Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration.

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would.

Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig.

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells.

Transmembrane TNF-dependent uptake of anti-TNF antibodies.

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.

Generation and characterization of ABBV642, a dual variable domain immunoglobulin molecule (DVD-Ig) that potently neutralizes VEGF and PDGF-BB and is designed for the treatment of exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis.

Identification of Anti-EGFR and Anti-ErbB3 Dual Variable Domains Immunoglobulin (DVD-Ig) Proteins with Unique Activities.

Epidermal growth factor receptor (EGFR) and receptor tyrosine-protein kinase 3 (ErbB3) are two well-established targets in cancer therapy. There is significant crosstalk among these two receptors and others. To block signaling from both EGFR and ErbB3, we generated anti-EGFR and anti-ErbB3 DVD-Ig proteins.

Generation and characterization of ABT-981, a dual variable domain immunoglobulin (DVD-Ig(TM)) molecule that specifically and potently neutralizes both IL-1α and IL-1β.

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.

Cutting Edge: DNase II deficiency prevents activation of autoreactive B cells by double-stranded DNA endogenous ligands.

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice.

Identification of anti-ErbB2 dual variable domain immunoglobulin (DVD-Ig™) proteins with unique activities.

Inhibiting ErbB2 signaling with monoclonal antibodies (mAbs) or small molecules is an established therapeutic strategy in oncology. We have developed anti-ErbB2 Dual Variable Domain Immunoglobulin (DVD-Ig) proteins that capture the function of a combination of two anti-ErbB2 antibodies. In addition, some of the anti-ErbB2 DVD-Ig proteins gain the new functions of enhancing ErbB2 signaling and cell proliferation in N87 cells.

The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen.

A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules.

Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule.

Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously.

Design and generation of DVD-Ig™ molecules for dual-specific targeting.

The dual variable domain immunoglobulin (DVD-Ig™) protein is a new type of dual-specific IgG. As a novel therapeutic class, the great potential of the DVD-Ig protein is to simultaneously target two mediators of disease by a single pharmaceutical entity. The molecule contains an Fc region and constant regions in a configuration similar to a conventional IgG; however, the DVD-Ig protein is unique in that each arm of the molecule contains two variable domains (VDs).

Generation of dual-variable-domain immunoglobulin molecules for dual-specific targeting.

Bispecific antibodies may be used to improve clinical efficacy by targeting two disease mechanisms for the treatment of complex human diseases in a single agent. Bispecific antibodies also hold promise for certain therapeutic applications difficult to achieve by single-targeting monospecific antibodies, such as immune (T cell or NK) cell retargeting, site-specific targeting, enabling therapeutics to cross the blood-brain barrier, and unique receptor modulation. Although the history of bispecific antibody research is almost as long as hybridoma technology, it is not until recent that bispecific antibodies have made substantial breakthrough, thanks to promising clinical trial results of a few bispecific antibodies and the development of new formats which largely ease manufacturing and physicochemical property challenges encountered by early bispecific antibody formats.

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design.

The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain.

Rationale and development of multispecific antibody drugs.

Bispecific antibodies (bsAbs) have been on the scene for decades and represent the next generation of antibody-based therapeutics. Unlike monospecific, monoclonal antibodies (mAbs), bsAbs can target two or more disease mechanisms as a single agent and can offer certain unique therapeutic strategies that are difficult to acheive with mAbs. The lessons learned during the past 35 years of mAb development and 25 years of bsAbs experience are shaping development of the next generation of bsAbs and multispecific antibody-based drugs.

Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules.

Signal transduction through the interleukin-1 receptor (IL-1R) pathway mediates a strong pro-inflammatory response, which contributes to a number of human diseases such as rheumatoid arthritis. Within the IL-1 family, IL-1alpha and IL-1beta are both agonistic ligands for IL-1R, whereas IL-1 receptor antagonist (IL-1ra) is an endogenous antagonist that binds to IL-R, but does not signal. Therefore, the ideal therapeutic strategy would be blocking both IL-1alpha and IL-1beta, but not IL-1ra.

Unusual water-mediated antigenic recognition of the proinflammatory cytokine interleukin-18.

The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies.

The Bcl-2 family antagonist ABT-737 significantly inhibits multiple animal models of autoimmunity.

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases.

Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin.

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility.

Expression and alternative processing of IL-18 in human neutrophils.

Interleukin-18 (IL-18), a member of the IL-1 cytokine superfamily, is an important regulator of both innate and acquired immune responses. We demonstrate here constitutive expression of IL-18 by human neutrophils. Unexpectedly, we observed that neutrophils from peripheral blood or rheumatoid synovial compartments contained not only pro and mature IL-18, but also several novel smaller-molecular-weight IL-18-derived species.

Interleukin-1alphabeta gene-deficient mice show reduced nociceptive sensitivity in models of inflammatory and neuropathic pain but not post-operative pain.

The pro-inflammatory cytokine interleukin-1 (IL-1) has been implicated in both inflammatory processes and nociceptive neurotransmission. To further investigate the role of IL-1 in different pain states, gene-disrupted mice lacking both IL-1alpha and IL-1beta genes (IL-1alphabeta (-/-)) were characterized in inflammatory, neuropathic, and post-operative pain models. IL-1alphabeta (-/-) mice showed normal sensorimotor function as measured by the rotorod assay compared to control mice (BALB/c).

Role of interleukin-18 in experimental group B streptococcal arthritis.

Objective: To assess the role of interleukin-18 (IL-18) in the evolution of septic arthritis induced by group B streptococci (GBS) in mice.

Methods: CD1 mice were inoculated intravenously with 8 x 10(6) colony-forming units (CFU) of type IV GBS (strain 1/82), and administered intraperitoneally 1 hour before infection with anti-IL-18 monoclonal antibodies (0.25 mg/mouse).

IL-18 receptor beta-induced changes in the presentation of IL-18 binding sites affect ligand binding and signal transduction.

IL-18 is a pleiotropic proinflammatory cytokine that is involved in induction of inflammatory mediators, regulation of the cytotoxic activity of NK cells and T cells, and differentiation and activation of both Th1 and Th2 cells. IL-18 signals through its specific cell surface receptor IL-18R, which comprises two subunits: IL-18R alpha and IL-18R beta. IL-18R alpha alone has a weak affinity for IL-18 binding, while the IL-18R alpha/beta complex has a high affinity.

Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset.

During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56(bright) and CD56(dim)). In this report, it is shown that CD56(bright) NK cells produce significantly greater levels of interferon-gamma, tumor necrosis factor-beta, granulocyte macrophage-colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56(dim) NK cells, which produce negligible amounts of these cytokines.