Semirational design of a potent, artificial agonist of fibroblast growth factor receptors.

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs.

Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation.

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis.

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated.

Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation.

Identification of functional and structural amino-acid residues by parsimonious mutagenesis.

For in vitro evolution of protein function, we previously proposed using parsimonious mutagenesis (PM), a technique where mutagenic oligodeoxynucleotides (oligo) are designed to minimize coding sequence redundancy and limit the number of amino acid (aa) residues which do not retain parental structural features. For this work, PM was used to increase the affinity of C6.5, a human single-chain Fv (scFv) that binds the glycoprotein tumor antigen, c-erbB-2.

Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection.

The use of antibodies to target tumor antigens has had limited success, partially due to the large size of IgG molecules, difficulties in constructing smaller single chain Fv (scFv) antibody fragments, and immunogenicity of murine antibodies. These limitations can be overcome by selecting human scFv directly from non-immune or semi-synthetic phage antibody libraries; however, the affinities are typically too low for therapeutic application. For hapten antigens, higher-affinity scFv can be isolated from phage antibody libraries where the VH and VL genes of a binding scFv are replaced with repertoires of V genes (chain shuffling).

Affinity, specificity, and kinetics of the interaction of the SHC phosphotyrosine binding domain with asparagine-X-X-phosphotyrosine motifs of growth factor receptors.

The phosphotyrosine binding (PTB) domain specifically binds to tyrosine-phosphorylated proteins, but differs in structure and mechanism of action from the SH2 domain family. We quantitated the affinity, specificity, and kinetics of the interaction of the SHC PTB domain with a sequence motif, asparagine-X-X-phosphotyrosine (NXX(pY)), found in several receptor tyrosine kinases and oncogenic proteins. PTB domain-mediated interaction with the NXX(pY) motif of c-ErbB2 was characterized by similar overall affinity but slower kinetics than that reported for SH2 domains.

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library.

Background: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv.

Tumor targeting in a murine tumor xenograft model with the (sFv')2 divalent form of anti-c-erbB-2 single-chain Fv.

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific cross-linking of this sFv' to make divalent (sFv')2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv' and (sFv')2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv')2 demonstrated by its complex formation with ECD.

Cytotoxicity against human tumor cells mediated by the conjugate of anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain.

We have produced monoclonal antibodies against the epidermal growth factor (EGF) receptor which bind to the receptor with high affinity, compete with EGF for binding, block EGF-induced tyrosine kinase activity, and activate internalization and down-regulation of the receptor. These antibodies are cytostatic against cultured A431 cells at concentrations of 5-20 nM. In addition, they prevent the growth of A431 tumor xenografts in athymic mice.

Heterogeneity of a murine B cell lymphoma. Isolation and characterization of idiotypic variants.

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy.

Urinary excretion of modified nucleosides in patients with acquired immune deficiency syndrome (AIDS) and individuals at high risk of AIDS.

Patients with certain malignant diseases excrete in their urine elevated levels of modified nucleosides originating from breakdown of transfer RNA (tRNA). A high incidence of acquired immune deficiency syndrome (AIDS), often associated with rapidly progressing Kaposi's sarcoma (KS), is currently being observed in many countries. Male homosexuals are considered to be at highest risk of developing these disorders.

Transfer RNA breakdown products in the urine of asbestos workers.

Patients with malignant mesothelioma, a neoplasia strongly associated with previous asbestos exposure, excrete in the urine high levels of modified purines, pyrimidines, and their ribosides, breakdown products of transfer RNA. The urinary excretion levels of modified nucleosides were measured in 47 male insulation workers with long term exposure to asbestos and, therefore, at high neoplastic risk. The nucleoside levels of 44 male control subjects were used for comparison.

Biliproteins of cyanobacteria and Rhodophyta: Homologous family of photosynthetic accessory pigments.

Amino-terminal sequence determinations are reported of the subunits of biliproteins of prokaryotic unicellular and filamentous cyanobacteria and of eukaryotic unicellular red algae. The biliproteins examined, allophycocyanin, C-phycocyanin, R-phycocyanin, b-phycoerythrin, and phycoerythrocyanin, vary with respect to the chemical nature and the number and distribution of the bilin chromophores between the two dissimilar subunits. The amino-terminal sequences fall into two classes, "alpha-type" and "beta-type", with a high degree of homology within each class.