Surg Oncol Clin N Am 11 (2002) 645660

Dendritic cell gene therapy

Mark Onaitis, MD, Matthew F. Kalady, MD, Scott Pruitt, MD, PhD, Douglas S. Tyler, MD*
Department of Surgery, Surgical Oncology Section, Duke University Medical Center 3118, Durham, NC 27710, USA

Dendritic cells (DCs) are specialized antigen-presenting cells located throughout the human body. Adept at antigen uptake, processing, and presentation on major histocompatibility complex (MHC) class I and class II molecules, DCs display molecules such as CD80 and CD86, which are thought to present costimulatory signals to CD8-positive cytotoxic T lymphocytes (Fig. 1). Once cytotoxic T-lymphocytes (CTLs) are activated after interaction with a DC exhibiting the appropriate tumor-associated peptide antigen and class I molecule, they may seek out and kill other cells that express these molecules (such as tumor cells) (Fig. 2). This simple biology forms the basis of all attempts at vaccination of cancer patients with DC vaccines. As oncogenic transformation proceeds, genetic changes occur within previously normal cells. One of the results of this series of mutations is expression of novel proteins. Some of these new proteins have not been encountered by the human immune system before and are recognized as nonself. These proteins, after immunologic processing and presentation as peptides on MHC class I molecules, lead to targeting of these cells by CD8-positive killer T cells with subsequent tumor cell destruction. This process, which has evolved to protect the organism from intracellular pathogens as well as tumorigenesis, should kill nascent tumors at early stages. Methods by which tumor cells may evade immune destruction have also developed over time, however. One of these methods is via down-regulation of MHC class I molecules resulting in inability of CTLs to see the tumor cell. Another is altered cytokine microenvironment near tumor cells leading to inability of immune cells to carry out their normal duties. Cells may also divide and mutate quickly enough that these mutant tumor-associated
MFK is supported by a National Research Service Award; DST is supported by a Lustgarten Foundation grant and a NIHK08 Award. * Corresponding author. E-mail address: tyler002@acpub.duke.edu (D.S. Tyler). 1055-3207/02/$ - see front matter 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 3 2 0 7 ( 0 2 ) 0 0 0 2 7 - 3

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Fig. 1. Schematic of DC-CTL interaction.

antigens are expressed only transiently. Finally, tumors may secrete factors that are systemically immunosuppresive. Recognition of these evasive modalities employed by tumor cells has allowed strategies for overcoming them to be introduced. Some have pursued methods of improving either local or systemic cytokine levels to up-regulate

Fig. 2. Tumor cell recognition and destruction by an activated CD8 CTL.

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class I expression and to activate CTLs. Others have endeavored to isolate and expand tumor-inltrating CTLs specic for a certain antigen or antigens. These are then reinfused into the patient with hopeful tumor regression. But these cells, which are isolated from the tumor itself, may or may not possess tumoricidal ability. Still others have sought to activate the immune system to better recognize and kill cells expressing tumor-associated antigens. This may be performed in several ways. Attenuated tumor cells may be infused into patients as vaccines. Tumor-associated antigen peptides may be injected into patients with or without immune adjuvants. Finally, various antigens may be loaded in any of several ways onto dendritic cells, which are professional antigen-presenting cells 1000-fold more ecient stimulators of resting T cells than other antigen-presenting cells [1]. The recent increase in our understanding of dendritic cells and our ability to utilize them in a variety of novel vaccine strategies has rekindled enthusiasm in the possibility that the dendritic cell-based vaccines may have a future role in cancer treatment. This article will focus on the current status of dendritic cell-based vaccines, beginning with an overview of dendritic cell physiology, next discussing methods of loading/types of dendritic cell vaccines, and nally analyzing the results of phase I/II clinical trials published to date. DC physiology Two subsets of DCs have been identied, with the main distinction between them being functional. DC1s, when presenting antigen to CD4 helper T cells, secrete interleukin-12, which promotes T-helper-1 (Th1) responses. Th1 responses involve production of interferon-gamma by CD4 cells leading to a strong CD8 killer T cell response. This type of response is desirable in induction of antitumor immunity. DC2s promote T-helper2 (Th2) responses, in which CD4 T cells produce interleukin-4. This induces primarily a B-cell immune response. DC1s also express myeloid markers such as CD33 and CD13 and mature when encountering inammatory stimuli, whereas DC2s express lymphoid markers and mature after exposure to interleukin-3 and CD40 ligand. As briey stated above, eective Th1 immune responses require DCs, CD4 T cells, and CD8 T cells. DCs acquire antigens in various ways. When encountering exogenous antigens, DCs may perform macropinocytosis, phagocytosis, and receptor-dependent mechanisms. Some of these receptors include Fc receptors (gamma and epsilon) [2,3], macrophage mannose receptor for uptake of glycosylated proteins [4], and DC-SIGN for acquisition of HIV [5]. Any of these processes leads to intracellular localization and breakdown in lysoendosomes. These lysoendosomes then merge with other endosomes, where antigenic peptides are loaded onto class II molecules. Tracking of these peptide-class II complexes to the cell membrane is followed by presentation to nave CD4 T cells. These CD4 T cells are

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important in activating CD8 T cells directly and in stimulating DCs to activate CD8 T cells. Endogenous antigens are also encountered by dendritic cells after infection by various pathogens. Proteins produced in the cytoplasm are broken down by proteosomes into peptides. These peptides are transported into the endoplasmic reticulum, where they are loaded onto MHC class I molecules. The peptide-class I complexes are then transported to the cell membrane so that presentation to CD8 T cells may occur. In addition to this presentation of endogenous antigens onto class I molecules, investigators have found that exogenous antigens may also be presented on class I molecules [6]. This cross-presentation allows ecient immunologic responses against pathogens that do not reach the cytoplasm eciently. Although the exact mechanisms of this phenomenon are unclear, much work has focused on ingestion of necrotic or apoptotic cells by DCs. In fact, apoptotic cells expressing microbial antigens, tumor antigens, and self antigens have been shown to be cross-presented to CD8 T cells [7]. More information regarding the mechanisms of cross-presentation needs to be gathered. Another important consideration in dendritic cell physiology is maturation of DCs. DCs in the periphery are immature cells, specialized for uptake of antigen via the processes described above. Exposure to a variety of stimuli, including various microbial products and inammatory cytokines, leads to changes in DCs that are characterized as maturation. During this process, MHC class I; MHC class II; the costimulatory molecules CD80, CD86, and CD40; and the DC-specic markers CD83, p55, and LAMP are up-regulated [8]. In addition, the DCs become specialized for production of the cytokines interleukin-12, interleukin-15, interleukin-18, and other chemokines that are essential for stimulation of CD4 and CD8 T cells [9]. At maturity, presentation to T cells in lymph nodes may take place with resultant ecient immune responses. Vaccine development The development of dendritic cell vaccines has benetted from research into methods of generating large numbers of clinical-grade DCs, as less than 1% of circulating white blood cells are DCs. The two main sources of DCs used for vaccine development have been circulating monocyte precursors in the blood and hematopoietic progenitor cells in the bone marrow or cord blood. The latter of these involves use of antibody against CD 34 to isolate the hematopoietic precursors. These precursors are cultured in granulocyte/ macrophage colony stimulating factor and tumor necrosis factor-alpha, resulting in a population of immature DCs [10]. The former method involves either leukapheresis of patients or phlebotomy followed by adherence to plastic. Monocyte precursors adhere to the plastic and are then cultured for 57 days in granulocyte/macrophage colony stimulating factor and interleukin-4. Large numbers of immature DCs can be cultured using this

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method. These DCs can then be loaded with antigens. This antigen loading may take the form of peptide pulsing, pulsing with tumor cell lysates, infection with viral vectors, direct nucleic acid loading, or ingestion/fusion with tumor cells. Peptides Pulsing DCs with small peptides is the easiest method of delivering antigen to DCs. Tumor-associated antigens have been analyzed in terms of amino acid sequence such that the immunodominant peptides for some tumor-associated antigens are known for given HLA types. Large-scale production of these peptides has been possible for the last several years. In 1995, using mice, bone marrow-generated dendritic cells were shown to elicit strong antigen-specic CTL responses in vivo. Using a peptide from the OVA murine antigen, CD8 cells specic for OVA were elicited [11]. At about the same time, another group independently revealed that peptide-pulsed DCs could elicit a strong CTL response but also demonstrated that administration of such a vaccine would protect against a subsequent tumor challenge [12,13]. These experiments revealed that vaccination with peptide-pulsed DCs could lead to tumor immunity in mice. Evaluation of whether this sort of immunity could also be elicited in humans has also been studied extensively. Several groups have shown that human dendritic cells, when pulsed with synthetic peptides in vitro, can elicit strong antigen-specic CTL responses [1416]. Although these studies proved the in vitro feasability of using peptide-pulsed DCs to stimulate nave T cells, further studies would have to involve in vivo application of the technology. The group at Rockefeller University has used the inuenza matrix protein HLA-A2-restricted peptide to elicit long-lasting immune responses. Mature, monocyte-derived DCs were pulsed with the relevant peptide and were administered to healthy volunteers subcutaneously. Subsequent injection elicited a strong memory response that was characterized by increased avidity [17,18]. These studies have been followed by several small clinical studies using peptide-pulsed DC vaccines in cancer patients. The rst of these was performed at Stanford University by injecting monocyte-derived DCs incubated with idiotype proteins derived from B cell lymphomas [19]. Four patients had their lymphoma cells fused with hybridomas and the resulting idiotype proteins puried. These DCs were then incubated with idiotype protein for 24 hours, washed, and injected intraveneously into patients. This initial injection was followed by two subcutaneous booster injections of only protein 2 weeks later. The cycle of a pulsed DC injection followed by two booster injections was repeated at 4 weeks and at 6 weeks and at 56 months after lekapheresis. Keyhole limpet hemocyanin (KLH) was used as a control using the same protocol in each patient. All patients had prior chemotherapy. All four patients had impressive humoral and cellular immune

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responses against both KLH and the respective idiotype proteins. One patient had a complete clinical response with two others having partial responses to the vaccine. Although not strictly a peptide-pulsed DC trial, one must assume that immudominant peptides were selected by the DCs themselves so that antigen-specic CTLs were induced. Such an approach may be unique to tumors in which monoclonal cells produce one dominant protein that can be produced in vitro. A slightly dierent, peptide-based approach was used in patients with malignant gliomas. The group at Cedars-Sinai in Los Angeles treated 9 patients with gliomas with autologous DCs loaded with eluted MHC class I-binding peptides from autologous tumor cells that had been established in culture [20]. This phase I trial called for administration of three vaccinations subcutaneously at biweekly intervals. No autoimmune side eects were noted after vaccinations. Of these patients, seven completed the vaccinations and were monitored immunologically. Four of the seven demonstrated signicant cytotoxic activity against their primary tumors after vaccination. Four patients also developed new areas of gadolineum enhancement on MR imaging and were resected. Of these, two patients demonstrated a robust CD8/CD45RO memory T cell inltration at the tumor site. Finally, a signicant survival advantage was seen when comparing these 7 patients with 42 control patients who were matched for age, gender, and tumor bulk (median 455 days vs. 257 days). These data must be further veried by phase II trials with larger numbers of patients, but the results are encouraging. Another pilot peptide-based vaccine trial used a cocktail of immunodominant peptides or a tumor lysate to pulse the dendritic cells. The background of this study was identication of numerous melanoma-associated antigens such as melanoma antigen (MAGE), Mart/MelanA, tyrosinase, and gp100 after analysis of tumor-inltrating lymphocytes in melanoma patients. In this study performed at the University of Zurich, 16 patients with known metastatic disease were injected with monocyte-derived DCs pulsed with either tumor lysate or a cocktail consisting of Mart, tyrosinase, and gp100 peptides if the patient was HLA-A2 or a cocktail consisting of MAGE 1 and MAGE 3 peptides if the patient was HLA-A1 [21]. KLH was used as a helper antigen in order to stimulate CD4 T cells as well as to serve as an immunologic marker. These DCs were injected into uninvolved inguinal lymph nodes every week for 4 weeks followed by a fth vaccination at week 6. Vaccinations then occurred every month for a total of 10 vaccinations. The results of this peptide cocktal study demonstrated no overt toxicity in any patient, although three patients revealed development of asymptomatic anti-TSH receptor antibodies and one patient developed asymptomatic antinuclear antibodies. Delayed-type hypersensitivity (DTH) was used to measure immunologic response to the vaccines. All patients had strong DTH responses to KLH-pulsed DCs injected subcutaneously. Five of the 12 patients who were HLA-A1 or -A2 had signicant DTH responses to melanoma antigen peptide-pulsed DC injected subcutaneously with 4 of them

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exhibiting major clinical responses. Two of the four patients who received a DC-lysate vaccine had DTH responses to DCs pulsed with tumor lysate, with both exhibiting clinical responses. In addition, lymphocytes from DTH response sites were isolated and used in chromium-release assays to lyse peptide-pulsed T2 cells. Mart and gp100-labelled T2 cells were lysed eectively above baseline in these assays. This landmark study is important for several reasons. First, it revealed that peptide-pulsed DC vaccines were safe in that autoimmunity did not develop. Also, marked immunologic responses to the antigenic peptides occurred. Finally, a signicant clinical response was seen in several patients. Melanoma is known, however, to be a highly immunogenic tumor, and anecdotal responses had been demonstrated by others. Larger numbers of patients in melanoma and trials with other tumor systems are needed to validate this approach. Because of the availability of several known tumor antigens, other have also focused work on melanoma. Dr. Schulers group in Germany has studied responses of stage IV melanoma patients to various regimens of injection of mature DCs pulsed with MAGE 3 peptides restricted to HLA-A1 or HLA-A2 [22,23]. In the groups rst article detailing proof of concept, 13 patients were vaccinated with DCs matured with monocyte-conditioned medium pulsed with an A1-resticted MAGE 3 peptide. The patients received three intradermal or subcutaneous vaccinations followed by two intravenous vaccinations. Eleven of the thirteen received all biweekly vaccinations and were immunologically and clinically evaluated. The vaccines resulted in no autoimmunity, and 7 of the 11 patients had DTH responses to DCs pulsed with MAGE 3 peptide, although many had responses to unpulsed DCs as well. Eight of the 11 patients demonstrated signicant increases in MAGE 3-specic CTL precursors as measured by a semiquantitative microculture assay. But analysis of fresh peripheral blood mononuclear cells (PBMCs) from the patients using an enzyme-linked immunospot assay for interferongamma revealed that only 2 of the 11 patients had signicant increases after 1 or 2 vaccinations. Clinical responses were seen in 6 of 11 patients. The second study again used MAGE 3, but this time 12 HLA-A2 advanced-stage melanoma patients were studied. Also, the DCs were pulsed with inuenza matrix peptide as well as the MAGE 3 peptide. Eight of these patients completed all ve and were evaluable. Again, no signicant toxicity was noted. All eight patients had increases in numbers of both circulating CD8-positive T cells specic for inuenza matrix protein and those specic for MAGE 3. In addition, in the two patients with enough cells to complete the assays, lytic and tetramer-binding analyses were performed, revealing increased numbers of MAGE 3 CTL precursors postimmunization. The explanation for the impressive clinical but disappointing immunologic results seen in one study and the impressive immunologic but disappointing clinical response in the second is unclear. Possible reasons include dierent immunogenicity of the two epitopes in terms of production of avid killer T cells, slightly dierent schedules and doses of vaccination, and dierences in patient populations.

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A second tumor system in which a tumor antigen has been eectively isolated and sequenced is prostate cancer. The group at Pacic Northwest Cancer Foundation in Seattle has conducted a phase II clinical trial in which 33 HLA-A2 patients with hormone-refractory metastatic prostate cancer were infused with DCs pulsed with 2 prostate-specic membrane antigen (PMSA) peptides at 6-week intervals for a total of 6 injections [24]. Twenty-ve patients were evaluated after nishing at least one injection. Of these, 2 patients had complete responses, 6 patients had partial responses, 1 patient had no change, and 16 patients had progression of disease as measured by National Prostate Cancer Project criteria, plus a 50% decrease in serum prostate specic antigen (PSA), or signicant improvements in ProstaScint scan. Disappointingly, these responses did not correlate well with DTH responses. Further immunologic readouts such as lyphoproliferative assays, quantication of antigen-specic T cells, and cytotoxicity assays are lacking in this study. Despite these shortcomings, the study did show objective clinical responses in a minority of patients. Another tumor in which a known tumor antigen has been targeted by a peptide-pulse DC vaccine is colorectal cancer. Investigators at Duke University undertook a phase I clinical trial in order to test safety of a DC vaccine pulsed with an HLA-A2-restricted peptide from the carcinoembryonic antigen [25]. In this study, subgroups of 21 patients with metastatic carcinoembryonic antigen (CEA)-expressing tumors were injected with escalating doses of cryopreserved peptide-pulsed DCs in four weekly or biweekly immunizations. Nineteen of the patients were evaluable for clinical response, and 15 were evaluable for immunologic response. These vaccinations were deemed safe as no patient had an adverse reaction to intravenous infusion of the DCs, and no autoimmune manifestations occurred. DTH responses were dicult to evaluate, but four of eight patients who received intradermal injections of DCs loaded with the peptide had areas of induration. But 20 of the 21 patients had measurable pretreatment DTH responses to the peptide alone. Biopsies of skin of the former revealed pleomorphic perivascular inltrates on microscopic examination. Clinical responses were disappointing with one patient having a minor response and one patient having stable disease. Unfortunately, immunologic readouts performed on the post-treatment patients are not reported in this study. There are several reasons for the relatively disappointing results from the CEA-pulsed dendritic cell clinical study. Among them may be that CEA is a poorly immunogenic antigen, that the patient population chosen here exhibited relatively more advanced disease than that chosen in similar studies, that the DCs used here were somehow dierent from those generated in other centers, that they were delivered dierently (intravenously as opposed to intranodally in the Zurich study), or that only one antigenic peptide was used rather than a cocktail of peptides which may produce a stronger, multivalent response. Regardless of the degree of success of peptide-based DC vaccines, some have focused on other limits of such a system. In using

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dened peptide antigens, the HLA type of the recipient must be known. If it is not known, eluted peptides may be used if autologous tumor can be cultured or tumor lysates may be used. These are limited, however, by availability of adequate amounts of tumor. In addition, delivery of entire antigenic proteins to the cytoplasm may allow a multivalent immune response to occur. Nucleic acids For all of the above reasons, there has been increasing focus on the use of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to transfect dendritic cells. The ease of using the polymerase chain reaction (PCR) or the reverse transcription-polymerase chain reaction (RT-PCR) allows amplication of antigen from small tumor samples in which expressed antigens are unknown. Also, methods exist for isolation and amplication of total tumor RNA or DNA in order to provide as yet undened antigens to the dendritic cell. First, DNA had been explored as a source of antigen for dendritic cells. Experiments in mice revealed that retrovirally delivered tumor-associated antigens yield eective CTLs and tumor immunity [26,27]. Using both Mart and alpha-fetoprotein (AFP) constructs in adenovirus, the groups at UCLA revealed that DCs are transduced eectively and can elicit strong CTL responses in vitro [28,29]. None of these techniques has been published in clinical trials in humans, however. Others have investigated use of RNA to deliver antigen to dendritic cells. Much in vitro work in this area has been performed at Duke University by the laboratories of Drs. Johannes Vieweg and Eli Gilboa. The technique of RNA transfection of dendritic cells was rst performed using liposomal delivery agents in mice resulting in antigen-specic CTLs and tumor immunity [30]. These data were followed up by studies using human cells in which liposomal CEA RNA was used to transfect both normal volunteer DCs as well as DCs of CEA-expressing cancer patients. These DCs were able to stimulate strong, specic CTL responses in vitro. In addition, genetic modication of the RNA using a lysosomal targeting signal allowed greater stimulation of CD4 T cells [31]. Recently, DC transfection of RNA by electroporation has been shown to be an eective transfection method that results in increased DC antigen processing and presentation [32,33]. Further in vitro experiments using RNA-transfected DCs have demonstrated eectiveness against both prostate cancer and renal cell cancer. In the rst case, PSA RNA was delivered to DCs via naked RNA incubation with the cells, during which the RNA is thought to be pinocytosed. Transfection of DC via this method followed by in vitro stimulation of nave T cells resulted in strong cytolytic responses against PSA-transfected targets regardless of the patients HLA haplotype [34]. One important aspect of this study is that DC stimulation is able to overcome tolerance in that PSA is expressed on normal prostatic cells. Finally, both prostate and renal cell cancer total tumor RNA

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was used by these investigators to transfect dendritic cells, which were then used to generate strong cytotoxic T lymphocyte responses against autologous tumors and cells expressing known tumor antigens [35,36]. Both unamplied and amplied total tumor RNA were used to produce strongly immunogenic DCs. All of the DCs used for these experiments were immature DCs.
Table 1 Summary of DC-based clinical trials Author Hsu et al DC vaccine type B cell lymphoma idiotype proteins Eluted glioma peptides Type of DC Imm No. patients 4 Immune response 100% humoral/ cellular responses against proteins 57% CTL responses against culture tumor 43% DTH responses to peptide-pulsed DCs 64% DTH responses to peptide-pulsed DCs/72% increases in MAGE-3 specic CTL precursors 100% increases in specic precursors N/A N/A N/A Clinical response 25% CR 50% PR

Yu et al

Imm

Survival advantage

Nestle et al

Thurner et al

Melanoma peptide cocktail KLH MAGE-3 peptide

Imm

16

37.5% major clinical responses 55% PR

Mat

13

SchulerThurner et al

Murphy et al Morse et al Rains et al

MAGE-3/ Infuenza matrixpeptide Prostate PMSA peptide CEA peptide Total tumor RNA KLH Autologous tumor cellDC hybrids

Mat

12

None

Imm Imm Imm

33 21 15

Kugler et al

Mat

17

65% DTH responses to autologous tumor cells

8% CR 24% PR 5% PR 47% with decreases in serum CEA level 24% CR 18% CR

Abbreviations: CEA, carcinoembryonic antigen; CR, complete response; CTL, cytotoxic Tlymphocyte; DTH, delayed-type hypersensitivity; KLH, Keyhole limpet hemocyanin; MAGE, melanoma antigen; PMSA, prostate-specic membrane antigen; PR, partial response; RNA, ribonucleic acid.

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Published clinical trials using DCs transfected with RNA are rare (Table 1). In New Zealand, a phase I/II trial was recently conducted on 15 patients with metastatic colorectal cancer [37]. These patients received four weekly infusions of immature DCs that had been incubated with autologous naked total tumor RNA and the neoantigen KLH. Three patients died from progression of disease before receiving all vaccinations. No autoimmune complications or serious toxicity occurred. Eleven patients had positive DTH reactions to KLH after vaccination. Seven patients had a fall in CEA level after vaccination. No clinical responses were seen on serial CT scans, however. Unfortunately, no other immune readouts were performed in these patients. Other methods Other methods of DC pulsing have also proven eective in vitro. These include delivery of antigens to DCs via other cells or via fusions of cells. A group from France has experimented with exosomes, antigen presenting vesicles containing MHC class I and MHC class II which are secreted from DCs. In a murine model, exosomes were pulsed with peptides and injected, leading to induction of specic immune responses and tumor immunity [38]. This cell-free vaccination technique is promising, although further trials with human cells/subjects are required. Another novel method involves fusion of DCs with cancer cells. Human breast cancer cells were incubated with breast cancer cells in the presence of polyethylene glycol (PEG), resulting in the production of heterokaryons [39]. After one stimulation in vitro with these heterokaryons, impressive specic cytotoxicity was seen. One clinical trial has been performed using this approach in renal cell cencer [40]. Vaccination of 17 metastatic renal cell carcinoma patients with DCautologous tumor cell electrofusions resulted in no toxicity. Eleven patients developed DTH responses to irradiated autologous tumor cells. Seven patients developed either complete or partial clinical responses. This technique allows presentation of as yet unknown antigens and is certainly of further study. Finally, much work has focused on the ability of DCs to ingest apoptotic or necrotic tumor cells. This is an ecient method of generating a broad CD8 response in vitro [41,42]. This response relying on multivalence as the response to a single tumor-associated antigen is not as strong as that induced with peptide-pulsed DC in melanoma [43]. Again, these impressive laboratory data will need to be tested in clinical trials to conrm their ecacy.

Summary All of these studies taken together highlight key areas that must be addressed in the future in order for the eld to continue to move forward.

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These issues are many, including but not limited to method of delivery of dendritic cells to patients, maturation status of the dendritic cells, and methods of monitoring responses to these vaccines. Each of these requires some comment. Dierent strategies of immunization were used in these studies. DCs were injected at various times and in various locations, including intradermally/subcutaneously, intranodally, and intravenously. Investigation of the pattern of spread of subcutaneously injected uorescently labeled DCs in the chimpanzee was studied at the University of Pittsburgh. Although rodent DCs had previously been shown to remain at the site of injection, these immature primate DCs migrated to draining lymph nodes and interact appropriately with T cells for as long as 5 days after administration. Data not shown in the same study reveal that intravenously administered DCs were undetectable in draining lymph nodes. Two groups have undertaken evaluation of route of administration of DCs in humans. The rst of these examined migration of immature, indium111labeled dendritic cells after RNA-loading in metastatic cancer patients [44]. The DCs were injected either intravenously, subcutaneously, and intradermally. Only DCs injected intradermally were cleared from the injection site with migration to regional lymph nodes. The immunologic signicance of these ndings is unclear, however. Another study examined this issue by studying prostatic acid phosphatase (PAP) protein-loaded mature DCs injected intravenously, intradermally, and intralymphatically in prostate cancer patients [45]. Regardless of route of administration, T cell responses were induced as measured by proliferation when PBMCs in vitro were stimulated with the PAP protein. Cytokine analysis of the patients revealed that the majority of patients undergoing either intralymphatic or intradermal injection had increases in measurable interferon-gamma but that none of the intravenously-injected patients did. The intralymphatic and intradermal routes thus seem to lead to stronger Th1 responses. But no data was presented regarding the numbers of PAP precursors induced by vaccination nor their specicity/cytotoxicity. Another issue in DC administration that should also aect route of administration is maturation status of the dendritic cells. Many of the studies used immature dendritic cells to immunize patients whereas others used mature cells. A number of studies have demonstrated that maturation signals such as inammatory cytokines and CD40 ligation lead to down-regulation of antigen processing and up-regulation of the chemokine receptor CCR7, which leads to homing to lymph nodes [46] as well as the MHC molecules, costimulatory molecules, and maturation markers [8,47,48]. In addition, different maturation agents and sequences of addition of these maturation agents may lead to dierences in functions of dendritic cells [4851]. Others have found that injection of immature DCs pulsed with inuenza matrix peptide and KLH, and lead to greater numbers of inuenza-specic T cells, but these cells had reduced interferon-gamma production and lacked killer activity [52]. Perhaps the most impressive results in a clinical trial, however, were

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gained by injecting similar cells loaded with melanoma peptides [21]. In addition, sequence of loading and maturation may be important in creating vaccines. One study using CEA peptides and CEA RNA found that optimal T cell presentation occurs when peptides are loaded after maturation with CD40 ligand and when RNA is transfected before maturation with CD40 ligand [53]. As all of these studies reveal, more investigation into the role of DC maturation as well as its timing and sequence is needed. Finally, a multitude of methods to detect response to vaccination have been attempted in all of the above studies. Many use DTH responses, but these may measure CD4 T cells instead of CD8 T cells. The availability of tetramers allows easier quantication of CTL precursors, but they provide no assessment of the function of these T cells. Enzyme-linked immunospot assays allow identication and quantication of numbers of cells producing cytokines such as interferon-gamma when encountering target antigens, but cytokine production may not correlate with tumor cell killing. Chromium release assays or ow cytometric assays for molecules such as perforin may be used to validate killing, but inability of many tumors to grow in vitro precludes direct assessment of tumor cell killing via this method. Other responses in human subjects may also be measured. Some of the cited studies yielded clinical responses that could be measured via physical examination or radiologic study. This is the exception rather than the rule, however. Others have monitored the decrease in serum tumor markers such as PSA or CEA. But these may not correlate directly with tumor burden. Indirect calculation of tumor burden by using quantitative PCR to estimate the number of circulating tumor cells in peripheral blood may be promising in this regard. Despite the lack of consensus as to what constitutes an eective response, most would agree that monitoring of these patients should include measures of both immunologic response and clinical tumor eect. All of this leads to the conclusion that DC-based cancer vaccines have progressed a great deal but that much work still needs to be done. Only rigorous benchtop experimentation followed by careful patient selection and vaccine administration, and then by meticulous patient monitoring, will lead to advances in the eld.

References
[1] Bhardwaj N, Young JW, Nisanian AJ, et al. Small amounts of superantigen, when presented on dendritic cells, are sucient to initiate T cell resonses. J Exp Med 1993;178:633. [2] Maurer D, Fiebiger E, Ebner C, et al. Peripheral blood dendritic cells express FceR1 as a complex composed of FceR1a- and FceR1 gamma-chains and can use this receptor for IgE-mediated allergen presentation. J Immunol 1996;157:607. [3] Sallusto F, Lanzavecchia A. Ecient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994; 179:1109.

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