Kana Hasegawa

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19+ clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19+ B cells. We found that in vitro MM colony-forming cells were enriched in CD138−CD19−CD38++ plasma cells, while CD19+ B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138−CD19−CD38++ plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19+ B cells was detected. In 4 out of 9 cases, CD138+ plasma cells propagated MM, although more slowly than CD138− cells. Finally, we transplanted CD19+ B cells from 13 MM patients into NOD/SCID IL2Rγc−/− mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138− and CD138+ cells have the potential to propagate MM clones in vivo in the absence of CD19+ B cells.

Inhibitory Roles of Signal Transducer and Activator of Transcription 3 in Antitumor Immunity during Carcinogen-Induced Lung Tumorigenesis

Stat3 mediates a complex spectrum of cellular responses, including inflammation, cell proliferation, and apoptosis. Although evidence exists in support of a positive role for Stat3 in cancer, its role has remained somewhat controversial because of insufficient study of how its genetic deletion may affect carcinogenesis in various tissues. In this study, we show using epithelium-specific knockout mice (Stat3(Δ/Δ)) that Stat3 blunts rather than supports antitumor immunity in carcinogen-induced lung tumorigenesis. Although Stat3(Δ/Δ) mice did not show any lung defects in terms of proliferation, apoptosis, or angiogenesis, they exhibited reduced urethane-induced tumorigenesis and increased antitumor inflammation and natural killer (NK) cell immunity. Comparative microarray analysis revealed an increase in Stat3(Δ/Δ) tumors in proinflammatory chemokine production and a decrease in MHC class I antigen expression associated with NK cell recognition. Consistent with these findings, human non-small cell lung cancer (NSCLC) cells in which Stat3 was silenced displayed an enhancement of proinflammatory chemokine production, reduced expression of MHC class I antigen, and increased susceptibility to NK cell-mediated cytotoxicity. In addition, supernatants from Stat3-silenced NSCLC cells promoted monocyte migration. Collectively, our findings argue that Stat3 exerts an inhibitory effect on antitumor NK cell immunity in the setting of carcinogen-induced tumorigenesis.

Maintenance of complete remission after allogeneic stem cell transplantation in leukemia patients treated with Wilms tumor 1 peptide vaccine

The prognosis of patients after allogeneic hematopoietic stem cell transplantation (HSCT) is still not satisfactory because, while treatment-related mortalities have decreased, relapse after HSCT remains a major concern. The effectiveness of allogeneic HSCT for hematological malignancies is the result of immunologic rejection of recipient leukemia cells by donor T cells, known as the graft-versus-leukemia (GVL) effect.1 It is thus obviously important to be able to exploit the GVL effect while minimizing graft-versus-host disease (GVHD). A targeted anti-leukemic immunotherapy, such as use of a leukemia vaccine,2 is a promising strategy to boost the GVL effect. Wilms tumor1 (WT1) protein is one of the best targets for leukemia vaccines. Overexpression of the wild-type WT1 gene has been detected in all types of human leukemia.3, 4, 5 We performed a phase I clinical study of immunotherapy targeting the WT1 protein in patients with leukemia, and were able to show that WT1 vaccination was safe and could induce WT1-specific cytotoxic T lymphocyte (CTL).6 Furthermore, reduction of minimal residual disease and long-lasting complete remission (CR) was observed in some leukemia patients who were given the WT1 vaccine.7 This report presents the results of phase I clinical study of WT1 vaccination for HLA-A*2402-positivie post-HSCT patients who were at high risk of relapse (HSCT in non-CR and 2nd HSCT for post-transplant relapse) or had already relapsed. The HLA-A*2402-restricted modified 9-mer WT1 peptide (amino acids 235–243 CYTWNQMNL)8 was emulsified with Montanide ISA51 adjuvant. Patients were intradermally injected with 1.0 mg (three patients: UPNs 1, 4 and 6) or 3.0 mg (other six patients) of WT1 peptide four times weekly. When no adverse effects and no obvious disease progression were observed after the fourth injection, further WT1 vaccinations at 2-week intervals were administered. Nine patients (five with acute myeloid leukemia (AML), one each with acute lymphoblastic leukemia, chronic myelomonocytic leukemia, multiple myeloma and T-cell lymphoblastic lymphoma) were enrolled in this study (Supplementary Tables 1 and 2). Local inflammatory response was observed at the vaccine injection sites of all patients. One patient (UPN5) suffered mild hypoxia (PaO2 65 mm Hg at room air) and restrictive pulmonary dysfunction (FEV1.0 40%) 65 days after the start of WT1 vaccination (day 199 after HSCT; Figure 1a). He was diagnosed with bronchioleitis obliterans (BO), which was a symptom of chronic GVHD. The patient recovered soon after administration of inhaled steroids. While early and sudden discontinuation of prednisolone and tacrolimus (day 103 after HSCT) were considered to be the reason for development of BO, the possibility of an association between BO and WT1 vaccination cannot be entirely ruled out. In other eight patients, no severe toxicities related to WT1 vaccine were observed (Table1). Figure 1 Clinical course of patients who attained CR after the start of WT1 peptide vaccination. (a) Clinical course of UPN5 who achieved CR after administration of WT1 vaccine but stopped vaccination because of the development of bronchioleitis obliterans. ( ... Table 1 Patient outcomes Three AML patients (UPN1–3), who had undergone HSCT in non-CR, started WT1 vaccine in CR (Supplementary Tables 1 and 2). They started WT1 vaccination on post-HSCT days 141, 76 and 93 and have remained in CR for 1038, 973 and 662 days, respectively (as of 8 April 2013; Table1), suggesting the potential of WT1 vaccination as a maintenance therapy after HSCT. Six patients started WT1 vaccination in non-CR and two of them became CR after WT1 vaccination. One B-ALL patient (UPN4) with MLL-AF4 underwent bone marrow transplantation from an HLA-matched unrelated donor during the first CR. On post-HSCT day 111, MLL-AF4 and WT1 mRNA in peripheral blood (PB) had increased to 16 000 and 15 000 copies/μg RNA, indicating that the disease had relapsed. Tacrolimus and prednisolone doses were tapered off to induce GVL effects. The expression levels of MLL-AF4 and WT1 mRNA in PB had decreased to 2700 and 190 copies/μg RNA by day 132, and WT1 vaccination was started on day 133. MLL-AF4 mRNA had become undetectable by day 146, and had never appeared until post-HSCT day 1312 (day 1179 after the start of WT1 vaccination as of 8 April 2013; Figure 1b). Skin tumors appeared in UPN5 (AML-M5) on post-HSCT day 103 and was diagnosed by biopsy as leukemia relapse. Tacrolimus was discontinued on day103, and WT1 vaccination was started on day 130. Cutaneous tumors had regressed 2 weeks after the start of WT1 vaccination, but vaccination was terminated after the second injection because of the development of BO as described earlier (Figure 1a). This patient has been remained in CR until post-HSCT day 972 (day 842 after the start of WT1 vaccination at 8 April 2013). While the exact contribution of the vaccination effect to the disease remission in addition to the GVL effect was unclear, the fact that both of these two patients still have remained in CR until now is encouraging to continue this trial. In the following phase II trials, the enumeration of WT1-specific CTLs should be performed more frequently after the start of vaccination to clarify the relationship between the effect of WT1 peptide vaccination and leukemia regression. WT1 (a natural 9-mer WT1 peptide) HLA-A*2402 tetramer assays could be performed with peripheral blood mononuclear cell in seven of the nine patients to determine whether WT1235 peptide-specific CD8+ T cells had increased after WT1 vaccination. The gates for WT1 tetramer+ cells were drawn as <0.1% of CD8+ T cells were included in the tetramer-positive gate in multiple healthy individuals (Supplementary Figure 1A). WT1235 tetramer+ cells increased after the start of vaccination in three (UPNs1, 2 and 4) of the four patients who have remained in CR (Figure 1b and Supplementary Figure 1B). In the cases with progressive disease, continuous increase in the frequencies of WT1235 tetramer+ cells was not observed (Supplementary Figure 1B). Our results suggest that WT1 vaccination should be started when the leukemia burden is minimal. The timing of the start of WT1 vaccination may be also important. For the cases with good outcomes, WT1 vaccination was started 76–140 days after transplantation (UPNs1–5), and at later times (days 299–1815) for PD cases (UPNs 6–9). A lymphopenic environment a few months after transplantation may be favorable for rapid and extensive expansion of tumor antigen-specific CTLs. In summary, this report suggests that WT1 vaccine can be safely administrated for post-HSCT patients with hematological malignancies and has potential as a maintenance therapy. Clinical benefit of WT1 vaccination for post-HSCT patients will be evaluated in the subsequent phase II trials.

The translation elongation factor eEF2 is a novel tumor‑associated antigen overexpressed in various types of cancers

Recent studies have shown that cancer immunotherapy could be a promising therapeutic approach for the treatment of cancer. In the present study, to identify novel tumor-associated antigens (TAAs), the proteins expressed in a panel of cancer cells were serologically screened by immunoblot analysis and the eukaryotic elongation factor 2 (eEF2) was identified as an antigen that was recognized by IgG autoantibody in sera from a group of patients with head and neck squamous cell carcinoma (HNSCC) or colon cancer. Enzyme-linked immunosorbent assay showed that serum eEF2 IgG Ab levels were significantly higher in colorectal and gastric cancer patients compared to healthy individuals. Immunohistochemistry experiments showed that the eEF2 protein was overexpressed in the majority of lung, esophageal, pancreatic, breast and prostate cancers, HNSCC, glioblastoma multiforme and non-Hodgkin’s lymphoma (NHL). Knockdown of eEF2 by short hairpin RNA (shRNA) significantly inhibited the growth in four eEF2-expressing cell lines, PC14 lung cancer, PCI6 pancreatic cancer, HT1080 fibrosarcoma and A172 glioblastoma cells, but not in eEF2-undetectable MCF7 cells. Furthermore, eEF2-derived 9-mer peptides, EF786 (eEF2 786–794 aa) and EF292 (eEF2 292–300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. These results indicated that the eEF2 gene is overexpressed in the majority of several types of cancers and plays an oncogenic role in cancer cell growth. Moreover, the eEF2 gene product is immunogenic and a promising target molecule of cancer immunotherapy for several types of cancers.

The activated conformation of integrin β7 is a novel multiple myeloma-specific target for CAR T cell therapy

Cancer-specific cell-surface antigens are ideal targets for monoclonal antibody (mAb)-based immunotherapy but are likely to have previously been identified in transcriptome or proteome analyses. Here, we show that the active conformer of an integrin can serve as a specific therapeutic target for multiple myeloma (MM). We screened >10,000 anti-MM mAb clones and identified MMG49 as an MM-specific mAb specifically recognizing a subset of integrin β7 molecules. The MMG49 epitope, in the N-terminal region of the β7 chain, is predicted to be inaccessible in the resting integrin conformer but exposed in the active conformation. Elevated expression and constitutive activation of integrin β7 conferred high MMG49 reactivity on MM cells, whereas MMG49 binding was scarcely detectable in other cell types including normal integrin β7+ lymphocytes. T cells transduced with MMG49-derived chimeric antigen receptor (CAR) exerted anti-MM effects without damaging normal hematopoietic cells. Thus, MMG49 CAR T cell therapy is promising for MM, and a receptor protein with a rare but physiologically relevant conformation can serve as a cancer immunotherapy target.

In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2−/−γc−/− mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2−/− mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2−/− recipients and inhibited by transfer of NK cells into Rag2−/−γc−/− recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2−/− recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells

Leukemia differs substantially with respect to stromal milieu from tumors that progress locally as solid masses, and the physiological importance of immunosurveillance in leukemia remains unclear. However, currently available mouse leukemia models have critical limitations in the context of analyzing immunological regulation of leukemia development. In this study, we transferred mouse MLL/AF9 leukemia-initiating cells into immunocompetent recipient mice without any pre-conditioning such as irradiation, and then analyzed the spontaneous T cell response to an immunogenic antigen expressed in leukemia cells. When the minimum numbers of leukemia-initiating cells for engraftment were transferred, leukemia cells were eradicated by the adaptive immune response in most, if not all, wild-type mice, but not in Rag2 -/- recipient mice, which lack adaptive immunity. By contrast, mice transplanted with larger numbers of leukemia cells always developed leukemia. In mice with advanced leukemia, antigen-specific CTLs were also expanded, but were unresponsive to antigen stimulation and expressed high levels of PD-1 and LAG-3. These results provide the first clear demonstration that the spontaneous CTL response to a tumor-cell antigen has the potential to eradicate leukemia, whereas antigen-specific CTLs are exhausted in animals with advanced leukemia. This immunocompetent mouse leukemia model provides a useful platform for developing effective immunotherapies against leukemia.

Wilms tumour 1 peptide vaccine as a cure-oriented post-chemotherapy strategy for patients with acute myeloid leukaemia at high risk of relapse

Sally B. Killick Nick Bown Jamie Cavenagh Inderjeet Dokal Theodora Foukaneli Peter Hillmen Robin Ireland Austin Kulasekararaj Ghulam Mufti John A Snowden Sujith Samarasinghe Anna Wood Judith C.W. Marsh on behalf of the British Society for Standards in Haematology AA Guidelines Committee The Royal Bournemouth and Christchurch Hospitals NHS Foundation Trust, Bournemouth, Northern Genetics Service, Newcastle upon Tyne, St Bartholomew’s Hospital, Barts Health NHS Trust, Barts and The London School of Medicine and Dentistry, Queen Mary University of London and Barts Health NHS Trust, London, Addenbrooks Hospital, University of Cambridge, Cambridge, Leeds Teaching Hospitals, Leeds, Kings College Hospital NHS Foundation Trust, London, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, Great Ormond Street Hospital for Children NHS Foundation Trust, London, and West Hertfordshire NHS Trust, Watford, UK. E-mail: sally.killick@rbch.nhs.uk

WT1 peptide-based immunotherapy for advanced thymic epithelial malignancies: WT1 peptide vaccine for thymic malignancies

Thymic epithelial tumors are rare malignancies, and no optimal therapeutic regimen has been defined for patients with advanced disease. Patients with advanced thymic epithelial tumors, which were resistant or intolerable to prior therapies, were eligible for this study. Patients received 9 mer‐WT1‐derived peptide emulsified with Montanide ISA51 adjuvant via intradermal administration once a week as a monotherapy. After the 3‐month‐protocol treatment, the treatment was continued mostly at intervals of 2–4 weeks until disease progression or intolerable adverse events occurred. Of the 15 patients enrolled, 11 had thymic carcinoma (TC) and 4 had invasive thymoma (IT). Median period from diagnosis to the start of treatment was 13.3 and 65.5 months for TC and IT, respectively. No patients achieved a complete or partial response. Of the 8 evaluable TC patients, 6 (75.0%) had stable disease (SD) and 2 had progressive disease (PD). Of the 4 evaluable IT patients, 3 (75.0%) had SD and 1 (25.0%) had PD. Median period of monotherapy treatment was 133 and 683 days in TC and IT patients, respectively. No severe adverse events occurred during the 3‐month‐protocol treatment. As adverse events in long responders, thymoma‐related autoimmune complications, pure red cell aplasia and myasthenia gravis occurred in two IT patients. Cerebellar hemorrhage developed in a TC patient complicated with Von Willebrand disease. Induction of WT1‐specific immune responses was observed in the majority of the patients. WT1 peptide vaccine immunotherapy may have antitumor potential against thymic malignancies.

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54high or B2high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.