ALBERT

Description: The transcription factor Wilms tumor protein (WT) 1 belongs to a new generation of tumor antigens, which are essential for tumor cell proliferation. WT1 is highly expressed in AML and in MDS upon appearance of blasts. A phase II trial of vaccination with the HLA-A2-restricted WT1.126–134 peptide was performed in patients with AML and MDS and overexpression of WT1 to determine immunogenicity and clinical activity. Patients received vaccinations with 0.2 mg WT1.126–134 peptide (day 3), 62.5 mcg dendritic cell-stimulating adjuvant GM-CSF (days 1–4) and 1 mg T helper protein keyhole limpet hemocyanin (day 3). The initial 13 patients were to receive 4 biweekly and subsequent 4-weekly vaccinations, the subsequent 13 patients were continuously vaccinated biweekly. Vaccination was continued in absence of overt disease progression. WT1 levels were assessed by quantitative RT-PCR and WT1-specific T cell responses by tetramer analyses and cytokine flow cytometry. Response assessment following IWG-MDS criteria was used, capturing stable disease and hematologic improvement. A duration of 8 weeks was required for stable disease. Enrolment was completed in June 2006 with 24 patients with AML and 2 with MDS (RAEB). Of the 24 AML patients, 16 had 〉 5% marrow blasts at study onset (8 without prior chemotherapy, 4 with disease persistence following chemotherapy, 4 with PR), and 8 were in CR at high risk for relapse. A median of 10 (range 4 – 23) vaccinations was administered with 8 patients currently still under treatment. No significant toxicity occurred. To date, 22 patients are evaluable for clinical response. Overall, 8/16 patients with 〉 5% marrow blasts at study onset displayed clinical efficacy of vaccine treatment (SD or better). One AML patient achieved CR for 12 months after brief initial progression, and 7 patients had disease stabilization (2, 2+, 3, 3, 6, 10+, 14 months). One of these patients with RAEBII had a major response of neutrophils and platelets, and one AML patient had initial progression and subsequent transient complete clearance of peripheral blasts. WT1 transcripts as molecular disease marker decreased at least 3-fold (range 3-fold - 〉50-fold, median 〉10-fold) in 12 of 20 currently evaluated patients, including all 8 patients with evidence of clinical efficacy and 4 of 5 AML patients vaccinated in CR. The generation of a WT1-specific T cell response in peripheral blood and bone marrow was detected in 12 of 16 evaluated patients including all 6 of these 16 patients with evidence for clinical efficacy. This study shows that WT1 vaccination has promising antileukemia activity. A multicenter comparative WT1 vaccination study in CR patients at high risk of relapse is currently initiated.

Description: Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore, 100 bone marrow samples from patients with acute lymphoblastic leukemia (ALL) were cultured in the presence of TNFα, GM-CSF, c-kit ligand and FLT3 ligand with or without interleukin-3 and –4, and after administration of the HdI valproic acid (VAL), suberoylanilide hydroxamic acid (SAHA), isobutyramid or trichostatin A. Twenty-six of these ALL samples were positive for the t(12;21) translocation encoding the fusion gene TEL/AML1. SAHA increased CD83 expression of TEL/AML1- positive blasts in conditions without interleukins (36±6.5% versus 26.3±7.1%, p〈 0.05), while SAHA and VAL increased the number of CD86(+)80(−) cells in the presence of interleukins (12.5±2.8 ×104 versus 6.3±1.1 ×104 cells). VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive leukemic DC (48.5±6.4 ×103cpm and 50.3±7.1 ×103cpm) and reduced those of TEL/AML1-negative DC (41.8±6.8 ×103 cpm versus 52.8±11.2 ×103cpm, p= 0.05). Cytotoxic T-cells sensitized with leukemic DC produced more INFγ and TNFα upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of non-differentiated blasts, which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. In conclusion, the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended.

Description: Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2–positive AML patients by interferon-γ (IFN-γ) ELISPOT assay and flow cytometry for intracellular IFN-γ and in 3 additional patients by flow cytometry only. T cells producing IFN-γ in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3+CD8+CD45RA+ CCR7−T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B+. These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.

Description: The induction of a graft versus leukaemia (GvL) effect following allogenic hematopoietic stem cell transplantation (HSCT) is critical for the outcome in patients with hematological malignancies. Tumor-specific antigens as WT-1 are expressed on leukemic blasts and are major targets of the GvL effect. Therefore the generation of a WT-1 specific immune response through peptide vaccination could enhance the antileukemic effects after HSCT. In a phase I study five HLA A*0201 positive patients with high-risk AML (median age 64 yrs, range 33–67yrs) were vaccinated with the WT-1 derived peptide RMFPNAPYL (126–134) beginning at day 21 after HSCT. The vaccination protocol consisted in four biweekly vaccinations with the peptide in a dose of 0,2mg administered i.d. and s.c. with 1mg of keyhole limpet hemocyanin (KLH) as adjuvant. Concomitantly, patients received daily doses of GM-CSF (75μg/d) for four days beginning two days before vaccination. After the first four cycles, vaccine was administered monthly. When patients showed signs of GvHD or infection vaccination was discontinued. WT-1-specific T cell responses were monitored in peripheral blood using tetramer analysis. No severe acute toxicity attributable to the vaccination was observed. In all patients a local inflammatory response at the site of injection was detected, which resolved fast after ending of the vaccination cycle. In some patients a systemic inflammatory response with fever and increase of the leukocyte count were observed, which also resolved a few days after vaccination. Three patients achieved a complete remission of the AML after transplantation and did not relapse. One patient developed an intraneural relapse of the AML in the N. medianus of the left arm, which was successfully treated by irradiation. Though, she did not show a relapse in the bone marrow and maintains a full donor chimerism in bone marrow and peripheral blood 600 days after transplantation. One patient relapsed and died four months after transplantation. Two patients developed a grade I GvHD of the skin shortly after the first vaccination. Therefore vaccination was discontinued and resumed two weeks after resolution of the GvHD. No GvHD signs were observed after the subsequent vaccination cycles. One patient developed a grade III GvHD of the skin and gut after the 3rd vaccination. In this patient GvHD proved to be resistant to the common immunosuppressive agents and the patients died of septicaemia four months after transplant. One patient developed a three-system grade IV GvHD after the 2nd vaccination. Vaccination was then resumed after resolution of the GvHD. He unfortunately died in complete remission because of systemic aspergillosis ten months after transplant. In three patients WT-1 specific T cell responses were monitored in peripheral blood at different time points prior and after vaccination by tetramer analysis. Prior to vaccination none of the patients showed a positive response to WT1. In all these patients CD8+/WT1-specific T cells were detected after one (1/3, 0,66% tetramer binding CD8+ cells) or two vaccinations (2/3, 0,99% and 0,81% tetramer binding CD8+ cells, respectively). In accordance with our observations, WT1 vaccination could contribute to the maintenance of a complete remission in patients with high-risk AML after HSCT. However, it could enhance GvH reactions because of the adjuvants used. Therefore further clinical observations in a larger number of patients are needed.

Description: Background: Recent data suggest that NK cell mediated antibody dependent cellular cytotoxicity (ADCC) is a major mechanism of action of the anti-CD20 monoclonal antibody (mAb) rituximab and the anti-CD52 mAb alemtuzumab, which are frequently applied in patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. However, the exact mechanisms leading to NK cell activation are not completely understood and the cytotoxic subpopulation of peripheral blood NK cells mediating ADCC remains to be defined. In order to quantify and characterize the NK cells mediating ADCC, we used a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation. Methods: PBMCs from healthy individuals were coincubated at 37°C for 3 h with different human leukemia and lymphoma cell lines. In each tube, containing 200μl effector/target suspension (4x105 cells), 15μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. To assess antibody dependent cellular cytotoxicity (ADCC) saturating concentrations (10μg/ml) of rituximab or alemtuzumab were used. After the first 1 h 5μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3, NKG2D, CD69, CD94, NKp30, NK46) for flow cytometry. NK cell-mediated cytotoxicity (specific lysis) was analyzed by flow cytometric detection of propidium iodide uptake. Results: After coincubation with NK sensitive K562 cells up to 6% of CD56+ cells expressed CD107a, indicating that a subpopulation of NK cells releases cytotoxic granules after contact with these target cells. In contrast, coincubation with NK-resistant leukemia cells (ML2, EHEB, DAUDI, RAJI, AM0-1, YT-1) was not followed by an increased surface expression of CD107a. However, when rituximab was added to CD20+ lymphoma or leukemia cells (EHEB, DAUDI, RAJI) we observed that up to 15% of NK cells expressed CD107a after coincubation. In contrast no increased CD107a surface expression was observed when rituximab was added to the CD20− cell lines AMO-0 and YT-1, which excludes unspecific NK cell activation. When alemtuzumab was added to the CD52+ cell lines AMO-1, DAUDI, EHEB, RAJI and YT-1, surface expression of CD107a on NK cells was increased considerably. The majority of degranulating NK cells had the phenotype: CD56dim, CD69+, NKG2D+, NKp30−, NKp46− and CD94−. Furthermore we found that the CD107a assay can also visualize ADCC under clinical conditions as we observed increased numbers of NK cells degranulating in response to CD20+ lymphoma cell lines in patients with non-Hodgkin’s lymphoma treated with rituximab. The number of degranulating NK cells was closely related to the concentration of rituximab and the effector:target ratio, showing a maximum at a ratio of 1:1 and concentrations above 5μg/ml. CD107a surface expression and specific lysis demonstrated a strong positive correlation (r2 = 0.99), confirming that NK cell cytotoxicity can be assessed by this method. Conclusion: The CD107a assay represents a promising new method not only for assessment of natural cytotoxicity on a single cell level but also for determination of ADCC in vitro and in patients treated with mAb. In clinical practice, it may help to find optimal doses and time schedules for the treatment with different mAbs.

Description: This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)–peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer+ T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.

Description: BACKGROUND: The transcription factor Wilms tumor protein 1 (WT1) holds great promise for immunotherapy of leukemia. WT1 is strongly expressed in the majority of leukemic blasts, is essential for blast proliferation, and is spontaneously immunogenic. METHODS: In the present phase II trial, 12 HLA-A2+ patients with AML without curative treatment option, were vaccinated with WT1.126–134 peptide mixed with adjuvant KLH as T-helper protein and GM-CSF 4 times bi-weekly, then monthly. RESULTS: Patients characteristics, immune responses and clinical outcome are shown in table 1. Patient characteristics, immunologic response, and clinical outcome Pat FAB/caryotype previous chemotherapy disease status at study onset no. of vaccinations clinical outcome WT1Tetr+ T cells in PB after vaccination WT1Tetr+ T cells in BM after vaccination *PB, peripheral blood; BM, bone marrow; MDS, myelodysplastic syndrome; MPD, myeloproliferative disease. 1 M4 yes 2.PR 15 CR 12 months 0.49% 0.87% 2 M2 11q23 yes 1.CR 18 cCR 30+ months 0.43% 0.91% 3 M2 no PD 4 SD 3 months 0.42% 0.80% 4 M6 yes PD 4 PD neg. neg. 5 M2 yes 1.PR 6 PD 0.37% 0.51% 6 M1 yes 2. PR 9 PD 0.43% 0.40% 7 M2 yes 2.PR 9 PD 2.00% 1.36% 8 M7 yes PD 4 PD neg. neg. 9 M5b yes 2.CR 12 cCR 8+ months 0.44% 0.33% 10 sAML from MDS no PD 12 SD 8 months 0.23% 0.13% 11 sAML from MPS no PD 12 SD 9+ months 0.22% 0.53% 12 M4 no PD 8 SD 3 months 1.11% 1.35% WT1-specific T cells could be detected in 3 patients before vaccination. An induction or enhancement of a T cell response against WT1 was observed in 10 of 12 patients after 2 – 6 vaccinations ranging from 0.22 to 2.00% (median 0.43%) in peripheral blood and from 0.33 to 1.36% (median 0.80%) in bone marrow as analysed by tetramer and cytokine staining. At study onset 6 patients had progressive AML (PD) with 40 – 90% marrow blasts, 4 patients partial remission (PR) following chemotherapy and two patients complete remission (CR) at high risk for relapse. Four of the 6 patients with progressive AML had disease stabilization for 3, 3, 8 and 9 months, which is ongoing in the latter patient. Disease stabilization was accompanied by a decrease/normalization of peripheral blasts in two patients and a 〉50% decrease in RBC transfusion requirements in a patient with AML evolved from MDS. One patient with PR at study onset had an early relapse and then achieved CR for 12 months (patient 1). Both patients vaccinated in CR are in continuous hematological CR (cCR) for 8+ and 30+ months (patient 2 and 9). The remaining 5 patients had PD after 4 – 9 vaccinations. Bone marrow WT1 RNA levels as molecular disease marker paralleled the clinical course as they decreased 1 – 2 logs in the 3 patients with CR or cCR after 6 vaccinations (Fig. 1A), stabilized or decreased in all 4 patients with SD (Fig. 1B), and increased 1 – 2 logs in 4 of the 5 patients with PD (Fig. 1B). No significant toxic effects were observed. CONCLUSION: WT1 peptide vaccination can efficiently induce a specific immune response and has clinical activity in the absence of significant toxicity. These results warrant further studies of WT1 vaccination in AML patients at high risk for relapse. Fig. 1 WT1 levels in bone marrow before and after 6 vaccinations in patients with CR or cCR (A), SD (B) or PD (C) after vaccination. Fig. 1. WT1 levels in bone marrow before and after 6 vaccinations in patients with CR or cCR (A), SD (B) or PD (C) after vaccination.