We have identified an amino acid sequence within the E peptide of the insulin-like growth factor IB (IGF-IB) precursor that is biologically active and designated this peptide insulin-like growth factor IB-(103-124) E1 amide (IBEl). Its existence was predicted by a flanking Gly-Lys-LysLys, a signal sequence for sequential proteolytic cleavage and peptidyl C-terminal amidation. A synthetic analog of the predicted IBE, peptide, designated Y-23-R-NH2, was generated with tyrosine added at position 0. This peptide at 2-20 nM had growth-promoting effects on both normal and malignant human bronchial epithelial cells. Y-23-R-NH2 bound to specific high-affinity receptors (Kd = 2.8 ± 1.4 x 10-11 M) present at 1-2 x 104 binding sites per cell. Ligand binding was not inhibited by recombinant insulin or recombinant IGF-I. Furthermore, a monoclonal antibody antagonist to the IGF-I receptor (aIR3) did not suppress the proliferative response induced by Y-23-R-NH2. In addition, C-terminal amidation was shown to be important in receptor recognition since the free-acid analog of IBE1 (Y-23-R-OH) did not effectively compete for binding and was not a potent agonist of proliferation. Immunoblot analysis of human lung tumor cell line extracts using an antibody raised against Y-23-R-NH2 detected a low molecular mass band of -5 kDa, implying that a protein product is produced that has immunological similarity to IBE1.Extracts of human, mammalian, and avian livers analyzed on an immunoblot with the anti-Y-23-R-NH2 antibody contained proteins of :21 kDa that were specifically recognized by the antiserum and presumably represent an IGF-I precursor molecule. This implies that in species where an IGF-I mRNA with homology to the human IGF-IB E domain has not yet been described, an alternate mRNA must be produced that contains a sequence similar to that of the midportion of the human IGF-IB E domain. Our rmdings demonstrate that IBE1 is a growth factor that mediates its effect through a specific highaffinity receptor and is most likely conserved in many species.

Most drugs currently used for human therapy interact with proteins, altering their activity to modulate the pathological cell physiology. In contrast, 2-hydroxy-9-cis-octadecenoic acid (Minerval) was designed to modify the lipid organization of the membrane. Its structure was deduced following the guidelines of the mechanism of action previously proposed by us for certain antitumor drugs. The antiproliferative activity of Minerval supports the above-mentioned hypothesis. This molecule augments the propensity of membrane lipids to organize into nonlamellar (hexagonal H II ) phases, promoting the subsequent recruitment of protein kinase C (PKC) to the cell membrane. The binding of the enzyme to membranes was marked and significantly elevated by Minerval in model (liposomes) and cell (A549) membranes and in heart membranes from animals treated with this drug. In addition, Minerval induced increased PKC␣ expression (mRNA and protein levels) in A549 cells. This drug also induced PKC activation, which led to a p53-independent increase in p21 CIP expression, followed by a decrease in the cellular concentrations of cyclins A, B, and D3 and cdk2. These molecular changes impaired the cell cycle progression of A549 cells. At the cellular and physiological level, administration of Minerval inhibited the growth of cancer cells and exerted antitumor effects in animal models of cancer without apparent histological toxicity. The present results support the potential use of Minerval and related compounds in the treatment of tumor pathologies.

Immunotoxins were made using five different murine monoclonal antibodies to the human erbB2 gene product and LysPE40, a 40-kDa recombinant form ofPseudomonas exotoxin (PE) lacking its cell-binding domain. All five conjugates were specifically cytotoxic to cancer cell lines overexpressing erbB2 protein. The most active conjugate was e23-LysPE40, generated by chemical crosslinking of anti-erbB2 monoclonal antibody e23 to LysPE40. In addition, a recombinant immunotoxin, e23(Fv)PE40, was constructed that consists of the light-chain variable domain of e23 connected through a peptide linker to its heavy-chain variable domain, which in turn is fused to PE40. The recombinant protein was made in Escherichia coli, purified to near homogeneity, and shown to selectively kill cells expressing the erbB2 protooncogene. To improve the cytotoxic activity of e23(Fv)PE40, PE40 was replaced with a variant, PE38KDEL, in which the carboxyl end of PE is changed from Arg-Glu-Asp-Leu-Lys to Lys-Asp-GluLeu and amino acids 365-380 of PE are deleted. The e23(Fv)PE38KDEL protein inhibits the growth of tumors formed by the human gastric cancer cell line N87 in immunodeficient mice.

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using '25I-labeled IGF