Levels Of Donor Chimerism Research Articles

Galectin-1 is associated with hematopoietic cell engraftment in murine MHC-mismatched allotransplantation.

Haploidentical hematopoietic cell transplantation (haplo-HCT) is associated with an increased risk of allograft rejection. Here, we employed a major histocompatibility complex (MHC)-mismatched allogeneic HCT (allo-HCT) murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected or engrafted based on donor chimerism levels. We noted significantly higher frequencies of CD4+ T cells, CD8+ T cells, natural killer cells, IFN-γ and TNF-α producing CD4+ T cells, and IFN-γ producing dendritic cells and macrophages in rejected mice. Conversely, we found significantly increased frequencies of regulatory T cells (Tregs), predominantly Helios+, IL-10-producing CD4+ T cells, type 1 regulatory (Tr1) cells, and the proportion of Tr1+Gal-1+ cells in engrafted mice. Further, Gal-1 specific blockade in Tregs reduced suppression of effector T cells in engrafted mice. Lastly, effector T cells from engrafted mice were more prone to undergo apoptosis. Collectively, we have shown that Gal-1 may favor HSC engraftment in an MHC-mismatched murine model. Our results demonstrate that Gal-1-expressing Tregs, especially at earlier time points post-transplant, are associated with inducing immune tolerance and stable mixed chimerism after HCT.

Hematopoietic cell transplantation for DOCK8 deficiency: Results from a prospective clinical trial

DOCK8 deficiency is a primary immunodeficiency in which allogeneic hematopoietic cell transplantation (HCT) represents the only known cure. We tested the ability of a busulfan-based regimen to achieve reliable engraftment and high levels of donor chimerism with acceptable toxicity in a prospective clinical trial in DOCK8 deficiency. To both evaluate the ability of HCT to reverse the clinical phenotype and to correct the immunologic abnormalities by 1 year post HCT. We conducted a prospective HCT trial for recipients with DOCK8 deficiency. Subjects were recruited from October 5, 2010, to December 30, 2022. Donor sources included fully matched related and unrelated donors and haploidentical donors. The reduced toxicity, myeloablative conditioning regimen contained no serotherapy. Graft-versus-host disease (GVHD) prophylaxis included either a calcineurin inhibitor with methotrexate or post-HCT cyclophosphamide (PT/Cy) followed by tacrolimus and mycophenolate mofetil. The trial was later amended to study PT/Cy in all patients. (Pilot Study of Reduced-Intensity Hematopoietic Stem Cell Transplant of DOCK8 [NCT01176006].) RESULTS: Thirty-six subjects, both children and adults (median age 16.4 years), underwent HCT for DOCK8 deficiency. Most patients, 33 of 36 (92%), achieved full (≥98%) donor chimerism in whole blood as early as day+30. With a median potential follow-up of 7.4 years, 29 (80.6%) were alive with no evidence of new DOCK8 deficiency-related complications. PT/Cy was effective in reducing the risk of acute GVHD in patients who had received matched unrelated donor and haploidentical transplants, but it was associated with transient delays in immune-reconstitution and hemorrhagic cystitis. A busulfan-based HCT regimen using PT/Cy for GVHD prophylaxis and a broad range of donor types and hematopoietic cell sources were well tolerated, leading to the reversal of the clinical immunophenotype.

Azathioprine/hydroxyurea preconditioning prior to nonmyeloablative matched sibling donor hematopoietic stem cell transplantation in adults with sickle cell disease: A prospective observational cohort study.

Nonmyeloablative, matched sibling donor hematopoietic stem cell transplantation with alemtuzumab/total body irradiation (TBI) conditioning is a curative therapy with low toxicity for adults with sickle cell disease (SCD). However, relatively low donor chimerism levels and graft rejection remain important challenges. We hypothesized that adding azathioprine/hydroxyurea preconditioning will improve donor chimerism levels and reduce graft failure rate. In this prospective cohort study, we enrolled consecutive adult patients with SCD undergoing matched sibling donor transplantation at the Amsterdam UMC. Patients received azathioprine 150 mg/day and hydroxyurea 25 mg/kg/day for 3 months prior to alemtuzumab 1 mg/kg and 300 cGy TBI conditioning. Twenty patients with SCD (median age 26 years [range 19-49], 13 females) were transplanted. Median follow-up was 46.0 months (IQR 21.8-57.9). One-year overall survival and event-free survival (graft failure or death) were both 95% (95% confidence interval 86-100). Mean donor myeloid and T-cell chimerism 1-year post-transplant were 95.2% (SD ±10.6) and 67.3% (±15.3), respectively. One patient (5%) experienced graft failure without autologous regeneration, resulting in infections and death. All other patients had a corrected SCD phenotype and were able to discontinue sirolimus. Three patients were successfully treated with alemtuzumab (1 mg/kg) after the transplant because of declining donor chimerism and cytopenias to revert impending graft rejection. Toxicity was mostly related to sirolimus and alemtuzumab. One patient developed steroid-responsive grade II intestinal acute graft-versus-host disease. Collectively, preconditioning with azathioprine/hydroxyurea prior to nonmyeloablative matched sibling donor transplantation resulted in excellent event-free survival and robust donor T-cell chimerism, enabling the successful withdrawal of sirolimus. ClinicalTrials.gov: NCT05249452.

Persistent or New Cytopenias Predict Relapse Better than Routine Bone Marrow Aspirate Evaluations After Hematopoietic Cell Transplantation for Acute Leukemia or Myelodysplastic Syndrome in Children and Young Adult Patients

The clinical value of serial routine bone marrow aspirates (rBMAs) in the first year after allogeneic hematopoietic cell transplantation (alloHCT) to detect or predict relapse of acute leukemia (AL) and myelodysplastic syndrome (MDS) in pediatric and young adult patients is unclear. The purpose of this analysis was to determine if assessment of minimal residual disease (MRD) by multiparameter flow cytometry (MFC, MFC-MRD) or donor chimerism (DC) in rBMAs or serial complete blood counts (CBCs) done in the year after alloHCT predicted relapse of AL or MDS in pediatric and young adult patients. We completed a retrospective analysis of patients with AL or MDS who had rBMAs performed after alloHCT between January 2012 and June 2018. Bone marrow (BM) was evaluated at approximately 3, 6, and 12 months for disease recurrence by morphology, MFC-MRD, and percent DC by short tandem repeat molecular testing. CBCs were performed at every clinic visit. The main outcome of interest was an assessment of whether MFC-MRD or DC in rBMAs or serial CBCs done in the year after alloHCT predicted relapse in AL or MDS pediatric and young adult patients. A total of 121 recipients with a median age of 13 years (range 1 to 32) were included: 108 with AL and, 13 with MDS. A total of 423 rBMAs (median 3; 0 to 13) were performed. Relapse at 2 years was 23% (95% CI: 16% to 31%) and at 5 years 25% (95% CI: 18% to 33%). One hundred fifty-four of 157 (98%) rBMAs evaluated for MRD by MFC were negative and did not preclude subsequent relapse. Additionally, low DC (<95%) did not predict relapse and high DC (≥95%) did not preclude relapse. For patients alive without relapse at 1 year, BM DC (P = .74) and peripheral T-cell DC (P = .93) did not predict relapse. Six patients with low-level T-cell and/or BM DC had a total of 8 to 20 BM evaluations, none of these patients relapsed. However, CBC results were informative for relapse; 28 of 31 (90%) relapse patients presented with an abnormal CBC with peripheral blood (PB) blasts (16 patients), cytopenias (9 patients), or extramedullary disease (EMD, 3 patients). Two patients with BM blasts >5% on rBMA had circulating blasts within 5 weeks of rBMA. Neutropenia (ANC <1.5 K/mcl) at 1 year was predictive of relapse (P = .01). Neutropenia and thrombocytopenia (<160 K/mcl) were predictive of disease-free survival (DFS) with inferior DFS for ANC <1.5 K/mcl, P = .001, or platelet count <160 K/mcl (P = .04). These results demonstrate rBMAs after alloHCT assessed for MRD by MFC and/or for level of DC are poor predictors for relapse in pediatric and young adult patients with AL or MDS. Relapse in these patients presents with PB blasts, cytopenias, or EMD. ANC and platelet count at 1-year were highly predictive for DFS.

Low dose post-transplant cyclophosphamide and sirolimus induce mixed chimerism with CTLA4-Ig or lymphocyte depletion in an MHC-mismatched murine allotransplantation model

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.

Improved T-Cell Chimerism and Successful Withdrawal of Immunosuppression after Non-Myeloablative Stem Cell Transplantation for Sickle Cell Disease with Azathioprine/Hydroxyurea Preconditioning Added to Alemtuzumab/TBI Conditioning

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only established curative treatment option for sickle cell disease (SCD). In adult SCD patients, myeloablative conditioning is associated with significant toxicity, primarily because of cumulative organ damage. Recently, matched sibling donor (MSD) transplantation with non-myeloablative conditioning (alemtuzumab/3 Gy total body irradiation (TBI)) showed promising outcomes in adult SCD patients. This chemotherapy-free regimen, typically resulting in mixed chimerism, is associated with mild toxicity with no reports of graft-versus-host disease (GvHD). Despite mixed chimerism, the red blood cell (RBC) phenotype is completely donor-derived. However, in a large series, 13% of patients experienced graft failure. Another 8.2% of patients could not stop their immunosuppression (sirolimus) after one year, because of low donor T-cell chimerism (<50%). Reported mean donor T-cell chimerism at 12-months post-transplantation was 48% (Br J Haematol. 2021 Feb;192(4): 761-768). We hypothesized, that the addition of azathioprine and hydroxyurea as preconditioning to the alemtuzumab/TBI conditioning regimen will reduce the risk of graft failure and improve donor T-cell chimerism, enabling successful withdrawal of sirolimus in all patients. Aim: To investigate the effects of adding azathioprine/hydroxyurea preconditioning to alemtuzumab/TBI conditioning on donor chimerism levels and the occurrence of graft failure in adult SCD patients receiving non-myeloablative MSD-HSCT. Methods: Adult SCD patients with an available HLA-identical sibling donor were eligible for this treatment. All patients received three months of azathioprine 150mg qd and hydroxyurea 25mg/kg qd preconditioning. After exchange transfusion (target HbS <30%), conditioning with alemtuzumab 1mg/kg total dose and 3Gy TBI started on day -7, followed by unmanipulated peripheral hematopoietic stem cell infusion on day 0. Sirolimus as GvHD and rejection prophylaxis started on day -1. Results: Twenty SCD patients (median age 26 (range 19-49) years) were transplanted with a median follow-up of 24.5 months (range 1-52 months). Patient and transplant characteristics are shown in Table 1. Median donor myeloid and T-cell chimerism of engrafted patients was 100% (range 87-100%) and 68.5% (range 37-89%) at 12-months post-transplantation and 94% (range 86-100%) and 78.5% (range 68-88%) at 18-months post-transplantation, respectively (Figure 1). These donor T-cell chimerism percentages are higher than previously reported with alemtuzumab/TBI conditioning only. All engrafted patients had a corrected SCD phenotype with normalized hemoglobin levels. Importantly, all patients with a follow-up of ≥ 12 months (n=12) could successfully taper and stop sirolimus without decreases in donor chimerism. One year estimated disease-free survival and overall survival were 94.7%. and 93.3%, respectively. One patient (5%) experienced graft failure without autologous regeneration and subsequently died after a series of complications, including EBV reactivation, post-transplantation lymphoproliferative disease, and systemic aspergillosis. Acute gastrointestinal GvHD grade 2 occurred in one patient (5%) and resolved quickly after treatment with prednisolone. Summary/Conclusion: Azathioprine/hydroxyurea preconditioning prior to alemtuzumab/TBI resulted in improved donor T-cell chimerism, potentially reducing the risk of graft failure after non-myeloablatieve MSD transplantation in SCD patients. Importantly, all engrafted patients reached donor T-cell chimerism >50% and were able to stop immunosuppressives as scheduled. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia.

The current global platelet supply is often insufficient to meet all the transfusion needs of patients, in particular for those with alloimmune thrombocytopenia. To address this issue, we have developed a strategy employing a combination of approaches to achieve more efficient production of functional megakaryocytes (MKs) and platelets collected from cord blood (CB)-derived CD34+ hematopoietic cells. This strategy is based on ex-vivo expansion and differentiation of MKs in the presence of bone marrow niche-mimicking mesenchymal stem cells (MSCs), together with two other key components: (1) To enhance MK polyploidization, we used the potent pharmacological Rho-associated coiled-coil kinase (ROCK) inhibitor, KD045, resulting in liberation of increased numbers of functional platelets both in-vitro and in-vivo; (2) To evade HLA class I T-cell-driven killing of these expanded MKs, we employed CRISPR-Cas9-mediated β-2 microglobulin (β2M) gene knockout (KO). We found that coculturing with MSCs and MK-lineage-specific cytokines significantly increased MK expansion. This was further increased by ROCK inhibition, which induced MK polyploidization and platelet production. Additionally, ex-vivo treatment of MKs with KD045 resulted in significantly higher levels of engraftment and donor chimerism in a mouse model of thrombocytopenia. Finally, β2M KO allowed MKs to evade killing by allogeneic T-cells. Overall, our approaches offer a novel, readily translatable roadmap for producing adult donor-independent platelet products for a variety of clinical indications.

Erythroid Chimerism Measurement As a Predictive Tool of Bone Marrow Transplantation Outcome in Sickle Cell Disease

Allogeneic bone marrow transplantation (BMT) is the only curative treatment available for sickle cell disease (SCD). The effectiveness of the treatment is primarily assessed by the chimerism rate, which is the fraction of cells derived from the donor's hematopoietic stem cells (HSCs) relative to the recipient's cells. New less toxic conditioning agents, described as reduced, nonmyeloablative (as opposed to myeloablative conditioning agents) are now used for BMT with sibling donors. Moreover, most sibling donors are sickle cell trait carriers (heterozygous A/S genotype). In the absence of a test performed on erythroid cells, the rate of chimerism is currently assessed either on different subpopulations of myeloid cells by molecular biology techniques or on hemoglobin measurement by chromatography (HPLC). However, the percentage of myeloid chimerism is rarely representative of the percentage of erythroid chimerism in these patients and HPLC cannot allow analysis at the single cell level.An easy-to-implement method for accurate measurement of fetal hemoglobin (HbF) content in individualized red blood cells (RBCs) has recently been developed (Hebert, et. al, AJH 2020). This method relies on flow cytometry for cell individualization and intracellular labeling of HbF with a fluorescent monoclonal antibody for quantification. Based on the same approach, we propose the use of monoclonal antibodies directed against adult hemoglobins (HbA0 and HbA2) or hemoglobin S (HbS). This strategy allows to discriminate, from a blood sample, the different RBC subpopulations coming from either the recipient or the donor, or from the transfusion associated with this procedure, thus allowing us to precisely determine the rates of erythroid chimerism in patients.As a proof of concept, we show here the evaluation of the percentage of erythroid chimerism during a longitudinal follow-up performed on 3 patients with SCD (P1 - 18 months, P2 - 4 months, and P3 - 4 months). The 3 patients received allogeneic BMT from a sibling A/S donor following nonmyeloablative conditioning consisting of the administration of an anti-CD52 antibody associated with radiotherapy (300 cGy TBI - total-body irradiation). We observed for P1 and P2 donor erythroid chimerism >90% and >95%, 1 and 2 months after BMT, respectively (measured after exclusion of transfused RBCs). These results are in accordance with the presence of most RBCs HbA- and HbS-positive, as determined by flow cytometry. In addition, the analysis of the reticulocyte (retic) subpopulations (CD71-positive cells) of these patients provides precise information on the RBC production of the bone marrow following transplantation. Retic donor chimerism levels were >99% for both P1 and P2, 1 month after BMT procedure. P1 and P2 showed stable donor reticulocyte and donor erythroid chimerism levels during the follow-up. On the contrary, P3 presented a different response with 43%, 41% and 5% donor erythroid chimerism 1, 2 and 3 months after BMT, respectively. Retrospectively, this bad response in P3 would have been predicted by low donor reticulocyte chimerism levels (5%, 6% and 0.5% at 1, 2 and 3 months after BMT, respectively). Our erythroid specific chimerism results were also compared to the assessment of chimerism performed on either total white blood cells (WBCs) or lineage specific subpopulations after sorting. Over 18 months after BMT in P1, the correlation between retic chimerism and total WBCs or T lymphocytes (CD3-positive cells) chimerism were not statistically significant (P = 1 and P = 0.833, respectively - Spearman test) (Figure 1).This new tool to monitor BMT outcome to treat SCD shows great precision and potentially a stronger predictive capability than total WBCs or lineage specific chimerism assessed by molecular biology. Our results highlight the importance of analyzing reticulocyte population to better assess BMT efficacy and predict the outcome a few days after the transplant. Its use could also be considered in the context of the various gene therapy strategies currently under development using modified β-globin gene addition or direct mutation correction through CRISPR/Cas9 and homologous recombination system. [Display omitted] DisclosuresBencheikh: Innovhem: Current Employment. Bartolucci: Innovhem: Current holder of individual stocks in a privately-held company.

Fludarabine–Cyclophosphamide-Based Conditioning with Antithymocyte Globulin Serotherapy Is Associated with Durable Engraftment and Manageable Infections in Children with Severe Aplastic Anemia

Severe aplastic anemia (SAA) is a bone marrow failure syndrome that can be treated with hematopoietic cell transplantation (HCT) or immunosuppressive (IS) therapy. A retrospective cohort of 56 children with SAA undergoing transplantation with fludarabine–cyclophosphamide–ATG-based conditioning (FluCyATG) was analyzed. The endpoints were overall survival (OS), event-free survival (EFS), cumulative incidence (CI) of graft versus host disease (GVHD) and CI of viral replication. Engraftment was achieved in 53/56 patients, and four patients died (two due to fungal infection, and two of neuroinfection). The median time to neutrophil engraftment was 14 days and to platelet engraftment was 16 days, and median donor chimerism was above 98%. The overall incidence of acute GVHD was 41.5%, and that of grade III-IV acute GVHD was 14.3%. Chronic GVHD was diagnosed in 14.2% of children. The probability of 2-year GVHD-free survival was 76.1%. In the univariate analysis, a higher dose of cyclophosphamide and previous IS therapy were significant risk factors for worse overall survival. Episodes of viral replication occurred in 33/56 (58.9%) patients, but did not influence OS. The main advantages of FluCyATG include early engraftment with a very high level of donor chimerism, high overall survival and a low risk of viral replication after HCT.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant Using a Human Antibody Drug Conjugate Targeting C-KIT

For patients undergoing hematopoietic stem cell transplantation (HSCT), conditioning remains a primary source of transplant related morbidity and mortality and limits the broader application of this life-saving therapy. Current conditioning regimens, including total body irradiation and high dose chemotherapy, cause acute toxicity, cytokine storm, increased graft-versus-host disease, and organ damage. In addition, these regimens are also associated with the development of secondary malignancies, autoimmunity, and infertility.To address this significant unmet medical need, we have developed a conditioning regimen using an antibody drug conjugate (ADC) targeting C-KIT (CD117) to specifically deplete host hematopoietic stem cells. CD117 is highly expressed on hematopoietic stem and progenitor cells (HSPCs) and binding to its ligand, stem cell factor, promotes hematopoiesis, self-renewal, and rapid internalization of the receptor. The rapid internalization and selective expression of CD117 on HSPCs make it an ideal target for an ADC-based approach to depleting HSPCs, while limiting systemic toxicity. In addition, CD117 is frequently overexpressed on malignant cells in patients with acute myelogenous leukemia. Thus, an ADC targeting CD117 may provide an opportunity for disease control prior to or concomitant with an HSCT. Previous studies have demonstrated that an antagonistic antibody against CD117 lead to high levels of HSPC depletion and donor engraftment in immunocompromised mice, but failed to facilitate this effect in immunocompetent hosts. To overcome the engraftment barrier, additional studies were conducted with an anti-CD117 antibody conjugated to the ribosome inactivating toxin, saporin, which allowed for robust HSPC depletion and high levels of congenic donor chimerism in immunocompetent mice (Czechowicz et.al., Blood 2016, 128:493).To advance this technology toward the clinic, we have developed a fully human anti-CD117 antibody conjugated to a small molecule toxin capable of depleting proliferating and quiescent cells (CD117-ADC), that demonstrated greater than 90% killing of the leukemia cell line Kasumi-1, and was equally effective at killing primary human CD34+ bone marrow cells during in vitro culture. Humanized NSG mice treated with CD117-ADC had greater than 90% depletion of human HSPCs in the bone marrow, 21 days following a single administration of the ADC. The specificity of CD117-ADC for HSPCs was confirmed by the presence of stable peripheral human lymphocyte populations, and absence of human myeloid cells; indicating a lack of stem and progenitor cells capable of replenishing these short-lived cells. In addition to the ability of CD117-ADC to deplete HSPCs in humanized mice, preliminary data suggests that CD117-ADC is also effective at reducing tumor burden in mice challenged with CD117 expressing tumor cells.Eliminating HSPCs from the stem cell niche with a targeted ADC has the promise to enable the engraftment of donor HSCs, while preventing the systemic toxicity associated with irradiation and chemotherapy. We have demonstrated that an ADC targeting CD117 is capable of depleting human HSPCs in vitro and in vivo and has the potential to reduce tumor burden in mice. Ultimately, this novel approach for safer conditioning prior to HSCT could greatly increase the number of patients eligible for transplant and significantly reduce the damaging side effects associated with current protocols and may provide effective non-genotoxic conditioning for all patients suffering from malignant or non-malignant diseases who require a HSCT. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

Treosulfan-based conditioning for inborn errors of immunity.

Inborn errors of immunity (IEI) are inherited disorders that lead to defects in the development and/or function of the immune system. The number of disorders that can be treated by haematopoietic stem-cell transplantation (HSCT) has increased rapidly with the advent of next-generation sequencing. The methods used to transplant children with IEI have improved dramatically over the last 20 years. The introduction of reduced-toxicity conditioning is an important factor in the improved outcome of HSCT. Treosulfan has myeloablative and immunosuppressive properties, enabling engraftment with less toxicity than traditionally used doses of busulfan. It is firmly incorporated into the conditioning guidelines of the Inborn Errors Working Party of the European Society for Blood and Marrow Transplantation. Unlike busulfan, pharmacokinetically guided dosing of treosulfan is not part of routine practice, but data are emerging which indicate that further improvements in outcome may be possible, particularly in infants who have a decreased clearance of treosulfan. It is likely that individualized dosing, not just of treosulfan, but of all agents used in conditioning regimens, will be developed and implemented in the future. This will lead to a reduction in unwanted variability in drug exposure, leading to more predictable and adjustable exposure, and improved outcome of HSCT, with fewer late adverse effects and improved quality of life. Such conditioning regimens can be used as the basis to study the need for additional agents in certain disorders which are difficult to engraft or require high levels of donor chimerism, the dosing of individual cellular components within grafts, and effects of adjuvant cellular or immunotherapy post-transplant. This review documents the establishment of treosulfan worldwide, as a safe and effective agent for conditioning children with IEI prior to HSCT.

First Report of Non-Genotoxic Conditioning with JSP191 (anti-CD117) and Hematopoietic Stem Cell Transplantation in a Newly Diagnosed Patient with Severe Combined Immune Deficiency

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A&amp;gt;G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

5-Azacytidine Depletes Hematopoietic Stem Cells and Synergizes with an Anti-CD117 Antibody to Augment Engraftment of Donor Stem Cells in Immunocompetent Mice

Monoclonal antibody (mAb)-targeting of CD117 (c-Kit) present on hematopoietic stem cells (HSC) is an emerging conditioning strategy for hematopoietic cell transplantation (HCT) to replace standard-of-care alkylator and radiation therapy used to deplete recipient HSC and permit donor HSC engraftment. In preclinical and clinical studies anti-CD117 mAbs, which inhibit stem cell factor (SCF) from binding to CD117, have shown single agent efficacy in permitting donor HSC engraftment in immunocompromised recipients. However, preclinical data suggest that anti-CD117 mAb, ACK2, used as a single agent is insufficient to enable HSC engraftment in immunocompetent mice. Hence, there is a desire to potentiate the effect of naked anti-CD117 mAbs to permit targeted elimination of host HSC for a range of immune competent bone marrow (BM) states, from monogenic disorders, e.g., hemoglobinopathies, to BM clonal malignant diseases. Here, we show that ACK2 synergizes with the widely used hypomethylating agent, 5-Azacytidine (AZA), to deplete HSC and enable robust long-term HSC engraftment in immunocompetent mice. The cell depleting effect of AZA or ACK2+AZA was first tested on mature and primitive hematopoietic cells of C57BL/6 (B6) mice. Mice received AZA at 5mg/kg i.p. for 5 days either alone or combined with ACK2 at 500 µg i.v. 5 days prior to AZA. BM and spleen were harvested days 6, 10 and 20 after the first AZA dose and analyzed by extended immunophenotype. For HCT studies both congenic [B6 (H-2b, Thy1.1, CD45.1) into B6 recipients (H-2b, Thy1.1, CD45.1/CD45.2)] and allogeneic [B6 (H-2b, Thy1.1, CD45.1) into BALB.B recipients (H-2b, Thy1.2, CD45.2)] transplantation mouse models were tested. Single agent AZA induced rapid depletion of CD117+ cells in the BM, including LT-HSC (Lin-Sca+Kit+[LSK]CD150+CD48-) and ST-HSC (LSKCD150-CD48-). In vivo HSC depletion by AZA has not been previously reported, and was surprising given that most HSC are quiescent, and non-proliferating cells are known to be insensitive to the toxicities of AZA. Examination of HSC populations in the spleen did not suggest that mobilization of LT- or ST-HSC to secondary organs caused the decrease in BM. Rather, AZA-induced myelosuppression was followed by rapid expansion of myeloid-biased MPP and increased HSC proliferation as assessed by Ki67 intracellular protein expression. We hypothesized, that the in vivo HSC-depletive effects of AZA might result from increased sensitivity of proliferating HSC to AZA. Hence, the in vitro effects of different AZA concentrations were tested on proliferating HSC. In both mouse and human HSC cultures, supplemented with SCF and TPO, we observed that AZA reduced the number of cells in a dose dependent manner and decreased cell viability at concentrations of ≥ 0.5 µg/ml. In vivo, combined ACK2+AZA led to more robust HSC depletion and prolonged time to HSC recovery compared to AZA alone. We then evaluated if blockade of CD117 with its ligand SCF by ACK2 was the cause of this prolonged HSC-depletion. 2B8 mAb, which also binds mouse CD117, but only partially inhibits CD117/SCF signaling, did not result in augmented in vivo HSC depletion (Figure 1A). We further tested if immunocompetent mice treated with ACK2+AZA could be "rescued" with exogenous recombinant SCF. SCF-treatment resulted in faster recovery of LT-HSC. Together these studies suggest that the synergy of ACK2+AZA on the depth and duration of HSC-depletion depends of the disruption of CD117/SCF binding. Finally, we tested ACK2+AZA conditioning on donor stem cell engraftment in immunocompetent mice. Transplantation of 15-20x106 WBM cells following ACK2+AZA conditioning led to significantly increased stable mean multilineage donor chimerism of &amp;gt;60% in the congenic (Figure 1B) and &amp;gt;20% in the allogeneic setting. Single agent AZA enabled sustained donor engraftment with levels of myeloid donor chimerism of 10-15%, serving as proof of principle that AZA treatment can indeed clear HSC niches and permit engraftment of true HSC. Similar to WBM grafts, 5x104 purified HSC (LSK) engrafted successfully in the ACK2+AZA group, reaching long-term (26 weeks) mean donor myeloid chimerism &amp;gt;25% in both congenic and allogeneic settings. Our studies reveal a previously unknown effect of AZA on BM HSC and provide a platform for the clinical use of a novel less toxic non-myeloablative conditioning strategy for HCT, which is aimed at the rapid translation into clinical practice. Disclosures Pang: Jasper Therapeutics, Inc: Current Employment, Current equity holder in private company. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Peripheral Blood CD34+ Donor Chimerism Is Superior to CD3+ Donor Chimerism for Predicting Relapse Following Allogeneic Stem Cell Transplantation for Myeloid Malignancies

Background: Disease relapse remains the main cause of mortality after allogeneic stem cell transplantation (allo-SCT) for patients with myeloid malignancies. Loss of donor chimerism (DC) is commonly used as a biomarker of impending relapse, which allows the initiation of pre-emptive therapies such as withdrawal of immunosuppression or donor lymphocyte infusion (DLI). Surprisingly, there are few if any direct comparisons of peripheral blood CD34+ and CD3+ DC as biomarkers to predict relapse. We hypothesized that loss of CD34+ DC may be a more direct measure of impending relapse given most myeloid malignancies express CD34. Methods: We prospectively measured peripheral blood CD34+ and CD3+ DC on days 30, 60, 90, 120 and 180 following allo-SCT for patients with AML (n=113) or MDS (n=23) transplanted at a single centre between July 2011 and November 2019. Chimerism analysis was performed using purified cell subsets isolated from 60 mL peripheral blood using PCR-based amplification of short tandem repeats (STRs). The goal of this retrospective analysis was to compare the value of CD3+ and CD34+ DC for predicting relapse. Institutional practice for CD34+ DC below 80% included a bone marrow biopsy to identify morphologic relapse and donor lymphocyte infusion. Statistical analysis was performed with R 3.5.2 (The R project for Statistical computing) or GraphPad (v8.2.0). Results: Overall, 41 of 136 (30%) patients had morphologic relapse at a median time of 153 days after allo-SCT (range 50-1742). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CD3+ and CD34+ donor chimerism for morphologic relapse is shown in Figure 1A. CD34+ DC outperformed CD3+ DC in all criteria, irrespective of the percentage chosen. Furthermore, a concurrent reduction in CD34+ DC was seen in almost all patients (14/16 at the 80% level) that had a fall in CD3+ DC. A fall in CD34+ DC below 80% was highly predictive for relapse, with a 5 year relapse free survival of only 17% compared with 80% for those that maintained DC &amp;gt; 80% for the first 180 days (Figure 1B). To determine the clinical utility of CD34+ DC, we measured the time to relapse in those patients that had a fall in DC without morphologic relapse at the time of DC measurement (Figure 1A). Based upon our institutional trigger of CD34+ DC &amp;lt; 80%, the median time to relapse was 49 days in 22 patients. In contrast, a CD34+ DC &amp;lt; 90% had a much longer time to relapse (71 days). However, this longer lead time would come at the price of unnecessary intervention in almost half of all patients as the PPV for CD34+ DC &amp;lt; 90% was only 55%. Loss of CD3+ DC had the longest lead time (&amp;gt; 70 days), however it was only useful up to 10 (24%) of all relapses. Finally, DLI administered for CD34+ DC &amp;lt; 80% maintained durable remission (&amp;gt; 12 months) in only 2 of 19 patients. Conclusion: This is the largest comparison of peripheral blood CD34+ and CD3+ DC following allo-SCT. Our results show that monthly monitoring of CD34+ DC in the first 6 months after allo-SCT is a more useful biomarker than CD3+ DC for predicting relapse in patients allografted for AML or MDS. The level of CD34+ DC chosen to trigger intervention should be guided by characteristics of the intervention such as toxicity and expected response time. Given the relative ineffectiveness of DLI for CD34+ &amp;lt; 80%, we suggest that 90% may provide greater time for immunologic responses. Figure Legend (A) Characteristics of different levels of CD3 and CD34 DC at any time in the first 180 days post-allo-SCT. Number of patients that fulfil the level in the total cohort of 136 patients. Number of patients before relapse indicates the number that do not have morphologic relapse at the time of DC measurement. Median days before relapse is shown for those patients. (B) Relapse free survival of patients according to CD34+ DC &amp;gt; 80% (blue line) or &amp;lt; 80% (yellow line) in the first 180 days. Disclosures Spencer: AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; Celgene, Janssen and Takeda: Speakers Bureau; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria. Wei:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; MacroGenics: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Walter and Eliza Hall Institute: Other: former employee and receives a fraction of its royalty stream related to venetoclax; Pfizer: Honoraria; Genentech: Honoraria; Astra Zeneca: Honoraria, Research Funding; AbbVie Inc.: Consultancy, Honoraria, Research Funding.

Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p&amp;lt;0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p&amp;lt;0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Low Level Donor Chimerism of CD19 CAR-T Cells Returned to Complete Donor Chimerism in Patients with Relapse After Allo-Hematopoietic Stem Cell Transplant.

PurposeTo investigate the donor chimerism changes and curative effects associated with the use of autologous anti-CD19 chimeric antigen receptor (CAR) T cells with B-cell acute lymphoblastic leukemia (B-ALL) presenting with a low donor chimerism level and relapse after allogeneic hematopoietic stem cell transplant (allo-HSCT).MethodsNine patients with B-ALL showing low donor chimerism level and relapse after allo-HSCT were enrolled. Patients 1–3 received CD19 CAR-T cell therapy using cells derived from autologous peripheral blood mononuclear cells (PBMCs) (comprising a mixture of patient and original donor cells) as their donors could not provide PBMCs. Samples from the other six patients (Patients A–F) were investigated only in vitro. The changes in the degree of donor chimerism, function of the CD19 CAR-T cells and T cells in all nine patients were analyzed in vitro. The therapeutic effects and adverse events (AEs) were also evaluated in Patients 1–3.ResultsThe CAR-T cells and T cells in all nine patients showed complete donor chimerism restoration following a 12-day culture period in vitro. These CD19 CAR-T cells demonstrated strong cytotoxicity towards Nalm 6 cells in vitro except in patients 3 and D. In the latter patients, the absolute numbers of all subsets, especially the CD8 + T-cell absolute numbers in peripheral blood were very low. Patients 3 and D showed relatively short durations from transplant to recurrence and received chemotherapy after relapse. In the patients receiving CD19 CAR-T cell therapy, the most commonly observed AE was grade 1 to 2 cytokine release syndrome. None of the cases showed acute graft-versus-host disease during treatment. Patients 1 and 2 achieved complete response with complete restoration of donor chimerism. Patient 3, who received the same CD19 CAR-T cell therapy, did not respond to this therapy.ConclusionCD19 CAR-T cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for salvage therapy and could restore to complete donor chimerism after 12 days’ culture in vitro.Trial RegistrationHumanized CD19 CAR-T cell therapy for relapse or refractory B-cell lymphoma or acute B lymphocytic leukemia, ChiCTR1800019622, Registered 24 November 2018, http://www.chictr.org.cn/index.aspx.

Impact of early chimerism status on clinical outcome in children with acute lymphoblastic leukaemia after haematopoietic stem cell transplantation

BackgroundThe significance of very early chimerism assessment before day + 28, which is considered the moment of engraftment, is still unclear. In this retrospective study, we evaluated the clinical impact of very early chimerism on the clinical outcome after allogeneic haematopoietic stem cell transplantation (allo-HSCT) in children with acute lymphoblastic leukaemia (ALL).MethodsThe study group included 38 boys and 18 girls. Very early chimerism was evaluated on days + 7, + 14, + 21 and + 28 after the transplant. Short tandem repeat polymerase chain reaction (STR PCR) was used to analyse chimerism.ResultsOverall survival (OS) and event-free survival (EFS) were 84 and 80%, respectively. The OS in the group of 24 patients with complete donor chimerism on day + 14 was 83%, and it did not differ statistically compared to the 32 patients with mixed chimerism on day + 14 (OS was 84%). In our cohort of patients, the matched unrelated donor, male gender of donor, number of transplanted cells above 4.47 × 106 kg and no serotherapy with anti-thymocyte globulin (ATG) were statistically related to a higher level of donor chimerism. The immunophenotypes of disease, age of patient at time HSCT, recipient sex, stem cell source (peripheral blood/bone marrow) and conditioning regimen had no impact on early chimerism. Acute graft versus host disease grades II-IV was diagnosed in 23 patients who presented with donor chimerism levels above 60% on day 7.ConclusionsThe data presented in this study provide valuable insight into the analysis of very early chimerism in children with ALL treated with HSCT.

Anti-CD19-CAR T-Cell from Patients Relapsed after Allo-HSCT with a Low Level of Donor Chimerism Restored to Complete Donor Chimerism: Successful Cases and Analysis

Purpose: To study the efficacy of anti-CD19-CAR T-cell therapy and the changes of donor chimerism in CAR-T cells in vitro culture for patients who relapsed after allo-HSCT with a low level of donor chimerism. Methods: Two patients relapsed after allo-HSCT with a low level of donor chimerism (11.22% and 16.08%). Their autologous PBMCs were selected for anti-CD19-CAR T-cell therapy because their sibling or unrelated donor could not provide PBMC cells. The adverse events (AEs) were observed after CAR T-cell infusion. The cytokine release syndrome (CRS), the expansion of anti-CD19-CAR T-cells, the DNA level of anti-CD19-CAR gene and the changes of the donor chimerism were detected after CAR T-cell infusion. Then we selected other 7 acute leukemia patients relapsed after allo-HSCT with a low level of donor chimerism to detect the changes of donor chimerism in their T cells and anti-CD19-CAR T-cells in vitro culture. Results: Their notable AEs were grade 2 CRS. They had no aGVHD during anti-CD19-CAR T-cell therapy. They achieved CR 14 days after anti-CD19-CAR T-cell therapy with a restoration of complete donor chimerism (98.61% and 98.48%). To date, they had leukemia-free survival (LFS) and complete donor chimerism for 8 and 7 months. It was very interesting and fortunate that the CAR-T cells infused into the two patients were restorated to complete donor chimerism (99.2% and 99.4%). Then we found that complete donor chimerism were restored after 12 days culture in vitro in all the T cells and anti-CD19-CAR T-cells in the other 7 patients relapsed after allo-HSCT with a low level of donor chimerism. Conclusions: Anti-CD19-CAR T-cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for this salvage therapy and could restorate to complete donor chimerism after 12 days culture in vitro. Disclosures No relevant conflicts of interest to declare.

Recipient Age and Marked Thrombocytopenia Are Associated with Increased Mortality in Patients with Poor Graft Function Post Allogeneic Transplantation

Introduction: Poor graft function (PGF) post allogeneic transplantation is defined as peripheral blood cytopenias in the presence of donor chimerism and is increasingly recognised as a specific clinical entity. The criteria for PGF diagnosis are variable with regards to level of donor chimerism level and duration of cytopenias. Patients with this condition are at high risk of complications from bone marrow failure and need to be differentiated from patients with persisting cytopenias that spontaneously recover without serious complications. We retrospectively analysed patients treated at our centre with complete donor chimerism and cytopenias in order to identify markers of poor prognosis and define an at-risk group that will form the basis of further clinical studies. Method: Patients who underwent allogenic stem cell transplant (alloHSCT) at our centre between 2000-2016 were included in this retrospective analysis after fulfilling the criteria for PGF (1) cytopenias in at least 2 lineages (defined as platelets &lt;100x109/L, neutrophils &lt;1.0x109/L, haemoglobin &lt;100g/L)sustained for 30 days and (2) myeloid chimerism &gt;95%. Patients with relapse of their haematological malignancy within 100 days post-alloSCT were excluded. The primary outcome variables were overall survival (OS) and death from PGF. OS was evaluated in landmark analyses from day 100 (D100) post-alloSCT. Variables were investigated for associations with OS using cox proportional hazards models. Relapse and death without PGF were treated as competing risks for the outcome of death from PGF; associations with PGF were investigated using the method of Fine and Gray. Variables statistically significant at α&lt;0.05 were included in a forward step-wise fashion in multivariate models. Previous studies have demonstrated an excess of early mortality from PGF and hence patients were censored if alive at 24 months. Results: Of 908 transplants performed between 2000-2016, 152 patients (16%) fulfilled our criteria for PGF and were alive at day 100 post transplantation. The overall survival of the PGF cohort at 24 months was 65%. There were 102 patients alive at 24 months vs 50 who died within the 24 months period. The early mortality group had a median survival of 6 months (Range 3.3-23). Demographic details of the early mortality vs those who survived to 24 months are listed in Table 1. Univariate analysis of the PGF cohort identified the following variables were significantly associated with inferior OS: non-CMV viral infection, ICU admission during initial 30 days, age as a continuous variable, grade 2-4 acute GVHD and platelet count ≤40x109 /L (P40) at D100. Age and P40 remained significantly associated with OS on multivariate analysis. Hazard ratios and P values are summarised in Table 2. Competing risk analysis revealed, Age, non-CMV viral infections, ICU admission, MPN as an indication for transplantation, P40 at D100 and grade 2-4 GVHD as significant associations for death from PGF. Age, GVHD and P40 remained significantly associated with death from PGF in multivariate analysis. Hazard ratios and P values are summarised in Table 2. Conclusion: Increasing patient age and marked thrombocytopenia at D100 are associated with inferior survival in patients with PGF. These factors identify patients at higher risk of death from PGF which inform a stricter definition of the syndrome and provide a population for future interventional studies. Disclosures Ritchie: Sanofi: Honoraria; Novartis: Honoraria; Imago: Research Funding; Beigene: Research Funding; Takeda: Research Funding; BMS: Research Funding; Pfizer: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Koldej:NanoString Technologies: Other: Travel grant.