OR.93. Superagonistic Ex Vivo and In Vivo Stimulation of T Lymphocytes by Novel Human Cd28-specific Monoclonal Antibodies

Abstracts cells down-regulated T-cell proliferation and cytokine production in a syngenic or allogenic co-culture system, suggesting an immunoinhibitory effect of this single-chain anti-CTLA-4 Fv during T-cell response in vitro. However, transgenic NOD mice overexpressing this single-chain antibody on their h cells dramatically accelerated the development of autoimmune diabetes. Compared to non-transgenic littermates starting disease after 12 weeks old, they developed diabetes as early as 3 to 4 weeks old. We hypothesize that pancreatic expression of transgene, instead of transducing inhibitory signal through CTLA-4, masks the original interaction between B7 and CTLA-4, breaks down the tolerance and thus accelerates the disease process. To test this hypothesis, we currently cross these single-chain anti-CTLA-4 transgenic mice to another transgenic mice generated in our lab in which overexpressing PDL1 on their h cells. We assume that additional inhibitory signal provided by transgenic PD-L1 in double transgenic mice may rescue the accelerated autoimmune phenotype caused by losing the original tolerance maintained by B7 and CTLA-4 in single-chain anti-CTLA-4 Fv transgenic mice. doi:10.1016/j.clim.2006.04.396

OR.92. Ligand Independent Form of CTLA-4 Induced By Antisense Exon Skipping Alters T-Cell Activity and Inhibits Autoimmune Diabetes in NOD Mice. Dan Mourich,1 Marta Johnson,1 Shannon Oda,1 Brandon Manly.1 1Immunology, AVI BioPharma , Corvallis, OR; 2 Microbiology, Oregon State University, Corvallis, OR. The cytotoxic lymphocyte-associated antigen-4 (CTLA-4) is expressed primarily on T-cells and transduces a negative co-stimulatory signal regulating T-cell responses. Single nucleotide polymorphisms (SNPs) in the CTLA-4 gene that disrupt alternative splicing have been shown to be associated with the occurrence of autoimmune disease in humans and mice. We employed antisense exon skipping technology to induce a ligand independent form of CTLA-4 (CTLA-4li) in mouse T-cells by interfering with the splice acceptor sequence domain of exon 2. This alternative splice form does not naturally occur in NOD mice and was readily induced in vitro with 0.1 uM antisense oligomer synthesized on a phosphorodiamidate morpholine (PMO) backbone. Tcell adhesion, activation and proliferation were also examined after treatment with various antisense oligomers to determine if induction of different CTLA-4 splice forms would influence T-cell activity. T-cells induced to express CTLA-4li exhibited reduced activation and proliferation and increased adhesion to ICAM-1 similar to T-cells treated with Mab antibody to CTLA-4. In vivo studies were conducted using the NOD mouse to determine if the exon skipping antisense technology would influence the onset of diabetes. Mice received either a prophylactic (prior to evaluated blood sugar) or therapeutic treatment of antisense PMO that targeted either the induction of CTLA-4li, a soluble form CTLA-4(s) or direct inhibition of CTLA-4 expression. Animals were treated for ten days and then monitored for blood sugar levels twice weekly to 30 weeks age. Animals receiving PMO to induce CTLA-4li exhibited a significant reduction in

S39 disease incidence and time to onset of disease compared to controls. doi:10.1016/j.clim.2006.04.397

OR.93. Superagonistic Ex Vivo and In Vivo Stimulation of T Lymphocytes By Novel Human CD28-Specific Monoclonal Antibodies. Christine Guntermann,1 Martin Trischler,1 Niklas Beyersdorf,2 Peter Mueller,1 Thomas Kerkau,2 Thomas Huenig,2 Thomas Hanke.1 1Research and Development, TeGenero AG, Wuerzburg, Germany; 2Institute for Virology and Immunobiology, University of Wuerzburg, Wuerzburg, Germany. The frequent association of T lymphopenia with autoimmune manisfestations indicate an intimate linkage of T-cellmediated immunocompetence and tolerance. Consequently, restoration of a functional and tolerant T lymphocyte compartment is a major therapeutic goal in a broad panel of oncological as well as autoimmune indications. Recently, superagonistic CD28-specific antibodies with the capacity to both accelerate T-cell reconstitution and successfully suppress autoimmune diseases have been described in the rat model system. Here, we report the initial functional characterization of two novel humanized anti-CD28 antibodies specific for human and non-human primate CD28. Fine-specificity for the CD28 CU D loop as well as their ability to stimulate ex vivo the polyclonal expansion, activation and cytokine secretion of human T-cells and to induce proliferation of both naRve and memory CD4+ as well as CD8+ T-cells independently of T-cell antigen receptor stimulation characterizes the antibodies as btrueQ CD28 superagonists. Importantly, human CD28 superagonists potently expanded functional human CD4+CD25+ regulatory Tcells known to be key mediators of T-cell tolerance. In macaques, human CD28 superagonists elicited a welltolerated and transient in vivo T-cell expansion. Together, these findings highlight the quantitatively and qualitatively unique T-cell stimulating properties of human CD28 superagonists and underline their potential as future therapeutics in a cornucopia of indications with high unmet medical need. doi:10.1016/j.clim.2006.04.398

OR.94. Regulated Surface Expression of Programmed Cell Death-1 Discriminates CD4+CD25+Foxp3+ Resting Regulatory T-Cells from Activated T-Cells and Allows Identification of Antigen-Specific Regulatory T-Cells. Giorgio Raimondi, William Shufesky, Daisuke Tokita, Adrian Morelli, Angus Thomson. Surgery, TE Starzl Transplantation Institute-University of Pittsburgh, Pittsburgh, PA. Better characterization of naturally-arising CD4+CD25+ regulatory T-cells (Treg) will improve purification protocols and the application of Treg in the clinic. We have