Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs.

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.

Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131).

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.

High viral eradication with a daily 12-week natural interferon-beta treatment regimen in chronic hepatitis C patients with low viral load. IFN-beta Research Group.

Virological sustained response (SR) is achieved in 31-49% of patients with chronic hepatitis C with combination therapy using interferon (IFN)-alpha and ribavirin for 24-48 weeks. However, as a period of 24-48 weeks is a burden for patients, we investigated the effect of daily intravenous administration of natural IFN-beta for 12 weeks in this study. In all, 112 patients were enrolled and received a daily administration of 6 MU of natural IFN-beta intravenously for 12 weeks. Serum HCV-RNA before treatment was assessed by the competitive reverse-transcription polymerase chain reaction assay. The patients were divided into two groups according to pretreatment viral load: the low viral load group (N = 25, <6.3 x 10(5) copies/ml), and the high viral load group (N = 87, > or =6.3 x 10(5) copies/ml) who were additionally administered IFN-beta thrice weekly for subsequent 14 weeks at the patients' request. Virological SR was obtained in 37% (41/112) of all the patients; 88% of those with a low viral load, and 22% of patients with a high viral load. Virological SR was attained in 21% of patients with HCV subtype 1, and in 67% of those with subtype 2a. In patients with HCV subtype 1b, virological SR was obtained in patients with the mutant-type (> or =4 amino acid mutations in the NSSA2209-48) having a low viral load (4/4), but not in those having a high viral load (0/3). The results suggest that a daily intravenous administration of natural IFN-beta for 12 weeks showed high therapeutic efficacy in patients with a low viral load despite the shorter treatment period and that the therapeutic efficacy depends on viral load rather than on the number of NS5A2209-48 amino acid mutations.

A clinicopathological study of lung cancer patients with occupational exposure to chrysotile asbestos fibers.

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.

A study of the growth changes in the maxillofacial skeleton of females with reversed occlusion: investigation by means of lateral x-ray cephalogram.

321 females with reversed occlusion from the ages of 6 to 20 were classified into 15 groups according to chronological age, and the changes of maxillofacial skeleton accompanying growth were investigated. Lateral cephalometric roentgenogram before treatment of the subjects were used for analyses and measurements of maxillofacial skeleton and overjet. The cephalometric analysis was standardized on the S-N plane, and 5 angular items and 6 linear distances were examined. The results of the investigation are summarized as follows: (1) angle ANB, the relationship between the upper and lower jaws, deteriorated to a minus degree from the age of 9 and became -3.3 degrees at the age of 14. Skeletal convexity became negative from the same age and was -7.3 degrees at the age of 14. Thus, the abnormal skeletal features progressed in this condition. (2) The Ar-Gn distance measurement gradually increased from the age of 6 to 18. The increase was 16.2 mm from 7 to 12, an increase of 3.2 mm yearly. (3) A high correlation (r = 0.99) was observed between angle ANB and Convexity. As well, a high coefficient of correlation (r = 0.87) was observed between skeletal convexity and soft tissue convexity. The change in soft tissue convexity may occur 3-4 years later than that of skeletal convexity. (4) The growth curve of Ar-Gn became a logistic curve with the point of deflection at the age of 10.87. The growth curves of S-Gn and N-Me became logistic curves in the same way.

[A case of von Recklinghausen's disease associated with large cell carcinoma of the lung].

A 75-year-old man, a heavy smoker with a history of exposure to dust, was admitted to our hospital because of right back pain. Since café au lait spots, diffuse pigmentation and multiple neurofibromatosis were observed on the skin, he was considered to have von Recklinghausen's disease. Chest X-ray film showed a tumor shadow in the right S2, and chest CT demonstrated that the tumor involved the chest wall. Histopathological examination of the percutaneous lung biopsy specimen demonstrated large cell carcinoma. Following radiotherapy to the tumor, he underwent right upper lobectomy with chest wall resection. Macroscopic examination of the resected specimen revealed invasion of tumor into the external intercostal muscles, although microscopically all cancerous tissue was replaced by necrotic tissue and no viable tumor cells remained. The patient survived for three years after treatment, and subsequently died of respiratory failure caused by pneumonia.

[Argyrophilic nucleolar organizer region proteins (Ag-NORs) in human central nervous tumors].

A silver colloid staining technique for identifying nucleolar organizer region-associated proteins (Ag-NORs) was applied to 17 primary gliomas and 16 meningiomas. These comprised 8 glioblastomas, one pleomorphic xanthoastrocytoma, 8 benign astrocytoma, 10 nonrecurrent meningiomas and 6 recurrent meningiomas, in which the mean number of Ag-NORs' per nuclei, were 3.37, 2.34, 2.57, 2.16 and 2.80 respectively, identifying significant differences among these groups. Ag-NORs of four giant cell glioblastomas showed low numbers of Ag-NORs, suggesting better prognosis. In spite of the time consuming and complicated counting method Ag-NORs is reproductive and useful as a tool for estimating the proliferating potential of various brain tumors, and appears to widely applicable in clinical laboratories.

[A case of prolonged eosinophilic pneumonia with pulmonary emphysema and bronchial asthma diagnosed by open lung biopsy].

A 65-year-old man was admitted to our hospital complaining of productive cough, dyspnea and stridor. Chest X-ray disclosed overinflation with micronodular infiltrates. Blood examination showed mild eosinophilia and IgE elevation. Pulmonary function test disclosed severe airway obstruction and diffusion capacity impairment. Although clinical improvement was achieved after bronchodilator therapy, laboratory abnormalities continued. Open lung biopsy demonstrated mononuclear cellular and eosinophilic infiltration at alveolar lumen and vessel walls without prominent fibrosis, which was compatible for prolonged eosinophilic pneumonia. From above findings, this case was thought as a prolonged eosinophilic pneumonia combined with pulmonary emphysema and bronchial asthma.

[Clinical study on asbestos pleurisy and review of literature].

Seven asbestos workers with asbestos pleurisy were investigated in terms of clinical symptoms, findings of pleural biopsies and course of chest roentgenograms. All cases were male and smokers. Duration of asbestos exposure ranged from 3 to 33 years (mean: 20.6 years). Duration between initial exposure and onset of the pleurisy ranged from 22 to 34 years (mean: 28.7 years). Five pleural effusions were aseptic and exudative, and one had sanguineus and eosinophilic effusion. According to the findings of pleural biopsies, our cases were classified into 2 groups. Group A (4 cases) had "basket-weaving formation" on the parietal side and inflammatory changes on the visceral side. No circulating autoantibodies were found in group A. Group B (3 cases) had no "basket-weaving formation" but only intensive inflammatory changes. These 3 cases were respectively complicated with anti-GBM antibody mediated glomerulonephritis, with acute interstitial pneumonia, and with rheumatoid arthritis and acute interstitial pneumonia. They all had circulating autoantibodies. In the course of chest roentgenograms, 6 of the 7 cases finally showed diffuse pleural thickenings which involved obliterations of costo-phrenic angles. Among them, one case developed into progressive pleural fibrosis. It was concluded that asbestos pleurisy can be classified into 2 groups according to the mechanism of occurrence, and immunological changes should be recognized as one of the mechanisms of asbestos pleurisy.

[An operative case of chronic constrictive pericarditis with silicosis and lumbar caries].

We reported a case of chronic constrictive pericarditis complicated with silicosis and lumbar caries, who was improved by the operation. The patient was a 65 year old man whose past occupation was a mason. He was admitted to our hospital with chronic heart failure on March, 1986. Atypical silicosis was diagnosed from the occupational history and the histopathological silicotic changes in mediastinal lymph nodes and fibrosis of alveolar wall. The diagnosis of chronic constrictive pericarditis was made from chest roentgenogram and intracardiac catheterization. The symptoms of chronic constrictive pericarditis was improved by the pericardial resection. The exact pathogenesis of the chronic constrictive pericarditis could not be identified from the histology of pericardial tissue, but tuberculosis was suspected because of the past history of tuberculous pleurisy and the recurrence of lumbar caries.

[Establishment of a simple fluorescent microscopy method for measurement of the CD4/CD8 ratio in BALF].

The authors established an easy fluorescent microscopy method to measure the ratio of CD4 (helper/inducer T lymphocytes) to CD8 (suppressor/cytotoxic T lymphocytes) (CD4/CD8 ratio) in bronchoalveolar lavage fluid (BALF) in patients with diffuse pulmonary diseases and measles pneumonia. The CD4/CD8 ratio determined by this method strongly correlated with the CD4/CD8 ratio obtained by flow cytometry in both peripheral blood lymphocytes and BALF lymphocytes. When the proportion of lymphocytes in BALF nuclear cells is more than 6%, the CD4/CD8 ratio in BALF was measurable. The CD4/CD8 ratio in peripheral blood was not significantly different among hypersensitive pneumonitis (HP), sarcoidosis (SAR), collagen vascular disease (CVD), idiopathic interstitial pneumonitis (IIP), and measles pneumonia (MP). However, the BALF CD4/CD8 ratio was 0.61 +/- 0.37 in HP, 6.83 +/- 2.73 in SAR, 0.77 +/- 0.40 in CVD, 1.49 +/- 0.29 in IIP. These results were consistent with previously published data. The CD4/CD8 ratio in patients with MP was as low as 0.39 +/- 0.34. In patients with summer type HP, this ratio was low in the acute phase, but became within normal range after moving or reconstructing their homes, accompanied with the improvement of subjective symptoms and the disappearance of inflammation. These data indicate that our method is very simple and useful in the diagnosis and the monitoring of diffuse pulmonary diseases.

[Clinical study of taste abnormalities in advanced lung cancer cases].

Taste recognition thresholds for four basic tastes, sweet, salty, and bitter, have been examined in 36 advanced lung cancer patients to identify the presence or absence of taste abnormalities in such patients. Twenty-six untreated lung cancer patients, or 65.3%, demonstrated an abnormal taste threshold for at least one of the 4 taste, a result varying significantly from suitable controls (p less than 0.05). Among the total 36 lung cancer patients, the number of those showing a threshold abnormality was no different between the smoker and non-smoker groups, though abnormalities were higher in the low serum zinc level group than in the normal serum zinc level group (p less than 0.05). We also observed transient threshold elevations after chemotherapy including Cisplatin. Thus, it is thought that taste abnormalities are more prevalent in advanced lung cancer patients.

[A case of multiple small pulmonary arteriovenous fistulas with chronic hepatitis and abdominal vessel abnormalities].

A 63-year-old female was admitted with dyspnea on exertion. She had previously suffered from a large amount of epistaxis. Chest X-ray showed the bilateral diffuse granular infiltrates. Arterial blood analysis revealed severe hypoxemia and pulmonary function test showed reduced function of diffusing capacity. Dynamic radionuclide perfusion scintigraphy performed with 99mTc-labeled MAA demonstrated passage of microspheres through the pulmonary vasculature into the systemic circulation, indicating a right-to-left shunt. Although selective pulmonary wedge angiography did not directly demonstrate pulmonary arteriovenous fistulae, the histopathological findings of TBLB specimens revealed multiple dilated alveolar vessels that microspheres could pass through. In addition, chronic hepatitis, dilatation of splenic vein and spleno-renal anastomosis had been present. This type of pulmonary arteriovenous fistula is said to be a rare dispersed telangiectasic type, associated with a part of Rendu-Osler-Weber syndrome and a cause of hypoxemia in liver cirrhosis. We consider that in this case, the chronic hepatitis may have played an important role of developing multiple small pulmonary arteriovenous fistulas.

[A case of eosinophilic pneumonia with acute progressive dyspnea and diffuse small nodular shadows on chest X-ray film].

A 52-year-old man was admitted to our hospital with high-grade fever, dry cough and severe dyspnea. Chest X-ray films revealed diffuse small nodular shadows in all lung fields, prominently in the middle and lower lung fields. On examination, peripheral blood eosinophilic leukocytosis and severe hypoxemia were demonstrated. His clinical condition improved during five days after the admission without any therapy but oxygen inhalation. The abnormal shadows decreased in several days, and disappeared completely in thirty days. Bronchoalveolar lavage fluid revealed increased total cell counts, mostly with eosinophils (60%) and lymphocytes (21%). Histologically, the transbronchial lung biopsy specimen showed that the walls of pulmonary arteries and bronchioli were markedly infiltrated with eosinophils, and that alveolar septa were edematous with mononuclear and eosinophilic cells infiltration. There were no prominent changes in the alveolar lumen except few macrophage exudates. Our case was of importance for two reasons: first, eosinophilic pneumonia could take the form of interstitial pneumonia both roentgenologically and histologically; second, Crofton's classification of P.I.E. could not be applied to this case.

[Squamous cell carcinoma of the lung metastasized solitary cystic formation in the cerebellum].

A 54-year-old male previously diagnosed as having a lung squamous cell carcinoma T2N2M0 in stage III A was readmitted to our hospital, as he was complaining of a gait disturbance. A brain CT, an MRCT scan, and vertebral arteriography revealed that the lung carcinoma had metastasized as a solitary cystic formation in the right cerebellum. Thus, a craniotomy, a cyst fluid aspiration, and tumoral resection was performed. Histopathological findings of the cyst wall indicated a squamous cell carcinoma, and the protogram of the cyst fluid found it similar to that of the serum. These findings suggest that destruction of the blood-brain-barrier by the invasion of the carcinoma caused an exudation of serum, resulting in the accumulation of cyst fluid.

[Study of internal reaction on the maxilla by orthopedic force].

For the purpose of analyzing deformations and movements in the maxilla loaded by orthopedic force to the maxillary first molar, a three dimensional finite element model of the maxilla was constructed. For modelling of the maxilla, the right side of an adult Indian skull was used. Constructed model was composed of 1,108 solid elements and 1,195 nodes. Materials of this model was composed of compact bone, cancellous bone, teeth and sutures. Constraint condition was fixed adjacent bone side surrounded by sutures. Load was directed from upward to downward 30 degree, changing every 30 degree reference to occlusal plane. Volume of load were weighed at 1 kg for each directions. Results 1. At upward load, stress was transmitted through the lateral wall of the maxillary sinus. The posterior portion of the maxilla was displaced more upward than anterior. Each process of the maxilla and the maxillary sinus was deformed, however, there was no stress on the orbitary surface. 2. At upward 60 degree load, the maxilla was compressed simply to the direction of load. At the upward 30 degree load, it was compressed backward-downward and rotated clockwise together with bending. 3. At backward load, the maxilla was moved backward and rotated clockwise. At downward 30 degree load, the maxilla was moved backward-downward and rotated clockwise. Both loads bended anterior portion of the maxilla and compressed posterior portion. The center of rotation or bending fell on the line joining the frontal process and the zygomatic process. 4. Of all five directions, backward load showed the maximum value of displacement and upward showed the minimum. Concerning with the absolute value of principal stress, backward load showed the maximum and upward 60 degree load showed the minimum. 5. Concerning with the principal stress value of sutures, compressive stress were distributed equally at upward load. However, as the load went to downward direction, it caused stress concentration. Especially, compressive stress in the pterygopalatine suture and transverse palatine suture, extensive stress in the midpalatal suture were especially great.

[The effects of passive smoking in the development of female lung cancer in the Nara district].

Passive smoking in the development of lung cancer has been investigated in 25 female lung cancer patients in a case control study based on their histological type. Active smoking was notable squamous and small cell carcinoma, whereas passive smoking was notable in female lung cancer patients as a whole and current passive smoking being affected was the largest effect, but histological type was not notable. Adenocarcinoma was notable in cancer family aggregation. The female lung cancer patients as a whole was found the effect in the combination with passive smoking in each stage and cancer family aggregation.