1 Department of Haematology and Oncology, Hannover Medical School, Hannover, Germany; 2 Department of Medical Oncology, Centre René Gauducheau, Centre Regional de Lutte Contre le Cancer Nantes-Atlantique, Nantes, France; 3 EORTC New Drug Development Office (NDDG), Brussels, Belgium; 4 Department for Oncology, University Clinic Innsbruck, Innsbruck, Austria; 5 VISCUM AG, Bergisch Gladbach, Germany
* Correspondence to: Professor Dr P. Schöffski, Department of Oncology, Universitair Ziekenhuis Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel. +32-16-346900/16; Fax +32-16-346901; Email: patrick.schoffski{at}uz.kuleuven.ac.be
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Abstract |
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Patients and methods: Aviscumine was given twice weekly as a 1 h central i.v. infusion in patients with advanced, refractory progressive, solid malignant tumors who had not been previously exposed to natural mistletoe preparations. They had histologically or cytologically verified disease, were 18 years old, had an Eastern Cooperative Oncology Group performance status
2 and adequate bone marrow, liver and renal function. DLT was defined as any non-hematological grade 34 toxicity (National Cancer Institute Common Toxicity Criteria version 2.0), neutrophil count <500/µl for
7 days, febrile neutropenia or thrombocytopenia grade 4. The MTD was defined as the dose at which >20% of patients experienced DLT during the first treatment cycle. The Continual Reassessment Method was used to determine the number of patients required per dose level.
Results: Forty-one fully eligible patients (19 male, 22 female) with a median age of 56 years (range 3774) were enrolled. Colorectal, ovarian, renal cell and breast cancer were the most common tumor types. Dose levels of aviscumine ranged from 10 to 6400 ng/kg. The median number of cycles was two (range one to eight). Common clinical toxicities in cycle 1 were fatigue, fever, nausea, vomiting and allergic reactions. Fatigue grade 3 was dose limiting in one of six patients at 4000 ng/kg and reversible grade 3 liver toxicity (elevation in alkaline phosphatase, transaminases and/or -glutamyltransferase) occurred in one of 10 patients at 4800 ng/kg and in two of five patients at 6400 ng/kg. The best response (RECIST criteria) was stable disease in 11 patients, lasting for two to eight cycles. The pharmacokinetic evaluation revealed a short
half-life of 13 min and linear kinetics on dose levels
1600 ng/kg. Aviscumine stimulated the immune system with a release of cytokines such as interleukin (IL)-1ß, IL-6 and interferon-
, and induced immunoglobulin (Ig) G- and/or IgM-anti-aviscumine antibodies of uncertain clinical relevance.
Conclusions: The recommended dose for further clinical trials is 5600 ng/kg twice weekly. Based on the short half-life of the recombinant protein observed in this trial, the exploration of prolonged infusion schedules of aviscumine is warranted.
Key words: aviscumine, CD75s, phase I study, recombinant mistletoe lectin, ribosome-inactivating proteins, solid tumors
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Introduction |
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The 1923-nucleotide ML-I gene was sequenced in 1999 and the existence of a single intron-free gene was demonstrated. The production of pure, biochemically defined ML-I was then achieved by recombinant cloning and separate expression of the A- and B-chain in Escherichia coli BL21DE3 by recombinant DNA technique, yielding the active heterodimeric protein rViscumin (INN; aviscumine) [9, 10
]. The expression vectors contain the exact coding sequence of the genomic DNA fragments of the subunits A (26 kDa) and B (30 kDa) as found in European mistletoe. The recombinant drug is obtained by a single processing step folding of the solubilized subunits and linking by a disulfide bond. Purification of the protein is achieved by two chromatographical steps yielding >98% homogeneous material. The manufacturing process finally results in an aqueous, buffered solution of aviscumine, which is stable at pH 79 (Figure 1) [10
].
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The availability of the pure, homogeneous protein was a prerequisite for reproducible structural and mechanistic studies, in order to gain a better insight into the mode of action of this product on tumor and immune cells, as the isolation of the glycosylated proteins from natural mistletoe only yields inhomogeneous material, probably due to post-translational modifications. Therefore, the recombinant drug was studied extensively in preclinical models. It was found to possess impressive antineoplastic and immunomodulatory properties in vitro and in vivo, and was tolerated well in animals [1619
].
Furthermore, the cellular receptor involved in the binding of the heterodimer could be identified [20, 21
]. Aviscumine was shown to bind preferentially to terminally
2-6-sialylated ganglioside structures. In contrast to another well-known ribosome-inactivating agent, ricin, aviscumine was therefore characterized as a sialic acid-specific type II ribosome-inactivating protein. Neolacto-series gangliosides with a Neu5Ac
2-6Galß1-4GlcNAc-terminus (CD75s), rather than galactose-specific, were defined as the aviscumine receptors for the B-chain, leading to internalization of the holoprotein [20
].
In further mechanistic studies, the A-chain was confirmed to be a potent hydrolase that selectively cleaves the N-glycosidic bond of the adenine-4324 residue in eukaryotic 28S ribosomal RNA. This effect leads to a catalytical inactivation of ribosomes, and thereby inhibits translation and protein synthesis as the presumed mode of action [7, 9
].
In vitro, aviscumine was found to be a very potent cytotoxic agent, which induced apoptosis in a low concentration range (fM to pM) and necrosis at higher concentrations (nM to mM). The mean aviscumine concentration required to eliminate 70% of human tumor cell lines in vitro (Freiburg tumor panel) was 0.4 ng/ml, and was 2 ng/ml to inhibit tumor xenograft colony formation by 70%. On a molar basis, aviscumine was 5000 times more potent than the standard therapeutic agent doxorubicin and 1500 times more potent than paclitaxel against some human tumor cell lines. Aviscumine was more active against doxorubicin-resistant breast tumor cells and vindesine-resistant pleural mesothelioma cells than against the respective drug-sensitive parental cell lines, indicating that the drug is not affected by common resistance mechanisms. Interactions between aviscumine and conventional anticancer drugs were investigated in vitro. Aviscumine was capable of enhancing the cytotoxic effects of vincristine, mafosfamide, idarubicin and cisplatin in the human leukemia cell lines K562 and KG1a.
The intraperitoneal (i.p.), subcutaneous (s.c.) or intravenous (i.v.) administration of the recombinant protein was shown to have growth inhibitory action in various heterotopic tumors and metastasis mouse models, including the C8 colon 38-carcinoma, Lewis lung sarcoma, Renca renal carcinoma, F9 testicular carcinoma, RAW 117-H10 P lymphosarcoma, L-1 sarcoma and B16 melanoma models [18]. A significant inhibitory effect on experimental urothelial carcinogenesis was seen after intravesical instillation into rats. Intratumoral treatment of human ectopic CXF 280, and i.p. administration in 60444 and 5776 colon cancers in immunodeficient mice, led to a strong, dose-dependent inhibition of tumor progression. Combination therapy of aviscumine with doxorubicin was found to be superior to the effect of each single agent alone. The i.p. administration of aviscumine into human ovarian cancer SoTü3-bearing SCID mice led to a significant prolongation of survival [17
].
Immunological studies in vitro showed that aviscumine stimulated the release of interleukin (IL)-1 and IL-6 from human keratinocytes and fibroblasts, the release of IL-12, interferon (IFN)-
and tumor necrosis factor (TNF)-
from peripheral blood mononuclear cells, as well as the expression of the IL-2 receptor
chain and HLA-DR on peripheral blood T lymphocytes. Additionally, aviscumine increased the activity of human natural killer cells against lymphoma cells ex vivo [16
]. In tumor-bearing mice, aviscumine was found to enhance the number or to induce the activated state of some blood cell subpopulations (CD4/8 + CD45+ cells, activated MAC3 + CD45+ cells).
Toxicology studies have shown that aviscumine can be safely applied in animals and lacks relevant genotoxic or mutagenic effects up to dosages of 1000 ng/kg i.v. Repeated i.v. or s.c. dosing did not reveal any specific target organ toxicity in rats and dogs up to 1000 ng/kg. Bleeding complications were observed on much higher doses, i.e. at 4000 ng/kg in rats and at 10 000 ng/kg in dogs after 1 week of daily i.v. application. Local reactions at s.c. injection sites were observed at concentrations from 50 ng/ml upwards.
The promising preclinical profile of the recombinant ribosome-inactivating protein was the scientific basis for designing this very first i.v. dose-finding trial of aviscumine in patients with refractory solid tumors.
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Patients and methods |
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Patient selection criteria
Patients with a histologically or cytologically confirmed diagnosis of a progressive solid malignant tumor were eligible for the trial, provided the disease was assumed to be refractory to further conventional treatment. Standard selection criteria included age 18 years, Eastern Cooperative Oncology Group (ECOG) performance status 02, life expectancy of
3 months, no central nervous system involvement, adequate bone marrow (white blood cell count
3 x 109/l, neutrophils
1.5 x 109/l, platelets
100 x 109/l), liver [serum bilirubin within 1.5x the upper limit of normal (ULN), serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase <2.5x ULN if no liver metastases, or <5x ULN in case of liver metastases] and renal (serum creatinine <120 µmol/l) function. The study excluded patients with previous exposure to natural mistletoe preparations to avoid interference with secondary trial end points. Prior chemotherapy, hormone and/or radiation treatment had to be completed at least 4 weeks prior to study entry. Patients were not allowed to receive any immunostimulating substances, biological response modifiers, colony-stimulating factors, systemic steroids or monoclonal antibodies within 4 weeks prior to and during the study. Patients had to use effective contraception if of reproductive potential and were not allowed to be pregnant or lactating. Written informed consent was obtained from all participating patients according to applicable German and French laws and regulations. Patient insurance was provided by the European Organization for Research and Treatment of Cancer (EORTC). The study was conducted in accordance with the Good Clinical Practice Guidelines as issued by the International Conference on Harmonization and the Declaration of Helsinki.
Study logistics
After obtaining informed consent, patients were screened for potential trial participation. Eligible candidates were registered by a telephone procedure at the EORTC Data Center prior to the start of treatment after verification of the eligibility criteria and selection of the target lesions. The treatment started within 1 week after registration. Aviscumine was given twice weekly until one of the following withdrawal criteria occurred: disease progression, unacceptable toxicity, patient's refusal or patient's best interest according to the treating physician.
Study treatment
Aviscumine concentrate was supplied in 10 ml vials by the sponsor (VISCUM AG, Bergisch Gladbach, Germany) and kept in the hospital pharmacy at 28°C. The drug was formulated as a pyrogen-free, phosphate-buffered solution at pH 8.0, and contained sodium chloride, polysorbate 80 and glutamic acid. The appropriate volume of aviscumine was calculated according to the body weight of the patient, diluted with physiological saline up to a total volume of 1000 ml and administered within 8 h after preparation of the infusion. Aviscumine was given twice weekly (days 1 and 4) as a 1-h i.v. infusion through a central line, to avoid the potential risk of local vascular effects in peripheral veins. In most cases, port catheters were used for this purpose. One cycle was arbitrarily defined as three consecutive weeks of treatment (six infusions). The treatment was given continuously without interruption between the treatment cycles. After the end of each cycle, the safety and tolerability of the treatment was assessed in detail. Patients with progressive disease were removed from the study; patients achieving stable disease or an objective response could remain on treatment. Supportive care was at the discretion of the treating physicians, but corticosteroids, immunostimulating agents, other anticancer drugs, monoclonal antibodies or hematopoietic growth factors were prohibited.
Dose escalation procedure
The dose escalation procedure in this phase I study was guided by the Continual Reassessment Method (CRM) [22, 23
]. Dose escalation was done on an inter-patient basis. The aim of using the CRM was to decrease the number of patients on low, non-toxic dose levels in this trial. The protocol allowed the evaluation of intermediate dose levels. The number of dose levels was not fixed in advance. The initial version of the protocol foresaw the following steps: 10, 20, 40, 100, 200, 400, 800 and 1600 ng/kg per administration of aviscumine. The protocol was later amended, as the treatment was found to be well tolerated up to the dose of 1600 ng/kg, and higher dose levels were allowed.
Definition of DLT, MTD and recommended dose
DLTs were defined as any non-hematological grade 34 toxicity (with the exclusion of nausea, vomiting or fever), an absolute neutrophil count <500/µl lasting for 7 days, febrile neutropenia (defined as an absolute neutrophil count <500/µl lasting for
3 days associated with fever
38.5°C for 24 h) or thrombocytopenia of National Cancer Institute Common Toxicity Criteria NCI CTC grade 4. The MTD for this study was defined as the highest dose at which a maximum of 20% of the patients of a certain dose level experienced DLT during the first treatment cycle.
Clinical evaluation, laboratory tests and follow-up
Within 2 weeks prior to study treatment, the following baseline tests were obtained: tumor evaluation, tumor markers (if applicable), complete medical history, ECG and pregnancy test in fertile female patients. Within 7 days, the following parameters were determined: weight, height, complete blood count with differential and platelet count, serum chemistry (blood urea nitrogen, creatinine, total protein, albumin, bilirubin, lactate dehydrogenase, creatin kinase, alkaline phosphatase, ALT, AST and electrolytes) and coagulation panel (PTT and PT). Immediately prior to the first administration of aviscumine, the following parameters were obtained: physical examination, ECOG performance status, temperature (°C) and vital signs. Vital signs were repeated at 15, 30, 60 and 120 min, and then 4 and 8 h, after the first i.v. infusion. On a weekly basis during all treatment cycles, the following parameters were assessed: physical examination, toxicity assessment, ECOG performance status, vital signs, complete blood count, serum chemistry and coagulation panel. A detailed toxicity evaluation according to NCI CTC was performed prior to each treatment cycle. The tumor imaging and marker assessment was repeated every 6 weeks.
Criteria for evaluation of toxicity and response
All adverse events were graded according to the NCI CTC (version 2.0). The causality of clinical events was categorized as unrelated, unlikely, possible, probable, definitely related or not assessable. Diseases and clinical symptoms already known at the beginning of the study were not classified as adverse events if they were encountered again at subsequent examinations, except in the case of a relevant increase in intensity or incidence. All patients who had started the treatment were included in the overall toxicity assessment. Serious adverse events were defined according to the ICH Good Clinical Practice Guidelines and reported within 24 h to the EORTC. This included all events occurring during or within 30 days of termination of the study treatment. The response to the treatment with aviscumine was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria [24].
Statistical considerations
The experimental design for this study was an adaptation of the CRM, which is based on the concept of entering every patient at the best current statistical estimation of the MTD given the previous observations. The trial was conducted in two consecutive steps: step 1, until mild toxicity was observed; and step 2, toxicity evaluation regarding the DLT and MTD. Cohorts of only one patient were to be treated per dose level, as long as there was no toxicity exceeding grade 1. New patient inclusion was blocked until the tolerance of the patient to the first cycle was fully documented. In the event of moderate drug-related toxicity (grade 2), the cohort size was increased from one to three patients for all further groups and the dose escalation was slowed down. At that point, at each dose level, after the first patient had been registered, new patient inclusion was blocked until the tolerance of the first patient to the first cycle was fully documented. In order to proceed to a next dose level, none of three patients treated on that dose level was allowed to have experienced a grade 3 or 4 toxicity related to the treatment during the first cycle.
As soon as a DLT was observed, the second stage started. In this stage, the dose escalation strategy was based on a one-parametric statistical model, based on the relationship between the dose level di and the probability of DLT at that level, (
i, a)=
ia, where
1=0.04,
2=0.07,
3=0.20,
4=0.35,
5=0.55,
6=0.70 was a recoding of the dose levels di. Using a maximum likelihood estimator, estimates of the probability of toxicity at each available dose level were updated after every event. Given these estimates, the next patient was included at the dose level whose estimate was closest to the targeted percentile, 20%. After each estimate a confidence interval of the probability of toxicity at the current recommended dose was calculated.
Pharmacokinetic analysis
The translational research component of the protocol included plasma sampling for pharmacokinetics and immunological effects of aviscumine. Pharmacokinetic sampling was carried out during and after the first and 11th administration of aviscumine. Samples were taken 15 min prior to the i.v. infusion and 1 h, 1 h 15 min, 1 h 30 min, and 2, 4, 8 and 24 h after the administration. The samples were shipped on dry-ice to Chimera Biotech (Dortmund, Germany) for analytical purposes. Pharmacokinetic analyses were performed using an immuno-PCR method [25].
Patient plasma samples were analyzed directly or diluted prior to analysis with blank plasma samples to an aviscumine concentration of up to 6100 pg/ml. Quantitation was carried out in comparison with an aviscumine reference curve in each individual patient's blank plasma. Plasma samples were transferred into 96-well titre plates with capture antibody (polyclonal rabbit anti-aviscumine antibody; VISCUM AG), and aviscumine was detected with a polyclonal antibody rabbitbiotin conjugate and streptavidineDNA conjugate (Chimera Biotech). PCR was carried out with Taq polymerase (bioTaq; Biomaster GmbH, Köln, Germany) and digoxigenin-ddUTP (Roche, GrenzachWyhlen, Germany), and a specific competitor (Chimera Biotech) for 28 cycles. Digoxigenin was determined with anti-digoxigenin alkaline phosphatase conjugate and Atto-Phos Substrate Kit (Roche) by fluorogenic detection.
For the detection of anti-avicumine antibodies in plasma, an enzyme-linked immunosorbent assay (ELISA) was used with the murine monoclonal anti-aviscumine B-chain antibody 36-2-0 (VISCUM AG) at a concentration of 5 µg/ml plus aviscumine at a concentration of 5 µg/ml as antigen. Plasma samples were diluted to 1:100, 1:1000 and 1:10 000 with phosphate-buffered saline. Detection of the IgM and IgG titre was carried out with anti-human IgM (µ-chain specific) POD conjugate and with anti-human IgG (Fc specific) both from goat (Sigma-Aldrich, Munich, Germany). This analysis was carried out by PHAST (Homburg, Germany).
Cytokine studies involved the repeated determination of plasma IL-1ß, -6, -10, -12, IFN- and TNF-
. The samples were shipped to the Department of Internal Medicine at University Clinic of Innsbruck, where commercially available ELISAs were used to determine the plasma levels. The results of the translational research were analyzed by descriptive and exploratory methods.
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Results |
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The dose levels visited ranged from 10 to 6400 ng/kg. Only one patient per cohort was required on dose levels 10, 20, 40, 100, 200, 400, 800, 1600 and 2400 ng/kg, respectively, as toxicity in cycle 1 did not exceed grade 1. The CRM allowed these nine dose levels to be visited within only 7 months, and to escalate the dose 240-fold in only nine patients without compromising patient safety. With the occurrence of grade 2 toxicity from dose level 3200 ng/kg onwards, the dose escalation was slowed down according to the model. The observation of a sporadic grade 3 event (fatigue in a patient with renal cell carcinoma and advanced lung metastasis) during the first treatment cycle with 4000 ng/kg aviscumine, triggered the statistical model to evaluate six patients on this dose level, but no further DLTs were observed in this cohort. Another 10 patients were treated with the 4800 ng/kg with the occurrence of one DLT at this dose level, and seven patients were exposed to 5600 ng/kg without occurrence of a DLT. Further dose-limiting events were then observed in two out of five patients treated with a dose of 6400 ng/kg. The majority of patients discontinued the treatment due to disease progression; three patients were taken off the study due to liver toxicity (Table 2).
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Discussion |
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The administration of a twice weekly 1-h i.v. infusion of aviscumine was found to be safe up to a dose of 5600 ng/kg body weight. The MTD was 6400 ng/kg, and was associated with reversible hepatotoxicity as dose-limiting event in two out of five patients. The toxicity profile in this clinical trial was quite acceptable, and according to the adverse events data it was concluded that the recommended dose for further clinical trials should be 5600 ng/kg.
Aviscumine was found to stimulate the immune system with a secretion of cytokines in plasma, independent of the dose of the drug administered. Furthermore, the recombinant drug induced anti-aviscumine antibodies in plasma in doses above 3200 ng/kg body weight. These findings are of unclear clinical relevance, and should be studied further in clinical trials.
The pharmacokinetic data revealed linear kinetics of the recombinant protein on the higher dose levels, 3200 ng/kg body weight. The initial half-life of the drug of 13 min indicates a fast degradation of the compound. This corresponds to in vitro studies which revealed that degradation could be expected due to a proteolytic process. Distribution volumes remained in a low range, especially not exceeding the patients weight for the extracellular body fluid (
18% of total body weight), indicating a lack of accumulation. The mean IC70 against the growth of tumor xenograft in vitro models was 2 ng/ml. This concentration could be exceeded during and after the infusion for a mean duration of 2.37.5 h with acceptable tolerability. This duration above plasma levels of 2 ng/ml and the short T1/2
of only 13 min led us to the conclusion that the 1-h infusion twice weekly is probably not the optimal dosing regimen with regard to the exposure of the tumor to adequate drug levels. Therefore, the investigation of prolonged infusion schedules seems to be more appropriate and warrants further investigation in clinical trials, which are already ongoing. Based on preclinical data and the observed toxicity profile of the agent, combination trials with conventional chemotherapeutic agents should also be considered.
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Acknowledgements |
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Received for publication June 9, 2004. Accepted for publication July 9, 2004.
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References |
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