Biomedical subjects

R Bordens

Publications and source records attributed to R Bordens.

7 recordsLinked to original sources

PubMed2006

Biological activities of a recombinant adenovirus p53 (SCH 58500) administered by hepatic arterial infusion in a Phase 1 colorectal cancer trial.

The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.

Adenoviridae↗

PubMed2001

Structural and biologic characterization of pegylated recombinant IFN-alpha2b.

The type I interferon-alpha (IFN-alpha) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-alpha2b (Intron A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-alpha2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-alpha2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed that approximately 50% of PEG Intron was monopegylated on the His(34) residue of the IFN-alpha2b protein. The highest antiviral activity of the pegylated positional isomers for PEG Intron was associated with the His(34) pegylated isomer. The specific activity for PEG Intron in an antiviral cytopathic protection assay was 28%, relative to Intron A. However, the potency of PEG Intron, defined as bioactivity independent of protein concentration, was comparable to Intron A at both the molecular and cellular levels in a battery of in vitro assays. Equivalent units of PEG Intron and Intron A were indistinguishable for the induction of several key IFN-induced genes, including 2',5'-oligoadenylate synthetase (2',5'-OAS) and protein kinase R (PKR), in Molt 4 cells. The antiviral dose-response curves revealed that there were no significant differences between PEG Intron and Intron A. This demonstrated that the introduction of more IFN-alpha2b protein associated with equivalent unit dosing of PEG Intron did not create any antagonism or agonism in the antiviral assay. In assays for the immune response, PEG Intron and Intron A displayed comparable potency for both natural-killer (NK) and lymphokine-activated killer (LAK) cell cytolytic activity and for the induction of class I major histocompatibility protein. These results demonstrate that PEG Intron maintains an in vitro biologic potency profile for both antiviral and immunotherapeutic activity that is highly comparable to that of Intron A.

Antineoplastic Agents↗

PubMed1999

Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector.

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.

Adenovirus E1A Proteins↗

PubMed1998

Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons.

IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago. Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases. Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine. Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction. Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes. We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans. These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products. Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized.

Antineoplastic Agents↗

PubMed1998

Review of recent developments in the molecular characterization of recombinant alfa interferons on the 40th anniversary of the discovery of interferon.

Recombinant alfa interferons (IFN-alpha s) are approved worldwide for the treatment of a variety of cancers and diseases of virologic origin. A series of recent advances in the molecular characterization of recombinant IFN-alpha s have allowed the determination of the three-dimensional IFN-alpha 2b structure by high-resolution x-ray crystallography. We review here recent developments in our understanding of the molecular and physicochemical properties of recombinant IFN-alpha, including our current state of knowledge of the IFN-alpha gene family and the multiple species of human leukocyte IFN. Based on the reported three-dimensional structure of IFN-alpha 2b, we propose a molecular model for the IFN-alpha 2b receptor complex and predict models for the naturally occurring subtypes IFN-alpha 1 and IFN-alpha 8, as well as the synthetic, non-naturally occurring consensus IFN. Such models provide molecular insights into the mechanism of action of IFN-alpha.

Humans↗

PubMed1997

Validation parameters for a novel biosensor assay which simultaneously measures serum concentrations of a humanized monoclonal antibody and detects induced antibodies.

This report documents the validation of an assay using BIAcore 2000 that, with a single injection of mouse serum, allows the quantitation of a humanized monoclonal antibody and can also detect the presence of antibodies directed against this humanized antibody. The important components required for the validation of biosensor assays including precision, accuracy, linearity, stability of the immobilized ligand, specificity and sensitivity are addressed. The tandem assay that is used as a model for biosensor validations is accomplished by flowing each sample across the surface of two flowcells in sequence. The first flowcell has the antigen that the humanized mAb was generated against immobilized while the humanized mAb itself is immobilized on the second flowcell. The quantitation component of this assay is precise and accurate with a limit of quantitation of 1 microg/ml in mouse serum samples. Any antibodies directed against the humanized mAb can be detected and also characterized as to isotype. This unique assay can be performed with as little as 10 microl of serum which is then diluted ten-fold prior to analysis. The small sample requirement allows analysis of individual mouse serum samples rather than requiring the use of pooled samples from multiple mice. A further advantage of this assay is the automation capability which allows unattended operation.

Animals↗

PubMed1997

Molecular and biologic characterization of recombinant interferon-alpha2b.

Recombinant IFN-alpha2b (Intron A, Schering-Plough Corp, Kenilworth, NJ), derived from Escherichia coli, is currently approved worldwide for the therapy of various cancers and chronic hepatitis B and C. We describe here the purity and identity tests for both physicochemical properties and bioactivity that have been developed for the characterization of highly purified IFN-alpha2b with a specific activity of 2.5 x 10(8) IU/mg protein. The data indicate that a product of high purity can be consistently produced without DNA contamination. The antiviral bioassay chosen to measure potency is based on the ability of IFN-alpha2b to protect human foreskin fibroblast FS-71 cells from the cytopathic effects of encephalomyocarditis virus. Various immunoassays are described that have been used to quantitate the presence of binding and neutralizing antibodies in patients treated with IFN-alpha2b. Overall, the available data indicate a very low incidence of neutralizing antibody formation. We also summarize the current state of knowledge with respect to IFN reference standards for the biologic assay as well as recommendations for the calculation of antibody neutralization titers.

Antiviral Agents↗