ABSTRACT
The potential allergenicity of Bar, Barnase, and Barstar recombinant proteins expressed in genetically engineered mustard for pollination control in plant breeding was evaluated for regulatory review. To evaluate the potential allergenicity of the Bar, Barnase and Barstar proteins amino acid sequence comparisons were made to those of known and putative allergens, and search for published evidence to the sources of the genes using the AllergenOnline.org database. Initial comparisons in 2012 were performed with version 12 by methods recommended by the Codex Alimentarius Commission and the Indian Council of Medical Research, Government of India. Searches were repeated with version 15 in 2015. A literature search was performed using PubMed to identify reports of allergy associated with the sources of the three transgenes. Potential open reading frames at the DNA insertion site were evaluated for matches to allergens. No significant sequence identity matches were identified with Bar, Barnase or Barstar proteins or potential fusion peptides at the genomic-insert junctions compared to known allergens. No references were identified that associated the sources of the genes with allergy. Based on these results we conclude that the Bar, Barnase and Barstar proteins are unlikely to present any significant risk of food allergy to consumers.
Subject(s)
Allergens/adverse effects , Bacterial Proteins/adverse effects , Food, Genetically Modified/adverse effects , Models, Molecular , Mustard Plant/metabolism , Seeds/metabolism , Acetyltransferases/adverse effects , Acetyltransferases/chemistry , Acetyltransferases/genetics , Acetyltransferases/metabolism , Allergens/chemistry , Allergens/genetics , Allergens/metabolism , Amino Acid Sequence , Antigens, Plant/adverse effects , Antigens, Plant/chemistry , Antigens, Plant/genetics , Antigens, Plant/metabolism , Bacillus/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Computational Biology , Databases, Protein , Dietary Proteins/adverse effects , Dietary Proteins/chemistry , Dietary Proteins/metabolism , Food Hypersensitivity/etiology , Food Hypersensitivity/prevention & control , Humans , India , Mustard Plant/enzymology , Mustard Plant/genetics , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Pollination , Recombinant Proteins/adverse effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonucleases/adverse effects , Ribonucleases/chemistry , Ribonucleases/genetics , Ribonucleases/metabolism , Risk Assessment , Seeds/enzymology , Seeds/genetics , Sequence Alignment , Streptomyces/enzymologyABSTRACT
BACKGROUND: The most prevalent phenotype of asthma is characterized by eosinophil-dominated inflammation that is driven by a type 2 helper T cell (Th2). Therapeutic targeting of GATA3, an important transcription factor of the Th2 pathway, may be beneficial. We evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 messenger RNA (mRNA). METHODS: We conducted a randomized, double-blind, placebo-controlled, multicenter clinical trial of SB010 involving patients who had allergic asthma with sputum eosinophilia and who also had biphasic early and late asthmatic responses after laboratory-based allergen provocation. A total of 40 patients could be evaluated; 21 were assigned to receive 10 mg of SB010, and 19 were assigned to receive placebo, with each study drug administered by means of inhalation once daily for 28 days. An allergen challenge was performed before and after the 28-day period. The primary end point was the late asthmatic response as quantified by the change in the area under the curve (AUC) for forced expiratory volume in 1 second (FEV1). RESULTS: After 28 days, SB010 attenuated the mean late asthmatic response by 34%, as compared with the baseline response, according to the AUC for FEV1, whereas placebo was associated with a 1% increase in the AUC for FEV1 (P=0.02). The early asthmatic response with SB010 was attenuated by 11% as measured by the AUC for FEV1, whereas the early response with placebo was increased by 10% (P=0.03). Inhibition of the late asthmatic response by SB010 was associated with attenuation of allergen-induced sputum eosinophilia and with lower levels of tryptase in sputum and lower plasma levels of interleukin-5. Allergen-induced levels of fractional exhaled nitric oxide and airway hyperresponsiveness to methacholine were not affected by either SB010 or placebo. CONCLUSIONS: Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma. Biomarker analysis showed an attenuation of Th2-regulated inflammatory responses. (Funded by Sterna Biologicals and the German Federal Ministry of Education and Research; ClinicalTrials.gov number, NCT01743768.).
Subject(s)
Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , DNA, Catalytic/therapeutic use , GATA3 Transcription Factor/metabolism , RNA, Messenger/metabolism , Ribonucleases/therapeutic use , Administration, Inhalation , Adult , Anti-Asthmatic Agents/adverse effects , Area Under Curve , Asthma/metabolism , Biomarkers/blood , DNA, Catalytic/adverse effects , Double-Blind Method , Forced Expiratory Volume , GATA3 Transcription Factor/genetics , Humans , Interleukin-5/blood , Male , Middle Aged , Phenotype , Ribonucleases/adverse effects , Th2 Cells/metabolism , Young AdultABSTRACT
Ranpirnase [Onconase] is an amphibian oocyte/early embryo ribonuclease (RNase) of 105 amino acids in length that is capable of controlling tumour growth by degrading RNA within cancer cells, resulting in inhibition of protein synthesis and arresting mitosis in G(1 )phase. It represents the first successful isolation, purification and characterisation of the oocytic/early embryonic factor that is capable of controlling cell growth activities of the early embryonic tissues. Alfacell Corporation is currently conducting clinical trials of ranpirnase in patients with unresectable malignant mesothelioma and non-small-cell lung cancer. The company may initiate phase II clinical trials in breast cancer and oesophageal cancer in 2006. Alfacell expanded a research agreement with the National Cancer Institute in September 2002, allowing the NCI to examine the effects of ranpirnase as a radiation enhancer. However, investigation in this use of ranpirnase now appears to be discontinued. Alfacell is conducting a confirmatory phase IIIb registration trial of ranpirnase plus doxorubicin versus doxorubicin alone in more than 360 patients with unresectable malignant mesothelioma, and will assess survival as the primary endpoint. The targeted treatment group in this trial represents 90% of malignant mesothelioma patients at the time of diagnosis. The trial is being conducted in the US, Canada, Poland, Italy, Germany, Australia, New Zealand, Russia, Romania, Mexico and Brazil. In April 2006, a total of 210 events (patient deaths) was reached, representing two-thirds of the required events for the study. Results from the protocol-specified first interim analysis based on one-third of the required events have been reported and the company has the option to conduct a second interim analysis of the data at any point after 210 events. A final analysis will be undertaken at 316 events. Alfacell completed a phase III trial of single-agent ranpirnase in patients with unresectable malignant mesothelioma in April 1999. The efficacy of ranpirnase was compared with that of doxorubicin (head-to-head). The primary objectives were overall survival, progression-free survival and quality of life. In preclinical studies, ranpirnase demonstrated significant activity against neuroblastoma, rhabdomyosarcoma and chemotherapy-resistant variants of these cancer cells. Development for these indications has been discontinued. Preclinical investigations conducted by Alfacell showed synergistic antitumour effects between ranpirnase and proteasome inhibitors. However, development is this area has been discontinued. Alfacell announced in May 2003 that it would be providing ranpirnase to the federal severe acute respiratory syndrome (SARS) testing programme for evaluation against the human coronavirus implicated in the disease. No further development has been reported. Alfacell has received nine US and four European patents for ranpirnase. Patents issued in the US range from the 1996-issued patent (No. 5 559 212) covering the amino acid sequence of ranpirnase, to the patent (No. 6 175 003 B1) issued in January 2001 protecting the gene sequences of the compound plus another genetically engineered variant, effectively protecting the company's proprietary technology. In August 2002, Alfacell received a US patent (No. 6 423 515 B1) entitled 'Methods of Making Nucleic Acids Encoding Ribonucleases'. This patent is effective until 2020.
Subject(s)
Antineoplastic Agents/pharmacology , Antiviral Agents/pharmacology , Ribonucleases/pharmacology , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Antiviral Agents/adverse effects , Antiviral Agents/therapeutic use , Humans , Neoplasms/drug therapy , Ribonucleases/adverse effects , Ribonucleases/therapeutic use , Virus Diseases/drug therapyABSTRACT
Cytotoxic ribonucleases (RNases), such as ranpiranase, represent a novel mechanism-based approach to anticancer therapy. These relatively small proteins selectively attack malignant cells, triggering apoptotic response and inhibiting protein synthesis. Ranpirnase, originally isolated from oocytes of Rana pipiens, is a member of a family of endoribonucleases. The anticancer effects of ranpiranase have been documented in both in vitro and in vivo experimental tumor models. The effects of ranpiranase appear to be selective for cancer cells. Based on Phase I study data, the maximum tolerated dose (MTD) was 960 microg/m2, with the dose-limiting toxicity (DLT) characterized by proteinuria with or without azotemia, peripheral edema, and fatigue. Ranpirnase did not induce myelosuppression, mucositis, alopecia, cardiotoxicity, coagulopathy, hepatotoxicity, or adverse metabolic effects. Phase II tumor-specific trials investigated the activity of ranpirnase in malignant mesothelioma, breast cancer, non-small cell lung cancer, and renal cell cancer. A Phase III randomized study in malignant mesothelioma patients compares the combination of ranpirnase plus doxorubicin to doxorubicin monotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Ribonucleases/pharmacology , Ribonucleases/therapeutic use , Animals , Antineoplastic Agents/adverse effects , Clinical Trials as Topic , Humans , Maximum Tolerated Dose , Neoplasms/drug therapy , Rana pipiens , Ribonucleases/adverse effectsABSTRACT
PURPOSE: A multicenter phase II trial of ranpirnase (Onconase; Alfacell Corp, Bloomfield, NJ) as a single agent was conducted to further assess the safety and clinical efficacy of this novel antitumor ribonuclease. Patients with unresectable and histologically confirmed malignant mesothelioma (MM) were eligible. PATIENTS AND METHODS: One hundred five patients with Eastern Cooperative Oncology Group performance status 0 to 2 were enrolled onto the study. Thirty-seven percent of patients had not responded to prior chemotherapy. The primary end point of the study was survival. Tumor responses and time to progression were also assessed. The Cancer and Leukemia Group B (CALGB) prognostic group criteria were used to define a treatment target group (TTG). Both the intent-to-treat (ITT) and the TTG populations were analyzed for survival. RESULTS: Median survival times of 6 months for the ITT and 8.3 months for the TTG populations were observed. The 1- and 2-year survival rates were 34.3% and 21.6% for ITT, respectively, and 42% and 26.8% for TTG, respectively. Among the 81 patients assessable for tumor response, four had partial responses, two had minor regressions, and thirty-five experienced stabilization of previously progressive disease. Patients with responses and stable disease demonstrated markedly prolonged survival. Ranpirnase was well tolerated in the majority of patients, and there were no drug-related deaths. CONCLUSION: Ranpirnase demonstrated activity and a tolerable toxicity profile in patients with unresectable MM. The prognostic value of the CALGB groups was confirmed.
Subject(s)
Antineoplastic Agents/therapeutic use , Mesothelioma/drug therapy , Ribonucleases/therapeutic use , Aged , Antineoplastic Agents/adverse effects , Female , Humans , Infusions, Intravenous , Male , Mesothelioma/mortality , Middle Aged , Multivariate Analysis , Proportional Hazards Models , Ribonucleases/adverse effects , Statistics, Nonparametric , Survival RateABSTRACT
Ranpirnase (Onconase) is the first ribonuclease to enter cancer clinical trials. In prior phase II trials, responses were seen in mesothelioma and other solid tumors. This phase II trial tested ranpirnase (480 microg/m2/w) in 14 patients with refractory advanced renal cell cancer. The median performance status was zero and the median age was 55. All patients had prior immunotherapy and three had prior chemotherapy. No responses were seen in 14 patients. The median survival from on study was 16 months (range two to 28 months). At this dose and schedule ranpirnase has minimal activity in metastatic renal cell cancer.