We identified a novel cDNA (IG20) that is homologous to cDNAs encoding a protein differentially expressed in normal and neoplastic cells (DENN-SV) and human MADD (MAPK-activating death domain-containing protein). Furthermore, we show that the above variants most likely result from alternative splicing of a single gene. Functional analyses of these variants in permanently transfected HeLa cells revealed that IG20 and DENN-SV render them more susceptible or resistant to tumor necrosis factor ␣ (TNF-␣)-induced apoptosis, respectively. All variants tested could interact with TNF receptor 1 and activate ERK and nuclear factor B. However, relative to control cells, only cells expressing IG20 showed enhanced TNF-␣-induced activation of caspase-8 and -3, whereas cells expressing DENN-SV showed either reduced or no caspase activation. Transfection of these cells with a cDNA encoding CrmA maximally inhibited apoptosis in HeLa-IG20 cells. Our results show that IG20 can promote TNF-␣-induced apoptosis and activation of caspase-8 and -3 and suggest that it may play a novel role in the regulation of the pleiotropic effects of TNF-␣ through alternative splicing.

The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a singlechain human insulin to human serum albumin. Albulin showed an elimination t 1/2 of ϳ7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes. Diabetes 54:251-258, 2005

We identified seven putative splice variants of the human IG20 gene. Four variants namely, IG20, MADD, IG20-SV2 and DENN-SV are expressed in human tissues. While DENN-SV is constitutively expressed in all tissues, expression of IG20 appears to be regulated. Interestingly, overexpression of DENN-SV enhanced cell replication and resistance to treatments with TNFa, vinblastine, etoposide and c-radiation. In contrast, IG20 expression suppressed cell replication and increased susceptibility to the above treatments. Moreover, cells that were resistant and susceptible to TNFa-induced apoptosis exclusively expressed endogenous DENN-SV and IG20, respectively. When PA-1 ovarian cancer cells that are devoid of endogenous IG20 variant, but express higher levels of DENN-SV, were transfected with IG20, they showed reduced cell proliferation and increased susceptibility to apoptosis induced by TNFa, TRAIL and c-radiation. This indicated that overexpression of IG20 can override endogenous DENN-SV function. CrmA reversed the effects of IG20, but not DENN-SV. In contrast, dominant-negative-I-kappa B reversed the effects of DENN-SV, but not IG20, and showed that DENN-SV most likely exerted its effects through NFjB activation. Together, our data show that IG20 gene can play a novel and significant role in regulating cell proliferation, survival and death through alternative mRNA splicing.

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.

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.