Tomomi Sakai

B7‐H1 expression is regulated by MEK/ERK signaling pathway in anaplastic large cell lymphoma and Hodgkin lymphoma

B7‐H1 is a member of the B7 family that inhibits the function of T‐cells through its receptor programmed death‐1 (PD‐1). We examined B7‐H1 expression in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) and found that it was constitutively expressed in both clinical samples and cell lines. In anaplastic lymphoma kinase–positive (ALK+) ALCL cells, B7‐H1 expression was suppressed by the blocking of extracellular signal‐regulated kinase (ERK) signaling and upregulated by the augmentation of ERK activity by phorbol 13‐myristate 12‐acetate stimulation, suggesting that B7‐H1 expression is regulated by ERK signaling pathway in ALCL. ERK is one of the downstream mediators of nucleophosmin (NPM)/ALK signaling in ALK+ALCL, and pharmacological inhibition of ALK was shown to dephosphorylate ERK and down‐regulate B7‐H1. The involvement of NPM/ALK in B7‐H1 expression was also demonstrated by introducing the construct into human non‐ALCL lymphoid cell lines, which resulted in B7‐H1 expression. In the case of HL, B7‐H1 expression was shown to be dependent on the ERK and p38 mitogen‐activated protein kinase (MAPK) signaling pathways. These results suggest that B7‐H1 expression is controlled by common ERK signaling pathways in ALCL and HL cells. Our findings provide a potentially effective immunotherapeutic strategy for these B7‐H1‐expressing tumors. (Cancer Sci 2009)

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. Human 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. Fig 1 KM2760-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 ... Next, 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. Fig 2 Granulocyte 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 ... In 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.

Serological identification of adult T-cell leukaemia-associated antigens.

Adult T‐cell leukaemia (ATL) is a peripheral T‐cell neoplasm caused by human T‐cell leukaemia virus type I (HTLV‐I). Several clinical observations suggest that some tumour‐associated antigens in ATL may be recognised by the immune system. In this study, we performed the serological screening of an expression library to identify ATL‐associated antigens by using materials from a unique ATL patient with long‐term stable disease. Among five distinct genes isolated, serine/arginine protein kinase 1 (SRPK1), which has been reported to have a restricted normal tissue distribution, was found to be overexpressed in most acute type ATL samples, but not in chronic type ATL or in normal peripheral blood mononuclear cells by real‐time reverse transcription polymerase chain reaction. Interestingly, the overexpression of SRPK1 in aggressive types of ATL was more exclusively observed at the protein level than at the mRNA level. Autologous antibody to SRPK1 was confirmed in the ATL patient using Western blot analysis with plasma, but not detected in asymptomatic HTLV‐I carriers or in healthy volunteers. These results indicate that SRPK1 may be useful for the development of therapeutic and diagnostic methods for patients with ATL.

Identification of APOBEC3B as a potential target for the graft-versus-lymphoma effect by SEREX in a patient with mantle cell lymphoma.

Recently, a graft‐versus‐lymphoma (GVL) effect has been shown in patients allografted for mantle cell lymphoma (MCL), but the antigenic targets of this response remain unclear. We screened an MCL cDNA expression library with sera at GVL response after allogeneic haematopoietic stem cell transplantation and isolated five genes including APOBEC3B. Antibody responses to APOBEC3B were correlated with clinical response. Furthermore, APOBEC3B‐specific T‐cell response was induced with one of the HLA‐A*0201 binding peptides. In addition, APOBEC3B mRNA appeared to be upregulated in some MCL. These findings warrant further investigation of APOBEC3B as a candidate gene eliciting an effective immune response against MCL.

Antibody Responses Associated with the Graft-versus-Leukemia Effect in Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The prognosis of ATL, especially the acute and lymphoma subtypes, is poor with conventional and high-dose chemotherapy. The effectiveness of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for ATL has been reported, suggesting the presence of a graft-versus-leukemia (GVL) effect against this malignancy.To identify the target antigens associated with tumor rejection, we used SEREX (serological identification of antigens by recombinant cDNA expression cloning) to screen ATL complementary DNA expression libraries with sera from an ATL patient who had a GVL response after allo-HSCT. Among the isolated clones, autocrine motility factor receptor (AMFR), which encodes a glycosylated transmembrane protein, was found to have selective reactivity with the sera obtained during tumor regression. Real-time reverse transcription polymerase chain reaction analysis for AMFR showed highest expression in the testis among normal tissues. Furthermore, aberrant AMFR expression was found in at least some ATL patients.Taken together, these findings suggest that AMFR may be one of the GVL antigens that provoke effective antitumor immunity against ATL in allogeneic settings.

Distinctive cell properties of B cells carrying the BCL2 translocation and their potential roles in the development of lymphoma of germinal center type

The BCL2/IGH translocation is a hallmark of follicular lymphoma and germinal center B‐cell type diffuse large B‐cell lymphoma. Although a strong determinant of these histological subtypes, this translocation is insufficient by itself for lymphomagenesis, so that other genetic alterations are required. To clarify how the BCL2 translocation contributes to the development of specific lymphoma subtypes, we used chimeric mouse models and a bone marrow transplantation system to examine the biological features of BCL2‐overexpressing B cells. These cells showed a cell‐autonomous differentiation preference for follicular B cells. Their cell cycle progression was enhanced in wild‐type but not in Eμ‐BCL2 transgenic mice, indicating that the low proliferative activity of B cells in Eμ‐BCL2 transgenic mice is partly due to their specific microenvironment, which is caused by the abnormal B cells themselves. Moreover, in vitro experiments demonstrated that Eμ‐BCL2+ B cells have reduced responsiveness to terminal differentiation stimulation. According to these results, we hypothesize that B cells that have undergone BCL2/IGH translocation might possibly be forced to localize in follicles, and accumulate genetic abnormalities by being subjected to recurrent stimulation. Our findings lead us to propose that B cells carrying the BCL2/IGH translocation comprise a distinctive cell population that leads to the development of germinal center B‐cell type lymphoma. (Cancer Sci 2009; 100: 2361–2367)