Hiroki Yano

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

PurposeThere are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials.Experimental designNOD/Shi-scid, IL-2Rγnull (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors.ResultsHumanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice.ConclusionsAnti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.

Regulatory T-cell function of adult T-cell leukemia/lymphoma cells.

Adult T‐cell leukemia/lymphoma (ATLL) patients are highly immunocompromised, but the underlying mechanism responsible for this state remains obscure. Recent studies demonstrated that FOXP3, which is a master control gene of naturally occurring regulatory T (Treg) cells, is expressed in the tumor cells from a subset of patients with ATLL. Since most ATLL cells express both CD4 and CD25, these tumors might originate from CD4+CD25+FOXP3+ Treg cells, based on their phenotypic characteristics. However, whether ATLL cells actually function as Treg cells has not yet been clearly demonstrated. Here, we show that ATLL cells from a subset of patients are not only hypo‐responsive to T‐cell receptor‐mediated activation, but also suppress the proliferation of autologous CD4+ non‐ATLL cells. Furthermore, ATLL cells from this subset of patients secrete only small amounts of IFN‐γ, and suppress IFN‐γ production by autologous CD4+ non‐ATLL cells. These are the first data showing that ATLL cells from a subset of patients function as Treg cells in an autologous setting. The present study provides novel insights into understanding the immunopathogenesis of ATLL, i.e., how HTLV‐1‐infected cells can survive in the face of host immune responses. It also adds to our understanding of ATLL patients severely immunocompromised state.

Bortezomib-induced apoptosis in mature T-cell lymphoma cells partially depends on upregulation of Noxa and functional repression of Mcl-1

Bortezomib, a proteasome inhibitor that was originally developed as an inhibitor of nuclear factor‐κB pathways, is currently used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). The mechanisms of action of this antitumor agent have been studied by several investigators. Here, we explore the underlying mechanisms of bortezomib‐induced apoptosis in cutaneous T‐cell lymphoma (CTCL) and adult T‐cell leukemia/lymphoma (ATLL) at the level of mitochondrial membrane injury. In all cell lines including (KMS‐12‐PE [MM], HUT78 [CTCL], ATN1 [ATLL], and MT4 [ATLL]), antiapoptotic factors such as c‐Flip and XIAP were downregulated after exposure to bortezomib, probably via inhibition of nuclear factor‐κB signaling. In addition, among the members of the BH3‐only family, upregulation of Noxa was consistently seen at both the transcriptional and protein levels in a p53‐independent manner after exposure to bortezomib. Repression of Noxa by small interfering RNA partially rescued CTCL and ATLL cells from bortezomib‐induced apoptosis. Immunoprecipitation assays indicated time‐dependent binding of Noxa and Mcl‐1 in all cell types, suggesting that functional repression of Mcl‐1 led to the loss of mitochondrial outer membrane potential. Similar results were also obtained in primary tumor cells from patients with ATLL. Taken together, we conclude that bortezomib‐induced apoptosis in ATLL and CTCL cells at least partly depends on the upregulation of Noxa and functional repression of Mcl‐1, as is also the case in MM and malignant melanoma. (Cancer Sci 2009; 100: 341–348)

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.

The Asn505 mutation of the c-MPL gene, which causes familial essential thrombocythemia, induces autonomous homodimerization of the c-Mpl protein due to strong amino acid polarity

We previously reported that a dominant-positive activating mutation (Asn505) in the transmembrane domain (TMD) of c-MPL, which encodes the thrombopoietin receptor, caused familial essential thrombocythemia. Here, we show that the Asn505 mutation induces both autonomous dimerization of c-Mpl and signal activation in the absence of its ligand. Signal activation was preserved in a truncated mutant of Asn505 that lacked the extracellular domain of c-MPL. We also found that the substitution of the amino acid (AA) residue at position 505 with others of strong polarity (Glu, Asp, or Gln) also resulted in activated dimerization without ligand stimulation. Overall, these data show that the Asn505 mutation transduced the signal through the autonomous dimerization of the c-MPL protein due to strong AA polarity. This finding provides a new insight into the mechanism of disease causation by mutations in the TMD of cytokine/hematopoietic receptors.

Expression of the ULBP ligands for NKG2D by B-NHL cells plays an important role in determining their susceptibility to rituximab-induced ADCC.

Antibody‐dependent cellular cytotoxicity (ADCC) is a major antitumor mechanism of action of therapeutic monoclonal antibodies (mAbs). The aim of this study was to identify tumor‐associated factors which determine susceptibility to rituximab‐induced ADCC. Thirty different CD20+ non‐Hodgkin lymphoma cell lines were phenotyped for characteristics such as level of expression of NKG2D ligands, and the influence thereof on susceptibility to rituximab‐induced ADCC was established. The present study demonstrated that tumor cell susceptibility to rituximab‐induced ADCC was determined by 3 major tumor‐associated factors: (i) the amount of the target molecule, CD20; (ii) the amount of the ligands for inhibitory killer Ig‐like receptors, major histocompatibility complex class I; and (iii) the amounts of some of the NKG2D ligands, especially UL16‐binding protein (ULBP) 1–3. The importance of the ULBPs was confirmed using antibody blockade. In conclusion, this is the first report to show the importance for rituximab‐induced ADCC of ULBPs expressed on tumor cells. The ULBPs could be valuable diagnostic biological markers and significant targets for immunotherapy to improve efficacy not only of rituximab but also of other therapeutic mAbs.

Augmentation of antitumour activity of defucosylated chimeric anti‐CCR4 monoclonal antibody in SCID mouse model of adult T‐cell leukaemia/lymphoma using G‐CSF

Adult T-cell leukaemia/lymphoma (ATLL) has a very poor prognosis (Uchiyama et al, 1977; Taylor & Matsuoka, 2005). Because tumour cells from most ATLL patients express CCR4 (Ishida et al, 2003), we postulated that this molecule might represent a novel molecular target for immunotherapy and developed a chimeric anti-CCR4 monoclonal antibody (mAb), KM2760 (Niwa et al, 2004). Antibody-dependent cellular cytotoxicity (ADCC) is one of the most important mechanisms of action of therapeutic mAb (Ishida & Ueda, 2006). Although ADCC depends on the cytotoxic activity of effector cells, such as natural killer (NK) cells, monocytes/macrophages and neutrophils, these cells are commonly qualitatively suppressed and quantitatively reduced in cancer patients. To overcome this problem, the Fc region of KM2760 is defucosylated, which results in enhanced ADCC (Niwa et al, 2004). We have previously reported that a robust KM2760-induced ADCC mediated by autologous effector cells were triggered in some ATLL patients (Ishida et al, 2004), whereas little autologous KM2760-induced ADCC was observed in other ATLL patients, especially those with few effector cells in their peripheral blood mononuclear cells (data not shown). These findings prompted us to seek ways in which to augment KM2760-induced ADCC using the immunomodulatory agent granulocyte colony-stimulating factor (G-CSF; Nartograstim, Kyowa Hakko Kogyo Incorporation, Tokyo, Japan), which is commonly used clinically. n nHuman G-CSF increased granulocyte counts in a dose-dependent manner in severe-combined immunodeficient (SCID) mice (Fig 1A). Subsequently, we evaluated KM2760-induced antitumour activity in the ATLL tumour-bearing mouse model with or without G-CSF in the disseminated setting. The ATLL cell line S-YU (Imada et al, 1996) was positive for CCR4 by flow cytometric analysis (data not shown). 1·5 × 107 S-YU cells were inoculated s.c. into SCID mice. The tumour-bearing mice were divided into four groups of five each for treatment with KM2760 or control (saline), KM2760 plus G-CSF or G-CSF alone, to ensure that the mean tumour volumes were same in each group. KM2760 (20 mg/kg) or saline injections into the tail veins of the mice were started 12 d after tumour inoculation, and continued weekly for 4 weeks. The significance of differences in the tumour volume among the groups was estimated by the Mann–Whitney U-test. In this study, P < 0·05 was considered significant. G-CSF (10 μg/mouse) was injected s.c. into SCID mice on days 0–6 and 14–20 from the start of KM2760 treatment. Mean tumour volumes in KM2760 recipients and control mice 27 d after the start of KM2760 treatment were 1174 ± 409 and 5015 ± 1033 mm3 respectively (P = 0·0090). KM2760 combined with G-CSF treatment yielded an even more robust antitumour effect, with all recipients achieving complete remission, i.e. subcutaneous S-YU tumours completely vanished. The difference between the tumour volume in animals treated with KM2760 plus G-CSF and those with KM2760 alone was highly significant (P = 0·0053) (Fig 1B and C). None of the mice in the present study showed evidence of toxicity that could be attributed to the KM2760 and/or the G-CSF injections. These findings indicate that human G-CSF significantly augments the antitumour activity of KM2760 in the ATLL tumour-bearing mouse model. n n n nFig 1 n nKM2760-induced potent antitumour activity in the Adult T-cell leukaemia/lymphoma (ATLL) tumour-bearing mouse model in the therapeutic setting (A) Total white blood cell, granulocyte and monocyte counts in blood of mice injected with 1, 3 or 10 μg ... n n n nNext, we explored the mechanisms whereby human G-CSF augments the anti-ATLL activity of KM2760 in vivo. Because KM2760 mediates only ADCC and not complement-dependent cytotoxicity or direct anti-proliferative activity (Ishida et al, 2004), it was hypothesized that G-CSF augmented KM2760-induced ADCC in vivo. Standard 4-h 51Cr-release assays were performed using pooled granulocytes from 14 G-CSF-injected or 15 naive SCID mice as effector cells (E) and S-YU cells as targets (T) at E/T ratios of 50:1, 100:1 and 200:1. However, KM2760 (10 μg/ml) failed to induce any ADCC against S-YU cells in the presence of granulocytes from either G-CSF-injected or naive SCID mice at any E/T ratio (data not shown). We next investigated the phagocytic activity of G-CSF-primed monocytes/macrophages, because we recently reported the importance of the innate monocyte/macrophage network, not the NK cell network, to deplete target cells through FcR-dependent pathways during antibody-based immunotherapy in mice (Uchida et al, 2004; Ishida et al, 2006). Monocytes/macrophages were prepared from peritoneal exudate cells of 14 SCID mice receiving 10 μg G-CSF twice (at 20 h and 5 h before testing) or 15 naive SCID mice. As S-YU does not express CD20, rituximab (Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) was used as a control mAb for KM2760. PKH26 (Sigma-Aldrich, St Louis, MO, USA) labelled S-YU (1 × 104) and pooled monocytes/macrophages from 14 G-CSF-injected or 15 naive SCID mice (5 × 104) were co-cultured with or without KM2760 or rituximab at a final concentration of 10 μg/ml for 1 h. The cells were stained with phycoerythrin cyanin 5 (PECy5)-conjugated anti-F4/80 mAb (Serotec Ltd, Oxford, UK), which specifically binds mouse monocytes/macrophages, and analysed by BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) as previously described (Ishida et al, 2006). The percentage of spontaneous, rituximab-induced, and KM2760-induced phagocytosis of S-YU mediated by pooled monocytes/macrophages from 15 naive SCID mice was 8·6%, 13·4% and 21·5% respectively, whereas these values from 14 G-CSF primed SCID mice were 19·2%, 17·8% and 30·6% respectively (Fig 2A and B). The formula used to calculate the percentage of phagocytosis of S-YU is shown in Fig 2C. These findings indicate that human G-CSF augments not only natural phagocytosis but also KM2760-induced phagocytosis of S-YU by mouse monocytes/macrophages, although the underlying mechanisms require further clarification. The data presented here are the first to address the potential G-CSF-induced augmentation of monocytes/macrophages in the antitumour activity of a therapeutic mAb in an in vivo animal model. n n n nFig 2 n nGranulocyte colony-stimulating factor (G-CSF)-enhanced KM2760-induced phagocytosis mediated by mouse monocytes/macrophages (A) Mouse monocytes/macrophages phagocytosis of S-YU cells in the presence or absence of KM2760. Rituximab was the control chimeric ... n n n nIn conclusion, the present study is the first to report that antitumour activity induced by next-generation defucosylated therapeutic mAb (Carter, 2006; Ishida et al, 2006) was strongly augmented by the immunomodulatory agent G-CSF in vivo. This finding has encouraged us to conduct of a phase-I clinical trial of the completely defucosylated anti-CCR4 mAb in patients with CCR4-positive T-cell lymphomas including ATLL (ClinicalTrials.gov Identifier: NCT00355472) to develop a novel treatment strategy for patients with ATLL. In the near future, the efficacy of combination treatments including anti-CCR4 mAb and immunomodulatory agents against refractory ATLL will be assessed in clinical trials.