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PC3 cells stably expressing different levels of MSLN

Agonistic CD40 monoclonal antibodies (mAb) have demonstrated some clinical activity, but with dose-limiting toxicity. To reduce systemic toxicity, we developed a bispecific molecule that was maximally active in the presence of a tumor antigen and had limited activity in the absence of the tumor antigen. LB-1 is a bispecific molecule containing single-chain Fv domains targeting mouse CD40 and the tumor antigen mesothelin. LB-1 exhibited enhanced activity upon binding to cell-surface mesothelin but was less potent in the absence of mesothelin binding. In a mouse model implanted with syngeneic 4T1 tumors expressing cell-surface mesothelin, LB-1 demonstrated comparable antitumor activity as an agonistic CD40 mAb but did not cause elevation of serum cytokines and liver enzymes, as was observed in anti-CD40–treated mice. The results from our study of LB-1 were used to develop a human cross-reactive bispecific molecule (ABBV-428) that targeted human CD40 and mesothelin. ABBV-428 demonstrated enhanced activation of antigen-presenting cells and T cells upon binding to cell-surface mesothelin, and inhibition of cultured or implanted PC3 tumor cell growth after immune activation. Although expression of cell-surface mesothelin is necessary, the bispecific molecules induced immune-mediated antitumor activity against both mesothelin+ and mesothelin− tumor cells. ABBV-428 represents a class of bispecific molecules with conditional activity dependent on the binding of a tumor-specific antigen, and such activity could potentially maximize antitumor potency while limiting systemic toxicity in clinical studies.

Binding affinity of LB-1

Table S1: ABT-165 binding affinity; Table S2: ABT-165 in vitro potency; Table S3: Effect of VEGF on anti-DLL4 cellular potency of ABT-165; Table S4: Summary of in vivo efficacy; Table S5: Key safety findings of ABT-487 and ABT-165; Figure S1: Serum concentration-time profiles of ABT-165 in cynomolgus monkeys; Figure S2: Effect of antibody or antibody fragment valency on anti-DLL4 cellular potency; Figure S3: Effect of VEGF on the activity of DLL4 mAb and ABT-165 to downregulate DLL4 protein; Figure S4: Plasma levels of total soluble DLL4 and VEGF in cynomolgus monkeys after ABT-165 treatment.

This file describes the binding assay details of surface plasmon resonance technology, ligand and receptor binding competition assays, western blot detection of DLL4 protein down-regulation, and the methods to measure total circulating soluble DLL4 and VEGF in cynomolgus monkey plasma.

Antiangiogenic therapy is a clinically validated modality in cancer treatment. To date, all approved antiangiogenic drugs primarily inhibit the VEGF pathway. Delta-like ligand 4 (DLL4) has been identified as a potential drug target in VEGF-independent angiogenesis and tumor-initiating cell (TIC) survival. A dual-specific biologic targeting both VEGF and DLL4 could be an attractive strategy to improve the effectiveness of anti-VEGF therapy. ABT-165 was uniquely engineered using a proprietary dual-variable domain immunoglobulin (DVD-Ig) technology based on its ability to bind and inhibit both DLL4 and VEGF. In vivo, ABT-165 induced significant tumor growth inhibition compared with either parental antibody treatment alone, due, in part, to the disruption of functional tumor vasculature. In combination with chemotherapy agents, ABT-165 also induced greater antitumor response and outperformed anti-VEGF treatment. ABT-165 displayed nonlinear pharmacokinetic profiles in cynomolgus monkeys, with an apparent terminal half-life > 5 days at a target saturation dose. In a GLP monkey toxicity study, ABT-165 was well-tolerated at doses up to 200 mg/kg with non-adverse treatment–related histopathology findings limited to the liver and thymus. In summary, ABT-165 represents a novel antiangiogenic strategy that potently inhibits both DLL4 and VEGF, demonstrating favorable in vivo efficacy, pharmacokinetic, and safety profiles in preclinical models. Given these preclinical attributes, ABT-165 has progressed to a phase I study. Mol Cancer Ther; 17(5); 1039–50. ©2018 AACR.

Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.

Background Limited data describe cognitive deficits in patients with Cushing syndrome (CS). The impact of mild autonomous cortisol secretion (MACS) on cognition is unknown. Objective To determine the impact of MACS on cognitive function. Methods Single-center cross-sectional study of adults with MACS and age and sex-matched volunteers. MACS was defined as cortisol >1.8 mcg/dL following the dexamethasone suppression test (DST) in patients with an adrenal incidentaloma and no features of CS. We measured cognitive function through the NIH toolbox cognition battery (7 standardized tests of attention, episodic and working memory, language, executive function, and processing speed). T-scores corrected for age, sex, education, and race were used for analysis. All patients completed the SF-36 questionnaire. The frailty index was calculated as previously reported (Singh S. JCEM 2020). Results Participants included 50 patients with MACS (median age 61, range 54-68) and 50 volunteers (median age 60 years, range 54-68), with 60% women in both groups. In patients with MACS, median post-DST morning cortisol was 3.6 (range, 2.4-6.2), and median corticotropin was 8.5 (range, 5.9-13) pg/mL (normal 6-60). Patients with MACS had a higher median frailty index (median of 0.26 vs. 0. 05 in volunteers, p Conclusion Compared to volunteers, patients with MACS demonstrate cognitive impairment, particularly in the executive domain, which closely mimics changes observed with aging. These deficits do not correlate with the degree of cortisol excess. Presentation: No date and time listed

The impact of stress in persons with multiple sclerosis (MS) is an area of ongoing inquiry. A limited number of published systematic reviews have reported an association between stress and MS onset. In addition to reviewing more recently published evidence, this review expands upon existing systematic reviews by considering the timing of stress exposure (childhood or adulthood).A review of human-subjects literature published in English after 2010 was conducted between April 2020 and May 2020. In total, 6 databases were searched using the terms 'allostatic load', 'multiple sclerosis (MS)', 'chronic stress', and 'stress', and 13 relevant studies were analyzed. Each article was read by at least two of the authors to confirm its relevance. Studies were categorized based on the timing of stress exposure (childhood or adulthood). Study designs included longitudinal cohort, cross-sectional cohort, cross-sectional case-control, and longitudinal case-control studies. The NIH study quality assessment tools and Strengthening The Reporting of OBservational studies in Epidemiology (STROBE) checklist were used to assess study quality. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews were followed.Manuscripts reviewed included 8 studies on MS onset, 2 studies on MS relapse, and 3 studies on MS onset and relapse. In total, 5 studies examined childhood stress, 7 studies examined adulthood stress, and 1 study examined both childhood and adulthood stress in 2 respective cohorts. Studies of varying design consistently reported an association between MS relapse and stress, regardless of the timing of stress exposure. However, the link between stress and MS onset was conflicting for both childhood and adult stress exposures.This systematic review is the first to report the strong association between stress and MS relapse, and, contradictory to past reports, an inconsistent relationship between stress and MS onset. Notably, the timing of stress (childhood or adulthood) seems to have little effect on the occurrence of either MS onset or relapse, though no study examined both childhood and adult stress within the same participant. Most studies were conducted in small, homogeneous samples using self-reported stress measures, which ultimately limits the generalizability of the findings. Overall, the state of the science remains tentative for all areas examined in this review, necessitating future research.