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.

Effective interaction energy of water dimer at room temperature: an experimental and theoretical study.

Buffer-gas pressure broadening for the nu(1)+nu(3) band of H(2)O at 1.34-1.44 mum for a variety of buffer gases was investigated at room temperature using continuous-wave cavity ring-down spectroscopy. The effective interaction energy of water dimer under room temperature conditions was evaluated from the pressure broadening coefficients for rare gases using Permenter-Seaver's relation. Monte Carlo simulations were performed using ab initio molecular orbital calculations to evaluate the interaction energies for the water dimer at 300 K. In this theoretical calculation, the orientations of the two water molecules were statistically treated.

Comprehensive allelotyping of human intrahepatic cholangiocarcinoma.

We performed a genome-wide scan for loss of heterozygosity (LOH) in 22 intrahepatic cholangiocarcinoma (ICC) cases using 168 polymorphic microsatellite markers throughout all of the human chromosomes and 48 markers of which LOH is reportedly characteristic of hepatocellular carcinoma (HCC). Markers with LOH in more than 30% of informative cases were observed at 21 loci. Among these, eight markers on 6q (three loci), 4q (two loci), 9q, 16q, and 17p shared high frequencies of LOH with HCC in our previous study. As for gross appearance, mass-forming type tumors showed higher frequency of LOH (P < 0.001) compared with other types. Compared by tumor size (< or =5 cm versus >5 cm), number (multiple versus solitary), and the International Union Against Cancer TNM classification (stage IVB versus II-IVA), LOH was observed more frequently in advanced stages (P < 0.01, respectively). However, LOH frequency does not differ regardless of lymph node status (pN0 versus pN1). Frequent LOH on 1p36 including the p73 locus was noted in large tumors without lymph node metastasis. These suggest that ICC shares some common carcinogenic steps with HCC such as LOH of 4q and 6q and that inactivation of tumor suppressor genes on chromosome 1p36 contributes to progression of ICC but not to metastatic traits.

Increased expression of cytosolic chaperonin CCT in human hepatocellular and colonic carcinoma.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTbeta in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTbeta antibody. The levels of CCTbeta were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTalpha gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.

Comprehensive allelotype study of hepatocellular carcinoma: potential differences in pathways to hepatocellular carcinoma between hepatitis B virus-positive and -negative tumors.

To examine the role of the loss of heterozygosity (LOH) in hepatitis-related carcinogenesis, we performed a genome-wide scan of LOH in 44 tumors of hepatocellular carcinoma (HCC) using 216 microsatellite markers throughout all human chromosomes. A high frequency of LOH (>30% of informative cases) was observed at 33 loci on chromosome arms 4q, 6q, 8p, 8q, 9p, 9q, 13q, 16p, 16q, 17p, and 19p. LOH on 19p has not yet been reported, and that appears to be a new candidate in the search for tumor suppressor genes. High rates of LOH are correlated with hepatitis B virus (HBV) positivity, poorly differentiated tumors, vascular invasion, and intrahepatic metastasis (P <.0001). LOH on 13q and 16q occurred more frequently in HBV(+) patients (P <.0001), and LOH on 6q occurred more frequently in virus-negative patients (P <.001). The frequency of LOH on 4q and 13q was significantly lower in well-differentiated tumors than in moderately and poorly differentiated tumors (P <.01). In contrast, LOH on 6q was frequently detected in well-differentiated tumors compared with other histological subclasses (P <.001). Our results suggest that LOH on 6q may play an important role in the early stage of hepatocarcinogenesis in virus-negative patients, but different mechanisms might underlie the initial step to carcinogenesis in HBV(+) patients. LOH on 13q and 16q may play an essential role in the progression of HBV(+) tumors. Further studies of fine deletion mapping on chromosomes 13q and 16q are required to define the genomic segments on which putative tumor suppressor genes responsible for HBV(+) tumors exist.

Molecular design of antibrowning agents.

Tyrosinase inhibitory and antioxidant activity of gallic acid and its series of alkyl chain esters were investigated. All inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase. However, gallic acid and its short alkyl chain esters were oxidized as substrates yielding the colored oxidation products. In contrast, the long alkyl chain esters inhibited the enzyme activity without being oxidized. This indicates that the carbon chain length is associated with their tyrosinase inhibitory activity, presumably by interacting with the hydrophobic protein pocket in the enzyme. On the other hand, the esters, regardless their carbon chain length, showed potent scavenging activity on the autoxidation of linoleic acid and 1,1-diphenyl-2-p-picryhydrazyl (DPPH) radical, suggesting that the alkyl chain length is not related to the activity. The effects of side-chain length of gallates in relation to their antibrowning activity are studied.

Analysis of genetic changes in intrahepatic cholangiocarcinoma induced by thorotrast.

Thorotrast, a colloidal suspension of radioactive (232)ThO(2) that emits alpha particles, was used as a radiographic contrast agent in the 1930s-1950s. Several decades after injection, Thorotrast causes liver cancers, among which intrahepatic cholangiocarcinoma (ICC) is prominent. We investigated mutations of the RAS and the TP53 genes in archival sections of ICC induced by Thorotrast. Compared to ICC that was not associated with Thorotrast, the frequency of mutation of the KRAS gene was lower, while that of the TP53 gene was more than two times higher. The most common mutation of the TP53 gene was A-G transitions. Interestingly, TP53 mutations were also found in noncancerous areas of livers in which Thorotrast had been deposited. Furthermore, mutations tended to accumulate in tissues from more advanced tumors. These results suggest that deposited Thorotrast continuously damages DNA in liver cells in some way, resulting in A-G transitions of the TP53 gene. However, we have not been able to rule out the possibility that genetic insults occur indirectly in the proliferating cells adjacent to the necrosis rather than being a direct effect of alpha particles.

Expression of the thymidine phosphorylase gene in epithelial ovarian cancer.

Thymidine phosphorylase (TP) is associated with angiogenesis and the progression of solid tumours. High intracellular levels of this enzyme indicate increased chemosensitivity to pyrimidine antimetabolites. TP gene expression in 56 cases of epithelial ovarian cancer (27 of serous, 10 mucinous, 12 endometrioid, five clear cell and two undifferentiated) were analysed by polymerase chain reaction of RNA after reverse transcription. These included eight of low malignant potential. Twenty were stage I, four stage II, 27 stage III and five stage IV. The level of TP gene expression was presented by the relative yield of the TP gene to the beta2-microglobulin gene. TP gene expression ranged from 0.19 to 5.38 (median 0.93). The value of TP gene expression in stage III-IV was significantly higher than that of TP gene expression in stage I-II (P = 0.0005). Histological grade significantly associated with TP gene expression (P = 0.008), but histological subtype did not (P = 0.166). A follow-up study of 34 cases after complete resection of the primary tumours by surgical operation was performed. TP gene expression of the cases with recurrence showed significantly higher levels compared to cases without recurrence (P = 0.049). Survival data were available for 47 of the 56 patients. The prognosis of the patients with high TP gene expression (equal to, or greater than, median) was to be significantly worse than patients with low TP gene expression (less than median) (P = 0.021). The TP gene expression level may play one of the key roles in the biology of ovarian epithelial cancer and define a more aggressive tumour phenotype. A new therapeutic intervention mediated by TP protein activity is anticipated.

Sulfated glycoglycerolipid from archaebacterium inhibits eukaryotic DNA polymerase alpha, beta and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity.

A sulfated glycoglycerolipid, 1-O-(6'-sulfo-alpha-D-glucopyranosyl)-2,3-di-O-phytanyl- sn-glycerol (KN-208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) alpha and pol beta in vitro among 5 eukaryotic DNA polymerases (alpha, beta, gamma, delta, and epsilon). It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E. coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT). The mode of inhibition of these polymerases was competitive with the DNA template primer and was non-competitive with the substrate dTTP. KN-208 inhibited pol beta most strongly, with a Ki value of 0.05 microM, 10-fold lower than that for pol alpha (0.5 microM) and 60- or 140-fold lower than that for HIV RT (3 microM) or for E. coli pol I (7 microM), respectively. The loss of sulfate on the 6'-position of glucopyranoside of this compound completely abrogated inhibition. However, the hydrophilic part of KN-208, glucose 6-sulfate alone, showed no inhibition. Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition. Our results suggest that the whole molecular structure of KN-208 is required for inhibition. KN-208 was shown to be modestly cytotoxic for the human leukemic cell line K562. Interestingly, a subcytotoxic dose of KN-208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin.