Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF

Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB-231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment.     

Glycolipid-anchored IL-12 expressed on tumor cell surface induces antitumor immune response

Systemic or local administration of cytokine has been used as a mode to enhance the antitumor immune response induced by many cancer vaccines. We have investigated whether the expression of cytokines on the tumor cell surface as a glycolipid (GPI)-anchored form will be effective in inducing antitumor immune response using a GPI-anchored interleukin (IL)-12 (GPI-IL-12) as a model. GPI-IL-12-induced the proliferation of concanavalin A-activated T cells and induced IFN-gamma secretion by activated and allogeneic T cells, indicating that the membrane-expressed IL-12 can stimulate T cells. GPI-IL-12 expressed on the tumor cell surface prevented tumor growth in mice in a highly tumorigenic murine mastocytoma model. These results suggest that the cell surface-expressed GPI-IL-12 can be effective in inducing antitumor immune response, and GPI-anchored cytokines expressed on the tumor cell surface may be a novel approach to deliver cytokines at the immunization site during vaccination against cancer. Furthermore, purified GPI-anchored cytokines can be used to quickly modify tumor membranes by the protein transfer method to express the desired cytokines for vaccine development.     

Effective treatment of preexisting melanoma with whole cell vaccines expressing alpha(1,3)-galactosyl epitopes

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.c. and pulmonary alphaGal-negative melanoma (B16Null) tumors in the alpha(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16alphaGal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-alpha. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16alphaGal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing alphaGal epitopes. Vaccination with B16alphagal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.     

Cyclophosphamide, doxorubicin, and paclitaxel enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2/neu tolerized mice

Tumor-specific immune tolerance limits the effectiveness of cancer vaccines. In addition, tumor vaccines alone have a limited potential for the treatment of measurable tumor burdens. This highlights the importance of identifying more potent cancer vaccine strategies for clinical testing. We tested immune-modulating doses of chemotherapy in combination with a granulocyte/macrophage-colony stimulating factor (GM-CSF)-secreting, HER-2/neu (neu)-expressing whole-cell vaccine as a means to treat existing mammary tumors in antigen-specific tolerized neu transgenic mice. Earlier studies have shown that neu transgenic mice exhibit immune tolerance to the neu-expressing tumors similar to what is observed in patients with cancer. We found that cyclophosphamide, paclitaxel, and doxorubicin, when given in a defined sequence with a GM-CSF-secreting, neu-expressing whole-cell vaccine, enhanced the vaccine's potential to delay tumor growth in neu transgenic mice. In addition, we showed that these drugs mediate their effects by enhancing the efficacy of the vaccine rather than via a direct cytolytic effect on cancer cells. Furthermore, paclitaxel and cyclophosphamide appear to amplify the T helper 1 neu-specific T-cell response. These findings suggest that the combined treatment with immune-modulating doses of chemotherapy and the GM-CSF-secreting neu vaccine can overcome immune tolerance and induce an antigen-specific antitumor immune response. These data provide the immunological rationale for testing immune-modulating doses of chemotherapy in combination with tumor vaccines in patients with cancer.     

Cancer vaccines

Although cancer immunotherapy was initiated by William Coley more than a century ago, the field of cancer vaccines is in an early stage of development. Only recently, major advances in cellular and molecular immunology have allowed a comprehensive understanding of the complex and high rate of interactions between the immune system and tumor cells. We have learned that these tumor-immune system interactions may result either in strong immune antitumor response or tolerance to tumor-associated antigens. This article will discuss the profound interest in cancer vaccines derived from their potential to induce antitumor responses in vivo. Substantial data from several preclinical models and early human clinical trials have confirmed the ability of cancer vaccines to induce immune responses that are tumor-specific and, in some cases, associated with clinical responses. One future challenge will be to determine how to appropriately stimulate the pathways leading to effective interaction among antigen-presenting cells, T lymphocytes, and tumor cells. It also is critical to develop monitoring strategies that may allow the identification of patients who may benefit from cancer vaccines.     

Failure of cancer vaccines: the significant limitations of this approach to immunotherapy

Immunotherapy has always represented a very attractive fourth-modality therapeutic approach, especially in light of the many shortcomings of conventional surgery, radiation, and chemotherapies in the management of cancer. Subsets of neoplastically transformed cells have been shown to (re-)express on their surface molecules which are not typically present on the surface of neighboring normal cells. In some instances, especially in malignant melanomas, cytotoxic T lymphocytes (CTLs) directed against such tumor associated antigens (TAAs) have been isolated. The cancer vaccine approach to therapy is based on the notion that the immune system could possibly mount a rejection strength response against the neoplastically transformed cell conglomerate. However, due to the low immunogenicity of TAAs, downregulation of MHC molecules, the lack of adequate costimulatory molecule expression, secretion of immunoinhibitory cytokines, etc., such expectations are rarely fulfilled. Various approaches have been explored ranging from the use of irradiation inactivated whole-cell vaccines derived from both autologous and allogeneic tumors (even tumor cell lines), and genetically modified versions of such cellular vaccines which aim at correcting costimulatory dysfunction or altering the in situ humoral milieu to aid immune recognition and activation. Anti-idiotype vaccines, based on cancer cell associated idiotypes, have also been explored which aim at increasing immunogenicity through in vivo generation of vigorous immune responses. Dendritic cell (DC) vaccines seek to improve the presentation of TAAs to naive T lymphocytes. Unfortunately, there is always the possibility of faulty antigen presentation which could result in tolerance induction to the antigens contained within the vaccine, and subsequent rapid tumor progression. The theoretical basis for all of these approaches is very well founded. Animal models, albeit highly artificial, have yielded promising results. Clinical trials in humans, however, have been somewhat disappointing. Although general immune activation directed against the target antigens contained within the cancer vaccine has been documented in most cases, reduction in tumor load has not been frequently observed, and tumor progression and metastasis usually ensue, possibly following a slightly extended period of remission. The failure of cancer vaccines to fulfill their promise is due to the very relationship between host and tumor: through a natural selection process the host leads to the selective enrichment of clones of highly aggressive neoplastically transformed cells, which apparently are so dedifferentiated that they no longer express cancer cell specific molecules. Specific activation of the immune system in such cases only leads to lysis of the remaining cells expressing the particular TAAs in the context of the particular human leukocyte antigen (HLA) subclass and the necessary costimulatory molecules. The most dangerous clones of tumor cells however lack these features and thus the cancer vaccine is of little use. The use of cancer vaccines seems, at present, destined to remain limited to their employment as adjuvants to both traditional therapies and in the management of minimal residual disease following surgical resection of the primary cancer mass.     

Generation of effective antitumor vaccines using photodynamic therapy

Preclinical studies have shown that photodynamic therapy (PDT) of tumors augments the host antitumor immune response. However, the role of the PDT effect on tumor cells as opposed to the host tissues has not been determined. To test the contribution of the direct effects of PDT on tumor cells to the enhanced antitumor response by the host, we examined the immunogenicity of PDT-generated murine tumor cell lysates in a preclinical vaccine model. We found that the PDT-generated tumor cell lysates were potent vaccines and that PDT-generated vaccines are more effective than other modes of creating whole tumor vaccines, i.e., UV or ionizing irradiation, and unlike other traditional vaccines, PDT vaccines do not require coadministration of an adjuvant to be effective. PDT vaccines are tumor specific and appear to induce a cytotoxic T-cell response. We have demonstrated that although both UV and PDT-generated tumor cell lysates are able to induce phenotypic DC maturation, only PDT-generated lysates are able to activate DCs to express IL-12, which is critical to the development of a cellular immune response. Our results show that PDT effects on tumor cells alone are sufficient to generate an antitumor immune response, indicating that the direct tumor effects of PDT play an important role in enhancing that host antitumor immune response. These studies also suggest that in addition to the role of PDT as a therapeutic modality, PDT-generated vaccines may have clinical potential as an adjuvant therapy.     

Immune cells and cytokines - their role in cancer-immunotherapy (review)

Conventional cancer therapies such as surgery, chemotherapy and/or radiotherapy, are not always successful in providing long-term survival for cancer patients. One of the major problems with conventional cancer therapies is their inability to eradicate residual/metastatic tumor cells that are resistant to therapy. Therefore, it is necessary to develop new methods for treating such cancer cells in order to improve the clinical outcome of these patients. Despite antitumor effector mechanisms working against cancer cells in the host's body, tumor-cell-induced immunosuppression and or antigenic modulation by the tumor cells often help tumor cells escape host defense mechanisms. Therefore, one approach for treating residual cancer would be to enhance the host's own immunological/antitumor defense mechanisms. Immune cells that have a significant role in mediating antitumor responses include: T lymphocytes; natural killer (NK) cells; macrophages; and B lymphocytes. The ability of these immune cells to effectively destroy malignant cells is carefully governed by chemical mediators in the form of proteins otherwise known as cytokines. Many cytokines (interleukins, interferons, and tumor necrosis factor) have been shown to enhance in vitro and in vivo effector cell antitumor cytotoxic activities. Utilization of cytokines in conjunction with effector cells can also mediate significant antitumor responses in both animal models and cancer patients. One of the major problems associated with systemic treatment with cytokines is the development of dose limiting toxicities. Currently, attempts to reduce this problem include developing techniques to allow for the preferential release of cytokines in proximity to the tumor cell. In this regard, effector cells or tumor cells that have been genetically engineered to secrete cytokine(s) may be useful in localizing an immune response, preferably at the tumor site. Clinical trial using cytokine gene transfected cells for treating cancer are currently under investigation. With the availability of recombinant lymphokines and with our ability to genetically modify effector cells and tumor cells this hopefully will allow us to improve current therapeutic modalities for treating cancer.     

Cytotoxic T lymphocytes: role in immunosurveillance and in immunotherapy

Experiments in mice and recent human clinical studies have clearly shown the contribution of CD8+ T lymphocyte in the control of tumor development. CD8+ T lymphocytes are a constitutive component of the immune response during the development of cancer. In murine models, the efficiency of various cancer vaccines mainly depends on their ability to induce CD8+ T lymphocytes. Clinical responses in immunotherapy treated cancer patients have been associated with the presence of antitumor specific CD8+ T lymphocytes. In spontaneous regressive melanomas, intratumor antigen specific CD8+ cytotoxic T cells were expanded suggesting their involvement in the tumor shrinkage. Administration of antitumor specific cytotoxic T clones in mice resulted in antitumor responses which directly demonstrated the therapeutic efficiency of these cells. However, in most cases during cancer progression, the presence of antitumor CD8 T lymphocyte is not associated with clinical responses. Intrinsic functional abnormalities of these cells or a defect of CD8+ T cell migration to the tumor may in part explain their failure to inhibit tumor development. On the other hand, tumors also develop immune escape mechanisms (down modulation of tumor antigens, secretion of immunosuppressive factors, expression of anti-apoptotic molecules by the tumors, or pro-apoptotic factors inducing T cell death) to resist to the CD8+ T cell attack. To circumvent these tumor escape mechanisms, efficient cancer vaccines will have to recruit CD8+ T cells associated with other immune effectors.     

Hyperthermia enhances tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells

There is growing body of evidence linking the cellular response to heat stress with the response of the immune system to cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range. In addition, the heat shock proteins (hsp) which are produced in abundant quantities in cells exposed to heat are potent immune modulators and can lead to stimulation of both the innate and adaptive immune responses to tumors. Immunostimulation by hyperthermia involves both direct effects of heat on the behavior of immune cells as well as indirect effects mediated through hsp release. In addition, the hsp can be deployed as components of antitumor vaccines in protocols that do not include hyperthermia. Understanding these process may permit the effective deployment of hyperthermia and hsp based vaccines in tumor treatment.     

Cancer cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer as vaccines for the treatment of genitourinary malignancies

When irradiated and administered intradermally as vaccines, cancer cells engineered to secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) by gene transfer elicit potent anticancer immune responses in a variety of animal tumor models. Upon vaccination, antigens present in the cancer cells are phagocytosed and processed by skin dendritic cells. These dendritic cells then prime anticancer immune responses by presenting antigenic peptides to T cells. The immune responses generated are capable of eradicating small but lethal cancer cell inocula with minimal toxicity in preclinical animal tumor studies. To develop this vaccination strategy for the treatment of human genitourinary cancers, we have conducted phase I clinical trials using human genitourinary cancer cells as sources of cancer cell antigens. In the first human clinical trial of genetically engineered cancer cell vaccines, a phase I clinical trial of kidney cancer cell vaccines (n=18), kidney cancer cells were removed at surgery, propagated briefly in vitro, and then genetically modified to secrete high levels of GM-CSF via ex vivo transduction with the retrovirus MFG-GM-CSF. After irradiation, the kidney cancer cells were administered as vaccines to 18 patients with advanced kidney cancers. Vaccine treatment, which caused few side effects, nonetheless appeared to trigger anticancer immune responses manifest as conversion of delayed-type hypersensitivity (DTH) skin responses against irradiated autologous cancer cells after vaccination. Biopsies of vaccine sites yielded findings reminiscent of biopsies from preclinical animal model studies, with evidence of vaccine cell recruitment of dendritic cells, T cells, and eosinophils. One patient with measurable kidney cancer metastases treated at the highest vaccine dose level experienced a partial treatment response. The bioactivity of GM-CSF-secreting autologous cancer cell vaccines was confirmed in a phase I clinical trial for prostate cancer (n=8). Vaccine cells were prepared from surgically harvested prostate tumors by ex vivo transduction with MFG-GM-CSF in a manner similar to that used for the kidney cancer trial. Vaccine treatment was well tolerated and associated with induction of anticancer immunity as assessed using DTH skin testing. In addition, new antiprostate cancer cell antibodies were detected in serum samples from treated men as a consequence of vaccination. These first clinical trials of GM-CSF-secreting cancer cell vaccines for the treatment of genitourinary cancers have demonstrated both safety and bioactivity, in that very few side effects have been seen and anticancer immune responses have been detected. Future clinical studies will be required to assess vaccine treatment efficacy, refine vaccination dose and schedule, define the appropriate clinical context for the use of such vaccines, and ascertain optimal combinations involving vaccines and other local or systemic anticancer treatments.     

How is the immune response affected by hyperthermia and heat shock proteins?

There is growing body of evidence linking the cellular response to heat stress with the response of the immune system to cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range. In addition, the heat shock proteins (hsp) which are produced in abundant quantities in cells exposed to heat are potent immune modulators and can lead to stimulation of both the innate and adaptive immune responses to tumors. Immunostimulation by hyperthermia involves both direct effects of heat on the behavior of immune cells as well as indirect effects mediated through hsp release. In addition, the hsp can be deployed as components of antitumor vaccines in protocols that do not include hyperthermia. Understanding these process may permit the effective deployment of hyperthermia and hsp based vaccines in tumor treatment.     

Cryoimmunology for malignant bone and soft-tissue tumors

Several new methods have recently been developed for the treatment of malignant bone and soft-tissue tumors, and many of these targeted therapies have yielded promising initial results in clinical settings. As more sarcomas become amenable to effective molecular-targeting therapy, the need to evaluate the synergistic effects of combination therapies with anticancer drugs will grow. Other immunologic therapies have also been reported, such as exogenous cytokines, dendritic cell (DC) therapy and peptide vaccines. Cryoimmunology has shown promising results in some malignant tumors after cryosurgery and is expected to influence the next generation of tumor immunotherapy. In this report, we describe the induction of a systemic antitumor immune response following liquid nitrogen cryotreatment of a destructive murine osteosarcoma. Combining tumor cryotreatment with DCs to promote tumor-specific immune responses enhanced systemic immune responses and inhibited metastatic tumor growth. We also describe the induction of a systemic antitumor immune response following reconstruction for malignant bone tumors using frozen autografts treated with liquid nitrogen.     

Immunotherapy opportunities in ovarian cancer

Ovarian cancer is responsible for the majority of gynecologic cancer deaths and despite the highest standard of multimodality therapy with surgery and cytotoxic chemotherapy, long-term survival remains low. With compelling evidence that epithelial ovarian cancer is an immunogenic tumor capable of stimulating an antitumor immune response, renewed efforts to develop immune therapies to augment the efficacy of traditional therapies are underway. Current immunotherapies focus on varied modes of antitumor vaccine development, particularly with the use of dendritic cell vaccines, effective methods for adoptive T-cell transfer and combinatorial approaches with immune modulatory therapy subverting natural tolerance mechanisms or boosting effector mechanisms. Additional combinatorial approaches include the use of cytokines and/or chemotherapy with immune therapy.     

Immunotherapy opportunities in ovarian cancer

Ovarian cancer is responsible for the majority of gynecologic cancer deaths and despite the highest standard of multimodality therapy with surgery and cytotoxic chemotherapy, long-term survival remains low. With compelling evidence that epithelial ovarian cancer is an immunogenic tumor capable of stimulating an antitumor immune response, renewed efforts to develop immune therapies to augment the efficacy of traditional therapies are underway. Current immunotherapies focus on varied modes of antitumor vaccine development, particularly with the use of dendritic cell vaccines, effective methods for adoptive T-cell transfer and combinatorial approaches with immune modulatory therapy subverting natural tolerance mechanisms or boosting effector mechanisms. Additional combinatorial approaches include the use of cytokines and/or chemotherapy with immune therapy.     

Cancer stem cell vaccination confers significant antitumor immunity

Most studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosuppressed mice, preventing an assessment on the immunologic interactions and effects of CSCs. In this study, we examined the vaccination effects produced by CSC-enriched populations from histologically distinct murine tumors after their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. Immune sera from CSC-vaccinated hosts contained high levels of IgG which bound to CSCs, resulting in CSC lysis in the presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established that CSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity. Together, these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based on the development of CSC vaccines that can specifically target CSCs.     

Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo

Introduction of genes encoding immuno-stimulatory cytokines into cancer cells is known to enhance antitumor immunity. CD40 ligand (CD40L, CD154) and fms-like tyrosine kinase 3 ligand (Flt3L) are recently identified cytokines, which have been demonstrated to stimulate antitumor immunity in several cancer models. However little is known about antitumor activity of Ftl3L and CD40L against hepatocellular carcinoma (HCC). In the present study, we constructed replication-defective adenoviruses expressing Flt3L and CD40L and examined their therapeutic efficacy on mouse HCC, MH134 cells. Subcutaneous injection of MH134 cells genetically engineered to express Flt3L and/or CD40L developed tumors in all the syngeneic immunocompetent mice, but tumor growth was significantly delayed as compared to control mice. Partial inhibition of this antitumor effect in athymic nude mice suggests that both innate and adaptive immunity appear to play a role. It was shown by immunodepletion of NK cells with anti-asialo-GM1 antibody that the effector cells involved in innate immunity are NK cells. In a therapeutic setting, however, injection of adenovirus expressing Flt3L or CD40L into pre-established MH134 tumors exhibited no efficacy. These data demonstrate that Flt3L and CD40L induce significant, but only weak, antitumor immunity against MH134 cells presumably through both innate and adaptive immunity. Our results suggest that immuno-gene therapy with Flt3L and CD40L may need adjuvant modalities to achieve strong immune response.     

Targeting glycosylated antigens on cancer cells using siglec-7/9-based CAR T-cells

Chimeric antigen receptor (CAR) T-cells treatment demonstrate the increasing and powerful potential of immunotherapeutic strategies, as seen mainly for hematological malignancies. Still, efficient CAR-T cell approaches for the treatment of a broader spectrum of tumors are needed. It has been shown that cancer cells can implement strategies to evade immune response that include the expression of inhibitory ligands, such as hypersialylated proteins (sialoglycans) on their surface. These may be recognized by sialic acid-binding immunoglobulin-type lectins (siglecs) which are surface receptors found primarily on immune cells. In this regard, siglec-7 and -9 are found on immune cells, such as natural killer cells, T-cells, and dendritic cells and they can promote immune suppression when binding to sialic acids expressed on target cells. In the present study, we hypothesized that it is possible to use genetically engineered T-cells expressing siglec-based CARs, enabling them to recognize and eliminate tumor cells, in a non-histocompatibility complex molecule restricted way. Thus, we genetically modified human T-cells with different chimeric receptors based on the exodomain of human siglec-7 and -9 molecules and selected optimal receptors. We then assessed their antitumor activity in vitro demonstrating the recognition of cell lines from different histologies. These results were confirmed in a tumor xenograft model exemplifying the potential of the present approach. Overall, this study demonstrates the benefit of targeting cancer-associated glycosylation patterns using CAR based on native immune receptors and expressed in human primary T-cells.     

Interleukin-21 augments the efficacy of T-cell therapy by eliciting concurrent cellular and humoral responses

Interleukin (IL)-21 modulates T-cell-associated, B-cell-associated, and natural killer cell-associated immunity. However, the potential of IL-21 to simultaneously stimulate cellular and humoral antitumor responses and the mechanisms involved have not yet been adequately explored. In this report, we examined the immune-modulating effect of IL-21 when used in vitro and its adjuvant effects when administrated concomitantly with T-cell transfer for cancer therapy. Use of IL-21 in concert with IL-2 in culture up-regulated both type 1 and type 2 cytokine production of activated tumor-draining lymph node cells and enhanced their therapeutic efficacy. Administration of IL-21 and IL-2 as an adjuvant to T-cell transfer resulted in simultaneously elicited cellular and humoral responses. This concurrent response has led to effective regression of established pulmonary metastatic tumors and s.c. tumors. T-cell transfer plus IL-21/IL-2 administration conferred systemic immunity to the treated hosts. This was evident by the induction of protective immunity against tumor rechallenge, expansion of memory T cells, and significantly elevated serum levels of IFN gamma and IL-10. Furthermore, we observed significantly enhanced tumor-associated antibody response after T-cell + IL-2 + IL-21 therapy. Cytotoxic antibody subclass IgG2b increased strikingly in the sera of treated animals; they bound specifically to MCA205 tumor cells, and such immune sera mediated tumor cell lysis in the presence of complement. Use of B-cell-deficient mice provided direct evidence that humoral responses contribute to T-cell + IL-2 + IL-21-elicited antitumor immunity. Collectively, these findings provide a rationale to evaluate the use of IL-21 in T-cell therapy of human cancers.