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Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

We report the identification of rat and human cDNAs for a type 1 membrane protein that contains a novel six-cysteine immunoglobulin-like domain and a mucin domain; it is named kidney injury molecule-1 (KIM-1). Structurally, KIM-1 is a member of the immunoglobulin gene superfamily most reminiscent of mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Human KIM-1 exhibits homology to a monkey gene, hepatitis A virus cell receptor 1 (HAVcr-1), which was identified recently as a receptor for the hepatitis A virus. KIM-1 mRNA and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney. In situ hybridization and immunohistochemistry revealed that KIM-1 is expressed in proliferating bromodeoxyuridine-positive and dedifferentiated vimentin-positive epithelial cells in regenerating proximal tubules. Structure and expression data suggest that KIM-1 is an epithelial cell adhesion molecule up-regulated in the cells, which are dedifferentiated and undergoing replication. KIM-1 may play an important role in the restoration of the morphological integrity and function to postischemic kidney.

The members of the tumor necrosis factor (TNF) family play pivotal roles in the regulation of the immune system. Here we describe a new ligand in this family, designated TWEAK. The mouse and human versions of this protein are unusually conserved with 93% amino acid identity in the receptor binding domain. The protein was efficiently secreted from cells indicating that, like TNF, TWEAK may have the long range effects of a secreted cytokine. TWEAK transcripts were abundant and found in many tissues, suggesting that TWEAK and TRAIL belong to a new group of widely expressed ligands. Like many members of the TNF family, TWEAK was able to induce interleukin-8 synthesis in a number of cell lines. The human adenocarcinoma cell line, HT29, underwent apoptosis in the presence of both TWEAK and interferon-gamma. Thus, TWEAK resembles many other TNF ligands in the capacity to induce cell death; however, the fact that TWEAK-sensitive cells are relatively rare suggests that TWEAK along with lymphotoxins alpha/beta and possibly CD30L trigger death via a weaker, nondeath domain-dependent mechanism.

The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells. The signaling function of this receptor-ligand pair is not precisely known but is believed to be involved in the development of the peripheral lymphoid organs. To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired. The LT-beta molecule was engineered into a secreted form and co-expressed with LT-alpha using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-alpha3, LT-alpha2/beta1, and LT-alpha1/beta2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-alpha3-like components in the LT-alpha1/beta2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors. Heteromeric LT complexes were prepared with this mutant LT- alpha form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors. A LT-alpha3 based cytotoxic activity was used to show that the LT-alpha1/beta2 form cannot readily scramble into a mixture of forms following various treatments and storage periods. This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analysis of the specificity of the LT-bet a and TNF receptors for the various LT forms.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. VCAM1 cDNAs cloned from mouse, rat, rabbit, and human libraries contain six, seven, or eight extracellular Ig-like domains generated by alternate splicing, but to date shorter forms have not been found. We have cloned a novel cDNA encoding only the three N-terminal domains of murine VCAM1 followed by a unique C-terminal tail generated by alternate splicing of a previously undescribed exon. This truncated form of murine VCAM1 (3D-VCAM1) is expressed in COS cells as a functional adhesion molecule which is lost from the cell surface following treatment with phosphatidylinositol-specific phospholipase C. 3D-VCAM1 is found only in endotoxin-treated but not control murine and rat tissues. Thus in rodents alternate splicing of the VCAM1 gene generates a unique truncated inflammation-specific phosphatidylinositol-linked form of VCAM1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.

Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.

Purification of the dihydrofolate reductases overproduced by 16 independently-derived antifolate-resistant sublines of Chinese hamster lung cells and their drug-sensitive parental cell line, DC-3F, has confirmed our original observation that two molecular weight classes of this enzyme, viz. Mr = 21,000 can be overproduced by drug-resistant cells (Melera, P. W., Wolgemuth, D., Biedler, J. L., and Hession, C. (1980) J. Biol. Chem. 255, 319-322). Of the 16 drug-resistant sublines analyzed, 12 overproduce the 21,000-dalton enzyme, while 4 overproduce the 20,000-dalton enzyme. Both molecular weight classes are found in the drug-sensitive parental line. Two-dimensional polyacrylamide gel electrophoresis of the two molecular weight classes of enzyme has shown that each is composed of two isoelectric forms, and that the relative distributions of the isoelectric forms within a molecular weight class are maintained during overproduction. In vitro translation experiments have demonstrated that the synthesis of these isoelectric forms and their relative distributions are mediated by mRNA. Finally, evidence is presented for the existence of two sets of three dihydrofolate reductase mRNAs in overproducing sublines. Both sets, within a subline, encode the same molecular weight class of dihydrofolate reductase, while each set directs the synthesis of only one of the isoelectric forms within that class. Although the genetic basis for these observations is not yet understood, it is clear that the production of dihydrofolate reductase by Chinese hamster lung cells is complex and probably involves the expression of multiple dihydrofolate reductase genes and/or alleles.

We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.

Purification of a cDNA probe specific for dihydrofolate reductase mRNA has allowed the relative quantification of dihydrofolate reductase gene numbers and mRNA levels between a line of Chinese hamster lung fibroblasts sensitive to the antifolate drugs methotrexate and methasquin and sublines which by virtue of their overproduction of the target enzyme dihydrofolate reductase are cross-resistant to these site-specific inhibitors. Previous results have shown that resistant sublines could overproduce one of two molecular weight forms of dihydrofolate reductase, one parental-like (Mr = 21,000) and one nonparental-like (Mr = 20,000), each encoded by a different mRNA present in elevated amounts in resistant cells (Melera, P. W., Wolgemuth, D., Biedler, J. L., and Hession, C. (1980). J. Biol. Chem. 255, 319--322). The cDNA-RNA and cDNA-DNA hybridization experiments reported here confirm the elevated levels of dihydrofolate reductase-specific mRNA in resistant cells and show that the overproduction of either molecular weight form of dihydrofolate reductase is accompanied by amplification in dihydrofolate reductase gene number.

A comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins synthesized by antifolate-sensitive (DC-3F) and -resistant (DC-3F/A3 and DC-3F/MQ19) Chinese hamster lung fibroblast cells has shown that an apparent molecular weight difference exists between the dihydrofolate reductases overproduced by the resistant sublines. Translation in a rabbit reticulocyte lysate system of polysomal and cytoplasmic mRNAs from the three sublines has shown that the overproduction of dihydrofolate reductase by resistant cells is accompanied by increased levels of dihydrofolate reductase-specific mRNA and that the different molecular weight forms of dihydrofolate reductase are encoded by their respective mRNAs.