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Myostatin, a member of the transforming growth factor-b superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via “sweeping antibody technology”, GYM329 reduces or “sweeps” myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.

Monoclonal antibodies are currently the most attractive therapeutic modality in a broad range of disease areas, including infectious diseases, autoimmune diseases, and oncology. Fc engineering is one attractive application to maximize the value or overcome the drawbacks of monoclonal antibodies for therapeutic use. With the Fc region, antibodies bind to several types of receptors, such as Fc gamma receptors, a complement receptor, and a neonatal Fc receptor. Through this interaction with the receptors, antibodies demonstrate unique functions, such as antibody-dependent cellular cytotoxicity, antibody- dependent cellular phagocytosis, complement dependent cytotoxicity, agonistic activity, and endosomal recycling. Fc engineering technology is conducted mainly to maximize the receptor-mediated functions of antibodies. Moreover, Fc engineering of the two heavy chains to facilitate heterodimerization is indispensable for generating IgG-like bispecific antibodies that are asymmetric. Fc engineering is also conducted to avoid the undesired properties, such as cytokine release and protease-mediated cleavage of the hinge region, of wild-type antibodies, as well as providing additional functions. Thus, Fc engineering technology is an attractive approach for maximizing the potency and convenience of therapeutic antibodies. This review will cover a variety of Fc engineering technologies that improve the functions of therapeutic antibodies.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

Enhancing the effector function by optimizing the interaction between Fc and Fcγ receptor (FcγR) is a promising approach to enhance the potency of anticancer monoclonal antibodies (mAbs). To date, a variety of Fc engineering approaches to modulate the interaction have been reported, such as afucosylation in the heavy chain Fc region or symmetrically introducing amino acid substitutions into the region, and there is still room to improve FcγR binding and thermal stability of the CH2 domain with these approaches. Recently, we have reported that asymmetric Fc engineering, which introduces different substitutions into each Fc region of heavy chain, can further improve the FcγR binding while maintaining the thermal stability of the CH2 domain by fine-tuning the asymmetric interface between the Fc domain and FcγR. However, the structural mechanism by which the asymmetrically engineered Fc improved FcγR binding remained unclear. In order to elucidate the mechanism, we solved the crystal structure of a novel asymmetrically engineered Fc, asym-mAb23, in complex with FcγRIIIa. Asym-mAb23 has enhanced binding affinity for both FcγRIIIa and FcγRIIa at the highest level of previously reported Fc variants. The structural analysis reveals the features of the asymmetrically engineered Fc in comparison with symmetric Fc and how each asymmetrically introduced substitution contributes to the improved interaction between asym-mAb23 and FcγRIIIa. This crystal structure could be utilized to enable us to design a more potent asymmetric Fc.

Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIa(R131) allotype to a similar degree because FcγRIIb and FcγRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIa(R131) and FcγRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the C(H)2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T(M)) of the C(H)2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T(M) of the C(H)2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.

Homologous recombination is almost the only way to modify the genome in a predetermined fashion, despite its quite low frequency in mammalian cells. It has been already reported that the frequency of this biological process can be notably increased by inducing a double strand break (DSB) at target site. This article presents completely chemistry-based artificial restriction DNA cutter (ARCUT) for the promotion of homologous recombination in human cells. This cutter is composed of Ce(IV)/EDTA complex (molecular scissors) and two strands of peptide nucleic acid (PNA), and contains no proteins. Its scission site in the genome is determined simply by Watson-Crick rule so that ARCUT for desired homologous recombination is easily and straightforwardly designed and synthesized. The site-specificity of the scission is high enough to cut human genome at one target site. The DSB induced by this cutter is satisfactorily recognized by the repair system in human cells and promotes the targeted homologous recombination.

The final cut. Two types of artificial tools (artificial restriction DNA cutter and zinc finger nuclease) that cut double-stranded DNA through hydrolysis of target phosphodiester linkages, have been recently developed. The chemical structures, preparation, properties, and typical applications of these two man-made tools are reviewed.Two types of artificial tools that cut double-stranded DNA through hydrolysis of target phosphodiester linkages have been recently developed. One is the chemistry-based artificial restriction DNA cutter (ARCUT) that is composed of a Ce(IV)-EDTA complex, which catalyses DNA hydrolysis, and a pair of pseudo-complementary peptide nucleic acid fragments for sequence recognition. Another type of DNA cutter, zinc finger nuclease (ZFN), is composed of the nuclease domain of naturally occurring FokI restriction endonuclease and a designed zinc finger DNA-binding domain. For both of these artificial tools, the scission site and specificity can be freely chosen according to our needs, so that even huge genomic DNA sequences can be selectively cut at the target site. In this article, the chemical structures, preparation, properties, and typical applications of these two man-made tools are described.

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

An artificial restriction DNA cutter (ARCUT), which was composed of Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acid, was used for construction of recombinant plasmid vector. The plasmid vector was cut by ARCUT only at target site, and resultant specific overhangs were blunted by treatment with Mung Bean Nuclease and Klenow fragment. This fragment having blunt ends at both termini was ligated with the gene of enhanced green fluorescent protein (EGFP) prepared by PCR. The resultant recombinant vector was translated and the fluorescent protein was successfully expressed in cells. Throughout the manipulation, neither natural restriction enzymes nor homologous recombination was employed. Thus, this strategy has promising applicability to construction of still larger vectors.

The structures of Ce(4+) complexes that are active for DNA hydrolysis were determined for the first time by X-ray crystallography. The crystals were prepared from a 1:2 mixture of Ce(NH(4))(2)(NO(3))(6) and dipicolinic acid (2,6-pyridinedicarboxylic acid). Depending on the recrystallization conditions, three types of crystals were obtained. Some of the Ce(4+) ions in these complexes have enough coordinated water molecules that can directly and indirectly participate in the catalysis. The distances between the Ce(4+) and the dipicolinate ligand are considerably shorter than those in the corresponding La(3+) and Ce(3+) complexes. On the other hand, the distances between the Ce(4+) and its coordinated water are similar to those for the La(3+) and Ce(3+) complexes. In a proposed mechanism of DNA hydrolysis, the scissile phosphodiester linkage is notably activated by coordination to Ce(4+) and attacked by the Ce(4+)-bound hydroxide. The process is further assisted by acid catalysis of Ce(4+)-bound water.

Objective To summarize the features of asbestos-related lung cancer. Patients Thirty-one lung cancer patients with occupational exposure to chrysotile asbestos fibers. They worked or had worked in one asbestos factory or its subcontracters. Result All patients were male with mean age of 60.6 when diagnosed, and all except one were current or ex-'heavy' smokers. Histological types were fairly evenly divided into adeno-, squamous and small cell carcinoma and 24 (78%) of patients showed 'peripheral type' lung cancers. Regarding clinical stages, 20 patients (65%) were classified as III or IV (advanced stage). Tumor shadow(s) was detected on chest X-ray in 22 patients (71%), and in 5 patients with 'negative' chest X-ray, chest CT was necessary to recognize a primary tumor. Seventeen patients (55%) did not undergo periodical check-ups. Conclusion Occupational asbestos exposure is interpreted as one of the important risks for lung cancer and frequent and accurate observation is necessary.(Internal Medicine 38: 780-784, 1999)

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

By using an artificial restriction DNA cutter which is composed of Ce(IV)/EDTA and two pseudo-complementary peptide nucleic acid strands (pcPNAs), only one target site in the whole genome of human beings (one site in the X chromosome) was selectively hydrolyzed.

The final cut. Two types of artificial tools (artificial restriction DNA cutter and zinc finger nuclease) that cut double-stranded DNA through hydrolysis of target phosphodiester linkages, have been recently developed. The chemical structures, preparation, properties, and typical applications of these two man-made tools are reviewed.Two types of artificial tools that cut double-stranded DNA through hydrolysis of target phosphodiester linkages have been recently developed. One is the chemistry-based artificial restriction DNA cutter (ARCUT) that is composed of a Ce(IV)-EDTA complex, which catalyses DNA hydrolysis, and a pair of pseudo-complementary peptide nucleic acid fragments for sequence recognition. Another type of DNA cutter, zinc finger nuclease (ZFN), is composed of the nuclease domain of naturally occurring FokI restriction endonuclease and a designed zinc finger DNA-binding domain. For both of these artificial tools, the scission site and specificity can be freely chosen according to our needs, so that even huge genomic DNA sequences can be selectively cut at the target site. In this article, the chemical structures, preparation, properties, and typical applications of these two man-made tools are described.

We report here a new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment. By using caged primers in PCR, unnatural sticky-ends of any length and sequence are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in desired arrangement, tightly enough to be repaired and ligated in competent cells. We have successfully constructed a recombinant vector based on pBR322 and coding GFP gene by applying this simple and effective system.

Control of the terminal structures of PCR products is crucially important to facilitate molecular biology and biotechnology. Here, we report a new method to prepare PCR products having desired sticky ends directly after thermal cycles. When a pair of caged primers is used, polymerase reaction is site-selectively terminated in front of the caged nucleotide, and the 5'- portion of the primer remains single-stranded throughout the reaction. Successive removal of the photo-cleavable protecting group gives restriction-enzyme free sticky ends on the product. We succeeded in applying this technique to make a recombinant vector bearing GFP gene.

Two of three amino acid residues, which compose the chromophore of the enhanced green fluorescent protein (EGFP), were converted to others by using artificial restriction DNA cutter (ARCUT). The vector prepared by ARCUT was easily connected with the insert by using oligonucleotide additive and resultant fluorescent protein such as blue fluorescent protein (BFP) was successfully expressed in cells.

A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

症例は, 70才男性, 咳漱, 呼吸困難多量の漿液性喀疾を主訴とし当科入院.胸部X線上両側肺野に浸潤陰影を伴う多発性空洞陰影を認めた.剖検にて, 乳頭型腺癌, 細気管支肺胞型と判明した.剖検肺には両側に粘液様物質を貯留する円型薄壁空洞が多発していた.空洞はチェックバルブ作用によるtension cavitiesと考えられた.又他の部位には壁の厚い空洞も認められ, 空洞形成の機序には腫瘍細胞の粘液過剰産生, 易剥脱性, 間質の脆弱性等が関与していると推定された.

Summary The carcinogenic effect of N-bis(2-hydroxypropyl)nitrosamine (BHP) was studied in newborn Wistar rats. Tumors were induced in the lung (adenoma), pancreas (adenoma), kidney (nephroblastoma), and ovary (benign granulosa cell tumor) at 24 weeks after the treatment and in the lung (adenoma and adenocarcinoma), liver (hemangioma, hemangioendothelioma, and hepatocellular carcinoma), pancreas (adenoma), kidney (renal cell carcinoma and nephroblastoma), thyroid (adenocarcinoma) and ovary (benign and malignant granulosa cell tumors) at 52 weeks. The highest incidence of tumor was seen in the liver of rats applied BHP within 24 hours after birth. These results indicate that newborn Wistar rats are more susceptible to BHP than are adult rats.

The effects of chrysotile asbestos on lung and pleural carcinogenesis by N-bis(2-hydroxypropyl)nitrosamine (DHPN) in male Wistar rats were studied. Chrysotile, 30 mg per rat, was injected into the left pleural cavity and 3 g/kg body wt. DHPN was injected once into the abdominal cavity. Lung tumors (adenoma, adenocarcinoma, squamous cell carcinoma, and combined carcinoma) occurred at the highest incidence (100%). Adenocarcinoma was seen in 4 of 11 (36%) rats killed at 35 weeks and in 6 of 12 (50%) rats killed at 52 weeks, squamous cell carcinoma occurred in 1 of 11 (9%) rats killed at 35 weeks and 3 of 12 (25%) rats killed at 52 weeks, and mixed carcinoma was seen in 1 of 12 (8%) rats killed at 52 weeks, which received chrysotile and DHPN. Adenocarcinoma was seen in 9 of 11 (82%) rats which received DHPN only and killed at 52 weeks. Mesotheliomas were seen in 2 of 11 (18%) rats, killed at 35 weeks, and 3 of 12 (25%) rats, killed at 52 weeks, which received chrysotile and DHPN. Hyaline thickening of the pleura was seen in 100% of rats receiving chrysotile. Mesothelial cell hyperplasia and adenomatous and/or fibromatous growth of the mesothelium were seen in the pleura on both sides, ranging from 36% to 50% and 31% to 64% in rats receiving chrysotile and DHPN, respectively. Asbestos bodies were seen in the pleura on both sides and in the lung.

肺癌未治療患者58例について, NK活性とDNCB反応を検索し, 両者の組合わせによる免疫スペクトルを4群に分け, 免疫能と臨床病態との関係を検討した.A群は一般状態 (PS) が良好で, 遠隔転移が少なく, 82%の高率で治療が期待できるが, BないしC群, D群と免疫能の低下に伴って, PSの悪化と病期の進展がみられ, D群は全例治療不可能であった.この新しい免疫スペクトルは, 患者の予後の推則と治療方針の決定に有用である.

71才, 男性. 胸部X線にて右上野に腫瘤陰影を指摘され, 諸検査より肺扁平上皮癌 (T3N2Mx) と診断. 化学療法・放射線療法にてMinor Responseを得た. 約1ヶ月後・顔面・上肢の浮腫, 呼吸困難が出現, 放射線療法にて改善するも約1ヶ月後両側乳糜胸水出現, 上記症状の増強をみた.静脈撮影より上大静脈症候群が確認された. 胸水排液後, 放射線療法, 胸膜癒着術を施行するも約1ヶ月で死亡. 上大静脈症候群に両側乳糜胸水を合併した肺癌は稀である.

症例は57才男性で, 健康診断にて右下肺野の孤立性輪状影を指摘された. この陰影が同じ形態を保ったまま次第に増大するため手術を施行し, 気管支腺由来の腺癌と診断した. 肺原発の腺癌で孤立性薄壁空洞を呈することは極めて稀であり, 薄壁空洞の形成機序を考える上でも興味深い症例である. 本症例では, 交通気管支のvalvularobstructionにcheckvalvemechanismが加わって孤立性薄壁空洞が形成されたものと推測した.

Mechanisms of the defence of the airway was studied with 54 cases of sinobronchial syndrome (SBS) by means of trans-tracheal aspiration (TTA) and aerosol inhalation cine scintigraphy (AICS).1. TTA obtained from the lower airway of cases of SBS revealed continuous infection with organisms usually observed in the normal upper airway such as Haemophilus influenzae and others. We estimated that the clinical course of SBS took a turn of worse with continuous infection.2. Damages of mucocilliary transport in lower airways were clearly observed by AICS in cases of SBS.We strongly suggested that continuous infection due to the damage of the mucocilliary transport in the lower airway was the main factor causing symptoms observed in cases of SBS.

A 64-year-old man was reffered to the clinic with the chief complant of a tumorous lesion in the extracted cavity immediately after the extraction of the decayed 5. Prior to the tooth extraction the patient had a right upper lobectomy for lung cancer a month before.The specimen from the gingiva indicated histologically large cell carcinoma the same as seen in the lung lesion. The patient died of heart failure 2 months later.Autopsy findings revealed that the tumor at the tail portion of pancreas was the primary one, which metastasized to the lung and the gingiva.

症例は73歳男性.右側胸部痛で受診, 胸部X線上右下葉無気肺を指摘.3年後右完全無気肺となり, 右主気管支にポリープ様腫瘍を認め, 生検でカルチノイドと診断した.カルチノイド症候群として顔面の毛細血管拡張および皮膚潮紅と下痢を呈し, 血中serotonin, 尿中5-HIAAの上昇がみられた.剖検でえた腫瘍組織のGrimelius染色で細胞内に嗜銀顆粒を, 電顕にて神経内分泌顆粒を確認し, 抗serotonin抗体を用いたABC法でserotoninの存在を証明できた.

症例は, 80才, 男性.胸部X線写真で, 右中肺野に腫瘤影を指摘され, 右上葉切除施行した.摘出標本より, 間葉性軟骨肉腫と扁平上皮癌の重複癌と診断され, 術後約6ヶ月後, 脳転移により死亡した.剖検で, 問葉性軟骨肉腫の多臓器に及ぶ転移と多発性神経線維腫症が認められた.肺原発の間葉性軟骨肉腫は, 極めて稀で, 扁平上皮癌との合併はみられない.