Ray Rosa

Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4+ CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%–50% for over 2 weeks, despite its relatively short terminal half-life (t1/2 = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Antibody-mediated blockade of the CXCR3 chemokine receptor results in diminished recruitment of T helper 1 cells into sites of inflammation

Naïve T cells, when activated by specific antigen and cytokines, up‐regulate adhesion molecules as well as chemokine receptors on their surface, which allows them to migrate to inflamed tissues. Human studies have shown that CXCR3 is one of the chemokine receptors that is induced during T cell activation. Moreover, CXCR3‐positive T cells are enriched at inflammatory sites in patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this study, we use a mouse model of inflammation to demonstrate that CXCR3 is required for activated T cell transmigration to inflamed tissue. Using an anti‐ mCXCR3 antibody, we have shown that in vitro‐differentiated T helper (Th) 1 and Th2 cells up‐regulated CXCR3 upon stimulation with specific antigen/major histocompatibility complex. However, only Th1 cells, when adoptively transferred to syngeneic recipients, are efficiently recruited to the peritoneum in an adjuvant‐induced peritonitis model. Furthermore, the neutralizing anti‐mCXCR3 antibody profoundly inhibits the recruitment of Th1 cells to the inflamed peritoneum. Real‐time, quantitative reverse transcriptase‐polymerase chain reaction analysis demonstrates that the CXCR3 ligands, interferon (IFN)‐inducible protein 10 (CXCL10) and IFN‐inducible T cell α chemoattractant (CXCL11), are among the many chemokines induced in the adjuvant‐treated peritoneum. The anti‐mCXCR3 antibody is also effective in inhibiting a delayed‐type hypersensitivity response, which is largely mediated by enhanced trafficking of activated T cells to peripheral inflammatory sites. Collectively, our results suggest that CXCR3 has a critical role in T cell transmigration to sites of inflammation and thus, may serve as a molecular target for anti‐inflammatory therapies.

Fine Mapping, Gene Content, Comparative Sequencing, and Expression Analyses Support Ctla4 and Nramp1 as Candidates for Idd5.1 and Idd5.2 in the Nonobese Diabetic Mouse

At least two loci that determine susceptibility to type 1 diabetes in the NOD mouse have been mapped to chromosome 1, Idd5.1 (insulin-dependent diabetes 5.1) and Idd5.2. In this study, using a series of novel NOD.B10 congenic strains, Idd5.1 has been defined to a 2.1-Mb region containing only four genes, Ctla4, Icos, Als2cr19, and Nrp2 (neuropilin-2), thereby excluding a major candidate gene, Cd28. Genomic sequence comparison of the two functional candidate genes, Ctla4 and Icos, from the B6 (resistant at Idd5.1) and the NOD (susceptible at Idd5.1) strains revealed 62 single nucleotide polymorphisms (SNPs), only two of which were in coding regions. One of these coding SNPs, base 77 of Ctla4 exon 2, is a synonymous SNP and has been correlated previously with type 1 diabetes susceptibility and differential expression of a CTLA-4 isoform. Additional expression studies in this work support the hypothesis that this SNP in exon 2 is the genetic variation causing the biological effects of Idd5.1. Analysis of additional congenic strains has also localized Idd5.2 to a small region (1.52 Mb) of chromosome 1, but in contrast to the Idd5.1 interval, Idd5.2 contains at least 45 genes. Notably, the Idd5.2 region still includes the functionally polymorphic Nramp1 gene. Future experiments to test the identity of Idd5.1 and Idd5.2 as Ctla4 and Nramp1, respectively, can now be justified using approaches to specifically alter or mimic the candidate causative SNPs.

Plasma lipid profiling across species for the identification of optimal animal models of human dyslipidemia.

In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.

An Anti-PCSK9 Antibody Reduces LDL-Cholesterol On Top Of A Statin And Suppresses Hepatocyte SREBP-Regulated Genes

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

Identification and validation of genes affecting aortic lesions in mice

Atherosclerosis represents the most significant risk factor for coronary artery disease (CAD), the leading cause of death in developed countries. To better understand the pathogenesis of atherosclerosis, we applied a likeli-hood-based model selection method to infer gene-disease causality relationships for the aortic lesion trait in a segregating mouse population demonstrating a spectrum of susceptibility to developing atherosclerotic lesions. We identified 292 genes that tested causal for aortic lesions from liver and adipose tissues of these mice, and we experimentally validated one of these candidate causal genes, complement component 3a receptor 1 (C3ar1), using a knockout mouse model. We also found that genes identified by this method overlapped with genes progressively regulated in the aortic arches of 2 mouse models of atherosclerosis during atherosclerotic lesion development. By comparing our gene set with findings from public human genome-wide association studies (GWAS) of CAD and related traits, we found that 5 genes identified by our study overlapped with published studies in humans in which they were identified as risk factors for multiple atherosclerosis-related pathologies, including myocardial infarction, serum uric acid levels, mean platelet volume, aortic root size, and heart failure. Candidate causal genes were also found to be enriched with CAD risk polymorphisms identified by the Wellcome Trust Case Control Consortium (WTCCC). Our findings therefore validate the ability of causality testing procedures to provide insights into the mechanisms underlying atherosclerosis development.

Biphenyl-Substituted Oxazolidinones as Cholesteryl Ester Transfer Protein Inhibitors: Modifications of the Oxazolidinone Ring Leading to the Discovery of Anacetrapib

The development of the structure-activity studies leading to the discovery of anacetrapib is described. These studies focused on varying the substitution of the oxazolidinone ring of the 5-aryloxazolidinone system. Specifically, it was found that substitution of the 4-position with a methyl group with the cis-stereochemistry relative to the 5-aryl group afforded compounds with increased cholesteryl ester transfer protein (CETP) inhibition potency and a robust in vivo effect on increasing HDL-C levels in transgenic mice expressing cynomolgus monkey CETP.

siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE–/– and low density lipoprotein receptor (LDLr)–/– mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP+/– hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP+/– mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.

Small molecule activation of lecithin cholesterol acyltransferase modulates lipoprotein metabolism in mice and hamsters.

The objective was to assess whether pharmacological activation of lecithin cholesterol acyltransferase (LCAT) could exert beneficial effects on lipoprotein metabolism. A putative small molecule activator (compound A) was used as a tool compound in in vitro and in vivo studies. Compound A increased LCAT activity in vitro in plasma from mouse, hamster, rhesus monkey, and human. To assess the acute pharmacodynamic effects of compound A, C57Bl/6 mice and hamsters received a single dose (20 mg/kg) of compound A. Both species displayed a significant increase in high-density lipoprotein cholesterol (HDLc) and a significant decrease in non-HDLc and triglycerides acutely after dosing; these changes tracked with ex vivo plasma LCAT activity. To examine compound As chronic effect on lipoprotein metabolism, hamsters received a daily dosing of vehicle or of 20 or 60 mg/kg of compound A for 2 weeks. At study termination, compound treatment resulted in a significant increase in HDLc, HDL particle size, plasma apolipoprotein A-I level, and plasma cholesteryl ester (CE) to free cholesterol ratio, and a significant reduction in very low-density lipoprotein cholesterol. The increase in plasma CE mirrored the increase in HDL CE. Triglycerides trended toward a dose-dependent decrease in very low-density lipoprotein and HDL, with multiple triglyceride species reaching statistical significance. Gallbladder bile acids content displayed a significant and more than 2-fold increase with the 60 mg/kg treatment. We characterized pharmacological activation of LCAT by a small molecule extensively for the first time, and our findings support the potential of this approach in treating dyslipidemia and atherosclerosis; our analyses also provide mechanistic insight on LCATs role in lipoprotein metabolism.