High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin-4 competing with the neuromyelitis optica autoantibody, NMO-IgG.

BACKGROUND AND PURPOSE: Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO-IgG, which recognizes the extracellular domains of the water channel, aquaporin-4. Binding of NMO-IgG to aquaporin-4 expressed in end-feet of astrocytes leads to complement-dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO-IgG to aquaporin-4, using high-avidity, non-pathogenic-chimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies. EXPERIMENTAL APPROACH: cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin-4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Igκ respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies. KEY RESULTS: Original monoclonal antibodies showed high avidity binding to human aquaporin-4, as determined by ELISA. Live imaging using Alexa-Fluor-555-labelled antibodies revealed that the antibody D15107 more rapidly bound to cells expressing human aquaporin-4 than others and strongly enhanced endocytosis of this water channel, while D12092 also bound rapidly to human aquaporin-4 but enhanced endocytosis to a lesser degree. Chimeric D15107 prevented complement-dependent cytotoxicity induced by NMO-IgG from patient sera in vitro. CONCLUSIONS AND IMPLICATIONS: We have established non-pathogenic, high-avidity, chimeric antibodies against the extracellular domains of human aquaporin-4, which provide a novel therapeutic option for preventing the progress and recurrence of NMO/NMO spectrum disorders.

The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis.

Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.

A novel monoclonal antibody against the C-terminal region of aquaporin-4.

Aquaporin-4 (AQP4), the most abundant water channel in the brain, plays a central role in water homeostasis, neuronal activity, and migration of astrocytes in the central nervous system. Recent studies have demonstrated that AQP4 is a target of an autoantibody specifically detected in an autoimmune neurologic disease called neuromyelitis optica. Here we have generated a monoclonal antibody (MAb) against the C-terminal region of AQP4 using a baculovirus expressing mouse AQP4 as an immunogen. This antibody (clone E5206) recognized both human and mouse AQP4s in a denaturing condition and was able to precipitate AQP4 from cell lysates of CHO cells stably expressing AQP4. Western blot analysis using deletion mutants revealed that the epitope was located within a region between Asp(303) and Leu(320) in the C-terminal tail of AQP4. Although clone E5206 could not be used for immunostaining when cells or tissues were fixed with 4% paraformaldehyde or 10% formalin, it could be used when cells were fixed with 10% trichloroacetic acid and when a formalin-fixed tissue section was pretreated with antigen-retrieval reagents. This MAb can be a valuable tool for analysis of AQP4 in a variety of physiological and pathophysiological contexts, in human tissues and organs as well as in rodent models, both in vitro and in vivo.

Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4.

Neuromyelitis optica is a demyelinating disease characterized by a disease-specific autoantibody designated as NMO-IgG that specifically recognizes aquaporin-4, and the binding of NMO-IgG to AQP4 causes the progress of the disease. Prevention of the binding of NMO-IgG, therefore, may alleviate the disease. Here we have developed monoclonal antibodies against AQP4 with a baculovirus display system in order to obtain high affinity monoclonal antibodies against the extracellular domains of AQP4. Our monoclonal antibodies can block the binding of NMO-IgG in spite of their heterogeneity. Taken together, we propose that our monoclonal antibodies can be applied in clinical therapy for NMO patients.

Functional reconstitution of olfactory receptor complex on baculovirus.

Despite that recent progress in genomics has elucidated the genomic structure of the olfactory receptors (ORs), most of them are still orphan receptors. The low expression level of ORs in heterologous cells has hampered many attempts to establish cell biological OR assay systems. Recently, we demonstrated that certain G protein-coupled receptors, such as the leukotriene B4 receptor or the dopamine D1 receptor, were efficiently reconstituted on baculovirus budding from infected Sf9 cells. The budded virus (BV) was shown to be mostly free of exogenous proteins other than those related to viral infection, resulting in low-noise assay conditions. Taking advantage of these conditions, we attempted to reconstitute OR complexes on BV. Sf9 cells were coinfected with recombinant baculoviruses harboring the cDNAs encoding adenylyl cyclase, trimeric G-protein, and the receptor: mOR-EG or S6. The coexpression of these proteins was detected by western blot, and the agonist- or antagonist-dependent receptor response was confirmed using ligand-dependent cyclic AMP production. These results demonstrated the successful reconstitution of functional OR complex on BV. Additionally, the expression of OR8B3 on BV, one of human orphan ORs, was also confirmed. This BV expression system is expected to be a highly effective tool for screening unknown ligands for ORs.

A method of generating antibodies against exogenously administered self-antigen by manipulating CD4+CD25+ regulatory T cells.

The generation of antibodies against self-antigens or antigens having a high degree of structural homology with self-antigens is a difficult task because of immunological tolerance. CD4(+)CD25(+) regulatory T cells play an important role in maintaining peripheral tolerance. Sakaguchi et al. previously reported that the transfusion of CD25(+) cell-depleted mouse splenocytes into syngeneic nude mice results in a breaking of peripheral tolerance that leads to the development of autoimmunity. In this study, we attempted to apply this mouse model to the generation of antibodies against self-antigens. We depleted CD25(+) cells from BALB/c mouse splenocytes and transferred the rest of the cells into syngeneic nude mice. The animals were immunized with mouse thyroglobulin. We observed a significant increase of the anti-mouse thyroglobulin antibody titer in the group of mice immunized twice within 10 days after the cell transfer (P<0.05). From these mice, we established hybridoma cell lines producing anti-mouse thyroglobulin monoclonal antibodies, including a clone with a dissociation constant of 10(-8)M. Control nude mice which received CD25(+) cell-containing BALB/c splenocytes did not produce anti-mouse thyroglobulin antibodies. When the CD25(-)cell-transferred nude mice were immunized with mouse Gα12, another self-antigen, anti-Gα12 antibodies were produced in the sera. This method should prove highly useful in the generation of antibodies against self-antigens or antigens for which the structure is highly conserved across species.

A novel screening system for claudin binder using baculoviral display.

Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

A simple detection method for low-affinity membrane protein interactions by baculoviral display.

BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV). In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA). Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L), and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand (GITRL). Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system.

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice.

A combination of genetic and environmental factors can cause autoimmune disease in animals. SKG mice, which are genetically prone to develop autoimmune arthritis, fail to develop the disease under a microbially clean condition, despite active thymic production of arthritogenic autoimmune T cells and their persistence in the periphery. However, in the clean environment, a single intraperitoneal injection of zymosan, a crude fungal beta-glucan, or purified beta-glucans such as curdlan and laminarin can trigger severe chronic arthritis in SKG mice, but only transient arthritis in normal mice. Blockade of Dectin-1, a major beta-glucan receptor, can prevent SKG arthritis triggered by beta-glucans, which strongly activate dendritic cells in vitro in a Dectin-1-dependent but Toll-like receptor-independent manner. Furthermore, antibiotic treatment against fungi can prevent SKG arthritis in an arthritis-prone microbial environment. Multiple injections of polyinosinic-polycytidylic acid double-stranded RNA also elicit mild arthritis in SKG mice. Thus, specific microbes, including fungi and viruses, may evoke autoimmune arthritis such as rheumatoid arthritis by stimulating innate immunity in individuals who harbor potentially arthritogenic autoimmune T cells as a result of genetic anomalies or variations.

Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9.

Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono- and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.

Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells.

Naturally arising CD25(+)CD4(+) regulatory T (T(R)) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25(+)CD4(+) T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25(+)CD4(+) T(R) cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T(R) cells, which became CD25(low) in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T(R) cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T(R) cells derived from originally transferred CD25(+)CD4(+) T(R) cells evoked graft rejection in the long-term tolerant mice, indicating that any T(R) cells deriving from CD25(-)CD4(+) naive T cells minimally contribute to graft tolerance and that natural T(R) cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T(R) cells can also be achieved in vitro by stimulating naturally present CD25(+)CD4(+) T cells with alloantigen in the presence of IL-2. The expanded CD25(+)CD4(+) T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3(+)CD25(+)CD4(+) T(R) cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.

Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice.

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. Here we show that a spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA.

Identification of gene expression profile in tolerizing murine cardiac allograft by costimulatory blockade.

The induction of specific tolerance would be the ultimate achievement in transplant immunology, but the precise mechanisms of immunologic tolerance remain largely unknown. Here, we investigated global gene expression analysis in tolerizing murine cardiac allografts by means of oligonucleotide microarrays. Tolerance induction was achieved in cardiac allografts from BALB/c to C57BL/6 mice by daily intraperitoneal injection of anti-CD80 and anti-CD86 monoclonal antibodies (mAbs). Comparative analysis revealed 64 genes to be induced more extensively in the tolerizing than in the syngeneic isografts, and 16 genes than in the rejecting allografts. Two genes were specifically upregulated in the tolerizing allografts. In the tolerizing allografts there were induced marked expressions of a number of genes for pro-inflammatory factors, including interferon-gamma-inducible cytokines and chemokines, as well as apoptosis-related genes, which were also upregulated in the rejecting allografts. Moreover, these gene expression patterns continued to be upregulated more than 70 days posttransplant. These results provide evidence that immunologic tolerance can be induced and maintained in the presence of prominent pro-inflammatory gene expression in vivo.

A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor.

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.