TRAIL-R2 Superoligomerization Induced by Human Monoclonal Agonistic Antibody KMTR2.

The fully human monoclonal antibody KMTR2 acts as a strong direct agonist for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), which is capable of inducing apoptotic cell death without cross-linking. To investigate the mechanism of direct agonistic activity induced by KMTR2, the crystal structure of the extracellular region of TRAIL-R2 and a Fab fragment derived from KMTR2 (KMTR2-Fab) was determined to 2.1 Å resolution. Two KMTR2-Fabs assembled with the complementarity-determining region 2 of the light chain via two-fold crystallographic symmetry, suggesting that the KMTR2-Fab assembly tended to enhance TRAIL-R2 oligomerization. A single mutation at Asn53 to Arg located at the two-fold interface in the KMTR2 resulted in a loss of its apoptotic activity, although it retained its antigen-binding activity. These results indicate that the strong agonistic activity, such as apoptotic signaling and tumor regression, induced by KMTR2 is attributed to TRAIL-R2 superoligomerization induced by the interdimerization of KMTR2.

The novel CA IX inhibition antibody chKM4927 shows anti-tumor efficacy in vivo.

Carbonic anhydrase IX (CA IX) is an attractive target for cancer therapy. Many anti-CA IX antibodies have been reported but few have been shown to possess inhibition activity. Furthermore, effective use of CA IX-inhibition antibodies for cancer immunotherapy has not been well-validated since data are mainly limited to in vitro assays. In this study, we established that chKM4927, an anti-CA IX chimeric antibody, recognizes CA IX and has CA IX-specific inhibition activity. ChKM4927 also retains antibody-dependent cellular cytotoxicity (ADCC) activity against CA IX-expressing cancer cells. Compared to controls, chKM4927 treatment (10 mg/kg) showed anti-tumor activity in the VMRC-RCW xenograft model in vivo. ChKM4927-attenuated ADCC activity showed equally effective anti-tumor activity. These results suggest that the CA IX-inhibition antibody chKM4927 has an anti-tumor effect in the VMRC-RCW xenograft model via an ADCC-independent mechanism.

Switching constant domains enhances agonist activities of antibodies to a thrombopoietin receptor.

We enhanced the activities of two agonist antibodies specific for the thrombopoietin receptor (c-MPL) by switching domains within their constant regions to those of different antibody isotypes. Our results suggest the importance of the hinge region in modulating agonist activity. The antibodies' thrombopoietin-like activity in vitro and in vivo, as well as the desirable pharmacokinetic profile conferred by retaining the whole-IgG structure, suggests that they provide a valuable option for treating thrombocytopenia.

Efficient antibody production upon suppression of O mannosylation in the yeast Ogataea minuta.

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

Antibody expression in protease-deficient strains of the methylotrophic yeast Ogataea minuta.

When human antibody genes were expressed in the methylotrophic yeast Ogataea minuta, the secreted antibody became partially degraded. To suppress the degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the vacuolar protease-deficient strain, the secreted antibody still became partially degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains.

Distinct function of monoclonal antibody to TRAIL-R2 as potentiator or inhibitor of the ligand TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) specifically induces apoptosis in tumor cells but may be toxic to human hepatocytes. Although hepatocytes are susceptible to apoptotic signals mediated by TRAIL-receptor 2 (TRAIL-R2), we previously reported that some anti-TRAIL-R2 monoclonal antibodies (mAbs) produce little hepatocyte toxicity. Those mAbs neutralized the cytotoxic activity of TRAIL by inhibiting receptor-ligand binding. The hepatocyte-toxic mAbs did not compete with TRAIL for binding to TRAIL-R2, and potentiated ligand activity in both cancer cells and hepatocytes. A neutralizing antibody to TRAIL inhibited hepatocyte death by anti-TRAIL-R2 mAbs, suggesting that the toxicity may reflect their ability to potentiate membrane-bound TRAIL on hepatocytes.

Enhanced apoptosis and tumor regression induced by a direct agonist antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2.

PURPOSE: Substantial evidence indicates that supraoligomerization of the death receptors for Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is necessary for efficient activation of the apoptotic pathway. Bivalent IgG antibodies can induce the efficient apoptosis by mimicking the natural ligands but only after these antibodies are further oligomerized by cross-linking. In this study, we generated a novel agonist antibody to TRAIL receptor 2 (TRAIL-R2) capable of inducing apoptosis without cross-linking and elucidated its mode of action and efficacy. EXPERIMENTAL DESIGN: A fully human antibody to TRAIL-R2, KMTR2, was generated from KM Mouse immunized with TRAIL-R2 ectodomain. Apoptosis-inducing activities of unfractionated or purified monomeric IgG of KMTR2 was evaluated in the presence or absence of cross-linkers, secondary antibodies or Fc receptor-expressing effector cells, against human colorectal adenocarcinoma Colo205. Oligomerization of TRAIL-R2 was analyzed by size exclusion chromatography and confocal microscopy, and in vivo efficacy was examined in Colo205 xenograft model. RESULTS: KMTR2 specifically recognized TRAIL-R2 and induced apoptosis with or without cross-linking. Size exclusion chromatography showed that the apoptosis activity coeluted with monomeric IgG and was effective independent of secondary antibody or Fc receptor-expressing effector cells. The antibody formed supracomplexes with soluble recombinant and membrane-anchored TRAIL-R2 and enhanced clustering of TRAIL-R2 on cell surface without cross-linking. KMTR2 was dramatically efficacious in reducing established human tumor. CONCLUSION: Our findings indicate that novel agonist antibody KMTR2 can direct antibody-dependent oligomerization of TRAIL-R2 and initiates efficient apoptotic signaling and tumor regression independent of host effector function. Thus, the direct agonist would be a lead candidate for cancer therapeutics.

Essential role of bystander cytotoxic CD122+CD8+ T cells for the antitumor immunity induced in the liver of mice by alpha-galactosylceramide.

We recently reported that NK cells and CD8(+) T cells contribute to the antimetastatic effect in the liver induced by alpha-galactosylceramide (alpha-GalCer). In the present study, we further investigated how CD8(+) T cells contribute to the antimetastatic effect induced by alpha-GalCer. The injection of anti-CD8 Ab into mice 3 days before alpha-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-gamma production nor did it reduce the NK activity of liver mononuclear cells after alpha-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after alpha-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8(+) T cells did. CD122(+)CD8(+) T cells in the liver increased after alpha-GalCer injection, and antitumor cytotoxicity of CD8(+) T cells in the liver gradually increased until day 6. These CD8(+) T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122(+)CD8(+) T cells. The critical, but bystander role of CD122(+)CD8(+) T cells was further confirmed by adoptive transfer experiments into CD8(+) T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/alpha-GalCer injection for the mice to generate CD8(+) T cells that can reject s.c. rechallenged B16 cells. These findings suggest that alpha-GalCer activates bystander antitumor CD122(+)CD8(+) T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8(+) CTLs.

Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro.

We recently reported that the direct antitumor effectors in the liver induced by alpha-galactosylceramide (alpha-GalCer) are NK cells that are activated by the IFN-gamma produced from NK1.1 Ag(+) T cells (NKT cells) specifically stimulated with alpha-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the alpha-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-gamma and IL-4 concentrations as well as alanine aminotransferase levels after the alpha-GalCer injection increased in an age-dependent manner. An alpha-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of alpha-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-gamma and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1(+) cells. Furthermore, liver mononuclear cells from old mice stimulated with alpha-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most alpha-GalCer-injected old mice, but no young mice, died, while anti-IFN-gamma Ab pretreatment completely inhibited mouse mortality. However, alpha-GalCer-induced hepatic injury did not improve at all by anti-IFN-gamma Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-gamma is responsible for alpha-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-gamma.