Novel anti-GARP antibody DS-1055a augments anti-tumor immunity by depleting highly suppressive GARP+ regulatory T cells.

Regulatory T (Treg) cells, which are essential for maintaining self-tolerance, inhibit anti-tumor immunity, consequently hindering protective cancer immunosurveillance, and hampering effective anti-tumor immune responses in tumor-bearing hosts. Here, we show that depletion of Treg cells via targeting glycoprotein A repetitions predominant (GARP) induces effective anti-tumor immune responses. GARP was specifically expressed by highly suppressive Treg cells in the tumor microenvironment (TME) of multiple cancer types in humans. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation. DS-1055a, a novel afucosylated anti-human GARP monoclonal antibody, efficiently depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FoxP3+CD4+ T cells in the TME and exhibited remarkable anti-tumor activity in humanized mice bearing HT-29 tumors. We propose that DS-1055a is a new Treg-cell-targeted cancer immunotherapy agent with augmentation of anti-tumor immunity.

Inhibition of ergosterol synthesis by novel antifungal compounds targeting C-14 reductase.

The limited number of clinically available antifungal drugs for life-threatening fungal infections has produced an increased demand for new agents. In the course of our screening for novel antifungals, we identified aminopiperidine derivatives which exhibit antifungal activities against the major pathogenic yeasts. Thin layer chromatography (TLC) analysis of the extracted non-saponifiable lipids from Candida albicans showed that these compounds inhibited the ergosterol production in the late step of the synthesis pathway. The results of an LC/Q-Tof MS analysis showed that abnormal sterols including predicted ignosterol, which is known to be accumulated in C. albicans ERG24 deleted mutant, were accumulated in C. albicans treated with one of these derivatives (Compound 1b). Furthermore, the partial disruption of the cell membrane of C. albicans treated with compound 1b was observed by electron microscopy analysis, suggesting its inhibition of ergosterol synthesis. Additionally, a genetic approach demonstrated that ERG24 gene would be responsible for the resistance of Saccharomyces cerevisiae against Compound 1b, strongly indicating that the enzyme targeted by Compound 1b is Erg24p. From all these data, we concluded that these aminopiperidine derivatives are novel antifungal compounds inhibiting C-14 reduction in the ergosterol synthesis pathway.

In vitro and in vivo antifungal activities of aminopiperidine derivatives, novel ergosterol synthesis inhibitors.

Aminopiperidine derivatives, Compound 1a and 1b, are novel small molecules that inhibit C-14 reduction catalyzed by Erg24p in ergosterol synthesis of Candida albicans. We evaluated the properties of the in vitro and in vivo activities of these compounds against pathogenic fungi and compared their activities with those of fluconazole. Compound 1a and 1b exhibited potent in vitro activities against clinically important fungi such as Candida species, including both of fluconazole-resistant strains of C. albicans and non-albicans Candida, Aspergillus fumigatus, and Cryptococcus neoformans. Against C. albicans, its mode of action was fungistatic. Furthermore, orally administered Compound 1b clearly prolonged the survival of infected mice in systemic lethal infection caused by C. albicans. These results suggest that aminopiperidine derivative is a promising lead compound for an orally available novel antifungal drug with a broad spectrum.

In vitro antifungal activities of D11-2040, a beta-1,6-glucan inhibitor, with or without currently available antifungal drugs.

We recently reported our discovery of small molecule beta-1,6-glucan inhibitor named D75-4590 and the anti-Candida activity of its derivatives D11-2040 and D21-6076. In this study, we further evaluated the antifungal profile of D11-2040. It alone strongly inhibited the vegetative growth and/or hyphal development of various Candida species, but no significant activity was observed against Cryptococcus neoformans or any of the filamentous fungi tested. Synergism was detected for C. albicans in the interaction of D11-2040 and caspofungin by the chequerboard method and in that of D11-2040 and fluconazole by the time-kill method. Slight but positive interactions were observed in several combinations for C. neoformans and Aspergillus fumigatus as well. These results suggested that beta-1,6-glucan inhibitors have promising potential as single drugs as well as concomitants.

Discovery of a small-molecule inhibitor of {beta}-1,6-glucan synthesis.

It is possible that antifungal drugs with novel modes of action will provide favorable options to treat fungal infections. In the course of our screening for antifungal compounds acting on the cell wall, a pyridobenzimidazole derivative with unique activities, named D75-4590, was discovered. During treatment of Saccharomyces cerevisiae with D75-4590, (i) incorporation of [(14)C]glucose into the beta-1,6-glucan component was selectively reduced, (ii) proteins released from the cell had lost the beta-1,6-glucan moiety, and (iii) cells tended to clump, resulting in impaired cell growth. Genetic analysis of a D75-4590-resistant mutant of S. cerevisiae indicated that its primary target was Kre6p, which is considered to be one of the beta-1,6-glucan synthases. These results strongly suggest that D75-4590 is a specific inhibitor of beta-1,6-glucan synthesis. D75-4590 showed potent activities against various Candida species. It inhibited hyphal elongation of C. albicans as well. KRE6 is conserved in various fungi, but no homologue has been found in mammalian cells. These lines of evidence indicate that D75-4590 is a promising lead compound for novel antifungal drugs. To our knowledge, this is the first report of a beta-1,6-glucan inhibitor.

Effect of beta-1,6-glucan inhibitors on the invasion process of Candida albicans: potential mechanism of their in vivo efficacy.

Beta-1,6-glucan is a fungus-specific cell wall component that is essential for the retention of many cell wall proteins. We recently reported the discovery of a small molecule inhibitor of beta-1,6-glucan biosynthesis in yeasts. In the course of our study of its derivatives, we found a unique feature in their antifungal profile. D21-6076, one of these compounds, exhibited potent in vitro and in vivo antifungal activities against Candida glabrata. Interestingly, although it only weakly reduced the growth of Candida albicans in conventional media, it significantly prolonged the survival of mice infected by the pathogen. Biochemical evaluation of D21-6076 indicated that it inhibited beta-1,6-glucan synthesis of C. albicans, leading the cell wall proteins, which play a critical role in its virulence, to be released from the cell. Correspondingly, adhesion of C. albicans cells to mammalian cells and their hyphal elongation were strongly reduced by the drug treatment. The results of the experiment using an in vitro model of vaginal candidiasis showed that D21-6076 strongly inhibited the invasion process of C. albicans without a significant reduction in its growth in the medium. These evidences suggested that D21-6076 probably exhibited in vivo efficacy against C. albicans by inhibiting its invasion process.