Comprehensive

Review

Immunologic Therapies for Gastrointestinal Cancers

Benjamin Levy, John F. Deeken, Greg Holt, John L. Marshall
Abstract
The treatment of solid tumors has shown a dramatic change in recent years. This revolution has come from an improved understanding of the mechanisms of tumor growth and the relationship of cancers to their microenvironments.As part of this revolutoin, we have had a dramatic increase in our understanding of the immune system and the role it could play in the management of cancers.This article reviews the current data supporting the use of cancer immunologic therapies for gastrointestinal tract cancers. The scope is focused on the variety of approaches that have been used, citing the clinical data that have been obtained and reviewing future trials.
Clinical Colorectal Cancer, Vol. 5, No. 1, 37-49, 2005 Key words: Adenovirus, Anti-idiotype antibodies, Carcinoembryonic antigen, Dendritic cells, Monoclonal antibodies, Recombinant viral vaccines, Whole-cell vaccines

Introduction
Gastrointestinal (GI) malignancies as a group, led by colorectal, pancreatic, gastric, esophageal, and hepatocellular cancers, are the leading cause of cancer mortality in the United States.1 More specifically, colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States and has been projected to claim > 56,000 lives in 2005. Despite recent advances in treatments, the overall survival rate for patients with advanced locoregional disease remains < 50%.2 Because of the prevalence and aggressive nature of these cancers, much attention has been dedicated to finding new treatment modalities. The need for more effective treatment is critical. Gastrointestinal cancers offer an ideal setting in which to explore vaccine therapy. Not only are these diseases common, but they carry a grave prognosis, creating an urgent need for more effective agents. In addition, there are many clinical situations in which immune-based therapy would be logical (eg, adjuvant therapy for stage II/III disease, postoperative therapy after resection of metastases, and in combination with chemotherapy for advanced disease). Finally, GI cancers express
Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC Submitted: Nov 1, 2004; Revised: Feb 7, 2005; Accepted: Feb 7, 2005 Address for correspondence: John L. Marshall, MD, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20007 Fax: 202-444-1229; e-mail: marshalj@georgetown.edu

several tumor-specific antigens (TSAs) that have been well characterized and have high potential for immune targeting. In the past decade, there have been significant advances in the field of tumor immunotherapy. The concept of immunotherapy is grounded in the idea that the immune system can selectively recognize and subsequently destroy cancer cells. Many strategies have been employed that attempt to target and elicit an immune response against tumor cells. These strategies include nonspecific and specific immunotherapy as well as antibody-mediated immunotherapy. Recent clinical studies that have incorporated these novel approaches in the treatment of patients with GI malignancies will be discussed further in this review.

Nonspecific Immunotherapy
Early attempts at immunotherapy focused mostly on the enhancement of the immune response nonspecifically by the addition of agents such as interferon, interleukin (IL)2, levamisole, and bacillus Calmette-Gurin (BCG). Nonspecific immunotherapy relies on the existence of immune effector cells (T and B cells) that have previously recognized and processed the tumor antigens as foreign. Then, by enhancing the overall immune response, the intent is to enhance the tumor-specific elements to create an antitumor response. Unfortunately, attempts to augment the immune system nonspecifically have yielded poor clinical outcomes and have led clinical researchers to move toward active, specific immunotherapeutic approaches.

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(1) antibodies that have been conjugated to immunotoxins and Mouse Antibodies Used to Generate Human Antibodies Against the Intended Target radionucleotides; (2) MoAbs used alone that are selective for growth Human Antibody Mouse Antibody Anti-Idiotype factor receptors, thereby decreasing Made by Patients cellular signaling; and (3) the use of Mouse Antibody Immune Response Therapy anti-idiotype antibodies. When an MoAb is administered, it can serve as an antigen itself. Antibodies can then be generated against the idiotype represented by the Human Antibody Human Antibody MoAb. This process, called the idiotype Idiotype Human Cell Idiotype network, was originally hypothesized by Jerne (Figure 1).5 The generation of Targeted Approach: Tumoranti-idiotype antibodies begins with the immunization of an animal with a TAA of choice (eg, CEA) and the subsequent Associated Antigens and production of an antibody against the TAA (named Ab1). Ab1 Costimulation is then used to generate a series of anti-idiotype antibodies One of the most significant advances in the field of tumor called Ab2. Selected Ab2 antibodies effectively mimic the 3immunology in the past 10 years has been the isolation and dimensional structure of the original TAA, which can then be molecular characterization of tumor-associated antigens used as a surrogate for the TAA as an immunogen, with the (TAAs) recognized by T cells.2 These peptides are broadly final result of generating antianti-idiotype antibodies. categorized in 3 groups: unique antigens specific for an Although this may seem like a long way to go to get back to individual tumor, overexpressed peptides that are found to a the original antigen, there are significant advantages to this variable degree on normal tissue, and antigens shared among strategy. The primary advantage is that the anti-idiotype different tumors but confined to malignancy.2,3 Examples of represents an exogenous protein that expresses the target these TAAs in CRC include overexpressed peptides such as antigen, whereas the original antigen itself is a native (ie, self) carcinoembryonic antigen (CEA) and epithelial cell mucin protein. Therefore, the native antigen has been converted to a (ie, MUC-1) as well as TSAs such as mutant transforming foreign protein that will be endocytosed by APCs, processed, growth factor receptor II and mutant adenomatous and presented to T cells to activate the immunologic activities polyposis coli protein. of CD4 and CD8 T cells. Via this APC presentation to T cells, In addition to the discovery of TAAs, the use of the production of endogenous cytokines increases, further costimulatory molecules has further enhanced targeted tumor stimulating the response.6 This would not be expected from immunotherapy. It is established that inciting an immune simply vaccinating with the TAA itself. This process of response by antigen presentation involves 2 signals. The first inducing anti-idiotype immunity has been the subject of signal involves interaction between a protein expressed by a significant basic and clinical research during the past decade. major histocompatibility complex (MHC) molecule on Several anti-idiotype antibodies are being tested in clinical antigen-presenting cells (APCs) and a T-cell ligand. The trials against colon cancer, including CEA, CD55, 17-1A, second signal is mediated through costimulatory molecules Lewis Y, and mutant p53. expressed on APCs that interact with other T-cell surface receptors. Thus, successful priming of an effective antitumor Carcinoembryonic Antigen Anti-Idiotype Antibody T-cell response requires presentation of TAA with appropriate Foon et al studied an anti-idiotype antibody directed stimulatory molecules.2 Strategies that use this concept of against CEA called CEAVac.7 By using this compound in costimulation include antibody-mediated blockade patients with various stages of colon cancer, they mechanisms as well as the insertion of costimulatory demonstrated that patients generate high polyclonal anti-CEA molecules into the vaccine in use.4 responses and idiotype-specific T-cell responses, 75% of Antibody Therapy: Monoclonal which were CEA-specific. Several patients received 5Antibodies and Anti-Idiotype fluorouracil (5-FU)based chemotherapy during the Antibodies vaccinations, and responses were not adversely affected. The use of monoclonal antibodies (MoAbs) to treat Finally, they have demonstrated the ability to boost the malignancy has been clinically proven to generate an antibody response with monthly injections of CEAVac. antitumor response. Their use involves distinct advantages Although nothing definitive can be said about these patients including ease of administration, low toxicity profile, and the clinical responses given the variability in stage and small ability to specifically target tumor cells while sparing normal sample, 7 of 8 patients with resected stage IV disease all host tissue.1 The use of antibodies in the field of remained on study without evidence of recurrence after 12-33 immunotherapy employs 3 different approaches and includes months of follow-up.7
Figure 1 Schematic Mechanism of Anti-Idiotype Cascade

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A randomized double-blind, placebo-controlled phase III study compared the use of CEAVac versus placebo in patients with stage IV CRC also receiving 5-FU/leucovorin (LV).8 Endpoints were survival, time to progression, and anti-CEA immunity. The study enrolled 630 patients. Patients were treated for a median time of 31 weeks and received a median of 5 cycles of 5-FU/LV. More than 75% of the treated patients developed CEA antibodies, but there were no significant differences in median survival or time to progression. Patients who received a prospectively defined priming regime of 6 vaccination cycles showed a trend toward improved survival (19.1 months vs. 17.1 months; P = 0.1), a trend that was strengthened in those patients who received 8 cycles (21.3 months vs. 18.5 months; P = 0.04). In a related study, 32 patients with advanced colon cancer were treated with 3H1 (ie, TriAb), an antibody that targets human milkfat globule. All patients generated antibody and CD4 responses; 75% were CEA-specific.9 Currently, CEAVac and TriAb, which is also highly expressed in colon cancer, are being tested together in a phase II trial by Cancer and Leukemia Group B (CALGB). The patients selected for this trial have stage IV colon cancer with isolated liver metastases that have been removed. have been ineffective in this group of patients. Another study by this group used the 105AD7 anti-idiotype vaccine as neoadjuvant therapy in patients with CRC. They compared their cohort of 35 patients versus matched controls. Based on preclinical data that compared the anti-idiotype amino acid sequences with the CD55 antigen and predicted that patients with human leukocyte antigen (HLA) haplotypes DR1, DR3, and DR7 would show CD4+ T-cell responses, the authors analyzed the results based on whether patients expressed these 3 haplotypes. Eighty-three percent of the patients who expressed these haplotypes exhibited T-cell responses. Eighty-eight percent who did not have these HLA haplotypes did not exhibit a response. A further phase I trial of 12 patients with CRC confirmed this finding.14 Eleven of the patients expressed HLA haplotypes DR1, DR3, or DR7 and had a CD4 and CD8 T-cell response. The one patient who did not respond did not have a DR1, DR3, or DR7 haplotype. Interestingly, only 1 of the 11 patients who exhibited a T-cell response showed a sustained memory response.

CD17-1A
The most extensively studied anti-idiotype compound is 17-1A, a murine immunoglobulin (Ig) G2a MoAb that targets a 26-kd polypeptide tumor-associated cell-surface glycoprotein, GA733-2. This glycoprotein is also known as CD17-1A, EGP, KSI-4, KSA, and Ep-CAM.15 Although this agent may function in MoAb-mediating complementdependent cytolysis, antibody-dependent cell-mediated cytotoxicity (ADCC), and apoptosis, it also may induce antiidiotype immune responses. As a single agent in advanced colon cancer, a 5% response rate was observed with little toxicity. It has also been tested in combination with granulocyte-macrophage colony-stimulating factor (GMCSF).16 This agent was tested as adjuvant therapy for patients with stage III colon cancer in a small randomized trial (N = 189) comparing 5 months of 17-1A injections with observation alone.17 The primary endpoints of the trial were overall survival (OS) and disease-free survival (DFS). The results were initially presented after 5 years and re-presented after 7 years; they continued to show a 32% reduced mortality rate and a 23% reduced recurrence rate in the treatment arm. The reduction in metastatic disease was significant, whereas local recurrences were not altered between the 2 arms. This latter difference could be explained by the fact that 11 patients in the control arm received radiation, whereas none of the patients in the treatment arm did. It could also be explained by some unknown difference in the biology of local and metastatic recurrences. Eighty percent of the patients developed human antimouse antibodies after the second or third infusion. Regardless, these data were compelling enough for the agent to gain approval in Germany, and 2 large randomized trials were launched enrolling patients with colorectal disease. These 2 trials used 5-FU/LV with or without 17-1A in stage III disease and 17-1A versus observation in stage II

CD55
The anti-idiotype MoAb 105AD7 mimics the TAA 791Tgp72 (ie, CD55), which is expressed in 70%-80% of CRCs.10 CD55 plays a role in signaling between the innate and adaptive immune responses. Absence of the molecule makes the tumor cell susceptible to complement, resulting in cytolysis, whereas overexpression results in the antigen being a target for T-cell immunotherapy. A phase I trial in advanced CRC showed the anti-idiotype vaccine to be nontoxic with a suggestion of improved survival among vaccinated patients compared with historical paired controls.11 This effect is thought to be mediated by antitumor T-cell responses. To test this theory, 19 patients with CRC were immunized before resection of the primary tumors. Tumor samples were found to have a significantly higher activated lymphocyte infiltration compared with nonvaccinated patients. Further analysis of the infiltrating cells showed them to contain a high level of natural killer and CD4 cells as well as evidence of CD8 activation.12 This evidence supports further study with this vaccine in CRC and demonstrates the value of translational studies in early clinical trials. Maxwell-Armstrong et al conducted a randomized, doubleblind phase II survival study with the 105AD7 anti-idiotypic MoAb.13 Although patient characteristics and baseline disease were similar between the treatment and placebo arms, a decreased mean survival was seen in the treatment arm (124 days vs. 184 days). Compliance with the scheduled vaccinations was poor in the study, with only 50% of patients receiving the 3 planned vaccinations. The negative finding may have been caused by this lack of compliance or the lack of efficacy resulting from the high disease burden in the enrolled patients. It is also a possibility that this vaccine could

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disease. In the first trial, patients who received anti-idiotype therapy had a modest but statistically significant improvement in OS (81.6% vs. 78.9% at 3 years).18 Patients in the cooperative group phase III trial (CALGB 9581) had stage II disease and received placebo or MoAb 17-1A. Final accrual totaled 1738 of a planned size of 2100; the study closed early in 2002 because the manufacturer discontinued making the drug. Endpoints were OS and failure-free survival (FFS). With a mean follow-up of 32 months, there were no significant differences in the treatment arms. Although the median OS and FFS were not reached, MoAb 17-1A did not appear to have a clinical benefit. The data presented did not differentiate between patients who were found to have an antibody response and patients who did not.19 In an earlier study of 10 patients with CRC by Fagerberg et al, the investigators measured the anti-idiotype (Ab2) and the antianti-idiotype (Ab3) antibodies as well as the corresponding T cells (T2 and T3, respectively).20 Interestingly, all 10 patients developed Ab2 and Ab3 antibodies, but only 5 developed the corresponding T3 cells directed against the tumor antigen. In addition, only 4 of these 5 patients with T3 cells responded to the in vitro stimulation. Only these 4 patients had a clinical response during the trial, raising the question of whether antigen-directed T cells are more important than antibody-mediated cytotoxicity in antiidiotype therapy. This was the only trial of 17-1A we found that investigated T-cell response in addition to antibody and anti-idiotype response. The role of direct antibody effect from vaccination versus the induction of an anti-idiotype immune response was recently reported. IGN101 is the murine MoAb 17-1A absorbed by aluminum, which targets GA733-2. Sixty patients in a phase I trial with epithelial cancers (mainly colon) were given vaccinations over 2 months. Sera were analyzed, and the investigators found that vaccinations led to a dramatic decrease in Ep-CAMpositive circulating cells.21 Interim results from a follow-up randomized, placebocontrolled phase II trial of 240 patients with stage III epithelial tumors (mainly colorectal but also upper GI tract and nonsmall-cell lung cancers) showed a positive survival benefit in patients who exhibited an immune response.22 Another phase II study of IGN101 analyzed the immunogenicity of the vaccine when given concurrently with chemotherapy.23 Compared with patients from the phase I trial, patients required more vaccinations to seroconvert (4 vs. 23 vaccinations), and mean titers were lower in the group that received chemotherapy. However, the mean immunoglobulin G (IgG) concentration was higher in the patients who received chemotherapy. Although some clinical activity has been seen with the use of CD-171A, the only phase III study with this vaccine has not yet shown clinical benefit. A viable area of research would seem to be available in looking at which patients do and do not exhibit so-called T3-cell responses and correlating that with other markers of immunologic as well as therapeutic responses.

Lewis Y
Lewis Y is another antigen expressed in a majority of cancer cells but with limited expression in nonmalignant tissue. In a phase I study, Thakkar et al used IGN 301, a monoclonal anti-idiotype antibody mimicking Lewis Y.24 An interim analysis of 20 patients, 6 of whom had colon cancer, from a planned group of 42 was recently reported. In the trial, patients were randomized to receive the vaccine plus GM-CSF, IFN2, or just the vaccine alone. This study did analyze T-cell response. Of 14 evaluated patients, 10 exhibited antibody responses by day 29, and 10 of 10 patients exhibited antibody responses by day 71. T-cell response was detected in only 1 of 11 analyzed patients. Whether T-cell response compared with anti-idiotype response alone correlates with clinical response has not yet been reported by these investigators.

Mutant p53
Lomas et al used a vaccine with 8 peptides representing the complimentarity-determining regions (CDRs) of human antip53 antibodies.25 In preclinical animal testing, this vaccination approach overcame tolerance and induced an anti-idiotype response. In a phase I trial of 24 patients with incurable disease, 5 of whom had CRC, the mutant p53 vaccine was given in 4 monthly injections with GM-CSF. Six of the 14 patients completed the vaccination schedule. The vaccine induced Ab2 antibodies specific for 3 of the peptides in 4 of the 6 patients. None of these patients exhibited a T-cell response against p53. Clinical responses were not noted. It is clear from this line of basic and clinical research that anti-idiotype immune therapy can induce a T-cell response, and clinical activity has been observed at times. Many aspects of this response are yet to be elucidated. Additional trials are ongoing, but, given the more significant results seen in other lines of immune therapy research, it remains unclear how prominent this line of therapy will be in treating CRC in the future.

Active Specific Immunotherapy

In addition to antibody therapy, other approaches have been used in clinical trials to induce an immune response. Like antibody therapy, the goal of these approaches is to evoke a tumor-specific immune response, resulting in destruction of tumor cells, as well as memory against relevant antigens.26 The remaining approaches, termed active specific therapy, also rely on TAAs and their unique presentation to naive, tumor antigenspecific T cells. Treatment modalities involving active, specific immunotherapy include whole-cell tumor vaccines, recombinant viral vaccines, peptide vaccines, and dendritic cell (DC) vaccines.

Whole-Cell Vaccines
One method of vaccinating patients against their tumor involves the inoculation of actual tumor cells treated with certain adjuvant agents to augment their immunogenicity. Theoretically, the injected tumor cells are destroyed, and their

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proteins are taken up by professional APCs for the presentation to and subsequent activation of immune effector cells. The strength of this method is its ability to incorporate the entire scope of known and unknown TAAs and TSAs into a comprehensive vaccine. Additionally, the frequent use of autologous tumor cells for immunization relatively assures the vaccine will specifically cover the antigens needed for each individuals cancer. Finally, as is the case in many colorectal tumors, surgical resection of the neoplasm provides ample tissue for cryopreservation with subsequent preparation of inoculum. Unfortunately, this method is not without its weaknesses. Although surgical resection provides ample numbers of tumor cells, it requires the patient to undergo surgery, which is not without its morbidity and mortality. Use of primary cultures of tumor cells is also fraught with problems, because, as they are a nonrenewable resource, in vitro infections and accidental interruption of refrigeration can lead to irretrievable loss. Use of allogeneic cell lines derived from similar cancers would obviate this problem but introduces new complexities of potential loss of tumor antigens through repetitive cell culture, difference in antigens in the individual cancer versus the cell line, and injection of other alloantigens with potential autoimmune consequences. carcinomas after resection. Although the patient numbers were small and quite lopsided (9 in the BCG group vs. 57 in the NDV group), patients vaccinated with autologous tumor cells with NDV as the adjuvant agent had a 97.9% survival rate at 2 years, as opposed to a 66.7% survival rate for those vaccinated with BCG.31 More recently, the use of NDV as an adjuvant agent for an autologous tumor cell vaccine was revisited by Liang et al, in whose study patients with digestive tract cancers after surgical resection were immunized with the NDV/autologous tumor vaccine. These patients had significant increases in mean and median survival times compared with patients treated with resection alone.32 This study also demonstrated an improvement in survival times in patients with a documented positive delayed-type hypersensitivity (DTH) response to the vaccine.

Allogeneic Tumor Vaccines

In a slightly different approach, an allogeneic colon carcinoma cell line that expresses known TAAs from colon, CEA, Ep-CAM/KSA, MUC1, HER2/neu, and MAGE was tested for its ability to induce tumor-specific immunity in patients with colon cancer. Instead of using a microorganism as an adjuvant agent, the researchers fused the cell line with a cytokine-secreting fibroblast cell line to ideally create an environment suitable for initiation and activation of an immune system response. From these patients, cytotoxic T lymphocyte (CTL) cell line clones were grown in culture from peripheral blood mononuclear cells that showed an HLA-restricted specificity for their autologous tumor cells without cross-reactivity with the fibroblast line.33 Overall, the use of whole cancer cells as a vaccine, whether autologous or allogeneic, represents one method of immunization with case reports of positive results. The unfortunate reality of the difficulties of acquisition, propagation, and the individualized nature of this vaccination route as detailed in this section makes it hard to believe it will be feasible for clinical use. Should another vaccine have similar successes and an easier preparation, whole-cell vaccines will be abandoned.

Autologous Tumor Vaccines

Work in this area has focused predominantly on the use of autologous cells, with minimal work with allogeneic cancer cell lines. In 1985, Hoover et al performed a randomized, controlled, prospective study comparing recurrence and survival data for patients with Dukes stage B2-C3 colorectal carcinoma with surgical resection versus resection plus vaccination with autologous tumor cell admixed with BCG.27 Initial results revealed a statistically significant reduction in both parameters in vaccinated patients that was not seen on longer follow-up.28 A subset analysis of those with colon cancer revealed a significant improvement in recurrence rates and survival data. Ultimately, a larger Eastern Cooperative Oncology Group study with more patients analyzed on an intent-to-treat principle revealed no improvement in recurrence or survival using similar vaccination methods with BCG-admixed autologous tumor cells.29 Capitalizing on the confusing data of these studies, Vermorken et al repeated these studies but asked whether the distinct stage of a cancer at vaccination altered response to the autologous tumor cell/BCG vaccine.30 In their study, patients with stage II/III colon cancer did not benefit from immunization with BCG-admixed autologous tumor cell vaccines. However, when patients were grouped according to stage, those with stage II cancer had a statistically significant 61% risk reduction for recurrence, increased recurrence-free survival, and a trend toward increased OS. In an attempt to augment the responses seen previously, an alternate adjuvant agent, Newcastle Disease Virus (NDV), was used and directly compared in a head-to-head study with BCG in the vaccination of patients with colorectal

Recombinant Viral Vaccines

To take advantage of the usual strong immunity produced from viral infections, recombinant DNA technology has allowed the creation of new viruses that retain their normal outside capsule but with a newly engineered genome. The recombinant virus retains its natural tropism and infects cells as it would normally. When inside, its DNA is programmed to express the desired TAA or TSA for processing in the MHC class I or class II pathway of peptide presentation for subsequent initiation and activation of TAA/TSAspecific immune effector cells. Additionally, these new viruses are engineered with defective replication systems to prevent rampant infection (Figure 2). The poxviruses have justifiably received significant research attention as a result of many desirable qualities. These viruses are capable of enclosing larger genomes that allow the

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Figure 2 Life Cycle of a Dendritic Cell
DC Progenitor DC Progenitor

overcoming potentially complicating issues of tolerance and anergy.

Step 1: Virus infects APC and produces messenger RNA Step 2: Full-length protein is made Step 3: APC presents peptides to T cells

Avipox

Macrophage

Eosinophil

Antigen

NK
Inflamed Vessels Mature DC Antigen Presentation

inclusion of more genes encoding other TAAs/TSAs, costimulatory molecules, or cytokines. They do not incorporate into the genome of their host cell, which prevents the small but alarming chance that their use could transform cells through alteration of proto-oncogenes or tumor-suppressor genes. Their DNA promoters are highly active, producing greater quantities of the desired proteins. Neutralizing antibodies to the vaccinia virus from previous smallpox vaccines or with the initial vaccine inoculation have been circumvented by use of alternative poxviruses or poxviruses with less immunogenicity, such as the carnarypox virus.34 For the most part, these viral vectors, including vaccina, canarypox virus, and adenovirus, have been well tolerated in clinical trials, as detailed later, with the majority of reactions pertaining to injection-site tenderness, injection-site reactions, and mild flulike symptoms. Adenoviruses have been tested extensively in US Army recruits and have shown a proven safety record,35 whereas vaccinia has been associated with encephalopathy and postvaccinal encephalitis in rare cases.36 Should a vaccine successfully eliminate a patients cancer with a vaccinia vector, this benefit would have to be weighed against the risk of such adverse central nervous system side effects.

Vaccinia
The vaccinia virus, engineered to express the CEA, a TAA of colon cancer, was initially evaluated for its ability to induce specific immune effector cells in patients with colon cancers that expressed the CEA antigen. In 1995, Tsang et al generated specific anti-CEA CTLs in patients with metastatic carcinomas that were able to lyse CEA targets in an HLA-restricted manner.37 In 2000, Conry et al vaccinated similar patients with vaccinia/CEA and found the induction of a specific anti-CEA humoral response.38 These studies, in addition to proving the potency of the vaccine, furthered evidence that it would be possible to induce an immunity against a self-protein, thus

As a result of the inability of the avipox virus to replicate in human hosts and the rare induction of neutralizing antibody responses, the ALVAC (fowlpox) virus was used in later trials. Its initial clinical trial involved the vaccination of patients Immature Antigen-Capturing with advanced colon cancer with DC intramuscular injections of ALVAC/CEA. Although no objective clinical responses were seen, 1 patients CEA normalized after the fourth vaccination, and 4 had progression-free disease for 5-9 months. Importantly, specific antiCEA T cells were found after vaccination and were able to lyse autologous and CEA-positive allogenic targets. Noted side effects included mild skin reactions and injection-site soreness.39 In 2000, Zhu et al corroborated these results in a similar experiment with the finding of CEA-specific T cells.40 With use of an alternate digestive tract TAA, p53, incorporated into an ALVAC vector, van der Burg et al found humoral and cellular immune responses to p53 when patients with advanced CRC were vaccinated with ALVAC/p53, further legitimizing the method of vaccination as a potentially efficacious antitumor therapy.41 With the increased understanding of the immune system (specifically T-cell activation pathways), costimulatory molecules were added to viral vaccines and tested for their ability to augment previously seen responses. Specifically, a T-cell costimulatory molecule, B7.1, was added to ALVAC/CEA,42 and 2 experiments documented stabilization of disease in a small but significant number of patients. In 2000, von Mehren et al43 and Horig et al44 reported on the vaccination of patients with the ALVAC/CEA viral vector now engineered to also express B7.1 and found stable disease in 27% and 16% of patients, respectively. Horig et al also went on to report that the 3 patients with stable disease were the only patients to have a measurable T-cell response.44 Another adjuvant therapy tested for its ability to augment previously seen immune responses is GM-CSF, a cytokine important in creating the proper milieu for T-cell initiation and activation. Patients with metastatic carcinoma were vaccinated with ALVAC/CEA B7.1 with localized injections of GM-CSF, with the finding of 10 of 27 patients (37%) with stable disease 13 months.45 More recently, Ullenhag et al vaccinated 12 colorectal carcinoma patients with ALVAC/KSA, with half additionally receiving GM-CSF.46 They observed greater T-cell responses in patients given GMCSF than in those given placebo, identifying GM-CSF as a potent adjuvant therapy.

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Prime/Boost Phenomenon
Figure 3 Mechanism of Viral-Mediated Cancer Vaccines

In 1997, preclinical data from Hodge et al revealed a 4-fold CEA protein in cytosol CD8 augmentation of a vaccines induction of a T-cell response when Proteasome CD8+ cleaves CEA protein cytotoxic vaccinia virus was used to prime the T cell response, and the avipox virus was Translation used to boost the response.47 This TAP was translated into the clinical realm in 2000, when a vaccination schedule that mimicked the preclinical data Retroviral CEA and targeted CEA was found to be superior in T-cell induction compared T-cell receptor with an alternative arrangement in which avipox was used first followed by vaccinia.48 Although no clinical Viral CEA gene objective responses were noted, the promotor Golgi apparatus patient population enrolled had advanced disease with high tumor Transcription burdens. Follow-up work found that the addition of GM-CSF to the mRNA prime/boost strategy increased the numbers of CEA-specific T-cell Endoplasmic reticulum DNA precursors, but the addition of IL-2 in 49 addition to GM-CSF did not. Further prime and boost methods have been incorporated in recent phase I trials involving CEA, MUC-1 (a glycoprotein and IL-2 for induction of the T-cell responses. discussed later), and 3 costimulatory molecules (B7.1, In a phase I study, Marshall et al were able to achieve ICAM-1, and LFA-3). In one study, 22 patients with stabilization of CEA-expressing carcinoma in 21 of 58 patients advanced pancreatic cancer (stage III) were initially evaluated.53 In this study, 3 broad groups were studied: (1) vaccinated with the vaccinia vaccine followed by 3 boost avipox/CEA with TRICOM, (2) vaccinia/CEA plus TRICOM doses of fowlpox.50 Although the median OS cannot yet be followed by avipox/CEA plus TRICOM, and (3) vaccinia/CEA calculated because of the continued survival of the patients, it plus TRICOM followed by avipox/CEA plus TRICOM with currently stands at 7.3 months. These data are encouraging GM-CSF. The results did not prove the superiority of the because the OS is more than double the OS of historical prime/boost method nor did they firmly establish the role of controls. It must be noted that such comparisons are of cytokines in augmenting the response. Evaluation of the patients limited value in small studies with the potential for selection expressing the HLA-A*0201 MHC molecules did reveal CEA bias and must be confirmed with future randomized peptidespecific CTL responses restricted to this haplotype. controlled trials. Adenovirus Based on these recent studies, the hope is that, by using 2 Although not specifically targeting a TAA or TSA, work different TAAs (MUC-1 in combination with CEA), a greater with adenoviruses has produced encouraging results for their clinical benefit can be generated. This approach could help possible use in anticancer vaccines. One adenovirus, ONYXovercome the relatively high level of tumor resistance 015 (ie, CI-1042), has a specific deletion of parts of the E1B observed during earlier clinical trials. protein of the virus necessary for cellular transformation after TRICOM infection. The result of this mutation is a select ability to lyse In more preclinical work, Hodge et al discovered that the cells that do not express p53 tumor suppressor activity, which addition of 3 costimulatory molecules, B7.1, ICAM-1, and is a theme common to many cancers of the digestive tract.54 LFA3 (TRICOM), produced greater T-cell responses than the Although only early studies have been reported so far, Hecht addition of 1 or 2 of the molecules alone (Figure 3).51 These et al reported good safety data for injection into pancreatic data were also translated into clinical investigations with the carcinomas55 and Hamid et al reported that 7 patients with finding of CEA-specific T cells after vaccination with a colorectal carcinoma exhibited stable disease.56 Although similar prime/boost algorithm with use of TRICOM as the limited in its current clinical results, this adenovirus could adjuvant agent packaged within the vaccinating viral have potential as an antitumor immune therapy for tumors genome.52 They also reported on the requirement of GM-CSF that lack p53 activity.

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Viral vectorbased vaccines engineered to express specific tumor antigens represent a promising method of immunization that, with continued experimentation and augmentation, should improve anticancer immunotherapies. Possible limitations to their use remain in preexisting neutralizing antibodies; however, this problem can seemingly be overcome with the use of different viral vectors or vectors with low immunogenicity. Additionally, the potential side effects of each individual vector may have to be tolerated if the benefit of the method outweighs the risks.

CEA
Additional trials using peptide vaccines have been reported. Ullenhag et al used recombinant CEA in 24 patients with CRC in a phase I trial. Half the patients received GMCSF as well.60 All 12 patients who received GM-CSF exhibited a T-cell and IgG response to CEA. In the arm not treated with GM-CSF, only 9 of 12 showed a T-cell response, and 8 had an IgG response. No dose response was seen in 4 different dose levels administered. T-cell and IgG levels were present at 12- and 24-month follow-ups. Survival was found to correlate with anti-CEA IgG titers.

Peptide Vaccines: ras, CEA, SART3, bHCG, Gastrin, and MUC-1

Tumors express peptides, often oncogenes, that are unique to the malignant cells. Typically, these proteins are mutated forms of the native protein. Although variation in the specific mutations would make immunologic targeting difficult across a patient population, several oncogenes share significant homology among patients and have been the targets of peptide vaccine development. These include ras, CEA, and SART3, as well as MUC-1, gastrin, and bHCG.

SART3
Miyagi et al used peptide vaccines that targeted the tumorrejection antigen SART3 at 2 antigenic epitopes.61 Twelve patients with advanced CRC were given vaccinations. Patients exhibited cellular immune responses to the colon cancer cells (7 of 11 analyzed) and the vaccinated peptide (7 of 10 analyzed).

bHCG
bHCG is another peptide tested as a target for vaccine therapy for GI cancers. bHCG is a glycoprotein hormone that has been found by immunohistochemistry in as many as 52% of those with CRC, and increased serum levels have been measured in 41% of patients. In addition, earlier studies have demonstrated that bHCG is detected more frequently in those tumors that are less differentiated and more aggressive.62 Unlike CEA, bHCG does not appear to be found in normal host tissue cells, making it an attractive protein for immune stimulation. CTP37-DT, the vaccine used, is a synthetic peptide composed of the terminal peptide of bHCG conjugated to diphtheria toxoid. Initial studies with the vaccine demonstrated its ability to elicit a humoral and cellular immune response. Subsequent phase I/II trials have been completed. The first was a trial of 77 patients with metastatic colon cancer who were randomly assigned to 1 of 2 doses of vaccine.63 The vaccines were well tolerated and anti-HCG antibodies were induced in most patients. Interestingly, there was an association between antibody response and survival. However, there was no OS advantage for those who were vaccinated. The second study was a phase II randomized trial of vaccine alone compared with vaccine plus gemcitabine in 55 patients with untreated metastatic pancreatic cancer.64 The results demonstrated that gemcitabine had little influence on whether patients had an antibody response, but it did negatively alter the titer. The group receiving vaccine and gemcitabine had a longer median survival (6.6 months vs. 4.7 months). Most recently, monoclonal antibodies to 2 epitopes of bHCG have been tested in patients with metastatic cancer.65

Mutant ras
The ras oncogene produces an abnormal protein compared with the wild-type gene, which is processed and presented by HLA molecules on tumor cells. Mutant ras is present in 90% of pancreatic cancers and 40% of CRCs. The ras oncogene can have different mutations, including in the coding region that is presented by HLA molecules. A group of investigators used the region in codon 12 to develop a mutant ras peptide vaccine. In an initial phase I trial, Behrens et al used a 13-mer mutated ras peptide that corresponded with the mutated version of the patients tumors. All 3 patients treated exhibited a CD4+ and CD8+ T-cell response.57 In a follow-up phase I trial, 18 patients with recurrent and metastatic disease, including 15 with CRC, were given the mutant ras vaccine along with GM-CSF and IL-2.58 Toxicities were mainly associated with IL-2. Seven patients received < 3 vaccinations as a result of advancing disease and worsening performance status. Of the 11 patients who completed 3 vaccinations, median progression-free survival was 14 months. One patient with metastatic CRC had complete resolution of his lung metastases, and 5 patients had no disease progression for 12 months. The same group of investigators also completed a phase II pilot using the mutant ras peptide vaccine in resected stage II/III pancreatic cancer and Dukes stage C and D CRC.59 Again, in this trial, patients tumors were analyzed for which ras mutation they had, and vaccinations were tailored to their specific type (termed ras/ASP, ras/VAL, and ras/CYS). The peptide was mixed with Detox adjuvant and given subcutaneously. Of 12 patients, no significant toxicities were observed except for a grade 2 rash in 1 patient. The 5 patients with pancreatic cancer had an average DFS of 22 months, whereas the 7 with CRC had a mean DFS of 14.6 months.

Gastrin
G17DT is a gastrin immunogen that stimulated antibodies that neutralize gastrin-stimulated growth. Gastrin acts like a growth factor for several GI tumors including pancreatic,

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gastric, and colon cancer.66 It has Figure 4 Evolution of TRICOM Vaccines been demonstrated to promote Pox: Antigen Alone TRICOM Antigen cellular proliferation indirectly through gene transcription as well as APC T Cell APC T Cell reduce apoptosis.67 Phase I, II, and III MHC + Peptide MHC + Peptide T-Cell Response T-Cell Response trials have been completed that show Costimulatory Ligand promising data with the use of this Molecules CD28 B7-1 CTLA-4 vaccine. Initial phase I/II trials were conduced in patients with advanced ICAM-1 LFA-1 end-stage CRC. In these studies, 40 LFA-3 CD2 patients immunized with G17DT had a median survival of 338 days, Antigen = CEA, CEA 6D, PSA, MUC-1 compared with 184 days for the 68,69 Phase II/III trials placebo group. have also been conducted in patients rectum. The results showed large amounts of IgG1 and with pancreatic cancer. In a phase II trial, 20 of 30 patients antiMUC-1 antibodies produced in 13 of 25 patients. T-cell immunized with G17DT were shown to have an immune proliferation was found in 4 of 15 patients, and CTL responses response to the vaccine. Furthermore, antibody responders were found in 2 of 10 patients. Therefore, this MUC-1based demonstrated significantly greater survival than construct was found to be highly immunogenic in patients with nonresponders (217 days vs. 121 days).70 This study was cancer, with dose responses observed for antibody production followed by a European phase III trial in which patients with (increased dose led to higher antibody titers).75 A phase II stage IV pancreatic cancer who had received no previous study using MUC-1 peptide admixed with BCG was performed treatment were immunized with G17DT or a control. Patients in 30 patients with advanced colon cancer. The observed treated with G17DT had a median survival time that was 53% systemic symptoms and injection-site reactions suggested an longer than that of the patients in the control arm. In addition, activation of the immune system, and 7 of 22 patients tested 25% of patients immunized with G17DT had a survival time demonstrated a 2-4fold increase in CTLs.76 that was 106% longer than that of the control arm. This trial was a randomized, double blind, controlled, statistically powered phase III trial.71,72
Figure 5 Maturation of Dendritic Cells

MUC-1
MUC-1, a heavily glycosylated transmembrane protein, is another attractive peptide for immunotherapy. Expressed on many epithelial adenocarcinomas including breast, ovary, prostate, and colon, the protein functions as an antiadhesion molecule that could facilitate metastasis.73 Whereas mucin proteins are found throughout normal ductal epithelium, MUC-1 expression on malignant cells is greatly upregulated and undergoes changes in glycosylation and distribution.74 These modifications in the protein allow for immune recognition and subsequent T-cell proliferation. Furthermore, it has been found that the protein might be recognized by a specific T-cell receptor, independent of any MHC restricting component. The role of MUC-1 as a potential tumor antigen has been most extensively studied in breast cancer, and its use in GI malignancy is currently being reviewed. Initial phase I studies involving the MUC-1 fusion protein were carried out in 25 patients with advanced metastatic carcinoma of the breast, colon, stomach, or
CD14+/CD11C+

Subsets of Human Dendritic Cells

?
Monocytes CD14/CD11C+ CD14/CD11C

GM-CSF/TNF/IL-4

TGF-

IL-3

T T T

T T

B T T

B T

Interstitial DC

Langerhans DC

Plasmacytoid DC

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Vaccines for Gastrointestinal Cancers

Figure 6 Methods of Vaccination Against Cancers
B
Tumor Lysate Viral Vector Peptides

Vector Encoding A Immunostimulatory Cytokines

C
Plasmid DNA

Viral Vector

Peptides

Tumor Cells

DCs

Direct Injection Antigen-Loaded DCs

Epidermis Attraction of DCs Dermis CD4+ T Cell MHCPeptide Complex LN

Local TAA Expression

Tumor

Help CD8+ CTL Tumor Cell Killing CD8+ T Cell

A. Cytokine stimulation. B. Ex vivo DC stimulation. C. In vivo DC stimulation.

nonactivated or improperly activated DCs could induce T-cell tolerance to tumor antigen.78 Another approach to produce mature, activated DCs is to fuse immature DCs with tumor cells to create DCs full of tumor antigen ready for internal processing (Figure 6). When reinfused, local or systemic cytokines have been administered in various trials to further induce a DC-mediated T-cell response. The main difficulty with DC vaccine therapy development has been the fact that preparing sufficient numbers of mature DCs from patients peripheral blood is costly and time-consuming.77 As with other immune therapy approaches to CRC, investigators have used different antigens as targets for DC processing, including CEA, MAGE, MUC-1, and p53.

Peptide vaccines as a whole have been proven to elicit a strong immune response as evidenced by measured T cells and immunoglobulins. In addition, several small trials have illustrated a clinical response with these vaccines, as described earlier. These vaccines offer advantages such as ease of construction, chemical stability, and limited sideeffect profile. It is hoped that immunogenicity will be enhanced through improved modifications of the peptide (ie, addition of adjuvant agents and direct modification of the peptide itself) and that peptide vaccines will continue to play a role in the treatment of GI malignancies.

Carcinoembryonic Antigen
Itoh et al used CEA652, a 9-mer peptide derived from the CEA protein. They pulsed patients DCs with CEA652 and then reinfused them along with IFN and TNF. Delayedtype hypersensitivity reactions were found in 2 of 10 patients when challenged with the peptide. These 2 patients had stable disease for 6 and 9 months.79 Morse et al have reported80 and then updated81 a phase I study using ex vivo stimulated DCs with a recombinant fowlpox vector coding for CEA along with the costimulatory molecules B-7, ICAM, and LFA-3 (the same TRICOM combination mentioned earlier). Thus far, 14 patients have been enrolled in 2 cohorts, and 10 of 12 patients who completed the vaccination schedule showed an immune response in CD4+ and CD8+ cells. One patient had a decrease in serum CEA levels and a minor regression in lymph node metastatic disease, and 3 others had stable disease through at least one 3-month cycle of vaccination. Fong and colleagues administered Flt-3 ligandmobilized DCs loaded with the modified CEA peptide CEA610D to patients with CEA-expressing malignancies, predominantly colon cancer.82 The immunizations resulted in 2 of 12 patients with tumor regression, 1 patient with a mixed response, and 2 with stable disease. Carcinoembryonic antigenspecific T cells identified by tetramer analysis were expanded in several patients and correlated with clinical outcome. Finally, Morse and colleagues demonstrated the feasibility of administering CEA peptide and CEA messenger RNAloaded DCs to patients with CEA-expressing malignancies, again predominantly colon cancer.83

Dendritic Cell Vaccines

Dendritic cells are the most potent APCs in the immune system. Immature DCs take up and process antigen in the peripheral circulation, mature to activated DCs, migrate to draining lymph nodes, present antigens on MHC I and II molecules, stimulate T cells, and thereby activate CD8+ cells for an effective immune response (Figures 4 and 5). Dendritic cells are more effective APCs than macrophages and B cells because of their higher expression of MHC molecules and costimulatory molecules as well as their efficient ability to capture and process antigen and migrate to lymph nodes.77 In creating DC vaccines, investigators typically isolate immature DCs from patients peripheral blood, mature those cells in the presence of cytokines (usually GM-CSF and IL-4, as well as tumor necrosis factor [TNF], IL-1, IL-6, and prostaglandin E-2), and then culture or pulse these DCs in the presence of antigen. This loading of DCs with antigen produces mature, antigen-directed DCs, which are then reinfused into patients. This, in turn, it is thought, leads to a T-cellactivated (mainly CD8+) immune response directed at tumor cells. Dendritic cells must be activated before reinfusion, because

MAGE
Another DC vaccine target has been the cancer antigen MAGE. In one study, MAGE was expressed by one third of

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colon cancers.84 In a larger study, MAGE expression was exclusive to the tumor tissue, with 1 of 10 MAGE antigens tested being present on 70 of the 80 samples. MAGE-3 expression was associated with increased metastatic potential.85 Sandanaga et al demonstrated the feasibility and safety of treating patients with GI cancer with MAGE-3pulsed DCs.86 Twelve patients with GI malignancies were treated (6 stomach, 3 esophagus, and 3 colon). Eight of the 12 patients had peripheral blood mononuclear cells leukapheresed and analyzed before and after vaccination (3 did not complete 2 vaccinations, and a leukapheresis was not performed on a fourth). Of those 8, 4 were shown to develop CD8+ T-cell responses. Tumor markers (cancer antigen 19-9, CEA, and squamous cell carcinoma antigen) decreased in 7 of the 12 patients on study, including 3 of the 4 who had detectable CD8+ T-cell responses. Minor tumor regressions were seen in 3 patients, all of whom had a decrease in tumor markers, and 1 who had a detectable T-cell response.86 One final approach used by Nesselhut et al was the culturing of immature DCs with the serum of patients with cancer, along with IL-4 and GM-CSF, with the aim of inducing an antitumor response from circulating tumor cell proteins.90 Twenty-three patients with GI malignancies were treated with this approach; 6 of the 23 had clinical responses.

Summary
The work that is summarized herein represents several decades of research and improved understanding of the immune system and its role in cancer therapy. It is important to note, however, that some of the research reviewed involves small studies in which little if any clinical response is demonstrated. Although negative results from these trials could be attributed to technical or patient selection issues, there also exists the possibility that these vaccines were merely ineffective. However, we are now seeing more vaccine-generated clinical responses in GI cancer, increased recognition of the importance of chemotherapy on immune response, and consistent data from different approaches suggesting that immune response correlates with clinical outcome. Because of a greater understanding and characterization of tumor-associated antigens, as well as development of different types of vaccines, immunotherapy is rapidly emerging as a potential treatment option for GI malignancies. Although the role of immunotherapy in the treatment of GI malignancies is not yet clearly defined, it is our belief that, in the next decade, vaccine approaches in GI cancers will be clinically established.

MUC-1
Clinical trials involving MUC-1pulsed DC vaccines are now under way. In a study still being conducted, 8 patients with resected pancreatic or biliary tumors have been administered MUC-1 peptide pulsed with immature DC in a series of vaccinations. Preclinical data show vaccine administration to be safe, with preliminary evidence of immune activity.87

Mutant p53
Ibrahim et al used mutant p53 as a DC vaccine target. Twenty-four patients with advanced malignancies, including 10 with CRC, were given their DCs cultured in the presence of mutated p53 along with IL-2. Overall progression-free survival and post-vaccination survival were longer than expected (12.5 months and 27.2 months, respectively) in this group of patients despite their advanced disease and poor immune status. Of 20 evaluated patients, 9 showed a statistically significant immune response.88 Inducing an immune response via DC activation is a powerful tool in our growing arsenal of immune therapies. This field within immune therapy is active and growing, with preclinical and phase I, II, and III trials ongoing. Although it remains difficult to imagine how this therapy could be integrated into general clinical oncology practice given the difficulty and expense in treating individual patients in clinical trials, this area of immunotherapy research remains promising and will likely continue to yield scientific insights and clinical benefit.

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As mentioned previously, some investigators have fused DCs with tumor cells to induce an immune response to more than one tumor antigen. Okinaga et al recently reported on their vaccination in 9 patients with GI malignancies using this approach.89 Six of 9 patients had positive DTH skin test results after vaccinations. Five of 9 patients showed clinical response with stable disease at the time the initial results were reported.

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