Cancer vaccines based on dendritic cells loaded with tumor-associated antigens

Dendritic cells (DCs) can be used as an antigen presentation platform for vaccination against cancer. In this approach, DCs are expanded in vitro from monocyte-derived progenitors, and subsequently loaded with well-characterized tumor-associated antigens (TAAs). TAAs can be incubated with DCs in various forms, including peptides, recombinant proteins, plasmid DNA, formulated RNA, or recombinant viruses. Advantages and limitations of DC-based cellular vaccines against cancers, as well as preliminary results of clinical studies already performed in humans, are discussed. Importantly, significant advances in our understanding of the biology of DCs can be used to support the design of new vaccines or adjuvants in order to elicit T_H1 cellular immune responses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chaperone-rich cell lysates, immune activation and tumor vaccination

We have utilized a free-solution-isoelectric focusing technique (FS-IEF) to obtain chaperone-rich cell lysates (CRCL) fractions from clarified tumor homogenates. The FS-IEF technique for enriching multiple chaperones from tumor lysate is relatively easy and rapid, yielding sufficient immunogenic material for clinical use. We have shown that tumor-derived CRCL carry antigenic peptides. Dendritic cells (DCs) uptake CRCL and cross-present the chaperoned peptides to T cells. Tumor-derived CRCL induce protective immune responses against a diverse range of murine tumor types in different genetic backgrounds. When compared to purified heat shock protein 70 (HSP70), single antigenic peptide or unfractionated lysate, CRCL have superior ability to activate/mature DCs and are able to induce potent, long lasting and tumor specific T-cell-mediated immunity. While CRCL vaccines were effective as stand-alone therapies, the enhanced immunogenicity arising from CRCL-pulsed DC as a vaccine indicates that CRCL could be the antigen source of choice for DC-based anti-cancer immunotherapies. The nature of CRCL's enhanced immunogenicity may lie in the broader antigenic peptide repertoire as well as the superior immune activation capacity of CRCL. Exongenous CRCL also supply danger signals in the context of apoptotic tumor cells and enhance the immunogenicity of apoptotic tumor cells, leading to tumor-specific T cell dependent long-term immunity. Moreover, CRCL based vaccines can be effectively combined with chemotherapy to treat cancer. Our findings indicate that CRCL have prominent adjuvant effects and are effective sources of tumor antigens for pulsing DCs. Tumor-derived CRCL are promising anti-cancer vaccines that warrant clinical research and development.     

Antigen loading of DCs with irradiated apoptotic tumor cells induces improved anti-tumor immunity compared to other approaches

Dendritic cells (DCs) serve as central regulators of adaptive immunity by presenting antigens and providing necessary co-signals. Environmental information received by the DCs determines the co-signals delivered to the responding adaptive cells and, ultimately, the outcome of the interaction. DCs loaded with relevant antigens have been used as therapeutic cellular vaccines, but the optimal antigen loading method has not been determined. We compared different methods to load class I and class II epitopes from the male antigenic complex, HY, onto DCs for the potency of the immune response induced in vivo. Co-incubation of female DCs with HY peptides, RNA or cell lysate from HY expressing tumor induced immune responses equivalent to male DCs. In contrast, female DCs incubated with irradiated, apoptotic HY expressing tumor cells (or male B cells) generated a stronger immune response than male DCs or female DCs loaded using any of the other methods. DC loading with apoptotic tumor resulted in complete protection against high dose HY-expressing tumor challenge whereas 100% lethality was observed in groups receiving DCs that were loaded with peptides, RNA, or lysate. We conclude that signals provided to the DCs by apoptotic cells substantially augment the potency of DC vaccines.     

Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy

Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.     

Combinational adenovirus-mediated gene therapy and dendritic cell vaccine in combating well-established tumors

Recent developments in tumor immunology and biotechnology have made cancer gene therapy and immunotherapy feasible. The current efforts for cancer gene therapy mainly focus on using immunogenes, chemogenes and tumor suppressor genes. Central to all these therapies is the development of efficient vectors for gene therapy. By far, adenovirus （AdV）-mediated gene therapy is one of the most promising approaches, as has confirmed by studies relating to animal tumor models and clinical trials. Dendritic cells （DCs） are highly efficient, specialized antigen-presenting cells, and DC- based tumor vaccines are regarded as having much potential in cancer immunotherapy. Vaccination with DCs pulsed with tumor peptides, lysates, or RNA, or loaded with apoptotic/necrotic tumor cells, or engineered to express certain cytokines or chemokines could induce significant antitumor cytotoxic T lymphocyte （CTL） responses and antitumor immunity. Although both AdV-mediated gene therapy and DC vaccine can both stimulate antitumor immune responses, their therapeutic efficiency has been limited to generation of prophylactic antitumor immunity against re-challenge with the parental tumor cells or to growth inhibition of small tumors. However, this approach has been unsuccessful in combating well-established tumors in animal models. Therefore, a major strategic goal of current cancer immunotherapy has become the development of novel therapeutic strategies that can combat well-established tumors, thus resembling real clinical practice since a good proportion of cancer patients generally present with significant disease. In this paper, we review the recent progress in AdV-mediated cancer gene therapy and DC-based cancer vaccines, and discuss combined immunotherapy including gene therapy and DC vaccines. We underscore the fact that combined therapy may have some advantages in combating well-established tumors vis-a-vis either modality administered as a monotherapy.     

Optimizing Dendritic Cell-Based Approaches for Cancer Immunotherapy

Dendritic cells (DC) are professional antigen-presenting cells uniquely suited for cancer immunotherapy. They induce primary immune responses, potentiate the effector functions of previously primed T-lymphocytes, and orchestrate communication between innate and adaptive immunity. The remarkable diversity of cytokine activation regimens, DC maturation states, and antigen-loading strategies employed in current DC-based vaccine design reflect an evolving, but incomplete, understanding of optimal DC immunobiology. In the clinical realm, existing DC-based cancer immunotherapy efforts have yielded encouraging but inconsistent results. Despite recent U.S. Federal and Drug Administration (FDA) approval of DC-based sipuleucel-T for metastatic castration-resistant prostate cancer, clinically effective DC immunotherapy as monotherapy for a majority of tumors remains a distant goal. Recent work has identified strategies that may allow for more potent “next-generation” DC vaccines. Additionally, multimodality approaches incorporating DC-based immunotherapy may improve clinical outcomes.     

Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting

The realization that dendritic cells (DCs) orchestrate innate and adaptive immune responses has stimulated research on harnessing DCs to create more effective vaccines. Early clinical trials exploring autologous DCs that were loaded with antigens ex vivo to induce T-cell responses have provided proof of principle. Here, we discuss how direct targeting of antigens to DC surface receptors in vivo might replace laborious and expensive ex vivo culturing, and facilitate large-scale application of DC-based vaccination therapies.     

The role of dendritic cells in tumor microenvironments and their uses as therapeutic targets

Dendritic cells (DC), which consist of several different subsets, specialize in antigen presentation and are critical for mediating the innate and adaptive immune responses. DC subsets can be classified into conventional, plasmacytoid, and monocyte-derived DC in the tumor microenvironment, and each subset plays a different role. Because of the role of intratumoral DCs in initiating antitumor immune responses with tumor-derived antigen presentation to T cells, DCs have been targeted in the treatment of cancer. By regulating the functionality of DCs, several DC-based immunotherapies have been developed, including administration of tumor-derived antigens and DC vaccines. In addition, DCs participate in the mechanisms of classical cancer therapies, such as radiation therapy and chemotherapy. Thus, regulating DCs is also important in improving current cancer therapies. Here, we will discuss the role of each DC subset in antitumor immune responses, and the current status of DC-related cancer therapies.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Exploiting dendritic cells for cancer immunotherapy: genetic modification of dendritic cells

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct characteristics and possible advantages and disadvantages in respect to their use in DC-based immunotherapy.     

Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells

BACKGROUND: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. METHODS: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta). RESULTS: mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. CONCLUSIONS: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.     

CD40-targeted adenoviral cancer vaccines: the long and winding road to the clinic

The ability of dendritic cells (DCs) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DCs for clinical administration, their loading with tumor associated antigens (TAAs) and their activation, is laborious and expensive, and, as a result of inter-individual variability in the personalized vaccines, remains poorly standardized. An attractive alternative approach is to load resident DCs in vivo by targeted delivery of TAAs, using viral vectors and activating them simultaneously. To this end, we have constructed genetically-modified adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAAs to the CD40 receptor on DCs. Pre-clinical human and murine studies conducted so far have clearly demonstrated the suitability of a 'two-component' (i.e. Ad and adaptor molecule) configuration for targeted modification of DCs in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in the clinical translation of this approach.     

The performance and perspectives of dendritic cell vaccines modified by immune checkpoint inhibitors or stimulants

Therapeutic dendritic cell (DC) vaccines stimulate the elimination of tumor cells by the immune system. However, while antigen-specific T cell responses induced by DC vaccines are commonly observed, the clinical response rate is relatively poor, necessitating vaccine optimization. There is evidence that the suppression of DC function by immune checkpoints hinders the anti-tumor immune responses mediated by DC vaccines, ultimately leading to the immune escape of the tumor cells. The use of immune checkpoint inhibitors (ICIs) and immune checkpoint activators (ICAs) has extended the immunotherapeutic range. It is known that both inhibitory and stimulatory checkpoint molecules are expressed by most DC subsets and can thus be used to manipulate the effectiveness of DC vaccines. Such manipulation has been investigated using strategies such as chemotherapy, agonistic or antagonistic antibodies, siRNA, shRNA, CRISPR-Cas9, soluble antibodies, lentiviruses, and adenoviruses to maximize the efficacy of DC vaccines. Thus, a deeper understanding of immune checkpoints may assist in the development of improved DC vaccines. Here, we review the actions of various ICIs or ICAs shown by preclinical studies, as well as their potential application in DC vaccines. New therapeutic interventional strategies for blocking and stimulating immune checkpoint molecules in DCs are also described in detail.     

Regulatory perspective on in vitro potency assays for human dendritic cells used in anti-tumor immunotherapy

Dendritic cells (DCs) are key connectors between the innate and adaptive immune system and have an important role in modulating other immune cells. Therefore, their therapeutic application to steer immune responses is considered in various disorders, including cancer. Due to differences in the cell source and manufacturing process, each DC medicinal product is unique. Consequently, release tests to ensure consistent quality need to be product-specific.Although general guidance concerning quality control testing of cell-based therapies is available, cell type-specific regulation is still limited. Especially guidance related to potency testing is needed, because developing an in vitro assay measuring cell properties relevant for in vivo functionality is challenging. In this review, we provide DC-specific guidance for development of in vitro potency assays for characterisation and release. We present a broad overview of in vitro potency assays suggested for DC products to determine their anti-tumor functionality. Several advantages and limitations of these assays are discussed. Also, we provide some points to consider for selection and design of a potency test. The ideal functionality assay for anti-tumor products evaluates the capacity of DCs to stimulate antigen-specific T cells. Because this approach may not be feasible for release, use of surrogate potency markers could be considered, provided that these markers are sufficiently linked to the in vivo DC biological activity and clinical response. Further elucidation of the involvement of specific DC subsets in anti-tumor responses will result in improved manufacturing processes for DC-based products and should be considered during potency assay development.     

Plant-derived polysaccharides activate dendritic cell-based anti-cancer immunity

Today, cancers pose a major public health burden. Although a myriad of cancer treatments are available, only a few have achieved clinical efficacy. This is partly attributed to cancers capability to evade host immunity by converting dendritic cells (DCs) from potent stimulators to negative modulators of immunity. Dendritic cell-based immunotherapy attempts to resolve this problem by manipulating the functional characteristics of DCs. Plant-derived polysaccharides (PDPs) can stimulate the maturation of DCs conferring on them the capacity to present internalised tumorigenic antigens to naïve T cells and subsequently priming T cells to eliminate tumours. PDPs have been used as immune modulators and later as anti-cancer agents by Traditional Chinese Medicine practitioners for centuries. They are abundant in nature and form a large group of heterogeneous though structurally related macromolecules that exhibit diverse immunological properties. They can induce antigen pulsed DCs to acquire functional characteristics in vitro which can subsequently be re-introduced into cancer patients. They can also be used as adjuvants in DC-based vaccines or independently for their intrinsic anti-tumour activities. Clinically, some in vitro generated DCs have been shown to be both safe and immunogenic although their clinical application is limited in part by unsatisfactory functional maturation as well as impaired migration to draining lymph nodes where T cells reside. We review the relative potencies of individual PDPs to induce both phenotypic and functional maturation in DCs, their relative abilities to activate anti-cancer immunity, the possible mechanisms by which they act and also the challenges surrounding their clinical application.     

Exploiting dendritic cells for active immunotherapy of cancer and chronic infections

Dendritic cells (DCs) are important antigen-presenting cells (APCs) that can prime naive T cells and control adaptive immune responses with respect to magnitude, memory and self-tolerance. Understanding the biology of these cells is central to the development of new generation immunotherapies for cancer and chronic infections. This review presents a brief overview of DC biology and of the preparation and use of DC-based vaccines.     

Chemically engineered glycan-modified cancer vaccines to mobilize skin dendritic cells

Dendritic cell (DC)–targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and presentation to CD4⁺ and CD8⁺ T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node–resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use.     

树突状细胞肿瘤疫苗的研究进展

树突状细胞(DC)是目前发现的抗原提呈功能最强的专职抗原提呈细胞。大量实验已证明DC疫苗在抗肿瘤免疫中发挥着重要的作用。随着分子生物学、免疫学、基因工程技术的发展,各种DC疫苗和DC细胞的治疗应运而生,发展迅速。简要综述了DC的生物学特性、DC与肿瘤发生发展的关系、DC抗原的负载方法及其在临床实验中的应用,以为进一步的基础研究和临床实验提供参考。     

Loading DCs with Ag

Understanding the biology of the DC and its pivotal role in immune response initiation and regulation has enabled new effective anti-cancer therapies to be developed for treatment of a wide range of tumor types. Many studies on DC-based cancer vaccine development have focused on the development of methods that can effectively deliver tumor Ags to DCs. Numerous methods have been developed or evaluated for the delivery of defined and undefined tumor Ags to DCs for processing and presentation to T lymphocytes. This review provides a brief summary of these methods, the techniques that are used in each, how they have -- or could -- be applied clinically, as well as the advantages and disadvantages of each method.     

Large-scale production and directed induction of functional dendritic cells ex vivo from serum-free expanded human hematopoietic stem cells

BackgroundDendritic cells (DCs) that are derived from hematopoietic stem cells (HSCs) are the most potent antigen-presenting cells and play a pivotal role in initiating the immune response. Hence, large-scale production and direct induction of functional DCs ex vivo from HSCs are crucial to HSC research and clinical potential, such as vaccines for cancer and immune therapy.MethodsIn a previous study, we developed a serum-free HSC expansion system (SF-HSC medium) to expand large numbers of primitive HSCs ex vivo. Herein, a DC induction and expansion medium (DC medium) was proposed to further generate large numbers of functional DCs from serum-free expanded HSCs, which were developed and optimized by factorial design and the steepest ascent method.ResultsThe DC medium is composed of effective basal medium (Iscove's modified Dulbecco's medium [IMDM]) and cytokines (2.9 ng/mL stem cell factor [SCF], 2.1 ng/mL Flt-3 ligand, 3.6 ng/mL interleukin [IL]-1β, 19.3 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF] and 20.0 ng/mL tumor necrosis factor-α [TNF-α]). After 10-day culture in DC medium, the maximum fold expansion for accumulated CD1a⁺CD11c⁺ DCs was more than 4000-fold, and the induced DCs were characterized and confirmed by analysis of growth kinetics, surface antigen expression, endocytosis ability, mixed lymphocyte reaction, specific cytokine secretion and lipopolysaccharide stimulation.DiscussionIn conclusion, the combination of DC medium and SF-HSC medium can efficiently induce and expand a large amount of functional DCs from a small scale of HSCs and might be a promising source of DCs for vaccine and immune therapy in the near future.