GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cross-presentation of epitopes on virus-like particles via the MHC I receptor recycling pathway

Effective vaccines and immunotherapies against cancer require professional antigen-presenting cells to cross-present exogenous antigen to initiate cytotoxic T-cell responses to destroy tumors. Virus-like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross-presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP-derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen-coupled VLPs and murine ovalbumin-specific and human melanoma-associated antigen recognized by T cells (MART-1)-specific CD8(+) T cells were used to demonstrate cross-presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter-associated with antigen presentation. Finally, we found that cross-presentation of VLPs in vivo was not confined to CD8α(+) DC subsets. These data define the cross-presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.     

Liposomal vaccines--targeting the delivery of antigen

Vaccines that can prime the adaptive immune system for a quick and effective response against a pathogen or tumor cells, require the generation of antigen (Ag)-specific memory T and B cells. The unique ability of dendritic cells (DCs) to activate na?ve T cells, implies a key role for DCs in this process. The generation of tumor-specific CD8(+) cytotoxic T cells (CTLs) is dependent on both T cell stimulation with Ag (peptide-MHC-complexes) and costimulation. Interestingly, tumor cells that lack expression of T cell costimulatory molecules become highly immunogenic when transfected to express such molecules on their surface. Adoptive immunotherapy with Ag-pulsed DCs also is a strategy showing promise as a treatment for cancer. The use of such cell-based vaccines, however, is cumbersome and expensive to use clinically, and/or may carry risks due to genetic manipulations. Liposomes are particulate vesicular lipid structures that can incorporate Ag, immunomodulatory factors and targeting molecules, and hence can serve as potent vaccines. Similarly, Ag-containing plasma membrane vesicles (PMV) derived from tumor cells can be modified to incorporate a T cell costimulatory molecule to provide both TCR stimulation, and costimulation. PMVs also can be modified to contain IFN-gamma and molecules for targeting DCs, permitting delivery of both Ag and a DC maturation signal for initiating an effective immune response. Our results show that use of such agents as vaccines can induce potent anti-tumor immune responses and immunotherapeutic effects in tumor models, and provide a strategy for the development of effective vaccines and immunotherapies for cancer and infectious diseases.     

Emerging evidence indicates that physiologically relevant thermal stress regulates dendritic cell function

Summary Elevations in temperature that are associated with inflammation or fever have been linked to improved survival from infections, enhanced immunological functions, and increased control of tumor growth. Over the past few years, several groups have begun to explore the possible linkage among these observations and have tested the hypothesis that various immune cells are especially sensitive to thermal stimulation. However, relatively little is known regarding the effects of thermal stimulation on antigen presenting cells (APCs), such as dendritic cells (DCs). Very recently, several groups have begun to examine the ability of thermal stimuli to regulate the function of these cells which are known to play a pivotal role in the efficacy of vaccines and other immunotherapies. In this review, we summarize what has been discovered about the role of mild thermal stress in regulating various Dendritic cell (DC) activities. Excitingly, it appears that mild elevations of temperature have the potential to enhance antigen uptake, activation associated migration, maturation, cytokine expression and T cell stimulatory activity of DCs. While these studies reveal that the timing, temperature and duration of heating is important, they also set the stage for essential questions that now need to be investigated regarding the molecular mechanisms by which elevated temperatures regulate DC function. With this information, we may soon be able to maximize the strategic use of thermal therapy as an adjuvant, i.e., combining its use with cancer immunotherapies such as vaccines, which depend upon the function of DCs. Several possible strategies and timepoints involving the clinical application of hyperthermia in combination with immunotherapy are presented. This article forms part of the Symposium in Writing “Thermal stress-related modulation of tumor cell physiology and immune responses”, edited by Elfriede Noessner.     

Engineering T cells for immunotherapy of primary human hepatocellular carcinoma

Liver cancers, majority of which are primary hepatocellular carcinoma(HCC), continue to be on the rise in the world. Furthermore, due to the lack of effective treatments, liver cancer ranks the 4~(th) most common cause of male cancer deaths. Novel therapies are urgently needed. Over the last few years,immunotherapies, especially the checkpoint blockades and adoptive cell therapies of engineered T cells,have demonstrated a great potential for treating malignant tumors including HCC. In this review, we summarize the current ongoing research of antigen-specific immunotherapies including cancer vaccines and adoptive cell therapies for HCC. We briefly discuss the HCC cancer vaccine and then focus on the antigen-specific T cells genetically engineered with the T cell receptor genes(TCRTs) and the chimeric antigen receptor genes(CARTs). We first review the current options of TCRTs and CARTs immunotherapies for HCC, and then analyze the factors and parameters that may help to improve the design of TCRTs and CARTs to enhance their antitumor efficacy and safety. Our goals are to render readers a panoramic view of the current stand of HCC immunotherapies and provide some strategies to design better TCRTs and CARTs to achieve more effective and durable antitumor effects.     

Addressing cancer immunotherapy research in Iran: adoptive cell therapy on the horizon

Recent advances in immunotherapeutic modalities have profoundly changed the prospect of cancer treatment. These modalities mainly focus on modulating the immune response toward tumor cells by using monoclonal antibodies, cancer vaccines, adoptive cell transfer or combination of these methods. In the last few years, Iranian scientists have conducted several projects in these arenas. Here, we provide an overview of these studies and analyze the quality and trend of publications in each sub-specialty of the field. In addition, the contribution of different universities and scientific institutes is assessed. This study may benefit scientific community and policymakers to plan future cancer immunotherapies in Iran and other countries.     

Immunotherapeutic strategies employing RNA interference technology for the control of cancers

Summary The human immune system is comprised of several types of cells that have the potential to eradicate tumors without inflicting damage on normal tissue. Over the past decade, progress in the understanding of tumor biology and immunology has offered the exciting possibility of treating malignant disease with vaccines that exploit the capacity of T cells to effectively and selectively kill tumor cells. However, the immune system frequently fails to mount a successful defense against cancers despite vaccination with tumor-associated antigens. The ability of these vaccines to generate an abundant supply of armed effector T cells is often limited by immunoregulatory signaling pathways that suppress T cell activation. In addition, many tumors create a local microenvironment that inhibits the function of T cells. The attenuation of these pathways, which facilitate the evasion of tumors from immune surveillance, thus represents a potentially effective approach for cancer immunotherapy. Specifically, it may be of interest to modify the properties of dendritic cells, T cells, and tumor cells to downregulate the expression of proteins that diminish the immune response to cancers. RNA interference (RNAi) techniques have developed into a highly effective means of intracellular gene ‘knockdown’ and may be successfully employed in this way to improve cancer immunotherapies. This strategy has recently been explored both in vitro and in vivo, and has generated significantly enhanced antitumor immunity in numerous studies. Nevertheless, several practical concerns remain to be resolved before RNAi technology can be implemented safely and efficiently in humans. As novel developments and discoveries in molecular biology rapidly continue to unfold, it is likely that this technology may soon translate into a potent form of gene silencing in the clinic with profound applications to cancer immunotherapy.     

B7-H1 signaling is integrated during CD8+ T cell priming and restrains effector differentiation

A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines with the goal of activating anti-tumor T cell responses. Current formulations for dendritic cell-based immunotherapies have limited effects on patient survival, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Using a brief in vitro priming model, we have found that B7-H1 expressed by activated dendritic cells is integrated during priming of naïve CD8⁺ T cells and functions to limit the differentiation of effector T cell responses. CD8⁺ T cells primed by B7-H1-deficient dendritic cells exhibit increased production of IFN-γ, enhanced target cell killing, and improved anti-tumor activity. Additionally, enhanced memory populations arise from CD8⁺ T cells primed by B7-H1-deficient dendritic cells. Based on these findings, we suggest that early blockade of B7-H1 signaling should be investigated as a strategy to improve dendritic cell-based anti-tumor immunotherapy.     

High lipid content of irradiated human melanoma cells does not affect cytokine-matured dendritic cell function

Gamma irradiation is one of the methods used to sterilize melanoma cells prior to coculturing them with monocyte-derived immature dendritic cells in order to develop antitumor vaccines. However, the changes taking place in tumor cells after irradiation and their interaction with dendritic cells have been scarcely analyzed. We demonstrate here for the first time that after irradiation a fraction of tumor cells present large lipid bodies, which mainly contain triglycerides that are several-fold increased as compared to viable cells as determined by staining with Oil Red O and BODIPY 493/503 and by biochemical analysis. Phosphatidyl-choline, phosphatidyl-ethanolamine and sphingomyelin are also increased in the lipid bodies of irradiated cells. Lipid bodies do not contain the melanoma-associated antigen MART-1. After coculturing immature dendritic cells with irradiated melanoma cells, tumor cells tend to form clumps to which dendritic cells adhere. Under such conditions, dendritic cells are unable to act as stimulating cells in a mixed leukocyte reaction. However, when a maturation cocktail composed of TNF-alpha, IL-6, IL-1beta and prostaglandin E2 is added to the coculture, the tumor cells clumps disaggregate, dendritic cells remain free in suspension and their ability to efficiently stimulate allogeneic lymphocytes is restored. These results help to understand the events following melanoma cell irradiation, shed light about interactions between irradiated cells and dendritic cells, and may help to develop optimized dendritic cell vaccines for cancer therapy.     

Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.     

Carbohydrate antigens as targets for active specific immunotherapy

 Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewis^y and globo-H (neutral glycolipids and glycoproteins), and Tn, TF and sTn (glycoproteins) are overexpressed in a variety of cancers. Antibodies against several of these carbohydrate antigens have been detected in sera from patients treated with cancer vaccines, and have been associated with a more favorable prognosis. Clinical responses have been reported after treatment with monoclonal antibodies against some of these antigens. Hence cell-surface carbohydrate antigens have been identified as suitable targets for immune attack by both active and passive immunotherapies. Different approaches have been adopted to induce immune responses against these carbohydrate antigens. These includes vaccination with whole or lysed tumor cells, purified or synthetic carbohydrates, immunogenic carbohydrate derivatives, or carbohydrates conjugated with immunogenic carriers and administered with immunological adjuvants. In the case of gangliosides, immunization with either whole tumor cells or cell lysates has only occasionally induced responses against carbohydrate antigens, and the antibodies were generally IgM antibodies of low titer. Compared with other methods of vaccination, conjugate vaccines have consistently induced the highest titer of IgM and IgG antibodies against gangliosides and other carbohydrate antigens. Preclinical and clinical studies with conjugate carbohydrate vaccines have induced IgM and IgG antibody responses capable of inducing complement-mediated cytotoxicity of tumor cells in vitro and associated with prolonged disease-free and overall survival in patients. Received: 6 August 1996 / Accepted: 20 September 1996     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells     

Dendritic cell delivery of plasmid DNA

Positive human clinical data using biolistic-mediated gene transfer (i.e., gene gun) to administer a nucleic acid-based Hepatitis B vaccine has validated genetic immunization as an effective clinical vaccine modality. Although the precise mechanism of action has yet to be determined, preclinical studies using jet injection have indicated that direct targeting of resident antigen presenting cells (Langerhan's cells) in the skin as the primary immunological driving force for the potent and long-lived immune response. Moreover, positive results with topical delivery of genetic vaccines and ex vivo loading of dendritic cells with antigen has strengthened the movement toward directly targeting antigen presenting cells as a means to amplify, control, and mediate the immunological consequences of prophylactic and/or therapeutic genetic vaccines. Despite these encouraging results with the gene gun, it is unclear whether this technology will translate into commercially available vaccines due to potential product development barriers such as cost and convenience. It is clear that safety concerns in using genetic approaches to treat and prevent disease have highlighted the need for strict product requirements for genetic vaccines. A plausible strategy to meet these requirements is to combine controlled plasmid delivery systems with tissue-specific gene expression systems.     

Cross-reactive CD4+ T cells against one immunodominant tumor-derived epitope in melanoma patients

TCRs exhibit a high degree of specificity but may also recognize multiple and distinct peptide-MHC complexes, illustrating the so-called cross-reactivity of TCR-peptide-MHC recognition. In this study, we report the first evidence of CD4(+) T cells recognizing the same tumor peptide-epitope from NY-ESO-1, in the context of multiple HLA-DR and HLA-DP molecules. These cross-reactive CD4(+) T cells recognized not only autologous but also allogenic dendritic cells previously loaded with the relevant protein (i.e., the normally processed and presented epitope). Using clonotypic real-time RT-PCR, we have detected low frequencies of CD4(+) T cells expressing one cross-reactive TCR from circulating CD4(+) T cells of patients with stage IV melanoma either spontaneously or after immunization but not in normal donors. The maintenance of cross-reactive tumor Ag-specific CD4(+) T cells in PBLs of cancer patients required the presence of tumor Ag/epitope in the context of the MHC molecule used to prime the Ag-specific CD4(+) T cells. Our findings have significant implications for the optimization of TCR gene transfer immunotherapies widely applicable to cancer patients.     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBodyTM, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBodyTM expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBodyTM are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBodyTM vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.     

Cellular magnetic resonance imaging of monocyte-derived dendritic cell migration from healthy donors and cancer patients as assessed in a scid mouse model

BACKGROUND AIMS. The use of dendritic cells (DC) as an adjuvant in cell-based immunotherapeutic cancer vaccines is a growing field of interest. A reliable and non-invasive method to track the fate of autologous DC following their administration to patients is required in order to confirm that clinically sufficient numbers are reaching the lymph node (LN). We demonstrate that an immunocompromised mouse model can be used to conduct translational studies employing cellular magnetic resonance imaging (MRI). Such studies can provide clinically relevant information regarding the migration potential of clinical-grade DC used in cancer immunotherapies. METHODS. Human monocyte-derived dendritic cells (mo-DC) were generated from negatively selected monocytes obtained from either healthy donors or cancer patients. DC were labeled with superparamagnetic iron oxide (SPIO) nanoparticles in order to track them in vivo in a CB17scid mouse model using cellular MRI. SPIO did not have any adverse effects on DC phenotype or function, independent of donor type. Cellular MRI readily detected migration of SPIO-loaded DC in CB17scid mice. No differences in migration were observed between DC obtained from healthy donors and those obtained from donors undergoing autologous stem cell transplant for cancer therapy. CONCLUSIONS. Cellular MRI provided semi-quantitative image data that corresponded with data obtained by digital morphometry, validating cellular MRI's potential to assess DC migration in DC-based cancer immunotherapy clinical trials.     

Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.     

Gene therapy meets vaccine development

Therapeutic vaccines such as those used to combat cancer or persistent viral infection are required to reprogramme a downregulated immune system. This presents a difficult challenge for vaccine design and merits the development of novel immunization protocols. Currently, we know that mobilization of dendritic cells (DCs) to present antigens to T lymphocytes is crucial for effective immunization. Our increasing understanding of DC biology, coupled with the growing sophistication of viral vectors developed for gene therapy, makes more rational vaccine design an exciting possibility. Here we propose that engineering viral vectors to express antigens in activated DCs will provide the most effective vaccines for priming an immune response.     

Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181-188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.     

基因疫苗导入技术研究进展

基因疫苗积极的临床结果证明了,基因免疫是一种有效的临床免疫模式。虽然,喷射注射法的精确作用机制还不太清楚,但临床前研究表明,在皮肤内直接打靶抗原呈递细胞可有效地增强免疫反应。另外,局部给药法和树突细胞体外加载抗原的实验结果显示,直接打靶抗原呈递细胞可放大、控制和调节预防及治疗性基因疫苗的免疫结果。尽管基因枪有许多令人鼓舞的优点,但由于价格和便利性的障碍,它是否能商业化还不能确定。利用基因法治疗和预防疾病所涉及的安全性对基因疫苗要求更严格。这要有可控的质粒导入系统和组织特异性表达系统。