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A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method. [ABSTRACT FROM AUTHOR]

Abstract: The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichia coli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions. [Copyright &y& Elsevier]

Examines the structural consequences of target epitope-directed functional alteration of an antibody. Comparison of the structure of a mutated antigen-mutant antibody complex with a wild-type antigen-wild type antibody complex; Role of plasticity of loops in the complementarity-determining region; Importance of the plasticity of the interaction between the variable regions of immunoglobulin chains.

We have been able to isolate several species of 5-S ribosomal RNA from Eschzerichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92. The consequent G U base pair, formed in a normally highly stable G. C-rich region. is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates laster in electrophoresis under denaturing conditions. This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired. [ABSTRACT FROM AUTHOR]

Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies. • A masking sequence, PAS, was used to inhibit antibody binding. • Fusion of PAS inhibited anti-CD3 scFv binding in a length-dependent manner. • Fusion of PAS inhibited the functions of T cell-engaging bispecific antibodies. • Antibody function was recovered by removing the PAS sequence. • Masking procedure using PAS was successfully applied to another scFv. [ABSTRACT FROM AUTHOR]

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or l-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6 M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by l-arginine. It is noteworthy that l-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2 M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and l-arginine may be used to recover the proteins in the native form from such insoluble particles. [Copyright &y& Elsevier]

Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inclusion bodies, (2) change in conformation and flexibility or solubility of proteins during refolding upon reduction of denaturant concentration, and (3) the effect of small molecule additives on refolding and aggregation of the proteins. [Copyright &y& Elsevier]

Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically. BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E. coli by using the avidin–biotin–peroxidase complex method with electron microscopy. Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space. Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes. The periplasmic spaces in some infected cells were expanded and filled with reaction products. Some cells showed wavy lines of positive staining in the periplasmic space. BIO-phages were detected as thick filaments or clusters covered with reaction products. The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane. These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection. [Copyright &y& Elsevier]

Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR–528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations. [ABSTRACT FROM AUTHOR]

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential. [ABSTRACT FROM AUTHOR]

A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents. • Backbone cyclization of bispecific VHHs has been constructed. • The cyclized bispecific VHHs, termed Cyclobody, show bispecificity. • The new format will expand the construction strategy of antibody engineering. [ABSTRACT FROM AUTHOR]

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies. [ABSTRACT FROM AUTHOR]

N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody. In contrast, lectin binding assays, which enable the total amounts of specific sugar residues to be detected, showed that both galactose and fucose residues in dimers and large aggregates were reduced to 70–80% of the amount in monomers. These results strongly suggest that the lack of N-linked oligosaccharides, a result of deglycosylation or aglycosylation, occurred in a proportion of the dimeric and large aggregated components. The present study demonstrates that glycosylation heterogeneities are a potential cause of antibody aggregation in cell culture of Chinese hamster ovary cells, and that the lack of N-glycosylation promotes the formation of dimers and finally results in large aggregates. [Copyright &y& Elsevier]

The aggregation of therapeutic antibodies during the manufacturing process is problematic because of the potential risks posed by the aggregates, such as an unexpected immune response. One of the hallmark effects of trehalose, a disaccharide consisting of two alpha-glucose units, is as a chemical chaperone with anti-aggregation activity. In this study, Chinese hamster ovary (CHO) cell line producing a diabody-type bispecific antibody were cultured in medium containing trehalose and the aggregation of the secreted proteins during the culture process was analyzed. An analysis of the various forms of the antibody (monomeric, dimeric, and large aggregates) showed that trehalose decreased the relative content of large aggregates by two thirds. The aggregation kinetics indicated that trehalose directly inhibited the polymerization and aggregation steps in a nucleation-dependent aggregation mechanism. Moreover, both specific and volumetric antibody production were increased in CHO cells cultured in trehalose-containing medium. Thus, the addition of trehalose to recombinant CHO cell cultures would offer a practical strategy for quality improvement in the production of therapeutic antibodies. [Copyright &y& Elsevier]

As a complementary tool to nanofluidics, biomolecular-basedtransportis envisioned for nanotechnological devices. We report a new methodfor guiding microtubule shuttles on multi-walled carbon nanotube tracks,aligned by dielectrophoresis on a functionalized surface. In the absenceof electric field and in fluid flow, alignment is maintained. Thedirected translocation of kinesin propelled microtubules has beeninvestigated using fluorescence microscopy. To our knowledge, thisis the first demonstration of microtubules gliding along carbon nanotubes. [ABSTRACT FROM AUTHOR]

To improve the efficiency of conventional gene amplification systems, the effect of cell cycle modification during the gene amplification process on IgG production was investigated in Chinese hamster ovary (CHO) cells. The full-length cDNA of CHO cell division cycle 25 homolog A (Cdc25A) was introduced into CHO DG44 cells and the effects of CDC25A overexpression on the cell cycle, transgene copy number and IgG productivity were examined. Both wild-type and mutated CDC25A-overexpressing CHO cells showed a rapid increase in transgene copy number compared with mock cells during the gene amplification process, in both cell pools and individual clones. High-producing clones were obtained with high frequency in CDC25A-overexpressing cell pools. The specific production rate of the isolated clone CHO SD-S23 was up to 2.9-fold higher than that of mock cells in the presence of 250 nM methotrexate (MTX). Cell cycle analysis revealed that the G2 to M phase transition rate was increased ∼1.5-fold in CDC25A-overexpressing CHO cells under MTX treatment. Our results show the improvement of conventional gene amplification systems via cell cycle engineering at an early stage of cell line development. [ABSTRACT FROM AUTHOR]

The construction of antibody fragments has the potential to reduce the high cost of therapeutic antibody production, but the structures of these fragments, with monovalency and the lack of an Fc region, can lead to reduced function. Multimerization is one strategy for recovering function that also yields better tumor-to-blood ratios than IgGs or monomeric antibody fragments because of rapid tumor uptake and clearance. Here, we constructed single-chain variable fragment (scFv) multimers by modifying the linker length and domain order of humanized anti- (epidermal growth factor receptor) IgG 528 (h528) and tested their ability to inhibit tumor growth. h528 scFv multimers, expressed using a bacterial expression system, were successfully fractionated and inhibited cancer growth in a multimerization-dependent manner, whereas the h528 scFv monomer showed no inhibition. h528 scFv trimers with variable heavy-light domain order and no linkers showed the highest in vitro and in vivo antitumor effects, which were comparable with those of the approved anti-(epidermal growth factor receptor) therapeutic IgG Cetuximab and Panitumumab. The trimers were also structurally stable in vitro and in vivo, which may be attributable to a strong interaction between the variable heavy and variable light domains of h528 Fv. Thus, h528 scFv multimers, especially trimers, are attractive as the next generation of anti-(epidermal growth factor receptor) therapeutic IgG and offer the possibility of low-cost production. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types. [ABSTRACT FROM AUTHOR]

Molecular evolution was used to generate capping molecules that selectively bound to the noncellulose components in cellulosic biomass and facilitated access of cellulolytic enzymes to the substrate components. The peptides, which were selected by means of a phage-display method, strongly promoted the enzymatic degradation of cellulose components in the biomass. [ABSTRACT FROM AUTHOR]

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr52 to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen–antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody. [ABSTRACT FROM AUTHOR]

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies. [ABSTRACT FROM AUTHOR]

Abstract: We report that several tryptophan-rich peptides exhibit an affinity for a hydrophobic ionic liquid (IL) (1-ethyl-3-methylimidazolium bis-trifluoromethanesulfonyl imide), and that green fluorescent protein (GFP) fused to a peptides, “SSSWWSWWWW” (SW1) or “SWWWWSWWWW” (SW2), containing serine (S) and tryptophan (W) at the C terminus localized at the IL/water interface. While GFPs without W-rich peptide distributed only in water phase, SW1- and SW2-GFPs were accumulated at the interface. The localization of SW1-GFP showed biphasic behavior, and most distinctive localization was observed at 7.1μM. The localization of SW2-GFP presumably occurred at largely lower concentration (≤0.5μM) than that of SW1-GFP, which difference was due to the higher hydrophobicity of SW2 peptide. [Copyright &y& Elsevier]

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure. [ABSTRACT FROM AUTHOR]

A plasmonic biosensor chip of silver-coated PMMA grating with a zinc oxide (ZnO) overlayer is fabricated for surface plasmon field-enhanced fluorescence (SPF) detection of Cy5-labeled green fluorescent protein (GFP). A bispecific antibody (anti-GFP x anti-ZnO antibody) prepared in our lab is densely immobilized on the sensor chip for GFP detection. The sensitivity of the plasmonic biosensors is improved due to densely packed antibodies and ZnO-coating that suppresses nonspecific protein adsorption and fluorescent quenching. With the ZnO-coated plasmonic chip, Cy5-labeled GFP of 10 pM can be detected through SPF. This sensitivity is 100 higher compared with the normal fluorescent detection on a ZnO-coated glass slide. [ABSTRACT FROM AUTHOR]

Abstract: Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD–EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism. [Copyright &y& Elsevier]

Abstract: Recently, a zinc oxide (ZnO)-binding peptide (ZnOBP) has been identified and has been used to assist the synthesis of unique crystalline ZnO particles. We analyzed the influence of ZnOBP on the crystal growth of ZnO structures formed from zinc hydroxide. The addition of ZnOBP in the hydrothermal synthesis of ZnO suppressed [0001] crystal growth in the ZnO particles, indicating that the specificity of the material-binding peptide for specific inorganic crystal faces controlled the crystal growth. Furthermore, the dipeptides with a partial sequence of ZnO-binding “hot spot” in ZnOBP were used to synthesize ZnO particles, and we found that the presence of these dipeptides more strictly suppressed (0001) growth in ZnO crystals than did the complete ZnOBP sequence. These results demonstrate the applicability of dipeptides selected from material-binding peptides to control inorganic crystal growth. [ABSTRACT FROM AUTHOR]

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling. [ABSTRACT FROM AUTHOR]

Abstract: Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and molecular/cellular biotechnology. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addition, frequently used bacterial alkaline phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here we tried to express the fusion proteins of antibody variable region(s) and secreted human placental alkaline phosphatase (SEAP) using mammalian cell culture. As a result, functional VH-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid production of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent VH/VL interaction for antigen quantitation. [ABSTRACT FROM AUTHOR]

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin-transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state. [Copyright &y& Elsevier]

A novel method that enables antibody fragments to be immobilized on a sensor substrate with a high binding capability using molecular recognition has been developed. Using genetic engineering, we fabricated bispecific recombinant antibody fragments, which consist of two kinds of antibody fragments: a gold antibody fragment and a target molecule antibody fragment. Surface plasmon resonance (SPR) analysis indicated that these gold-binding bispecific antibody fragments bind directly to the gold substrate with high affinity (KD ∼ 10-9 M). About 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target protein-binding efficiency. The Sips isotherm was used to assess the heterogeneity in antibody affinity for the bispecific antibody fragments. The results showed that the immobilized bispecific antibody fragments exhibited an increased homogeneity of affinity (KD) to target molecules when compared with monospecific antibody fragments immobilized by conventional methods. The use of bispecific antibody fragments to directly immobilize antibody fragments on a solid-phase substrate offers a useful platform for immunosensor applications [ABSTRACT FROM AUTHOR]

Antibodies and their fragments are attractive binding proteins because their high binding strength is generated by several hypervariable loop regions, and because high-quality libraries can be prepared from the vast gene clusters expressed by mammalian lymphocytes. Recent explorations of new genome sequences and protein structures have revealed various small, nonantibody scaffold proteins. Accurate structural descriptions of protein–protein interactions based on X-ray and NMR analyses allow us to generate binding proteins by using grafting and library techniques. Here, we review approaches for generating binding proteins from small scaffold proteins on the basis of tertiary structural information. Identification of binding sites from visualized tertiary structures supports the transfer of function by peptide grafting. The local library approach is advantageous as a go-between technique for grafted foreign peptide sequences and small scaffold proteins. The identification of binding sites also supports the construction of efficient libraries with a low probability of denatured variants, and, in combination with the design for library diversity, opens the way to increasing library density and randomized sequence lengths without decreasing density. Detailed tertiary structural analyses of protein–protein complexes allow accurate description of epitope locations to enable the design of and screening for multispecific, high-affinity proteins recognizing multiple epitopes in target molecules. [ABSTRACT FROM AUTHOR]

Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications. [ABSTRACT FROM AUTHOR]

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface. [ABSTRACT FROM AUTHOR]

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombinant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. Here, we propose a novel method for preparing refined, functional, tag-free bi-specific diabodies from IgG-like bi-specific antibodies (BsAbs) in a mammalian expression system. We created an IgG-like BsAb in which bi-specific diabodies were fused to the human Fc region via a designed human rhinovirus 3C (HRV3C) protease recognition site. The BsAb was purified by protein A affinity chromatography, and the refined tag-free hEx3-Db was efficiently produced from the Fc fusion format by protease digestion. The tag-free hEx3-Db from the Fc fusion format showed a greater inhibition of cancer growth than affinity-tagged hEx3-Db prepared directly from Chinese hamster ovary cells. We also applied our novel method to another small recombinant antibody fragment, hEx3 single-chain diabody (hEx3-scDb), and demonstrated the versatility and advantages of our proposed method compared with papain digestion of hEx3-scDb. This approach may be used for industrial-scale production of functional tag-free small therapeutic antibodies. [ABSTRACT FROM AUTHOR]

The glycosylation pattern of a humanized anti-EGFR×anti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody. [ABSTRACT FROM AUTHOR]

In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel β-sheet, designated as a ‘platform’, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)5. A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)3 was bound in the A–B–C subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these ‘platform’ residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis. [ABSTRACT FROM PUBLISHER]

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry. [ABSTRACT FROM AUTHOR]

Abstract: Although adoptive transfer of tumor-specific T cells is a plausible approach for cancer immunotherapy, the therapeutic application was hampered due to severe side effects caused by administration of high-dose interleukin (IL)-2, which was used for long-lasting maintenance of tumor-specific T cells in vivo. To solve this problem, here we propose to use an antibody/IL-2 receptor chimera, which can transduce a growth signal in response to a cognate antigen. As a model system, VH or VL region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was tethered to extracellular D2 domain of erythropoietin receptor and transmembrane/cytoplasmic domains of IL-2 receptor β or γ chain. When the pairs of chimeric receptors (VH-IL-2Rβ and VL-IL-2Rγ, or VH-IL-2Rγ and VL-IL-2Rβ) were expressed in IL-3-dependent pro-B cell line Ba/F3 and IL-2-dependent T cell line CTLL-2, the cognate antigen HEL induced selective expansion of gene-modified cells in the absence of IL-3 and IL-2, respectively. Growth assay revealed that the combination of VH-IL-2Rβ and VL-IL-2Rγ transduced a more stringent HEL-dependent growth signal, indicating some conformational effects of the chimeras. Furthermore, STAT3, STAT5 and ERK1/2, which are hallmarks for IL-2R signaling, were all activated by the antibody/IL-2R chimeras. These results clearly demonstrate that the antibody/IL-2R chimeras could substantially mimic the wild-type IL-2R signaling, suggesting the potential application in expansion of gene-modified T cells. [Copyright &y& Elsevier]

Abstract: Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials. [Copyright &y& Elsevier]

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized. [ABSTRACT FROM AUTHOR]

Abstract: We surveyed IL-21 receptor (IL-21R) in leukemia and lymphoma and found that follicular lymphoma cells showed exceptionally high IL-21R expression. Notably, IL-21 showed divergent effects depending on the cell origin: growth stimulation in Burkitt lymphoma cell lines and adult T cell leukemia/lymphoma cell lines but induction of apoptosis in B lymphoma cell lines with t(14;18)(q32;q21), a marker karyotype of follicular lymphoma. IL-21 activated caspase-8 and -3 and reduced mitochondrial membrane potential. More importantly, IL-21 decreased Bcl-2 expression but increased Bax expression. These results support a new therapeutic approach using the IL-21/IL-21R system in follicular lymphoma. [Copyright &y& Elsevier]

We previously reported the marked in vitro and in vivo anti-tumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgGl (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1–100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced anti-tumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense anti-tumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed. [ABSTRACT FROM AUTHOR]

We have previously designed antibody-cytokine receptor chimeras that could respond to a cognate antigen. While these chimeric receptors were functional, it has not been investigated exactly how they mimic signal transduction through corresponding wild-type receptors. In this study, we compared the growth properties and the phosphorylation status of intracellular signal transducers between the erythropoietin receptor (EpoR)- or gp130-based chimeric receptors and wild-type EpoR or EpoR-gp130 chimera, respectively. Expression plasmids, encoding VH or VL region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domain of EpoR and transmembrane/cytoplasmic domains of either EpoR or gp130, were constructed, and pairs of chimeric receptor combinations (VH-EpoR and VL-EpoR, VH-gp130 and VL-gp130, VH-EpoR and VL-gp130, VH-gp130 and VL-EpoR) were expressed in an IL-3-dependent myeloid cell line, 32D. Growth assay revealed that the transfectants all grew in a HEL-dependent manner. As for phosphorylation of Stat3, Stat5, ERK and Akt, the chimeric receptors showed similar activation pattern of signalling molecules with wild-type receptors, although the chimeric receptors showed ligand-independency and a little lower maximal phosphorylation than the corresponding wild-type receptors. The results demonstrate that antibody-receptor chimeras could substantially mimic wild-type receptors. [ABSTRACT FROM PUBLISHER]

Tyrosine is an important amino acid in protein-protein inter- action hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions. [ABSTRACT FROM AUTHOR]

HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte Ig-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including β2-microglobulin (132m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the β2m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the β2m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-Å resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCl recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G α3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/β2m) and free forms of β2m. Binding studies using β2m -free MHCls revealed differential β2m -dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCls, which may in turn regulate immune suppression. [ABSTRACT FROM AUTHOR]

HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys42-Cys42 disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed. [ABSTRACT FROM AUTHOR]

The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHCI-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCI recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell–cell recognition receptors. Thermodynamic analyses showed that LILRB1–MHCI interactions are entropically driven (−TΔS=−9.4∼−6.6kcalmol−1) with low heat capacity changes (ΔC p=−0.22∼−0.10kcalmol−1 K−1). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility. Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1–MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from “induced-fit” binding, which is associated with large reductions in conformational flexibility, and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells. [Copyright &y& Elsevier]

Abstract: The electromotility of outer hair cells (OHCs) is believed to be a major factor in cochlear amplification that enables the high sensitivity of hearing in mammals. This motility is thought to be based on voltage-dependent conformational changes of a motor protein embedded in the lateral wall of the OHC. In 2000, this motor protein was identified and termed prestin. To obtain knowledge on the function of prestin, research at the molecular level is necessary. For this purpose, a method of obtaining a large amount of prestin is required. In this study, an attempt was therefore made to construct an expression system for prestin. Prestin cDNA was introduced into Escherichia coli (E. coli), insect cells and Chinese hamster ovary (CHO) cells, and the expression of prestin was examined by Western blotting. As CHO cells expressed prestin well, we generated prestin-expressing cell lines using CHO cells by limiting dilution cloning. The stable expression and the activity of prestin in generated cell lines were then confirmed. Finally, to obtain prestin from these cell lines efficiently, culture conditions of the cells were examined, and it was clarified that cells should be cultured in serum-free medium and harvested around 48h after passage. [Copyright &y& Elsevier]