Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker.

Named-Entity-Recognition-Based Automated System for Diagnosing Cybersecurity Situations in IoT Networks.

The aim of this paper was to enhance the process of diagnosing and detecting possible vulnerabilities within an Internet of Things (IoT) system by using a named entity recognition (NER)-based solution. In both research and practice, security system management experts rely on a large variety of heterogeneous security data sources, which are usually available in the form of natural language. This is challenging as the process is very time consuming and it is difficult to stay up to date with the constant findings in the areas of security threats, vulnerabilities, attacks, countermeasures, and risks.

Nicotinamide Phosphoribosyltransferase Inhibitor as a Novel Payload for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are a novel modality that allows targeted delivery of potent therapeutic agents to the desired site. Herein we report our discovery of NAMPT inhibitors as a novel nonantimitotic payload for ADCs. The resulting anti-c-Kit conjugates ( and ) demonstrated efficacy in the c-Kit positive gastrointestinal stromal tumor GIST-T1 xenograft model in a target-dependent manner.

The molecular mode of action and species specificity of canakinumab, a human monoclonal antibody neutralizing IL-1β.

Interleukin-1β (IL-1β) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1β antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1β from mouse, rat, rabbit, or macaques.

Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies and antibody drug conjugates.

Antibody engineering technologies are constantly advancing to improve the clinical effectiveness of monoclonal antibodies (mAbs). Effector functions may be modified by engineering the Fc region, for example to improve or reduce binding to Fc gamma receptors (FcγRs) or complement factors. Other examples for Fc engineering include modification of the half-life of immunoglobulin G (IgG); various studies have shown that half-life can be prolonged by increasing the affinity of Fc for the Fc neonatal receptor (FcRn).

The second-generation active Aβ immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects.

Immunization against amyloid-β (Aβ) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer's disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aβ1-6 coupled to the virus-like particle Qβ.

Rapid cerebral amyloid binding by Aβ antibodies infused into β-amyloid precursor protein transgenic mice.

Background: Passive immunization for the treatment of Alzheimer's disease (AD) was rapidly translated into clinical trials. However, basic mechanisms of AD immunotherapy remain only partially understood.

Methods: We analyzed the dynamic changes of amyloid-β (Aβ) levels in plasma, brain, and cerebrospinal fluid (CSF) as well as cerebral amyloid binding by Aβ antibody after a single β1-antibody infusion into APP(Swedish) and APP(wildtype) transgenic mice at preplaque and plaque-bearing age.

Cloning and pharmacological characterization of CCR7, CCL21 and CCL19 from Macaca fascicularis.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play an important role in lymphocyte homing and have also been associated with inflammatory, allergic and lung disorders. Cloning of the cynomolgus monkey genes encoding CCR7, CCL19 and CCL21 revealed 93-97% sequence identity of the deduced proteins with their respective human homologs. In chemotaxis assays, B300-19 cells transfected with the cynomolgus (c) CCR7 receptor migrated in response to cCCL19 and cCCL21 in a dose-dependent manner with EC(50) values of 324+/-188nM and 247+/-29nM, respectively.

Assessing the function of human UNC-93B in Toll-like receptor signaling and major histocompatibility complex II response.

The high sequence identity observed between UNC-93B of mouse and human imply common evolutionary ancestors and a conserved function. A nonconservative point mutation in the mouse Unc93b1 gene has been associated with defective Toll-like receptor (TLR) signaling and impaired major histocompatibility complex (MHC) I and II restricted antigen responses. Like murine UNC-93B, the human homologue is predicted to form 12 transmembrane domains, and it localizes to the endoplasmic reticulum.

Potent and selective xanthine-based inhibitors of phosphodiesterase 5.

Inhibitors of PDE5 are useful therapeutic agents for treatment of erectile dysfunction. A series of novel xanthine derivatives has been identified as potent inhibitors of PDE5, with good levels of selectivity against other PDE isoforms, including PDE6. Studies in the dog indicate excellent oral bioavailability for compound 21.

Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation.

Background: Anti-CD154 monoclonal antibodies (mAbs) cause long-term graft survival in preclinical allotransplantation experiments. This is the first report on the efficacy and safety of ABI793, a novel human anti-human CD154 mAb, in Cynomolgus renal transplant recipients.

Methods: ABI793 (human immunoglobulin-G1:kappa) was derived from a hybridoma generated after immunization of human immunoglobulin transgenic mice (HuMAb-Mouse, Medarex Inc.

Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies.

Culture conditions for successful amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with (15)N-phenylalanine, (15)N-glycine, (15)N-tyrosine or (15)N-valine. For the essential amino acids phenylalanine, tyrosine and valine high (15)N-label incorporation rates of >/=90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine.

Anti-Nogo-A antibody infusion 24 hours after experimental stroke improved behavioral outcome and corticospinal plasticity in normotensive and spontaneously hypertensive rats.

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti-Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks).

8-Aryl xanthines potent inhibitors of phosphodiesterase 5.

In clinical studies, several inhibitors of phosphodiesterase 5 (PDE5) have demonstrated utility in the treatment of erectile dysfunction. We describe herein a series of 8-aryl xanthine derivatives which function as potent PDE5 inhibitors with, in many cases, high levels of selectivity versus other PDE isoforms.

Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein.

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin-based anti-(human CD3) immunotoxin, DT389-scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1-389 of diphtheria toxin, scFv is single-chain variable-region antibody fragment and UCHT1is an anti-(human CD3) monoclonal antibody].

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity.

Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines.

Coordinate activation of endogenous p38alpha, beta, gamma, and delta by inflammatory stimuli.

The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines.

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus.

Use of organic solvents and small molecules for locating binding sites on proteins in solutions.

Application of a modified ePHOGSY and other NMR experiments to an H2O-DMSO solution of the protein FKBP12 identified the presence of one molecule of DMSO bound in the substrate binding site. It occupies the same spatial region occupied by the pipecolidine moiety of the immunosuppressive drugs FK506 and Rapamycin complexed to the protein. The binding constant K(D) for ths DMSO molecule was only 275 mM.

Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1).

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding.

Isolation, identification and immunosuppressive activity of a new IMM-125 metabolite from human liver microsomes. Identification of its cyclophilin A-IMM-125 metabolite complex by nanospray tandem mass spectrometry.

The isolation from human liver microsomes and identification by electrospray mass spectrometry and tandem mass spectrometry of a new metabolite of IMM-125 resulting from the biotransformation of the amino acid 1 vinylic methyl group to a carboxylic acid, called the IMM-125-COOH metabolite, is described. It was found that the complex of this new metabolite with cyclophilin A is formed less easily than the corresponding cyclophilin A-IMM-125-CH2OH main metabolite and cyclophilin A-IMM-125 complexes. However, when formed, the IMM-125-COOH metabolite-cyclophilin A complex requires more collision-induced dissociation (CID) to dissociate the complex than the complexes formed with the two other ligands.

Crystal structure of the SH2 domain from the adaptor protein SHC: a model for peptide binding based on X-ray and NMR data.

Src homology 2 domains (SH2) are protein molecules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl (pY)-containing protein sequences. We report here for crystal structure of the SH2 domain from the adaptor protein SHC (Shc), which has been refined by restrained least-squares methods to an R-factor of 17.3% to 2.