Phage Display Selection of scFv to Murine Endothelial Cell Membranes

Abstract

The diversity of endothelial cells is becoming more apparent and more important in defining vessel systems that supply blood to normal organs and to tumors. Reagents that identify expression of cell surface determinants on these cells are crucial for differentiating among different vessel types. As a first step in this process we have selected a panel of 25 scFvs from a phage display library that bind to the endothelial cell line LEII. The scFvs are of high affinity and bind to some tumor cells as well as to the target endothelial cell. The scFvs can be divided into 8 epitope groups by use of competition binding studies. DNA sequencing of the members of these groups generally support the classification. This work shows that phage display is a rapid and efficient method for identification of reagents for cell surface molecules.

Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

“…Soluble scFv fragments protein expression levels for these clones were in the range of 0.044 to 1.8 mg/liter of culture. These yields are in general agreement with published yields in the range of 0.1 to 5.0 mg per liter of E. coli culture for clones isolated from the Tomlinson libraries (Kennel et al, 2004; Wu et al, 2007; Lobova et al, 2008). SDS-PAGE analysis for these four clones show bands corresponding approximately to 26–28 kD molecular weight when compared to the 25 kD band of the protein standard ladder run in parallel with the samples (Fig.…”

Biomedical aspects of targeted delivery of drugs to pulmonary endothelium

“…This method uses repetitive cycles of intravenous injections of phages encoding stochastic peptides, which may bind to sites accessible to the circulation, followed by the identification of specific phages homing into selected tissues and the eventual isolation of the homing determinants [151]. This method becomes more popular in the analysis of specific endothelial surface determinants, especially those characteristic of pathologically altered endothelium in tumours and atherosclerotic lesions, both in cell cultures and in vivo [55,[152][153][154][155][156][157]. Some of the affinity ligands defined by this methodology, including those binding to previously identified endothelial determinants including adhesion molecules and ectopeptidases, are currently being explored for vascular targeting of imaging probes and drugs in animals [158].…”

Identification of an Antilaminin-1 scFv That Preferentially Homes to Vascular Solid Tumors

“…Mice were sacrificed after 1 hour and biodistribution was determined. 8,24 Immunohistochemistry After deparaffinization, slides of 5-m-thick tumor sections were deparaffinized and treated for antigen retrieval with citrate buffer pH 6.0 (for vWF and CD34 staining) and pepsin (Biogenex; San Ramon, CA) digestion for laminin staining. Slides were blocked prior to the primary antibody incubation.…”

“…We have tested several libraries for the identification of specific scFvs. 24 The Tomlinson I and J scFv phage libraries were selected for this study, as they have a stable framework, have been preselected for properly folded scFv, and employ a trypsin-sensitive site as a specific means to elute selected phage from their target. In this study, we identified 17 antilaminin-1 scFvs, representing at least four epitope groups.…”