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1.
MAbs ; 10(8): 1168-1181, 2018.
Article in English | MEDLINE | ID: mdl-30199322

ABSTRACT

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method's high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.


Subject(s)
Antibodies/immunology , Histocompatibility Antigens Class II/immunology , Peptides/immunology , Proteomics/methods , Adalimumab/immunology , Amino Acid Sequence , Animals , Antibodies/metabolism , Antibodies/therapeutic use , Antibody Affinity/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Infliximab/immunology , Mutation , Peptides/genetics , Peptides/metabolism , Protein Binding , Reproducibility of Results
2.
Exp Dermatol ; 27(1): 14-21, 2018 01.
Article in English | MEDLINE | ID: mdl-27714851

ABSTRACT

Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Interleukins/pharmacology , Pruritus/chemically induced , Receptors, Interleukin/metabolism , A549 Cells , Animals , CHO Cells , Cell Line , Cricetulus , DNA, Complementary/metabolism , Humans , Kinetics , Macaca fascicularis , Male , Mice , Pruritus/metabolism , Signal Transduction , Skin/immunology , Skin/pathology , Skin Diseases/immunology , Skin Diseases/pathology
3.
Sci Rep ; 7(1): 1080, 2017 04 24.
Article in English | MEDLINE | ID: mdl-28439081

ABSTRACT

Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.


Subject(s)
Antibodies, Neutralizing/immunology , Complement C5/antagonists & inhibitors , Complement C5/immunology , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Complement C5/chemistry , Crystallography, X-Ray , Hemoglobinuria, Paroxysmal/drug therapy , Humans , Macaca fascicularis , Protein Binding , Protein Conformation
4.
MAbs ; 9(5): 844-853, 2017 07.
Article in English | MEDLINE | ID: mdl-28387635

ABSTRACT

Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases.


Subject(s)
Histocompatibility Antigens Class I/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Receptors, Fc/immunology , Rheumatoid Factor/immunology , Amino Acid Substitution , Animals , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Macaca fascicularis , Mice , Receptors, Fc/chemistry , Receptors, Fc/genetics , Rheumatoid Factor/chemistry
5.
Drug Metab Pharmacokinet ; 31(2): 123-32, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26944099

ABSTRACT

Previously, we have reported novel engineered antibody with pH-dependent antigen-binding (recycling antibody), and with both pH-dependent antigen-binding and increased FcRn-binding at neutral pH (sweeping antibody). The purpose of this study is to perform PK/PD predictions to better understand the potential applications of the antibodies as therapeutics. To demonstrate the applicability of recycling and sweeping antibodies over conventional antibodies, PK/PD analyses were performed. PK/PD parameters for antibody and antigen dynamics were estimated from the results of a pharmacokinetic study in human FcRn transgenic mice. A simulation study was performed using the estimated PK/PD parameters with various target antigen profiles. In comparison to conventional antibody, recycling antibody enhanced antibody-antigen complex clearance by 3 folds, while sweeping antibody accelerated antigen clearance by 10 folds in a pharmacokinetic study. Simulation results showed that recycling and sweeping antibodies can improve dosage frequency and reduce the required dose for target antigens with various clearances, plasma concentrations or binding kinetics. Moreover, importance of the association rate constant to enhance the beneficial effect of antibodies was shown. These results support the conclusion that recycling and sweeping antibodies can be applied to various target antigens with different profiles, and expand the number of antigens that antibodies can target.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Antigens/chemistry , Antigens/immunology , Animals , Antibody Specificity , Binding Sites, Antibody , Humans , Hydrogen-Ion Concentration , Mice , Mice, Transgenic , Models, Animal , Protein Binding
6.
J Immunol ; 195(7): 3198-205, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26320252

ABSTRACT

Fc engineering can modulate the Fc-FcγR interaction and thus enhance the potency of Abs that target membrane-bound Ags, but it has not been applied to Abs that target soluble Ags. In this study, we revealed a previously unknown function of inhibitory FcγRII in vivo and, using an Ab that binds to Ag pH dependently, demonstrated that the function can be exploited to target soluble Ag. Because pH-dependent Ab dissociates Ag in acidic endosome, its Ag clearance from circulation reflects the cellular uptake rate of Ag/Ab complexes. In vivo studies showed that FcγR but not neonatal FcR contributes to Ag clearance by the pH-dependent Ab, and when Fc binding to mouse FcγRII and III was increased, Ag clearance was markedly accelerated in wild-type mice and FcR γ-chain knockout mice, but the effect was diminished in FcγRII knockout mice. This demonstrates that mouse FcγRII efficiently promotes Ab uptake into the cell and its subsequent recycling back to the cell surface. Furthermore, when a human IgG1 Fc variant with selectively increased binding to human FcγRIIb was tested in human FcγRIIb transgenic mice, Ag clearance was accelerated without compromising the Ab half-life. Taken together, inhibitory FcγRIIb was found to play a prominent role in the cellular uptake of monomeric Ag/Ab immune complexes in vivo, and when the Fc of a pH-dependent Ab was engineered to selectively enhance human FcγRIIb binding, the Ab could accelerate soluble Ag clearance from circulation. We assume such a function would enhance the therapeutic potency of Abs that target soluble Ags.


Subject(s)
Antigen-Antibody Complex/blood , Antigen-Antibody Reactions/immunology , Antigens/blood , Immunoglobulin G/immunology , Receptors, IgG/immunology , Animals , Antibodies/blood , Antibodies/immunology , Antigen-Antibody Complex/immunology , Antigens/immunology , Humans , Immunoglobulin G/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, IgG/genetics
7.
PLoS One ; 8(5): e63236, 2013.
Article in English | MEDLINE | ID: mdl-23667591

ABSTRACT

Monoclonal antibodies are widely used to target disease-related antigens. However, because conventional antibody binds to the antigen but cannot eliminate the antigen from plasma, and rather increases the plasma antigen concentration by reducing the clearance of the antigen, some clinically important antigens are still difficult to target with monoclonal antibodies because of the huge dosages required. While conventional antibody can only bind to the antigen, some natural endocytic receptors not only bind to the ligands but also continuously eliminate them from plasma by pH-dependent dissociation of the ligands within the acidic endosome and subsequent receptor recycling to the cell surface. Here, we demonstrate that an engineered antibody, named sweeping antibody, having both pH-dependent antigen binding (to mimic the receptor-ligand interaction) and increased binding to cell surface neonatal Fc receptor (FcRn) at neutral pH (to mimic the cell-bound form of the receptor), selectively eliminated the antigen from plasma. With this novel antigen-sweeping activity, antibody without in vitro neutralizing activity exerted in vivo efficacy by directly eliminating the antigen from plasma. Moreover, conversion of conventional antibody with in vitro neutralizing activity into sweeping antibody further potentiated the in vivo efficacy. Depending on the binding affinity to FcRn at neutral pH, sweeping antibody reduced antigen concentration 50- to 1000-fold compared to conventional antibody. Thereby, sweeping antibody antagonized excess amounts of antigen in plasma against which conventional antibody was completely ineffective, and could afford marked reduction of dosage to a level that conventional antibody can never achieve. Thus, the novel mode of action of sweeping antibody provides potential advantages over conventional antibody and may allow access to the target antigens which were previously undruggable by conventional antibody.


Subject(s)
Antibodies, Monoclonal/metabolism , Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Protein Engineering/methods , Receptors, Fc/metabolism , Animals , Antigens/blood , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Protein Binding/immunology , Signal Transduction/immunology
8.
Pharm Res ; 28(2): 405-12, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20953676

ABSTRACT

PURPOSE: Niemann-Pick C1-like 1 (NPC1L1), a pharmacological target of ezetimibe, is responsible for cholesterol absorption in enterocytes and hepatocytes. In the present study, the involvement of peroxisome proliferator-activated receptor α (PPARα) and its cofactor, PPARγ coactivator 1α (PGC1α) in the transcriptional regulation of human NPC1L1 was analyzed. METHODS: Reporter gene assays and electrophoretic mobility shift assays (EMSAs) were performed with the 5'-flanking region of the human NPC1L1 gene and the effect of siPPARα was examined. RESULTS: PPARα-mediated transactivation was observed with human NPC1L1 promoter constructs. Detailed analyses using deletion- and mutated-promoter constructs revealed the presence of a functional PPARα-response element (PPRE) upstream of the human NPC1L1 gene (-846/-834), a direct binding of PPARα and RXRα to which was confirmed by EMSAs. Moreover, PPARα-specific knockdown resulted in a significant decrease in the endogenous expression of NPC1L1 mRNA and protein in human-derived HepG2 cells. Furthermore, cotransfection of PGC1α stimulated the SREBP2/HNF4α- and PPARα/RXRα-mediated activation of the human NPC1L1 promoter. CONCLUSIONS: We found that PPARα positively regulates human NPC1L1 transcription via direct binding to a PPRE. Additionally, PGC1α stimulates the SREBP2/HNF4α- and PPARα/RXRα-mediated transactivation of human NPC1L1. These findings may provide new insights into the close relationship of glucose, fatty acids and cholesterol homeostasis.


Subject(s)
Intestinal Mucosa/metabolism , Membrane Proteins , PPAR alpha/metabolism , PPAR gamma/metabolism , Azetidines/pharmacology , Cholesterol/genetics , Cholesterol/metabolism , Electrophoretic Mobility Shift Assay , Enterocytes/metabolism , Ezetimibe , Fenofibrate/pharmacology , Gene Expression Regulation , Hep G2 Cells , Humans , Intestinal Absorption/drug effects , Intestinal Absorption/genetics , Intestines/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/metabolism , PPAR alpha/genetics , PPAR gamma/genetics , Protein Binding , Response Elements/genetics
9.
Pharm Res ; 25(5): 1134-41, 2008 May.
Article in English | MEDLINE | ID: mdl-18080173

ABSTRACT

PURPOSE: Niemann-Pick C1-like 1 (NPC1L1) has been identified as a target of ezetimibe and found to be responsible for intestinal cholesterol absorption. Although, it was recently demonstrated that sterol responsive element binding protein 2 (SREBP2) is responsible for the cholesterol-dependent down-regulation of NPC1L1, the molecular mechanism of NPC1L1 expression is not fully understood. In the present study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4alpha), a key modulator of lipid metabolism, in the transcriptional regulation of human NPC1L1 gene. METHODS: Reporter gene assays and EMSAs were performed using human NPC1L1 promoter constructs and the effect of siHNF4alpha was examined. RESULTS: Transfection of SREBP2 induced the transcriptional activities of NPC1L1 and additional transfection of HNF4alpha results in a marked stimulation of the activities. Studies with deletion mutants indicated that important elements are located within 264 nt upstream in the human NPC1L1 promoter. In addition, studies with mutations in putative binding sites of HNF4alpha indicated the existence of binding sites in -209 to -197 and -52 to -40. Moreover, HNF4alpha knockdown resulted in the reduced expression and regulation by cholesterol. CONCLUSIONS: It is concluded that HNF4alpha plays a crucial role in the expression and regulation of human NPC1L1 gene.


Subject(s)
Cholesterol/physiology , Hepatocyte Nuclear Factor 4/physiology , Membrane Proteins/physiology , 5' Flanking Region/genetics , Binding Sites , Cell Line , Cloning, Molecular , Electrophoretic Mobility Shift Assay , Gene Deletion , Gene Expression Regulation/physiology , Genetic Vectors , Hepatocyte Nuclear Factor 4/metabolism , Humans , Luciferases/genetics , Luciferases/metabolism , Membrane Proteins/genetics , Membrane Transport Proteins , Promoter Regions, Genetic/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 2/genetics , Sterol Regulatory Element Binding Protein 2/physiology , Transcriptional Activation , Transfection
10.
Pharm Res ; 24(2): 390-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17177110

ABSTRACT

PURPOSE: Recently identified organic solute transporter (Ost) alpha and beta are located on the basolateral membrane of enterocytes and may be responsible for the intestinal absorption of many substrates including bile acids. In the present study, the mechanism governing the transcriptional regulation of their expression was investigated. METHODS AND RESULTS: To clarify the transcriptional regulation of Osts, reporter gene assays were performed using mouse Ostalpha/beta promoter-luciferase reporter constructs. Co-transfection of the constructs with farnesoid X receptor (FXR) and retinoid X receptor alpha (RXRalpha) or liver X receptor alpha (LXRalpha) and RXRalpha into Caco-2 cells induced the transcriptional activities of both Ost alpha and beta and further increases were observed following treatment with each agonist. Sequence analyses indicated the presence of IR-1 regions in Ostalpha and Ostbeta promoters, which was confirmed by the finding that the deletion of IR-1 sequences abolished the response to FXR and LXRalpha. Furthermore, mutations in IR-1 reduced the FXR- and LXRalpha-dependent transactivation of Ostalpha/beta. Together with the detection of direct binding of FXR/RXRalpha and LXRalpha/RXRalpha to the IR-1 elements, the presence of functional FXRE/LXRE was revealed in the promoter region of both Ostalpha and Ostbeta. In addition, the stimulatory effect of FXR/RXRalpha and LXRalpha/RXRalpha on Ostalpha, but not on Ostbeta, was further enhanced by HNF-4alpha. CONCLUSIONS: It was concluded that LXRalpha/RXRalpha transcriptionally regulate mouse Ostalpha/beta via IR-1 elements shared with FXR/RXRalpha. Exposure to FXR/LXRalpha modulators may affect the disposition of Ostalpha/beta substrates.


Subject(s)
DNA-Binding Proteins/genetics , Membrane Transport Proteins/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Repetitive Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Transcriptional Activation/genetics , 5' Flanking Region/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophoretic Mobility Shift Assay , Genes, Reporter/genetics , Genetic Vectors/genetics , Hepatocyte Nuclear Factor 4/genetics , Liver X Receptors , Luciferases/genetics , Mice , Molecular Sequence Data , Mutation/physiology , Orphan Nuclear Receptors , Reverse Transcriptase Polymerase Chain Reaction
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