A Phase I Trial of Repeated Intrapleural Adenoviral-mediated Interferon-β Gene Transfer for Mesothelioma and Metastatic Pleural Effusions

Abstract

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-β (Ad.IFN-β) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-β vector would be superior. Ten patients with MPM and seven with MPE received two doses of Ad.IFN-β through an indwelling pleural catheter. Repeated doses were generally well tolerated. High levels of IFN-β were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector. Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days.

Introduction

Malignant pleural mesothelioma (MPM) is usually advanced at the time of diagnosis, with the tumor growing in a locally invasive fashion, often invading thoracic structures such as chest wall, pericardium, and great vessels. The median survival for patients with MPM at the time of diagnosis is ~12 months.¹ Therapy for MPM has included three modalities that have been used individually or in combination: surgery, irradiation, and chemotherapy. Unfortunately, to date, none of these modalities has proven curative. The most effective combination chemotherapy, pemetrexed/cisplatin, has reported response rates of up to 40%, but with overall median survival improvement of only 3.5 months compared with cisplatin alone.² It is clear that novel therapeutic approaches are desperately needed in this disease.¹

Malignant pleural effusions (MPE) are a common and frequently troublesome complication of advanced malignancy resulting from hematogenous spread or direct tumor extension to the pleural lining. An MPE, in general, portends advanced disease and poor prognosis with median patient survival estimated at 6 months.³ Frequently, therefore, the primary therapeutic goal in MPE is effective palliation of associated dyspnea and chest discomfort. They too would thus benefit substantially from novel therapeutic approaches.

We previously conducted a phase I dose-escalation trial of single-dose intrapleural administration of a recombinant adenoviral vector carrying the human interferon-β (IFN-β) transgene (Ad.IFN-β/BG00001) in patients with MPM and MPE.^4,5 Ten patients were treated in this trial to a maximal tolerated dose of 3.0 × 10¹² viral particles (vp) with evidence of reasonable patient tolerance, measurable intrapleural IFN-β gene transfer, significant humoral antitumor immune responses, and some clinical responses. Of particular interest were antibody responses against the SV40 large T-antigen (Tag) and mesothelin, as well as anecdotal clinical responses, including dramatic positron emission tomography (PET) responses in two patients and disease stability in four heavily pretreated patients.

Given these encouraging results, we aimed at augmenting these immunologic and clinical responses. Preclinical studies in mouse models of MPM suggested two primary strategies: administration of repeat doses of Ad.IFN-β⁶ and/or combination with chemotherapy.⁷ In order to identify the dosing schema that could be used in future Ad.IFN-β/chemotherapy trials, we therefore designed this phase I clinical trial to examine the safety, toxicity, and efficacy of repeated intrapleural administration of Ad.IFN-β/BG00001. Because of potential safety concerns, we began the trial using a dose interval of 14 days. After showing no additional toxicity, additional patients were treated with 7-day dosing interval. We sought to answer three questions: (i) Would multiple administrations of intrapleural Ad.IFN-β be safe and well tolerated? (ii) Would multiple administrations of intrapleural Ad.IFN-β result in gene transfer after the second dose of Ad.IFN-β despite induction of neutralizing antibodies (Nabs)? and (iii) Would our immunologic or clinical responses be improved after giving two doses?

Results

Patient characteristics

This study enrolled a total of 17 patients. Details are shown in Table 1. There were nine males and eight females enrolled, with an age range from 50 to 87 (median age 64 years). Underlying cancer diagnoses in the patients with MPE included three with carcinoma of the lung, two with ovarian cancer, and two with breast cancer. The remaining 10 subjects had MPM. Thirteen patients had received prior chemotherapy (Table 1). The first subject was dosed with BG00001 on 9 March 2006.

Table 1.

Clinical data

On study days 1 and 15 (for patients 201–215) and on study days 1 and 8 (for patients 216–219), a dose of Ad.huIFN-β (BG00001), diluted in 50 cc of sterile normal saline, was instilled via the catheter into the pleural space. All patients were treated as inpatients in the Clinical and Translational Research Center of the University of Pennsylvania Medical Center. A total of 13 subjects were enrolled in the initial 14-day dosing interval portion of the trial. In a modified 3+3 dose-escalation schema, four subjects received two vector doses of 3 × 10¹¹ vp; three subjects received two doses of 1 × 10¹² vp; and six subjects received two vector doses of 3 × 10¹² vp. An additional four patients were enrolled in an amended protocol that allowed for a decreased dosing interval of 7 days. The first three patients in this protocol received two doses of 1.5 × 10¹² vp and the last patient received two vector doses of 3 × 10¹² vp. The trial was stopped at this point due to reaching the expiration date of BG00001 and the decision of Biogen Idec (who supplied the vector) to discontinue the Ad.IFN-β program.

Safety and toxicity

No maximum tolerated dose was reached. Most patients experienced the expected mild-to-moderate adenoviral vector–related adverse events. The most common adverse events were as follows: lymphopenia, hypoalbuminemia, hypotension, anemia, hypocalcemia, and a mild cytokine release syndrome characterized by fever, rigors/chills, nausea, and tachycardia (see Supplementary Table S1). These side effects were seen at all dose levels.

There were two unusual toxicities: patient 205 received the first dose of BG00001 and tolerated dosing well, but did not meet eligibility criteria for the second dose due to an elevated partial thromboplastin time. On continued long-term follow-up, no abnormal bleeding, clotting, or other systemic or constitutional symptoms developed. Additional workup showed an elevated anticardiolipin antibody level. Because patient 205 did not receive the two doses of BG00001, an additional subject was treated in the first cohort (P206).

The second toxicity was a study-related serious adverse event identified in the third dosing cohort in an older male patient (P210) with metastatic pleural adenocarcinoma that occurred ~14 days following the second dose of BG00001. The patient presented with increasing dyspnea and vomiting with a transthoracic echocardiogram demonstrating pericardial tamponade. He was hospitalized and a pericardiocentesis removed of 620 ml of sanguineous fluid. Fluid cytology was negative for malignant cells, and pericardial fluid testing for adenovirus vector (via PCR and viral cultures) was also negative. No IFN-β was detected in the pericardial fluid. The subject was discharged home in stable condition with two repeat echocardiograms demonstrating a smaller, but persistent, pericardial effusion. We presume the development of the significant pericardial effusion in patient 210 after the second dose of vector was related to antitumor inflammatory responses in the pericardium or possibly to nonspecific inflammation induced by repeat instillation of adenoviral vector into the pleura. As a result of this serious adverse event, three additional subjects, for a total of six, were dosed at the third cohort, and a change was made to the enrollment eligibility to exclude subjects with baseline pericardial effusions.

To monitor systemic antiadenoviral inflammatory responses, serum IL-6 was measured before and for 2 days after each dose of vector. We saw virtually no systemic inflammatory responses after the first vector dose, and although there were slightly higher levels after the second vector dose, no patient's serum level went above 40 pg/ml. The one exception was P212 (a lung cancer patient) who had a baseline level of IL-6 of 55 pg/ml. This increased to 100 pg/ml after his first dose of vector and then dropped to baseline 3 days later (data not shown).

Gene transfer assessment

Gene transfer was assessed by measuring IFN-β protein levels in the pre- and postvector instillation serum samples and pleural fluid after each dose. The pleural measurements should be considered semiquantitative because the volume of pleural fluid varied and likely affected the final concentration. In two patients (patients 206 and 207), pleural lavages with 50 cc of sterile saline were performed to obtain samples.

No patients had detectable levels of IFN-β protein (<0.2 ng/ml) in their serum. As summarized in Table 2, levels of IFN-β in pleural fluid after the first dose ranged from undetectable to 430 ng/ml. Detectable levels after the first dose were not found in 5 of the 17 patients (two of which required pleural lavages). The time course of intrapleural transgene expression is depicted in Figure 1a,b: IFN-β was detectable for at least 3 days in most patients with no obvious dose–response relationship.

Table 2.

Intrapleural IFN-β levels and serum adenovirus neutralizing antibody titers

Figure 1.

Pleural gene transfer and serum adenoviral neutralizing antibody (Nab) data. (a,b) Levels of IFN-β protein (ng/ml) in pleural fluid samples removed via the tunneled pleural catheter (PleurX) (y axis) were measured by enzyme-linked immunosorbent assay and plotted versus the time after Ad.IFN-β instillation (arrows). Day 1 shows levels of preadministration. a shows data from patients given a second dose on day 14. b shows data from patients given a second dose on day 7. (c) Adenovirus neutralizing antibodies. c plots the inverse titer of Nab versus time (in weeks) after the first Ad.IFN-β instillation. Virtually every patient rapidly developed high titers (>1:1,000) of anti-Ad Nab within 1 week. (d,e) Relationship between Nab titers and gene transfer. d plots the relationship between the pleural fluid IFN-β level (in ng/ml on the y axis) versus the serum adenovirus neutralizing antibody titer (expressed as 1/titer on the x axis). A value of 10 signifies a titer of 1:10 or less. e plots the percentage of patients who showed detectable levels of IFN-β after gene transfer versus their Nab titers at the time of the instillation of Ad.IFN-β. Data from both the first and second viral instillation are graphed.

In contrast, levels of IFN-β were markedly lower after the second dose of vector delivered either 2 weeks (13 patients) or 1 week (4 patients) after the first dose (Figure 1, Table 2). Only 3 of 17 patients had detectable pleural IFN-β levels after their second dose, and these levels were 10- to 285-fold lower (ranging from 200 to 800 pg/ml) than those after the first dose.

Antiviral immune responses

Serum anti-Ad Nab titers were measured before and after gene transfer (Figure 1c). As shown in Table 2, baseline titers of anti-Ad Nab ranged from <1:10 (arbitrarily called 1) to 1:3,600. Nine of seventeen patients had baseline titers <1:100. Only three patients had baseline titers above 1:1,000. All tested patients demonstrated markedly increased anti-Ad Nab titers by 8 weeks postvector instillation. Only one of the initial 13 patients tested had an 8-week titer <1:1,000. Upregulation was very rapid after the first dose administered; most patients had large increases in Nab titer after only 1-week postvector delivery. Pleural fluid titers of anti-Ad Nab were measured in most early samples and found to be virtually identical to serum titers (data not shown).

Relationship between gene transfer and antiadenoviral Nabs

Correlation of serum anti-Ad Nab titers with IFN-β gene transfer delineated a clear inverse relationship (Figure 1d,e). Whereas >90% of the patients with a Nab titer of <1:100 showed measurable gene transfer, only about 15% of patients with titers >1:1,000 showed detectable IFN-β levels in their pleural fluids.

Antitumor immunologic responses

Humoral responses to known MPM tumor antigens. In 9 of the 10 MPM patients, humoral responses to two defined mesothelioma-associated antigens were evaluated by using patient sera from before and after gene transfer to immunoblot purified proteins (Table 3). Two patients had some baseline reactivity against purified SV40 virus large T-antigen (SV40 Tag protein). Both of these patients (P211 and P213) showed increased reactivity after gene transfer (data from P213 are shown in Figure 2a). Two patients had some baseline level of staining against mesothelin. One patient showed increased reactivity after vector instillation (P203) and one maintained the same level of reactivity (P206) (data not shown).

Table 3.

Humoral antitumor immune responses

Figure 2.

Antitumor immune responses. Antibody responses against tumor antigens were visualized on immunoblots using patient serum (diluted 1:1,500) before and after gene transfer. (a) Response of mesothelioma patient 213 to purified mesothelin (meso) and SV40 large T-antigen (SV40). Purified mesothelin and SV40 protein were run on SDS-PAGE gels, transferred to nitrocellulose, and immunoblotted with diluted pre- and 6-week postgene transfer serum. Blots were stripped and blotted with commercial anti-SV40 Tag and antimesothelin antibodies to show equal loading of SV40 Tag and mesothelin (data not shown). (b) Immunologic response of patient 214 to an autologous breast cancer cell extract. Extracts from a breast cancer cell line established from this patient's own pleural fluid were run on an SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with diluted pre- and postgene transfer serum.

Humoral responses to tumor antigens on cell line extracts. We also used pre- and postgene transfer serum for immunoblotting to identify new or increased intensity bands on extracts of mesothelioma, lung cancer, breast, or ovarian cancer cell lines (Table 3). Patient 205 received only one dose of vector and was not included in the analysis. Of the remaining 16 patients, the sera from 12 patients recognized one or more new antigens in the post-treatment samples (Table 3). Most patients (11 of 12) had new or increased bands seen on multiple cell lines. Figure 2b shows an example from P214 (metastatic breast cancer) of strong new bands at 48 and 53 kd in the 6-week postvector serum when reacted against an autologous breast cancer cell line established from the patient's own pleural fluid.

Radiographic and clinical evaluation. Radiologic evaluation of therapeutic response was evaluated at 2 months (except patient 215 who died at 1 month) using computed tomography (CT) and PET imaging (Table 1). By modified Response Evaluation Criteria in Solid Tumors (RECIST) criteria, there was one partial response (>30% decrease in tumor measurements from baseline), four stable disease, and nine progressive disease patients (>25% increase in tumor measurements over baseline), and two patients with no measurable disease. The waterfall plot demonstrates these responses (Figure 3a). Note that patient 215 who died at 1 month and the two patients (211 and 219) with no measurable disease are not included on this graph).

Figure 3.

Clinical responses. (a) Waterfall plot of tumor responses determined by CT scan using modified RECIST criteria. The plot shows the change in tumor size comparing pretreatment to 2 months postvector measurements for each patient. By modified RECIST criteria, >30% reduction in tumor size defines a partial response, >25% increase in size defines progressive disease, and change in tumor size from <30% reduction to <25% increase is defined as stable disease. Data from patient 203 (outside films were not available for quantification) and patients 211 and 219 (whose tumor was not measurable) are not included. (b) CT scans and FDG-PET scans from patient 218 prevector instillation (left panels), 3 months after vector instillation (middle panels), and 6 months post-therapy (right panels) show a mixed response. PET scan identifies a right apical lesion (solid white arrows) that does not change over time (upper two rows) and a basilar lesion (dotted white arrow) pretherapy that demonstrated decreased FDG uptake at the 3- and 6-month time point. The patient received no other treatment. (c) A Kaplan–Meier plot of survival of the 17 patients with malignant pleural mesothelioma treated with Ad.IFN-β in our original one dose trial⁵ and the current study. The median survival (arrow) is 22 months. Three patients remain alive at 42, 39, and 18 months after vector administration (as of October 2009). CT, computed tomography; FDG, fluorodeoxyglucose; PET, positron emission tomography; RECIST, Response Evaluation Criteria in Solid Tumors.

Using fluorodeoxyglucose (FDG)-PET SUVmax (maximum standardized uptake value) as a semiquantitative measure of metabolic activity on FDG-PET, there were 2 mixed responses, 11 with stable disease, and 3 with progressive disease (one patient did not have a follow-up PET).

Therapeutic response evaluation by imaging was also carried out at 6 months (relative to baseline) for seven patients based on availability of data for review. By CT-modified RECIST criteria, there were two stable disease and two progressive disease. By PET, there was one mixed response, four stable disease, and three progressive disease. Figure 3b demonstrates an example of mixed therapeutic response by imaging. CT and PET images reveal that patient 218 had complete regression of his right basilar tumor with no additional treatment; however, there was simultaneous slight progression of a right apical tumor.

Seven of the seventeen patients enrolled with advanced pleural malignancies had survival times of >18 months, and four patients are still alive at 18+, 28+, 39+, and 42+ months.

Discussion

We previously reported the results of several phase I clinical trials of single-administration adenoviral-mediated gene delivery into the pleural space of patients with MPM and MPE. With single intrapleural delivery of the suicide gene HSVtk, and with a single dose of a recombinant adenoviral vector expressing the human IFN-β gene (BG00001), we were able to demonstrate successful gene transfer and transgene protein-product expression.^4,5,8 With these single administrations, we showed safety, and even observed some unexpected significant clinical responses, which in two cases were durable for >10 years. Given these positive results and based on preclinical data, we hypothesized that repeated administration of adenoviral vectors carrying immunostimulatory transgenes might prove even more beneficial to patients with MPM and MPE.

Safety issues

In general, this study confirmed the safety of repeated administration of recombinant adenoviral vectors into the pleural space. We saw no strong inflammatory reactions and only minimal elevations in serum IL-6 levels, a measurement thought to be a good indication of Ad-induced systemic activation.⁹ This is consistent with the overall good safety record of adenoviral vectors injected via the intravenous, intratumoral, and intracavitary routes for a variety of cancers.^{10,11,12,13,14,15,16}

The most serious adverse event seen in this clinical trial was the development of pericardial tamponade in patient 215 ~2 weeks after administration of the second BG00001 dose. Interestingly, the pericardial fluid cytology showed no malignancy, but did show acute and chronic inflammatory cells suggestive of an induced intrapericardial immune response postvector administration. Pericardial fluid viral culture and PCR failed to reveal evidence of recombinant vector or replication-competent adenovirus, making it difficult to implicate direct vector dissemination as the cause of the pericardial tamponade. Nonetheless, this patient's complication illustrates the potential risks of inducing antitumor immune responses in “vulnerable locations” such as the pericardial space.

Gene transfer issues

An important issue in the field of human gene therapy has been the documentation of efficiency and length of gene transfer. Relatively little data exist about the efficacy of transgene expression after initial and subsequent doses of Ad vectors. Given that most human gene transfer studies utilizing Ad vectors have demonstrated rapid rises in serum anti-Ad Nab titers, successful gene transfer after repeated Ad vector dosing is a challenging objective. Although clinical data are scant, based on preclinical studies,¹⁷ most investigators agree that high levels of Nab rapidly inactivate Ad vectors introduced systemically. In contrast, a number of studies using intratumoral injection of Ad vectors suggest that successful gene transfer can occur in patients with high baseline Nab. For example, both Tong et al.¹⁸ and Fujiwara et al.¹² showed that intratumoral injections of Ad.mda-7 or Ad.53 (respectively) led to staining of transgene protein in tumors that was not correlated with subject Nab titers. It is postulated that this intratumoral gene expression occurs due to poor penetration of antibodies into tumors or because very high local concentrations of vector can overwhelm the limited amounts of antibody in the tumor microenvironment.

The situation in the pleura or peritoneum has not yet been studied directly. High titers of Nab in the serum appear to correlate with levels in pleural^5,19 or peritoneal fluid.²⁰ However, a murine study suggested that intraperitoneal delivery of an Ad vector containing the suicide gene HSV.tk was still effective despite the presence of Nabs.⁶

In this study, we were able to evaluate both the levels and kinetics of pleural space gene transfer because patients had indwelling pleural catheters to facilitate both vector delivery and serial pleural fluid withdrawal for research and clinical purposes. Gene transfer efficacy, as measured in pleural fluid specimens, appeared to be significantly impaired after a second intrapleural dose of BG00001 at 2 weeks (Figure 1a) or even 1 week (Figure 1b) after the initial dosing in inverse relationship to levels of serum anti-Ad Nabs (Figure 1d,e, Table 2). In addition, we were able to demonstrate that patients who had pre-existent or induced anti-Ad Nab titers >1:1,000 did not have evidence of successful gene transfer as evidenced by the absence of pleural fluid IFN-β (Figure 1d,e). In fact, Figure 1e is strikingly similar to data presented by Harvey et al.²¹ showing a correlation between serum Nab titers and CFTR bronchial gene expression data after instillation of Ad.CFTR into patients with varying levels of serum anti-Ad Nab. These data suggest that Ad-mediated gene transfer in the pleural cavity (and likely peritoneal space) will be more similar to the blood compartment than the tumor compartment.

Immunologic responses

In cancer immunotherapy trials, it has been very difficult to identify intermediate immunological end points that demonstrate proof of principle, especially when not immunizing against a specific tumor antigen. Based on our inability to detect frequent cellular immune responses in our first trial, we did not focus on this area. In collaboration with scientists at Cerus, Concord, CA, however, we did assess the peripheral blood mononuclear cells isolated pre- and postdosing from MPM patients for mesothelin-specific cellular immune responses using peptide libraries to induce cytokine production. We found that mesothelin-specific T-cell responses were not detectable ex vivo (data not shown) suggesting that mesothelin-specific T-cell frequencies were either very low in our patient population, or that T cells were anergic and unable to produce IFN-γ or TNF-α after stimulation with recombinant mesothelin protein. It is also possible that sampling of peripheral blood mononuclear cells may not accurately reflect immunological activity within the tumor microenvironment.

We did, however, demonstrate strong humoral antitumor immune responses. As in our first trial, we consistently saw that Ad.IFN-β induced the generation of antibodies directed against known antigens (mesothelin or SV40 Tag in MPM patients) and/or unknown tumor antigens (Table 3, Figure 2). This strong reactivity against tumor lysates was true for both the MPM patients and the advanced MPE patients. It was interesting that four patients developed new antibodies against a cellular protein with an apparent molecular mass of 53 kd. Using purified protein, we found that these antibodies did not identify the classic p53 tumor-suppressor protein (data not shown), and we are pursuing other possible targets.

Radiographic and clinical responses

Unlike most solid tumors, where CT scanning provides a relatively straightforward method to measure tumor burden, evaluation of disease regression or progression is much more challenging for pleural malignancies, even using the modified RECIST criteria;¹ however, using the modified RECIST criteria, we observed one partial response and saw lack of disease progression at 2 months in a subset of patients (Figure 3). Because of these limitations, FDG-PET may provide a useful functional indicator of pleural tumor by measuring tumor glucose metabolism with the realization that specificity may be limited by activity from pleural inflammation. Although FDG-PET imaging is not part of the standard RECIST or modified RECIST criteria, it is increasingly recognized as an early signal of tumor response to therapy, and is evolving as a measure of treatment efficacy in MPM patients.^22,23 When one examines the 10 patients who lived ≥1 year, none had PD at 2 months by FDG-PET scanning, whereas by CT scanning, 5 had progressive disease. These data support a hypothesis that PET responses may precede CT responses and may indicate tumor response even in the setting of anatomic stability on CT scan, particularly given the degree of stromal tissue found in malignant mesothelioma that may not respond to antitumor therapy. Clearly, more work in this area is needed. Finally, it should be emphasized that although disease stability does not qualify as a “response,” it has substantial clinical benefit and is becoming increasingly recognized as an important outcome in biologic therapies.^24,25

Clearly, the ultimate indicator of clinical success is survival. Seven of seventeen patients in this trial lived beyond 18 months. Given the heterogeneity of underlying diagnoses (MPM versus MPE from various types of cancer), we have combined data from just the MPM patients in the one-dose⁵ and two-dose Ad.IFN-β trials, and plotted survival (Figure 3c). Median survival of this group of only MPM patients was 22 months, with three patients still alive at 18+, 39+, and 42+ months. This compares quite favorably to the 12- to 14-month median survival reported in the pivotal pemetrexed/cisplatin trial² and dozens of other trials over the past 15 years (summarized by Fennell et al.¹). Of course, this is phase I trial data and is subject to several caveats including the following: selection bias; administration of varying doses and schedules of Ad.IFN-β for the extensive pretreatment of most patients with chemotherapy, radiotherapy, and/or palliative surgery; and the fact that many patients received additional therapies after Ad.IFN-β. Nonetheless, we believe that our data support the concept that Ad.IFN-β has single agent activity in pleural malignancies, primarily as an agent that can forestall disease progression with minimal toxicity.

Conclusions and future directions

This trial supports our previous observation that Ad.IFN-β induces consistent antitumor immune responses and has some single agent activity in MPM and MPE. Compared with a single dose of vector, however, we found no solid evidence that two doses of vector, given either 1 or 2 weeks apart, increased duration of IFN-β gene expression, altered humoral antitumor immune responses, or changed radiographic/clinical responses. We believe this lack of increased efficacy after the second dose of vector was due to rapid induction of antiadenoviral Nab that minimized target cell transduction and IFN-β gene expression. Based on these results, in our current ongoing trial, we are administering a second dose of rAd-IFN vector 3 days after the first dose, prior to the expected peak of Nab production. Preliminary results suggest we are indeed seeing augmented gene transfer after this earlier second administration of vector. We next plan to combine Ad.IFN gene transfer with chemotherapy and surgery. This idea is based on our preclinical animal model data that have shown markedly enhanced antitumor efficacy of Ad.IFN-β when combined with chemotherapy (Suzuki et al.⁷) or with debulking surgery (Kruklitis et al.²⁶).

Materials and Methods

Vector. The vector used in this trial, Ad.huIFN-β virus (BG00001), was a GMP grade, E1/E3 deleted replication-incompetent adenovirus with insertion of the human IFN-β gene in the E1 region of the adenoviral genome. The transgene was driven by a human cytomegalovirus promoter. The vector was provided by Biogen Idec (Cambridge, MA and San Diego, CA) and used in our previous trial.⁵

Patients. Patients were eligible for these studies based upon the following criteria: (i) a pathologically confirmed diagnosis of MPM or MPE; (ii) an ECOG performance status of 0 or 1; and (iii) an accessible pleural space for instillation of vector. Exclusion criteria included prior surgical resection, successful pleurodesis, recent chemotherapy or radiotherapy, inadequate pulmonary function, or significant cardiac/hepatic/renal disease. Presence of a pericardial effusion was added as an additional exclusion criterion after an adverse event occurred in patient 210 (see below).

Protocol summary. The protocol received full approval by the University of Pennsylvania Medical Center Review Board, the Food and Drug Administration, and the Recombinant DNA Advisory Committee of the National Institutes of Health. Written informed consent, in accordance with the Declaration of Helsinki protocols, was obtained from each patient at the time of enrollment.

Eligible patients were recruited and staged radiographically. Enrolled MPM/MPE patients then underwent insertion of a tunneled intrapleural catheter under local anesthesia as an outpatient. If this was not technically feasible, a pleural catheter was inserted via thoracoscopy under general anesthesia by thoracic surgery or via image-guided placement by interventional radiology. At the time of insertion, pleural fluid was collected for analyses. Pretreatment serum and peripheral blood lymphocytes were also saved for similar testing in comparison with post-treatment samples.

On study days 1 and 15 (for patients 201–215) and on study days 1 and 8 (for patients 216–219), a dose of Ad.huIFN-β (BG00001), diluted in 50 cc of sterile normal saline, was instilled via the catheter into the pleural space. Patients were monitored as inpatients for ~48 hours after each dose and then followed as outpatients for toxicity assessment and laboratory testing with serum and pleural fluid obtained serially for IFN-β quantification, as well as antibody and lymphocyte responses. One month after vector instillation, the pleural catheter was removed under local anesthesia, unless still needed for control of symptomatic MPE. Patients were assessed for antitumor responses ~60 days after initial treatment using chest CT scans and ¹⁸FDG-PET scans (see below for details). Patients were monitored closely as outpatients after treatment through day 190 (~6 months). Eight patients had radiographic evaluations at that time. If progressive disease was documented any time after 2 months, patients proceeded with other antitumor therapies, as desired.

Radiographic response assessment. Radiographic analysis was performed by an experienced thoracic radiologist (S.I.K.) blinded to the patient name, medical history, and other clinical trial results (i.e., gene transfer and immune response analyses). Routine FDG-PET and FDG-PET/CT exams were performed on either the HUP Philips Gemini TF (Philips Healthcare, Andover, MA) or Philips Allegro scanner (Philips Healthcare), using routine departmental protocol including 15 mCi FDG IV and static image acquisition at 60-minute radiotracer uptake time. Imaging data were reconstructed into 8 mm slices and analyzed on a TeraRecon 3D workstation (TeraRecon, San Mateo, CA) allowing for multidimensional PET measurements. SUVmax, the value chosen to measure therapeutic response on PET, reflects the highest value SUV among the reconstructed slabs.

Assessment of response to therapy by CT was performed by simultaneous display of serial patient exams on high-resolution four-monitor displays attached to a GE Centricity PACS clinical workstation (GE Healthcare–Dynamic Imaging Solutions, Allendale, NJ). Modified RECIST measurements were meticulously acquired for each exam in the manner described by Byrne and Nowak.²⁷ Imaging data were then analyzed on the Excel Spreadsheet Data Analysis Toolpak.

Modified RECIST measurements were performed in the manner described by Byrne and Nowak.²⁷ Statistical analysis for radiographic analysis was performed utilizing the Excel Spreadsheet Data Analysis Toolpak (Microsoft, Redmond, WA).

Assessment of gene transfer and cytokines. Commercial cytokine enzyme-linked immunosorbent assays were used to measure the levels of IFN-β (PBL Biomedical Labs, Piscataway, NJ) and IL-6 (BD Biosciences, San Jose, CA) in pleural fluid and/or serum.

Immunoblots. To detect IFN-β induced humoral responses against tumor antigens, immunoblotting against purified proteins and extracts from commercially available mesothelioma, lung cancer, breast cancer, and ovarian cancer cell lines was performed. Purified SV40 large Tag protein was purchased from Chimerx (Milwaukee, WI). Purified mesothelin was provided by Mitchell Ho and Ira Pastan (NCI). In some cases, cell lines were derived from patient pleural fluid samples and were grown in culture as previously described.⁵ Extracts from cells or purified proteins were prepared and immunoblotted with patient serum (diluted at 1:1,500) from time points before treatment, and 6 weeks to 6 months after treatment as previously described.⁵ See Supplementary Materials and Methods for details.

Adenovirus serotype 5 Nab levels. Serum adenoviral Nabs were evaluated as described in the Supplementary Materials and Methods.

Funding. This trial was funded by grants from the National Cancer Institute (NCI PO1 CA66726) and from Biogen Idec.

Statistical analyses. The primary end point of the trial was safety. We used a standard 3+3 design to determine the maximum tolerated dose, with an implicit 50% chance of further dose escalation after achievement of a toxicity rate of 30%. Immunologic responses (cytokine levels, lymphocyte response to tumor antigens, levels of antibodies in tumor lysates, and fractions of lymphocytes measured by flow cytometry) and overall tumor response rates were secondary end points.

SUPPLEMENTARY MATERIALTable S1. Adverse events.Materials and Methods.

Supplementary Material

Table S1.

Adverse events.