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We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 μg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.

Summary Transplantation of T- and B-cell depleted allografts from haploidentical family donors was evaluated within a prospective phase II trial in children with acute lymphoblastic leukaemia, acute myeloid leukaemia and advanced myelodysplastic syndrome (n = 46). 20 patients had active disease; 19 patients received a second or third stem cell transplantation (SCT). Toxicity-reduced conditioning regimens consisted of fludarabine or clofarabine (in active disease only), thiotepa, melphalan and serotherapy. Graft manipulation was carried out with immunomagnetic microbeads. Primary engraftment occurred in 88%, with a median time to reach >1·0 × 109/l leucocytes, >20 × 109/l platelets and >0·1 × 109/l T-cells of 10, 11 and 50 days, respectively. After retransplantation, engraftment occurred in 100%. Acute graft-versus-host disease (GvHD) grade II and III-IV occurred in 20% and 7%, chronic GvHD occurred in 21%. Both conditioning regimens had comparable toxicity. Transplant-related mortality (TRM) was 8% at one year and 20% at 5 years. Event-free survival at 3 years was: 25% (whole group), 46% (first, second or third complete remission [CR], first SCT) vs. 8% (active disease, first SCT) and 20% (second or third SCT, any disease status). This approach allows first or subsequent haploidentical SCTs to be performed with low TRM. Patients in CR may benefit from SCT, whereas the results in patients with active disease were poor.

In acute myeloid leukemia (AML), the leukemia-initiating cell is found within the CD34(+)/CD38(-) cell compartment. Over the last years evidence grew that AML is initiated and propagated by leukemic stem cells (LSCs). Conceivably, these most immature leukemia cells are more resistant to therapy and subsequently initiate relapse. The authors studied 17 patients with childhood AML treated according to the AML-BFM 98/04 protocol. At diagnosis, the authors determined the characteristic immunophenotype of the leukemic cells by flow cytometry and investigated the expression of CD34, CD38, and CD45 to define a population of immunophenotypically immature cells (CD34(+)/CD38(-)/CD45(-/low)) enriched for LSCs in many cases of AML. The authors compared the fraction of this population of all myeloid cells at diagnosis with event-free survival. Kaplan-Meier analysis revealed significant higher event free survival of patients with low CD34(+)/CD38(-)/CD45(-/low) cell proportion (0.68%) compared to patients with high burden of this population (0.83%; log-rank P.04). This correlation was not found for the total number of CD34(+) cells. This is the first study to show that a higher proportion of immature CD34(+)/CD38(-)/CD45(-/low) blasts at diagnosis correlates with unfavorable prognosis in childhood AML. The results suggest that a large CD34(+)/CD38(-)/CD45(-/low) population reflects a higher fraction of LSCs, leading to increased chemotherapy resistance and elevated relapse rate. Thus the initial frequency of CD34(+)/CD38(-)/CD45(-/low) cells may serve as a prognostic marker in pediatric AML. Future treatment in childhood AML should specifically target this immature population as well as the mature blast population.

Haploidentical hematopoietic cell transplantation (HHCT) using CD34 selected grafts is complicated by slow engraftment and immune reconstitution. Engraftment and immune reconstitution might be improved using CD3/CD19-depleted grafts and reduced intensity conditioning (RIC). We report on 28 patients after HHCT with CD3/CD19-depleted grafts using RIC, which were prospectively evaluated for engraftment and immune reconstitution. Engraftment was rapid with full chimerism reached on day +15 after HHCT. T-cell reconstitution was delayed with a median of 205 CD3+ cells/μl, 70 CD3+CD4+ cells/μl and 66 CD3+ CD8+ cells/μl on day +100, respectively. A skewed T-cell receptor-Vβ repertoire with oligoclonal T-cell expansions to day +100 and normalization after day +200 was observed. B-cell reconstitution was slow with a median of 100 CD19+ CD20+ cells/μl on day +150. Natural killer (NK) cell engraftment was fast reaching normal values on day +20. An increased natural cytotoxicity receptor and NKG2A, but decreased NKG2D and KIR expressions were observed on NK cells until day +100. We observed a positive impact of donor lymphocyte infusions on immune reconstitution. In conclusion, after HHCT, using CD3/CD19-depleted grafts and RIC, T- and B-cell reconstitution is delayed, whereas NK-cell reconstitution occurs early and fast.

Background Natural killer cells have been demonstrated to exert remarkable graft-versus-leukemia effects after haploidentical transplantation. Acquisition of both, inhibiting and activating, receptors on developing natural killer cells is an important step in their functional maturation. Here, we report on the reconstitution of natural killer receptors after haploidentical transplantation of T-and B-cell (CD3/CD19) depleted grafts with co-transfusion of natural killer cells in children and its influence on natural killer cell activity and clinical outcome.Design and Methods We analyzed reconstitution patterns of natural killer receptors at different time intervals after haploidentical transplantation by multi-color flow cytometry. Natural killer cell activity and antibody-dependent cellular cytotoxicity was tested against cell lines and leukemic blasts in vitro. Survival was analyzed using Kaplan-Meier estimates.Results Recovery of CD56+/CD16+ cells was fast with high cytolytic activity against K562 and strong antibody-dependent cellular cytotoxicity activity against neuroblastoma and leukemic blasts as early as day 14 posttransplant. KIR reconstitution showed a predominance of KIR negative natural killer cells early after transplantation and an early reconstitution of CD158b compared to CD158a and CD158e. These differences were independent of presence or absence of the corresponding KIR ligands in donors or recipients. This reconstitution pattern was associated with a higher relapse probability of patients homozygous for HLA-C1-alleles compared to patients homozygous or even heterozygous for HLA-C2-alleles.Conclusions Our results indicate a fast recovery of functional and alloreactive natural killer cells with a constant KIR pattern after haploidentical transplantation with T- and B-cell depleted grafts. Moreover, these natural killer cells can mediate antibody-dependent cellular cytotoxicity and therefore may allow for an early use of antibodies against residual malignant cells.

Graft failure is a life-threatening complication after transplantation of hematopoietic stem cells. We report a cohort of 11 pediatric patients with leukemias ( n = 8) and severe aplastic anemia ( n = 3) who experienced graft rejection after myeloablative transplantation from mismatched related donors ( n = 6) or after cord blood or matched unrelated donor transplantation ( n = 5). In the latter, the original donor was not available anymore. All patients were re-transplanted with CD34 + selected or CD3/CD19 depleted stem cells from a second, haploidentical donor. Median time span from diagnosis of rejection to second transplant was 9 days. Reconditioning regimens comprised total lymphoid irradiation, thiotepa, fludarabine, ATG/OKT3 and were well tolerated. A median number of 23.5 × 10 6 /kg stem cells with 95,000/kg residual T-cells were infused. Sustained engraftment of neutrophiles/platelets and complete donor chimerism was achieved in all patients (ANC > 500/μl: 9 (11–32) days). No GvHD > grade II was observed. 8/11 patients are disease free (median follow up 1.9 years; 1 year-EFS = 72%). Causes of death were: pneumonitis, infection, relapse. Thus, haploidentical transplantation represents a realistic option to rescue patients with graft failure within a short time span, for whom a second donation from the original donor is not available. The use of different donors may contribute to avoid a second rejection.

The impact of human leucocyte antigen (HLA) class I expression intensity and killer-cell immunoglobulin-like receptor (KIR)-mismatch was investigated on natural killer (NK)-cell mediated lysis of B-lineage leukaemic blasts from 21 paediatric patients in vitro. Blast susceptibility to standardised NK-activity and HLA-expression differed widely. A clear association between HLA-molecules/cell (range 442 000-13 000) and specific lysis (mean 59%, range 13-98%) was observed (r(2) = 0.68). A compound model incorporating HLA-expression and KIR-ligand-mismatch provided an even stronger association (r(2) = 0.87), whereas KIR-ligand-mismatch alone was not significant. Assessment of these factors might identify patients who could benefit from NK-mediated graft-versus-leukaemia effects after mismatched stem cell transplantation.

Transplantation of HLA-mismatched stem cells may allow determination of chimerism status of single cells by differential expression of HLA molecules. Monoclonal antibodies against HLA antigens can be used to determine the HLA type of sub-populations by standard flow cytometry. Blood samples from 23 patients transplanted from HLA-mismatched family donors were monitored using HLA-specific antibodies. Suitable antibodies could be found for all donor recipient pairs by using differences in HLA Bw4 and Bw6 groups or other serological antigens. Pretransplant controls of donor and recipient were used to correct for variable fluorescence intensities of the antibodies and sub-populations. Owing to the high sensitivity, cell populations with a minimum frequency of 0.1% were detectable. Flow-cytometric analysis was confirmed by chimerism analysis of immunomagnetically isolated T cells by standard PCR technique. In addition to chimerism evaluation, HLA antibodies improved the detection of leukemic cells after transplantation with aberrant phenotype. In conclusion, flow cytometry using antibodies against HLA antigens is an interesting tool for determination of chimerism and minimal residual disease after HLA-mismatched transplantation. Information about the chimerism status is given on a single-cell level and allows fast and convenient analysis of sub-populations.

Donor choice in haploidentical stem cell transplantation: fetal microchimerism is associated with better outcome in pediatric leukemia patients

Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, myelodysplastic syndrome and nonmalignant diseases, who received αβ T- and B-cell-depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius. Graft manipulation was carried out with anti-TCRαβ and anti-CD19 Abs and immunomagnetic microbeads. The γδ T cells and natural killer cells remained in the grafts. Primary engraftment occurred in 88%, acute GvHD (aGvHD) grades II and III-IV occurred in 10% and 15%, respectively. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F (n=19). Median time to reach >100 CD3+ cells/μL, >200 CD19+ cells/μL and >200 CD56+ cells/μL for the whole group was 13, 127 and 12.5 days, respectively. Compared with a historical control group of patients with CD34+ selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs 27 and 397 vs 163 cells/μL), for CD3+4+ at day +30 (58 vs 11 cells/μL) and for CD56+ at day +14 (622 vs 27 cells/μL). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multicenter trial.

Summary A recombinant bispecific single-chain fragment variable antibody (bsscFv), directed against the B-cell antigen CD19 and the low affinity Fc-receptor FcγRIII (CD16), was designed for use in the treatment of patients with leukaemias and lymphomas. The Fc-portions of whole antibodies were deliberately eliminated in this construct to avoid undesired effector functions. A stabilised bsscFv, ds[CD19 × CD16], was generated, in which disulphide bonds bridging the respective variable light (VL) and variable heavy (VH) chains were introduced into both component single-chain (sc)Fvs. After production in 293T cells and chromatographic purification, ds[CD19 × CD16] specifically and simultaneously bound both antigens. The serum stability of ds[CD19 × CD16] was increased more than threefold when compared with the unstabilised counterpart, while other biological properties were not affected by these mutations. In antibody-dependent cellular cytotoxicity experiments, ds[CD19 × CD16] mediated specific lysis of both CD19-positive malignant human B-lymphoid cell lines and primary tumour cells from patients with B-cell chronic lymphocytic leukaemia or B-cell acute lymphoblastic leukaemia. Natural killer cells, mononuclear cells (MNCs) from healthy donors and, in some instances, MNCs isolated from patients after allogeneic stem cell transplantation, were used as effectors. Thus, ds[CD19 × CD16] holds promise for the treatment of CD19+ B-lineage malignancies.

Relapse is a major problem after allogeneic transplantation in children with acute B-lineage lymphoblastic leukemias (ALL) and lymphomas and additional therapeutic strategies are needed to increase graft versus leukemia effects without inducing graft versus host disease (GvHD). Several studies have shown the efficacy of a humanized CD20 antibody (rituximab) for treatment of CD20+ malignancies together with conventional chemotherapy but less is known about its post transplant usefulness. We studied the ability of rituximab to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with effector cells and complement from patients who were transplanted with T-cell-depleted grafts from unrelated or mismatched related donors. Highest lytic activity (ADCC) was observed against leukemia-derived MHH4 cells and Burkitt's lymphoma-derived Raji cells in the first months after transplantation, corresponding to the high percentage of regenerating CD56+ CD16+ cells. Moreover, primary cryopreserved ALL-blasts from a pediatric patient were also efficiently lysed. Increased lysis was obtained after stimulation with interleukin-2. Combination of ADCC and CDC had additive effects. These findings encourage clinical trials on the use of rituximab for improving minimal residual disease control and relapse prevention after allogeneic high-risk transplantation in the small group of pediatric patients with CD20+ leukemias/lymphomas.

Relapse is a major problem after transplantation in children with acute B-lineage leukemias, and new therapies are needed to increase graft-versus-leukemia (GvL) effects without inducing graft-versus-host disease (GvHD). Here, we studied the ability of effector cells recovered from patients after transplantation with positive-selected stem cells from alternative donors to induce antibody-dependent cellular cytotoxicity (ADCC). For this purpose, a chimeric CD19 antibody, CD19-4G7chim, was generated. This antibody efficiently mediated ADCC against primary acute lymphoblastic leukemia (ALL) blasts by using purified natural killer (NK) cells from healthy donors or mononuclear cells from patients as effector cells. Increased lysis was obtained after stimulation of effector cells with interleukin-2 (IL-2). ADCC was not prevented by inhibitory effects mediated by HLA class I. We propose that treatment with chimeric CD19 antibodies leading to ADCC by donor-derived NK cells may become a therapeutic option for the post-transplantation treatment of minimal residual B-lineage ALLs.

Extracorporeal photopheresis (ECP) is beneficial in patients with T-cell-mediated disorders, including GvHD, but the underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells characterized by their capacity to suppress T-cell proliferation. We quantified MDSCs by flow cytometry in peripheral blood from patients after BMT with GvHD before and after ECP treatment, patients after BMT but without GvHD and age-matched healthy controls. MDSC functionality was analyzed using T-cell proliferation, cytokine release and arginase activity. GvHD patients showed increased baseline percentages of neutrophilic MDSCs (PMN-MDSCs) compared with healthy controls and patients after BMT without GvHD. ECP treatment in GvHD patients rapidly increased circulating percentages of PMN-MDSCs. Functionally, PMN-MDSCs efficiently dampened Th1 and Th17 responses and were paralleled by an increase of cellular and extracellular arginase activity. Following ECP longitudinally over 16 weeks, two GvHD responder subgroups were identified, with group one continuously increasing PMN-MDSCs and group two with stable or decreasing PMN-MDSCs over time. This study demonstrates for the first time that ECP increases T-cell-dampening PMN-MDSCs in GvHD patients, a finding that should be confirmed in larger series of GvHD patients.

IL-15-stimulated CD3/CD19-depleted stem-cell boosts in relapsed pediatric patients after haploidentical SCT

BACKGROUND T-cell-depleted allografts may exhibit delayed T-cell recovery, severe infections, and relapse after haploidentical hematopoietic stem cell transplantation (HSCT). Required donor lymphocyte infusions containing nonalloreactive cells may transfer immune function without causing graft-versus-host disease. METHODS We developed an ex vivo approach for the immunomagnetic depletion of alloreactive CD25+, CD69+, and HLA-DR+ T-cells. To achieve highest rates of alloantigen expression, we cocultured peripheral blood mononuclear cells (PBMNCs) with PBMNCs (A/B*), dendritic cells (A/B* DCs), or cytokine-pretreated PBMNCs (A/B* cyt cells). Functional analyses were performed after depletion. RESULTS After coculture with PBMNCs (A/B* cells), 29% of T-cells became CD25+, CD69+, and HLA-DR+. In modified mixed lymphocyte reactions (MLR) (A/B* cyt cells and A/B* DCs), 35% and 37% of T-cells became CD25+, CD69+, and HLA-DR+. Alloactivation was confirmed by interferon gamma release and proliferation. Immuno-magnetic depletion produced

Delayed immune reconstitution is 1 of the major contributions to the morbidity and mortality after haploidentical transplantation. Patients with a slow recovery of the innate and especially of the adaptive immune system are at high risk for severe and often lethal infections. The reason for delayed immune reconstitution after haploidentical transplantation include the T cell depletion (TCD) of the graft, the thymic dysfunction induced by pretransplant chemotherapies and by the conditioning regimens, and the occurrence of graft-versus-host disease (GVHD) and its treatment. The detailed analysis, understanding, and manipulation of the reconstitution of the cellular immune system will be of utmost importance to overcome the posttransplant immunodefcient status, and should result in a reduced risk of severe and overwhelming infections and hopefully also to a reduced risk of relapse through better immunological control of residual malignant cells.

We present an update of our results with transplantation of highly purified stem cells from one to three loci mismatched parental donors. Sixty-three pediatric patients with acute lymphoblastic leukemias (n = 32), acute myeloid, chronic myeloid and myelomonocytic leukemias (n = 13), myelodysplastic syndromes (n = 4), lymphomas (n = 4), and various nonmalignant diseases (n = 10) underwent transplantation. Mobilized peripheral-blood stem cells were selected with either anti-CD34- or anti-CD133-coated microbeads. Patients received a median of 19.5 x 10(6) purified cells and

GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Haploidentical hematopoietic stem cell transplantation (hHSCT) from a parent to a child has become well established for pediatric patients with high risk leukemia in the absence of an HLA-matched donor. Donor choice in hHSCT has been an issue addressed by several groups. Some showed a beneficial impact of certain Killer immunoglobulin-like receptor (KIR)/KIR ligand constellations in different transplantation settings. Others demonstrated an improved outcome after mother-to-child transplantations compared to father-to-child transplantations in terms of lower transplant-related mortality and lower incidence of graft-versus-host disease (GvHD). Obviously, pregnancy leaves an imprint on the mother’s immune system, which is beneficial in hHSCT from mother to child. During pregnancy, a fetal microchimerism (FM) is established in the mother by fetal cells crossing the placental barrier dividing maternal and fetal circulation. In some mothers, fetal cells can be detected in peripheral blood for more than three decades after birth. Yet, not all mothers develop a persisting FM. CD56brightNK cells make up approximately 70% of the lymphocytes in the decidua during the first trimester of pregnancy and represent the major cell subset interacting with cells of the developing fetus. Not surprisingly, certain maternal KIR/fetal KIR ligand combinations are associated with recurrent spontaneous abortions or preeclampsia. We hypothesized that (i) FM plays a role in hHSCT, and that (ii) FM may be influenced by the KIR pattern on maternal NK cells and KIR ligands on the child’s cells. In a multicenter retrospective study, we analyzed 46 pediatric patients from 6 German transplantation centers (Tübingen, Frankfurt, Düsseldorf, Heidelberg, Jena, and Hamburg) who underwent first T-cell depleted hHSCT from a parent following conditioning with fludarabine, thiotepa, melphalan and ATG for standard indications: acute lymphoblastic leukemia (n=10), acute myeloid leukemia (n=10), juvenile myelomonocytic leukemia (n=1), lymphoproliferative disease (n=2), myelodysplastic syndrome (n=4), hemophagocytic lymphohistiocytosis (n=3), immune deficiencies (n=9), and others (n=7). Excess DNA of the donors extracted from peripheral blood prior to transplantation was assayed for fetal DNA (in mother-to-child transplantations), and for KIR genotype by real-time PCR. Mothers who had at least one fetal cell within 250,000 maternal cells could be detected were considered as FM+. Although we confirmed that, overall mothers as a group are better donors than fathers, in closer analysis this held only true for FM+ mother. Overall survival was significantly higher in the group of patients receiving stem cells from their FM+ mother compared to the patients grafted from their FM¯ mother (72% vs. 29), or father (72% vs. 50%). Most interestingly, looking at the minimal residual disease (MRD) negative acute leukemia patients undergoing first allogeneic HSCT for standard indications, overall survival was also significantly higher after hHSCT from FM+ mothers than from FM¯ mothers (66% vs. 26%) and there was a trend towards lower risk of relapse in the FM+ group (25% vs. 52%). There was no case of grade IV acute GvHD and only one or no case of acute GvHD grade III in all groups. Percentages of grade I + II acute GvHD did not differ significantly among FM+and FM¯ groups. Reasons for persistence of fetal cells in some, but not all mothers are controversial. While fetal microchimerism is detected during pregnancy and early after birth in almost all mothers, it is reported to decrease below the detection limit in many mothers over time. In our cohort, we found no correlation of FM with age or gender of the respective child. Since a poor outcome after hHSCT has been reported for patients that are homozygous for HLA-C1, we also grouped our cohort into a HLA-C1 heterozygous and a homozygous group. Interestingly, not only an improved outcome, but also a higher average FM could be seen in the HLA-C1 heterozygous group compared to the C1 homozygous group. Furthermore, mothers expressing KIR2DS1 showed a significantly higher level of FM compared to those negative for this activating KIR. These findings suggest a certain role for KIR and HLA-C on the persistence of fetal microchimerism in mothers. In conclusion, detection of fetal microchimerism may contribute to identification of the most promising parental donor in haploidentical stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

We present a clinical scale method for immunomagnetic separation of CD56+ donor natural killer cells for adoptive immunotherapy of pediatric leukemias after allogeneic transplantation. This time-saving and partially automated procedure employed CD56+ selection followed by CD3+ depletion, resulting in a median purity of 98.6% NK cells and a four-log depletion of T cells. The enriched NK cells demonstrated high cytotoxic activity against K562 target cells and fresh leukemic blasts with low HLA class I expression, which could be further enhanced by IL-2 stimulation. Lysis of NK-insensitive leukemic cells with high HLA class I expression could also be demonstrated via ADCC. Due to the high degree of T cell depletion, alloreactive proliferation in mixed lymphocyte cultures and response to T cell-specific mitogen stimulation was profoundly decreased. Our results suggest that, even in the case of mismatched donors, infusions of donor NK cells with extremely low T cell content may be a promising treatment option for leukemic minimal residual disease after allogeneic transplantation without risk of inducing severe GVHD.

Allogeneic stem cell transplantation is still the curative treatment option for the majority of patients with severe aplastic anemia and refractory cytopenias. However, a HLA-matched donor is not available for all patients. Alternative donor transplantation has been an experimental treatment option, limited by high rejection rates and transplant related mortality. We performed a prospective clinical trial to evaluate the safety and feasibility of haploidentical stem cell transplantation, since haploidentical family members are always available donors. We investigated a cohort of 10 pediatric patients with severe aplastic anemia or myelodysplatic syndrome transplanted (refractory cytopenia) with T-cell depleted grafts between 2004 and 2011. 7 patients had myelodyslastic syndrome with refractory cytopenia (MDS-RC), 3 had severe aplastic anemias (SAA) refractory to immunosuppresive treatment. 3 patients received a 2nd SCT after rejecting the graft from matched donors. Median age was 11.4 years. Standard conditioning regimen consisted of Fludarabin 3-4x40mg/m2, Thiotepa 1-3x5mg/kg, Melphalan 2x70mg/m2 (n=8) and serotherapy using OKT3 (n=5) and ATG (n=5). 8 patients received additional total lymphoid irradiation (TLI 7 Gy) to prevent graft rejection. In vitro graft manipulation was carried out by direct depletion using antiCD3/19 magnetic microbeads. A median number of 10.1x106 CD34+ progenitor cells and 27x103 T-cells/kg body weight (BW) were transfused. Pharmacological GvHD prophylaxis (graft vs. host disease) was carried out with Mycofenolate until day 60, if residual T-cells in the graft exceeded 25000/kg BW. Primary engraftment occurred in all patients Median time to reach 500/µl neutrophiles was 9 days (9-11). Independence from platelet substitution was reached after 13 days (8-16). Three patients rejected the graft later on. 6/10 patients had no signs of acute GvHD or GvHD grade I, 2 patients had GvHD grade II. TRM at day +100 and after 1 year was 0% and 20%, respectively. Event free survival (EFS) at 3 years was 80%. Conclusions Haploidentical SCT with T-cell depleted grafts is a therapeutic option for refractory cytopenias and severe aplastic anemia after nonresponse to immunosuppressive treatment if no HLA-matched donor is available. Recovery of neutrophiles and platelets were fast and TRM was low, even if retransplantation was necessary. Since spontaneous outcome of these conditions are poor, alternative donor SCT is a realistic option for these patients. Disclosures: No relevant conflicts of interest to declare.

T-cell depletion of the graft is an effective method to prevent or completely avoid Graft-versus-Host Disease (GvHD) in haploidentical stem cell transplantation. In order to increase the T-cell depletion efficacy while maintaining the anti-tumor and anti-infectious properties of the graft, we have investigated a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, Natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of posttransplant EBV-associated lymphoproliferative disease. The CliniMACS system was used for manipulation of peripheral stem cell grafts from full haplotype mismatched family donors in 35 patients. Results The overall depletion of αβ+ T-cells was highly effective with 4.6 log (range 3.8–5.0). Patients received a median number of only 14 x 103/kg residual αβ+ T-cells. Recovery of CD34+ stem cells was 72%, and the median number of infused CD34+ stem cells was 12 x 106/kg (range 5-38 x 106/kg). Additionally, the patients received 2 types of potential antileukemic effector cells: 107 x 106/kg (range 35 -192 x 106/kg) CD56+ NK-cells and 11 x 106/kg (range 5–30 x 106/kg) γδ+ T-lymphocytes. Diagnoses were ALL (n=20), AML/MDS/JMML (n=9), nonmalignant diseases (n=4), solid tumors (n=2); disease status: CR2-CR6 (n=17), active disease (n=18). 23 patients received a second or third SCT (65%). A toxicity reduced conditioning regimen (fludarabin 40mg/m² or clofarabin 50mg/m² (day -8 to d -5), thiotepa 10mg/kg (d -4), melphalan 70mg/m² (d -3 and d -2) was used. The anti CD3 specific OKT3 antibody was used as rejection prophylaxis from day -8 to day -1 without affecting cotransfused effector cells because of its short half-life period in the first 7 patients. However, due to its restricted availability, the substance was substituted since 2011 by a reduced ATG-F dose (15mg/kg) given at start of the conditioning regimen in order not to impair NK and γδ+ T-cells of the grafts (1 mg/kg d -12, 4 mg/kg d -11, 5 mg/kg d -10 and -9; n=28 patients). Short course MMF (until day +30) was given in 25 patients. Graft rejection occurred in 14% of the patients. However, after reconditioning and second stem cell donation, final engraftment was achieved in all patients. The median time to reach neutrophil and platelet recovery in patients with primary engraftment was 10 and 11 days respectively. All patients showed a rapid immune reconstitution with 250 (OKT3 conditioning) and 273 (ATG conditioning) CD3+ T-cells/µl, 30 (OKT3) and 47 (ATG) CD3+4+/µl and 300 (OKT3) and 382 (ATG) CD56+ NK-cells/µl at day +30 posttransplant. γδ+ T-cells started to expand faster than αβ+ T-cells in the early post-transplant period (156 vs. 82 cells/µl at day +30) whereas at day +90, αβ+ T-cells were predominant (170 vs. 134 cells/µl). Acute GvHD grade 0-I occurred in 25 patients (71%); 6 patients had GvHD II (17%), 3 patients had GvHD III (9%) and one patients experienced GvHD grade IV (3%). 3 patients experienced chronic GvHD (8%). Incidence of acute GvHD was not influenced by the number of residual T cells or by the type of serotherapy. 1 year EFS for patients with acute leukemias was 66% (any CR) and 14% (active disease).TRM at 1 year was 20%. Conclusions These data indicate that transplantation of TcR αβ+/CD19 depleted cells from a haploidentical donor results in sustained engraftment, remarkably fast immune reconstitution and low incidence of both acute and chronic GvHD. OKT3 could be substituted by ATG without negative effects. The anti-leukemic efficacy of this approach in comparison to other methods of T-cell depletion needs to be evaluated with a longer patient follow-up. Disclosures: No relevant conflicts of interest to declare.

Abstract 4141 T cell depletion with magnetic microbeads can effectively reduce GvHD rates after stem cell transplantation from both mismatched related donors as well as from matched or partially matched unrelated donors. However, T cell recovery is markedly delayed after this procedure and T cell mediated antileukemic effects may be reduced. Thus, we focused on the rapidly regenerating donor derived NK cell system and addressed the question, whether its functional activity would influence the probability of relapse in a long term analysis. Temporal development of NK cell activity was monitored in 47 pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n=18) and mismatched related (haploidentical, n=29) donors with a median follow up of 7.4 years (2.1–12). 38 patients had CR1-3, 9 patients had active disease at time of transplantation. EFS and relapse rate at 5 years for the entire group were 36% and 40%, respectively (EFS and relapse rate for ALL patients in CR1-3: 50% and 36%; EFS and relapse rate for AML/MDS patients: 22% and 30%). CD34+ selection with magnetic microbeads resulted in 8×103/kg residual T cells. No posttransplant immune suppression was given. 89% of the patients had no GvHD, 9% had GvHD grade I and only 2 patients had GvHD grade II or III. NK cells recovered rapidly after transplantation (300 CD56+/μL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/μL at day 90). NK activity was measured as specific lysis of K562 targets several times after transplantation (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 5 years, p Disclosures: No relevant conflicts of interest to declare.

Abstract 1956 Graft failure is a rare but life-threatening complication after hematopoetic stem cell transplantation. Treatment comprises immunoablative reconditioning regimens and a second stem cell donation as soon as possible to minimize the time of pancytopenia and its sequelae. We report a cohort of 21 pediatric patients with acute leukemias (complete remission (CR)1-2 n= 7, active disease n=4; lymphatic n=7, myeloic n=4), myelodysplastic syndrome (n=2), immunodeficiencies (n=3), aplastic anemias (n=3), and hemoglobinopathies (n=2) who experienced graft failure (nonengraftment n=1; rejection n=20) after busulphan or melphalan based myeloablative transplantation from mismatched related donors (n=14) or matched unrelated or related donors (n=7). All patients were retransplanted with T cell depleted grafts (CD34 positive selection n=3 or CD3/CD19 depleted grafts n=18) from a haploidentical donor. Median time from diagnosis of graft rejection to second transplantation was 19 days. Reconditioning regimens consisted of irradiation (total lymphnode irradiation 7Gray (Gy) or TBI 2 Gy), fludarabine (120mg/m2) in combination with thiotepa (5mg/kg) or cyclophosphamide (60mg/kg body weight (bw)) and ATG/OKT3. A median number of 15×106/kg of body weight stem cells with 54×103/kg residual T cells were infused. Mofetilmycophenolat was given as Graft vs. Host Disease (GvHD) prophylaxis, if residual T cells exceeded 25 000/kg bw. Sustained engraftment was achieved in 20 out of 21 patients. One patient died before engraftment. Median time to absolute neutrophile counts above 500/μl was 9 (9–24) days. Independence from platelet substitution was reached at a median time of 10 (8–22) days. Only 1 patient developed GvHD °III, 10% developed GvHD °II, 33% of all patients developed GvHD °I, and 52% had no signs of GvHD. T cell recovery was delayed, however no lethal viral infection occurred. Severe organ toxicity was observed in 5 patients (bronchiolitis obliterans n=1, hemorrhagic cystitis n=2, leukencephalopathy n=2). Event free survival (EFS) of all patients at 5 years was 71%. 5 year EFS of patients with leukemias in complete remission was 83%. Patients with non-malignant diseases had 5 year EFS of 80%. Transplant related mortality at one year was 10%. Causes of death were: multi organ failure (n=1), fungal infection (n=1) and bronchiolitis obliterans organizing pneumonia, 4 patients with acute leukemias relapsed. None of the patients rejected the second graft. Thus, transplantation of stem cells from haploidentical donors after reconditioning is a realistic option to rescue patients with graft failure within a short time span. Graft rejection itself may have an antileukemic effect since patients with malignant diseases showed a favourable EFS. The use of a different donor in combination with irradiation based conditioning regimens may help to avoid a second rejection and results in a robust engraftment and low TRM. Disclosures: No relevant conflicts of interest to declare.

Abstract 4133 The Killer immunoglobulin-like receptor (KIR) gene family consists of 17 genes (15 KIR genes and 2 pseudogenes) encoding for the different receptors and is a key regulator of Natural Killer (NK) cells. Their diversity is similar to the HLA-system, however they segregate independently from the HLA-system and every human being possesses an individual pattern of KIR genes which can be assigned to either KIR haplotype A or B. Haplotype A is characterized by a relatively fixed gene content of inhibitory receptors. The presence of at least one activating KIR-receptor is necessary for defining haplotype B. The importance of the KIR system has been demonstrated in haploidentical as well as in HLA-matched transplantation in adult patients with AML, but not with ALL. Patients who received a haploidentical transplant from a KIR ligand-ligand mismatched donor had a significant lower risk of relapse. In more recent studies in adult patients with AML but not with ALL, a significant influence of the donor KIR haplotype was demonstrated after allogeneic transplantation from HLA-matched sibling or unrelated donors, and patients who received grafts from haplotype B donors had a significant lower risk of relapse. In contrast to the results in adult patients, it has been shown in pediatric ALL that children who received an haploidentical transplant from KIR receptor-ligand mismatched donors had a better event-free survival. The influence of the donor KIR haplotype in pediatric haploidentical transplantation in patients with ALL has not yet been investigated sofar. We have therefore analyzed the influence of the donor KIR haplotype on the risk of relapse in 48 children with very high risk ALL, who received an allogeneic transplant from a haploidentical donor at our center. The presence or absence of the 15 KIR genes was determined by real-time PCR as described by Alves et al. (Tissue Antigens 2008) and donors were assigned the A/A or B/x haplotype. Genotypes for the centromeric (Cen) or telomeric (Tel) parts of the KIR locus were assigned according to the absence or presence of one or more B haplotype defining KIR genes. Most of the patients (n=28) were conditioned with a non-TBI-based reduced intensity regimen, whereas 20 patients received a TBI-based preparative regimen. At time of transplant, 38 patients were in remission and 10 children had refractory leukaemia. The graft comprised mobilised peripheral stem cells depleted ex-vivo from T-cells by either CD34+ positive selection or CD3/19 negative depletion. Of the 48 donors, 12 had KIR haplotype A and 36 KIR haplotype B. Patients grafted with a KIR haplotype A donor were more likely to relapse (odds ratio 4.90; p=0.02), whereas patients who received a graft from a KIR haplotype B donor had a five-fold lower risk of relapse. We also analysed the risk of relapse according to the haplotype B-content-score system (from 0 to 4) described by Cooley et.al (Blood 2010). Briefly, the B-content score is calculated by adding the number of CenB and/or TelB motifs in each genotype.We found a reduced risk of relapse with an increasing B-content score, with 58% of relapse for a score of 0 (n=12), 31% for a score of 1 (n=16), 20% for a score of 2 (n=15) and 0% for a score of 3 (n=5). In addition, the risk of relapse was much lower in patients grafted with a donor TelB haplotype (odds ratio 7.3, p We conclude from our results that a donor KIR B haplotype with a high B-content score should be favored as haploidentical donor and that KIR genotyping should be implemented in the donor selection algorithm in haploidentical transplantation for children with ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1728 Cell surface glycoconjugates have important functions in the modulation of immune cell maturation, activation and homeostasis. Glycans on cell surfaces are generally sialylated. Sialic acids are predominantly found on the non-reducing end of oligosaccharide chains of glycoconjugates. They are recognized by sialic acid-binding immunoglobulin-like lectins (siglecs). CD33-related siglecs are receptors predominantly expressed on immune cells. Most of the CD33-related siglecs display a tyrosine-based inhibitory motif (ITIM) known to downregulate effector cell functions. So far, identification of ligands for siglecs was hampered by low affinity between proteins and glycans, but by glycan array analysis α2,8-, α2,3- as well as α2,6-linked sialic acids were identified as the carbohydrate part of the ligand. In order to analyse the expression of physiological siglec ligands on different lymphocyte subpopulations, the extracellular three domains of siglec-7 were cloned. To allow for mammalian glycosylation, the protein was expressed in 293T cells. To overcome the low affinity of lectins, siglec-7 was enzymatically biotinylated and oligomerized using streptavidin in analogy to HLA tetramers. Flow cytometric analysis of lymphocytes revealed reproducible binding patterns of siglec-7 tetramers. Specificity was ensured by treatment of PBMC with neuraminidase, specifically cleaving terminal sialic acids in α2,3, α2,6 and α2,8 linkages. On T cells, the density of siglec-7 ligands increased 3.5-fold during activation. This is contradictory to results using plant lectins, like Maackia amurensis lectin and Sambucus nigra lectin, which indicated an 1.8- and 1.5-fold decrease, respectively. Activation of NK cells did not affect the siglec-7 ligand expression. Interestingly, B cells could be divided in a siglec-7 ligand positive and a siglec-7 ligand negative population. This may reflect different differentiation or activation stages, which were not found with any antibody combination directed against typical protein B cell markers. Malignant transformation is often associated with aberrant cell surface glycosylation. In acute lymphoblastic leukemia (ALL), blasts of the more aggressive T-ALL express 17 fold higher amounts of siglec-7 ligand than blasts of the B cell lineage (cALL), where the siglec-7 ligand was nearly not detectable. These findings may contribute to the poor prognosis of T-ALL by a possible immune escape achieved by siglec-ligand binding induced inhibition. Since immune evasion mechanisms are also described for other malignancies, we additionally analysed rhabdomyosarcoma (RMS) cells. Siglec-7 ligand could be found on all tested RMS cell lines in different expression levels, independent of the classification. Siglec-7 is a known inhibitory receptor expressed by NK cells and T cells. The effect of siglec-7 ligand expressed by malignant cells on NK cell cytotoxicity was analysed. By the removal of all terminal sialic acids on the surface of RMS cells by neuraminidase treatment, the NK cell-induced cytotoxicity could be increased by 20%. Coating of siglec-7 ligands on target cells by preincubation with recombinant siglec-7 protein also resulted in an increased cytotoxicity. These results show that sialic acids play an important role in the immune system as physiologic ligands. However, the expression of siglec-7 ligands on malignant cells may contribute to immune evasion mechanisms. Disclosures: No relevant conflicts of interest to declare.

Abstract 4652 Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation and allows to minimize GvHD despite HLA incompatibility. However, posttransplant recovery of donor derived T cells is delayed after various graft manipulation procedures and may result in severe infections. Methods to improve this recovery are of great importance. Here we present immune reconstitution data in patients who received CD3/CD19 depleted stem cells in combination with melphalan based or standard conditioning regimens. 32 patients with ALL (n=14), AML/MDS (n=17), CML (n=1) were included. T and B cells were directly depleted using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACSTM device. The patients received either TBI or Bu i.v. and OKT3 (n=9) or a reduced intensity conditioning (“RIC”: Mel 140mg/m2, Flud 160mg/m2, TT 10mg/kg, OKT3, n= 23). Absolute numbers of lymphocyte subsets per microliter on day 90 were compared within these both groups and with a historical control group (patients with leukemias who received CD34 selected grafts and TBI or Bu based standard conditioning regimen in combination with ATG, n=28). CD3+4+, CD3+8+ and total numbers of CD3+ of patients after CD3/CD19 depletion were significantly higher in the RIC-group than in the TBI/Bu-group (mean numbers: 85.83 vs. 38.84; 133.46 vs. 19.69; 270.27 vs. 63.99; p Comparison with the whole CD34 historical group showed a faster recovery of CD3+4+ in patients with CD3/19 depletion and RIC (104.26 vs. 54.22; p= 0,034) but no significant difference in CD3+8+ and CD3+. Furthermore, subgroups of the CD34 historical population were compared: patients with CD3/19 depletion and RIC had a significantly faster recovery of CD3+4+, CD3+8+ and CD3+ than CD34 patients with TBI (104.26vs. 25.48; 133.46vs. 43.17; 270.27 vs. 65.86; p Conclusions: the type of graft manipulation appeared to have an influence on the speed of CD4+ recovery (CD3/19 depletion > CD34 selection). Moreover, the use of TBI had a clear negative impact on all T cell subsets: patients with TBI had a slower recovery than patients with non-TBI conditioning, independent from graft manipulation procedures and probably due to thymic damage. Thus, the use of RIC-protocols in combination with CD3/CD19 depletion may help to speed up the immune recovery after haploidentical transplantation. Further studies are warranted to evaluate the risk of relapse. Disclosures: No relevant conflicts of interest to declare.

39 pedatric patients with acute leukemias (ALL (n=19), AML (n=14) and MDS (n=6)) received T and B cell depleted grafts from full haplotype mismatched related donors. Depletion of the G-CSF stimulated leukapheresis products was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS device and resulted in a median number of 15.9×106 CD34 (2.5–41) stem cells, 147×106 CD56 NK-cells (9–552) and 413×106 CD14 monocytes (101–1100) per kg body weight. Median numbers of residual T and B cells were 56 000 (10 000–192 000) and 26 000 (2000–149 000) respectively. A reduced intensity regimen (melphalan (140mg/m2), thiotepa (10mg/kg), fludarabine (160mg/m2), OKT3 (0.1mg/kg)) was given in most patients. Co-transfused, HLA mismatched NK cells were traced in peripheral blood of 26 patients starting on day +1 with flow cytometry and appropriate HLA antibodies. Mean numbers of donor derived CD56+ cells/μl were: 3 (day 1), 22 (d 3), 17 (d 7), 75 (d 10), 197 (d 14). Theoretically, the mean absolute number of 4.8×106 co-transfused NK cells should have resulted in a mean number of 2000 cells/μl in peripheral blood of the patients. Comparison of this expected amount with the mean number of NK cells measured within the first week postransplant (25/μl, n=17 data points) showed, that only 1.2% of the cells remained in circulating blood. Thus, the majority of donor NKs did not circulate and probably homed to other compartments (bone marrow, lymph nodes). The number of NK cells cotransfused at day 0 partially influenced the speed of NK cell recovery: patients, who received > 100×106 donor NK cells/kg had significantly higher amounts of circulating cells at day 14 than patients, who received

We report the results of a pilot study with a clofarabine containing conditioning regimen for allogeneic stem cell transplantation. Several studies have already shown the effectivity of clofarabine in adult patients and children with acute leukemias, resistant to standard treatment protocols. Thus, we replaced fludarabine in a melphalan based myeloablative regimen by clofarabine for pediatric patients with refractory disease after standard treatment (n=7) or after previous transplantation (n=3). A total of 10 pediatric patients with acute lymphatic leukemia (non-remission (NR) 1=3, NR 2=3), acute myeloic leukemia, (NR 1=2) and metastatic rhabdomyosarcoma/neuroblastoma (partial remission (PR) =2) received clofarabine 4×50mg/m2 (day -8 to -5), thiotepa 10mg/kg (day -4), melphalan 2×70mg/m2 (day -3 to -2) and OKT3 (0, 1 mg/kg day -8 to +14; n=9) or ATG (10mg/kg; n=1), followed by infusion of full haplotype mismatched peripheral stem cells (n= 9) or bone marrow from matched unrelated donors (n=1). In mismatched family donors, depletion of T and B cells was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS® device. A median number of 16×106/kg stem cells (5.6–28) with 90 000/kg residual T cells was infused. Primary engraftment was observed in 9/10 patients. One patient experienced graft rejection but was successfully retransplanted with stem cells from a second parental donor. Thus, sustained engraftment could be obtained in all patients. Median time to ANC >500/μl with G-CSF stimulation was 10 days (9–11). Independence from platelet transfusion was reached after 9 days. Median T cell count at day 100 was 138 cells/μl (n=5). 7/10 patients are disease free with a median follow up of 100 days. Single cause of death was relapse of the underlying disease (median time to relapse: 90 days). The regimen was well tolerated without severe side effects. No TRM and no toxicity grade 4 according to NCI-CTC grading system occurred apart from mucositis. The profile of toxic side effects was as follows: gastrointestinal: diarrhea grade 0–2 (n=10), stomatitis grade 3–4 (n=10); skin: none; pulmonary: hypoxia and pneumonitis grade 3 in 2 and 1 patient, respectively; cardiac: none; hepatic: GOT/GPT or bilirubin elevation grade 1–2 in 4 patients, grade 3 in 6 patients; renal: creatinine (-clearance) grade 1 in 1 patient; neurological: none; infection: grade 2 (n=8), grade 3 (n=2). Conclusions: clofarabine was well tolerated as part of a conditioning regimen for allogeneic transplantation. Compared to our standard regimen with fludarabine, thiotepa, melphalan, the introduction of clofarabine did not result in any unexpected toxicity. Immunosuppressive effects appeared to be similar to those of fludarabine, since primary engraftment was observed in 90%. Further studies are warranted to evaluate, whether clofarabine can contribute to reduce the risk of relapse in patients with refractory disease.

Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation. We present our preliminary results with a direct depletion procedure using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACS™ device. Only patients with high risk malignancies (n=25, most of them had active disease or relapsed after previous trp.) or with nonmalignant diseases and high risk of graft failure (n=2) were included. The diagnoses were: AML/MDS/CML (n=13), ALL(n=6), relapsed Neuroblastoma/Ewing-/Rhabdomyosarcoma (n=6), SAA, PNH (n=2); Remission status: NR=8, 2nd transplantation=10, CR/CP1=3, CR2–4=6. The patients received either TBI or Bu based standard conditioning regimens (n=9) or a toxicity reduced protocol (Flud, TT, Mel, OKT3, MMF, n=18). The grafts comprised a median number of 16x106/kg (7–41) stem cells as well as high amounts of NK-cells (137x106 (9–550)) and monocytes/granulocytes (6x108 (0.6–13)) with 49x103/kg (7–200) residual CD3+ cells and 500 neutrophiles/μl and independence from platelet substitution was 10 and 9 days respectively. Recovery of CD3+ cells was favorable (d90: 320/-l, d180: 600/μl). Acute GvHD grade 0–1 occurred in 74%, 26% had GvHD grade II. Limited chronic GvHD occurred in 4 patients. No transplant related mortality (TRM) and, in particular, no lethal infections were observed. 13/27 patients (49%) were alive with a median follow up of 0.8 years (3 months – 2.1 years). Single cause of death was relapse. Disease status was predictive: 8/9 patients with leukemias and active disease relapsed, whereas only 2/10 patients in CR relapsed. 2/6 patients with solid tumors are alive. Recovery of platelets was faster in patients with CD3/CD19 depleted grafts than in a historical control group of patients (n=53) transplanted with haploidentical positive CD34+ selected standard grafts (23 vs. 9 days, p Conclusions: our preliminary results indicate that CD3/CD19 selected grafts can improve immunoreconstitution and TRM. Engraftment rates similar to that of patients with myeloablative standard conditioning regimens and positive selected stem cells could be achieved, although most patients of the study group received an intensity reduced regimen. However, the outcome was poor in patients with active disease. Thus, further options have to be investigated to increase the potential antileukemic activity of donor derived effector cells.

Changes in the glycosylation pattern of cell surface proteins occur during early stages of malignant transformation. These alterations in the glycan repertoire may contribute to numerous processes of tumor cell survival such as adhesion, migration and immune evasion. Sialic acid is the most common terminal carbohydrate moiety of glycan structures in humans binding to a family of eleven receptors termed sialic acid binding Ig-like lectins (siglecs). Siglecs are expressed on virtually all effector cells of the immune system and several members function as inhibitory receptors. Siglec-7 is known as an inhibitory receptor expressed by NK cells and T cells. Therefore, siglec-7 ligands may contribute to immune evasion of malignant cells. In order to analyze the expression of siglec-7 ligands by malignant cells, we cloned the extracellular domain of human siglec-7. The recombinant protein was expressed in 293 cells to allow glycosylation, which is supposed to be important for specificity. As the interaction of a single glycan and lectin is of low affinity, we tetramerized siglec-7 in analogy to MHC-tetramers to increase avidity. This strategy allowed for reliable use of siglec-7 fusion protein in flow cytometry. Analysis of PBMC from healthy donors revealed that siglec-7 ligands are expressed on all subpopulations of PBMC: expression was detected on CD4+ and CD8+ T-cells as well as on monocytes and NK cells. B cells could be subdivided into a positive and negative population after staining with siglec-7 tetramers. Subsequently, we analyzed primary lymphatic blasts from childhood leukemias. Interestingly, T-ALL blasts expressed ligands for siglec-7, whereas these ligands were absent from cALL blasts. As cALL blasts are known to be better targets for NK cell-mediated cytotoxicity than T-ALL blasts, we hypothesized that siglec-7 ligands on the surface of leukemic blasts inhibit NK cells. To test the functional relevance of siglec-7 ligand expression for NK cell-mediated cytotoxicity, we used soluble monomeric siglec-7 as competitive inhibitor in cytotoxicity assays. In these competition assays, NK cell-mediated specific lysis of leukemic cells increased roughly twofold in the presence of soluble siglec-7. These results show that engagement of inhibitory siglecs on the surface of effector cells might contribute to immune escape mechanisms of malignant cells. It underlines the function of siglec-7 as a non-MHC-specific inhibitory receptor on NK cells and the important role of altered glycans expression on malignant cells in tumor immunology.

There is still a significant risk of GvHD in transplantation of peripheral stem cells from matched unrelated donors, despite the use of ATG and pharmacological prophylaxis. Especially in the case of young pediatric patients and adult donors, grafts with high numbers of stem cells but also with extreme amounts of T cells can be obtained. We present an update of a clinical study with T cell reduced grafts in 22 patients, which offer both large stem cell numbers and clearly defined amounts of T cells. Positive selection of peripheral stem cells was done with CD34 or CD133 coated magnetic microbeads and the CliniMACS™ device. Afterwards, an unselected aliquot containing 10x106/kg T cells was added to the purified stem cells and infused on day 0, resulting in a median T cell depletion of 2 log. The diagnoses were: acute lymphatic leukemia (n=9; CR1=4, CR2=5), NHL (n=1) acute (n=5; CR1=1, CR2=4) and chronic myeloic leukemias (n=3), MDS (n=3) and Wiskott-Aldrich (n=1). Median age was 10 years (0.5–18). The donors were matched for HLA-A/B (medium resolution typing) and DRB1/DQB1 (high resolution). A short course of MTX (2–3x10 mg/m2) and CSA (2–3 mg/kg, adjusted to blood levels) until day 100 were given. 21/22 patients had primary engraftment with a median time to ANC >500 of 18 days (12–60). No G-CSF was given. One patient rejected his graft and was successfully retransplanted from another MUD. Platelet recovery was fast (median time to reach independence from substitution: 20 days). Mean numbers of CD3+, CD3+CD4+ and CD3+CD8+ on day 180 (365) were 259/μl (868/μl), 100/μl (400/μl) and 146/μl (456/μl). 19/22 patients had GvHD grade 0–I (86%), 1 and 2 patients had grade II and III, (5%, 10%) respectively. Chronic GvHD occurred in 2 patients (10%). 15/22 patients are alive with a median follow up of 3 years (1.5–5). 3 year EFS was 64% (all patients), 50% (ALL/NHL) and 73% (myeloic leukemias/MDS). Relapse probability at 2 years was 25%, probability of TRM at 2 years was 15%.Causes of death were relapse (n=4) and infections (n=3). Thus, stable and favourable survival rates with a low incidence of GvHD were achieved. The method allows to administer clearly defined T cell numbers independent from the size of the grafts. This may be of advantage in particular in small children and if the donor does not accept a bone marrow harvest: all available stem cells can be infused without the limitation of unacceptable high T cell numbers.

Relapse after allogeneic stem cell transplantation of patients with acute lymphoblastic leukemia (ALL) remains a major problem. A beneficial impact of alloreactive NK cells has been reported for myeloid malignancies, but has been questionable for B-lineage-ALL. Here we show that the majority of NK cell clones from healthy donors could effectively lyse primary blasts from a representative childhood precurser-B-ALL and demonstrate the relevance of differential surface expression of KIRs (killer cell immunglobulin like receptors CD158a, CD158b and CD158e) for antileukemic alloreactivity. More than 79% of clones exerted specific lysis >40% against primary ALL blasts. Mismatch of effector/target HLA-C type (KIR-ligand incompatibility) did not correlate with antileukemic alloreactivity, although non-malignant lymphoblastoid cell lines were lysed according to KIR-ligand incompatibility. In contrast differential surface expression of the three major KIRs showed significant impact on the antileukemic activity against precursor-B-ALL blasts. NK clones with none of the three KIRs or a single KIR that recognized no ligand, were not inhibited by the targets and exerted higher lysis (p =

Relapses represent a major problem after transplantations in children with ALL. Natural Killer (NK) cells have been shown to exert remarkable Graft versus Leukemia effects after mismatched stem cell transplantation in myeloic leukemia, whereas the efficacy against lymphatic leukemia is still unclear. We therefore measured intensity of HLA class I expression on leukemic blasts by quantitative FACS analysis and investigated the impact of quantitative HLA class I expression, of several adhesion molecules and of KIR-mismatch on NK cell mediated lysis of the leukemic blasts from 21 pediatric patients with ALL. Expression of HLA class I molecules differed widely from patient to patient (range 5000–500000) and was reduced in comparison to B cells from healthy donors in 70% of cases. NK cells killed leukemic blasts very heterogeneously but a clear association between number of HLA class I molecules per cell and specific lysis (range 13–98%) was found (r2=0.68, p

Activating and inhibitory cell surface receptors regulate natural killer (NK) cell effector functions. The extent of expression of their activating ligands on target cells probably plays a critical role in tumor immune surveillance, but the data of this expression on blasts of leukemia patients are still poor. We examined blasts of children with ALL for the activating human NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3, and for the ligands of the activating human natural cytotoxic receptors (NCRs) NKp30, NKp44 and NKp46. Using ligand specific mouse monoclonal antibodies and flow cytometry, we screened 24 children (23 common-ALL, one pre-T-ALL) for the expression of NKG2D ligands and 15 children (all common-ALL) for NCR ligands. In 13 patients (all common-ALL), also the density of the NKG2D ligands was determined by quantitative flow cytometry. Considering cells positive for a particular ligand in case of a two fold increase of median fluorescence above negative control, 38 percent of the patients were positive for one or more NKG2D ligands, while only 13 percent expressed one or more NCR ligands at significant levels. ULBP1 was most frequently expressed (29 percent of patients positive), while no patients were positive for ULBP2 and NKp44 ligands. ULBP3 was positive in 17 percent of the patients, NKp30 and NKp46 ligands in 13 percent, MICA and MICB in 4 percent. The patient with pre-T-ALL was positive only for ULBP1. So ULBP1 was expressed more frequently, while the other NKG2D ligands were expressed less frequently in children than reported for adult leukemia patients before (Salih et al. Blood.2003;102:1389–1396.). The density of detected NKG2D ligand molecules was always rather low. For MICA the maximum were 1700 molecules per cell in a single patient, for MICB 900, for ULBP1 1100, and for ULBP3 1000 molecules per cell. In summary, blasts of pediatric ALL patients displayed low or negative surface levels of ligands for the human activating NK cell receptors NKG2D, NKp30, NKp44 and NKp46. QUALITATIVE LIGAND EXPRESSION QUALITATIVE LIGAND EXPRESSION QUANTITATIVE LIGAND EXPRESSION QUANTITATIVE LIGAND EXPRESSION

In allogeneic transplantation it is a major goal to maintain a stable long term engraftment. However, several factors like viral infections, drug toxicity or low stem cell numbers may hamper hematopoietic recovery or may even cause a late graft failure after successful initial engraftment. Hematopoietic growth factors or additional stem cell donations (boosts) may help to overcome this problem. In this retrospective analysis, the efficacy of boosts with highly purified peripheral stem cells either from closely matched unrelated or from haploidentical related donors was evaluated in pediatric patients with stagnant hematopoiesis (10x106 (unrelated donors) or >20x106/kg BW CD34+ cells, surplus stem cells were purified with an immunomagnetic selection method using antiCD34 or antiCD133 coated microbeads and cryopreserved. Three haploidentical donors were asked for a second donation. Our aim was to maintain stable and complete hematopoiesis after transplantation without inducing acute or chronic GvHD. A total of 18 boosts were given between 30 and 290 days after transplantation with a median number of 5 (0.6–34) x 106 CD34+/CD133+ progenitor cells/kg and only 2200 (100–25000) residual T-cells/kg. All patients had a complete donor chimerism and no reconditioning or posttransplant pharmacological immunosuppression was administered. Patients who received G-CSF were excluded. Due to the low number of T cells, acute GvHD >grade I and also chronic GvHD could be completely avoided even in haploidentical donors. Leukocyte counts, neutrophile counts, lymphocyte counts (and platelet counts in patients with thrombocytopenia) within the week before infusion of the boosts were compared with cell counts 4 and 8 weeks after infusion, using the paired T-test. A significant increase of all cell counts was observed (medina numbers pre boosting: 900/μl, 312/μl, 142/μl; 4 weeks post boosting: 2065/μl, 1264/μl, 317/μl; 8 weeks post boosting:2570/μl, 1780/μl, 385/μl; p Conclusions: Boosts with purified stem cells and low T-cell contamination (

Transplantation of haploidentical, positive selected grafts with TBI or Bu based conditioning regimens can result in significant toxicity and TRM. New graft manipulation methods and a melphalan based intensity reduced regimen decreased these complications. We present our results with a direct depletion procedure using antiCD3/antiCD19 coated magnetic microbeads. Melphalan (2×70mg/m2), fludarabine (4×40mg/m2), thiotepa (10mg/kg) and OKT3 (0.1mg/kg) was used as preparative regimen in 27 pediatric patients. All patients underwent intensive pretreatment according to current study protocols; 11/27 already received previous autologous or allogeneic transplantations (median time between 1st and 2nd trp: 2.3 years). Diagnoses were: AML/MDS (n=9), ALL (n=5), relapsed neuroblastoma/Ewing-/rhabdomyosarcoma (n=11), SAA (n=2); remission status: CR2=6, CR3=3, NR/PR=16. Donors were full haplotype mismatched parents. The grafts comprised a median number of 14×106/kg stem cells and 139×106/kg NK-cells with 55×103/kg residual CD3+ cells and 24×103/kg CD20+ cells. Primary engraftment occurred in 85%. After TLI based reconditioning and second haploidentical stem cell donation, final engraftment was achieved in 100%. Median time to reach >500 neutrophiles/μl and independence from platelet substitution was 10 (9–12) and 8 (6–188) days respectively. GvHD grade 0–1 occurred in 63%. 30% and 7% had grade II and III, respectively. Chronic GvHD was observed in 3 patients. Transplant related mortality (TRM) occurred only in 1/27 patients (4%) with a median follow up of 0.8 years (3 months-3.0 years). In general, the regimen was well tolerated without severe side effects. The profile of toxic side effects according to NCI-CTC grading system was: gastrointestinal: diarrhea grade 0–2 (n=25), grade 3 (n=2); stomatits grade 3–4 (n=27); skin: grade 0–2 (n=27); pulmonary: hypoxia grade 0 (n=24), grade 3 (n=3); pneumonitis (n=1); cardiac: lethal myocardiopathy (n=1), none (n=26); hepatic: GOT/GPT or bilirubin elevation grade 0–2 (n=19), grade 3 (n=4), grade 4 (n=4), VOD: none; renal: creatinine grade 0–2 (n=27); neurological: none; infection: grade 0–2 (n=17), grade 3 (n=7), grade 4 (n=3), EBV-LPD: none. Overall survival at 1 year was 47% for the whole group and 40% for patients with solid tumors. Causes of death were myocardiopathy (n=1) and relapse (n=13). Pretransplant disease status was predictive: 1 year EFS in patients with leukemias in CR and SAA was 83%, whereas all patients with active disease relapsed (median time to relapse: 0.3 years). Conclusions: transplantation of CD3/CD19 depleted haploidentical stem cells with our melphalan based reduced intensity regimen resulted in a fast recovery of neutrophiles and platelets. Engraftment rates similar to that of patients with standard conditioning regimens and positive selected grafts were achieved, possibly due to graft facilitating effects of cotransfused NK-cells. The regimen helped to minimize side effects and TRM. No lethal infections occurred despite delayed T cell recovery. Thus, our approach forms the basis for haploidentical transplantation with low toxicity even in intensively pretreated patients(including previous transplantations). However, relapse remains an unsolved problem in patients with active disease. In those patients, further therapeutic elements have to be evaluated, like additional cytoreductive drugs immediately before transplantation as well as posttransplant immunotherapy.

Abstract 3548 T and B cell depleted haploidentical grafts and a melphalan based intensity reduced regimen result in low toxicity and stable event free survival in patients with leukemias in CR1-3. However, patients with active disease or with second or subsequent transplantation show unacceptable relapse rates. In an ongoing study, 36 pediatric patients with acute leukemias and advanced MDS (median age: 11 years) received melphalan (2×70mg/m2), fludarabine (4×40mg/m2) or clofarabine (4×50mg/m2), thiotepa (10mg/kg) and OKT3 (0.1mg/kg). T and B cells were depleted by antiCD3/antiCD19 coated magnetic microbeads, whereas NK cells remained into the grafts (median number= 120×106/kg). Remission status was: CR1-3=18, NR=18, 18/36 already received previous allogeneic transplantations. Relapse rates at 2 years were 20% (CR patients) and 73% (active disease patients or 2nd trp). Thus, we investigated options to reduce the risk of relapse by increasing antileukemic activity of donor NK cells in the grafts in vitro. Over night incubation with Interleukin 15 increased NK activity most effectively (specific lysis at E:T=20:1 against K562: 28% prior to and 71% after stimulation, n=10). After additional IL2 stimulation a 22 fold increase in thymidine uptake indicated proliferation of NK cells (n=5). Due to the profound depletion, no T cell proliferation was detectable. Based on these results, we started a pilot study with ex vivo IL15 stimulated grafts in 4 patients at very high risk (ALL, 3rd relapse, active disease (n=1); ALL 2nd relapse, remission (n=1); AML 1st relapse, active disease (n=2)). All patients received a backbone of unstimulated cells at day 0 to ensure engraftment. Additionally, parts of the grafts were incubated over night, washed four times and afterwards infused at day +1 (median numbers: CD56+CD3- =9.4×106/kg (range 3.7–24.4); CD34=1.5×106/kg; CD14=34×106/kg, CD3=0.01×106/kg). No acute side effects occurred. All patients engrafted within 12 days. 3 patients had acute GvDH grade 0-I, 1 patient had GvHD grade III. Recovery of NK cells was remarkably fast (526 CD56+/μl at day 14 posttransplant versus 256 CD56+/μl in patients without IL15 stimulation (means)). After additional administration of IL2 in vivo (1×106 Units/m2/day s.c.) high NK activity (specific lysis>90% against K 562, E:T=20:1) was detectable in peripheral blood. Two patients are disease free (day 154 and 674 posttransplant), 2 patients died from relapse (day 56 and 64). Conclusions: ex vivo stimulation with IL15 strongly increases cytotoxic activity of NK cells in T and B cell depleted grafts from haploidentical donors and can counterbalance G-CSF mediated inhibitory effects. Those grafts were infused without any acute side effects and resulted in a fast recovery of functional donor NK cells. Potential interactions with stem cells and stem cell derived NK reconstitution have to be investigated. Further studies have to evaluate if this approach might contribute to reduce relapse rates in high risk patients. Disclosures: No relevant conflicts of interest to declare.