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Biomedical subjects

David Vogel

Publications and source records attributed to David Vogel.

6 recordsLinked to original sources
PubMed2006

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

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PubMed2006

Antibodies neutralizing hepsin protease activity do not impact cell growth but inhibit invasion of prostate and ovarian tumor cells in culture.

Hepsin is a type II transmembrane serine protease that is expressed in normal liver, and at lower levels in kidney, pancreas, and testis. Several studies have shown that hepsin mRNA is significantly elevated in most prostate tumors, as well as a significant fraction of ovarian and renal cell carcinomas and hepatomas. Although the overexpression of mRNA in these tumors has been extensively documented, there has been conflicting literature on whether hepsin plays a role in tumor cell growth and progression. Early literature implied a role for hepsin in human tumor cell proliferation, whereas recent studies with a transgenic mouse model for prostate cancer support a role for hepsin in tumor progression and metastases. To evaluate this issue further, we have expressed an activatable form of hepsin, and have generated a set of monoclonal antibodies that neutralize enzyme activity. The neutralizing antibodies inhibit hepsin enzymatic activity in biochemical and cell-based assays. Selected neutralizing and nonneutralizing antibodies were used in cell-based assays with tumor cells to evaluate the effect of antibodies on tumor cell growth and invasion. Neutralizing antibodies failed to inhibit the growth of prostate, ovarian, and hepatoma cell lines in culture. However, potent inhibitory effects of the antibodies were seen on invasion of ovarian and prostate cells in transwell-based invasion assays. These results support a role for hepsin in tumor cell progression but not in primary tumor growth. Consistent with this, immunohistochemical experiments with a mouse monoclonal antibody reveal progressively increased staining of prostate tumors with advanced disease, and in particular, extensive staining of bone metastatic lesions.

Amino Acid Sequence↗
PubMed2006

Anti-EphA2 antibodies decrease EphA2 protein levels in murine CT26 colorectal and human MDA-231 breast tumors but do not inhibit tumor growth.

The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb) that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of EphA2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose cross reactivity with murine EphA2 is unknown, we generated a mAb (Ab20) that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to approximately 12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7). No inhibition of humanMDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies.

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PubMed2004

Interferon receptor expression regulates the antiproliferative effects of interferons on cancer cells and solid tumors.

In addition to antiviral effects, Type I interferons (IFN) have potent antiproliferative and immunomodulatory activities. Because of these properties IFNs have been evaluated as therapeutics for the treatment of a number of human diseases, including cancer. Currently, IFNs have been shown to be efficacious for the treatment of only a select number of cancers. The reason for this is unclear. Recent evidence has demonstrated that some cancer cell types seem to be defective in their ability to respond to IFN. It has been suggested that defects in IFN signaling is one mechanism by which cancer cells escape responsiveness to Type I IFNs and growth control in general. We report that transfection and enhanced expression of the Type I IFN receptor chain (IFNAR2c) in 3 different human cancer cell lines markedly increases the sensitivity of these cells to the antiproliferative effects of IFNs. In cancer cells transfected with IFNAR2c, dose response curves demonstrate a significant decrease in the concentrations of IFN required to achieve maximum cell death. Furthermore, in these transfected cells, we observe a significant increase in the number of cells undergoing apoptosis, as measured by DNA fragmentation and Caspase 3 activation. In addition, using an in vivo xenograft tumor model we show an increase in the effectiveness of systemically delivered Betaseron in decreasing tumor burden in animals in which solid tumors were generated from IFNAR2c transfected cells. These data show that specific regulation of IFN receptor expression can play a major role in determining the clinical outcome of IFN-based cancer therapeutics by regulating the relative sensitivity of cancer cells to IFN-dependent growth control.

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PubMed2001

Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

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