During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24 -197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH 2 terminus of the protein on the ␣-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell-based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 Å resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin-7-yl-cyclopentane at 1.6 Å resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

The lymphotoxin-B receptor (LTBR) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LTBR monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LTBR caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LTBR activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LTBR with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

SummarySurface lymphotoxin (LT) is a heteromeric complex of LT-c~ and LT-[3 chains that binds to the LT-I3 receptor (LT-I3-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.g., the TNF and Fas receptors, it is important to determine if similar signaling events can be communicated via the LT-13-tL. A soluble form of the surface complex was produced by coexpression of LT-c~ and a converted form of LT-[3 wherein the normally type II LT-[3 membrane protein was changed to a type I secreted form. Recombinant LT-oq/[32 was cytotoxic to the human adenocarcinoma cell lines HT-29, WiDr, MDA-MB-468, and HT-3 when added with the synergizing agent interferon (IFN) y. When immobilized on a plastic surface, anti-LT-[3-tL monoclonal antibodies (mAbs) induced the death of these cells, demonstrating direct signaling via the LT-I3-R. Anti-LT-[3-R mAbs were also identified that inhibited ligand-induced cell death, whereas others were found to potentiate the activity of the ligand when added in solution. The human WiDr adenocarcinoma line forms solid tumors in immunocompromised mice, and treatment with an anti-LT-~-R, antibody combined with human IFN-~/arrested tumor growth. The delineation of a biological signaling event mediated by the LT-I3-R opens a window for further studies on its immunological role, and furthermore, activation of the LT-I3-R may have an application in tumor therapy.T he TNF family ofligands and receptors is a set of regulatory elements in the immune system (t). TNF was discovered as a cytolytic agent circulating in the blood of endotoxin-stimulated animals (2-4). Originally cloned in the expectation that TNF would be a novel antitumor agent, it was later shown that its primary physiologic function lies in initiating the inflammatory cascade underlying the host's immediate defensive response to infection or stress. More complex immunological functions have been described (5,6). Lymphotoxin (LT) 1 e~ (also called TNF-I3) is a similar cytokine secreted by activated lymphocytes (7) and was originally characterized as having the same functions as TNF. Later, activated T and B cells were found to display LT-c~ on their surfaces in an unusual form compIexed with another member of the TNF family called LT-13 in an . A complex with an apparent 1 Abbreviations used in this paper: LT, lymphotoxin; LT-I3-1L, LT-[3 receptor; MTT, 3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase UTP nick-end labeling; VCAM, vascular cell adhesion molecule.LT-c~2/I31 stoichiometry is also present, but only in minor amounts on human lymphocytes. The major LT-cq/f3 z form does not bind to the known TNF receptors, referred to here as TNF-R55 and TNF-R.75, but rather interacts with another receptor in the TNF family called the LT-[3 receptor 14).Currently, the function of the LT system is poorly character...

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membranebound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPIanchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.The Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) 2 family of genes, including mammalian Cripto-1 (CR-1/Tdgf1), has been shown to play an important role in vertebrate development and in tumor progression (1). EGF-CFC proteins are indispensable for early embryonic development, being involved in the formation of the primitive streak, patterning of the anterior/posterior axis, mesoendoderm formation, and establishment of left-right asymmetry (2). CR-1 is an oncofetal gene, which is expressed during early developmental stages and is re-expressed in several types of human carcinomas (3). In fact, CR-1 protein is highly expressed in a number of human carcinomas, including breast (75-82%), colon (67-84%), and gastric cancer (33-47%), where it is being assessed as a tumor-specific target for immunotherapy (3, 4). Mice that overexpress a human CR-1 transgene selectively in mammary epithelium develop mammary hyperplasia and adenocarcinomas (5-7). Conversely, blockade of CR-1-mediated signaling suppresses tumor growth (4, 8, 9).CR-1 functions through at least three different signaling pathways: 1) as a co-receptor for the transforming growth factor ␤-related proteins Nodal and growth and differentiation factors 1 and 3 (10 -12), 2) as a ligand for glypican-1/c-Src/ MAPK/PI3K-Akt signaling (13), and 3) as an inhibitor for activin/transforming growth factor-␤ signaling (8,14). Nodal requires EGF-CFC proteins as co-receptors to bind the activin type I receptors (activin-like kinases 4 and 7) and activin type II receptor (ActRII). Embryological defects in CR-1-null mice are lethal mainly due to a disruption of Nodal-dependent signaling (15). CR-1 can also function independently of Nodal as a ligand for glypican-1, which can act...

Cripto, a cell surface-associated protein belonging to the EGF-CFC family of growth factor-like molecules, is overexpressed in many human solid tumors, including 70-80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-β ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-β ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti-CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin Binduced growth suppression by blocking Cripto's association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development.

Cripto, a cell surface-associated protein belonging to the EGF-CFC family of growth factor-like molecules, is overexpressed in many human solid tumors, including 70-80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-β ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-β ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti-CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin Binduced growth suppression by blocking Cripto's association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.