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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.

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.

Interleukin-1 (IL-1) cytokines such as IL-1α, IL-1β, and IL-1Ra contribute to immune regulation and inflammatory processes by exerting a wide range of cellular responses, including expression of cytokines and chemokines, matrix metalloproteinases, and nitric oxide synthetase. IL-1α and IL-1β bind to IL-1R1 complexed to the IL-1 receptor accessory protein and induce similar physiological effects. Preclinical and clinical studies provide significant evidence for the role of IL-1 in the pathogenesis of osteoarthritis (OA), including cartilage degradation, bone sclerosis, and synovial proliferation. Here, we describe the generation and characterization of ABT-981, a dual variable domain immunoglobulin (DVD-Ig) of the IgG1/k subtype that specifically and potently neutralizes IL-1α and IL-1β. In ABT-981, the IL-1β variable domain resides in the outer domain of the DVD-Ig, whereas the IL-1α variable domain is located in the inner position. ABT-981 specifically binds to IL-1α and IL-1β, and is physically capable of binding 2 human IL-1α and 2 human IL-1β molecules simultaneously. Single-dose intravenous and subcutaneous pharmacokinetics studies indicate that ABT-981 has a half-life of 8.0 to 10.4 d in cynomolgus monkey and 10.0 to 20.3 d in rodents. ABT-981 exhibits suitable drug-like-properties including affinity, potency, specificity, half-life, and stability for evaluation in human clinical trials. ABT-981 offers an exciting new approach for the treatment of OA, potentially addressing both disease modification and symptom relief as a disease-modifying OA drug.

The first generation anti-angiogenic drugs designed to block the VEGF/VEGFR pathway lend modest clinical benefit for cancer patients. Other than VEGF, DLL4 is the only known angiogenic factor with a haploinsufficiency phenotype, underscoring its essential role in vascular function. Indeed, both the VEGF/VEGFR and the DLL4/Notch signaling axes are known to cooperate during pathological angiogenesis. DLL4 is also implicated in VEGF-independent pathways, cancer stem cell survival, and immune suppression that could collectively contribute to tumor cell resistance. Given both intrinsic and acquired patient resistance mechanisms exist, targeting the DLL4/Notch pathway represents a unique opportunity for a combination strategy to improve upon current VEGF/VEGFR pathway inhibitor therapies. To this end, ABT-165 was developed as a first-in-class dual specific biologic using AbbVie's proprietary dual-variable domain immunoglobulin (DVD-IgTM) technology. ABT-165 is capable of simultaneously binding to DLL4 and VEGF with nanomolar affinities and blocking the cognate ligand-receptor interactions that result in the potent inhibition of DLL4-mediated Notch1 activation and VEGF-stimulated endothelial cell proliferation. ABT-165 is functionally superior in vitro compared to the combination of parental anti-VEGF and anti-DLL4 antibodies. In human tumor xenograft models, ABT-165 induced significant inhibition of tumor growth and survival benefit compared to single anti-DLL4 or anti-VEGF antibody treatments at equivalent doses. Mechanistically, this enhancement of anti-tumor efficacy is due in part to the disruption of new tumor vasculature coupled with blockade of vessel perfusion. Furthermore, ABT-165 in combination with cytotoxic chemotherapy agents induced tumor regression, which outperformed bevacizumab plus chemotherapy in both human breast and colon xenograft models. ABT-165 displays non-linear, dose-dependent pharmacokinetic profiles in mice and cynomolgus monkeys, with an apparent terminal half-life > 5 days in both species at a target saturation dose. In a GLP monkey toxicity study, ABT-165 at doses up to 200 mg/kg was well-tolerated with non-adverse treatment-related histopathology findings limited to the liver and thymus. In contrast, adverse and non-adverse findings were observed in the hearts of rats and monkeys, respectively, with an in-house proprietary anti-DLL4 antibody. Given that coupling of anti-DLL4 with anti-VEGF activities into a DVD-Ig may lend improved safety and/or efficacy profiles compared to antibodies, ABT-165 was advanced into a Phase 1 clinical trial. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Yingchun Li, Jonathan Hickson, Dominic Ambrosi, Deanna Haasch, Kelly Foster-Duke, Lucia Eaton, Fang Jiang, Surekha Akella, Wenqing Gao, Sherry Ralston, Jijie Gu, Susan Morgan-Lappe. ABT-165 is a first-in-class therapeutic Dual Variable Domain Immunoglobulin (DVD-IgTM) that targets DLL4 and VEGF for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 867.

Mouse embryonic stem cells (ESCs) proliferate with rapid cell cycle kinetics but without loss of pluripotency. The histone methyltransferase Dot1L is responsible for methylation of histone H3 at lysine 79 (H3K79me). We investigated whether ESCs require Dot1L for proper stem cell behavior. ESCs deficient in Dot1L tolerate a nearly complete loss of H3K79 methylation without a substantial impact on proliferation or morphology. However, shortly after differentiation is induced, Dot1L-deficient cells cease proliferating and arrest in G2/M-phase of the cell cycle, with increased levels of aneuploidy. In addition, many aberrant mitotic spindles occur in Dot1L-deficient cells. Surprisingly, these mitotic and cell cycle defects fail to trigger apoptosis, indicating that mouse ESCs lack stringent cell cycle checkpoint control during initial stages of differentiation. Transcriptome analysis indicates that Dot1L deficiency causes the misregulation of a select set of genes, including many with known roles in cell cycle control and cellular proliferation as well as markers of endoderm differentiation. The data indicate a requirement for Dot1L function for early stages of ESC differentiation where Dot1L is necessary for faithful execution of mitosis and proper transcription of many genes throughout the genome. Disclosure of potential conflicts of interest is found at the end of this article.

Recent experiments demonstrate that somatic nuclei can be reprogrammed to a pluripotent state when fused to ESCs. The resulting hybrids are pluripotent as judged by developmental assays, but detailed analyses of the underlying molecular-genetic control of reprogrammed transcription in such hybrids are required to better understand fusion-mediated reprogramming. We produced hybrids of mouse ESCs and fibroblasts that, although nearly tetraploid, exhibit characteristics of normal ESCs, including apparent immortality in culture, ESC-like colony morphology, and pluripotency. Comprehensive analysis of the mouse embryonic fibroblast/ESC hybrid transcriptome revealed global patterns of gene expression reminiscent of ESCs. However, combined analysis of variance and hierarchical clustering analyses revealed at least seven distinct classes of differentially regulated genes in comparisons of hybrids, ESCs, and somatic cells. The largest class includes somatic genes that are silenced in hybrids and ESCs, but a smaller class includes genes that are expressed at nearly equivalent levels in hybrids and ESCs that contain many genes implicated in pluripotency and chromatin function. Reprogrammed genes are distributed throughout the genome. Reprogramming events include both transcriptional silencing and activation of genes residing on chromosomes of somatic origin. Somatic/ESC hybrid cell lines resemble their pre-fusion ESC partners in terms of behavior in culture and pluripotency. However, they contain unique expression profiles that are similar but not identical to normal ESCs. ESC fusion-mediated reprogramming provides a tractable system for the investigation of mechanisms of reprogramming. Disclosure of potential conflicts of interest is found at the end of this article.

Advances in mammalian cloning prove that somatic nuclei can be reprogrammed to a state of totipotency by transfer into oocytes. An alternative approach to reprogram the somatic genome involves the creation of hybrids between somatic cells and other cells that contain reprogramming activities. Potential fusion partners with reprogramming activities include embryonic stem cells, embryonic germ cells, embryonal carcinoma cells, and even differentiated cells. Recent advances in fusion-mediated reprogramming are discussed from the standpoints of the developmental potency of hybrid cells, genetic and epigenetic correlates of reprogramming, and other aspects involved in the reprogramming process. In addition, the utility of fusion-mediated reprogramming for future cell-based therapies is discussed.