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1.
MAbs ; 12(1): 1709322, 2020.
Article in English | MEDLINE | ID: mdl-31924119

ABSTRACT

CX3CR1 has been identified as a highly attractive target for several therapeutic interventions. Despite this potential, no potent antagonists, either small molecule or monoclonal antibody, have been identified. Here we describe the lead finding and engineering approach that lead to the identification of BI 655088, a potent biotherapeutic antagonist to CX3CR1. BI 655088 is a potent CX3CR1 antagonist that, upon therapeutic dosing, significantly inhibits plaque progression in the standard mouse model of atherosclerosis. BI 655088 represents a novel and highly selective biotherapeutic that could reduce inflammation in the atherosclerotic plaque when added to standard of care treatment including statins, which could result in a significant decrease in atherothrombotic events in patients with existing cardiovascular disease.


Subject(s)
Atherosclerosis/pathology , CX3C Chemokine Receptor 1/antagonists & inhibitors , Single-Domain Antibodies/pharmacology , Animals , Disease Progression , Humans , Macaca fascicularis , Mice
2.
Arthritis Res Ther ; 17: 135, 2015 May 20.
Article in English | MEDLINE | ID: mdl-25994180

ABSTRACT

INTRODUCTION: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. METHODS: ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. RESULTS: ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. CONCLUSIONS: ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.


Subject(s)
Antibodies, Bispecific/pharmacology , Antirheumatic Agents/pharmacology , Arthritis, Experimental/drug therapy , Receptors, Interleukin-6/antagonists & inhibitors , Single-Domain Antibodies/pharmacology , Animals , Arthritis, Rheumatoid/drug therapy , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Half-Life , Humans , Immunoglobulin Heavy Chains/immunology , Interleukin-6/immunology , Macaca fascicularis , Macaca mulatta , Serum Albumin/immunology
3.
Eur J Haematol ; 91(5): 399-410, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23952536

ABSTRACT

BACKGROUND: c-MET is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF-c-MET signaling is involved in many human malignancies, including multiple myeloma (MM). Recently, multiple agents have been developed directed to interfere at different levels in HGF-c-MET signaling pathway. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy-chain-only antibodies. In this study, we wanted to determine the anticancer effect of a novel anti-c-MET Nanobody in MM. METHODS: We examined the effects of an anti-c-MET Nanobody on thymidine incorporation, migration, adhesion of MM cells, and osteoblastogenesis in vitro. Furthermore, we investigated the effects of the Nanobody on HGF-dependent c-MET signaling by Western blotting. RESULTS: We show that the anti-c-MET Nanobody effectively inhibited thymidine incorporation of ANBL-6 MM cells via inhibition of an HGF autocrine growth loop and thymidine incorporation in INA-6 MM cells induced by exogenous HGF. HGF-induced migration and adhesion of INA-6 were completely and specifically blocked by the Nanobody. Furthermore, the Nanobody abolished the inhibiting effect of HGF on bone morphogenetic protein-2-induced alkaline phosphatase activity and the mineralization of human mesenchymal stem cells. Finally, we show that the Nanobody reduced phosphorylation of tyrosine residues in c-MET, MAPK, and Akt. We also compared the Nanobody with anti-c-MET monoclonal antibodies and revealed the similar or better effect. CONCLUSIONS: The anti-c-MET Nanobody inhibited MM cell migration, thymidine incorporation, and adhesion, and blocked the HGF-mediated inhibition of osteoblastogenesis. The anti-c-MET Nanobody might represent a novel therapeutic agent in the treatment of MM and other cancers driven by HGF-c-MET signaling.


Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Signal Transduction/drug effects , Single-Domain Antibodies/pharmacology , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Hepatocyte Growth Factor/pharmacology , Humans , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Osteogenesis/drug effects , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , Signal Transduction/genetics , Thymidine/metabolism
4.
Vet Immunol Immunopathol ; 124(3-4): 253-63, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18466981

ABSTRACT

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.


Subject(s)
Antigens, Protozoan/immunology , Protozoan Proteins/immunology , Protozoan Vaccines/pharmacology , T-Lymphocytes, Cytotoxic/immunology , Theileria parva/immunology , Theileriasis/immunology , Animals , Antibodies, Protozoan/blood , Antibody Formation/immunology , Antigens, Protozoan/genetics , Cattle , Cytotoxicity Tests, Immunologic/veterinary , Female , Flow Cytometry/veterinary , Histocompatibility Antigens Class I/immunology , Immunization/veterinary , Lymphocyte Activation , Protozoan Proteins/genetics , Protozoan Vaccines/immunology , Protozoan Vaccines/therapeutic use , Theileriasis/parasitology , Theileriasis/prevention & control , Vaccines, DNA/immunology , Vaccines, DNA/pharmacology , Vaccines, DNA/therapeutic use
5.
J Agric Food Chem ; 55(19): 7682-8, 2007 Sep 19.
Article in English | MEDLINE | ID: mdl-17715986

ABSTRACT

To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.


Subject(s)
Endo-1,4-beta Xylanases/antagonists & inhibitors , Enzyme Inhibitors/analysis , Enzyme-Linked Immunosorbent Assay/methods , Plant Proteins/analysis , Triticum/chemistry , Carrier Proteins/analysis , Edible Grain/chemistry , Intracellular Signaling Peptides and Proteins
6.
Am J Physiol Regul Integr Comp Physiol ; 291(1): R138-47, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16455759

ABSTRACT

Cerulenin, a natural fatty acid synthase (FAS) inhibitor, and its synthetic analog C75 are hypothesized to alter the metabolism of neurons in the hypothalamus that regulate ingestive behavior to cause a profound decrease of food intake and an increase in metabolic rate, leading to body weight loss. The bulk of data exclusively originates from mammals (rodents); however, such effects are currently lacking in nonmammalian species. We have, therefore, addressed this issue in broiler chickens because this species is selected for high growth rate and high food intake and is prone to obesity. First, we demonstrate that FAS messenger and protein are expressed in the hypothalamus of chickens. FAS immunoreactivity was detected in a number of brain regions, including the nucleus paraventricularis magnocellularis and the nucleus infundibuli hypothalami, the avian equivalent of the mammalian arcuate nucleus, suggesting that FAS may be involved in the regulation of food intake. Second, we show that hypothalamic FAS gene expression was significantly (P < 0.05) decreased by overnight fasting similar to that in liver, indicating that hypothalamic FAS gene is regulated by energy status in chickens. Finally, to investigate the physiological consequences of in vivo inhibition of fatty acid synthesis on food intake, we administered cerulenin by intravenous injections (15 mg/kg) to 2-wk-old broiler chickens. Cerulenin administration significantly reduced food intake by 23 to 34% (P < 0.05 to P < 0.0001) and downregulated FAS and melanocortin receptors 1, 4, and 5 gene expression (P < 0.05). However, the known orexigenic (neuropeptide Y, agouti gene-related peptide, orexin, and orexin receptor) and anorexigenic (pro-opiomelanocortin and corticotropin-releasing hormone) neuropeptide mRNA levels remained unchanged after cerulenin treatment. These results suggest that the catabolic effect of cerulenin in chickens may be mediated through the melanocortin system rather than the other neuropeptides known to be involved in food intake regulation.


Subject(s)
Appetite Depressants/pharmacology , Cerulenin/pharmacology , Chickens/physiology , Fatty Acid Synthases/metabolism , Receptors, Melanocortin/metabolism , Animals , Body Weight/drug effects , Fatty Acid Synthases/antagonists & inhibitors , Feeding Behavior , Gene Expression Regulation, Enzymologic/drug effects , Genotype , Hypothalamus/cytology , Hypothalamus/enzymology , Neuropeptides/metabolism , RNA, Messenger/metabolism , Receptors, Melanocortin/genetics
7.
Avian Pathol ; 34(3): 264-72, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16191712

ABSTRACT

The aim of this study was to investigate whether vaccination with the sugar-binding domain of FimH (FimH156) was able to protect chickens against avian pathogenic Escherichia coli (APEC). FimH156 was expressed and purified using Ni-NTA affinity chromatography. Binding of FimH156 to mannosylated bovine serum albumin demonstrated that the protein retained its biological activity. Moreover, anti-FimH156 antisera were able to inhibit in vitro binding of E. coli to mannosylated bovine serum albumin. In a first vaccination experiment, FimH156 was administered intramuscularly as a water-in-oil emulsion to specific pathogen free broiler chicks. A predisposing infection with the Newcastle disease virus strain Lasota was administered 3 weeks later, followed 3 days later by an aerosol challenge with the virulent APEC strain CH2. A good anti-FimH156 immunoglobulin (Ig)G immune response was detected in serum, but no protective effects of FimH156 against APEC were seen. In a second experiment, SPF chicks were vaccinated intramuscularly or intranasally with FimH156. Booster vaccinations were administered 20 days later. While the intramuscular immunization yielded a strong IgG response in the serum and trachea, no significant IgA response could be detected in tracheal washes. Intranasal immunization did not yield a significant IgG or IgA response in serum and trachea. No protective effects of the FimH156 could be detected, confirming the results of the first experiment. Thus, although the FimH156 induced a strong immune response, it was unable to protect chickens against APEC.


Subject(s)
Adhesins, Escherichia coli/immunology , Chickens/immunology , Escherichia coli Infections/veterinary , Escherichia coli Vaccines , Fimbriae Proteins/immunology , Poultry Diseases/prevention & control , Animals , Antibodies, Bacterial/blood , Chickens/microbiology , Escherichia coli Infections/microbiology , Escherichia coli Infections/prevention & control , Immunization/veterinary , Immunoglobulin G/blood , Poultry Diseases/microbiology , Protein Binding , Specific Pathogen-Free Organisms
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