IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFß.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

Imaging the Alternatively Spliced D Domain of Tenascin C in a Preclinical Model of Inflammatory Bowel Disease.

PURPOSE: To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR)-labeled targeted molecular imaging agents. PROCEDURES: A human IgG1 with nanomolar binding affinity specific to the alternatively spliced D domain of tenascin C was generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix protein in the colon of mice given dextran sulfate sodium in the drinking water. The antibody reagent was labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate in vivo targeting specificity. Increasing doses of imaging agent were given to estimate the saturating dose. RESULTS: The NIR-labeled proteins successfully targeted colonic lesions in a murine model of colitis. Co-administration of a molar excess competing unlabeled dose reduced normalized uptake in diseased colon by > 70%. Near infrared ex vivo images of colon resected from diseased animals showed saturation at doses exceeding 1 nmol and was confirmed with additional quantitative ex vivo biodistribution. Cellular-level specificity and protein stability were assessed via microscopy. CONCLUSIONS: Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.

Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain.

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.

Generation and characterization of ABBV642, a dual variable domain immunoglobulin molecule (DVD-Ig) that potently neutralizes VEGF and PDGF-BB and is designed for the treatment of exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis. Preclinical studies and early phase clinical trials suggest that combined suppression of VEGF and platelet-derived growth factor-BB (PDGF-BB) provides better outcomes than suppression of VEGF alone, due to more frequent regression of neovascularization (NV) and suppression of subretinal fibrosis. We generated a dual variable domain immunoglobulin molecule, ABBV642 that specifically and potently binds and neutralizes VEGF and PDGF-BB. ABBV642 has been optimized for treatment of exudative AMD based on the following design characteristics: 1) high affinity binding to all VEGF-A isoforms and both soluble and extracellular matrix (ECM)-associated PDGF-BB; 2) potential for extended residence time in the vitreous cavity to decrease the frequency of intraocular injections; 3) rapid clearance from systemic circulation compared with molecules with wild type Fc region for normal FcRn binding, which may reduce the risk of systemic complications; and 4) low risk of potential effector function. The bispecificity of ABBV642 allows for a single injection of a single therapeutic agent, and thus a more streamlined development and regulatory path compared with combination products. In a mouse model of exudative AMD, ABBV642 was observed to be more effective than aflibercept. ABBV642 has potential to improve efficacy with reduced injection frequency in patients with exudative AMD, thereby reducing the enormous disease burden for patients and society.

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule.

Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.

Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase and TNF-alpha production.

COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production of TNF-alpha and other pro-inflammatory cytokines such as IL-1beta via the ERK/MAP kinase pathway. As TNF-alpha and IL-1beta are clinically validated targets for therapeutic intervention in rheumatoid arthritis (RA), blocking COT provides a potential avenue for amelioration of disease. Herein we describe identification of a cellular active selective small molecule inhibitor of COT kinase.

A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors.

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin.

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification and characterization.

Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.

Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105.

Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT.