 receptor signaling by host tumor-infiltrating lymphocytes may have been the result of suppression by a soluble product produced by renal tumors.27

Alterations in the production of growth factors can play a double role, affecting either the homeostatic control of cell growth or the progression to malignancy. Angiogenic molecules, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, are involved with early tumorigenesis; and it has been demonstrated that they are involved with regulating cell growth, differentiation, migration, and extracellular matrix28, 29 as well as possibly affecting the production of granulocyte-macrophage–colony stimulating factor (GM-CSF).30

Other biologic factors play a role in disease course as well. Elevated levels of cytokines, such as IL-6 and IL-10, that can lead to suppression of cellular immune responses have been associated with the prognosis of patients with a number of solid tumors31–33 and patients with chronic lymphocytic leukemia.34 Alterations in receptor ligand pairs, such as Fas (CD95):Fas ligand, which are responsible for the regulation of apoptosis in a variety of cell types, have been implicated in tumorigenesis and metastasis.35 Duhe et al. have reported that the constitutive up-regulation of Janus kinase signaling molecules may be responsible for the induction of a number of leukemias that may respond to treatment by biologic response modifiers.36

Abnormalities in cell-mediated immunity can show improvement after successful antitumor treatment. In one dramatic example, Yoo et al. reported that the increased production of T-helper type 2 (Th2) cytokines and decreased levels of Th1 cytokines found in patients with advanced cutaneous T-cell lymphoma was normalized after therapy with biologic response modifiers.37 Therefore, the administration of cytokines that modulate immune cell function could be an effective part of clinical treatment if such treatment resulted in breaking immune tolerance to tumor antigens.

IL-2 and IFN Therapy

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Initial approaches using cytokines as immunotherapy for patients with malignant disease focused on agents that acted directly on T-cells, especially IL-2 and IFN. It was found that RCC was particularly susceptible to high-dose IL-2 therapy,38 and long-term follow-up of combined IFN and IL-2 therapy for patients with metastatic RCC indicated that approximately 9% of patients remained in remission for > 10 years.39 It has been found that the combination of high-dose IL-2 and IFN is quite toxic, and IFN does not appear to add much to the efficacy of IL-2. Lower doses of monotherapy IL-2 resulted in long-term remissions in 5% of patients with metastatic RCC.40 Largely due to concerns of toxic side effects noted in early studies, few Phase III clinical trials have been conducted exploring treatments with IL-2 and IFNs. However, it has been reported that inhaled IL-2 was tolerated well and prevented the progression of metastases in patients with RCC, breast carcinoma, ovarian carcinoma, and melanoma.41 European Phase II trials reported by the Cancer Renal Cytokine Study explored the survival characteristics of patients with metastatic RCC who received continuous infusion IL-2 alone, IFN alone, or IL-2 and IFN in combination. The therapeutic benefit generally was limited to those patients who achieved a complete response, and the 5-year survival rate for patients with metastatic disease was 8%. No benefit was conferred to patients who received combination treatment.42

Vaccination Strategies

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Initial efforts to produce tumor specific immunity have used TAA from allogeneic and autologous tumor cells, soluble proteins, peptides, and DNA as immunogens. Genetically modified allogeneic tumor cells as well as recombinant viruses and bacterial genes have served as vectors. Although many of the preliminary studies seemed promising, clinical studies using these vaccination schemes have had variable and sometimes frustrating results. In most studies, TAAs alone appeared to be insufficient to break tolerance. However, in recent years, some DNA and RNA vectors have been designed to express TAA in combination with costimulatory molecules and cytokines. Gene therapy studies by Hersh and Stopeck reported moderate intratumoral T-cell and antitumor responses.43 In a study of patients with colorectal carcinoma, Sobol et al. reported five-fold increases in tumor specific cytotoxic precursors in two of six patients after immunization with IL-2-expressing autologous fibroblasts mixed with autologous tumor cells.44 In other studies, irradiated melanoma cells that were engineered to express GM-CSF and were used as a potent vaccine to induce strong Th1 and Th2 responses and destroy metastatic lesions.45, 46

Dendritic Cell Therapy

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Dendritic cell (DC) therapy, a form of adoptive cellular immunotherapy, provides a promising alternative to direct vaccination strategies. It is believed that DCs are the immune system's most effective antigen-presenting cells. These cells are found in their immature state in the peripheral tissues, where they encounter and process antigens that are then transported to secondary T-cell-rich lymph organs and are displayed to T-cells. The interaction between activated, antigen-loaded, myeloid-type DCs and T-cells is a critical event in eliciting a cellular immune response to tumor antigens. DCs also may be responsible for enhancing the survival of CTLs recruited to the tumor site by protecting these effector cells from tumor-induced apoptosis.47 However, this protective effect may not extend to all DC subsets. Although donor DC subsets appear to have an important regulatory role in graft-versus-leukemia responses after allogeneic bone marrow transplantation (BMT) and also may be important in autologous antitumor responses, when high numbers of CD4 positive DCs are derived from lymphocyte precursors (lymphoid-type DCs) in bone marrow allografts, increased rates of recurrence have been reported in human leukemic antigen (HLA)-matched siblings in patients with leukemia and lymphoma after allogeneic BMT.48

Role of Cytokines in DC Therapy

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Immune inflammatory responses release an array of cytokines that are responsible for inducing the migration, maturation, and regulation of DCs and that also may play a role in inducing the expression of MHC and costimulatory ligands on the antigen-presenting cell.49, 50 Migratory effects have been demonstrated by GM-CSF, which has been shown to recruit mature myeloid-type DCs into primary and metastatic tumor sites51, 52 as well as into normal skin.53 Likewise, combination treatment with GM-CSF and TNF has resulted in increased levels of both hematopoietic progenitor cells and mature DCs in the skin.54 It is known that the maturation of DCs is stimulated by TNF or CD40L; and other cytokines, such as IFN-α, also may play an important role.55 Chen et al. reported that in vitro combination treatment of antigen-presenting cells using IFN and GM-CSF resulted in a marked stimulation of antileukemic CTLs in patients with chronic myelogenous leukemia (CML).56 By contrast, patients undergoing immunotherapy for metastatic RCC had no change in DC numbers as a result of treatment with IL-2 plus IFN or IL-12,57 and it has been shown that the presence of VEGF suppressed the maturation of progenitor DCs.28

Clinical Studies with Hemapoietic Cytokines that Act on DCs

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Stimulators of hematopoiesis, such as GM-CSF and granulocyte-colony stimulating factor (G-CSF), are used commonly to mobilize peripheral blood progenitor cells for collection prior to transplantation and to help restore normal hematopoiesis after ablative chemotherapy. The predominant clinical data available for stimulatory cytokines indicate that, of G-CSF and GM-CSF, both of which act on myeloid hematopoietic lineages, GM-CSF has the major effect on DC maturation and commitment to different pathways of differentiation.58 Furthermore, GM-CSF stimulates the function of myeloid-type CD11c positive DCs, enhancing antigen processing and presentation.59 It has been shown that combination treatment with G-CSF and GM-CSF was more effective in mobilizing peripheral blood progenitor cells compared with G-CSF alone,60 did not effect the risk of recurrence or the incidence of graft-versus-host disease (GVHD),61 and stimulated the generation of more CD14 positive DCs.58

Phenotypic and functional analyses have demonstrated two general types of DCs, type 1 (CD11c positive; DC1) and type 2 (CD123 positive; DC2), that differ in surface marker expression as well as their functional effect on cognate T-cells (see Fig. 1). Studies of cytokines used to mobilize peripheral blood hematopoietic progenitors have indicated the presence of increased numbers of DC1 cells after administration of Flt3 and GM-CSF, whereas increased numbers of DC2 cells have been noted after treatment with G-CSF.62 Interaction of DC1 cells with T-cells results in polarization of the T-cells toward Th1 immune responses, characterized by the generation of Tc1 cytotoxic effectors and the production of IL-12, TNF, and IFNγ. In contrast, interaction of DC2 cells with T-cells leads to the generation of effector cells that facilitate humoral immune responses, secrete IL-4 and IL-10, and suppress cytotoxic Th1 immune responses.63

thumbnail image

Figure 1. Schematic presentation of the interactions of cytokines in the ontogeny and function of type 1 (CD11c positive) dendritic cells (pDC1) and type 2 (CD123 positive) dendritic cells (pDC2). G-CSF: granulocyte colony stimulating factor; GM-CSF: granulocyte-macrophage–colony stimulating factor; HPC: hematopoietic progenitor cell; IFN-α, IFN-β, IFN-γ: interferon α, β, and γ; IL1I–IL12: interleukins 1–12; SCF: stem cell factor; Th1 and Th2: type 1 and type 2 T-helper cells; TNF: tumor necrosis factor.

Download figure to PowerPoint

Maturation of CD34 positive pluripotent hematopoietic progenitors into DC progenitors has been induced in culture with mixtures of interleukins, stem cell factor (SCF), and Flt-3.64 Whereas immature CD34 negative-CD1a negative-CD14 positive populations result from the treatment of CD34 positive cord blood cells with Flt-3 and erythropoietin, and the addition of SCF induced marked proliferation, the addition of GM-CSF and TNF resulted in generation of DCs with a mature phenotype (CD1a positive, CD14 negative, CD83 positive) without additional cell expansion.55 Arpinati et al. found that donor cells mobilized with G-CSF stimulated the expansion of DC2 populations without affecting DC1 cells. Because the DC2 subset is associated with Th2 activation, those researchers concluded that patients who undergo transplantation and receive G-CSF-stimulated peripheral blood stem cells should have a reduced incidence of GVHD.62 Recipients of T-cell-depleted, haplomismatched, allogeneic blood hematopoietic progenitor cell transplantation who received posttransplantation G-CSF had increased numbers of circulating T-cells with Th2 polarization compared with similar patients who did not receive G-CSF, potentially exacerbating the risk for opportunistic infections.65 Chang et al. demonstrated that autologous, pulsed DCs that were cultured in GM-CSF and IL-4 were capable of inducing a Th1-directed immune response in vitro.66

Clinical Trials of Antigen-Primed DCs

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Clinical trials using DC vaccines to elicit antitumor responses have shown mixed results (see Table 1). In a vaccination study using DC hybrid cells that were created by fusion of autologous tumor cells with allogeneic DCs, Kugler et al. treated 17 patients with metastatic RCC. At 13 months, 4 patients had complete responses, and three other patients had partial responses.67 In contrast, by vaccinating patients with irradiated autologous melanoma cells engineered to express GM-CSF, Soiffer et al. demonstrated a vigorous antitumor response in 11 of 16 patients with unresected metastatic melanoma. All 21 patients evaluated showed evidence of immune cell infiltration at the sites of immunization.45 When a DC vaccine using an HLA-A2 specific, prostate specific membrane antigen peptide was administered to patients with prostate carcinoma, no benefit was conferred by the addition of GM-CSF to the regimen;68 however, the coexpression of GM-CSF with tumor antigen was a more effective vaccine strategy. In patients with prostate carcinoma (n = 13 men) who were vaccinated with autologous DCs that were pulsed with a fusion protein comprised of GM-CSF and prostatic acid phosphatase (PAP), the response rate was 25% using prostate specific antigen (PSA) as an endpoint, and the 12 evaluable patients demonstrated T-cell specific responses to PAP and to GM-CSF.69 Among commercially available, U.S. Food and Drug Administration-approved cytokines, GM-CSF appears to have the most significant clinical effect on mobilizing DC1 cells and enhancing Th1/Tc1 cellular immune responses.

Table 1. Published Reports of Clinical Trials in Dendritic Cell Therapy
DiseaseAntigen source for pulsed DCsImmune responsesClinical responsesReference
  1. DCs: dendtitic cells; HLA; human leukocyte antigen; DTH: delayed type hypersensitivity; KLH: keyhole limpet hemocyanin; CR: complete response; PR: partial response; PSMA: prostate specific membrane antigen; CTL: cytotoxic T-lymphocytes; OR: objective response; CEA: carcinoembryonic antigen; MR: mixed/minor response; SD: stable disease; NHL: non-Hodgkin lymphoma.

Solid tumors    
 Metastatic melanomaHLA-restricted peptides In 12 patients and tumor lysate in 4 patientsDTH to peptides in 10 patients, to tumor lysate in 4 patients, and to KLH in all patients2 CR, 3 PRNestle et al.116
 Metastatic prostate carcinomaHLA-A0201 specific PSMA peptides in 33 patientsT-cell proliferation to antigen in HLA-A2 patients2 CR, 6 PRMurphy et al.117
 Metastatic melanomaMAGE-3A1 and tetanus toxoid or tuberculin protein in 11 patientsRecall antigen boosted in all patients, CTL to antigen in 8 of 11 patients6 ORThurner et al.118
 CEA and metastatic carcinomaCAP-1 peptide of CEA in 21 patientsNone consistent1 MR, 1 SDMorse et al.119
 Metastatic renal cell carcinomaElectrofusion of allogenic DCs and autologous tumor cells in 17 patientsInduction of tumor specific CTL, DTH in 11 patients4 CR, 2 PR, 1 MRKugler et al.67
Hematologic malignancies    
 B-cell NHLIdiotypic proteinSpecific cellular immunity in 4 patients, cellular and humoral immunity to KLH in 4 patients1 CR, 1 MR,Hsu et al.120
 MyelomaIdiotypic protein in 6 patientsCellular aned humoral immunity to idiotypic protein in all 6 patients6 SDLim and Bailey-Wood121

Antitumor Activity of GM-CSF Therapy Alone

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Reports of the dramatic effects of GM-CSF on DC maturation and function have led to a variety of studies exploring the antitumor effects of this cytokine in hematologic and nonhematologic malignancies.

Hematologic malignancies

GM-CSF can be used to stimulate the differentiation of some leukemic cells to phenotypically, morphologically, and functionally defined DCs. In a recent study that compared the effects of G-CSF and GM-CSF on the outcomes of patients who underwent stem cell transplantation for AML, the recovery of hematopoiesis was faster for patients who received G-CSF, and the risk of recurrence was lower for patients who received GM-CSF.70 Myeloid progenitors obtained from patients with AML differentiated to enhanced antigen-presenting cells when treated in vitro with GM-CSF, TNF, IL-4, CD40 ligand, Flt3, or SCF.71 Likewise, others have reported similar results after GM-CSF, TNF, and IL-4 culture of cells obtained from patients with AML and CML;72, 73 and Coleman et al. reported the restoration of costimulatory molecules on GM-CSF and IL-4-incubated T-cells obtained from patients with CML.74 In a series of interrelated trials reported by Bedi et al. and Jones et al., CML progenitor cells were eliminated preferentially from culture through terminal differentiation in the presence of hematopoietic growth factors. Subsequent allogeneic and autologous transplantation using these cultured cells, followed by posttransplantation treatment with GM-CSF, demonstrated an antileukemic effect by restoring normal hematopoiesis in the evaluable patients.75, 76 These findings were supported by a report that morbidity rates posttransplantation were reduced by treatment with GM-CSF.77 However, studies of GM-CSF treatment that were conducted in the absence of BMT produced variable results. Although high-risk patients with multiple myeloma who did not undergo transplantation experienced less neutropenia after treatment with GM-CSF, neither morbidity nor mortality rates were improved.78 By contrast, in an earlier report of a trial in patients with AML, chemotherapy followed by consolidation treatment that included GM-CSF resulted in improved remission rates, fewer treatment-related toxicities, and improved survival.79

Nonhematologic malignancies

Nonhematologic malignancies that have resisted standard treatments also may be targeted successfully by means of DC vaccines and/or stimulation of effector cells by cytokines that act on DCs. Studies of the antitumor effects of GM-CSF in patients with melanoma, breast carcinoma, prostate carcinoma, and RCC are underway, and some encouraging findings have been reported.

Melanoma.

Trials of GM-CSF in patients with advanced melanoma have been promising. In a nonrandomized, Phase II study reported by Spitler et al., survival was prolonged significantly by adjuvant treatment with GM-CSF after patients underwent surgical resection of Stage III and IV disease compared with a group of historically matched controls.80 Vaughn et al. have reported a preliminary study of GM-CSF combined with biochemotherapy for patients with metastatic melanoma that has suggested some benefits that may be dose-related.81 Additional observations supporting the role of GM-CSF in recruiting DCs were obtained from studies that explored intratumoral injection of GM-CSF. The studies by Nasi et al. and Si et al. showed that direct treatment with GM-CSF stimulated high levels of DC and T-cell infiltrates at the site of the tumor.53, 82 It also has been shown that perilesional intradermal treatment with GM-CSF increases the number of infiltrating monocytes found in noninjected bystander lesions, suggesting a systemic activation of the immune system.83

Breast carcinoma.

Two recent studies exploring the use of combination regimens for the treatment of patients with high-risk breast carcinoma have yielded promising results. Women with locally advanced breast carcinoma have responded to treatment with prolonged neoadjuvant chemotherapy (six cycles) that employed doxorubicin, cyclophosphamide, and GM-CSF prior to surgery plus postoperative radiotherapy. This regimen resulted in increased levels of DCs in the draining lymph nodes and a significant improvement in overall survival rates compared with standard, three-cycle neoadjuvant therapy. Pinedo and colleagues attributed this success to the prolonged interaction between GM-CSF-stimulated DCs, the tumor antigen, and the intact draining lymph node.84 A Phase III randomized trial is being pursued as a result of this work. Compared with an historic control group, Lum and Sen also observed improved cytokine production, T-cell activity, and survival in a similar patient group after treatment with IL-2, GM-CSF, and activated T-cells armed with anti-CD3 × anti-HER2/neu bispecific antibody.85

Prostate carcinoma.

Evaluation of GM-CSF for the treatment of patients with prostate carcinoma has been assessed by changes in PSA levels. James et al. reported a > 50% reduction in serum PSA levels in 35% of evaluable patients (n = 20 men) after treatment with GM-CSF and a bispecific antibody directed against HER2. Toxic side effects were acceptable, and 58% of patients experienced decreased pain.86 Dreicer et al. reported only moderate decreases in PSA levels during treatment with GM-CSF alone after failure on hormonal regimens in patients with advanced prostate carcinoma. The decreases in PSA levels were transient, and levels rose again at the termination of treatment.87 Two studies by Small et al. revealed a saw-tooth pattern of PSA response to GM-CSF treatment in patients with progressive, hormone-refractory prostate carcinoma. Based on imaging data, those researchers concluded that GM-CSF may have had an antitumor effect in their study population; however, in that study, no GM-CSF effect could be attributed to changes in PSA levels, thus raising concerns that treatment with GM-CSF may provide a confounding variable for the assessment of disease status using this measure.88 In a randomized study of two different doses of thrice-weekly GM-CSF, a correlation between increasing GM-CSF dose and the magnitude of reduction in PSA was observed, further supporting the hypothesis of a dose response correlation between GM-CSF administered systemically and immune-mediated antitumor effects.89 In other work, DCs have been used to deliver prostate specific membrane antigens through a systemic infusion with or without subcutaneous administration of GM-CSF; however, no enhancement of immune response was detected as a result of that method.68

RCC.

Compared with melanoma studies, efforts to improve outcomes for patients with RCC using GM-CSF have been disappointing. Antitumor effects of GM-CSF have been low, with few partial responses, although it was reported that these responses were slightly better in patients who were tumor free at the time of treatment90 and among patients who had not received prior therapy.91 In a recent Phase I trial by de Gast et al., combination therapy for patients with progressive, metastatic RCC with GM-CSF, IL-2, and IFN-γ resulted in significant stimulation of effector cells and complete responses in three of eight patients.92 In other work, an autologous tumor vaccine administered with GM-CSF followed by systemic GM-CSF demonstrated enhanced peripheral blood antitumor cytotoxic T-cell precursor ratios,93 suggesting that GM-CSF administered with the appropriate signals will enhance anti-RCC T-cell responses.

Future Directions for Antitumor Studies Using Cytokines

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES

Future clinical studies of the antitumor effects of immune-modulating cytokines, such as GM-CSF, will determine whether possible applications for this immunotherapeutic strategy translate into reproducible clinical benefit. This review has focused on roles for hematopoietic cytokines that are in addition to their use in supporting patients after chemotherapy-induced myelosuppression. Potential roles for cytokines that stimulate DC maturation and activation, such as Flt-3 and GM-CSF, include their use in combination therapies, in enhancing tumor cell antigenicity, in carrying protein targeted to antigen presenting cells, and in combination with other cytokines or biotherapies to recapitulate the physiologic pathways of antigen presentation and T-cell activation.

Combination therapy

The addition of GM-CSF to serve as an immune adjuvant for cytoreductive therapies is now being explored. In addition to the above-mentioned studies by Spitler et al. and Vaughn et al., Pinedo et al. reported increased DC levels in draining lymph nodes and improved survival after neoadjuvant biochemotherapy, surgical resection, and postoperative radiotherapy in 42 patients with locally advanced breast carcinoma.84 Those studies support the concept of cytoreduction of bulky carcinoma with chemotherapy, radiation, or surgery followed by immunotherapy and provide an attractive approach that warrants further study in randomized clinical trials.

Enhancing antigenicity

It has been shown that transfection of tumor cells with GM-CSF genes is an effective approach for the construction of vaccines that have demonstrated enhanced immune responses and possible clinical benefit in the treatment of patients with prostate carcinoma,94 pancreatic carcinoma,95 RCC,96 and melanoma.45, 97 Many research groups are investigating the feasibility of antitumor therapies using peptide-pulsed DCs in which maturation has been stimulated by GM-CSF. Promising early studies have been conducted in patients with melanoma,98–100 RCC,101 and multiple myeloma.102 Antigenicity also may be enhanced through an alternate technique that would not require genetic engineering methods. Recent studies have shown that using a combination of GM-CSF and other immune cytokines can stimulate myeloid leukemia cells to differentiate into cells with an antigen-presenting cell phenotype that are effective in alloantigen presentation.71, 103, 104

GM-CSF as a carrier protein

Another approach is the use of a cytokine as a carrier protein to direct tumor antigens to immune cells, thereby providing antigen and cytokine stimuli. G250 is a widely expressed renal carcinoma antigen. A G250-GM-CSF fusion protein was constructed that was capable of up-regulating DC MHC Class I and II molecules and enhancing T-helper cell-supported, G250-targeted, and CD8 positive-mediated antitumor response.105 Using a similar concept, antibody directed at tumor antigens can be fused with cytokines. An anti-HER2/neu immunoglobulin G3-(GM-CSF) fusion protein was constructed and, in animal studies, was capable of enhancing both Th1-mediated and Th-2-mediated immune responses and retarding the growth of HER2/neu-expressing tumors.106 Other carrier molecules also have been investigated.107 Several recent in vitro studies have explored the immune effects of GM-CSF fused to a diphtheria toxin (DT) carrier protein. Senchenkov et al. have demonstrated that DT(388)-GM-CSF stimulates ceramide formation, which, in turn, induces the apoptosis of human leukemia cells.108 Other preliminary work by Kim et al. resulted in similar findings, suggesting a synergistic apoptotic response of DT(388)-GM-CSF combined with cytosine arabinoside.109

Recapitulation and regulation of immune physiology

The long-term goal for immunotherapy is to manipulate cellular and humoral immune responses selectively to break tolerance against TAAs in a manner that results in clinically significant antitumor immune responses. Combining systemic cytokine administration with DC-based cellular immunotherapy may be one way to enhance these responses. In this approach, the physiologic pathways of antigen presentation are recapitulated ex vivo using enriched DC populations and antigen loading under defined laboratory conditions. Shimizu et al. have shown that systemic IL-2 therapy enhances the therapeutic efficacy of tumor lysate-pulsed DC therapy in animal models.110 Schwaab et al. have demonstrated that combining autologous tumor vaccine and IFN-α or INF-γ in patients with metastatic RCC can stimulate RCC specific T-cell subsets, supporting the hypothesis that appropriate cytokine signals are important in enhancing and amplifying tumor specific immune responses generated by DC therapies.111

In an attempt to recapitulate the physiology of antigen presentation, investigators also have studied the effect of different routes for cytokine administration. Aerosolized GM-CSF and IL-2 have been employed as a means of delivering cytokine to the effector cells in the regional organ. Using this delivery approach, cytokines can be administered safely. Immune regulation within the lung can be modulated, and clinical responses have been reported.112, 113

Another approach for cytokine therapies in regulating immune responses has been to develop fusion proteins of different cytokines, thereby delivering two or more signals simultaneously to the appropriate regulatory cells. GM-CSF has been linked to IL-3, resulting in a fusion protein, PIXY321.114 In Clinical trials have shown that PIXY321 allows bone marrow recovery and fewer episodes of neutropenia, although a higher incidence of thrombocytopenia was noted.115 These promising findings provide encouragement for the further exploration of the immunomodulatory properties of these molecules.

Cytokine-based immunotherapies hold much promise in the treatment of patients with malignant disease, and the recent successes in this area of research may help to broaden the range of tools available to clinicians in the treatment of patients for whom other treatment modalities have failed. Methods like those described herein, as they are developed more fully, also may be used as valuable neoadjuvant therapies. The availability of GM-CSF and the low toxicities associated with GM-CSF treatment, combined with the ability of this immune cytokine to enhance the efficacy of targeted vaccine therapies, should lead to randomized, placebo-controlled, immune adjuvant trials in patients who are not good candidates for more cytotoxic regimens. Novel approaches that activate and mature DCs to enhance antitumor responses can be tested as additional cytokines that act on DCs become available.

REFERENCES

  1. Top of page
  2. Abstract
  3. Defective Cellular Immunity
  4. Abnormal Patterns of Immune Cytokines
  5. IL-2 and IFN Therapy
  6. Vaccination Strategies
  7. Dendritic Cell Therapy
  8. Role of Cytokines in DC Therapy
  9. Clinical Studies with Hemapoietic Cytokines that Act on DCs
  10. Clinical Trials of Antigen-Primed DCs
  11. Antitumor Activity of GM-CSF Therapy Alone
  12. Future Directions for Antitumor Studies Using Cytokines
  13. Acknowledgements
  14. REFERENCES
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