Fumiko Mori

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.

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.

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.

Reactivation of hepatitis B virus in HBsAg-negative patients with multiple myeloma: two case reports

It was recently reported that hepatitis B virus (HBV) reactivation had occurred in HBsAg-negative lymphoma patients who received rituximab plus steroid combination chemotherapy. HBV reactivation in myeloma patients have not been reported extensively. We describe here two cases of HBV reactivation in HBsAg-negative myeloma patients receiving systemic chemotherapy: one from the medical records of 40 patients and another from 61 patients with prospective HBV-DNA monitoring. In the first case positive for anti-HBs, HBV reactivation was diagnosed when hepatitis developed during conventional chemotherapy such as MP and MCP regimen in a relapsed patient after autologous stem cell transplantation (APBSCT); in the second case positive for anti-HBc and anti-HBs, elevation of HBV-DNA was recognized by serial HBV-DNA monitoring performed prospectively following APBSCT. Interestingly, these two cases had the reduction of the titer of anti-HBs during the treatment, followed by HBV reactivation. These clinical data suggest that the HBV-DNA monitoring is necessary for not only HBsAg-positive but also HBsAg-negative myeloma patients with anti-HBc-positive and/or anti-HBs-positive following transplantation and after conventional chemotherapy in the salvage setting. Establishment of a standard strategy to prevent HBV reactivation is important for myeloma patients receiving systemic chemotherapy.

Tax is a potential molecular target for immunotherapy of adult T-cell leukemia/lymphoma

We expanded CTL specific for Tax (a human T‐lymphotropic virus type‐1‐encoded gene product) in vitro from PBMC of several adult T‐cell leukemia/lymphoma (ATL) patients, and document its potential significance as a target for ATL immunotherapy. Tax‐specific CTL responses against tumor cells were restricted by Tax‐expression and the appropriate human leukocyte antigen (HLA) type. Tax‐specific CTL recognized HLA/Tax‐peptide complexes on autologous ATL cells, even when their Tax expression was so low that it could only be detected by RT‐PCR but not by flow cytometry. Recognition resulted in interferon gamma (IFN‐γ) production and target cell lysis. This would be the first report that Tax‐specific CTL from ATL patients specifically recognized and killed autologous tumor cells that expressed Tax. The Tax‐specific CTL responded to as little as 0.01 pM of the corresponding peptide, indicating that their T‐cell receptor avidity was much higher than that of any other CTL recognizing viral or other tumor antigens. This is presumably the reason why the Tax‐specific CTL recognized and killed autologous ATL cells despite their very low Tax expression. In addition, cell cycle analyses and experiments with primary ATL cell‐bearing mice demonstrated that ATL cells present at the site of active cell proliferation, such as in the tumor masses, expressed substantial amounts of Tax, but it was minimally expressed by the tumor cells in a quiescent state, such as in the blood. The present study not only provides a strong rationale for exploiting Tax as a possible target for ATL immunotherapy but also contributes to our understanding of the immunopathogenesis of ATL.

Overexpression of carboxylesterase‐2 results in enhanced efficacy of topoisomerase I inhibitor, irinotecan (CPT‐11), for multiple myeloma

Multiple myeloma (MM) remains an incurable disease and further development of novel agents is needed. Because constitutive expression of topoisomerase I (TopoI) in MM cells and the efficacy of SN‐38, an active metabolite of irinotecan (CPT‐11), have been reported, we investigated the therapeutic potential of CPT‐11. Of the eight MM cell lines analyzed, four showed 50% inhibitory concentration values of less than 2 µg/mL for CPT‐11 and less than 2 ng/mL for SN‐38. This efficacy was partly explained by the high expression level of human carboxylesterase‐2 (hCE‐2) in MM cells. Interestingly, high expression of hCE‐2 represented the nature of normal plasma cells, suggesting that hCE‐2 could efficiently generate SN‐38 within the plasma cells. As expected, higher sensitivity to CPT‐11 was observed in hCE‐2‐overexpressing U266 cells than mock U266 cells. On the other hand, the expression levels of hCE‐1, TopoI, UGT1A and ABCG2 did not seem to be associated with the sensitivity of MM cells to CPT‐11. In a murine xenograft model inoculated s.c. with RPMI8226 cells, administration of CPT‐11 alone significantly reduced the tumor volume. When a combination of CPT‐11 and bortezomib was administered, the subcutaneous tumors completely disappeared. Thus, clinical trials on CPT‐11 in patients with relapsed or refractory MM are warranted. (Cancer Sci 2008; 99: 2309–2314)