Optimizing dendritic cell-based immunotherapy for

Perspective

Optimizing dendritic cell-based immunotherapy for cancer Hua Zhong, Michael R Shurin† and Baohui Han

Five-year view

Expert Rev. Vaccines 6(3), xxx–xxx (2007)

DC-based immunotherapy Efficacy of dendritic cell-based immunotherapy

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Standartization & optimization of dendritic cell-based immunotherapy

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CONTENTS

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Expert commentary

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells and are unique in their capability to initiate, maintain and regulate the intensity of primary immune responses, including specific antitumor responses. Development of practical procedures to prepare sufficient numbers of functional human DC in culture, from the peripheral blood precursors, paved the way for clinical trials to evaluate various DC-based strategies in patients with malignant diseases. However, no definite conclusions regarding the clinical and even immunological efficacy of DC vaccination can be stated, despite the fact that 12 years have passed since the first clinical trial utilizing DCs in cancer patients. Many unanswered questions hamper the development of DC-based vaccines including the source of DC preparation and protocols for DC generation, activation and loading with tumor antigens, source of tumor antigens, route of vaccine administration and methods of immunomonitoring. Fortunately, in spite of the many obstacles, DC vaccines continue to hold promise for cancer therapy.

Key issues References

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Affiliations

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells and are characterized by a unique capability to initiate, maintain and regulate the intensity of primary immune responses, including specific antitumor responses. DCs loaded ex vivo with tumor antigen(s) to be administered to cancer patients in an effort to induce or boost a therapeutically meaningful antitumor immune response is currently the most common approach utilizing DCs. Examples of loading DCs include DCs pulsed with defined peptides, proteins, or tumor cell lysates or exosomes; DCs fed with apoptotic or necrotic tumor cells/bodies; DCs genetically modified to express tumor antigens; and DCs fused with tumor cells. Numerous preclinical studies proved that DC-based therapy is a potent antitumor approach, able to initiate tumor antigen-specific immunity leading to both prophylactic protection from tumor development and therapeutic eradication of the tumor in a variety of primary and metastatic tumor models, including orthotopic and spontaneous tumor models. Development of feasible procedures to prepare a large amount of human

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Author for correspondence 5725 Children’s Hospital of Pittsburgh – Main Tower, 200 Lothrop St., Pittsburgh, PA 15213, USA Tel.: +1 412 648 9841 Fax: +1 412 647 7741 [email protected]

KEYWORDS: cancer, clinical trials, cytokines, dendritic cells, immune escape, immunosuppression, immunotherapy, vaccine

www.future-drugs.com

10.1586/14760584.6.3.xxx

DCs in cultures from peripheral blood monocytes and CD34+ hematopoietic precursor cells supported wide expansion of clinical trials, testing different DC-based approaches in patients with malignant diseases. This article focuses on the analysis of the dozens of reviews devoted to numerous clinical protocols utilizing DC vaccine,s with an attempt to picture the progress and promising directions regarding the development of the field. DC-based immunotherapy

Immediately after the results of the first clinical trials, utilizing patients’ DCs loaded with tumor antigens, were reported by Mukherji and colleagues in melanoma patients [1], Hsu and colleagues in B-cell lymphoma patients [2] and Murphy and colleagues in prostate cancer patients [3], a first editorial review appeared and opened the avenue for multiple discussions of a novel therapeutic approach for cancer, its advantages and disadvantages and its further developments. The first conclusion was pretty optimistic and was based on the fact that the majority of patients treated developed T-cell-mediated antitumor immune responses

© 2007 Future Drugs Ltd

ISSN 1476-0584

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allow a detailed historical analysis of clinical employment of DC vaccines. A very crude estimation of the numbers of reviews focusing on DC-based therapy for cancer suggests that, in 2005–2006, a new review appeared every 1–2 weeks (FIGURE 2). Surprisingly, these numbers nearly exceed the numbers of original publications on the results of actual DC-based clinical trials. This probably reflects our still unwaning interest in DC vaccines, as well as the hope and optimism regarding their efficacy and potency. Efficacy of dendritic cell-based immunotherapy

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Virtually hundreds of different reviews estimated the efficacy of DC vaccines in patients with malignant diseases during the last 10 years. Some of these reviews are based on careful evaluation and analysis of clinical data presented in original publications. Others simply cite the original conclusions or repeat previous arguments and discussions. However, in spite of different styles and methodological approaches, all reviews, with only a very few exceptions, are almost similar in their conclusions regarding the efficiency of clinical trials utilizing autologous DCs in cancer patients (TABLE 1). Basically, all of the authors agree that many, especially early, data on the clinical efficacy of DC vaccines were quite promising since many treated patients demonstrated detectable development or upregulation of antitumor immunity with some patients experiencing complete or partial clinical responses. For instance, from the end of the 1990s until the middle of the first decade of the 21st Century, the following conclusions were common and typical: DCs are an attractive target for therapeutic manipulation of the human immune system and for enhancement of insufficient immune responses in cancer [10]. In clinical trials, DC-based vaccination of patients with advanced cancer has in many cases led to immunity and in selected patients, to tumor regression [11]. Among the many developmental vaccination strategies, DC-based immunization appears to be one of the most promising, to date [12]. Thus, from these and many other reviews, it became clear that administration of autologous DCs loaded with tumor antigens in cancer patients is a safe and nontoxic procedure, which might result in partial or complete clinical responses in certain individuals. However, in spite of these optimistic and promising results, recent reviews began to express a significant skepticism regarding the clinical effectiveness of the administration of autologous tumor antigen-loaded DCs in patients with different malignancies. For instance, O’Neill and colleagues concluded in 2004 that “published reports of DC-based vaccine trials in humans have yet to demonstrate improved potency of DC vaccines over more traditional vaccine preparations” [13]. Similarly, Figdor and colleagues stated “in most DC trials, clinical responses are not reported using specific, well-defined and generally accepted criteria for the codification of tumor responses” [14] and Soruri and colleagues stated “unequivocal proof for the clinical efficiency of monocyte-derived DC-based antitumor vaccinations is still missing” [15]. Furthermore, Vieweg and colleagues concluded that “despite these encouraging results, the vaccine-induced immune responses achieved to date are not yet sufficient to

Mukherji et al. [1] Hsu et al. [2] Murphy et al. [3]

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and some of the patients experienced tumor regression [4,5]. Those results suggested that DC-based immunotherapy was a potentially useful approach to B-cell lymphoma, melanoma and prostate adenocarcinoma and raised the possibility that the mode may also be useful in the treatment of other tumors. DC-based therapy is considered as a cell-based approach to vaccination against cancer, that is, to exploit DCs as ‘nature’s adjuvants’ and actively immunize cancer patients with a sample of their own DCs loaded with tumor antigens [6–8]. A decade of developing and testing DC vaccines included a variety of pilot and Phase I–III clinical trials in patients with melanoma, B-cell lymphoma, lung, kidney, colon, breast and prostate carcinomas, multiple myeloma, brain tumors and other malignancies. In 2003, Ridgway pointed out that, despite the technical encumbrances of this treatment, we had reached the ‘millennium’ of trials in search of feasible modifications of a DC-based approach [9]. Since the first published clinical trial of DC vaccination in 1995, 98 studies describing more than 1000 patients have been published in peer-reviewed medical journals or presented at the annual meetings of the American Society for Clinical Oncology, the American Association of Cancer Research or the American Society of Hematology. Trials have been performed in 15 countries and included patients with more than 24 tumor types (FIGURE 1). Based on growing interest in DC vaccines and improved understanding and appreciation of the role that the DC system plays in tumor development and control, it can roughly be estimated that the number of treated patients and clinical trials evaluating DC vaccines might triple by 2008. A constantly growing quantity of medical center-based and industry-based clinical trials utilizing administration of autologous DCs in patients with cancer requires frequent analyses of their feasibility, safety, as well as the immunologic and clinical effects. A large number of formal and full-scale reviews emerging in parallel with the original description of clinical data

2002

>100 clinical trials >1000 immunized patients 15 countries

2008

>300 clinical trials >3000 immunized patients a20–25 countries

Figure 1. An intensive development of dendritic cell (DC)-based protocols for cancer therapy resulted in an astounding number of treated patients in numerous clinical trials in many countries worldwide. Starting from the first trials reported by Mukherji and colleagues [1] and Hsu and colleagues [2], tens of DC-based vaccines were developed and tested in many clinics every year with a first enumeration attempt by Ridgway in 2003 [9]. The number of immunized patients and trials by 2008 is a crude estimation from the analysis of numerous reviews and extrapolation from available data.

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Expert Rev. Vaccines 6(3), (2007)

Optimizing dendritic cell vaccines

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Review numbers

attain a robust and durable therapeutic 30 effect in the cancer patient. Therefore, further improvements are required to 25 enhance vaccine potency and optimize the potential for clinical success” [12]. Why are 20 there different opinions coming from ana15 lyzing limited numbers of published results of using DC vaccines? It appears 10 that the main reasons relate to increasing differences in the trial evaluation criteria. 5 Although the majority of clinical trials are 0 still in Phase I, interpretations are hampered by marked variation in study design related to methodological aspects of DC Year generation, administration and schedule, monitoring of immune response, and clin- Figure 2. Approximate annual numbers of reviews devoted to the analysis of DC-based clinical ically relevant end points, including toxic- trials in cancer, their efficacy, pitfalls, solutions and future improvements. These numbers were ity and response analysis [11]. Tens of varia- obtained using PubMed search engine and utilizing keywords ‘dendritic cells’, ‘cancer’, ‘tumor’, ‘immunotherapy’, ‘vaccine’ and ‘review’. Additional search included the analysis of publications of specific tions make it quite difficult to compare authors. It is estimated that the real numbers of related reviews could be 10–15% higher than shown in feasibility and efficacy of DC-based clini- this figure. cal trials carried out in different institu- DC: Dendritic cell. tions worldwide. Furthermore, the analyFortunately, it is becoming increasingly clear that further sis of conclusions provided by numerous reviews (TABLE 1) suggests that, although immunomonitoring of patients receiving progress in developing DC vaccines will be impossible without DC vaccines has shown some success in the induction or stimu- an adequate and sufficient comparison of the trials’ efficacy, lation of antitumor immunity, clinical response rates remain which, in turn, is impractical without reaching a consensus unsatisfactory and inconsistent. Thus, despite eliciting robust with respect to criteria for DC validation, standardized immune responses, to date, most DC vaccines have had limited immune monitoring techniques and a comprehensive narrative clinical impact justifying the rationale for developing alternative of clinical responses, including a long-term outcome. Although DC-based immunotherapeutic strategies. our knowledge of standardization and development of quality The following study illustrates a complex design of typical clin- criteria for DC vaccination is just beginning to grow, certain ical protocol and demonstrates potential difficulties associated progress in this direction give us reasonable hope that the with the interpretation of results of clinical trials testing DC vac- design of novel DC-based trials will soon reach a new level. cines in cancer patients. Between March 1999 and May 2000, 18 HLA-A*0201+ patients with metastatic melanoma were enrolled Standartization & optimization of dendritic in a Phase I trial using a DC vaccine generated by culturing cell-based immunotherapy CD34+ hematopoietic progenitors, including Langerhans’ cells In spite of a 12-year expansion and exploration of DC-based [16]. DCs were loaded with four melanoma peptides (MARTvaccination for cancer treatment, the fundamental optimization 1/MelanA, tyrosinase, MAGE-3, and gp100), influenza matrix problems have not really been solved. Which DC subtypes peptide (Flu-MP), and keyhole limpet hemocyanin. Ten patients should be used? How should DCs be matured? How to load received eight vaccinations, one patient received six vaccinations, DCs with tumor antigens? What is the best route of DC adminone patient received five vaccinations and six patients received istration? Even though these and related technical questions four vaccinations. Melanoma-specific immunity was measured have been asked repeatedly in all reviews discussing DC-based by interferon (IFN)-γ production and tetramer staining of clinical trials [6–8,17,18], they are still waiting to be answered. For peripheral blood mononuclear cell (PBMC). As of August 2005, example, it has been repeated recently that the type of human four patients were alive with three of them having had no addi- DCs, the mode of their activation and the strategy for delivery tional therapy. Patients who survived longer were those who of antigen(s) are three critical factors for efficient stimulation of mounted melanoma peptide-specific immunity to at least two tumor-specific CD8+ and CD4+ T cells [19,20]. Despite numerous discussions on how to improve the generamelanoma peptides. The authors concluded that their results justified the design of larger follow-up studies to assess the immuno- tion of DCs for patient vaccination [21–23], there is no common logical and clinical outcomes in patients with metastatic protocol for preparing, maturing and pulsing DCs ex vivo. The melanoma vaccinated with peptide-pulsed CD34-derived DC main reasons for our incapability to agree on an optimal proto[16]. This report thus exemplifies the complexity of analyzing the col for generating and injecting DCs in patients have been efficacy of DC-based clinical trials since different schemas were revealed repeatedly and discussed frequently. This includes the utilized in a single trial. complexity of the regulatory issues associated with adoptive


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Table 1. Analysis of clinical efficacy of dendritic cell-based vaccines in clinical trials . Year

Ref.

The majority of patients with low-grade malignant B-cell lymphoma, who were treated with DCs pulsed with tumor-derived idiotype protein, developed T-cell-mediated anti-idiotype immune responses and some of the patients experienced tumor regression

1997

[5]

The results of pilot clinical trials indicated that DC vaccines can induce efficacious tumor-specific immune responses

1997

[68]

The initial results of DC-based clinical trials revealed effective antitumor responses even in heavily pretreated patients bearing advanced cancers; however, further clinical trials are required to validate the efficacy of vaccination with tumor-antigen-loaded DCs

1998

[69]

Administration of antigen-loaded DCs in cancer patients with metastatic renal carcinoma, melanoma and B-cell lymphoma induced some lasting remissions with no or minor side effects

1999

[17]

Using DCs as cancer vaccines may lead to measurable immune responses and, in a few cases, to complete disease responses in patients with B-cell lymphoma and melanoma

1999

[70]

The earliest attempts at DC vaccination for cancer immunotherapy have demonstrated efficacy against several human cancers. However, a large number of technical variables of DC vaccines await in vivo testing before this approach is optimized.

1999

[34]

Application of DCs as adjuvants for cancer immunotherapy has demonstrated its unique clinical capacity, including durable complete responses

2000

[38]

Clinical studies using DCs demonstrated induction of objective immune responses

2000

[71]

The analysis of reported studies suggest that clinically relevant immune responses may be induced against some types of malignancies

2000

[20]

Although the results of the various DC trials are provocative, these vaccines are not yet broadly applicable to clinical practice

2000

[72]

Clinical trials reported responses in the minor fraction of the vaccinated cancer patients. Although the new results with antigen-loaded DCs are encouraging, DC vaccination is at an early stage

2001

[8]

Specific antitumor immune responses have occurred in many DC vaccination studies and some clinically meaningful responses have also been reported. However, it is unclear whether these clinical response rates are better than might be achieved by direct vaccination approaches

2001

[73]

The results of the Phase I clinical trials indicated that little is still known regarding the exact mechanisms by which DCs modulate tumor immunity. The concern is that premature clinical trials might be harmful to the concept of DC-based tumor immunotherapy

2001

[74]

Initial clinical studies of human DC vaccines demonstrated induction of tumor-specific immune responses and tumor regression. Nevertheless, much work is still needed to address several variables that are critical for optimizing this approach

2001

[75]

DC vaccines appear to generate a detectable CTL response; however, clinical responses, except for the response to B-cell lymphoma, remain rather low

2002

[76]

The results of the initial clinical trials suggest that DC vaccination has distinct potential for inducing immune responses and might have considerable therapeutic potential. However, they are difficult to compare because of the variability in DC quality, study design, immunomonitoring and the patient populations

2003

[6]

There are a lot of questions regarding the best way to exploit DC vaccines, including the degree of the DC maturity, choice of priming antigens, route and schedule of administration, targeting to lymphoid tissue and use of additional adjuvants

2003

[77]

The development of DC cancer vaccines is still in the exploratory stage at each of several dozen investigational centers

2003

[9]

Even though many advances have been achieved over the past years, DC vaccines are still far from a unifying therapy choice

2003

[78]

Although induction of CTL and clinical responses have been observed in vaccinated cancer patients, DC-based therapy is still in its infancy and there are many issues to be addressed

2003

[79]

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Conclusions

CTL: Cytotoxic T lymphocyte; DC: Dendritic cell.

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Table 1. Analysis of clinical efficacy of dendritic cell-based vaccines in clinical trials (cont.). Year

Ref.

In spite of the several unclear aspects regarding the optimal modalities for preparing and using DC vaccines, the research progress in this field is rapid and one can predict that in the near future DCs will represent a valuable treatment option for certain patients with cancer

2003

[80]

DC-based therapy is still in its infancy and two-arm trials are required for optimizing their use and assessing their efficacy in comparison with other immunotherapies

2004

[13]

The studies have demonstrated that DCs pulsed with tumor antigen can be safely administered and could produce antigen-specific immune responses; however, clinical responses have been observed in a minority of patients

2004

[81]

Although injection of DCs can prime and boost antigen-specific CTL, protocols must be optimized further to fully harness the adjuvant properties of DCs

2004

[37]

In initial clinical trials, DC were largely suboptimal, yet encouraging results have been seen

2004

[82]

Several of the early studies were inadequate in their design and interpretation and quality control of the DC vaccines has been largely lacking in many studies. In most DC trials, clinical responses are not reported

2004

[14]

The first RNA-transfected DC-based clinical studies have indicated their feasibility and safety and, in some cases, clinical responses were observed even in patients heavily pretreated with standard chemotherapy and/or radiotherapy approaches

2005

[24]

While the rate of objective tumor regression is limited, it warrants further exploration to establish the therapeutic value of vaccination with DCs

2005

[35]

Although early DC trials established evidence of vaccine safety, immunological stimulation and even clinical responses in selected subjects, the vaccine-induced immune responses achieved to date are not yet sufficient to attain a robust and durable therapeutic effect in cancer patients

2005

[12]

Even though some clinical trials have established the feasibility and safety of DC vaccines and have been associated with immune responses and clinical responses, others have been the targets of major ethical and scientific concerns. For unknown reasons, the results of some clinical trials remained unpublished, raising further doubts on the original enthusiastic reports

2005

[15]

Although the data from published studies are encouraging, whether or not DC vaccination can provide a significant, long-term clinical benefit in cancer patients remains to be seen

2006

[26]

In spite of immunological and clinical responses in selected patients, mechanisms involved in DC-based cancer immunotherapy are still poorly understood, therefore, requiring a standardized study design and small pilot trials

2006

[83]

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Conclusions

CTL: Cytotoxic T lymphocyte; DC: Dendritic cell.

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transfer of patients’ cells, financial, ethical, statistical and legal problems related to designing protocols allowing systematic comparison of three or more arms in one trial, and financial and lawful concerns linked to utilizing reagents from different manufacturers in one clinical trial. All authors analyzing DC-based clinical protocols and attempting to compare and contrast the efficacy of DC immunotherapy entirely concur that, in order to improve the clinical usefulness and further development of DC tumor vaccination trials, several different dilemmas need to be considered and several problems to be solved [20]. Among them, first of all, are a number of choices, which should be made rationally for an essential optimization of clinical protocols (BOX 1). Therefore, in order to improve the clinical efficacy of DC vaccination trials and optimize clinical protocols, different points need to be considered. More importantly, all of these procedures need standardization to allow comparison of clinical outcomes in further in vivo studies [15,24,25]. First, standard


protocol(s) for DC generation and activation should be developed to improve the reproducibility of the vaccination procedure and allow a comparison of the results from different studies. Secondly, the search for highly immunogenic antigens should be intensified not only to possibly improve clinical benefit, but also to monitor the immunological response [26]. Furthermore, some believe that for optimization of DC vaccination, it is now a priority to address the many variables and potential improvements suggested by emerging insights into DC biology in small two-armed trials using immune monitoring to define the end point [6]. Thus, again as has been repeated tens of times, “DC vaccines and clinical studies must be standardized” [14]. However, it might be more prudent to standardize the outcome of trials – immune monitoring and tumor monitoring – but not only the form or other particulars of the DC vaccine. In addition, useful standardization will be impossible without understanding the mechanisms responsible for limited efficacy of modern DC vaccines.

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Box 1. Required choices for the essential optimization of clinical protocols . Source of DC precursors: • Monocyte obtained from PBMC versus isolated CD34+ hematopoietic precursors • Agents upregulating precursor numbers (e.g., granulocyte–macrophage colony-stimulating factor and FLT3L) • Whole blood versus leukopheresis Method of isolation of DC precursors: • Gradient centrifugation, elutriation, adherence to plastic surface and isolation with magnetic beads • Types of magnetic beads and means of their harvesting; positive versus negative selection • Markers for DC or monocyte isolation (e.g., CD83 versus CD1a versus CD11c; CD14 and others) • Storage medium, conditions and duration; cryopreservation; transportation

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DC culture conditions:

• IL-4, -13 or -15 for monocyte-derived DCs; FLT3L or stem cell factor for CD34-derived DCs • Number of additions of cytokines to DC cultures; duration of cultures DC maturation: • Mature versus immature

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• Concentrations of cytokines and growth factors

• Timing of maturation in cultures • Maturation in cultures versus in situ maturation DC protection:

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• Maturation with TNF-α, IFN-α, IFN-γ, CD40L, IL-1β, IL-6, TLR ligands (e.g., microbial products, such as LPS or dsRNA [poly-I:C], which signal via TLR4 and TLR3, respectively), PgE2 or various combinations

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• IL-2, -12, -15 (protection from tumor-induced apoptosis or functional inhibition or polarization in vivo) Analysis of DC prior to administration:

• Phenotyping (markers), morphology (criteria), function (e.g., motility, antigen processing/presentation capacity and cytokine production)

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• Sensitivity to stimulation; level of viability

• Elimination of dead cells; contamination with other cell populations DC administration:

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• Dose, frequency and number of injections

• Route (intravenous, intradermal, subcutaneous, intralymphatic, intranodal, intratumoral) • Prime–boosting regime; combinations Tumor antigens:

• Antigenic peptides and their mixtures versus whole proteins versus RNA coding for defined antigens versus peptide analog with increased affinity to major histocompatibility complex class I molecules (known as heteroclitic peptides) • Plasmid DNA • Tumor lysates (different methods of tumor destruction and isolation of antigenic fractions) • Apoptotic versus necrotic tumor cells • Exosomes; antigens in capsules, liposomes, biodegradable microspheres or bound to immunoglobulins or immune complexes • Use of adeno-like viruses versus vaccinia viruses versus lentiviruses • DC–tumor cell hybrids (fusion) • Autologous tumor cells versus allogeneic tumor cell lines CTL: Cytotoxic T lymphoctye; DC: Dendritic cell; IFN: Interferon; IL: Interleukin; PBMC: Peripheral blood mononuclear cell; TLR: Toll-like receptor; TNF: Tumor necrosis factor.

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Box 1. Required choices for the essential optimization of clinical protocols (cont.). Monitoring of immune responses: • Tetramers/pentamers, CTL assay, ELISPOT and others Objective tumor responses: • For example, RECIST. CTL: Cytotoxic T lymphoctye; DC: Dendritic cell; IFN: Interferon; IL: Interleukin; PBMC: Peripheral blood mononuclear cell; TLR: Toll-like receptor; TNF: Tumor necrosis factor.

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be mentioned and taken into account [33]. Many of these problems were and are under consideration and some of them were partly solved. However, the complexity of the DC system necessitates its rational manipulation to achieve therapeutic immunity [10]. Expert commentary

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It is difficult to overestimate the role of basic science in leading the progress of clinical trials and opening new opportunities for increasing the efficacy of DC vaccinations. This was already apparent after several initial publications and confirmed in later reports. In fact, Timmerman and Levy stressed that further integration of fundamental tumor immunology research with well-designed clinical trials of DC-based vaccination promised to improve our methods for generating clinically effective antitumor immunity [34]. Banchereau and Palucka emphasized that a better understanding of how DCs regulate immune responses would allow us to better exploit these cells to induce effective antitumor immunity [35]. Integration of growing knowledge on DC biology into clinically meaningful vaccines stimulated the development of both different immunotherapeutic modalities and academic research on DC behavior and function in health and diseases. Indeed, questioning DC longevity after administration in tumor-bearing hosts moved forward the analysis of DC–tumor cell interactions and inspired studies focusing on protection of the DC vaccine from tumor-mediated suppression and polarization. Hopefully, there has been a growing realization that it is precisely these tumor-mediated immunosuppressive and tolerizing mechanisms that must be overcome for an immunotherapeutic strategy to be successful. Although, there remains a lot to be understood regarding the effects of tumors on DCs before we can expect to benefit from DC-based tumor immunotherapy of cancer patients [36], “with research continuing at its current pace, it is reasonable to expect that substantial progress with DC-based cancer vaccines can be achieved in the near future” [12]. There is no doubt that DC vaccines can prime and boost antigen-specific T-cell responses in patients and, thus, hold the promise of a treatment modality that is tumor-specific, bears little toxicity and, once fully developed, could have a long-lasting effect. The results of various DC-based clinical trials provided initial indications of their potent antitumor capacity associated with tumor regression in selected cancer patients. However, at present, no solid conclusions regarding the clinical efficacy of DC-based vaccination can be drawn. Many continuous

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Despite numerous data suggesting that cell-mediated immune responses are imperative in controlling tumor growth in cancer patients, there are several explanations for a limited efficacy of modern DC-based therapies. For instance, limited longevity and function of injected DCs in the tumor environment might be due to tumor-derived factor-mediated induction of DC apoptosis or suppression of antigen processing, presentation and cell motility [27,28]. Inhibited generation and function of endogenous DC in cancer patients [29] may markedly attenuate the efficacy of exogenous DCs, as the interaction between endogenous and administered DCs and antigen exchange between these cell populations has been shown recently to play an important role in the activation of T cells and induction of antigen-specific immune response [30]. Finally, dysbalanced cytokine and chemokine networks in the tumor microenvironment, including both involved and noninvolved regional lymph nodes could also downregulate the efficiency of antigen presentation to T cells and induction of maintenance of antitumor immune response [31]. Furthermore, another layer of problems relates to the effect of conventional cancer therapies on the immune system and, particularly, the immune effectors. For instance, the gold standard treatment for locally advanced cervical cancer is primary radiation therapy combined with chemotherapy. A potential drawback of this potentially curative treatment is a profound and long-lasting negative effect on the immune system. Thus, treatment-induced immunosuppression combined with tumor-induced subversion of the immune system may, therefore, impose severe limitations on the efficacy of conventional vaccination strategies in late-stage cervical cancer patients [32]. All of the abovementioned scenarios may explain some of the shortcomings in the development of effective DC vaccines. However, the use of ex vivo generated autologous DCs that are loaded with tumor antigen(s) and protected from tumor-mediated dysfunction under controlled conditions might permit to establish the parameters for optimal vaccination against cancer. In addition, there are common problems for all immunotherapeutic approaches, which are in focus of many laboratories optimizing these strategies. These include weak immunogenicity of many human tumors, tumor-mediated immune tolerance, unresponsiveness and immunosuppression, antigenic drift, biophysical barrier in solid tumors, and developing resistance to immune attack by tumor cells (immunoediting). Furthermore, age-dependent alterations of the DC system functioning and its relevance to the DC vaccine efficacy should also


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of DC vaccine immunotherapy” [14]. This wish is still mostly accepted as an inclination today and will not, unfortunately, be truly materialized for many years ahead. Five-year view

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Although numerous suggestions for the optimization and standardization of DC vaccines migrate from one review to another without any significant input on ongoing clinical trials, novel ideas suggested in many excellent reviews were partly tested in clinical settings and, thus, significantly supported the progress in the field (TABLE 2). For example, regardless of a wide skepticism, vaccination studies using RNAtransfected DCs should be evaluated further and critically analyzed as showing relatively high levels of clinical and immunological responses. In fact, after the first vaccination Phase I clinical trial utilizing prostate-specific antigen (PSA) RNA-transfected DCs in patients with metastatic prostate cancer, which demonstrated appearance of PSA-reactive IFNγ-secreting T cells with the ability to lyse PSA-expressing cells [39], other clinical trials in patients with renal cell carcinoma, colon cancer and other malignancies confirmed potential efficacy of DCs transfected with RNA coding for tumor antigen(s) [24,40]. An important and interesting finding of some studies was the induction of both CD4+ and CD8+ T-cellmediated responses. Moreover, although recent studies revealed that interaction between DCs and natural killer (NK) cells plays an essential role in antitumor immunity, approaches for improving DC vaccine interaction with NK cells has not yet been implied in clinical practice. Interestingly, analysis of NK cell responses in a Phase I clinical trial of a vaccine consisting of autologous DCs loaded with tumor antigens revealed that NK cell responses following DC vaccination correlated more closely with clinical outcome of the patients than did antigen-specific T-cell responses [41]. Development of combination chemoimmunotherapy is another perspective direction in enhancing efficacy of DC vaccines. DC-based immunotherapy might cooperate with chemotherapy to augment tumor cell killing or indirectly generate additional proinflammatory signals [42]. Interestingly, DCs respond differentially to killed tumor cells, depending upon the mechanism of DNA damage and consequent cell death. Tumor cells killed by alkylating agents, such as melphalan and chlorambucil, are more effective at activating DCs and antigen presentation, when compared with tumor cells killed by antimetabolites or freeze thaw [43]. In addition, some cytotoxic agents (e.g., cyclophosphamide) may neutralize the inhibitory arm of the immune system and enhance antitumor responses [44]. One of the means to conquer the problem of identifying and introducing suitable tumor antigens in DCs is not to rely on antigen loading into DCs in vitro but on that in situ by intratumoral injection of ex vivo-generated DCs [45]. Several lines of evidence suggest that intratumoral DCs play an imperative role in antitumor immune responses and there is a strong rationale for exploitation of intralesional administration of DC vaccines

…DCs are an attractive target for therapeutic manipulation…

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attempts from 1995 until now concentrated on the construction of the DC-based vaccines, which would produce measurable antitumor immunity and modified stages and stimuli of DC differentiation, means for antigen loading, routes and schedules of DC administration, as well as the type of used tumor antigens. The standard and universal conclusions from all clinical trials are summarized in FIGURE 3 and basically suggests that more trials are needed. Even though the analysis of published results reveals no explicit advantages in using DC vaccines over other strategies, we want to believe that the full potential of these cells has not yet been fully investigated. Given the still developmental stage of DC-based immunotherapy, the lack of confirmable clinical benefits should not warrant the premature abandonment of DC-based vaccine strategies. To draw definitive conclusions, the field would benefit from clinical trials comparing DCs with other vaccination strategies in patients with similar stages of disease and using standardized clinical protocols [12,37]. Nestle, 7 years ago, concluded his review on therapeutic use of DCs with the following: “The challenge of the future will be to extend these early results to a well defined and reproducible vaccination strategy according to current standards of good clinical practice. The history of cell therapy in the treatment of cancer has demonstrated that this is a difficult endeavor. Only the concerted efforts of academic centers, regulatory authorities and the pharmaceutical industry will be able to drive this expensive development process”. [38]. Figdor and colleagues repeated this suggestion several years later: “Collaborative efforts between biotech companies, blood banks and academic institutions will enhance the development

…DC vaccine-induced immune responses achieved to date are not yet sufficient to attain a robust and durable therapeutic effect in cancer patients…

…further improvements are required to enhance DC-vaccine potency and optimize the potential for clinical success…

Figure 3. A decade of improving and testing DC vaccines in cancer clinics from 1995/1996 until now resulted in hundreds of original publications and associated reviews; however, three general conclusions remain almost identical with a very few variations. DC: Dendritic cell.

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Year

Ref.

Vaccination with total-tumor-derived material as source of tumor antigens DC loaded with unfractionated tumor-derived antigens in the form of RNA

1998

[84]

Immunization with cytokine-transduced tumor cells In vivo antigen delivery to DCs may be through the injection of plasmid DNA into skin or muscle Expansion of DC in vivo with FLT3L Coordination of DC-based immunotherapies with other treatment modalities, such as cytokines or neutralization of immunosuppressive factors (IL-10, VEGF) or surgery and chemotherapy

1999

[34]

Genetic modification of DC Utilization of CD34+ precursors Direct delivery of DC to tumors Application of tumor lysates or apoptotic cells as sources of additional, as yet undefined, antigens

1999

[85]

In vivo expansion of DC using specific growth factors, such as FLT-3L Combinations with cytotoxic chemotherapy might be envisaged Combination of antigen specific immunotherapy with target oriented molecular therapies

2000

[38]

Using of combinations of antigens to reduce the risk of generating antigen-loss variants Administration of cytokines or other DC activators in combination with DC vaccination Optimization of approaches to assess immune responses in patients undergoing immunotherapy

2000

[72]

Concurrent administration of cytokines with DC vaccination Optimization of reliable methods of measuring patient responses to DC vaccines

2001

[86]

Evaluation of cytokines, such as Flt3-L, IL-2 or IL-12, for optimizing the efficacy of DC-based therapies Focusing on patients with minimal residual disease Optimization of adjuvant settings

2001

[87]

Optimization of antigen loading onto DC Evaluation of danger signals recruiting, activating or conditioning DC, such as TLR, scavenger receptor, Fc receptor, or inducible costimulatory molecule signaling

2001

[88]

Adequate activation of DC Optimization of antigen choice and their delivery

2002

[89]

Small, preferably two-armed, immunogenicity trials to optimize DC vaccination Combination of DC with other therapies, such as cytokine administration (IL-2, IL-15), CTLA-4 blockade and removal or blockade of Treg cells Development of commercial DC generation kits

2003

[6]

Comparison of distinct ex vivo-generated DC subsets activated via different pathways Identification of the parameters for DC targeting in vivo

2003

[90]

Definition of standardized procedures for both DC generation and cell quality controls Systematic comparative studies in clinical trials

2003

[80]

Supplementary co-administration of cytokines, such as interferon or IL-2, or other DC activators Treatment of patients with minimal residual disease Standardization of therapeutic regimens

2003

[11]

Standardization of DC vaccines

2004

[14]

Concomitant administration of cytokines or immunogenic factors, such as IL-2, IL-12 or CpG dinucleotides Novel methods of antigen loading

2004

[81]

DC vaccination should only be evaluated in the setting of clinical trials with standardized immunological and clinical read-out Prospective DC vaccination trials could be offered to patients with minimal residual disease

2004

[91]

A combination approach (i.e., vaccination with antiangiogenic therapy or removal of Treg) Early disease stage

2005

[25]

Focus on patients with minimal residual disease

2005

[24]

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Table 2. Proposed perspective developments of dendritic cell-based immunotherapy for cancer .

CTLA: Cytotoxic T lymphocyte associated; DC: Dendritic cell; IL: Interleukin; Treg: T regulatory cell; VEGF: Vascular endothelial growth factor.


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Table 2. Proposed perspective developments of dendritic cell-based immunotherapy for cancer (cont.). Future directions

Year

Ref.


2005

[35]

A combination approach that includes multiple therapeutic steps

2005

[12]


2006

[92]

Inclusion of modern techniques that allow quantification and characterization of tumor antigen-specific T helper cells and cytotoxic T cells Necessity to correlate clinical effects with sophisticated immunological methods in order to define important parameters, such as the number of T cells sufficient to induce tumor regression Improvement of ex vivo strategies should help to identify the parameters for in vivo targeting of DC

2006

[26]

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Thus, intratumoral approaches are appealing because they may direct antigen-specific responses back to the tumor site, exploit the presence of multiple undefined tumor antigens present in the endogenous tumor, and would be expected to reduce systemic toxicity [45]. However, clinical efficacy of intratumoral delivery of DCs might be limited by suppression of DC longevity and function in the tumor environment, which are the two main mechanisms mediating suppression of the DC system in the local tumor microenvironment. Therefore, in the intratumoral DC delivery strategy, for the purpose of enhancing antigen-specific antitumor immunity in the absence of defined tumor antigens, it is important to increase antigen processing in DCs and prolong survival of DCs at the local tumor sites. Several different approaches can be used to increase the antitumor efficacy of intratumorally delivered DC vaccines. It has been reported that transfection of DC with the antiapoptotic gene bcl-xl significantly increases their resistance to prostate cancer-induced apoptosis and is associated with a significant antitumor potential upon intratumoral delivery in vivo in animal models [54]. Interestingly, pretreatment of DC with certain cytokines, including tumor necrosis factor-α, IL-12 and IL-15, also protect DCs from tumor-induced apoptosis and suppression of DC function [28,64–67]. Thus, several strategies could be suggested for increasing efficacy of modern DC vaccines utilized for the treatment of cancer patients: • Protection of DCs from functional inhibition in the tumor environment • Prolongation of DC survival in the tumor environment • Recovery and/or improvement of antigen-processing cells and effector T and NK cells in tumor-bearing hosts for an efficient interaction with DC vaccines • Controlling of DC function as inducers/regulators of T regulatory cells • Redirection of DC migration in tumor-bearing hosts • Regulation of DC maturation and function (such as cytokine production) in situ

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in different tumors, such as melanoma, squamous cell carcinoma of the head and neck, and, probably, other solid tumors including prostate, breast and lung cancer. First, increased numbers of tumor-infiltrating DC are associated with better outcome in cancer patients with a variety of tumors [29,46]. Second, we have reported recently that the lost expression of chemokine CXCL14 in human tumors may be associated with decreased attraction of DCs to the tumor site and, thus, deficient induction of antitumor immune responses [47]. Using two murine tumor models, we have demonstrated that attraction of DCs into CXCL14-transfected tumors is associated with inhibition of tumor growth. In agreement, overproduction of CCL20 (MIP-3α) by tumor cells caused the local accumulation of DC and activation of tumor specific cytotoxic T lymphocytes (CTLs) in four murine tumor models [48]. Together with clinical evidence demonstrating that infiltration of the tumor bed by DCs is associated with a better patient survival, these results suggest that administration of DC vaccines in the tumor site might provoke proficient antitumor immune responses. Third, the clinical benefits from intratumoral administration of DCs, including tumor rejection, prolonged survival and induction of immune memory, were reported in mice and rats [49–55]. A pilot study in patients with melanoma and breast carcinoma demonstrated a marked antitumor potential of intratumoral delivery of DCs without addition of tumor antigens [56]. Other clinical trials, evaluating intralesional administration of interleukin (IL)-12-transfected DCs in patients with hepatocellular carcinomas and metastatic pancreatic and colorectal malignancies, concluded that intratumoral injection of DCs is feasible and well tolerated [57,58]. Similarly, injection of autologous immature DCs into tumor under radiotherapy in advanced hepatoma patients revealed its safety and induction of tumorspecific and innate immunity [59]. Evidence has been obtained showing that intratumoral DCs can capture and process tumor antigens to be presented to T lymphocytes [60]. Interestingly, DCs were recently found to be cytotoxic for several tumor cell lines suggesting that this may have important consequences for their ability to stimulate tumor-specific CTL [61–63].

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CTLA: Cytotoxic T lymphocyte associated; DC: Dendritic cell; IL: Interleukin; Treg: T regulatory cell; VEGF: Vascular endothelial growth factor.

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• Combination of DC vaccine with other therapeutic modalities, including surgery, radiation therapy and chemotherapy and stimulation of recovery of the immune system after conventional therapies.

Acknowledgements

Some of the studies referred to in this manuscript were, in part, supported by grants from the NCI (2RO1 CA84270 to MRS) and DoD (PC050252 to MRS).

Key issues • Dendritic cells (DCs) are the most powerful professional antigen-presenting cells and are characterized by a unique capability to initiate, maintain and regulate the intensity of primary immune responses, including specific antitumor responses. • The results of various DC-based clinical trials provided initial indications of their potent antitumor capacity associated with tumor regression in selected cancer patients. However, at present, no solid conclusions regarding the clinical efficacy of DC-based vaccination can be drawn.

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• In spite of a 12-year expansion and exploration of DC-based vaccination for cancer treatment, the fundamental optimization problems have not yet been solved. • It might be more prudent to standardize the outcome of DC-based clinical trials – immune monitoring and tumor monitoring – but not only the form or other particulars of the DC vaccine.

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• Useful standardization will be impossible without understanding the mechanisms responsible for limited efficacy of modern DC vaccines.

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Affiliations •

Hua Zhong, MD Research and Clinical Fellow, Shanghai Jiao Tong University, Shanghai Chest Hospital, 241 Huaihai Road (w), Shanghai 200030, China Tel.: +86 216 282 1990 Fax: +86 216 280 1105 [email protected]

•

Michael R Shurin, MD, PhD Associate Professor of Pathology and Immunology, Associate Director, Clinical Immunopathology, 5725 Children’s Hospital of Pittsburgh – Main Tower, 200 Lothrop St. Pittsburgh, PA 15213, USA Tel.: +1 412 648 9841 Fax: +1 412 647 7741 [email protected]

•

Baohui Han, MD Chief Director, Division of Pulmonology, Shanghai Jiao Tong University, Shanghai Chest Hospital, 241 Huaihai Road (w), Shanghai 200030, China Tel.: +86 216 282 1990 Fax: +86 216 280 1105 [email protected]

Turtle CJ, Hart DN. Dendritic cells in tumor immunology and immunotherapy. Curr. Drug Targets 5, 17–39 (2004). Beautiful review with many interesting suggestions on how to improve DC vaccines for cancer therapy.

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