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Access to HLA-matched unrelated donors (MUD) has long been a barrier to hematopoietic stem cell transplantation (HSCT). In more recent years however, the development and optimization of alternate d...

As one of the three successful approaches for haplo-identical stem cell transplantation (haplo-SCT) in recent decades, "Beijing protocol" was based on immune tolerance induced by granulocyte colony-stimulating factor plus anti-thymocyte globulin, which included individualized conditioning regimens, novel donor selection algorithm, and risk-directed strategies for GvHD and relapse, etc. Haplo-HSCT following "Beijing protocol" demonstrated similar efficacy to HLA-matched SCT, which has become the predominant strategy for allogeneic SCT in China as well as inspiration for refinement of global practice. This review will focus on recent progressions and prospective of this approach in (1) the indications from hematological malignancies to non-malignant diseases; (2) microenvironment for hematopoietic recovery; and (3) improving T cells function.

Post-transplant cyclophosphamide (PTCy) has become the most popular approach in haplo-identical hematopoietic cell transplant (haplo-HCT). Although there are reports of a small number of adult patients in the literature who experienced graft failure and were re-transplanted with a haploidentical donor with PTCy prophylaxis, there is still insufficient guidance for patients with specific contraindications/complications to cyclophosphamide and virtually no data in the pediatric setting. Abatacept (Aba), a T cell co-stimulation blockade, has been shown in previous studies to prevent severe acute graft-versus-host disease (GVHD) with minimal toxicity and durable engraftment. We report the efficacy of Aba-based GVHD prophylaxis in four pediatrics patients (ages 2-12 years) who received a haplo-HCT with peripheral blood stem cells (PBSC). Three patients had previous transplants. One patient developed acute GVHD of skin stage 3 and one patient had both stage 3 skin and stage 1 GI acute GVHD. Two patients had mild chronic skin GVHD. All 4 patients are alive with full donor chimerism and without disease at 7-23 months follow up, weaning or off immunosuppressive agents with no complications. Successful haplo-HCT utilizing an Aba- based regimen can result in reliable engraftment and acceptable GVHD. However, our small sample size limits generalizability and encourages the consideration of a larger prospective trial to validate these results in the haplo-HCT setting.

Patients with relapsed/refractory early T-cell precursor lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) respond poorly to traditional therapy and have dismal prognosis. CD7 expresses in almost all blasts of T-cell lymphoma/leukemia and represents one of the most promising therapeutic targets for T-ALL/LBL by CD7 targeted chimeric antigen receptor modified T cell therapy (CD7-CART). Because of shared CD7 expression in the majority of normal T-cell surfaces, we utilized an non-gene editing strategy by co-transducing CAR-T cells with a CD7 protein expression blocker (PEBL), and successfully overcame the fratricide as well as maintain the proliferation and cytotoxicity of CD7-CART-cells. Here, we presented the efficacy and safety results of CD7-CART therapy in a pediatric patient with TP53 mutated ETP-ALL/LBL. The patient was diagnosed with ETP-ALL/LBL at 2016, achieved and maintained complete remission (CR) for 2 years with traditional chemotherapy. The disease relapsed at a month after discontinuation of chemotherapy. He underwent haploidentical HSCT at the second CR, but suffered relapse again 2 years post haplo-HSCT. TP53 mutation（VAF:96.5%） and extensive extramedullary infiltration was detected at relapse. The patient was resistant to venetoclax combined with decitabine, homoharringtonine, aclarubicin, cytarabine and granulocyte colony stimulating factor (G-CSF), high-dose cytarabine combined with cladribine, G-CSF, chidamide and CD38 CART therapy. Nanobody derived CD7-CART cells were manufactured from lymphocytes of the donor. The CART cells were negative for CD7, CD223 and CD279. 70.5% of blasts in the bone marrow aspirates were observed prior to CAR T-cells infusion. A total of 5×10 6/kg CD7-CART-cells were infused. CR was confirmed at day 30 bone marrow evaluation and maintained at the last followup at day 91. Partial remission was achieved as evaluated by PET-CT scan at day 93. Persistence of CD7-CART-cells can be detected with flowcytometry until day 96 post CAR T-cells infusion. Grade 3 cytokine release syndrome with high fever and hypotension were observed, which was relived by tocilizumab and dexamethosone. No organ dysfuction and immune effector cell-associated neurotoxicity syndrome were observed. In general, we showed for the first time that the nanobody derived CD7-CART with PEBL technology was a potent and safe salvage therapy in a relapsed/refractory ETP-ALL/LBL patient with high tumor burden. Disclosures No relevant conflicts of interest to declare.

Background: The use of post-transplant cyclophosphamide (PTCy)/tacrolimus/mycophenolate mofetil (MMF) for GVHD prophylaxis has improved outcomes in haploidentical hematopoietic cell transplantation (haplo-HCT). PTCy is now being evaluated in matched-related (MRD) and matched-unrelated (MUD) allo-HCT. Previous studies demonstrated improved GVHD-free/relapse-free survival (GRFS) when PTCy was combined with two immunosuppressive agents and PTCy has also been associated with better relapse-free survival (RFS) as demonstrated in De Jong et al 2019, though only one immunosuppressive agent was used. Currently, there is limited published data comparing outcomes using PTCy/tacrolimus/MMF to standard MRD/MUD GVHD prophylaxis of methotrexate (MTX)/tacrolimus. The importance of studying this comparison may help to improve GVHD outcomes in MRD and MUD allo-HCT. Methods: We retrospectively analyzed adult patients at USC Norris Cancer Hospital (age ≥ 19) who received allo-HCT from 2018 to 2020. The primary end-points assessed were incidence and severity of 1-year aGVHD and cGVHD. Secondary end-points included day+100 mortality, 1-year overall survival (OS), 1-year RFS, 1-year transplant-related mortality (TRM), and 1-year GRFS, defined as grade 3-4 acute GVHD, systemic therapy-requiring chronic GVHD, relapse, or death in the 1-year post-HCT period. Results: A total of 65 adult MRD and MUD allo-HCT recipients and 53 haplo-HCT patients were reviewed. Of the MRD/MUD patients evaluated, approximately 51% (n = 33) were female and 49% (n = 32) were male. The age range was 20-69 years old (median = 46), and the most common diseases included ALL (46%), AML (31%), MDS (11%), and others (i.e. lymphoma, aplastic anemia (AA), myelofibrosis) (12%). 34% (n = 22) of patients received PTCy on D+3 and D+4 with tacrolimus/MMF/ on D+5 as GVHD prophylaxis and 66% (n = 43) of patients received MTX/tacrolimus on D+1, +3, +6, and +11 as GVHD prophylaxis. All haplo-HCT patients received standard PTCy/tacrolimus/MMF. Stem cell source was primarily PBSC except in HLH and AA patients. The PTCy group had more MUD allo-HCT (64%), degree of antigen mismatch (56%), and median age of 50.5 years compared with the MTX group at 44%, 47%, and 44 years respectively. 70% in the MTX group received MAC compared with 45% in the PTCy group. The haplo group had similar demographics to the MTX group. The mean CD34 cell doses in the PTCy, MTX, and haplo groups were 4.87, 5.36, and 7.24x106 cells/kg respectively. Incidences of total GVHD, aGVHD, and aGVHD grade 3 or 4 in the PTCy group were 55%, 50%, and 4.5% respectively compared with 65%, 35%, and 7% in the MTX group, though not significant. The haplo group had 68%, 55%, and 1.9% respectively. Incidence of total cGVHD and cGVHD requiring systemic therapy in the PTCy group was 4.5% and 0% respectively compared with 30% (p = .02) and 23% (p =.01). The haplo group had 13% and 1.9% respectively. Day+100 mortality, 1-year OS, 1-year RFS, 1-year TRM, and 1-year GRFS in the PTCy group were 0%, 80%, 60%, 0%, and 64% respectively compared with 7%, 88%, 90%, 7.3%, and 59%. The haplo group had 3.8%, 86%, 89%, 14%, and 66%. In a univariate analysis, factors significantly associated with GVHD were disease status (p = .0.12) and CD34 dose (p = 0.015) and antigen mismatch (p = 0.04) was associated with increased mortality. Discussion: Our results demonstrate improved overall and extensive cGVHD outcomes in the PTCy group and thus an improvement in 1-year GRFS. Furthermore, incidence and severity of 1-year cGVHD in this group are improved when compared with previously reported outcomes. 1-year GRFS reported in De Jong et al 2019 was 45% and 1-year GRFS reported for all groups in our study is higher at 66%, 64%, and 59% for the haplo, PTCy, and MTX groups respectively. Although this was not significant, it may be clinically meaningful given the significant improvement in extensive GVHD and improvement in all other secondary end-points except 1-year OS and RFS. Furthermore, the PTCy group had a higher percentage of mismatched antigens yet demonstrated superior outcomes. 1-year OS and RFS were superior in the MTX group however this is likely due to sample size differences. The improved extensive cGVHD and GRFS outcomes observed using PTCy/tacrolimus/MMF in the MRD/MUD setting should continue to be evaluated and currently there is an ongoing prospective, randomized study to further investigate. Disclosures Yaghmour: Jazz: Consultancy, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Haplo-identical transplant is being increasingly used in patients who do not have a readily available matched related or unrelated donor. Post-transplant cyclophosphamide's use due to its simplicity and documented efficacy has made this approach readily employable across diverse transplant centres across the globe. The outcomes of regimens used for conditioning in recipients of bone marrow are at times in variance to that from more commonly employed G-CSF mobilised peripheral stem cell (PBSC). This review highlights various conditioning regimens used in PBSC recipients, with emphasis on toxicities, practicalities and transplant related outcomes of relapse, non-relapse mortality and graft versus host disease.

Key Clinical Message The principal limitation of allogeneic hematopoietic stem cell transplantation except relapse remains the transplant-related mortality (TRM). In addition to graft-versus-host disease (GvHD), infections contribute to TRM in many patients. We describe herein a case of an adult patient presenting 5 months after haplo-identical transplantation an isolated fulminant hepatitis due to adenovirus.

Purpose: Even if the romanian population is ethnically compact caucasian-type population, many of the patients referred for bone marrow transplantation lack a suitable donor. In order to expand the donor pool and the accesibility to transplant for those who have indications it is necessary to perform haplo-identical bone marrow transplant procedure in Fundeni Clinical Institute. Since 2009 Romania established a National Volunteer Stem Cell Donor Registry (RNDVCSH), the goal was to enlarge the possibility to find HLA-matched unrelated donors (MUD) for patients. This approach offered transplant for only up to 60%patients referred for transplantation in 2014, even we chose one HLA-mismatch donors. The haploidentical transplant protocol proposed for our institution is based on Sidney Kimmel Comprehensive Cancer Center protocol from Johns Hopkins University School of Medicine. The major milestones of this protocol include: patient eligibility, donor selection criterias, evaluation of the haplo donor, the conditioning regimen plan and additional supportive care, the bone marrow harvest, prophylaxis of graft versus host disease, assessment during and after the transplant. Donor must be HLA-haploidentical first-degree relatives of the patient with signed consent. The patient, parents and children are typed at the allelic level for HLA-A, -B,-C,-DRB1 and -DQB1. They will perform also de HLA-antibody search using cross-match test in complement-dependent cytotoxicity. The conditioning regimen is composed by Fludarabine 30 mg/m2/day (from day -6 to day -2) combined with Cyclophosphamide 14,5 mg/kgIBW/day (from day -6 to day-5) and TBI at 2 Gyîn day -1. In case of lacking TBI procedure at 2 Gy dose in day -1, it could be replaced by two dose of Busulfan iv in day -3 and day-2 (dose=3,2 mg/kgIBW/day) for those with acute and chronic leukemias. The donor will have general anesthesia,the target yield of marrow is 4 x 10^8 total nucleated cells/kg recipient using his IBW. The GVHD prophylaxis consisted of post-transplant Cyclophosphamide (PTCy) of 50mg/kgIBW/day in IV administration in day +3 and +4, followed by tacrolimus and mycophenolatemofetil (MMF) beginning day +5. The MMF will be stopped at day+35, the tacrolimus will continue till 6 months after the transplantation. Conclusion: One of the most important factors affecting transplantation outcome is proper timing.Therefore, donor availability is an crucial issue. Haploidentical related donors are available for almost all patients, so the use of those donors is a viable alternative.

Background: Significant advances have been achieved in the outcomes of patients with myelodysplastic syndromes (MDS) after both HLA-matched sibling donor transplants (MSDT) and non-MSDT in the past few years, the latter including HLA-matched unrelated donor (MUDT) and haplo-identical donor transplants (HIDT). Methods: We retrospectively analyzed the data of 85 consecutive patients with MDS who received allogeneic HSCT between Dec 2007 and Apr 2014 in our center. These patients comprised 38 (44.7%) who received MSDT, 29 (34.1%) MUDT, and 18 (21.2%) HIDT. Results: The median follow-up was 30.0 months. Over all, the median overall survival (OS) was 60.2 months and the probabilities of OS at the first, second and fifth year were 63%, 57% and 48%, respectively. Median OS post transplant (OSPT) was 57.2 months and the probabilities of OSPT at the first, second and fifth year were 58%, 55% and 48%, respectively. Relapses occurred in 16 patients (18.8%), and the cumulative incidence of relapse at the first, second and third year were 14%, 23% and 27%, respectively. The survival of patients receiving non-MSDT was superior to that of MSDT, median OSPT being 84.0 months and 23.6 months, respectively (P=0.042); And the similar result was observed when we analyzed the OS of patients who underwent non-MSDT or MSDT (median OS: 120 months vs. 27.0 months; P=0.028). We also found that using antithymocyte globulin (ATG) in conditioning regimens significantly improved survival after non-MSDT, with better OS and OSPT (P=0.016 and P=0.025). Furthermore, we compared OS or OSPT of patients who received non-MSDT with ATG (n=38) and MSDT without ATG (n=37). Our results showed that using ATG in conditioning regimen significantly improved survival of non-MSDT, with better OSPT (median OS: 84.0 months vs. 23.0 months; P=0.026) and OS (median OS: 120 months vs. 25.1 months; P=0.016). Conclusions: These data suggest that, non-MSDT could be a valid alternative when MSDT is not available, and ATG may improve the survival of MDS patients after non-MSDT. Disclosures No relevant conflicts of interest to declare.

Invasive fungal infections (IFIs) such as candidiasis and mold infections have caused significant morbidity and mortality among immunocompromised patients in recent years. Micafungin, a new echinocandin, inhibits fungal cell wall β-glucan synthesis, with potent activity against most species of Candida and Aspergillus. The aim of this observational study was to investigate the efficacy and safety of micafungin in prophylaxis of IFIs in 26 high-risk adult patients with various hematological diseases receiving haplo-identical Allo-SCT. Only two patients had a history of possible aspergillosis before transplant treated by voriconazole. The patients received a median of four lines (2-7) of treatment before Allo-SCT. Thirteen patients (50%) received at least one prior Auto-SCT; and eight patients (31%) received a previous Allo-SCT. Patients received a median of 29 infusions (range, 15-85) of micafungin (50 mg/day i.v. as a 1-h infusion). The treatment was initiated at the beginning of the transplant conditioning regimen until the hospital discharge. None of our patients discontinued the treatment for drug-related adverse events. Micafungin was not associated with any hepatotoxicity. Only one patient (4%) discontinued the treatment because of early disease progression. In all patients no Candida and/or Aspergillus species was documented after 3 and 6 months from transplant. None of our patients presented a positive galactomannan antigenemia0.5. Nine patients (35%) presented a CMV reactivation. Four patients presented an acute GVHD grade II and two patients presented a chronic GVHD. The median follow-up was 11 months (3-23). At the last follow-up, there were 20 patients (77%) who were alive; four patients (12%) died because of disease progression and two patients because of graft failure. Micafungin has a good safety and tolerability profile, with an efficacy in preventing IFI in this high-risk population. Our data provide support for an efficacy study in a prophylaxis setting, but prospective and comparative clinical trials using micafungin are urgently needed to define the role of this drug in prophylaxis after haplo-identical Allo-SCT.

Background : While dCBT is associated with high rates of sustained donor engraftment, delayed neutrophil recovery in adults is frequent and can contribute to extended hospitalization and early transplant-related mortality. Methods : We investigated engraftment after myeloablative dCBT supplemented with CD34+ selected haploidentical PBSC (haploCD34+) in patients (pts) with high risk hematologic malignancies or aplastic anemia in a phase II clinical trial. The aim was to abrogate neutropenia (ANC >/= 500 within 14 days) with a haplo myeloid bridge prior to CB engraftment. Pts did not receive ATG due to the increased mortality risk reported in adult CBT. Double unit CB grafts allowed comparison to dCBT controls without haploidentical graft supplementation. Results : 78 adult pts [median age 48.5 years (range 21-68), median weight 82 kgs (range 48-138), 44 (56%) CMV+, 3 with prior allografts] underwent haplo-dCBT between 9/2012-12/2017. Diagnoses included 54 (69%) acute leukemias, 10 (13%) MDS/ MPN (all ≤ 10% blasts at work-up), 10 (17%) NHL/ HD and 1 aplastic anemia. Conditioning was myeloablative (1 Cy 120/ Flu 75/ TBI 1375, 77 intermediate intensity Cy 50/ Flu 150/ Thio 5-10/ TBI 400) with CSA/ MMF. CB units had a median infused TNC of 2.3 (range 1-5.7) x 107/kg/unit & median infused viable CD34+ cell dose of 1.1 (range 0.1-3.1) x 105/kg/unit with a median 5/8 (range 2-7) unit-recipient HLA-allele match. Haplo CD34+ grafts [procured from children (46%), siblings (31%), parents (13%) or extended family (10%)] had a median infused CD34+ dose of 5.2 x 106/kg (range 1.1-16.8) and a median infused CD3+ dose of 1.6 x 103/kg (range 0.3-13.7). Sixty-one (78%) haplos were 4/8 and 17 (22%) were 5-7/8 HLA-matched to the pt. In 77 evaluable pts (1 pt died on day 14), 4 engraftment patterns were observed (Table 1). All but 2 pts had sustained CB engraftment with either an optimal haplo-bridge (Gp. 1, 34/77, 44%), a transient bridge with a second nadir preceding CB engraftment (Gp. 2, 20/77, 26%), or no bridge (Gp. 3, 21/77, 27%). The 2 remaining pts had CB/ haplo graft failure (Gp. 4, 2/77, 3%); both were successfully re-transplanted with single CB units. While there was no difference in the day 100 TRM in the 34 optimal bridge pts vs pts with transient or no bridge [9% (95%CI 2-21) vs 15% (95%CI 6-27), p = 0.388], optimal bridge pts had faster platelet recovery [19 (range 14-41) vs 44.5 days (range 14-67)] and earlier hospital discharge [28.5 (range 20-60) vs 36 days (range 28-98)]. Similar to dCBT alone, chimerism analysis revealed sustained engraftment in haplo-dCBT is mediated by a "winning" CB unit. This was heralded by winning unit-derived T-cells seen as early as day +28. Although universal, the speed of haplo rejection varied, and a high haplo chimerism percentage early post-transplant did not guarantee successful bridging. Analysis of factors potentially predicting an optimal bridge is shown in Table 2. The median winning CB unit-haplo 8-allele HLA-match was 3/8 (range 1-7/8). In univariate analysis, higher haplo CD34+ dose/kg, > 4/8 haplo-recipient HLA-match and ≥ 3/8 winning unit-haplo HLA-match were associated with a higher likelihood of bridging. Haplo CD34+ dose and winning unit-haplo HLA-match remained significant in multivariate analysis. Conclusions: While haplo-dCBT can be associated with enhanced neutrophil recovery, this platform does not guarantee a successful myeloid bridge due to early haplo rejection by the winning CB unit. This universal haplo rejection highlights the importance of the CB graft dose and quality with this ATG-free strategy as the CB will mediate sustained engraftment. Our findings have significance for strategies that combine unmanipulated CB with any third-party or ex vivo expanded T-cell depleted product given higher product CD34+ cell dose and better HLA-match to the unmanipulated CB unit could improve the likelihood of successful myeloid bridging. The data also support alternative approaches to improve myeloid recovery after CBT such as optimized unit selection and vivo expansion. Disclosures O'Reilly: Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding. Perales:Takeda: Other: Personal fees; Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Merck: Other: Personal fees; Abbvie: Other: Personal fees. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Shah:Janssen: Research Funding; Amgen: Research Funding.

Unmanipulated HLA-haplo identical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) has been developed as an alternative transplantation strategy for patients without an HLA-matched related or unrelated donor. In this transplantation setting, the cost and outcome of stem cell collections have not been defined completely. The aim of this study was to compare the cost and outcome of stem cell collection in HLA-haplo identical/mismatched related transplantation with combined G-PBSCs and G-BM to the HLA-identical/matched transplantation with G-PBSCs alone for patients with hematologic malignancies. Hundred and fifty-two healthy donors received twice-daily granulocyte-colony stimulating factor (G-CSF) subcutaneously for 5 days. The PBSCs were collected on day 4 and 5 of G-CSF treatment for HLA-identical/matched transplantation from unrelated/related donors. The PBSC collections and BM harvests was performed on day 4 and 5 of G-CSF treatment for HLA-haplo identical/mismatched related transplantation from related donors, respectively. There was no difference in the major characteristics between groups. More stem cells were harvested in HLA-haplo identical/mismatched related donors than that of HLA-identical/matched donors and a lower cost was seen in the former. The HLA-haplo identical/mismatched related transplantation with combined G-PBSCs and G-BM was a feasible approach with high cell harvest and low cost of stem cell collection for patients with hematologic malignancies.

Allogeneic bone marrow transplant is a life-saving procedure for adults and children that have high-risk or relapsed hematological malignancies. Incremental advances in the procedure, as well as expanded sources of donor hematopoietic cell grafts have significantly improved overall rates of success. Yet, the outcomes for patients for whom suitable donors cannot be found remain a significant limitation. These patients may benefit from a hematopoietic cell transplant wherein a relative donor is fully haplotype mismatched. Previously this procedure was limited by graft rejection, lethal graft-versus-host disease, and increased treatment-related toxicity. Recent approaches in haplo-identical transplantation have demonstrated significantly improved outcomes. Based on years of incremental pre-clinical research into this unique form of bone marrow transplant, a range of approaches have now been studied in patients in relatively large phase II trials that will be summarized in this review.

A new method of graft manipulation based on physical removal of αβ+ T cells and CD19+ B cells, leaving mature NK cells and γδ T cells in the graft, has been recently developed for HLA-haploidentical HSCT. We demonstrated that γδ T cells collected from transplanted patients are endowed with capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in patients receiving this type of graft, may boost γδ T cell cytotoxic activity against leukemia cells. Thirty-three patients were treated with ZOL every 28 days at least twice. γδ T cells before and after ZOL treatments were studied till at least 7 months after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. Proteomic analysis of γd T cells purified from patients showed that, starting from the first infusion, ZOL caused up-regulation of proteins involved in activation processes and immune response, paralleled by down-regulation of proteins involved in proliferation. These findings are consistent with an induction of Vδ2 cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts. Furthermore, a proteomic signature was identified for each individual ZOL treatment. Patients given 3 or more ZOL infusions had a better probability of survival in comparison to those given 1 or 2 treatments. In conclusion,ZOL influences Vδ2 cell activity, determines a specific proteomic signature and enhances anti-leukemia activity, this potentially resulting into an increased anti-tumor effect. Disclosures No relevant conflicts of interest to declare.

Background aims. Immunomagnetic cell selection (ICS) of CD34 � cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection. Methods . The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The effi cacy of the automated procedure was compared with the control group, where washing were performed using a standard method. Results . The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34 � cell recovery of 84.87% (71.80 – 105, SD 8.62; range, standard deviation) and 83.45% (47 – 109, SD 16.12), respectively. The NC and CD34 � cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36 – 97.76, SD 22.5) and 61.51% (30.87 – 81.79, SD 19.3), respectively. The CD34 � cell recovery after ICS was 51.27% (13.77 – 98.82, SD 24.97) and 48.89% (15.57 – 88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4 – 96.10, SD 13.07) and in group 2 84.97% (58.1 – 97.8, SD 15.58). Conclusions. The effi cacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.

Temporal relationship between HHV 6 and graft vs host disease in a patient after haplo-identical SCT and severe T-cell depletion

Background aims: Immunomagnetic CD34 cell selection (ICS) is utilized in autologous and allogeneic transplants. In the fi rst case it is used to reduce the neoplastic contamination of concentrates, while in the second case it is needed to carry out a T-depletion of cell concentrates in order to reduce the incidence of graft-versus-host disease (GvHD) in patients who have undergone haplo-identical transplants. Methods: The effi cacy of CliniMACS technology, after reduction of platelet contamination, incubation of monoclonal antibodies (MAb) and successive washings of concentrates, performed in 16 ICS using the standard method without reducing platelet content, was compared with the use of the automated system CytoMate, which was carried out in 46 ICS. Results: In the group of ICS carried out after automatic manipulation, a signifi cant statistical difference in purity was noted (91.39% versus 83.57, P 0.017) compared with the group of ICS carried out with the standard procedure. The same signifi cant difference was noted in relation to the remaining percentages of CD3 and CD19 cells (2.31% versus 5.68%, P 0.012, and 1.58% versus 2.71%, P 0.014, respectively). Recovery of CD34 cells overlapped in the two groups (70.49% versus 68.39%, P 0.774). Conclusions: Immunomagnetic selection carried out using the automated procedure was more effi cient, producing a purer sample, more effi cient T-depletion and optimal reduction of B cells, without infl uencing cell recovery. Furthermore, conforming to good manufacturing practice (GMP) guidelines, the entire procedure with CytoMate took place in a contamination-controlled environment.

Haplo-identical HSCT may resolve the shortage of sufficient available HLA-matched donors for treatment of end-stage blood-cancer patients with a HSCT. Yet, current techniques do not allow for such procedure as presence of T-cells will cause severe GvHD and absence of T-cells (i.e. naked Haplo) will lead to occurrence of opportunistic infections. Kiadis Pharma is developing ATIR, a personalized T-cell immunotherapy based on a donor lymphocyte preparation selectively depleted of host alloreactive T-cells, through use of photo-dynamic therapy. Briefly, recipient-reactive T-cells from donor-origin are activated in a unidirectional mixed lymphocyte reaction (MLR). Thereafter the cells are treated with the proprietary compound TH9402 which is selectively retained in activated T-cells. Subsequent light-exposure eliminates the activated recipient-reactive T-cells but preserves the other T-cells. In a dose-finding phase I/II clinical study (CR-GVH-001), the product was shown to enable safe and efficacious haplo-identical HSCT with doses up to 5x106 viable T-cells/kg. Currently, a subsequent phase II clinical study (CR-AIR-007) on ATIR (2x106/kg) is ongoing. Figure 1 Figure 1. Using recently developed analytical methods, the ATIR product is extensively characterized showing that recipient-alloreactive T-cells were selectively depleted from the product while retaining reactivity against unrelated 3rdparty antigens and general potent T-cells. In the figure above, a typical example of the ATIR responsiveness profile is shown when analyzing the cells in a CFSE-dilution based proliferation assay. Clearly, the cells in ATIR no longer proliferate to recipient antigens, but do still propagate when co-incubated with unrelated 3rd party cells or the poly-clonal T-cell stimulus anti-CD3/28. Generally, almost all recipient-responsiveness is depleted, while 3rdparty responsiveness remains unchanged. The ATIR proliferation profile is corroborated by examination of interferon-γ production at day 5: the original donor cells produced a median of 775 pg/ml IFN-γ when stimulated with recipient cells, whereas ATIR produced a median of 1 pg/ml IFN-γ. Also less IFN-γ was produced by ATIR when co-incubated with 3rd party cells: 127 pg/ml vs 1106 pg/ml. However, ATIR produced more IFN-γ when polyclonally stimulated with PMA+ionomycin: 4075 pg/ml vs 2580 pg/ml. TableNaïve (%)Effector (%)CM (%)EM (%) CD4 T-cellsDonor4840210ATIR4435714 CD8 T-cellsDonor59161114ATIR6911911 Additionally, ATIR was shown to have retained the presence of memory and naïve T-cells (see table), viral-specific T-cells as assessed by multimers staining and showed responsiveness to pathogens. Thereby, ATIR will provide mature immune cells that offer immune protection without eliciting severe GvHD. The in vitro characterization data are supported by clinical data showing absence of TRM during 5-year follow-up (CR-GVH-001) over a broad dose range and no occurrence of grade 2 or higher GvHD in the ongoing clinical study (CR-AIR-007). Together, these data show that using ATIR as an adjunctive medication, haplo-identical HSCT can be safe and efficacious. Disclosures No relevant conflicts of interest to declare.

Epstein-Barr virus (EBV) related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation (SCT) and solid organ transplantation. In immune suppressed transplant recipients the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. This is however both time-consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific Tcells, treated and cured a patient suffering from PTLD with multiple associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within days treat patients with lifethreatening EBV-associated malignancies.

Ultraviolet B (UV-B) irradiation of BM cells (BMC) prior to transplantation into lethally gamma irradiated allogeneic rats prevents graft-versus-host disease (GVHD), induces stable allogeneic chimerism and specific tolerance to donor-type allografts. This study evaluated the role of UV-B modulation of bone marrow transplant (BMT) in the prevention of GVHD in the parent (P)-to-F1 hybrid rat combination of Lewis-to-Lewis × Brown Norway F1 (LBNF1) and of subsequent tolerance to small bowel transplantation (SBT). Lethally gamma irradiated (10.5 Gy) LBNF1 recipients of unmodified Lewis BMT (admixture of 108 BMC and 5 × 106 spleen cells) developed lethal GVHD and died within 55 days. Similarly, groups of lethally irradiated LBNF1 recipients of Lewis BMT treated with 100–300 J/m2 UV-B developed GVHD and died within 47 days. All F1 hybrid recipients of Lewis BMT treated with 700 or 900 J/m2 of UV-B permanently accepted their BM grafts, gained weight, showed no clinical evidence of GVHD and survived for > 300 days. These stable chimeras expressed 94–98% donor T-cells in their lymph nodes and became tolerant to BM donor-type (Lewis) SBT when grafted 180 days after BMT. In contrast, similarly prepared F1 recipients rejected BN SBT, thus demonstrating donor specificity. The chimeric rats were specifically unresponsive to donor (Lewis) Ag in MLR while they maintained normal alloreactivity to BN stimulators. These results suggest that UV-B irradiation of BMT offers a promising approach to overcoming the limitations of T-cell depletion of BMT and may offer a useful approach for recipient conditioning for induction of transplantation tolerance.

Immunosuppression with ALG has become a new alternative in the treatment of SAA [19]. In the original reports ALG was followed by a BM infusion from a family donor [12, 15]. In subsequent experimental and clinical trials ALG has been effective with and without BM infusion [16, 17, 19, 20, 21]. This modality is applicable in the HLA-nonidentical setting; it is not followed by GvHD and leads to autologous BM recovery in more than 50% of patients with SAA. It is still an unsolved problem whether ALG followed by BM is more or less effective than ALG alone.

Introduction Allogeneic stem-cell transplantation (SCT) is curative therapy in AML by providing intensive chemotherapy and enhancing a graft-versus-leukemia (GVL) effect. The GVL effect is usually closely associated with GVHD. The use of haploidentical SCT (haploSCT) is rapidly increasing due to the introduction of non-T depleted methods, in particular with post-transplant cyclophosphamide (PTCy), with similar outcomes as following other donor sources. There is no data whether GVL after haploSCT is associated with GVHD as in matched donor SCT. Methods We assessed the impact of acute and chronic GVHD on SCT outcomes following non-T depleted haploSCT with PTCy, by using a series of landmark analyses. Results The study included 605 patients with AML in CR1 (73%) or CR2 (27%) after haploSCT with PTCy, given during the years 2009-2016. The median age was 53 years (18-76). The overall rate of acute GVHD grade II-IV and III-IV was 28.4% and 8.0%, respectively. The rates of chronic GVHD all grades and extensive were 32.7% and 12.3%, respectively. The 2-year leukemia-free survival (LFS) was 59.9%. 509 patients were alive and leukemia-free 100 days after SCT; 366 had no prior acute GVHD at this landmark, 107 had acute GVHD grade II and 36 had grade III-IV. The subsequent relapse rate was 20.3%, 18.3% and 11.9%, respectively (P=0.60). The subsequent non-relapse mortality (NRM) rate was 10.3%, 19.0% and 35.7%, respectively (P Conclusions Acute and chronic GVHD of any grade were not associated with subsequent relapse. Acute GVHD grade III-IV and extensive chronic GVHD were associated with higher NRM and lower LFS. GVL is thus not closely associated with GVHD after non T-depleted haploSCT with PTCy. Future novel strategies for prevention of significant GVHD are warranted.