Vaccine and gene therapy of renal cell carcinoma

The concept of tumor vaccines is not new. However, advances in gene transfer technology, tumor immunology, molecular biology, and methods of monitoring antitumor response, have allowed for novel, more specific vaccine approaches. For example, first-generation tumor vaccines were composed of whole inactivated cancer cells, or tumor lysates (Tuly) given together with immune adjuvants like bacillus Calmette-Guerin (BCG). Current strategies include tumor cells modified with genes encoding molecules necessary to stimulate a cytotoxic T cell response, such as cytokine genes, foreign HLA genes, tumor-associated antigen (TAA) genes, and even costimulatory molecules. Activation of cellular immunity requires at least three synergistic signals including presentation of specific tumor antigens, costimulatory signals (B7 molecules), and propagation of the immune response via cytokine release. In general, tumor cells often fail to demonstrate any of these immunostimulatory properties. Dendritic cell-based vaccines are gaining popularity as these cells can properly present TAA to the immune system, thus circumventing the poor antigen-presenting qualities of tumor cells. Dendritic cells can be "loaded" with TAA or other molecules either by their natural endocytotic capabilities, or by genetic modification.     

Vaccination with dendritic cells pulsed with apoptotic tumors in combination with anti-OX40 and anti-4-1BB monoclonal antibodies induces T cell-mediated protective immunity in Her-2/neu transgenic mice

Tumor cells express tumor-associated antigens (TAAs), which can serve as targets for the immune system. However, the majority of TAAs are overexpressed products of normal cellular genes; as such, self-tolerance mechanisms have hindered their use for the induction of effective antitumor responses. One such normal self-protein is the growth factor receptor Her-2/neu, which is overexpressed in 25-35% of all mammary carcinomas in humans. In previous studies, we have demonstrated that Her-2/neu mice are functionally tolerant to neu antigens and contain only a low avidity T-cell repertoire to neu antigens. However, this residual low-avidity T-cell repertoire has antitumor activity. In this study, we compared the immune responses of Her-2/neu mice immunized with dendritic cells (DCs) pulsed with soluble neu protein or with apoptotic tumor cells. Analysis of the antitumor response shows that Her-2/neu mice vaccinated with DCs pulsed with Her-2/neu antigens retard tumor growth; however, vaccination with DCs pulsed with apoptotic tumor cells induces a stronger antitumor effect. Administration of multiple immunizations in combination with the costimulatory agonist anti-OX40 or anti-4-1BB MAb significantly enhanced the immune responses in these mice, resulting in complete tumor rejection if the tumor burden was small and substantial tumor reduction with a larger tumor burden. These results have important implications for the design of tumor vaccination strategies, suggesting that the use of vaccines that stimulate a broad immune response in combination with costimulatory molecules as immunomodulators could significantly improve the antitumor immune response in tolerant hosts.     

Pox viral vaccine approaches

Recent advances in understanding tumor-specific immunity have introduced new excitement in the clinical development of vaccines for the treatment of cancer. A better understanding of basic immunologic principles has led to a variety of techniques for enhancing tumor-specific immunity through vaccination. Approaches to antigen-specific immunotherapy have included: (1) peptides, usually in combination with various immunological adjuvants; (2) soluble proteins; (3) dendritic cells pulsed with specific antigens; (4) monoclonal antibodies; (5) recombinant plasmid DNA; (6) autologous and allogeneic tumor cells; and (7) recombinant viral vectors. This review will focus on the use of viral vectors, which offer unique advantages as both gene delivery vectors and as agents supplying additional adjuvant activity for vaccination. Viral vectors are particularly attractive for immunotherapy since they mimic natural infection and can induce potent immune responses. Replicating and nonreplicating members of the poxvirus family have been widely studied for expression of tumor antigens and other immunomodulatory genes, such as cytokines and costimulatory molecules. Although a large number of TAAs are available for insertion into viral vectors, this review will discuss the preclinical and clinical development of prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA) poxviral vaccines, as models of the pox viral vaccine approach.     

Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain

The specificity and potency of the immune system make immunotherapy a potential strategy for the treatment of cancer. To exploit this potential, we have developed cell-based cancer vaccines consisting of tumor cells expressing syngeneic MHC class II and costimulatory molecules. The vaccines mediate tumor regression in mice and activate human CD4+ T cells in vitro. Previous vaccines were generated by transducing MHC II negative tumor cells with a single HLA-DR allele. Because expression of multiple MHC II alleles would facilitate presentation of a broader repertoire of tumor antigens, we have now transduced tumor cells with the MHC class II transactivator (CIITA), a regulatory gene that coordinately increases expression of all MHC II alleles. Previous studies in mice indicated that coexpression of the MHC II accessory molecule invariant chain (Ii) inhibited presentation of endogenously synthesized tumor antigens and reduced vaccine efficacy. To determine if Ii expression affects presentation of MHC class II-restricted endogenously synthesized tumor antigens in human tumor cells, HLA-DR-MCF10 breast cancer cells were transduced with the CIITA, CD80 costimulatory molecule gene, and with or without small interfering RNAs (siRNA) specific for Ii. Ii expression is silenced >95% in CIITA/CD80/siRNA transductants; down-regulation of Ii does not affect HLA-DR expression or stability; and Ii(+) and Ii(-) transductants activate human CD4+ T cells to DRB1*0701-restricted HER-2/neu epitopes. Therefore, tumor cells transduced with the CIITA, CD80, and with or without Ii siRNA present endogenously synthesized tumor antigens and are potential vaccines for activating tumor-specific CD4+ T cells.     

therapeutic vaccines for colorectal cancer

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

Pitfalls or promise in prostate cancer immunotherapy-which is winning?

PURPOSE: Immunotherapy with vaccines, cytokines, and monoclonal antibodies against checkpoint molecules has been introduced into the clinical arena. Although all have demonstrated safety in clinical trials in patients with castrate metastatic prostate cancer, no one approach has been able to provide improved overall survival. This article is a review of the current issues and potential resolutions as to how we might go forward in developing and interpreting immunologic trials. DESIGN: Phase I, II, and III trials showed that immunologic tolerance can be abrogated against specific tumor-associated antigens, but the immunologic readouts are suboptimal in determining whether a trial can go forward in its development. RESULTS: Combinatorial approaches appear to be necessary for inducing immunogenicity and antitumor effects. Strategies include irradiated tumor cells lines, costimulatory molecules, or immune checkpoint inhibitors, which are in trials and are under intense scrutiny as to their impact on clinical end points such as time to disease progression and survival. DISCUSSION: Strategies to enhance immunogenicity of vaccines and reassess how to effectively establish interpretable immunologic end points are under development and appear to be successful in affecting how these trials go forward.     

Cancer vaccines.

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.     

Immunotherapy of murine prostate cancer using whole tumor cells killed ex vivo by herpes simplex viral thymidine kinase/ganciclovir suicide gene therapy

Whole cell cancer vaccines are currently under clinical evaluation. Their immunogenicity may depend on the mode of death of the vaccine cells prior to uptake by professional antigen-presenting cells and crosspriming of T cells. Destruction of tumor in vivo by genetic prodrug activation therapy leads to a marked local and systemic immune response, local T-cell infiltration and the establishment of T-cell memory. We postulated that this immunostimulation may be due to induction of danger signals and the inherent immunogenicity of products of HSVtk/ganciclovir kill. Using established models of murine prostate cancer, we have evaluated the efficacy of anti-tumor vaccines comprising irradiated allogeneic or autologous whole cells expressing HSVtK, which are first killed in vitro by prodrug activation using ganciclovir. HSVtk/ganciclovir-induced cell kill was through the induction of apoptosis. The vaccine was found to be effective in both models and superior to traditional irradiated whole tumor cells even after single doses. Protection against tumor challenge was associated with marked proliferative and Th1 cytokine responses. This approach would be applicable clinically in terms of ease of vaccine production, safety, storage and avoidance of potential toxicities of in vivo gene transfer.     

Challenges to the development of antigen-specific breast cancer vaccines

Continued progress in the development of antigen-specific breast cancer vaccines depends on the identification of appropriate target antigens, the establishment of effective immunization strategies, and the ability to circumvent immune escape mechanisms. Methods such as T cell epitope cloning and serological expression cloning (SEREX) have led to the identification of a number target antigens expressed in breast cancer. Improved immunization strategies, such as using dendritic cells to present tumor-associated antigens to T lymphocytes, have been shown to induce antigen-specific T cell responses in vivo and, in some cases, objective clinical responses. An outcome of successful tumor immunity is the evolution of antigen-loss tumor variants. The development of a polyvalent breast cancer vaccine, directed against a panel of tumor-associated antigens, may counteract this form of immune escape.     

An effective tumor vaccine optimized for costimulation via bispecific and trispecific fusion proteins

T cell costimulation has great therapeutic potential if it can be optimized and controlled. To achieve this, we engineered T cell-activating fusion proteins and immunocytokines that specifically attach to viral antigens of a virus-infected tumor vaccine. We employed the avian Newcastle Disease Virus because this agent is highly efficient for human tumor cell infection, and leads to introduction of viral hemagglutinin-neuraminidase (HN) molecules at the tumor cell surface. Here, we demonstrated the strong potentiation of the T cell stimulatory activity of such a vaccine upon attachment of bispecific or trispecific fusion proteins which bind with one arm to viral HN molecules of the vaccine, and with the other arm either to CD3 (signal 1), to CD28 (costimulatory signal 2a), or to interleukin-2 receptor (costimulatory signal 2b) on T cells. A vaccine with a combination of all three signals triggered the strongest activation of na?ve human T cells, thereby inducing the most durable bystander antitumor activity in vitro. Adoptive transfer of such polyclonally activated cells into immunodeficient mice bearing human breast carcinoma caused tumor regression. Furthermore, tumor-reactive memory T cells from draining lymph nodes of carcinoma patients could be efficiently reactivated in a short-term ELISpot assay using an autologous tumor vaccine with optimized signals 1 and 2, but not with a similarly modified vaccine from an unrelated tumor cell line. Our data describe new bioactive molecules which in combination with an established virus-modified tumor vaccine greatly augments the antitumor activity of T cells from healthy donors and cancer patients.     

Current developments in cancer vaccines and cellular immunotherapy.

This article reviews the immunologic basis of clinical trials that test means of tumor antigen recognition and immune activation, with the goal to provide the clinician with a mechanistic understanding of ongoing cancer vaccine and cellular immunotherapy clinical trials. Multiple novel immunotherapy strategies have reached the stage of testing in clinical trials that were accelerated by recent advances in the characterization of tumor antigens and by a more precise knowledge of the regulation of cell-mediated immune responses. The key steps in the generation of an immune response to cancer cells include loading of tumor antigens onto antigen-presenting cells in vitro or in vivo, presenting antigen in the appropriate immune stimulatory environment, activating cytotoxic lymphocytes, and blocking autoregulatory control mechanisms. This knowledge has opened the door to antigen-specific immunization for cancer using tumor-derived proteins or RNA, or synthetically generated peptide epitopes, RNA, or DNA. The critical step of antigen presentation has been facilitated by the coadministration of powerful immunologic adjuvants, the provision of costimulatory molecules and immune stimulatory cytokines, and the ability to culture dendritic cells. Advances in the understanding of the nature of tumor antigens and their optimal presentation, and in the regulatory mechanisms that govern the immune system, have provided multiple novel immunotherapy intervention strategies that are being tested in clinical trials.     

The first 1000 dendritic cell vaccinees

Dendritic cells (DCs) are potent antigen-presenting cells that have the ability to stimulate primary T cell antitumor immune responses in animals and humans. Since the first published clinical trial of dendritic cell vaccination in 1995, 98 studies describing more than 1000 vaccinees have been published in peer-reviewed medical journals or presented at the annual meetings of the American Society for Clinical Oncology, the American Association of Cancer Research, or the American Society of Hematology. Trials have been performed in 15 countries. Trials included patients with more than two dozen tumor types; most trials studied patients with malignant melanoma, prostate cancer, colorectal carcinoma, or multiple myeloma, using autologous DCs pulsed with synthetic antigens or idiotype antibodies. The DC vaccines were also prepared by pulsing DCs with tumor lysates or RNA, by transfection with tumor DNA, or by creating tumor cell/DC fusions. Various approaches to vaccine cell numbers, length of vaccine program, site of vaccination, frozen preservation of vaccine, and use of a maturation step for DCs were used. Adverse effects associated with DC vaccination were uncommon; most were mild and self-limited and none were serious. Clinical responses were observed in approximately half the trials. The DC vaccination may provide a safe approach to cancer immunotherapy that can overcome the limited reach and immunogenicity of peptide vaccines.     

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models

The ideal cancer vaccine should work regardless of MHC types but currently the barrier generated by MHC specificity hampers the development of human cancer vaccines, requesting to identify strong immunogenic molecules that can induce anti-cancer immune responses without being affected by MHC polymorphism. Tumor-derived exosomes are small membrane vesicles containing tumor antigens as well as other immunologically important molecules such as MHC molecules and heat shock proteins (HSPs). Because of their potential immunogenicity, the plausible utility of tumor-derived exosomes as an MHC independent cancer vaccine was proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce stronger immunogenicity as compared to normal tumor-derived exosomes in autologous as well as allogeneic murine models in vitro and in vivo. Western blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS Exo triggered the increased expressions of MHC class II molecules. Crucially, HS Exo performed greater therapeutic capability in regressing pre-established MHC type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited Th1-polarized immune responses defined by the increased productions of IgG2a and IFN-γ. In summary, the Hsp70-enriched exosomes extracted from heat-treated tumors induced strong Th1 immune responses, resulting in eliminating cancer cells in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC independent cell-free cancer therapeutic agent that can be developed for clinical trials.     

Vaccination against human cancers (review)

Classical and molecular immunological means of active tumor-specific immunization against human cancers yielded whole cell or tumor cell lysate vaccines of preventive value (reduced relapse rates) and dendritic cell-peptide or genetically engineered vaccines that may induce remissions even in metastatic disease. Active tumor-specific immunization was often successful in the past 50 years against experimental tumors maintained in the laboratory. During the epochs of classical and molecular immunology several vaccines were generated and used for the reduction of relapse rates of human cancer after surgical removal of the primary or metastatic tumors. Whole cell vaccines consist of X-irradiated autologous or allogeneic tumor cells coadministered with immunostimulants (BCG, Detox). Tumor cells haptenized biologically (as in viral oncolysates) or chemically were also used. Dendritic cell vaccines are prepared by transfection or transduction with tumor antigen-encoding DNA or by pulsing the cells with antigenic peptides in vitro; or collecting dendritic cells that engulfed apoptotic tumor cell DNA and/or peptide antigens in vivo for reinjection into the patient. Genetically engineered tumor cells are prepared in vitro to express MHC and peptides, costimulatory molecules (B7.1) and cyto- or lymphokines (interferons, interleukins, hematopoietic growth factors) for vaccination of patients. Antibody- and immune T cell-mediated immune reactions to autologous tumor cells are newly generated and/or quantitatively increased in immunized patients but do not always correlate with clinical response. Most vaccines are claimed to have reduced relapse rates presumably by inducing effective host immunity against micrometastases. Dendritic cell-peptide vaccines could induce partial or occasionally complete remissions in metastatic disease. The wrong antigenic presentation may result in tolerance induction toward the tumor; occasionally tumor enhancement may occur. Human tumor antigens when presented appropriately (with costimulatory molecules and with IL-2, IL-12) break the host's natural tolerance toward its tumor and induce rejection strength immune reactions even in patients with metastatic disease. Immune T cells thus generated could be collected for adoptive immunotherapy. For successful active specific immunization against human cancers the understanding of the immunoevasive maneuvers of the tumor cell (through FasL --> Fas; TRAIL; CD40L --> CD40; TGFbeta etc. systems) is essential.     

Targeted cancer therapy with gonadotropin-releasing hormone chimeric proteins

Tumor-associated antigens (TAAs) have been identified mainly to determine cancer prognosis. In the past few years, TAAs have been used in the development of treatment modalities such as tumor vaccination. This review describes an additional application of TAAs: as a target for specific antitumor treatment. Since TAAs are overexpressed on the tumor cell surface, they can be targeted to deliver drugs directly to cancer cells. One such delivery system exploits chimeric proteins. Chimeric proteins are a class of targeted molecules designed to recognize and specifically destroy cells that overexpress specific receptors. These molecules, designed and constructed by gene fusion techniques, comprise both cell-targeting and cell-killing moieties. The authors’ laboratory has developed a number of chimeric proteins using gonadotropin-releasing hormone (GnRH) as the targeting moiety. These chimeras recognize a GnRH binding site that is expressed on adenocarcinoma cells. GnRH was fused to a large number of killing moieties, including bacterial and human proapoptotic proteins. All GnRH-based chimeric proteins selectively killed adenocarcinoma cells both in vitro and in vivo. Utilizing chimeric proteins for targeted therapy represents a new and exciting therapeutic modality for the treatment of cancer in humans.     

Targeted cancer therapy with gonadotropin-releasing hormone chimeric proteins

Tumor-associated antigens (TAAs) have been identified mainly to determine cancer prognosis. In the past few years, TAAs have been used in the development of treatment modalities such as tumor vaccination. This review describes an additional application of TAAs: as a target for specific antitumor treatment. Since TAAs are overexpressed on the tumor cell surface, they can be targeted to deliver drugs directly to cancer cells. One such delivery system exploits chimeric proteins. Chimeric proteins are a class of targeted molecules designed to recognize and specifically destroy cells that overexpress specific receptors. These molecules, designed and constructed by gene fusion techniques, comprise both cell-targeting and cell-killing moieties. The authors' laboratory has developed a number of chimeric proteins using gonadotropin-releasing hormone (GnRH) as the targeting moiety. These chimeras recognize a GnRH binding site that is expressed on adenocarcinoma cells. GnRH was fused to a large number of killing moieties, including bacterial and human proapoptotic proteins. All GnRH-based chimeric proteins selectively killed adenocarcinoma cells both in vitro and in vivo. Utilizing chimeric proteins for targeted therapy represents a new and exciting therapeutic modality for the treatment of cancer in humans.     

Cancer cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer as vaccines for the treatment of genitourinary malignancies

When irradiated and administered intradermally as vaccines, cancer cells engineered to secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) by gene transfer elicit potent anticancer immune responses in a variety of animal tumor models. Upon vaccination, antigens present in the cancer cells are phagocytosed and processed by skin dendritic cells. These dendritic cells then prime anticancer immune responses by presenting antigenic peptides to T cells. The immune responses generated are capable of eradicating small but lethal cancer cell inocula with minimal toxicity in preclinical animal tumor studies. To develop this vaccination strategy for the treatment of human genitourinary cancers, we have conducted phase I clinical trials using human genitourinary cancer cells as sources of cancer cell antigens. In the first human clinical trial of genetically engineered cancer cell vaccines, a phase I clinical trial of kidney cancer cell vaccines (n=18), kidney cancer cells were removed at surgery, propagated briefly in vitro, and then genetically modified to secrete high levels of GM-CSF via ex vivo transduction with the retrovirus MFG-GM-CSF. After irradiation, the kidney cancer cells were administered as vaccines to 18 patients with advanced kidney cancers. Vaccine treatment, which caused few side effects, nonetheless appeared to trigger anticancer immune responses manifest as conversion of delayed-type hypersensitivity (DTH) skin responses against irradiated autologous cancer cells after vaccination. Biopsies of vaccine sites yielded findings reminiscent of biopsies from preclinical animal model studies, with evidence of vaccine cell recruitment of dendritic cells, T cells, and eosinophils. One patient with measurable kidney cancer metastases treated at the highest vaccine dose level experienced a partial treatment response. The bioactivity of GM-CSF-secreting autologous cancer cell vaccines was confirmed in a phase I clinical trial for prostate cancer (n=8). Vaccine cells were prepared from surgically harvested prostate tumors by ex vivo transduction with MFG-GM-CSF in a manner similar to that used for the kidney cancer trial. Vaccine treatment was well tolerated and associated with induction of anticancer immunity as assessed using DTH skin testing. In addition, new antiprostate cancer cell antibodies were detected in serum samples from treated men as a consequence of vaccination. These first clinical trials of GM-CSF-secreting cancer cell vaccines for the treatment of genitourinary cancers have demonstrated both safety and bioactivity, in that very few side effects have been seen and anticancer immune responses have been detected. Future clinical studies will be required to assess vaccine treatment efficacy, refine vaccination dose and schedule, define the appropriate clinical context for the use of such vaccines, and ascertain optimal combinations involving vaccines and other local or systemic anticancer treatments.     

Heat-shock protein vaccines as active immunotherapy against human gliomas

Modern advances in cancer immunotherapy have led to the development of active immunotherapy that utilizes tumor-associated antigens to induce a specific immune response against the tumor. Current methods of immunotherapy implementation are based on the principle that tumor-associated antigens are capable of being processed by antigen-presenting cells and inducing an activated cytotoxic T-lymphocyte-specific immune response that targets the tumor cells. Antigen internalization and processing by antigen-presenting cells, such as dendritic cells, or macrophages results in their surface association with MHC class I molecules, which can be recognized by an antigen-specific cytotoxic T-lymphocyte adaptive immune response. With the aim of augmenting current immunotherapeutic modalities, much effort has been directed towards enhancing antigen-presenting cell activation and optimizing the processing of tumor-associated antigens and major histocompatibility molecules. The goal of these immunotherapy modifications is to ultimately improve the adaptive specific immune response in killing of tumor cells while sparing normal tissues. Immunotherapy has been actively studied and applied in glioblastomas. Preclinical animal models have shown the feasibility of an active immunotherapy approach through the utilization of tumor vaccines, and recently several clinical studies have also been initiated. Recently, endogenous heat-shock proteins have been implicated in the mediation of both the adaptive and innate immune responses. They are now being investigated as a potential modality and adjuvant to immunotherapy, and they represent a promising novel treatment for human glioblastomas.     

Fusion of B lymphoblastoid and tumor cells expressing different antibiotic resistance genes facilitates selection of stable hybridomas

Abstract The development of tumor vaccines or generation of tumor-specific cytotoxic T lymphocytes (CTL) is limited by the fact that many tumor cells downregulate the expression of major histocompatibility complex (MHC) Class I and II molecules, as well as key co-stimulatory molecules such as CD80 and CD86. An immune response to a vaccine or in vitro stimulation of tumor-specific CTL requires antigen-presenting cells conveying tumor antigens in the context of a host's MHC antigens. We have used a retroviral vector (murine stem cell virus) encoding neomycin resistance to transduce three pediatric tumor cell lines (two neuroblastoma, one neuroepithelial tumor). An EBV transformed B lymphoblastoid cell line (BLCL) was transduced with a separate vector encoding puromycin resistance and green fluorescent protein, individual tumor lines were fused with the BLCL, and the resulting hybridomas were selected using both antibiotics. The resulting hybridoma cells expressed the neural antigen GD2 as well as MHC Class I, Class II, CD 80, and CD86. A similar strategy could be used to produce stable hybridomas for either vaccination or for CTL expansion.     

IFN-gamma and b7 as costimulators of antitumor immune-responses

We transfected the mouse IFN-gamma and/or the mouse B7 (T cell costimulatory molecule) cDNAs into B16 melanoma cells to study the effects of local constitutive expression of these molecules on the tumorigenicity and immunogenicity of this aggressive tumor. Cells expressing IFN-gamma (B16.IFN-gamma), B7 (B16.B7), B7 and IFN-gamma (B16.IFN-gamma/B7), and parental cells were injected subcutaneously (s.c.) into syngeneic C57BL/6 mice to compare their in vivo growth. We report that IFN-gamma secretion significantly reduced the tumorigenicity of B16 cells. These effects were related to the direct action of secreted IFN-gamma since i) in vivo injection of antiserum to IFN-gamma accelerated tumor growth, ii) development of tumor correlated with loss of IFN-gamma production, and iii) B16.IFN-gamma cells were tumorigenic in IFN-II receptor (IFN-gamma R) knockout mice, but not in parental mice. We propose that immune mechanisms are being activated by IFN-gamma since i) immune effector cells were recruited to the injection site, ii) expression of MHC class I and class II antigens was increased on cells secreting IFN-gamma and, iii) B16.IFN-gamma tumors appeared earlier in athymic mice than in immunocompetent mice. Since the in vivo growth of B16.IFN-gamma cells was not completely abolished, we studied the effect of co-expression of IFN-gamma and the T cell costimulatory molecule B7 on the tumorigenicity of B16 cells. We report that B16.IFN-gamma/B7 cells, which also express increased levels of MHC class I and class II molecules as compared to parental cells, had a dramatically suppressed tumorigenicity, while B16 cells expressing the B7 molecule only (B16.B7) were as tumorigenic as the parental cells. B16.IFN-gamma/B7 cells induced specific immune responses since all of the protected mice were able to reject challenges with parental cells. Results indicate that co-expression of two molecules which are involved in the activation of immune responses and in antigen presentation can influence the ability of the immune system to recognize and eliminate both transfected as well as parental tumor cell inocula and suggest that vaccines consisting of such cells may be used for the immunotherapy of cancer.