Stable Chimerism Research Articles

Outcomes from hematopoietic stem cell transplantation following treosulfan-based conditioning: A clinical and pharmacokinetic analysis.

The aims of this study are to report our experience with treosulfan-based conditioning regimens for patients with non-malignant hematologic conditions, correlating clinical outcomes at different time points post-transplant with treosulfan exposure (AUC). This study was a single-center observational study investigating overall survival (OS), disease-free survival (DFS), and event-free survival (EFS) end-points post-transplant. The consequences of treosulfan AUC with respect to toxicity, correction of underlying disease, and long-term chimerism were also explored using pharmacokinetic analysis. Forty-six patients received 49 transplants with treosulfan and fludarabine-based conditioning between 2005 and 2023. Twenty-four patients also received thiotepa. Donor chimerism was assessed on either whole blood or sorted cell lines at different time points post-transplant. Thirty-nine patients received treosulfan pharmacokinetic assessment to evaluate cumulative AUC, with five infants receiving real-time assessment to facilitate daily dose adjustment. OS, DFS, and EFS were 87%, 81%, and 69%, respectively. Median follow-up was 32.1 months (range 0.82-160 months) following transplant. Lower EFS was associated with patient age (<1 year; p = .057) and lower cumulative treosulfan dose (<42 g/m2; p = .003). Stable donor chimerism in B-cell, NK-cell, and granulocyte lineages at 1-year post-transplant were more prevalent in patients receiving thiotepa conditioning. Two infants required daily dose adjustment to treosulfan to avoid high AUC. Excellent clinical outcomes and stable chimerism were observed in this patient series. The addition of thiotepa conferred no significant toxicity and trended toward sustained ongoing donor engraftment. Correlating treosulfan AUC with long-term patient outcomes is required.

HLA molecular mismatches and induced donor-specific tolerance in combined living donor kidney and hematopoietic stem cell transplantation.

We investigated the potential role of HLA molecular mismatches (MM) in achieving stable chimerism, allowing for donor-specific tolerance in patients undergoing combined living donor kidney and hematopoietic stem cell transplantation (HSCT). All patients with available DNA samples (N=32) who participated in a phase 2 clinical trial (NCT00498160) where they received an HLA mismatched co-transplantation of living donor kidney and facilitating cell-enriched HSCT were included in this study. High-resolution HLA genotyping data were used to calculate HLA amino acid mismatches (AAMM), Eplet MM, three-dimensional electrostatic mismatch scores (EMS-3D), PIRCHE scores, HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence MM, and KIR ligands MM between the donor and recipient in both directions. HLA MM were analyzed to test for correlation with the development of chimerism, graft vs. host disease (GvHD), de novo DSA, and graft rejection. Follow-up time of this cohort was 6-13.5 years. Of the 32 patients, 26 developed high-level donor or mixed stable chimerism, followed by complete withdrawal of immunosuppression (IS) in 25 patients. The remaining six of the 32 patients had transient chimerism or no engraftment and were maintained on IS (On-IS). In host versus graft direction, a trend toward higher median number of HLA-DRB1 MM scores was seen in patients On-IS compared to patients with high-level donor/mixed chimerism, using any of the HLA MM modalities; however, initial statistical significance was observed only for the EMS-3D score (0.45 [IQR, 0.30-0.61] vs. 0.24 [IQR, 0.18-0.36], respectively; p=0.036), which was lost when applying the Bonferroni correction. No statistically significant differences between the two groups were observed for AAMM, EMS-3D, Eplet MM, and PIRCHE-II scores calculated in graft versus host direction. No associations were found between development of chimerism and GvHD and non-permissive HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence, and KIR ligands MM. Our results suggest an association between HLA-DRB1 molecular mismatches and achieving stable chimerism, particularly when electrostatic quality of the mismatch is considered. The non-permissive HLA-DPB1 T-cell epitope group, HLA-B leader sequence, and KIR ligands MM do not predict chimerism and GvHD in this combined kidney/HSCT transplant patient cohort. Further work is needed to validate our findings. https://clinicaltrials.gov/study/NCT00498160, identifier NCT00498160.

Allogeneic Bone Marrow Transplantation from HLA-Identical or Haploidentical Donors for Children and Adolescents with Sickle Cell Disease: A Feasible Curative Option

The only curative option widely available for sickle cell disease (SCD) is bone marrow transplantation (BMT). Since few patients have a healthy matched sibling donor (MSD), haploidentical (haplo) donors have expanded the donor pool to approximately 90% of the patients. The addition of thiotepa to the Johns Hopkins conditioning therapy backbone dor haplo BMT showed durable engraftment and excellent overall survival. However, graft rejection has remained an important obstacle. Our challenge in these patients has been to augment the conditioning therapy to provide adequate engraftment, while maintaining a low transplant-related mortality. Methods: Retrospective data of all 26 patients with SCD followed by our group after MSD (n=7) and haplo (n=19) BMT performed in four different institutions. With a MSD, conditioning was rATG 4.5mg/kg, Busulfan (Bu, AUC 4,500), Fludarabine (Flu, 120mg/m²) or rATG, Bu, Cyclophosphamide (Cy, 200mg/kg) and with haplo donors, rATG 4.5mg/kg, Thiotepa 10mg/kg, Flu 150mg/m², Cy 29 mg/kg and TBI 2Gy. Patients were prepared to BMT with intense hypertransfusion/bleeding or erythrocytapheresis to keep HbS&amp;lt;30 and Hb~10g/dL, reticulocytes &amp;lt;10% for 2-3 months prior to BMT, with intensive chelation to keep ferritin &amp;lt;1,000ng/mL. The presence of donor specific antibodies would exclude the patient from undergoing BMT. Due to two consecutive secondary graft failures (no detectable donor cells despite initial engraftment) and two falling mixed chimerism among the first 13 patients undergoing haplo, the doses of the conditioning therapy were increased to Cy 50 mg/kg and TBI 4Gy in a single fraction, as previously used in our patients with aplastic anemia undergoing BMT. Six consecutive patients received this augmented conditioning therapy. All patients had serial chimerism measured by STR. The graft-versus-host disease (GVHD) prophylaxis included Cyclosporine - Methotrexate in MSD and post-transplant Cy/mesna, mycophenolate mofetil and sirolimus in haplo. Results: Between September 2016 and April 2023, 26 patients underwent BMT, with a median age of 11 years (4-20), half of them females, 7 with a MSD and 19 an haplo (4 siblings, 6 fathers, and 9 mothers); 77% of the donors had sickle cell trait. The graft was bone marrow in all but one patient with Sb thal and Hodgkin's lymphoma. The median follow-up was 15.5 months. All patients engrafted. BMT with MSD had excellent outcomes and stable chimerism. Among the first 13 patients undergoing haplo, secondary graft failure occurred very suddenly in two patients (CD34 cell doses of 2 and 8x10 6 CD34/kg), both male teenagers; two additional patients had a significant drop in chimerism up to 30% donor cells but responded to serial donor leukocyte infusions 1-5 x10 6 CD3/kg (2 and 6) and developed very mild skin GVHD. The other patients maintained &amp;gt; 89-100% donor cells. Thereafter, with the augmented conditioning regimen, all six consecutive patients have stable 100% donor chimerism. Acute II-III GVHD was observed in 34% and chronic GVHD in 15% of the patients. Viral reactivations (CMV, HHV6, BKV, HHV7 and EBV) were observed in all patients. Overall survival is 100% and rejection-free survival is 83% at 1 year (Figures 1, 2). Conclusions: Allogeneic BMT with an HLA-identical or haploidentical family donor was feasible to treat SCD with an excellent overall survival. With a larger cohort and longer follow-up, the long-term effects of this strategy will be thoroughly studied.

Randomized Trial of Prophylactic Donor Lymphocyte Infusion with Positively Selected CD4+ T Cells after HLA-Identical Allogeneic Stem Cell Transplantation

Introduction Allogeneic stem cell transplantation (alloSCT) can cure patients with hematological malignancies by T-cell mediated alloreactivity against patient hematopoiesis, the graft-versus-leukemia (GvL) effect. GvL can coincide with graft-versus-host disease (GvHD) when donor T cells target non-hematopoietic tissues. Increase of donor chimerism in bone marrow (BM) or peripheral blood (PB) after alloSCT indicates GvL response. After T-cell depleted alloSCT, donor lymphocyte infusion (DLI) can restore GvL. Dose and timing of DLI influence the degree of alloreactivity. Low dose of unmodified DLI as low as 0.3x10 6 T cells/kg 3 months after alloSCT appeared not to cause GvHD. (Zouwen et al. Ann Hematol, 2023) We hypothesized that infusion of a higher dose of purified CD4+ donor T cells would not cause GvHD, while inducing GvL by selective targeting of the MHC class II positive hematopoietic cells. Method A randomized controlled phase 2 trial was conducted in patients with hematological malignancies after HLA-identical sibling alloSCT with alemtuzumab added to the graft. Patients without GvHD or relapse were randomized at 2.5 months for prophylactic DLI with 1.0x10 6/kg CD4+ T cells (CD4+ DLI) 3 months after alloSCT or conventional care without DLI (CC). Both groups were eligible for prophylactic unmodified DLI at 6 months. Cumulative incidence (CI) of GvHD requiring immunosuppressive treatment 3 months after randomization was the primary endpoint. Changes in the trajectory of chimerism in BM and subsets in PB (CD4+ T cells, CD8+ T cells, NK cells, B cells and monocytes) were compared by means of mixed effects beta regression as secondary endpoints. Results 66 patients were included (table 1). CI of GvHD did not differ between groups (CD4+DLI: 6.1% vs CC: 9.1%, Gray's test p&amp;gt;0.9). Relapse was comparable (CD4+DLI: 15% vs CC: 12%), although heterogeneity of diseases prevented a formal comparison. In the CD4+ DLI group, BM chimerism was full donor (FD, &amp;gt;95% donor) at baseline in 23/33 patients, while 10/33 had mixed chimerism (MC, ≤95%). In the CC group, 23/33 were FD and 10/33 MC. Some patients with MC at baseline converted to FD in both groups (CD4+ DLI: 1/10 vs CC: 1/10). Changes in BM chimerism did not differ between CD4+ DLI and CC (p = 0.9). Chimerism of B cells, NK cells and monocytes in PB resembled bone marrow chimerism with most patients being FD at baseline. Changes in chimerism did not differ between CD4+ DLI and CC (p = 1.0, 0.5 and 0.9 for B cells, NK cells and monocytes respectively). In the CD4+ DLI group, 25/30 evaluable patients were MC in the CD4+ T-cell subset in PB at baseline. 3 month after randomization 7/25 had converted to FD, 17/25 remained MC and 1/25 was unevaluable. Of the 17 patient who remained MC, CD4+ donor chimerism increased (absolute increase &amp;gt;5%) in 13, decreased (&amp;gt;5%) in 2 and was stable in 2. In the CC group, 26/32 were MC in CD4+ T cells at baseline, of which 6 converted to FD, 18 remained MC and 2 were unevaluable. Of the 18 patients remaining MC, CD4+ chimerism increased in 5, decreased in 9 and remained stable in 4. The CD4+ donor chimerism increased significantly after CD4+ DLI compared to CC (p = 0.01) (Figure 1). In the CD4+ DLI group, 23/29 evaluable patients were MC in the CD8 subset at baseline. 7/23 converted to FD, 15/23 remained MC and 1 was unevaluable. Of these 15 MC patients at 3 months, 9 had increasing, 5 stable and 1 decreasing donor chimerism after 3 months. In CC, 22/31 were MC at baseline. 4/22 converted to FD at 3 months and 17/22 remained in MC. Of the 17 MC patients, 6 had increasing, 3 decreasing and 8 stable chimerism. The CD8+ donor chimerism increased significantly in the CD4+ DLI group compared to CC (p = 0.03). Conclusion CD4+ selected DLI at a dose of 1x10 6 CD4+/kg 3 months after HLA identical T-cell depleted alloSCT did not cause GvHD. Chimerism in BM leukocytes, B cells, NK cells or monocytes did not indicate GvL mediated by CD4+ DLI, possibly because most patients were already FD at baseline. CD4+ and CD8+ T-cell donor chimerism increased significantly after CD4+ DLI, suggesting a GVL-effect - earlier demonstrated in vitro for 4 selected patients. (Van Balen et al. Front Immunol, 2018) However, most patients did not convert to FD in the T-cell compartment, suggesting the GvL-effect of CD4+ DLI was limited. We considered this GvL-effect insufficient to prevent relapse and stress the importance of CD8+ donor T cells as main drivers of alloreactivity after DLI following HLA-identical alloSCT.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant through Engineered Stem Cell Antibody Paired Evasion (ESCAPE)

While ex vivo gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) offers potentially curative therapies for patients with serious diseases, such as sickle cell anemia, significant autologous hematopoietic stem cell (HSC) transplant-related challenges remain, notably the requirement of myeloablative conditioning using chemotherapeutic agents such as busulfan. To address this challenge, we have developed a non-genotoxic conditioning strategy with our Engineered Stem Cell Antibody Paired Evasion (ESCAPE) approach where we aim to engineer the epitope of the HSC surface protein CD117 (cKIT) using base editors, in such a way that the engineered protein retains the normal CD117 receptor function, but escapes recognition by a cognate monoclonal antibody that recognizes the wildtype CD117 protein. The ESCAPE-CD117 edit can be combined with a therapeutic target edit to generate a multiplex-edited engineered HSC (eHSC). We previously reported that through large parallel antibody and antigen screening efforts, we could identify mAb: edit pairs in which the engineered epitope evades mAb-mediated blocking of the WT CD117 receptor-ligand interaction while maintaining normal CD117 receptor biology and HSPC function and differentiation potential in vitro and in vivo. To further characterize binding properties of our lead CD117 variant to the natural ligand SCF, we determined the 3-dimensional structure of the receptor-ligand complex using cryo-EM. Structural overlay between engineered CD117:SCF and WT CD117: SCF complexes showed high similarity, with root mean square deviation (RMSD) of 0.848-Å, indicating that our epitope engineering did not affect the structure of CD117 nor its binding to its ligand. We have previously demonstrated in vivo proof-of-concept data supporting selection of engrafted multiplex-edited eHSCs after a single dose of mAb in non-competitive and competitive transplant settings in multiple rodent xenotransplantation models. We next sought to examine whether mAb conditioning could achieve long-term, multilineage hematopoietic reconstitution of multiplex-edited eHSCs and potentially therapeutic mixed chimerism levels. To this end, we humanized an immunocompromised mouse model with unedited mobilized peripheral blood (mPB) hCD34 + cells to achieve stable human bone marrow chimerism and then administered a single dose of mAb followed by a second transplant from the same donor of eHSCs harboring both ESCAPE and therapeutic edits targeting the HBG1/2 promoter. Animals receiving CD117 mAb demonstrated significant engraftment by cells harboring our ESCAPE multiplex edits as detected by NGS of bulk BM (~50% multiplex editing) and within the sorted marrow CD34 + cell population (~60% multiplex editing), while the isotype control treated animals only showed 2-5% multiplex editing within the said compartments. We also isolated CD15 + myeloid cells and GlyA + erythroid cells from the bone marrow of the mAb treated animals, which also harbored &amp;gt;50% multiplex editing as detected by NGS. Furthermore, we could detect what is believed to be therapeutically relevant levels of HbF induction (&amp;gt;30%) in human GlyA + sorted bone marrow cells, suggesting that we could achieve therapeutic benefit with a single dose of mAb as the conditioning agent. In conclusion, our structural data showed a high level of structural similarity between our engineered CD117 epitope and the WT protein. Our in vitro assays and in vivo rodent xenotransplantation models showed enrichment of engineered HSCs upon mAb treatment. Altogether, our data suggest the feasibility of achieving therapeutically relevant levels of mixed bone marrow chimerism with our non-genotoxic conditioning ESCAPE platform. This approach may remove the need for patients to undergo systemic genotoxic conditioning prior to transplant and further unlock the promise of base editing therapies for the treatment of patients suffering from serious hematologic diseases.

Effective Prevention of Steroid-Requiring Chronic Graft-Vs.-Host Disease with B Cell Depletion: A Randomized, Placebo-Controlled Trial

Introduction: The pathophysiology of chronic graft-vs.-host disease (cGVHD) is complex, with many cell types playing critical roles. B cells are involved in cGVHD pathobiology, and strategies that eliminate or inhibit B cells (rituximab, ibrutinib) can be therapeutic in advanced disease. We previously demonstrated effective prevention of cGVHD in a phase II trial of B cell depletion with rituximab. Obinutuzumab (Obin, Genentech) is a 2 nd generation monoclonal anti-CD20 antibody with enhanced B cell depletion activity over rituximab. Here we present the results of a randomized trial of Obin for the prevention of cGVHD. Methods: We performed a randomized double-blind, placebo-controlled trial of B cell depletion using 4 doses of Obin to determine if steroid-requiring cGVHD (SR-cGVHD) could be prevented 12 months from HSCT (NCT# 02867384). Eligible subjects previously underwent myeloablative or reduced intensity 7-8/8 HLA-matched, related or unrelated HSCT using peripheral blood stem cells (PBSC) and tacrolimus-based primary GVHD prophylaxis without T cell depletion. Subjects without evidence of cGVHD, active Gr II-IV acute GVHD, or malignancy were enrolled between 60-90 days following HSCT. Eligibility included adequate hematopoiesis (ANC &amp;gt; 1000/mL, plt &amp;gt; 50 000/mL) and stable donor chimerism (&amp;gt; 80%). All subjects provided informed consent. Obin (1000 mg iv) or placebo was administered at 3, 6, 9 and 12 months after HSCT (+/- 10 days) with acetaminophen and diphenhydramine prior to each study drug dose. The primary endpoint was the cumulative incidence (CI) of SR-cGVHD, which was felt to be a more objective measurement of cGVHD severity than NIH cGVHD stage alone. Results: 181 subjects were enrolled (92 Obin, 89 placebo). 167 subjects who have been followed for &amp;gt;1 year from HSCT (84 Obin, 83 placebo) are included in this analysis. Baseline clinical characteristics, including age, recipient/donor gender, donor type/match, primary malignancy/DRI, conditioning intensity and HCT-CI, and prior Gr II-IV GVHD were balanced. The median number of study drug doses was equivalent (median 2.5 vs. 2 for Obin and placebo groups). For the entire cohort, the median follow-up time among survivors was 24 months (range 4, 74). The primary endpoint, the CI of SR-cGVHD at 12 months from HSCT, was significantly reduced with Obin - 11% (95% CI 5.8-20) in comparison with placebo - 38% (95% CI 28-49) (p=0.008, Fig 1). The corresponding rates of NIH Moderate-Severe cGVHD at 12 months were 20% vs. 35% (p=0.069). The rate of SR-cGVHD by 2 years remained lower in the Obin group (28% vs. 44%). In multivariable modeling, only study arm (Obin vs. placebo) was associated with SR-cGVHD (p=0.01). Obin was safe and well tolerated with only 5 Gr 3-4 infusion reactions (placebo - 0). There was more Gr 3-4 neutropenia (32.1% vs. 4.8%) but only 1 case of neutropenic fever in the Obin group. Neutropenia responded to G-CSF, administered at investigator discretion. There were 9 non-relapse related deaths in the Obin arm (COVID 3, infection 2, pulmonary 2, acute GVHD 1, unknown 1) compared with 3 in the placebo arm (unknown 2, pulmonary 1) (p=NS). There was no difference in the CI of relapse at 2 years (20% vs. 28%, p=NS). The SR-cGVHD-free, relapse-free survival in the Obin group was superior (CRFS, 43% vs. 30%, p=0.009, Fig 2) without a change in overall survival 82% vs. 86%, p=NS). CD19 + B cell depletion was complete in the Obin arm through 1 year (median cell count 0) while B cell recovery was normal in the placebo arm (p&amp;lt;0.0001 at 3, 6, 9, 12 months) and remained lower at 24 and 36 months (36 month median 55.5 vs. 484.4 cells/mL, p=0.02). Similar findings were noted for CD19 +CD27 + memory B cells and CD19 +CD27 - naïve B cells. No differences in T cells were noted, but NK cells were reduced in the Obin arm at late time points. H-Y antibody studies in F to M donor/recipient pairs are underway. Conclusions: In subjects undergoing well-matched PBSC HSCT using tacrolimus-based GVHD prophylaxis, a strategy of B cell depletion with 4 doses of Obin significantly reduces the requirement for corticosteroids for treatment of cGVHD and significantly increases CRFS. Obin is well tolerated and is associated only with transient neutropenia. Further studies may determine whether prolonged B cell depletion may be of greater utility, however this double-blind, placebo-controlled randomized trial supports practice changing cGVHD prophylaxis in tacrolimus-based HSCT.

Minimal dose of hematopoietic stem cell transplantation without myelosuppressive conditioning for T-B+NK- severe combined immunodeficiency

We reviewed the medical records of five patients with T-B+NK- severe combined immunodeficiency (SCID) who received minimal dose allogeneic hematopoietic cell transplantation (HCT) (total nucleated cell count (TNC) lower than 1.0 × 108/kg). Patients were administered a median of 5.0 mL of bone marrow or peripheral blood without conditioning (in four) or with anti-thymocyte globulin alone (in one). Three patients received HCT from a matched sibling donor, one from unrelated donor, and one from familial mismatched donor. The median TNC and CD34+ cells were 0.54 (0.29–0.84) × 108/kg and 0.61 (0.35–0.84) × 106/kg, respectively. Engraftment was achieved in all. Total T cell, CD4+ cell, and CD8+ cell recovery was obtained within a year in four, and immunoglobulin replacement was discontinued in all. All patients survived, exhibiting stable donor chimerism. We obtained sufficient therapeutic effects with minimal dose transplantation without intensive conditioning in patients with T-B+NK- SCID.

PLX-R18 (Avoplacel), a Placental Expanded Mesenchymal-like Stromal Cell Therapy Shows Favorable Safety and Appears to Improve Incomplete Hematopoietic Recovery after Hematopoietic Cell Transplantation (HCT)

Introduction: Persistent cytopenia in the post-HCT setting remains a challenge in some recipients despite good engraftment of donor cells. PLX-R18 is a placental-derived mesenchymal-like cell product, expanded ex vivo in a 3-dimentional environment according to Good Manufacturing Practices. PLX-R18 cells secrete a large array of hematopoietic factors including stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 and interleukin-11, which promote regeneration, maturation, and differentiation of hematopoietic cells in bone marrow and stimulate their migration to peripheral blood. Reported herein are the final encouraging safety and efficacy results from a phase 1 study (NCT03002519). Methods: 21 patients > 18 years who experienced incomplete hematopoietic recovery at least 3 months post-HCT, were enrolled into the study. Eligibility criteria were defined as meeting at least one of the following: neutrophil count (ANC) < 1000/μL, platelet (PLT) < 50,000/μL or hemoglobin (HGB) < 8 g/dL. Patients had no evidence of other causes for cytopenia such as an active infection or malignancy or severe acute or chronic Graft vs. Host Disease. For allogeneic (allo) HCT, complete, stable donor chimerism was required. Safety of PLX-R18 therapy was the primary objective. PLX-R18 was administered via multiple IM injection in 2 treatment sessions one week apart. Patients were treated in 3 cohorts: 1 million cells/kg (n=3); 2 million cells/kg (n=6); and 4 million cells/kg (n=12). Safety: 19 of 21 treated patients (90%) received an allo-HCT; for 5 patients (24%) this was a repeat HCT. The median time from HCT to PLX-R18 administration was ~8 months (236 days). Acute lymphocytic leukemia (ALL) was the most common underlying malignancy (33%) followed by acute myeloid leukemia (14%) and myelofibrosis (14%). The most common adverse events (AEs) were local injection-site reactions (pain), observed in 85.7% of patients, and were transient and of mild to moderate severity. No clinically meaningful changes were observed in vital signs, ECG and blood counts, liver and kidney function or coagulation laboratory results. A transient and reversible elevation in inflammatory markers, high sensitivity C-reactive protein (hsCRP) and complement factor 5a (C5a), were observed on the day following PLX-R18 administration and returned to normal levels thereafter. One patient developed severe febrile neutropenia two days after the second infusion (PLX-R18 4 million cells/kg) which was considered related to PLX-R18 and resolved 2 days later. Four AEs were associated with a fatal outcome (1 sudden death, 2 events of sepsis and 1 event of ALL recurrence); none were considered by the Investigator, or the Sponsor, as related to PLX-R18. One relapse was reported, an ALL patient who relapsed 296 days following study treatment and passed away 2 weeks later. No other events of relapse or recurrence were reported. Efficacy: For all PLX-R18 doses, a positive mean change from baseline in PLT and HGB was observed starting as early as 1 month after PLX-R18 administration and lasting up to 12 months following treatment (p<0.05). Positive mean change from baseline in ANC was observed in all doses as early as one month, peaking at 6 months for the 4 million cells/kg dose (p=0.062). Among patients who received blood transfusions at any time during follow up, a meaningful reduction in the mean number of transfused units per month, for both PLT and RBC transfusions, was observed as early as 3 months following PLX-R18 treatment and lasted throughout the 12-month follow up period. For PLT, the mean monthly number of transfused units dropped from 5.09 at baseline to 0.55 at 12 months (p<0.05); for RBC, from 2.91 at baseline to 0.00 at 12 months (p=0.0005). Conclusion: PLX-R18 therapy appears to be safe and well tolerated and shows promise in improving incomplete hematopoietic recovery after HCT. Further clinical studies are warranted to optimize the efficacy of PLX-R18 in treatment of cytopenia in the post-HCT setting and expand the potential to other marrow failure disorders as well as cytopenia associated with certain treatment modalities such as CAR-T therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bodyweight and Absolute Lymphocyte Count Based Dosing of Rabbit Anti-Thymocyte Globulin Results in Early CD4+ Immune Reconstitution in Patients with Inborn Errors of Metabolism Undergoing Umbilical Cord Blood Transplantation

Background Rabbit anti-thymocyte globulin (rATG) is used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft failure through depletion of host T cells and decrease the risk of graft-versus-host disease (GVHD) through partial depletion of donor T cells. However, it has highly variable pharmacokinetics. Previous reports suggest rATG dosing should be based on recipient bodyweight and absolute lymphocyte count (ALC) prior to initiating rATG. This new dosing nomogram reduces rATG overexposure, potentially leading to faster immune reconstitution and subsequently superior overall outcomes. Methods Since January 12, 2017, we use a previously described rATG dosing nomogram based on bodyweight and ALC before administering rATG in patients with inborn errors of metabolism receiving an umbilical cord blood transplant at the University of Minnesota Masonic Children's Hospital. The proposed rATG dosing varied from 2 to 10 mg/kg based on bodyweight and recipient ALC prior to initiating rATG, starting 8 or 9 days before HCT. The myeloablative conditioning regimen was Busulfan based, with addition of Fludarabine and Cyclophosphamide for lymphodepletion, as well as Rituximab and/ or Bortezomib for prevention of immune-mediated cytopenia. For GVHD prophylaxis cyclosporin A or tacrolimus were used with methylprednisolone. The primary endpoint was CD4+ immune reconstitution (>50 CD4+ T-cells/mL) within 100 days after transplantation. Secondary endpoints included overall survival, graft failure, acute and chronic GVHD, myeloid and T cell donor chimerism at one year post transplant, time to neutrophil engraftment and viral reactivations of cytomegalovirus, adenovirus and Epstein Barr virus. The study protocol was approved by the Institutional Review Board of University of Minnesota. Results Between February 20, 2017, and June 28, 2021, 27 patients were included in the study. Median follow-up time was 31 months (IQR 22.0 to 38.5) and median age was 1.45 years (IQR 0.7 to 3.9). Ten (37%) of 27 patients were female. The baseline characteristics are presented in Table 1. rATG dose was adjusted below 10 mg/kg recipient body weight in 7 (26%) of 27 patients on average by 29% (SD 14.9%). The CD4+ recovery at 100 days post HCT was reached in 22 (85%) of 26 patients (Figure 1A). Overall, four (15%) patients died resulting in overall survival of 83% (95%CI 67% to 100%). Causes of death were intracranial bleeding due to immune-mediated thrombocytopenia (this patient was transplanted before introduction of bortezomib and rituximab for immune-mediated cytopenia prevention), diffuse alveolar hemorrhage, respiratory syncytial virus infection and disease progression. Median time to neutrophil engraftment was 14 days (IQR 12.5 to 18.0), and no graft failure was observed. All surviving patients had full myeloid chimerism and 14 of 23 (61%) of patients had full T cell donor chimerism at one year post HCT. Two (7%) patients developed acute GVHD grade II to IV (Figure 1B), and no patients had chronic GVHD. Six patients (22%) had cytomegalovirus viremia, five of them were seropositive before transplant, one patient was seronegative as was the mother of the donor. One patient, who was seronegative before transplant, had Epstein-Barr virus reactivation requiring treatment, no patient had adenovirus reactivation. Conclusion In pediatric patients with inborn errors of metabolism individualized dosing of rATG was associated with a robust and early CD4+ immune reconstitution, with low therapy related mortality, low GVHD incidence compared to historical findings and stable full myeloid chimerism. These results are in line to the findings of the PARACHUTE trial previously published. However, the PARACHUTE trial included only small number of patients with inborn errors of metabolism. Table 1. Baseline characteristicsFigure 1A. Cumulative incidence of CD4+ immune reconstitutionFigure 1B. Cumulative incidence of acute Graft-versus-host disease Grade 2 to 4Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SiRNA against CD40 delivered via a fungal recognition receptor ameliorates murine acute graft-versus-host disease.

Acute graft‐versus‐host disease (aGvHD) remains a major threat to a successful outcome after allogeneic hematopoietic stem cell transplantation (HSCT). Although antibody‐based targeting of the CD40/CD40 ligand costimulatory pathway can prevent aGvHD, side effects hampered their clinical application, prompting a need for other ways to interfere with this important dendritic T‐cell costimulatory pathway. Here, we used small interfering RNA (siRNA) complexed with β‐glucan allowing the binding and uptake of the siRNA/β‐glucan complex (siCD40/schizophyllan [SPG]; chemical modifications called NJA‐312, NJA‐302, and NJA‐515) into Dectin1+ cells, which recognize this pathogen‐associated molecular pattern receptor. aGvHD was induced by the transplantation of splenocytes and bone marrow cells from C57BL/6J into CBF1 mice. Splenic dendritic cells retained Dectin1 expression after HSCT but showed lower expression after irradiation. The administration of siCD40/SPG, NJA‐312, and NJA‐302 ameliorated aGvHD‐mediated lethality and tissue damage of spleen and liver, but not skin. Multiple NJA‐312high injections prevented aGvHD but resulted in early weight loss in allogeneic HSCT mice. In addition, NJA‐312 treatment caused delayed initial donor T and B‐cell recovery but resulted in stable chimerism in surviving mice. Mechanistically, NJA‐312 reduced organ damage by suppressing CCR2+, F4/80+, and IL17A‐expressing cell accumulation in spleen, liver, and thymus but not the skin of mice with aGvHD. Our work demonstrates that siRNA targeting of CD40 delivered via the PAMP‐recognizing lectin Dectin1 changes the immunological niche, suppresses organ‐specific murine aGvHD, and induces immune tolerance after organ transplantation. Our work charts future directions for therapeutic interventions to modulate tissue‐specific immune reactions using Pathogen‐associated molecular pattern (PAMP) molecules like 1,3‐β‐glucan for cell delivery of siRNA.

A CD45-targeted antibody-drug conjugate successfully conditions for allogeneic hematopoietic stem cell transplantation in mice

AbstractAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment of patients with nonmalignant or malignant blood disorders. Its success has been limited by graft-versus-host disease (GVHD). Current systemic nontargeted conditioning regimens mediate tissue injury and potentially incite and amplify GVHD, limiting the use of this potentially curative treatment beyond malignant disorders. Minimizing systemic nontargeted conditioning while achieving alloengraftment without global immune suppression is highly desirable. Antibody-drug-conjugates (ADCs) targeting hematopoietic cells can specifically deplete host stem and immune cells and enable alloengraftment. We report an anti-mouse CD45-targeted-ADC (CD45-ADC) that facilitates stable murine multilineage donor cell engraftment. Conditioning with CD45-ADC (3 mg/kg) was effective as a single agent in both congenic and minor-mismatch transplant models resulting in full donor chimerism comparable to lethal total body irradiation (TBI). In an MHC-disparate allo-HSCT model, pretransplant CD45-ADC (3 mg/kg) combined with low-dose TBI (150 cGy) and a short course of costimulatory blockade with anti-CD40 ligand antibody enabled 89% of recipients to achieve stable alloengraftment (mean value: 72%). When CD45-ADC was combined with pretransplant TBI (50 cGy) and posttransplant rapamycin, cyclophosphamide (Cytoxan), or a JAK inhibitor, 90% to 100% of recipients achieved stable chimerism (mean: 77%, 59%, 78%, respectively). At a higher dose (5 mg/kg), CD45-ADC as a single agent was sufficient for rapid, high-level multilineage chimerism sustained through the 22 weeks observation period. Therefore, CD45-ADC has the potential utility to confer the benefit of fully myeloablative conditioning but with substantially reduced toxicity when given as a single agent or at lower doses in conjunction with reduced-intensity conditioning.

Nonmyeloablative HLA-Identical Sibling Donor Transplantation for Children and Young Adults with Sickle Cell Disease: Interim Results of the SUN Multicenter Phase II Trial

Background: HLA-identical sibling hematopoietic stem cell transplant (HSCT) using myeloablative chemotherapy conditioning is a proven cure for sickle cell disease (SCD), but is associated with serious short and long-term toxicities. Additionally graft versus host disease (GVHD) can complicate care post-HSCT and contribute to mortality. Given these concerns many pediatric hematologists and families are reluctant to pursue HSCT for SCD. Nonmyeloablative HSCT resulting in stable mixed chimerism has been demonstrated to abrogate the SCD phenotype in adults. Data on outcomes of this approach to decrease toxicities and achieve cure in children remains scarce.Objective: To evaluate event-free survival (EFS), toxicity, health-related quality of life (HRQL), and transfusion burden among pediatric patients with SCD using a chemotherapy-free nonmyeloablative regimen.Methods/design: Children and young adults with SCD were prospectively enrolled in the Sickle transplant Using a Nonmyeloablative approach (SUN) multicenter clinical trial (NCT03587272). Six Sickle Cell Transplant Advocacy and Research Alliance (STAR) sites in the United States and Canada enrolled patients. The conditioning regimen consisted of alemtuzumab IV (0.03mg/kg on Day -7, 0.1mg/kg on Day -6, 0.3mg/kg on Days -5 to -3), low-dose total body irradiation with gonadal shielding (300 cGY on day -2), peripheral blood stem cells from an HLA-identical sibling, and sirolimus for at least one year. Baseline HRQL was compared with scores at day +30 and day +100 post-HSCT using the PedsQL and PROMIS measures. EFS was defined by any of the following events: death, graft failure (myeloid donor chimerism <10%), or GVHD.Results: All 30 of the patients planned for the primary endpoint of EFS have been enrolled. As of 7/20/2021, 24 patients are greater than 100 days post-HSCT with a median follow-up of 365 days (range 189-1105). The median age of this group at time of transplant was 13.5 years (range 2-22). All patients had neutrophil recovery (ANC >500/ul x 3 days) at a median of 21.5 days (range 0-29). Eleven patients (46%) did not require any platelet transfusions. Median transplant hospitalization was 16.5 days (range 14-43). Seven patients (29%) were again hospitalized (median 4 days, range 1-26). Three patients had secondary graft rejection at Day +71, +91, and +92 with no major complications and autologous recovery resulting in an EFS of 87.5% (Figure 1). Three additional patients have stable low donor chimerism (myeloid chimerism 11%, 38%, and 47%). The remaining 18 patients (75%) are disease-free and have robust donor engraftment (72-100% myeloid chimerism). Nine of these patients are >1 year post-HSCT and off sirolimus. Overall survival is 100% with no acute or chronic GVHD. Figure 2 shows total PedsQL scores during the peri-transplant time period. The median change in day +30 scores compared to baseline was 0 (IQR -7, +10), p=0.66. The median change in day +100 scores compared to baseline was +5 (IQR 0, +10), p=0.02.Conclusions: Outcomes of pediatric nonmyeloablative HLA-identical sibling HSCT for SCD appear similar to the larger adult experience with a GVHD-free, rejection-free survival of >80%. Importantly, patients have no worsening of their quality of life in the first month post-HSCT and already an improvement at day +100, suggesting that the short-term toxicities experienced by patients transplanted with this regimen are minimal. The majority of patients achieve disease resolution with no GVHD, but graft failure incidence appears higher than with myeloablative HSCT. Given the low intensity of this approach, a reasonable strategy may be to reserve myeloablation for a second transplant in the minority of patients who experience graft failure. [Display omitted] DisclosuresRangarajan: Medexus (Treosulfan): Consultancy, Honoraria. Guilcher: BlueBirdBio: Research Funding; Project Sickle Cure Study: Other: Principal Investigator, Research Funding. OffLabel Disclosure:alemtuzumab and sirolimus were used off-label as part of the studied transplant regimen

HLA-Matched Related Hematopoietic Stem Cell Transplantation in Adolescents and Adults with Sickle Cell Disease: Comparison of Myeloablative Versus Non Myeloablative Approaches. Report from the Société Francophone De Greffe De Moelle Et De Thérapie Cellulaire

IntroductionAllogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for sickle cell disease (SCD) allowing a SCD-free survival over 95% among children with matched HLA-identical donor (Gluckman E. Blood, 2017; Bernaudin F. Haematologica, 2020). In adults, myeloablative HSCT is associated with more graft-versus host disease (GVHD) and higher toxicity (Cappelli B. Haematologica, 2019). More recent approaches using non myeloablative (NMA) conditioning regimen (3 gray (Gy) total body irradiation (TBI) plus alemtuzumab) followed by HLA-identical peripheral blood stem cells (PBSC) and post-transplant sirolimus appear safe in adults (Hsieh MM. JAMA, 2014), but incidence of graft failure might be higher than the one reported after myeloablative conditioning (MAC) (Alzahrani M. Br J Haematol, 2021). Here, we compare outcomes after MAC or NMA conditioning regimens in patients over 15 years old transplanted from a matched related donor (MRD).Patients and methodsAll consecutive patients transplanted for SCD from a MRD from January 2015 to October 2020 were eligible if they were over 15 years old and received a busulfan-based MAC conditioning regimen or a NMA conditioning regimen.Chimerism was studied by analyzing various polymorphisms after polymerase chain reaction (PCR) amplification of DNA obtained from whole blood cells. Rejection was defined as donor chimerism <5%. Event-free survival (EFS) was defined as the probability to be alive with blood donor chimerism over 20%.ResultsThirty-four patients were included: 20 in the MAC and 14 in the NMA groups. Median age at transplant was 17 years (range 15-46) without difference between groups. Forty four percent of patients had a history of cerebral vasculopathy, 79% of recurrent vaso-occlusive crises and 76% of acute chest syndrome. ABO major incompatibility was present in 15% of patients. There was no difference in patient characteristics according to the conditioning regimen group except for pre-transplant cerebral vasculopathy, more frequently reported in the MAC group: 70% versus 6% in the NMA group (p<0.001).In the MAC group, conditioning regimen associated busulfan (12.8 mg/kg IV), cyclophosphamide (N=17) or fludarabine (N= 3) and anti-thymoglobulin (ATG, mostly 20mg/kg). Stem cell source was bone marrow and post-transplant immunosuppression combined cyclosporine and mycophenolate or methotrexate. In the NMA group, conditioning regimen associated 3Gy TBI and alemtuzumab (1mg/kg), followed by PBSC and post-transplant sirolimus.All patients engrafted and no secondary graft failure was observed. One MAC group patient died from GVHD. The 2-year overall and EFS were 95% (CI 95%: 85.9-100) in the MAC group (median follow-up of 39 months (range 8-63)) and 100% (CI 95%: 100-100) in the NMA group (median follow-up of 17 months (range 9-39)). Incidence of grade II-IV acute GVHD was 0% in the NMA group versus 20% in the MAC group (p=0.12). Incidence of chronic GVHD was 0% in the NMA group versus 25% in the MAC group (p=0.06). From the 27 patients with follow-up>12 months, 17/17 discontinued immunosuppressive therapy in the MAC group versus 8/10 in the NMA group. Hematopoietic recovery was faster in the NMA group with less platelet and red blood cell units transfused (Table). Throughout the follow-up, median donor chimerism was higher after MAC transplantation than after NMA transplantation: 98% (range 69-100) and 86 % (range 50-97) respectively at 1 year (p=0.017). Donor chimerism remained above 50% throughout the follow-up in all patients except 1 of the NMA group who displayed stable chimerism between 40 and 50%. All patients achieved HbS level close to the one of their donors. There was no difference between the 2 groups regarding occurrence of infections. MAC transplant was more often associated with hypertension, neurological complications, severe mucositis and need of enteral or parenteral nutrition. Moreover, duration of hospitalization was longer in MAC group: 54 days (range 39-192) versus 35 days in the NMA group (range 21-52) (p<0.001) (table).ConclusionIn this series of adolescents and adults transplanted for SCD, the survival without SCD was excellent with a faster hematological recovery and a lower toxicity after NMA HSCT. Longer follow-up is required to confirm stable mixed donor chimerism and the cure of SCD after NMA approach. [Display omitted] DisclosuresJoseph: bluebird bio: Consultancy. Boissel: Servier: Consultancy, Honoraria; JAZZ Pharma: Honoraria, Research Funding; Incyte: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; SANOFI: Honoraria; CELGENE: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; PFIZER: Consultancy, Honoraria. Pondarré: ADDMEDICA: Honoraria.

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.

Promising Results at 1 Year Follow-up Following Familial Haploidentical (FHI) T-Cell Depleted (TCD) with CD34 Enrichment and T-Cell (CD3) Addback Allogeneic Stem Cell Transplantation in Patients with High-Risk Sickle Cell Disease (SCD) (IND 14359)

AlloSCT from HLA-matched MSD has been successful for high-risk SCD, and is the only known curative therapy (Talano/Cairo et al EJH, 2014). We have recently demonstrated 100% EFS and absence of sickle cell symptoms following reduced toxicity conditioning in HLA matched sibling donor (MSD) or cord blood AlloSCT (Bhatia/Cairo et al BMT, 2014). However, 5 out of 6 children lack an HLA MSD without SCD. Only 19% of recipients of African descent will identify a well-matched unrelated donor (MUD) and results after unrelated UCBT are poor (Radhakrishnan/Cairo et al BBMT, 2013). Recent results utilizing matched unrelated donors in a multi-center trial showed unacceptable rates of cGVHD at 62% (95% CI 41-77) (Shenoy et al Blood, 2016). We demonstrated CD34+ selection followed by T-cell addback from MUD in pediatric recipients led to 100% engraftment with minimal aGVHD (Geyer/Cairo et al BJH, 2011). Limitations in the past of various T-cell depletion methods have included a higher incidence of graft failure, delayed immune reconstitution, opportunistic fungal infection and higher incidence of cGVHD. FHI TCD AlloSCT utilizing the approach of CD34+ enrichment and T-cell addback could expand the donor pool and improve outcomes for patients with high risk SCD and have similar outcomes to AlloSCT from HLA MSD.This SCD consortium trial (www.sicklecelltransplantconsortium.org) (NCT01461837) is investigating the safety, feasibility, EFS, donor chimerism, graft failure, aGVHD and cGVHD after FHI TCD AlloSCT in high-risk SCD patients.High risk included: ≥1 CVA, ≥2 ACS, ≥3 VOC in past 2 years, or 2 abnormal TCDs. Patients (2-<21 yrs) who have ≥1 high-risk SCD features were eligible. Patients received hydroxyurea and azathioprine, day -59 - day -11, fludarabine (150mg/m2), busulfan (12.8mg/kg) (targeted trough Css of 600-900), thiotepa (10 mg/kg), cyclophosphamide (200mg/kg), R-ATG (8mg/kg), and TLI (500cGy) followed by FHI T-cell depleted AlloSCT. AGVHD prophylaxis: tacrolimus. We utilized the CliniMACS to enrich for peripheral blood HPC's; target dose of 10 x 106 CD34+ cells/kg with a fixed dose of 2 x 105 CD3+ T cells/kg. The Child Health Rating Inventories (CHRIs) was used to serially collect parent proxy-rated health-related quality of life (HRQL).Twenty-one patients have been enrolled. Nineteen patients have received AlloSCT to date. Fifteen patients utilized maternal donors and 4 patients utilized paternal donors. Two donors encountered thrombocytopenia that resolved spontaneously. The mean ± SD CD34+ enrichment prior to cryopreservation was 12.66 ± 3.3 x 106/kg and log T-cell depletion was 4.8 ± 0.58. Evaluable pts had early neutrophil engraftment (median day +9), and ≥95% whole blood and RBC (CD71) donor chimerism at 1yr. Probability of aGVHD n=17, 7.1% (CI95: 0-70.0); cGVHD n=17, 20% (CI95: 0.7-59.6) (Figure 1A and 1B). Probability of 1-Year Overall Survival (OS) n=17; 88.2% (CI95: 60.6-96.9) (Figure 1C). Immune reconstitution was robust with 1 yr NK, CD3, CD4 and CD8 204±37 cells/μL, 795±168 cells/μL, 408±102 cells/μL and 375±90 cells/μL, respectively. Two-yr PFT results were also robust with % predicted FVC, FEV1, TLC and DLCO of 89%, 84%, 82%, and 68%, respectively. All survivors are free of SCD symptoms and without signs of progression of multi-organ toxicity. One patient developed late hepatic SOS and died at day +59; one patient died at day +390 due to complications of CGVHD, and one patient died at day +141 of steroid refractory aGVHD, the remainder are alive and free of SCD symptoms with a median follow-up of +711 days (range, +231 to +1684 days). Most importantly, mean + SD HRQL score (0-100) reported at 1 yr for emotional, physical functioning, role, and global scores were 82.9±9.7 points, 82.8±15.8 points, 89.6±13.3 points, and 86±8.2 points, respectively.Early results indicate promising results in 1 yr OS, stable donor chimerism, and low incidence of acute and/or cGVHD after FHI HCT utilizing CD34 enrichment and CD3/MNC addback in high-risk SCD patients who lack a MSD or URD. A longer term follow-up is needed to assess long-term safety and outcomes. This research was supported by RO1FD004090-01A1. [Display omitted] DisclosuresWalters:AllCells, Inc: Other: Medical Director; bluebird bio: Research Funding; Sangamo Therapeutics: Consultancy; ViaCord Processing Lab: Other: Medical Director. Cairo:Jazz Pharmaceuticals: Speakers Bureau.

Modulation of Donor Graft By the Steroidal Lactone Withaferin A Alleviates Post-Transplant Acute GVHD Via Nrf-2 Independent T-Reg Induction

Acute Graft Versus Host Disease (aGVHD), resulting from an immune mediated attack of donor cells against recipient's healthy tissues remains the bugbear of allogeneic Bone Marrow Transplantation (allo-BMT). Considering the heterogeneous nature of aGVHD, associated high incidence of morbidity and mortality, severe side effects and limited success of current immunosuppressive prophylactic regimens, we were encouraged to develop a novel, safe and easy-to-use intervention for prophylaxis of aGVHD.Our group, for the first time ever, employed an approach of donor graft modulation with Withaferin A (WA) prior to allogeneic transplantation to decrease the allo-reactivity of donor lymphocytes for mitigating post-transplant aGVHD. WA is a steroidal lactone isolated from the medicinal plant Withania somnifera . Our previous studies have shown the immune-suppressive efficacy of WA on T lymphocytes in vitro {Gambhir et al Toxicol Appl Pharmacol 2015; 289: 297}. Extending those findings, we demonstrate the anti-GVHD potential of WA in a murine model of aGVHD.Transient (2h) treatment of lymphocytes of C57Bl/6 mice with 1µM WA inhibited mitogen induced activation, proliferation and cytokine secretion (Figure 1A) in vitro without inducing significant cell death (≤10% at 1µM WA). In order to evaluate graft modulatory potential of WA for alleviating aGVHD in vivo, we validated and characterized a MHC mismatched and High Dose Myeloablative Chemo-Conditioning (HDMCC) based C57BL/6-to-BALB/c aGVHD model earlier reported by Sadeghi et al {Bone Marrow Transplant. 2008;42:807}, except we increased the splenocyte dose to 42*106 per recipient to intensify aGVHD manifestation. In this model, we observed early mortality with a median survival of 9 days (Figure 1B), clinically and histologically relevant characteristics of aGVHD accompanied by cytokine storm in recipients of HDMCC + allo-graft (n=33) compared to HDMCC alone (n=35).Infusion of donor graft incubated with 1µM WA in vitro for 2 h prior to transplantation abrogated the post-transplant cytokine storm. On day +3, levels of IFN-g, IL-1b, IL-2, IL-6, TNF-a and IL-17 were suppressed by 3.21, 3.18, 1.57, 3.80, 1.70 and 4.32 folds respectively in WA group compared to Vehicle Control (VC) group. Conversely levels of the anti-inflammatory cytokine, IL-10, increased by 1.86 fold in WA group as compared to VC group. The levels of IFN-g, IL-1b and IL-2 continued to be low on day +7 in WA group compared to VC group by 4.53, 1.39 and 1.93 folds respectively.Further,survival was significantly improved in recipients of allo-graft modulated with WA compared to VC [Hazard Ratio = 11.86 (7.164 to 50.06)] (Figure 1C). GVHD associated tissue damage was also markedly reduced in the WA group as compared to VC group (Figure 1D) . At the same time reconstitution of lymphoid, myeloid and erythroid lineages was also achieved in peripheral blood with complete and stable donor chimerism observed even at day +69. Clinical scoring of aGVHD using minor modification of Hill's method {Blood 1997; 90:3204} showed that mice from VC group had a median score of 11 (10-12) on a scale of 15, with moderate to severe clinical GVHD features on day +15. In contrast, mice from WA group, scored 1 (1-2) with mild clinical GVHD features (Figure 1E).In order to elucidate the cellular mechanism of action of WA, we studied its effect on T cell differentiation. We found that WA induced T-regulatory (Treg) differentiation in CD4+ T helper cells in vitro . WA is known to activate the transcription factor Nuclear Respiratory Factor-2 (Nrf-2) in a Keap1-independent and Pten/Pi3k/Akt-dependent mechanism. Using Nrf-2 knockout mice, we found that Treg induction by WA was independent of Nrf-2 (Figure 1F) .In conclusion, we are the first group to show pre-clinical evidence for therapeutic efficacy of WA by in vitro modulation of donor graft prior to transplantation. WA suppressed two causal events in GVHD pathophysiology namely, ‘immune-priming’ and ‘release of myriad cytokines’. In addition, WA also induced Treg differentiation in donor T cells resulting in improved overall survival and reduction in aGVHD associated morbidity without compromising the reconstitution of host immune system. We are currently investigating the GVL effect of WA modulated graft in order to successfully transfer this novel approach to the clinics.This work was supported by the Department of Biotechnology (6242 P61/RGCB/PMD/DBT/ARMR2015). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Relationship Between Busulfan Exposure and Achievement of Sustained Donor Myeloid Chimerism in Patients with Non-Malignant Disorders

The overall objective of allogeneic hematopoietic cell transplantation (HCT) in patients with non-malignant conditions involves replacing a dysfunctional or absent cell or gene product for disease correction. It is unclear whether lower busulfan exposure may be sufficient in this population to facilitate durable myeloid engraftment and limit toxicity. Given that neither the ideal level of mixed myeloid chimerism for specific non-malignant diseases nor how to condition a patient to achieve stable mixed myeloid chimerism is fully known, we sought to analyze the relationships among busulfan exposure, myeloid chimerism, and outcomes in patients with non-malignant conditions receiving busulfan as a part of combination pretransplant conditioning at our institution. This was a single-center, retrospective study including pediatric patients with a variety of non-malignant disorders who underwent allogeneic HCT at the University of California San Francisco Benioff Children's Hospital from March 2007 to June 2018. The busulfan cumulative area under the curve (cAUC) was estimated using a validated population pharmacokinetic model and nonlinear mixed effects modeling. Median busulfan cAUC for all patients was 70 mg·h/L (range, 53 to 108). All of the 29 patients with a busulfan cAUC of ≥70 mg·h/L achieved long-term disease correction with full or stable mixed (>20%) myeloid chimerism, compared to 78.5% (22/28) of patients with a cAUC of <70 mg·h/L (P=.01). Overall ksurvival was evaluated up to 3 years and was identical in patients with busulfan cAUC < 70 mg·h/L and patients with busulfan cAUC ≥70 mg·h/L (96% versus 93%; P=.92). Only three patients died, at days 65, 164 and 980 days post-HCT. Severe busulfan-related toxicities and graft-versus-host-disease (GVHD) were rare, with veno-occlusive disease occurring in four patients (7%), acute respiratory distress syndrome in three patients (5%), and GVHD in five patients (9%). These results demonstrate excellent outcomes and extremely low rates of toxicity across our entire cohort. Based on the results of this study, we recommend a busulfan exposure target of 75 mg·h/L (range, 70 to 80) in all non-malignant patients receiving allogeneic HCT to ensure optimal exposure for achievement of high-level stable myeloid chimerism.

Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

Outcome of Hematopoietic Stem Cell Transplantation in Children with Primary Hemophagocytic Lymphohistiocytosis: A Single Institution Experience Using Reduced Intensity Conditioning

Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for primary hemophagocytic lymphohistiocytosis (HLH). Conventional myeloablative conditioning regimen using busulfan, cyclophosphamide with or without etoposide was associated with high rate of transplant-related mortality (Horne et al. BJH 2005). Reduced intensity conditioning (RIC) is used in HLH to minimize transplant-related toxicities. The use of alemtuzumab, fludarabine, and melphalan in HLH was associated with low mortality; however, mixed donor chimerism and graft failure were frequent (Allen et al. Blood 2018). A recent report in 25 HLH patients who underwent HSCT from HLA matched donors using targeted busulfan (45-65 mg/L X h), fludarabine, and serotherapy resulted in adequate stable donor chimerism, no graft rejection, and no mortality (Felber et al. Blood Adv. 2020). In this study, we reviewed the outcome of 16 HLH patients who underwent HSCT from HLA matched donors using RIC. All patients were initially treated using HLH-2004 protocol. They subsequently underwent HSCT from 10/10 HLA matched related or unrelated donor using RIC regimens. We initially used AFM conditioning regimen that included alemtuzumab (1 mg/kg), fludarabine (150 mg/m2), and melphalan (140 mg/m2) with or without thiotepa (10 mg/kg). GVHD prophylaxis included cyclosporine (CSA) and steroids. Given the high rate of low donor chimerism with AFM regimen, we subsequently used BF conditioning regimen that included busulfan (weight-based dosing for total of 16 doses) and fludarabine (160 mg/m2). Therapeutic drug monitoring of busulfan was performed. Thymoglobulin (10 mg/kg) was added in matched unrelated donor (MUD). GVHD prophylaxis included CSA and steroids in matched related donors (MRD) and CSA and methotrexate in MUD. Bone marrow was the stem cell source in all patients except one who underwent second transplant using peripheral blood stem cells. Supportive care was consistent among all patients. Defibrotide prophylaxis was administered to prevent sinusoidal obstruction syndrome (SOS). Donor chimerism &amp;lt;5% from whole blood or &amp;lt;20% in T-cell, graft failure, and death from any cause were considered as events. A total of 16 HSCT transplants were performed, 5 received AFM regimen and 11 received BF regimen. Patient and transplant characteristics are shown in Table. All patients had successful neutrophil and platelet engraftments. Among the five patients who underwent AFM conditioning, one patient with SH2D1A mutation is 8 years post transplant with stable full donor chimerism and another patient with PRF1 mutation who received AFM with thiotepa has stable sufficient donor chimerism 3 years post transplant. Both patients did not require donor leukocyte infusions (DLI). The remaining 3 patients had declining donor chimerism beyond the first year of transplant despite the frequent use of DLI, one of them had disease reactivation and underwent second HSCT using BF regimen. Event free survival (EFS) at 3 years was 60% and overall survival (OS) was 100%. Among the 11 patients who underwent BF regimen, 7 were transplanted using MRD and 4 MUD. The median cumulative area under the curve of busulfan was 64 mg/L X h (range, 56-73). Median donor myeloid chimerism was 97% (range, 83-100) and median T-cell chimerism was 78% (range, 40-93). One patient who underwent MUD had grade III acute GVHD and none had chronic GVHD. Two patients had mild SOS. There were no events among BF group with median follow up duration of 393 days (151-2080). The use of BF regimen in HLH was associated with excellent outcome and stable donor chimerism with no graft failure in comparison to AFM regimen. Longer follow up is needed to confirm our findings. Disclosures No relevant conflicts of interest to declare.

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.