Long-term survival of dogs with advanced malignant melanoma after DNA vaccination with xenogeneic human tyrosinase: a phase I trial.

PURPOSE: Canine malignant melanoma (CMM) is a spontaneous, aggressive, and metastatic neoplasm. Preclinical mouse studies have shown that xenogeneic DNA vaccination with genes encoding tyrosinase family members can induce antibody and cytotoxic T-cell responses, resulting in tumor rejection. These studies provided the rationale for a trial of xenogeneic DNA vaccination in CMM using the human tyrosinase gene. EXPERIMENTAL DESIGN: Three cohorts of three dogs each with advanced (WHO stage II, III, or IV) CMM received four biweekly i.m. injections (dose levels 100, 500, or 1500 micro g, respectively/vaccination) of human tyrosinase plasmid DNA i.m. via the Biojector2000 delivery device. RESULTS: Mild local reactions at injection sites were the only toxicities observed, with no signs of autoimmunity. One dog with stage IV disease had a complete clinical response in multiple lung metastases for 329 days. Two dogs with stage IV disease had long-term survivals (421 and 588+ days) in the face of significant bulky metastatic disease, and two other dogs with locally controlled stage II/III disease had long-term survivals (501 and 496 days) with no evidence of melanoma on necropsy. Four other dogs were euthanized because of progression of the primary tumor. The Kaplan-Meier median survival time for all nine dogs was 389 days. CONCLUSIONS: The results of this trial demonstrate that xenogeneic DNA vaccination of dogs with advanced malignant melanoma is a safe and potentially therapeutic modality. On the basis of these results, additional evaluation of this novel therapeutic is warranted in locally controlled CMM and advanced human melanoma.     

Modified vaccinia virus ankara recombinants are as potent as vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses

Cancer vaccine regimens use various strategies to enhance immune responses to specific tumor-associated antigens (TAAs), including the increasing use of recombinant poxviruses [vaccinia (rV) and fowlpox (rF)] for delivery of the TAA to the immune system. However, the use of replication competent vectors with the potential of adverse reactions have made attenuation a priority for next-generation vaccine strategies. Modified vaccinia Ankara (MVA) is a replication defective form of vaccinia virus. Here, we investigated the use of MVA encoding a tumor antigen gene, carcinoembryonic antigen (CEA), in addition to multiple costimulatory molecules (B7-1, intercellular adhesion molecule-1, and lymphocyte function-associated antigen-3 designated TRICOM). Vaccination of mice with MVA-CEA/TRICOM induced potent CD4+ and CD8+ T-cell responses specific for CEA. MVA-CEA/TRICOM could be administered twice in vaccinia na?ve mice and only a single time in vaccinia-immune mice before being inhibited by antivector-immune responses. The use of MVA-CEA/TRICOM in a diversified prime and boost vaccine regimen with rF-CEA/TRICOM, however, induced significantly greater levels of both CD4+ and CD8+ T-cell responses specific for CEA than that seen with rV-CEA/TRICOM prime and rF-CEA/TRICOM boost. In a self-antigen tumor model, the diversified MVA-CEA/TRICOM/rF-CEA/ TRICOM vaccination regimen resulted in a significant therapeutic antitumor response as measured by increased survival, when compared with the diversified prime and boost regimen, rV-CEA/TRICOM/rF-CEA/TRICOM. The studies reported here demonstrate that MVA, when used as a prime in a diversified vaccination, is clearly comparable with the regimen using the recombinant vaccinia in both the induction of cellular immune responses specific for the "self"-TAA transgene and in antitumor activity.     

Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects

Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conventional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule transgenes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowlpox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccinations with rF-CEA or two vaccinations with rF-CEA/B7-1. Moreover, up to four vaccinations with rF-CEA/TRICOM induced greater CEA-specific T-cell responses with each vaccination. (b) A diversified prime and boost strategy using a prime with a recombinant vaccinia vector expressing CEA and the triad of costimulatory molecules (designated rV-CEA/TRICOM) and a boost with rF-CEA/TRICOM was more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA/TRICOM alone. (c) The addition of granulocyte macrophage colony-stimulating factor (GM-CSF) to the rF-CEA or rF-CEA/TRICOM vaccinations via the simultaneous administration of a rF-GM-CSF vector enhanced CEA-specific T-cell responses. These strategies (TRICOM/diversified prime and boost/GM-CSF) were combined to treat CEA-expressing carcinoma liver metastases in CEA-transgenic mice; vaccination was initiated 14 days posttumor transplant. Antitumor effects in terms of survival and CD8(+) and CD4(+) responses specific for CEA were also observed in this CEA-transgenic mouse model. These studies demonstrate that the use of cytokines and diversified prime and boost regimens can be combined with the use of recombinant vectors expressing signal 1 and multiple costimulatory molecules to further amplify T-cell responses toward more effective vaccine strategies.     

Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy

Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed.     

Efficient induction of T-cell responses to carcinoembryonic antigen by a heterologous prime-boost regimen using DNA and adenovirus vectors carrying a codon usage optimized cDNA

The immunogenic properties of plasmid DNA and recombinant adenovirus (Ad) encoding the carcinoembryonic antigen (CEA) were examined in mice by measuring both the amplitude and type of immune response, and the immunogenicity of codon usage optimized cDNA encoding CEA (CEAopt) was assessed both in C57Bl/6 and CEA transgenic mice. Vectors were injected into quadriceps muscle either alone or in combination, and plasmid DNA was electroporated to enhance gene expression efficiency and immunogenicity. Injection of plasmid pVIJ/CEA followed by Ad-CEA boost elicited the highest amplitude of both CD4+ and CD8+ T-cell response to the target antigen, measured by both IFNgamma-ELIspot assay and intracellular staining. Vectors carrying cDNA of CEAopt expressed a greater amount of the CEA protein than their wild-type counterparts, and this enhanced expression was associated with greater immunogenicity. Both CD4+ and CD8+ T-cell epitopes were mapped in the C-terminal portion of the protein. In CEA transgenic mice, only immunization based on repeated injections of pVIJ/CEAopt followed by Ad-CEAopt was able to elicit a CEA-specific CD8+ T-cell response, whereas the wild-type vectors did not break tolerance to this target antigen. MC38-CEA tumor cells injected s.c. in CEA transgenic mice vaccinated with CEAopt vectors exhibited delayed growth kinetics. These studies demonstrate that this type of genetic vaccine is highly immunogenic and can break tolerance to CEA tumor antigen in CEA transgenic mice.     

Update on immunotherapy for melanoma

Several experimental immunotherapy approaches and standard therapy with high doses of interleukin (IL)-2 can cause prolonged objective responses in some patients with metastatic melanoma. Experimental immunotherapy approaches in clinical development include 1) cytokines such as IL-7 and IL-21, 2) cytokine-antibody fusion proteins or immunocytokines, 3) whole tumor cell vaccines, 4) genetically modified tumor cells, 5) heat shock protein vaccines, 6) peptide vaccines, 7) dendritic cells pulsed with tumor antigens, 8) tumor antigen-naked DNA vectors, 9) recombinant viral vectors (either alone or in a prime boost schedule), 10) adoptive transfer of cloned tumor antigen-specific T cells, 11) Toll-like receptor ligands, 12) antagonistic antibodies to the cytotoxic T-lymphocyte antigen 4 (CTLA4, CD152), and 13) activating antibodies to CD40 and CD137 (41-BB). These improved approaches to induce cytotoxic T-cell responses to tumors are based on a more detailed understanding of the immune system activation and regulation. The higher response rates with modern immunotherapy approaches may allow exploration of the molecular mechanisms that make tumor targets sensitive or resistant to immunotherapy.     

DNA vaccines: an active immunization strategy for prostate cancer

Immunotherapy is currently being investigated as a treatment for patients with asymptomatic, recurrent prostate cancer manifested only by a rising prostate-specific antigen (PSA) level. Several different approaches to active immunization against antigens found on cancer cells have been explored. Immunization with DNA overcomes many of the obstacles noted in previous studies. Injection of plasmid DNA encoding a xenogeneic differentiation antigen (prostate-specific membrane antigen [PSMA]) is a potent means to induce antibody and T-cell responses to these otherwise poorly immunogenic self proteins. Use of the xenogeneic DNA (ie, human PSMA DNA injected into mouse) has been shown to be an absolute requirement to overcome immunologic tolerance. We are currently conducting a phase I trial of human and mouse PSMA DNA vaccines in patients with recurrent prostate cancer, based on preclinical experiments described below.     

Induction of an antigen cascade by diversified subcutaneous/intratumoral vaccination is associated with antitumor responses.

PURPOSE: Cancer vaccines targeting tumor-associated antigens are being investigated for the therapy of tumors. Numerous strategies, including the direct intratumoral (i.t.) vaccination route, have been examined. For tumors expressing carcinoembryonic antigen (CEA) as a model tumor-associated antigen, we previously designed poxviral vectors that contain the transgenes for CEA and a triad of T-cell costimulatory molecules, B7-1, intercellular adhesion molecule-1, (ICAM-1), and leukocyte function associated antigen-3 (LFA-3) (CEA/TRICOM). Two types of poxvirus vectors were developed: replication-competent recombinant vaccinia and replication-defective recombinant fowlpox. We have shown previously that a vaccine regimen composed of priming mice s.c. with recombinant vaccinia-CEA/TRICOM and boosting i.t. with recombinant fowlpox-CEA/TRICOM was superior to priming and boosting vaccinations using the conventional s.c. route in inducing T-cell responses specific for CEA. These studies also showed that CEA was needed to be present both in the vaccine and in the tumor for therapeutic effects. EXPERIMENTAL DESIGN: To determine specific immune responses associated with vaccination-mediated tumor regression, CEA-transgenic mice bearing CEA(+) tumors were vaccinated with the CEA/TRICOM s.c./i.t. regimen, and T-cell immune responses were assessed. RESULTS: In CEA(+) tumor-bearing mice vaccinated with the CEA/TRICOM s.c./i.t. regimen, T-cell responses could be detected not only to CEA encoded in vaccine vectors but also to other antigens expressed on the tumor itself: wild-type p53 and an endogenous retroviral epitope of gp70. Moreover, the magnitude of CD8(+) T-cell immune responses to gp70 was far greater than that induced to CEA or p53. Finally, the predominant T-cell population infiltrating the regressing CEA(+) tumor after therapy was specific for gp70. CONCLUSION: These studies show that the breadth and magnitude of antitumor immune cascades to multiple antigens could be critical in the therapy of established tumors.     

In vivo effects of vaccination with six-transmembrane epithelial antigen of the prostate: a candidate antigen for treating prostate cancer

Immunotherapy may provide an alternative treatment for cancer patients, especially when tumors overexpress antigens that can be recognized by immune cells. The identification of markers and therapeutic targets that are up-regulated in prostate cancer has been important to design new potential treatments for prostate cancer. Among them, the recently identified six-transmembrane epithelial antigen of the prostate (STEAP) is considered attractive due to its overexpression in human prostate cancer tissues. Our study constitutes the first assessment of the in vivo effectiveness of STEAP-based vaccination in prophylactic and therapeutic mouse models. Two delivery systems, cDNA delivered by gene gun and Venezuelan equine encephalitis virus-like replicon particles (VRP), both encoding mouse STEAP (mSTEAP) and three vaccination strategies were used. Our results show that mSTEAP-based vaccination was able to induce a specific CD8 T-cell response against a newly defined mSTEAP epitope that prolonged the overall survival rate in tumor-challenged mice very significantly. This was achieved without any development of autoimmunity. Surprisingly, CD4 T cells that produced IFNgamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2) played the main role in tumor rejection in our model as shown by using CD4- and CD8-deficient mice. In addition, the presence of high IL-12 levels in the tumor environment was associated with a favorable antitumor response. Finally, the therapeutic effect of STEAP vaccination was also assessed and induced a modest but significant delay in growth of established, 31 day old tumors. Taken together, our data suggest that vaccination against mSTEAP is a viable option to delay tumor growth.     

Enhancement of tumor-specific immune response with plasmid DNA replicon vectors

To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.     

Phase II randomized study of vaccine treatment of advanced prostate cancer (E7897): a trial of the Eastern Cooperative Oncology Group.

PURPOSE: A phase II clinical trial was conducted to evaluate the feasibility and tolerability of a prime/boost vaccine strategy using vaccinia virus and fowlpox virus expressing human prostate-specific antigen (PSA) in patients with biochemical progression after local therapy for prostate cancer. The induction of PSA-specific immunity was also evaluated. PATIENTS AND METHODS: A randomized clinical trial was conducted by the Eastern Cooperative Oncology group and 64 eligible patients were randomly assigned to receive four vaccinations with fowlpox-PSA (rF-PSA), three rF-PSA vaccines followed by one vaccinia-PSA (rV-PSA) vaccine, or one rV-PSA vaccine followed by three rF-PSA vaccines. The major end point was PSA response at 6 months, and immune monitoring included measurements of anti-PSA and anti-vaccinia antibody titers and PSA-specific T-cell responses. RESULTS: The prime/boost schedule was well tolerated with few adverse events. Of the eligible patients, 45.3% of men remained free of PSA progression at 19.1 months and 78.1% demonstrated clinical progression-free survival. There was a trend favoring the treatment group that received a priming dose of rV-PSA. Although no significant increases in anti-PSA antibody titers were detected, 46% of patients demonstrated an increase in PSA-reactive T-cells. CONCLUSION: Therapy with poxviruses expressing PSA and delivered in a prime/boost regimen was feasible and associated with minimal toxicity in the cooperative group setting. A significant proportion of men remained free of PSA and clinical progression after 19 months follow-up, and nearly half demonstrated an increase in PSA-specific T-cell responses. Phase III studies are needed to define the role of vaccination in men with prostate cancer or those who are at risk for the disease.     

Intratumoral vaccination and diversified subcutaneous/ intratumoral vaccination with recombinant poxviruses encoding a tumor antigen and multiple costimulatory molecules.

PURPOSE: Intratumoral (i.t.) vaccination represents a potential modality for the therapy of tumors. Previous i.t. vaccination studies have focused on the efficacy of i.t. vaccination alone. There are no reports that clearly compared i.t. vaccination with systemic vaccination achieved by s.c., intradermal, or i.m. injection, or combining both modalities of systemic and i.t. vaccination. Here, we compared the antitumor effects induced by a systemic vaccination regimen (s.c.) and i.t. vaccination, and a sequential s.c/i.t. vaccination regimen. In this study, we used a recombinant vaccinia virus containing the transgenes for carcinoembryonic antigen (CEA) and a triad of T-cell costimulatory molecules (B7-1, ICAM-1, and LFA-3; designated rV-CEA/TRICOM) for s.c. priming and a replication defective avipox (fowlpox) virus containing the same four transgenes (designated rF-CEA/TRICOM) for i.t. vaccination or s.c. booster vaccinations. EXPERIMENTAL DESIGN: Vaccination was started on day 8 after s.c. implantation with CEA-positive tumors. We compared the antitumor activity induced by these vaccines when administered via the i.t. route versus the s.c. route. Subsequent therapy studies examined the sequential combination of these routes, s.c. priming with rV-CEA/TRICOM followed by i.t. boosting with rF-CEA/TRICOM. Initial studies were conducted in conventional mice to define optimal vaccine regimens and then in CEA-transgenic mice that expressed CEA as a "self" antigen in a manner similar to that of an advanced colorectal cancer patient. RESULTS: The results demonstrate that the antitumor activity induced by i.t. vaccination is superior to that induced by s.c. vaccination. For more advanced tumors, a s.c. priming vaccination, followed by i.t. boosting vaccinations was superior to either s.c. or i.t. vaccination alone. Both of these phenomena were observed in tumor models where the tumor-associated antigen is a foreign antigen and in a CEA-transgenic tumor model where the tumor-associated antigen is a self-antigen. The cytokine, granulocyte macrophage colony-stimulating factor admixed in vaccines, was shown to be essential in inducing the antitumor activity. CONCLUSIONS: These studies demonstrate that the diversified vaccine regimens that consisted of s.c. prime and i.t. boosts with CEA/TRICOM vectors could induce antitumor therapy superior to that seen by either route alone.     

Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity

Immunization of mice with plasmids encoding xenogeneic orthologues of tumor differentiation antigens can break immune ignorance and tolerance to self and induce protective tumor immunity. We sought to improve on this strategy by combining xenogeneic DNA vaccination with an agonist anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) monoclonal antibody (mAb), DTA-1, which has been shown previously both to costimulate activated effector CD4(+) and CD8(+) T cells and to inhibit the suppressive activity of CD4(+)CD25(+) regulatory T cells. We found that ligation of GITR with DTA-1 just before the second, but not the first, of 3 weekly DNA immunizations enhanced primary CD8(+) T-cell responses against the melanoma differentiation antigens gp100 and tyrosinase-related protein 2/dopachrome tautomerase and increased protection from a lethal challenge with B16 melanoma. This improved tumor immunity was associated with a modest increase in focal autoimmunity, manifested as autoimmune hypopigmentation. DTA-1 administration on this schedule also led to prolonged persistence of the antigen-specific CD8(+) T cells as well as to an enhanced recall CD8(+) T-cell response to a booster vaccination given 4 weeks after the primary immunization series. Giving the anti-GITR mAb both during primary immunization and at the time of booster vaccination increased the recall response even further. Finally, this effect on vaccine-induced CD8(+) T-cell responses was partially independent of CD4(+) T cells (both helper and regulatory), consistent with a direct costimulatory effect on the effector CD8(+) cells themselves.     

Induction of P815 tumor immunity by DNA-based recombinant Semliki Forest virus or replicon DNA expressing the P1A gene

AIM: To compare the prophylactic and therapeutic effects of alphaviruses in the same tumor model, we used a DNA-based approach to generate a replicon DNA and recombinant Semliki Forest virus (rSFV) particles expressing P1A, the P815 mastocytoma tumor associated antigen, and compared the immune effect of each vaccine. METHODS: Six to eight-week-old female DBA/2 mice were inoculated with P1A plasmid or viral vaccines. Spleen cells were assayed for antigen-specific cytotoxic T cell activity. Tumor growth or survival rate was observed in preventive and therapeutic experiments, respectively. RESULTS: We found that the rSFV particles prevented tumor growth when delivered prior to innoculation of mice with P815 cells, and more importantly, improved survival when delivered after the initiation of tumor growth. Naked P1A replicon DNA also functioned as a protective and therapeutic vaccine, although with less potency than rSFV particles. Virus particles also elicited a stronger cellular immune response as measured by target cell lysis. CONCLUSION: rSFV particles have stronger specific prophylactic and therapeutic immune effects in mice than replicon DNA-based DNA vaccines, though the latter is more effective than traditional plasmid vectors (e.g. pCI-neo vector).     

DNA vaccines against the human papillomavirus type 16 E6 or E7 oncoproteins

DNA vaccines expressing the E6 or E7 oncoproteins of human papilloma virus type 16 (HPV-16) in either their wild-type form or fused to sequences that affect intracellular trafficking were tested for induction of protective immunity against tumor cell challenge in two models based on BALB/c and C57Bl/6 mice. The DNA vaccines to E7 gave uniformly disappointing results, while the DNA vaccine that expressed E6 linked to a viral leader sequence protected BALB/c mice against tumor cell challenge given before or after vaccination. The efficacy of this vaccine could be enhanced by a DNA vector prime/viral vector boost regimen. In contrast, priming of mice with the DNA vaccines to E7 reduced the efficacy of a viral vector expressing the same antigen.     

Recombinant adenovirus vaccine encoding a chimeric T-cell antigen receptor induces protective immunity against a T-cell lymphoma

Vaccination using recombinant tumor-derived T-cell antigen receptor (TCR) protein induces a protective, idiotype-specific immune response against a murine T-cell tumor. However, the technically demanding task of producing patient-specific, recombinant TCR protein restricts the translation of TCR vaccines for clinical use. We report here the development of an effective recombinant TCR adenovirus vaccine. Individual adenoviruses were constructed to encode a chimeric TCR derived from either tumor Valpha or Vbeta regions fused to xenogeneic human constant regions. Coinjection of the chimeric alpha- and the beta-TCR adenoviruses protected mice against tumors. The level of protection was comparable to that achieved by an optimized regimen of recombinant TCR protein vaccines. Tumor immunity induced by TCR adenoviruses required the xenogeneic constant regions and was mediated by CD8+ T cells. Independent vaccines consisting of adenovirus expressing either chimeric alpha- or beta-TCR chain also stimulated a protective immune response. Immunization with TCR adenovirus may offer a new efficacious, protein-free vaccination approach for the treatment of T-cell malignancies.     

therapeutic vaccines for colorectal cancer

A number of cancer vaccine strategies for the treatment of colorectal cancer have entered clinical trials. Whole tumor cell vaccines have been developed from both patients’ autologous tumor cells as well as established allogeneic tumor cell lines. A vaccine consisting of autologous tumor cells along with bacillus Calmette-Guerin (BCG) has shown a potential clinical benefit in patients with stage II colon cancer. Other approaches using autologous tumor cells have involved transfection of primary tumor cells with cytokine genes. Allogeneic tumor cell vaccines have also been modified to express cytokine genes. Vectors have been studied extensively as a means of vaccine strategy. One tumor-associated antigen (TAA) that has been extensively studied in viral vector vaccines is carcinoembryonic antigen (CEA). A recombinant vaccinia virus containing the CEA transgene (rV-CEA) has been shown to elicit CEA-specific immune responses in advanced carcinoma patients. However, patients receiving multiple vaccinations had limited increases in CEA-specific responses by the third vaccination. This problem may be overcome by the use of non-replicating poxviruses, which have been shown in clinical trials to be safe and to elicit CEA-specific responses. However, recent clinical studies have shown that the optimal use of poxviruses is to prime with vaccinia, followed by boosts with avipox vectors. A recent randomized clinical trial showed that patients primed with rV-CEA and boosted with avipox-CEA had greater immune responses compared with patients receiving three 1-monthly avipox-CEA vaccinations followed by an rV-CEA vaccination. Furthermore, a statistically significant survival advantage was noted in the prime/boost arm. Ongoing studies are now incorporating the genes for costimulatory molecules along with TAA in these vectors. Another vaccine strategy involving TAA that is currently in clinical trials for colorectal cancer is the peptide vaccine. Dendritic cells (DCs) are considered to be the most potent antigen-presenting cell, thus providing an attractive modality for cancer vaccines. In addition to using DCs for peptide-based vaccines, a number of other strategies, including transfection with messenger RNA, have produced specific T-cell responses in clinical trials. In addition, several clinical trials using murine anti-idiotype antibodies as vaccines for patients with advanced colorectal cancer have shown both immunologic responses as well as clinical responses.     

Skewing the T-cell repertoire by combined DNA vaccination, host conditioning, and adoptive transfer

Approaches for T-cell-based immunotherapy that have shown substantial effects in clinical trials are generally based on the adoptive transfer of high numbers of antigen-specific cells, and the success of these approaches is thought to rely on the high magnitude of the tumor-specific T-cell responses that are induced. In this study, we aimed to develop strategies that also yield a T-cell repertoire that is highly skewed toward tumor recognition but do not rely on ex vivo generation of tumor-specific T cells. To this end, the tumor-specific T-cell repertoire was first expanded by DNA vaccination and then infused into irradiated recipients. Subsequent vaccination of the recipient mice with the same antigen resulted in peak CD8(+) T-cell responses of approximately 50%. These high T-cell responses required the presence of antigen-experienced tumor-specific T cells within the graft because only mice that received cells of previously vaccinated donor mice developed effective responses. Tumor-bearing mice treated with this combined therapy showed a significant delay in tumor outgrowth, compared with mice treated by irradiation or vaccination alone. Furthermore, this antitumor effect was accompanied by an increased accumulation of activated and antigen-specific T cells within the tumor. In summary, the combination of DNA vaccination with host conditioning and adoptive transfer generates a marked, but transient, skewing of the T-cell repertoire toward tumor recognition. This strategy does not require ex vivo expansion of cells to generate effective antitumor immunity and may therefore easily be translated to clinical application.