TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside ‘reverse’ signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.

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 VEGFindependent 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.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries.

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<p>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.</p>

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 &gt; 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.

<p>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.</p>

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