Although genetically engineered adenoviruses hold promise for the treatment of cancer, clinical trial reports have utilized intratumoral injection to date. To determine the feasibility of intravenous delivery of ONYX-015, an E1B-55kD gene-deleted replication selective adenovirus with demonstrated clinical safety and antitumoral activity following intratumoral injection, we performed a clinical trial in patients with metastatic solid tumors. ONYX-015 was infused intravenously at escalating doses of 2 × 1010 to 2 × 1013 particles via weekly infusion within 21-day cycles in 10 patients with advanced carcinoma metastatic to the lung. No dose-limiting toxicity was identified. Mild to moderate fever, rigors and a dose-dependent transient transaminitis were the most common adverse events. Neutralizing antibody titers significantly increased within 3 weeks in all patients. IL-6, γ-IFN, TNF-α and IL-10 increased within 24 h following treatment. Evidence of viral replication was detectable in three of four patients receiving ONYX-015 at doses ⩾2 × 1012 particles and intratumoral replication was confirmed in one patient. In conclusion, intravenous infusion of ONYX-015 was well tolerated at doses up to 2 × 1013 particles and infection of metastatic pulmonary sites with subsequent intratumoral viral replication was seen. The intravenous administration of genetically altered adenovirus is a feasible approach.
Despite the successes of cytotoxic chemotherapeutics, new anticancer agents with improved safety and efficacy are still needed. Adenoviruses were initially engineered as replication-incompetent vectors whose only role was to deliver exogenous therapeutic genes.1,2 A contrasting strategy has now emerged whereby replication-selective viruses, including adenoviruses, are themselves the therapeutic agent.3,4,5,6 Proof of principle for this concept comes from animal studies where such viruses are found to replicate directly in cancer cells and destroy them, without the need for exogenous genes.3,4,5,6 Eventually, superior efficacy may be achieved by combining these two strategies and arming replication-selective viruses with therapeutic transgenes.7 However, to date, published clinical trials have been limited to intratumoral or intracavitary injections, and no data are as yet available on the safety or feasibility of intravenous adenovirus delivery to distant tumor metastases. Indeed, the recent death of a patient being treated with an adenoviral delivery vector for ornithine transcarbamylase deficiency, has raised concerns about the feasibility and safety of intravenous adenovirus administration.8 Here we detail, for the first time, the initial results of a dose-escalation trial using the intravenous delivery of a replication-selective adenovirus in the treatment of cancer and showing it to be both well tolerated and feasible.
ONYX-015 is a replication-selective adenovirus with a deletion in the E1B-55kD gene region. Since the 55kD protein is necessary for inhibition of wild-type p53 function, as are similar proteins of other DNA viruses,9,10 deletion of this gene should prevent efficient viral replication in cells with normal p53 function,11 but allow replication in tumor cells harboring a p53 gene mutation or otherwise lacking functional p53. The p53 protein functions as part of a DNA repair mechanism in addition to its growth arrest and apoptosis induction functions, so that its loss plays a major role in tumorigenesis.12 P53 also serves to protect the normal cell from viral replication if the cells should become infected by any of a number of viruses.9,10,11 Sixty percent of human solid tumors contain mutations of the p53 gene, and many of the tumor cells with wild-type p53 gene sequences will have other abnormalities at the protein level.13 Preclinical data, in general, confirm the modulation of ONYX-015 replication and oncolysis in tumor cell lines, although results have varied based on the cell system and endpoints studied.11,14,15,16,17,18 Selection in clinical trials has been defined.
Phase I and II investigations in refractory head and neck cancer patients treated by intratumoral injection of ONYX-015 further support the selective nature of ONYX-015 replication in p53 mutant tumor tissue (58% necrosis rate for p53 mutant tumors versus 0% in tumors with normal p53 genome; P = 0.017) (Ganly et al, unpublished data). Adverse effects in normal tissue were not demonstrated despite injection of normal peritumoral tissue. A potentially synergistic interaction between chemotherapy and replication-selective adenovirus has also been discovered.13,17,19,20 Chemotherapy-naive head and neck cancer patients treated with combination cisplatin, 5-fluorouracil (5-FU) and ONYX-015 had a response rate of 63% and minimal toxicity.20 This compares favorably with the historical response rate of 35% for a similar population of patients. In light of these results, a phase III randomized trial is currently underway.
Intratumoral injection therapy, however, is limited to needle-accessible disease sites, whereas intravenous administration allows the potential for viral infection of even non-injectable disease sites. Intravenous administration of ONYX-015 to athymic mice bearing subcutaneous tumor xenografts supported the feasibility of this approach in mice.18 Approximately 90% of ONYX-015 was recovered from the liver 3–6 h after a single infusion of 2 × 1010 particles; the liver had over 250 times more infectious virus particles per gram of tissue than tumor masses in the same mice. However, following tumor-selective replication for 3 days, no infectious virus was recovered from liver while titers had increased 100-fold in tumor tissue. In situ hybridization of the tumor tissue also showed focal areas of ONYX-015 replication within tumor margins and adjacent to feeding blood vessels. These results, therefore, documented the feasibility of adenoviral delivery to distant tumor masses following intravenous administration and established an LD50 in mice of ∼2 × 1010 particles.18 This dose is equivalent to 2 × 1013 particles in humans based on allometric scaling on a particle/g comparison of mice and human livers. Significantly, no additional toxicity was observed in animal models when i.v. ONYX-015 was combined with chemotherapy (5-FU, cisplatin, taxol).13,19
Although these preclinical mouse model results are encouraging, several problems exist with this model for use with replication-selective adenoviruses. First, normal murine tissues are not fully permissive for adenoviral replication, and therefore the rapid clearance of ONYX-015 from mouse liver may not occur in humans. Second, T cell-dependent immune responses are absent in athymic mice; the role of T cell-dependent humoral, cellular and cytokine-mediated immunity is therefore unclear. The intact immune response that adenovirus will encounter in patients may lead to more rapid clearance and/or inflammation-related toxicities. Finally, safety concerns have been raised following fatal hematologic and hepatic complications in a patient receiving an adenoviral gene delivery vector (replication-incompetent) via the bloodstream.8
To address these issues, we initiated a phase I dose-escalation trial to determine safety, pharmacokinetics, and feasibility of tumor uptake and replication of ONYX-015 administered intravenously alone, as well as in combination with carboplatin and paclitaxel, in patients with end-stage refractory carcinoma metastatic to the lung.
The age, sex, Karnofsky performance status, site of disease, histologic tumor type, prior treatment and p53 mutation status of the 10 treated patients are shown in Table 1. The 10 patients described in this article received 126 intravenous infusions (42 cycles) of ONYX-015 (Table 2). Nine patients were entered into protocol, and one patient (C) was treated in nearly identical fashion on a compassionate basis following FDA and IRB approval (the patient had previously participated in a trial in which intratumoral injection of ONYX-015 virus at a dose of 2 × 1011 particles was administered for an initial 5 consecutive days of a 28-day cycle).20 All patients were treated at the Mary Crowley Medical Research Center, Sammons Cancer Center, Baylor University (Dallas, TX, USA) from 11 January 1999 to 13 December 1999.
Three patients had grade 3 toxic events requiring hospitalization. All were classified as serious adverse events. None were related to the study agent. Grade 3 and 4 toxicity identified in patient C was related to tumor progression (dehydration and left carotid artery body compression related to tumor mass effect induced periodic hypotensive episodes). Grade 3 toxicity in patient 0003 was due to gram-negative sepsis from an infected indwelling catheter, and grade 3 toxicity in patient 0009 was due to a gram-positive intravenous central catheter infection. Neither of the bacterial infection events were related to study medication and completely resolved following antibiotics. Grades 2–4 clinical adverse events and relationship to study drug are shown in Table 3. Additional grade 3 toxic events not requiring hospitalization and not related to study medication occurred in patients 0004 and 0008 (Table 3).
The most common toxicities related to the administration of ONYX-015 were fever and rigors. All patients developed fever during either cycle 1 or 2 of ONYX-015. Fever most commonly developed within 4–8 h after infusion of ONYX-015 in cycle 1. Patients who received ⩾2 × 1012 particles of ONYX-015 (n = 5) also developed transient mild elevations (1.5–4.0 times the upper limit of normal) in AST and/or ALT during cycle 1. Patient 0007, who received 2 × 1013 particles, had transient ALT elevation approximately four times the upper limit of normal in cycle 1. Patients receiving less than 2 × 1012 particles in cycle 1 did not show any elevation of ALT or AST related to study agent, although a nonspecific elevation correlating with progression of malignancy within the liver was observed (n = 2 patients). There was no evidence of progressive clinical hepatotoxicity in any patient. Similarly, a transient elevation in circulating lymphocytes was observed in nine of 10 patients during cycles 1 and 2, but returned to baseline with continued ONYX-015 infusion (data not shown).
Evidence of viral genome could be detected for a minimum of 90 min after the end of ONYX-015 infusion in nine of nine patients at the initiation of the first and second cycle (ie fourth infusion) of therapy. One of four patients who received ONYX-015 at ⩽6 × 1011 particles had detectable viral genome at 6 h after infusion (patient 0001, 2.11 × 105 particles/ml) in cycle 1, whereas all five patients receiving ⩾2 × 1012 particles had minimally detectable ONYX-015 at 6 h after the end of infusion (patient 0005, 17.3 × 105; patient 0006, 19.9 × 105; patient 0007, 14.7 × 105; patient 0008, 2.2 × 105; patient 0009, 2.5 × 105 particles/ml). All patients, except 0001, had detectable ONYX-015 genome at 6 h after the end of ONYX-015 infusion at initiation of cycle 2.
Plasma titers of viral DNA were independent of dose over the initial dose range of 2 × 1010 to 6 × 1011 particles. At 2 × 1012 particles and above, there appeared to be a greater plasma PCR signal, but it was not statistically significant (Figure 1). DNA titers declined with a half-life of about 20 min over the first 2 h following dosing (Figure 2), then did not change as a function of time from 2 to 6 h after treatment. Higher doses yielded higher levels within this time-window. There were no observable differences between cycle 1 and cycle 2 in dose or time dependencies or in individual systemic exposure to the ONYX-015 genome following equivalent single doses.
Evidence for viral replication
Detection of ONYX-015 genome in plasma by quantitative PCR on day 7 after the end of infusion is summarized in Table 4. Day 7 plasma sample positivity was more common at doses ⩾2 × 1012 (13/27, 48% versus 7/21, 33%) particles. Furthermore, persistent viral genome at day 2 and day 7 after a virus infusion was shown in 18 of 34 (53%) patient samples receiving ⩾2 × 1012 particles compared with seven of 24 (29%) patient samples at the dose of ⩽6 × 1011 particles. Overall, 46% (17 of 37) of all plasma samples had detectable ONYX-015 genome in cycle 1, and 38% (eight of 21) of plasma samples were positive in cycle 2.
In order to confirm that replication was occurring at doses ⩾2 × 1012 particles, viral genome concentration from blood drawn 48 h after infusion of virus was compared to the 6-h viral genome blood concentration. Three of four patients with samples drawn had an increase in viral concentration (2.5 to 10-fold) between 6 and 48 h (one patient missed the blood draw). Viral genome increased from 4.7 × 106 to 8.6 × 106 genomes/ml in patient 0007, from 2.2 × 105 to 8.9 × 106 genomes/ml in patient 0008, and from 2.5 × 105 to 1.7 × 106 genomes/ml in patient 0009.
Once viral replication had been documented, attempts were made to confirm that viral replication was occurring within the tumor tissue as predicted. Because tumor tissue was non-evaluable in most cases (due to necrosis and/or inaccessibility), only one tumor biopsy sample was evaluable for immunohistochemical staining for replication and/or for ONYX-015-specific PCR (to confirm the virus identity). Immunohistochemical staining of adenoviral capsid antigen confirmed intratumoral replication (Figure 3), and quantitative PCR testing (Figure 4) confirmed that this was ONYX-015 (ie not wild-type adenovirus) at a concentration of 5.91 × 104 ONYX-015 genomes/μg of DNA analyzed. This patient (0008) had multiple positive day 7 plasma PCR samples and was one of the three patients in whom quantitative PCR indicated increasing plasma genome levels at 48 h and day 7 in comparison to the 6-h plasma genome level. Some capsid antigen expression can also be identified in adjacent bronchial alveolar cells; this appears to have been due to phagocytosis of adenoviral protein by adjacent pneumocytes. Surrounding pulmonary tissue did not show immunohistochemical evidence of adenovirus replication or histologic evidence of toxicity. Patient C underwent autopsy analysis 134 days following his last ONYX-015 infusion. No evidence of ONYX-015 replication was identified in normal organ tissues or malignant tissue by quantitative PCR.
The role of neutralizing antibodies in blocking infection could not be assessed. All high-dose patients were antibody negative at baseline.
Neutralizing antibody response
Two patients (Nos 0003 and C) had elevated neutralizing antibody titers at baseline (Table 5). Patient C had previously received ONYX-015 via intratumoral injection. All patients showed marked elevation in neutralizing antibody titers within 2–3 weeks of the first injection (Table 5).
Cytokines IL-6, IL-1α, IL-1β, TNF-α, γ-IFN, IL-10 and IL-2 were assessed in patients who received ONYX-015 at doses ⩾2 × 1012 particles (patients 0005–0009) (Figure 5a, b). Significant elevation (>25% of baseline value) of IL-6, IL-10, TNF-α, and γ-IFN, were observed in the majority of patients tested after infusion on days 1 to 3 of cycle 1 and days 1 to 3 of cycle 2. Patient 0006 was unavailable for cycle 2 due to inability to acquire the follow-up samples after cycle 2 baseline assessment. We did not detect any significant increase in IL-1α, IL-1β or IL-2 levels in patient serum collected throughout the course of study. However, an early rise with subsequent drop back to baseline within the first 24 h could have been missed; this pattern of rise and fall was demonstrated for IL-1 following intra-arterial administration of ONYX-015 on another trial (Kirn et al, unpublished data). Elevated levels of serum IL-6 from 128% to 834% over baseline (from 5.4 to 50.6 pg/ml; No. 0006) were observed in five of five patients within 24 h after infusion at day 1, cycle 1 and probed within 6 h of infusion. A similar increase of 28% to 586% was seen in three of four patients (0005, 0007, 0009) ⩽24 h at cycle 2. This increase subsided by days 3–4 and returned to baseline before the next infusion of ONYX-015, 7 days later in all patients. Concurrent or delayed increase in serum IL-10 (five of five patients tested; 70–6259% increase) and γ-IFN (four of five patients; 865–3321% increase) were observed, peaking at days 2 (γ-IFN) and 3–4 (IL-10) of cycle 1. Markedly higher increases of IL-6 and IL-10 but not γ-IFN were seen at cycle 2. There were relatively modest increases (26–170%; 22–42 pg/ml) of TNF-α in four of five patients at days 2–5 of treatment in cycle 1. Only one patient (0007) was found to increase TNF-α at cycle 2. Impressive increases were observed at the 6-h time-point only with IL-6 suggesting a possible correlation of IL-6 with initial fever commonly observed 4–8 h after administration of cycles 1 and 2. All but one instance was correlated with a >25% increase of IL-6. Patients 0005, 0006, 0007, 0008 and 0009 all had transient fever ⩽10 h from initial infusion of ONYX-015.
Tumor response is shown in Table 6. Eight patients had stable disease at the 2-month assessment, one a mixed response, and one progressive disease. Time to progression and survival is also shown in Table 6. Although no objective responses were demonstrated with ONYX-015 alone in this safety study, tumor stabilization of 6.5+ months was demonstrated in two patients with ONYX-015 and carboplatin/paclitaxel. One (No. 0005) of the two patients had previously failed prior carboplatin/taxol treatment.
We demonstrate, for the first time, that an attenuated adenovirus can be administered safely as a single intravenous infusion to a dose of 2 × 1013 particles and as weekly infusions at doses up to 2 × 1012 particles. No dose-limiting toxicities were identified; mild to moderate fever, rigors and mild transient transaminitis (following high doses only) were the most common toxicities. This lack of significant toxicity is remarkable given evidence for ongoing viral replication and shedding into the circulation for several weeks in high-dose patients. Toxicity was not altered when ONYX-015 was administered in combination with carboplatin and taxol. These results suggest that future therapeutic designs can safely incorporate administration of intravenous ONYX-015 in humans. Importantly, viral infection of metastatic tumor deposits was documented at these well-tolerated doses of ⩾2 × 1012 particles. Both acute inflammatory cytokine-mediated immunity (primarily IL-6, IL-10, interferon-gamma, tumor necrosis factor) and humoral immunity were induced rapidly following intravenous administration. A correlation between IL-6 induction and the initial febrile response was suggested. This is consistent with toxicity attributed to IL-6 during preclinical and clinical investigation of recombinant human (rh)IL-6 protein,21,22 and observations in other trials involving adenoviral vectors.8 Up-regulation of IL-6, which we observed, is consistent with an acute phase reaction that peaked within 6 h of initial viral infusion and first of the host's triphasic immune response.23 Elevated γ-IFN levels that peaked at 24–48 h after infusion are indicative of NK, and subsequently TH1-mediated immune activation events. Maximal IL-10 levels at 72–96 h after infusion may reflect collateral TH2 activation, although IL-4 and IL-5 determinations are needed to confirm this hypothesis. Similarly, others have shown adenoviral antigen stimulation of TH1 and TH2 response.24,25,26 Our findings with a limited number of patients also suggest preferential TH2 activation at the time of viral reinfusion at cycle 2, based on the marked increase in IL-2 and IL-10 within 72 h.
Our results demonstrate that infection of, and replication within, tumors by ONYX-015 is feasible following intravenous administration. More than half of the plasma samples from patients receiving doses greater than 2 × 1012 particles showed persistence of viral genome in blood more than 24 h after administration (nearly half at 7 days). Without replication, the viral genome is not observed in circulation for more than 24 h in animal models (and usually not more than 6 h) after infusion. Interestingly, the levels of viral genome detected in the plasma of three patients 48 h after infusion were 2.5- to 10-fold higher than levels in the plasma at 6 h. Therefore, significant on-going replication was taking place. Immunohistochemistry and quantitative PCR analysis of tumor tissue was used to confirm intratumoral viral in the one patient with an accessible and evaluable tumor. We cannot rule out viral replication at sites outside the tumor, as well, but even if this did occur, it was not associated with any clinically significant toxicity.
Although it is possible that antibody binding will decrease the efficiency of tumor injection, viral pharmacokinetics were not demonstrably altered by antibody formation. Future studies will need to address this issue. If necessary, approaches to block antibody development can be tested.
Although primarily a safety study, we did confirm intratumoral delivery and replication in at least one patient using this systemic approach. If intravenous delivery of virus is to evolve into an effective therapy, consistent delivery to, and uptake by, the tumor will be necessary. Several approaches may be taken to improve the efficacy of future intravenous treatments. One is to combine the favorable attributes of replication-selective viral therapy with gene delivery;6,7 replication-selective viruses can be engineered to express therapeutic genes (eg cytokines, prodrug-activating enzymes) and thereby improve tumor destruction as compared with the virus alone. Inhibition of immune-mediated viral clearance may also be possible through the use of immunosuppressive regimens, particularly in light of the safety demonstrated on this trial. It is also clear that replication-selective adenoviruses are effective and potentially synergistic in combination with standard chemotherapeutics.19,20,27 Since the mechanisms of action of adenoviral therapy and chemotherapy are very different, and overlapping toxicities are not apparent, this approach has great promise.
Future studies may test approaches to reduce hepatic clearance of therapeutic adenoviruses. Preclinical studies indicate that more than 80% of ONYX-015 and other adenoviruses are taken up by the liver following intravenous administration.27 Thus, the delivery of adenovirus to metastatic tumor deposits, and the resulting antitumoral activity, should be improved if hepatic uptake is inhibited.28,29,30 Furthermore, approaches which decrease hepatocyte and Kupffer cell uptake such as infusion of silica and/or multi-lamellar lyposomes (Cl2MBP lyposomes),31,32,33,34,35,36,37,38,39,40,41,42,43,44 have been shown to delay elimination of adenoviral vectors significantly. A second important factor influencing internalization of adenovirus and induction of actin cytoskeleton reorganization for endocytosis of the viral particle to take place is the expression of coxsackie-adenovirus receptor (CAR) on target cells which serves as a primary receptor for adenovirus.45 Interaction with a second receptor, αV-β3,5 integrin, facilitates virus internalization into cells.46 Internalization probably occurs by clathrin-mediated endocytosis,47,48 an actin-dependent process, under the control of the PI3K pathway,49 and may be therapeutically modulated by blocking of the Rho family of small GTP-binding proteins that serve as regulators of PI3K activation.45,50,51,52,53 Strategies to modulate the expression levels or function of these receptors in tumor tissues and/or normal organs are being developed and might eventually be tested in clinical trials.
Another major factor influencing the efficacy and toxicity of adenoviral agents is the immune response to adenovirus administration and replication.29,46,54,55,56,57,58,59,60 Adenovirus-mediated gene transfection in immunodeficient mice24,25,61,62,63,64,65,66,67 or immunocompetent animals treated with immunosuppressive agents67,68 has resulted in prolonged transgene expression.25,26,64,66,67,69,70,71,72,73,74,75,76,77 Future studies exploring intravenous infusion of ONYX-015 may involve testing of transient immunosuppression.
The clearance of adenovirus is also affected by the expression of immunomodulatory gene products from the virus itself. For example, the E1B 19kD gene is responsible for blocking fas and TNF receptor-induced apoptosis, whereas the adenovirus early transcription unit (E3) encodes for polypeptides (14.7k, 10.4, 14.5),78,79,80,81,82 which function to block directly TNF-α activation as well as apoptotic pathways shared by TNF-α and fas.78,80,83 The E3 gp-19k protein functions to bind and retain MHC class I molecules within the endoplasmic reticulum, thus preventing surface presentation of viral antigens, thereby limiting class I-restricted CTL clearance of virally infected cells.79,82,84,85,86,87 The E3b region of ONYX-015 is deleted and this virus may therefore be hypersensitive to clearance by fas and/or TNF. Replacement of this gene region may, therefore, decrease viral clearance, increasing expression of viral genes that suppress immune recognition which may improve replication.17,78,88
In conclusion, results of this trial demonstrate that ONYX-015 can be safely administered to refractory cancer patients as a single agent or in combination with chemotherapy. These results open the door to innovative therapeutic approaches utilizing attenuated replication-selective adenoviruses as a new therapeutic modality via intravenous delivery in cancer patients with epithelial or even hematologic malignancy.89 Methods of modulating adenoviral clearance, uptake, tropism for malignant cells and functional activity need to be further investigated. Future directions will include the use of replication-selective viruses in combination with standard oncologic treatment regimens, as well as the expression of exogenous therapeutic genes.7,90
ONYX-015 is a chimeric human group C adenovirus (Ad2 and Ad5), originally constructed in the laboratory of Arnold Berk, that does not express the 55kD product of the E1B gene.91 The virus contains a deletion between nucleotides 2496 and 3323 in the E1B region encoding the 55kD protein. In addition, a C to T transition at position 2022 in E1B generates a stop codon at the third codon position of the protein. These alterations eliminate expression of the E1B 55kD gene in ONYX-015 infected cells. ONYX-015 was grown and titered on the human embryonic kidney cell line HEK293 as previously described.13
This was an open-label phase I, dose-escalation study. Patients received ONYX-015 on days 1, 8 and 15 of each 21-day cycle. Dosage cohorts were as follows (one patient at each dose level): (1) 2 × 1010 particles; (2) 6 × 1010 particles; (3) 2 × 1011 particles; (4) 6 × 1011 particles; (5) 2 × 1012 particles; and (6) 6 × 1012 particles for the first dose, then 2 × 1012 particles for subsequent doses; (7) 2 × 1013 particles for the first dose, then 2 × 1012 particles for subsequent doses; (8) 2 × 1012 particles with chemotherapy starting cycle 1, day 8. Carboplatin (AUC 2) and taxol (80 mg/m2) were administered in combination with ONYX-015 starting with cycle 3 (continued for all subsequent cycles) in cohorts 1–7 and starting with cycle 1, day 8 in cohort 8. A single patient was treated on a compassionate use protocol testing intra-patient dose escalation as follows: 2 × 1010; 6 × 1010; 2 × 1011; 6 × 1011; 2 × 1012 particles. Treatment was continued as long as patients maintained a status of stable disease and did not develop significant toxicity.
ONYX-015 handling and processing
ONYX-015 is formulated as a sterile viral solution in TRIS buffer (10 mM TRIS pH 7.4, 1 mM MgCl2, 150 mM CaCl, 10% glycerol). The solution is supplied frozen (−20°C) in single-use, plastic screw-cap vials. Each vial contains 0.5 ml of virus solution at a specified viral titer. Vialed virus solution was warmed and diluted to the appropriate titer for dosing. Following warming, ONYX-015 was maintained at 2° to 8°C during dilution and handling, except for warming to room temperature immediately before administration.
Drug preparation was performed within 10 min before tumor infusion, using plastic syringes. Vials of ONYX-015 were opened and the appropriate volume of virus diluted to 10 ml with normal saline in a biologic safety cabinet. All waste items were disposed of in biohazard containers and were autoclaved and incinerated. For dose levels of 3 × 1011 and 1 × 1012 only, the prepared dose of ONYX-015 was injected into a 100 cc bag of normal saline for infusion.
ONYX-015 intravenous infusion procedure
A single infusion of 10 ml of ONYX-015 with normal saline was administered over approximately 3–5 min, as specified for each cohort in the product information sheet through a peripheral or central intravenous line (22-gauge catheter). For dose levels of 6 × 1012 and 2 × 1013, a 100 cc bag of normal saline with the prepared dose of ONYX-015 was administered via infusion pump over a 10-min period.
Dose-escalation scheme – cycle 1
One patient was entered per dose level until the highest dose was shown to be safe; at this point three additional patients were treated at the highest dose level. Patients were enrolled sequentially into treatment cohorts with at least a 2-week interval between each cohort. Dose escalation was based on the toxicity seen with the first cycle of therapy. If a DLT related to ONYX-015 was observed in a cohort related to ONYX-015, up to six patients were to be enrolled at that dose level. If a DLT, felt not to be related to ONYX-015 occurred, that patient was replaced. Patients were enrolled until a second DLT occurred (which defined the toxic dose) or until six patients were enrolled at that dose level. The MTD was defined as the dose immediately below the dose at which two patients experienced a DLT after their first treatment with ONYX-015. DLT was defined as grade 4 toxicity of any duration and grade 3 toxicity lasting more than 5 days. A minimum of a 1-week interval between treatment of each of these patients was required unless toxicity was observed at >1 week in which case the duration between patients was a minimum of the duration of time at which the toxicity was observed from the treatment date.
A combination of carboplatin (i.v. dose at AUC of 2 per standard practice) and paclitaxel (80 mg/m2 i.v. per standard practice) was administered weekly concurrently with systemic virus administration. Pre-medications for chemotherapy were given per standard of practice for the clinic. They involve dexamethasone 20 mg/12 h then 2 h before dosing; diphenhydramine 25 mg and ranitadine 50 mg i.v.; then odensatron 1 mg and dexamethasone 10 mg i.v. just before dosing.
Patients with advanced metastatic cancer who failed one prior therapeutic regimen and for whom a curative therapy is not available were eligible. Patients were required to have histologically or cytologically confirmed carcinoma, tumor not curable with surgery or other standard modalities, and a documented p53 gene mutation. For dose levels greater than 6 × 1012 particles, patients were required to have disease involving the lung. Other eligibility criteria included a Karnofsky performance status of ⩾90%, life expectancy of ⩾3 months, age ⩾18, absolute neutrophils >1500/μl, hemoglobin >9 mg/dl, platelets >75000/μl, creatinine <2.0 mg/dl, AST and ALT >3.0-fold upper limit of normal, PTT within normal limits, and total bilirubin <2.0 mg/dl, and consent for study participation on an IRB-approved informed consent form. Women and men of reproductive potential were required to use a reliable form of contraception.
Patients with the following were excluded from study: >50% liver replacement by tumor as determined radiographically; ongoing active infection (including human immunodeficiency virus); viral syndrome diagnosed within the last 2 weeks; chemotherapy within the last 3 weeks (6 weeks for nitrosourea or mitomycin-C); radiotherapy to tumor site within the last 4 weeks (if this is the only site of disease); treatment with any other investigation therapy within the last 4 weeks; concomitant hematological malignancy (ie chronic lymphocytic leukemia, non-Hodgkin's lymphoma); and current requirement for chronic immunosuppressive medication including glucocorticoid or cyclosporin, or chronic use of any such medication within the last 4 weeks. Pregnant or lactating females were excluded, and patients previously participating in research protocols involving administration is adenovirus vectors were excluded (with the exception of one compassionate use protocol patient).
Up to five masses were measured for response assessment. A complete response (CR) was defined as complete disappearance of all tumors at the assessed site, partial response (PR) was defined as regression of the tumor mass by ⩾50% but <100% with no growth of any individual lesion, stable disease (SD) was defined as tumor decrease by <50% or increase in size by <25%, and progressive disease (PD) was defined as a ⩾25% increase in tumor size. Tumor response was assessed by CT scan of relevant organs each odd cycle. CT confirmation of all PRs and CRs 4 weeks after initial documentation of the response was required.
Adverse event definition
Severity of adverse events were assigned according to the NCIC-CTG Expanded Common Toxicity Criteria. A grade 1 or mild was considered a noticeable adverse event, but one which does not interfere with normal daily activities; a grade 2 or moderate adverse event is one which is sufficient to interfere with normal daily activities; a grade 3 or severe adverse event is incapacitating and causes the patient to be unable to perform daily activities (hospitalization may or may not be required); a grade 4 or life-threatening adverse event poses an immediate risk of death as it occurs; and a grade 5 adverse event is fatal. If severe toxicity attributable to active viral replication had occurred, initiation of intravenous ribavirine therapy was recommended at 35 mg/kg divided into three doses given every 8 h (the first day) followed by 25 mg/kg divided into three daily doses and given every 8 h; no patients required ribavirine therapy.
Screening and pretreatment evaluation
CT scans, chest radiographs or ultrasound evaluations were completed within 21 days before treatment. All other examinations and procedures were completed within 14 days before the initial treatment with ONYX-015. The biopsies documenting carcinoma and evaluation of p53 status were taken before enrollment or treatment with ONYX-015. Screening and pretreatment evaluation included a signed IRB-approved informed consent form, CT-scan evaluation of the chest and/or liver/abdomen with tumor measurements, chest X-ray (PA/lateral), 12-lead electrocardiogram (ECG), complete medical history including secondary diagnosis, complete physical examination, medications within the last 2 months, previous anticancer treatments, Karnofsky performance status score determination. Screening laboratory assessment included hematological tests, including prothrombin time (PT), partial thromboplastin time (PTT), serum chemistry tests, neutralizing antibody to ONYX-015, ONYX-015 DNA in plasma, urinalysis, urine or serum pregnancy test, and a paraffin tumor block. Plasma samples were also archived.
Treatment evaluation on days 1, 8 and 15
For administration of ONYX-015, patients were treated at the Mary Crowley Medical Research Center of US Oncology located in the Sammons Cancer Center at Baylor University Medical Center, Dallas, TX, USA. Vital sign assessments were taken at baseline (45 min before ONYX-015 infusion ± 15 min). In addition, vital signs were monitored just before infusion, at the conclusion of the infusion and every 15 min thereafter for a total of 2 h. If vital signs had changed ±20% from baseline after 2 h, patients were to be retained, examined and had vital signs taken until they returned to baseline values.
Procedures during the ONYX-015 treatment period
Day 1 (±1 day) procedures included a brief physical examination directed to relevant signs, symptoms, vital signs and weight, hematological tests, serum chemistry tests including liver function, cytokine analysis (baseline and 6 h, ±1 h, following injection of the virus) for cycles 1 and 2 only. On day 2 (±1 day), cytokine analysis (24 h, ±3 h, following injection of the virus) was done on cycles 1 and 2. On day 3 (±1 day), serum liver function tests were repeated, plasma for PCR (polymerase chain reaction) and antibody titer (only cycles 1 and 2) was obtained, and cytokine analysis (48 h, ±3 h, following injection of the virus) was done in cycles 1 and 2 only. On day 4 (±1 day), cytokine analysis (48 h, ±3 h, following injection of the virus) was done in cycles 1 and 2. On day 5 (±1 day), serum liver function tests were repeated, and three or more biopsies of tumor and normal tissue (liver, pulmonary, skin) on cycle 1 only were done to assess for replicating ONYX-015. Cytokine analysis (96 h, ±3 h, following the first injection of each cycle of the virus) was also done in cycles 1 and 2. On days 8 to 15 (±2 days) of the second and third infusion of ONYX-015, hematological tests, serum chemistry tests including liver function, and physical examination were repeated. Changes in concomitant medications and reporting of adverse events were summarized. A plasma sample was archived (cycles 1 and 2 only). Neutralizing antibody to ONYX-015 (only cycles 1 and 2), plasma for PCR, ONYX-015 titer (only cycles 1 and 2), and cytokine analysis (168 h, ±3 h, following the first injection of each cycle the virus) were done in cycles 1 and 2.
Pharmacokinetic venous blood draws
All patients had pharmacokinetic blood draws taken on day 1 of cycles 1 and 2. Blood was drawn at the start of the ONYX-015 infusion and at 5, 10, 30, 60, 90, 120, 180 and 360 min. Pharmacokinetics were repeated for the first dose of cycle 2 at time 0 before ONYX-015 infusion and at 30, 90, 120 and 360 min. Samples were also drawn before ONYX-015 infusion on days 8 and 15.
Off study assessment
Patients who completed their final cycle of ONYX-015 treatment or came off study for other reasons were withdrawn from study and had the following assessments completed: baseline physical examination, Karnofsky performance status, labs for hematology and chemistry, urinalysis, adverse events, and concomitant medications.
If patients went off study with an objective tumor response or stable disease (ie without tumor progression), an abdominal CT scan or ultrasound was taken every 6–8 weeks to follow for disease progression. All patients who went off study for any reason were followed for survival.
Cytokines IL-6, IL-1α, IL-1β, TNF-α, IL-10, IL-12, and γ-IFN were assayed from patient serum. ELISA assays (R+D Quantikine kits; Minneapolis, MN, USA) were carried out to quantify cytokine levels. Post-treatment cytokine levels were compared with baseline levels harvested before infusion 1 of cycle 1.
Patient peripheral blood was collected by venipuncture into Vacutainer tubes without anticoagulant. Serum samples were extracted from clotted samples and stored at –80°C. Multiple serum samples collected over each patient's treatment course were analyzed simultaneously (in triplicate), using cytokine-specific immunoassay reagents according to the manufacturer's protocols. The colorimetric reaction was quantified as a function of OD absorbance at 540 nm (SpectraMax 340; Molecular Devices, Sunnyvale, CA, USA). Cytokine concentration was calculated according to a reference standard curve generated with four parameter logistic (4-PL) curve fit and OD values of known, graded concentrations of recombinant cytokine. The lowest concentration of each cytokine's standard curve consistently yielded an OD values that are ⩾3-fold higher than the ODbackground (no recombinant cytokine or serum added), and are considered to be the minimal detectable level (IL-6: 3.1 pg/ml; IL-1α/IL-1β: 3.9 pg/ml; TNF-α: 15.6 pg/ml; IL-10: 7.8 pg/ml; IL-2: 31.2 pg/ml; IFN-γ: 15.6 pg/ml). Post-treatment cytokine levels were compared with baseline levels harvested before infusion 1 of cycle 1 by the formula: % increase over baseline = 100% × (CT − C0)/C0; where CT and C0 represent the mean calculated cytokine concentration at time-point T, and before treatment, respectively.
The study included seven treatment cohorts, with one to six patients in each cohort. The data for each cohort were summarized with descriptive statistics, frequency tabulations, graphs, and data listings as appropriate. The data from the treatment cohorts were combined for selected data displays. Parametric or non-parametric methods were used to compare dose groups, as appropriate.
P53 gene sequencing
Pretreatment tumor biopsies were taken for p53 sequencing from the recurrent tumor mass that was to be injected (sequencing performed at the John T Mallams Laboratory, supported by the Mary C Crowley Foundation). DNA was extracted directly from sections of formalin-fixed paraffin-embedded needle biopsies. If necessary, tumor cells were micro-dissected from sections of paraffin-embedded tissue. DNA was isolated from tumor cells using phenol/chloroform extraction and ethanol precipitation, and the p53 gene was amplified in several fragments by PCR. The amplicons were purified by gel electrophoresis and analyzed using either the DNA Sequencing Kit (Boehringer Mannheim, Indianapolis, IN, USA), or the ABI Model 310 automated sequencer. Accurate reading of the sequencing gels and the sequencer printout was confirmed by National Biosciences (Plymouth, MN, USA).
Quantitative PCR of ONYX-015
To quantify the amount of ONYX-015 virus genome in human plasma, viral DNA was extracted from patient samples, standard and controls using a QIAamp DNA mini kit (Valencia, CA, USA) then quantified by real-time PCR using assay-specific primer and probe as described below. All tissue samples were analyzed independently by Althea Technologies (San Diego, CA, USA). In order to monitor recovery during extraction, patient samples were spiked with exogenous DNA. As PCR tumor tissue analysis had not been previously studied with other patient tumor samples, a TaqMan-based assay was developed using a primer based on a 92 nucleotide (nt) sequence from nt 2453 to nt 2544. This amplicon overlaps the E1B region deletion and includes the 8bp Puc-derived linker insert. The central region of the TaqMan probe is homologous to the linker insert, making the probe specific to ONYX-015.
For quantification, a standard curve was constructed by assaying serial dilutions of ONYX-015 virus ranging from 2 × 109–1.05 × 104 vp/ml. Negative controls consist of a plasma control without virus and a type D adenovirus wild-type control. All samples were done with 1 μg of human placental DNA as background. The correlation coefficient of the standard curve was 1.00. The presence of PCR inhibitors in any sample was detected by the results of an independent PCR reaction using a primer specific for 18s rDNA.
Using this assay, the lower limits of detection of ONYX-015 particles in plasma was 1.05 × 104 particles/ml with a lower limit of quantitation of 4.2 × 104 particles/ml of plasma. In tissue, the lower limit of detection was 13 copies of ONYX-015 genome/μg with a quantitation limit of 23 copies/μg.
Histochemical assessment of adenoviral capsid antigen
Immunohistochemical studies for adenovirus were performed using a mouse monoclonal antibody against adenoviral capsid proteins (adenovirus clone 20/11 and 2/6; Cell Marrque, Austin, TX, USA). Formalin-fixed, paraffin-embedded sections were studied using an automated Ventana 320ES system (Tucson, AZ, USA) and a 1:10 dilution of the primary antibody. For detection, an avidin–biotin complex secondary antibody technique was used. Adequate negative and positive controls were included.
Determination of ONYX-015 neutralizing antibody titers
Patient and control samples were incubated at 55°C for 30 min to inactivate complement. Clinical plasma samples previously determined to produce high, midrange and negative titers were designated as plasma controls. Each dilution was mixed with adenovirus stock at a titer pre-qualified to produce 15–20 plaques per well of a 12-well dish in DMEM growth medium. The patient's samples and controls were inoculated for 1 h at room temperature, and applied to 70–80% confluent JH393 cells in 12-well dishes. After 2 h of incubation at 37°C, 5% CO2 plasma-virus mix was removed and 2 ml of 1.5% agarose in DMEM was added to each well. Plates were read on day 7 after inoculation by counting the number of p.f.u.s per well. The titer of neutralizing antibody for each sample was reported as the dilution of plasma that reduced the number of plaques to 60% of the number of plaques in the virus control without antibody.
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We wish to thank Ana Petrovich for manuscript preparation, Sherry Toney for extensive time and effort in coordinating study samples and results as well as editorial proofing of the manuscript, and George Smith and Carrie LeDuc from Althea for providing extensive assistance in determining ONYX-015 DNA analysis.
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Nemunaitis, J., Cunningham, C., Buchanan, A. et al. Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity. Gene Ther 8, 746–759 (2001). https://doi.org/10.1038/sj.gt.3301424
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