Humanization and simultaneous optimization of monoclonal antibody.

Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in the light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial.

Reduced elimination of IgG antibodies by engineering the variable region.

Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.

MR and CT findings of leiomyomatosis peritonealis disseminata with emphasis on assisted reproductive technology as a risk factor.

Leiomyomatosis peritonealis disseminata (LPD) is a rare benign smooth muscle tumour located in the peritoneal cavity. Increased oestrogen exposure appears to be an aetiological factor for LPD. We report two cases of LPD after leiomyomectomy and assisted reproductive technology pregnancy, which can cause a high serum concentration of oestrogen. CT and MR scanning demonstrate many intraperitoneal well-demarcated nodules of varying size that mimic widespread intraperitoneal malignancy.

High incidence of silent venous thromboembolism before treatment in ovarian cancer.

Venous thromboembolism (VTE) such as deep-vein thrombosis (DVT) and pulmonary thromboembolism (PTE) often occurs after surgery and rarely occurs even before surgery in patients with ovarian cancer. It is well known that levels of plasma D-dimer (DD) before treatment in most ovarian cancer patients are increased. This study therefore examined whether increased levels of DD are associated with presence of VTE before treatment of ovarian cancer. Between November 2004 and March 2007, DD levels prior to initial treatment were measured in 72 consecutive patients with presumed epithelial ovarian cancer (final diagnosis: epithelial ovarian cancer, n=60; and epithelial ovarian borderline malignancy, n=12). Venous ultrasound imaging (VUI) of the lower extremity was conducted for all patients except for two patients in whom DVT was detected by pelvic computed tomography (CT). When DVT was found, pulmonary scintigraphy was subsequently performed to ascertain presence of PTE. D-dimer levels were above the cut-off value (0.5 microg ml(-1)) in 65 of 72 patients (90.2%). Venous ultrasound imaging or CT revealed DVT in 18 of 72 patients (25.0%) and pulmonary scintigraphy found PTE in 8 patients (11.1%). All patients with VTE were asymptomatic when VTE was found. D-dimer levels were associated with incidence of VTE (0-1.4 microg ml(-1); 0 of 26 (0%), 1.5-7.4 microg ml(-1); 9 of 30 (30%) and > or =7.5 microg ml(-1); 9 of 16 (56.3%), P for trend=0.0003). However, even if 1.5 microg ml(-1) was used as a cut-off value, this had low specificity and positive predictive value (47.2, 38.3%), though it had high sensitivity and negative predictive value (100, 100%). Therefore, ovarian cancer patients with DD level > or =1.5 microg ml(-1) should be examined using VUI to detect silent DVT. Patients with VTE underwent preventive managements including anticoagulant therapy before initial treatment, chemotherapy or surgery, and after surgery. There was no clinical onset of postoperative VTE in all 72 patients. Measurement of DD levels and subsequent ultrasonography revealed that silent or subclinical VTE frequently occurs before surgery in ovarian cancer. The usefulness of preoperative assessment of VTE needs further confirmation in randomised controlled trials.

Tissue factor expression as a possible determinant of thromboembolism in ovarian cancer.

Ovarian cancer, and clear cell carcinoma in particular, reportedly increases the risk of venous thromboembolism (VTE). However, the mechanisms remain unclear. Tissue factor (TF) supposedly represents a major factor in the procoagulant activities of cancer cells. The present study examined the involvement of TF expression in VTE for patients with ovarian cancer. Subjects comprised 32 consecutive patients (mean age 49.8 years) with histologically confirmed ovarian cancer. Presence of VTE was examined using a combination of clinical features, D-dimer levels and venous ultrasonography. Immunohistochemical analysis was used to evaluate TF expression into 4 degrees. Venous thromboembolism was identified in 10 of the 32 patients (31%), including five of the 11 patients with clear cell carcinoma. Tissue factor expression was detected in cancer tissues from 24 patients and displayed significant correlations with VTE development (P=0.0003), D-dimer concentration (P=0.003) and clear cell carcinoma (P<0.05). Multivariate analysis identified TF expression as an independent predictive factor of VTE development (P<0.05). Tissue factor (TF) expression is a possible determinant of VTE development in ovarian cancer. In particular, clear cell carcinoma may produce excessive levels of TF and is more likely to develop VTE.

The advantage of using carbon nanotubes compared with edge plane pyrolytic graphite as an electrode material for oxidase-based biosensors.

Carbon nanotubes (CNTs) are promising materials for use in amperometric biosensors. The defect sites at their ends, and on their sidewalls, are considered to be edge plane-like defects and show high electrocatalytic activity toward several biological molecules. However, electrocatalytic activity toward H(2)O(2) has not been compared among bamboo-structured CNTs (BCNTs), which have many defect sites; hollow-structured CNTs (HCNTs), which have few defect sites; edge plane pyrolytic graphite (EPG); and traditional glassy carbon (GC). The advantages of using CNTs in electrodes for biosensors are still equivocal. To confirm the utility of CNTs, we analyzed the electrochemical performance of these four carbon electrodes. The slope of the calibration curve for H(2)O(2) at potentials of both +0.6 V and -0.1 V obtained with a BCNT paste electrode (BCNTPE) was more than 10 times greater than the slopes obtained with an HCNT paste electrode and a GC electrode, reflecting the BCNT's larger number of defect sites. Although the slope with the EPG electrode (EPGE) was about 40 times greater than that with BCNTPE at +0.6 V, the slopes with these two carbon electrodes were nearly equivalent at -0.1 V. EPGE demonstrated excessive electrochemical activity, detecting currents on the basis of consumption of oxygen and oxidation of ascorbic acid, even at -0.1 V. In contrast, BCNTPE could dominantly detect a cathodic current for H(2)O(2) at -0.1 V, even when interfering molecules were added. BCNTPE possesses appropriate electrochemical activity and is an effective electrode materials for developing interference-free oxidase-based biosensors operated by the application of an appropriate potential.

Pelvic imaging: multicystic uterine cervical lesions. Can magnetic resonance imaging differentiate benignancy from malignancy?

PURPOSE: To evaluate whether magnetic resonance imaging (MRI) can be used to differentiate between malignant and benign multicystic uterine cervical lesions. MATERIAL AND METHODS: Twenty-two patients with cervical adenocarcinomas, including minimal deviation adenocarcinoma, and five patients with benign multicystic lesions were classified as a percentage of solid components on MRI. Cystic components were further classified by average cyst diameter and signal intensity of the cyst fluid on T1WI. RESULTS: All nine of the entirely solid lesions were malignant. In contrast, two of the entirely cystic lesions were benign. Ten of the ordinary adenocarcinomas had both solid and cystic components. However, three of the 16 solid and cystic lesions were benign. Lesions composed of cysts smaller than 5mm tended to be malignant; however, some lesions composed of larger cysts were also malignant. Three of 5 lesions with low-signal and 7 of 10 with intermediate-signal fluid were malignant. CONCLUSION: The malignancy potential was higher in the lesions with a higher percentage of solid components. However, determining whether multicystic lesions were benign or malignant based on the existence of solid components, the average cyst size, and the signal intensity of cyst fluid was impossible. Although a multicystic lesion with solid components in the deep cervical stroma had been reported as a MR finding of a minimal deviation adenocarcinoma, this does not appear to be pathognomonic.

Ultrasound diagnosis of uterine arteriovenous fistula associated with placental site trophoblastic tumor.

We report a case of a woman with abnormal vaginal bleeding who had a placental site trophoblastic tumor (PSTT) detected following hysterectomy. Surgery was performed because of a large uterine arteriovenous fistula detected by transvaginal color and pulsed Doppler sonography. Color Doppler sonography revealed a lacunar-type lesion with a marked increase in uterine vascularity, and pulsed Doppler sonography demonstrated a low resistance index. This vascular pattern indicated the formation of blood lacunae and arteriovenous shunts caused by PSTT within the uterine myometrium. This is the first report to describe the ultrasound findings in a case of PSTT complicated by a uterine arteriovenous fistula.

Use of small pelvic field instead of whole pelvic field in postoperative radiotherapy for node-negative, high-risk stages I and II cervical squamous cell carcinoma.

We investigated whether a small pelvic (SP) field that covers primarily the pericervical regions in postoperative radiotherapy for cervical squamous cell carcinoma is adequate for a subgroup of node-negative patients. Of 84 patients with stage I-II disease treated with postoperative radiotherapy due to pathologic risk factors, 42 node-negative patients received SP-field radiotherapy, whereas remaining 42 node-positive patients were treated with a conventional whole pelvic (WP) field that also covered pelvic lymph nodes, both with 50.0-50.4 Gy/25-28 fractions. The pathologic risk factors included positive nodes, deep stromal invasion (>/=2 /3 thickness), parametrial extension, and positive or close surgical margin. Recurrence was identified for 20 patients: three in the SP group and 17 in the WP group. Intrapelvic recurrence accounted for all three recurrences in the SP group and for four in the WP group; 5-year pelvic-control rate did not differ significantly between the SP (93%) and WP (90%) groups. Extrapelvic recurrence (n = 11) was identified exclusively in the WP group. Patterns of recurrence indicate that use of an SP field instead of a WP field may be adequate in postoperative radiotherapy for a subgroup of node-negative, high-risk patients.

Possible involvement of p38 mitogen-activated protein kinase in decidual function in parturition.

We designed the present study to elucidate the molecular mechanism for parturition, focusing on p38 mitogen-activated protein kinase (p38). The kinase activity of p38 in mouse uterus was gestation stage-dependent, and was markedly increased on day 19 of gestation and during labor. Immunohistochemical examination with anti-phospho p38 antibody revealed that activated p38 was predominantly localized in decidual stromal cells stained with anti-prolactin antibody. In human primary cultured decidual cells, a p38 inhibitor, SB202190, significantly inhibited both prostaglandin F(2alpha) production and COX-2 expression induced by stimulation with IL-1beta. These results suggest that the p38 signaling pathway is involved in decidual function at the late stage of gestation and may contribute to parturition.

Surgical management of a penetrated greenfield inferior vena cava filter.

This is a case report of an IVC filter penetration identified during lymph-node dissection for endometrial carcinoma. Although the spread strut put the adjacent abdominal aorta in danger of penetration, surgical removal of the filter could not be performed because there was still an increased risk of pulmonary embolism. Instead of IVC interruption, we used a wrapping technique as a prophylaxis against major bleeding complication due to IVC filter dislocation and recurrent pulmonary embolism.

Topical application of cyclosporin A induces rapid-remodeling of damaged anagen hair follicles produced in cyclophosphamide administered mice.

Adult C3H mice which had either anagen IV or anagen VI hair follicles were given the anti-tumor drug cyclophosphamide, and cyclosporin A or minoxidil were topically applied to the mice daily from the 4th day after cyclophosphamide administration. In the mice that had anagen IV-hair follicles, 0.5% cyclosporin A induced very thick and long hairs after 21 days of cyclophosphamide administration, while vehicle and 1% minoxidil induced sparsely visible, short hairs. In the mice which received cyclosporin A, the injured hair follicles seemed to remodel themselves into intact anagen hair follicles and restart the production of hairs, instead of shifting to telogen. In the mice that had anagen VI-hair follicles at the time of cyclophosphamide administration, complete alopecia occurred within the first 7 days in all groups. After 14 days of cyclophosphamide administration, hair regrowth was observed in both the 0.5% cyclosporin A-group and the 1% minoxidil- group with the predominant effect over the vehicle. This study shows that anagen hair follicles respond to cyclophosphamide in different ways depending on their stages (IV and VI), and that the damaged anagen IV hair follicles have the potential of remodeling themselves, which is promoted by topical cyclosporin A administration.

Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro.

PURPOSE: Some studies have suggested that computer-aided sperm analysis (CASA) estimates of concentration and movement characteristics of progressively motile spermatozoa are related to fertilization rates in vitro. However, it has also been suggested that the greater number of motility parameters assessed by CASA does not imply more precision in predicting fertility. This study was carried out to investigate the relationships between the CASA estimates and fertilization rates in vitro. METHODS: Semen quality analysis was performed using CASA in 136 in vitro fertilization-embryo transfer (IVF-ET) cycles with at least 3 oocytes collected. The CASA estimates before and after swim-up were compared between 108 cycles with fertilization rate > 50% ("good" group) and 28 cycles with fertilization rate < or = 50% ("poor" group). RESULTS: Before swim-up, there were significant correlations between fertilization rates and CASA estimates, including amplitude of lateral head displacement (ALH) (r = .269), curvilinear velocity (VCL) (r = .297), straight line velocity (VSL) (r = .266), and rapid sprm movement (Rapid) (r = .243). There was also a significant correlation between the fertilization rates and straightness (STR) after swim-up (r = -0.178). As for sperm movement characteristics, there were significant differences of ALH (p < .005), VCL (p < .001), VSL (p < .005), and Rapid (p < .01) between "good" and "poor" groups before swim-up. After swim-up, there were significant differences of VCL (p < .005), average path velocity (VAP) (p < .005), and Rapid (p < .05) between the two groups. CONCLUSIONS: These results indicate that some of the CASA estimates provide reliable estimation of the fertilizing ability of human sperm. There were significant differences of the two sperm movement characteristics, including VCL and Rapid (before and after swim-up), indicating that the total distance traveled by rapid sperm movement might be important in human sperm fertilizing abilities.

Highly sensitive and rapid method for determination of fluoride ion concentrations in serum and urine using flow injection analysis with a fluoride ion-selective electrode.

An apparatus for flow injection analysis (FIA) was developed to measure very low fluoride ion concentrations (<1 micromol/l). The analytical conditions of the apparatus were investigated, and the instrument was used to determine fluoride ion concentrations in serum and urine. All interferences caused by serum and urine matrices were eliminated using the proposed method. The recovery was almost 100.0% for serum and urine samples. The precision was within 4%. The results of determination of fluoride ion concentrations in the NIST Standard Reference Material of urine, SRM 2671a, agreed with the certified values. The detection limits in serum and urine were 0.016 and 0.16 micromol/l, respectively. The assay throughput was 15 samples/h in serum and 24 samples/h in urine. The mean fluoride ion concentrations in serum and urine samples from 53 young Japanese women were 0.383+/-0.158 micromol/l and 0.207+/-0.103 mg/g Cr, respectively. There was a significant correlation (r=0.39, p<0.01) between serum and urine fluoride ion concentrations.

Expression of glutathione S-transferase pi (GST-pi) in human malignant ovarian tumors.

OBJECTIVES: In recent years, glutathione S-transferase pi (GST-pi) has attracted much attention and has been studied as a mechanism of multidrug resistance of tumors to anticancer drugs. In the present study, we immunohistologically measured the expression of GST-pi in tumor tissues using surgical specimens obtained from patients with malignant ovarian tumors. METHODS: Of 137 patients with malignant ovarian tumors treated and managed during a period of 20 years since the establishment of Tsukuba University Hospital, 117 patients were selected as subjects because of the presence of complete data on their clinical courses as well as paraffin blocks preserved in a good condition. GST-pi in these specimens was immunohistochemically stained to determine the correlation between GST-pi stainability and clinical outcomes. Stainability was graded as 0 when GST-pi was completely absent, 1 when less than 20% of tumor cells were stained, 2 when 20--60% were stained, and 3 when more than 60% were stained. RESULTS: When the correlation between stainability and clinical outcomes was analyzed with Kaplan--Meier method, excluding stage Ia cases that did not receive adjuvant chemotherapy at our hospital, significantly better clinical outcomes were observed in the low stainable group, compared with the high stainable group (P<0.01--0.05, Cox--Mantel test, Wilcoxon's test). CONCLUSION: Since the stainability for GST-pi was high in tumors of histological types with strong resistance to anticancer drugs, and better clinical outcomes were observed in cases having a lower stainability score, the expression of GST-pi was thought to play some role in the resistance of malignant ovarian tumors to anticancer drugs.

[Dose finding study of paclitaxel and carboplatin for ovarian cancer (JKTB)].

We conducted a dose-finding study for combination therapy of paclitaxel (Taxol; TXL) and carboplatin (Paraplatin; CBDCA). TXL is a novel plant-derived anticancer agent that is a diterpene derivative possessing the taxane ring. The subjects were patients with ovarian carcinoma, who were evaluated by a modified Fibonacci method. The dosage of TXL was 150 to 180 mg/m2. CBDCA was administered by dose escalation from AUC = 4 to 7. The administration schedule was as follows. Pre-medication was administered before TXL was given. TXL was then administered by intravenous infusion over 3 hours, followed by CBDCA. The dose of CBDCA was determined using the Calvert formula: [AUCX (GFR + 25)]. GFR was calculated with the Jelliffe equation. The non-hematological toxicities observed in 15 eligible cases were mainly grade 1, with no grade 3 or above, and no increase in severity was observed with stepping up. The hematological toxicities were grade 3 leukopenia in 5 of 15 cases, neutropenia in 5 cases and thrombocytopenia in 0 cases. No grade 4 toxicity was observed. The lowest counts of leukocytes and neutrophils were reached after 10.8 and 11.7 days, respectively. The toxicities were reversible in most cases with subsequent recovery. The above findings indicate that the recommended dosages for TJ therapy for Japanese ovarian cancer patients should be TXL 180 mg/m2 and CBDCA at a target of AUC = 6.

Prediction of unexpectedly poor fertilization and pregnancy outcome using the strict criteria for sperm morphology before and after sperm separation in IVF-ET.

This study was performed to investigate if unexpectedly poor fertilization and in-vitro fertilization (IVF) outcome could be predicted using sperm morphology as diagnosed by the strict criteria. Sperm morphology was assessed in 137 IVF-ET cycles with at least three oocytes collected. The lowest amount of normal forms was 5% in 137 samples, indicating there were no patients belonging to 'poor prognosis' (<5% normal forms). Treatment using intracytoplasmic sperm injection (ICSI) was also excluded. Before sperm separation by the swim-up method, sperm morphology demonstrated a significant correlation with the fertilization rate (p < 0.0001). The fertilization rate (80.5%) in 110 'normal' samples (>14% normal forms) was significantly higher (p < 0.01) than that (55.4%) in 27 samples with 'good prognosis' (those with 5--14% normal forms). No embryo was available for transfer (ET) in 4 (3.6%) of 110 'normal' cycles and in 3 (11.1%) of 27 'good prognosis' cycles (not significant). Fresh ET was intentionally cancelled to avoid severe ovarian hyperstimulation syndrome (OHSS) in six of 110 'normal' cycles as well as in one of 27 'good prognosis' cycles. The pregnancy rate per ET was 31.0% (31/100) in the former group, while it was 26.1% (6/23) in the latter group. There was no difference between the two groups. In the post swim-up evaluation of sperm characteristics, morphology was significantly correlated with the fertilization rate in IVF-ET (p < 0.05) while other sperm parameters were not. When the cut off level for the post swim-up sperm morphology was set at 25%, there was a significant difference in the fertilization rates between patients (78.6%) with post-swim-up >25% and those (55.0%) with post-swim-up < or =25% (p < 0.01). Taken together, a relative indication for ICSI using sperm morphology before and after swim-up was established. Category A includes < or =14% normal forms in the ejaculate and post-swim-up < or =25%, while Category B includes < or =14% in the ejaculate and post-swim-up >25%. There was a significant difference in the fertilization rates between patients (47.2%) in Category A and those (60.2%) in Category B (p < 0.05). The clinical pregnancy rate was 11.1% for patients in Category A compared with 35.7% for patients in Category B. However, there was no significant difference between the two categories. These results indicate that the strict criteria provide a reliable estimation of the fertilizing ability of human spermatozoa. ICSI might be considered in Category A patients to avoid poor fertilization and pregnancy outcome.

Uterine inversion caused by uterine sarcoma: a case report.

Uterine inversion caused by uterine sarcoma is a rare condition with 12 reported cases to date according to a MEDLINE search. We report two cases of this rare condition. A 71- and a 72-year-old woman presented with uterine sarcomas rapidly extruded into the vagina. In both cases, magnetic resonance imaging (MRI) scans showed U-shaped uterine cavities and the pedicles of these tumors were attached to the uterine fundi. Pathological examination confirmed a leiomyosarcoma and a heterologous carcinosarcoma. Uterine inversion can occur when uterine sarcoma rapidly increases in size and extrudes into the vagina. MRI should be performed in the diagnosis of this rare combination.

Non-invasive lobular carcinoma within a fibroadenoma, a preoperatively diagnosed case.

Breast cancer within a fibroadenoma is rare and usually diagnosed postoperatively from pathological specimens. This paper reports a 54-year-old female with non-invasive carcinoma within a fibroadenoma, diagnosed preoperatively. She underwent a medical examination and mastopathy was suspected. On physical examination a mass 2 cm in diameter was palpated in the left breast. Ultrasonography showed a mass with smooth margins and uniform internal echoes, but cytology showed malignancy. Mammography showed a round mass with distinct margins and no calcification. As fibroadenoma, diagnosed by ultrasonography and mammography, and breast cancer, diagnosed by cytology, were not consistent results several core biopsies were performed. Needle biopsy showed proliferation of atypical epithelial cells; breast cancer within a fibroadenoma was diagnosed. MRI showed a circular mass with distinct, smooth margins and in a dynamic study, the mass showed irregular staining and the presence of early staining. Left lumpectomy and dissection of the left axillary lymph nodes was performed. Histological examination showed non-invasive lobular carcinoma occurring within a fibroadenoma.