Hisashi Takino

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.

Primary cutaneous marginal zone B-cell lymphoma: a molecular and clinicopathological study of cases from Asia, Germany, and the United States

Primary cutaneous marginal zone B-cell lymphoma is considered the cutaneous counterpart of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Although its molecular pathogenesis is currently unknown, an etiological link with Borrelia burgdorferi infection has been identified in European, but not in American or Asian cases. To better understand the pathogenesis and the geographical differences of cutaneous marginal zone B-cell lymphoma, 60 cases from the East Asia, Germany, and the United States at their initial presentation were subjected to the following analyses; (1) clinicopathological comparison between the geographical regions, (2) detection of B. burgdorferi DNA, (3) detection of the API2–MALT1 fusion transcript, a gene alteration specific to mucosa-associated lymphoid tissue lymphoma, and (4) inactivation of tumor suppressor genes (death-associated protein kinase (DAPK), p16INK4a, p14ARF, MGMT, TIMP3, CDH1, and RARB) by hypermethylation of the CpG islands. Cases from the three geographical regions showed similar clinicopathological features. However, moderate/marked tissue eosinophilia was found in 9/25 Asian cases, but only 1/23 German cases (P=0.011) and 0/12 American cases (P=0.015). All 60 cases were negative for either Borrelia DNA or API2–MALT1 fusion. Tumors from the three regions were highly methylated for DAPK (38–50% of the cases, mean 43%) and p16INK4a (42–70%, mean 49%), and the positivities were significantly higher than those of nonneoplastic skin (8%, P=0.0010 and 14%, P=0.0032, respectively). Methylation of these genes had no significant association with progressive features of the tumor. Primary cutaneous marginal zone B-cell lymphomas from the three geographical regions have common clinicopathological features, however, moderate/marked tissue eosinophilia is a feature found almost exclusively in Asian cases. Borrelia infection and API2–MALT1 fusion are not significant in this tumor. Methylation of DAPK and p16INK4a genes is a frequent event in this lymphoma at its initial presentation, but may not be associated with tumor progression.

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-scid, IL-2Rγnull Mice In Vivo

There is a lack of suitable small animal models to evaluate human Ab-dependent cellular cytotoxicity (ADCC) in vivo, because of the species incompatibility between humans and animals or due to nonspecific allogeneic immune reactions. To overcome these problems, we established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T cell leukemia/lymphoma (ATLL) cells and a therapeutic chimeric anti-CCR4 mAb, the Fc region of which is defucosylated to enhance ADCC. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in Ab-based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future.

Warthin-like Mucoepidermoid Carcinoma: A Combined Study of Fluorescence In Situ Hybridization and Whole-slide Imaging.

There has been some debate as to whether a subset of metaplastic Warthin tumors (mWTs) harbor the mucoepidermoid carcinoma (MEC)-associated CRTC1-MAML2 fusion. We analyzed 15 tumors originally diagnosed as mWT (mWT-like tumors), 2 of which had concurrent MECs. We looked for the CRTC1/3-MAML2 fusion transcripts and performed immunohistochemistry for p63 and fluorescence in situ hybridization (FISH) for the MAML2 split. To localize MAML2 split–positive cells at the cellular level, whole tumor tissue sections were digitalized (whole-slide imaging [WSI]). The CRTC1-MAML2, but not CRTC3-MAML2 was detected in 5/15 mWT-like tumors. FISH-WSI results showed that all epithelial cells harbored the MAML2 split in fusion-positive mWT-like tumors and were totally negative in fusion-negative mWT-like tumors. A review of the hematoxylin and eosin–stained slides showed that morphology of the “metaplastic” epithelium was virtually indistinguishable between fusion-positive and fusion-negative tumors. However, oncocytic bilayered tumor epithelium, characteristic to typical WT, was always found somewhere in the fusion-negative tumors but not in the fusion-positive tumors. This distinguishing histologic finding enabled 5 pathologists to easily differentiate the 2 tumor groups with 100% accuracy. The age and sex distribution of fusion-positive mWT-like tumor cases was similar to that of fusion-positive MEC cases and significantly different from those of fusion-negative mWT-like tumor and typical WT cases. In addition, only fusion-positive mWT-like tumors possessed concurrent low-grade MECs. In conclusion, a subset of mWT-like tumors were positive for the CRTC1-MAML2 fusion and had many features that are more in accord with MEC than with WT. The term Warthin-like MEC should be considered for fusion-positive mWT-like tumors.

p16/INK4a gene methylation is a frequent finding in pulmonary MALT lymphomas at diagnosis

p16/INK4a gene alterations have been associated with tumor progression in lymphoid malignancies. However, their significance in mucosa-associated lymphoid tissue (MALT) lymphoma is unclear. We investigated p16 gene methylation and mutation in a large series of untreated cases of pulmonary MALT lymphoma and diffuse large B-cell lymphoma (DLBL), and correlated p16 gene alterations with a MALT lymphoma-specific API2–MALT1 fusion and the clinicopathologic features of MALT lymphoma. The API2–MALT1 fusion was detected by multiplex reverse transcription polymerase chain reaction in 25/60 (42%) cases of MALT lymphoma, but none of 11 DLBLs. Methylation-sensitive single-strand conformation analysis showed that p16 gene methylation was frequently detected in 36/60 (60%) cases of MALT lymphoma. The gene was similarly methylated in DLBL cases (6/11, 55%). A p16 gene mutation was found in one (p16 gene-methylation) of 44 MALT lymphomas and in none of six diffuse large B-cell lymphomas. Statistical analysis showed that the p16 gene methylation status did not correlate with API2–MALT1 fusion or any of the clinicopathologic factors including serum LDH, clinical stage, and increased large cells. These findings suggest that p16 methylation is not associated with tumor progression, but may be an early event in MALT lymphomagenesis that might be maintained through the progression of the tumor.

Analysis of API2-MALT1 fusion, trisomies, and immunoglobulin VH genes in pulmonary mucosa-associated lymphoid tissue lymphoma.

Pulmonary mucosa-associated lymphoid tissue lymphoma is unique in that chronic inflammation is rare and that API2-MALT1 fusion, resulting from t(11;18)(q21;q21), occurs frequently. In this study, we examined 20 cases for API2-MALT1 fusion using the multiplex reverse-transcription polymerase chain reaction and looked for trisomy 3, trisomy 18, and abnormalities of MALT1 and IGH genes using fluorescence in situ hybridization. In addition, we analyzed VH genes by subcloning of the monoclonal polymerase chain reaction products. Of 20 cases studied, we detected gene abnormalities in 16: API2-MALT1 fusion in 9, trisomy 3 in 5, trisomy 18 in 4, MALT1 abnormality in 13, and IGH abnormality in 1. MALT1 gene abnormalities were concordant with API2-MALT1 fusion or trisomy 18. One case showed API2-MALT1 fusion and trisomy 3. On detection of API2-MALT1 fusion and trisomies, we were able to divide our cases into 3 groups, API2-MALT1 positive, trisomy positive, and no detectable gene abnormality, suggesting that tumor development had processed along different genetic pathways. All 20 cases were analyzed for VH genes. Most of the VH genes selected by the lymphomas belonged to the VH3 family, but there was no restriction to any particular VH fragment. Of interest, VH genes were unmutated in 7 cases, suggesting that T-cell-independent extrafollicular B-cell maturation may be important in the development of this lymphoma. In addition, both mutated and unmutated tumor cases were found to carry the API2-MALT1 fusion and trisomy 3. This observation suggests that these gene abnormalities may occur in microenvironments found before or outside of follicular germinal centers.

Prognostic Significance of Tryptophan Catabolism in Adult T-cell Leukemia/Lymphoma.

Purpose: Indoleamine 2,3-dioxygenase 1 (IDO1: IDO), an enzyme catabolizing tryptophan (Trp) into the kynurenine (Kyn) pathway, is increasingly being recognized as an important microenvironmental factor suppressing antitumor immune responses. The purpose of the present study was to determine the prognostic significance of Trp catabolism in adult T-cell leukemia/lymphoma (ATL). Experimental Design: We quantified serum Trp and Kyn in 96 ATL patients, 38 human T-cell lymphotropic virus type-1 asymptomatic carriers (HTLV-1 ACs), and 40 healthy adult volunteer controls. The relationships between various clinical parameters including overall survival were analyzed. IDO expression was evaluated in the affected lymph nodes of ATL patients. Results: Serum Kyn concentrations and Kyn/Trp ratios were significantly higher in HTLV-1 ACs than healthy controls. Both increased significantly with progression from HTLV-1 AC to ATL. However, there were no significant differences in the serum Trp concentrations between ATL patients, HTLV-1 ACs, and controls. IDO was possibly produced by ATL and/or cells of the microenvironment. Multivariate analyses demonstrated that a high serum Kyn/Trp ratio and high Kyn level, but not a high Trp level, were significantly independent detrimental prognostic factors in ATL, as well as in that subset of patients with aggressive variant ATL. Conclusions: Quantification of serum Kyn and Trp is useful for predicting prognosis of an individual ATL patient. Furthermore, ATL, especially in patients with a high serum Kyn/Trp ratio, is an appropriate disease for testing novel cancer immunotherapies targeting IDO. Clin Cancer Res; 21(12); 2830–9. ©2015 AACR.

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4+ cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL.

Forkhead box P1 overexpression and its clinicopathologic significance in peripheral T-cell lymphoma, not otherwise specified

Forkhead box P1 protein is a transcription factor involved in cell signaling and regulation of gene expression and is essential for B-cell development. Forkhead box P1 overexpression has been associated with a worsened prognosis in some B-cell lymphomas. However, little is known about the clinicopathologic significance of forkhead box P1 in T-cell malignancies. In this study, immunohistochemistry for forkhead box P1 was performed in peripheral T-cell lymphoma, not otherwise specified, cases (n = 41), which were then divided into lower (n = 15) and higher (n = 26) forkhead box P1 expressers. Results of real-time quantitative reverse transcriptase polymerase chain reaction for forkhead box P1 messenger RNA supported the data on immunohistochemical forkhead box P1 expression. Forkhead box P1 overexpression in lymphoma cells was inversely associated with proliferation activity as evaluated by Ki-67 expression. Double immunostain for forkhead box P1 and a T-cell marker in normal lymph nodes showed forkhead box P1 signals in many of nonneoplastic T cells. Prognostic analysis showed that forkhead box P1 overexpression was associated with an improved overall survival of the patients with peripheral T-cell lymphoma, not otherwise specified, and was independent of the International Prognostic Index in multivariate analysis. Forkhead box P1 overexpression may be associated with less activated phenotype of the tumors and with a better prognosis in patients with peripheral T-cell lymphoma, not otherwise specified. The clinicopathologic significance of forkhead box P1 overexpression in peripheral T-cell lymphoma, not otherwise specified, may be different from that in B-cell lymphomas.

A complement-dependent cytotoxicity-enhancing anti-CD20 antibody mediating potent antitumor activity in the humanized NOD/Shi-scid, IL-2Rγ(null) mouse lymphoma model.

Engineering the Fc region of monoclonal antibodies (mAb) in order to enhance effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC) is likely to a be promising approach for next-generation mAb therapy. Here, we report on such an antibody, 113F, a novel CDC-enhancing variant of rituximab, and determine the tumor-associated factors influencing susceptibility to 113F-induced CDC. The latter included the quantity of complement inhibitors present, such as CD55 and CD59. We report that compared to rituximab, 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells in vitro. Currently, a major problem in the field of immunotherapy research is the lack of suitable small animal models to evaluate human CDC in vivo. Therefore, we established a novel human tumor-bearing NOD/Shi-scid, IL-2Rγnull mouse model, in which human complement functions as the CDC mediator. We demonstrated that rituximab exerted significant antitumor effects via human CDC in this humanized mouse. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes was consistent with the observation that human CDC indeed contributed to the antitumor effect in this model. Moreover, 113F exerted significantly more potent antitumor effects than rituximab in this in vivo model. The detection of more abundant dense signals from C1q using 113F compared to rituximab was consistent with the concept that this reagent represented a CDC-enhancing mAb. In the near future, the efficacy of this type of CDC-enhancing antibody will be determined in clinical trials in humans.