Genetically Modified Dendritic Cells for Cancer Immunotherapy

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Genetically Modified Dendritic Cells for Cancer Immunotherapy. Antoni Ribas*. Assistant Professor of Medicine and Surgery, Divisions of Hematology-Oncology ...

Current Gene Therapy, 2005, 5, 000-000

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Genetically Modified Dendritic Cells for Cancer Immunotherapy Antoni Ribas* Assistant Professor of Medicine and Surgery, Divisions of Hematology-Oncology and Surgical-Oncology, University of California at Los Angeles, CA 90095-1782, USA Abstract: The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposomeencoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.

Keywords: Dendritic cells, genetic immunotherapy, gene therapy. DENDRITIC THERAPY

CELLS

FOR

CANCER

IMMUNO-

The majority of cancer vaccine strategies developed in the past decades have a final common pathway, the generation of a pool of tumor antigen in an immunostimulatory environment that attracts host antigen-presenting cells (APC), which process these antigens for T cell cross-priming in lymph nodes. Vaccines composed of lysates of autologous or allogeneic tumor cells, intratumoral injection of bacillus Calmette-Guérin (BCG), vaccination with cytokine genemodified tumor cells or in vivo vaccination with tumor antigen naked DNA and viral vectors, or with heat shock proteins require host APC for antigen cross-presentation (for review see [Ribas et al., 2003]). Among host APC, the best equipped for antigen crosspresentation is the dendritic cell (DC), the most efficient cell type for processing exogenous antigen to major histocompatibility complex (MHC) class I and II pathways. Like other APC, DC can process foreign antigen to MHC class II pathway for CD4+ helper T cell activation, while they are unique in the ability to also present antigen through MHC class I for CD8+ cytotoxic T cell recognition and activation [Mellman and Steinman, 2001]. The cognate antigen presentation by the same APC allows an efficient CD4+ T cell/DC and CD8+ T cell/DC crosstalk leading to the activation of a powerful T cell adaptive immune response. Additionally, DC *Address correspondence to this author at the 11-934 Factor Building, UCLA Medical Center, 10833 Le Conte Avenue, Los Angeles, CA 900951782, USA; Tel: 310-206-3928; Fax: 310-825-7575; E-mail: [email protected] 1566-5232/05 $50.00+.00

have been shown to activate natural killer (NK) cells through unknown ligands [Fernandez et al., 1999], and have high level of CD1 expression for antigen presentation to natural killer T (NKT) cells, therefore also being able to activate innate immune responses [Porcelli and Modlin, 1999] (Fig. 1). Their superior antigen processing machinery is coupled with high surface density of antigen presenting molecules (MHC, CD1, costimulatory and adhesion molecules) and production of immunostimulatory factors (cytokines and chemokines). Taken together, DC represent specialized bone marrow-derived leukocytes specialized in orchestrating immune responses [Banchereau and Steinman, 1998]. The ability to grow DC in vitro in culture systems containing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) [Caux et al., 1992; Inaba et al., 1992; Romani et al., 1994] not only has allowed a further understanding of their immunobiology, but also the testing of their role as adjuvants for tumor antigen presentation and stimulation of tumor immunity. DC directly injected into tumors to exploit their ability to take up foreign antigens and present them to T cells have limited immune stimulatory activity. Therefore, ex vivo antigen loading and/or the addition of immune-stimulating molecules is critical for their use as cancer vaccines [Ribas et al., 2002b]. DC can be loaded with whole tumor protein in form of tumor lysates, by macropinocytosis of apoptotic bodies or by DC-tumor cell fusion. These approaches have been shown to stimulate antitumor immune responses in preclinical models [Mayordomo et al., 1997] and in human clinical trials [Rosenberg, 2001; Timmerman and Levy, 1999]. They have © 2005 Bentham Science Publishers Ltd.

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Fig. (1). Activation of Immune Effector Cells by Genetically Modified Dendritic Cells:

also been demonstrated to be central players in tolerance to self antigens and suppress immune responses [Steinman and Nussenzweig, 2002]. In an attempt to further enhance the ability to present tumor antigens, in vitro-generated DC can also be genetically modified with tumor antigen encoding RNA or DNA. GENETICALLY MODIFIED DENDRITIC CELLS ARE POWERFUL VACCINES IN PRECLINICAL MODELS. Ex vivo tumor antigen genetic modification of DC bypasses the need for host APC uptake of tumor antigens. Animal models have shown that the vaccinating genemodified DC, as opposed to host APC, are the ones directly interacting with host T cells [Ribas et al., 2000a]. Therefore, they bypass a critical step in the mechanism of immune activation of other genetic immunization strategies like direct intramuscular or intradermal injection of naked DNA or viral vectors, which may provide a theoretical advantage for genetically modified DC. Similarly, cytokine gene-modified tumor cells, the most powerful being GM-CSF-modified tumor cell vaccines, requires host APC processing and crosspresentation of tumor antigens. Animal models have repeatedly demonstrated that genetically modified DC are superior to these three forms of immunization [Ashley et al., 1997; Boczkowski et al., 1996; Brossart et al., 1997; Cao et al., 1999; Kaplan et al., 1999; Klein et al., 2000; Ribas et al., 1997; Vollmer et al., 1999; Wan et al., 1997; Wan et al., 1999a; Wan et al., 1999b; Yang et al., 1999]. However, when tested in vitro in T cell culture systems, a bell-shaped immunological activity has been noted, where high concentrations of genetically modified DC respect to responder T

cells leads to tolerance to the transgenes expressed by the DC [Jonuleit et al., 2000; Tuettenberg et al., 2004]. Genetically modified DC allows antigen processing and presentation through MHC class I and II molecules, with continuous presentation of both dominant and subdominant epitopes with endogenous MHC restriction [Perez-Diez et al., 1998; zum Buschenfelde et al., 2001]. This provides a theoretical advantage over immunization with DC pulsed with MHC class I–binding epitopes. When these forms of DC-based immunization have been compared side-by-side, again genetically modified DC have shown superiority [Ashley et al., 1997; Boczkowski et al., 1996; Brossart et al., 1997; Schnell et al., 2000; Sonderbye et al., 1998; Specht et al., 1997; Tuting et al., 1997]. Feeding DC with whole tumor antigen protein, either synthetic or in the form of tumor lysates or apoptotic bodies, would also allow endogenous antigen presentation of multiple MHC-restricted epitopes. However, their expression would be limited to the available cytosolic or vesiclecontained protein antigen, as opposed to continuous protein translation from RNA or DNA transfection. In fact, animal models have repeatedly shown superiority for gene-modified DC compared to DC fed with proteins [Ashley et al., 1997; Miller et al., 2000]. METHODS OF ENGINEERING

DENDRITIC

CELL

GENE

Although DC were initially shown to be difficult to genetically engineer [Arthur et al., 1997], expression of foreign genetic material in DC has been achieved using different approaches, like transfection of plasmid DNA either coated

Genetically Modified Dendritic Cells for Cancer Immunotherapy

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by liposomes or propelled by a helium gene-gun, by transfecting tumor-derived mRNA alone or coated in liposomes, or by viral transduction using replication-incompetent or attenuated viral vectors (Table 1, for further review see [Jenne et al., 2001; Kirk and Mule, 2000; Morse and Lyerly, 2002; Ribas et al., 2002b; Wysocki et al., 2002]). DC transfection with tumor antigen mRNA or naked DNA, either alone, with liposome coating, electroporation, or using a gene gun leads to undetectable or very low level or transgene expression [Arthur et al., 1997; Boczkowski et al., 2000]. However, this seems to be enough to stimulate T cells, possibly because the assays for testing transgene expression are less sensitive than antigen-specific T cells [Gilboa et al., 1998]. The T cell receptor (TCR) is a high affinity receptor for MHC-antigen complexes, and low-level antigen expression can be recognized and lead to T cell activation. Animal models have clearly shown that these nonviral methods of DC transfection can lead to protective immunity to a tumor challenge expressing the same antigen (for review see [Kirk and Mule, 2000; Ribas et al., 2002b]). Viral vector-mediated transduction approaches are more efficient than several physical methods of DC transfection [Arthur et al., 1997], leading to higher levels of transgene expression, and may be a preferred method of DC gene modification [Kirk and Mule, 2000; Wysocki et al., 2002]. Several types of virus are very efficient in infecting DC and, once internalized, have evolved to use the host cell machinery to their advantage by tricking the host cell to express the viral genetic material. These viruses can be genetically engineered to carry foreign genes (encoding tumor antigens or immune stimulatory molecules), while at the same time are rendered replication deficient by deleting critical genes that allow progeny virion generation. Additionally, virus that can infect but do not replicate in mammalian cells (like avian pathogen viruses or highly attenuated mammalian pathogen viruses) can be used as vectors for transgene expression in DC. Viral vectors used for gene modification are listed in Table 1. Each vector has theoretical advantages and disadvantages, and have been extensively reviewed recently [Jenne et Table 1.

al., 2001; Kirk and Mule, 2000; Morse and Lyerly, 2002; Ribas et al., 2002b; Wysocki et al., 2002]. The most widely used have been replication-deficient adenoviral vectors, able to infect differentiated DC leading to transient transgene expression. Retroviral vectors have also been extensively tested in preclinical models. Retroviral vectors require cell division for target cell transduction. Therefore, they need to be used early in the DC culture from bone marrow precursors, being unable to genetically modify differentiated DC. Lentivirus, poxvirus, herpes virus and AAV vectors can infect resting cells, an important feature since human DC are usually differentiated from monocytes without cell division [Romani et al., 1996]. VECTOR-INDUCED ADVERSE AND BENEFICIAL EFFECTS OF DENDRITIC CELLS GENE ENGINEERING Attempts at transgene expression in DC may interfere with DC function (Table 2). Some physical methods, like electroporation, have been shown to be directly toxic to DC [Arthur et al., 1997]. Also, viral vectors may induce a cytopathic effect in DC. Adenoviral vectors used at extremely high ratios lead to DC death, but at optimized multiplicity of infection (MOI) do not result in DC cytopathic effect [Arthur et al., 1997]. Other vectors invariably result in eventual DC cytotoxicity and cell death, like the use of attenuated poxviruses. The window of time between the initiation of transgene expression and cytopathic changes in DC may be enough to provide appropriate signals for T cell activation in a strong immune activating environment [Kim et al., 1997]. In an attempt to shield themselves from immune recognition, viruses have evolved to express genes specifically targeted to interfere with the host cell antigen processing and expressing machinery. For example, E3 and E4 gene regions in adenovirus interfere with MHC class I expression. However, transgene expression in earlier gene regions has reproducibly shown ability to stimulate MHC-restricted T cell responses even when using vectors with intact MHC pathway inhibiting genes, therefore suggesting that this represents a relative but not absolute limitation for the use of viral vectors in DC gene engineering [Ribas et al., 2002b].

Methods of Dendritic Cell Genetic Modification

Genetic Information RNA

Type of Vector Synthetic Oligonucleotides Tumor-derived mRNA

DNA

Plasmid + Liposomes Viral Vectors

Adenovirus Retrovirus Lentivirus Poxvirus: Vaccinia, Avipox, Fowlpox Herpesvirus Adeno-associated Virus

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Table 2.

Antoni Ribas

Effect of the Vector on Dendritic Cell Biology

Vector

Dendritic Cell Cytopathic Effect*

Dendritic Cell Maturation

RNA

No

Yes

Plasmid DNA

No

No

Adenovirus

No

Yes

Retrovirus

No

No

Lentivirus

No

No

Poxvirus

Yes

No

Herpesvirus

Yes

No

Adeno-associated virus

No

Yes

*Cytopathic effect only considered at optimized vector to DC ratios.

An additional potential adverse effect of viral vector transduction of DC would be the presentation of highly immunogenic virally-derived epitopes, leading to a powerful host immune response that may damage the DC vaccines before they stimulate T cell responses. Viral vectors like retroviruses and lentiviruses have low immunogenicity, while adenoviruses and poxviruses are highly immunogenic. Due to the well-recognized inherent immunogenicity of adenoviral vectors, third generation adenoviral vectors devoid of most virally-encoded genes but retaining the ability to express transgenes have been produced [Mitani et al., 1995], which are encapsulated with the help of wild type adenoviruses (“gutless” or helper-dependent vectors). These third generation adenoviral vectors can be used to gene-modify DC. When directly compared, DC gene-modified with third generation helper-dependent and E1-deleted first generation adenoviral vectors had equal ability to stimulate T cells to adenoviral epitopes [Roth et al., 2002]. The reason was that the vector capside, and not the adenoviral genes, contain the epitopes responsible for vector immunogenicity. Therefore, one of the initial conceptual advantages for the use of helperdependent adenoviral vectors for genetic immunization was shown to be incorrect [Roth et al., 2002]. On the contrary, gutless adenoviral vectors were able to overcome the immune suppressive effects of high concentrations of adenovirally-transduced DC in T cell culture systems [Tuettenberg et al., 2004]. This strongly suggests that some adenovirallyderived genes have a direct immune suppressive effect, and further testing of gutless vectors is warranted. Most vectors used for DC gene engineering are foreign to mice. Therefore, the relevant testing of a potential adverse effect of pre-existing host immunity to the viral vector should be carried out in mice that have previously been exposed to type-matched viruses and have developed neutralizing antibodies and viral-specific T cell responses. Several animal models have demonstrated that highly immunogenic adenoviral vector gene-modified DC induce tumor antigenspecific T cell responses in mice with pre-existing high titers of neutralizing antibodies [Brossart et al., 1997; Ribas et al., 2000b].

Conversely, the immunogenicity of viral vectors may not be detrimental. Viral epitopes may further attract immune cells and create an immune stimulatory milieu enhancing the ability of genetically modified DC to activate immune responses. Furthermore, certain methods of DC gene engineering, like RNA transfection and adenoviral or AAV transduction result in DC maturation in a process that is dependent on nuclear factor κB (NF κB) activation, leading to enhanced ability to stimulate T cell responses [Heiser et al., 2002; Hirschowitz et al., 2000; Morelli et al., 2000; Schumacher et al., 2004]. GOALS OF FICATION

DENDRITIC

CELL

GENE

MODI-

Genetic modification of DC can be aimed at expressing tumor antigens or changing their biology to increase or decrease their ability to present antigens (Table 3). In the great majority of published studies, the aim of DC gene modification has been to load them with tumor antigens, taking advantage of the high efficiency of antigen presentation and T cell activation by DC. This can be achieved by defined tumor antigen transfection in form of synthetic RNA or DNA, or by uncharacterized whole tumor antigen mRNA, allowing the transfected DC to endogenously process and present antigenic epitopes through MHC class I and II molecules. Additionally, foreign genetic material can be transferred with the goal of enhancing DC functions, through the supraphysiological expression of additional molecules (soluble cytokines, chemotactic factors, surface receptors or ligands, and antiapoptotic molecules). On the contrary, some reports have transferred transgenes that lead to potentiate the immune suppressive effects of DC. This approach has been used to tolerize to self-antigens in allogeneic organ transplants and not the treatment of cancer [Lu et al., 1999; Takayama et al., 2001], therefore being beyond the scope of this review. 1. Antigen Processing and Presentation by Tumor Antigen Genetically Modified Dendritic Cells Immune subset depletion and the use of knock-out mice allow direct testing of the role of different cell subsets in the protective responses generated by genetically modified DC.

Genetically Modified Dendritic Cells for Cancer Immunotherapy

Table 3.

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Goals of Dendritic Cell Genetic Modification Approach

Examples

Requirement for Characterized Tumor Antigen

Synthetic tumor antigen

MART-1, gp100, PSA, CEA, AFP, telomerase, HPV E6/E7

Yes

Uncharacterized tumor antigens

Tumor-derived RNA

No

Cytokines

IL-2, IL-7, IL-12, IL-18, GM-CSF, IFN, TNF-α

No

Chemokines

Lymphotactin, SCL (CCL21)

No

Costimulatory molecules

B7.1, B7.2, CD40

No

Adhesion molecules

ICAM, LFA-3

No

Pro-inflammatory genes

RHO

No

Anti-apoptotic molecules

Bcl-XL

No

Immune suppressive molecules

IL-10, TGF-β, CTLA-4

No

Abbreviations: MART-1: melanoma antigen recognized by T cells-1; PSA: prostate-specific antigen; CEA: carcino-embryonic antigen; AFP: alpha fetoprotein; HPV: human papilloma virus; GM-CSF: granulocyte-macrophase colony stimulating factor; IL: interleukin; TGF: transforming growth factor; IFN: interferon; RHD: Rel homology domain; SLC: secondary lymphoid tissue chemokine; CTLA: cytotoxic T lymphocyte antigen.

Depletion of CD4+ or CD8+ T cell subsets leads to complete abrogation of gene-modified DC-induced immune responses in the majority of models (reviewed in [Ribas et al., 2002b]), confirming that both immune cell subsets are required for the response. The requirement of CD4+ T cells can be bypassed by CD40 crosslinking of the gene-modified DC, and the immune response in a CD4-null environment generated by CD40-engaged DC is abrogated when CD8+ T cells are depleted [Ribas et al., 2001]. Therefore, it is likely that tumor antigen gene-modified DC present antigen through MHC class II to CD4+ T helper cells, which engage the CD40 receptor on the DC leading to activation of effector CD8+ CTL recognizing cognate antigen presented by MHC class I molecules. Several recent publications have clearly documented a NK-DC cross talk both in murine [Fernandez et al., 1999] and human systems [Zitvogel, 2002]. The contribution of NK cells to the protective response after tumor antigen genemodified DC vaccines has not been frequently reported, although it has been noted that NK1.1 depletion partially abrogated protective responses in three models [Miller et al., 2002; Wan et al., 2000; Wargo et al., 2005]. Therefore, a two or three cell interaction may be involved in the antitumor response generated by gene-modified DC. There are no published reports on the contribution of CD1-restricted NKT cells to the protective responses generated by genetically modified DC. In our unpublished experience, immunization of NKT-deficient C57BL/6/Vα14 mice, or immunization of wild type mice with CD1-deficient DC did not abrogate responses by melanoma antigen genemodified DC (Ribas, A., Economou, J.S. unpublished). It is likely that other hematopoietic cells (monocytes, macrophages, neutrophils, eosinophils) may contribute to tumor regression responses in vivo by being attracted into tumor cell deposits, guided by cytokines and chemokines released after tumor antigen recognition by innate or adaptive immune cells.

2. Dendritic Cells Genetically Modified to Enhance their Immune Stimulatory Properties DC genetically modified to express immune stimulatory molecules usually are injected intratumoraly to take advantage of the high ability of DC to uptake foreign antigens. Tumors grow in an immune suppressive environment, producing multiple molecules that interfere with T cell and DC function. Production of vascular endothelial growth factor (VEGF) inhibits DC differentiation, and tumor-derived transforming growth factor beta (TGF-β) renders tumorinfiltrating DC inactive or even tolerizing [Gabrilovich et al., 1996; Gorelik and Flavell, 2001]. Additionally, tumorproduced IL-10 or prostaglandin E2 (PgE2), among multiple other tumor-produced molecules, interfere with T cell function [Kiertscher et al., 2000; Sharma et al., 1999]. Therefore, DC need to maintain their functional activity in this unfriendly environment to function as effective tumor antigen cross-presenters. With this goal, DC can be engineered to express immune stimulatory cytokines like IL-2, IL-7, IL-12, IL-18 or interferon (IFN)-α [Miller et al., 2000; Tüting et al., 1998], which directly activate T cells that recognize DC-presented antigens. Although the majority of reports suggest that forced continuous type 1 cytokine expression by DC enhances immune activation, cytokine transduction may alter a tightly controlled process resulting in an opposite effect [Ribas et al., 2002a]. The DC growth and differentiation factor GM-CSF has also been expressed in DC, leading to enhanced antigen presentation ability and improved migration to T cell areas of lymph nodes [Curiel-Lewandrowski et al., 1999]. Another approach has been to genetically engineer DC to express genes that enhance the expression of multiple proinflammatory molecules. This can be achieved by transferring genes that enhance NFκB expression. When DC were infected with an adenoviral vector expressing the Rel homology domain (RHD) of NFκB, it resulted in increased DC

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activation and the production of several immune stimulatory cytokines (IL-1β, IL-2, IL-6, IL-12) and upregulation of surface activation and costimulatory markers [Lee et al., 2002]. Chemokines are secreted molecules used by immune system cells to attract or repel other cells. Genetic modification of DC with chemokines that enhance their ability to interact with T cells may enhance their function. DC modified to express lymphotactin or secondary lymphoid tissue chemokine (SLC or CCL-21) have been shown to enhance tumor immunity generated by antigen-loaded DC or when injected intratumorally to allow uncharacterized tumor antigen uptake and cross-presentation to T cells [Cao et al., 1998; Kirk et al., 2001]. Costimulatory and adhesion molecules are key in the interaction between DC and T cells, allowing adequate T cell anchoring and providing critical immunological signals that enhance T cell recognition of MHC-antigen complexes. Although DC are the cell type with the highest level of surface expression of these molecules, additional expression by DC gene engineering has further enhanced their ability to generate tumor immune responses [Hodge et al., 1999; Kikuchi et al., 2000]. The short half-life of DC in lymph nodes (2 to 6 days) is due to apoptotic cell death. Protection from DC apoptosis may enhance immune stimulation by prolonging the interaction between DC and immune effector cells. DC transduced with the antiapoptotic gene Bcl-XL enhanced their survival when injected intratumorally, leading to superior ability to treat established tumors [Pirtskhalaishvili et al., 2000]. CLINICAL TRIALS USING MODIFIED DENDRITIC CELLS

GENETICALLY

The promising preclinical data has provided a strong rationale for the translation of this vaccine approach to the clinical setting (Table 4). The logistic and regulatory hurdles are significant. Opening a clinical trial using genetically modified DC requires the certification of a gene therapy vector for its use with an unstable cell-based vaccine. The minimal regulatory oversight for trial approval in the United States is the local Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC), and the federal Food and Drug Administration (FDA) and Recombinant Advisory DNA Committee (RAC). After gene therapy vector certification for identity (nucleic acid sequence and adequate protein translation) and purity (free from known and unrecognized contaminating agents), regulatory agencies require assays for immunological potency. This usually involves repeating critical preclinical studies with the final, clinicalgrade vector, to demonstrate that it has the same ability as the non-clinical laboratory vector to activate immune responses. Once approved to initiate subject accrual, conduct of the trial requires close oversight best achieved by prospective trial monitoring and frequent data safety monitoring (DSM) review.

There were no responses (defined using the response evaluation criteria for solid tumors – RECIST - criteria [Therasse et al., 2000]) in the 6 evaluable patients. Immunological monitoring clearly demonstrated the stimulation of MHC class I-restricted CD8+ T cell responses, while response to hole PSA protein in form of IFN-γ production (likely mediated primarily by CD4+ T cells) was also evident. The same group of investigators conduced a phase I trial of whole tumor mRNA pulsed DC administered to patients with metastatic renal cell carcinoma [Su et al., 2003]. There was evidence of immune activation to whole tumor mRNA and also to defined antigens expressed by kidney tumors, but there were no objective clinical responses. A phase I/II trial of DC transfected with carcinoembryonic antigen (CEA) also proved to be safe, but devoid of tumor responses by objective criteria, in 24 evaluable patients within the phase I part of the study [Morse et al., 2003]. Thirteen patients were entered after resection of CEA positive tumors in the phase II part of this study, of which 9 had relapsed after a mean follow up of 6 months. Dendritic cells pulsed with tumor-derived RNA were administered to 7 pediatric and young adults with a variety of brain tumors within a phase I trial [Caruso et al., 2004]. Vaccine administrations were well tolerated. One patient with a pleomorphic xanthoastrocytoma had an objective response. There was no evidence of cellular immune response detected when tested using cytokine production and proliferation assays. The same group conducted a study of tumorderived RNA pulsed DC in patients with neuroblastoma [Caruso et al., 2005]. Vaccines were administered six weeks after high dose chemotherapy with stem cell support. There was no evidence of specific immune activation by the RNA loaded DC, most likely due to the prior cytotoxic regimen. None of the 4 patients with measurable disease at the time of vaccine administration had an objective response. A clinical trial administering DC transfected with human telomerase mRNA by electroporation to patients with prostate cancer has been reported [Su et al., 2005]. This study demonstrated CD8+ T cell responses in most patients, while CD4+ T cell responses were stronger in a subset of patients who received a chimeric mRNA containing lysosome-associated membrane protein-1 (LAMP) fused to telomerase. The rationale for using LAMP was based on preclinical data from this same group demonstrating that LAMP redirects antigen expression to the MHC class II pathway, resulting in stronger CD4+ T cell responses [Su et al., 2005]. There were no objective responses, although there were changes in the slope of PSA progression. Two ongoing experiences from the same group have been communicated in abstract form1. Twenty patients with biochemical relapse (increase in PSA) while on androgen ablation therapy for prostate cancer received i.d. or intranodal (i.n.) DC pulsed with allogeneic prostate cancer mRNA derived from 3 established cell lines. Twelve patients 1

Clinical Trials with RNA Transfected Dendritic Cells Immunization with PSA RNA-transfected DC was proven to be safe, with only minimal injection site erythema and occasional flu-like symptoms [Heiser et al., 2002].

Duneland, S., Mu, L., Kvalheim, G., Hauser, M., Waehre, H., Aamdal, S., and Gaudernack, G. Dendritic cells transfected with allo-tumor RNA as cancer vaccine in treatment of hormone resistant prostate cancer patients. Proc Am Soc Clin Oncol. Abstract 2541, (2005). Aamdal, S., Kyte, J., Duneland, S., Mu, L., Gullestad, H., Ryder, T., Hauser, M., Kvalheim, G., Saeboe-Larsen, S., and Gaudernack, G. Phase I/II trial of vaccine therapy with tumor-RNA transfected dendritic cells in patients with advanced malignant melanoma. Proc Am Soc Clin Oncol. Abstract 2540, (2005).

Genetically Modified Dendritic Cells for Cancer Immunotherapy

Table 4.

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Published and Ongoing Genetically Modified Dendritic Cell Clinical Trials

Reference

Principal Investigators

Vector

Transgene

Immune Responses

Clinical Responses

[Su et al., 2003]

Vieweg, J. Gilboa, E.

mRNA

Whole tumor RNA

CD8 and CD4

No O.R.

[Caruso et al., 2005; Caruso et al., 2004]

Ashley, D.M.

mRNA

Whole tumor RNA

NA

1 P.R.

2

Gaudernak, G.

mRNA

Whole tumor RNA

NA

NA

[Heiser et al., 2002]

Vieweg, J. Gilboa, E.

mRNA

PSA

CD8 and CD4

No O.R.

[Morse et al., 2003]

Lyerly, H.K. Morse, M.A. Gilboa, E.

mRNA

CEA

CD8

No O.R.

[Pecher et al., 2002]

Thiel, E.

cDNA

MUC1

CD8

No O.R.

[Tsao et al., 2002]

Haluska, F.G.

Adenovirus

gp100 and MART-1

NA

NA

3

Antonia, S. Gabrilovich, D.

Adenovirus

p53

NA

NA

[Morse et al., 2005]

Lyerly, H.K. Morse, M.A.

Fowlpox

CEA-B7-ICAM-LFA

CD8 and CD4

No O.R.

[Di Nicola et al., 2004]

Di Nicola, M. Anichini, A. Gianni, A.M. Parmiani, G.

Vaccinia

Tyrosinase

CD8 and CD4

1 P.R.

Abbreviations: NA: Not available; PSA: prostate-specific antigen; CEA: carcino-embryonic antigen; AFP: alpha fetoprotein; OR: overall response; P.R.: partial response.

had evidence of immune activation and 10 had evidence of a change in the slope of PSA increase, but none had a decrease in PSA levels. In the second trial, 19 patients with metastatic melanoma received i.d. or i.n. DC loaded with autologous tumor mRNA. Nine had evidence of immune activation but none had an objective clinical response. Clinical Trials with DNA Transfected Dendritic Cells A preliminary report of a clinical trial administrating autologous DC genetically modified with type 2 adenoviral vectors expressing the melanoma antigens gp100 and MART-1 showed evidence of autoimmune depigmentation in three subjects [Tsao et al., 2002]. Immunization with DC gene modified with naked DNA expressing MUC1 using liposomes was devoid of major toxicities. Immunological monitoring using IFN-γ intracellular cytokine staining demonstrated MHC class I-restricted antigen-specific responses 2

Duneland, S., Mu, L., Kvalheim, G., Hauser, M., Waehre, H., Aamdal, S., and Gaudernack, G. Dendritic cells transfected with allo-tumor RNA as cancer vaccine in treatment of hormone resistant prostate cancer patients. Proc Am Soc Clin Oncol. Abstract 2541, (2005). Aamdal, S., Kyte, J., Duneland, S., Mu, L., Gullestad, H., Ryder, T., Hauser, M., Kvalheim, G., Saeboe-Larsen, S., and Gaudernack, G. Phase I/II trial of vaccine therapy with tumor-RNA transfected dendritic cells in patients with advanced malignant melanoma. Proc Am Soc Clin Oncol. Abstract 2540, (2005). 3 Gabrilovich, D.I., Mirza, N., Chiappori, A., Dunn, M., Willis, M., Janssen, W., Smilee, R., Menader, K., Chada, S., and Antonia, S.J. Initial results of a Phase II trial of patients with extensive stage small cell lung cancer (SCLC) immunized with dendritic cells (DC) transduced with wild-type p53. Proc Am Soc Clin Oncol. Abstract 2543, (2005).

in a subset of patients with mucine positive adenocarcinomas of the breast or pancreas [Pecher et al., 2002]. A phase I study of CD34-derived DC transduced with a modified vaccinia vector expressing the melanoma antigen tyrosinase proved safe in 6 patients with metastatic melanoma [Di Nicola et al., 2004]. Antigen-specific CD8+ T cell responses were demonstrated in all tested patients, and antigen-specific CD4+ T cell responses were noted in one out of 2 tested patients. One patient with a single nodal metastasis from melanoma achieved a partial response. In addition, the administration of autologous DC tranduced with an adenoviral vector expressing wild type p53 has been communicated in abstract form4. Twenty-two patients with small cell lung cancer with a stable disease or minimal progression to first line chemotherapy were vaccinated. Eleven had evidence of immune responses, but none had an objective response. CONCLUSIONS Genetic engineering provides a powerful tool to modify the immune activating effects of DC. Direct comparisons in preclinical models suggest that genetically modified DC vaccines are among the most powerful vaccine approaches to stimulate tumor protective responses. The variety of methods 4

Gabrilovich, D.I., Mirza, N., Chiappori, A., Dunn, M., Willis, M., Janssen, W., Smilee, R., Menader, K., Chada, S., and Antonia, S.J. Initial results of a Phase II trial of patients with extensive stage small cell lung cancer (SCLC) immunized with dendritic cells (DC) transduced with wild-type p53. Proc Am Soc Clin Oncol. Abstract 2543, (2005).

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to genetically modify DC have different effects on DC biology, but they all have shown to efficiently prime immune cells to reject tumors. These approaches are now being tested in pilot clinical trials for several tumors, with very limited evidence of antitumor activity thus far. Limitations to the further clinical development of this approach includes the requirement for specialized facilities and highly skilled personnel for the generation of these genetically modified personalized vaccines, and the possibility of inducing unwanted responses to the transgenes, gene therapy vectors or unrecognized self antigens presented by the DC. However, the ability of genetically modified DC vaccines to induce T cell responses to tumor antigens in patients with cancer provides a rationale for their continued testing beyond feasibility trials. In this regard, cancers with well characterized tumor responsiveness, like malignant melanoma, renal cell carcinoma, chronic myelogenous leukemia or low grade lymphomas, as well as cancers with currently limited treatment options like brain tumors, hormone refractory prostate cancer or advanced epithelial cancers, may be amenable for continued clinical testing. ACKNOWLEDGEMENTS This work was supported by a Career Development Award from Stop Cancer, a UCLA Human Gene Medicine Seed Grant and K23 CA93376. ABBREVIATIONS DC

=

Dendritic cells

APC

=

Antigen-presenting cells

AAV

=

Adeno-associated virus

BCG

=

Bacillus Calmette-Guérin

MHC

=

Major histocompatibility complex

NK

=

Natural killer cells

NKT

=

Natural killer T cells

GM-CSF =

Granulocyte-macrophase colony stimulating factor

IL

=

Interleukin

MOI

=

Multiplicity of infection

NFκB

=

Nuclear factor κB

VEGF

=

Vascular endothelial growth factor

TGF-β

=

Transforming growth factor beta

PgE2

=

Prostaglandin E2

IFN

=

Interferon

RHD

=

Rel homology domain

SLC

=

Secondary lymphoid tissue chemokine

IRB

=

Institutional Review Board

IBC

=

Institutional Biosafety Committee

FDA

=

Food and Drug Administration

RAC

=

Recombinant Advisory DNA Committee

DSM

=

Data safety monitoring

Antoni Ribas

PDQ

=

Physician Data Query

PSA

=

Prostate-specific antigen

NA

=

Not available

AFP

=

Alpha fetoprotein

OR

=

Overall response

P.R.

=

Partial response

RECIST

=

Response evaluation criteria for solid tumors

CEA

=

Carcinoembryonic antigen.

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Received: ???????????

Accepted: September 07, 2005

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