Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" receptor tyrosine kinases (RTKs) in which the ligands are unknown. Ligand-mediated activation of RTKs is important throughout development. ALK is particularly relevant to the development of the nervous system. Increased activation of RTKs by mutation, genetic amplification, or signals from the stroma contributes to disease progression and acquired drug resistance in cancer. Aberrant activation of ALK occurs in subsets of lung adenocarcinoma, neuroblastoma, and other cancers. We found that heparin is a ligand that binds specifically to the ALK extracellular domain. Whereas heparins with short chain lengths bound to ALK in a monovalent manner and did not activate the receptor, longer heparin chains induced ALK dimerization and activation in cultured neuroblastoma cells. Heparin lacking N- and O-linked sulfate groups or other glycosaminoglycans with sulfation patterns different than heparin failed to activate ALK. Moreover, antibodies that bound to the extracellular domain of ALK interfered with heparin binding and prevented heparin-mediated activation of ALK. Thus, heparin and perhaps related glycosaminoglycans function as ligands for ALK, revealing a potential mechanism for the regulation of ALK activity in vivo and suggesting an approach for developing ALK-targeted therapies for cancer.

ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.cancer | surface receptor | cell signaling | therapeutic antibodies | crystal structure T he EGF receptor (EGFR)/ErbB family of receptor tyrosine kinases (RTKs) participates in a multitude of roles during embryonic development and in adult homeostasis. In healthy tissues, ErbB signal transduction is initiated through ligandinduced homo-or heterodimerization of receptor extracellular domains leading to the stimulation of tyrosine kinase activity and autophosphorylation of several tyrosine residues in the cytoplasmic domain followed by recruitment and activation of multiple downstream signaling pathways (1). In contrast, unregulated ErbB signaling through activating mutations, receptor overexpression, or aberrant autocrine ligand signaling loops can lead to cellular transformation and tumorigenesis (2). Thus, members of the ErbB receptor family (in particular EGFR and ErbB2) have become well-validated targets for the development of anticancer therapeutics, resulting in a number of Food and Drug Administration-approved and marketed monoclonal antibodies and small-molecule tyrosine kinase inhibitors used for treatment of different cancers (3).The activity of ErbB3 (also designated HER3) is normally regulated by the neuregulin (NRG) family of growth factors (4), but, unlike other members of the family, ErbB3 functions as an obligate heterodimer with other ErbB receptors because its cytoplasmic domain contains a pseudo kinase in place of a tyrosine kinase domain (5, 6). ErbB3 therefore takes part in heterodimer formation through ligand binding, whereas its coreceptor (most often ErbB2) provides enzymatic activity to phosphorylate multiple tyrosine residues located primarily in the ErbB3 C-terminal tail. The role of ErbB3 in cancer has been fully appreciated only within the last decade and has prompted the development of several monoclonal antibodies geared at inhibiting its action in solid tumors. ErbB3 phosphorylation and subsequent signaling have been associated wi...

Objective. To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents.Methods. Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression.Results. Treatment of HSE with interleukin-la (IL-la) or tumor necrosis factor a (TNFa) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-y (IFNy) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFNy and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, tluorescenceactivated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN y also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNFPllymphotoxin. an identical manner. Unexpectedly, IFNy alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE.Conclusion. These studies indicate that IFNy plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

Head and neck squamous cell carcinoma (HNSCC) accounts for 3–5% of all tumor types and remains an unmet medical need with only two targeted therapies approved to date. ErbB3 (HER3), the kinase-impaired member of the EGFR/ErbB family, has been implicated as a disease driver in a number of solid tumors, including a subset of HNSCC. Here we show that the molecular components required for ErbB3 activation, including its ligand neuregulin-1 (NRG1), are highly prevalent in HNSCC and that HER2, but not EGFR, is the major activating ErbB3 kinase partner. We demonstrate that cetuximab treatment primarily inhibits the ERK signaling pathway and KTN3379, an anti-ErbB3 monoclonal antibody, inhibits the AKT signaling pathway, and that dual ErbB receptor inhibition results in enhanced anti-tumor activity in HNSCC models. Surprisingly, we found that while NRG1 is required for ErbB3 activation, it was not sufficient to fully predict for KTN3379 activity. An evaluation of HNSCC patient samples demonstrated that NRG1 expression was significantly associated with expression of the EGFR ligands amphiregulin (AREG) and transforming growth factor α (TGFα). Furthermore, NRG1-positive HNSCC cell lines that secreted high levels of AREG and TGFα or contained high levels of EGFR homodimers (H11D) demonstrated a better response to KTN3379. Although ErbB3 and EGFR activation are uncoupled at the receptor level, their respective signaling pathways are linked through co-expression of their respective ligands. We propose that NRG1 expression and EGFR activation signatures may enrich for improved efficacy of anti-ErbB3 therapeutic mAb approaches when combined with EGFR-targeting therapies in HNSCC.