Novel Anti-CD70 Antibody Drug Conjugate for the Treatment of Adult T-Cell Leukemia (ATL).

BACKGROUND/AIM: Adult T-cell leukemia (ATL) is a hematological malignancy caused by infection with human T-cell leukemia virus type 1 (HTLV-1). Chemotherapy, antibody therapy, and bone marrow transplantation are used to treat this disease, however, median survival time has not been significantly improved. Our aim was to develop and evaluate a novel antibody-drug conjugate (ADC) with regards to cell cytotoxicity and target specificity. MATERIALS AND METHODS: In this study, we have constructed a novel ADC, which is composed of an anti-CD70 single chain Fv-Fc antibody conjugated with the anticancer agent emtansine using a novel antibody modification method. Cell cytotoxicity and target specificity were assessed using a cell proliferation assay. RESULTS: The anti-CD70 ADC selectively killed HTLV-1-infected cells and ATL cells without affecting other cells. CONCLUSION: The anti-CD70 ADC offers some chemotherapeutic potential for the treatment of ATL.

Synthesis and structure-activity relationship studies of IgG-binding peptides focused on the C-terminal histidine residue.

Currently, IgG-binding peptides are widely utilized as a research tool, as molecules that guide substrates to the Fc site for site-selective antibody modification, leading to preparation of a homogeneous antibody-drug conjugate. In this study, a structure-activity relationship study of an IgG-binding peptide, 15-IgBP, that is focused on its C-terminal His residue was performed in an attempt to create more potent peptides. A peptide with a substitution of His17 by 2-pyridylalanine (2-Pya) showed a good binding affinity (15-His17(2-Pya), K d = 75.7 nM). In combination with a previous result, we obtained 15-Lys8Leu/His17(2-Pya)-OH that showed a potent binding affinity (K d = 2.48 nM) and avoided three synthetic problems concerning the p-hydroxybenzyl amidation at the C-terminus, the difficulty associated with coupling at the His7 position and the racemization of 2-Pya.

Kinetics-Based Structural Requirements of Human Immunoglobulin G Binding Peptides.

Currently, antibodies are widely used not only in research but also in therapy. Hence, peptides that selectively bind to the fragment crystallizable site of an antibody have been extensively utilized in various research efforts such as the preparation of antibody-drug conjugates (ADC). Consequently, appropriate peptides that bind to immunoglobulin G (IgG) with a specific K d value and also k on and k off values will be useful in different applications, and these kinetic parameters have been perhaps overlooked but are key to development of peptide ligands with advantageous binding properties. We prepared structural derivatives of IgG-binding peptide 1 and evaluated the binding affinity and kinetic rates of the products by surface plasmon resonance assay and isothermal titration calorimetry to obtain novel peptides with beneficial antibody binding properties. In this way, 15-Lys8Leu with fast-binding and slow-release features was obtained through a shortened peptide 15-IgBP. On the other hand, we successfully obtained distinctive peptide, 15-Lys8Tle, with a similar K d value but with k on and k off values that were as much as six-fold different from those of 15-IgBP. These new peptides are useful for the elucidation of kinetic effects on the function of IgG-binding peptides and various applications of antibody or antibody-drug interactions, such as immunoliposome, ADC, or half-life extension strategy, by using a peptide with the appropriate kinetic features.

Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments.

To design an affinity ligand for purification of antigen-binding fragment (Fab) antibody, variable domain of heavy chain antibody (VHH) phage libraries were constructed from Fab-immunized Alpaca and subjected to biopanning against Fabs. To find the specific binders, we directly applied high-throughput sequencing (HTS) analysis of the VHH sequences in the panned phages on next-generation sequencer. The efficiently enriched sequences were aligned for construction of the phylogenetic tree to be categorized into five groups. VHHs from three major groups were first selected to analyze their properties as an affinity ligand. However, those VHHs were not suitable as an affinity ligand because of lack of resistance against alkaline pH and/or difficulty in acidic elution from the affinity column. So, we further searched the candidates from minor group sequences. Among five, one VHH showed the binding ability but with low affinity against Fabs. Therefore, we improved its affinity-by-affinity maturation through error-prone PCR library techniques. The final designed VHH showed highly alkaline pH resistance and easy acidic elution together with high affinity to Fabs. These results indicate that HTS techniques combined with biopanning and followed by error-prone PCR library techniques is powerful in designing specific binders with desired properties.

A new technology for increasing therapeutic protein levels in the brain over extended periods.

Effective delivery of protein therapeutics into the brain remains challenging because of difficulties associated with crossing the blood-brain barrier (BBB). To overcome this problem, many researchers have focused on antibodies binding the transferrin receptor (TfR), which is expressed in endothelial cells, including those of the BBB, and is involved in receptor-mediated transcytosis (RMT). RMT and anti-TfR antibodies provide a useful means of delivering therapeutics into the brain, but the anti-TfR antibody has a short half-life in blood because of its broad expression throughout the body. As a result, anti-TfR antibodies are only maintained at high concentrations in the brain for a short time. To overcome this problem, we developed a different approach which slows down the export of therapeutic antibodies from the brain by binding them to a brain-specific antigen. Here we report a new technology, named AccumuBrain, that achieves both high antibody concentration in the brain and a long half-life in blood by binding to myelin oligodendrocyte glycoprotein (MOG), which is specifically expressed in oligodendrocytes. We report that, using our technology, anti-MOG antibody levels in the brains of mice (Mus musculus) and rats (Rattus norvegicus) were increased several tens of times for a period of one month. The mechanism of this technology is different from that of RMT technologies like TfR and would constitute a breakthrough for central nervous system disease therapeutics.

Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format.

Artificially modified IgG molecules are increasingly utilized in industrial and clinical applications. In the present study, the method of chemical conjugation by affinity peptide (CCAP) for site-specific chemical modification has been developed by using a peptide that bound with high affinity to human IgG-Fc. This method enabled a rapid modification of a specific residue (Lys248 on Fc) in a one-step reaction under mild condition to form a stable amide bond between the peptide and Fc. The monovalent peptide-IgG conjugate not only maintained complete antigen binding but also bound to Fc receptors (FcRn, FcγRI, and FcγRIIIa), indicating that it is a suitable conjugate form that can be further developed into highly functional antibody therapeutics. CCAP was applied for the preparation of an antibody-drug conjugate and a bispecific antibody to demonstrate the usefulness of this method.

Prostaglandin E2 potentiates interferon-γ-induced nitric oxide production in cultured rat microglia.

Prostaglandin E2 (PGE2 ) plays crucial roles in managing microglial activation through the prostanoid EP2 receptor, a PGE2 receptor subtype. In this study, we report that PGE2 enhances interferon-γ (IFN-γ)-induced nitric oxide production in microglia. IFN-γ increased the release of nitrite, a metabolite of nitric oxide, which was augmented by PGE2 , although PGE2 by itself slightly affects nitrite release. The potentiating effect of PGE2 was positively associated with increased expression of inducible nitric oxide synthase. In contrast to nitrite release induced by IFN-γ, lipopolysaccharide-induced nitrite release was not affected by PGE2 . An EP2 agonist, ONO-AE1-259-01 also augmented IFN-γ-induced nitrite release, while an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, or an EP4 agonist, ONO-AE1-329, did not. In addition, the potentiating effect of PGE2 was inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3-240, or an EP4 antagonist, ONO-AE3-208, at 10-6  M. Among the EP agonists, ONO-AE1-259-01 alone was able to accumulate cyclic adenosine monophosphate (AMP), and among the EP antagonists, PF-04418948 was the only one able to inhibit PGE2 -increased intracellular cyclic AMP accumulation. On the other hand, IFN-γ promoted phosphorylation of signal transducer and activator of transcription 1, which was not affected by PGE2 . Furthermore, other prostanoid receptor agonists, PGD2 , PGF2α , iloprost, and U-46119, slightly affected IFN-γ-induced nitrite release. These results indicate that PGE2 potentiates IFN-γ-induced nitric oxide production in microglia through the EP2 receptor, which may shed light on one of the pro-inflammatory aspects of PGE2 .

Formation of three-way scanning electron microscope moiré on micro/nanostructures.

Three-way scanning electron microscope (SEM) moiré was first generated using a designed three-way electron beam (EB) in an SEM. The spot-type three-way SEM moiré comes from the interference between the three-way EB and the specimen grating in which the periodic cells are arranged in a triangular manner. The deformation and the structure information of the specimen grating in three directions can be simultaneously obtained from the three-way SEM moiré. The design considerations of the three-way EB were discussed. As an illustration, the three-way SEM moiré spots produced on a silicon slide were presented. The proposed three-way SEM moiré method is expected to characterize micro/nanostructures in triangular or hexagonal arrangements in three directions at the same time.

Spot moiré fringes: determination of domain boundaries and structural parameters in ordered nanoporous structures.

Spot moiré fringes are generated by the superposition between a nanoporous structure and a digital three-way grating. The spot moiré fringes are useful for the characterization of the domain boundaries and structural parameters in ordered nanoporous materials. The pitches and the orientations of the nanopore arrays in three directions can be simultaneously determined in a large view field.

Formation of secondary Moiré patterns for characterization of nanoporous alumina structures in multiple domains with different orientations.

We first report the formation of secondary Moiré patterns from electron Moiré fringes to characterize nanostructures in multiple domains with different orientations. The pitches and the orientations of the nanoporous alumina arrays in several domains are simultaneously measured using only one electron Moiré image.

Three-directional structural characterization of hexagonal packed nanoparticles by hexagonal digital moiré method.

We present the hexagonal digital moiré method for three-directional structural characterization of hexagonal packed nanostructures. A mismatch between a three-way grating and a nanoparticle assembly is shown to produce hexagonal moiré fringes due to the interference between three groups of parallel moiré patterns. The measuring principles of the pitches and the orientations of the three 1D arrays of the nanoparticles are presented. The structural information on a silica nanoparticle assembly is analyzed.

The exploration of domain sizes and orientation directions in ordered assembled nanoparticles with electron Moiré fringes.

Structural information such as domain sizes and orientation directions in ordered assembled nanoparticles can be simultaneously obtained by using generated electron Moiré fringes, without direct observation of the nanoparticles.

Fabrication of micromodel grid for various moiré methods by femtosecond laser exposure.

The femtosecond laser exposure system was used to fabricate model grids for the charge-coupled device (CCD) moiré method, scanning laser moiré method, and electron moiré method for microstrain deformation measurements. The femtosecond laser exposure produces mesoscopic variation patterns on the surface. These variation patterns make the grid in the scanning laser microscope and CCD images darker and make the grid in the scanning electron microscope image brighter. The CCD moiré fringe, scanning laser moiré fringe, and electron moiré fringe consisting of bright and dark lines were generated. As a demonstration, microstrain distribution of the three-point bending tested specimen was measured.

Study of the surface structure of butterfly wings using the scanning electron microscopic moiré method.

Scanning electron microscopic (SEM) moiré method was used to study the surface structure of three kinds of butterfly wings: Papilio maackii Menetries, Euploea midamus (Linnaeus), and Stichophthalma howqua (Westwood). Gratings composed of curves with different orientations were found on scales. The planar characteristics of gratings and some other planar features of the surface structure of these wings were revealed, respectively, in terms of virtual strain. Experimental results demonstrate that SEM moiré method is a simple, nonlocal, economical, effective technique for determining which grating exists on one whole scale, measuring the dimension and the whole planar structural character of the grating on each scale, as well as characterizing the relationship between gratings on different scales of each butterfly wing. Thus, the SEM moiré method is a useful tool to assist with characterizing the structure of butterfly wings and explaining their excellent properties.