David W. Lafleur

Tumor Necrosis Factor (TNF) Receptor Superfamily Member TACI Is a High Affinity Receptor for TNF Family Members APRIL and BLyS

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-κB responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.

Regulatory Effect of IFN-κ, A Novel Type I IFN, On Cytokine Production by Cells of the Innate Immune System

IFN-κ is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-κ on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-κ or two recombinant IFN subtypes, IFN-β and IFN-α2a. Although IFN-α2a failed to stimulate monocyte cytokine secretion, IFN-κ, like IFN-β, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-κ was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-β, IFN-κ did not induce release of IFN-γ by PBL. Expression of the IFN-κ mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-γ stimulation in monocytes, while IFN-β mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-κ was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-κ, like IFN-β, binds strongly to heparin (Kd: 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-κ in monocytes, coupled with the unique induction of IFN-κ mRNA by IFN-γ, indicates a potential role for IFN-κ in the regulation of immune cell functions.

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis.

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.

Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.

Modification of pharmacokinetic and abuse-related effects of cocaine by human-derived cocaine hydrolase in monkeys.

Although substantial research effort has focused on developing pharmacological treatments for cocaine abuse, no effective medications have been developed. Recent studies show that enzymes that metabolize cocaine in the periphery, forestalling its entry into the brain, can prevent cocaine toxicity and its behavioral effects in rodents. Here we report on effects of one such enzyme (Albu‐CocH) on the pharmacokinetic and behavioral effects of cocaine in squirrel monkeys. Albu‐CocH was developed from successive mutations of human butyrylcholinesterase (BChE) and has 1000‐fold greater catalytic activity against cocaine than naturally occurring BChE. Pharmacokinetic studies showed that Albu‐CocH (5 mg/kg) had a half‐life of 56.6 hours in squirrel monkeys. In these studies, plasma levels of cocaine following i.v. 1 mg/kg cocaine were reduced 2 hours after administration of Albu‐CocH, whereas plasma levels of the cocaine metabolite ecgonine methyl ester were increased. These effects were still evident 72 hours following Albu‐CocH administration. In behavioral experiments in monkeys, pre‐treatment with 5 mg/kg Albu‐CocH dramatically decreased self‐administration of a reinforcing dose of i.v. cocaine (30 µg/kg/injection) for over 24 hours. Pre‐treatment with 5 mg/kg Albu‐CocH also attenuated the reinstatement of extinguished cocaine self‐administration by an i.v. priming injection of cocaine (0.1 or 0.3 mg/kg) and, in separate studies, attenuated the discriminative‐stimulus effects of cocaine. The ability of Albu‐CocH to attenuate the abuse‐related effects of cocaine in squirrel monkeys indicates that further investigation of BChE mutants as potential treatment for cocaine abuse and toxicity is warranted.