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[CANCER RESEARCH 62, 4968 – 4976, September 1, 2002]
Phase I Study of Replication-competent Adenovirus-mediated Double Suicide Gene Therapy for the Treatment of Locally Recurrent Prostate Cancer1 Svend O. Freytag,2 Mark Khil, Hans Stricker, James Peabody, Mani Menon, Mariza DePeralta-Venturina, Daniel Nafziger, Jan Pegg, Dell Paielli, Steve Brown, Ken Barton, Mei Lu, Estuardo Aguilar-Cordova, and Jae Ho Kim Departments of Radiation Oncology [S. O. F., M. K., J. Peg., D. P., S. B., K. B., J. H. K.], Urology [H. S., J. Pea., M. M.], Pathology [M. D-V.], Infectious Disease [D. N.], and Biostatistics [M. L.], Henry Ford Health System, Detroit, Michigan 48202, and Department of Cell and Gene Therapies, Baylor College of Medicine, Houston, Texas 77030 [E. A-C.]
ABSTRACT Adenovirus-mediated suicide gene therapy may hold promise in the treatment of human cancer. We have developed a novel approach that utilizes a lytic, replication-competent adenovirus (Ad5-CD/TKrep) to deliver a cytosine deaminase/herpes simplex virus-1 thymidine kinase fusion gene to tumors. The cytosine deaminase and herpes simplex virus-1 thymidine kinase suicide genes render malignant cells sensitive to specific pharmacological agents and, importantly, sensitize them to radiation. The Phase I study described here represents the first gene therapy trial in which a replication-competent virus was used to deliver a therapeutic gene to humans. The indication is local recurrence of prostate cancer after definitive radiation therapy. An escalating dose (1010, 1011, and 1012 viral particles) of the Ad5-CD/TKrep virus was injected intraprostatically under transrectal ultrasound guidance into 16 patients in four cohorts. Two days later, patients were given 5-fluorocytosine and ganciclovir prodrug therapy for 1 (cohorts 1–3) or 2 (cohort 4) weeks. There were no doselimiting toxicities, and the maximum tolerated dose of the Ad5-CD/TKrep vector was not defined. Ninety-four percent of the adverse events observed were mild or moderate (grade 1/2) in nature. Seven of 16 (44%) patients demonstrated a >25% decrease in serum prostate-specific antigen, and 3 of 16 (19%) patients demonstrated a >50% decrease in serum prostatespecific antigen. Transgene expression and tumor destruction at the injection site were confirmed by sextant needle biopsy of the prostate at 2 weeks. Two patients were negative for adenocarcinoma at 1 year followup. Although Ad5-CD/TKrep viral DNA could be detected in blood as far out as day 76, no infectious adenovirus was detected in patient serum or urine. Together, the results demonstrate that intraprostatic administration of the replication-competent Ad5-CD/TKrep virus followed by 2 weeks of 5-fluorocytosine and ganciclovir prodrug therapy can be safely applied to humans and is showing signs of biological activity.
INTRODUCTION Approximately half of the estimated 198,000 men diagnosed with prostate cancer this year will receive EBRT3 as their primary treatment. Although prolonged survival is common, disease control after EBRT is only modest for locally aggressive but nonmetastatic cancers (stage T2⫺T4), as documented by clinical examination, prostate biopsy and serum PSA (1–5). Using rigorous PSA criteria, only 40% of patients with stage T3/T4 tumors are disease-free at 10 years (5). Clinical local failure, which significantly underestimates tumor local Received 4/1/02; accepted 7/10/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA75456, DK57833, and CA85551 and by an award from the RAID Program (to S. O. F.). 2 To whom requests for reprints should be addressed, at Molecular Biology Research, Henry Ford Health System, One Ford Place, Wing 5D, Detroit, MI 48202-3450. Phone: (313) 876-1949; Fax: (313) 876-1950; E-mail: [email protected] 3 The abbreviations used are: EBRT, external beam radiation therapy; GCV, ganciclovir; CD, cytosine deaminase; HSV-1, herpes simplex virus-1; TK, thymidine kinase; 5-FC, 5-fluorocytosine; vp, viral particle(s); TRUS, transrectal ultrasound; PSA, prostatespecific antigen; DLT, dose-limiting toxicity; MTD, maximum tolerated dose; AST, aspartate aminotransferase; NAB, neutralizing antibody; pfu, plaque-forming unit(s); AE, adverse event; GI, gastrointestinal.
control, occurs in about 30 –50% of patients at 5 years and in up to 75% of patients at 10 years (5). The observation that ⬎50% of distant recurrences occur concomitantly with local recurrence highlights the importance of local tumor control as a clinical end point (6). Although tumor control in locally advanced prostate cancer increases with radiation dose (7), unfortunately, so does toxicity. Prescription doses beyond 70 Gy are associated with increased long-term complications including grade ⱖ2 rectal complications (bleeding and/or mucous discharge requiring medical treatment) as high as 30% (8). Even these considerable doses appear to be inadequate at eradicating tumor in patients with bulky disease. Thus, it would seem that increasing tumor cell killing by biochemical, rather than technical, means may be a better approach to improve the effectiveness of EBRT as a cancer treatment. Indeed, the gain achieved by biochemical enhancement may be many times greater than that achieved by increasing the prescription radiation dose. With this goal in mind, we have pioneered the concept of using gene therapy as a means to improve the effectiveness of EBRT. We have developed a novel, trimodal approach involving oncolytic viral, double suicide gene, and EBRT (9, 10). Our approach utilizes a modified, replication-competent adenovirus (Ad5-CD/TKrep) to deliver a pair of therapeutic suicide genes to tumors. The Ad5-CD/ TKrep virus itself generates a potent antitumor effect by replicating in and destroying cancer cells. The therapeutic effect of the Ad5-CD/ TKrep virus can be significantly enhanced by invoking two suicide gene systems (CD/5-FC and HSV-1 TK/GCV), which render malignant cells sensitive to specific pharmacological agents and sensitize them to radiation (9 –20). Using a variety of tumor models, we demonstrated previously that Ad5-CD/TKrep viral and double suicide gene therapies markedly increase the effectiveness of EBRT when used in an adjuvant setting (9, 10, 20). These preclinical studies provided the scientific basis for the Phase I study described here in which the safety of arms 1 (Ad5-CD/TKrep viral therapy) and 2 (double suicide gene therapy) of our trimodal approach was evaluated in patients with locally recurrent prostate cancer. The clinical trial described here represents the first in which a replication-competent virus was used to deliver a therapeutic gene to humans. It builds upon and significantly extends previous studies that used replication-defective adenoviruses containing therapeutic genes (21) or more recent trials involving replication-competent adenoviruses lacking therapeutic genes (22–26). As described here, these approaches, overall, have been associated with little toxicity in humans and have shown signs of biological activity when used alone or in combination with conventional cancer therapies. Based on these encouraging clinical data, it would seem that further investigation of such novel approaches is fully warranted. MATERIALS AND METHODS
Study Design. The primary objective of this Phase I study was to determine the DLTs and MTD of intraprostatic administration of the replicationcompetent Ad5-CD/TKrep adenoviral vector concomitant with 5-FC and GCV 4968
prodrug therapy. All patients were treated in the Department of Radiation Oncology at the Henry Ford Hospital. The original study called for three cohorts of 4 patients/cohort to be treated with an escalating dose (1010, 1011, and 1012 vp) of the replication-competent Ad5-CD/TKrep adenovirus on day 1, followed by 1 week (days 3–9) of 5-FC (150 mg/kg/day) and GCV (10 mg/kg/day) prodrug therapy (Table 1). If none of the patients experienced a DLT (defined as any irreversible grade 3 or 4 toxicity), the study proceeded to the next cohort. Secondary objectives included evaluation of antitumor activity as demonstrated by a decrease in serum PSA, histological evidence of tumor destruction in prostate biopsies, and histochemical evidence of therapeutic gene expression. After completion of cohort 3, the study was expanded to include a fourth cohort of four patients who received 1012 vp of Ad5-CD/ TKrep vector followed by 2 weeks (days 3–16) of 5-FC and GCV prodrug therapy (Table 1). Patient Selection. All patients were required to have (a) biopsy-proven local recurrence of prostate cancer at least 1 year after the completion of definitive radiation therapy, (b) evidence of biologically active disease as demonstrated by an unequivocally rising serum PSA level on at least three separate occasions at least 2 weeks apart with a serum PSA below 20 ng/ml, and (c) possess the ability to give informed consent and express a willingness to meet all of the expected requirements of the protocol for the duration of the study. Patients could not have any evidence of metastatic disease, as evaluated by bone scan and computed tomography scan of the abdomen and pelvis. Suspicious abnormal areas on bone scans were evaluated by magnetic resonance imaging. Patients could not have received prior hormonal therapy. Patients were required to have adequate baseline organ function, as assessed by the following laboratory values, before initiating the protocol: (a) adequate renal function with serum creatinine ⱕ 1.5 mg/dl or creatinine clearance ⱖ 45 ml/min/m2; (b) platelet count ⱖ 100,000/mm3; (c) absolute neutrophil count ⱖ 1,000/mm3; (d) hemoglobin ⱖ 8.5 mg/dl; (e) normal partial thromboplastin time and prothrombin time; and (f) bilirubin ⱕ 1.5 mg/dl, and AST and alanine aminotransferase ⬍ 2.5 times the upper limit of normal. Patients with acute infection (any viral, bacterial, or fungal infection that required specific therapy), HIV-positive tests, or a history of liver disease were excluded from the study. Pretreatment Planning and Injection of Ad5-CD/TKrep Adenovirus. Pretreatment planning included TRUS and sextant needle biopsy of the prostate. Transverse images of the prostate were obtained every 0.5 cm from base to apex. Ultrasound images were examined for hypoechoic regions and “areas of suspicion.” Three-dimensional reconstruction of the prostate was performed using treatment planning system. Needle biopsies were obtained from the base, mid, and apex regions of the left and right lobes. Ad5-CD/TKrep vector was injected into those regions thought to contain the majority of cancer based on the combined results of the TRUS and sextant needle biopsy. If a patient’s biopsy showed adenocarcinoma in only one lobe, then only that lobe was
injected. If a patient’s biopsy showed adenocarcinoma in both lobes, then both lobes were injected. This was done to concentrate the virus in the lobe that contained most of the cancer. Injection of the Ad5-CD/TKrep adenovirus was performed on an outpatient basis on day 1. The viral vector was diluted to the proper concentration with sterile saline in a final volume of 2 ml. With patients in the extended lithotomy position, the viral vector was injected through the perineum with TRUS guidance to aid in the placement of the injection needles. The virus was deposited in multiple aliquots (4 – 8 deposits; 0.25– 0.5 ml/deposit) divided from the original dose through two separate injection sites using 20-gauge needles. The injection needles were placed in a pattern to expose as much of the tumor as possible to the adenoviral vector. After each deposit, the needle was withdrawn, the tip was positioned into the next injection area, and the next aliquot of virus was delivered. This was repeated until all of the virus was delivered. Administration of Prodrugs and Patient Monitoring. Patients were admitted to the hospital on day 3 and remained hospitalized during the first week of prodrug therapy. In patients receiving prodrug therapy for 2 weeks (cohort 4), the second week of prodrug therapy was administered on an outpatient basis. 5-FC (Ancobon; Roche Laboratories) was administered p.o. beginning on day 3 and continued for 7 (cohorts 1–3) or 14 (cohort 4) days. A total of 150 mg/kg/day was given in four equally divided doses. GCV (Cytovene; Roche Laboratories) was administered i.v. beginning on day 3 and continued for 7 (cohorts 1–3) or 14 (cohort 4) days. A total of 10 mg/kg/day was given in two equally divided doses over 1 h every 12 h. For each dose, the lyophilized powder (500 mg supplied in a glass vial) was dissolved in 10 ml of sterile water to give a solution of 50 mg/ml. The following evaluations were conducted every day during the viral/ prodrug therapy course (days 1–9): (a) physical exam; (b) serum PSA; (c) presence of infectious adenovirus in serum and urine; and (d) presence of Ad5-CD/TKrep viral DNA in blood. Blood chemistries were monitored on days 3, 5, and 8 during the first week of prodrug therapy (cohorts 1–3), on days 11 and 14 during the second week of prodrug therapy (cohort 4), and at all routine follow-up visits. Toxicities were graded using the National Cancer Institute’s Common Toxicity Criteria. NABs to adenovirus were monitored on days 9 (end of prodrug therapy period), 14, and 30. In selected patients, NABs were monitored more frequently during the viral/prodrug therapy course to correlate development of NABs with PSA response. Because no patient received any hormonal therapy before or during treatment, it was not deemed necessary to monitor serum testosterone levels. Prothrombin time and partial thromboplastin time were monitored throughout. Fibrinogen and fibrin degradation products were not monitored. After treatment, patients received standard urologic care. The following evaluations were performed at 1, 2, 3, 6, and 9 months and 1 year after injection of virus: (a) physical exam; (b) serum PSA; (c) blood chemistries; (d)
Table 2 AEs occurring in two or more patients Cohort Body system No. of patients treated No. of patients with any AE No. of all AEs No. of treatment-related SAEsa No. of grade 1 events No. of grade 2 events No. of grade 3 events No. of grade 4 events Body as a whole
Total Dizziness Fatigue Headache Pain-perineum Pain-back Total Anemia Lymphopenia Monocytosis Neutropenia Thrombocytopenia Total Diarrhea GI discomfort Nausea Total AST increased Total BUN increased CO2 decreased CPK increased Hypercalcemia Hyperglycemia Hypermagnesemia Hypernatremia Hypochloremia Hypomagnesemia Hyponatremia Total Cold symptoms Dyspnea Total Bacteriuria Creatinine increased Hematuria Ketosuria Nocturia Proteinuria
presence of infectious adenovirus in serum and urine; and (e) presence of Ad5-CD/TKrep viral DNA in blood. TRUS of the prostate was performed at 6 weeks; 3, 6, and 9 months; and 1 year. Sextant needle biopsy of the prostate was performed at 2 weeks (cohorts 2– 4), 3 months (cohort 1), and 1 year (all cohorts). Manufacturing of Ad5-CD/TKrep Adenovirus. Clinical grade good manufacturing practice (GMP) Ad5-CD/TKrep adenovirus was manufactured at the Baylor College of Medicine Gene Vector Laboratory (Houston, TX). The virus was supplied as a sterile, clear, frozen liquid in vials containing 1.0 –1.3 ml at three different concentrations ranging from 3.3 ⫻ 1010 to 1.87 ⫻ 1012 vp/ml. The vp:pfu ratio of the undiluted, final product was 13. Just before each patient injection, the viral vector was diluted to the proper concentration with sterile saline in a final volume of 2 ml. The Master Viral Bank was subjected to the following safety tests: (a) in vitro, general sterility, adventitious virus, adeno-associated virus (AAV), cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), human T-cell lymphotrophic virus type I, human T-cell lymphotrophic virus type II, and Parvovirus B19; and (b) in vivo, serial injection into hen eggs via two routes (allantoic, yolk sac) and serial injection into adult and suckling mice. All safety tests were negative. The final vector product was assayed for general sterility, titer (both vp and pfu), endotoxin, identity (DNA sequencing and PCR), transgene expression, and potency testing. All safety tests were negative. All identity and potency tests were com-
parable with the original Ad5-CD/TKrep viral stock. Based on a quantitative PCR assay that can discern between wild-type adenovirus and Ad5-CD/TKrep, the final vector product contained less than 1 vp of wild-type adenovirus per 109 Ad5-CD/TKrep vp. Detection of Ad5-CD/TKrep Viral DNA in Blood. The persistence of Ad5-CD/TKrep viral DNA in patient blood was determined using a PCRSouthern blot assay (27). The PCR assay generates a 427-bp product that is unique to the CD/HSV-1 TK fusion gene contained in Ad5-CD/TKrep. The PCR-Southern blot assay can detect at least 2 vp Ad5-CD/TKrep per ml of human blood. A standard curve was generated by spiking 107 vp of CsCl gradient-purified Ad5-CD/TKrep into 1 ml of heparinized whole human blood and preparing serial, 10-fold dilutions of the stock using whole human blood as diluent. Aliquots (0.5 ml) of each dilution were processed on DNA purification spin columns (Qiagen), and DNA was eluted in 0.2 ml of distilled water. Patient samples were processed in exactly the same manner. Twenty l of each standard dilution and patient sample were added to a 25-l PCR reaction, and 10 l of the reaction products were applied to a 2% agarose gel for Southern blotting. A standard curve representing 2.5 ⫻ 107 to 2.5 vp/ml was run on every gel. The Southern filter was probed with the 32P-labeled 427-bp PCR product. All patient samples were analyzed by PCR for the human ␤-actin gene to assess the integrity of the genomic DNA. The 5⬘ and 3⬘ primers were obtained from R&D Systems (Minneapolis, MN). The PCR conditions for the ␤-actin gene were the same as those used for the CD/HSV-1 TK fusion gene.
Shedding of Infectious Ad5-CD/TKrep Virus in Blood and Urine. Shedding of infectious Ad5-CD/TKrep virus was examined in patient serum and urine using a cytopathic effect assay with A549 (human lung adenocarcinoma) cells. These determinations were performed daily during the treatment period and at routine follow-up visits (1, 2, 3, 6, 9, and 12 months). Three hundred l of serum or urine (neutralized) were added to near confluent A549 monolayers, and cells were monitored daily for cytopathic effect for 12 days. The assay can detect at least 100 infectious particles of Ad5-CD/TKrep per ml of fluid. NABs to Adenovirus. The titer of NABs to adenovirus was determined in serum samples taken before and after treatment using a plaque reduction assay. Serial dilutions of patient serum (heat-inactivated for 30 min at 56°C) were incubated with 200 pfu of the Ad5-CD/TKrep virus for 1 h at 37°C. After incubation, samples were immediately titered on HEK 293 cells. Controls included no serum and serum from an untreated human with a low NAB titer. The dilution that inhibited Ad5-CD/TKrep infectivity by 50% was used as an end point. Detection of CD/HSV-1 TK Transgene Expression in Prostate Biopsies. Expression of the CD/HSV-1 TK transgene was detected in prostate biopsies by indirect immunofluorescence (9). Biopsy cores were frozen, sectioned (10 m), fixed in 3.7% formalin, permeabilized with methanol, and washed with PBS. Samples were reacted with a polyclonal antibody to Escherichia coli CD (supplied by Cynthia Richards; Glaxo Wellcome Laboratories, Research Triangle Park, NC) for 1 h at 37°C. Background immunofluorescence was assessed in duplicate samples leaving out the primary antibody. Samples were washed with PBS and reacted with a goat antirabbit secondary conjugate (Alexa Flour 594; Molecular Probe, Eugene, OR) for 1 h at 37°C. Samples were washed with PBS, stained with 4⬘,6-diamidino-2-phenylindole, and photographed using an Olympus fluorescent BX40 microscope.
RESULTS Baseline Patient Characteristics. A total of 16 patients in four cohorts were treated between January 2000 and December 2001. Baseline patient characteristics are listed in Table 1. The median age of patients was 75 years, with a range of 63–90 years. The preradiation disease stage ranged from T1c to T3c. The median preradiation Gleason score was 7, with a range of 4 –9. The median dose of radiation used in the initial management of these patients was 6600 cGy. The median time between radiation therapy and gene therapy was 74 months (6.3 years), with a range of 21–201 months. The median pre-gene therapy serum PSA level was 8.0 ng/ml, with a range of 3.4 –14.3 ng/ml (Table 3). The most common comorbidities included hypertension (56%), diabetes (38%), and coronary arterial disease (25%). The median follow-up was 15 months. Treatment-related Toxicities. Table 2 summarizes all AEs reported in two or more men. The vast majority (94%) of AEs were mild (82% were grade 1) or moderate (12% were grade 2) in nature. The most frequent AEs, in descending order, were increase in creatine phosphokinase (CPK) (81%), decreased blood CO2 (63%), lymphopenia (56%), hematuria (56%), hyperglycemia (50%), anemia (44%), thrombocytopenia (38%), cold symptoms (38%), diarrhea (31%), hypermagnesemia (31%), pain at the injection site (25%), and elevation in AST (25%). Many of the AEs were expected and could be attributed to the injection procedure (elevation in CPK, pain at injection site, hematuria, and bacteriuria), possible dissemination of the adenoviral vector to collateral tissues (cold symptoms, fatigue, and elevation in AST), immune response to the adenoviral vector (monocytosis), and the 5-FC ⫹ GCV prodrug therapy (anemia, lymphopenia, neutropenia, thrombocytopenia, diarrhea, GI discomfort, and nausea). All patients exhibiting hyperglycemia and ketosuria were diabetic. The causes for the decrease in blood CO2 and hypermagnesemia are unknown. All hematological events occurred during administration of the
5-FC ⫹ GCV prodrugs and resolved shortly after completion of the prodrug therapy course. Only one event required medical intervention. In patient 12 (1012 vp, 1 week of prodrug therapy), platelet levels dropped to a low of 94,000/l on day 4. Both the 5-FC and GCV prodrugs were reduced to 75% of their original dose level until platelet levels exceeded 100,000/l, which occurred on day 6 (107,000/l). Prodrugs were returned to their original dose level on day 7. Platelet levels continued to increase thereafter and were within normal range (⬎150,000/l) upon completion of the prodrug therapy course (day 9). Four patients exhibited a transient, grade 1 elevation in AST that occurred on day 3. This condition resolved by day 5 in three patients. In one patient (patient 12, 1012 vp, 1 week of prodrug therapy), AST levels remained elevated until day 8 and were accompanied by a grade 1 elevation in gamma-glutamyltransferase (GGT) and mild pain in the upper right quadrant of the abdomen. An ultrasound showed mild coarsening of the hepatic echotexture that may have been related to hepatocellular disease or fatty infiltration. All other liver function tests (alanine aminotransferase, bilirubin, alkaline phosphatase, and albumin) in all patients were within normal limits. Patients were monitored carefully for the following urinary symptoms: bacteriuria; dysuria; hematuria; nocturia; urinary incontinence; urinary retention; urinary frequency; and pain. Although some patients experienced hematuria (n ⫽ 9), bacteriuria (n ⫽ 3), and nocturia (n ⫽ 2; Table 2), there was only one event of urinary incontinence, and there were no events of urinary retention, urinary frequency, or pain. No patient required a Foley catheter. There were no treatment-related serious AEs. There were nine grade 3 events (6%) including four events of hyperglycemia and one event each of cardiac ischemia, dyspnea, hypermagnesemia, lymphopenia, and neutropenia. All patients exhibiting grade 3 hyperglycemia were diabetics. The grade 3 events of cardiac ischemia (occurred in a patient with a history of heart disease), hypermagnesemia, lymphopenia, and neutropenia were transient (lasting 1 day) and required no medical intervention. The one event of grade 3 dyspnea occurred on day 12 in a patient with a history of chronic obstructive pulmonary disease. His condition improved after oral administration of Serevent and albuterol. In a routine follow-up visit (day 14), the patient was found to be slightly febrile (99.7°C) and exhibited grade 1 tachycardia and cough. A chest X-ray revealed no acute thoracic processes and was unchanged from pretreatment evaluations. The patient was treated with antibiotics, and the condition resolved 3 days later. The Cochran-Armitage test was used to determine a possible correlation between the frequency and/or severity of any AE and Ad5-
CD/TKrep viral dose level and the duration of prodrug administration. Spearman correlation coefficients were calculated. There was no significant correlation between Ad5-CD/TKrep viral dose and the incidence or severity of any AE. Likewise, there was no significant
correlation between the duration of prodrug administration and the incidence or severity of any AE, although there was a 58% correlation coefficient between duration of prodrug administration and grade 3 lymphopenia (P ⫽ 0.21).
Fig. 1. Serum PSA and presence of Ad5-CD/TKrep viral DNA in blood. A, patient 1 representing cohort 1; B, patient 5 representing cohort 2; C, patient 12 representing cohort 3; D, patient 14 representing cohort 4; E, patient 11 representing a nonresponder from cohort 3. The dashed line represents the upper limit of normal serum PSA (4 ng/ml). The intense band on the left side of each Ad5-CD/TKrep PCR assay is a positive control and represents 1 ⫻ 105 vp/ml. PCR for ␤-actin was included to evaluate DNA integrity. The solid black bar indicates the viral/prodrug period. The hatched bar indicates the period in which Ad5-CD/TKrep viral DNA was detected in blood, but prodrugs were not being administered. The day at which Ad5-CD/TKrep viral DNA was no longer detected in the patient’s blood is indicated.
Table 4 Comparison of rate of PSA decrease before and after prodrug therapy Rate of PSA decrease (ng/ml/day)
During prodrug therapya
After prodrug therapyb
1 5 6 7 10 12 14
0.34 0.65 0.00 0.31 0.40 0.59 0.21
0.01 0.01 0.03 0.08 0.01 0.03 0.03
26 85 0 4 40 21 6
a The rate of PSA decrease during prodrug therapy was calculated by subtracting the PSA level at the end of the prodrug therapy course (day 9 for cohorts 1–3, day 16 for cohort 4) from the pretreatment PSA and dividing by the number of days. b The rate of PSA decrease after prodrug therapy was calculated by subtracting the PSA level at the nadir from the PSA level at day 9 (cohorts 1–3) or day 16 (cohort 4) and dividing by the number of days.
Post-Gene Therapy PSA Kinetics and Presence of Ad5-CD/ TKrep Viral DNA. Table 3 summarizes the changes in serum PSA after treatment. Seven of 16 (44%) patients exhibited a reduction of serum PSA of ⱖ25% from pretreatment levels. Three of 16 patients (19%) achieved a partial response as defined by a reduction in serum PSA of ⱖ50% for at least 4 weeks. These partial responses occurred in patients 1 (1010 vp, 1 week of prodrug therapy), 12 (1012 vp, 1 week of prodrug therapy), and 14 (1012 vp, 2 weeks of prodrug therapy). The maximum duration of the response was 4 months. Posttreatment PSA kinetics of four responders (patients 1, 5, 12, and 14; Fig. 1, A–D) representing each of the four cohorts and one nonresponder (patient 11, Fig. 1E) are shown in Fig. 1. The presence of Ad5-CD/TKrep viral DNA in each patient’s blood is also shown for comparison. In six of seven patients that responded to treatment as defined by a ⱖ25% drop in PSA, PSA levels decreased rapidly during the Ad5-CD/TKrep viral/prodrug therapy course, with 60 –100% of the drop occurring during this period (Fig. 1, A–D). In a minority of patients (e.g., patient 5, Fig. 1B), a transient rise in PSA was observed shortly after injection of the virus and was probably attributable to prostatic manipulation. Upon completion of the prodrug therapy course (day 9, cohorts 1–3; day 16, cohort 4), serum PSA either stabilized abruptly (e.g., patient 5, Fig. 1B) or continued to drop, but at a much reduced rate (patients 1, 12, and 14; Fig. 1, A, C, and D). In the seven “responders,” the rate of PSA decrease was, on average, 26 times greater during the viral/prodrug therapy period than it was
during the following period in which Ad5-CD/TKrep viral DNA was detected in patient’s blood but prodrugs were not being administered (Table 4). After injection, Ad5-CD/TKrep viral DNA was detected in the blood of all patients of cohorts 2– 4 (Figs. 1, B–E) but was undetectable in patients of cohort 1 (Fig. 1A). In cohorts 2– 4, there appeared to be a trend between PSA response and the persistence of high levels of Ad5-CD/TKrep viral DNA throughout the prodrug therapy course. In patients that exhibited ⬍25% drop in PSA, Ad5-CD/TKrep viral DNA peaked between days 2 and 4, declined rapidly thereafter, and was undetectable by the end of the prodrug therapy course (e.g., patient 11, Fig. 1E). In patients that exhibited a 25–50% drop in PSA, Ad5-CD/TKrep viral DNA levels peaked between days 2 and 4 and began declining shortly after initiation of prodrug therapy. However, in all of these patients, Ad5-CD/TKrep viral DNA persisted throughout and beyond the prodrug therapy course. In some patients, Ad5CD/TKrep viral DNA persisted at a reduced level relative to the peak (patient 5, Fig. 1B), whereas in others, a second peak of viral DNA was observed (data not shown). In patients that showed a ⱖ50% drop in PSA and in whom viral DNA could be detected in blood (patients 12 and 14, Fig. 1, C and D), Ad5-CD/TKrep viral DNA persisted at a high steady state throughout and beyond the entire prodrug therapy course. In these patients, Ad5-CD/TKrep viral DNA remained at peak levels for at least 16 (patient 12, Fig. 1C) and 37 days (patient 14, Fig. 1D). In all responders in whom viral DNA could be detected in blood, treatment failure (rising PSA) correlated with the absence of Ad5CD/TKrep viral DNA (Fig. 1, B–D). Ad5-CD/TKrep viral DNA was detected in the blood of patients as far out as day 76. Post-Gene Therapy Prostate Biopsies. Posttreatment sextant needle biopsies were obtained at 3 months (cohort 1), 2 weeks (cohorts 2– 4), and 1 year (all cohorts) for assessment of therapeutic gene expression and tumor destruction. In 4 of 12 patients biopsied at 2 weeks, expression of the CD/HSV-1 TK transgene was detected by indirect immunofluorescence (Fig. 2). In all cases, transgene expression was confined to the sextant of the injection site and was observed in ⬍100 cells. Importantly, several posttreatment prostate biopsies showed clear evidence of tumor destruction as demonstrated by extensive coagulative necrosis (Fig. 3). The posttreatment prostate biopsies of two patients were negative for adenocarcinoma at 1 year.
Fig. 2. Detection of transgene expression in 2 week prostate biopsy. The results shown were obtained from patient 7, who was injected in the left base and left midregions only. LB, left base; RB, right base. Positive cells are red, and nuclei are blue. Duplicate samples leaving out the primary antibody gave a low level of background fluorescence. Similar results were observed in three other patients.
Fig. 3. Histopathological evidence of tumor destruction in 2 week prostate biopsy. The results shown were obtained from patient 12. Both pre- and posttreatment biopsies shown were obtained from the left base region, which is where the Ad5-CD/TKrep vector was injected. The pretreatment biopsy shows fused prostatic acini with a Gleason score of 8. The posttreatment biopsy shows extensive tumor destruction and coagulative necrosis.
NABs to Adenovirus. The titer of NABs to adenovirus was determined before and after treatment using a plaque reduction assay. Fifty percent of patients had preexisting NABs (titer ⱖ 1:20) to adenovirus before treatment (Table 5). All patients exhibited an increase in NAB titer after treatment, ranging from 4- to 1700-fold (Table 5). There was no correlation between baseline NAB titer and PSA response. The development of NABs to adenovirus was measured frequently in two of the best responders (patients 12 and 14) during the period in which they exhibited a rapid drop in PSA. These patients exhibited a 750and 1700-fold increase in NAB titer by day 14, which coincided with a 40% drop in PSA (data not shown). Shedding of Infectious Ad5-CD/TKrep in the Blood and Urine. Shedding of infectious Ad5-CD/TKrep virus in the blood and urine was examined daily after injection (see “Materials and Methods”). Although Ad5-CD/TKrep viral DNA was detected in the blood of patients as far out as day 76, no infectious adenovirus was detected in the blood or urine of any patient at any time point. DISCUSSION This Phase I study represents the first Food and Drug Administration-approved gene therapy trial in which a replicationTable 5 NAB titers to adenovirus before and after treatment NAB titer
competent virus was used to deliver a therapeutic gene to humans. The primary objective was to determine the DLTs and MTD of intraprostatic administration of the replication-competent Ad5-CD/ TKrep adenoviral vector concomitant with 5-FC and GCV prodrug therapy. Escalation to 1012 vp and 2 weeks of 5-FC ⫹ GCV prodrug therapy resulted in no DLTs, and the MTD of the Ad5CD/TKrep adenoviral vector was not defined. Remarkably, 94% of the AEs observed were mild/moderate (grade 1/2) in nature, and only one (grade 1 thrombocytopenia) required modification of the treatment plan. Of the remaining 6%, all were grade 3 and selflimiting, and most were attributable to comorbid conditions. Many AEs could be attributed to the injection procedure or possible dissemination of the adenoviral vector beyond the prostate gland or are known side effects of the 5-FC and GCV prodrugs. The results demonstrate that intraprostatic administration of an attenuated, replication-competent adenovirus containing two therapeutic suicide genes concomitant with 2 weeks of 5-FC and GCV prodrug therapy can be applied safely to humans. One of the major concerns of all gene therapy trials, particularly suicide gene therapy, is dissemination of the recombinant vector to vital organs such as the liver. The immune response (both innate and specific) to the adenoviral vector and cytotoxic effects of the CD/5-FC and HSV-1 TK/GCV suicide gene systems can result in severe hepatotoxicity and death (28, 29). Remarkably, only 4 of 16 patients (25%) exhibited any evidence of hepatotoxicity. All events were transient, grade 1 elevations in AST that occurred on day 3 and resolved by days 5 (n ⫽ 3) and 8 (n ⫽ 1). Although Ad5-CD/TKrep viral DNA was detected in the blood of all patients receiving ⱖ1011 vp, no infectious adenovirus was detected in the blood or urine of any patient at any time point (limit of detection, ⬃100 vp/ml fluid). The lack of infectious Ad5-CD/TKrep virus in blood may indicate that (a) with the procedures and viral dose levels used here, significant amounts of the virus do not disseminate beyond the prostate gland or (b) the Ad5-CD/TKrep virus does not replicate efficiently in the human prostate. We consider the latter possibility somewhat unlikely, given that the Ad5-CD/TKrep virus replicates very efficiently in human DU145 and LNCaP adenocarcinoma cells in vitro (9, 20), and we have strong evidence that the Ad5-CD/TKrep virus replicates in
the dog prostate in vivo.4 In a previous study, infectious adenovirus was detected in the urine of 11 of 19 patients 2 days after intraprostatic injection of the replication-competent CV706 adenovirus (26). However, it should be noted that both the injection procedure (up to 40 needles and 80 deposits) and vector dose level (up to 1013 vp) differed significantly from those used here. The presence of Ad5-CD/ TKrep viral DNA, but not infectious virus, in the blood may reflect the release of inactive vp or unpackaged viral DNA from damaged prostate tissue. A secondary study objective was to examine whether the Ad5-CD/ TKrep viral and double suicide gene therapy combination showed any signs of biological activity. Two standard end points were used: (a) serum PSA, which is a widely accepted surrogate marker for disease burden and activity; and (b) histological evidence of tumor destruction in prostate needle biopsies (30 –34). Forty-four percent of patients exhibited a ⱖ25% reduction in serum PSA from pretreatment levels, and 19% exhibited a ⱖ50% reduction in serum PSA from pretreatment levels. These results are encouraging, given the fact that the PSA level of all patients was rising at the time of treatment. Thus, even minor reductions (ⱕ25%) or stabilization of serum PSA may be indicative of biological activity. That most of the PSA responses were short-lived indicates that additional improvements or therapies (e.g., EBRT) will be needed for this approach to have value in the clinic. As was observed by others (26), it is likely that more durable responses will be observed with higher viral dose levels (beyond 1012 vp) and by depositing the Ad5-CD/TKrep viral vector throughout the entire prostate gland. Indeed, the latter approach makes much sense, given the multifocal nature of prostate cancer. Importantly, histological evidence of tumor destruction was confirmed by needle biopsy in some patients. The observation that the prostate biopsy of patient 1 was negative for adenocarcinoma at 1 year follow-up and was accompanied by a sustained 37% drop in PSA (nadir of 53%) raises the possibility that tumor may have been eradicated from the prostate gland of this patient. Such results must be interpreted with caution, for it is well-documented that the false negative incidence of sextant prostate biopsies is high (35–37). Nevertheless, taken together, the results indicate that the combination of Ad5-CD/TKrep viral and double suicide gene therapies is showing signs of biological activity and warrants further investigation. It six of the seven patients that responded to therapy as defined by a ⱖ25% decrease in serum PSA, the rate of PSA decrease was greatest during the viral/prodrug therapy period. Upon completion of the prodrug therapy course, PSA levels either stabilized abruptly or continued to drop (but at a much reduced rate) as long as Ad5-CD/TKrep viral DNA was detected in blood. Patient 1 showed a good PSA response during and after treatment, even though no Ad5-CD/TKrep viral DNA was detected in blood. This patient had a well-defined tumor that was clearly recognizable by TRUS, which facilitated accurate placement of the Ad5-CD/TKrep viral vector. Interestingly, in cohorts 2– 4, there was an excellent correlation between the absence of Ad5-CD/TKrep viral DNA in blood and biochemical failure (rising PSA). The observation that the rate of PSA decrease changed dramatically upon completion of the 5-FC and GCV prodrug therapy course may indicate that much of the observed activity is attributable to the destructive effects of the CD/5-FC and HSV-1 TK/GCV suicide gene systems. However, in several patients, the change in PSA kinetics was also accompanied (albeit not perfectly) by reduced levels of Ad5-CD/ TKrep viral DNA in blood. If we assume that the presence of Ad5-CD/TKrep viral DNA in blood reflects the persistence of virus in the prostate, then the observed change in PSA kinetics might also
reflect the elimination of active virus from the prostate gland by the immune system. Only in patient 14 (and possibly patient 12) did Ad5-CD/TKrep viral DNA in blood remain at peak levels throughout and well beyond the prodrug therapy course. In this patient, the rate of PSA decrease was 6-fold greater during the viral/prodrug therapy period than after its completion, even though Ad5-CD/TKrep viral DNA persisted in the blood at high levels up to day 64. This anecdotal evidence would seem to support the thesis that much of the observed activity is due to the destructive effects of the CD/5-FC and HSV-1 TK/GCV gene systems. Although not addressed here, it is also likely that the immune response to the adenoviral vector and suicide gene therapy is contributing significantly to the observed biological activity (38, 39). Indeed, it is very difficult to discern among these possibilities in small human trials such as that described here. Regardless of which thesis is correct, the data are consistent with the notion that both the suicide gene systems and lytic activity of the replication-competent Ad5-CD/TKrep virus are contributing to the observed biological activity. Fifty percent of patients had preexisting NABs (titer ⱖ 1:20) to adenovirus before treatment, and all patients exhibited an increase in NAB titer posttreatment. To ascertain whether the development of high NAB titers to adenovirus impacted biological activity after a single intraprostatic injection, NAB titers were examined in two of the best responders (patients 12 and 14) during the period in which they exhibited marked drops in serum PSA. Both patients exhibited dramatic drops in PSA (40% by day 14), despite the fact that the NAB titer increased 750- and 1700-fold over the same time period. Our results are consistent with other studies that failed to find a correlation between baseline NAB titer or the development of high NAB titer and biological response after a single administration of vector (21–26). However, it is possible and likely that high NAB titers may dampen or even preclude the biological activity of repeated vector administration. The Ad5-CD/TKrep viral and double suicide gene therapy combination was not designed to be used as a primary cancer treatment. Although we are very encouraged by the results of this study, we believe the greatest potential of this multimodal approach stems from the fact that both oncolytic viral and suicide gene therapies have been demonstrated to be effective adjuvants to EBRT in preclinical models. Using a variety of tumor models, we (10, 40) and others (41) have demonstrated that the interaction between oncolytic viral therapy and EBRT is at least additive and that the interaction between the CD and HSV-1 TK suicide gene systems and EBRT is synergistic (i.e., radiosensitization; Refs. 9 –20). For example, using a C33A human tumor xenograft model, the addition of Ad5-CD/TKrep viral and double suicide gene therapies with EBRT (trimodal therapy) markedly improved tumor control and cure relative to EBRT alone (10). Similar results have been obtained with i.m. DU145 and intraprostatic LNCaP C4-2 tumor xenograft models (20). Given that suicide gene radiosensitization has been observed in vivo at prodrug doses below those required for their chemotherapeutic effect (10), we believe that even greater biological activity will be observed when the Ad5-CD/TKrep viral and double suicide gene therapy combination is used in an adjuvant setting with EBRT. Modifications in the parental Ad5-CD/ TKrep vector that increase the catalytic efficiency of the CD/HSV-1 TK fusion gene and prolong the duration of therapeutic gene expression in vivo should further enhance the effectiveness of this multifaceted approach to a point where it may be a safe and effective adjuvant to EBRT in the clinic. ACKNOWLEDGMENTS
K. Barton, D. Tyson, H. Stricker, Y. Lew, G. Heisey, S. Koul, S. Jhiang, J. H. Kim, S. O. Freytag, and S. L. Brown, unpublished results.
We thank Sweaty Koul for performing the viral potency assays.
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Phase I Study of Replication-competent Adenovirus-mediated Double Suicide Gene Therapy for the Treatment of Locally Recurrent Prostate Cancer Svend O. Freytag, Mark Khil, Hans Stricker, et al. Cancer Res 2002;62:4968-4976.
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