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Originally published as JCO Early Release 10.1200/JCO.2005.00.463 on December 14 2004 © 2005 American Society of Clinical Oncology. Intratumoral Injection of Dendritic Cells Engineered to Secrete Interleukin-12 by Recombinant Adenovirus in Patients With Metastatic Gastrointestinal Carcinomas
From the Division of Hepatology and Gene Therapy and Departments of Radiology, Pathology, Gastroenterology, Pharmacology, Hematology, Clínica Universitaria/School of Medicine, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain Address reprint requests to Guillermo Mazzolini MD, PhD, Clínica Universitaria/School of Medicine, University of Navarra, c/Irunlarrea, s/n, 31008 Pamplona, Spain; e-mail: gmazzolini{at}unav.es.
PURPOSE: To evaluate the feasibility and safety of intratumoral injection of autologous dendritic cells (DCs) transfected with an adenovirus encoding interleukin-12 genes (AFIL-12) for patients with metastatic gastrointestinal carcinomas. Secondarily, we have evaluated biologic effects and antitumoral activity. PATIENTS AND METHODS: Seventeen patients with metastatic pancreatic (n = 3), colorectal (n = 5), or primary liver (n = 9) malignancies entered the study. DCs were generated from CD14+ monocytes from leukapheresis, cultured and transfected with AFIL-12 before administration. Doses from 10 x 106 to 50 x 106 cells were escalated in three cohorts of patients. Patients received up to three doses at 21-day intervals. RESULTS: Fifteen (88%) and 11 of 17 (65%) patients were assessable for toxicity and response, respectively. Intratumoral DC injections were mainly guided by ultrasound. Treatment was well tolerated. The most common side effects were lymphopenia, fever, and malaise. Interferon gamma and interleukin-6 serum concentrations were increased in 15 patients after each treatment, as well as peripheral blood natural killer activity in five patients. DC transfected with AFIL-12 stimulated a potent antibody response against adenoviral capsides. DC treatment induced a marked increase of infiltrating CD8+ T lymphocytes in three of 11 tumor biopsies analyzed. A partial response was observed in one patient with pancreatic carcinoma. Stable disease was observed in two patients and progression in eight patients, with two of the cases fast-progressing during treatment. CONCLUSION: Intratumoral injection of DC transfected with an adenovirus encoding interleukin-12 to patients with metastatic gastrointestinal malignancies is feasible and well tolerated. Further studies are necessary to define and increase clinical efficacy.
Surgical resection is the only curative treatment available for gastrointestinal carcinomas. Unfortunately, a high proportion of patients are diagnosed when current therapies are largely ineffective. Dendritic cells (DCs)1,2 are attractive adjuvants for therapeutic immunization of cancer patients.1,3 Immature DCs take up and process antigens4,5 and, upon maturation/activation, migrate to draining lymph nodes where they can prime tumor-antigen–specific CD4+ and CD8+ T cells.2,4,6 The DC maturation program includes: downregulation of their capacity to acquire antigens, increased expression of major histocompatibility complex and co-stimulatory molecules, changes in chemokine receptors,6,7 and enhanced production of key cytokines such as interleukin (IL) -2, IL-12, and IL-15.8-10 Besides, mature DCs have the ability to activate both natural killer (NK)11 and NK T cells.12 For immunotherapy, tumor antigens are provided to DCs in different forms such as incubating DCs with synthetic or native peptides, tumor tissue lysates,13,14 purified proteins,15 tumor-derived total RNA,16 or by fusing malignant cells and DCs.17 Furthermore, the efficacy of DC-based immunotherapy may also be increased by transduction with either cytokine genes or co-stimulatory molecules (IL-2, IL-7, IL-12, secondary lymphoid-tissue chemokine, CD40-L) into DCs.18-24
IL-12 is a potent immunostimulatory cytokine, produced physiologically by DCs, which has shown properties as an anticancer agent in experimental tumors, as a recombinant protein,25 or in gene therapy approaches including delivery with transfected fibroblasts.26,27 Recently, we have reported that intratumoral injection of an adenovirus encoding for human IL-12 (AFIL-12) to 21 patients with advanced digestive malignancies was feasible and well tolerated.28 Up to 3 x 1012 viral particles exert mild antitumor effect (one partial response and six stable disease cases). Biologic effects included increases in serum interferon gamma (IFN- We have previously demonstrated that repeated intratumor injections of murine bone-marrow–derived DC transfected with an adenovirus encoding IL-12 induce a potent antitumoral response.21,29 Importantly, this strategy not only eradicates the directly injected tumors, but also distant disease demonstrating systemic immunity.29 The activity of CD8+ T cells is crucial to achieve such a therapeutic effect.21,22,30 We decided clinical translation of this strategy with intratumoral maturing/semimature DCs,31 despite the potential risk of inducing immunity to self antigens shared by normal and cancer tissues.32 Both our preclinical data and reports from several phase I/II studies were encouraging and demonstrated the safety of immunotherapy with DCs.3,33,34 Another potential drawback of this intratumoral route of administration is the presence of immunosuppressive factors at the tumor microenvironment that could endanger the performance of DCs even if transfected to produce IL-12.35-37 Recent observations have highlighted that under certain conditions tumor can exploit intratumoral DCs for their own growth.38 This article describes the first clinical experience of intratumoral administration of DCs engineered to produce IL-12 by recombinant adenovirus, thus combining gene and cellular immunotherapy.
Study Design This was an open-label, nonrandomized, dose-escalation phase I trial in which DCs transfected with AFIL-12 were administered intratumorally to patients with metastatic gastrointestinal carcinomas. Objectives The primary end point was to assess both feasibility and safety of direct intratumoral injections of DCs. Secondary end points were biologic effect and antitumor activity.
Patient Selection and Enrollment
AFIL-12 and Generation of Monocyte-Derived DCs
Isolation of PBMC and Clinical-Grade Purification of CD14+ Cells
DC Cultures and Transfection
Treatment Plan
Patient Evaluation
Immunologic Monitoring Cytokine measurement DCs (106/mL) were assayed for IL-12 p70 production 24 and 48 hours after AFIL-12 infection using an enzyme-linked immunoabsorbent assay (ELISA) kit in the culture supernatant (Pharmingen, San Diego, CA). Simultaneous measurement of IL-6, IL-10, TNF–  , and IL-12 in serum samples at baseline and on days 0, 1, 2, and 3 after each dose of DCs from patients was analyzed by microparticle-based flow cytometry according to manufacturer's instructions (BD Bioscience, San Jose, CA). IFN- levels were monitored by ELISA in serum samples at baseline and on days 0, 1, 2, and 3 after each dose of DCs (Pharmingen). NK activity Cytotoxic NK activity against K562 cells was measured by standard 5-hour sodium 51chromate-release assay as described.30 Antiadenovirus antibodies measurement Total and neutralizing antibody titers against adenovirus were monitored by ELISA and interference with infection by an adenovirus encoding luciferase, respectively, at baseline and just before the second and the third dose of DCs (manuscript in preparation). Delayed-type hypersensitivity test Delayed-type hypersensitivity (DTH) skin tests were used to assess in vivo immune reaction against autologous tumor antigens. Tumor tissue was placed in phosphate-buffered saline and a single-cell suspension was obtained. Cells were lysed by three to four freeze-and-thaw cycles and debris was removed by centrifugation for 10 min at 2,000 rpm. Supernatants were collected, irradiated at 5,000 rads and aliquots stored at –80°C. GM-CSF 50 µg were added to each lysate solution. Skin tests were performed by 50 µl intradermal injection of this tumor lysate when indicated in Figure 1. A solution containing AFIL-12 inactivated by ultraviolet light were injected to assess reactivity against viral antigens. Pathologic studies Tumor samples obtained at baseline and at day 21 were histopathologically analyzed as described.28 Surface area calculations were computer-assisted by image analysis software and positive cells were manually counted by a pathologist blinded to the identity of the samples.
Patient Characteristics Seventeen patients were enrolled between May 2002 and March 2004 (Table 1). Mean age was 56 years. Eight patients had hepatocellular carcinoma (HCC), three had pancreatic cancer (PC), five had colorectal cancer (CRC), one had cholangiocarcinoma (ChC), and all of them had metastatic disease. Six of the eight patients with HCC had underlying liver cirrhosis due to hepatitis C virus. All cirrhotic patients had portal hypertension and a fairly preserved liver function (five cases of Child-Pugh class A and one case of class B). Patients with PC and CRC had had prior standard chemotherapy, whereas most HCC patients received multimodal therapy including surgery, transarterial embolization, or radiofrequency ablation.
Generation of DCs Results of leukapheresis Autologous DCs were differentiated in serum-free medium (AIM-V) from CD14+ monocytes. The immediate yield of thawed cells alive was above 90% and ranged from 40% to 78% (mean, 62%) after 24 hours back in culture. The number of DCs injected was > 75% of the planned dose in all patients. After 8 days of culture, harvested cells were consistently 100% CD11c bright, being nearly 50% CD1a+, and expressed high levels of HLA-DR (75%; Table 2) and intermediate levels of CD80 and CD86 (41% and 44%, respectively). These findings were consistent with a phenotype of intermediate maturity or "in process of maturation/activation."31
Production of IL-12 In vitro transfected DCs with AFIL-12 proved able to produce high amounts of IL-12 p70 at 24 and 48 hours (Table 3).
Treatment Procedure Intratumoral injection of DCs was feasible in 100% of cases. As shown in Table 4, 43 injections were administered to the 17 patients guided by either US (n = 38), endoscopy US (n = 3), or CT scan (n = 2). Eleven patients underwent all of the three planned doses, with four patients receiving two injections and two patients receiving only one injection.
Toxicity A total of 118 adverse events were recorded throughout the follow-up period (Table 5). In general, DC administration was well tolerated and dose-limiting toxicity was not reached. None of the patients withdrew from the study because of adverse events. Mild to moderate fever, responsive to common antipyretics, was observed 24 to 48 hours after DC injection in 65% of patients, irrespective of the dose of DCs. Temperature increase was associated frequently with malaise and occasionally with sweating.
Three patients experienced pain at the site of injection that resolved in all cases with analgesics. Five patients (29%) had grade 1 vomiting after the treatment. One patient experienced an episode of confusion in association with a high dose of narcotic analgesics due to chronic pain. Another patient developed progressive encephalopathy due to portosystemic shunt and died 40 days after the first DC injection. Six patients developed hypoalbuminemia after treatment that could be associated with fluid therapy and also to the concomitant cirrhosis. Grade 1/2 lymphopenia was the most frequent adverse event, appearing in 14 patients (76%). Four grade 3 lymphopenia events were also documented. Grade 1/2 lymphopenia before treatment was common, but lymphocyte count almost invariably decreased at day 1 and returned to basal values by day 3. Mild neutropenia was observed in three patients. No significant deterioration in hepatic function was observed in cirrhotic patients (n = 8). However, five patients (four with HCC) had a transient, mild hyperbilirubinemia shortly after treatment. Among patients receiving multiple doses, side effects usually recurred, but cumulative toxicity was not observed. In addition, there was no clinical evidence of long-term toxicity among patients followed for more than 6 months. No clinical manifestations of autoimmune reactions were observed with the exception of patient 5, who developed two small vitiligo lesions in the forehead after the third dose of DCs. However, serum autoantibodies became detectable or increased their titer in five cases (Table 6).
Biologic Response Systemic production of cytokines There was no significant increase in serum IL-12 after treatment (not shown). In contrast, serum IFN- increased after each dose of DCs reaching the maximum level at day 1/2 after treatment (Fig 2). IFN- concentrations appeared to be dose dependent, ranging from 100 pg/mL in cohort 1 to 500 pg/mL in cohort 3. Although it is possible that released IFN- could be the result of an unspecific immune response against adenovirus, part of the IFN- detected systemically could be the consequence of the expression of IL-12 as a transgene, because DCs were injected when they were producing high amounts of IL-12 (Table 3). There was a great heterogeneity in the production of IL-12 in vitro by transfected DCs and, intriguingly, there was no correlation between the amount of IL-12 produced in vitro and the serum concentration of IFN-.
In addition, there was a dose dependent systemic production of IL-6 in the majority of patients (Fig 3). It is well known that IL-6 is produced in the context of an antiadenovirus immune response, but IL-6 is also induced by IL-12 and could contribute to enhance immune responses.42
NK-cell activity Before DC administration, NK activity was low or undetectable (Fig 4). Five patients developed increases of peripheral blood NK activity after treatment. Among patients who showed increased NK activity, one patient achieved a partial response and another patient experienced a clear stabilization of her disease (patient No. 15).
Antiadenovirus antibodies To explore the possibility that DCs infected with an adenovirus encoding IL-12 gene may immunize against viral antigens, we measured antibodies against adenovirus. All patients had high pre-existing levels of total and neutralizing antiadenovirus antibodies (Fig 5). The great majority of patients increased the titer of antibodies against adenovirus after DC injection in a dose-dependent fashion. It should be noted that DCs were extensively washed before injection and, accordingly, only internalized or strongly DC-absorbed viral particles should have remained in the injected doses. These results suggest an adjuvant activity of DCs for boosting antibody production.
DTH tests DTH skin test reactivity towards tumor extracts were negative in 13 patients assessed, the only exception being a weak positive reaction in patient No. 10 that declined 1 week later. However, the reaction against inactivated AFIL-12 was positive in all patients after therapy. Pathologic study A tumor sample was obtained at day 21 in all but three patients. The quality of samples permitted comparison of the two specimens in 11 patients. In five of these cases (patients 10, 11, 13, 15, and 16; all of them had HCC), there was a substantial augmentation of the tumor infiltration by lymphocytes, with increases in CD3 and CD8 cells ranging from 51% to 344% and from 28% to 325%, respectively (Fig 6A). Importantly, patients 10 and 15 experienced stabilization of their metastatic disease. As an example, Figure 6B shows immunohistochemical staining for CD8+ lymphocytes in tumor tissue sections from patient 10. In the remaining six patients, there were no significant changes in the level of lymphocyte infiltration (not shown).
Antitumor Response Response to therapy was evaluated at day 63 in 11 of 17 patients. Two patients with CRC, patients 7 and 8, withdrew from the study due to rapid progression of their liver metastases before receiving second and third dose of DCs, respectively. One patient with PC (patient 12) developed peritoneal carcinomatosis and tense ascitis and was excluded from the study shortly after the second dose of DCs. Another patient with metastatic ChC died prematurely after a sudden development of a Budd-Chiari syndrome and liver failure due to an aggressive procoagulant state. Patient 6 was not evaluated because he died before the administration of the third dose of DC due to encephalopathy and noninfectious refractory fever. Hence, only 11 patients received complete treatment and were assessed for antitumoral response. Two patients with metastatic HCC showed stable disease and eight patients showed tumor progression. A partial response was observed in patient 1, who had metastatic PC that had progressed even after radiochemotherapy. He received three doses of 10 x 106 DCs into primary tumor mass and a reduction in the number of noninjected liver metastases was obtained. Importantly, the response persisted more than 3 months. Figure 7 shows abdominal CT scans from this patient taken before and after three injections of DCs. Remission lasted 3 months but duodenal obstruction due to primary tumor progression developed at that time.
Patient 4 with CRC showed a significant decrease in serum levels of carcinoembryonic antigen without CT scan evidence of response or stabilization. In contrast, DC treatment was followed by an increase of carcinoembryonic antigen in patients 7 and 8 with progression of their liver metastases from CRC.
In this study we demonstrate for the first time that intratumoral administration of DCs engineered to produce IL-12 by recombinant adenovirus is both feasible and safe in humans. We, and others, have previously shown that intratumoral injection of bone marrow–derived dendritic cells engineered to produce IL-12 by means of viral vectors21,22 induces a potent immune response in mice. In these animals, neither toxicity nor any sign of autoimmunity were ever found.21,22,29 We have recently finished a phase I study in which patients with advanced digestive carcinomas safely received direct intratumoral injections of AFIL-12.28 Hence, this previous study provided us with key information about the safety of the viral vector at much higher doses than those used to transfect DC.28 Clinical trials with DCs loaded with peptide or tumor lysate, that include experience with gastrointestinal carcinomas, show increases in T-cell–mediated specific immunity against the tumor antigens used in these vaccines.14,34,43-46 The intratumoral route of administration sounds attractive because T-cell–defined tumor antigens have been identified for only a minority of human cancers47 and because in certain tumors, such as digestive tumors, it is difficult to obtain sufficient malignant cells as a source of antigen. The idea of injecting DCs intratumorally pursues artificial tumor antigen cross-priming,4,5 resembling physiologic antigen presentation.6 However, exposure of DCs to the suppressive local environment might be a serious pitfall. In fact, local effects of transforming growth factor beta, vascular endothelial growth factor, IL-10, and prostaglandins on injected DCs might seriously damage their functional capabilities.35,36,48 Nonetheless, results obtained in mice strongly suggest that intratumoral administrations might be more efficacious than all the intravenous and subcutaneous routes when tested in parallel.49,50 Unfortunately, those studies did not include direct comparisons with intranodal release of DCs, as it is being performed in various clinical trials.14,31,34 Despite the purity of magnetic bead–selected monocytes, the resulting phenotype of DCs after 7 days of culture in the presence of IL-4 and GM-CSF was heterogeneous with regard to CD1a and other surface markers, indicating individual variability in the differentiation culture from monocytes even in defined serum-free medium. Moreover, there was a remarkable heterogeneity in the production of IL-12 after transfection with AFIL-12. This could be explained by an individual susceptibility of DCs to adenoviral transduction that can be genetically determined.51
Adenovirus transfection is known to elicit partial maturation of DCs,52,53 but we sought to further push the maturation program of the DCs with the TNF-
DC doses elicited systemic production of IFN- We found a substantial increase in the amount of both CD3+ and CD8+ T lymphocytes infiltrating the tumor in four of 11 patients after a single dose of DCs. Although two of these patients experienced stabilization of their tumor lesions, further studies will address whether infiltrating lymphocytes were specific for any tumor antigen and whether they are endowed with cytolytic effector machinery.
A comparison of these biologic effects with those observed upon intratumoral injection of AFIL-12 indicate that there is an additional effect mediated by DCs, at least in terms of IFN-
Evaluation of the T-cell immunity against tumors was an extremely difficult task due to the limited access to biopsy antigenic material. In two cases, there are data showing increased ELISPOT reactivity to tumor lysate. However, the limited amount of this tumor material precludes repetition and extension of these observations to a sufficient number of patients as to draw firm conclusions. On the contrary, reactivity to adenoviral capsides was evident in all eight studied cases both by DTH and by IFN- Patients increased antibody titers against adenovirus, most of which were able to neutralize infection by this viral vector. This fact further supports experiments in rodents suggesting that DCs carrying absorbed viral particles act as adjuvant for antibody responses.56 In our case, adenoviral transfected DCs carry much less viral particles than those used in the previous trial and, by contrast, the increases in antibody titer are higher, indicating adjuvant activity of DCs. Our studies offer solid ground to support that intratumoral injection of up to 5 x 107 DCs in patients with metastatic disease, including patients with advanced cirrhosis, is safe. Of note, one patient developed mild vitiligo that was not associated with any other sign of autoimmunity or any rise in the titer of autoantibodies. In this regard, increments in the titer of antismooth muscle antibodies, antinuclear antibodies, or rheumatoid factor were observed in five patients, but no clinical signs of autoimmunity have been recorded, as it has been the case in other DC-based protocols.3 The preliminary appraisal of clinical efficacy included one partial response and two cases of stable disease. If compared with the previous clinical trial using intratumoral AFIL-12, the clinical results were comparable, although more cases showed tumor stabilizations in the previous one. However, the second study recruited patients with more advanced and metastatic disease. Two lines of research can be fruitful to improve these effects: (1) introducing changes to fight the immunosupressive tumor microenvironment, such as local radiotherapy57 or molecular interference with DC-suppressing factors (TGF-ß, IL-8, vascular endothelial growth factor); and (2) to further enhance the elicited immune response with cytokines58 or immunostimulating monoclonal antibodies.29,59,60 Better clinical results would probably be seen in patients with resectable tumors and microscopic residual disease at high risk of recurrence.
The authors indicated no potential conflicts of interest.
We thank Maria Eugenia Cornet, Maria del Mar Municio, and Elena del Corral for their work in trial monitoring, and Blanca Larrea for providing nursing care for patients. G. Gonzalez-Aseguinolaza, J.J. Lasarte, P. Sarobe, and J. Larrache are acknowledged for critical reading of the manuscript, as are Cell Therapy Unit staff for technical assistance.
Supported by grants from Ministerio de Ciencia y Tecnologia (SAF 02/0373), Fondo de Investigaciones Sanitarias (PI031253), Redes Temáticas de Investigación Cooperativa (C03/10 and C03/02), Departamento de Salud y Departamento de Educación del Gobierno de Navarra and "UTE-project CIMA". Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
18 years; (2) metastatic disease; (3) histologic diagnosis of primary liver cancer, colorectal cancer, or pancreatic cancer; (4) an Eastern Cooperative Oncology Group performance status 
3; and (5) an accessible tumor mass. Exclusion criteria included: (1) pregnancy or lactation; (2) neutrophil count 
