Asahi Ito

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.

Bortezomib-resistant myeloma cell lines: a role for mutated PSMB5 in preventing the accumulation of unfolded proteins and fatal ER stress

Bortezomib is an effective agent for treating multiple myeloma (MM). To investigate the underlying mechanisms associated with acquired resistance to this agent, we established two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, the 50% inhibitory concentration values of which were respectively 24.7- and 16.6-fold higher than their parental cell lines. No activation of caspase and BH3-only proteins such as Noxa was noted in bortezomib-resistant cells after exposure to the drug. The accumulation of polyubiquitinated proteins was reduced in bortezomib-resistant cells compared with the parental cells, associated with avoidance of catastrophic ER stress as assessed by downregulation of CHOP expression. These resistant MM cells have a unique point mutation, G322A, in the gene encoding the proteasome β5 subunit (PSMB5), likely resulting in conformational changes to the bortezomib-binding pocket of this subunit. KMS-11 parental cells transfected to express mutated PSMB5 also showed reduced bortezomib-induced apoptosis compared with those expressing wild-type PSMB5 or the parental cells. Expression of mutated PSMB5 was associated with the prevention of the accumulation of unfolded proteins. Thus, a fraction of MM cells may acquire bortezomib resistance by suppressing apoptotic signals through the inhibition of unfolded protein accumulation and subsequent excessive ER stress by a mutation of the PSMB5 gene.

Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Stevens–Johnson Syndrome associated with mogamulizumab treatment of adult T-cell leukemia / lymphoma

We report an adult T‐cell leukemia/lymphoma patient suffering from Stevens–Johnson Syndrome (SJS) during mogamulizumab (humanized anti‐CCR4 monoclonal antibody) treatment. There was a durable significant reduction of the CD4+CD25highFOXP3+ regulatory T (Treg) cell subset in the patients PBMC, and the affected inflamed skin almost completely lacked FOXP3‐positive cells. This implies an association between reduction of the Treg subset by mogamulizimab and occurrence of SJS. The present case should contribute not only to our understanding of human pathology resulting from therapeutic depletion of Treg cells, but also alert us to the possibility of immune‐related severe adverse events such as SJS when using mogamulizumab. We are currently conducting a clinical trial of mogamulizumab for CCR4‐negative solid cancers (UMIN000010050), specifically aiming to deplete Treg cells.

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.

Cancer/testis antigens are novel targets of immunotherapy for adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an intractable hematologic malignancy caused by human T-lymphotropic virus type 1 (HTLV-1), which infects approximately 20 million people worldwide. Here, we have explored the possible expression of cancer/testis (CT) antigens by ATLL cells, as CT antigens are widely recognized as ideal targets of cancer immunotherapy against solid tumors. A high percentage (87.7%) of ATLL cases (n = 57) expressed CT antigens at the mRNA level: NY-ESO-1 (61.4%), MAGE-A3 (31.6%), and MAGE-A4 (61.4%). CT antigen expression was confirmed by immunohistochemistry. This contrasts with other types of lymphoma or leukemia, which scarcely express these CT antigens. Humoral immune responses, particularly against NY-ESO-1, were detected in 11.6% (5 of 43) and NY-ESO-1-specific CD8(+) T-cell responses were observed in 55.6% (5 of 9) of ATLL patients. NY-ESO-1-specific CD8(+) T cells recognized autologous ATLL cells and produced effector cytokines. Thus, ATLL cells characteristically express CT antigens and therefore vaccination with CT antigens can be an effective immunotherapy of ATLL.

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 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.

Autologous Tax-Specific CTL Therapy in a Primary Adult T Cell Leukemia/Lymphoma Cell–Bearing NOD/Shi-scid, IL-2Rγnull Mouse Model

We expanded human T-lymphotropic virus type 1 Tax-specific CTL in vitro from PBMC of three individual adult T cell leukemia/lymphoma (ATL) patients and assessed their therapeutic potential in an in vivo model using NOG mice bearing primary ATL cells from the respective three patients (ATL/NOG). In these mice established with cells from a chronic-type patient, treatment by i.p. injection of autologous Tax-CTL resulted in greater infiltration of CD8-positive T cells into each ATL lesion. This was associated with a significant decrease of ATL cell infiltration into blood, spleen, and liver. Tax-CTL treatment also significantly decreased human soluble IL-2R concentrations in the sera. In another group of ATL/NOG mice, Tax-CTL treatment led to a significant prolongation of survival time. These findings show that Tax-CTL can infiltrate the tumor site, recognize, and kill autologous ATL cells in mice in vivo. In ATL/NOG mice with cells from an acute-type patient, whose postchemotherapeutic remission continued for >18 mo, antitumor efficacy of adoptive Tax-CTL therapy was also observed. However, in ATL/NOG mice from a different acute-type patient, whose ATL relapsed after 6 mo of remission, no efficacy was observed. Thus, although the therapeutic effects were different for different ATL patients, to the best of our knowledge, this is the first report that adoptive therapy with Ag-specific CTL expanded from a cancer patient confers antitumor effects, leading to significant survival benefit for autologous primary cancer cell–bearing mice in vivo. The present study contributes to research on adoptive CTL therapy, which should be applicable to several types of cancer.