The selective adhesion molecule inhibitor Natalizumab decreases multiple myeloma cell growth in the bone marrow microenvironment: therapeutic implications.

Recent advances regarding the introduction of anti-adhesion strategies as a novel therapeutic concept in oncology hold great promise. Here we evaluated the therapeutic potential of the new-in-class-molecule selective-adhesion-molecule (SAM) inhibitor Natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin-α4, in multiple myeloma (MM). Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non-cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, Natalizumab blocked both the proliferative effect of MM-bone marrow (BM) stromal cell interaction on tumour cells, and vascular endothelial growth factor (VEGF)-induced angiogenesis in the BM milieu. Moreover, Natalizumab also blocked VEGF- and insulin-like growth factor 1 (IGF-1)-induced signalling sequelae triggering MM cell migration. In agreement with our in vitro results, Natalizumab inhibited tumour growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment. Importantly, Natalizumab not only blocked tumour cell adhesion, but also chemosensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM-stroma cell co-culture system model. Our data therefore provide the rationale for the clinical evaluation of Natalizumab, preferably in combination with novel agents (e.g. bortezomib) to enhance MM cytotoxicity and improve patient outcome.

CRIPTO3, a presumed pseudogene, is expressed in cancer.

Cripto is a cell surface protein highly expressed in certain solid tumors, and overexpression of Cripto protein is oncogenic. Cripto-1 protein is encoded by CRIPTO1 gene. CRIPTO3, a presumed pseudogene, has an open reading frame with six amino acid differences from Cripto-1. We show that CRIPTO3 mRNA is the CRIPTO message expressed in many cancer samples. A CRIPTO3 SAGE tag was found in several cancer SAGE libraries, while the CRIPTO1 tag was found in ES cell libraries. In vitro experiments indicate both Cripto-1 and Cripto-3 proteins are functional in the Nodal-dependent signal pathway. Our data indicate that CRIPTO3 is an expressed gene, particularly in certain cancers, and suggest a potentially novel mechanism of oncogenesis through activation of a retrogene.

Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo.

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-beta ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-beta 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 B-induced 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.

Anti-alpha4 integrin monoclonal antibody inhibits multiple myeloma growth in a murine model.

In a syngeneic murine model of multiple myeloma with many of the characteristics of the human disease, a monoclonal antibody (mAb) to the integrin very late antigen-4 (VLA-4), given after the myeloma has already homed to and begun to establish itself within the bone marrow compartment, produces statistically significant effects on multiple disease variables. These include reductions in circulating levels of IgG2b; percentage of IgG2b-positive myeloma cells circulating in blood; spleen weight; and myeloma cell burden in spleen, bone marrow, and liver. mAb therapy had no effect on nonmalignant hematopoietic cells. An acute 6-day regimen of mAb treatment, initiated very late in disease to avoid mAb elimination in the immunocompetent animals, still significantly reduced spleen and blood myeloma cell burden. The ability of the (VLA-4) mAb to affect multiple variables in this model, even as monotherapy, suggests this pathway plays a central role in disease progression.

An antibody-cytotoxic conjugate, BIIB015, is a new targeted therapy for Cripto positive tumours.

BIIB015 is an immunoconjugate created for the treatment of solid tumours and is currently in Phase I of clinical evaluation. BIIB015 consists of a humanised monoclonal antibody against the Cripto protein carrying a payload, via a hindered disulphide linker, of the maytansinoid derivative, DM4. Cripto is a GPI-linked protein required for signal transduction of the TGF-beta ligand, Nodal. Cripto has been previously described as an oncogene and fits the classic pattern of an embryonic gene that is re-expressed in a transformed tumour cell. Cripto expression is highly prevalent on a number of solid tumours, including greater than 75% of breast, lung, and colorectal tumours. Our report documents for the first time that targeting the cell surface Cripto protein with an anti-Cripto antibody-cytotoxic conjugate is an effective means of inhibiting or regressing growth of Cripto positive tumours. BIIB015 which utilises a 'cleavable' linker containing a disulphide bond exhibits superior activity when compared to huB3F6 mAb conjugates with different linker systems, including one with a 'non-cleavable' linker. BIIB015 displays specificity for Cripto in both in vitro and in vivo experiments. In human xenograft models originating from lung (Calu-6), colon (CT-3), testicular (NCCIT) and breast (MDA-MB-231) tumour samples, BIIB015 shows robust activity with results ranging from >50% tumour inhibition to complete tumour regression. The efficacy seen in the MDA-MB-231 model, a triple negative (-HER2, -ER, and -PR) tumour, is particularly exciting since there is currently no approved therapy for this indication. In addition, BIIB015 can be combined with standard of care chemotherapeutics for enhanced efficacy.

Disruption of hedgehog signaling reveals a novel role in intestinal morphogenesis and intestinal-specific lipid metabolism in mice.

BACKGROUND & AIMS: The hedgehog (hh) signaling pathway has been shown to play crucial roles in the development of embryonic gut. However, its role in intestinal development and function beyond the embryonic stage is still undefined. METHODS: Expression of hh and its receptor, Patched, were examined by Western blot and X-gal staining. An anti-hh monoclonal antibody was administered into developing embryos or postnatal mice and histologic analyses were performed. Effects on lipid metabolism were examined by Oil Red O and Sudan III stainings, messenger RNA (mRNA) analysis, and electron microscopy. Serum apolipoprotein IV level, a marker for lipid absorption, was quantified by Western blot. RESULTS: Mice receiving anti-hh monoclonal antibody in utero or after birth exhibited progressive runting and died before weaning. Histology revealed hyperproliferation of intestinal crypt epithelial cells and disorganization of the villi with prominent vacuolation and accumulation of neutral lipid. Fecal fat microscopy revealed numerous large fat droplets. Intestinal mRNA abundance of 2 candidate genes involved in lipid transport, mtp and apob, was unchanged, although serum levels of apolipoprotein A-IV were reduced. CONCLUSIONS: Abnormal villus structure, lipid-filled enterocytes, and fatty stools in anti-hh monoclonal antibody-treated mice indicate a novel role for hh signaling in intestinal morphogenesis and lipid transport in postnatal mice.

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.