Automated production of specific T cells for treatment of refractory viral infections after allogeneic stem cell transplantation.

Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.

Clearance of Treatment Refractory Adenoviremia via Adenovirus-specific Donor T-Cell Transfer During Aplasia After αßTCR-CD19-Depleted Stem Cell Transplantation.

Here, we report the case of severe adenoviremia in a 7-year-old boy with highly-resistant, acute leukemia. A combined approach of αßTCR-CD19-depleted stem cell transplantation, enabling immunosuppression-free post-transplant care, and early transfer of adenovirus-specific donor T cells during aplasia resulted in rapid and complete clearance of the treatment-refractory adenoviremia.

Adoptive T-cell therapy with hexon-specific Th1 cells as a treatment of refractory adenovirus infection after HSCT.

Hematopoietic stem cell transplantation (HSCT) has improved over the last few decades. However, viral infections are often refractory to pharmacologic treatment and require alternative treatment strategies such as immunotherapy. Adenovirus (AdV) is th predominant disease-causing pathogen in pediatric HSCT. In a clinical trial, we analyzed safety and efficacy of ex vivo adoptive T-cell transfer (ACT) with hexon-specific T cells, predominantly of the T-helper cell 1 (Th1) phenotype, in 30 patients with AdV disease or viremia. ACT was feasible with no acute toxicities or significant onset of graft-versus-host disease. ACT led to in vivo antiviral immunity for up to 6 months with viral control, resulting in complete clearance of viremia in 86% of patients with antigen-specific T-cell responses. After ACT and a follow-up of 6 months, overall survival was markedly increased in responders (mean, 122 days; 15 survivors) compared with nonresponders who all died shortly after ACT (mean, 24 days; no survivors). AdV-related mortality was 100% in nonresponders compared with 9.5% in responders (≥1 log reduction of DNA copies per milliliter after ACT). In summary, ex vivo ACT of AdV-specific Th1 cells was well tolerated and led to successful and sustained restoration of T-cell immunity correlated with virologic response and protection from virus-related mortality. This cellular immunotherapy is a short-term available and broadly applicable treatment. The study is registered at European Union Clinical Trials Register as 2005-001092-35.

Reduction of Minimal Residual Disease in Pediatric B-lineage Acute Lymphoblastic Leukemia by an Fc-optimized CD19 Antibody.

Prognosis of primary refractory and relapsed pediatric B-lineage acute lymphoblastic leukemia (ALL) is very poor. Relapse rates significantly correlate with persistent minimal residual disease (MRD). In MRD, favorable effector-target ratios prevail and thus this situation might be optimally suited for immunotherapy with antibodies recruiting immunological effector cells. We here report on the generation, preclinical characterization and first clinical application in B-lineage ALL of an Fc-optimized CD19 antibody. This third-generation antibody (4G7SDIE) mediated enhanced antibody-dependent cellular cytotoxicity (ADCC) against leukemic blasts with effector cells from healthy volunteers and B-lineage ALL patients. The antibody was produced in a university-owned production unit and was applied on a compassionate use basis to 14 pediatric patients with refractory and relapsed B-lineage ALL at the stage of MRD. In 10/14 patients, MRD was reduced by ≥ 1 log or below the patient-individual detection limit, and 5/14 patients have achieved ongoing complete molecular remission with a median leukemia-free survival of 428 days. Two additional patients died in complete molecular remission due to complications not related to antibody therapy. Besides profound in vivo B-cell depletion, side effects were negligible. A clinical phase 1/2 study to further assess the therapeutic activity of 4G7SDIE is in preparation.

De novo PIK3R1 gain-of-function with recurrent sinopulmonary infections, long-lasting chronic CMV-lymphadenitis and microcephaly.

PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1) gain-of-function has recently been described in patients with recurrent sinopulmonary infections, chronic CMV-/EBV-infections, lymphoproliferation, and hypogammaglobulinemia. Here we report a 15-year-old boy with treatment refractory CMV lymphadenitis, severe combined immunodeficiency, microcephaly and a severe developmental defect of Th17 cells. To avoid poor outcome, hematopoietic stem cell transplantation (HSCT) was performed. Subsequently, whole exome sequencing revealed a de novo heterozygous G-to-C mutation (chr5: 5:67,589,663: G>C) at the splice donor site of the PIK3R1 gene. Our data suggest that PIK3R1 gain-of-function leads to developmental defects in helper and regulatory T-cell subsets, the latter expanding the immunological features of PIK3R1 gain-of-function. T-cell subsets play a critical role in the regulation of immune response against infectious agents and of autoimmunity and thus may be particularly accountable for the clinical phenotype of affected patients.

Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon adoptive immunotherapy.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Immunotherapy with the trifunctional anti-CD20 x anti-CD3 antibody FBTA05 (Lymphomun) in paediatric high-risk patients with recurrent CD20-positive B cell malignancies.

Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow-up of ten patients diagnosed with relapsed or refractory mature B-cell Non Hodgkin Lymphoma (B-NHL), Burkitt leukaemia (B-AL) or pre B-acute lymphoblastic leukaemia (pre B-ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti-CD3 x anti-CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo-SCT, n = 6) to induce sustained long-term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo-SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow-up. The other patients still maintain CR with a current overall survival of 874-1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients.

Influence of ATLG serum levels on CD3/CD19-depleted hematopoietic grafts and on immune recovery in pediatric haplo-HSCT.

ABSTRACT: Anti-T lymphocyte globulin (ATLG) significantly reduces the risk of engraftment failure in allogeneic hematopoietic stem cell transplant (HSCT) but hampers posttransplant immune reconstitution. We hypothesized that in patients receiving haploidentical CD3/CD19-depleted grafts, these double-edged effects could be better balanced by attaining high ATLG serum concentrations before transplant but as low as possible on the day of transplant. Therefore, we moved the start of ATLG application to day -12 and determined serum concentrations of T-cell-specific ATLG in pediatric patients treated with 3 established dosing regimens (15, 30, or 60 mg/kg). Corresponding mean T-cell-specific ATLG serum concentrations at day 0 were 1.14, 2.99, or 12.10 µg/mL, respectively. Higher ATLG doses correlated with higher peak levels at days -8 and -7 and reduced graft rejection, whereas lower ATLG doses correlated with significantly faster posttransplant recovery of T and natural killer cells. The rate of graft-versus-host disease remained low, independent of ATLG doses. Moreover, in vitro assays showed that ATLG concentrations of 2.0 µg/mL and lower only slightly reduced the activity of natural killer cells, and therefore, the function of such effector cells might be preserved in the grafts. Pharmacokinetic analysis, compatible with linear first-order kinetics, revealed similar half-life values, independent of ATLG doses. Hence, the day on which a desired ATLG serum level is reached can be calculated before HSCT. Our retrospective study demonstrates the relevance of dosing and time of administration of ATLG on engraftment and immune recovery in ex vivo CD3/CD19-depleted haploidentical HSCT.

Anti-leukaemic activity of a novel haploidentical-transplantation approach employing unmanipulated bone marrow followed by CD6-depleted peripheral blood stem cells in children with refractory/relapsed acute leukaemia.

The treatment outcome of children with refractory acute leukaemia or relapse post-stem cell transplantation is dismal. We report 10 children (non-remission n = 7) who underwent a new haploidentical transplant approach utilizing unmanipulated bone marrow followed by CD6-depleted peripheral blood stem cells. Nine patients had successful engraftment and no evidence of leukaemia. Acute and chronic graft-versus-host-disease was observed in five and three patients, respectively; two patients died of treatment-related toxicity. Seven patients relapsed after 7 (range 3-34) months, however two patients are alive at 6·5 and 7·0 years. This approach provides anti-leukaemic activity even in heavily pre-treated children but long-term disease control requires further intervention.

Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a ß chain CDR3 sequence associated with hepatitis-induced pathogenesis.

Current diagnostic approaches that characterize T-cell deficiency by analyzing diversity of T-cell receptor sequences effectuate limited informational gain about the actual restrictiveness. For deeper insight into T-cell receptor repertoires we developed next-generation-sequencing-spectratyping, which employs high coverage Roche/454 sequencing of T-cell receptor (ß)-chain amplicons. For automated analysis of high-throughput-sequencing data, we developed a freely available software, the TCR profiler. Gene usage, length, encoded amino acid sequence and sequence diversity of the complementarity determining region 3 were determined and comprehensively integrated into a novel complexity score. Repertoires of CD8(+) T cells from children with idiopathic or hepatitis-induced very severe aplastic anemia (n=7), children two months after bone marrow transplantation (n=7) and healthy controls (children n=5, adults n=5) were analyzed. Complexity scores clearly distinguished between healthy and diseased, and even between different immune deficiency states. The repertoire of aplastic anemia patients was dominated by public (i.e. present in more than one person) T-cell receptor clonotypes, whereas only 0.2% or 1.9% were public in normal children and adults, respectively. The CDR3 sequence ASSGVGFSGANVLT was highly prevalent in 3 cases of hepatitis-induced anemia (15-32% of all sequences), but was only low expressed in idiopathic aplastic anemia (2-5%, n=4) or healthy controls (<1%). Fifteen high frequent sequences were present exclusively in aplastic anemia patients. Next-generation-sequencing-spectratyping allows in-depth analysis of T-cell receptor repertoires and their restriction in clinical samples. A dominating clonotype was identified in hepatitis-induced anemia that may be associated with disease pathogenesis and several aplastic-anemia-associated, putatively autoreactive clonotypes were sequenced.

Anti-GD2 Antibody Dinutuximab Beta and Low-Dose Interleukin 2 After Haploidentical Stem-Cell Transplantation in Patients With Relapsed Neuroblastoma: A Multicenter, Phase I/II Trial.

PURPOSE: Patients with relapsed high-risk neuroblastoma (rHR-NB) have a poor prognosis. We hypothesized that graft-versus-neuroblastoma effects could be elicited by transplantation of haploidentical stem cells (haplo-SCT) exploiting cytotoxic functions of natural killer cells and their activation by the anti-GD2 antibody dinutuximab beta (DB). This phase I/II trial assessed safety, feasibility, and outcomes of immunotherapy with DB plus subcutaneous interleukin-2 (scIL2) after haplo-SCT in patients with rHR-NB. METHODS: Patients age 1-21 years underwent T-/B-cell-depleted haplo-SCT followed by DB and scIL2. The primary end point 'success of treatment' encompassed patients receiving six cycles, being alive 180 days after end of trial treatment without progressive disease, unacceptable toxicity, acute graft-versus-host-disease (GvHD) ≥grade 3, or extensive chronic GvHD. RESULTS: Seventy patients were screened, and 68 were eligible for immunotherapy. Median number of DB cycles was 6 (range, 1-9). Median number of scIL2 cycles was 3 (1-6). The primary end point was met by 37 patients (54.4%). Median observation time was 7.8 years. Five-year event-free survival (EFS) and overall survival from start of trial treatment were 43% (95% CI, 31 to 55) and 53% (95% CI, 41 to 65), respectively. Five-year EFS among patients in complete remission (CR; 52%; 95% CI, 31 to 69) or partial remission (44%; 95% CI, 27 to 60) before immunotherapy were significantly better compared with patients with nonresponse/mixed response/progressive disease (13%; 95% CI, 1 to 42; P = .026). Overall response rate in 43 patients with evidence of disease after haplo-SCT was 51% (22 patients), with 15 achieving CR (35%). Two patients developed GvHD grade 2 and 3 each. No unexpected adverse events occurred. CONCLUSION: DB therapy after haplo-SCT in patients with rHR-NB is feasible, with low risk of inducing GvHD, and results in long-term remissions likely attributable to increased antineuroblastoma activity by donor-derived effector cells.

Fatal outcome despite full lympho-hematopoietic reconstitution after allogeneic stem cell transplantation in atypical ataxia telangiectasia.

Allogeneic hematopoietic stem cell transplantation (HSCT) has not been a therapeutic option in ataxia telangiectasia (AT) due to overwhelming toxicity of conditioning in the context of the global DNA repair deficiency. Furthermore HSCT is unable to cure neurological involvement of AT. We report on a Turkish child with a Hyper IgM phenotype disorder, in which clinical aspects of AT were absent and thus, AT not diagnosed. He was transplanted with a reduced toxicity, but full intensity conditioning regimen comprising treosulfan, fludarabine and ATG. The peritransplant period was uneventful and the patient was discharged at day +57. 8 months after HSCT, the patient developed hepatopathy with monoclonal gammopathy of unclear significance and died due to hepatic failure and encephalopathy at the age of 32 months. Post mortem high throughput sequencing revealed a mutation in the ATM gene.

Adoptive transfer of pp65-specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation.

Cytomegalovirus (CMV) disease and infection refractory to antiviral treatment after allogeneic stem cell transplantation (allo-SCT) is associated with a high mortality. Adoptive transfer of CMV-specific T cells could reconstitute viral immunity after SCT and could protect from CMV-related complications. However, logistics of producing virus-specific T-cell grafts limited the clinical application. We treated 18 patients after allo-SCT from human leukocyte antigen-mismatched/haploidentical or human leukocyte antigen-matched unrelated donors with polyclonal CMV-specific T cells generated by ex vivo stimulation with pp65, followed by isolation of interferon-γ-producing cells. Patients with CMV disease or viremia refractory to antiviral chemotherapy or both were eligible for adoptive T-cell transfer and received a mean of 21 × 10³/kg pp65-specific T cells. In 83% of cases CMV infection was cleared or viral burden was significantly reduced, even in cases of CMV encephalitis (n = 2). Viral control was associated with in vivo expansion of CMV-specific T lymphocytes in 12 of 16 evaluable cases, resulting in reconstitution of antiviral T-cell responses, without graft-versus-host disease induction or acute side effects. Our findings indicate that the infusion of low numbers of CMV-specific T cells is safe, feasible, and effective as a treatment on demand for refractory CMV infection and CMV disease after allo-SCT.

CD34+ gene expression profiling of individual children with very severe aplastic anemia indicates a pathogenic role of integrin receptors and the proapoptotic death ligand TRAIL.

UNLABELLED: BACKGROUND Very severe aplastic anemia is characterized by a hypoplastic bone marrow due to destruction of CD34(+) stem cells by autoreactive T cells. Investigation of the pathomechanism by patient-specific gene expression analysis of the attacked stem cells has previously been impractical because of the scarcity of these cells at diagnosis. DESIGN AND METHODS: Employing unbiased RNA amplification, patient-specific gene expression profiling was carried out for CD34(+) cells from patients newly diagnosed with very severe aplastic anemia (n=13), refractory anemia (n=8) and healthy controls (n=10). These data were compared to profiles of myelodysplastic disease (n=55), including refractory anemia (n=18). To identify possible targets of autoimmune attack, presence of autoreactive antibodies was tested in pre-therapeutic sera of patients with very severe aplastic anemia (n=19). RESULTS: CD34(+) gene expression profiling distinguished between healthy controls, children with aplastic or refractory anemia and clonal disease. Interferon stimulated genes such as the apoptosis inducing death ligand TRAIL were strongly up-regulated in CD34(+) cells of patients with aplastic anemia, in particular in patients responding to immunosuppressive treatment. In contrast, mRNA expression of integrin GPVI and the integrin complexes GPIa/IIa, GPIIb/IIIa, GPIB/GPIX/GPV was significantly down-regulated and corresponding antibodies were detected in 7 of 11 profiled patients and in 11 of 19 aplastic anemia patients. CONCLUSIONS As a potential diagnostic tool, patient-specific gene expression profiling of CD34(+) stem cells made it possible to make the difficult differential diagnosis of most patients with aplastic and refractory anemia. Profiling indicated a prognostic correlation of TRAIL expression and patient benefit from immunosuppressive therapy. Downregulation of integrin expression and concurrent presence of autoreactive anti-integrin-antibodies suggested a previously unrecognized pathological role of integrins in aplastic anemia.

Adoptive antifungal T cell immunotherapy--into the clinic?

The morbidity and mortality rates of invasive fungal infection in allogeneic stem cell recipients are still unacceptably high and have not been significantly improved by alternative antifungal strategies to date. Over the last few years, rapid methods for the clinical-scale generation of functionally active and well characterized antifungal T(H)1 cells have become available. In addition, current data on the use of donor-derived virus-specific T cells in allogeneic stem cell transplantation suggest that the risk of severe adverse events, in particular the risk of graft-versus-host disease, is negligible. Therefore, adoptive antifungal immunotherapeutic strategies should be evaluated in clinical trials. However, one has to recognize that these trials are only meaningful with sufficiently large and homogenous cohorts of patients and if the settings of adoptive antifungal immunotherapy are comparable. Ultimately, the strategy of adoptively transferring antifungal immune responses might improve the outcome in hematopoietic stem cell recipients suffering from invasive fungal infection.

Mucormycosis in paediatric patients: demographics, risk factors and outcome of 12 contemporary cases.

Mucormycosis is associated with high morbidity and mortality and is perceived as an emerging fungal infection. However, contemporary paediatric data are limited. We present a series of paediatric cases of mucormycosis reported from Germany and Austria collected within a voluntary epidemiological survey through standardised, anonymized case report forms. Twelve cases were reported between January 2004 and December 2008 (six men; mean age: 12.6 years, range: 0.1-17 years). Mucormycosis was proven in nine, and probable in three cases. Isolates included Lichtheimia (syn. Absidia pro parte, Mycocladus) (five), Rhizopus (three) and Mucor (one) species. Infection was limited to soft tissue in three cases, the lung in two cases, and an infected thrombus in one case; rhinocerebral disease was found in three cases, and pulmonary-mediastinal, pulmonary-cerebral and soft tissue-cerebral involvement in one case each. All three patients with isolated soft tissue infection were cured, whereas seven of the remaining patients died (one patient without follow-up). The overall mortality rate was 67%. While these data cannot provide conclusive data on incidence and disease burden of mucormycosis in paediatric patients, they reflect the continuing threat of these infections to immunocompromised patients and the need for improved diagnosis and management.

Expression profiles of HMGB1 on B-CLL related leukocytes contribute to prediction of relapse.

BACKGROUND: The High Mobility Group Box 1 (HMGB1) is a nuclear protein that is frequently overexpressed in hematologic diseases and might be of relevance in immunogenic cancer control thus correlating with patients' (pts.) prognosis in diseases such as acute myeloid, acute lymphatic and chronic lymphocytic leukemia. MATERIALS AND METHODS: Expression profiles of blasts from AML (n = 21), ALL (n = 16) and of B-lymphocytes of CLL (n = 9) pts. were analyzed for surface expression of HMGB1 using flow cytometry. Expression was quantified and correlated with clinically and prognostically relevant markers. RESULTS: Expression profiling of HMGB1 in blasts of AML and ALL subtypes did not show differences between primary vs. secondary disease development and gender related differences. In ALL pts. however, age groups at initial diagnosis between ≥20 vs. <20 years were compared and showed significant differences (≥20 vs. <20 years; 89% vs. 49%, p  <0.05) with higher expression in higher age. In AML and CLL these differences were not visible. To evaluate the prognostic significance of HMGB1 expression, expression quantity was correlated with established and prognostic classification systems (in AML ELN, in ALL GMALL) and probability to relapse. No significant correlation was seen in these entities. However, when AML pts. were analyzed for remission rates after first anthracycline based induction therapy, in those who did not experience a complete remission significantly enhanced HMGB1 surface expression was seen (98 vs. 94%; p < 0.05; n = 20). Furthermore, for CLL it was shown that higher HMGB1 expression was found in pretreated patients with relapsed or/and refractory disease (1 vs. more relapses; 94 vs. 98%; p  <0.05; n = 9). CONCLUSION: HMGB1 is frequently expressed in hematologic malignancies. In this study it was shown that HMGB1 surface expression on AML blasts can be used as predictors for treatment response. In CLL it may be a marker for advanced disease. In order to implement this marker in FACS routine it could be a useful and practical tool for prognostic assessment and treatment planning.

Expression of Surface-associated 82 kDa proMMP-9 in Lymphatic Leukemia Blast Cells Differentially Correlates With Prognosis.

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) degrade extracellular matrix and process regulatory proteins. Recently, a membrane-bound 82kDa variant of proMMP-9 identified on myeloid blasts was shown to be associated with prognosis. PATIENTS AND METHODS: To investigate the role of 82kDa proMMP-9 with acute lymphoblastic leukemia (ALL) and chronic lymphoid leukemia (CLL), we performed flow-cytometry analysis of expression on ALL blasts (n=18) and CLL lymphocytes (n=21) from blood and correlated data with clinical parameters. RESULTS: In ALL, mature B-linear blasts expressed higher levels of 82kDa proMMP-9 compared to T-linear blasts. Elevated levels of 82kDa proMMP-9 were found in elderly patients and at patients with relapse. No correlation was observed on blood cells and extramedullary disease. In CLL, the 82kDa proMMP-9 expression did not correlate with any of the clinical parameters. CONCLUSION: Our findings suggest that higher levels of 82kDa proMMP-9 expression on blast cells may correlate with a more unfavorable ALL-subtype. Further studies are required to clarify the prognostic role of the 82kDa pro-MMP-9 expression.