Novel Vaccines for the Treatment of Gastrointestinal Cancers

Continuing advances in immunology and molecular biology during the past several decades have provided optimism that immunomodulatory strategies may be clinically useful in patients with cancer. Key advances have included: (1) recognition of the critical role of the antigen-presenting cell and greatly improved understanding of antigen processing and presentation, including the molecular interactions between HLA molecules and antigenic epitopes on the antigen-processing cell and the receptors on T cells, and (2) the roles of costimulatory molecules such as B7.1, ICAM-1, and LFA-3 in the induction and maintenance of an immune response. In addition, new techniques have allowed us to identify immunogenic antigenic determinants, alter their binding affinities, and evaluate the overall success of the intervention through both in vivo and in vitro assays. Carcinoembryonic antigen (CEA) is overexpressed in a large number of gastrointestinal, lung, and breast cancers. Clinical trials have established treatment protocols using viral vectors to immunize patients to CEA without producing deleterious autoimmune phenomena. By combining various vectors to include MUC-1 and/or CEA plus costimulatory molecules in a prime-and-boost regimen, we are beginning to see signs that this intervention can not only produce changes in immune function but also potentially improve clinical outcomes. Phase III studies to test these hypotheses are under way.

In the late 19th century, William Coley at Memorial Sloan-Kettering Cancer Center in New York reported spontaneous regression of sarcomas following severe bacterial infections.[1] However, attempts to reproduce these results with bacterial extracts met with limited success.[2] Subsequently, attention shifted from infectious particles to antigens specific for tumor cells recognized by the immune system in a manner that causes the rejection of the tumor as a foreign tissue or non-self (tumorspecific transplantation antigens). For example, in mice, immunity to methylcholanthrene- induced malignancies could be produced by injecting genetically identical animals with tumor cell lysates.[3] This observation appeared to reflect the tumor-specific nature of the antigens induced by chemical mutagens. In the early 1960s, investigators were encouraged by reports that malignant cells from different tumors share common tumor-associated antigens.[ 4] The results of these studies, coupled with advances in immunology, led to the concept of immune surveillance- a process in which immunocompetent hosts are protected from the emergence of malignant clones, which are ostensibly identified as non-self, by elements of the innate and adaptive immune system. [ 5] These observations were exciting and provided hope that stimulating an active, specific immune response might cure cancer, but subsequent basic science and clinical studies were disappointing. For example, the concept of immunosurveillance was questioned when athymic nude mice were shown to have the same incidence of tumors as wild-type animals, and results of early clinical trials with various approaches to stimulating an active antitumor immune response were not encouraging.[6] More recent work has greatly expanded our understanding of the interactions between elements of the immune system and cancer cells and has renewed hope that therapeutic cancer vaccines and other immunomodulatory approaches may, in fact, become valuable tools for clinical on- cologists. Cytotoxic T lymphocytes have been shown to kill HLA-matched tumor cells in vitro, and immunogeninduced upregulation of humoral immunity has been shown to be associated with improved outcomes in patients with breast cancer or melanoma.[ 7-9] The success of donor lymphocyte infusions in patients with hematologic malignancies has also provided empiric evidence that immunomodulatory therapy can be an important clinical tool in patients with advanced malignancies.[10,11]

Advances in Therapeutic Cancer Vaccines It is a key tenet of tumor immunology that tumor cells express antigens and the uniqueness of these antigens makes them potential targets of innate and adaptive immune elements. Thus, it is no surprise that clinical failures of therapeutic cancer vaccines may be linked to a failure of antigen recognition by the immune system. With the advent of sensitive assay systems, investigators have been able to demonstrate the presence of naturally occurring T cells directed against tumor-associated antigens (TAAs).[12] Gilboa has categorized TAAs that may function in tumor rejection as two types: (1) patient-specific, mutated self-antigens; and (2) shared, nonmutated self-antigens.[2] Mutated self-antigens are the result of the genetic instability of an individual tumor and are generally incidental to tumor pathophysiology. While they would be anticipated to be potent immunogens, identification and isolation of these unique (private) antigenic determinants would be impractical. The significance of mutated self-antigens to successful immunotherapy was illustrated in a series of experiments by Boon and colleagues. These investigators were able to easily transplant cell lines from teratocarcinoma and Lewis lung carcinoma into syngeneic mice. If, however, the malignant cell line was first mutated by exposure to N-methyl-N'-nitro-Nnitrosoguanidine, development of cellline- specific mutated self-antigens elicited an immune response that protected the mice from the transplanted tumor cells.[13-15] Unlike vaccines to private antigens, vaccines to shared (public) antigens as off-the-shelf therapeutics have the potential to benefit many patients. In most instances, these shared antigens correspond to normal gene products expressed on tumor cells, so patients may be expected to be tolerant to many of them. Other shared tumor antigens, however, are fetal gene products not expressed in adult tissues or are common molecules related to malignancy and found only in tumor cells. By virtue of their limited distribution in either time or space, oncofetal or sequestered antigens, respectively, have the potential to be targets for immunotherapy. Recently reported examples of oncofetal tumor antigens include survivin, an inhibitor of apoptosis expressed during fetal development and in many types of cancer cells,[13] and OFA, the precursor to the mature laminin receptor found in fetal cells.[16,17] In addition, some proteins have been identified as common to certain types of tumor cells but absent from normal tissues. Human telomerase reverse transcriptase may be an example of a common tumor- specific antigen since it is overexpressed in many tumors but absent from normal cells.[18] Optimizing Antigenicity
The process of effective recognition of antigens and the development of an adaptive immune response is complex. Tumor antigens must be taken up by phagocytic cells such as dendritic cells, processed into immunogenic fragments of defined size, and assembled into complexes with the appropriate major histocompatability complex (MHC) molecule-class I for presentation to CD8 T cells and class II for presentation to CD4 T cells. These complexes are then displayed on the surface of the dendritic cells.[19] MHC molecules derive their name from their role in transplant rejection. In response to differentiating elements released by inflammatory cells and the pathologic process itself, dendritic cells differentiate into potent antigen- presenting cells and migrate to lymphoid tissue, where they encounter the T cells. T cells become activated when their antigen-specific T-cell receptor binds the antigen-MHC complex presented on the surface of the antigen-presenting cell. T cells only bind an antigen-MHC complex if the complexed antigen is recognized by its T-cell receptor (cognate antigen). The ability of an antigen to elicit an immune response is partially a function of the number of cognate reactive (vs tolerant) T cells and the binding affinity between the responsive T cell and the antigen-presenting cell.[2] However, the potency of an immune response also depends on other factors such as the interactions between the MHC complex and CD4 and CD8 molecules on T cells and the generation of costimulatory signals between the antigen-presenting cell and T cells. Costimulatory molecules are expressed on the surface of antigen-presenting cells and are recognized by corresponding receptors on T cells. Tumor cells generally lack costimulatory molecules and, therefore, function very poorly as antigen-presenting cells. Moreover, when antigen is presented in the absence of costimulation, T cells tend to become anergic, which may be a mechanism for protection of normal cells against an autoimmune response and also is a proposed mechanism of tumor escape. The most widely studied costimulatory interaction is through B7.1 (CD80) on antigen-presenting cells and the molecule CD28 on T cells (Figure 1).[2] For the T cells to survive, proliferate, and form memory T cells, additional costimulatory interactions are required, such as the interaction between CD40 on the activated antigenpresenting cell and CD40 ligand on the T cell. Thus, to optimize antigenicity, it is reasonable for therapeutic cancer vaccine developers to employ strategies similar to those observed in vivo-ie, to maximize the immunologic response by presenting antigen in the context of costimulatory molecules. The importance of costimulatory molecular signals may have been reflected in the observations by Coley more than a century ago that immune responses against weak antigens require additional signals (eg, such as those released during infection).[1] In Vitro Quantification of Immune Responses
While experiments can be conducted in syngeneic animal models or with standardized cell lines, it is difficult to quantify responses of patients to novel immunotherapies. Although two patients may both have the same type, grade, and stage of solid tumor, they may differ in age, comorbidities, tumor antigenicity, tumor suppressor gene status, HLA type, dendritic cell function, T- and B-cell numbers and function, response to chemotherapy, and so forth. Consequently, it is important to develop quantitative methods to evaluate the magnitude and durability of immunologic responses to cancer vaccines.[20] In vivo assays have been available for some time and include delayed-type hypersensitivity skin reactions, depigmentation of the tumor in the case of melanoma vaccines, and quantification of tumor infiltrating lymphocytes. In addition, a variety of new in vitro analytic tools have been developed. These in vitro methods have been important in the continuous improvement of vaccine strategies and in the selection of strategies to move to clinical trials. Ultimately, an in vitro assay may serve as a surrogate indicator of therapeutic vaccine efficacy if the measured immune responses can be shown to correlate with clinical outcome. These in vitro assay systems include enzyme-linked immunospot (ELISpot) for the measurement of antigen- specific T-cell responses, flow cytometric measurement of intracellular cytokine production, and MHCpeptide tetramer analysis. ELISpot employs antibodies in a sandwich assay with signal amplification to enumerate cytokine-secreting cells. When automated, the results are reliable and reproducible. Cloned peptide-MHC complexes can be stabilized and tetramerized with streptavidin labeled with phycoerythrin. When incubated with mononuclear cells, the ability of tetramers to stain and activate CD8 T cells is strongly dependent on binding of CD8 to the same class I molecule engaged by the T-cell receptor. Peptide-specific T cells can then be enumerated in a flow cytometer. All three methods are very sensitive, but the peptide-MHC tetramer analysis appears to be the most sensitive. It is also the most technically demanding assay and, at this time, is not sufficiently robust for broad clinical application.[21] Development of CEA-Based Vaccines in GI CancersCEA as a Target of Immunotherapy
Carcinoembryonic antigen (CEA), reported in 1965, is one of the first oncofetal tumor antigens to be discovered. It is normally expressed in fetal colon. Although its expression is restricted in adult life, CEA is present in saliva, feces, serum, colonic mucosa, and fluid from colonic lavages in adults.[22] CEA is overexpressed in a high percentage of adenocarcinomas, particularly those of endodermal origin (eg, non-small-cell lung, stomach, colon, rectum, and pancreas). As such, it is considered a shared (public), nonmutated oncofetal self-antigen and its concentration in the circulation is widely used as a serologic marker of malignancy. CEA is a member of the immunoglobulin superfamily with a molecular weight of 180 to 200 kDa.[23] The gene for CEA codes for a 70-kDa protein and is located on chromosome 19. The difference in molecular weight between the gene product and the final antigen is secondary to extensive post-translational glycosylation.[ 24] In normal colonic epithelium, CEA is localized to the luminal surface-an arrangement that suggests that it contributes to spatial orientation of colonocytes and that it may also function to preserve the adult gut's mucosal barrier. In tumor cells, however, CEA is irregularly distrib- uted throughout the cell membrane. As an intercellular adhesion molecule, CEA may contribute to the formation of metastasis.[22] There is a correlation between serum CEA in patients with cancer and the incidence of hepatic metastases, but this could also simply reflect tumor burden.[24] Due to its association with malignancy and wide distribution in human tumors, CEA has the characteristics of an ideal target for vaccine therapy. However, there are several potential problems that must be resolved. Since CEA is also normally expressed in adult life, the immune system is normally tolerant to CEA.[22] The tolerance develops in utero with deletion of autoreactive clones of T cells through a process of negative selection within the thymus. Small numbers of CEA-reactive T cells may still reach the periphery. In general, tolerance is induced in these clones by some combination of clonal inactivation, clonal deletion, and/or cytokinedependent suppression and immune deviation. Thus, strategies employed to overcome tolerance to CEA might overcome tolerance in these clones and elicit an immune response that would not only target the tumor but could also induce autoimmune side effects. Experimentally, tolerance to CEA can be overcome without induction of adverse autoimmune events. Investigators have vaccinated mice transgenic for human CEA and generated anti-CEA antibodies, immunoglobulin class switching, T_H1-type CEA-specific CD4+ cells, and CD8+ cellular cytotoxicity.[25,26] CEA Vaccine Strategies: Peptide Modification
As described above, T cells only recognize antigen after it has been processed into fragments (epitopes) composed of 8 to 10 amino acids (CD8+ cells) or longer (CD4+ cells) and presented on the surface of the antigen-presenting cell complexed with MHC class I or II molecules, respectively.[24] T-cell epitopes from tumor antigens such as CEA have been identified by either identifying the target of an existing T-cell response or, more commonly, using modeling to predict the binding affinity of a particular amino acid sequence to a specific MHC class I or II molecule.[27] At present, a number of CEA-associated peptides have been identified and have been used to generate T-cell responses. The majority have been restricted to the (MHC class I type) HLA-A2, the supertype present in 40% to 50% of humans.[28]. HLA-A3- restricted peptides have also now been identified and shown to be immunogenic.[ 28] While less attention has been focused on MHC class II-restricted peptides for numerous reasons, evolving understanding of the role of CD4+ cells in modulating antigen-specific immune responses has led to increased emphasis on these epitopes.[24] As an antigen, CEA is weakly immunogenic. It is reasonable to assume that at least part of the explanation for the low immunogenicity of native CEA peptides is their low affinity for MHC molecules. Therefore, to develop a more effective therapeutic cancer vaccine, strategies have been employed to increase the immunogenicity of CEA. If, in fact, binding affinity is the problem, amino acid substitutions of key residues that anchor the peptide to the MHC molecule might be used to increase the peptide's affinity for MHC molecules. This strategy has been employed effectively to improve T-cell responses to several melanoma antigens.[29] Another successfully used approach to enhance the T-cell response is to modify the residues recognized by the T-cell receptor. The HLA-A2- restricted 9-amino acid peptide, CEAassociated peptide 1 (CAP-1), was modified by replacing the asparagine at position 6 with an aspartic acid. In in vitro assays, this modified peptide, CAP-1-6D, was more effectively recognized by T cells than the native CAP-1 peptide[30] and significantly upregulated the production of granulocyte macrophage colony-stimulating factor (GM-CSF) and interferon- gamma by the target cell.[31] Gene array studies demonstrated that, compared with the native CAP-1 peptide, CAP-1-6D stimulates the expression of different genes and gene clusters in CD8+ T cells than the native peptide. Expression of lymphotactin and granzyme B were increased many times over the levels in CD8+ T cells stimulated with CAP-1.[32] Lymphotactin recruits T and NK cells into the region, and granzyme B is transferred from cytotoxic T lymphocytes to target cells during the process of granule- mediated cytotoxicity.[33,34] Viral Vectors for Antigen Delivery
Recombinant viruses have been used for both gene therapy and as vectors for immunotherapy.[35] When used as immunotherapeutics, genes for a tumor antigen can be introduced into a virus that directs the expression of the antigen and stimulates a strong immune response to the specific tumor antigen. In the development of vaccine therapy, the choice of a vector is as important as the selection of the tumor antigen. Potential viruses include adenoviruses, retroviruses, and poxviruses, and each has its advantages and disadvantages. For example, while the adenoviruses are effective vectors and have been widely used in experimental studies, they infect nondividing cells and require repetitive dosing because their genes are only transiently expressed.[36] In addition, adenoviruses generate an immune response or enhance the immune response in the approximately 80% of humans who already have neutralizing antibodies to these viruses.[ 37] Retroviruses integrate into the host genomes and, therefore, bring the risk of insertional mutagenesis.[36]

• Poxviruses- Poxviruses such as the vaccinia virus are particularly well suited as vectors for therapeutic cancer vaccines. They are easily engineered, accommodate large inserts of foreign DNA, allow post-translational modification of expressed proteins, replicate accurately without helper viruses, stimulate potent immune responses, and have been extensively tested in humans as smallpox prophylaxis.[ 24] Recombinant vaccinia (rV) viruses that express human CEA have been developed.[38] Incorporating the gene for CEA into the virus involves inserting the nucleic acids for the gene's start site immediately adjacent to the transitional start site of a vaccinia gene promoter.[39]

Following transfection, the rV- CEA infected cells express a protein product recognized by monoclonal antibodies to CEA.[38] CEA transgenic mice, which express human CEA as a self-antigen and are immunologically unresponsive to CEA administered as a whole protein, respond to immunization with rV-CEA with TH1-type CEA-specific CD4+ responses and CEA peptide-specific cytotoxicity. Immunization with rV-CEA protected these mice from challenge with CEA-expressing tumor cells.[25] Vaccination with rV-CEA is also immunogenic in humans. Following vaccination with the viral construct, previously immuno-unreactive peripheral blood lymphocytes from patients with metastatic carcinoma were able to mount a cytolytic T-cell response to specific CEA epitopes in vitro.[40] Vaccinia virus has many properties desired in a vector. Although its intense immunogenicity potentially could limit its repeated use, this concern is not clinically significant since current strategies only employ it as a vector to prime the immune system.[ 41] Vaccinia proteins are highly immunogenic; consequently, it is likely that vaccinia can only be administered once or twice before a patient will develop high titers of neutralizing antibodies that will limit expression of recombinant genes. To circumvent this problem, researchers have studied potential alternative vectors such as attenuated vaccinia strains or avian pox viruses such as the canarypox virus.[42,43] An advantage of canarypox virus is that it replicates only in avian species. When administered to humans, it expresses its transgene products for 2 to 3 weeks in the infected mammalian cells but does not replicate and is unable to infect other cells, thus minimizing the likelihood that an enhanced immune response will neutralize the vaccine.[22] The protective and antitumor activity of canarypox virus has been demonstrated in preclinical models. [ 41] The first phase I trial of a fowlpox recombinant CEA vaccine was conducted in 1999 by Marshall et al.[44] Although no objective antitumor response was observed in patients with measurable disease during the trial, vaccination resulted in statistically significant increases in cytotoxic T-cell precursors specific for CEA. In preclinical studies, Hodge et al[35] showed that a combination vaccination strategy with rV-CEA administered first followed by fowlpox-CEA could generate a more vigorous Tcell response than either vaccine alone.[41] This approach was subsequently validated in a phase I study in patients with advanced CEA-positive malignancies.[45] The study confirmed that immunization was most effective when rV-CEA was the primer and the fowlpox-CEA used to boost the immune response. In addition, this study showed that further increases in cytotoxic T cells could be produced by administering GM-CSF (Leukine) with subsequent vaccinations.[46]

Dendritic Cells and CEA
Dendritic cells are the most potent type of antigen-presenting cells and are essential to prime the adaptive immune response.[47] Although rare in the peripheral blood, dendritic cells can be harvested from the blood and expanded in vitro. Alternatively, administration of the hematopoietic growth factor flt3 ligand can dramatically mobilize these cells into the peripheral blood, increasing numbers by as much as 20-fold.[48] When exposed for short periods to high concentrations of antigen, dendritic cells are said to be "pulsed." Pulsing has been used to generate more effective antigen-presenting cells. Dendritic cells pulsed with an HLA class I-restricted CEA peptide epitope have been shown to generate cytotoxic T lymphocytes specific for CEA.[49,50] In a phase I study of patients with advanced CEA-positive malignancies, Morse et al administered autologous dendritic cells pulsed with CEA mRNA.[51] There were no significant treatment-related toxicities. Of 24 evaluable patients, 1 showed a complete response, 2 had minor responses, 3 exhibited stable disease, and 18 had progressive disease.

Costimulatory MoleculesT cells require at least two signals for activation. The first is delivered when the antigen binds to the T-cell receptor. The second is a costimulatory signal delivered by receptorligand interactions between the T cell and the antigen-presenting cell (eg, CD28 and B7.1).[59] Binding of CD28 to B7.1 upregulates the production of multiple cytokines by CD8+ and CD4+ cells, including IL-2 and interferon-gamma. Von Mehren et al demonstrated that a fowlpox- CEA B7.1 vaccine was well tolerated and induced CEA-specific T-cell responses in patients with advanced re- current adenocarcinomas.[60] Thus, it was logical to explore the potential of vaccination with multiple costimulatory molecules.

Hodge et al reported that murine cells infected with a recombinant virus expressing genes for three costimulatory molecules (eg, B7.1 [CD80], ICAM-1 [CD54], A-3 [D58] referred to as the TRIad of COstimulatory Molecules [TRICOM]), activated T cells to a far greater extent than cells infected with either one or two of the costimulatory molecules.[61] This was the first experimental evidence that vectors could be used to transfect cells with three costimulatory molecules; in addition, the TRICOM strategy activated both CD8+ and CD4+ cells. Direct intralesional injection of TRICOM into metastatic melanoma has been shown to alter the tumor's microenvironment with a resultant local tumor regression.[62] Subsequently, the gene for CEA has been incorporated along with the TRICOM molecules to provide a single vector that can transfect cells with genetic elements supplying both signals necessary for T-cell activation, thereby amplifying T-cell responses.[ 63-65] With this approach, therapeutic vaccines could alter the phenotype of an infected cell by converting any cell into an antigenpresenting cell expressing both costimulatory molecules and antigen- MHC complexes on its surface. This approach appears to be augmented when the prime-and-boost administration strategy is employed, and is facilitated by coadministration of GMCSF and/or IL-2.[66] Studies by Marshall et al have demonstrated that rV-CEA and fowlpox- CEA (rF-CEA) are well tolerated and have clinical activity with a correlation between CEA-specific immune reactivity and survival.[67,68] The investigators recently published the results of a phase I study of a primeand- boost strategy with rV-CEA/ fowlpox-CEA plus TRICOM in 58 patients with advanced CEA-expressing cancers.[69] Patients were accrued to eight cohorts that involved vaccinations with the following: fowlpox- CEA-TRICOM; primary vaccination with rV-CEA-TRICOM plus fowlpox- CEA-TRICOM booster vaccinations; and rV-CEA-TRICOM and then fowlpox-CEA-TRICOM plus GM-CSF with vaccines, or with divided doses of vaccine with GM-CSF (Table 1). The therapeutic cancer vaccines were administered for six doses at 28-day intervals and then once every 3 months. If, in the judgment of the investigator, patients progressed on the 3-month schedule, they were allowed to revert to 28-day treatment intervals. The vaccines were well tolerated. One patient (2%) had a histologically documented, complete response. Twenty-three patients (40%) had stable disease for at least 4 months, with 14 of these patients having stable disease for 6 months or longer. Eleven patients (19%) had decreasing or stable serum CEA. The ELISpot assay was used to assess interferon-gamma released by peripheral blood mononuclear cells in response to CEA peptide exposure. The majority of the patients demonstrated enhanced CEAspecific T-cell responses. Interestingly, several patients had preexisting CEA-specific T-cell responses prior to vaccination-presumably reflecting an endogenous, but suboptimal, response to their tumor. The investigators concluded that not only is the vaccine well tolerated and able to generate significant CEA-specific immune responses, but the vaccines also appeared to clinically benefit some patients. Addition of MUC-1
Alterations in mucin are common in colonic tumors.[70] Mucins are high-molecular-weight, carbohydrate- rich proteins. cDNA sequences of genes coding for mucin protein have identified a number of mucin genes. They have been designated based upon their chronologic order of identification as MUC-1 through MUC- 17. However, the nomenclature is somewhat convoluted since additional studies have demonstrated mucins originally attributed to one gene that are actually products of two separate genes and two named mucins that may actually be the product of two closely related genes or a single gene. In addition to CEA, mucin-associated antigens (particularly the MUC-1 gene product) are overexpressed on the surface of many human malignancies including adenocarcinomas of the gastrointestinal system, prostate, and breast, and hematologic malignancies including non-Hodgkin's lymphoma and multiple myeloma.[71] Experimentally, MHC-restricted T cells have been shown to recognize epitopes in the MUC-1 protein. Thus, employment of MUC-1 epitopes in vaccine therapy in a fashion similar to that described for CEA offers the potential of additional hope to future patients with a variety of cancers. Autoimmunity Use of autoantigens raises the possibility that vaccination with therapeutic vaccines expressing the CEA or MUC-1 antigens might precipitate the development of autoimmune side effects such as enteritis or colitis. However, studies of mice transgenic for human CEA have demonstrated that significant antitumor activity can be produced by vaccination strategies without upregulation of autoreactive T cells.[23] In a murine intestinal tumor model, Greiner et al were able to use a CEA-based vaccine to dramatically reduce the number of malignant tumors. Histopathologic study of normal intestinal tissues from the animals demonstrated preservation of the normal architecture and CEA expression. Since CEA is normally expressed on the luminal surface of the colonic epithelium, it may be able to escape immune detection. Thus, CEA may be an optimal target for therapeutic antitumor vaccines. Poxvirus-Based Vaccine Safety Issues Between 1991 and 2003, recombinant poxvirus vaccines have been administered to over 700 patients participating in 29 clinical trials.[72] Targets of the recombinant virus vaccines have included CEA, MUC-1, prostate-specific antigen (PSA), gp100, MART-1, and tyrosinase. The vaccines have been administered through intradermal, intralesional, subcutaneous, intramuscular, intravenous, and intrathecal routes. Administration has been generally well tolerated. The most common adverse events are flu-like symptoms. Phase I Studies in Pancreatic Cancer Schuetz et al recently reported the preliminary results of two phase I studies of vaccines targeting CEA and MUC-1 in 22 patients with stage III or IV pancreatic cancer.[73] While the first study tested an earlier generation of the vaccines (N = 12), the second study tested a prime-and-boost strategy with rV-CEA-MUC-1-TRICOM genes plus fowlpox-CEA-MUC-1-TRICOM genes (N = 10). All patients had received prior therapy and 20 (91%) had metastatic disease. Patients received a "prime" dose of vaccinia on day 0, followed by the "boost" doses of fowlpox on days 14, 28, and 42. GMCSF (100 μg) was administered to all patients following each vaccination for 4 days. All patients who were stable and able to participate received vaccinations on a monthly basis. Safety parameters included vital signs, physical examination, laboratory tests, and adverse events. All subjects were followed for survival. The most common vaccine-related adverse event was injection-site reaction (grade 1); flu-like symptoms were also common. A single patient reported grade 3 fever. Among the 10 patients enrolled in the second study, the median survival was 6.3 months, compared to historical controls of approximately 3 months; 1-year survival was 30%. The investigators concluded that the vaccination strategy was well tolerated, and, although the study was small and uncontrolled, the results suggested that therapeutic vaccination had improved median overall survival. Realizing the Promise: Current and Future Studies Recent research suggests that other interventions can alter the phenotype of tumor cells, thereby lessening their immune tolerance. Chakraborty et al, for example, have demonstrated that when sublethal doses of radiation are combined with vaccination using a prime-and-boost strategy, dramatic and significant cure rates can occur in mice transgenic for human CEA with a murine carcinoma transfected with CEA.[74] Within 72 hours of irradiation, multiple human cancer cell lines show alterations in expression of Fas and other cell-surface antigens that make them more susceptible to cytotoxic T cells.[75] Other strategies that may also contribute to immunotherapy and improve clinical outcomes include use of adjuvants such as CpG oligonucleotides, cytokine combinations to improve harvest of antigenpresenting cells or stimulate in vivo immunity, and additional studies of the phenotypic and genotypic properties of tumor-infiltrating T cells.[76] Potential Limitations Despite the advances described above, a number of issues remain and present potential obstacles to implementing vaccine strategies in patients with cancer. Disease burden is a significant issue. At some point, the tumor mass may become so large that it over whelms the immune system; alternatively, the antigen burden of the tumor might render the patient anergic to the tumor antigens. While tumor bulk can be reduced prior to immunotherapy in patients with localized disease, the issue of management of metastatic disease in candidates for immunotherapy remains unresolved. Although vaccinations clearly modulate the function of lymphocytes, it is important to evaluate various therapeutic interventions to maximize the results of the immune intervention. For example, different routes of administration should be evaluated. The complex nature of the immune system consists of a series of regulatory and counterregulatory processes finely balanced to discriminate self from not-self and eliminate the latter. Therefore, additional work is needed to modulate the activities of inhibitory T cells and cytokines during vaccine therapy. Lastly, malignant cells have demonstrated remarkable evolutionary capacities. Selection of one or more clones by tumoricidal interventions allows resistant clones to emerge and become dominant. Therefore, additional studies should assess means to counteract immune evasion. While these limitations might seem daunting, our progress from the time of Dr. Coley offers encouragement.

Disclosures:

Dr. Marshall is a consultant and researcher for Therion Biologics Corporation.

References:

1. Coley WB: A report of recent cases of inoperable sarcoma successfully treated with mixed toxins of erysipelas and Bacillus prodigiosus. Surg Gynecol Obstet 13:174-190, 1911.
2. Gilboa E: The promise of cancer vaccines. Nat Rev Cancer 4:401-411, 2004.
3. Prehn RT, Main JM: Immunity to methylcholanthrene- induced sarcomas. J Natl Cancer Inst 18:769-778, 1957.
4. Tian J, Olcott AP, Kaufman DL: Antigenbased immunotherapy drives the precocious development of autoimmunity. J Immunol 169:6564-6569, 2002.
5. Burnet FM: The concept of immunological surveillance. Prog Exp Tumor Res 13:1-27, 1970.
6. Alexander P: Innate resistance and specific immunity in host control of cancer. J Med 8:279- 285, 1977.
7. Mukherji B, Chakraborty NG, Sivanandham M: T-cell clones that react against autologous human tumors. Immunol Rev 116:33- 62, 1990.
8. von Menbrsdorff-Pouilly S, Verstraeten AA, Kenemans P, et al: Survival in early breast cancer patients is favorably influenced by a natural humoral immune response to polymorphic epithelial mucin. J Clin Oncol 18:574-583, 2000.
9. Livingston PO, Wong GY, Adluri S, et al: Improved survival in stage III melanoma patients with GM2 antibodies: A randomized trial of adjuvant vaccination with GM2 ganglioside. J Clin Oncol 12:1036-1044, 1994.
10. Bethge WA, Hegenbart U, Stuart MJ, et al: Adoptive immunotherapy with donor lymphocyte infusions after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning. Blood 103:790- 795, 2004.
11. Cheson BD: What is new in lymphoma? CA Cancer J Clin 54:260-272, 2004.
12. Nagorsen D, Scheibenbogen C, Marincola FM, et al: Natural T cell immunity against cancer. Clin Cancer Res 9:4296-4303, 2003.
13. Boon T, Kellermann O: Rejection by syngeneic mice of cell variants obtained by mutagenesis of a malignant teratocarcinoma cell line. Proc Natl Acad Sci U S A 74:272-275, 1977.
14. Boon T, Van Pel A: Teratocarcinoma cell variants rejected by syngeneic mice: Protection of mice immunized with these variants against other variants and against the original malignant cell line. Proc Natl Acad Sci U S A 75:1519- 1523, 1978.
15. Van Pel A, Georlette M, Boon T: Tumor cell variants obtained by mutagenesis of a Lewis lung carcinoma cell line: Immune rejection by syngeneic mice. Proc Natl Acad Sci U S A 76:5282-5285, 1979.
16. Tsuruma T, Hata F, Torigoe T, et al: Phase I clinical study of anti-apoptosis protein, survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer. J Transl Med 2:19, 2004.
17. Zelle-Rieser C, Barsoum AL, Sallusto F, et al: Expression and immunogenicity of oncofetal antigen-immature laminin receptor in human renal cell carcinoma. J Urol 165:1705- 1709, 2001.
18. Minev B, Hipp J, Firat H, et al: Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc Natl Acad Sci U S A 97:4796-4801, 2000.
19. Caligiuri MA, Velardi A, Scheinberg DA, et al: Immunotherapeutic approaches for hematologic malignancies. Hematology (Education Program Book) 337-353, 2004.
20. Lyerly HK: Quantitating cellular immune responses to cancer vaccines. Semin Oncol 30(3 suppl 8):9-16, 2003
21. Whiteside TL, Zhao Y, Tsukishiro T, et al: Enzyme-linked immunospot, cytokine flow cytometry, and tetramers in the detection of Tcell responses to a dendritic cell-based multipeptide vaccine in patients with melanoma. Clin Cancer Res 9:641-649, 2003.
22. Marshall J: Carcinoembryonic antigenbased vaccines. Semin Oncol 30(suppl 8):30-36, 2003.
23. Greiner JW, Zeytin H, Anver MR, et al: Vaccine-based therapy directed against carcinoembryonic antigen demonstrates antitumor activity on spontaneous intestinal tumors in the absence of autoimmunity. Cancer Res 62:6944-6951, 2002.
24. Horig H, Medina FA, Conkright WA, et al: Strategies for cancer therapy using carcinoembryonic antigen vaccines. Expert Rev Mol Med 2000:1-24, 2000.
25. Kass E, Schlom J, Thompson J, et al: Induction of protective host immunity to carcinoembryonic antigen (CEA), a self-antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus. Cancer Res 59:676-683, 1999.
26. Mizobata S, Tompkins K, Simpson JF, et al: Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen. Cancer Immunol Immunother 49:285-295, 2000.
27. Stevanovic S: Identification of tumourassociated T-cell epitopes for vaccine development. Nat Rev Cancer 2:514-520, 2002.
28. Kawashima I, Tsai V, Southwood S, et al: Identification of HLA-A3-restricted cytotoxic T lymphocyte epitopes from carcinoembryonic antigen and HER-2/neu by primary in vitro immunization with peptide-pulsed dendritic cells. Cancer Res 59:431-435, 1999.
29. Parkhurst MR, Salgaller ML, Southwood S, et al: Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues. J Immunol 157:2539-2548, 1996.
30. Zaremba S, Barzaga E, Zhu M, et al: Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Res 57:4570-4577, 1997.
31. Salazar E, Zaremba S, Arlen PM, et al: Agonist peptide from a cytotoxic t-lymphocyte epitope of human carcinoembryonic antigen stimulates production of tc1-type cytokines and increases tyrosine phosphorylation more efficiently than cognate peptide. Int J Cancer 85:829-838, 2000.
32. Palena C, Schlom J, Tsang KY: Differential gene expression profiles in a human T-cell line stimulated with a tumor-associated self-peptide versus an enhancer agonist peptide. Clin Cancer Res 9:1616-1627, 2003.
33. Peterson FC, Elgin ES, Nelson TJ, et al: Identification and characterization of a glycosaminoglycan recognition element of the C chemokine lymphotactin. J Biol Chem 279:12598-12604, 2004.
34. Waterhouse NJ, Sedelies KA, Clarke CJ: Granzyme B; the chalk-mark of a cytotoxic lymphocyte. J Transl Med 2:36, 2004.
35. Kerr D: Clinical development of gene therapy for colorectal cancer. Nat Rev Cancer 3:615-622, 2003.
36. Johnson LG: Gene therapy for cystic fibrosis. Chest 107(2 suppl):77S-83S, 1995.
37. Eto Y, Gao JQ, Sekiguchi F, et al: Neutralizing antibody evasion ability of adenovirus vector induced by the bioconjugation of methoxypolyethylene glycol succinimidyl propionate (MPEG-SPA). Biol Pharm Bull 27:936- 938, 2004.
38. Kaufman H, Schlom J, Kantor J: A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA). Int J Cancer 48:900-907, 1991.
39. Mackett M, Smith GL, Moss B: General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol 49:857-864, 1984.
40. Tsang KY, Zaremba S, Nieroda CA, et al: Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 87:982-990, 1995.
41. Hodge JW, McLaughlin JP, Kantor JA, et al: Diversified prime and boost protocols using recombinant vaccinia virus and recombinant non-replicating avian pox virus to enhance Tcell immunity and antitumor responses. Vaccine 15:759-768, 1997.
42. Tartaglia J, Perkus ME, Taylor J, et al: NYVAC: A highly attenuated strain of vaccinia virus. Virology 188:217-232, 1992.
43. Taylor J, Meignier B, Tartaglia J, et al: Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species. Vaccine 13:539- 549, 1995.
44. Marshall JL, Hawkins MJ, Tsang KY, et al: Phase I study in cancer patients of a replication- defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J Clin Oncol 17:332-337, 1999.
45. Marshall JL, Hoyer RJ, Toomey MA, et al: Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses. J Clin Oncol 18:3964-3973, 2000.
46. Marshall J, Tsang KY, Arlen P, et al: Phase I trial to determine the toxicity and immunologic efficacy of the addition of GM-cSF and IL-2 to the combination of vaccinia-CEA and ALVAC-CEA administered as a prime and boost in patients with advanced CEA-bearing cancers (abstract 1087). Proc Am Soc Clin Oncol 20:272a, 2001.
47. Song SY, Kim HS: Strategies to improve dendritic cell-based immunotherapy against cancer. Yonsei Med J 45(suppl):48-52, 2004.
48. Fong L, Hou Y, Rivas A, et al: Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc Natl Acad Sci U S A 98:8809-8814, 2001.
49. Alters SE, Gadea JR, Philip R: Immunotherapy of cancer. Generation of CEA specific CTL using CEA peptide pulsed dendritic cells. Adv Exp Med Biol 417:519-524, 1997.
50. Alters SE, Gadea JR, Sorich M, et al: Dendritic cells pulsed with CEA peptide induce CEA-specific CTL with restricted TCR repertoire. J Immunother 21:17-26, 1998.
51. Morse MA, Nair SK, Mosca PJ, et al: Immunotherapy with autologous, human dendritic cells transfected with carcinoembryonic antigen mRNA. Cancer Invest 21:341-349, 2003.
52. Disis ML, Bernhard H, Shiota FM, et al: Granulocyte-macrophage colony-stimulating factor: An effective adjuvant for protein and peptide- based vaccines. Blood 88:202-210, 1996.
53. Jager E, Ringhoffer M, Dienes HP, et al: Granulocyte-macrophage-colony-stimulating factor enhances immune responses to melanomaassociated peptides in vivo. Int J Cancer 67:54- 62, 1996.
54. Dranoff G, Jaffee E, Lazenby A, et al: Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A 90:3539-3543, 1993.
55. Nemunaitis J, Sterman D, Jablons D, et al: Granulocyte-macrophage colony-stimulating factor gene-modified autologous tumor vaccines in non-small-cell lung cancer. J Natl Cancer Inst 96:326-331, 2004.
56. Ueda Y, Itoh T, Nukaya I, et al: Dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen-derived, HLA-A24- restricted CTL epitope: Clinical outcomes of 18 patients with metastatic gastrointestinal or lung adenocarcinomas. Int J Oncol 24:909-917, 2004.
57. Ullenhag GJ, Frodin JE, Jeddi-Tehrani M, et al: Durable carcinoembryonic antigen (CEA)- specific humoral and cellular immune responses in colorectal carcinoma patients vaccinated with recombinant CEA and granulocyte/macrophage colony-stimulating factor. Clin Cancer Res 10:3273-3281, 2004.
58. Kass E, Panicali DL, Mazzara G, et al: Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant. Cancer Res 61:206-214, 2001.
59. Hodge JW, McLaughlin JP, Abrams SI, et al: Admixture of a recombinant vaccinia virus containing the gene for the costimulatory molecule B7 and a recombinant vaccinia virus containing a tumor-associated antigen gene results in enhanced specific T-cell responses and antitumor immunity. Cancer Res 55:3598-3603, 1995.
60. von Mehren M, Arlen P, Gulley J, et al: The influence of granulocyte macrophage colony-stimulating factor and prior chemotherapy on the immunological response to a vaccine (ALVAC-CEA B7.1) in patients with metastatic carcinoma. Clin Cancer Res 7:1181-1191, 2001.
61. Hodge JW, Sabzevari H, Yafal AG, et al: A triad of costimulatory molecules synergize to amplify T-cell activation. Cancer Res 59:5800- 5807, 1999.
62. Hesdorffer C, Hoerig H, Deraffele G, et al: Manipulating the local tumor microenvironment with vaccinia virus expressing costimulatory molecules for the treatment of melanoma (abstract 7504). J Clin Oncol 23(14S):708, 2004.
63. Grosenbach DW, Barrientos JC, Schlom J, et al: Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects. Cancer Res 61:4497-4505, 2001.
64. Slavin-Chiorini DC, Catalfamo M, Kudo- Saito C, et al: Amplification of the lytic potential of effector/memory CD8+ cells by vectorbased enhancement of ICAM-1 (CD54) in target cells: Implications for intratumoral vaccine therapy. Cancer Gene Ther 11:665-680, 2004.
65. Morse MA, Clay TM, Hobeika AC, et al: Phase I study of immunization with dendritic cells modified with recombinant fowlpox encoding carcinoembryonic antigen and the trial of costimulatory molecules CD54, CD58, CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies (abstract 2508). J Clin Oncol 22(14S):165, 2004.
66. Aarts WM, Schlom J, Hodge JW: Vectorbased vaccine/cytokine combination therapy to enhance induction of immune responses to a selfantigen and antitumor activity. Cancer Res 62:5770-5777, 2002.
67. Marshall J, Arlen P, Rizvi N, et al: A phase I study of sequential vaccinations with fowlpox- CEA (6D)-TRICOM (B7/ICAM/LFA3) alone, and in combination with vaccinia-CEA (6D)- TRICOM and GM-CSF in patients with CEA expressing carcinomas (abstract 24). Proc Am Soc Clin Oncol 21:7a, 2002.
68. Marshall J, Odogwu L, Hwang J, et al: A phase I study of sequential vaccinations with fowlpox-CEA (6D)-TRICOM (B7/ICAM/ LFA3) alone, and in combination with vaccinia- CEA (6D)-TRICOM and GM-CSF in patients with CEA expressing carcinomas (abstract 662). Proc Am Soc Clin Oncol 22:165, 2003.
69. Marshall JL, Gulley JL, Arlen PM, et al: Phase I study of sequential vaccinations with fowlpox-CEA(6D)-TRICOM alone and sequentially with vaccinia-CEA(6D)-TRICOM, with and without granulocyte-macrophage colonystimulating factor, in patients with carcinoembryonic antigen-expressing carcinomas. J Clin Oncol 23:720-731, 2005.
70. Byrd JC, Bresalier RS: Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev 23:77-99, 2004.
71. Tsang KY, Palena C, Gulley J, et al: A human cytotoxic T-lymphocyte epitope and its agonist epitope from the nonvariable number of tandem repeat sequence of MUC-1. Clin Cancer Res 10:2139-2149, 2004.
72. Kaufman H, Dipaola R, von Mehren M, et al: Safety profile of therapeutic pox virusbased vaccines for cancer (abstract 2513). J Clin Oncol 23(14S):166, 2004.
73. Schuetz T, Marshall J, Kaufman H, et al: Two phase I studies of prime-boost vaccinations with vaccinia-fowlpox vaccines expressing CEA, MUC-1, and TRICOM costimulatory molecules (B7.1/ICAM-1/LFA-3) in patients with advanced pancreatic cancer (abstract 2564). J Clin Oncol 22(14S):179, 2004.
74. Chakraborty M, Abrams SI, Coleman CN, et al: External beam radiation of tumors alters phenotype of tumor cells to render them susceptible to vaccine-mediated T-cell killing. Cancer Res 64:4328-4337, 2004.
75. Garnett CT, Palena C, Chakraborty M, et al: Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes. Cancer Res 64:7985-7994, 2004.
76. Belardelli F, Ferrantini M, Parmiani G, et al: International meeting on cancer vaccines: how can we enhance efficacy of therapeutic vaccines? Cancer Res 64:6827-6830, 2004.