Identification of a new endothelial cell growth factor receptor tyrosine kinase.

A new growth factor receptor tyrosine kinase (RTK) gene (designated KDR) has been cloned from a human endothelial cell cDNA library. The gene was identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers complementary to conserved tyrosine kinase domains that flank the insert domain, characteristic of known type III RTKs [e.g. platelet-derived growth factor receptor (PDGF-R), colony-stimulating-1 receptor (CSF-1-R), fibroblast growth factor receptor (FGF-R) and ckit]. The DNA product from PCR was then used as a probe to isolate larger DNA segments encoding the receptor from the cDNA library. The predicted amino acid sequence contained multiple characteristics (i.e. an ATP-binding site, a membrane-spanning region, split tyrosine kinase regions) typical of a type III receptor tyrosine kinase. The KDR gene is expressed as a 7.0 kb transcript, and is localized to human chromosome 4.

Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs.

The adenovirus coat protein, penton base, contains the peptide motif RGD which mediates binding to the integrin cell surface receptors alpha v beta 3 and alpha v beta 5. These integrins then mediate adenovirus internalization. We have developed penton base chimeras that recognize tissue-specific integrin receptors by replacing the wild-type RGD peptide motif with alpha v beta 3- or alpha 4 beta 1-specific peptide motifs. In one chimera the original haiRGDtfa motif was replaced with the peptide motif eiLDVpst which mediated chimera binding to the integrin alpha 4 beta 1. This integrin is expressed at high levels on lymphocytes and monocytes but is not expressed on epithelial or endothelial cells. In a second chimera the wild-type sequences flanking the RGD motif were altered to abrogate its interaction with alpha v beta 5 while retaining its specificity for alpha v beta 3. The integrin alpha v beta 5 is expressed primarily on epithelial cells whereas the integrin alpha v beta 3 is normally expressed on endothelial cells. The integrin alpha v beta 3 is also aberrantly expressed on certain metastatic melanomas and glioblastomas. A deletion mutant lacking the RGD sequence did not bind to any integrins. Such chimeras incorporated into adenovirus virions may be useful in targeting specific tissues in adenovirus-mediated gene delivery.

The KDR gene maps to human chromosome 4q31.2----q32, a locus which is distinct from locations for other type III growth factor receptor tyrosine kinases.

KDR (kinase insert domain receptor), a new type III receptor tyrosine kinase gene, maps to human chromosome 4q31.2----q32 by fluorescence in situ hybridization. This differs from the chromosomal locations of other members of this gene family.

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer.