Originally published as JCO Early Release 10.1200/JCO.2004.01.227 on January 15 2004

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TNFerade Biologic, an Adenovector With a Radiation-Inducible Promoter, Carrying the Human Tumor Necrosis Factor Alpha Gene: A Phase I Study in Patients With Solid Tumors

From US Oncology, Dallas, TX; Albert Einstein College of Medicine, Bronx, NY; University of South Florida, Tampa, FL; GenVec Inc, Gaithersburg, MD; Department of Medicine, Dana-Farber Cancer Institute, Boston, MA; University of Chicago Medical Center, Chicago, IL; and the Department of General Surgery, University of Kentucky Medical Center, Lexington, KY

Address reprint requests to Nader Hanna, MD, Assisstant Professor of Surgery, University of Kentucky Medical Center, Lexington, KY; e-mail: nhanna1{at}pop.uky.edu.

	ABSTRACT

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PURPOSE: TNFerade is a replication deficient adenovector that expresses human tumor necrosis factor alpha under control of the radiation-inducible Egr-1 promoter. The goals of this study were to determine the safety and toxicity of TNFerade in combination with radiation therapy.

PATIENTS AND METHODS: TNFerade was administered by intratumoral administration, weekly for 6 weeks with concomitant radiation (30 to 70 Gy). Seven dose levels were studied (4 x 10⁷ particle units [pu] to 4 x 10¹¹ pu) in patients with solid tumors being treated with radiation.

RESULTS: Thirty-six patients were assessable for toxicity and 30 for tumor response. Most frequent TNFerade-related toxicities were fever (22%), injection site pain (19%), and chills (19%). No dose-limiting toxicities were observed. Overall, 21 of 30 patients (70%) demonstrated objective tumor response (five complete responses, nine partial responses, and seven minimal responses). In four of five patients with synchronous lesions, a differential response between lesions treated with TNFerade + radiation compared with radiation only was observed.

CONCLUSION: This is the first human study with TNFerade and radiation. The integrated treatment was well tolerated in patients with predominantly prior treatment-refractory solid tumors. Controlled prospective clinical trials have been initiated to more fully define the therapeutic contribution of TNFerade.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Tumor necrosis factor alpha (TNF) is a multifunctional cytokine with potent anticancer properties, as demonstrated with the recombinant protein in numerous preclinical models [1-4] and in clinical trials [5-6]. However, severe systemic toxicity has limited the clinical development of TNF [7-9]. Selected patients with extremity soft tissue sarcoma (or extensive in-transit melanoma metastases), either unresectable or without a locoregional alternative to amputation or mutilating surgery, are candidates for the TNF treatment. In this setting, TNF is administered with chemotherapy (± interferon gamma) during isolated limb perfusion to limit systemic toxicity [10,11]. Although this approach is used in a limited number of cancer patients, these studies have demonstrated that TNF, if administered such that systemic toxicity can be controlled, is a useful anticancer agent that induces objective tumor responses rates and improves local control rates [11].

A gene therapy approach, using intratumoral injections of an adenovector expressing TNF, is one way to potentially maximize local antitumoral activity and minimize systemic toxicity. The TNFerade construct represents such an approach. TNFerade was constructed as a second generation (E1-, partial E3-, and E4-deleted) adenovector, expressing the human TNF cDNA. To further optimize local effectiveness and minimize the systemic toxicity seen in a preclinical model with a constitutive promoter [12-13], the radiation-inducible immediate response Egr-1 (early growth response) gene promoter was ligated upstream to the transcriptional start site of the human TNF cDNA. This vector was constructed to ensure that maximal TNF gene expression with subsequent protein secretion is constrained by the spatial and temporal parameters of focused ionizing radiation [6,14]. Furthermore, the approach capitalizes on the known therapeutic synergy between radiation and TNF [6].

The activity of TNFerade in combination with radiation has been evaluated in a number of different human xenograft models, including human prostate cancer [15], human malignant glioma [16], radioresistant human laryngeal carcinoma [14], and human esophageal adenocarcinoma [17]. In these models, the combined effect of TNFerade and radiation was significantly greater than the effect of either modality alone. Furthermore, the combination of TNFerade and radiation was effective even against tumors that were resistant to radiation or TNF [14].

TNFerade has been evaluated in three different toxicology studies: two in nude mice and one in immunocompetent (BALB/c) mice [18]. TNFerade in combination with radiation was well tolerated in these studies and demonstrated a large therapeutic window. In this regard, the serum-TNF levels remained low even after administration of relatively high viral doses [18]. The dose-limiting toxicity was local ulceration and necrosis at viral doses of 4 x 10¹¹ particle units (pu) in immunocompetent mice.

This phase I clinical study of TNFerade was conducted to evaluate the safety, toleration, and feasibility of intratumoral administration of TNFerade in conjunction with radiation in patients with various solid tumors. Furthermore, antitumor activity as judged by tumor shrinkage was assessed in patients with measurable disease.

	PATIENTS AND METHODS

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Patient Selection
Patients were included who met the following criteria: (1) histologically confirmed advanced cancer resistant or refractory to standard therapy or for which no standard therapy exists; (2) lesions accessible for repeated injections; (3) radiation indicated for palliation; (4) age older than 18 years; (5) Karnofsky performance status 60 or above; (6) negative pregnancy test and use of effective means of contraception; (7) measurable disease; and (8) life expectancy greater than 3 months. The following exclusion criteria applied: (1) Liver enzymes more than 3 x upper limit of normal (AST, ALT, and bilirubin); (2) hemoglobin less than 9 g/dL or platelet count less than 50,000/µL; (3) evidence of active infection of any type, including adenovirus, hepatitis or HIV; and (4) chemotherapy or experimental medications within the last 4 weeks before day 1.

If patients had more than one lesion that required radiation for palliation, one lesion was injected and irradiated (the index lesion), and the other lesion would be irradiated only and used as a control lesion. The index as well as the control lesion would be assessed and compared for tumor response. All patients provided signed informed consent. The Institutional Review Boards as well as the Institutional Biosafety Committees in all participating institutions approved the protocol. The protocol was also reviewed by the Recombinant DNA Advisory Committee to the Director of the National Institutes for Health in accordance with its guidelines for research involving recombinant DNA molecules, as well as by the US Food and Drug Administration as part of an investigational new drug application.

Treatment Plan
The trial was an open-labeled dose-escalation study of TNFerade in combination with radiation. TNFerade was administered by intratumoral injection twice weekly during weeks 1 to 2, then once weekly from week 3 to week 6. During each treatment, a clockwise scheme was used to distribute the vector. For the first TNFerade injection, the drug was injected at sites corresponding to 12, 3, 6 and 9 o'clock; for the next treatment, the injections were rotated to sites at 1, 4, 7 and 10 o'clock. With each subsequent treatment, the site was rotated in a clockwise manner. Between one and five needle passes were delivered per injection session, using a 21- to 22-gauge needle, depending on tumor size and accessibility; a volume of 1 mL was given per injection (with a minimum total volume of 2 mL). For tumors difficult to access in deep visceral organs (eg, pancreas or biliary tract) only one needle pass was attempted per treatment. In these cases, a total volume of 2 mL was administered. Three to seven patients were to be treated per dose level, starting with a dose of 4 x 10⁹ pu and escalating in 0.5 log increments to a maximal dose of 4 x 10¹¹ pu or until the maximum-tolerated dose (MTD) was reached. After completion of the initial five dose levels, the protocol was amended to include two lower doses, 4 x 10⁷ pu and 4 x 10⁸ pu, to determine response probability at maximum serum concentrations shown clinically to be achievable with systemic administration at vector doses of 2 x 10¹² pu [19].

All patients received concomitant external beam radiation; radiation dose and therapy parameters were determined by a radiation oncology consultation before entry onto the study and depended on tumor type, site, previous irradiation (if any), and the performance status of the patient. Radiation was typically given in 1.8 to 2.0 Gy single-daily fractions, 5 days per week, for up to 5 weeks. The total dose ranged from 20 to 66.6 Gy.

Patients were assessed twice weekly during week 1 to week 2, once weekly for week 3 to week 6, and at 2 and 4 weeks post-treatment. Physical examination, symptom assessment, and laboratory safety tests were used during the study and protocol specified follow-up period.

Definition of Dose-Limiting Toxicities (DLTs) and Schedule for Dose Escalation
Patients were carefully monitored with regard to safety and tolerability using the National Cancer Institute Common Toxicity Criteria. Initially, three patients were to be included at each dose level. If no DLT developed, dose escalation would continue until reaching the highest dose of 4 x 10¹¹ pu. If one of three patients developed DLT, another three would be enrolled at that dose level. If at least two of three patients or at least two of six patients developed DLT, the previous dose level would be classified as the MTD without further dose escalation. If no more than one of six patients developed DLT, dose escalation would proceed. In situations in which an eligible patient could not wait for the next dose level to start because of disease progression, the patient could be enrolled at the previous dose level at the discretion of the investigator. In the event four patients were enrolled, one DLT would prompt enrollment of two additional patients for a total of six; if at least two of four patients developed DLTs, the previous lower dose was the MTD.

There was no intrapatient dose escalation. Once three patients completed treatment (defined as completion of TNFerade as well as radiation) and had been observed for at least 2 weeks for acute toxicity, patients were allowed to start treatment at the next dose level. DLT was defined as (1) any grade 3 or higher nonhematologic toxicity (except alopecia, nausea, vomiting, or diarrhea) possibly, probably, or definitely related to TNFerade or radiation occurring during the combined modality treatment and up to 2 weeks after; (2) grade 4 thrombocytopenia, neutropenia, or anemia; and (3) grade 3 or higher nausea, vomiting, or diarrhea, despite maximal antiemetic and/or antidiarrheal agents occurring in the combined modality treatment period and up to 2 weeks after.

Although this is a phase I study, standard criteria were used to evaluate target tumor response: complete response (CR), complete disappearance of targeted tumor; partial response (PR), greater than 50% reduction in tumor (calculated as the product of the tumor's greatest diameter and its perpendicular measurement); minimal response (MR), a reduction of 25% to 50%; stable disease (SD), reduction of 25% to progression of less than 25% (all parameters sustained for a minimum of 4 weeks); and progressive disease (PD), tumor expansion to 25% of prestudy volume.

Special Pharmacodynamic and Safety Measures
Serum TNF levels were monitored at baseline, and on study days 1, 2, 4, and 5 during the first week, days 2 and 5 during week 2, and on day 2 during weeks 3 to 6. An enzyme-linked immunoabsorbent assay kit (Quantikine High Sensitive) with a level of detection of 0.18 pg/mL was used. Neutralizing antibody titers against adenovirus was monitored at baseline, on day 5 during week 1, at the end of treatment, and 4 weeks after completion of the study, using a standard assay. Cultures from blood, sputum, and urine were taken at baseline and at the end of the study and analyzed for the presence of adenovirus. In addition, the protocol allowed for optional biopsies to be taken at the discretion of the investigator and after specific consent by the patient before, during, and after TNFerade and radiation treatment.

Construction of TNFerade
TNFerade was constructed using GenVec's AdFAST technology as described [20]. Briefly, TNFerade is a human adenovirus serotype 5 (Ad5) deleted of early regions 1A, 1B, and 4 and partially of early region 3 and with a transcriptionally inert spacer sequence inserted into the E4 region [20]. The TNF expression cassette contained the nucleotides 1-455 of Ad2, the mouse Egr-1 promoter (including the radiation inducible CArG elements), a splice acceptor and splice donor from SV40 late genes, the complete TNF cDNA, and an SV40 poly A site [20]. TNFerade was propagated and expanded in 293orf6 cells as described [20]. Similar methods were used for production in accordance with good manufacturing practices. The 293orf6 cells were plated in serum-containing media, infected with the adenovector, and treated with ZnCl₂ to induce E4-ORF6 expression. Adenovirus particles were purified using three successive rounds of CsCl gradient centrifugation. The adenovector was assayed for potency, purity, sterility, and absence of replication competent adenovirus.

Statistical Analyses
All data are presented as mean ± SE unless otherwise indicated. No formal statistical testing was carried out in this phase I study.

	RESULTS

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Patient Characteristics
Thirty-nine patients were enrolled onto this trial from four institutions. Three patients were not treated with TNFerade, either because they withdrew consent before dosing or because their condition deteriorated rapidly before dosing. Thirty-six patients thus underwent at least one injection of TNFerade. Of these, three patients only received one or two injections of TNFerade (week 1) and never started radiation. One patient with metastatic breast cancer had a significant pleural effusion at entry, which prevented her from lying horizontally and making radiation treatment impossible. Another patient with pancreatic cancer and pronounced peritoneal carcinomatosis deteriorated rapidly and was deemed too sick to undergo radiation. The third patient had recurrent metastatic esophageal cancer with a big lesion at the base of the tongue. The first two injections of TNFerade were given, but the lesion was considered too difficult to access repeatedly, and the patient withdrew after the first week of treatment. Of the thirty-three patients assessable for safety analysis, three were not assessable for tumor response measurements. One patient was not assessable for response because she did not have bi-dimensionally measurable disease at entry. The other two withdrew from the study before the end of treatment. One of these, a patient with metastatic sarcoma, developed brain metastases that required gamma knife surgery, and was withdrawn from the study after 2 and a half weeks; the other, a patient with squamous cell carcinoma in the oropharynx, withdrew after 1 and a half weeks of radiation because of radiation toxicity. Hence, 30 patients were assessable for tumor response. Demographic details of all 36 patients who received at least one dose of TNFerade are provided in Table 1.

View larger version (31K): [in this window] [in a new window]   Fig 1. Serum TNF- levels. Pu, particle units; BL, baseline; SCR, screen; Wk, week; EOT, end of treatment; W, week. Values plotted as means.

	DISCUSSION

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The clinical application of recombinant TNF protein to patients with cancer has been limited by severe systemic side effects [7-9]. Gene therapy, however, represents an approach by which high intratumoral levels of TNF can be achieved with acceptable systemic toxicity [6]. Preclinical studies have shown that gene therapy with adenovectors expressing TNF under control of a constitutive promoter is effective in producing partial and complete tumor regressions but also results in significant systemic toxicity [12-13]. Radiation therapy is a frequently used and widely applicable locoregional therapeutic modality associated with the activation of numerous radiation-inducible genes [21-24] and proteins [25]. Therefore, it was hypothesized that incorporating a radiation inducible promoter [24] upstream to the transgene could optimize the therapeutic index of TNF. This approach would allow for maximal TNF gene expression and subsequent protein secretion under the regulatory control of locally targeted radiation therapy. Radiation-inducible transcriptional activation of TNF production would then be constrained within a specific volume for a defined period of time. This approach also incorporates the potential for a supra-additive interaction between TNF and ionizing radiation [26-28]. The available data support the hypothesis that the effector pathways constituting the interactive networking of these two therapeutic modalities converge in two primary functional target streams: direct cellular cytotoxicity and endothelial vasculopathy.

The mechanisms of cytotoxic action and the signaling pathways making up the effector network of TNF have recently been reviewed [29-30]. Both apoptosis and cellular necrosis have been shown to be involved [31]. Mauceri et al [32] confirmed the association between the specific and differential production of TNF and the selective effect of combined modality radiation and intratumoral adenoviral Egr-TNF vector on the tumor neovasculature. The enhanced production of angiostatin after such combined-modality therapy, at least in part, mediates the antiangiogenic effects [33]. In addition, there is evidence supporting a critical role for the sphingomyelin-ceramide signal transduction pathway in both TNF and radiation mediated endothelial apoptosis [34-35]. Although not addressed in this study, the possible mechanistic contribution of TNF-mediated immune stimulation has been previously discussed [14] and is the subject of ongoing evaluation [36]. However, no abscopal objective responses were detected in this study.

The present phase I trial shows that TNFerade (up to 4 x 10¹¹ pu repeated up to eight times over a 6-week period) is well tolerated without significant systemic or local toxicities. Some of the toxicities (fever, chills, and fatigue) have been associated with systemic TNF exposure, but they could also be explained by the adenovector itself or be the result of an advanced underlying malignancy. Although the low serum TNF levels argue against a causal relationship between TNF and the observed toxicities, it is conceivable that the time points selected for quantitative TNF assessment were not frequent enough to detect nonsustained increases in serum levels, recognizing that the half-life of the protein is short (20 to 30 minutes) [7].

We did not find a correlation between toxicity and either the presence of pre-existing levels of neutralizing antibodies or a vector-induced increase in antibody titers. These findings are consistent with other gene therapy studies [37-38]. No ascribable toxicity was observed even in the one patient previously treated with TNFerade and radiation in a companion protocol, who had high pre-existing adenoviral neutralizing antibody titers at the time of entry onto this study.

One of the key issues in interpreting the results of this study is assessment of the contribution of TNF to the effectiveness of the combined modality regimen. In preclinical studies, interactive antitumor activity was produced by combining the adenoviral Egr-TNF construct with ionizing radiation in treatment of the radioresistant human SQ-20B xenograft [39] and the radio- and TNF-resistant P4L tumor [14]. Likewise, the functionality of the Egr-TNF expression cassette has been demonstrated in both in vitro and in vivo models [18]. In the present open-labeled study, such analytic separation is difficult, and a final answer will require both quantitative assessment of TNF expression in clinical material as well as randomized trials. The differential response patterns observed in four of five patients with uninjected control lesions are suggestive but clearly not definitive. Of potential significance was the observation of objective responses in patients with tumor types that usually show a limited objective response to radiation alone (adenocarcinoma of the pancreas [40], large soft tissue sarcoma [41], and large melanomas [42-43]). For example, two of four patients receiving 4 x 10⁹ pu with pancreatic cancer showed a partial response, which is infrequently documented after 50 Gy of radiation alone [40]. Very large melanoma lesions typically respond poorly to monomodal radiation [42-43], yet we observed complete pathologic responses in all three melanoma patients, including one patient (patient 13; Table 4) with a very large tumor (36 cm²) at baseline, despite the fact that this patient's disease had previously failed to respond to radiation in another lesion.

Sustained tumor regression was seen with greater reductions at 4 weeks and 3 months post-treatment than at the end of treatment. This response pattern is consistent with the proposed biologic effect of TNF in combination with radiation, where the key mechanism of action appears to be a selective destruction of tumor vasculature, leading to tumor necrosis [4-6]. Subsequent resorption of the necrotic tissue takes time, which is why a volume-reduction end point as assessed by CT scan is not immediate (patient 3 is a good example; Table 7). This pattern occurred consistently across a range of different tumor types.

Considering the substantial toxicity historically associated with systemic and local administration of the TNF, the limited side-effect profile observed in this study was reassuring. No serious adverse events attributable to TNFerade were observed, a finding consistent with measured plasma TNF levels, which did not exceed 50 pg/mL in any patient and were significantly below the peak plasma concentrations of TNF (approximately 10,000pg/mL) at MTD documented in clinical studies using the recombinant protein [5,8,45-46]. However, in an open-labeled phase I study in patients with predominantly refractory end-stage metastatic disease, it may be difficult to detect subtle TNFerade-induced incremental toxicity, whether systemic or locoregional, especially as all patients in the phase I study received simultaneous radiation.

In summary, the present study demonstrates that repeated intratumoral injections of TNFerade in combination with radiation are feasible and well tolerated. Randomized studies permitting statistical evaluation of the therapeutic role of TNFerade and demonstration of gene expression and/or TNF production are required to support the hypothesis that gene therapy, using a radiation-inducible promoter, in combination with irradiation, represents an effective way to optimize the anticancer activity of TNF while minimizing systemic toxicity. In this context, such studies are underway in patients with locally advanced pancreatic cancer, esophageal and rectal cancer.

	Authors' Disclosures of Potential Conflicts of Interest

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The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Owns stock (not including shares held through a public mutual fund): Neil Senzer, GenVec Inc. Henrik Rasmussen, Karen Chu, and Michelle Gillen are employees of GenVec; Ralph Weichselbaum and Donald Kufe are paid consultants to GenVec and own equity in the company. Camilla Rasmussen is a consultant to GenVec. Ralph Veichselbaum also receives research support from GenVec. He did not participate in the care of patients or the evaluation of any primary patient data.

	Acknowledgment

 
We thank Donna Washington for her competent and knowledgeable assistance in the preparation of this manuscript. Also, our deepest appreciation is extended to Elena Smidt for playing a large role in the data management for this study.

	NOTES

 
Authors' disclosures of potential conflicts of interest are found at the end of this article.

Supported by a grant from GenVec Inc, Gaithersburg, MD.

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Submitted January 30, 2003; accepted October 30, 2003.

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