Functional ␥-secretase inhibitors (FGSIs) can block the cleavage of several transmembrane proteins including amyloid precursor protein (APP), and the cell fate regulator Notch-1. FGSIs, by inhibiting APP processing, block the generation of amyloid ␤ (A␤) peptides and may slow the development of Alzheimer's disease. FGSIs used to inhibit APP processing may disrupt Notch processing, thus interfering with cell fate determination. Described herein is a FGSI-mediated gastrointestinal toxicity characterized by cell population changes in the ileum of rats, which are indicative of Notch signaling disruption. Microarray analysis of ileum from FGSItreated rats revealed differential expression responses in a number of genes indicative of Notch signaling perturbation, including the serine protease adipsin. We were able to show that FGSI-treated rats had elevated levels of adipsin protein in gastrointestinal contents and feces, and by immunohistochemistry demonstrated that adipsin containing ileum crypt cells were increased in FGSI-treated rats. The mouse Adipsin proximal promoter contains a putative binding site for the Notchinduced transcriptional regulator Hes-1, which we demonstrate is able to bind Hes-1. Additional studies in 3T3-L1 preadipocytes demonstrate that this FGSI inhibits Hes-1 expression while up-regulating adipsin expression. Overexpression of Hes-1 was able to down-regulate adipsin expression and block pre-adipocyte differentiation. We propose that adipsin is a Hes-1-regulated gene that is de-repressed during FGSI-mediated disruption of Notch/Hes-1 signaling. Additionally, the aberrant expression of adipsin, and its presence in feces may serve as a noninvasive biomarker of gastrointestinal toxicity associated with perturbed Notch signaling.The small intestine can be a site of injury associated with drug treatment (1-3). Tissue organization within the small intestine relies upon a small number of stem cells in the intestinal crypts to continuously produce several types of differentiated cells that together comprise the villous epithelium (enterocytes, goblet cells, paneth cells, and enteroendocrine cells) (4). This rapid maturation, transport, and cell loss make the small intestine particularly susceptible to toxicants that affect cell differentiation and proliferation (5, 6). The process by which dividing intestinal epithelial stem cells in the crypt produce differentiated progeny requires the transcriptional regulation of genes necessary for cell fate determination. The control of this cell fate determination pathway is dependent on a number of positive and negative transcription factors that operate in undifferentiated precursor cells of the crypt (6 -8). For example, the bHLH transcriptional repressor protein Hairy and Enhancer of split homologue-1 (Hes-1) 1 has been shown to be important in determining whether differentiating intestinal epithelial stem cells adopt an exocrine/secretory (goblet cell, enteroendocrine cell, paneth cell) fate or an absorptive (enterocyte) fate (9). Expression of Hes-1 is kn...

(2016) Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys, mAbs, 8:5, 969-982, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACT Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgGscFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.

Angiogenesis plays an important role in tumor growth. Angiogenic growth factors may be useful as biomarkers of antiangiogenic activity since their plasma concentrations correlate with the efficacy of treatments directed toward angiogenic targets. SW2 small-cell lung carcinoma (SCLC), Caki-1 renal cell carcinoma and HCT-116 colon carcinoma tumors produce measurable plasma VEGF, bFGF and TGFbeta in nude mice. Mice bearing these human tumor xenografts were treated orally twice daily with the PKCbeta inhibitor, LY317615 (days 14-30 for SW2 and HCT116, and days 21-39 for Caki-1). Plasma was collected every 3 days from control and treated mice. LY317615 significantly decreased plasma VEGF levels in mice bearing SW2 SCLC and Caki-1 renal cell carcinoma compared to control plasma concentrations beginning 5-7 days after initiating therapy. VEGF plasma levels remained suppressed after termination of LY317615 treatment and for the duration of the study (an additional 2 to 3 weeks). Plasma VEGF levels in mice bearing HCT116 xenografts were not altered by LY317615 treatment and plasma bFGF and TGF-beta were not altered by LY317615 in any of the animals. As shown by CD31 immunohistochemical staining, LY317615 decreased intratumoral vessel density by nearly 40% in all three tumors. Only the Caki-1 tumor responded to single-agent LY317615 therapy with a measurable tumor growth delay. Thus, unexpectedly inhibition of PKCbeta in vivo led to decreased VEGF production that persisted after therapy as well as to decreased intratumoral vessels. Plasma VEGF was a weak marker of response to LY317615, and plasma bFGF and TGFbeta were not markers of LY317615 activity.

Treatment of bone disease in multiple myeloma (MM) has largely focussed on the osteoclast axis while the osteoblast axis has been underexploited. Dkk1, an inhibitor of the wingless int (wnt) pathway, is important in osteoblastogenesis. Increased expression of Dkk1 in a subset of MM patients and its association with lytic bone disease opens up the potential of targeting the osteoblast axis. The aim of this study was to test the effect of a Dkk-1 neutralizing chimeric antibody (Mab B3) on osteoblasts (OB), osteoclasts (OC) and MM cells in the context of the bone microenvironment. First, we tested the expression of Dkk1 in plasma and bone marrow of 16 MM patients and 10 MM cell lines. Dkk1 levels were >18 ng/mL in 2 out of 16 patients; levels were comparable in blood and bone marrow plasma. In contrast, very little Dkk1 (2–9 ng/ml) was produced by bone marrow stromal cells (BMSC). One out of 10 MM cell lines (INA-6) expressed low concentrations of Dkk1 in the supernatant. Next, we tested the effect of Mab B3 on MM cell lines, in the presence or absence of BMSC, and on OB and OC from MM patient derived bone marrow. The effects on OC were evaluated by TRAP staining and pit formation. Effects on OB were assayed by alkaline phosphatase staining and alizarin red assays for calcium deposition. Mab B3 treatment did not demonstrate direct cytotoxic effects on MM cell lines negative for Dkk1. Mab B3, however, enhanced OB differentiation and calcium deposition in a dose dependent manner and inhibited OC differentiation and function, as evidenced by a decrease in number of multinucleated TRAP+ cells and a decrease in pit formation. Ongoing studies are addressing the effect of Mab B3 on MM cells in the context of OC and OB. Mab B3 is also undergoing in vivo testing in a SCID-hu model bearing INA-6 MM cells. These studies and the underlying mechanism of action of Mab B3 will be presented. Our preliminary data suggests that Mab B3 has anabolic bone effects; a corresponding human monoclonal antibody may be useful for the treatment of MM related bone disease. Future studies will evaluate Mab B3 in combination with catabolic agents such as bisphosphonates with the goal of restoring normal bone homeostatsis.

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