Here, we have studied the activity of a novel proteintyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colonystimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.
CD28 and CTLA-4 are related members of a family of T lymphocyte cell surface receptors that function to regulate T cell activation. We have found that the cytoplasmic domains of both CTLA-4 and CD28 can associate with members of the PP2A family of serine/threonine phosphatases. The association of PP2A with CD28 was negatively regulated by tyrosine phosphorylation of the CD28 cytoplasmic domain. Inhibition of PP2A activity in Jurkat leukemia T cells by treatment with okadaic acid or by expression of a dominant-negative mutant enhanced T cell activation induced by CD28 engagement. Interactions between cell surface receptors such as CTLA-4 and CD28 and serine/threonine phosphatases may represent a novel mechanism for modulating the intracellular signal transduction pathways associated with cell activation.
Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.
The expression of endogenous retrotransposable elements including Long Interspersed Nuclear Element-1 (LINE-1 or L1) and Human Endogenous Retrovirus (HERV)-K accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. Here we address the safety and immunogenicity of vaccination with these elements. We employed immunohistochemical analysis and literature-precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 Open Reading Frame-2 (L1O2) induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human L1O2 (96% identity with macaque), and Simian ERV (SERV)-K Gag and Env induced polyfunctional T cell responses to all antigens, and antibody responses to SERV-K Env. There were no adverse safety or pathology findings related to vaccination. These studies provide the first evidence that immune responses can be induced safely against this class of self antigens, and pave the way for their investigation as HIV- or tumor-associated targets.
CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4 nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.
Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.-Zhu, J., J. Gardner, C. R. Pullinger, J. P. Kane, J. F. Thompson, and O. L. Francone. Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1. J. Lipid Res. 2009Res. . 50: 1330Res. -1339 Supplementary key words apoAI synthesisDyslipidemia is a major risk factor for premature development of cardiovascular disease. A high ratio of atherogenic lipoproteins (VLDL, LDL, and IDL) to HDL leads to endothelial dysfunction, focal inflammation, and oxidative stress resulting in the initiation and progression of atherosclerosis. Plasma levels of HDL are inversely correlated with the onset of coronary artery disease as attested to by the outcome of several large prospective epidemiological studies (1, 2). The protective effect of HDL is due, at least in part, to the role of this class of lipoproteins in modulating inflammation and mediating cholesterol transport from peripheral cells to sites of reutilization and degradation (3). Apolipoprotein AI (apoAI); the main protein moiety of HDL; drives the formation and speciation of HDL resulting in a very heterogeneous class of particles, particularly with respect to particle size, density, protein content, and surface charge, which determines their functional role and metabolic fate (4). Understanding the processes involved in the synthesis and maturation of HDL species will ultimately provide the knowledge required to understand and appreciate the functional significance of each of the HDL subpopulations and their impact in the development of atherosclerosis.The importance of HDL-C in disease makes it amenable to candidate-gene an...
2539 Background: CD40 is expressed on B-cells, monocytes, dendritic cells, other normal tissues and tumors. Previous studies showed that CD40 stimulation enhances antigen presentation, breaks tolerance, bypasses T-cell help, and induces apoptosis in CD40pos tumor cells. We report the in vitro activity and primate pharmacokinetics of a human anti-CD40 agonist antibody, CP-870,893, currently in clinical trials for the treatment of cancer. Methods: CP-870,893 was identified as a CD40 agonist antibody by screening lead molecules generated through the Abgenix Xenomouse® platform. Agonist activity was determined using upregulation of B-cell and monocytes derived dendritic cell surface markers, as well as dendritic cell IL-12 induction. BIAcore and equilibrium binding were utilized to determine affinity, and competition studies with CD40L were conducted on BIAcore. CP-870,893 was administered to cynomolgus monkeys i.v. at various doses, serum antibody levels were evaluated over time in an ELISA assay, and B-cell markers were monitored by FACS. Results: CP-870,893 (IgG2, kappa) binds CD40 with sub-nanomolar affinity, and does not block binding of CD40L. When human whole blood is incubated with CP-870,893, upregulation of key surface molecules involved in antigen presentation (MHC Class II, CD80, CD86, CD23 and ICAM-1) is observed with an EC50 of 5–50 ng/ml. Human monocytes derived dendritic cells, when stimulated with CP-870,893, upregulate activation markers (MHC Class II, CD80 and CD83) with an EC50 of 100–300 ng/ml, and secrete high levels of IL-12p40. In the presence of a second stimulus, such as LPS, human dendritic cells also secreted bioactive IL12-p70 when stimulated with CP-870,893 (EC50 ∼ 150 ng/ml). In addition, a CD40 positive human B-cell tumor line, when stimulated with CP-870,893, becomes susceptible to killing by human CTLs. In cynomolgus monkey studies, the clearance of CP-870,893 decreased with increasing dose. Circulating B-cell numbers decreased, and surface molecules were upregulated on B-cells. Conclusions: These data support the potential utility of CP-870,893 as an immune enhancing agent in cancer immunotherapy, by activating antigen presenting cells, and by enhancing the immunogenicity of CD40 positive tumor cells. [Table: see text]
SUMMARYAntigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4 þ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.
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