Relapse Of Hematologic Malignancies Research Articles

Prospects and Potential for Chimerism Analysis after Allogeneic Hematopoietic Stem Cell Transplantation.

Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1-5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated.

EHealth mindfulness-based music therapy for patients undergoing allogeneic hematopoietic stem cell transplantation: A pilot randomized controlled trial protocol

BackgroundAllogeneic stem cell transplantation (allo-SCT) is the preferred therapy for patients with high-risk or relapsed hematologic malignancies, but may be complicated by psychological distress (e.g., depression, anxiety) and symptom burden (e.g., fatigue, pain). Mindfulness-based music therapy (MBMT), a relatively novel integrative medicine intervention that draws from mindfulness and music therapy principles, has shown promise in improving psychosocial outcomes and symptom burden in cancer patients. We outline an eHealth-based MBMT (eMBMT) intervention protocol examining: (1) feasibility, acceptability, and intended effects of eMBMT in improving HRQOL, symptom burden, and clinical markers of disease activity (e.g., infections), and (2) the extent to which eMBMT music therapy component-associated improvements in HRQOL, symptom burden, and disease activity are mediated by improvements in psychosocial and physiological (e.g., systemic inflammation, immune recovery) adaptation. MethodsParticipants (n = 60) with a hematologic malignancy undergoing allo-SCT will be randomized to receive eMBMT or an eHealth-based mindfulness meditation (eMM) intervention. eMBMT includes eight 60-min sessions facilitated by a music therapist focusing on mindfulness and music therapy. eMM includes eight 60-min self-led MM practices. ResultsFeasibility, acceptability, HRQOL, symptom burden, disease activity, and mediation effects of psychosocial and physiological adaptation will be assessed at baseline, pre-infusion, and post-engraftment with blood collection at baseline and post-engraftment. ConclusionThe current pilot RCT is the first eMBMT intervention to address the HRQOL and symptom burden of patients who are undergoing allo-SCT. Results will inform a fully powered RCT to establish preliminary efficacy of eMBMT on improvements in HRQOL, symptom burden, and disease activity.

Childhood leukemias in Mexico: towards implementing CAR-T cell therapy programs.

Leukemias are the most common type of pediatric cancer around the world. Prognosis has improved during the last decades, and many patients are cured with conventional treatment as chemotherapy; however, many patients still present with a refractory disease requiring additional treatments, including hematopoietic stem cell transplantation. Immunotherapy with monoclonal antibodies or cellular therapy is a promising strategy for treating refractory or relapsed hematological malignancies. Particularly, CAR-T cells have shown clinical efficacy in clinical trials, and different products are now commercially approved by regulatory agencies in the USA and Europe. Many challenges still need to be solved to improve and optimize the potential of these therapies worldwide. Global access to cell therapy is a significant concern, and different strategies are being explored in the middle- and low-income countries. In Mexico, leukemias represent around 50% of total cancer diagnosed in pediatric patients, and the rate of relapsed or refractory disease is higher than reported in other countries, a multi-factorial problem. Although significant progress has been made during the last decades in leukemia diagnosis and treatment, making new therapies available to Mexican patients is a priority, and cell and gene therapies are on the horizon. Efforts are ongoing to make CAR-T cell therapy accessible for patients in Mexico. This article summarizes a general landscape of childhood leukemias in Mexico, and we give a perspective about the current strategies, advances, and challenges ahead to make gene and cell therapies for leukemia clinically available.

Increased CAR-T Infusions Occur in Conjunction with Decreased Hematopoietic Stem Cell Transplantation

Background: Chimeric Antigen Receptor T-cell (CAR-T) therapies were first approved by the FDA in 2017, and multiple commercial products are currently available. Hematopoietic stem cell transplantation (HSCT) is another treatment strategy often utilized for patients with advanced and/or relapsed hematologic malignancies. Both CAR-T and HSCT require significant, overlapping resources for their collection, storage, and processing. Understanding the impact of evolving standards of care in clinical practice is necessary in the planning of resource allocation and laboratory expansion. We aimed to characterize the trends of CAR-T and HSCT infusions over a five-year period. Methods: The processing lab database was reviewed to compile a list of patients that received either CAR-T or HSCT (autologous or allogeneic) infusions, between 01/01/2018 and 12/12/2022. The number of individual HSCTs and CAR-T infusions were plotted over time. We also calculated the percent contribution of both HSCT and CAR-T to the total number of products infused per year. We subsequently examined these parameters, within the specific diagnoses of diffuse large B-Cell lymphoma/large cell lymphoma (DLBCL/LCL, including double and triple hit DLBCL and High-Grade lymphoma) and multiple myeloma (MM), including MM with amyloid and MM with plasma cell leukemia/plasmacytoma. Simple linear regression was used to calculate statistical significance to determine if the slopes were non-zero, and the direction of change over time. Results: Overall, our study included 210 CAR-T infusions and 2366 HSCTs. 89/210 (42%) patients who received a CAR-T product also received HSCT between 2018 and 2022, with 85/89 receiving HSCT prior to CAR-T infusion and 4/89 receiving CAR-T prior to HSCT, including two patients who received HSCT before and after CAR-T. The median interval between the most recent HSCT and subsequent CAR-T was 37 months. DLBCL/LCL and MM were the disease indications for which CAR-T was most utilized, accounting for 87.6% of the 210 procedures. The total number of CAR-T procedures increased significantly over the five-year period, from 24 in 2018 to 74 in 2022 (Slope=13.8; p=0.02), accompanied by a marked decrease in the number of HSCT from 502 in 2018 to 393 in 2022 (Slope=-26.6; p=0.06). The overall number of infusions (calculated as number of HSCT + number of CAR-T) declined slightly, though this was not statistically significant (mean 511 + 30.6 per year; Slope=-12.8; p=0.22). When expressed as a percentage of the total number of infusions, we observed a significant increase in the proportion of CAR-T, from 4.6% in 2018 to 15.8 % in 2022, (Slope=2.98; p=0.02), and a significant decrease in the proportion of infusions that were HSCT, from 95.4% in 2018 to 84.2% in 2022 (Slope=-2.98; p=0.02). In DLBCL, we observed a slight decrease in the number of overall infusions, from 71 in 2018 to 58 in 2022 (Slope=-3.5; p=0.18) and a significant decrease in HSCT from 47 in 2018 to 26 in 2022 (Slope=-6.0; p=0.0172), while the number of CAR-T infusions remained stable (mean=27.4 + 5.3 per year; Slope=2.5; p=0.14). In MM, we observed a marked increase in CAR-T infusions, from none in 2018 to 33 in 2022 (p=0.0544), while the total number of infusions remained stable (Mean 231.8 + 16.54; slope=-3.5; p=0.58) and HSCT slightly decreased, from 228 in 2018 to 184 in 2022, though this was not statistically significant (Slope=-11.5; p=0.18). We observed a non-significant increase in the percent contribution of CAR-T to the total number of infusions performed for MM, from 0% in 2018 to 15.3% in 2022 (Slope=3.62; p=0.06) while in DLBCL we observed a significant increase, from 33.8% in 2018 to 55.2% in 2022 (slope=6.42; p=0.017). Conclusion: The findings from this study suggest a gradual trend in the clinical utilization of human cellular therapy products over time. Since many of the resources and personnel involved in the collection, storage, and processing of these products are shared, an increase in CAR-T is an important observation. These findings underscore key issues in lab management for cell manufacturing facilities when considering resource allocation and infrastructure development. More analysis is needed to determine if the decrease in HSCT and the increase in CART are related.

Donor Hematopoietic Cell Telomere Length and Magnitude of Post-HCT Shortening Predict Survival in Early Stage Leukemia and Myelodysplastic Syndrome

Introduction. Telomere length (TL) reflects cell replicative history and residual replicative potential. Telomeres in donated hematopoietic cells (HCs) shorten considerably after hematopoietic cell transplant (HCT) because of the extensive proliferation required to reconstitute the recipient hematopoietic system. Longer telomeres in donor HCs are associated with better survival post-HCT in patients with severe aplastic anemia (Gadalla et al., JAMA 2015; Blood 2018). However, it is unclear whether donor HC TL affects survival of patients receiving HCT for hematological malignancies. Methods. We identified 384 patients with leukemia or myelodysplastic neoplasms who received HCT between 2013 and 2016 at 1 st complete remission/early disease stage, and had post-HCT blood samples at day 90 (standard deviation: SD=14 days) in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) protocol 1202 repository. Pre-HCT matched donor samples were available at the Center for International Blood and Marrow Transplant Research (CIBMTR) biorepository. TL was measured by Southern blotting of the terminal restriction fragments. We used Cox proportional hazard models to evaluate the associations between HCT outcomes and (i) long donor HC telomeres (defined as ≥6.7 kb), and (ii) low post-HCT telomere shortening of donor HC TL (defined as <230 bp and calculated as the difference between pre-HCT donor HC TL and that in the recipient post-HCT). The telomere thresholds were determined by exploratory analyses relating HC TL parameters with recipients' outcomes. Study outcomes are 2-year overall survival (OS), relapse risk, and non-relapse mortality (NRM). We also calculated the adjusted cumulative risks of all-cause and cause-specific mortality associated with the two HC TL parameters using cause-specific competing risk methods. Follow-up started at day 100 post-HCT. Results. Patients' mean age was 49.6 years (SD=18) and that of the donors was 37.9 years (SD=15). About half (54.7%) of the patients received HCT from unrelated donors, and 9% from haplo-identical related donors. The 2- year OS probabilities were higher in patients receiving HCT from donors with HC TL ≥6.7 kb compared to those receiving HCT from donors with HC TL below this threshold (76% and 63%, respectively; p log-rank = 0.009). Similarly, recipients with telomere shortening of donated HCs ≥230 bp post-HCT compared with those below this threshold experienced better 2-year OS(78% and 62%, respectively; p=0.003). These associations remained significant after adjusting for donor age and predictive clinical factors (HR= 0.60, p = 0.03 for donor HC TL ≥ vs. < 6.7 kb; and HR = 1.95, p = 0.003 for donated HC telomere shortening post-HCT below vs. ≥ 230 bp). The leading cause of excess mortality differed by HC TL parameters. Donor HC TL of ≥6.7 kb was associated with lower risk of NRM (HR= 0.45, 95% CI = 0.22 - 0.93, p = 0.03; 2-year NRM risk of 9.0% vs. 15%). However, post-HCT telomere shortening of <230 bp was associated with higher risk of relapse (HR=1.82, 95% CI = 1.11 - 1.82, p=0.02; 2-year relapse-mortality risk of 28% vs. 12%) (Figure 1A). When considering the combined effect of donor HC TL and magnitude of TL shortening in recipient, the adjusted cumulative risk of all-cause mortality dropped in half (from 41% in patients with donor HC TL <6.7 kb and telomere shortening <230 bp to 21% in those with donor HC TL ≥6.7 kb and telomere shortening ≥230 bp (Figure 1B) Conclusions. Short telomeres in donated HCs and their limited telomere shortening after HCT are associated with poor survival in patients receiving HCT for hematological malignancies at 1st complete remission/early disease stage. The study suggests that TL in donors prior to HCT may be a useful marker for improved donor selection independent of age. Less HC telomere shortening in the recipients during the first 90 days might identify patients at higher risk for relapse of hematological malignancies.

Circulating T cell profiles associate with enterotype signatures underlying hematological malignancy relapses

Early administration of azithromycin after allogeneic hematopoietic stem cell transplantation was shown to increase the relapse of hematological malignancies. To determine the impact of azithromycin on the post-transplant gut ecosystem and its influence on relapse, we characterized overtime gut bacteriome, virome, and metabolome of 55 patients treated with azithromycin or a placebo. We describe four enterotypes and the network of associated bacteriophage species and metabolic pathways. One enterotype associates with sustained remission. One taxon from Bacteroides specifically associates with relapse, while two from Bacteroides and Prevotella correlate with complete remission. These taxa are associated with lipid, pentose, and branched-chain amino acid metabolic pathways and several bacteriophage species. Enterotypes and taxa associate with exhausted T cells and the functional status of circulating immune cells. These results illustrate how an antibiotic influences a complex network of gut bacteria, viruses, and metabolites and may promote cancer relapse through modifications of immune cells.

CARs and Drugs: Pharmacological Ways of Boosting CAR-T-Cell Therapy.

The development of chimeric antigen receptor T cells (CAR-T cells) has marked a new era in cancer immunotherapy. Based on a multitude of durable complete remissions in patients with hematological malignancies, FDA and EMA approval was issued to several CAR products targeting lymphoid leukemias and lymphomas. Nevertheless, about 50% of patients treated with these approved CAR products experience relapse or refractory disease necessitating salvage strategies. Moreover, in the vast majority of patients suffering from solid tumors, CAR-T-cell infusions could not induce durable complete remissions so far. Crucial obstacles to CAR-T-cell therapy resulting in a priori CAR-T-cell refractory disease or relapse after initially successful CAR-T-cell therapy encompass antigen shutdown and CAR-T-cell dysfunctionality. Antigen shutdown predominately rationalizes disease relapse in hematological malignancies, and CAR-T-cell dysfunctionality is characterized by insufficient CAR-T-cell proliferation and cytotoxicity frequently observed in patients with solid tumors. Thus, strategies to surmount those obstacles are being developed with high urgency. In this review, we want to highlight different approaches to combine CAR-T cells with drugs, such as small molecules and antibodies, to pharmacologically boost CAR-T-cell therapy. In particular, we discuss how certain drugs may help to counteract antigen shutdown and CAR-T-cell dysfunctionality in both hematological malignancies and solid tumors.

Haploidentical Hematopoietic Stem Cell Transplant Plus Add-Back NK Cells in Pediatric High-Risk ALL

Introduction: Natural killer (NK) lymphocytes are cells of innate immunity that protect against tumor and infections. NK cells recognize self and non-self HLA class I ligands, are regulated by a balance between activator and inhibitory signals through surface receptors, and can interact with HLA class I molecules on target cells. Killer-cell Immunoglobulin-like Receptors (KIRs) are inhibitory molecules expressed on NK cells and belong to the immunoglobulin superfamily. KIRs function to "educate" NK cells in effective defense against unhealthy self/non-self cells and tolerance against healthy self cells. NKs are thought to play a key role in preventing early relapse of hematologic malignancies, virus infection and graft versus host disease (GvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT).In B-/T-cell alpha/beta depleted haplo-HSCT, donor-versus-recipient allo-reactive NK cells can exert an efficient graft versus leukemia (GvL) effect, mediated by inhibitory KIRs where the HLA ligand is missing on recipient cells. Donor NK cells after haplo-HSCT are "immature" and not effective as the mature NKs. These immature NK cells are CD56bright, are derived from CD34+ stem cells, and mature to a CD56dim phenotype. The mature CD56dim NK cells are characterized by acquisition of KIRs, CD16 and CD57 in mature stages, which have functional significance in targeting and medicating their cytolytic activity. Based on these considerations, we performed haplo-HSCT with donor NK cell add-back to provide mature, functional immunological surveillance against tumor (MRD) and virus reactivation, which can frequently occur in manipulated haplo-HSCT for pediatric acute lymphoblastic leukemia (ALL). Methods: We evaluated the alloreactive effect of donor-derived NK cells in three pediatric high-risk ALL who underwent B-/T-cell alpha/beta depleted haplo-HSCT followed by donor NK infusion. Patients' characteristics are summarized in the Table. All 3 patients were male; median age was 13 months. Patients 1 and 2 had infant ALL, and patient 3 had refractory-relapsed (r/r) ALL. All patients received a median of 3 lines chemotherapy. Patient 1 received a prior allo-HSCT. Patients 1 and 2 received chemo-based conditioning regimens consisting of thyotepa, and fludarabine; In addition, patient1received treosulfan, and patient 2 received busulfan. Treosulfan was given to patient 1, due to less toxicity profile given prior allo-HSCT. Patient 3 received Total Body Irradiation (TBI), thyotepa, and fludarabine. All patients received rituximab and letermovirfor EBV and CMV prophylaxis, respectively, for all patients. Mother was Donor for all patients. Median CD34+, CD19+, CD3+-alpha/beta cell dose was 8.8, 0.05, 0.41 x 106/kg, respectively. NK cell allo-reactivity was tested by evaluating KIR genotyping and KIR/KIR-ligand mismatch in graft-versus-host direction. All three patients present KIR/KIR-ligand mismatch. Results: Engraftment was day +13, +14 and +19 for ANC, and +14, +18 and +17 for PLT (for both >20K and >50K), for patients 1, 2 and 3, respectively. NK cell number were monitored on day +7, +14, +21 and +28 (data pending). Patients received NK cell infusion on day 30 +/- 2 days. No infusion reactions were observed. MedianNK infused were 256x106/kg. None of the patients developed CMV, EBV or ADV reactivation. Patient 2 developed Grade 2 acute GvHD (skin) treated with steroids. No Grade GvHD 3-4 was observed. Median follow up was 58 days. All patients had full donor chimerism at day 30 ±2. All patients are currently in hematological CR; patient 1 converted to positive MRD at nine months from transplant. Conclusion: Alloreactive NK cell add-back infusion after B-/T-cell alpha/beta depleted haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation. These NK cells may improve efficacy by enhancing GvL and could contribute to maintain the "disease control" through immunologic surveillance in high-risk ALL. Indeed, NK killing, which is higher in mature NK cells, depends on acquisition of sequential phenotypic maturation. Thus, infusion of mature donor NK cells may help to maintain control of MRD and virus reactivation through immunologic surveillance. Although our preliminary observations seem to be promising, this hypothesis should be confirmed in clinical trial of a larger number of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Total marrow irradiation (TMI): Addressing an unmet need in hematopoietic cell transplantation - a single institution experience review.

PurposeTMI utilizes IMRT to deliver organ sparing targeted radiotherapy in patients undergoing hematopoietic cell transplantation (HCT). TMI addresses an unmet need, specifically patients with refractory or relapsed (R/R) hematologic malignancies who have poor outcomes with standard HCT regimens and where attempts to improve outcomes by adding or dose escalating TBI are not possible due to increased toxicities. Over 500 patients have received TMI at this center. This review summarizes this experience including planning and delivery, clinical results, and future directions.MethodsPatients were treated on prospective allogeneic HCT trials using helical tomographic or VMAT IMRT delivery. Target structures included the bone/marrow only (TMI), or the addition of lymph nodes, and spleen (total marrow and lymphoid irradiation, TMLI). Total dose ranged from 12 to 20 Gy at 1.5-2.0 Gy fractions twice daily.ResultsTrials demonstrate engraftment in all patients and a low incidence of radiation related toxicities and extramedullary relapses. In R/R acute leukemia TMLI 20 Gy, etoposide, and cyclophosphamide (Cy) results in a 1-year non-relapse mortality (NRM) rate of 6% and 2-year overall survival (OS) of 48%; TMLI 12 Gy added to fludarabine (flu) and melphalan (mel) in older patients (≥ 60 years old) results in a NRM rate of 33% comparable to flu/mel alone, and 5-year OS of 42%; and TMLI 20 Gy/flu/Cy and post-transplant Cy (PTCy) in haplo-identical HCT results in a 2-year NRM rate of 13% and 1-year OS of 83%. In AML in complete remission, TMLI 20 Gy and PTCy results in 2-year NRM, OS, and GVHD free/relapse-free survival (GRFS) rates of 0%, 86·7%, and 59.3%, respectively.ConclusionTMI/TMLI shows significant promise, low NRM rates, the ability to offer myeloablative radiation containing regimens to older patients, the ability to dose escalate, and response and survival rates that compare favorably to published results. Collaboration between radiation oncology and hematology is key to successful implementation. TMI/TMLI represents a paradigm shift from TBI towards novel strategies to integrate a safer and more effective target-specific radiation therapy into HCT conditioning beyond what is possible with TBI and will help expand and redefine the role of radiotherapy in HCT.

Comparison of Outcomes of Haploidentical Peripheral Blood Stem Cell Transplantation Supported by Third-Party Cord Blood Versus Human Leukocyte Antigen-Matched Sibling Peripheral Blood Stem Cell Transplantation in Hematologic Malignancy Patients.

Recent studies have shown that haploidentical hematopoietic stem cell transplantation supported by third-party cord blood (haplo-cord-HSCT) results in rapid hematopoietic recovery, low incidences of graft-versus-host disease (GVHD), and relapse of hematologic malignancies. However, few reports on haploidentical peripheral blood stem cell transplantation supported by third-party cord blood (haplo-cord-PBSCT) have been published. To evaluate the outcomes of patients who underwent haplo-cord-PBSCT or human leukocyte antigen (HLA)-matched sibling donor peripheral blood stem cell transplantation (MSD-PBSCT), we retrospectively reviewed the clinical data of patients with hematologic malignancies who underwent haplo-cord-PBSCT (n = 93) or MSD-PBSCT (n = 72) in our hospital from March 2017 to December 2020. In the haplo-cord-PBSCT and MSD-PBSCT groups, the median time for neutrophil and platelet engraftment was 13 vs. 12 days (p = 0.07) and 16 vs. 13 days (p = 0.06), respectively. The 30-day cumulative incidences of neutrophil engraftment were 100.0% and 98.6% (p = 0.12). The 100-day cumulative incidences of platelet engraftment were 96.8% and 98.6% (p = 0.01). The 100-day cumulative incidences of grade II–IV and grade III–IV acute GVHD were 29.1% vs. 23.6% (p = 0.42) and 9.7% vs. 4.2% (p = 0.18). The cumulative incidences of total and moderate/severe chronic GVHD at 1 year were 26.5% vs. 17.4% and 8.1% vs. 4.5%, respectively, and at 3 years were 34.7% vs. 34.3% (p = 0.60) and 13.6% vs. 10.6% (p = 0.49), respectively. The cumulative incidences of relapse at 1 year were 9.3% and 7.2% and at 3 years were 17.0% and 17.0% (p = 0.98). Non-relapse mortality (NRM) at 1 year was 14.6% and 8.6% and at 3 years was 17.4% and 8.6% (p = 0.13) in two groups. The probabilities of overall survival (OS), disease-free survival (DFS), and GVHD-free/relapse-free survival (GRFS) at 1 year were 81.7% vs. 88.6%, 76.1% vs. 84.2%, and 71.7% vs. 79.7%, respectively, and at 3 years were 78.7% vs. 79.0%, 65.6% vs. 74.4%, and 55.5% vs. 63.6%, respectively, in the corresponding group, p > 0.05. In conclusion, for patients with acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) and acute lymphoid leukemia (ALL), haplo-cord-PBSCT results in similar outcomes compared with MSD-PBSCT, and it may be a valid alternative transplantation method.

A retrospective single-center analysis of G-CSF-mobilized donor lymphocyte infusion in hematologic malignancies after unmanipulated allogenic PBSCT.

To explore the efficacy and safety of G-SCF-mobilized donor lymphocyte infusion (DLI) for treatment of relapse of hematologic malignancies after allogeneic peripheral blood stem cell transplantation, we performed a retrospective analysis in a cohort of patients with morphologic (n = 36) or molecular (n = 22) relapse post transplantation. The 3-year post-DLI survival rates for therapeutic and preemptive DLI recipients were 16.7% and 33.3%, respectively. The occurrence of DLI-associated acute graft-versus-host disease predicted longer survival, whereas diagnosis of T cell acute lymphoblastic leukemia/lymphoma or myelodysplastic syndromes or early relapse after transplant (< 6months) predicted shorter survival after therapeutic DLI. Cumulative incidence of progression to hematologic relapse and non-relapse mortality after preemptive DLI were 46.8% and 29.1%, respectively. Active disease prior to transplant and early molecular relapse after transplant (< 4months) were the strongest predictors of non-relapse mortality after preemptive DLI. In conclusion, although therapeutic DLI had limited efficacy against T cell acute lymphoblastic leukemia/lymphoma or myelodysplastic syndromes or early post-transplant relapse, patients who developed DLI-associated acute graft-versus-host disease would benefit from this procedure in the setting of G-SCF-mobilized DLI. Furthermore, preemptive DLI could protect half of patients from hematologic relapse after transplantation with acceptable toxicity.

A Metabolic Reprogramming Amino Acid Polymer as an Immunosurveillance Activator and Leukemia Targeting Drug Carrier for T-Cell Acute Lymphoblastic Leukemia.

Compromised immunosurveillance leads to chemotherapy resistance and disease relapse of hematological malignancies. Amino acid metabolism regulates immune responses and cancer; however, a druggable amino acid metabolite to enhance antitumor immunosurveillance and improve leukemia targeting‐therapy efficacy remains unexplored. Here, an l‐phenylalanine polymer, Metabolic Reprogramming Immunosurveillance Activation Nanomedicine (MRIAN), is invented to effectively target bone marrow (BM) and activate the immune surveillance in T‐cell acute lymphoblastic leukemia (T‐ALL) by inhibiting myeloid‐derived suppressor cells (MDSCs) in T‐ALL murine model. Stable‐isotope tracer and in vivo drug distribution experiments show that T‐ALL cells and MDSCs have enhanced cellular uptake of l‐phenylalanine and MRIANs than normal hematopoietic cells and progenitors. Therefore, MRIAN assembled Doxorubicin (MRIAN‐Dox) specifically targets T‐ALL cells and MDSCs but spare normal hematopoietic cells and hematopoietic stem and progenitor cells with enhanced leukemic elimination efficiency. Consequently, MRIAN‐Dox has reduced cardiotoxicity and myeloablation side effects in treating T‐ALL mice. Mechanistically, MRIAN degrades into l‐phenylalanine, which inhibits PKM2 activity and reduces ROS levels in MDSCs to disturb their immunosuppressive function and increase their differentiation toward normal myeloid cells. Overall, a novel amino acid metabolite nanomedicine is invented to treat T‐ALL through the combination of leukemic cell targeting and immunosurveillance stimulation.

Outcomes with Natural Killer Cells Infusion after Allogeneic Hematopoietic Stem Cell Transplantation: A Systematic Review and Meta-Analysis

Background:Natural killer cells (NK) are known to be the first cells that recover after allogeneic hematopoietic stem cell transplant (HCT). NK cells have anti-leukemic and anti-viral properties and are also implicated in the graft versus leukemia (GVL) effect without causing graft versus host disease (GVHD). There is emerging evidence supporting the use of NK cells post-transplant as adoptive immunotherapy to prevent or treat the relapse of hematologic malignancies. Here, we present a systematic review and meta-analysis aimed to investigate the outcomes with NK cells infusion after HCT.Methods:A detailed literature search was conducted for the systematic review and meta-analysis according to the guidelines mentioned in the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). Population, intervention, comparison, and outcome (PICO) table was developed and three electronic databases (PubMed, Cochrane Library, and ClinicalTrials.gov) were searched through January 2021 using MeSH terms and keywords for “hematologic malignancy and natural killer cells” and “hematopoietic stem cell transplantation and natural killer cells”. No filters or publication time limits were applied for the search. A total of 988 records were identified through database searching. After excluding duplicates, review, and non-relevant articles, we selected 9 studies that reported outcomes with NK cells infusion after HCT. Quality evaluation was done using the NIH quality assessment tool and Inter-study variance was calculated using the Der Simonian-Laird Estimator. Pooled analysis was done using the ‘meta’ package (Schwarzer et al, R programming language) and proportions with 95% confidence intervals (CI) were computed.Results:We identified 9 studies with a total of 149 participants. Out of these, 53 patients were from 4 studies using NK cells for the treatment of relapsed disease after HCT (therapeutic studies) and 96 patients were from 5 studies using NK cells to prevent relapse after HCT (pre-emptive studies).Therapeutic use of NK cells for relapsed disease after HCT: A total of 53 patients from 4 studies were evaluated. The median age of patients was 44.5 (1.9-69) years, and 40% patients were male. The median follow-up was 20 (1-69) months. Overall survival (OS) was reported by two studies at 2 years and was 36.3% and 55%. The pooled complete response (CR) was 18% (95% CI 0.06-0.33, I 2 =0%, n=37) and overall response rate (ORR) was 45% (95% CI 0.20-0.71, I 2 =71%, n=53). The pooled relapse rate was 65% (95% CI 0.32-0.92, I 2 =75%, n=42). The pooled incidence of acute GVHD and chronic GVHD was 15% (95% CI 0.03-0.31, I 2 =41%, n=53) and 8% (95% CI 0.01-0.18, I 2 =0%, n=53) respectively.Pre-emptive use of NK cells to prevent relapse after HCT: A total of 96 patients from 5 studies were evaluated. The median age of patients was 33.5 (2-75) years, and 48% patients were males. The median follow-up was 23.1 (8-81.6) months. The median OS was 1 (0.63-5) years. The pooled CR was 45% (95% CI 0.21-0.70, I 2 =76%, n=83) and ORR was 89% (95% CI 0.57-1.00, I 2 =77%, n=42). The pooled relapse rate was 35% (95% CI 0.20-0.52, I 2 =55%, n=96). The pooled incidence of acute GVHD and chronic GVHD was 30% (95%CI 0.14-0.48, I 2 =61%, n=96) and 8% (95%CI 0.00-0.24, I 2 =73%, n=96) respectively.Conclusion:Infusion of NK cells after HCT to prevent relapsed disease results in favorable outcomes with an acceptable toxicity profile. Optimal use of NK cells infusions after HCT is in a pre-emptive fashion to prevent relapsed disease and the efficacy of NK cells infusions after HCT in the setting of overt hematologic relapse is modest. Our findings suggest the need for large prospective clinical trials to establish the potential benefit of NK cells infusion after HCT to prevent relapse of hematologic malignancies without increased non-relapse mortality. [Display omitted] DisclosuresAbhyankar: Incyte/Therakos: Consultancy, Research Funding, Speakers Bureau. McGuirk: Bellicum Pharmaceuticals: Research Funding; Gamida Cell: Research Funding; Novartis: Research Funding; Novartis: Research Funding; EcoR1 Capital: Consultancy; Fresenius Biotech: Research Funding; Astelllas Pharma: Research Funding; Magenta Therapeutics: Consultancy, Honoraria, Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Allovir: Consultancy, Honoraria, Research Funding; Pluristem Therapeutics: Research Funding; Kite/ Gilead: Consultancy, Honoraria, Other: travel accommodations, expense, Kite a Gilead company, Research Funding, Speakers Bureau.

Complete Remissions Post Infusion of Multiple Tumor Antigen Specific T Cells for the Treatment of High Risk Leukemia and Lymphoma Patients after HCT

Background/Objectives: Relapse of hematologic malignancies after hematopoietic cell transplantation (HCT) confers high mortality because of progressive disease and graft-vs.-host disease (GVHD). Hence, we hypothesized that tumor-associated antigen-specific lymphocytes (TAA-L) would be specific, diminish immune escape, and could safely decrease cancer burden.Design/Methods: We expanded lymphocytes reactive to TAA: WT1, PRAME, Survivin, which are over-expressed and immunogenic in hematologic malignancies. On a prospective study: NCT002203902, eligible patients received TAA-L infusions at 4 sequential dose levels: 0.5 to 4.0e7/m2 per dose.Results: Thirty-two donor-derived TAA-L products were generated and lacked alloreactivity in vitro. TAA-L products comprised predominantly effector memory T-cells, with <15% of T-cells expressing exhaustion markers (PD1, TIM3, LAG3). Specificity of TAA-L showed greatest response to PRAME by ELIspot. Eleven HCT recipients (aged 9-70 years) with relapsed: Hodgkin's lymphoma (n=2), B-ALL (n=3), or AML (n=5), or high risk for relapse: AML post 2nd HCT (n=1)) received 1-3 doses of TAA-L. Patients had relapsed 2-12 months after HCT (9 allo, 1 auto) and all received therapy pre-TAA-L. Post infusion, there was only one adverse event possibly attributed to TAA-L (GVHD post viral infection on vidaza). One patient withdrew prior to study completion due to progressive disease with steroids for sepsis. Responses of evaluable patients (n=10) were: PD (n=2), PR (n=3), CR (n=5), with only 1 patient receiving adjunct therapy. Of the CR patients: AML (n=4); one patient who relapsed 10 months post HSCT is in a continuous CR for 314+ days after 3 doses of TAA-L only, one patient cleared peripheral blasts with TAA-L; one patient with Ph+-ALL achieved bcr-abl negativity post TAA-L only for the first time post-HSCT and has a continuous CR with 2 doses of TAA-L for 261 days. Immunosequencing of T-cell receptors showed expansion of TAA-L clones derived from the TAA-L product 4-5 weeks after infusion.Conclusion: This unique immunotherapeutic has been well tolerated without causing life-threatening cytokine release syndrome. Despite aggressive and multiply relapsed disease, 8/10 evaluable patients have demonstrated evidence of disease control after TAA-L, with 50% complete response, suggesting that TAA-L may have efficacy in relapsed high-risk hematological malignancies after HCT. DisclosuresBollard:Torque: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Neximmune: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Personalized Circulating Tumor DNA Dynamically Predicts Response and/or Relapse in Hematological Malignancies

Background: With the recent advent of molecular techniques such as droplet digital PCR (ddPCR), tumor specific genomic component in the plasma or serum, known as circulating tumor DNA (ctDNA), represents one of the most sensitive and noninvasive biomarkers in solid tumors. In the field of hematological malignancies, it remains to be elucidated whether ctDNA monitoring enables accurate prediction of clinical relapse in individual patients. To address this issue, we retrospectively investigated 17 patients with hematological malignancies who had achieved complete or partial remission after first-line induction therapy.Methods: Our cohort consisted of 8 patients with acute myeloid leukemia, 3 with multiple myeloma (MM), 2 with B cell non-Hodgkin's lymphoma, 2 with myelodysplastic syndromes, 1 with B cell acute lymphoblastic leukemia, and 1 with T cell non-Hodgkin's lymphoma. Serial biopsy and matched serum samples were collected at the time of diagnosis (termed “baseline”) and then throughout the treatment course. Cell-free DNA (cfDNA) was extracted from serum samples. We subjected DNA extracted from baseline tumor DNA and buccal swab to next generation sequencing (NGS), identifying one or two somatic driver mutations. We designed personalized ddPCR assays for each somatic mutation identified (n=16). Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. The first day of 1st line induction therapy was set to day 0 for estimation of the disease-free survival (DFS). For subset analysis of patients who relapsed during follow-up, the day of re-induction therapy initiation for relapse is re-set to day 0. Thereafter, the cumulative incidence of relapse, as assessed by ctDNA or by clinical parameters, was estimated and compared.Results: The amount of cfDNA at diagnosis was significantly higher compared with paired cfDNA at remission (p=0.037). NGS screening identified somatic driver mutations in all cases, with 2 mutations found in 2 individuals. Overall, we could construct ddPCR assay for all patients; these mutations included NRAS, KRAS, TP53, GATA2, STAG2, JAK3, MYD88, B2M, NPM1, DNMT3A, U2AF1, and SF3B1 . NGS and ddPCR assays had a good concordance in assessing the same tumor rich samples with regard to the variant allele frequency (VAF) (r2=0.96; p< 0.01). CtDNA could be detected in matched cfDNA from baseline serum. Moreover, the dynamic longitudinal changes of ctDNA VAF reflected the observed overall clinical response, as determined by clinical biomarker or imaging. 8 patients clinically relapsed at a median of 297 days post 1st line therapy (termed “relapsed group”), with the remaining 9 patients disease free at a median of 332 days post 1st line therapy (termed “remission group”). Notably, all of the patients in relapsed group became ctDNA detectable or had ctDNA increased at two consecutive points (termed “ctDNA-increase”). In marked contrast, all of the patients in remission group remained ctDNA undetectable or stable (termed “ctDNA-stable”). Patients with ctDNA-increased had a significantly inferior DFS: 2-year DFS 12.5% in patients with ctDNA-increased vs. 100% in ctDNA-stable, p=0.0115 (Figure 1A). Finally, we compared the timing of molecular relapse and clinical relapse, as assessed by ctDNA and by conventional biomarkers (flowcytometry in leukemia or serum free light chain in MM), respectively, in relapsed group. As a result, molecular relapse as assessed by ctDNA had a median of 30 days lead time (range, 4 to 119 days) over clinical relapse as assessed by conventional clinical parameters (Figure 1B).Conclusions: In summary, personalized ctDNA monitoring could identify patients with impending relapse in advance of established clinical parameters in hematological malignancies. This might allow for clinicians to initiating earlier therapeutic interventions for impending relapse. Further prospective studies in a much larger cohort of hematological malignancies are needed to confirm these findings. [Display omitted] DisclosuresNagamura-Inoue:Tsubakimoto Chain CO.: Research Funding; Rohto Pharmaceutical Co., Ltd.: Research Funding; AMED grant: Research Funding. Tojo:AMED grant: Research Funding.

Graft Versus Leukemia: Current Status and Future Perspectives.

Relapse of hematologic malignancy is the major reason for treatment failure after allogeneic stem cell transplantation. Graft-versus-leukemia (GVL) responses are critical to prevent relapse but may fail as a result of tumor immune evasion, for example through antigen loss or T cell dysfunction. In silico pipelines for the discovery of tumor-expressed antigens and novel therapies that target immune evasion mechanisms (small molecule, biological, immune cell transfer) are potential strategies to sustain or reimpose GVL. A key challenge will be how experimental therapies to enhance GVL can progress from first-in-human trials to prospective, controlled trials that evaluate efficacy and lead to regulatory approval.

Successful and Safe Treatment of Intestinal Graft-Versus-Host Disease (GvHD) with Pooled-Donor Full Ecosystem Microbiota Biotherapeutic: Results from a 29 Patient-Cohort of a Compassionate Use/Expanded Access Treatment Program

Introduction Intestinal graft-versus-host disease (GvHD), following allogeneic hematopoietic stem cell transplantation (allo-HSCT), has a high mortality rate and a reduced life-expectancy. In this context, failure to respond to steroid therapy is associated with limited further therapeutic options, thereby representing an unmet medical need. Aiming at restoring microbiome functions, fecal microbiota transfer (FMT) has proved to be a promising treatment modality in this challenging clinical setting, with recent studies reporting favorable results in steroid refractory-acute GVHD (SR-aGvHD) patients. Here we report on the use of a next-gen FMT product "MaaT013", a standardized, pooled-donor, high-richness microbiota biotherapeutic, used to treat 29 patients with intestinal aGvHD as part of a compassionate use/expanded access treatment program. Patients and methods Twenty-nine allo-HSCT recipients with steroid-dependent or SR intestinal GvHD (classical aGVHD n=22, late-onset aGVHD n=2; aGvHD with overlap syndrome n=5) were treated with MaaT013 biotherapeutic as part of a compassionate use/expanded access treatment program of the developer MaaT Pharma. These patients had previously received and failed 1 to 5 lines (median 3; 22/29 received ruxolitinib) of GvHD systemic treatments. Each patient received 1 to 3 doses (median: 3; total doses administered: 71) of MaaT013, a 150 mL bag, by enema (n=28) or nasogastric tube (n=1). GI-GvHD response was evaluated 7 days after each administration and 28 days after the first dose. Prepared under Good Manufacturing Practices, MaaT013 biotherapeutics are characterized by a highly consistent richness of 455 ±3% Operational Taxonomic Units and an Inverse Simpson index greater than 20. Batch release specifications are based on potency (viability), identity (diversity), and purity (microbiological safety testing and proportion of proinflammatory species), ensuring the desired consistency across batches. Results We observed a GI overall response rate of 59% (17/29) at day 28 after first dosing, including 9 complete response (CR), 6 very good partial response (VGPR), and 2 partial response (PR). Considering the best GI response achieved, 20/29 patients (69%) achieved at least a PR, with 9 CR, 8 VGPR and 3 PR. Among the 29 treated patients, 16 were still alive at last follow-up (median follow-up (FU): 131.5 days; range [28; 413]) and 6 months overall survival was 54%. Among the 9 patients achieving CR, all were still alive at last follow-up (median FU: 197 days; [49; 301]) and were able to taper or stop steroids and immunosuppressants. Among these 9 patients, three patients presented GI symptom recurrence between 2 and 3 months after dosing. In 2 patients, this recurrence occurred after heavy antibiotic treatment, one of them received an additional MaaT013 dose and achieved a second CR. Of note, among these 9 patients, mild chronic mucosal GvHD symptoms persisted in one patient, and molecular relapse of hematologic malignancy was observed in another. The safety of the MaaT013 microbiota biotherapeutic was satisfactory for all patients. One patient developed "possibly related" sepsis one day after the third dosing but no pathogen was identified in blood cultures, and the patient recovered after a course of antibiotics. Another patient developed C. difficile diarrhea 24 hours post-administration. This was not causally related to MaaT013, as C. difficile testing was negative in the MaaT013 lot. Nosocomial transmission was suspected as the cause as several patients with C. difficile infection were hospitalized in the same unit during this period. Conclusions We herein report for the first time the treatment of 29 patients with steroid-dependent or SR intestinal aGvHD using a full ecosystem, standardized, pooled-donor, high-richness biotherapeutic. The overall response rate was 59% with the off-the-shelf MaaT013 product, which was shown to be safe and effective in these heavily pre-treated and immunocompromised patients, warranting further exploration of the full ecosystem microbiota restoration approach. Disclosures Malard: Astellas: Honoraria; JAZZ pharmaceutical: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Keocyt: Honoraria; Theralos/Mallinckrodt: Honoraria; Biocodex: Honoraria. Plantamura:MaaT Pharma: Current Employment. Mohty:BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract 3252: OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell anti-tumor efficacy and durability of response

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against relapsed and refractory hematological malignancies, such as B cell non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Despite these advances, prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence prevent CAR T cell therapies from reaching their full curative potential. Interleukin-2 (IL-2) is a potent stimulator of CD4 and CD8 effector T cell proliferation, survival, and cytotoxic function, thereby making it an attractive molecule to support CAR T cell therapy. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations, including non-tumor reactive T cells and NK cells. To facilitate selective in vivo expansion of engineered T cells we have developed an orthogonal (ortho) ligand/receptor system consisting of a pegylated, IL-2 mutein (STK-009) and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that selectively bind each another, but do not interact with their wild type receptor and cytokine counterparts. This system allows for in-vivo IL-2 signaling in engineered adoptive cell therapies that express the orthoIL-2Rβ while avoiding signaling in non-tumor reactive T cells and NK cells. Here, we demonstrate the ability of STK-009/orthoIL-2Rβ pair to selectively potentiate human orthoIL-2Rβ expressing CD19 CAR T cells in vitro and in vivo. We incorporated orthoIL-2Rβ into a CD19 directed CAR lentiviral construct utilizing a T2A peptide linker, allowing the use of a single lentiviral plasmid to generate orthoCAR T cells. Transduction of donor T cells with the CAR+orthoIL-2Rβ lentivirus and the use of STK-009 during in vitro manufacturing enabled selective proliferation and enrichment of orthoCAR transduced T cells. orthoCAR T cells grown in STK-009 maintained a similar immunophenotype and cytotoxic function compared to T cells manufactured with wild type IL-2. Subcutaneous administration of STK-009 dramatically enhanced the expansion (&amp;gt;100-fold) and efficacy of orthoCAR T cells in an aggressive pre-clinical mouse model of B cell malignancy (Raji), demonstrating 100% durable complete responses, compared to 50% relapsed and refractory lymphoma in CD19 CAR alone. Immunoprofiling of in vivo orthoCAR T cells revealed that STK-009 treatment significantly increased both naïve and effector memory RA (TEMRA) populations. RNAseq and IHC analysis also showed that STK-009 treatment increases expression of cytotoxic molecules such as Granzyme B and perforin specifically in the CAR T cells, without inducing GranzymeB in other cells. These findings validate a potent platform that selectively harnesses the potent anti-tumoral T cell effector functions of IL-2 to improve the efficacy and durability CAR T cell therapy. Citation Format: Paul-Joseph Aspuria, Michele Bauer, Sandro Vivona, Steven E. Kauder, Scott McCauley, Romina Riener, Rene De Waal Malefyte, Navneet Ratti, Deepti Rokkam, Jan Emmerich, Patrick J. Lupardus, Rob A. Kastelein, Martin Oft. OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell anti-tumor efficacy and durability of response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3252.

Reconstitution of T Cell Subsets Following Allogeneic Hematopoietic Cell Transplantation.

Allogeneic (allo) hematopoietic cell transplantation (HCT) is the only curative treatment option for patients suffering from chemotherapy-refractory or relapsed hematological malignancies. The occurrence of morbidity and mortality after allo-HCT is still high. This is partly correlated with the immunological recovery of the T cell subsets, of which the dynamics and relations to complications are still poorly understood. Detailed information on T cell subset recovery is crucial to provide tools for better prediction and modulation of adverse events. Here, we review the current knowledge regarding CD4+ and CD8+ T cells, γδ T cells, iNKT cells, Treg cells, MAIT cells and naive and memory T cell reconstitution, as well as their relations to outcome, considering different cell sources and immunosuppressive therapies. We conclude that the T cell subsets reconstitute in different ways and are associated with distinct adverse and beneficial events; however, adequate reconstitution of all the subsets is associated with better overall survival. Although the exact mechanisms involved in the reconstitution of each T cell subset and their associations with allo-HCT outcome need to be further elucidated, the data and suggestions presented here point towards the development of individualized approaches to improve their reconstitution. This includes the modulation of immunotherapeutic interventions based on more detailed immune monitoring, aiming to improve overall survival changes.

Manufacturing A Donor Lymphocyte Product Depleted Of TCR-αβ T Cells And CD19+ B Cells For Prophylactic Infusion Following Allogeneic Stem Cell Transplantation

Background & Aim Background In allogeneic stem cell transplantation (HSCT), relapse of hematologic malignancies is a leading cause of post-transplant morbidity. In the early post-transplant period, when immune reconstitution is pending, residual leukemic clones can proliferate, resulting in disease relapse with bleak prognosis and limited treatment options. Donor lymphocyte infusions (DLI) are often used as treatment of post-transplant leukemia relapse but this practice is limited by graft versus host disease (GVHD) as a complication. Adoptive transfer of selected lymphocyte subsets that effect tumor cytotoxicity without inducing GVHD, such as natural killer (NK) cells and TCRγδ T cells, may provide early donor-derived surveillance for residual or early relapse disease after HSCT. With this background, a Phase I clinical trial (CASE1Z19) was designed to explore the prophylactic administration of donor lymphocytes depleted of TCR-αβ T cells and CD19+ B cells early in the post-transplant course. A maximum threshold of Methods, Results & Conclusion Methods Three qualification runs were conducted using the Miltenyi CliniMACS Plus system to deplete TCR-αβ T and CD19+ B cells from non-mobilized, peripheral blood mononuclear cells (PBMC) in leukapheresis products sourced by a commercial vendor. Each product was analyzed for lymphocyte subset composition and stability post-processing. Results The mean starting total white blood cell count was 6.4 × 109 cells from which TCR-αβ T cells and CD19+ B cells were maximally depleted by >99.8% with mean Log10 reductions of 3.10 and 3.06 respectively (Table1). The TCR-αβ T cell content in products generated from each run were 0.2-0.5 × 105/kg, based on an average 60kg adult. The yield of NK cells and TCRγδ T cells from each manufacturing ranged from 50-97% and 86-100% respectively. All products were stable post-processing, at room temperature for up to 6 hours, with a mean cell viability of 95.1%. Conclusion A donor lymphocyte product sufficiently purified of TCRαβ T and CD19+ B cells with subsequent enrichment of NK cells and TCR-γδ T cells can consistently be generated for prophylactic adoptive transfer in the post-transplant setting. Additional analysis characterizing NK and TCR-γδ T cell subsets for function and activation is underway.