Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study.

BACKGROUND: In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). STUDY DESIGN AND METHODS: B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. RESULTS: Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. DISCUSSION: We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.

A Proximity-Induced Fluorogenic Reaction Triggered by Antibody-Antigen Interactions with Adjacent Epitopes.

Proximity-induced chemical reactions are site-specific and rapid by taking advantage of their high affinity and highly selective interactions with the template. However, reactions induced solely by antibody-antigen interactions have not been developed. Herein, we propose a biepitopic antigen-templated chemical reaction (BATER) as a novel template reaction. In BATER, reactive functional groups are conjugated to two antibodies that interact with two epitopes of the same antigen to accelerate the reaction. We developed a method for visualizing the progress of BATER using fluorogenic click chemistry for optimal antibody selection and linker design. The reaction is accelerated in the presence of a specific antigen in a linker length-dependent manner. The choice of the antibody epitope is important for a rapid reaction. This design will lead to various applications of BATER in living systems.

The Kg-antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn.

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible Peptide, Strengthening the Binding Affinity for an Antibody.

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein-protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.

Structural behavior of keratin-associated protein 8.1 in human hair as revealed by a monoclonal antibody.

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.

Click conjugation of a binuclear terbium(III) complex for real-time detection of tyrosine phosphorylation.

Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.

Conjugation-free, visual, and quantitative evaluation of inhibitors on protein tyrosine kinases and phosphatases with a luminescent Tb(III) complex.

A straightforward and visual method to assess inhibitors on protein tyrosine kinases (PTKs) and phosphatases (PTPs) has been developed. These enzymes play critical roles in a number of diseases and, thus, their inhibitors are important for effective therapy. With the use of the long-life luminescence emitted from a binuclear Tb(III) complex, enzymatic reactions of PTKs and PTPs were monitored in real-time, and the inhibitor activity was quantitatively evaluated in terms of the decrease in the rate of luminescence change. No conjugation of the probe to a substrate peptide was necessary. The IC50 values of four inhibitors on three kinds of PTKs [Src, Fyn, and epidermal growth factor receptor (EGFR)] were determined. For example, gefitinib, which is a selective inhibitor on EGFR, inhibited this PTK with IC50 of 22 nM. Towards Src and Fyn (non-targeted PTK), however, IC50 of this inhibitor was greater than 20 µM as expected. Inhibition of two kinds of PTPs (Shp-1 and PTP1B) by two inhibitors was also assayed, providing completely consistent results on their known selectivity. Furthermore, the system where both PTK and PTP are active was monitored and the reactions were visualized with the present Tb(III) complex-based method. High potential of the present method to a variety of systems has been evidenced.

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

Epitope-dependent thermodynamic signature of single-domain antibodies against hen egg lysozyme.

A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft or to a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing.

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Optimization of anti-ADAMTS13 antibodies for the treatment of ADAMTS13-related bleeding disorder in patients receiving circulatory assist device support.

ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motif 13)-related bleeding disorder has been frequently observed as a life-threatening clinical complication in patients carrying a circulatory assist device. Currently, treatment modalities for the bleeding disorder are very limited and not always successful. To address the unmet medical need, we constructed humanized antibodies of mouse anti-ADAMTS13 antibody A10 (mA10) by using complementarity-determining region (CDR) grafting techniques with human antibody frameworks, 8A7 and 16E8. The characteristics of the two humanized A10 antibodies, namely A10/8A7 and A10/16E8, were assessed in vitro and in silico. Among the two humanized A10 antibodies, the binding affinity of A10/16E8 to ADAMTS13 was comparable to that of mA10 and human-mouse chimeric A10. In addition, A10/16E8 largely inhibited the ADAMTS13 activity in vitro. The results indicated that A10/16E8 retained the binding affinity and inhibitory activity of mA10. To compare the antibody structures, we performed antibody structure modeling and structural similarity analysis in silico. As a result, A10/16E8 showed higher structural similarity to mA10, compared with A10/8A7, suggesting that A10/16E8 retains a native structure of mA10 as well as its antigen binding affinity and activity. A10/16E8 has great potential as a therapeutic agent for ADAMTS13-related bleeding disorder.

Novel neutralizing human monoclonal antibodies against tetanus neurotoxin.

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)-reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix-reactive antibodies provided enhanced protection compared to the use of each antibody alone.

Binuclear terbium(III) complex as a probe for tyrosine phosphorylation.

By using the luminescence from binuclear complexes of Tb(III) (Tb(2)-L(1) and Tb(2)-L(2)), phosphorylated Tyr residue in peptides was selectively detected in neutral aqueous solutions. Neither the non-phosphorylated Tyr, pSer, pThr, nor the other phosphate-containing biomolecules tested affected the luminescence intensity to any notable extent. Upon the binding of the pTyr to these Tb(III) complexes, the luminescence from the metal ion was notably promoted, as the light energy absorbed by the benzene ring of pTyr is efficiently transferred to the Tb(III) center. The binding activity of the binuclear Tb(III) complexes towards pTyr is two orders of magnitude larger than that of the corresponding mononuclear complex. These binuclear complexes were successfully used for real-time monitoring of enzymatic phosphorylation of a peptide by a tyrosine kinase.

Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher.

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.

64Cu-labeled minibody D2101 visualizes CDH17-positive gastric cancer xenografts with short waiting time.

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.

GPC1 specific CAR-T cells eradicate established solid tumor without adverse effects and synergize with anti-PD-1 Ab.

Current xenogeneic mouse models cannot evaluate on-target off-tumor adverse effect, hindering the development of chimeric antigen receptor (CAR) T cell therapies for solid tumors, due to limited human/mouse cross-reactivity of antibodies used in CAR and sever graft-versus-host disease induced by administered human T cells. We have evaluated safety and antitumor efficacy of CAR-T cells targeting glypican-1 (GPC1) overexpressed in various solid tumors. GPC1-specific human and murine CAR-T cells generated from our original anti-human/mouse GPC1 antibody showed strong antitumor effects in xenogeneic and syngeneic mouse models, respectively. Importantly, the murine CAR-T cells enhanced endogenous T cell responses against a non-GPC1 tumor antigen through the mechanism of antigen-spreading and showed synergistic antitumor effects with anti-PD-1 antibody without any adverse effects in syngeneic models. Our study shows the potential of GPC1 as a CAR-T cell target for solid tumors and the importance of syngeneic and xenogeneic models for evaluating their safety and efficacy.

111In-labeled anti-cadherin17 antibody D2101 has potential as a noninvasive imaging probe for diagnosing gastric cancer and lymph-node metastasis.

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.

Dual-Sensitive Nanomicelles Enhancing Systemic Delivery of Therapeutically Active Antibodies Specifically into the Brain.

Delivering therapeutic antibodies into the brain across the blood-brain barrier at a therapeutic level is a promising while challenging approach in the treatment of neurological disorders. Here, we present a polymeric nanomicelle (PM) system capable of delivering therapeutically effective levels of 3D6 antibody fragments (3D6-Fab) into the brain parenchyma for inhibiting Aß aggregation. PM assembly was achieved by charge-converting 3D6-Fab through pH-sensitive citraconylation to allow complexation with reductive-sensitive cationic polymers. Brain targeting was achieved by functionalizing the PM surface with glucose molecules to allow interaction with recycling glucose transporter (Glut)-1 proteins. Consequently, 41-fold enhanced 3D6-Fab accumulation in the brain was achieved by using the PM system compared to free 3D6-Fab. Furthermore, therapeutic benefits were obtained by successfully inhibiting Aß1-42 aggregation in Alzheimer's disease mice systemically treated with 3D6-Fab-loaded glucosylated PM. Hence, this nanocarrier system represents a promising method for effectively delivering functional antibody agents into the brain and treating neurological diseases.

Effect of allotypic variation of human IgG1 on the thermal stability of disulfide-linked knobs-into-holes mutants of the Fc for stable bispecific antibody design.

BACKGROUND: Disulfide-linked knobs-into-holes (dKiH) mutation is a well-validated antibody engineering technique to force heterodimer formation of different Fcs for efficient production of bispecific antibodies. An artificial disulfide bond is created between mutated cysteine residues in CH3 domain of human IgG1 Fc whose positions are 354 of the "knob" and 349 of the "hole" heavy chains. The disulfide bond is located adjacent to the exposed loop with allotypic variations at positions 356 and 358. Effects of the variation on the biophysical property of the Fc protein with dKiH mutations have not been reported. METHODS: We produced dKiH Fc proteins of high purity by affinity-tag fusion to the hole chain and IdeS treatment, which enabled removal of mispaired side products. Thermal stability was analyzed in a differential scanning calorimetry instrument. RESULTS: We firstly analyzed the effect of the difference in allotypes of the Fcs on the thermal stability of the heterodimeric Fc. We observed different melting profiles of the two allotypes (G1m1 and nG1m1) showing slightly higher melting temperature of G1m1 than nG1m1. Additionally, we showed different characteristics among heterodimers with different combinations of the allotypes in knob and hole chains. CONCLUSION: Allotypic variations affected melting profiles of dKiH Fc proteins possibly with larger contribution of variations adjacent to the disulfide linkage.

Computer-guided library generation applied to the optimization of single-domain antibodies.

Computer-guided library generation is a plausible strategy to optimize antibodies. Herein, we report the improvement of the affinity of a single-domain camelid antibody for its antigen using such approach. We first conducted experimental and computational alanine scanning to describe the precise energetic profile of the antibody-antigen interaction surface. Based on this characterization, we hypothesized that in-silico mutagenesis could be employed to guide the development of a small library for phage display with the goal of improving the affinity of an antibody for its antigen. Optimized antibody mutants were identified after three rounds of selection, in which an alanine residue at the core of the antibody-antigen interface was substituted by residues with large side-chains, generating diverse kinetic responses, and resulting in greater affinity (>10-fold) for the antigen.