Establishment of humanized anti-interleukin-5 receptor alpha chain monoclonal antibodies having a potent neutralizing activity.

Human interleukin-5 is the key cytokine involved in regulating the production and function of human eosinophils. IL-5 binds to its specific receptor composed of two heterogeneous alpha and beta polypeptide chains (hIL-5Ralpha and betac) that are expressed on the cell surface. The hIL-5Ralpha specifically binds IL-5 without involvement of the betac. It has been suggested that neutralizing antibodies to hIL-5Ralpha could serve as a therapeutic agent in eosinophil-associated diseases. We describe here the creation and biologic activities of a mouse monoclonal antibody against hIL-5Ralpha that blocks the following IL-5 dependent activities (a) binding of the IL-5 ligand to its receptor, (b) IL-5 dependent growth of hIL-5R expressing cells, and (c) IL-5-induced adhesion of human eosinophils. We also describe the process for humanization of the mouse Mab towards development of a therapeutic MAb. The humanized version of the monoclonal antibody also displayed potent neutralizing activity against IL-5 dependent activities.

A novel steroidal selective steroid sulfatase inhibitor KW-2581 inhibits sulfated-estrogen dependent growth of breast cancer cells in vitro and in animal models.

We screened a series of 17beta-(N-alkylcarbamoyl)-estra-1,3,5(10)trine-3-O-sulfamate derivatives, and describe here a potent and selective steroid sulfatase (STS) inhibitor with antitumor effects in breast cancer models in vitro and in vivo. In biochemical assays using crude enzymes isolated from recombinant Chinese hamster ovary cells expressing human arylsulfatses (ARSs), one of the best compounds, KW-2581, inhibited STS activity with an IC(50) of 4.0 nM, while > 1000-fold higher concentrations were required to inhibit the other ARSs. The failure to stimulate the growth of MCF-7 human breast cancer cells as well as in uteri in ovariectomized rats indicated the lack of estrogenicity of this compound. In MCF-7 cells transfected with the STS gene, termed MCS-2 cells, KW-2581 inhibited the growth of cells stimulated by estrone sulfate (E1S) but also 5-androstene-3beta, 17beta-diol 3-sulfate (ADIOLS) and dehydroepiandrostenedione 3-sulfate. We found that oral administration of KW-2581 inhibited both E1S- and ADIOLS-stimulated growth of MCS-2 cells in a mouse hollow fiber model. In a nitrosomethylurea-induced rat mammary tumor model, KW-2581 induced regression of E1S-stimulated tumor growth as effectively as tamoxifen or another STS inhibitor, 667 Coumate. Dose-response studies in the same rat model demonstrated that more than 90% inhibition of STS activity in tumors was necessary to induce tumor shrinkage. STS activity in tumors has well correlated with that in leukocytes, suggesting that STS activity in leukocytes could be used as an easily detectable pharmacodynamic marker. These findings demonstrate that KW-2581 is a candidate for development as a therapeutic agent for the treatment of hormone receptors-positive breast cancer.

The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.

Reconstitution of telomerase activity utilizing human catalytic subunit expressed in insect cells.

Telomerase is a specialized reverse transcriptase responsible for maintaining the termini of linear chromosomes. The human enzyme is a ribonucleoprotein complex minimally comprising a catalytic protein moiety (hTERT) and an RNA subunit (hTR) which acts as the template for the reverse transcriptase reaction. Here we report expression of recombinant hTERT protein in insect cells utilizing a baculovirus expression system. The recombinant hTERT protein reconstitutes telomerase activity in the presence of hTR, either when co-expressed in insect cells or when added in vitro. Reconstitution of telomerase activity using this system will facilitate further analysis of the biochemical and biophysical properties of this enzyme.

N-terminal domain of eotaxin-3 is important for activation of CC chemokine receptor 3.

Eotaxin-3 belongs to the CC chemokine family, and specifically recognizes CC chemokine receptor (CCR) 3 that is expressed on eosinophils, basophils and helper T type 2 cells. The three-dimensional structure of eotaxin-3 determined by nuclear magnetic resonance has revealed that the N-terminal nine residues preceding the first cysteine comprise an unstructured domain, which is also observed in other chemokine molecules. In order to determine the function of the N-terminal domain of eotaxin-3, we constructed various N-terminal-deletion mutants, and then examined their binding and chemotactic activities toward eosinophils in vitro. Competitive binding studies showed that the binding affinity of truncated mutant toward CCR3 was almost the same as that of wild-type eotaxin-3 even though the N-terminal truncation involved the first through to the ninth residues. In contrast, the chemotactic activity gradually decreased with extension of the N-terminal deletion, and when the deletion extended to the eighth residue, the activity was not detected at all. Thus, the N-terminal nine residues are not critical for binding but the N-terminal eight residues are essential for activation of CCR3. The truncated eotaxin-3 proteins lacking the N-terminal eight or nine residues inhibited the chemotactic activity of chemokines that recognize CCR3. The truncated mutants can possibly be used for anti-allergic and anti-HIV-1 therapy.