[Show abstract][Hide abstract] ABSTRACT: Knowledge of the accurate copy number of HLA class I presented ligands is important in fundamental and clinical immunology. Currently, the best copy number determinations are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amount of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurification and filtration steps, are not well defined and must, therefore, be estimated. In addition, such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepared and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate determination of specific MHC class I-presented ligands. Our study pinpoints the immunopurification step as the origin of the rather extreme losses during sample pretreatment and offers a solution to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy number and thus allows improvement of vaccine design and strategies for immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Patients after allogeneic stem cell transplantation are at risk for invasive aspergillosis, especially during neutropenia. Recent data suggest that an impaired T-cell immune reconstitution post-transplantation plays an important role in this increased risk. In this study we investigated whether Aspergillus-specific T-cells are involved in the recovery from invasive aspergillosis by analyzing the Aspergillus-specific T-cell response in patients with invasive aspergillosis. In 9 patients with improvement of their aspergillus infection, we identified Crf1- or Catalase1-specific T-cells on the basis of CD154 expression and IFNγ production by stimulating with overlapping peptides of the A.fumigatus proteins Crf1 and Catalase1. These Aspergillus-specific T-cells were induced at the moment of regression of the aspergillus lesions. Crf1- and Catalase1-specific T-cells, sorted on the basis of CD154 expression at the peak of the immune response, had a Th1 phenotype and recognized a variety of T-cell epitopes. In contrast, in 2 patients with progressive invasive aspergillosis we were not able to identify any Crf1- or Catalase1-specific T-cells. These data indicate that the presence of Aspergillus-specific T-cells correlates with the clearance of aspergillus infection and suggest that adoptive immunotherapy may be beneficial in patients with invasive aspergillosis after stem cell transplantation.
[Show abstract][Hide abstract] ABSTRACT: In all, 661 of 680 centers in 48 countries reported 37 818 hematopoietic SCT (HSCT) in 33 678 patients (14 165 allogeneic (42%), 19 513 autologous (58%)) in the 2012 survey. Main indications were leukemias, 10 641 (32%; 95% allogeneic); lymphoid neoplasias, 19 336 (57%; 11% allogeneic); solid tumors, 1630 (5%; 3% allogeneic); and nonmalignant disorders, 1953 (6%; 90% allogeneic). There were more unrelated donors than HLA-identical sibling donors (54% versus 38% (8% being mismatched related donor HSCT)). Cord blood was almost exclusive in allogeneic transplants (5% of total). Since 2011, the highest increases in allogeneic HSCT were for AML in CR1 (12%) and for myeloproliferative neoplasm (15%). For autologous HSCT the main increases were for plasma cell disorders (7%), non-Hodgkin lymphoma (4%) and autoimmune disease (50%). There were 4097 pediatric patients <18 years of age receiving HSCT, 2902 received an allogeneic and 1195 an autologous HSCT. Overall, 69% of allogeneic and 64% of autologous HSCT were performed in dedicated pediatric centers and the remainder in combined adult and pediatric centers. Distributions of diseases, donor types and stem cell source for all patients and pediatric patients in particular are shown. A percentage of centers fulfilling the annual required criteria for patient numbers for JACIE accreditation are provided.Bone Marrow Transplantation advance online publication, 17 March 2014; doi:10.1038/bmt.2014.55.
Bone marrow transplantation 03/2014; · 3.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Following allogeneic stem cell transplantation (alloSCT) donor T-cells may recognize minor histocompatibility antigens (MiHA) specifically expressed on cells of the recipient. It has been hypothesized that T-cells recognizing hematopoiesis-restricted MiHA mediate specific graft-versus-leukemia (GvL) activity without inducing graft-versus-host-disease (GvHD), whereas T-cells recognizing ubiquitously expressed MiHA induce both GvL and GvHD-reactivity. Furthermore, alloreactive CD4 T-cells are hypothesized to be able to mediate specific GvL-reactivity due to the hematopoiesis-restricted expression of HLA-class-II. However, clinical observations suggest that an overt GvL-response, associated with expansion of T-cells specific for hematopoiesis-restricted antigens, is often associated with GvHD-reactivity. Therefore, we developed in-vitro models to investigate whether alloreactive T-cells recognizing hematopoiesis-restricted antigens induce collateral damage to surrounding non-hematopoietic tissues. We demonstrated that collateral damage to MiHA-negative fibroblasts was induced by misdirection of cytotoxic granules released from MiHA-specific T-cells activated by MiHA-positive hematopoietic cells resulting in granzyme-B-mediated activation of apoptosis in the surrounding fibroblasts. We demonstrated that direct contact between the activated T-cell and the fibroblast was a prerequisite for this collateral damage to occur. In conclusion, these data suggest that hematopoiesis-restricted T-cells actively participating in an overt GvL-response may contribute to GvHD via induction of collateral damage to non-hematopoietic targets.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 03/2014; · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematological malignancies often express surface HLA-class-II, making them attractive targets for CD4+ T-cell therapy. We previously demonstrated that HLA-class-II ligands can be divided into DM-resistant and DM-sensitive antigens. In contrast to DM-resistant antigens, presentation of DM-sensitive antigens is suppressed by HLA-DM, but can be rescued by HLA-DO. We also showed that HLA-DO expression remains low in non-hematopoietic cells under inflammatory conditions, suggesting that DM-sensitive antigens may be ideal T-cell targets with a low risk for GvHD. Here, we demonstrated that B-cell malignancies often express HLA-DO and that levels are in particular high in chronic lymphocytic leukemia. Moreover, we showed that surface presentation of DM-sensitive antigens is regulated by HLA-DO, and that DM-sensitive antigens are relevant T-cell targets for B-cell malignancies and especially chronic lymphocytic leukemia. These data open the perspective to target HLA-class-II ligands with specific processing and presentation behavior for CD4+ T-cell therapy of hematological malignancies.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 02/2014; · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI) can be applied as immunotherapeutic intervention to treat malignant diseases. Here, we describe a patient with progressive metastatic clear cell renal cell carcinoma (RCC) who was treated with T cell depleted non-myeloablative alloSCT and DLI resulting in disease regression accompanied by extensive graft versus host disease (GVHD). We characterized the specificity of this immune response, and detected a dominant T cell population recognizing a novel minor histocompatibility antigen (MiHA) designated LB-FUCA2-1V. T cells specific for LB-FUCA2-1V were shown to recognize RCC cell lines, supporting a dominant role in the graft versus tumor (GVT) reaction. However, coinciding with the gradual disappearance of chronic GVHD, the anti-tumor effect declined and 3 years after alloSCT the metastases became progressive again. To re-initiate the GVT reaction, escalating doses of DLI were given, but no immune response could be induced and the patient died of progressive disease 8.5 years after alloSCT. Gene expression studies illustrated that only a minimal number of genes shared expression between RCC and professional antigen presenting cells but were not expressed by non-malignant healthy tissues, indicating that in patients suffering from RCC, GVT reactivity after alloSCT may be unavoidably linked to GVHD.
PLoS ONE 01/2014; 9(1):e85198. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A two-step procedure in which T-cell depleted allogeneic stem cell transplantation is followed by sequential treatment with donor lymphocyte infusion at 6 months can significantly reduce the risk and severity of graft-versus-host disease, with postponed induction of the beneficial graft-versus-leukemia effect. However, patients with high-risk leukemia have a substantial risk of relapse early after transplantation, at a time when administration of donor lymphocyte infusion has a high likelihood of resulting in graft-versus-host disease, disturbing a favorable balance between graft-versus-leukemia effect and graft-versus-host disease. Therefore, new therapeutic modalities are required to allow early administration of T-cells capable of exerting graft-versus-leukemia effect without graft-versus-host disease. Here, we describe the isolation of virus-specific T-cells using the Streptamer-based isolation technology and subsequent transfer of the minor histocompatibility antigen HA-1-specific T-cell receptor using retroviral vectors. Isolation of virus-specific T-cells and subsequent transduction with HA-1-T-cell receptor resulted in rapid in vitro generation of highly pure dual-specific T-cells with potent anti-leukemic reactivity. Due to the short production procedure of only 10-14 days and the defined specificity of the T-cells, administration of virus-specific T-cells transduced with the HA-1-T-cell receptor as early as 8 weeks after allogeneic stem cell transplantation is feasible. (This clinical trial is registered at www.clinicaltrialsregister.eu as EudraCT number 2010-024625-20).
[Show abstract][Hide abstract] ABSTRACT: The prognosis of patients with relapsed acute myeloid leukemia after allogeneic transplant is poor. We hypothesized that initial disease control by effective cytoreduction, followed by rapid induction of a profound allo-immune response by donor-lymphocyte infusion during the neutropenic phase is essential for long-term survival. Additional interferon-α was administered in case no acute graft-versus-host-disease occurred within 3 weeks after donor-lymphocyte infusion. Overall, 44 patients with relapsed acute myeloid leukemia were assessed; 26 relapsed after myeloablative, and 18 after reduced-intensity conditioning. Of these, 7 patients were not eligible due to poor performance status (n=3) or severe graft-versus-host-disease (n=4) at time of relapse. Patients with smoldering relapses (n=5) received donor-lymphocyte infusion only. Thirty-two patients received cytoreductive treatment, followed by donor-lymphocyte infusion in 22 patients. Reasons for not receiving donor-lymphocyte infusion were chemotherapy-related death (n=1) and chemotherapy-refractory disease (n=9). Two-year overall survival after donor-lymphocyte infusion was 36% (95% confidence-interval: 16-57%). The impact of acute graft-versus-host-disease on survival was calculated with a Cox-regression model including onset of acute graft-versus-host-disease as a time-dependent variable. Development of grade 1-3, but not grade 4, acute graft-versus-host-disease, was associated with superior survival as compared to absence of graft-versus-host-disease (hazard ratio 0.22, p=0.03). In conclusion, efficient cytoreduction followed by donor-lymphocyte infusion and subsequent interferon-α leading to limited acute graft-versus-host-disease represents a potentially curative option for relapsed acute myeloid leukemia after allogeneic transplant.
[Show abstract][Hide abstract] ABSTRACT: It is well known that allo-reactive T-cells play a crucial role in graft-versus-leukemia and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Allo-reactive CD4+ T-cells can mediate direct cytolysis, but may also stimulate production of IgG antibodies as helper cells. Immune complexes may subsequently be processed and presented by professional antigen presenting cells and stimulate induction of specific CD8+ T-cells. As such, proteins targeted in coordinated T- and B-cell responses may represent a class of immunodominant antigens in clinical responses after alloSCT. We previously identified LB-PTK2B-1T as HLA class II restricted polymorphic antigen in a patient treated with donor lymphocyte infusion for relapsed chronic myeloid leukemia after HLA-matched alloSCT. Since PTK2B has also been described as antibody target, we here investigated whether a coordinated T- and B-cell response against PTK2B was induced. Patient serum before and after alloSCT and DLI was screened for antibodies, and we indeed observed development of a humoral immune response against PTK2B. Antibodies against PTK2B were only found after DLI and, in contrast to the CD4+ T-cells, recognized a monomorphic region of the protein. To our knowledge this is the first description of a coordinated allo-reactive CD4+ T-cell and auto-reactive antibody response against an autosomal antigen.
[Show abstract][Hide abstract] ABSTRACT: The prognosis of adult patients with ALL remains unsatisfactory. AlloSCT is associated with a beneficial GVL response mediated by donor T cells. However, GVHD results in substantial mortality and long-term morbidity. T-cell depletion (TCD) of the graft reduces the severity of GVHD, but is associated with an increased relapse rate after alloSCT. Therefore, early sequential donor lymphocyte infusion (DLI) is likely to be necessary for a successful GVL reaction. Twenty-five adult ALL patients (10 Ph(+)ALL) were eligible for early DLI after initial disease control with myeloablative TCD-alloSCT in first CR (CR1), if active GVHD was absent at 3-6 months after alloSCT. Patients with a sibling donor or an unrelated donor were scheduled for 3.0 × 10(6) CD3(+) cells/kg or 1.5 × 10(6) CD3(+) cells/kg, respectively, at 6 months after alloSCT. Three patients died before evaluation (one early relapse). Five patients had active GVHD. Fourteen of the remaining seventeen patients received DLI (median time-to-DLI: 185 days). Overall, only 17% required long-term systemic immunosuppression for GVHD. With a median follow-up after TCD-alloSCT of 50 months, 2-year survival probability was 68% (95% confidence interval (CI) 49-87%). In conclusion, myeloablative TCD-alloSCT with early sequential DLI is an efficient and safe post-remission treatment for adult ALL patients in CR1.Bone Marrow Transplantation advance online publication, 12 August 2013; doi:10.1038/bmt.2013.111.
Bone marrow transplantation 08/2013; · 3.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The low frequency of antigen-specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T-cell repertoire. Here we combine the generation of naïve repertoire-derived antigen-specific T-cell lines based on MHC-tetramer staining and magnetic-bead enrichment with in-depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T-cell lines were generated from seronegative individuals. Generated T-cell lines consisted of a variety of immunodominant CMV-epitope specific oligoclonal T-cell populations restricted to various HLA-molecules (HLA-A1, A2, B7, B8 and B40), and the functional and structural avidity of the CMV-specific T cells was studied. Although all CMV-specific T cells were isolated based on their reactivity towards a specific peptide-MHC complex, we observed a large variation in the functional avidity of the MHC-tetramer positive T-cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff -rate assay. Our data demonstrate that MHC-tetramer staining is not always predictive for the sensitivity of specific T-cell reactivity, and challenge the sole use of MHC-tetramers as an indication of the peripheral T-cell repertoire, independent of the analysis of functional activity or structural avidity parameters. This article is protected by copyright. All rights reserved.
European Journal of Immunology 07/2013; · 4.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD8+ T-cell depleted donor lymphocyte infusion (DLI) after T-cell depleted (TCD) allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of Graft-versus-Host Disease (GvHD) while preserving conversion to donor hematopoiesis and anti-tumor immunity, providing a rationale for exploring CD4+ T-cell based immunotherapy for hematological malignancies. Here, we analyzed the clinical course and specificity of T-cell immune responses in two patients with acute myeloid leukemia who converted to full donor chimerism, but developed severe acute GvHD after prophylactic CD4+ DLI following 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T-cells isolated during GvHD demonstrated allo-reactivity exerted by CD4+ T-cells directed against patient mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD4+ DLI, both patients contained residual patient-derived T-cells, including CMV-specific T-cells due to CMV-reactivations. Once activated by CMV-antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD4+ T cells, which in turn could target non-hematopoietic tissues in GvHD. In conclusion, our data demonstrate that GvHD following HLA-DPB1-mismatched CD4+ DLI can be mediated by allo-reactive HLA-DPB1-directed CD4+ T-cells and that ongoing viral infections inducing HLA-class-II expression on non-hematopoietic cells may increase the likelihood of GvHD development. (ISRTCN registered: 51398568, http://www.controlled-trials.com/ISRCTN51398568/LUMC).
[Show abstract][Hide abstract] ABSTRACT: Invasive aspergillosis is a serious infectious complication after allogeneic stem cell transplantation. One of the strategies to improve the management of aspergillosis is the adoptive transfer of antigen-specific T-cells, the success of which depends on the development of a broad repertoire of antigen-specific T-cells. In this study we identified CD4+ T-cells specific for the Aspergillus proteins Crf1 and Catalase1 in 18 of 24 healthy donors by intracellular staining for IFNγ and CD154. Crf1- and Catalase1-specific T-cells were selected on the basis of CD137 expression and single cell expanded. Aspergillus-specific T-cell clones mainly exhibited a Th1 phenotype and recognized a broad variety of T-cell epitopes. Five novel and 2 previously described Crf1 epitopes, and 30 novel Catalase1 epitopes were identified. Ultimately, by using overlapping peptides of A.fumigatus proteins, Aspergillus-specific T-cell lines with a broad specificity and favourable cytokine profile, suitable for adoptive T-cell therapy, can be generated in vitro.
The Journal of Infectious Diseases 05/2013; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In all, 651 from 680 centers in 48 countries reported 35 660 hematopoietic SCT (HSCT) in 32 075 patients (13 470 allogeneic (42%), 18 605 autologous (58%)) to the 2011 survey. Main indications were: leukemias; 10 113 (32%; 94% allogeneic); lymphoid neoplasias; non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasma cell disorders; 18 433 (57%; 12% allogeneic); solid tumours; 1573 (5%; 5% allogeneic); and non-malignant disorders; 1830 (6%; 92% allogeneic). There were more unrelated donors than HLA identical sibling donors (54% versus 39%); proportion of peripheral blood as stem cell source was 99% for autologous and 73% for allogeneic HSCT. Cord blood was only used in allogeneic transplants (6% of total). In the past 10 years, the overall number of transplants has increased by 53%. Allogeneic HSCT have doubled (from 7272 to 14 549) while, autologous have increased by 32% and continue to increase by about 1100 HSCT per year since 2001. In the past 2 years, an increase of >2000 HSCT per year was seen. Transplant activity is shown by team size. For allogeneic HSCT, we show use of reduced-intensity conditioning versus myeloablative conditioning across Europe and use of post-transplant donor lymphocyte infusions with considerable variation across different countries.Bone Marrow Transplantation advance online publication, 15 April 2013; doi:10.1038/bmt.2013.51.
Bone marrow transplantation 04/2013; · 3.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the revolutionary change in the prognosis of chronic myeloid leukemia (CML) patients with the introduction of imatinib, patients with resistant disease still pose a considerable problem. In this multicenter, randomized phase III trial, we investigate whether the combination of high-dose imatinib and intermediate-dose cytarabine compared to high-dose imatinib alone, improves the rate of major molecular response (MMR) in newly diagnosed CML patients. This study was closed prematurely because of declining inclusion due to the introduction of second generation tyrosine kinase inhibitors and only one third of the initially required patients were accrued. One hundred nine patients aged 18-65 years were randomly assigned to either imatinib 800 mg (n = 55) or to imatinib 800 mg in combination with two successive cycles of cytarabine 200 mg/m(2) for 7 days (n = 54). After a median follow-up of 41 months, 67 % of patients were still on protocol treatment. The MMR rate at 12 months was 56 % in the imatinib arm and 48 % in the combination arm (p = 0.39). Progression-free survival was 96 % after 1 year and 89 % after 4 years. Four-year overall survival was 97 %. Adverse events grades 3 and 4 were more common in the combination arm. The addition of intermediate-dose of cytarabine to imatinib did not improve the MMR rate at 12 months. However, the underpowering of the study precludes any definitive conclusions. This trial is registered at www.trialregister.nl (NTR674).
Annals of Hematology 04/2013; · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allo-HLA directed T-cell responses after HLA-mismatched allogeneic stem cell transplantation (alloSCT) and donor lymphocyte infusion (DLI) are typically regarded as detrimental responses mediating Graft-versus-Host Disease (GvHD). However, allo-HLA reactive T-cells with beneficial and selective Graft-versus-Leukemia (GvL) reactivity can also be identified within an HLA-mismatched context. Here, we investigated whether allo-HLA-class II directed T-cells with beneficial GvL reactivity induced in NOD/scid mice engrafted with human chronic myeloid leukemia in lymphoid blast crisis after treatment with DLI mediated detrimental xenogeneic GvHD as a result of broad off-target cross-reactivity. The results demonstrated that beneficial GvL reactivity and xenogeneic GvHD were mediated by separate T-cells. GvL reactivity was mediated by human T-cells recognizing allo-HLA-class II molecules, whereas xeno-reactivity was exerted by human T-cells recognizing H-2 molecules. In conclusion, our data show a limited risk for detrimental off-target effects by allo-HLA-class II directed T-cells, and therefore provide a basis for development of strategies for selection of allo-HLA restricted T-cells with selective GvL reactivity for adoptive transfer after HLA-mismatched alloSCT.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 03/2013; · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies on T cell action, and in cellular immunotherapy and transplantation. In this study we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA-ligandome of B-cells. This large number of identified ligands provides a general insight in the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA-ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes and post-translationally modified HLA ligands and can be a good starting point to solve a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes, not based on in silico predictions, but based on the bona fide eluted HLA-ligandome.
[Show abstract][Hide abstract] ABSTRACT: T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry-based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.
The Journal of Immunology 03/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T-cell receptor (TCR) gene transfer is an attractive strategy to equip T cells with defined antigen-specific TCRs using short-term in vitro procedures to target both hematological malignancies and solid tumors. TCR gene transfer poses different safety issues that might warrant the inclusion of a suicide gene. High affinity TCRs may result in on-target toxicity, and off-target reactivity directed against healthy tissue can be observed due to mixed TCR dimers. Inclusion of a suicide gene as a safety switch may abrogate these unwanted toxicities. Human CD20 has been proposed as a nonimmunogenic suicide gene targeted by widely used clinical-grade anti-CD20 antibodies that can additionally function as a selection marker. However, transduction of T cells with a multi-cistronic vector encoding both TCR and CD20 resulted in poor coexpression. In this study, we demonstrated that codon optimization of TCR and CD20 resulted in profound coexpression of both the preferentially expressed antigen in melanoma (PRAME)-TCR and CD20, allowing selective as well as efficient elimination of these engineered T cells in vitro. These results demonstrate the great potential of codon optimized CD20 to be broadly used in clinical trials as a safety switch.Gene Therapy advance online publication, 31 January 2013; doi:10.1038/gt.2013.4.
[Show abstract][Hide abstract] ABSTRACT: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.
We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.
Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.
Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.
PLoS ONE 01/2013; 8(11):e80070. · 3.73 Impact Factor