Coupling and uncoupling of tumor immunity and autoimmunity

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.     

Implicating a role for immune recognition of self in tumor rejection: passive immunization against the brown locus protein

The immune system can recognize differentiation antigens that are selectively expressed on malignant cells and their normal cell counterparts. However, it is uncertain whether immunity to differentiation antigens can effectively lead to tumor rejection. The mouse brown locus protein, gp75 or tyrosinase-related protein 1, is a melanocyte differentiation antigen expressed by melanomas and normal melanocytes. The gp75 antigen is recognized by autoantibodies and autoreactive T cells in persons with melanoma. To model autoimmunity against a melanocyte differentiation antigen, mouse antibodies against gp75 were passively transferred into tumor-bearing mice. Passive immunization with a mouse monoclonal antibody against gp75 induced protection and rejection of both subcutaneous tumors and lung metastases in syngeneic C57BL/6 mice, including established tumors. Passive immunity produced coat color alterations but only in regenerating hairs. This system provides a model for autoimmune vitiligo and shows that immune responses to melanocyte differentiation antigens can influence mouse coat color. Immune recognition of a melanocyte differentiation antigen can reject tumors, providing a basis for targeting tissue autoantigens expressed on cancer.     

Optimization of a self antigen for presentation of multiple epitopes in cancer immunity

T cells recognizing self antigens expressed by cancer cells are prevalent in the immune repertoire. However, activation of these autoreactive T cells is limited by weak signals that are incapable of fully priming naive T cells, creating a state of tolerance or ignorance. Even if T cell activation occurs, immunity can be further restricted by a dominant response directed at only a single epitope. Enhanced antigen presentation of multiple epitopes was investigated as a strategy to overcome these barriers. Specific point mutations that create altered peptide ligands were introduced into the gene encoding a nonimmunogenic tissue self antigen expressed by melanoma, tyrosinase-related protein-1 (Tyrp1). Deficient asparagine-linked glycosylation, which was caused by additional mutations, produced altered protein trafficking and fate that increased antigen processing. Immunization of mice with mutated Tyrp1 DNA elicited cross-reactive CD8(+) T cell responses against multiple nonmutated epitopes of syngeneic Tyrp1 and against melanoma cells. These multi-specific anti-Tyrp1 CD8(+) T cell responses led to rejection of poorly immunogenic melanoma and prolonged survival when immunization was started after tumor challenge. These studies demonstrate how rationally designed DNA vaccines directed against self antigens for enhanced antigen processing and presentation reveal novel self epitopes and elicit multi-specific T cell responses to nonimmunogenic, nonmutated self antigens, enhancing immunity against cancer self antigens.     

Protective immunization against melanoma by gp100 DNA-HVJ-liposome vaccine.

DNA-based vaccine immunization effectively induces both humoral and cell-mediated immunity to antigens and can confer protection against numerous infectious diseases as well as some cancers. Human and mouse melanomas consistently express the tumor-associated antigen interacted with the melanogenesis pathway. Gp100 is immunogenic and has been shown to induce both antibody and cytotoxic T cell (CTL) responses in humans. To explore the potential use of DNA immunization to induce melanoma-specific immune responses, we assessed HVJ-AVE liposome incorporated with plasmid DNA encoding human gp100. The gp100 DNA vaccine was used in a mouse melanoma model to assess immunity against the B16 melanoma of C57BL/6 mice. Intramuscular injection of the DNA-HVJ-AVE liposomes induced both anti-gp100 antibody and CTL responses. Gp100 DNA-HVJ-AVE liposome immunization significantly delayed tumor development in mice challenged with B16 melanoma cells. Mice immunized with gp100 DNA-HVJ-AVE liposomes survived longer compared with control mice immunized with HVJ-AVE liposome alone. These results indicate that immunization with human gp100 DNA by HVJ-AVE liposomes can induce protective immunity against melanoma in this pre-clinical mouse model. This strategy may provide an effective approach for vaccine therapy with tumor-associated antigens against human melanoma.     

Hydrodynamic limb vein delivery of a xenogeneic DNA cancer vaccine effectively induces antitumor immunity.

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.     

Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.     

Safety and immunogenicity of tyrosinase DNA vaccines in patients with melanoma.

Immunity to self antigens on cancer is constrained by tolerance/ignorance. DNA vaccines encoding xenogeneic differentiation antigens, such as tyrosinase (TYR), mediate tumor protection and regression in implantable mouse models, and dogs with spontaneous melanoma. We conducted a trial of mouse and human TYR DNA vaccines in stage III/IV melanoma patients. Eighteen human leukocyte antigen (HLA)-A*0201(+) melanoma patients were randomized as follows: one group received three mouse TYR DNA injections followed by three human TYR DNA injections; the other group received the same vaccines in opposite sequence. The study was conducted at three dose levels: 100, 500, and 1,500 microg DNA/injection, administered intramuscularly (IM) every 3 weeks. Most toxicities were grade 1 injection site reactions. Seven patients developed CD8(+) T-cell responses, defined by a >3 SD increase in baseline reactivity to TYR peptide in tetramer or intracellular cytokine staining (ICS) assays. There was found to be no relationship between dose, assigned schedule, and T-cell response. At a median of 42 months follow-up, median survival has not been reached. Mouse and human TYR DNA vaccines were found safe and induced CD8(+) T-cell responses in 7 of 18 patients. T cells recognizing a native TYR peptide had a phenotype consistent with that of effector memory cells.     

A novel DNA vaccine based on ubiquitin-proteasome pathway targeting 'self'-antigens expressed in melanoma/melanocyte

Cancer vaccine that targets 'self'-antigens expressed at high levels in tumor cells is a potentially useful immunotherapy, but immunological tolerance often defeats this strategy. Here, we describe the use of a naked DNA vaccine encoding a self tumor antigen, tyrosinase-related protein 2, to whose N-terminus ubiquitin is fused in a 'nonremovable' fashion. Unlike conventional DNA vaccines, this vaccine broke the tolerance and induced protective immunity to melanoma in C57BL/6 mice, as evaluated by tumor growth, survival rate and lung metastasis. The protective immunity was cancelled in the proteasome activator PA28alpha/beta knockout mice. Moreover, this vaccination exhibited therapeutic effects on melanoma implanted before vaccination. Our findings provide evidence for the first time that naked DNA vaccines encoding a ubiquitin-fused self-antigen preferentially induce the main effector CD8+ T cells through efficient proteolysis mediated by the ubiquitin-proteasome pathway, and lead the way to strategies aimed at targeting tissue differentiation antigens expressed by tumors.     

An optimum anti-melanoma response in mice immunized with fibroblasts transfected with DNA from mouse melanoma cells requires the expression of both syngeneic and allogeneic MHC-determinants

Most neoplasms do not induce antitumor immune responses that can control tumor growth. Tumor associated antigens (TAAs) are insufficiently immunogenic. A vaccine that augments the immunogenic properties of TAAs could be of importance in the treatment of cancer patients. In an animal model, we prepared a vaccine by transfection of highly antigenic allogeneic mouse fibroblasts (LM; H-2(k)) with DNA from B16 mouse melanoma cells. We then tested the transfected cells' immunogenic properties in C57BL/6 mice, syngeneic with the melanoma (H-2(b)). We hypothesized that the immunogenic properties of 'weak' TAAs formed by the neoplasm would be enhanced if they were expressed by highly antigenic cells. The results indicated that mice with melanoma treated by immunization with the DNA-transfected fibroblasts survived significantly longer than mice in various control groups. To investigate the contribution of MHC determinants expressed by the transfected cells to their immunogenic properties, we compared the antimelanoma responses in mice immunized with transfected cells that expressed allogeneic or syngeneic class I determinants. The results indicated that the immunogenic properties of the DNA-transfected cells were enhanced if the cells expressed allogeneic MHC determinants. The antimelanoma responses of greatest magnitude, however, mediated predominantly by CD8(+) T cells, were in mice immunized with transfected fibroblasts that expressed both syngeneic and allogeneic class I determinants.     

The dual nature of specific immunological activity of tumor-derived gp96 preparations

Mice immunized with optimal doses of autologous tumor-derived gp96 resist a challenge with the tumor that was the source of gp96. Immunization with quantities of gp96 5-10 times larger than the optimal dose does not elicit tumor immunity. This lack of effect is shown to be an active, antigen-specific effect, in that immunization with high doses of tumor-derived gp96, but not normal tissue-derived gp96, downregulates the antitumor immune response. Furthermore, immunization with fractionated doses of gp96 elicits the same kind and level of response as elicited by a single dose equivalent to the total of the fractionated doses. This is true of the tumor-protective doses as well as the high downregulatory doses of gp96. The downregulatory activity can be adoptively transferred by CD4(+) but not CD8(+) T lymphocytes from mice immunized with high doses of gp96. These observations indicate that immunization with gp96 induces a highly regulated immune response that, depending upon the conditions of immunization, results in tumor immunity or downregulation.     

Injection of DNA encoding granulocyte-macrophage colony-stimulating factor recruits dendritic cells for immune adjuvant effects

An important issue for effective vaccines is the development of potent adjuvants that can facilitate induction or augmentation of immunity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for myeloid progenitors of monocytes and dendritic cells (DC), which upon maturation are antigen-presenting cells (APC). The adjuvant effects of inoculation of DNA encoding GM-CSF into skin were studied. Initial experiments examined whether the GM-CSF gene injected into the skin of mice could affect the density of epidermal DC (Langerhans cells). DNA encoding GM-CSF delivered by particle bombardment into skin resulted in a significant increase of epidermal DC at the inoculation site. Kinetic analysis of epidermal recruitment after GM-CSF inoculation showed an increase in DC that peaked at seven days. This increase was accompanied by recruitment of DC into draining lymph nodes. The adjuvant effects of DNA encoding GM-CSF inoculated into skin were measured by the ability to augment antibody and T-cell responses against poorly immunogenic tumor antigens. Peptide immunization at skin sites containing epidermal DC newly recruited by GM-CSF DNA elicited T-cell responses against mutant p53, whereas peptide immunization of control skin sites did not elicit any detectable T-cell responses. Likewise, generation of antibodies following immunization with DNA encoding human gp75TRP1, a tyrosinase family member expressed by melanomas, was accelerated and protection from tumor challenge augmented by GM-CSF DNA.     

Prime-boost vaccines encoding an intracellular idiotype/GM-CSF fusion protein induce protective cell-mediated immunity in murine pre-B cell leukemia

Two vaccines against an intracellularly expressed B cell idiotype were assessed for their ability to induce protective immunity in mice against challenge with a pre-B cell leukemia. One vaccine was based on a plasmid expression vector and the other was a recombinant vaccinia virus; both vaccines expressed a polypeptide derived from the complementarity-determining regions (CDR(2)-CDR(3)) of the leukemic clone-specific immunoglobulin heavy chain (IgH), as a fusion product with mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF). Mice inoculated with either vaccine showed significantly higher survival rates than controls after challenge with leukemia cells. However, protection from tumor challenge was optimal when the DNA vaccine was used for priming, followed by a booster immunization with the vaccinia virus recombinant. This vaccination protocol induced resistance not only to the first tumor challenge given shortly afterwards, but also to a second challenge given months later. Both CD4(+) and CD8(+) T cells contributed to protection in vaccinated mice. These data suggest that such a vaccine regimen might reduce the incidence of recurrence in patients with minimal residual disease after conventional therapy.     

Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.     

Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy.

A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.     

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.     

Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

Dendritic cells (DCs) can initiate and shape host immune responses toward either immunity or tolerance by their effects on antigen-specific CD4(+) T cells. DC-asialoglycoprotein receptor (DC-ASGPR), a lectinlike receptor, is a known scavenger receptor. Here, we report that targeting antigens to human DCs via DC-ASGPR, but not lectin-like oxidized-LDL receptor, Dectin-1, or DC-specific ICAM-3-grabbing nonintegrin favors the generation of antigen-specific suppressive CD4(+) T cells that produce interleukin 10 (IL-10). These findings apply to both self- and foreign antigens, as well as memory and naive CD4(+) T cells. The generation of such IL-10-producing CD4(+) T cells requires p38/extracellular signal-regulated kinase phosphorylation and IL-10 induction in DCs. We further demonstrate that immunization of nonhuman primates with antigens fused to anti-DC-ASGPR monoclonal antibody generates antigen-specific CD4(+) T cells that produce IL-10 in vivo. This study provides a new strategy for the establishment of antigen-specific IL-10-producing suppressive T cells in vivo by targeting whole protein antigens to DCs via DC-ASGPR.     

AUTOSENSITIZATION IN VITRO

Autosensitization of rat or mouse lymphoid cells against syngeneic fibroblast antigens was induced in cell culture. Rat lymphoid cells autosensitized by this method were able to produce immunospecific lysis of syngeneic target fibroblasts in vitro or GvH reactions in newborn rats. Autosensitized mouse spleen cells mediated similar GvH reactions when injected into newborn mice. The nature of the system used to induce immunity in vitro appears to argue against the possibility that lymphocytes capable of reacting against self-antigens could arise by mutation in cell culture. Hence, it is likely that cells potentially reactive against self-antigens preexisted in the lymphoid cell donors. The ability of autosensitized cells to mediate immune reactions in vivo suggests that the immunogenic self-antigens present on sensitizing fibroblasts also were accessible in the intact animals. Loss of natural self-tolerance in vitro, therefore, can be explained most simply by the existence of lymphocytes which are reversibly tolerant to self. Hence, ontogenic elimination of potentially self-reactive cells may not be the only basis for natural tolerance. Regulatory mechanisms, such as antigen excess, may have to function in vivo to prevent differentiation of self-tolerant lymphocytes. These regulatory mechanisms appear to be annulled in the cell-culture system. The present system thus may offer a new approach to studies of tolerance and regulation of cellular immunity.     

DNA vaccination with CD25 protects rats from adjuvant arthritis and induces an antiergotypic response

Ab's to the alpha-chain of the IL-2 receptor (anti-CD25) are used clinically to achieve immunosuppression. Here we investigated the effects of DNA vaccination with the whole CD25 gene on the induction of rat adjuvant arthritis. The DNA vaccine protected the rats and led to a shift in the cytokine profile of T cells responding to disease target antigens from Th1 to Th2. The mechanism of protection was found to involve the induction of an antiergotypic response, rather than the induction of anti-CD25 Ab's. Antiergotypic T cells respond to activation molecules, ergotopes, expressed on syngeneic activated, but not resting, T cells. CD25-derived peptides function as ergotopes that can be recognized by the antiergotypic T cells. Antiergotypic T cells taken from control sick rats did not proliferate against activated T cells and secreted mainly IFN-gamma. In contrast, antiergotypic cells from CD25-DNA-protected rats proliferated against activated T cells and secreted mainly IL-10. Protective antiergotypic T cells were found in both the CD4+ and CD8+ populations and expressed alpha/beta or gamma/delta T cell receptors. Antiergotypic alpha/beta T cells were MHC restricted, while gamma/delta T cells were MHC independent. Thus, CD25 DNA vaccination may induce protection from autoimmunity by inducing a cytokine shift in both the antiergotypic response and the response to the antigens targeted in the disease.     

Frequent and specific immunity to the embryonal stem cell-associated antigen SOX2 in patients with monoclonal gammopathy

Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.     

Immunogene Therapy for Murine Melanoma Using Recombinant Adenoviral Vectors Expressing Melanoma-Associated Antigens

Adenoviral vectors expressing tumor-associated antigens can be used to evoke a specific immune response and inhibit tumor growth. In this study, we tested the efficacy of adenoviral vectors encoding human gp100 (Ad2/hugp100), murine gp100 (Ad2/mugp100), or murine TRP-2 (Ad2/muTRP-2) for their ability to elicit a specific cellular immune response and inhibit the growth of B16 melanoma tumor cells in the mouse. C57BL/6 mice were immunized with Ad2/hugp100, Ad2/mugp100, or Ad2/muTRP-2 either 2 weeks prior to B16-F10 tumor challenge (prophylactic treatment) or 3 days after tumor challenge (active treatment). Ad2/hugp100 and Ad2/muTRP-2 administered to two or more intradermal (id) sites inhibited subsequent subcutaneous tumor growth in ≥80% of the mice and elicited an antigen-specific cytotoxic T lymphocyte response, whereas other administration routes were not as effective. Ad2/mugp100 administered to two id sites did not inhibit tumor growth or provoke cellular immunity. Immunization was less effective with active treatment where tumor growth was not significantly inhibited by a single dose of either Ad2/muTRP-2 or Ad2/hugp100. However, increasing the number of intradermal immunization sites and the number of doses resulted in progressive improvements in protection from tumor growth in the active treatment model. In conclusion, breaking host tolerance to elicit protective immunity by using adenoviral vectors expressing melanoma-associated antigens is dependent upon the choice of antigen, the site of administration, and the number of doses. These observations provide insights into the clinical applicability of adenoviral vaccines for immunotherapy of malignant diseases.