ABSTRACT
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.
Subject(s)
Gastrointestinal Microbiome , Oxalates , Animals , Oxalates/chemistry , Bacterial Proteins/metabolism , Membrane Transport Proteins/metabolism , Biological Transport , Bacteria/metabolismABSTRACT
Angiotensin II (Ang II) type 2 receptor (AT2R) is one of the major components of the renin-angiotensin-aldosterone system. Nevertheless, the physiological role is not well defined compared to the understanding of the Ang II type 1 receptor (AT1R), which is a well characterized G-protein coupled receptor in the cardiovascular system. While the AT2R signaling pathway remains unclear, AT2 receptor interacting protein 1 (ATIP1) has been identified as a candidate molecule for interacting with the C-terminal region of AT2R. In this study, we investigated the ATIP1 dependent AT2R inducible genes in human umbilical vein endothelial cells (HUVECs). CGP42112A, an AT2R specific agonist, resulted in an upregulation of inflammatory genes in HUVECs, which were inhibited by knocking down ATIP1 with siRNA (siATIP1). Among them, we confirmed by quantitative PCR that the induction of COX-2 mRNA expression was significantly downregulated by siATIP1. COX-2 was also upregulated by Ang II stimulation. This upregulation was suppressed by treatment with the AT2R specific antagonist PD123319, which was not replicated by the AT1R antagonist telmisartan. These findings suggest that ATIP1 plays an important role in AT2R dependent inflammatory responses. This may provide a new approach to the development of cardio-protective drugs.
ABSTRACT
We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.
Subject(s)
Cisplatin/pharmacology , Neoplasms/therapy , Radioimmunotherapy , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms/pathology , Treatment OutcomeABSTRACT
Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.
Subject(s)
Aflatoxin B1/toxicity , Hepatocytes/drug effects , Activation, Metabolic , Aflatoxin B1/administration & dosage , Aflatoxin B1/pharmacokinetics , Animals , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/pathology , Hepatocytes/transplantation , Humans , In Vitro Techniques , Lethal Dose 50 , Liver Transplantation , Male , Mice , Mice, SCID , RNA, Small Interfering/genetics , Transplantation Chimera , Vacuoles/drug effects , Vacuoles/pathologyABSTRACT
OBJECTIVE: We previously reported In-labeled anti-cadherin17 (CDH17) IgG visualized CDH17-positive gastric cancer xenografts. Unfortunately, a long waiting time was required to obtain high-contrast images due to long blood retention (blood half-life: 26 h). To accelerate blood clearance, we have developed anti-CDH17 minibody (D2101 minibody) and evaluated the pharmacokinetics in gastric cancer mouse models. METHODS: Two different single chain Fvs (scFvs), D2101 mutant and D2111, were developed from each parental IgG. The binding ability to CDH17 and stability in plasma were evaluated. D2101 minibody, constructed based on D2101 mutant scFv, was labeled with Cu (Cu-D2101 minibody), and the in-vitro and in-vivo properties were evaluated by cell ELISA, biodistribution experiments, and PET imaging in mice bearing CDH17-positive AGS and CDH17-negative MKN74 tumors. RESULTS: D2101 mutant and D2111 scFvs showed similar affinities to CDH17. D2101 mutant scFv was more stable than D2111 scFv in plasma. No loss of binding affinity of the D2101 minibody by chelate conjugation and radiolabeling procedures was observed. The biodistribution of Cu-D2101 minibody showed high uptake in AGS tumors and low uptake in MKN74. The blood half-life of Cu-D2101 minibody was 6.5 h. Improved blood clearance of Cu-D2101 minibody provided high tumor-to-blood ratios compared with the previous results of parental IgG in AGS xenograft mice. PET studies showed consistent results with biodistribution studies. CONCLUSIONS: Cu-D2101 minibody exhibited higher tumor-to-blood ratios at earlier time points than those of the radiolabeled parental IgG. Cu-D2101 minibody has potential as an immunoimaging agent for CDH17-positive tumors.
Subject(s)
Cell Transformation, Neoplastic , Copper Radioisotopes , Immunoglobulin Fragments/chemistry , Stomach Neoplasms/diagnostic imaging , Stomach Neoplasms/pathology , Amino Acid Sequence , Animals , Cell Line, Tumor , Humans , Isotope Labeling , Mice , Positron-Emission Tomography , Time Factors , Tissue DistributionABSTRACT
Angiotensin II (AngII) is a peptide hormone that plays a key role in regulating blood pressure, and its interactions with the G protein-coupled receptors, AngII type-1 receptor (AT1R) and AngII type-2 receptor (AT2R), are central to its mechanism of action. We solved the crystal structure of human AT2R bound to AngII and its specific antibody at 3.2-Å resolution. AngII (full agonist) and [Sar1, Ile8]-AngII (partial agonist) interact with AT2R in a similar fashion, except at the bottom of the AT2R ligand-binding pocket. In particular, the residues including Met1283.36, which constitute the deep end of the cavity, play important roles in angiotensin receptor (ATR) activation upon AngII binding. These differences that occur upon endogenous ligand binding may contribute to a structural change in AT2R, leading to normalization of the non-canonical coordination of helix 8. Our results will inform the design of more effective ligands for ATRs.
Subject(s)
Molecular Docking Simulation , Receptor, Angiotensin, Type 2/chemistry , Angiotensin II/chemistry , Angiotensin II/metabolism , Animals , Binding Sites , HEK293 Cells , Humans , Protein Binding , Receptor, Angiotensin, Type 2/metabolism , Sf9 Cells , SpodopteraABSTRACT
Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially added to the antigen, their proximity on the antigen results in covalent bond formation and generation of a biparatopic antibody. We validated the system with purified recombinant antigen. Results in antigen-overexpressing cells were promising although further optimization will be required. Because this strategy results in high-affinity targeting with a bipartite molecule that has considerably lower molecular weight than an antibody, this technology is potentially useful for diverse applications.
ABSTRACT
OBJECTIVE: Cadherin-17 (CDH17) is a transmembrane protein that mediates cell-cell adhesion and is frequently expressed in adenocarcinomas, including gastric cancer. CDH17 may be an effective diagnostic marker for the staging of gastric cancer. Here, we developed an 111In-labeled anti-CDH17 monoclonal antibody (Mab) as an imaging tracer and performed biodistribution and single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies using mice with CDH17-positive gastric cancer xenografts. CDH17 expression in gastric cancer specimens was also analyzed. METHODS: The cross-reactivity and affinity of our anti-CDH17 Mab D2101 was evaluated by surface plasmon resonance analysis and cell enzyme-linked immunosorbent assay, respectively. Biodistribution and SPECT/CT studies of 111In-labeled D2101 (111In-D2101) were performed. CDH17 expression in gastric cancer specimens was evaluated by immunohistochemistry. RESULTS: Surface plasmon resonance analysis revealed that D2101 specifically recognizes human CDH17, but not murine CDH17. The affinity of D2101 slightly decreased as a result of the radiolabeling procedures. The biodistribution study revealed high uptake of 111In-D2101 in tumors (maximum, 39.2 ± 9.5% ID/g at 96 h postinjection), but low uptake in normal organs, including the stomach. Temporal SPECT/CT imaging with 111In-D2101 visualized tumors with a high degree of tumor-to-nontumor contrast. Immunohistochemical analysis revealed that, compared with HER2, which is a potential marker of N-stage, CDH17 had a higher frequency of positivity in specimens of primary and metastatic gastric cancer. CONCLUSION: Our 111In-anti-CDH17 Mab D2101 depicted CDH17-positive gastric cancer xenografts in vivo and has the potential to be an imaging probe for the diagnosis of primary lesions and lymph-node metastasis in gastric cancer.
Subject(s)
Cadherins/immunology , Immunoconjugates/chemistry , Immunoconjugates/immunology , Indium Radioisotopes/chemistry , Single Photon Emission Computed Tomography Computed Tomography/methods , Stomach Neoplasms/diagnostic imaging , Stomach Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic , Gene Expression Regulation, Neoplastic , Humans , Immunoconjugates/pharmacokinetics , Isotope Labeling , Lymphatic Metastasis , Mice , Neoplasm Staging , Tissue DistributionABSTRACT
To investigate favorable single amino acid substitutions that improve antigen-antibody interactions, alanine (Ala) mutagenesis scanning of the interfacial residues of a cancer-targeted antibody, B5209B, was performed based on X-ray crystallography analysis. Two substitutions were shown to significantly enhance the binding affinity for the antigen, by up to 30-fold. One substitution improved the affinity by a gain of binding enthalpy, whereas the other substitution improved the affinity by a gain of binding entropy. Molecular dynamics simulations showed that the enthalpic improvement could be attributed to the stabilization of distant salt bridges located at the periphery of the antigen-antibody interface. The entropic improvement was due to the release of water molecules that were stably trapped in the antigen-antibody interface of the wild-type antibody. Importantly, these effects of the Ala substitutions were caused by subtle adjustments of the binding interface. These results will be helpful to design high-affinity antibodies with avoiding entropy-enthalpy compensation.
Subject(s)
Alanine/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Neoplasms/immunology , Amino Acid Substitution , Antibodies, Monoclonal/metabolism , Antibody Affinity , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/genetics , Antigen-Antibody Complex/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Neoplasms/therapy , Protein Binding , Protein Conformation , Protein EngineeringABSTRACT
Small leucine-rich repeat proteoglycan (SLRP) proteins have an important role in the organization of the extracellular matrix, especially in the formation of collagen fibrils. However, the mechanism governing the shape of collagen fibrils is poorly understood. Here, we report that the protein Osteomodulin (OMD) of the SLRP family is a monomeric protein in solution that interacts with type-I collagen. This interaction is dominated by weak electrostatic forces employing negatively charged residues of OMD, in particular Glu284 and Glu303, and controlled by entropic factors. The protein OMD establishes a fast-binding equilibrium with collagen, where OMD may engage not only with individual collagen molecules, but also with the growing fibrils. This weak electrostatic interaction is carefully balanced so it modulates the shape of the fibrils without compromising their viability.
ABSTRACT
Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.
Subject(s)
Antibodies, Monoclonal/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Animals , Antibodies, Monoclonal/immunology , Blotting, Western , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/immunology , Humans , Immunohistochemistry , In Situ Hybridization , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sheep , Substrate Specificity , Surface Plasmon ResonanceABSTRACT
Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the 'core' binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the 'extended' binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.
Subject(s)
Angiotensin II/analogs & derivatives , Receptor, Angiotensin, Type 2/chemistry , Allosteric Site , Amino Acid Sequence , Angiotensin II/chemistry , Angiotensin II/metabolism , Crystallography, X-Ray , Endothelin-1/chemistry , Endothelin-1/metabolism , Humans , Immunoglobulin Fragments , Kinetics , Ligands , Models, Molecular , Protein Conformation , Receptor, Angiotensin, Type 2/genetics , Receptor, Angiotensin, Type 2/metabolism , Signal Transduction , Static ElectricityABSTRACT
Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody-antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol-1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role.
Subject(s)
Antibodies/chemistry , Peptides/chemistry , Antigen-Antibody Reactions , Calorimetry , Hydrogen Bonding , Models, Molecular , Peptides/isolation & purification , Protein RefoldingABSTRACT
Cadherin-17 (CDH17) is highly expressed in gastric cancer and is thus considered to be a good target for antibody therapy. CDH17 is classified as a nonclassical cadherin, in that it is composed of seven extracellular cadherin domains. We generated anti-CDH17 monoclonal antibodies (mAbs) which recognize the extracellular domain of CDH17. Competitive assay using AGS, a gastric cancer cell line, cells revealed that five selected anti-CDH17 mAbs recognize different epitopes on CDH17. As AGS cells were shown to exhibit broad expression pattern of CDH17 by flow cytometry, we separated three clones with a low (10,000/cell), medium (50,000/cell), and high (200,000/cell) expression level, designating them as AGSlow, AGSmed, and AGShigh, respectively. The mAbs, coupled with saporin, exhibited effective cytotoxicity to AGShigh, but poor cytotoxicity to AGSlow. By contrast, the immunotoxin cocktail using the three clones D2101, D2005, and D2008, which recognize different epitopes, exhibited efficient cytotoxicity, even to the AGSlow group. The effect of the immunotoxin cocktail is synergistic, as the combination index was demonstrated to be below 1.0, as calculated by the method of Chou and Talalay using CalcuSyn software. These results suggest that the immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which expand the applicable range of immunotoxin therapy for cancer.
Subject(s)
Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Cadherins/immunology , Drug Synergism , Epitopes/immunology , Immunotoxins/pharmacology , Stomach Neoplasms/pathology , Biomarkers, Tumor/metabolism , Cadherins/antagonists & inhibitors , Cadherins/metabolism , Humans , Stomach Neoplasms/immunology , Stomach Neoplasms/metabolism , Tumor Cells, CulturedABSTRACT
METTL20 is a seven-ß-strand methyltransferase that is localised to the mitochondria and tri-methylates the electron transfer flavoprotein (ETF) ß subunit (ETFB) at lysines 200 and 203. It has been shown that METTL20 decreases the ability of ETF to extract electrons from medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase in vitro. METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria, suggesting that METTL20-mediated ETFB methylation may also play a regulatory role in mitochondrial metabolism. In this study, we generated Mettl20 knockout (KO) mice to uncover the in vivo functions of METTL20. The KO mice were viable, and a loss of ETFB methylation was confirmed. In vitro enzymatic assays revealed that mitochondrial ETF activity was higher in the KO mice than in wild-type mice, suggesting that the KO mice had higher ß-oxidation capacity. Calorimetric analysis showed that the KO mice fed a ketogenic diet had higher oxygen consumption and heat production. A subsequent cold tolerance test conducted after 24 h of fasting indicated that the KO mice had a better ability to maintain their body temperature in cold environments. Thus, METTL20 regulates ETF activity and heat production through lysine methylation when ß-oxidation is highly activated.
Subject(s)
Fasting/metabolism , Ketone Bodies/metabolism , Methyltransferases/metabolism , Oxidation-Reduction , Thermogenesis , Animals , CRISPR-Cas Systems , Catalysis , Electron-Transferring Flavoproteins/metabolism , Fatty Acids/metabolism , Gene Editing , Humans , Loss of Function Mutation , Lysine/metabolism , Metabolomics/methods , Methylation , Methyltransferases/genetics , Mice , Mice, Knockout , Mitochondria/metabolism , Oxygen Consumption , Substrate SpecificityABSTRACT
Accumulated evidence suggests a physiological relationship between the transcription factor NRF3 (NFE2L3) and cancers. Under physiological conditions, NRF3 is repressed by its endoplasmic reticulum (ER) sequestration. In response to unidentified signals, NRF3 enters the nucleus and modulates gene expression. However, molecular mechanisms underlying the nuclear translocation of NRF3 and its target gene in cancer cells remain poorly understood. We herein report that multiple regulation of NRF3 activities controls cell proliferation. Our analyses reveal that under physiological conditions, NRF3 is rapidly degraded by the ER-associated degradation (ERAD) ubiquitin ligase HRD1 and valosin-containing protein (VCP) in the cytoplasm. Furthermore, NRF3 is also degraded by ß-TRCP, an adaptor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase in the nucleus. The nuclear translocation of NRF3 from the ER requires the aspartic protease DNA-damage inducible 1 homolog 2 (DDI2) but does not require inhibition of its HRD1-VCP-mediated degradation. Finally, NRF3 mediates gene expression of the cell cycle regulator U2AF homology motif kinase 1 (UHMK1) for cell proliferation. Collectively, our study provides us many insights into the molecular regulation and biological function of NRF3 in cancer cells.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Cell Cycle/genetics , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Aspartic Acid Proteases/genetics , Aspartic Acid Proteases/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , COS Cells , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Chlorocebus aethiops , Cullin Proteins/genetics , Cullin Proteins/metabolism , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation , Epithelial Cells/pathology , HCT116 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction , Stem Cell Factor/genetics , Stem Cell Factor/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolism , beta-Transducin Repeat-Containing Proteins/genetics , beta-Transducin Repeat-Containing Proteins/metabolismABSTRACT
Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.
Subject(s)
Antibodies, Bispecific/immunology , Antigens/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Antigens/chemistry , Epitopes/chemistry , Humans , Immunoglobulin Fragments/immunologyABSTRACT
Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.