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Abstract Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and T reg function, making T reg -specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rβγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D) 2. The reduced affinity of IgG-(IL-2N88D) 2 for the IL-2Rβγ receptor resulted in a T reg -selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D) 2 induced sustained preferential activation of T regs accompanied by a corresponding 10–14-fold increase in CD4+ and CD8+ CD25+FOXP3+ T regs ; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus T regs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D) 2 increased T regs while wild-type IgG-IL-2 increased NK cells in addition to T regs. The expanded human T regs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and T reg -selective IL-2 that activates and expands functional T regs in vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures. Highlights • A human IL-2 molecule mutated to decrease binding to the intermediate affinity IL-2 receptor preferentially activates Tregs. • Two IL-2 muteins fused to human IgG1 allow for sustained, preferential expansion of Tregs in cynomolgus and humanized mice. • As compared to the wild type IL-2 fusion protein, humanized mice expand fewer NK cells in response to the mutein. • The dynamic range of Treg increase based on dose suggests the ability to individualize dosing for particular diseases. [ABSTRACT FROM AUTHOR]

Human genome wide association studies (GWAS) have recently identified at least four new, non-MHC-linked candidate genes or gene regions causing type one diabetes (T1D), highlighting the need for functional models to investigate how susceptibility alleles at multiple common genes interact to mediate disease. Progress in Iocalizing genes in congenic strains of the nonobese diabetic (NOD) mouse has allowed the reproducible testing of gene functions and gene-gene interactions that can be reflected biologically as intra-pathway interactions, for example, IL-2 and its receptor CD25, pathway-pathway interactions such as two signaling pathways within a cell, or cell-cell interactions. Recent studies have identified likely causal genes in two congenic intervals associated with T1D, ldd3, and ldd5, and have documented the occurrence of gene-gene interactions, including "genetic masking", involving the genes encoding the critical immune molecules IL-2 and CTLA-4. The demonstration of gene-gene interactions in congenic mouse models of T1D has major implications for the understanding of human T1D since such biological interactions are highly likely to exist for human T1D genes. Although it is difficult to detect most gene-gene interactions in a population in which susceptibility and protective alleles at many loci are randomly segregating, their existence as revealed in congenic mice reinforces the hypothesis that T1D alleles can have strong biological effects and that such genes highlight pathways to consider as targets for immune intervention. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4 . [ABSTRACT FROM AUTHOR]

Abstract: Cholesteryl ester transfer protein (CETP) is a serum component responsible for both cholesteryl ester and triglyceride trafficking between high-density lipoprotein (HDL) and the apolipoprotein B (apoB)-containing very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). Several fluorescence-based assays that monitor these transfers have been reported, but to date such assays have suffered from a low signal/background (S/B) ratio and have been described for use only in relatively purified in vitro systems. We have modified the more advanced of these assays to incorporate a noninterfering, nondiffusable fluorescence quencher into previously described cosonicate particles, often referred to as microemulsions. This simple improvement resulted in particles that had an average threefold enhanced S/B window over particles without quenchers but that continued to show the essential properties of a catalytic assay, including catalysis to a single endpoint, excellent linearity with protein and particle concentration, and an appropriate sensitivity to inhibition. This reduced assay noise allowed the subsequent development of protocols for the direct measure of cholesteryl ester (CE) transfer activity resident in human and animal serum as well as the development of 384- and 3456-well screening protocols with good precision and accuracy. Thus, by expanding the dynamic response window of the assay, we have created an assay generalizable to many settings. [Copyright &y& Elsevier]

Abstract: The identification of causative genes for the autoimmune disease type 1 diabetes (T1D) in humans and candidate genes in the NOD mouse has made significant progress in recent years. In addition to sharing structural aspects of the MHC class II molecules that confer susceptibility or resistance to T1D, genes and pathways contributing to autoimmune pathogenesis are held in common by the two species. There are data demonstrating a similar need to establish central tolerance to insulin. Gene variants for the interacting molecules IL2 and CD25, members of a pathway that is essential for immune homeostasis, are present in mice and humans, respectively. Variation of two molecules that negatively regulate T cells, CTLA-4 and the tyrosine phosphatase LYP/PEP, are associated with susceptibility to human and NOD T1D. These observations underscore the value of the NOD mouse model for mechanistic studies on human T1D-associated molecular and cellular pathways. [Copyright &y& Elsevier]

Abstract: Idd9.3, a locus that determines susceptibility to the autoimmune disease type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse, has been mapped to the distal region of chromosome 4. In the current report we reduce the size of the Idd9.3 interval to 1.2Mb containing 15 genes, including one encoding the immune signaling molecule, 4-1BB, which shows amino acid variation between diabetes sensitive and resistant strains. 4-1BB, a member of the TNF receptor superfamily expressed by a variety of immune cells, mediates growth and survival signals for T cells. Functional analyses demonstrate that purified T cells from NOD congenic mice with the C57BL/10 (B10) allele at Idd9.3 produce more IL-2 and proliferate more vigorously in response to anti-CD3 plus immobilized 4-1BB ligand than T cells from NOD mice with the NOD allele at Idd9.3. In contrast, the response to anti-CD3 plus anti-CD28 costimulation was indistinguishable between the congenic strains, pinpointing the differences in NOD versus NOD.B10 Idd9.3 T cell responses to the 4-1BB costimulatory pathway. These data provide evidence in support of Idd9.3 as the locus encoding 4-1BB and suggest that the 4-1BB signaling pathway could have a primary function in the etiology of autoimmune disease. [Copyright &y& Elsevier]