Biomedical subjects

M Wittmer

Publications and source records attributed to M Wittmer.

13 recordsLinked to original sources

PubMed2003

Single-chain antibody and its derivatives directed against vascular endothelial growth factor: application for antiangiogenic gene therapy.

Single-chain antibodies (scFv) have an enormous potential for clinical application. However, rapid blood clearance and difficulties in large-scale production of active scFvs have limited the practical use of these antibody fragments. Recently, an anti-vascular endothelial growth factor (VEGF) scFv (scFv V65) was selected in our laboratory from a human antibody phage-display library. This antibody was able to reduce tumor growth in mice by approximately 50%. Here, we employ a gene therapy strategy for sustained in vivo expression of scFv V65 and its derivatives. scFv fusion proteins containing parts of the constant IgG1 region were generated (minibody and scFv V65-Fc) to increase the serum half-life of the scFv V65. Systemic administration of recombinant adenovirus encoding scFv V65 resulted in substantial tumor inhibition. This effect could be improved by multiple virus injections. We found that the efficacy of different scFv V65 formats was dependent on the mode of administration: whereas scFv V65-Fc was the most efficient when expressed locally, scFv V65 was superior when delivered systemically. Our results show that therapeutic levels of scFv V65 can be obtained by systemic injection of recombinant adenoviruses. Therefore, therapeutic gene delivery of scFv is a feasible strategy that overcomes several limitations of conventional antibody therapy.

Adenoviridae↗

PubMed2001

Signal-sequence trap in mammalian and yeast cells: a comparison.

Membrane associated and secreted proteins are translated as precursors containing a signal peptide that allows protein-insertion into the membrane of the endoplasmic reticulum and is co-translationally removed in the lumen. The ability of the signal peptide to direct a polypeptide into the secretory pathway is exploited in methods developed to select cDNAs encoding such proteins. Different strategies are known in which cDNA libraries can be screened for signal peptides by the ability of the latter to rescue the translocation of signal sequence-less proteins. In one method, a cDNA library is tested for interleukin 2 receptor alpha chain translocation to the membrane in COS cells, in another one for invertase secretion from yeast. In this work, we compared the two systems by testing six mouse signal peptides in COS and yeast cells. All of them were functional in the mammalian system, whereas only three of them in yeast. Two other sequences needed the 5' cDNA sequence flanking the ATG codon to be removed in order to enable protein translocation. Although the structure of signal sequences and the functioning of the secretory machinery are well conserved from prokaryotes to eukaryotes, it seems evident that not all signal peptides can be interchanged between different proteins and organisms. In particular, signal peptides that are functional in the mammalian system do not necessarily lead to protein translocation in yeast.

Animals↗

PubMed2000

Increased expression of H/T-cadherin in tumor-penetrating blood vessels.

Neovascularization is a prerequisite for tumor growth. Thus, selective destruction of the tumor vasculature should prevent tumor expansion. We have established a method to identify proteins that are specifically expressed on the surface of endothelial cells in tumors. CD31-positive endothelial cells were isolated from Lewis lung carcinoma lung metastases as well as from normal lung tissue. cDNAs derived from these cells were subjected to a subtractive hybridization procedure, and cDNAs overrepresented in tumor-derived endothelial cells were isolated; those encoding surface proteins were selected using a signal sequence trap assay. One isolated cDNA encoded H/T-cadherin. In this report, we show that mouse H/T-cadherin is overexpressed on endothelial cells of several tumors, whereas it is expressed only on a subset of endothelial cells in healthy organs. On the basis of the expression of H/T-cadherin in lung metastases of different tumors, we suggest that different tumors can have a differential influence on the expression of endothelial cell surface proteins.

Amino Acid Sequence↗

PubMed2000

Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor.

Monoclonal antibody (Ab) directed against the vascular endothelial growth factor, one of the major inducers of angiogenesis, can inhibit tumor growth in mice. Treatment of cancer patients with monoclonal Ab requires large-scale production of the clean Ab and frequent application of the Ab. This might be improved by using single-chain Ab fragments (scFvs), which can be produced in large quantities in bacteria and are attractive for gene therapeutic approaches. Here we describe anti-vascular endothelial growth factor scFvs derived from a human phage-display library able to block the vascularization of the chorioallantoic membrane of chick embryos and reduce the growth of s.c. tumors in nude mice. This work opens the way to develop gene therapy-based strategies using a scFv to treat angiogenesis-dependent diseases.

3T3 Cells↗

PubMed1993