Use of anti-inhibin monoclonal antibody for increasing the litter size of mouse strains and its application to in vivo-genome editing technology†.

The litter size of mouse strains is determined by the number of oocytes naturally ovulated. Many attempts have been made to increase litter sizes by conventional superovulation regimens (e.g., using equine or human gonadotropins, eCG/hCG but had limited success because of unexpected decreases in the numbers of embryos surviving to term. Here, we examined whether rat-derived anti-inhibin monoclonal antibodies (AIMAs) could be used for this purpose. When C57BL/6 female mice were treated with an AIMA and mated, the number of healthy offspring per mouse increased by 1.4-fold (11.9 vs. 8.6 in controls). By contrast, treatment with eCG/hCG or anti-inhibin serum resulted in fewer offspring than in nontreated controls. The overall efficiency of production based on all females treated (including nonpregnant ones) was improved 2.4 times with AIMA compared with nontreated controls. The AIMA treatment was also effective in ICR mice, increasing the litter size from 15.3 to 21.2 pups. We then applied this technique to an in vivo genome-editing method (improved genome-editing via oviductal nucleic acid delivery, i-GONAD) to produce C57BL/6 mice deficient for tyrosinase. The mean litter size following i-GONAD increased from 4.8 to 7.3 after the AIMA treatment and genetic modifications were confirmed in 80/88 (91%) of the offspring. Thus, AIMA treatment is a promising method for increasing the litter size of mice and may be applied for the easy proliferation of mouse colonies as well as in vivo genetic manipulation, especially when the mouse strains are sensitive to handling.

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3.

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.

Evaluation of expression and glycosylation status of UGT1A10 in Supersomes and intestinal epithelial cells with a novel specific UGT1A10 monoclonal antibody.

UDP-Glucuronosyltransferases (UGTs) are major phase II drug-metabolizing enzymes. Each member of the UGT family exhibits a unique but occasionally overlapping substrate specificity and tissue-specific expression pattern. Earlier studies have reported that human UGT1A10 is expressed in the gastrointestinal tract at the mRNA level, but the evaluation at the protein level, especially tissue or cellular localization, has lagged behind because of the lack of a specific antibody. In this study, we prepared a monoclonal antibody to UGT1A10 to elucidate the tissue/cellular distribution and interindividual variability of UGT1A10 protein expression. Western blot analysis revealed that the prepared antibody does not cross-react with any other human UGTs. Using this specific antibody, we observed that UGT1A10 protein is expressed in the small intestine but not in the liver or kidney. Immunohistochemical analysis revealed the expression of UGT1A10 protein in epithelial cells of the crypts and villi of the duodenum. In the small intestine microsomes from six individuals, the UGT1A10 protein levels exhibited 16-fold variability. Dopamine 3- and 4-glucuronidation, which is mainly catalyzed by UGT1A10 and by other UGT isoforms marginally, exhibited 50- to 65-fold variability, and they were not correlated with the UGT1A10 protein levels. Interestingly, the enzymatic activities of recombinant UGT1A10 in insect cells that were normalized to the UGT1A10 protein level were markedly lower than those in pooled human small intestine microsomes. Thus, the UGT1A10 antibody we generated made it possible to investigate the tissue/cellular distribution and interindividual variability of UGT1A10 protein expression for understanding the pharmacological and toxicological role of UGT1A10.

Preparation of a specific monoclonal antibody against human UDP-glucuronosyltransferase (UGT) 1A9 and evaluation of UGT1A9 protein levels in human tissues.

Glucuronidation is a major detoxification pathway of drugs and xenobiotics that are catalyzed by the UDP-glucuronosyltransferase (UGT) superfamily. Determination of the protein levels of the individual UGT isoforms in human tissues is required for the successful extrapolation of in vitro metabolic data to in vivo clearance. Most previous studies evaluating UGT isoform expression were limited to the mRNA level because of the high degree of amino acid sequence homology between UGT isoforms that has hampered the availability of isoform-specific antibodies. In this study, we generated a peptide-specific monoclonal antibody against human UGT1A9. We demonstrated that this antibody does not cross-react with the other UGT1A isoforms including UGT1A7, UGT1A8, and UGT1A10 and shows a high degree of amino acid sequence similarity with UGT1A9. Using this antibody, we found that UGT1A9 protein is expressed in the kidney and the liver but not in the jejunum or the ileum, consistent with previous reports of mRNA expression. In a panel of 20 individual human livers, the UGT1A9 protein levels exhibited 9-fold variability. It is noteworthy that the relative UGT1A9 protein levels were not correlated with the UGT1A9 mRNA level (r = -0.13), like other UGT isoforms reported previously, suggesting the importance of evaluating UGT isoform expression at protein levels. In conclusion, we generated a specific monoclonal antibody against UGT1A9 and evaluated the distribution and relative expression levels of the UGT1A9 protein in human tissues. This antibody may serve as a useful tool for further studies of UGT1A9 to evaluate its physiological, pharmacological, and toxicological roles in human tissues.

Clinical benefit of non-toxic therapy in patients with advanced cancer (opinion).

It has been widely recognized that chemotherapy of cancer has profound toxic side-effects and suffers limitations in efficacy but, as yet, no solution has been found to this conflict. Consequently, many patients with advanced cancer desire treatment by less toxic therapies even if the technique is not established as a conventional cancer therapy. Thus the establishment of less toxic therapies should be considered as a tentative requirement in clinical practice. Adoptive immunotherapy is one such therapy which has enjoyed some popularity in Japan. Clinical experience accumulated over the last 15 years indicates benefits to be gained from the therapy. In 1998, the Ministry of Health, Labor and Welfare of Japan (MHLW) approved portions of adoptive immunotherapy practices in their definition of highly-advanced medical technology. In April 1999, we established a private out-patient clinic specializing in adoptive immunotherapy; this clinic has administered 200 infusions of activated lymphocytes a month. Out of the 188 patients treated, we evaluated the tumors of 57 cases (2 CR, 4 PR, 20 SD and 31 PD). The response rate was 11%. It is noteworthy that in 11 of the 20 SD patients, the disease remained stable for more than six months. The follow-up periods for these 11 patients ranged from 11 to 24 months. During this period tumor progressed in 2, but the remaining 9 were still alive and without noticeable disease progression. Our present experience of adoptive immunotherapy administered to a large number of patients indicates that this non-toxic therapy has some beneficial role for the treatment of refractory cancer.