17 research outputs found
Assessing agonistic potential of a candidate therapeutic anti-IL21R antibody
<p>Abstract</p> <p>Background</p> <p>Selective neutralization of the IL21/IL21R signaling pathway is a promising approach for the treatment of a variety of autoimmune diseases. Ab-01 is a human neutralizing anti-IL21R antibody. In order to ensure that the activities of Ab-01 are restricted to neutralization even under <it>in vitro </it>cross-linking and <it>in vivo </it>conditions, a comprehensive assessment of agonistic potential of Ab-01 was undertaken.</p> <p>Methods</p> <p><it>In vitro </it>antibody cross-linking and cell culture protocols reported for studies with a human agonistic antibody, TGN1412, were followed for Ab-01. rhIL21, the agonist ligand of the targeted receptor, and cross-linked anti-CD28 were used as positive controls for signal transduction. <it>In vivo </it>agonistic potential of Ab-01 was assessed by measuring expression levels of cytokine storm-associated and IL21 pathway genes in blood of cynomolgus monkeys before and after IV administration of Ab-01.</p> <p>Results</p> <p>Using a comprehensive set of assays that detected multiple activation signals in the presence of the positive control agonists, <it>in vitro </it>Ab-01-dependent activation was not detected in either PBMCs or the rhIL21-responsive cell line Daudi. Furthermore, no difference in gene expression levels was detected in blood before and after <it>in vivo </it>Ab-01 dosing of cynomolgus monkeys.</p> <p>Conclusions</p> <p>Despite efforts to intentionally force an agonistic signal from Ab-01, none could be detected.</p
Development and application of a biomarker assay for determining the pharmacodynamic activity of an antagonist candidate biotherapeutic antibody to IL21R in whole blood
Discovery and implementation of transcriptional biomarkers of synthetic LXR agonists in peripheral blood cells
<p>Abstract</p> <p>Background</p> <p>LXRs (Liver X Receptor α and β) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds.</p> <p>Objective</p> <p>Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators.</p> <p>Methods</p> <p>Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRα, LXRβ, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated <it>ex vivo </it>with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623.</p> <p>Results</p> <p>A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRα and LXRβ, and all cell types significantly increased ABCA1 and ABCG1 expression upon <it>ex vivo </it>LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription.</p> <p>Conclusion</p> <p>Peripheral blood cells express LXRα and LXRβ, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.</p
Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3
Abstract The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody’s activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb
LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model
Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E2 (PGE2) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRα and -β) are expressed in cartilage, with LXRβ being the predominant isoform. Here we show that genetic disruption of Lxrβ gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE2 production in articular cartilage treated with IL-1β, indicating a protective role of LXRβ in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE2 production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE2 synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis
Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO
Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease