Development of XPA067.06, a potent high affinity human anti-gastrin monoclonal antibody.

The peptide hormone gastrin is a key factor in regulation of gastric acid secretion. It has also been implicated in the development or maintenance of various types of cancer, such as pancreatic and stomach carcinoma. Inhibition of gastrin activity has potential for therapeutic use as a suppressor of acid secretion as well as an inhibitor of gastrin-responsive tumors. XPA067.06 is an affinity matured, 30 pM fully human anti-gastrin monoclonal antibody that was generated. The antibody was tested in a mouse gastric pH model to determine its effect on acid secretion. In this model, animals were treated with human gastrin, XPA067.06, and H2R or M1 receptor antagonists. Gastric fluid was collected and acid output was measured as a function of pH. XPA067.06 was shown to significantly inhibit gastrin-17-stimulated acid output for at least 48h. These results demonstrate that XPA067.06 effectively binds and neutralizes human gastrin-17 in vivo with rapid onset and prolonged duration of efficacy.

ATR and ATM-dependent movement of BLM helicase during replication stress ensures optimal ATM activation and 53BP1 focus formation.

The BLM helicase, a deficiency that markedly increases cancer incidence in humans, is required for optimal repair during DNA replication. We show that BLM rapidly moves from PML nuclear bodies to damaged replication forks, returning to PML bodies several hours later, owing to activities of the DNA damage response kinases ATR and ATM, respectively. Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different sub-cellular compartments after replication stress. Unexpectedly, fibroblasts lacking BLM were deficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failed to form foci following replication stress. Expression of a dominant p53 mutant or helicase-deficient BLM restored replication stress-induced 53BP1 foci, but only mutant p53 restored optimal ATM activation. Thus, optimal repair of damaged replication fork lesions likely requires both ATR and ATM. BLM recruits 53BP1 to these lesions independent of its helicase activity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.

BACKGROUND: We previously used a three-dimensional (3D) reconstituted basement membrane (rBM) assay to demonstrate that tumorigenic HMT-3522 T4-2 human breast cells can be induced to form morphologically normal structures ("reversion") by treatment with inhibitors of beta1 integrin, the epidermal growth factor receptor (EGFR), or mitogen-activated protein kinase (MAPK). We have now used this assay to identify reversion and/or death requirements of several more aggressive human breast cancer cell lines. METHODS: Breast tumor cell lines MCF7, Hs578T, and MDA-MB-231 were cultured in 3D rBM and treated with inhibitors of beta1 integrin, MAPK, or phosphatidylinositol 3-kinase (PI3K). MDA-MB-231 cells, which lack E-cadherin, were transfected with an E-cadherin cDNA. The extent of reversion was assessed by changes in morphology and polarity, growth in 3D rBM or soft agar, level of invasiveness, and tumor formation in nude mice. RESULTS: All three cell lines showed partial reversion (MCF7 the greatest and Hs578T the least) of tumorigenic properties treated with a single beta1 integrin, MAPK, or PI3K inhibitor. Combined inhibition of beta1 integrin and either PI3K or MAPK resulted in nearly complete phenotypic reversion (MDA-MB-231, MCF7) or in cell death (Hs578T). E-cadherin-transfected MDA-MB-231 cells showed partial reversion, but exposure of the transfectants to an inhibitor of beta1 integrin, PI3K, or MAPK led to nearly complete reversion. CONCLUSION: The 3D rBM assay can be used to identify signaling pathways that, when manipulated in concert, can lead to the restoration of morphologically normal breast structures or to death of the tumor cells, even highly metastatic cells. This approach may be useful to design therapeutic intervention strategies for aggressive breast cancers.