This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Greten, T. F. Articles by Jaffee, E. M. Search for Related Content PubMed PubMed Citation Articles by Greten, T. F. Articles by Jaffee, E. M.

Cancer Vaccines

From the Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD.

Address reprint requests to Elizabeth M. Jaffee, MD, Department of Oncology, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Ross 350, Baltimore, MD 21205-2196; email ejaffee{at}welchlink.welch.jhu.edu

ABSTRACT

It has been more than 100 years since the first reported attempts to activate a patient's immune system to eradicate developing cancers. Although a few of the subsequent vaccine studies demonstrated clinically significant treatment effects, active immunotherapy has not yet become an established cancer treatment modality. Two recent advances have allowed the design of more specific cancer vaccine approaches: improved molecular biology techniques and a greater understanding of the mechanisms involved in the activation of T cells. These advances have resulted in improved systemic antitumor immune responses in animal models. Because most tumor antigens recognized by T cells are still not known, the tumor cell itself is the best source of immunizing antigens. For this reason, most vaccine approaches currently being tested in the clinics use whole cancer cells that have been genetically modified to express genes that are now known to be critical mediators of immune system activation. In the future, the molecular definition of tumor-specific antigens that are recognized by activated T cells will allow the development of targeted antigen-specific vaccines for the treatment of patients with cancer.

THE CLASSIC CONCEPT of the vaccine derives from the practice of immunizing against infectious agents to prevent disease by generating a humoral immunity. Individuals are immunized against viral or bacterial antigens before they encounter the pathogenic organisms. This strategy became rapidly successful for viruses because viral genes are relatively simple, possessing a limited number of defined antigens. However, in the case of most tumors, there is an unlimited number of potential antigens that can be the target of an immune response. In addition, it is likely that many of these antigens arise during or as a result of the tumorigenesis process. Therefore, when we talk about tumor vaccines, the most common clinical setting is one in which the induction of a systemic immune response by the vaccine occurs subsequent to, rather than before, the antigen insult. Finally, in contrast to prophylactic vaccines, the majority of cancer vaccines aim to induce a cellular, antigen-specific T-cell response.

The first reported cancer vaccine was tested over a century ago by William Coley, a New York surgeon, who used extracts of pyogenic bacteria to stimulate antitumor immune responses.¹ Numerous subsequent clinical trials have been conducted, particularly using irradiated whole tumor cells mixed with bacterial adjuvants such as Calmette-Guérin bacillus or Corynebacterium parvum. Studies of this approach for patients with malignant melanoma and renal cell and colorectal carcinomas have demonstrated small but significant clinical effects.^2-6 These early studies established a basis for using immune modulators in a paracrine fashion to generate antitumor immunity.⁷

ROLE OF T CELLS IN THE ANTITUMOR IMMUNE RESPONSE

Several preclinical studies have demonstrated that activation of both CD4⁺ and CD8⁺ T cells is critical for generating the most potent antitumor immune responses.^8,9 These antigen-specific T-cell responses are initiated by professional antigen-presenting cells (APCs) (Fig 1). Any protein in the tumor cell is a potential tumor antigen. These antigens are released by secretion, shedding, or tumor lysis and captured by APCs. Uptaken antigens are processed and presented by major histocompatibility complex (MHC) class I and MHC class II molecules for priming and activation of CD8⁺ and CD4⁺ T cells, respectively. Eight– to 10–amino acid peptide fragments are presented on MHC class I molecules, whereas peptides presented on class II molecules are between 12 and 20 amino acids long.¹⁰ Activated CD4⁺ T cells provide important costimulation via cytokine secretion, which can initiate and also amplify the CD8⁺ T-cell response. In addition, memory CD4⁺ T cells play a critical role in maintaining the protective immunity.^11,12 Ultimately, activated antigen-specific CD8⁺ T cells become cytotoxic and lyse tumor cells.¹³

View larger version (44K): [in this window] [in a new window]   Fig 1. Tumor cells present their antigens on the surface to CD8+ T cells or release the antigen so that it can be taken up by professional APCs. These cells process the antigen and present it to CD4+ and CD8+ T cells by a mechanism called cross-priming. CD4+ T cells provide cytokine help for CD8+ T cells.

If T cells have the ability to recognize and lyse tumors, then why doesn't the immune system naturally eradicate developing cancers? This question has perplexed tumor immunologists for years. It is now becoming clear that when the immune system encounters a new antigen, which in the case of tumor antigens might have arisen during the tumorigenesis process, the outcome may be tolerance rather than activation.¹⁴ The context in which the antigen is presented to the immune system seems to determine whether or not a T cell becomes activated.^15,16 In the absence of appropriate costimulatory signals, engagement of the T-cell receptor itself can lead to ignorance, anergy, or even apoptotic death of the T cell (Fig 2), scenarios that are the complete opposite of what one might wish for tumor antigen-specific T cells. There are many examples of these mechanisms of tolerance in animal tumor models.^17-19

View larger version (41K): [in this window] [in a new window]   Fig 2. T-cell tolerance occurs when tumor cells present tumor antigens on MHC molecules (1) to T cells without a second costimulatory signal (2). Genetically engineered tumor cells express either costimulatory molecules on their cell surface (A) to activate T cells directly or release cytokines (B) to attract professional APCs which subsequently activate antigen-specific T cells.

To overcome these mechanisms of tolerance, different vaccine approaches have been developed that use tumor cells (tumor cells provide the antigen source because most tumor antigens are still not known) that have been genetically modified to either express the critical surface costimulatory molecules (such as B7 and intercellular adhesion molecule) required for direct T-cell activation or express costimulatory cytokines that attract and activate bone marrow–derived professional APCs, which can themselves effectively process and present tumor antigens as well as constitutively express the appropriate surface costimulatory molecules required for T-cell activation (Fig 2). There are successful examples of both approaches in preclinical models, and both strategies are currently being tested in clinical trials (see Specific Vaccine Strategies, below). However, the recent advances in our understanding of the mechanism of T-cell activation strongly favor exploiting professional APCs because this strategy may be the most natural way of activating potent and long-lasting antitumor immunity.

In animal models, it has been shown that bone marrow–derived APCs are indeed able to endocytose tumor antigens and present them not only to CD4⁺ T cells but also to CD8⁺ T cells, a process called "cross-priming".²⁰ There is also evidence from in vitro experiments that dendritic cells efficiently present antigen derived from apoptotic cells stimulating class I–restricted CD8⁺ cytotoxic T cells (CTLs).²¹ The ability to target antigens to the MHC class I and class II pathway by a single APC, thereby presenting antigenic epitopes to both CD4⁺ and CD8⁺ T cells, seems to facilitate the provision of CD4⁺ T-cell "help" to CD8⁺ T cells. This occurs via CD4⁺ T cell–derived lymphokines and also through the APC itself via the interaction of CD40 ligand on T cells and CD40 on APCs.^22-24

The cross-priming mechanism suggests that the vaccinating tumor cells do not need to be HLA compatible with the host to generate specific antitumor immunity, thereby providing rationale for a more generalized and feasible whole-cell tumor vaccine approach. Two recent reports using murine cancer models, one evaluating an allogeneic tumor vaccine in which the antigen is not known²⁵ and one evaluating an allogeneic tumor vaccine that expresses the human papilloma virus (HPV) antigen E7,²⁶ support the feasibility of an allogeneic vaccine approach. Clinical trials have recently been initiated to test cytokine-secreting allogeneic vaccines in patients with various cancers.

TUMOR ANTIGENS RECOGNIZED BY T CELLS

Techniques are now available to molecularly define tumor-specific antigens that serve as T-cell targets (Fig 3). As a result, the field of tumor antigen identification has exploded. Eventually, the entire spectrum of tumor-specific antigens capable of being recognized by T cells should be identified. Identification of these tumor antigens that are T-cell targets provides the basis for antigen-specific immunotherapy.

View larger version (29K): [in this window] [in a new window]   Fig 3. Diagram shows the genetic and biochemical approaches currently used to identify MHC class I tumor antigens. Antigen-specific T cells are used to screen COS cells expressing a tumor-derived cDNA library (genetic approach) or peptides eluted from the tumor cells pulsed onto APCs (biochemical approach).

Two strategies, a genetic approach and a biochemical approach, are currently used to identify MHC class I-restricted tumor antigens. Both of these methods require the availability of tumor-specific MHC class I–restricted T-cell lines or clones to provide a readout of T-cell recognition of antigen (which can be either the measurement of target-cell lysis or cytokine release). Unlike strategies that search for evidence of immune responses to candidate antigens, such as the products of mutated oncogenes or tumor suppressor genes, these methods do not limit the search, but rather attempt to define the actual antigen recognized by CD8⁺ T cells obtained from patients. The genetic approach has been successful in identifying two murine tumor antigens^27,28 and over 10 human melanoma antigens.²⁹ The biochemical approach has been successfully adapted to identify two murine tumor antigens and one human melanoma antigen.^30-32 Recently, a third approach for identifying CTL target antigens, which uses the serum from patients with cancer to screen cDNA libraries generated from tumor cells, has been described.^33-37 This method has already identified two melanoma antigens (melanoma antigen 1 [MAGE-1] and tyrosinase), both originally identified by cloning the epitopes recognized by CTL, as well as a novel human esophageal cancer–associated antigen (NY-ESO-1). Identification of T-cell antigens with this method depends on high-titer antitumor immunoglobulin G antibody in the patient's serum. The development of such high-titer antibodies, however, requires the help of antigen-specific CD4⁺ T cells. In addition, a classical biochemical approach for protein purification has led to the identification of one murine MHC class II antigen.³⁸

Table 1 lists the categories of antigens that are potential in vivo tumor-associated antigens recognized by T cells. Examples of antigens in each category have already been identified. Malignant melanoma-associated antigens form the largest database of identified human tumor antigens recognized by T cells. So far, melanoma antigens fall into three of these categories. Currently, the largest group are the tissue-specific differentiation antigens. The identification of differentiation antigens, antigens that are present on normal melanocytes as well as on melanoma cells, was unexpected, because it was previously thought that T cells specific for a self-antigen would be deleted or at least functionally tolerized. The first differentiation antigen that was identified was tyrosinase, a central enzyme of melanin synthesis.³⁹ Others have subsequently been identified, including MART-1/Melan A,^40,41 gp100,^30,42 TRP-1,⁴³ and TRP-2.⁴⁴ These tissue-specific antigens are shared common antigens overexpressed by about 50% of malignant melanomas tested, making them excellent targets of immunization. Many of the identified antigenic peptides that derive from these proteins are HLA-A2 restricted^45,46 and are currently undergoing clinical testing in the first generation of antigen-specific vaccine studies.

Another important category of commonly expressed potential tumor antigens is represented by viral gene products. It is now known that a number of common human malignancies have viral associations and are indeed characterized by the expression of specific viral gene products. These include the Epstein-Barr virus gene products Epstein-Barr nuclear antigen 1, late membrane protein expressed by nasopharyngeal cancers and some Burkitt's and Hodgkin's lymphomas,⁵⁵ HPV E6 and E7 gene products expressed by cervical cancers,⁵⁶ and hepatitis B virus gene products associated with some hepatomas.⁵⁷ Under certain circumstances, both T-cell and antibody responses against these gene products can be identified.

So far, only one human MHC class II antigen has been identified by techniques that directly use CD4⁺ T cells. The antigen tyrosinase, which is expressed by more than 50% of human melanomas and encodes for MHC class I-bound epitopes, has been shown to also encode for two HLA-DR4–restricted epitopes.⁵⁸ Improved MHC class II antigen isolation methods are needed to facilitate the identification of other human MHC class II antigens. However, there is currently intense debate in the tumor immunology community as to whether the introduction of tumor-associated class II antigens to antigen-specific cancer vaccines will enhance the generation of antitumor immunity.

Significant progress has been made during this decade toward identifying human tumor antigens that are targets of T cells. However, this progress has also raised an important question: Which of the many identified antigens can serve as tumor-rejection antigens in patients? Specifically, the biologic roles of these antigens and their corresponding epitopes in the activation or tolerance induction of T cells and the in vivo rejection of tumors are still not clear. There is now evidence demonstrating that different antigenic epitopes that derive from the same cellular protein are differentially exposed or hidden from the immune system. Different mechanisms that define the degree of exposure include proteolytic processing, MHC binding on the side of the APC, and T-cell receptor interactions and the T-cell repertoire on the T-cell side. Because many tumor antigens arise endogenously and therefore are a form of self-antigen, those antigens that are most exposed to the immune system may induce the most profound tolerance, which would make them poor tumor-rejection antigens. Therefore, the most likely tumor antigens against which therapeutically relevant immune responses can be induced may be antigens that contain subdominant or cryptic epitopes. Their partially hidden nature may allow T cells specific for them to escape the most stringent forms of tolerance induction, such as anergy or deletion. If this hypothesis is shown to be true, it should even be possible to induce antitumor immunity against the large number of identified nonmutated melanoma antigens, if they are overexpressed and contain hidden epitopes. Thus, knowledge of the most immunologically relevant tumor antigens will have critical implications for the successful development of antigen-specific tumor vaccines.

PRINCIPLES OF CURRENT CANCER VACCINE STRATEGIES

Vaccines That Directly Activate T Cells
One approach to inducing tumor-specific immune responses is to genetically modify tumor cells to express MHC molecules and costimulatory molecules on their cell surface that are critical for T-cell activation. Modification of the vaccinating tumor cell in this way enhances the tumor's ability to directly stimulate T cells. For example, the B7 family of costimulatory molecules, originally described as an activation antigen on B cells, is constitutively expressed by most APCs and is now known to be the ligand for two receptors expressed on T cells. These are CD28 and the CTL-associated antigen-4.^59-64 CD28 is a critical receptor for generating the costimulatory signals in T-cell activation. Cross-linking of CD28 has been shown to enhance the level of lymphokine production by CD4⁺ T cells subsequent to antigen recognition. In animal studies, transfection of B7.1 into some tumors results in rejection of that tumor and generates systemic immunity against wild-type tumor challenges.⁶⁵ In other studies, cotransfection of B7.1 with either MHC class II,⁶⁴ interleukin (IL)-4,⁶⁶ or IL-7⁶⁷ is required to induce potent systemic immunity. The modification of tumor cells to function as professional APCs that directly activate T cells can be accomplished by both in vitro and in vivo gene-transfer strategies. This is also true for cancer vaccine approaches that manipulate professional APCs to enhance tumor-specific T-cell activation.

Cancer Vaccines That Attract Professional APCs as Facilitators of the Antitumor Immune Response
A second approach to inducing tumor-specific immune responses is to target the immunizing antigen to professional APCs, which can process and present the antigenic peptide to the T cell in the presence of constitutively expressed costimulatory molecules.⁶⁸ Depending on the type of vaccine, targeting of antigens to APCs can be accomplished through a number of different pathways. One central pathway for targeting of vaccinating antigens into bone marrow–derived APCs is the exogenous pathway of antigen uptake. The success of this pathway, operative in most cell-based as well as protein-based vaccines, depends largely upon the adjuvant with which the tumor cell or tumor antigen(s) is formulated. The adjuvant is a critical component of a vaccine. It is the vaccine component that attracts APCs to the sight of vaccination, where they then become activated and take up antigen for processing and presentation. Although extracts of pyogenic bacteria were used more than 100 years ago, the enhanced understanding of how particular cytokines and other signals mediate differentiation of APCs has led to the design of cancer vaccines that can activate more potent and qualitatively distinct immune responses.

Another pathway for introduction of antigens into appropriate bone marrow–derived APCs is via direct transduction. This can be achieved by certain recombinant bacterial and viral vaccines as well as nucleic acid vaccines, so-called "naked DNA" vaccines. In addition, antigenic peptides can be loaded directly onto empty MHC molecules on the surface of APCs either ex vivo or in vivo, thereby bypassing the processing steps.

Antibody-Directed Vaccines
Apart from direct treatment of cancer with tumor-specific antibodies that are often used to localize various toxins at the tumor site, different active vaccination strategies are being investigated to induce tumor-specific antibody responses. For example, vaccine strategies for the treatment of melanoma were tested using either GM2-expressing melanoma cells or vaccines containing GM2 conjugated to keyhole limpet hemocyanin. These studies demonstrated that vaccines containing purified GM2 ganglioside result in induction of GM2 antibodies, and high titers of GM2 antibodies were correlated with increased survival.^69,70 One idiotypic vaccine study, which involved the immunization of non-Hodgkin's B-cell lymphoma patients with the unique idiotype of the immunoglobulin expressed on the surface of their tumors, demonstrated a clinical benefit for patients with recurrent B-cell lymphomas.⁷¹ Furthermore, in a preclinical study, an idiotypic immunization with an antibody specific for mutated p53 prevented the development of a p53 mutation–bearing tumor cell line in mice.⁷² These encouraging results strongly support the further clinical development of these approaches.

SPECIFIC VACCINE STRATEGIES

Cell-Based Cancer Vaccines
Until the tumor antigens expressed by most tumors are identified, the tumor cell itself will continue to be the best source of immunizing antigens. With the development of improved genetic techniques, the concept of presenting immunologically defined "adjuvants" at the same site as tumor antigens in order to augment antitumor immunity has been tested more directly. In animal models, gene transfer of genes encoding MHC molecules, costimulatory molecules, and cytokines has been studied.^63-65 Early animal studies evaluated the immune effects of enhanced expression of both autologous and allogeneic MHC class I molecules on the tumor cell surface. Both ex vivo and in vivo gene transfers of these molecules to the tumor have been studied and have met with some success.^73-75 Similar vaccine strategies are undergoing clinical testing (discussed below).

Genes that encode cytokines are the most common types of genes that have been introduced into tumor cells. The cytokine is produced at very high concentrations at the vicinity of the tumor, thereby altering the local immunologic environment of the tumor cell so as to either enhance presentation of tumor-specific antigens to APCs or to enhance the activation of tumor-specific lymphocytes.⁷ Many cytokine genes have been introduced into tumor cells with varying effects on both tumorigenicity and immunogenicity.^8,9,76-82 Table 2 summarizes the most common cytokines that have been evaluated using this approach and describes the proposed immune mechanisms of tumor rejection generated by these cytokines. Varying results have been reported for certain cytokines, undoubtedly because of the large variations in other critical parameters of immunization. The level of cytokine expression, location of immunization, and challenge site are crucial parameters affecting vaccine efficacy for any form of genetically engineered tumor vaccine. Many of these cytokines have been studied as single cytokines in one or more tumor vaccine models. However, for a human tumor vaccine to be developed, it is critical that these cytokines be compared head to head to determine which cytokine or cytokines are most effective. Also, given that most mouse tumors show significant immunogenicity when simply irradiated, identification of genes that truly enhance the tumor's immunogenicity significantly above that of irradiated wild-type tumor cells is important. Only one study has directly compared multiple cytokines and other genes in murine tumor models.⁸ This study demonstrated, in a number of poorly and moderately immunogenic tumors, that immunization with the tumors transduced with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produced the greatest degree of systemic immunity, which was enhanced relative to irradiated nontransduced tumor cells. In vivo depletion of T-cell subsets demonstrated that this immunity was dependent on both CD4⁺ and CD8⁺ T cells, despite the fact that the tumors did not express MHC class II antigens. Importantly, tumor cells genetically altered to express GM-CSF were able to cure mice of pre-established small burdens of tumor.

Development and Testing of Human Gene–Transduced Tumor Vaccines
A number of critical immunologic concerns require consideration before human gene–transduced tumor vaccines can be developed. First, human tumors are heterogeneous and therefore probably express many potential tumor antigens. Therefore, one important question to consider is what is the best source of tumor cells that will express these antigens. The best source may be the primary tumor rather than a metastasis, because metastases likely develop from only one or a few clones of cells that arise from the primary tumor. If so, for treatment with an autologous vaccine approach, each patient will require surgical resection of their primary tumor, followed by in vitro expansion and genetic modification to express costimulatory molecules or cytokines. A completed phase I clinical trial evaluating an autologous GM-CSF–secreting tumor vaccine for the treatment of metastatic renal cell carcinoma showed promising results but uncovered a number of technical problems, including the requirement of labor-intensive procedures for production of an individualized vaccine and the difficulty of routinely expanding most primary tumors to the high numbers required for vaccination.⁸³ These technical limitations might be avoided by using cytokine-expressing bystander cells⁸⁴ or cytokine-secreting allogeneic vaccine cells that express shared tumor antigens.^25,26

A significant number of clinical studies aimed at augmenting antitumor immune responses against several different cancers have already been completed (Table 3 and the National Cancer Institute homepage for ongoing clinical trials [http://www.nci.nih.gov]). The main aim of these studies has been to demonstrate the safety of the genetically modified vaccine. These initial studies have demonstrated that genetically modified tumor vaccines are extremely safe.^83,85-89 In fact, the main side effect that has been described is self-limited grade I/II local inflammatory skin changes consisting of erythema and induration at the site of vaccination. The results of a few of these studies have been optimistic in that they have provided evidence of immune priming as measured by delayed-type hypersensitivity (DTH) reactions to autologous tumor cells.^83,85-89 In one study, postvaccination T-cell responses against autologous tumor- and melanoma-specific peptides were observed in vitro. In this same study, an increase in the postvaccination tumor-specific T-cell precursor frequency was also demonstrated.⁸⁸ In another study, antimelanoma immunoglobulin G antibody responses were correlated with tumor regression.⁸⁹ In a few studies, DTH responses against autologous tumor correlated with regression of distant metastases.^83,85,86 In one study, histologic analysis of the vaccine and autologous tumor DTH sites demonstrated immune infiltrates similar to what was seen in animal model testing of the vaccine.⁸³ These findings suggest a similar mechanism of immune activation against the human tumor as was seen for the murine tumors.

Antigen-Specific Cancer Vaccines
Several antigen-based vaccine strategies have already demonstrated their ability to augment antitumor immune responses in animal models and some are currently undergoing clinical testing. These strategies can be divided into four categories: (1) plasmid DNA–based vaccines that deliver the gene encoding the antigen; (2) recombinant viral and bacterial vaccines; (3) peptide- or protein-based vaccines that deliver the antigen mixed with adjuvants; and (4) antigen-pulsed dendritic cell vaccines.

DNA-Based Vaccines
There has been a renewed enthusiasm to study DNA vaccines because of the surprising finding that plasmid DNA is taken up and expressed by cells in vivo with much greater efficiency than was previously predicted. DNA vaccines are easy to produce in large quantities, thereby providing an advantage over other vaccine strategies. From an immunologic perspective, the unique ability of DNA to either integrate stably into the host's genome or be maintained in an episomal form long-term provides an opportunity for expressing the immunizing antigen for an extended period of time. Successful vaccination using naked DNA against a viral antigen⁹⁰ has encouraged an intense effort to explore the possibility of naked DNA as cancer vaccines. Specifically, DNA injection was shown to mediate antibody and cellular immune responses in mice.^91,92 DNA vaccines are already entering clinical studies for vaccination against infectious diseases⁹³ and are currently undergoing rigorous testing in animal tumor models. The exact mechanism by which DNA vaccines induce antitumor immunity has not yet been determined. Several studies suggest that immunity is generated by DNA transfection of muscle cells and keratinocytes.⁹⁴ More recent studies suggest that bone marrow–derived APCs also play a critical role in inducing antigen-specific immunity.^95,96

Recombinant Viral and Bacterial Vaccines
With the current knowledge of augmenting antitumor immune responses, the ideal antigen delivery vector should (1) directly infect APCs in vivo and (2) facilitate antigen delivery to both MHC class I and II antigen–processing pathways. Several recombinant viral vectors are currently undergoing rigorous testing for their ability to augment antitumor immune responses against model tumor antigens. Of particular interest are vaccinia and other pox viruses and Listeria monocytogenes.

Poxviruses are attractive candidates for the expression of tumor-associated antigens because heterologous proteins are delivered to the cytoplasm and, therefore, are directly targeted to the compartment in which processing of MHC class I antigens is initiated for presentation to CD8⁺ CTLs. Restifo et al have published several studies demonstrating the generation of antigen-specific immunity resulting in the protection against tumor challenges, using vaccinia and fowlpox constructs.^97-101 Wu et al more recently demonstrated the enhanced potency of a vaccinia vector carrying the HPV gene E7 fused with the LAMP1 gene, which targets E7 to the MHC class II antigen–processing pathway for presentation to CD4⁺ T cells.^102,103 In one study, the cure of a significant tumor burden was demonstrated.¹⁰³ A recombinant E6- and E7-expressing vaccinia virus has already been tested in eight patients with late-stage cervical cancer. No significant side effects were described. An HPV-specific CTL response was observed in one of the eight treated patients. However, all eight patients mounted an antivaccinia antibody response.¹⁰⁴ These neutralizing antivirus antibodies represent one of the major barriers to the use of viral cancer vaccines, which can be the result of either previous immunizations (as in the case with vaccinia) or exposure to cross-reactive viruses (as in the case with adenoviral vaccines). It is unlikely that recombinant viral vaccines such as vaccinia will reach broad clinical application until methods have been developed to eliminate neutralizing antibodies or new viral vectors are identified that do not induce significant antibody responses.

L monocytogenes is a novel tumor antigen delivery vector that is particularly interesting because of its two-phase intracellular life cycle,¹⁰⁵ which enables it to deliver antigens to the class I and class II pathways. Recent data have demonstrated the efficacy of using L monocytogenes as a live recombinant vaccine that is sufficiently potent to cause regression of established macrometastases.¹⁰⁶

Protein- and Peptide-Based Vaccines
The aim of protein- and peptide-based vaccine strategies is to administer high doses of peptide that can be loaded onto empty MHC molecules on APCs in vivo. One major advantage of peptide vaccination is that it removes the safety concerns associated with using live recombinant vaccines or DNA vectors. Whereas vaccination with some tumor-associated peptides and proteins has induced systemic antitumor immunity,^107,108 the administration of other peptides has led to the induction of tolerance rather than activation.¹⁰⁹

Phase I clinical studies of peptide vaccines have been initiated for the treatment of melanoma. As for whole-cell vaccines, the only toxicities observed have been grade I/II induration and erythema at the vaccine site. One trial of an HLA-A1–restricted MAGE3 peptide mixed with incomplete Freund's adjuvant (IFA) reported three partial remissions in 16 patients with metastatic melanoma at pulmonary and subcutaneous sites.¹¹⁰ In another study, all three patients with metastatic melanoma demonstrated partial remissions that were associated with DTH reactivity at the vaccine sites after vaccination with three melanoma-associated HLA-A2–restricted antigenic peptides (MART-1/Melan A, tyrosinase, and gp100/Pmel 17) given simultaneously with systemic GM-CSF.¹¹¹ A more recent study evaluated an HLA-A2–restricted gp100 peptide analog, which was modified at one of the MHC anchor residues (ThrMet at position 2) to improve its binding affinity to HLA-A2, in patients with melanoma.¹¹² Nine patients receiving the unmodified gp100 peptide mixed with IFA were compared with 11 patients treated with the anchor-modified peptide in IFA and 31 patients treated with the anchor-modified peptide in IFA plus systemic IL-2. Immunization with the anchor-modified peptide in IFA induced gp100– and HLA-A2–restricted melanoma CTLs in 10 of 11 patients. Similar in vitro responses were seen in 16% of patients who received the anchor-modified peptide in IFA together with systemic IL-2. Although clinical responses were not observed in patients who received peptide plus IFA without concomitant IL-2, there was a 42% response rate (1 complete remission, 12 partial remissions) among patients who received the same vaccine along with systemic IL-2.¹¹³ It is not clear from this initial study whether the peptide and IFA vaccination contributed to the clinical responses because an IL-2–only treatment group was not included. Furthermore, prospective randomized clinical studies comparing vaccinated patients with unvaccinated patients are needed to determine whether the responses seen in phase I testing are due to the vaccine or the natural biology of malignant melanoma.

A novel peptide vaccine approach uses heat shock proteins (HSPs). HSPs, which are found in most cells and can bind a wide variety of peptides, are being tested as natural adjuvants for their ability to elicit antitumor immune responses. Immunization with tumor-derived HSP96, an endoplasmic reticulum resident, and HSP70, a cytosolic HSP, generates tumor-specific antitumor immunity in mice.¹¹⁴ This tumor-specific immunity is mediated by specific peptides, which are found bound to HSPs.¹¹⁵ It is postulated that HSPs induce a specific CTL response by introducing antigens into the MHC class I and II pathways as well as by binding to macrophages, thereby inducing the production of proinflammatory cytokines.

Dendritic Cell Vaccines
Dendritic cells, which were originally isolated and characterized by Steinman and Cohn in 1973,¹¹⁶ are currently the most potent APCs that have been identified. Activated bone marrow–derived dendritic cells express high levels of MHC class I and II molecules on their cell surface as well as intercellular adhesion molecule and B7, two important adhesion and costimulatory molecules required for T-cell activation. Recently, several dendritic cell growth factors have been identified that have facilitated their in vitro expansion and activation, allowing for the in vitro priming of dendritic cells with specific antigens.^117-123 On the basis of these findings, several approaches using dendritic cells have been tested in animal models, including dendritic cells loaded in vitro with peptides^124,125 or protein,¹²⁶ dendritic cells fused with whole tumor cells,¹²⁷ and ex vivo transduced dendritic cells.^128-130 Most of these studies have demonstrated rejection of significant tumor burdens. The first clinical trial testing peptide– and tumor lysate–pulsed dendritic cells in 16 patients with metastatic melanoma has recently been completed.¹³¹ Regression of metastases in various organs (skin, soft tissue, lung, and pancreas) was observed in five patients. No physical signs of autoimmunity were detected and the vaccinations were well tolerated. Another clinical trial used dendritic cells pulsed with tumor-specific idiotype protein for patients with follicular B-cell lymphoma.¹³² All four patients in this study developed a measurable immune response, and one patient experienced tumor regression.

OUTLOOK

Recent advances have made possible the design of recombinant vaccines that more effectively augment antitumor immune responses in preclinical models. Many of these strategies are already undergoing initial clinical testing. The availability of tumor antigens defined at the genetic level will provide new opportunities for recombinant vaccine design. However, several issues require consideration during the early phases of clinical testing. First, although many promising vaccine strategies have been demonstrated in animal models, it seems inefficient and a waste of limited resources to rigorously test each one separately in patients. Most of these vaccines are expensive to produce and require labor-intense development for scale-up to treat large numbers of patients. A head-to-head comparison of vaccine strategies demonstrated to be most potent in animal models would help to limit the focus. Similarly, as has been demonstrated for melanoma, many tumor antigens can serve as T-cell targets expressed by tumors. Yet, it is a time-consuming process to test each antigen for its potential as an immunogen in patients with cancer. Preclinical models are needed to determine which antigens require expedited testing in clinical trials. Second, the optimal dose, route, and schedule of administration for each vaccine strategy is usually not known because it is difficult to address these questions in animal models in which the doubling time of a growing tumor mass is 48 to 72 hours. Therefore, these questions must be addressed in early clinical trials.

Several hurdles must be overcome. First, it has been demonstrated that cancer cells can undergo genetic alterations that result in their ability to evade immunologic recognition and eradication, either because of loss of antigen expression or loss of the ability to present the antigen to T cells. One possible solution to this problem is to design polyvalent vaccines that target immunity against several antigens. A second possibility is to treat patients with early stages of disease before the tumor population has a chance to undergo significant mutations. A third possibility is to combine vaccines with other cancer treatment modalities. Second, the identification of tissue-specific antigens as a major category of tumor antigens that serve as T-cell targets has generated significant concern that vaccination might result in significant autoimmune toxicities. However, as we learn more about the mechanisms of immune activation and regulation, it should be possible to incorporate strategies for immune regulation into the overall cancer vaccine treatment plan.

Although caution is needed, there are many promising vaccine approaches that require testing. Ultimately, the development of multiple strategies that can be applied in synergy will most likely be responsible for the major advances against cancer. It is too early to see what place immunotherapy in general will have in the fight against cancer. However, it is clear that it is now feasible to design vaccines that are based on rational immunologic principles.

ACKNOWLEDGMENTS

Supported by grant no. CA 62924 from National Cancer Institutes Gastrointestinal Specialized Programs of Research Excellence.

REFERENCES

1. Coley WB: The treatment of malignant tumors by repeated inoculations of erysipelas: With a report of ten original cases. Am J Med Sci 105:487-511, 1893

2. Hoover HJ, Surdyke M, Dangel RB, et al: Prospectively randomized trial of adjuvant active-specific immunotherapy for human colorectal cancer. Cancer 55:1236-1243, 1985[Medline]

3. Hoover HJ, Brandhorst JS, Peters LC, et al: Adjuvant active specific immunotherapy for human colorectal cancer: 6.5-Year median follow-up of a phase III prospectively randomized trial. J Clin Oncol 11:390-399, 1993[Abstract/Free Full Text]

4. Berd D, Maguire HCJ, McCue P, et al: Treatment of metastatic melanoma with an autologous tumor-cell vaccine: Clinical and immunologic results in 64 patients. J Clin Oncol 8:1858-1867, 1990[Abstract]

5. Lipton A, Harvey HA, Balch CM, et al: Corynebacterium parvum versus Bacille Calmette-Guérin adjuvant immunotherapy of stage II malignant melanoma. J Clin Oncol 9:1151-1156, 1991[Abstract]

6. McCune CS, O'Donnell RW, Marquis DM, et al: Renal cell carcinoma treated by vaccines for active specific immunotherapy: Correlation of survival with skin testing by autologous tumor cell—Bacillus Calmette-Guérin vaccine. Cancer Immunol Immunother 32:62-66, 1990[Medline]

7. Pardoll DM: Paracrine cytokine adjuvants in cancer immunotherapy. Annu Rev Immunol 13:399-415, 1995[Medline]

8. 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[Abstract/Free Full Text]

9. Golumbek PT, Lazenby AJ, Levitsky HI, et al: Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4. Science 254:713-716, 1991[Abstract/Free Full Text]

10. Germain RN: Immunology: The ins and outs of antigen processing and presentation. Nature 322:687-689, 1986[Medline]

11. Swain SL, Bradley LM, Croft M, et al: Helper T-cell subsets: Phenotype, function and the role of lymphokines in regulating their development. Immunol Rev 123:1115-1144, 1991

12. Mackay C: Immunological memory. Adv Immunol 53:217-265, 1993[Medline]

13. Boon T, Cerottini JC, Van den Eynde B, et al: Tumor antigens recognized by T lymphocytes. Annu Rev Immunol 12:337-365, 1994[Medline]

14. Jenkins MK, Schwartz RH: Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 165:302-319, 1987[Abstract/Free Full Text]

15. Schwartz RH: Costimulation of T lymphocytes: The role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71:1065-1068, 1992[Medline]

16. Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 12:991-1045, 1994[Medline]

17. Staveley-O'Carroll K, Sotomayor E, Montgomery J, et al: Induction of antigen-specific T cell anergy: An early event in the course of tumor progression. Proc Natl Acad Sci U S A 95:1178-1183, 1998[Abstract/Free Full Text]

18. Wick M, Dubey P, Koeppen H, et al: Antigenic cancer cells grow progressively in immune hosts without evidence for T cell exhaustion or systemic anergy. J Exp Med 186:229-238, 1997[Abstract/Free Full Text]

19. Speiser DE, Miranda R, Zakarian A, et al: Self antigens expressed by solid tumors do not efficiently stimulate naive or activated T cells: Implications for immunotherapy. J Exp Med 186:645-653, 1997[Abstract/Free Full Text]

20. Huang AY, Golumbek P, Ahmadzadeh M, et al: Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science 264:961-965, 1994[Abstract/Free Full Text]

21. Albert ML, Sauter B, Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells and induce class I restricted CTLs. Nature 392:86-89, 1998[Medline]

22. Schoenberger SP, Toes RE, van der Voort EI, et al: T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480-483, 1998[Medline]

23. Ridge JP, Di Rosa F, Matzinger P: A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474-478, 1998[Medline]

24. Bennett SR, Carbone FR, Karamalis F, et al: Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478-480, 1998[Medline]

25. Thomas MC, Greten TF, Pardoll DM, et al: Enhanced tumor protection by granulocyte-macrophage colony-stimulating factor expression at the site of an allogeneic vaccine. Hum Gene Ther 9:835-843, 1998[Medline]

26. Toes RE, Blom RJ, van Voort E, et al: Protective antitumor immunity induced by immunization with completely allogeneic tumor cells. Cancer Res 56:3782-3787, 1996[Abstract/Free Full Text]

27. De Plaen E, Lurquin C, Van Pel A, et al: Immunogenic (tum-) variants of mouse tumor P815: Cloning of the gene of tum-antigen P91A and identification of the tum- mutation. Proc Natl Acad Sci U S A 85:2274-2278, 1988[Abstract/Free Full Text]

28. Lurquin C, Van Pel A, Mariame B, et al: Structure of the gene of tum- transplantation antigen P91A: The mutated exon encodes a peptide recognized with Ld by cytolytic T cells. Cell 58:293-303, 1989[Medline]

29. Vandeneynde BJ, Vanderbruggen P: T cell defined tumor antigens. Curr Opin Immunol 9:684-693, 1997[Medline]

30. Cox AL, Skipper J, Chen Y, et al: Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 264:716-719, 1994[Abstract/Free Full Text]

31. Huang AYC, Gulden PH, Woods AS, et al: The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc Natl Acad Sci U S A 93:9730-9735, 1996[Abstract/Free Full Text]

32. Mandelboim O, Berke G, Fridkin M, et al: CTL induction by a tumor-associated antigen octapeptide derived from a murine lung carcinoma. Nature 369:67-71, 1994[Medline]

33. Sahin U, Tureci O, Schmitt H, et al: Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci U S A 92:11810-11813, 1995[Abstract/Free Full Text]

34. Sahin U, Tureci O, Pfreundschuh M: Serological identification of human tumor antigens. Curr Opin Immunol 9:709-716, 1997[Medline]

35. Jager E, Chen YT, Drijfhout JW, et al: Simultaneous humoral and cellular immune response against cancer-testis antigen Ny-Eso-1: Definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J Exp Med 187:265-270, 1998[Abstract/Free Full Text]

36. Chen YT, Scanlan MJ, Sahin U, et al: A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci U S A 94:1914-1918, 1997[Abstract/Free Full Text]

37. Chen YT, Gure AO, Tsang S, et al: Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library. Proc Natl Acad Sci U S A 95:6919-6923, 1998[Abstract/Free Full Text]

38. Monach PA, Meredith SC, Siegel CT, et al: A unique tumor antigen produced by a single amino acid substitution. Immunity 2:45-59, 1995[Medline]

39. Brichard V, Van Pel A, Wolfel T, et al: The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 178:489-495, 1993[Abstract/Free Full Text]

40. Coulie PG, Brichard V, Van Pel A, et al: A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 180:35-42, 1994[Abstract/Free Full Text]

41. Kawakami Y, Eliyahu S, Delgado CH, et al: Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci U S A 91:3515-3519, 1994[Abstract/Free Full Text]

42. Bakker AB, Schreurs MW, de Boer AJ, et al: Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med 179:1005-1009, 1994[Abstract/Free Full Text]

43. Wang RF, Robbins PF, Kawakami Y, et al: Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes [published erratum appears in J Exp Med 181:1261, 1995]. J Exp Med 181:799-804, 1995[Abstract/Free Full Text]

44. Wang RF, Appella E, Kawakami Y, et al: Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J Exp Med 184:2207-2216, 1996[Abstract/Free Full Text]

45. Wolfel T, Van Pel A, Brichard V, et al: Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes. Eur J Immunol 24:759-764, 1994[Medline]

46. Bakker AB, Schreurs MW, Tafazzul G, et al: Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1- restricted anti-melanoma CTL line. Int J Cancer 62:97-102, 1995[Medline]

47. Van der Bruggen P, Traversari C, Chomez P, et al: A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643-1647, 1991[Abstract/Free Full Text]

48. Boel P, Wildmann C, Sensi ML, et al: BAGE: A new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity 2:167-175, 1995[Medline]

49. Van den Eynde B, Peeters O, De Backer O, et al: A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J Exp Med 182:689-698, 1995[Abstract/Free Full Text]

50. Wolfel T, Hauer M, Schneider J, et al: A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269:1281-1284, 1995[Abstract/Free Full Text]

51. Peace DJ, Chen W, Nelson H, et al: T cell recognition of transforming proteins encoded by mutated ras proto-oncogenes. J Immunol 146:2059-2065, 1991[Abstract]

52. Jung S, Schluesener HJ: Human T lymphocytes recognize a peptide of single point-mutated, oncogenic ras proteins. J Exp Med 173:273-276, 1991[Abstract/Free Full Text]

53. Fuchs EJ, Bedi A, Jones RJ, et al: Cytotoxic T cells overcome BCR-ABL-mediated resistance to apoptosis. Cancer Res 55:463-469, 1995[Abstract/Free Full Text]

54. Theobald M, Biggs J, Dittmer D, et al: Targeting p53 as a general tumor antigen. Proc Natl Acad Sci U S A 92:11993-11997, 1995[Abstract/Free Full Text]

55. Ambinder RF, Robertson KD, Moore SM, et al: Epstein-Barr virus as a therapeutic target in Hodgkin's disease and nasopharyngeal carcinoma. Semin Cancer Biol 7:217-226, 1996[Medline]

56. Wu TC: Immunology of the human papilloma virus in relation to cancer. Curr Opin Immunol 6:746-754, 1994[Medline]

57. Chang MH, Chen CJ, Lai MS, et al: Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children: Taiwan Childhood Hepatoma Study Group. N Engl J Med 336:1855-1859, 1997[Abstract/Free Full Text]

58. Topalian SL, Rivoltini L, Mancini M, et al: Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proc Natl Acad Sci U S A 91:9461-9465, 1994[Abstract/Free Full Text]

59. Azuma M, Ito D, Yagita H, et al: B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366:76-79, 1993[Medline]

60. Freeman GJ, Borriello F, Hodes RJ, et al: Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice. Science 262:907-909, 1993[Abstract/Free Full Text]

61. Freeman GJ, Gribben JG, Boussiotis VA, et al: Cloning of B7-2: A CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 262:909-911, 1993[Abstract/Free Full Text]

62. Linsley PS, Brady W, Urnes M, et al: CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med 174:561-569, 1991[Abstract/Free Full Text]

63. Chen L, Ashe S, Brady WA, et al: Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 71:1093-1102, 1992[Medline]

64. Baskar S, Ostrand-Rosenberg S, Nabavi N, et al: Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules. Proc Natl Acad Sci U S A 90:5687-5690, 1993[Abstract/Free Full Text]

65. Townsend SE, Allison JP: Tumor rejection after direct costimulation of CD8+ T cells by B7- transfected melanoma cells [see comments]. Science 259:368-370, 1993[Abstract/Free Full Text]

66. Cayeux S, Beck C, Dorken B, et al: Coexpression of interleukin-4 and B7.1 in murine tumor cells leads to improved tumor rejection and vaccine effect compared to single gene transfectants and a classical adjuvant. Hum Gene Ther 7:525-529, 1996[Medline]

67. Cayeux S, Beck C, Aicher A, et al: Tumor cells cotransfected with interleukin-7 and B7.1 genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaccines. Eur J Immunol 25:2325-2331, 1995[Medline]

68. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392:245-252, 1998[Medline]

69. Kitamura K, Livingston PO, Fortunato SR, et al: Serological response patterns of melanoma patients immunized with a GM2 ganglioside conjugate vaccine. Proc Natl Acad Sci U S A 92:2805-2809, 1995[Abstract/Free Full Text]

70. Helling F, Zhang S, Shang A, et al: GM2-KLH conjugate vaccine: Increased immunogenicity in melanoma patients after administration with immunological adjuvant QS-21. Cancer Res 55:2783-2788, 1995[Abstract/Free Full Text]

71. Hsu FJ, Caspar CB, Czerwinski D, et al: Tumor-specific idiotype vaccines in the treatment of patients with B-cell lymphoma: Long-term results of a clinical trial. Blood 89:3129-3135, 1997[Abstract/Free Full Text]

72. Ruiz JP, Wolkowicz R, Waisman A, et al: Idiotypic immunization induces immunity to mutated p53 and tumor rejection. Nat Med 4:710-712, 1998[Medline]

73. Ostrand-Rosenberg S, Roby C, Clements VK, et al: Tumor-specific immunity can be enhanced by transfection of tumor cells with syngeneic MHC-class-II genes or allogeneic MHC-class-I genes. Int J Cancer Suppl 6:61-68, 1991[Medline]

74. Wallich R, Bulbuc N, Hammerling GJ, et al: Abrogation of metastatic properties of tumour cells by de novo expression of H-2K antigens following H-2 gene transfection. Nature 315:301-305, 1985[Medline]

75. Plautz GE, Yang ZY, Wu BY, et al: Immunotherapy of malignancy by in vivo gene transfer into tumors. Proc Natl Acad Sci U S A 90:4645-4649, 1993[Abstract/Free Full Text]

76. Pulaski BA, McAdam AJ, Hutter EK, et al: Interleukin 3 enhances development of tumor-reactive cytotoxic cells by a CD4-dependent mechanism. Cancer Res 53:2112-2117, 1993[Abstract/Free Full Text]

77. Gansbacher B, Zier K, Daniels B, et al: Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity. J Exp Med 172:1217-1224, 1990[Abstract/Free Full Text]

78. Asher M, Mule J, Kasid A: Murine tumor cells transduced with the gene for tumor necrosis factor-a. J Immunol 146:3227-3229, 1991[Abstract]

79. Gansbacher B, Bannerji R, Daniels B: Retroviral vector-mediated gamma-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity. Cancer Res 50:7820-7826, 1990[Abstract/Free Full Text]

80. Hock H, Dorsch M, Diamantstein T, et al: Interleukin 7 induces CD4+ T cell-dependent tumor rejection. J Exp Med 174:1291-1298, 1991[Abstract/Free Full Text]

81. Porgador A, Tzehoval E, Katz A, et al: Interleukin 6 gene transfection into Lewis lung carcinoma tumor cells suppresses the malignant phenotype and confers immunotherapeutic competence against parental metastatic cells. Cancer Res 52:3679-3686, 1992[Abstract/Free Full Text]

82. Blankenstein T, Qin Z, Uberla K: Tumor suppression after tumor cell-targeted tumor necrosis factor via gene transfer. J Exp Med 173:1047-1052, 1991[Abstract/Free Full Text]

83. Simons JW, Jaffee EM, Weber CE, et al: Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer. Cancer Res 57:1537-1546, 1997[Abstract/Free Full Text]

84. Golumbek PT, Azhari R, Jaffee EM, et al: Controlled release, biodegradable cytokine depots: A new approach in cancer vaccine design. Cancer Res 53:1-4, 1993[Abstract/Free Full Text]

85. Lotze MT, Zitvogel L, Campbell R, et al: Cytokine gene therapy of cancer using interleukin-12: Murine and clinical trials. Ann N Y Acad Sci 795:440-454, 1996[Medline]

86. Tepper RI, Mule JJ: Experimental and clinical studies of cytokine gene-modified tumor cells. Hum Gene Ther 5:153-164, 1994[Medline]

87. Cheson BD, Phillips PH, Sznol M: Clinical Trials Referral Resource: Trials using tumor vaccines. Oncology 11:81-82, 84, 90, 1997

88. Arienti F, Sule-Suso J, Belli F, et al: Limited antitumor T cell response in melanoma patients vaccinated with interleukin-2 gene-transduced allogeneic melanoma cells. Hum Gene Ther 7:1955-1963, 1996[Medline]

89. Abdel-Wahab Z, Weltz C, Hester D, et al: A phase I clinical trial of immunotherapy with interferon-gamma gene-modified autologous melanoma cells: Monitoring the humoral immune response. Cancer 80:401-412, 1997[Medline]

90. Wolff JA, Malone RW, Williams P, et al: Direct gene transfer into mouse muscle in vivo. Science 247:1465-1468, 1990[Abstract/Free Full Text]

91. Condon C, Watkins SC, Celluzzi CM, et al: DNA-based immunization by in vivo transfection of dendritic cells. Nat Med 2:1122-1128, 1996[Medline]

92. Tang DC, DeVit M, Johnston SA: Genetic immunization is a simple method for eliciting an immune response. Nature 356:152-154, 1992[Medline]

93. Donnelly JJ, Ulmer JB, Shiver JW, et al: DNA vaccines. Annu Rev Immunol 15:617-648, 1997[Medline]

94. Pardoll DM, Beckerleg AM: Exposing the immunology of naked DNA vaccines. Immunity 3:165-169, 1995[Medline]

95. Doe B, Selby M, Barnett S, et al: Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc Natl Acad Sci U S A 93:8578-8583, 1996[Abstract/Free Full Text]

96. Corr M, Lee DJ, Carson DA, et al: Gene vaccination with naked plasmid DNA: Mechanism of CTL priming. J Exp Med 184:1555-1560, 1996[Abstract/Free Full Text]

97. McCabe B, Irvine K, Nishimura M, et al: Minimal determinant expressed by a recombinant vaccinia virus elicits therapeutic antitumor cytolytic T lymphocyte responses. Cancer Res 55:1741-1757, 1995[Abstract/Free Full Text]

98. Restifo NP, Bacik I, Irvine KR, et al: Antigen processing in vivo and the elicitation of primary CTL responses. J Immunol 154:4414-4422, 1995[Abstract]

99. Irvine K, McCabe B, Rosenberg S, et al: Synthetic oligonucleotide expressed by a recombinant vaccinia virus elicits therapeutic CTL. J Immunol 154:4651-4657, 1995[Abstract]

100. Wang M, Bronte V, Chen PW, et al: Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen. J Immunol 154:4685-4692, 1995[Abstract]

101. Bronte V, Tsung K, Rao JB, et al: IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases. J Immunol 154:5282-5292, 1995[Abstract]

102. Wu TC, Guarnieri FG, Staveley-O'Carroll KF, et al: Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. Proc Natl Acad Sci U S A 92:11671-11675, 1995[Abstract/Free Full Text]

103. Lin KY, Guarnieri FG, Staveley-O'Carroll K, et al: Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 56:21-26, 1996[Abstract/Free Full Text]

104. Borysiewicz LK, Fiander A, Nimako M, et al: A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer [see comments]. Lancet 347:1523-1527, 1996[Medline]

105. Falkow S, Isberg RR, Portnoy DA: The interaction of bacteria with mammalian cells. Annu Rev Cell Biol 8:333-363, 1992

106. Pan ZK, Ikonomidis G, Lazenby A, et al: A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat Med 1:471-477, 1995[Medline]

107. Kast WM, Offringa R, Peters PJ, et al: Eradication of adenovirus E1-induced tumors by E1A-specific cytotoxic T lymphocytes. Cell 59:603-614, 1989[Medline]

108. Mandelboim O, Vadai E, Fridkin M, et al: Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides [see comments]. Nat Med 1:1179-1183, 1995[Medline]

109. Toes RE, Blom RJ, Offringa R, et al: Enhanced tumor outgrowth after peptide vaccination: Functional deletion of tumor-specific CTL induced by peptide vaccination can lead to the inability to reject tumors. J Immunol 156:3911-3918, 1996[Abstract]

110. Marchand M, Weynants P, Rankin E, et al: Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3. Int J Cancer 63:883-885, 1995[Medline]

111. Jager E, Ringhoffer M, Dienes HP, et al: Granulocyte-macrophage-colony-stimulating factor enhances immune responses to melanoma-associated peptides in vivo. Int J Cancer 67:54-62, 1996[Medline]

112. 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[Abstract]

113. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al: Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 4:321-327, 1998[Medline]

114. Tamura Y, Peng P, Liu K, et al: Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 278:117-120, 1997[Abstract/Free Full Text]

115. Blachere NE, Li Z, Chandawarkar RY, et al: Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med 186:1315-1322, 1997[Abstract/Free Full Text]

116. Steinman RM, Cohn ZA: Identification of a novel cell type in peripheral lymphoid organs of mice: I. Morphology, quantitation, tissue distribution. J Exp Med 137:1142-1162, 1973[Abstract]

117. Inaba K, Inaba M, Romani N, et al: Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176:1693-1702, 1992[Abstract/Free Full Text]

118. Caux C, Dezutter-Dambuyant C, Schmitt D, et al: GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 360:258-261, 1992[Medline]

119. Sallusto F, Lanzavecchia A: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necro