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Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Ne-(o-azidobenzyloxycarbonyl)- l -lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Photodynamic therapy (PDT) utilize a photosensitizing agent and light for cancer therapy. It exerts anti-cancer effect mainly by inducing vascular occlusion at the irradiated site. By controlling the irradiation area, PDT can be used in a tumor-specific manner. However, the non-specific cellular damage in the surrounding normal tissue is still a serious concern. Photoimmunotherapy (PIT) is a new type of targeted cancer therapy that uses an antibody-photon absorber conjugate (APC). The superiority of PIT to PDT is the improved target specificity, thereby reducing the damage to normal tissues. Here, we developed a novel APC targeting epithelial cell adhesion molecule (EpCAM) as well as a negative control APC that does not bind to the EpCAM antigen. Our in vitro analysis of APC cytotoxicity demonstrated that the EpCAM APC, but not the negative control, was cytotoxic to EpCAM expressing COLO 205 cells after photoirradiation, suggesting that the cytotoxicity is antigen-dependent. However, in our in vivo analysis using a mouse xenograft tumor model, decreased volume of the tumors was observed in all the mice treated with irradiation, regardless of whether they were treated with the EpCAM APC or the negative control. Detailed investigation of the mechanism of these in vivo reveal that both APCs induce vascular occlusion at the irradiation site. Furthermore, the level of vascular occlusion was correlated with the blood concentration of APC, not the tumor concentration. These results imply that, similar to PDT, PIT can also induce non-targeted vascular occlusion and further optimization is required before widespread clinical use.

We have developed 10-inch RGBW LCD with edge-lit 2D local dimming backlight system for automotive applications. This prototype system has power consumption reduction performance of 41.5% compared to a conventional RGB display without local dimming backlight. The performance and evaluation results are described in this paper.

Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Ne-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallogr...

We have developed a new 7-inch WQXGA RGBW mobile LCD with local dimming backlight technology. This panel achieves a 20% power reduction compared to that of the prior RGBW displays, with no image quality degradation. It also realizes contrast improvement and a total module thickness of 1.16mm.

Carcinoembryonic antigen (CEA) is a classic tumor‐specific antigen that is overexpressed in several cancers, including gastric cancer. Although some anti‐CEA antibodies have been tested, to the best of our knowledge, there are currently no clinically approved anti‐CEA antibody therapies. Because of this, we have created the novel anti‐CEA antibody, 15‐1‐32, which exhibits stronger binding to membrane‐bound CEA on cancer cells than existing anti‐CEA antibodies. 15‐1‐32 also shows poor affinity for soluble CEA; thus, the binding activity of 15‐1‐32 to membrane‐bound CEA is not influenced by soluble CEA. In addition, we constructed a 15‐1‐32‐monomethyl auristatin E conjugate (15‐1‐32‐vcMMAE) to improve the therapeutic efficacy of 15‐1‐32. 15‐1‐32‐vcMMAE showed enhanced antitumor activity against gastric cancer cell lines. Unlike with existing anti‐CEA antibody therapies, antitumor activity of 15‐1‐32‐vcMMAE was retained in the presence of high concentrations of soluble CEA.

Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC.

A high efficiency backlight for LCDs is proposed, in which p-polarized light is preferentially out-coupled by using the interface reflection of optical components. In our backlight the luminance for the p-polarized light has improved by 30% or more compared to a conventional backlight and can be produced using conventional processes.

The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C{sub 2}H{sub 2}-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.

The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides.

The total synthesis of a peptide segment corresponding to the DNA-binding segment of Sp1 (positions 532–623) using a native chemical ligation approach is described. The folding of the synthetic segment in the presence of Zn(II) gave a zinc-coordinated protein. The dissociation constant (KD) for the DNA binding of the resulting protein, determined by a gel mobility shift assay, was 130 nM, almost nine times higher than that of the genetically prepared protein. However, methylation interference assay showed an identical sequence specificity of both proteins in DNA recognition. The chemical ligation method to connect the respective zinc-finger units was also accomplished. Successive ligation between a cysteine-containing peptide segment and a chloroacetylated peptide segment gave an artificial three-finger protein, which corresponds to the above DNA-binding segment of Sp1. However, this protein failed to bind DNA, even at 1.25 mM. Assessment of their folding structure based on the absorption spectra of their Co(II) complexes showed that the linker design to connect the respective finger units is critical for the proper folding of the proteins as well as the occurrence of the DNA-binding function. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5′-(G/T)GGGCGG(G/A)(G/A)(C/T)-3′ (GC box). In order to determine whether the three-zinc finger peptide Sp1(530–623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5′-GGGTGGGCGTGGC-3′ (s-GC box), which is flanked by a novel conserved guanine triplet on the 5′-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530–623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5′-GGGGCGGGGC-3′ (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530–623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530–623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline–copper complex (OP-Cu), binding of Sp1(530–623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530–623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.

The unique linker sequence of the native nine zinc finger transcription factor IIIA (TFIIIA) appears to significantly affect its novel DNA recognition mode. An artificial new nine zinc finger peptide Sp1ZF9T has been created by connecting three units of the three zinc finger domains of Sp1 with the TFIIIA-type linker. The DNA-binding characteristics of Sp1ZF9T were evaluated by the gel mobility shift, DNase I footprinting, and methylation interference assays, and compared with those of the previous Sp1ZF9 with a Kruppel-type linker. The gel mobility shift assays revealed that Sp1ZF9T forms two complex species, a short-lived species (B-2) and a long-lived species (B-1), with GCIII DNA (5′-GGG GCG GGG GGG GCG GGG GGG GCG GGGCC-3′). The B-2 complex dissociated into the free peptide and DNA, whereas the B-1 complex was stable even after 72 h. The DNase I footprinting and methylation interference results indicated that 3′- and central portions of GCIII DNA are recognized by Sp1ZF9T in the B-1 complex. The present DNA binding mode of Sp1ZF9T is evidently different from that of Sp1ZF9. Namely, fingers 1–5 participate in the DNA contact of Sp1ZF9T, and fingers 1–9 in that of Sp1ZF9. Therefore, the linker sequence among the zinc finger domains has a significant effect on the specific DNA recognition by the multi-zinc finger proteins.

To understand the DNA recognition mechanism of zinc finger motifs of transcription factor Sp1, we have determined the solution structure of DNA-binding domain of the Sp1 by solution NMR techniques. The DNA-binding domain of Sp1 consists of three Cys(2)His(2)-type zinc finger motifs. They have typical betabetaalpha zinc finger folds and relatively random orientations. From DNA-binding analysis performed by NMR and comparison between structures determined here and previously reported structures of other zinc fingers, it was assumed that DNA recognition modes of fingers 2 and 3 would be similar to those of fingers of Zif268, in which each finger recognizes four base pairs strictly by using residues at positions -1, 2, 3, and 6 of the recognition helix. On the contrary, finger 1 can use only two residues for DNA recognition, Lys550 and His553 at positions -1 and 3 of the helix, and has more relaxed sequence and site specificity than other Cys(2)His(2) zinc fingers. It is proposed that this relaxed property of finger 1 allows transcription factor Sp1 to bind various DNA sequences with high affinity.

In the DNA recognition mode of C(2)H(2)-type zinc fingers, the finger-finger connection region, consisting of the histidine spacing (HX(3-5)H) and linker, would be important for determining the orientation of the zinc finger domains. To clarify the influence of spacing between two ligand histidines in the DNA binding, we exchanged the histidine spacing between Sp1 and GLI zinc fingers, which have an HX(3)H-TGEKK linker (typical) and an HX(4)H-SNEKP linker (atypical), respectively. A significant decrease in the DNA binding affinity and specificity is found in Sp1-type peptides, whereas GLI-type peptides show a mild reduction. To evaluate the effect of the linker characteristics, we further designed Sp1-type mutants with an SNEKP linker. As a result, the significant effect of the histidine spacing in Sp1-type peptides was reduced. These results demonstrate that (1) the histidine spacing significantly affects the DNA binding of zinc finger proteins and (2) the histidine spacing and the following linker regions are one effective target for regulating the DNA recognition mode of zinc finger proteins.

We developed a novel lenticular lens, whose edge lines were modulated with a sinusoidal form, for a luminance enhanced LCD to reduce moire fringe between lenses and prism sheets and demonstrated the moire reduction to 1/5 with the wobbled lenticular lens formed by a printing method.