ALBERT

Description: The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

Description: In acute-type leukemia, no method for the prediction of relapse following allogeneic stem cell transplantation based on minimal residual disease (MRD) levels is established yet. In the present study, MRD in 72 cases of allogeneic transplantation for acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia (accelerated phase or blast crisis) was monitored frequently by quantitating the transcript of WT1 gene, a “panleukemic MRD marker,” using reverse transcriptase–polymerase chain reaction. Based on the negativity of expression of chimeric genes, the background level of WT1 transcripts in bone marrow following allogeneic transplantation was significantly decreased compared with the level in healthy volunteers. The probability of relapse occurring within 40 days significantly increased step-by-step according to the increase in WT1 expression level (100% for 1.0 × 10−2-5.0 × 10−2, 44.4% for 4.0 × 10−3-1.0 × 10−2, 10.2% for 4.0 × 10−4-4.0 × 10−3, and 0.8% for 〈 4.0 × 10−4) when WT1 level in K562 was defined as 1.0). WT1 levels in patients having relapse increased exponentially with a constant doubling time. The doubling time of theWT1 level in patients for whom the discontinuation of immunosuppressive agents or donor leukocyte infusion was effective was significantly longer than that for patients in whom it was not (P 〈 .05). No patients with a short doubling time of WT1 transcripts (〈 13 days) responded to these immunomodulation therapies. These findings strongly suggest that the WT1 assay is very useful for the prediction and management of relapse following allogeneic stem cell transplantation regardless of the presence of chimeric gene markers.

Description: Introduction Allogeneic stem cell transplantation (allo-SCT) is a radical treatment for pediatric patients with relapsed and/or chemotherapy-resistant leukemia. Because these leukemia cells seem refractory to chemotherapy and irradiation, immunotherapy are needed. WT1 is highly expressed in pediatric leukemia and essential for leukemia cell growth, leading to the hypothesis that the WT1 protein-based immunization after allo-SCT may improve the clinical outcome of the SCT therapy. We investigated the clinical efficacy and immunological effect of immunotherapy targeting WT1 protein for pediatric patients after allo-SCT. Method Major inclusion criteria were as follows: Patients with HLA-A*2402 and/or HLA-*A0201 aged 20 years or younger and WT1 mRNA expression in leukemic cells; donors with HLA-A*2402 and/or HLA-*A0201. The HLA A*2402 restricted 9mer modified WT1 peptide or HLA A*0201 restricted natural WT1 peptide emulsified in Montanide ISA 51 adjuvant was injected intradermally. The dose of WT1 peptide depended on patient weight. The vaccinations were scheduled to be given weekly for 12 consecutive weeks, and if no recurrence and severe adverse effect were observed, vaccination was continued. Absolute number and frequency of WT1 specific cytotoxic T lymphocytes were analyzed by WT1 tetramer assay. Results A total of 18 patients with 9 AML/MDS, 8 ALL and 1 NHL were enrolled. Seven patients were those with high risk (HR) of relapse who had received SCT at 3rd CR or on disease. The other 11 patients were at standard risk (SR) but had either relapsed after SCT or shown induction failure. 16 had HLA-A*2402 and the other 2 had HLA-A*0201. 12 of the 18 patients received WT1 peptide vaccination monthly after the first 12-time consecutive vaccinations with one-week interval and are still in complete remission for 16-72 months (median 31 mo.). One patient discontinued the vaccination because of pancreatitis due to treatment with steroid for GVHD. One year probabilities of PFS is 66.7% in all, and it is notable that 4 of 7 HR patients are maintained in long-term CR. Although, of these 12 patients with persistence of CR, seven patients had high WT1 mRNA level before vaccination in peripheral blood (PB) or bone marrow (BM), WT1 mRNA levels decreased to within normal level after vaccination. WT1-specific CTLs in PB were detected and increased after vaccination in all cases and absolute numbers of these cells were significantly increased after vaccination in all cases (p〈 0.05). The fold increase of WT1-specific CTL numbers after the vaccination was significantly higher in cases which maintained CR (p〈 0.05). The WT1-specific CTLs with high tetramer staining were detected during the first 12-time vaccinations in 14 patients, and of these patients, 12 patients are still in CR. In the other 4 patients, only very few WT1 specific CTLs were detected, and three of the four patients showed recurrence. Five patients (1AMKL, 4 ALL) had recurrence. There was no significant difference in the fold increase of WT1-specific CTL numbers after the vaccination between cases with immunosuppressive agents for the prevention of GVHD and those without the agents. Discussion We reported the interim result of the phase II clinical study of WT1 peptide immunotherapy after SCT for pediatric patients, in which promising clinical efficacy of the therapy was suggested. In addition, we found that an increase in WT1-specific CTLs was associated with a decrease in WT1 mRNA, suggesting occurrence of WT1-driven graft versus leukemia (GVL) effect, which is consistent with strategies to enhance leukemia antigen-specific GVL response without deterioration of GVHD. The following three mechanisms are suggested. 1) After SCT, many kinds of cytokines are produced and proliferation of T lymphocytes occur; 2) lymphodepletion after SCT allows development of T cells specific to WT1 peptide; and resultantly, 3) efficient homeostatic expansion of WT1-specific CTLs is induced. Post-SCT vaccination with WT1 peptide can be a safe and effective strategy to prevent the recurrence of the disease without deterioration of GVHD in pediatric hematological malignancy. For avoiding late side effect due to preconditioning regimen and GVHD, effective post SCT immunotherapy is a promising strategy for pediatric patient. Larger studies are warranted on application of WT1 targeted immunotherapy to prevent recurrence after pediatric SCT Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3295 WT1 (Wilms' tumor gene 1) encodes a transcription factor, which regulates various types of genes and plays an important role in cell proliferation and differentiation. WT1 is highly expressed in leukemia and various types of solid tumors. In vivo immunization of mice with MHC class I-restricted 9-mer WT1 peptide elicited WT1-specific cytotoxic T lymphocytes (CTLs) and rejected WT1-expressing leukemia cells, demonstrating that WT1 is a tumor rejection antigen. Next, identification of human HLA class I-restricted WT1 peptides was performed, and WT1 235 (235-243 aa) and WT1 126 (126-134 aa) were determined as an HLA-A*2402- or HLA-A*0201- restricted WT1 peptide, respectively, by using in vitro WT1-specific CTL induction assay. On the basis of these fundamental experiments, we performed for the first time a phase I clinical trial of WT1 peptide immunotherapy with the approval of our ethical committee. A total of 26 patients with acute myeloid leukemia (AML), myelodysplastic syndrome, breast or lung cancer were intradermally injected with a HLA-A*2402-restricted natural or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0 or 3.0 mg at 2-week intervals. As shown in Table1, 12 AML patients were WT1-vaccinated and eight of the 12 AML patients were evaluable for clinical effect because of the existence of minimal residual disease (MRD) assessed by WT1 mRNA assay. Since four of the eight patients had obvious clinical response with the decrease in WT1 mRNA within three injections of WT1 vaccine, WT1 vaccination was repeated for them afterward (in the case that clinical response was not obvious within three injections of WT1 vaccine, WT1 vaccination was discontinued). However, one patient (No. 20) had re-increase in WT1 mRNA and WT1 vaccination was discontinued. The remaining three patients (Nos. 21, 23 and 24) have been repeatedly receiving WT1 vaccination (intervals were changed from 2 weeks to one month) over 7 years until now. WT1 mRNA levels, which directly reflected the amount of leukemic blast cells, were abnormally high and 102-103 copies/mg RNA in PB (normal level is 〈 50 copies/mg RNA) before WT1 vaccination in these three AML patients, showing that the patients were at molecular relapse with MRD. In nos. 21 and 23 patients, a decrease and increase in WT1 mRNA levels were repeated during early stages of WT1 vaccination. However, afterward, WT1 mRNA levels gradually decreased and reached normal level approximately three and a half years after the beginning of WT1 vaccination and have been maintained at normal level until now. In patient no. 24, high levels of WT1 mRNA (near 103 copies) gradually decreased by repeated WT1 vaccination and returned to normal level approximately 5 years after the beginning of WT1 vaccination although it was much delayed. Thus, these three patients had achieved continuous complete remission regardless of high risk for clinical relapse. WT1-specific immune responses were evaluated by WT1 tetramer assay. Frequencies of WT1-specific cytotoxic T lymphocytes (CTLs) (WT1 tetramer+ CD8+ T cells) in the patients were already higher before WT1 vaccination, compared to those in healthy volunteers, which was a frequent phenomenon in WT1-expressing tumor-bearing patients, but the WT1-specific CTLs were a IFN-g− inactivated form. After WT1 vaccination, a IFN- g+ activated from of WT1-specific CTLs appeared and was maintained until now. No adverse effects except local erythema and eruption at the injection sites of WT1 vaccine were observed regardless of repeated long-term WT1 vaccination over 7 years. These finding indicated that WT1-specific CTLs could attack WT1-expressing leukemic blast cells but ignored WT1-expressing normal cells such as podocytes of kidney and progenitor cells of various types of organs. In conclusion, we presented here the first report of promising clinical outcome of very long-term WT1 vaccination for AML patients with high risk for relapse. WT1 peptide immunotherapy should have good prospects as a cure-oriented therapy for AML and could be the first choice of the treatments for AML patients who had still MRD after induction chemotherapy, especially for elder patients who were intolerable to intensive chemotherapy. WT1 peptide immunotherapy may be also a better choice of treatments for AML patients who have high risk for relapse but are intolerable to allogeneic hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: AML1-ETO, a chimeric gene frequently detected in acute myelogenous leukemia (AML), inhibits the differentiation of myeloid progenitors by suppressing genes associated with myeloid differentiation and increases the replating ability of clonogenic myeloid progenitors. However, AML1-ETO alone cannot induce AML and thus additional genetic events are required for the onset of AML. The Wilms tumor gene (WT1), which has been identified as the gene responsible for Wilms tumor, is expressed at high levels in almost all human leukemias. In this study, we have generated transgenic mice (WT1-Tg) that overexpress WT1 in hematopoietic cells to investigate the effects of WT1 on AML1-ETO-associated leukemogenesis. AML1-ETO-transduced bone marrow (BM) cells from WT1-Tg mice exhibited inhibition of myeloid differentiation at more immature stages and higher in vitro colony-forming ability compared with AML1-ETO-transduced BM cells from wild-type mice. Most importantly, all of the mice that received a transplant of AML1-ETO-transduced BM cells from the WT1-Tg mice rapidly developed AML. These results demonstrate that AML1-ETO may exert its leukemogenic function in cooperation with the expression of WT1.

Description: Abstract 16 Immunization using tumor antigen-loaded dendritic cells (DC) holds promise for the adjuvant treatment of cancer to control residual disease. In a phase I/II trial, we investigated the effect of autologous DC vaccination in 17 patients with acute myeloid leukemia (AML) in remission but at high risk of full relapse. Wilms’ tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two out of 3 patients, who were in partial remission with morphologically demonstrable disease after chemotherapy, were brought into complete remission following 4 biweekly intradermal injections of WT1 mRNA-electroporated DC. In those 2 patients as well as in 7 other patients who were in complete remission but who had molecularly demonstrable residual disease, there was a return to normal of the AML-associated WT1 mRNA tumor marker following DC vaccination, compatible with clinical and molecular response in 9/17 patients. In 3 patients, the WT1 mRNA tumor marker returned to pathological values following normalization after initial DC vaccination and additional injections of DC were needed to bring back the tumor marker to normal. Of the 9 responders, 3 have relapsed and 2 have died. Of the 8 non-responders, 7 have relapsed and 7 have died. Of the 2 patients in partial remission who were brought into complete remission by the DC vaccination, 1 has relapsed and has died. Median overall survival was 52.0 months in responders as compared to 6.0 months in non-responders (P=0.0007). Median relapse-free survival was 47.0 months in responders and 3.0 months in non-responders (P smaller than 0.0001). Immunomonitoring performed on the first 10 patients, showed a significant increase in WT1-specific interferon-gamma+ CD8+ T cells and signs of general immune stimulation, such as a significant increase of plasma levels of interleukin 2 and of HLA-DR+ CD4+ T-cells. Clinical responses were correlated with elevated levels of activated natural killer cells post-vaccination. Long-term clinical responses, lasting for at least 3 years, were significantly correlated with an increase in polyepitope WT1-specific tetramer+ CD8+ T-cell frequencies. There was no significant change post-vaccination in WT1 antibody levels or of regulatory T lymphocytes. In conclusion, DC-based immunotherapy elicits both innate and adaptive cellular immune responses correlated with clinical benefit. WT1 mRNA-loaded DC emerge as a feasible and effective strategy to control residual disease in AML, in particular as a post-remission treatment to prevent full relapse. Disclosures: Berneman: Argos Therapeutics: Patents & Royalties. Van Tendeloo:Argos Therapeutics: Patents & Royalties.

Description: Cancer-specific cell surface antigens are ideal targets for therapies using monoclonal antibodies (mAbs) and their derivatives, such as chimeric antigen receptor (CAR)-T cells. However, such antigens are not likely to remain unidentified following extensive searching by transcriptome or proteome analyses. However, we hypothesized that cancer-specific antigens formed by post-translational events, such as glycosylation, complex formation, or conformational changes, might have been missed in previous screens. Such antigens could be discovered by thoroughly searching for cancer-specific mAbs and characterizing the antigens recognized by these mAbs. To test our hypothesis, we applied this strategy to identify novel therapeutic targets specific for multiple myeloma (MM), a major hematological cancer. We first identified two MM-specific mAbs designated as MMG49 or R8H283 after screening more than 10,000 anti-MM mAb clones. Then, we identified the antigens recognized by these mAbs by an expression cloning method. Interestingly, genes identified as antigens for both mAbs were not specific to MM cells, suggesting that both mAbs recognize MM-specific epitopes formed by post-translational events. Finally, we showed that these mAbs or CAR-T cells derived from them could reduce tumor burden in MM-xenograft models, but did not damage normal hematopoietic cells. These results not only demonstrate that these mAb or CAR-T cell therapy is promising for MM, but also suggest that MM-specific immunotherapetic target antigens formed by post translational events may be still missed. Disclosures Aoyama: Alexion: Honoraria.