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Quarta-feira, 30.04.14

ONCOLYTIC IMMUNOTHERAPY

 

Review ARTICLE

Front. Oncol., 10 April 2014 | doi: 10.3389/fonc.2014.00074

Oncolytic immunotherapy: dying the right way is a key to eliciting potent antitumor immunity

                        Zong Sheng Guo*, Zuqiang Liu and David L. Bartlett

Department of Surgery, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Oncolytic viruses (OVs) are novel immunotherapeutic agents whose anticancer effects come from both oncolysis and elicited antitumor immunity. OVs induce mostly immunogenic cancer cell death (ICD), including immunogenic apoptosis, necrosis/necroptosis, pyroptosis, and autophagic cell death, leading to exposure of calreticulin and heat-shock proteins to the cell surface, and/or released ATP, high-mobility group box 1, uric acid, and other damage-associated molecular patterns as well as pathogen-associated molecular patterns as danger signals, along with tumor-associated antigens, to activate dendritic cells and elicit adaptive antitumor immunity. Dying the right way may greatly potentiate adaptive antitumor immunity. The mode of cancer cell death may be modulated by individual OVs and cancer cells as they often encode and express genes that inhibit/promote apoptosis, necroptosis, or autophagic cell death. We can genetically engineer OVs with death-pathway-modulating genes and thus skew the infected cancer cells toward certain death pathways for the enhanced immunogenicity. Strategies combining with some standard therapeutic regimens may also change the immunological consequence of cancer cell death. In this review, we discuss recent advances in our understanding of danger signals, modes of cancer cell death induced by OVs, the induced danger signals and functions in eliciting subsequent antitumor immunity. We also discuss potential combination strategies to target cells into specific modes of ICD and enhance cancer immunogenicity, including blockade of immune checkpoints, in order to break immune tolerance, improve antitumor immunity, and thus the overall therapeutic efficacy.

Introduction

Oncolytic viruses (OVs) have been shown to be effective in treating cancer in preclinical models and promising clinical responses in human cancer patients (13). OV-mediated cancer therapeutic includes three major mechanisms. The first is the direct infection of cancer and endothelial cells in the tumor tissue leading to direct oncolysis of these cells. The second is necrotic/apoptotic death of uninfected cells induced by anti-angiogenesis and vasculature targeting of the OVs as shown in both animal models and human cancer patients (46). The last is the activated innate and adaptive tumor-specific immunity, which exert cytotoxicity to surviving cancer and stromal cells. A number of recent studies have demonstrated that the antitumor immunity has played an important role in the overall efficacy of oncolytic virotherapy, which has been shown to contribute to the efficacy of oncolytic virotherapy (714). In the case of oncolytic vesicular stomatitis virus (VSV), reovirus, and herpes simplex virus (HSV), the antitumor immune response is very critical to the overall efficacy of oncolytic virotherapy, sometimes even more important than that of direct oncolysis (7, 9, 11, 14).

Oncolytic viruses provide a number of potential advantages over conventional cancer therapies. First, OVs are tumor-selective antitumor agent, thus providing higher cancer specificity and better safety margin. Second, OV-mediated oncolysis not only leads to regression of tumor size, but this process provides key signals to dendritic cells (DCs) and other antigen presenting cells to initiate a potentially potent antitumor immune response. The immunogenic types of cell death induced by OVs provide danger signal (signal 0) and a natural repertoire of tumor-associated antigens (TAAs) to DCs, both required to trigger an adaptive immunity against cancer (1517). The danger signals include damage-associated molecular pattern (DAMP) and pathogen-associated molecular pattern (PAMP) molecules derived from the OVs. Therefore, this process could provide a highly favorable immunological backdrop for the host to respond and generate potent adaptive antitumor immunity. However, just like other immunotherapeutic regimens for cancer, a number of challenges remain for OVs-mediated immunotherapy. One is that relative inefficiency of delivering OVs to tumor nodules, viral replication within tumor mass, and spread to distant metastases dampens its overall efficacy. Second, most TAAs are self-antigens and thus weakly immunogenic. As we will discuss below, OVs may enhance tumor immunogenicity in many cases. Yet, this low immunogenicity still is a problem due to the highly immunosuppressive tumor microenvironment (TME). Third, a highly immunosuppressive TME in late stages of cancer often suppresses the activities of tumor-infiltrated lymphocytes (TILs) generated either spontaneously or by an immunotherapeutic regimen (18).

In this review, we will discuss different modes of cell death induced by various OVs, their potential effects on the subsequent antitumor immunity. Then we discuss rationales and strategies of inducing ideal types of cancer cell death by either genetic modification on OVs or by combination with specific antitumor agents that lead to specific mode of immunogenic cancer cell death (ICD). Finally, we provide some perspective on future combination strategies to improve antitumor immunity for enhanced overall efficacy of virotherapy.

OV: Tumor Selectivity and Relevance of Animal Model

Ideally, OVs selectively infect and replicate in cancer cells and cancer-associated endothelial cells, leading to direct oncolysis and subsequent antitumor activities without harming normal tissue (13). Some OVs display intrinsic tumor tropism (naturally occurring OVs), while others obtain their tumor selectivity through natural evolution or genetic engineering. The mechanisms underlying the tumor selectivity may include altered signaling pathways of ataxia telangiectasia mutated (ATM), epidermal growth factor receptor (EGFR), p53, PKR, Ras, RB/E2F/p16, Wnt, anti-apoptosis, or defects in cellular innate immune signaling pathways or hypoxia conditions in the TME (1, 3, 19, 20).

Viruses display strict viral tropism, specific for a cell type, tissue, or species. However, OVs often broaden their tropism to cancer cells from non-permissive species to various degrees. As an example, human adenovirus (Ad) does not infect normal murine cells, yet infect murine cancer cells even though the production of infectious virus progeny is often limited. A recent study may provide some answer to this phenomenon. McNeish et al. have found that murine cancer cells support viral gene transcription, mRNA processing, and genome replication of human Ad, but there is a profound failure of viral protein synthesis, especially late structural proteins with reduced loading of late mRNA onto ribosomes. Interestingly, in trans expression of the non-structural late protein L4-100K increases both viral mRNA loading on ribosomes and late protein synthesis, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy (21). The key point is that some OVs display aberrant, non-productive infection in non-native hosts such as mouse cells, leading to mode of cancer cell death different from the mode of cell death in native host. As we will discuss extensively later, the mode of cancer cell death dictates to a significant degree the subsequent antitumor immunity. As a consequence, the OV-elicited antitumor immunity in tumor models of syngeneic animals might not be relevant to the situation in human cancer patients. This is an often overlooked issue when tumor models in animals are chosen along with OVs as therapeutic models for human cancer.

Signal 0: DAMPs and PAMPs

PAMPs: Signal 0s from Pathogens

In the late 1980s, Charles Janeway proposed that the immune system protects the host against infectious pathogens by presenting the molecules as signal 0s, which is what now called PAMPs, to the antigen presenting cells (22, 23). PAMPs consist of essential components of microorganisms that direct the targeted host cells, key components in the innate immune arm, to distinguish “self” from “non-self,” and promote signals associated with innate immunity (24). Major PAMPs are nucleic acids (DNA, double-stranded RNA, single-stranded RNA, and 5′-triphosphate RNA), proteins (lipoproteins and glycoproteins), as well as other components of the cell surface and membrane (17, 25). Interestingly, defective viral genomes arising in vivo are a critical danger signal for triggering antiviral immunity in the lung (26).

This concept of PAMPs has been strongly supported by the discovery of several classes of pattern-recognition receptors (PRRs). These PRRs include the toll-like receptors (TLRs), retinoic acid-inducible gene-1 (RIG-1)-like receptors (RLRs), nucleotide oligodimerization domain (NOD)-like receptors (NLRs), AIM2-like receptors, and the receptor for advanced glycation end products (RAGE) (17, 27). It is now well accepted that both DAMPs and PAMPs stimulate the innate immune system through PRRs. DCs express a wide repertoire of these PRRs. The binding of PAMP to its receptors on the APC activates the DCs (28, 29).

DAMPs: Signal 0s from Host

Matzinger proposed what is known now as the “danger theory” in 1994 (30). In the theory, it proposed that the immune system can distinct self from non-self and dangerous from innocuous signals. In this model, APCs are activated by both PAMPs and DAMPs from distressed or damaged tissues or microbes. The theory has been well accepted in recent years, as we have learned more and more about how dying cells alert immune system to danger (31). Over the years, a number of endogenous danger signals have been discovered. For examples, it was shown that uric acid functions as a principal endogenous danger signal, which is released from injured cells (32).

Damage-associated molecular patterns are molecules derived from normal cells that can initiate and perpetuate immunity in response to cell stress/tissue damage in the absence of pathogenic infection. DAMPs vary greatly depending on the type of cell and injured tissue. They can be proteins, DNA, RNA, or metabolic products. Protein DAMPs include intracellular proteins, such as high-mobility group box 1 (HMGB1), heat-shock proteins (HSPs), and proteins in the intracellular matrix that are generated following injury, such as hyaluronan fragments (33). HMGB1 is one prototypic DAMP (34, 35). The protein DAMPs can be localized within the nucleus, cytoplasm, cell membrane, and in exosomes, the extracellular matrix, or as plasma components (17). Other types of DAMPs may include DNA, ATP, uric acid, and heparin sulfate. It is interesting to note that mitochondria are a rich and unique source of DAMPs, including formyl peptides, the mitochondrial DNA (mtDNA)-binding proteins, transcription factor TFAM, and mtDNA itself (36). Following interactions between DAMPs and PRRs on the target cells, the intracellular signaling cascades triggered by the interactions between DAMPs and PRRs lead to activation of genes encoding inflammatory mediators, which coordinate the elimination of pathogens, damaged, or infected cells (27). In cancer, chronic inflammation and release of DAMPs promotes cancer, while acute inflammation of release/presentation of DAMPs may induce potent antitumor immunity and helps in cancer therapy (35, 37). Based on the work in chemotherapy and radiation therapy, the concept of ICD of cancer cells has been established about 10 years ago (37, 38). As we will discuss below, this concept leads to development of novel strategies for cancer therapeutics.

OVs Induce Mostly Multimodality ICD and Release/Present Danger Signal Molecules

Investigators have long been interested in what defines the immunogenicity of cancer cells and how we can enhance the immunogenicity for the purpose of immunotherapy. Pioneering work by Lindenmann and Klein almost half a century ago demonstrated that viral oncolysis of cancer cells by influenza virus increases immunogenicity of tumor cell antigens (39). However, it was not clear how this immunogenicity was enhanced at the time. Over a decade ago, it was found that tumor immunogenicity is enhanced by cell death via induced expression of HSPs (40). A few years ago, investigators working on chemotherapy and radiation for cancer therapy have led to this new concept as they classify the types of cancer cell death by the immunological consequence, into “immunogenic cancer cell death” (ICD) and “non-immunogenic cancer cell death” (NICD) (4143). The original concept of ICD includes only “immunogenic apoptosis.” We and others have recently proposed that ICD includes not only immunogenic apoptosis, but also necroptosis, necrosis, autophagic cell death, and pyroptosis of cancer cells (Figure 1) (44, 45). Basically, cancer cells dying via ICD have the following common features as summarized by Tesniere, Zitvogel, Kroemer, and their colleagues (46). They stated that, “some characteristics of the plasma membrane, acquired at pre-apoptotic stage, can alarm immune effectors to recognize and then attack these pre-apoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response, elements of the endoplasmic reticulum stress response, as well as elements of the apoptotic response” (46). For cells undergoing pre-apoptotic phase, they may express “danger” and “eat-me” signals on the cell surface (calreticulin and HSPs) or can secrete/release immunostimulatory factors (cytokines, ATP, and HMGB1) to stimulate innate immune effectors (46). For other types of ICD, extracellular ATP, HMGB1, uric acid, other DAMPs, and PAMPs released in the mid or late phases functions as potent danger signals, thus making it highly immunogenic.

FIGURE 1

 

Figure 1. Four key modes of cancer cell death and their immunogenicity. In classic apoptosis, the retention of plasma membrane integrity and the formation of apoptotic bodies render it an immunologically silent death mode, or non-immunogenic cell death. However, recent studies have shown that cancer cells treated with certain cytotoxic agents (some chemotherapeutic agents and oncolytic viruses) lead to the cell surface exposure of calreticulin (ecto-CRT) and heat-shock proteins (HSPs) prior to apoptosis, and other DAMPs released in the later phase of apoptosis, danger signals to DCs. This is immunogenic apoptosis. Cancer cells dying by necrosis/necroptosis or pyroptosis secrete pro-inflammatory cytokines and release their cytoplasmic content, including DAMPs (ATP, HMGB1, and uric acid, etc.), into the extracellular space. Some DAMPs (such as HMGB1) can be secreted through non-classical pathways (25). These later modes of cancer cell death are ICD. Drawings are modified and reprinted from Lamkanfi and Dixit (47), copyright 2010, with permission from Elsevier.

Oncolytic viruses kill cancer and associated endothelial cell through a variety of types of cell death as classically defined by the morphological and ultrastructural changes of dying cells. These include apoptosis, necrosis, necroptosis, pyroptosis, and autophagic cell death, often with one as the predominant form of death for a particular OV. By the new definition, cancer cell death induced by OVs is mostly immunogenic (Table 1). Probably all oncolytic Ads induced autophagic cell death in cancer cells (4851). Coxsackievirus B3 (CVB3) induces immunogenic apoptosis in human non-small cell lung cancer cells (52). Measles virus (MV) causes ICD in human melanoma cells, because inflammatory cytokines and HMGB1 are released, and DCs are activated by MV-infected cancer cells (53). HMGB1 release often happens in late stage of apoptosis, during autophagy process and in necrotic cells infected with OVs. We first reported in 2005 that human cancer cells infected by an oncolytic poxvirus, led to necrotic/apoptotic death pathways and release of HMGB1 (54). Later studies have confirmed and extended the findings of HMGB1 release in cancer cells infected with Ads (12), CVB3 (52), an MV (53), vaccinia viruses (VVs) (5557), HSV (14, 58), and parvovirus H-1 (H-1PV) (59). Extracellular ATP is another potent danger signal released from OV-infected cancer cells (12, 52, 56, 60). The third danger signal molecule released from OV-infected cells is uric acid (61). Some OVs may induce cell death partly through pyroptosis, a caspase-1 dependent inflammatory form of cell death (62). Both necrotic cells and pyroptotic cells release ATP more efficiently than apoptotic cells do. Pyroptotic cells, just like apoptotic cells, actively induce phagocytosis by macrophages using “eat-me” and “find-me” signals (63). Cytolytic immune cells, elicited by OVs or other agents, kill additional cancer cells leading to release of DAMPs such as HMGB1 (64). In summary, most OVs induce ICD of cancer cells and present/release a number of potent danger signals, and TAAs to DCs to trigger adaptive immune response (Table 1).

TABLE 1

 

Table 1. Oncolytic viruses lead to specific mode of immunogenic cell death and exposure/release of DAMPs/PAMPs.

Cancer cell death induced by some OVs has not been examined for their direct features of ICD. However, other properties suggest that cancer cells infected by the OV are immunogenic, or the viruses themselves are highly immunogenic. Newcastle disease virus (NDV) is a well-studied virus for its virology and immunostimulatory properties (76). NDV induces cancer cells into apoptosis (70), with autophagy taking place during the process (71). Human cancer cells infected by NDV show upregulation of HLA class I and II antigens, and costimulatory molecule ICAM-1, as well as induction of IFNs, chemokines (IP10 and RANTES) before apoptosis (72). Moreover, the inflammatory conditions and type I IFNs inhibit Treg cells (73). With these potent immunostimulatory properties, local administration of oncolytic NDV overcomes systemic tumor resistance to immunotherapy by blockade of immune checkpoints (74). Another RNA virus, reovirus, also induces cancer cells into apoptosis (77, 78), with autophagy taking place in the process (7981). Melanoma cells infected with reovirus release a range of inflammatory cytokines and chemokines while IL-10 secretion is abrogated (82). These molecules may provide a useful danger signal to reverse the immunologically suppressive environment of this tumor. Even more interestingly, reovirus can also interact with DCs directly and matured DCs activate NK and T cells (75) (Table 1). Those activated NK and T cells exert innate killing of cancer cells. This innate effector mechanism may complement the virus’s direct cytotoxicity and thus induced adaptive antitumor immunity, potentially enhancing the efficacy of reovirus as a therapeutic agent (75).

OV-Induced Autophagy in Cancer Cells Promotes Cross-Presentation of TAAs and Elicits Stronger Antitumor Immunity

Autophagy mediates sequestration, degradation, and recycling of cellular organelles and proteins, and intracellular pathogens. It is not too surprising that autophagy plays roles in both innate and adaptive immunity (17, 83). A number of OVs, such as Ad (4851), encephalomyocarditis virus (84), HSV (62, 85, 86), influenza virus (87), NDV (71), reovirus (7981), and VSV (84), induce autophagy in infected cancer cells. Evidence shows that autophagy may enhance tumor immunogenicity. One mechanism is that autophagic cells selectively release DAMPs such as ATP (88, 89), HMGB1 (90), and uric acid (61). The other mechanism is that autophagy promotes antigen cross-presentation from cancer cells by DCs to naïve T cells. It stimulates antigen processing for both MHC class II (91), and MHC class I pathways. These have been demonstrated for endogenous viral antigens during HSV-1 infection (85), and for cross-presentation of TAAs from uninfected cancer cells (92), and influenza A virus-infected tumor cells (93). In other words, autophagy within the antigen donor cells facilitates antigen cross-priming to generate TAA-specific or virus-specific CD8+ T cells (9295). This property has been explored for cancer vaccines (96), and for enhanced OV-mediated antitumor effects in the future (97).

Viruses Often Encode Specific Genes to Modulate Apoptosis, Autophagy, Necroptosis, and Possibly Other Death Pathways

Successful viral replication requires the efficient production and spread of progeny virus, which can be achieved through efficient evasion of host defense mechanisms that limit replication by killing infected cells. Viruses have thus evolved to encode genes whose products function to block or delay certain cell death pathways until sufficient progeny have been produced (47). These gene-encode products target a variety of strategic points in apoptosis, necroptosis, autophagy, or other death pathways. Table 2 lists some examples of genes encoded by viruses especially OVs that can intervene apoptosis, autophagy, or necroptosis. The presence of these types of viral genes may skew the mode of infected cancer cells from one to another cell death pathway(s). OVs can be engineered genetically with deletion or insertion of such genes so that a desired mode of ICD would happen in the virus-infected cancer cells.

TABLE 2

 

Table 2. Examples of viruses and viral genes modulating apoptosis, autophagy, and necroptosis.

Cancer Cells Often Show Defects in Certain Cell Death Pathways

Every cell in a multicellular organism has the potential to die by apoptosis. However, cancer cells often have faulty apoptotic signaling pathways evolved during carcinogenesis. This property derives from the overexpression of anti-apoptotic genes, deficiency of pro-apoptotic genes, or both (121). These defects not only increase tumor mass, but also render the cancer resistant to therapy.

Evidence has also been accumulating that necroptosis can be impaired in cancer cells. Chronic lymphocytic leukemia cells have defects in signaling pathways involved in necroptosis regulation such as RIP3 and the deubiquitination cylindromatosis (CYLD), an enzyme directly regulating RIP1 ubiquitination (122). Skin cancer cells contain an inactivating CYLD mutation (123). Despite the fact some cancers are resistant to necroptosis due to genetic and epigenetic defects, necroptosis undoubtedly represents an important death pathway induced by many anticancer regimens, particularly important to those cancer resistant to apoptosis. In this case, investigators have found that some compounds can circumvent cancer drug resistance by induction of a necroptotic death (124).

The fact that cancer cells resist certain death pathways will dictate to a degree which types of drugs (including OVs) to be used in therapeutic regimens. As we stated before, a number of OVs, such as VVs, often induces cancer cells into necroptotic cell death (54, 56, 57), while other viruses such as oncolytic Ad often induce cancer cells into autophagic cell death. Appropriate OVs can be picked depending on the sensitivity of the cancer to certain death pathways, and the immunogenic consequence if it is combined for immunotherapy.

Strategies to Modulate the Mode of Cancer Cell Death for Enhanced Immunogenicity

We know now that immunogenic apoptosis, necrosis/necroptosis, and autophagic cell death are desired modes of cancer cell death because they are ICD. Is immunogenic apoptosis (the original form of ICD) better than other forms of ICD in the induction of antitumor immunity? We do not know for sure. This question needs to be addressed in the future. What we do know now is that there are strategies that can enhance the ICD and subsequent antitumor immunity. They can be classified into, genetic modification of OV vectors, combination with ICD inducers, and combination with specific immunostimulatory regimens.

Genetic Engineering of Viral Vectors

Cancer cells have usually accumulated a number of genetic mutations and epigenetic modifications that enable them to resist apoptosis. Based on this property, a number of OVs are built for high tumor selectivity by deleting viral genes encoding anti-apoptotic genes (see Table 2). These viruses can replicate in cancer cells but lead to rapid apoptosis in normal cells. For examples, the γ34.5 gene has been deleted in many oncolytic HSVs, including the T-VEC that is going through a successful phase III clinical trial (125). The adenoviral protein E1B-19K is a Bcl-2 homolog that blocks apoptosis induction via the intrinsic and extrinsic pathways, specifically including tumor necrosis factor (TNF)-mediated cell death. Liu et al. have demonstrated that an E1B-19K gene deletion mutant had TNF-enhanced cancer selectivity due to genetic blocks in apoptosis pathways in cancer cells (126). Similarly, a tumor-selective oncolytic vaccinia virus was constructed by deleting two serpin genes, SPI-1 and SPI-2 (54). Due to the deletion of viral anti-apoptosis genes, these mutant OVs display more potent oncolysis through apoptosis pathways when combined with appropriate apoptosis-inducing agents.

We believe that by arming OVs with necrosis and autophagy-promoting genes, it is possible that the desired cell death pathway can be activated in cancer cells when infected with such OVs, leading to more ICD. More future studies with this strategy are warranted.

Combination with ICD Inducer or Autophagy Inducer

In theory, OV in combination with an ICD inducer would provide more potent danger signals to DCs and potentially elicit stronger antitumor immunity. Workenhe et al. demonstrated in a recent study that such a strategy worked well indeed (127). HSV-1 ICP0 null oncolytic vectors possess antitumor activity, but the virus alone is insufficient to break immune tolerance. Thus, the authors hypothesized that combination therapy with an ICD-inducing chemotherapeutic drug might get the job done. Indeed, the combination of HSV-1 ICP0 null oncolytic virus with mitoxantrone, which induces ICD, provided significant survival benefit to the Balb/C mice bearing Her2/neu TUBO-derived mammary tumors. Increased infiltration of neutrophils and tumor antigen-specific CD8+ T cells into tumor tissues provide the protection, as depletion studies verified that CD8-, CD4-, and Ly6G-expressing cells are essential for the enhanced efficacy. Importantly, the combination therapy broke immune tolerance. In conclusion, this study suggests that such a combination can enhance the tumor immunogenicity, breaking immunologic tolerance established toward the tumor antigens, thus a promising novel strategy for cancer therapy (127).

As we stated earlier, the autophagy in antigen donor cells (cancer cells) promotes the cross-presentation of antigens from DCs to T cells. The autophagy could be induced by some OVs, or its inducer could be provided in trans. This strategy works in combination with oncolytic adenoviruses that induce autophagy by themselves (60, 128). However, it may not work with an oncolytic vaccinia virus that does not induce autophagy by itself (our unpublished data).

Armed Virus and Combination Strategies for Breaking Immune Tolerance and Enhancing Antitumor Immunity

In order to further enhance the antitumor immunity, OVs have been armed with TAAs, cytokines (e.g., GM-CSF), chemokines (such as CCL5), or other innovative and artificial genes. We have recently reviewed the promising strategies of OVs in combination with other immunotherapeutic regimens (44). As we mentioned, two OVs in the most advanced stages of clinical trials, T-VEC, and Pexa-Vec, are HSV and VV armed with GM-CSF (125, 129). An oncolytic VV expressing the 4-1BBL T cell costimulatory molecule (rV-4-1BBL) showed modest tumor regression in the poorly immunogenic B16 murine melanoma model. However, rV-4-1BBL injection with lymphodepletion promoted viral persistence by reducing antiviral antibody titers, and promoted MHC class I expression, and rescued effector-memory CD8+ T cells. This significantly improved the therapeutic effectiveness of the oncolytic virus (130). Similarly, an unarmed oncolytic virus combined with anti-4-1BB agonist antibody elicits strong antitumor immunity against established cancer (56). We have also shown that the chemokine CCL5-expressing oncolytic VV in combination with a cancer vaccine or activated T cells resulted in better therapeutic effect in a MC38 colon cancer model (131). Recently, our collaborators have made an oncolytic VV encoding a secretory bispecific T cell engager consisting of two single-chain variable fragments specific for CD3 and the tumor cell surface antigen EphA2 [EphA2-T cell engager-armed VV (EphA2-TEA-VV)] (132). This virus retains its normal oncolytic potency and the secreted molecule also activates T cells. The virus plus T cells had potent antitumor activity in a lung cancer xenograft model. Thus, arming oncolytic VVs with T cell engagers may represent a promising approach to improve oncolytic virotherapy. In the context of OV-mediated cancer immunotherapy, it is interesting to observe the dual effects of antiviral immunity on cancer therapy. On one hand, the antiviral immunity may attenuate the replication of an OV and thus diminish the effect of direct oncolysis; on the other hand, antiviral immunity plays a key role for the therapeutic success of oncolytic virotherapy in some cases (11, 133).

The tumor-associated immune tolerance is a big obstacle in cancer immunotherapy. Some armed OVs (such as a GM-CSF-armed oncolytic Ad) can break immune tolerance and generated antitumor immunity in at least some human cancer patients (134). In other cases, an OV alone is not enough to break the immune tolerance in highly immunosuppressive TME (127). In these cases, a combination with an ICD-inducing chemotherapeutic drug may break the immune tolerance (127). Alternatively, an OV can be combined with an immune checkpoint inhibitor to achieve the same effect. During the preparation of this review, a study has just been published on such a strategy with oncolytic NDV and systemic CTLA-4 blockade. This combination led to rejection of pre-established distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models (74). It showcases the promise of such a combination strategy.

Conclusion and Perspectives

The TME in the advanced stage of disease is highly immunosuppressive (18). This immunological property is a double-edged sword for OV-mediated cancer therapy: good for viral replication but bad for the antitumor immunity. The evidence is accumulating that OVs not only kill infected cancer cells and associated endothelial cells by direct and indirect oncolysis, but also release/present danger signals to DCs and other professional APCs to elicit both antiviral and antitumor immunity. It has been demonstrated for a number of OVs, that the virus-elicited antitumor immunity plays a critical role in the overall efficacy of oncolytic virotherapy. As we and other colleagues have realized, ICD is important to elicit antitumor immunity (44, 45, 135).

In order to improve the potency of antitumor immunity, one key step is the initial presentation of danger signal (signal 0) and cross-presentation of TAAs (signal 1). Recent studies demonstrated that ICD of cancer cells leads to potent danger signals, and autophagy in antigen donor cells, in this case cancer cells and associated endothelial cells, enhance the cross-presentation of TAAs to naïve T cells by DCs. Genetic engineering and combination strategies can skew the cancer cell death into modes of ICD and autophagy, leading to potent and sustained antitumor immunity and thus enhancing the efficacy of oncolytic immunotherapy. Which mode of ICD in the context of OVs is the most potent way to elicit antitumor immunity needs careful investigation in the near future. It is also important to keep in mind that oncolytic viruses modulate cancer immunogenicity through multiple mechanisms (136). Other than the induced danger signals, they are out of the scope of this review article and thus have not been discussed. Finally, we and others believe that it is important to further test the idea that combination of OV with blockade of immune checkpoints for potent and sustained antitumor immunity would enhance this novel form of immunotherapy for cancer. We look forward to more exciting development of both preclinical and clinical studies with OVs as tools for cancer immunotherapy.

Author Contributions

Zong Sheng Guo collected and read relevant papers; designed and drafted the manuscript. David L. Bartlett and Zuqiang Liu have made suggestions to the manuscript. All authors have read and approved the final manuscript.

Conflict of Interest Statement

David L. Bartlett is a scientific advisor for and has financial interest with Jennerex Biotherapeutics, a biopharmaceutical company developing oncolytic virotherapy. The other authors declare no conflict of interest.

Acknowledgments

This work has been supported by the grants P01CA132714 and R01CA155925 from the National Institutes of Health. Additional Support was provided by David C. Koch Regional Therapy Cancer Center. We would like to thank Ms. Roshni Ravindranathan for critical reading and comments on the manuscript.

Keywords: immunogenic cancer cell death, DAMPs, PAMP, autophagy, tumor-associated antigen, cross-presentation, immune tolerance, antitumor immunity

Citation: Guo ZS, Liu Z and Bartlett DL (2014) Oncolytic immunotherapy: dying the right way is a key to eliciting potent antitumor immunity. Front. Oncol. 4:74. doi: 10.3389/fonc.2014.00074

Received: 07 February 2014; Accepted: 24 March 2014;
Published online: 10 April 2014.

Edited by:

Philippe Fournier, DKFZ, Germany

Reviewed by:

William L. Redmond, Providence Portland Medical Center, USA
Volker Schirrmacher, DKFZ, Germany
Karen Mossman, McMaster University, Canada

Copyright: © 2014 Guo, Liu and Bartlett. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Zong Sheng Guo, Department of Surgery, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA e-mail: guozs@upmc.edu

Autoria e outros dados (tags, etc)

por cyto às 14:43

Domingo, 27.04.14

Dendritic Cells Activation

Sci. Signal., 22 April 2014
Vol. 7, Issue 322, p. ec109
[DOI: 10.1126/scisignal.2005399]

EDITORS' CHOICE

Immunology

L[i]nc to Dendritic Cell Activation

Kristen L. Mueller

Science, AAAS, Washington, DC 20005, USA

Long noncoding RNAs (lncRNAs) are important regulators of gene expression, but whether they are important regulators of the immune system is poorly understood. Wang et al. identify a lncRNA expressed exclusively in human dendritic cells (DC), called lnc-DC, that is required for optimal DC differentiation from human monocytes and that regulates DC activation of T cells. Lnc-DC interacts with the transcription factor STAT3, which is also required for DC development and function, to prevent interaction with and to block dephosphorylation by tyrosine phosphatase SHP1.

P. Wang, Y. Xue, Y. Han, L. Lin, C. Wu, S. Xu, Z. Jiang, J. Xu, Q. Liu, X. Cao, The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science 344, 310–313 (2014). [Abstract] [Full Text]

Citation: K. L. Mueller, L[i]nc to Dendritic Cell Activation. Sci. Signal. 7, ec109 (2014).


The editors suggest the following Related Resources on Science sites:

In Science Signaling

REVIEWS
Noncoding RNAs
Potential Pathological and Functional Links Between Long Noncoding RNAs and Hematopoiesis

Bo-Wei Han and Yue-Qin Chen (20 August 2013)
Sci. Signal. 6 (289), re5. [DOI: 10.1126/scisignal.2004099]
 |  Gloss »  |  Abstract »  |  Full Text »  |  PDF »

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por cyto às 13:37

Domingo, 27.04.14

Imunoterapia melanoma

Kathy Boltz, PhD

April 22, 2014

New immunotherapy promising for advanced melanoma

A new type of immunotherapy that directs patients' immune responses toward tumor cell killing, IMCgp100, was well-tolerated and showed efficacy in some patients with advanced melanoma. These phase I clinical trial results were presented at the American Association for Cancer Research 2014 Annual Meeting, in San Diego, California.

“The part of the immune system widely acknowledged for its cancer-killing abilities is a type of white blood cells called the T cell, and IMCgp100 is a novel type of cancer drug designed entirely on this cancer-killing component of the immune system,” said Mark Middleton, MD, PhD, of the University of Oxford in the United Kingdom. “When delivered to a cancer patient, IMCgp100 travels around the body in the patient's blood stream, finds and binds tightly to the cancer cells, activates any adjacent T cell to kill the cancer cells, and recruits other parts of the immune system to help clear the disease.”

The drug is made up of two components: The first recognizes cancer cells and binds the drug tightly to the cancer cell; the second component works by binding to a specific molecule on any nearby T cell. This binding causes a cascade of immune activation that leads to destruction of the target cancer cell and also to the release of a range of immune-activating molecules, which serve to recruit other parts of the immune system.

“The drug is well-tolerated in advanced melanoma patients, and we have seen clinical responses in some of them,” said Middleton. “The one aspect that did surprise us is the extent of tumor inflammation that is possible to achieve from just a single dose of the drug, because we thought it might take several weeks to get going.

“The ability of IMCgp100 to target one of a largely unexplored class of molecular targets, HLA-peptides, opens the door to treatment of many forms of cancer for which no antibody-applicable target has yet been identified,” Middleton added.

This phase I trial determined toxicity and maximum tolerated dose of the drug. A total of 31 patients (18 men and 13 women) with late-stage and unresectable melanoma, including 60% of whom had received prior therapies, were recruited. Patients were enrolled in eight cohorts to receive 1 of 8 escalating doses of the drug. Those who tolerated the drug on day 1 went on to receive repeated cycles of six weekly doses.

The maximum tolerated dose was found to be 600 ng/kg. Adverse events included rash, fever, skin inflammation, and tumor flare, which Middleton explained were all expected from the drug's mode of action.

Partial responses occurred in four patients, with two of them meeting RECIST criteria and two being smaller responses. The responses of two patients lasted for more than 9 months, and one of these patients continues to be asymptomatic.

Middleton concluded that an improved dosing regimen should increase the level of clinical activity. A phase IIa trial is testing a weekly dosing regimen.

 

 

Kathy Boltz, PhD

April 21, 2014

Biomarker identifies melanoma patients who may respond to immunotherapy

                       
Biomarker identifies melanoma patients who may respond to immunotherapy

Among melanoma patients treated with PD-1 inhibitor MK-3475, those whose tumors had the protein PD-L1 had better immune responses and higher survival rates. These research results were presented at the American Association for Cancer Research 2014 Annual Meeting, in San Diego, California.

When the protein PD-L1, which is present on some melanoma tumors, binds to PD-1, a protein present on T cells, brakes are applied to these T cells, preventing them from attacking the cancer cells. The immunotherapy MK-3475 blocks PD-1, releasing the brakes on T cells and enabling them to attack the cancer cells.

This study found that among patients with melanoma who received MK-3475, those whose tumors had PD-L1 had an overall response rate of 46%, whereas those whose tumors did not have PD-L1 had an overall response rate of 17%. At 6 months, 64% of the patients whose tumors were PD-L1-positive had no disease progression, compared with 34% of those whose tumors were PD-L1-negative. Similarly, 86% of the patients whose tumors were PD-L1-positive were alive after 1 year, compared with 72% of those whose tumors were PD-L1-negative.

“We found a major difference in the response rates between patients with PD-L1-positive and PD-L1-negative tumors treated with MK-3475,” said Adil I. Daud, MD, codirector of the University of California San Francisco (UCSF) Melanoma Center, and director of melanoma clinical research at the UCSF Helen Diller Family Comprehensive Cancer Center. 

“Data from this study identifies PD-L1 as a robust marker in determining which melanoma patients may be well served when treated with MK-3475. However, we are studying more samples from randomized trials of PD-1 inhibitor versus ipilimumab or chemotherapy to establish the validity of this marker,” added Daud.

RELATED: Biomarker may help predict melanoma survival

To evaluate the relationship between tumor PD-L1 expression and clinical outcome, Daud and colleagues studied tumor samples collected from 195 patients recruited to a phase I clinical trial testing MK-3475 at three different doses. All patients had late-stage melanoma, and some of them had received prior treatment with another immunotherapy drug called ipilimumab.

The investigators measured the amounts of PD-L1 in the tumor samples and considered them PD-L1-positive if at least one cell per 100 tumor cells had the protein. They found that, of the 125 evaluable tumor samples, 89 were PD-L1-positive and 36 were PD-L1-negative.

Patients with PD-L1-positive tumors had disease that did not progress for about 50 weeks, while disease progressed at about 12 weeks for those with PD-L1-negative tumors.

The investigators also found that among patients whose tumors were PD-L1-positive, overall response rates between those who had and had not received prior therapy with ipilimumab (44% vs 47%) were not significantly different. Similarly, among patients whose tumors were PD-L1-negative, overall response rates between those who had and had not received prior therapy with ipilimumab (14% vs 17%) were not significantly different.

 

 

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por cyto às 13:30

Segunda-feira, 21.04.14

NOVEL BISPECIFIC ANTIBODY

                     

2013 April 1; 2(4): e23639. Published online 2013 April 1. doi:  10.4161/onci.23639

PMCID: PMC3654588

A novel bispecific antibody recruits T cells to eradicate tumors in the “immunologically privileged” central nervous system

Bryan D. Choi, 1 , 2 , 3 Ira Pastan, 4 Darell D. Bigner, 1 , 2 , 3 and John H. Sampson 1 , 2 , 3 ,*

1Duke Brain Tumor Immunotherapy Program; Division of Neurosurgery; Department of Surgery; Duke University Medical Center; Durham, NC USA

2Department of Pathology; Duke University Medical Center; Durham, NC USA

3The Preston Robert Tisch Brain Tumor Center at Duke; Duke University Medical Center; Durham, NC USA

4Laboratory of Molecular Biology; Center for Cancer Research; National Cancer Institute; National Institutes of Health; Bethesda, MD USA

*Correspondence to: John H. Sampson, Email: john.sampson@duke.edu

Abstract

Bispecific T-cell engagers (BiTEs) may break multiple barriers that currently limit the use of immunotherapy in glioblastoma patients. We have recently described a novel BiTE specific for a mutated form of the epidermal growth factor receptor, EGFRvIII, that exerts potent antineoplastic effects against established invasive tumors of the brain.

Keywords: bispecific antibodies, central nervous system neoplasms, epidermal growth factor receptor, immunotherapy, T lymphocytes

Glioblastoma (GBM) is the most common and most aggressive primary malignant tumor of the brain. Despite advances in surgical resection, radiation and chemotherapy, the prognosis of GBM patients remains dismal, with an expected overall survival at diagnosis of less than 15 mo.1 Although current therapies provide modest benefits, they are frequently associated with incapacitating toxicity, owing to their collateral effects on normal healthy tissues. Thus, there is great need for the development of safer, more effective treatments for patients affected by GBM.

Immunotherapy has emerged as an innovative approach that promises to eliminate tumor cells with unparalleled potency and precision. In particular, T cells have been shown to mediate successful antitumor immune responses.2 However, while promising, current T cell-based approaches rely on the adoptive transfer of lymphocytes expanded ex vivo, a process that is laborious, inconsistent, and in some cases complicated by the need for retroviral transduction.

In contrast, by using recombinant technologies, it has become possible to create highly specific antibody-based molecules that activate T cells against tumors, without the complexities associated with cell-based therapy. Through a divalent, “bispecific” design, these constructs tether T lymphocytes to tumor cells, resulting in a highly localized and specific T-cell activation with concomitant tumor cell lysis. A leading format of such molecules is known as “bispecific T-cell engager” (BiTE). Unlike other bispecific antibodies, BiTEs are able to activate even formerly unresponsive lymphocytes against tumor cells without the need for additional immunostimulatory signals or conventional antigen presentation via MHC molecules.3

In a recent manuscript published in the Proceedings of the National Academy of Sciences,4 we have reported the development of a novel BiTE called bscEGFRvIIIxCD3, which was designed to specifically redirect T cells against tumors expressing a well-characterized, mutated form of the epidermal growth factor receptor (EGFR), EGFRvIII.

 

 

EGFRvIII is frequently expressed on the surface of GBM cells as well as by many other neoplasms, but not by normal healthy tissues.5 Because of its exclusive tumor-specific expression pattern, EGFRvIII represents an ideal target for immunotherapy. In our hands, the EGFRvIII-targeting BiTE, bscEGFRvIIIxCD3, successfully activated human T cells against EGFRvIII-expressing target cells, in the absence of any additional immunostimulatory signal, resulting in the secretion of TH1-associated cytokines and tumor-cell lysis. This EGFRvIII-targeting BiTE was similarly effective in vivo. Thus, the intravenous administration of bscEGFRvIIIxCD3 induced consistent antitumor responses in mice bearing established, late-stage intracerebral gliomas, rapidly achieving complete remission rates as high as 75% in the absence of apparent toxicity.4

Given the exquisite tumor-specificity of EGFRvIII, we believe that our EGFRvIII-targeting BiTE represents a critical conceptual advance in the safety profile of the BiTE therapeutic platform. On the contrary, BiTEs that target antigens characterized by a promiscuous systemic expression pattern have been shown to elicit unintended autoimmune responses.6 The most recent example of this problem was recorded in clinical trials testing a BiTE specific for the pan-B-cell marker CD19, wherein patients affected by B-cell malignancies experienced not only dramatic disease regression but also an unwarranted ablation of healthy circulating B cells. Thus, perhaps the most obvious drawback of the BiTE technology unveiled to date is represented by the activation of immune responses against antigens that are also expressed by non-malignant cells. To the best of our knowledge, our work focused is the first example of a BiTE that targets a truly tumor-specific antigen like EGFRvIII.

One unexpected finding of our preclinical study was the ability of our EGFRvIII-targeting BiTE administered i.v. to treat established invasive tumors located beyond the blood-brain barrier (BBB). In order for BiTEs to exert antineoplastic affects against brain tumors, both BiTEs and T cells need to efficiently access areas that have long been considered as immunoprivileged (Fig. 1).

While circulating naïve T cells do not typically penetrate the central nervous system (CNS), activated T cells are known to traffic frequently across the BBB and into the CNS.7 Moreover, particles bound to the surface of antigen-specific T cells have been shown to localize to neoplastic lesions.8 However, whether this mechanism contributes in any way to the intracerebral accumulation of macromolecules like BiTEs has not been elucidated to date.

Another leading hypothesis is that in the complete absence of cross-reactivity with systemic antigens, highly specific antibodies targeting brain tumors may penetrate the CNS and accumulate over time at therapeutically relevant amounts, simply owing to their relative affinity for different tissues.9

Previous research suggests that BiTEs may actually promote the localization and retention of effector T cells at intracerebral sites. This concept is supported by clinical studies on a CD19-targeting BiTE, reporting that the peripheral activation of circulating effector memory T cells is temporally associated with unexplained, but transient, CNS side effects in multiple patients.6,10 Consistent with this, we observed that the intravenous administration of our EGFRvIII-targeting BiTE promotes the diffuse infiltration of peripheral lymphocytes within EGFRvIII-expressing brain tumors.

 

Further studies are underway to investigate these mechanisms, which have critical implications in multiple fields of medical research for which the physiology of the BBB and the delivery of therapeutic agents into the CNS are relevant.

 

Figure 1. A bispecific T-cell engager targeting EGFRvIII triggers immune responses against brain tumors. The systemic administration of an EGFRvIII bispecific T-cell engager (BiTE) results in its successful localization to EGFRvIII-expressing ...

 

In summary, the results of our preclinical study demonstrate that the EGFRvIII-targeting BiTEs may provide a safe, highly effective therapueutic option for GBM patients. Future studies will determine whether these results can be recapitulated in the clinical setting and whether BiTEs favorably interact with other therapies that are currently employed as a standard-of-care for GBM patients.

Disclosure of Potential Conflicts of Interest

The authors have a patent pending for EGFRvIII as a tumor-specific target for bispecific antibody therapy.

Footnotes

Previously published online: www.landesbioscience.com/journals/oncoimmunology/article/23639

References

1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups. National Cancer Institute of Canada Clinical Trials Group Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96. doi: 10.1056/NEJMoa043330. [PubMed] [Cross Ref]

2. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol. 2012;12:269–81. doi: 10.1038/nri3191. [PubMed] [Cross Ref]

3. Choi BD, Cai M, Bigner DD, Mehta AI, Kuan CT, Sampson JH. Bispecific antibodies engage T cells for antitumor immunotherapy. Expert Opin Biol Ther. 2011;11:843–53. doi: 10.1517/14712598.2011.572874. [PubMed] [Cross Ref]

4. Choi BD, Kuan CT, Cai M, Archer GE, Mitchell DA, Gedeon PC, et al. Systemic administration of a bispecific antibody targeting EGFRvIII successfully treats intracerebral glioma. Proc Natl Acad Sci U S A. 2013;110:270–5. doi: 10.1073/pnas.1219817110. [PMC free article] [PubMed] [Cross Ref]

5. Choi BD, Archer GE, Mitchell DA, Heimberger AB, McLendon RE, Bigner DD, et al. EGFRvIII-targeted vaccination therapy of malignant glioma. Brain Pathol. 2009;19:713–23. doi: 10.1111/j.1750-3639.2009.00318.x. [PMC free article] [PubMed] [Cross Ref]

6. Bargou R, Leo E, Zugmaier G, Klinger M, Goebeler M, Knop S, et al. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science. 2008;321:974–7. doi: 10.1126/science.1158545. [PubMed] [Cross Ref]

7. Odoardi F, Sie C, Streyl K, Ulaganathan VK, Schläger C, Lodygin D, et al. T cells become licensed in the lung to enter the central nervous system. Nature. 2012;488:675–9. doi: 10.1038/nature11337. [PubMed] [Cross Ref]

8. Cole C, Qiao J, Kottke T, Diaz RM, Ahmed A, Sanchez-Perez L, et al. Tumor-targeted, systemic delivery of therapeutic viral vectors using hitchhiking on antigen-specific T cells. Nat Med. 2005;11:1073–81. doi: 10.1038/nm1297. [PubMed] [Cross Ref]

9. Scott AM, Lee FT, Tebbutt N, Herbertson R, Gill SS, Liu Z, et al. A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors. Proc Natl Acad Sci U S A. 2007;104:4071–6. doi: 10.1073/pnas.0611693104. [PMC free article] [PubMed] [Cross Ref]

10. Klinger M, Brandl C, Zugmaier G, Hijazi Y, Bargou RC, Topp MS, et al. Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood. 2012;119:6226–33. doi: 10.1182/blood-2012-01-400515. [PubMed] [Cross Ref]

 

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por cyto às 14:13

Segunda-feira, 21.04.14

EGFR VIII Vacination Therapy (GLIOMAS)

EGFRvIII-Targeted Vaccination Therapy of Malignant Glioma

Bryan D. Choi, AB,1,2 Gary E. Archer, PhD,1,2,4 Duane A. Mitchell, MD, PhD,1,4 Amy B. Heimberger, MD,3 Roger E. McLendon, MD,2,4 Darell D. Bigner, MD, PhD,2,4 and John H. Sampson, MD, PhD, MHSc1,2,4

1 Duke Brain Tumor Immunotherapy Program, Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710

2 Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

3 Department of Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

4 The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, North Carolina 27710

Corresponding author: Bryan D. Choi, AB, Duke Brain Tumor Immunotherapy Program, Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710 (Email: bryan.choi@duke.edu )

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The publisher's final edited version of this article is available at Brain Pathol

See other articles in PMC that cite the published article.

Abstract

Given the highly infiltrative growth pattern of malignant glioma and the lack of specificity associated with currently available treatment regimens, alternative strategies designed to eradicate cancer cells while limiting collateral toxicity in normal tissues remain a high priority. To this end, the development of specific immunotherapies against targeted neoplastic cells represents a promising approach.

The epidermal growth factor receptor class III variant (EGFRvIII), a constitutively activated mutant of the wild-type tyrosine kinase, is present in a substantial proportion of malignant gliomas and other human cancers, yet completely absent from normal tissues. This receptor variant consists of an in-frame deletion, the translation of which produces an extracellular junction with a novel glycine residue, flanked by amino acid sequences that are not typically adjacent in the normal protein.

In this review, both preclinical and early clinical development of a peptide vaccine directed against  this portion of the EGFRvIII antigenic domain are recapitulated. Following vaccination, our group has demonstrated potent, redirected cellular and humoral immunity against cancer cells expressing the mutant receptor without significant toxicity. Additionally, the corresponding therapeutic outcomes observed in these studies lend credence to the potential role of peptide-based vaccination strategies among emerging antitumor immunotherapies in patients with malignant glioma.

INTRODUCTION

Glioblastoma multiforme (GBM) is the most common primary malignant brain neoplasm, representing over 50% of all tumors in this category diagnosed each year (15). It is also one of the most aggressive and difficult cancers to treat; despite standard multimodal therapy, including surgical resection and radiotherapy plus temozolomide (TMZ), GBM remains uniformly lethal, with a median survival of less than 15 months from the time of diagnosis (96). Recurrent tumors exhibit an even poorer prognosis with a progression-free survival of less than 20 weeks following currently available salvage therapy (102). Furthermore, although these outcomes represent remarkable advancements in the treatment of GBM, conventional strategies are ultimately limited by significant morbidity associated with nonspecific damage to normal cells and tissues (47). As a result, there is a clear need for more effective therapies that enable precise targeting of tumor cells while preserving an otherwise healthy milieu.

 

 

 

 

To this end, the immune system has emerged as a particularly promising approach. Over a century ago, Ehrlich first proposed that the body’s natural immune system, with its inherent specificity and biologic efficiency, could be redirected to eradicate targeted neoplastic cells while reducing collateral toxicity (27). It has since been established in numerous studies that proper immunological manipulation, namely via vaccination against tumor antigens, can result in the regression of even bulky and invasive human cancers (80). While this type of manipulation has been shown to take many forms—including cellular, humoral and myriad passive, active and adoptive strategies—the discussion continues with regard to the precise interplay necessary to achieve the greatest antitumor response. This uncertainty notwithstanding, the prospect of harnessing the discriminatingly potent nature of the human immune system remains a high priority, and the understanding of its potential role in the treatment of GBM grows accordingly.

CNS IMMUNOPRIVILEGE

Antitumor immunotherapies, in the context of intracerebral tumors, encounter a distinct set of challenges, one of the most prominent being that of central nervous system (CNS) immune privilege. The first studies to suggest the concept of limited immune surveillance in the CNS and other select tissues were first reported in 1948 by Sir Peter Medawar, who showed that allogeneic tissue grafts transplanted into the brains of experimental animals were not rejected (64). Later research in the area of neuro-immunology would support this finding based on unique characteristics that are now generally associated with the CNS: the presence of a specialized blood-brain barrier (BBB) and the absence of conventional draining lymph nodes as well as resident antigen-presenting cells (APCs) within the brain (30, 40).

While the CNS certainly exhibits immune privilege to some degree, a growing body of data suggests that its isolation from the immune system is not as complete as once believed. For instance, despite the BBB, immune cells have been shown to traffic to the brain relatively frequently (29, 44, 74), and, contrary to what was previously thought, antigen egress via cerebrospinal fluid (CSF) compartments and cervical lymphatics also appears to occur (19, 38). Furthermore, it has been proposed that specialized microglia (35) along with astrocytes (1) and certain cells of the choroid plexus epithelium (92) are able to mediate human leukocyte antigen (HLA) presentation, thereby functioning as surrogate APCs within the CNS.

CROSSING THE BBB

As previously mentioned, immune cells, specifically activated T lymphocytes, have the ability to penetrate the BBB under normal physiological conditions. This was first appreciated when experimental animals were injected intravenously with radioactively labeled T cell blasts, which were subsequently tracked to the CNS (29). Naïve T lymphocytes, however, are significantly restricted from entering the CNS, suggesting that penetration past the BBB is possible only after activation takes place (65). When it does occur, lymphocyte extravasation into the brain parenchyma is a highly regulated process mediated by several well-characterized adhesion molecules and chemotactic factors (29). Once inside the CNS compartment, whether T lymphocytes proliferate and differentiate within the brain microenvironment has yet to be established, as previous studies differ on this point; nevertheless, it has been shown that these cells do remain in the CNS for longer periods of time if given the opportunity to interact with their cognate antigen (25, 62).

Central memory T lymphocytes that alternatively enter the CNS via the choroid plexus (79) flux continuously throughout subarachnoid spaces, and have purportedly significant roles in routine CNS immunosurveillance. Subarachnoid-space macrophages and pericytes associated with CNS microvasculature are both considered to be critical in the presentation of recall antigens to this T cell population (29). At any given time, T lymphocytes represent over 80% of the approximately 150 000 cells normally found in the CSF of healthy individuals (29). As an absolute number, this somewhat diminutive quantity of cells may not be particularly relevant to immune responses within the brain parenchyma; however, it seems that these cells are relatively CSF enriched, given that lymphocytes typically compose less than 5% of all leukocytes present in circulating blood.

In its intact state, the BBB is thought to be poorly permeable to antibodies. This assumption stems from the observation that CSF titers in normal individuals are relatively low, especially in comparison to those measured in peripheral blood. Generally, the rate of immunoglobulin diffusion into the CNS varies depending on the molecular weight of a given protein (75); as an example, the physiological CSF/serum ratios for IgM and IgG have been quoted to range from 0.005% to 0.025% and 0.16% to 0.32%, respectively, reflecting the difference in size between these molecules (7). Although these limited ratios undoubtedly evidence CNS immune privilege to some degree, classic animal experiments have verified that, after both active and passive immunizations, corresponding antibodies can be detected within the CNS, specifically the brain, spinal cord and CSF (33). However, the reported fraction eventually isolated from these areas was again notably small—0.1% to 1% of that found in serum.

The theoretical possibility that even small amounts of antibody can cross the BBB and have physiologically relevant effector functions in the CNS is supported by the recent development of promising vaccines for patients with Alzheimer’s disease (AD). These vaccines target amyloid-β (AB), the cleavage product of amyloid precursor protein (APP); mutations in which have been shown to lead to parenchymal amyloid plaque accumulation (39, 91) in addition to other pathological features and clinical manifestations of AD (53, 60). Initial experiments using transgenic mice expressing mutant APP have shown that active immunization with the AB peptide reduces plaque burden and improves behavioral end points (13, 51, 88). This provided the first evidence that an immune response can be used as a potential treatment for AD, in theory by preventing formation of amyloid deposits and mediating clearance of preexisting plaques. Subsequent studies confirmed that the therapeutic effects of the vaccine are, at least in part, due to an antibody-mediated mechanism. This was primarily demonstrated by animal experiments showing that peripherally administered AB-specific antibody enters the CNS, localizes to plaques and achieves amyloid clearance mimicking that observed in previous mouse studies employing active immunization strategies (3, 4). Several hypotheses have been offered regarding the mechanism behind antibody-mediated plaque clearance in AD (26, 94). Of these, one prominent theory states that passively administered antibody sequesters AB peptide in the periphery without crossing the BBB, thereby generating a concentration gradient favoring efflux out of the brain (24). Other suggested antibody mechanisms rely on passage of antibody across the BBB; these include direct plaque disaggregation (93) and Fc receptor-mediated microglial phagocytosis (4).

 

 

 

 

Given these conclusions, the concept of BBB permeability in the absence of frank inflammation appears to be garnering support. However, it has long been asserted and widely accepted that in the presence of neuroinflammatory disease states—including experimental autoimmune encephalitis, meningitis and cancer—the BBB undergoes changes that alter its ability to block the migration of leukocytes and serum proteins into the CNS (23). Furthermore, by virtue of their existence, paraneoplastic syndromes clearly demonstrate that such changes in the BBB occur, and that these changes are in fact clinically significant. Most paraneoplastic neurological disorders (PND) are likely immune mediated (21), as suggested by the demonstration of antineural antibodies in the CNS of patients with peripheral tumors. These antibodies represent the body’s natural immune reactivity against systemic tumor antigens, and cross-reactivity with neurological structures has been found to result in significant morbidity. A number of paraneoplastic antibodies have been cited in the involvement of PND pathogenesis; these include anti-Hu, anti-Yo, anti-Ri, anti-CV2/CRMP5, anti-Ma and anti-amphiphysin antibodies (21).

Because pathological antibodies have been shown to cross the BBB in the context of malignancy, it follows that peripherally administered therapeutic antibodies should also have access to the intracerebral environment with physiologically relevant outcomes. The development of radiolabeled monoclonal antibodies (MAbs) for the diagnosis and treatment of brain tumors was first explored by Day and coworkers in 1965 (22), and since then, numerous studies have supported that MAbs are capable of localizing to intracerebral malignancies. Using radioiodinated antitenascin MAb 81C6, our group has shown that not only does 81C6 exhibit therapeutic activity in mice with intracranial human glioma xenografts, but that selective tumor localization also occurs in patients with a variety of intracranial malignancies following peripheral administration of the antibody (109). However, tumor-specific uptake of 81C6 remained quite low at less than 5 × 10− 3% of the injected dose per gram, and nonspecific antibody accumulation also took place in other tissues besides the brain including the liver, spleen and bone marrow. In contrast to what was observed with 81C6, results from later human studies using radiolabeled chimeric ch806, a MAb specific for the epidermal growth factor receptor class III variant (EGFRvIII) tumor antigen, suggest that higher-percentage BBB penetration may be achieved in the absence of cross-reactivity with systemic antigens, effectively creating an intracerebral antibody sink at the tumor site (90). Using single-photon emission computed tomography, this potential effect was observed given the physiological localization of Indium-111-labeled ch806, which was noted to accumulate within intracranial target lesions without visual evidence of nonspecific, residual binding in normal tissues (Figure 1).

                       

Figure 1

Targeting of glioma by radiolabeled chimeric monoclonal antibody directed against the EGFRvIII tumor antigen. (A–C) Planar images of the head and neck obtained on day 0 (A), day 3 (B), and day 7 (C) after infusion of 111In-ch806. Initial blood ...

 

TUMOR-SPECIFIC REJECTION ANTIGENS

The landmark paper published over two decades ago by van Pel and Boon rekindled the then waning interest in cancer vaccine development when it suggested that even nonimmunogenic tumors display sufficiently “foreign,” and therefore immunologically susceptible antigen profiles (101). Since that time, a great deal of effort has gone toward characterizing a variety of human tumor antigens, the majority of which can now be placed into one of two main categories: those consisting of overexpressed normal gene products or, alternatively, those derived from mutations in somatic genes (36, 37).

Most well-characterized targeted tumor antigens isolated to date correspond to overexpressed proteins that are also present in normal cells, two examples of which include CD20 and erbB2, proteins associated with lymphoma and breast cancer, respectively. The ability of antigens in this category to mediate optimal tumor rejection, however, is often compromised by the fact that proteins that are also found on normal cells have the potential to trigger immunologic tolerance to varying degrees. Notable exceptions to this limitation include antigens associated with fetal gene products, such as carcinoembryonic antigen (50), or those expressed solely in immunoprivileged, tissue-specific sites like the testes. The latter group includes the melanoma MAGE, GAGE (100), and BAGE (11) family antigens, all of which, due to their limited expression, trigger little to no tolerance and should therefore make ideal tumor rejection antigens.

Cancer vaccination protocols that effectively target normal gene products invariably pose the risk of autoimmune toxicity (37). This untoward effect can be avoided to some extent by directing the immune response against a mutated protein specific only to tumor cells. As targets, these antigens have the advantage of avoiding central tolerance mechanisms, in theory, making them more suitable for tumor rejection. However, a limitation of these antigens is that they are generally patient specific as they often reflect random mutations associated with the inherent genetic instability of tumors (56, 59). Thus, to the extent that mutated gene products are incidental to the oncogenic process, they are conceivably restricted in their use as practical targets of widely applicable cancer vaccines. Conversely, although the majority of somatic mutations in tumors does appear to be sporadic (57), recent studies using high-throughput screening have suggested that several functional mutations associated with, rather than incidental to, the oncogenic process are, in fact, not random, and that these variants are consistently shared among patients (98). The challenge, then, is to isolate and target these ideal antigens: frequent, highly specific, oncogenic mutations that are also absent from normal tissues, thereby avoiding the risk of autoimmunity. To date, few such antigens are known, although their discovery represents a potential boon for the further development of effective antitumor immunotherapies.

EGFRVIII TUMOR-SPECIFIC ANTIGEN

Among the many antigens that have been shown to be overexpressed on tumor cells, the type I epidermal growth factor receptor (EGFR) represents one of the most frequently implicated cell-surface markers for a wide range of human malignancies. Functionally, the EGFR has well-characterized roles in oncogenesis and tumor progression, and as such, amplification and overexpression of the EGFR gene is considered a poor prognostic indicator (72).

 

 

 

Regarding intracerebral cancers in particular, the EGFR gene is amplified in up to 50% and overexpressed in over 90% of GBM specimens (28, 49), suggesting significantly augmented cellular activity of this receptor in these tumors.

The EGFR is a 170-kDa transmembrane glycoprotein, consisting of an extracellular ligand-binding domain and an intracellular region with tyrosine kinase functionality (95). Activation via stimulatory interactions with growth factors—including epidermal growth factor (EGF) and transforming growth factor-α—results in receptor dimerization and subsequent intracellular autophosphorylation on tyrosine residues, in turn leading to the activation of downstream molecules associated with cellular mitogenesis and survival (Figure 2) (14). Given the nature of these potentially oncogenic pathways, it was originally believed that the impact of EGFR on neoplastic processes was exclusively due to amplification of its corresponding gene. However, it is now clear that many tumors, including GBM, also express rearranged, aberrant forms of the EGFR gene that have significant physiological relevance (28, 32). Several of these mutations have been reported in the literature and are typically associated with tumors that also exhibit extensive wild-type gene amplification (58, 107).

 

Figure 2 EGFR downstream signaling in cancer cells. Figure reproduced with permission from reference (6).

The most common and well-characterized EGFR mutant was first identified in primary human GBM tumors and is commonly referred to as the EGFR class III variant (EGFRvIII). EGFRvIII is a constitutively active, ligand-independent form of the EGF wild-type receptor (5, 45), the expression of which has been shown to have tumorigenic effects, both augmenting proliferation and inhibiting apoptosis (5, 73). Specifically, EGFRvIII has also been shown to promote greater cellular motility (12, 76) as well as resistance to radiation and chemotherapy (54, 55, 68), characteristics often associated with highly malignant tumors.

A number of molecular mechanisms have been implicated in the oncogenic pathways coupled with EGFRvIII downstream signaling. In the absence of ligand binding and dimerization, for example, EGFRvIII has been observed to constitutively interact with adaptor proteins central to the Ras cascade (17, 77). Similarly, growth advantage in cells expressing EGFRvIII has been attributed at least in part to elevated phosphatidylinositol (PI) 3-kinase levels and consequent activation of the c-Jun N-terminal kinase pathway (2, 70). The respective involvement of, and interplay among, these signals in neoplastic processes have yet to be fully described; however, it has been shown that malignant cells become dependent on these pathways to some extent, and that removal of such stimulation results in reduced cell survival (103).

 

 

 

 

Structurally, EGFRvIII is an 801 base pair in-frame deletion of the wild-type receptor that corresponds to mRNA exons 2–7, the absence of which leads to the translation of a truncated extracellular domain (Figure 3). A consequence of this deletion–mutation is the fusion of two otherwise distant portions of the molecule, which in turn creates an antigenic junction characterized by a novel glycine residue, flanked by amino acid sequences that are not typically adjacent in the wild-type receptor (10, 58). This tumor-specific epitope has been shown to be present on the surface tumor cells, yet completely absent from any normal adult tissues (46).

 

Figure 3 Schematic diagram of the EGFR wild-type protein showing the area of in-frame deletion which forms EGFRvIII. During the deletion, amino acids 6 and 273 are split forming a novel glycine at the junction of amino acids 5 and 274. PEPvIII is a 13 amino acid ...

Immunohistochemical (IHC) analysis represents one of the most common assays used to identify the EGFRvIII mutant along with a number of second messenger molecules also expressed in malignant cells (Figure 4). Alternative approaches to IHC which employ molecular techniques such as Western blotting and reverse transcription-polymerase chain reaction assays are currently being explored and have confirmed the specific expression of EGFRvIII in human GBM specimens; to date, data derived from IHC studies have been shown to be consistent with results obtained using other methods (31). As evidenced by IHC, EGFRvIII is consistently expressed in a wide variety of cancers, and can be found in approximately 20% of GBM specimens (69). Within such tumor samples, the proportion of EGFRvIII-expressing cells has been shown to range from 37% to 86% (105), suggesting that cells within EGFRvIII-positive tumors may translate the variant receptor with at least some level of homogeneity. Thus, given its oncogenic properties, inherent tumor specificity and frequent expression in malignancy, the EGFRvIII mutation represents a particularly attractive tumor-specific target for the development of anticancer immunotherapies (52, 106).

 

Figure 4 A. High-grade astrocytoma used in subsequent immunohistochemical assays (hematoxylin–eosin, original magnification ×400). B. Antiepidermal growth factor receptor (anti-EGFR) wild-type immunohistochemistry showing strong diffuse cytoplasmic ...

 

 

 

 

NATURE OF THE PEPTIDE VACCINE

Generally, immunotherapy can be divided into either active or passive approaches (81). Active immunizations rely on the natural immune system to mount physiological responses against specific antigens that are either inoculated directly into the body or are instead presented on autologous APCs. Prior to vaccination, APCs are pulsed with the appropriate antigen, cancer cells or lysates thereof, and are thereby loaded with the immunogenic material of interest. Passive vaccination strategies involve either the direct infusion of antibodies or, alternatively, the adoptive transfer of antigen-specific T lymphocytes.

Currently, a number of immunotherapeutic approaches targeting the unique EGFRvIII antigen are under investigation. Given the technical difficulty and relatively high cost of dendritic cell (DC) vaccination therapy, the most promising and practical active vaccination format to date is a peptide derived from the novel fusion junction amino acid sequence. PEPvIII (H-Leu-Glu-Glu-Lys-Lys-Gln-Asn-Tyr-Val-Val-Thr-Asp-His-Cys-OH) (71) is a well-characterized, EGFRvIII-specific, 14-mer peptide that has been shown, when coupled to keyhole limpet hemocyanin (KLH), to elicit both humoral and cellular immune responses. Our group has extensive experience with PEPvIII-KLH, and we have clearly demonstrated the induction of considerable EGFRvIII-specific immune responses in both murine tumor models and early clinical trials.

EGFRVIII: PRECLINICAL STUDIES

Our group has shown that passive administration of EGFRvIII-specific antibodies Y10 and L8A4 (unarmed murine IgG2a and IgG1, respectively) leads to significant tumor growth inhibition in subcutaneous murine melanoma models. These studies, which use syngeneic tumors transfected with a murine homolog of the variant receptor (msEGFRvIII), have shown that while these two MAbs achieve therapeutic efficacy when given intraperitoneally, only those mice treated with Y10 exhibit lasting tumor-free survival after treatment is discontinued (83). Evidence from in vitro studies suggests that Y10 has the ability to mediate a wide range of effector functions when incubated with cells expressing msEGFRvIII. These functions include the inhibition of DNA synthesis and cellular proliferation, as well as the activation of autologous, complement-mediated and antibody-dependent cell-mediated cytotoxicity.

Active vaccination strategies targeted against msEGFRvIII in syngeneic murine tumor models have also been proven to be effective. Following intraperitoneal injection with DCs pulsed with PEPvIII-KLH, C3H mice that had previously been challenged with intracerebral tumors demonstrated a significant increase in median survival. Furthermore, all the mice in this study survived rechallenge with tumor, suggesting that immunization was sufficient to create long-term immunological memory against the msEGFRvIII antigen in this model system (42). Following this experiment, we conducted a similar study in which C3H mice were treated using a one-time vaccination, this time with PEPvIII-KLH in complete Freund’s adjuvant as opposed to the DC vaccine. This vaccine protocol also resulted in increased median survival and, ultimately, long-term survival in nearly half of the mice (43). Notably, mice with tumors that failed to exhibit responses to the PEPvIII-KLH vaccine were found to have IHC evidence of down-regulated or completely absent EGFRvIII expression, suggesting that antigen escape variants may be associated with failure to adequately treat some tumors.

 

 

EGFRVIII: CLINICAL STUDIES

Our group has also demonstrated, in clinical trials, induction of EGFRvIII-specific immunity with vaccines targeted against the EGFRvIII tumor-specific antigen. A number of EGFRvIII-derived cytotoxic T lymphocyte epitopes have been characterized to date (108), and previous data have shown that EGFRvIII-specific antibody titers, while absent in normal volunteers, may be detectable in patients with tumors expressing the mutant receptor (78). It is still unclear, however, whether cellular or humoral responses will ultimately provide the critical mediators for specific antitumor eradication using our approach.

Our first clinical study evaluating the toxicity and potential efficacy of EGFRvIII-based vaccinations began with a Phase I trial (VICTORI) (86) conducted at Duke University Medical Center (PI: John H. Sampson).

Fifteen adults with newly diagnosed GBM (WHO grade III or IV) were enrolled in the study; criteria for eligibility did not include EGFRvIII expression. Of the 15 patients, three did not ultimately receive vaccine due to progression of their tumors during external beam radiotherapy (EBRT). Following gross-total tumor resection and completion of EBRT, 12 patients underwent leukapheresis to obtain peripheral blood mononuclear cells in preparation for DC generation and immunologic monitoring. Prior to inoculation, DCs were pulsed for 2 hours with 500 μg PEPvIII peptide (Anaspec, San Jose, CA, USA) conjugated to KLH (Biosyn, Carlsbad, CA, USA). In total, patients received up to 1.1 × 108 DCs in three equal doses, injected intradermally every 2 weeks into the upper thigh, 10 cm below the inguinal ligament. Patients were followed for toxicity and evidence of radiographic or clinical progression.

Patients in the VICTORI trial did not suffer serious adverse events exceeding Grade II toxicity at any DC dose tested (National Cancer Institute Common Toxicity Criteria). Blood drawn from patients following vaccination showed ex vivo evidence of antigen-specific cellular and humoral immune responses. Median survival for the 12 patients was 18.7 months after vaccination (CI95 14.5, 25.6) and 22.8 months after histological diagnosis (CI95 17.5, 29). These outcomes improve on what would have been expected by chance, according to Curran’s recursive partition analysis (20). Eight of the 12 patients in this study belonged to group III, and the remaining four belonged to group IV, which have estimated survivals of 17.9 and 11.1 months, respectively. While nine of the 12 patients in our study surpassed these estimates, the increase in survival was not statistically significant (P = 0.083; binomial proportions), although these results may be negatively biased by the fact that EGFRvIII expression was not a criterion for inclusion in this Phase I toxicity trial.

Nevertheless, the outcomes associated with our DC-based, PEPvIII-specific vaccine were encouraging and warranted further testing at different centers; however, the inherent cost and variability associated with autologous DC manufacturing made this approach impractical on a large scale. Thus, given the success of our preclinical studies, we decided to proceed with a Phase II multicenter trial (ACTIVATE) (41) without the use of DCs, instead administering PEPvIII-KLH directly in combination with granulocyte macrophage-colony stimulating factor (GM-CSF).

 

 

 

ACTIVATE, a Phase II, multicenter clinical trial conducted at Duke (PI: John H. Sampson) and University of Texas, M.D. Anderson Cancer Center (PI: Amy B. Heimberger), enrolled 19 adults who all had EGFRvIII-expressing, newly diagnosed primary GBM (WHO Grade IV). Prior to receiving the KLH-conjugated peptide vaccines, patients underwent >95% volumetric tumor resection, along with standard of care radiation therapy with concurrent TMZ. Vaccinations consisted of intradermal injections with 500 μg PEPvIII-KLH (Anaspec) and GM-CSF, administered near the inguinal region in the upper thigh, on alternating sides. The first three vaccines were given biweekly, followed by monthly injections until radiographic evidence of tumor progression or death.

Similar to what was observed in VICTORI, patients participating in ACTIVATE did not experience serious adverse events aside from local reactions at the injection site. We demonstrated that this vaccine formulation elicits both humoral (89) and delayed-type hypersensitivity immune responses specific for PEPvIII and EGFRvIII in a number of patients, and that detection of these responses predicts greater median overall survival (OS). Median time-to-progression (TTP) following surgery in patients who received the vaccine is 12 months (n = 12), exceeding a median TTP of 7.1 months (n = 29) calculated from a historical matched unvaccinated control group (P = 0.0058). If and when tumors recurred, pathological samples were obtained and evaluated by IHC to determine EGFRvIII expression. Of the specimens examined in this trial, none were found to contain cells that display positive staining for EGFRvIII.

Following ACTIVATE, our lab initiated the ACT II trial (84), which enrolled a total of 21 patients who essentially followed the same treatment scheme as those in ACTIVATE, except for the addition of two different TMZ dosing schedules concurrent with vaccination cycles; patients either received 200 mg/m2 TMZ × 5/28 days [ACT IIA (n = 13)] or 100 mg/m2 TMZ × 21/28 days [ACT IIB (n = 8)]. While grade 2 TMZ-associated lymphopenia was observed in the majority of ACT II patients, we found that all immune responses were unexpectedly either sustained or enhanced with successive TMZ treatments. The seemingly paradoxical relationship between TMZ-induced lymphopenia and improved PEPvIII-KLH-specific immunogenicity is currently under further investigation.

In summary, these trials to date collectively show that vaccination with a peptide containing the PEPvIII tumor epitope safely elicits a specific immune response against EGFRvIII, and that this approach might be effective against cancers bearing the variant antigen. While our group has demonstrated significantly greater TTP and OS in GBM patients who have received the PEPvIII-KLH vaccine, definitive evidence for this promising effect will require confirmation from our ongoing randomized Phase III clinical trials.

DISCUSSION

While the mechanisms underlying the beneficial effects of our vaccine in patients with GBM are still unclear, it is our hope that additional experience with PEPvIII-KLH will elucidate our general understanding of various peptide vaccination strategies and their potential role in eliciting effective antitumor responses. Previous trials employing peptide vaccines have targeted a wide range of cancers including those of the colon, prostate, breast, cervix, pancreas and ovaries.

 

 

The most convincing evidence in favor of peptide vaccine efficacy comes from the melanoma literature, in which a number of tumor-associated antigens have been specifically targeted with relative success, some of which include MART-1 (16), MAGE-3 (61), tyrosinase (87), and NY-ESO-1 (48). Data from many early clinical vaccine trials for melanoma and other neoplasms have been fairly encouraging, although some have claimed that these results have been overly optimistic due to a reliance on subjective or “soft” end points. In 2004, Rosenberg et al suggested that criteria for clinical responses to cancer vaccines should only include objective measurements such as those denoting tumor size and volume. Taking these new parameters into account, he reassessed 35 National Cancer Institute trials involving a wide range of cancers and concluded that overall, there were only seven objective tumor responses out of a total of 175 patients (4%) who had received some form of peptide vaccine. At that time, the implications of these results could not be understated, and thus represented a turning point for the field of cancer immunotherapy. However, the conclusions drawn by Rosenberg and colleagues have since been challenged as excessively pessimistic and potentially misleading (67, 99). Opposing views suggest that the literature instead supports more favorable tumor response rates (10%) following treatment with peptide vaccines, and that this frequency may even be higher when employing the objective criteria as mandated by Response Evaluation Criteria in Solid Tumors (97). In addition, it was brought to attention that many patients receive vaccinations only after completion of standard chemotherapeutic regimens, conceivably making their tumors more aggressive and resistant to treatment when compared with those who had not received therapy. Finally, it has been suggested that a number of immunological variables (eg, adjuvants, HLA haplotypes) have yet to be explored in the context of peptide vaccines, knowledge of which might ultimately improve efficacy of existing therapeutic regimens. Regardless of the ongoing discussion on the future of active cancer vaccination strategies, Rosenberg et al and other authors agree that, at this stage, it would be unwise to interpret any lack of clinical evidence as an “investigational dead end,” insofar as present shortcomings may simply reflect the need for further exploration (82).

As the field of tumor immunology moves forward, in addition to the standardization of and adherence to objective end points, there are still a number of issues regarding the specific targeting of cancer epitopes by peptide vaccines that must be addressed. It is known, for example, that although a variety of tumors including malignant glioma have been found to express the EGFRvIII mutation, cells within these cancers often exhibit significant antigenic heterogeneity (8, 9, 104, 105). This confounds immunotherapeutic approaches designed to target single tumor-specific antigens, as even effective vaccines will fail to target those cells in the tumor that do not happen to express a given epitope. We observed the potential consequences of this issue in both murine studies (43) and clinical trials, in which the majority of recurrent tumors from vaccinated patients no longer expressed EGFRvIII. Thus, in the context of cancer vaccines, greater antitumor effects may be achieved with the development of multiantigenic vaccines that target the various aberrant biological processes often present in GBM and other tumors (34); the recently characterized expression of human cytomegalovirus antigens in GBM, for instance, may provide multiple targets for such an approach (18, 66). Alternatively, strategies that enrich EGFRvIII-positive cell populations prior to treatment may also prove to increase antitumor efficacy.

 

 

 

GBM is a devastating disease, and despite recent advances in antitumor therapy, there is still a pressing need for novel and effective approaches designed to potently eradicate tumor cells while minimizing toxicity to neighboring tissues. The potential ability to harness and redirect the cytotoxic power and inherent specificity of the immune system against neoplastic cells provides one such approach. As reviewed herein, our lab has shown that peptide vaccines can be employed to safely generate both B and T cell-mediated immune responses against tumor-specific epitopes such as the EGFRvIII antigen. Moreover, we have demonstrated that these responses are efficacious in murine models, and that similar therapeutic outcomes may be expected in humans with malignant glioma, even in the context of severe immunosuppression.

 

Autoria e outros dados (tags, etc)

por cyto às 14:12

Segunda-feira, 21.04.14

Cytokinas - DCs enriquecidas com anti-tumor T Cells

Cancer Immunol Immunother. 2013 Aug 27. [Epub ahead of print]

A cytokine cocktail directly modulates the phenotype of DC-enriched anti-tumor T cells to convey potent anti-tumor activities in a murine model.

Yang S, Archer GE, Flores CE, Mitchell DA, Sampson JH.

Source

Division of Neurosurgery, Department of Surgery, Brain Tumor Immunotherapy Program, Duke University Medical Center, 303 Research Drive, 220 Sands Building, DUMC 3050, Durham, NC, 27710, USA.

Abstract

Adoptive cell transfer (ACT) using ex vivo-expanded anti-tumor T cells such as tumor-infiltrated lymphocytes or genetically engineered T cells potently eradicates established tumors. However, these two approaches possess obvious limitations. Therefore, we established a novel methodology using total tumor RNA (ttRNA) to prime dendritic cells (DC) as a platform for the ex vivo generation of anti-tumor T cells. We evaluated the antigen-specific expansion and recognition of T cells generated by the ttRNA-DC-T platform, and directly modulated the differentiation status of these ex vivo-expanded T cells with a cytokine cocktail. Furthermore, we evaluated the persistence and in vivo anti-tumor efficacy of these T cells through murine xenograft and syngeneic tumor models. During ex vivo culture, IL-2 preferentially expanded CD4 subset, while IL-7 enabled homeostatic proliferation from the original precursors. T cells tended to lose CD62L during ex vivo culture using IL-2; however, IL-12 could maintain high levels of CD62L by increasing expression on effector T cells (Tem). In addition, we validated that OVA RNA-DC only selectively expanded T cells in an antigen-specific manner. A cytokine cocktail excluding the use of IL-2 greatly increased CD62Lhigh T cells which specifically recognized tumor cells, engrafted better in a xenograft model and exhibited superior anti-tumor activities in a syngeneic intracranial model. ACT using the ex vivo ttRNA-DC-T platform in conjunction with a cytokine cocktail generated potent CD62Lhigh anti-tumor T cells and imposes a novel T cell-based therapeutic with the potential to treat brain tumors and other cancers.

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por cyto às 14:06

Segunda-feira, 21.04.14

GENETERAPIA. ONDE ESTAMOS?

 

Gene Therapy for Glioma

Alex Tobias, Atique Ahmed, Kyung-Sub Moon, Maciej S LesniakJ Neurol Neurosurgery Psychiatry. 2013;84(2):213-222.  

 

  

 

Abstract and Introduction

Abstract

Glioblastoma multiforme (GBM) is a highly invasive brain tumor that is unvaryingly fatal in humans despite even aggressive therapeutic approaches such as surgical resection followed by chemotherapy and radiotherapy.

Unconventional treatment options such as gene therapy provide an intriguing option for curbing glioma related deaths. To date, gene therapy has yielded encouraging results in preclinical animal models as well as promising safety profiles in phase I clinical trials, but has failed to demonstrate significant therapeutic efficacy in phase III clinical trials. The most widely studied antiglioma gene therapy strategies are: suicide gene therapy, genetic immunotherapy and Oncolytic virotherapy, and we have attributed the challenging transition of these modalities into the clinic to four major roadblocks:

(1) anatomical features of the central nervous system,

(2) the host immune system, (

3) heterogeneity and invasiveness of GBM and

(4) limitations in current GBM animal models.

 

In this review, we discuss possible ways to jump these hurdles and develop new gene therapies that may be used alone or in synergy with other modalities to provide a powerful treatment option for patients with GBM.

Introduction

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults.[1] Today, the current standard of care consists of surgical resection followed by radiotherapy and chemotherapy.[2] However, the effectiveness of surgical resection is often compromised due to the lack of a defined tumor margin and a tumor burden located at a close proximity to vital anatomical structures in the brain. Moreover, due to the limitations associated with current standard therapeutic options as well as the presence of a chemo-resistant and radio-resistant glioma stem cell (GSC) population, which play a major role in initiating clinical relapse,[3] the median survival time for patients diagnosed with GBM is a meagre 12–18 months with only 3% of patients surviving longer than 5 years.[45]

 

 

 

 

These statistics highlight the urgency of developing novel and effective therapeutic strategies against this devastating and uniformly fatal disease. As such, glioma has attracted a large amount of research attention as a target for gene therapy. '

Gene therapy' as related to brain tumors can be defined as the targeted transfer of genetic material into tumor cells for therapeutic purposes[6] and has the ability to target invasive tumor cells that are resistant to conventional therapy and give rise to recurrent disease.

Although gene therapy has shown promise in preclinical applications, it has not met clinical expectations due to various impediments related to the nature of the type of tumor and its location.

The obstructions of gene therapy include:

- the anatomical barriers and physiological aspects of the brain that decrease transduction efficiency,

- tumor heterogeneity and invasiveness that challenge vector targeting and delivery,[6,7] as well as,

- a lack of a satisfactory preclinical model to study glioma.

Here, we review relevant gene therapy approaches for the treatment of glioma and discuss the pertinent shortcomings, modifications and future directions in the field.

Genetherapy Strategies for Glioma

In the last decade, efforts to develop more effective and innovative gene therapy to target GBM have led to the preclinical characterization of many promising gene therapy approaches. Many of these methods demonstrate therapeutic efficacy against glioma xenografts in an animal model and have been tested in clinical trials. Retroviral and adenoviral vectors have been the most widely used vectors for delivery of antiglioma therapeutic genes.[8] According to the Journal of Gene Medicine, replication-defective adenoviruses represent 23% (n=424) and replication-deficient retroviruses 20% (n=365) of all gene therapy clinical trials worldwide as of January 2012. In this section, we outline the most widely evaluated antiglioma gene therapy strategies which are discussed in figure 1.

                        (Enlarge Image)Figure 1.

Highlights the advantages and limitations of the most commonly studied antiglioma gene therapies:

(A) Suicide gene therapy inhibits cell division by blocking DNA replication. In this system, tumor cells are transfected by a gene that encodes for an enzyme that converts a systemically administered prodrug into an active drug toxic to glioma cells.

(B) Oncolytic viral therapy takes advantage of viral infection and selective replication of virus in tumor cells through various genetic alterations of the virus genome thereby rendering the virus tumor specific and Oncolytic.

(C) Immunomodulatory gene therapy induces a host immune response to counteract the immune privileged central nervous system and immunosuppressive tumor microenvironment through various strategies.

(D) Synthetic vectors such as nanoparticles are unique in their ability to be delivered systemically and cross the blood–brain barrier. This approach has been employed to deliver genetic material such as DNA plasmid, proteins, RNA interference (RNAi) and small interfering RNA (siRNA) that silence genes and provide the opportunity for the development of drugs against specific glioma targets.

(A) Suicide Gene Therapy

The most commonly used gene therapy approach against GBM, in the preclinical setting, as well as, in clinical trials, is the enzyme-prodrug suicide gene therapy system. In this approach, viral vectors or cell carriers are genetically modified to express genes for an enzyme that converts an inactive prodrug, when administered systemically into toxic metabolites at the tumor sites, resulting in tumor cell killing. Such targeted cytotoxic gene delivery approaches are designed to achieve highly selective tumor cell destruction while sparing normal central nervous system (CNS) tissue from toxicity. A large number of enzyme-prodrug systems have been evaluated in 17 different clinical trials ranging from phase I to phase III in the USA and Europe. In all 17 trials, adenoviral, retroviral or non-viral vector based delivery methods were used and modest to no increase in median survival was demonstrated (figure 2).[334] e briefly discuss some of the most commonly used suicide gene therapy systems against GBM.

(Enlarge Image)Figure 2.

 

 

An up-to-date overview of results obtained from glioma clinical trials that used virus.

(1)Replication incompetent viruses or non-replicating viruses bearing suicide transgenes have been extensively studied and applied in clinical trials. Retro-mediated and adenoviral-mediated herpes simplex type 1 thymidine kinase (HSV-tk) gene therapies are the most commonly studied in clinical trials. Retrovirus: Prados et al,9 Rainov,10 Shand et al,11 Palu et al,12 Klatzmann et al,13 Izquierdo et al 14 and Ram et al.15 Adenovirus: Trask et al,16 Sandmair et al,17 Smitt et al,18 Germano et al,19 Immonen et al 20 and Lang et al.21

HSV-tk System Herpes simplex type 1 thymidine kinase (HSV-tk) is the most extensively investigated suicide gene therapy system against GBM. HSV-tk converts the inactive prodrug ganciclovir (GCV) into a toxic metabolite called GCV-triphosphate.[35] Induction of the 'bystander effect' is thought to be one advantage of this therapy,[36] which can be observed when the toxic metabolite converted by HSV-tk is lethal to tumor cells at distant sites that were not originally transduced with the therapeutic gene. In a xenograft glioma model, significant therapeutic efficacy has been observed when only 10% of total tumor cells in the disease burden are transduced with HSV-tk.[17,37] In the clinic, successful delivery of the HSV-tk system into the tumor cavity has been achieved by replication-defective retrovirus (RV), adenovirus (Adv), cell carrier and reovirus packing cells. One of the largest phase III randomized clinical trials was conducted by Rainov where retroviral packing cells were used to deliver HSV-tk in the tumor bed of patients with glioma. This study recruited 248 total patients with newly diagnosed and previously untreated GBM who were treated with standard chemotherapy and radiotherapy (n=124) or standard therapy in combination with adjuvant retrovirus-mediated HSV-tk/GCV gene therapy (n=124).[10] Patients received a mean volume of 9.1 ml of retroviral producing cells into the margins of the tumor cavity at a concentration of 108cells/ml during the craniotomy. Even though the clinical trial proved that adjuvant gene therapy was safe, patient median survival was 365 days versus 354 days and the 12-month survival rates were 50% versus 55% in the gene therapy and control groups, respectively. These data showed no significant therapeutic benefit between both groups.[10] Sandmair et al reported a phase I clinical trial where 21 primary or recurrent GBM patients were injected with RV-mediated HSV-tk/GCV (n=7) or replication-defective adenovirus carrying HSV-tk/GCV (n=7) intraoperatively in the margins of the tumor cavity.[17] In this clinical trial, the mean survival of the group that received Adv-mediated HSV-tk/GCV was significantly higher (15 months, p<0.012) as compared with the group that was administered RV-mediated HSV-tk/GCV injection (7.4 months), indicating that the adenoviral vector may be better suited for antiglioma gene delivery. The HSV-tk system has also been shown to enhance sensitivity to conventional chemotherapy and radiotherapy, which opens the possibility of combining such an approach with the standard of care for GBM patients.[38,39] Chiocca et al recently reported a phase IB clinical trial with 13 newly diagnosed GBM patients and observed that Adv-mediated HSV-tk/valacyclovir therapy in combination with conventional surgery and chemotherapy-radiotherapy can be clinically safe with no dose-limiting or significant added toxicity.[40] The study also shed light on possible clinical efficacy in patients with an unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) promoter with one patient living up to 46.4 months. A phase II study is currently ongoing to further evaluate survival and MGMT independence trends.[40] Furthermore, it has been observed that combining HSV-tk with pharmacological drugs can alter the pharmacokinetics of the administered prodrugs, and has also been shown to increase therapeutic efficacy when used in conjunction with conventional therapy. One study showed that scopadulciol enhanced prodrug activity through a HSV-tk specific mechanism and increased tumor cell killing through the bystander effect of acyclovir and GCV prodrugs.[41]

CD/5-FC System The cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy system has also been extensively investigated in the preclinical setting.[42,43] This system is also capable of inducing a strong bystander effect; significant therapeutic efficacy has been observed in a xenograft tumor model when only 2%–4% of tumor cells are transduced.[44] A second generation non-lytic retroviral replicating vector (Toca 511) has demonstrated that stable delivery of CD resulted in long-term survival in two different immunocompetent brain tumor models.[45] Toca 511 is currently under phase I–II clinical investigation in combination with 5-FC in patients with recurrent high-grade glioma (NCT01156584). The CD/5-FC system has also been reported to enhance conventional radiotherapy against glioma in an animal model,[46] and a fusion gene of CD used in conjunction with HSV-tk has shown to provide an increased antiglioma effect when compared with each individual gene used alone.[47,48] Taken together, the antiglioma gene therapy approach using suicide genes is safe in treating patients with GBM, but has failed to achieve a consistent therapeutic benefit. These results can be attributed to limited spatial distribution of the viral vector, poor gene transfer efficiency into tumor cells and the inability to target disseminated tumor burden by the currently available gene transfer vectors. Moreover, with the exception of the Rainov trial,[10] most of the early clinical trials treated a small number of patients, sometimes even without a control group. Therefore, it has been difficult to analyze whether these trials provided therapeutic efficacy in treated patients. Further optimization of vectors used to deliver suicide gene therapy is essential for the improvement of clinical effectiveness. For the majority of antiglioma suicide gene therapy protocols, the short-term expression of therapeutic transgenes is sufficient to achieve tumor cell death. However, the restricted intratumoral distribution of the therapeutic payload still remains an issue for achieving optimal clinical efficacy. Greater viral vector stability as well as prolonged therapeutic transgene expression might result in more successful treatment of GBM. Thus, with use of adenovirus with superior glioma cell transduction capacity,[17] and gutless adenovirus with reduced immunogenicity,[49] conditionally replicating viral vectors might allow us to successfully translate antiglioma suicide gene therapy into the clinic because of their ability to amplify therapeutic transgenes via tumor-selective replication.

(B) Oncolytic Viral Therapy Replication competent Oncolytic virus, such as, conditionally replicating adenoviruses, herpes simplex virus (HSV) mutant vectors, Newcastle disease virus (NDV), and reovirus have all been tested in the clinical setting for treatment of glioma. HSV-1 (G207): Markert et al 22 and Markert et al.23 HSV-1 (1716): Papanastassiou et al, 24 Kesari et al 25 and Rampling et al.26 NDV (MTH-68/H): Wagner et al, 27 Csatary and Bakacs, 28 and Csatary et al.29 NDV (NDV-HUJ): Freeman et al.30 Reovirus: Forsyth et al.31 AdV (ONYX-015): Chiocca et al.32

In order to address the issue surrounding the transduction efficiency of gene therapy vectors, researchers have engineered tumor-selective and conditionally replicating viral vectors referred to as Oncolytic virus (OVs). OVs act by selective self-replication in tumor cells that leads to tumor cell lysis, as well as by amplifying therapeutic genes at tumor sites. It is evident from the current literature that tumor transduction efficiency is higher with replication competent viruses than with replication-deficient viruses, which highlights the potential of OVs as therapeutic gene delivery vehicles for anticancer gene therapy. Oncolytic herpes simplex virus (oHSV), conditionally replicating adenovirus (CRAd), reovirus, poliovirus, Newcastle disease virus and measles virus have all been evaluated or are currently being applied in antiglioma clinical trials (figure 2). Here, we describe some of the most commonly used antiglioma OV systems.

Oncolytic Herpes Simplex Virus oHSV was among the first OVs to be safely administered to patients with recurrent malignant glioma.[50] Because HSV is a human pathogen with neurotropic properties, a critical issue in designing oHSVs is to provide tumor selectivity with an adequate safety profile. Since the first reported clinical trials using oHSV for the treatment of glioma in the late 1990s,[51] at least eight different HSV-1 genes, including TK (UL23), ICP6 (UL39), γ34.5 and Us3, have been deleted/mutated to reduce neurovirulence and induce tumor selectivity.[52] The most widely tested OV in clinical trials for antiglioma therapeutics is the oHSV vector G207, which is a genetically engineered HSV-1 vector that has a deleted γ34.5 gene at both alleles and a lacZ gene insertion that blocks the expression of the UL39 gene.[53] Heretofore, three phase II and three phase I clinical trials have been conducted using the oHSV vector. Crusade Laboratories in Glasgow, Scotland, has begun a phase III clinical trial in Europe using HSV1716, an oHSV derived from the wild-type strain of 'F' containing attenuating mutation in both copies of the γ134.5 gene.[53] In a recently reported phase IB clinical trial, six patients with resectable GBM received two injections of G207 during presurgery and postsurgery. Viral replication was observed but with limited evidence of antitumor activity.[23] Results from early clinical trials have demonstrated high safety profiles of multiple oHSV vectors with no evidence of encephalitis but with limited therapeutic efficacy.[54] Second generation oHSV vectors are currently under preclinical development where researchers have implemented various strategies to enhance Oncolytic activity. Such strategies include those with a single copy of the γ34.5 gene reintroduced back into the vector that are genetically engineered to encode for therapeutic transgenes such as TNFα, vascular endothelial growth factor (VEGF) specific shRNA and the immunostimulatory gene interleukin (IL)-4.[55–58] Others include surface retargeted HSVs that target glioma cells overexpressing human epidermal growth factor 2[59] and transcriptional targeting oHSVs that use tumor-selective promoters such as the HIF-responsive promoter.[60] Development of new oHSVs provides optimism for the future.

Conditionally Replicating Adenovirus CRAds have also been extensively evaluated in both preclinical and clinical settings for antiglioma therapeutics, with ONYX-015 and Ad5-Delta24 being the most widely studied. These CRAds have been adapted to replicate and lyse tumor cells in different ways: ONYX-015 has a deletion in the E1B gene that permits its replication in tumors with a defective p53 pathway, while Ad5-Delta24 relies on a deletion in the retinoblastoma binding region of the EIA protein allowing the vector to replicate in GBM cells that have a defective retinoblastoma function.[61] A phase I clinical trial conducted by Chiocca and colleagues show that ONYX-015 is safe to administer into the tumor bed cavity postsurgical resection.[32] A phase I clinical trial is currently underway evaluating Ad5-Delta24 (NCT00805376). Our group is currently conducting a US Food and Drug Administration (FDA) guided preclinical study evaluating the CRAd-Survivin-pk7 vector, which contains a tumor specific surviving promoter that drives adenovirus E1A replication and an inserted pk7 fiber region that has a high affinity to heparin sulphate proteoglycans, which confers tumor-selective replication.[62–64] One important advantage of CRAd viruses is they are naturally non-neurotropic and thus may possess an enhanced safety profile over the oHSV vector.

Oncolytic Measles and Reovirus Vectors Oncolytic measles virus and reovirus vectors are currently under preclinical evaluation for GBM virotherapy. Tumor specific reovirus replication is dependent on hyperactive RAS signaling and has shown efficacy against GBM in an orthotropic animal model.[65] In a phase I clinical trial, reovirus was injected directly into the tumor of patients with glioma, and no participants showed any signs of clinical encephalitis.[31] Strains of the attenuated measles virus derived from the Edmonston vaccine lineage (MV-Edm) are also under preclinical development and have yielded positive results.[66] A phase I clinical trial for recurrent GBM patients using MV-CEA, a MV-Edm vector expressing the soluble peptide marker, carcinoembryonic antigen, is currently underway (NCT00390299).[67] Although conditionally replicating viruses represent a major advantage over non-replicative viruses in terms of transduction efficiency, the host antivector immune response remains as the major obstacle for the translation of OVs into the clinic.

(C) Immunomodulatory Gene Therapy

The objective of antiglioma Immunomodulatory gene therapy is to induce or augment the T cell-mediated immune response against GBM. During tumourigenesis, glioma cells evolve to evade the host immune system. Moreover, the distinct immune privileged nature of the CNS also poses issues for generating effective antiglioma immune responses.[68] Nevertheless, preclinical experimental evidence has demonstrated the feasibility of inducing immune responses against glioma cells as well as chemo-resistant and radio-resistant GSCs, which has laid the foundation for formulating antiglioma gene therapy based on immunomodulation. Such strategies include cytokine-mediated gene therapy, immune cell recruitment strategies and application of cell carriers expressing Immunomodulatory genes.

Cytokine-mediated Gene Therapy The rationale for cytokine gene therapy is that tumor-selective gene transfer and in situ expression of various immunostimulatory genes such as IL-2, -12, -4, interferon (IFN)-γ and IFN-β may induce potent immune responses restricted towards antigens specific to glioma cells, but not to normal brain tissue.[69–74] Moreover, cytokine-mediated gene therapy compared with systemic administration of suicide gene therapy and OV gene therapy may allow us to achieve higher local concentrations, longer therapeutic gene persistence and reduce systemic toxicity. Type 1 interferon genes including IFN-γ, IFN-β and IFN-ω are primarily produced by specialized antigen presenting cells such as dendritic cells (DCs) post viral infection and have been shown to elicit robust antitumor effects.[75] Among the IFN genes, the IFN-β gene has direct antiproliferative effects and has been the most extensively evaluated cytokine for anticancer gene therapeutics. A two stage phase I clinical trial in which the initial treatment of five patients with GBM comprised of tumor resection was followed by injection of cationic liposomes with the human IFN-β gene into the margin of the resection cavity reported minimal clinical toxicity with 50% reduction of tumor size in two patients.[76] Another dose-escalating phase I clinical trial of stereotactic injection of an adenovirus vector expressing the IFN-β gene in 11 patients with GBM recently demonstrated safety as well as possible therapeutic effects due to an increased level of apoptosis in glioma cells.[77]

Immune Cell Recruitment Strategies In the preclinical setting, Castro and her colleagues have used the Ad-Fms-like thyrosine kinase 3 ligand to recruit antigen presenting cells such as DCs into the brain tumor mass. Their strategy used DC recruitment combined with suicide gene therapy by simultaneously administrating a second adenovirus vector with the TK gene. In this approach, dying tumor cells release endogenous tumor associated antigen as well as the high mobility group box 1 protein that acts as an agonist to toll-like receptor 2 leading to DC recruitment and an antitumor immune response.[78,79] This gene therapy approach has demonstrated tumor regression and long-term survival through its ability to induce an antiglioma immune response and immunological memory in several transplantable, orthotropic syngeneic models of GBM. In 2011, a phase I clinical trial was launched using this genetic immunotherapy approach.[78]

Cell Carriers Expressing Immunomodulatory Genes for Antiglioma Gene Therapy Stem cells or progenitor cells (SCs) have been evaluated extensively as therapeutic vehicles for antiglioma therapy due to their inherent tumor tropic properties. In the context of glioma, three types of SCs have been explored for their therapeutic use and are currently in preclinical development: neural, embryonic and mesenchymal. Embryonic stem cells have been modified to express and deliver mda-7/IL-24 and cause apoptosis in malignant glioma cells.[80] Data also show similar apoptotic effects of embryonic stem cell-derived astrocyte-mediated delivery of TRAIL.[81] Mesenchymal stem cells have been used to deliver a plethora of therapeutics to glioma including prodrugs, virus, cytokines and antibodies. One specific application is the genetic modification of human mesenchymal stem cells to express a single-chain antibody on their surface against the tumor specific antigen EGFRvIII. EGFRvIII was selected based on data showing that about ~20%–30% of human GBM express this genetic alteration.[82] In an intracranial glioma xenograft model of U87-EGFRvIII, animals injected with human mesenchymal stem cells expressing the single-chain antibody against EGFRvIII showed a significant survival advantage when compared with mock animals.[83]

(D) Synthetic Vectors Such as Nanoparticles

Nanoparticles have been studied as a method to intravenously deliver vectors that can cross the blood–brain barrier. This gene therapy modality is based on coupling genetic material to nanoparticles or micro particles, and delivering genes to a targeted site by way of their size, charge, as well as high surface to volume ratio that provides a powerful force for diffusion.[84,85] Various genetic materials such as DNA plasmids, protein, RNA and siRNA have been conjugated onto or encapsulated inside nanoparticles to be delivered to tumor cells.[85–87] Liposomes, due to their organic makeup, are the most widely investigated nanoparticles, and have been used to form artificial vesicles that encapsulate and deliver therapeutic agents such as RNA interference and small interfering RNA (siRNA). RNA interference has been used to silence specific messenger RNA and have led to the development of drugs against specific disease targets. Synthetic siRNAs have been shown to silence genes in vivo that are important for the pathogenesis of GBM.[86] Therapeutics using siRNA represent a powerful tool for precise targeting of novel genes and have led to five different clinical trials that are currently ongoing.[88]

Challenges in Developing Effective Antiglioma Gene Therapeutics

Each described strategy above has its own distinct advantages and disadvantages. Despite encouraging results in preclinical animal models and established safety profiles in phase I clinical trials, none of the gene therapies have demonstrated significant benefits in phase II and III clinical trials. The barriers limiting the efficient transition of gene therapy into the clinic include: anatomical barriers of the CNS that decrease the spatial distribution of the administered therapy, GBM heterogeneity and their invasiveness, cancer SCs, immunogenicity and limitations of established preclinical GBM models. In the following section, we discuss the various roadblocks of translation of antiglioma therapy from a preclinical setting to the clinic, and how the field of gene therapy has attempted to address them ( Table 1 ).

Limited Spatial Distribution of the Therapeutic Payload

One of major hurdles for achieving clinically relevant therapeutic efficacy by antiglioma gene therapeutic approaches is the limited tissue penetration and spatial distribution of the therapeutic payload in GBM tissue. To achieve clinically relevant therapeutic efficacy, any given anticancer therapy must effectively access the tumor site and destroy as many tumor cells as possible without affecting the surrounding healthy tissue. Physiologically, the CNS is protected by a unique anatomical barrier, the blood–brain barrier, which has been considered the major impediment to any systemic treatment of CNS diseases including glioma.[97] Thus, most antiglioma gene therapeutic approaches are applied during craniotomy directly in the tumor bed or into the margins of the tumor cavity itself. Despite direct delivery, the transduction efficiency of glioma cells with the currently available viral and non-viral vectors remains poor. One reason contributing to the poor transduction efficiency is because only a small percentage of primary GBM cells express the cognate receptor for the viral vector that allow them to enter into the target tumor cells efficiently. For example, Ad5-based gene therapy for malignant glioma is limited due to the poor expression of the adenovirus entry receptor CAR on primary GBM.[98] To overcome this problem, researchers have developed retargeted gene therapy strategies, which use receptors that are only expressed in glioma cells but not in normal neural tissue. Our laboratory has been using a CRAd with a fiber modification containing an inserted polylysine (pk7) motif that binds with a high affinity to heparin sulphate proteoglycans which has shown to confer glioma-selective internalization. Another major limitation of gene therapy vectors is poor tissue penetration of the therapeutic virus after injection into glioma tissue. A clinical study demonstrated that the distribution of the viral vector was limited to an average range of 5 mm from the needle track.[21] Researchers have been exploring a new delivery method known as convection-enhanced delivery (CED),[99] which relies on continuous infusion of drugs and virus via intracranial catheters, enabling convective distribution of high virus/drug concentrations over large volumes of the targeted tissue.[100] CED has been applied in a glioma clinical trial to administer large molecules, including immunotoxins,[101] as well as to achieve enhanced transduction efficiency of the viral vector in a glioma xenograft model.[102] These studies have shown that CED has the potential to improve the therapeutic efficacy of antiglioma gene therapy, but the success of this approach remains to be resolved. Furthermore, in the majority of antiglioma gene therapy clinical trials viral vectors were administered into the resection cavity or remaining tumor bed by a single injection, through a catheter or by multiple injections of a rather small volume of vector suspensions.[18,20,21] Such injection protocols can be technically demanding, requiring precise estimation of the correct depth of the injection with respect to the extent of parenchyma-invading tumor cells. The accuracy and targeting capacity of therapeutic payload delivery protocols can be significantly improved if such injections are carried out with the help of robotic technology and guided by advanced imaging systems.

The Host Immune System and Targeting the Heterogenic and Invasive Properties of GBM

In theory, OVs should provide a solution for poor gene transfer efficiency as progeny released from the initial infected tumor cells should laterally spread to the tumor burden and amplify OV killing effects. However, results from early clinical trials using antiglioma OVs showed limited success due to the inability of currently available OVs to target disseminated glioma burdens as well as the host immune response interfering with viral vectors. The use of SCs has recently received a great deal of attention as possible cell carriers for targeted antiglioma therapy. In the last decade, many in vitro and in vivo studies demonstrated that SCs have unique inherent properties to migrate throughout the brain, target and home to metastatic invasive solid tumors, including gliomas.[89,103,104] Aboody and colleagues have used prodrug systems to modify HB1.F3 neural SC (NSC) lines and were able to show a 70%–80% decrease in tumor volume of mice bearing orthotopic gliomas or intracranial melanoma.[90] Based on the encouraging preclinical results, the FDA recently approved Aboody and colleagues to conduct the first clinical study of genetically modified neural SCs (HB1.F3-CD) for patients with recurrent high-grade glioma. This clinical trial began recruiting with the goal of enrolling 12–20 patients. Similarly, our lab has extensively investigated the possibility of using the inherent tumor tropic properties of NSCs to deliver glioma restricted Oncolytic adenovirus selectively to disseminated tumor burdens. Our recent data indicates that distant delivery of NSCs loaded with Oncolytic adenovirus significantly prolonged survival of animals in several orthotopic murine models of human glioblastoma when compared with mice treated with virus alone.[63,64] We proved that the increased survival was due to amplified therapeutic virus at distant tumor sites in the presence of NSCs. Also, we have reported that a bone marrow mesenchymal SC carrier was able to protect the Oncolytic viral therapeutic payload from the host immune system in a cotton rat model.[105] There is also an abundance of preclinical data that suggest that in vivo transplanted NSCs can act as an immunosuppressant.[106] It has been shown that NSCs lack the expression of major histocompatibility complex class II and express low levels of the co-stimulatory molecules CD80 and CD86 which provide them with protection from immune-mediated killing.[94] NSCs have also been shown to express immunosuppressive cytokines such as IL-10 in the context of OV infection/loading.[64] In the future, it will be crucial to gain a better understanding of the molecular mechanism underlying the tumor tropic properties of NSCs in order to increase their migratory capacity and improve the efficacy of this gene therapy system. Recent advancements in molecular imaging protocols using PET and/or MRI are providing us with the capacity to study SC migration in a non-invasive longitudinal manner, and may allow us to precisely delineate the mechanism of tumor tropism. Our lab has used ferumoxides-protamine sulphate labeled NSCs to visually track the migration of NSCs towards human glioma in an orthotropic mouse model. Information gathered from this technique may provide us with the insight to increase the migration of NSCs towards glioma in the future.

Another inherent characteristic of GBM is its heterogeneous makeup that exists due to the diverse genetic and epigenetic changes that accumulate in the pathogenesis of the different tissue subtypes found in GBM. This tumor property makes it exceptionally difficult to select one appropriate therapeutic approach against all tissue types in GBM.[107]

The use of drugs in combination with viral vectors has been applied to target multiple tumor cell types or tumor pathways to achieve a synergistic outcome. Bevacizumab (BEV) or Avastin, an antiangiogenic monoclonal antibody against VEGF, has been approved by the FDA for the treatment of GBM but has yielded no survival benefits in humans. Results of a study conducted by Zhang et al have shown that a local injection of G47Δ-mAngio, an HSV-derived OV expressing angiostatin, in conjunction with systemic administration of BEV increases virus spread throughout the brain, tumor killing and angiostatin inhibition of VEGF expression. Furthermore, this therapy synergizes BEVs inhibitory activity of invasion markers such as matrix metalloproteinases-2 (MMP-2), MMP-9 and collagen. This adjunct therapy has led to increased survival in an intracranial mouse model of human glioma (U87) through increasing antiangiogenesis and reducing the invasiveness of GBM.[108] Researchers are also using multiple viral vectors to target GBM heterogeneity and achieve therapeutic viral synergy. A current example of such is combining the vesicular stomatitis virus (VSV) with vaccine virus (VV). VSV and VV were shown to enhance viral replication and infiltration throughout tumor cells of one another. Boeuf et al observed a 10- to 10 000-fold increase in VSV titres following co-infection of tumor samples with VV in 33 out of 44 tumor samples.[109]

Other approaches that target GBM heterogeneity focus on targeting cells that make it uniquely invasive and resistant to conventional cancer therapies, when compared with other human cancers. Research has attributed GBM's resistance to treatment and high rate of recurrence to a small subpopulation of cells called GSCs. GSCs have unique phenotypic properties which include relative quiescents as well as an ability to differentiate, self-renew, and resist chemotherapy and radiotherapy.[110] Since a majority of investigated gene therapies focus on targeting properties retained in the main tumor bulk (i.e., rapidly dividing cells) and not specific GSC properties, GSCs survive therapy and give rise to new tumor formation and re-initiate the disease. By using SC specific promoters such as, Cox-2, hTERT and mdr, Bauerschmitz et al were able to show a reduction in breast cancer SC population after the treatment with Ad5/3-mdr-Δ24.[111] Research on brain specific cancer SCs has shown that tumor-selective Oncolytic adenovirus Delta-24-RGD replicates and induces cell death in GSCs. A phase I clinical trial for patients with malignant gliomas is currently underway.[112] oHSV has also been used to target GSCs. G47δ has been tested in combination with a low-dose etoposide and showed increased tumor cell apoptosis and increased survival of mice with etoposide-insensitive intracranial human GSC-derived tumors.[113] G47δ has also been shown to cooperate with temozolomide in killing GSCs through viral manipulation of DNA damage response pathways in preclinical models.[114] A modified oHSV, MG18L, containing a Us3 deletion and an inactivating LacZ insertion in UL39, replicates in GSCs and has antitumor activity in GBM cells in vivo. Furthermore, when MG18L was used in combination with phosphoinositide-3-kinase/Akt inhibitors, increased GSC and glioma apoptosis were observed and survival of GBM-bearing mice was prolonged when compared with treatment with either single therapeutic agent alone.[115] Other groups have used OV vectors carrying an exogenous Endo-Angio fusion gene (VAE) to infect and lyse GSCs and have shown the significance of this modality in vitro.[116]

 

 

 

Moreover, GSCs have been shown to overexpress ATP-binding cassette (ABC) transporters, especially ABCG2 that can pump out active prodrugs and resist suicide gene therapy.[117] Combining an ABCG2 blocker, such as Gefitinib, with other suicide gene therapy vectors may provide an opportunity to further target GSCs and improve the therapeutic efficacy of antiglioma suicide gene therapy systems.[117]

 

Preclinical Animal Model

The therapeutic efficacy of most antiglioma gene therapeutic approaches is commonly evaluated in immunocompromised animal models using xenogeneic cells for tumor implantation with only a short interval of time between engraftment and treatment. The circumstances in human GBM do not closely mimic those in animal models as tumor initiation is usually sporadic and clinical symptoms can be observed months to years after initial establishment, resulting in increased heterogeneity. Thus, for the successful investigation of gene therapies, it is essential to build good animal models that are both reliable and representative of human glioma. To date, many models exist including: implantation of rodent glioma cells into immunocompetent rodents, implantation of human GBM into immunocompromised nude mice and endogenous brain tumor animal models.[118] These models have been used due to their high level of reproducibility and characteristics that accurately recapitulate the tumor microenvironment, heterogeneity, growth pattern, histopathology and antitumor immune response represented in human GBM.[33] Although these models are widely used and have generated vast amounts of data to lead to the development of novel gene therapies, the failure of the studied therapies transition into the clinic can be partially attributed to a need for a superior glioma model. As one of the possible options, the spontaneous GBM model in the brachycephalic canine has been reported.[119] Canine GBM is highly invasive and mimics human GBM characteristics such as necrosis with pseudopalizading, neovascularisation and endothelial proliferation.[5] Stoica et al have reported that GSCs are present in dog GBM and have a high capacity for self-renewal, proliferation and differentiation similar to human GBM.[96] The most important aspect of the canine model is its comparable brain size to the human brain. This characteristic is essential for a good preclinical model in order to precisely assess such pharmacokinetic properties as toxicity, dosage, side effects, as well as measure delivery strategies. For example, NSCs have been shown to deliver gene therapies to targeted tumor sites beyond the primary tumor in small animal models. But can NSCs withstand the test of distance and deliver to metastatic sites far away from the site of injection in a human brain? The failures of gene therapy can be undoubtedly linked to the inaccessibility to animal models that recapitulate human GBM and therefore answer prudent questions about an antiglioma gene therapy before its translation into clinical trials. It is essential to collaborate with veterinarian institutions that receive glioma bearing canines and cancer gene therapy laboratories with a need for this model, in order to bring antiglioma gene therapy closer to achieving clinical relevance.

 

 

 

 

 

 

 

Conclusions and Future Direction

Although antiglioma gene therapies have demonstrated promising efficacy in preclinical glioma models with favorable safety profiles in phase I clinical trials, they have ultimately failed to provide significant benefits in both phase II and III clinical trials. Since gene therapy has demonstrated great promise in the preclinical setting, we must accept the initial discouraging outcomes of clinical trials with a grain of salt. A majority of antiglioma phase I clinical trials have been conducted on patients with advanced stage cancer, and this may contribute to their low success rate. In order to adequately judge efficacy, clinical trials need to be conducted on patients with earlier stages of cancer. Furthermore, many phase I clinical trials are designed to determine the safety profile of a treatment modality and not clinical efficacy. Others have suggested that the failure of phase III clinical trials can be attributed to the lack of 'preclinical robustness,' a term coined to describe the need for more stringent experimental protocols that address whether a therapy will be well translated into the clinical setting.[120] As the field of gene therapy moves forward, it is vital that we modify current gene therapy approaches and adopt new ways to overcome the formidable obstacles GBM has presented. A growing level of attention has been given to therapeutic synergies. Antiglioma gene therapies such as OVs and genetically modified SCs have the potential to cooperate with standard modes of treatment.[121] An optimal combination therapy would include a well-designed strategy that uses multiple therapies to target heterogeneous GBM.[91] Multiple treatment modalities will have the power to target different parts of the tumor such as the tumor bulk or GSCs, which address the importance of strategically targeting tumor heterogeneity. The synergistic advantages between multiple therapies need to be further evaluated to attain optimal results. Given the highly variable and evolving nature of GBM, advancements in non-invasive imaging protocols and cancer genomics will allow neuro-oncologists to acquire information such as the molecular, cellular, genetic and epigenetic makeup of a specific tumor. This information will provide the clinician with the powerful tools to continually provide personalized gene therapy treatment protocols that can be adjusted based on specific and real-time information gathered on an individual basis. Although the road ahead is challenging, if we can overcome the obstacles and ameliorate current antiglioma gene therapies, one day it may be possible that gene therapy can be used as the standard of care for GBM patients.

Autoria e outros dados (tags, etc)

por cyto às 12:59

Segunda-feira, 21.04.14

ICT-107

Existem fortes divergencias sobre estes ensaios.

Seguirão artigos sobre NW BIO e DENDREON

 

Novel Vaccine Dramatically Boosts Survival in Glioblastoma

Roxanne Nelson/December 05, 2013 

Brain Cancer News & Perspectives

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A novel therapy has extended survival in glioblastoma patients far beyond the current median. After undergoing surgical resection and standard treatment, half the patients treated with the experimental vaccine in a small phase 1 study survived more than 5 years.

The usual median survival in such patients is around 15 months, but in this study, 7 of the 16 participants are still alive, with survival ranging from 60.7 to 82.7 months after diagnosis.

The findings were published earlier this year (Cancer Immunol Immunother. 2013; 62:125 -135) and were presented last month at the 4th Quadrennial Meeting of the World Federation of Neuro-Oncology in San Francisco.

It is striking that this many patients are still alive, said study author Keith Black, MD, chair of the Department of Neurosurgery and director of the Neurosurgical Institute at Cedars-Sinai Medical Center in Los Angeles. "Some patients are approaching 7 years, and 4 are alive without any sign of recurrence and living normal lives," he told Medscape Medical News. "Another patient has some signs of recurrence but is stable."

"I am not aware of any other trial that has had such dramatic results for glioblastoma multiforme," Dr. Black said. However, a question remains: "Is this just a statistical fluke, or is the mechanism of action of this vaccine really providing such a robust increase in survival?"

 

I am not aware of any other trial that has had such dramatic results. Dr. Keith Black  

The experimental therapy, known as ICT-107, is an autologous vaccine that consists of a patient's dendritic cells pulsed with 6 peptides from tumor-associated antigens: AIM-2, TRP-2, HER2/neu, IL-13Ra2, gp100, and MAGE1. The vaccine is being developed by ImmunoCellular Therapeutics.

"One of the speculations is that the antigens are also targeting cancer stem cells, which may actually be part of the mechanism for the robustness of these results," Dr. Black explained. "We have also initiated 2 other vaccine trials at Cedars-Sinai targeting these cancer stem cells."

Survival Beyond 5 Years

The primary objective of the study was to determine the benefit of the ICT-107 vaccine in patients newly diagnosed with glioblastoma multiforme and to identify patients who survived longer than 5 years or who were disease free more than 5 years after receiving the vaccine.

The 16 study patients had undergone surgical resection and standard follow-up therapy with concurrent temozolomide and radiation. Only 4 patients did not undergo complete resection.

Patients were human leukocyte antigen serotype A1 or A2, and had at least 1 of the vaccine antigens present in their tumor. The vaccine was administered intradermally 3 times at 2-week intervals.

 

 

Median progression-free survival in the cohort was 16.9 months, and the 5-year rate of progression-free survival was 37.5%. Median overall survival was 38.4 months, and the 5-year rate of overall survival was 50%.

Eight of the 16 patients survived more than 5 years, and 7 are still alive (at 60.7, 65.1, 67.5, 67.4, 69.4, 77.9, and 82.7 months).

In 6 patients, progression-free survival has been more than 5 years. Four of the 6 continue to be disease-free at 65.1, 67.4, 77.9, and 82.7 months. One patient died of leukemia at 61 months, but the leukemia was not related to the therapy, Dr. Black explained. Another patient, who developed tumor progression at 62 months and underwent surgery and active treatment with temozolomide, is currently stable and doing well, he reported.

All of the long-term survivors had tumors with at least 5 antigens, and 75% percent had tumors with all 6 antigens. In addition, 100% expressed at least 4 cancer stem cell antigens (AIM2, TRP-2, HER2/neu, IL-13Ra2).

The secondary outcome of interferon-gamma response was significantly higher in those who survived at least 5 years than in those who did not (P = .0499).

Phase 2 Trial Underway

On the basis of this single-institution phase 1 trial, a phase 2 trial was initiated. "It is being conducted at 25 medical centers, and is randomized, placebo-controlled, and blinded," Dr. Black reported.

About 125 patients are now enrolled, and preliminary results will be released after 64 events, he explained.

"If the results are as good as the phase 1 trial, where we saw a very robust response, and if there is a greater than 9-month median survival increase, there will be a lot of interest in talking with the FDA about accelerated approval," Dr. Black said. "But if it is an intermediate increase, such as 3 to 4 months, we will be looking at a larger phase 3 trial."

Dr. Black reports owning stock in ImmunoCellular Therapeutics. Several members of the research team report ties to the company.

4th Quadrennial Meeting of the World Federation of Neuro-Oncology.

Presented November 23, 2013.

Autoria e outros dados (tags, etc)

por cyto às 12:55

Sábado, 19.04.14

IMUNOTERAPIA. O que é?

 

Immunotherapy

 

What is immunotherapy?

Immunotherapy is also sometimes called biologic therapy or biotherapy. It is treatment that uses certain parts of the immune system to fight diseases such as cancer. This can be done in a couple of ways.

 

  • · Stimulating your own immune system to work harder or smarter to attack cancer cells
  • · Giving you immune system components, such as man-made immune system proteins

 

For a long time doctors suspected that the immune system had an effect on certain cancers. Even before the immune system was well understood, William Coley, MD, a New York surgeon, first noted that getting an infection after surgery seemed to help some cancer patients. In the late 1800s, he began treating cancer patients by infecting them with certain kinds of bacteria, which came to be known as Coley toxins. Although he had some success, his technique was overshadowed when other forms of cancer treatment, such as radiation therapy, came into use.

Since then, doctors have learned a great deal about the immune system. This has led to research into how it can be used to treat cancer, using many different approaches. In the last few decades immunotherapy has become an important part of treating several types of cancer.

Immunotherapy includes a wide variety of treatments that work in different ways. Some seem to work by boosting the body’s immune system in a very general way. Others help train the immune system to attack cancer cells specifically.

Immunotherapy seems to work better for some types of cancer than for others. It is used by itself to treat some cancers, but for many cancers it seems to work best when used along with other types of treatment.

As researchers have learned more about the body’s immune system in recent years, they have begun to figure out how it might be used to treat cancer more effectively. Newer treatments now being tested seem to work better and will have a greater impact on the outlook for people with cancer in the future.

 

What the immune system does

Your immune system is a collection of organs, special cells, and substances that help protect you from infections and some other diseases. Immune system cells and the substances they make travel through your body to protect it from germs that cause infections. They also help protect you from cancer in some ways. It may help to think of your body as a castle. Think of viruses, bacteria, and parasites as hostile, foreign armies that are not normally found in your body. They try to invade your body to use its resources to serve their own purposes, and they can hurt you in the process.

 

 

 

 

 

In fact, doctors often use the word foreign to describe invading germs or other substances not normally found in the body. The immune system is your body's defense force. It helps keep invading germs out, or helps kill them if they do get into your body.

The immune system basically works by keeping track of all of the substances normally found in the body. Any new substance in the body that the immune system does not recognize raises an alarm, causing the immune system to attack it. Substances that cause an immune system response are called antigens. The immune response can lead to destruction of anything containing the antigen, such as germs or cancer cells.

Germs such as viruses, bacteria, and parasites have substances on their outer surfaces, such as certain proteins, that are not normally found in the human body. The immune system sees these foreign substances as antigens. Cancer cells are also different from normal cells in the body. They often have unusual substances on their outer surfaces that can act as antigens.

But the immune system is much better at recognizing and attacking germs than cancer cells. Germs are very different from normal human cells and are often easily seen as foreign, but cancer cells and normal cells have fewer clear differences. Because of this the immune system may not always recognize cancer cells as foreign. Cancer cells are less like soldiers of an invading army and more like traitors within the ranks of the human cell population.

Clearly the immune system’s normal ability to fight cancer is limited, because many people with healthy immune systems still develop cancer. The immune system may not see the cancer cells as foreign because the cancer cells (and their antigens) are not different enough from those of normal cells. Sometimes the immune system recognizes the cancer cells, but the response may not be strong enough to destroy the cancer. Cancer cells themselves may also give off substances that keep the immune system in check.

To overcome this, researchers have designed ways to help the immune system recognize cancer cells and strengthen its response so that it will destroy them.

 

Types of immunotherapy

There are many types of cancer treatments that could be thought of as immunotherapy.

Some work by stimulating your body's own immune system to fight the disease. This may be done by boosting the immune system in a very general way, or by training the immune system to attack some part of cancer cells specifically.

 

 

 

 

 

 

 

 

 

 

Other treatments sometimes considered immunotherapy use immune system components (such as proteins called antibodies) that are made in the lab. Some of these boost the immune system once they are in the body. Others don’t really affect the immune system much, if at all. Instead, the antibodies themselves target specific parts of cancer cells, stopping them from growing or making them die.

The main types of immunotherapy now being used to treat cancer are discussed in the following sections. They include:

 

Monoclonal antibodies: These are man-made versions of immune system proteins.

Antibodies can be very useful in treating cancer because they can be designed to attack a very specific part of a cancer cell.

Cancer vaccines: Vaccines are substances put into the body to start an immune response against certain diseases. We usually think of them as being given to healthy people to help prevent infections. But some vaccines may help prevent or treat cancer.

Non-specific immunotherapies: These treatments boost the immune system in a very general way, but this may still result in more activity against cancer cells.

 

Immunotherapy drugs are now used to treat a number of cancers, including cancers of the bladder, breast, colon, kidney, lung, and prostate, as well as leukemia, lymphoma, multiple myeloma, and melanoma. If you would like more information about immunotherapy as a treatment for a specific cancer, please see our detailed guide for that cancer.

Many other types of immunotherapy are now being studied for use against cancer. Some of these are discussed in the section “What’s new in immunotherapy research?”

Many types of immunotherapy work by targeting specific parts of cancer cells. As such, they can be thought of as a form of targeted therapy, which differs from less specific treatments like chemotherapy. But there are also other targeted treatments that zero in on parts of cancer cells that are not immunotherapies. For more information on targeted drugs, see our document, Targeted Therapy.

 

 

1-Monoclonal antibodies

One way the immune system normally attacks foreign substances in the body is by making large numbers of different antibodies. An antibody is a “sticky” protein that targets a specific antigen. Antibodies circulate in the body until they find and attach to the antigen. Once attached, they recruit other parts of the immune system to destroy the cells containing the antigen.

Many copies of a specific antibody can be made in the lab. These are known as monoclonal antibodies (mAbs or moAbs). These antibodies can be useful in fighting diseases because they can be designed specifically to only target a certain antigen, such as one that is found on cancer cells.

 

 

 

 

 

 

Monoclonal antibodies are now used to treat many diseases, including some types of cancer. A major advantage of these drugs is that because they are so specific, they may have only mild side effects, unlike some other cancer treatments. But researchers  first  have to identify the right antigen to attack. For cancer, this is not always easy, and so far mAbs have proven to be more useful against some cancers than others. Over the past 15years or so, the US Food and Drug Administration (FDA) has approved about a dozen mAbs to treat certain cancers.

As researchers have found more antigens that are linked to cancer, they have been able to make monoclonal antibodies against more and more cancers. Clinical trials of newer mAbs are now being done on many types of cancer.

 

Types of monoclonal antibodies

Two types of monoclonal antibodies are used in cancer treatments:

  • · Naked mAbsare antibodies that work by themselves. There is no drug or radioactive material attached to them. These are the most commonly used mAbs at this time.
  • · Conjugated mAbsare those joined to a chemotherapy drug, radioactive particle, or a toxin (a substance that poisons cells). These mAbs work, at least in part, by acting as homing devices to take these substances directly to the cancer cells.

 

Naked monoclonal antibodies

Most naked mAbs attach to antigens on cancer cells, but some work by binding to antigens on other, non-cancerous cells, or to even free-floating proteins.

Naked mAbs can work in different ways. Some may boost a person’s immune response against cancer cells. Others work by blocking specific proteins that help cancer cells grow. (Some may do both.)

Some naked MAbs attach to cancer cells to act as a marker for the body's immune system to destroy them. An example of this is Alemtuzumab (Campath®), which is used to treat some patients with chronic lymphocytic leukemia. Alemtuzumab is an antibody that binds to the CD52 antigen, which is found on immune cells called B cells and T cells.

Once attached, the antibody triggers the destruction of the cell by the immune system.

Some naked mAbs work mainly by attaching to and blocking specific antigens that are important signals for cancer cells (or other cells that help cancer cells grow or spread).

For example, Trastuzumab (Herceptin®) is an antibody against the HER2/neu protein. A large amount of this protein is present on the cells in some types of cancer. WhenHER2/neu is activated, it helps these cells grow. Trastuzumab stops these proteins from becoming active. It is used to treat breast and stomach cancers that have large amounts of this protein.

Conjugated monoclonal antibodies

Monoclonal antibodies attached to a radioactive substance, drug, or toxin, are called conjugated mAbs. The mAb is used as a homing device to take one of these substances directly to the cancer cells. The mAb circulates in the body until it can find and hook onto the target antigen.

 

 

 

 

 

It then delivers the toxic substance where it is needed most. This lessens the damage to normal cells in other parts of the body.

Conjugated mAbs are also sometimes referred to as tagged, labeled, or loaded antibodies. They can be divided into groups depending on what they are linked to.

  • · mAbs with radioactive particles attached are referred to as radio labeled, and treatment with this type of antibody is known as radio immunotherapy (RIT).
  • · mAbs with chemotherapy drugs attached are referred to as chemo labeled.
  • · mAbs attached to cell toxins are called immunotoxins.

Radiolabeled antibodies: Radio labeled antibodies have small radioactive particles attached to them. Ibritumomab Tiuxetan (Zevalin®) and Tositumomab (Bexxar®) are examples of radio labeled mAbs. Both of these are antibodies against the CD20 antigen, but they each have a different radioactive particle attached. They deliver radioactivity directly to cancerous B cells and can be used to treat some types of non-Hodgkin lymphoma.

Chemolabeled antibodies: These mAbs have powerful chemotherapy drugs attached to them. (The chemotherapy drug is often too powerful to be used on its own – it would cause too many side effects if not attached to a mAb.)

There are only 2 chemo labeled antibodies approved by the FDA to treat cancer at this time: Brentuximab Vedotin (Adcetris®) and Ado-trastuzumab Emtansine (Kadcyla™).

Brentuximab Vedotin is made up of an antibody that targets the CD30 antigen (found on B cells and T cells), attached to a chemo drug called MMAE. It is used to treat Hodgkin lymphoma and anaplastic large cell lymphoma that is no longer responding to other treatments. Ado-trastuzumab emtansine is made of an antibody that targets the HER2 protein attached to a chemo drug called DM1. It is used to treat advanced breast cancer in patients whose cancer cells have too much HER2.

Immunotoxins: These mAbs have cell poisons (toxins) attached to them, which makes them similar in many ways to chemo labeled mAbs. At this time no immunotoxins are approved to treat cancer, although many are being studied.

However, a related drug known as Denileukin diftitox (Ontak®) is being used to treat some cancers. It consists of an immune system protein known as interleukin-2 (IL2) attached to a toxin from the germ that causes diphtheria. Although it’s not an mAb, IL2 normally attaches to certain cells in the body that contain the CD25 antigen, which makes it useful for delivering the toxin to these cells. Denileukin diftitox is used to treat lymphoma of the skin (also known as cutaneous T-cell lymphoma). It is also being studied to be used against a number of other cancers.

 

Possible side effects of monoclonal antibodies

Monoclonal antibodies are given intravenously (injected into a vein). Compared with the side effects of chemotherapy, the side effects of naked mAbs are usually fairly mild and are often more like an allergic reaction.

 

 

 

 

 

 

These are more common while the drug is first being given. Possible side effects can include:· Fever· Chills· Weakness· Headache· Nausea· Vomiting· Diarrhea· Low blood pressure· Rashes

Some mAbs can also have other side effects that are related to the antigens they target.

For example, Bevacizumab (Avastin®), an mAb that targets tumor blood vessel growth, can cause side effects such as high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage.

Conjugated antibodies may pack more of a punch than naked mAbs, but they often cause more side effects. The side effects depend on which type of substance they're attached to.

 

2- Cancer vaccines

 

Most of us know about vaccines given to healthy people to help prevent infections, such as measles and mumps. These vaccines use weakened or killed germs like viruses or bacteria to start an immune response in the body. Getting the immune system ready to defend against these germs helps keep people from getting infections.

Most cancer vaccines work the same way, but they usually prime the immune system to attack cancer cells in the body. The goal is to help treat cancer or to help prevent it from coming back after other treatments. But there are some vaccines that may actually help prevent certain cancers.

 

Vaccines to help prevent cancer

Many people might not realize it, but some cancers are caused by viruses. Vaccines that help protect against infections with these viruses might also help prevent some of these cancers.

Some strains of the human papilloma virus (HPV) have been linked to cervical, anal, throat, and some other cancers. Vaccines against HPV may help protect against some of these cancers.

People who have chronic (long-term) infections with the hepatitis B virus (HBV) are at higher risk for liver cancer. Getting the HBV vaccine to help prevent this infection may therefore lower some people's risk of getting liver cancer.

These are traditional vaccines in that they target the viruses that can cause certain cancers. They may help protect against some cancers, but they don't target cancer cells directly. These types of vaccines are only useful for cancers known to be caused by infections.

But most cancers, such as colorectal, lung, prostate, and breast cancers, are not thought to be caused by infections.

 

 

 

 

 

 

Doctors are not yet sure if it will be possible to make vaccines to prevent these other cancers. Some researchers are now trying, but this research is still in very early stages. Even if such vaccines prove to be possible, it will be many years before they become available.

Vaccines to help treat cancer

Cancer treatment vaccines are different from the vaccines that work against viruses. These vaccines try to get the immune system to mount an attack against cancer cells in the body. Instead of preventing disease, they are meant to get the immune system to attack a disease that already exists.

A cancer treatment vaccine uses cancer cells, parts of cells, or pure antigens to increase the immune response against cancer cells that are already in the body. Vaccines are often combined with other substances or cells called adjuvants that help boost the immune response even further.

Cancer vaccines don't just boost the immune system in general; they cause the immune system to attack cells with one or more specific antigens. And because the immune system has special cells for memory, it's hoped that the drugs will help keep cancer from coming back.

Sipuleucel-T (Provenge®) At this time, this is the only vaccine approved by the US Food and Drug Administration (FDA) to help treat cancer. It is used to treat advanced prostate cancer that is no longer being helped by hormone therapy.

For this vaccine, immune system cells are removed from the patient's blood and sent to a lab. There they are exposed to chemicals that turn them into special cells called dendritic cells. They are also exposed to a protein called prostatic acid phosphatase (PAP), which should produce an immune response against prostate cancer.

The dendritic cells are then given back to the patient by infusion into a vein (IV). This process is repeated twice more, 2 weeks apart, so that the patient gets 3 doses of cells.

Back in the body, the cells help other immune system cells attack the patient's prostate cancer. Although the vaccine does not cure prostate cancer, it has been shown to help extend patient’s lives by several months on average. Studies to see if this vaccine can help men with less advanced prostate cancer are now being done.

Side effects are usually mild and can include fever, chills, fatigue, back and joint pain, nausea, and headache. A few men may have more severe symptoms, including problems breathing and high blood pressure.

Other cancer vaccines have shown some promise in clinical trials, but have yet to  be approved in the United States to treat cancer. Some of these are described in the section, “What’s new in immunotherapy research?”

 

Types of cancer treatment vaccines being studied

Several types of cancer vaccines are now being studied, with a few reaching late stage clinical trials.

Tumor cell vaccines: These vaccines are made from actual cancer cells that have been removed during surgery. The cells are treated in the lab, usually with radiation, so they cannot form more tumors.

 

 

 

 

 

 

 

In most cases, doctors also change the cells in certain ways, often by adding chemicals or new genes, to make them more likely to be seen as foreign by the immune system. The cells are then injected into the patient. The immune system recognizes antigens on these cells, then seeks out and attacks any other cells with these antigens that are still in the body.

Most tumor cell vaccines are autologous, meaning the vaccine is made from killed tumor cells taken from the same person in whom they will later be used. In other words, cells are taken from you (during surgery), the vaccine is made from them in a lab, and the cells are injected back into you.

Some vaccines are allogeneic, meaning the cells for the vaccine come from someone other than the patient being treated. Allogeneic vaccines are easier to make than autologous vaccines, but it is not yet clear if one type is more effective than the other.

 

Antigen vaccines: These vaccines boost the immune system by using only one antigen (or a few), rather than whole tumor cells that contain many thousands of antigens. The antigens are usually proteins or pieces of proteins called peptides.

Antigen vaccines may be specific for a certain type of cancer, but they are not made for a specific patient like autologous cell vaccines are. Scientists often combine several antigens in a vaccine to try to get a stronger immune response.

 

Dendritic cell vaccines: Dendritic cells are special immune cells in the body that help the immune system recognize cancer cells. They break down cancer cells into smaller pieces (including antigens), then hold out these antigens so other immune cells called T cells can see them. This makes it easier for the immune system cells to recognize and attack cancer cells.

Dendritic cell vaccines are autologous vaccines (made from the person in whom they will be used), and must be made individually for each patient. The process used to create them is complex and expensive. Doctors remove some immune cells from the blood and expose them in the lab to cancer cells or cancer antigens, as well as to other chemicals that turn them into dendritic cells and help them grow. The dendritic cells are then injected back into the patient, where they should provoke an immune response to cancer cells in the body. Sipuleucel-T (Provenge), which is used to treat advanced prostate cancer, is an example of a dendritic cell vaccine.

 

DNA vaccines: When tumor cells or antigens are injected into the body as a vaccine, they may cause the desired immune response at first, but they may become less effective over time. This is because the immune system recognizes them as foreign and quickly destroys them. Without any further stimulation, the immune system often returns to its normal (pre-vaccine) state of activity. To get around this, scientists have looked for a way to provide a steady supply of antigens to keep the immune response going.

 

 

 

 

 

 

 

DNA is the substance in cells that contains the genetic code for the proteins that cells make. Vectors (see next section) can be given bits of DNA that code for protein antigens.

When the vectors are then injected into the body, this DNA might be taken up by cells and can instruct them to make specific antigens, which would then provoke the desired immune response. These types of therapies are called DNA vaccines.

Vector-based vaccines: These vaccines use special delivery systems (called vectors) to make them more effective. They aren't really a separate category of vaccine; for example, there are vector-based antigen vaccines and vector-based DNA vaccines.

Vectors are special viruses, bacteria, yeast cells, or other structures that can be used to get antigens or DNA into the body. The vectors are often germs that have been altered to make sure they can no longer cause disease.

Vectors may be helpful in making vaccines for a number of reasons. First, they may be used to deliver more than one cancer antigen at a time, which may make the body's immune system more likely to mount a response. Second, vectors such as viruses and bacteria may trigger their own immune responses from the body, which may help make the overall immune response even stronger. Finally, these vaccines may be easier and less expensive to make than some other vaccines. (HCMV/EBV/NEWCASTLE Virus) (DCs Vaccine) (Autologous/Monocytos)

 

3-Non-specific immunotherapies and adjuvants

 

Non-specific immune therapies do not target a certain cell or antigen. They stimulate the immune system in a very general way, but this may still result in more activity against cancer cells.

Some non-specific immunotherapies can be given by themselves as cancer treatments.

Others are used as adjuvants (along with a main treatment) to boost the immune system to improve how well another type of immunotherapy (such as a vaccine) works. Some are used by themselves against some cancers and as adjuvants against others.

Cytokines Cytokines are chemicals made by immune system cells. They are crucial in controlling the growth and activity of other immune system cells and blood cells in the body.

Man-made versions of some cytokines can be given alone to boost the immune system, or they can be given along with tumor vaccines as adjuvants.

Some man-made cytokines are used to lessen the side effects of other treatments such as chemotherapy. They can help the bone marrow make more white blood cells, red blood cells, or platelets when their levels in the body have gotten too low. While this is important in cancer treatment, it isn't truly immunotherapy. Cytokines are injected, either under the skin, into a muscle, or into a vein. The most common ones are discussed here.

 

 

 

 

 

 

 

Interleukins Interleukins are a group of cytokines that act as chemical signals between white blood cells.

Interleukin-2 (IL-2) helps immune system cells grow and divide more quickly. When a man-made version of IL-2 was approved by the US Food and Drug Administration in 1992 to treat advanced kidney cancer, it became the first true immunotherapy approved to be used alone in treating cancer. Since that time, it has also been approved to treat people with metastatic melanoma.

IL-2 can be used as a single drug treatment for these cancers, or it may be combined with chemotherapy or with other cytokines such as interferon-alfa. (It is also being studied for use as an adjuvant along with some vaccines.) Using IL-2 with these treatments might help make them more effective against some cancers, but the side effects of the combined treatment are also increased.

Side effects of IL-2 may include flu-like symptoms such as chills, fever, fatigue, and confusion. Most people gain weight. Some have nausea, vomiting, or diarrhea. Many people develop low blood pressure, which can be treated with other medicines. Rare but potentially serious side effects include an abnormal heartbeat, chest pain, and other heart problems. Because of these potential side effects, if IL-2 is given in high doses, it must be done in the hospital (as an inpatient).

Other interleukins, such as IL-7, IL-12, and IL-21, are now being studied for use against cancer too, both as adjuvants and as stand-alone agents.

 

Interferons These cytokines, first discovered in the late 1950s, help the body resist virus infections and cancers. The types of interferon (IFN) are named after the first 3 letters f the Greek alphabet: IFN-alfa, IFN-beta, and IFN-gamma.

Only IFN-alfa is used to treat cancer. It boosts the ability of certain immune cells to attack cancer cells. It may also slow the growth of cancer cells directly, as well as the blood vessels that tumors need to grow.

The FDA has approved IFN-alfa for use against these cancers:

  • · Hairy cell leukemia · Chronic myelogenous leukemia · Follicular non-Hodgkin lymphoma · Cutaneous (skin) T-cell lymphoma · Kidney cancer · Melanoma · Kaposi sarcoma

Side effects of interferons may include flu-like symptoms (chills, fever, headache, fatigue, loss of appetite, nausea, vomiting), low white blood cell counts (which increase the risk of infection), skin rashes, and thinning hair. These side effects can be severe and can make treatment with interferon hard to tolerate for many people. Most side effects do not last long after the treatment stops, but fatigue can last longer. Other rare long-term effects include damage to nerves, including those in the brain and spinal cord.

 

 

 

 

 

 

 

 

Granulocyte-macrophage colony-stimulating factor Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that causes the bone marrow to make more of certain types of immune system cells and blood cells.

A man-made version (known as sargramostim or Leukine®) is often used to boost white blood cell counts after chemotherapy.

GM-CSF is also being tested against cancer as a non-specific immunotherapy and as an adjuvant given with other types of immunotherapies. Clinical trials of GM-CSF, alone or with other immunotherapies, are being done in people with many different types of cancer.

Common side effects of GM-CSF include flu-like symptoms (fever, headaches, muscle aches), rashes, facial flushing, and bone pain.

Other drugs boost the immune system. Some other drugs boost the immune system in a non-specific way, similar to cytokines. But unlike cytokines, these drugs are not naturally found in the body.

Thalidomide Thalidomide (Thalomid®) is used as a treatment for multiple myeloma and some other cancers. It is thought to work in a general way by boosting the immune system, although it’s not exactly clear how it does this.

Side effects of thalidomide can include drowsiness, fatigue, severe constipation, and neuropathy (painful nerve damage). The neuropathy can be severe, and may not go away after the drug is stopped. There is also an increased risk of serious blood clots (that start in the leg and can travel to the lungs). Because thalidomide causes severe birth defects if taken during pregnancy, this drug can only be obtained through a special program run by the drug company that makes it.

Lenalidomide Lenalidomide (Revlimid®) is a newer drug that is similar to thalidomide. It is used to treatmultiple myeloma and some other cancers.

The most common side effects of lenalidomide are low platelet and low white blood cell counts. It can also cause painful nerve damage. The risk of blood clots is not as great as that seen with thalidomide, but it is still increased. Like thalidomide, access tolenalidomide is tightly controlled out of concern about possible serious birth defects.

Bacille Calmette-Guérin Bacille Calmette-Guérin (BCG) is a germ related to the one that causes tuberculosis.

Unlike its bacterial "cousin," BCG does not cause serious disease in humans, but it does infect human tissues and helps activate the immune system. This makes BCG useful as a form of cancer immunotherapy. BCG was one of the earliest immunotherapies used against cancer and is still being used today.

BCG is FDA-approved for early stage bladder cancer. It is placed directly into the bladder through a catheter. The body's immune system cells are attracted to the bladder and activated by BCG, which in turn affects the bladder cancer cells. Treatment with BCG may cause symptoms that are like having the flu, such as fever, chills, and fatigue. It can also cause a burning feeling in the bladder. BCG may also be used to treat some melanoma skin cancers by injecting it directly into the tumors.

 

 

 

 

 

Imiquimod Imiquimod (Aldara®) is a drug that, when applied as a cream, stimulates a local immune response against skin cancer cells. It is used to treat some very early stage skin cancers (or pre-cancers), especially if they are on sensitive areas such as on the face. The cream is applied anywhere from once a day to twice a week for several months. Some people may have serious skin reactions to this drug.

 

 

What’s new in immunotherapy research?

 

Immunotherapy is a very active area of cancer research. Many scientists and doctors around the world are studying new ways to use immunotherapy to treat cancer. Some of these are discussed here.

 

a)-Newer monoclonal antibodies

Monoclonal antibodies (mAbs) have already become an important part of the treatment for many cancers. As researchers have learned more about what makes cancer cells different from normal cells, they have developed mAbs to exploit these differences. They have also developed newer forms of mAbs, attaching them to drugs or other substances to make them more powerful. New mAbs are now being studied for use against many types of cancer. A few are listed here.

Breast cancer A conjugated mAb known as trastuzumab-DM1 (or T-DM1) combines the trastuzumab (Herceptin) antibody, which targets the HER2/neu protein, with a chemo drug. It has shown promise in early studies of women whose breast cancer no longer responds totrastuzumab alone. Another mAb, pertuzumab, targets a different part of the HER2/neu protein. It may be helpful when used along with trastuzumab to treat certain breast cancers.

Leukemias and lymphomas Several newer mAbs are being studied in clinical trials for people with different types of leukemia and lymphoma.

Ovarian cancer An mAb that attaches to certain antigens on both ovarian cancer cells and to certain spots on T cells (a bi-specific antibody) has shown promise when used with interleukin-2 (IL-2). The antibody causes T cells to bind to and attack the cancer cells.

Early studies have shown that radiolabeled mAbs against ovarian cancer may help some women live longer.

Bevacizumab (Avastin), another mAb, slows the growth of tumor blood vessels by targeting the VEGF protein. It can slow the growth of advanced ovarian cancer, although it's not yet clear if it helps women live longer.

 

 

 

 

 

 

 

 

 

b)-Newer cancer vaccines

Vaccines are not yet considered a major treatment for cancer. But there are many different types of vaccines now being studied to treat a variety of cancers.

 

Breast cancer Early studies have found that autologous vaccine therapy may lengthen the remission and survival times of some women with early breast cancer. This approach is being studiedfurther.

A HER2/neu peptide, (a small part of the HER2/neu protein), used as the antigen in a vaccine, has been shown to cause an increased immune response against the ER2/neu receptor on cancer cells. It is being studied further.

Other specific antigen vaccines are also promising. These vaccines are almost always used after primary therapy (lumpectomy and radiation therapy, or mastectomy) and sometimes together with hormonal therapy or chemotherapy, to try to keep the cancer from coming back.

Cervical cancer While HPV vaccines already available may help prevent some of these cancers, other HPV vaccines that may help treat this cancer are now being tested in clinical trials. These vaccines try to cause an immune reaction to the parts of the virus that aid the growth of cervical cancer cells. This may kill the cancer cells or stop them from growing.

Colorectal cancer A number of autologous and allogeneic tumor cell vaccines have shown early promise in treating colorectal cancer, but so far none have been shown to lengthen survival time.

Some vaccines against the carcinoembryonic antigen (CEA) protein have improved the immune response in a large portion of colorectal cancer patients, but the studies have not been going on long enough to see whether this lengthens remission or survival times.

Kidney cancer Whole tumor cell vaccines given along with the adjuvant BCG have shrunk tumors in a small number of people with advanced kidney cancer in early studies.

Researchers are also studying DNA vaccines that insert genes (segments of DNA) into cancer cells, causing the cells to make cytokines. These cytokines help the immune system recognize the cancer cells and also help activate immune system cells to attack those cells.

Lymphoma Several vaccines have shown promising results in early clinical trials against B-cell non-Hodgkin lymphomas, but they are not yet US Food and Drug Administration (FDA)-approved.

Lung cancer Stimuvax® (BLP25) is a peptide vaccine that is encased in a fat droplet (liposome) to make it work better. A small study of patients with advanced non-small cell lung cancer suggested it might improve survival time. Larger studies are being done to try to confirm this.

 

 

 

 

 

 

 

 

Melanoma Although no melanoma vaccines are FDA-approved yet, recent studies have found that some autologous and allogeneic tumor cell vaccines, as well as antigen vaccines, have shrunk tumors and helped some patients live longer. Dendritic cell vaccines have also been shown to shrink tumors in some patients. Some newer studies combine vaccines with IL-2 or newer adjuvants to further stimulate the immune reaction. There is a lot of research going on in this area.

Pancreas cancer GVAX is a tumor cell vaccine. It is made by modifying pancreatic cancer cells in the lab to express GM-CSF (to help stimulate the immune system). The cells are irradiated so they can't grow any more. They are then injected into the patient to cause an immune response. In a small early study, patients who got the vaccine, combined with the mAbipilimumab (Yervoy®), which boosts the immune system, lived longer than expected.

This vaccine is now being looked at in larger studies.

Prostate cancer Many prostate cancer vaccines are designed to cause immune responses to antigens found only on prostate cells, such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA).

A version of the GVAX vaccine using prostate cancer cells has shown some promise in early studies. This vaccine is now being tested in larger studies of prostate cancer.

Another prostate cancer vaccine (PROSTVAC-VF) uses a virus that has been genetically modified to contain PSA. The patient's immune system should respond to the virus and begin to recognize and destroy cancer cells containing PSA. Early results with this vaccine have been promising.

 

C)-Other ways to boost the immune system.

Some other forms of immunotherapy are now being studied try to boost specific parts of the immune system. These types of treatments have shown promise, but they are complex and so far are available only through clinical trials being done at major medical centers.

Lymphokine-activated killer cell therapy Scientists can make large numbers of active, cancer-fighting T cells in the lab by treating a small number of a patient's T cells in a test tube with the cytokine interleukin-2 (IL-2).

After being returned to a patient's bloodstream, these special cells, now called lymphokine-activated killer cells (or LAK cells), are more effective against cancer cells.

Researchers are now testing several ways to use these very active cancer-fighting cells.

LAK cell therapy has shown promising results in animal studies, where it shrunk tumors in animals with lung, liver, and other cancers. Although clinical trials in humans have not yet been as successful, researchers are constantly improving LAK cell techniques. They are testing these newly improved methods against melanoma, brain tumors, and other cancers.

 

 

 

 

 

 

Tumor-infiltrating lymphocyte vaccine with interleukin-2 Researchers have found immune system cells deep inside some tumors and have named these cells tumor-infiltrating lymphocytes (TILs). These cells can be removed from tumor samples taken from patients and made to multiply in the lab by treating them with IL-2.

When injected back into the patient these cells can be active cancer fighters.

Treatments using TILs are being tested in clinical trials in people with melanoma, kidney cancer, ovarian, and other cancers. Early studies of this approach by researchers from the National Cancer Institute have been promising, but its use may be limited because doctors may not be able to get TILs from all patients.

 

D)-To learn more

More information from your American Cancer Society

Along with the American Cancer Society, other sources of information and support include:

National Cancer Institute

Toll-free number: 1-800-4-CANCER (1-800-422-6237)

Web site: www.cancer.gov

*Inclusion on this list does not imply endorsement by the American Cancer Society.

No matter who you are, we can help. Contact us anytime, day or night, for cancer related information and support. Call us at 1-800-227-2345 or visit www.cancer.org.

 

References about immunotherapy

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particle vaccine on risk of cervical intraepithelial neoplasia grade 2, grade 3, and adenocarcinoma in situ: A combined analysis of four randomised clinical trials. Lancet. 2007; 369:1861−1868

Bast RC, Zalutsky MR, Kreitman RJ, Frankel AE. Monoclonal serotherapy. In: Kufe DW, Bast RC, Hait WN, Hong WK, Pollock RE, Weichselbaum RR, Holland JF, Frei E, eds. Cancer Medicine 7. Hamilton, Ontario: BC Decker, Inc.; 2006:770−785.

Burris HA 3rd, Rugo HS, Vukelja SJ, et al. Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J Clin Oncol. 2011;29:398-405.

Chesney J, Rasku M, Clem A, Miller D. Denileukin diftitox depletes T regulatory cells and causes regression of melanoma metastases in humans [abstract]. Eur J Cancer Suppl. 2006; 4:12:84.

Disis ML, Grabstein KH, Sleath PR, Cheever MA. Generation of immunity to the HER- 2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide based vaccine. Clin Cancer Res. 1999; 5:1289−1297.

Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science.2002; 298:850−854.

 

 

 

 

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Last Medical Review: 5/9/2012

Last Revised: 2/22/2013

2012 Copyright American Cancer Society

Autoria e outros dados (tags, etc)

por cyto às 01:08

Sábado, 19.04.14

Celulas Dendriticas em imunoterapia oncologica

The use of dendritic cells in cancer immunotherapy

Gerold Schuler_y, Beatrice Schuler-Thurner_z and Ralph M Steinman

 

A novel approach to vaccination against cancer is to exploit dendritic cells (DCs) as ‘nature’s adjuvants’ and actively immunize cancer patients with a sample of their own DCs primed with tumor antigens. DC vaccination is, however, still at an early stage, slowed in part by the need to carry out research in humans.

Nevertheless, valuable proofs of concept have been obtained with respect to the capacity of DCs to expand cancer-directed immune responses. The methods for preparing DCs are being improved continuously, and there are many opportunities to improve efficacy at the level of DC biology. An increased number of Phase I, II and III studies will drive this new area of human research.

 

Addresses:

-Department of Dermatology, University Hospital of Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany e-mail: gerold.schuler@derma.imed.uni-erlangen.de e-mail: beatrice.thurner@derma.imed.uni-erlangen.de

-Laboratory of Cellular Physiology and Immunology and Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA e-mail: steinma@rockvax.rockefeller.edu

 

Abbreviations:

APC antigen-presenting cell

CTL cytotoxic T lymphocyte

DC dendritic cell

FcR Fc receptor

GM-CSF granulocyte-macrophage colony-stimulating factor

GMP good manufacturing practice

i.d. intradermal

IL interleukin

i.v. intravenous

KLH keyhole limpet hemocyanin

Mo-DC monocyte-derived DC

MCM monocyte-conditioned medium

PAP prostatic acid phosphatase

PGE2 prostaglandin E2

PSA prostate-specific antigen

s.c. subcutaneous

TNF tumor necrosis factor

TT tetanus toxoid

 

 

 

 

 

 

 

 

Introduction

 

The use of dendritic cells (DCs) as adjuvants in order to induce tumor-specific killer and helper T cells directly in the patient is supported by many animal experiments as well as initial human trials (reviewed in [1–3]). The potentially important variables in such preliminary investigations now require systematic human studies, which will be guided by our increasing insight into the biology of DCs. This review emphasizes some of the most important findings at this time and some of the outstanding research questions pertinent to the induction of tumor-specific T cells with injections of antigen loaded DCs. We will also briefly illustrate how DCs could be used to explore immune-mediated resistance to cancer in several other ways.

 

Important variables for dendritic cell-based vaccination

 

Dendritic cell subsets and maturation

The particular DC subsets and the maturation stimuli that are most effective in inducing anti-tumor immunity are not yet known. So far, evidence indicates that the use of immature DCs should be avoided for use in vaccination, as such DCs are poor immunogens and can also induce regulatory T cells [4__, 5].

The survival of DCs and methods to increase their viability are now also considered critical, as enhanced survival will promote immunity [6].

Additionally, the reprocessing of dying DCs in the T-cell area of lymph nodes by recipient DCs could lead to tolerance [7__].

The first DC vaccination study, which was published in1996 [8], used rare DCs directly isolated ex vivo from blood, which undergo spontaneous maturation during the isolation procedure.

A problem of this approach is the low yield of DCs (even from Flt3 ligand-stimulated blood;[9__]).

A further potential disadvantage of this technique is that human blood contains several DC subsets [10],which are probably present at widely varying percentages in disease states. However, effective and clinically applicable methods based on magnetic cell sorting techniques are now available, making it possible to compare the immunogenic properties of distinct DC subsets, such as plasmacytoid DCs, to the various myeloid DC subsets that have been used so far. DC vaccination has been facilitated by the development of methods to generate DCs from either rare, but proliferating, CD34þ precursors or from common, but mostly non-proliferating, CD14þ monocytes (so-called monocyte-derived DCs; Mo-DCs; reviewed in [2,3]).

 

 

 

 

 

 

 

Only two clinical trials have employed CD34þ-derived DCs so far. CD34þ-derived DCs contain a mixture of interstitial DCs and DCs of the Langerhans cell type. Some believe that the latter could be more potent than Mo-DCs in cytotoxic T lymphocyte (CTL) priming, and improved methods for producing large numbers of Langerhans cells [11] will facilitate further studies.

The biology of CD14þ Mo-DCs has been extensively studied by many groups (although most often using media containing fetal calf serum, which yields DC progeny different in some properties from those obtained in non-FCS-containing media), and most clinical studies have used Mo-DCs, as their generation is easier. Populations of immature Mo-DCs are obtained by exposing Monocytes to GM-CSF and IL-4 (or alternatively, IL-13) and the immature Mo-DCs can then be differentiated into homogeneously mature Mo-DCs by exposure to various stimuli, such as Toll-like receptor (TLR) ligands (e.g. microbial products such as lipo polysaccharide [LPS], monophosphoryl lipid [MPL] A or poly-I:C, which signal via TLR4, TLR2 and TLR3, respectively), inflammatory cytokines (such as TNF-a), monocyte-conditioned medium(MCM), the MCM mimic (IL-1b, TNF-a, IL-6 andPGE2) or CD40L. It is not clear which maturation stimulusis best for the induction of tumor-specific T cells in vivo; however, MCM or its mimic currently represent a standard, because Mo-DCs matured in this way are homogenous, have a high viability, migrate well to chemotactic stimuli, and induce CTLs both in vitro and in vivo. Quite surprisingly, MCM and the MCM mimic also induce T helper (Th) 1 responses in vivo (see the section below on human trials) despite their weak production of bioactive IL-12, which is caused by the PGE2 used for maturation. Interestingly, it has recently become evident that PGE2 has to be part of the Mo-DC maturation stimulus in order to obtain functional CC chemokine receptor (CCR) 7 expression. CCR7 probably guides DCs into the lymph node in response to CCL19 andCCL21 [12__, 13__].

It is of note that many trials employing Mo-DCs have not used such optimally matured DCs. Reproducible methods for generating large numbers of Mo-DCs (300–500 million mature DCs per apheresis) from adherent monocytes in semi-closed cell factories and a set of quality control criteria for this generation technique have recently been developed [14_,15_].

The combination of an optimized freezing procedure and the use of Mo-DCs matured by exposure to the MCM mimic (rather than other maturation stimuli) are the optimal conditions for the production of cryopreserved mature Mo-DC aliquots, which are highly viable upon thawing and present antigens that were loaded onto DCs before freezing [16].

Alternative methods for generating Mo-DCs from monocytes enriched by negative or positive magnetic selection are available, but the final yields of mature DCs, at least with a pheresis products as starting material, appear to be smaller [17].

Information on the quality, composition and maturational status of DCs has unfortunately been scarce in past trials, but will be required in future trials because standardized, validated DC vaccines will allow meaningful conclusions and direct comparisons of results.

 

 

 

 

 

Types of antigen and loading methods

In principle, either defined or undefined tumor-related antigens can be delivered to DCs by a variety of methods.MHC class I-restricted, 9–11 amino acid peptides derived from defined antigens (natural sequences or peptide analogues designed for enhanced binding to MHC or T cell receptor [TCR] molecules) have most often been used for DC vaccination, as they can be made available in good manufacturing practice (GMP) form and are easily standardized.

The recent availability of antibodies raised against MHC–peptide complexes [18__, 19__] should finally allow the validation of the peptide loading technique.

This is important as the number and quality of MHC–peptide complexes directly influences the immunogenicity of the DC [20].

Several melanoma peptides restricted by the same MHC molecule have either been pulsed onto separate DCs or pulsed simultaneously onto a single DC batch. Interestingly, even though the latter approach leads to competition for a given HLA molecule, immune responses to all of the melanoma peptides could be detected in a recent trial employing CD34þ DCs [21__].

A recent elegant study by Palmowski et al. [22__] demonstrates that the simultaneous presence of several immune dominant epitopes on an antigen presenting cell (APC) skews the immune response towards the single epitope for which CTL precursor frequencies are highest, unless the number of APCs is high enough to compensate for the competition of antigen recognition at the APC surface.

Prolonged presentation of MHC–peptide complexes is known to enhance immunogenicity [23__]. Recently, longer 30mer peptides have been used for DC pulsing; they appear to be taken up by APCs and to be processed not only by MHC class II but surprisingly also the MHC class I pathways [24__, 25__]. Although the longevity of MHC–peptide complex presentation has not yet been tested, this novel peptide-pulsing method is an interesting approach to be tested in the context of DC vaccination.

 

Another approach yet to be investigated in clinical trials is the delivery of antigens as immune complexes to Fc receptor (FcR)-bearing DCs, which results in the formation of both MHC class II and MHC class I peptide complexes, and in vitro can induce CTLs and Th cells[26__,27__]. It has been demonstrated in the mouse that immune complex delivery to DCs is particularly potent if inhibitory CD32b FcRs are circumvented [28__, 29 __].

Clearly, this antigen delivery method is promising as it has the potential to generate multiple epitopes for both MHC class I and II epitopes, thus lifting the restriction to HLA molecules and defined HLA-binding peptides. The transfection of RNA coding for defined antigens is also an attractive method of antigen delivery [30], particularly because of the development of effective electroporation protocols as a substitute for variable transfection with naked RNA [31_].

 

 

 

 

 

 

 

 

In addition, RNA transfection does not require costly production of GMP quality proteins and antibodies. RNA also appears advantageous over naked DNA or viral transfection, as RNA transfection leads to only transient antigen expression. This is, however, sufficient for antigen processing. RNA, in contrast to DNA, does not integrate into the genome, and therefore is much easier to work with from a regulatory point of view. RNA transfection provides an opportunity to test many defined tumor antigens (including universal ones, such as surviving and telomerase) [32__].

Several methods are also available for the exploration of the whole antigenic repertoire of a given tumor. DC–tumour cell hybrids [33] are a possibility; however, such fusion products are difficult to standardize and are also short-lived. DCs loaded with either necrotic or apoptotictumor cells can generate not only MHC class II but also MHC class I epitopes via cross-presentation [34–36].

Cross-presentation is promoted by, and possibly even critically dependent on, CD40 triggering on DCs orT-cell help (L Delamarre, H Holcombe, A Giodiniet al., personal communication; [37]). To the best of our knowledge, only trials employing DCs pulsed with necrotic cells or cell lysates, but not with apoptotic cells, have been performed to date. Unfortunately, in most trials data on the preclinical validation of the loading method are not provided.

Recently, Dhodapkar et al.[38__] made the exciting observation that antibody-coating of dying myeloma cells markedly enhances cross presentation and allows induction of highly lytic CTLs that effectively lyse freshly isolated myeloma cells, but not autologous or allogeneic myeloma lines. This indicates that it might indeed be crucial to explore the actual antigenic repertoire of a given tumor for effective immunotherapy. However, this can in principle be achieved only with certain select tumors, such as renal cell carcinoma or myeloma, in which large amounts of tumor material are readily available by the methods mentioned above. A more generally applicable approach for the exploration of the whole antigenic repertoire of a given tumor is the use of total or PCR-amplified tumor RNA, which has the potential to allow vaccination of virtually all cancer patients against their specific tumor (antigens)[39]. The various methods that can be used to explore the total antigenic repertoire of tumors will now have to be tested and compared in vitro as well as in vivo.

In many human trials, DCs have also been pulsed with keyhole limpet hemocyanin (KLH) or tetanus toxoid (TT) to provide a positive control antigen for the immunogenicity of the injected DCs. However, these proteins could also provide unspecific help for CTL induction [40].

The need for such unspecific helper proteins should be investigated further, although in addition to possible benefits there are potential drawbacks to the use of helper proteins, such as side-effects from delayed hypersensitivity o the protein [41], and antigenic competition at the level of the DC [22__].

 

 

 

 

Dose, frequency, route of dendritic cell delivery and dendritic cell migration The vaccination schedule and path of DC migration following vaccination have yet to be addressed and optimized in small immunogenicity trials. The optimal number of DCs will of course very much depend on the route and effectiveness of migration.

One DC vaccination study are primed to a xeno antigen via various (intradermal, intravenous and intralymphatic) routes, but Th1 responses developed only after intradermal DC administration [42_]. Tumor antigen-specific T cells were unequivocally demonstrated following injection of DCs into the skin (either intradermal [41, 42_] or subcutaneous [21__,41,43__]), intranodal [44,45_] and intravenous [9__] injection.

The value of direct intranodal delivery should be corroborated by immunomonitoring: the rationale is to circumvent the need for DC migration into the nodes. Alternatively, it would be valuable to improve migration of DCs from the skin to the nodes as, even in the case of MCM mimic-matured Mo-DCs, atmost 6% reach the nodes within 48 hours, whereas immature DC are essentially non-migratory (TG Berger, PKeikavoussi, I Haendle et al., personal communication).

 

Possible approaches to increase the migratory capacity of DCs following vaccination are the dispersion of DCs over a larger area in a larger volume, as well as pre-treatment of DCs, or the injection site, to increase responsiveness to, and stimulate the local production of, CCL19, respectively [46]. Most DC vaccination trials started with biweekly or monthly injections. Select mouse experiments show that more frequent injections might facilitate priming [47], however, as soon as sensitized T cells are present, frequent injections might actually be counterproductive and result in lysis of the DCs [48]. Following priming, both injected DCs and reactive T cells might get trapped in increasing delayed-type hypersensitivity (DTH) reactions at the vaccination sites [49__]. This might also be disadvantageous, and could possibly be circumvented by increasing injection intervals or changing to the intravenous route of administration.

 

Some results from human trials

Because of limited space only a few select examples from over 30 DC vaccination trials published in the past three years are discussed here. DC vaccination continues to appear safe and to produce encouraging clinical responses. As clinical efficacy requires confirmation in large, controlled trials, we focus here on immunogenicity trials. As in the past, most trials have employed Mo-DCs.

Studies using volunteers proved informative and demonstrated that a single deep dermal injection of only between two and four million Mo-DCs matured by140 MCM and pulsed simultaneously with KLH, TT and flumatrix peptide rapidly induced the proliferation of KLHspecificTh1 cells and expanded the flu-specific CD8þCTL population. Furthermore, a second booster injection, of mature DCs loaded with flupeptide alone, led to a strong expansion of CD8þ T cells, that were responsive to lower doses of peptide and were presumably of higher affinity [50]. In sharp contrast, immature DCs pulsed with flumatrix induced antigen-specific IL-10-producing regulatory T cells [4__].

 

 

 

 

 

Several pilot studies of DC vaccination were performed in melanoma, for which many immunologically relevant antigen shave been defined. An update of the first study by Nestle et al. [44], in which one million Mo-DCs loaded with KLH and either tumor peptides or tumor cell lysates were intranodally delivered, showed that most of the clinical responses were durable over several years (F Nestle, abstract 46: Vaccination of melanoma patients with peptide-or tumor lysate-pulsed dendritic cells: a follow-up. 6th International Symposium on Dendritic Cells, Port Douglas, Australia, May 2000).

A recent reanalysis by tetramer staining (for a review of the technique see the section below entitled ‘Immunomonitoring’) confirmed the strong induction of Mage-3.A1-specific CTL precursor sthat we had previously reported on the basis of conventional chromium release assays in advanced melanoma patients intracutaneously vaccinated with Mage-3.A1-peptide-loaded DCs matured by MCM [41].

 

In a follow-up study, patients were vaccinated with Mage-3A2.1 and flumatrix peptide-loaded DCs matured by MCM (five biweekly vaccinations; three vaccinations with six million DCs s.c., then two vaccinations i.v. at six and twelve million cells each) and rapid induction of ex vivo-detectable peptide-specific CD8þ T cells was observed [51]. By contrast, no clinical responses occurred, possibly because of the recently recognized poor presentation of the HLA-A2-restricted Mage-3 epitope by most melanoma cells. Jonuleit et al. [45_] directly compared the immunogenicity of immature DCs and DCs matured by MCM-mimic upon intranodal administration into opposite inguinal lymph nodes in eight patients. Mature DCs were clearly superior inducers of effector T cells, both with respect to the two recall antigens used (TT and purified protein derivative [PPD]/tuberculin) and, more importantly, to tumor peptides.

Recently, we reported that MCM-mimic-matured, cryopreserved Mo-DCs loaded with multiple (five HLA-1-, eight HLA-A2.1- and threeHLA-A3-restricted) MHC class I peptides (each peptide pulsed onto a separate batch of four million DCs to avoid uncontrollable competition at the level of a given MHC molecule) and MHC class II peptides (again loaded in anon-competing fashion) rapidly induced Mage-3.DR13and Mage-3.DP4 restricted, IFN-g-inducing type 1tumor-specific Th cells — both effectors detectable ex-vivo and proliferating memory cells [43__].

Analysis of class I responses has been initiated, notably inpatients showing clinical responses. Extensive analysis (by tetramer staining and TCR sequencing in blood and tissues) of the first patient who exhibited unequivocal immune and clinical responses showed that the same T-cell specificities and clones are found both in the blood and in regressing skin and in suprarenal metastases (TGBerger, I Haendle, M Lueftl et al., personal communication). Banchereau and colleagues [21__] pulsed CD34þ-derived DCs simultaneously with KLH and four HLAA2.1restricted melanoma antigens (melan A, tyrosinase, gp100 and Mage-3) and vaccinated 18 stage IV melanoma patients with either 0.1, 0.25, 0.5 or 1:0 _ 106 DCs/kg bodyweight s.c. (four times biweekly) and found that regression of >1 tumor metastasis was observed in 7out of 17 evaluated patients.

 

 

 

 

 

Interestingly, the overall immunity to melanoma antigens (detectable either ex vivo or as responses in cultured T cells) after DC vaccination was clearly associated with clinical outcome, whereas in other studies [41] this correlation was less evident.

It is notable that other studies show that the cessation of CTL responses is associated with disease progression [52_]. The study by Kugler et al. [33] aroused great interest because of the significant clinical responses observed after implementing vaccination using hybrids of allogeneic DCs and renal cancer cells. Doubt, however, was soon raised by several investigators as to whether the technique reported could yield the postulated fusion products.

Furthermore, questions regarding the quality of the evaluation of responses have been raised and are the subject of an investigation at the University of Gottingen [53_].

Holtl et al. [54_] used MCM-matured Mo-DCs pulsed with KLH as well as lysates of autologous tumor or a permanent renal cancer cell line to vaccinate patients suffering from metastatic renal cell carcinoma (at least three monthly vaccinations with either 3.2–15.5million DCs i.v. or 3.2–10.3 million DCs i.d.) and found clinical responses in 10 of the 17 evaluated patients (two with a complete response, one with a partial response and seven with stable disease) with a mean follow up of 32 months. Enhanced immune responses against oncofetal antigen expressed in renal cancer, was observed in five out of six patients tested.

Similar to renal cell cancer, prostate cancer has also continued to be the subject of further DC vaccination studies.

An interesting xenoantigen immunization approach was taken by Fong and colleagues [55_], who administered DCs isolated from unmobilized aphereses and pulsed with mouse prostatic acid phosphatase (PAP; two monthly vaccinations with a mean of 11.2 million DCs per vaccination, either i.v., i.d. or into a lymphatic vessel on the dorsal side of the foot). All 21 patients with metastatic prostate cancer developed Th1 responses to mouse PAP, 11 also developed responses to the homogenous self-antigen, and six of these had clinical stabilization of their progressing prostate cancer. Small et al. [56] treated 31patients suffering from hormone refractory prostate cancer with infusions of a cellular vaccine prepared by isolating a DC precursor-enriched fraction from apheresis products, which was then exposed for two days to a recombinant fusion protein consisting of PAP linked to GM-CSF (PA2024, Dendreon Corporation, Seattle, WA, USA).

The fusion protein presumably targets cells expressing the GM-CSF receptor, including DC precursors, which then undergo maturation in vitro. This cellular vaccine (termed APC8015 or Provenge TM, Dendreon Corporation, Seattle, WA,USA), which contains a variable percentage of candidate DCs (a mean of 123 million DCs at 18% purity)as well as monocytes, NK cells, T cells and B cells, was then infused intravenously over 30 minutes at weeks 0, 4, 8 and 24. All patients developed immune responses to the fusion protein, and 38% developed immune responses to PAP.

Burch et al. [57] treated 13 patients with just two infusions of the same vaccine, one month apart, followed by three monthly s.c. injections of PA2024 without cells. Again, T-cell proliferative responses to PAP were induced.

 

 

 

 

Dendreon Corporation has recently concluded a Phase III trial involving 127 men with hormone-resistant prostate cancer. This demonstrated, in a subgroup of patients with a Gleason score of _7 (i.e. intermediate histopathological grade), an effect on time to disease progression [58].

Vieweg and colleagues [59__] exposed immature Mo-DCs to 15 mg/ml naked RNA encoding for prostate-specific antigen (PSA) to transfect and induce slight maturation of the DCs, as defined by only marginal induction of theCD83 maturation marker. The DCs were then administered in escalating doses (10 million DCs i.d. and concomitantly 10, 30 or 50 million DCs i.v.) to a total 13 patients with metastatic prostate cancer, three times every two weeks. Surprisingly, all patients tested positive for IFN-g, using ex vivo ELISPOT, in response to PSA protein after vaccination, which is indicative of the induction of CD4þ T-cell responses. CTL responses were also demonstrable in several patients using PSA RNA-transfected DCs as surrogate markers. An impact on surrogate clinical endpoints (such as log slope PSA analysis and number of circulating tumor cells) was demonstrated in several patients. Fong et al. [9__] studied DC vaccination of patients suffering from colon cancer or non-small cell lung cancer using enriched immature DCs from aphereses. The cells were cultured in vitro for 36 hours to induce spontaneous maturation and subsequently exposed to KLH as well as to an analogue of carcinoembryonic antigen (CEA605–613;natural peptide YLSGANLNL, altered peptide YLSGADLNL in amino acid one-letter code), which was altered to induce enhanced binding to the TCR. The aphereses were in part performed after a 10-day course ofFlt3 ligand treatment, which led to a 20-fold increase in DC yields (without pre-treatment with Flt3 ligand <107DCs were available from one apheresis). Two i.v. infusions, one month apart, of DCs at four escalating cell doses (107, 108, 109 and <1010) were administered to 12patients suffering from colon (nine colon, one rectum) or non-small cell lung cancer (two patients). All patients developed proliferative responses to KLH after the first DC infusion, and after the second infusion, five of the twelve patients displayed a 1.9–14-fold expansion of CEA (both CEA and CEA analogue)–HLA-A2.1 tetramer positive CD8þ T cells with an effector phenotype (CD45RAþCD27_CCR7_). Increased CTL activity both in response to CEA-pulsed artificial targets and to CEA expressing tumor cell lines was evident in seven out of twelve patients. Remarkably, clinical responses occurred (two dramatic regressions, one mixed response and two stabilization’s), which correlated well with the expansion of CD8þ tetramer T cell population. Brossart et al. [60] observed the induction of specific CTL responses in five out of ten patients with advanced breast and ovarian cancer vaccinated with mature Mo-DCs pulsed with HER-2/neu- or MUC-1-derived peptides. Interestingly, in two patients there was also evidence of consecutive epitope spreading. Some areas of investigation Immunomonitoring I has not been extensive in most studies to date. The value of future trials is likely to be enhanced with modern techniques that allow quantitative and qualitative monitoring of antigen-specific T-cell responses to provide more information on their immunogenicity. MHC class I and II tetramers or multimers will be critical as they allow the direct flow cytometric enumeration of specific T cells, irrespective of their functional capacity, even in whole blood [61].

 

 

Moreover, by combining tetramers with appropriate antibodies (to detect surface molecules such as CD45, CCR7, CD27and CD28, or to detect intracellular cytokine production and the presence of perforin) it is possible to sub-classify specific T cells into naıve, intermediate (central memory) and effector cells. Following tetramer-based cell sorting, further characterization analyses can be performed (including the lytic capacity for tumor cells in comparison to peptide-loaded targets and characterization of TCRs).

Select examples of the study of natural and vaccine induced responses have illustrated the power of this approach [62_,63_].

It will be increasingly important for a complete understanding of any clinical effects to study other topics such as epitope spreading, serological responses and the presence of T cells in regressing metastases, for example by using improved in situ tetramer-staining procedures [49__].The use of tetramers is, however, restricted to known epitopes of defined antigens. Nevertheless, APCs presenting naturally processed epitopes of defined (following loading with long overlapping peptides, protein–antibody complexes, viral or RNA transfection of natural sequences or sequences containing endosomal targeting motifs) used in conjunction with cytokine detection assays(such as ELISPOT, intracellular fluorescence activated cell sorting [FACS] or MACS) can also be valuable techniques for monitoring DC vaccination. These assays can also be used to measure responses to the whole antigenic repertoire of tumors by using either, tumor cells, tumor cel lines, or DCs loaded with tumor material as targets.

 

Active immunization with dendritic cells to induce NK and NKT cells

Several recent results suggest that DCs, notably Mo-DCs, will be useful to directly trigger NK and NKT cells and to mobilize the additional power of the innate immune system to attack tumor cells. These innate arms of the immune system are potentially relevant to resistance against malignancy, but as with adaptive T-cell dependent immunity, it will be necessary to pay more attention to the role of APCs, and in particular DCs, given recent findings on their capacity to activate resting NK[64__] and NKT [65__] cells.

 

In vivo dendritic cell targeting and delivery of dendritic cells into tumors to induce immunity

Targeting of antigen to DCs and the induction of their maturation will be important for any future in situ DC vaccination approach. An expansion of our current knowledge of DC growth factors (e.g. Flt3 ligand), chemokines that regulate DC traffic, DC-specific surface molecules, DC-specific promoters and DC maturation stimuli (e.g. synthetic TLR ligands that specifically trigger certain DC subsets) is necessary firstly to increase DC numbers in situ and secondly to target antigen expression and induction of DC maturation and migration. Improved targeting of exosomes (which are small membrane vesicles of endocytic origin that are secreted and constitute both an antigen source and a delivery method, already undergoing testing in phase I clinical trials) to host DCs might increase their efficacy [66].

 

 

 

 

An interesting approach that has so far been used in mice has been antigen targeting to the DEC-205 receptor on DCs to induce either immunity or tolerance directly in vivo, either with or without a concomitant DC maturation stimulus [67__]. Further work should also explore the induced migration of DCs and T cells into tumors to induce immunity against the total tumor antigen repertoire. The principal effectiveness of this pathway has been recently demonstrated by injection of chemokine secreting DCs into tumors to prime T cells [68__].

 

Use of dendritic cells in adoptive or passive immunotherapy

DCs have proven effective for the in vitro priming of tumor-specific T cells, a population that can rapidly be expanded following tetramer-guided cloning [69]. As DCs can now be loaded with defined as well as undefined tumor antigens, the adoptive transfer of cytotoxic T cells could become not only more straightforward but also more broadly applicable. In principle, adoptive transfer of tumor-reactive CTLs appears to be clinically effective, particularly after non-myeloablative chemotherapy [70__].

 

Discussion and conclusions

The results of animal studies and the initial clinical trials suggest that DC vaccination has distinct potential for inducing immune responses and might have considerable therapeutic potential. The initial clinical trials, although promising, have, however, been difficult to compare because of the variability in DC quality, study design, immunomonitoring and the patient populations included.

It is, therefore, necessary to reach a consensus with respect to (minimal) criteria for DC validation, some standardized immune monitoring methods and a detailed description of clinical responses, notably beyond the short-term outcome. To optimize DC vaccination we believe that it is now a priority to address the many variables and potential improvements suggested by emerging insights into DC biology in small, preferably two armed, immunogenicity trials (i.e. addressing a given variable in two ways and using immune monitoring to define the endpoint). Such trials are presently facilitated by the fact that DC vaccination, notably with defined antigens, appears to be safe (side-effects have been restricted thus far to transient fever, local reactions and— upon use of DCs loaded with tumor cell lysates or melanoma differentiation antigens in melanoma patients—the induction of autoimmune vitiligo), so that patients with less advanced disease can be evaluated. In the next few years, the results of initial Phase III trials obtained with currently suboptimal DC vaccines will become available. Furthermore, information from studies that combine DCs with other therapies, such as cytokine administration (IL-2, IL-15; [71, 72_,73_]), CTLA-4 blockade and removal or blockade of regulatory T cells(to enhance the expansion and survival of activated T cells; [74__]) will become accessible. We estimate that within the next several years the ex vivo DC vaccination approach will have been improved and standardized (e.g.by the development of commercial DC generation kits) to an extent that there will be consensus on its potential and the need for more vigorous research programs.

 

 

 

 

 

Phase III studies and direct comparisons to other vaccination strategies should also be possible. In the long run, ex vivo DC vaccination may be the method of choice for treating tumor-bearing patients, whereas a vaccine targeting DCs directly in vivo will be the approach for adjuvant setting and possibly even as a prophylactic therapy in the future. 

 

References and recommended reading

Papers of particular interest, published within the annual period ofreview, have been highlighted as:

-          of special interest

-          of outstanding interest

 

1. Dallal RM, Lotze MT: The dendritic cell and human cancer vaccines. Curr Opin Immunol 2000, 12:583-588.

2. Banchereau J, Schuler-Thurner B, Palucka AK, Schuler G: Dendritic cells as vectors for therapy. Cell 2001, 106:271-274.

3. Steinman RM, Dhodapkar M: Active immunization against cancer with dendritic cells: the near future. Int J Cancer 2001,14:459-473.

4._Dhodapkar MV, Steinman RM: Antigen-bearing immature dendritic cells induce peptide-specific CD8(þ) regulatory T cells in vivo in humans. Blood 2002, 100:174-177.

This study demonstrates that the administration of immature DCs might be dangerous as it can induce CD8þ regulatory T cells and thus tolerance.

5. Roncarolo MG, Levings MK, Traversari C: Differentiation of T regulatory cells by immature dendritic cells. J Exp Med 2001, 193:F5-F9.

6. Josien R, Li HL, Ingulli E, Sarma S, Wong BR, Vologodskaia M, Steinman RM, Choi Y: TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo. J Exp Med 2000, 191:495-502.

7. Liu K, Iyoda T, Saternus M, Kimura Y, Inaba K, Steinman RM: Immune tolerance after delivery of dying cells to dendritic cells in situ. J Exp Med 2002, 196:1091-1097.

This work provides yet another reason (see also [6]) to consider the viability of DCs used for vaccination as critical, because dying antigen carrying DCs can be taken up by resident tolerogenic DCs.

8. Hsu FJ, Benike C, Fagnoni F, Liles TM, Czerwinski D, Taidi B, Engleman EG, Levy R: Vaccination of patients with B-celllymphoma using autologous antigen-pulsed dendritic cells. Nat Med 1996, 2:52-58.

9. Fong L, Hou Y, Rivas A, Benike C, Yuen A, Fisher GA, Davis MM, Engleman EG: Altered peptide ligand vaccination with Flt3ligand expanded dendritic cells for tumor immunotherapy. Proc Natl Acad Sci USA 2001, 98:8809-8814.This study is interesting for several reasons. First, the induction of ex vivo detectable CEA-specific effector CTLs is unequivocally demonstrated by multicolor tetramer staining, second, the induced CTL responses directly correlate with clinical responses.

10. Shortman K, Liu YJ: Mouse and human dendritic cell subtypes. Nat Rev Immunol 002, 2:151-161.

 

 

 

 

 

11. Gatti E, Velleca MA, Biedermann BC, Ma W, Unternaehrer J, Ebersold MW, Medzhitov R, Pober JS, Mellman I: Large-scale culture and selective maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34þ progenitors. J Immunol 2000, 164:3600-3607.

12.Luft T, Jefford M, Luetjens P, Toy T, Hochrein H, Masterman KA, Maliszewski C, Shortman K, Cebon J, Maraskovsky E: Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E(2) regulates the migratory capacity of specific DC subsets. Blood 2002, 100:1362-1372.See annotation to [13__].

13.Scandella E, Men Y, Gillessen S, Forster R, Groettrup M:Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells. Blood 2002, 100:1354-1361.These two references [12__,13__] demonstrate that, in the case of Mo-DCs, PGE2 has to be part of the maturation stimulus in order to yield migratory DCs that respond to CCR7 ligands. In retrospect, this observation provides yet another rationale for the use of MCM or its mimic, a mixture of IL-1 b, IL-6, TNF-a and PGE2, as a maturation stimulus for DCs.

14.Berger TG, Feuerstein B, Strasser E, Hirsch U, Schreiner D, SchulerG, Schuler-Thurner B: Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories. J Immunol Methods 2002, 268:131-140.In this manuscript, the authors describe their extensive experience of using ‘cell factories’ manufactured by Nunc (a multilayered communicating closed culture vessel offering a surface big enough to cultivate one leukapheresis product) for the standardized production of Mo-DCs for clinical application. In addition, the data (yield, phenotype etc.) provided are based on a large number of processed leukaphereses and are thus meaning full for establishing validation criteria.

15.Tuyaerts S, Noppe SM, Corthals J, Breckpot K, Heirman C, DeGreef C, Van Riet I, Thielemans K: Generation of large numbers of dendritic cells in a closed system using Cell Factories. J Immunol Methods 2002, 264:135-151.These authors demonstrate that the Mo-DCs generated in Nunc ‘cell factories’ display identical properties to those prepared in standard tissue culture vessels.

16. Feuerstein B, Berger TG, Maczek C, Roder C, Schreiner D, Hirsch U, Haendle I, Leisgang W, Glaser A, Kuss O et al.: A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use. J Immunol Methods2000, 245:15-29.

17. Pullarkat V, Lau R, Lee SM, Bender JG, Weber JS: Large-scale monocyte enrichment coupled with a closed culture system for the generation of human dendritic cells. J Immunol Methods2002, 267:173-183.

18. Chames P, Willemsen RA, Rojas G, Dieckmann D, Rem L, Schuler G, Bolhuis RL, Hoogenboom HR: TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity. J Immunol 2002, 169:1110-1118. See annotation to [19__].

 

 

 

 

 

 

 

19.Lev A, Denkberg G, Cohen CJ, Tzukerman M, Skorecki KL, Chames P, Hoogenboom HR, Reiter Y: Isolation and characterization of human recombinant antibodies endowed with the antigen specific, major histocompatibility complex-restricted specificity of T cells directed toward the widely expressed tumor T-cell epitopes of the telomerase catalytic subunit. Cancer Res 2002, 62:3184-3194.These two papers [18__, 19__] describe examples of the use of monoclonal antibodies to MHC–peptide complexes, which were selected from phage display libraries and in vitro affinity matured for enhanced binding to allow flow cytometric detection of MHC–peptide complexes. These reagents should prove valuable for the validation of DC vaccines, and also for an investigation of the relationship between MHC–peptide complex density and the quantity as well as quality of induced T-cell responses.

20. van der Burg SH, Visseren MJ, Brandt RM, Kast WM, Melief CJ: Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability. J Immunol1996, 156:3308-3314.

21.Banchereau J, Palucka AK, Dhodapkar M, Burkeholder S, Taquet N, Rolland A, Taquet S, Coquery S, Wittkowski KM, Bhardwaj Net al.: Immune and clinical responses in patients with metastatic melanoma to CD34(þ) progenitor-derived dendritic cell vaccine. Cancer Res 2001, 61:6451-6458.This clinical trial is particularly interesting, as clinical regressions occurred during the study, which correlated with induced immune responses to four melanoma antigens. Remarkably, the latter were not only detectable after re-stimulation of T cells, but also directly ex vivo.

22. Palmowski MJ, Choi EM, Hermans IF, Gilbert SC, Chen JL, Gileadi U, Salio M, Van Pel A, Man S, Bonin E et al.: Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol 2002, 168:4391-4398.This work unravels a potential complication when an APC simultaneously presents several immunodominant epitopes, in that only those CTL precursors present at the highest frequency are expanded. These findings have to be taken into account in the rational design of vaccines.

23.Wang RF, Wang HY: Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells. Nat Biotechnol 2002, 20:149-154.This paper is a direct demonstration that the short half-life of MHC–peptide complexes formed upon peptide pulsing is improved through loading DCs from the inside using a peptide linked to a cell-penetrating peptide. This technique also results in enhanced immunogenicity, which is important for optimizing DC vaccines.

24. Gnjatic S, Atanackovic D, Matsuo M, Sager EJ, Chen YT, Ritter G, Knuth A, Old LJ: Cross-presentation of HLA class I epitopes from exogenous NY-ESO-1 polypeptides by non-professional antigen presenting cells. J Immunol 2003, 170:1191-1196. The authors find that long 30mer peptides enter APCs, and are processed to short peptides leading to the formation of MHC class I–peptide complexes at the cell surface.

 

 

 

 

 

 

 

 

 

25.Zwaveling S, Ferreira Mota SC, Nouta J, Johnson M, Lipford GB, Offringa R, van der Burg SH, Melief CJ: Established human papilloma virus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J Immunol2002, 169:350-358. These authors provide direct evidence that vaccination with long peptides and DC-activating agents results in a superior induction of E7-specificCD8þ T cells, demonstrating that the in vitro observations (see [25__]) are highly relevant.

26.Berlyn KA, Schultes B, Leveugle B, Noujaim AA, Alexander RB, Mann DL: Generation of CD4(þ) and CD8(þ) T lymphocyte Mo-DCs with immune complexes leads to the formation of MHC class II and, via cross-presentation, also MHC class I complexes, whereas soluble protein only yields MHC class II peptide complexes, as expected. The loading of antigens via Fc receptors thus allows DCs to induce both helper and cytotoxic T cells.

27.Nagata Y, Ono S, Matsuo M, Gnjatic S, Valmori D, Ritter G, Garrett W, Old LJ, Mellman I: Differential presentation of a soluble exogenous tumor antigen, NY-ESO-1, by distinct human dendritic cell populations. Proc Natl Acad Sci USA 2002,99:10629-10634.This work confirms the findings made by Berlyn et al. [26__] using another antigen and, in addition, shows that, quite surprisingly, only Mo-DCs but not CD34þ DCs are capable of cross-presentation upon uptake of NYESO-1–antibody complexes.

28. Kalergis AM, Ravetch JV: Inducing tumor immunity through the selective engagement of activating Fcc receptors on dendritic cells. J Exp Med 2002, 195:1653-1659. See annotation to [29__].

29. Rafiq K, Bergtold A, Clynes R: Immune complex-mediated antigen presentation induces tumor immunity. J Clin Invest2002, 110:71-79. These two groups [28__,29__] independently, and by using different improvement of DC-based vaccination.

30. Nair SK, Boczkowski D, Morse M, Cumming RI, Lyerly HK, Gilboa E: Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat Biotechnol 1998, 16:364-369.

31.Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN: Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 2001, 98:49-56. The authors describe a method to reproducibly electroporate RNA into DCs as a substitute for the variable and inefficient loading with naked RNA.

32.Su Z, Vieweg J, Weizer AZ, Dahm P, Yancey D, Turaga V, Higgins J, Boczkowski D, Gilboa E, Dannull J: Enhanced induction oftelomerase-specific CD4(þ) T cells using dendritic cells transfected with RNA encoding a chimeric gene product. Cancer Res 2002, 62:5041-5048. This work is important as it shows that RNA-transfected DCs can also be used to induce CD4þ Th-cell responses. DCs transfected with mRNA encoding a chimeric hTERT/lysosome-associated membrane protein (LAMP-1) protein, carrying the endosomal / lysosomal sorting signal of the LAMP-1, induced both hTERT-specific CD8þ and CD4þ T-cell responses in vitro.

 

 

 

 

 

 

33. Kugler A, Stuhler G, Walden P, Zoller G, Zobywalski A, Brossart P, Trefzer U, Ullrich S, Muller CA, Becker V et al.: Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med 2000, 6:332-336.

34. Herr W, Ranieri E, Olson W, Zarour H, Gesualdo L, Storkus WJ: Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBVspecificCD4þ and CD8þ T lymphocyte responses. Blood 2000, 96:1857-1864.

35. Jenne L, Arrighi JF, Jonuleit H, Saurat JH, Hauser C: Dendritic cells containing apoptotic melanoma cells prime human CD8þ T cells for efficient tumor cell lysis. Cancer Res 2000, 60:4446-4452.

36. Berard F, Blanco P, Davoust J, Neidhart-Berard EM, Nouri-Shirazi M, Taquet N, Rimoldi D, Cerottini JC, Banchereau J, Palucka AK: Cross-priming of naıve CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 2000, 192:1535-1544.37.

37. Terheyden P, Straten P, Brocker EB, Kampgen E, Becker JC: CD40-ligated dendritic cells effectively expand melanoma specificCD8þ CTLs and CD4þ IFN-c-producing T cells from tumor-infiltrating lymphocytes. J Immunol 2000, 164:6633-6639.

38. Dhodapkar MV, Krasovsky J, Olson K: T cells from the tumor microenvironment of patients with progressive myeloma can generate strong, tumor-specific cytolytic responses to autologous, tumor-loaded dendritic cells. Proc Natl Acad SciUSA 2002, 99:13009-13013. This is a remarkable manuscript as it not only shows that DCs loaded with antibody-coated autologous myeloma cells are effective in inducing tumor-specific CTLs but also that these CTLs are specific for the ‘fresh’ tumor cells of the patient and recognize cultured myeloma cells less efficiently. This indicates that patient-specific antigens are relevant, and that it might be important to use fresh tumor cells rather than tumor cell lines or defined antigens as a source of tumor antigens for successful vaccination against certain cancers.

39. Boczkowski D, Nair SK, Nam JH, Lyerly HK, Gilboa E: Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res 2000, 60:1028-1034.

40. Lanzavecchia A: Immunology. Licence to kill. Nature 1998, 393:413-414.

41. Thurner B, Haendle I, Roder C, Dieckmann D, Keikavoussi P, Jonuleit H, Bender A, Maczek C, Schreiner D, von den Driesch Pet al.: Vaccination with Mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 1999, 190:1669-1678.

42. Fong L, Brockstedt D, Benike C, Wu L, Engleman EG: Dendritic cells injected via different routes induce immunity in cancer patients. J Immunol 2001, 166:4254-4259. In this clinical trial, evidence is provided that the cutaneous route of DC vaccination might promote Th1 immunity.

 

 

 

 

 

 

43.Schuler-Thurner B, Schultz ES, Berger TG, Weinlich G, Ebner S,Woerl P, Bender A, Feuerstein B, Fritsch PO, Romani N, Schuler G: Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells. J Exp Med 2002, 195:1279-1288. In this paper, it is shown for the first time that mature, peptide-loaded DCs are capable of rapidly inducing Th1 cells (both ex vivo effectors and proliferating precursors) specific for a tumor antigen, specifically severalMage-3 peptides.

44. Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998, 4:328-332.

45.Jonuleit H, Giesecke-Tuettenberg A, Tuting T, Thurner-Schuler B, Stuge TB, Paragnik L, Kandemir A, Lee PP, Schuler G, Knop J, EnkAH: A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection. Int J Cancer 2001, 93:243-251.The authors show that upon intranodal injection (to avoid possible differences in migration rates after injection into the skin) only mature, not immature, DCs effectively induce immunity. This finding is additional evidence for the use of mature rather than immature Mo-DCs (see also [4__]).

46. Robbiani DF, Finch RA, Jager D, Muller WA, Sartorelli AC, Randolph GJ: The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell 2000, 103:757-768.

47. Ludewig B, Odermatt B, Landmann S, Hengartner H, Zinkernagel RM: Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue. J Exp Med 1998, 188:1493-1501.

48. Hermans IF, Ritchie DS, Yang J, Roberts JM, Ronchese F: CD8þ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity. J Immunol 2000, 164:3095-3101.

49. Schrama D, Pederson LO, Keikavoussi P, Andersen MH, thorStraten P, Brocker EB, Kampgen E, Becker JC: Aggregation of antigen-specific T cells at the inoculation site of mature dendritic cells. J Invest Dermatol 2002, 119:1443-1448. The authors use novel in situ tetramer staining and find that at the inoculation site of vaccination with mature DCs, antigen-specific T cells get trapped. This trapping of T cells and DCs might be disadvantageous, as the proportion of DCs migrating to the regional lymph nodes will be diminished.

50. Dhodapkar MV, Krasovsky J, Steinman RM, Bhardwaj N: Mature dendritic cells boost functionally superior CD8(þ) T-cell in humans without foreign helper epitopes. J Clin Invest 2000, 105:R9-R14.

51. Schuler-Thurner B, Dieckmann D, Keikavoussi P, Bender A,Maczek C, Jonuleit H, Roder C, Haendle I, Leisgang W, Dunbar Ret al.: Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1þ melanoma patients by mature monocyte-derived dendritic cells. J Immunol 2000, 165:3492-3496.

 

 

 

 

 

 

52. Andersen MH, Keikavoussi P, Brocker EB, Schuler-Thurner B,Jonassen M, Sondergaard I, Straten PT, Becker JC, Kampgen E: Induction of systemic CTL responses in melanoma patients by dendritic cell vaccination: cessation of CTL responses is associated with disease progression. Int J Cancer 2001, 94:820-824. Because of the possibility of immune escape, the induction of tumor immunity might not always correlate with clinical regressions. In this paper, the reverse approach is taken, that is, it is observed that following stabilization disease progression occurs upon exhaustion of an antitumor response.

53. Birmingham K: Misconduct trouble brewing in Gottingen. Nat Med 2001, 7:875.

This work questions the reported clinical regressions that caused so much interest following the publication of [33].

54. Holtl L, Zelle-Rieser C,Gander H, Papesh C, Ramoner R, Bartsch G, Rogatsch H, Barsoum AL, Coggin JH Jr, Thurnher M: Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous dendritic cells. Clin Cancer Res 2002,   8:3369-3376.This clinical trial protocol is somewhat heterogeneous (e.g. large variations in the number of tumor lysate-pulsed DCs administered etc.), but the rate of clinical regressions is of interest, as is the demonstration of T-cell responses to oncofetal antigen (immature laminin receptor), which might be an interesting antigen to look at in further studies.

55.Fong L, Brockstedt D, Benike C, Breen JK, Strang G, Ruegg CL, Engleman EG: Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy. J Immunol 2001, 167:7150-7156. In an interesting approach, the authors used mouse PAP xenoantigen to load DCs and circumvent the low immunogenicity of tolerance to human PAP.

56. Small EJ, Fratesi P, Reese DM, Strang G, Laus R, Peshwa MV, Valone FH: Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells. J Clin Oncol 2000, 18:3894-3903.

57. Burch PA, Breen JK, Buckner JC, Gastineau DA, Kaur JA, Laus RL, Padley DJ, Peshwa MV, Pitot HC, Richardson RL et al.: Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer. Clin Cancer Res 2000, 6:2175-2182.

58. Dendreon Corporation: Dendreon announces preliminary analysis of its first Phase III ProvengeTM trial. URL: http://www.dendreon.com, News and Events, August 9, 2002.

59.Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD,Dahm P, Niedzwiecki D, Gilboa E, Vieweg J: Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest2002, 109:409-417. This is the first clinical trial that makes use of the novel approach of using RNA-transfected DCs as a vaccine. The induction of PSA-specific CD8þ CTL responses, and interestingly also CD4þ T cell responses, are reported.

 

 

 

 

 

 

 

60. Brossart P, Wirths S, Stuhler G, Reichardt VL, Kanz L, Brugger W: Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood 2000, 96:3102-3108.

61. Klenerman P, Cerundolo V, Dunbar PR: Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol 2002, 2:263-272.

62. Monsurro V, Nagorsen D, Wang E, Provenzano M, Dudley ME, Rosenberg SA, Marincola FM: Functional heterogeneity of vaccine-induced CD8þ T cells. J Immunol 2002, 168:5933-5942.See annotation to [63_].

63. Speiser DE, Lienard D, Pittet MJ, Batard P, Rimoldi D, Guillaume P, Cerottini JC, Romero P: In vivo activation of melanoma-specificCD8þ T cells by endogenous tumor antigen and peptide vaccines A comparison to virus-specific T cells. Eur J Immunol2002, 32:731-741. These references [62_, 63_] nicely illustrate the power of modern immune monitoring techniques.

64. Ferlazzo G, Tsang ML, Moretta L, Melioli G, Steinman RM, Munz C: Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells. J Exp Med 2002, 195:343-351.This manuscript demonstrates that mature Mo-DCs directly activate human NK cells, indicating that Mo-DCs might be utilized to activate the power of NK cells against cancer.

65.Fujii S, Shimizu K, Kronenberg M, Steinman RM: Prolonged IFN-c producing NKT response induced with a-galactosylceramide loaded DCs. Nat Immunol 2002, 3:867-874.The authors demonstrate that a-galactosylceramide-loaded mature Mo-DCs activate NKT cells to release IFN-g, but not IL-4. This suggests that such DCs will be useful for activating NKT cells in man for cancer immunotherapy, as this will avoid the unwanted IL-4 release by NKT cells, which might result from administration of soluble a-galactosylceramide alone.

66. Thery C, Zitvogel L, Amigorena S: Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002, 2:569-579.

67. Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M,Ravetch JV, Steinman RM, Nussenzweig MC: Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 2001, 194:769-779. In this paper, it is shown that targeting of antigen to the DEC-205 endocytic molecule, which is constitutively expressed on the surface of certain lymph node DCs, results in deletion of antigen-specific T cells, whereas the simultaneous delivery of a DC-activating stimulus leads to immunity. As the DEC-205 molecule is also identified in humans, these findings open up a novel approach to manipulating immunity by targeting DCs directly in vivo.

68. Kirk CJ, Hartigan-O’Connor D, Mule JJ: The dynamics of the T cell antitumor response: chemokine-secreting dendritic cell scan prime tumor-reactive T cells extra nodally. Cancer Res 2001, 61:8794-8802. This work provides an important proof of concept by showing that the injection of DCs into tumors can lead to immunity. This suggests that in the future one might be able to induce cancer resistance by guiding DCs to tumors and then activating them.

 

 

 

 

 

69. Dunbar PR, Chen JL, Chao D, Rust N, Teisserenc H, Ogg GS, Romero P, Weynants P, Cerundolo V: Cutting edge: rapid cloning of tumor-specific CTL suitable for adoptive immunotherapy of melanoma. J Immunol 1999, 162:6959-6962.

70.Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ, Topalian SL, Sherry R, Restifo NP, Hubicki AM et al.: Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002, 298:850-854. In this adoptive immunotherapy approach, high numbers of melanoma specific T cells were administered after non-myelo ablative chemotherapy (which, in addition to other side-effects, may also remove regulatory T cells) and were further expanded by i.v. IL-2 administration, resulting in impressive clinical regressions. This approach is not widely applicable, but provides important evidence that tumor-specific T cells can indeed mediate clinical regressions.

71. Shimizu K, Fields RC, Giedlin M, Mule JJ: Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines. Proc Natl Acad Sci USA 1999, 96:2268-2273.

72. Eggert AO, Becker JC, Ammon M, McLellan AD, Renner G, Merkel A, Brocker EB, Kampgen E: Specific peptide-mediated immunity against established melanoma tumors with dendritic cells requires IL-2 and fetal calf serum-free cell culture. Eur J Immunol 2002, 32:122-127.The experiments outlined in [71,72_] demonstrate that it might be worthwhile to explore the combination of DC vaccination with IL-2 administration in man, as the T-cell responses induced by DC vaccination appear enhanced and therapeutically more effective.

73. Rubinstein MP, Kadima AN, Salem ML, Nguyen CL, Gillanders WE, Cole DJ: Systemic administration of IL-15 augments the antigen-specific primary CD8þ T cell response following vaccination with peptide-pulsed dendritic cells. J Immunol2002, 169:4928-4935.This work suggests that DC vaccination in combination with IL-15 should be explored in man, as the priming of CD8þ T-cell responses is augmented.

74.Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, Toes RE, Offringa R, Melief CJ: Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25þ regulatory T cells in antitumor therapy reveals alternative pathways for suppression of auto reactive cytotoxic T lymphocyte responses. J Exp Med 2001, 194:823-832.This work provides evidence that the efficacy of vaccination will be markedly enhanced firstly by the blocking of CTLA-4 (which favors the expansion of high-affinity T cells), and, secondly, by the removal or blockade of CD25þ regulatory T cells, which appear to suppress immune responses. 

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