BACKGROUND:Acute graft-versus-host disease (aGvHD) remains a major complication after allogeneic hematopoietic cell transplantation (alloHCT). Adoptive regulatory T-cell (Treg) therapy may suppress alloreactive T-cell responses, but clinical implementation has been limited by donor-specific manufacturing, prolonged ex vivo expansion, and logistical complexity. OBJECTIVE:We developed ATreg, a cell therapy product consisting of gp120-activated, polyclonal Tregs derived from HLA-unmatched third-party donors. The primary objective was to assess the safety, tolerability and toxicity of ATreg, hypothesizing that this would be feasable and safe for aGvHD prevention early after alloHCT in patients with hematologic malignancies. STUDY DESIGN:ATreg-001 is a first-in-human, prospective, open-label, single-arm, multi-center phase 1/2 trial (EU CT number 2024-516599-14-00) conducted at four German centers (Mainz, Dresden, Münster, Dortmund). ATreg was generated from standard non-mobilized apheresis products by Treg isolation followed by 16 hours of gp120-mediated activation in the presence of IL-2, without ex vivo expansion, thereby enhancing suppressive function and (potentially) enabling a therapeutic effect at substantially lower Treg doses. Ten patients received ATreg at 0.1-1.0 × 10⁶ cells/kg body weight on day +10 ± 5 after alloHCT, in addition to standard GvHD prophylaxis, in a dose-escalation design across three cohorts. ATreg was administered within 24 hours after manufacturing. The primary endpoint was the type, incidence, and severity of ATreg-related serious adverse events within 14 days after administration. Secondary endpoints included manufacturing feasibility, aGvHD incidence/severity within 100 days, engraftment, and infections. RESULTS:ATreg administration was well tolerated, with no infusion-related toxicities or other safety signals attributable to ATreg. All treated patients achieved hematopoietic engraftment and full donor chimerism. Within 100 days after alloHCT, no grade 3-4 aGvHD occurred, the cumulative incidence of grade 2-4 aGvHD was 10%, and no non-relapse mortality was observed. CONCLUSION:These first clinical data support the feasibility and favorable safety profile of ATreg, a third-party, gp120-activated Treg product requiring no ex vivo expansion, and warrant further evaluation in larger prospective clinical trials. MAIN POINTS:

Introduction: In patients with a clinical indication for autologous hematopoietic stem cell transplantation (ASCT), sufficient mobilization of CD34+ precursor cells into peripheral blood is essential to ensure adequate hematopoietic stem cell (HSC) collection prior to intensive therapy. However, with standard granulocyte-colony stimulating factor (G-CSF)-based mobilization schemes, an important minority of patients fail to mobilize sufficient (e.g., >10/µL) CD34+ cell counts into the peripheral blood and are considered as poor mobilizers (PM). Because failure to achieve sufficient CD34+ cell mobilization can negatively affect important clinical treatment endpoints, the use of plerixafor (PLX) was approved to increase CD34+ mobilization in PM patients. Methods: The German non-interventional, multicenter, open-label, prospective OPTIMOB study evaluated HSC mobilization strategies prior to planned ASCT in adult patients with hematologic malignancies (lymphomas or multiple myeloma [MM]) focusing on PM patients. PM patients were defined as follows: (1) never achieving ≥20 CD34+ cells/µL before 1st apheresis, (2) receiving PLX at any timepoint of mobilization, (3) their initially planned stem cell yield had to be reduced, or (4) they had not received apheresis due to low CD34+ count in peripheral blood. Results: 168 of 475 MM patients (35%) participating in the OPTIMOB study were classified as PM, and 155 of them (92%) received PLX (PM+PLX) during the study. PM patients were 40–78 years old, slightly more often male (n = 97, 58%), mostly newly diagnosed (n = 146, 87%) and received highly individualized previous treatments. Ninety-four of the PMs underwent chemotherapy mobilization (65%), and 51 patients (35%) received steady-state mobilization with G-CSF only during 1st mobilization attempt. 92% of the total PM population (n = 155) underwent apheresis, 78% of them (n = 117) achieved >2.0 × 106 CD34+ cells/kg body weight on the 1st day of apheresis. PM+PLX had a higher median total collection result than those PM patients without PLX support (7.2 vs. 5.7 × 106 CD34+ cells/kg body weight). In total, ASCT was performed in 136 PM+PLX (88%) versus 8 PM−PLX patients (62%). Conclusion: The OPTIMOB study showed that a considerable proportion of adult MM patients in Germany are PMs. Even though most of PMs were supported with PLX in the OPTIMOB study, PM-PLX also successfully mobilized HSCs, allowing ASCT in majority of all PMs. However, further analyses are required for treatment optimization in PMs.

Introduction: Successful mobilization and collection of peripheral hematopoietic stem cells (HSCs) are necessary for lymphoma patients eligible for myeloablative chemotherapy with subsequent autologous stem cell transplantation (ASCT). Albeit G-CSF alone or combined with chemotherapy is well-established methods for HSC mobilization, up to 40% of the patients fail to mobilize (poor mobilizer, PM). Plerixafor (PLX) is commonly used in PM patients resulting in increased migration of HSCs into peripheral blood and thus improves the collection outcome. Methods: The prospective, multicenter, open-label, non-interventional OPTIMOB study assessed mobilization and collection parameter of patients with lymphoma or multiple myeloma to get deep insights in the treatment of those patients in clinical routine focusing on PM patients. PM was defined as follows: (1) no achievement of ≥20 CD34+ progenitor cells/µL before first apheresis, (2) PLX administration at any time point during the observational period, (3) reduction of the initially planned CD34+ progenitor cell yield as necessity due to failed mobilization or HSC collection, and (4) no performance of apheresis due to low CD34+ progenitor level. Primary objective of the study was to assess mobilization success by the proportion of PM patients achieving >2 × 106 CD34+ progenitor cells/kg body weight on the first day of apheresis. Here, the data of the lymphoma cohort are presented. Results: Out of 238 patients with lymphoma documented in the study, 32% were classified as PM. 87% of them received PLX. Demographic data revealed no obvious differences between PM and good mobilizing (GM) patients. All patients were treated highly individualized prior to mobilization. Majority of all PM patients were able to undergo apheresis (95%) and reached their individual requested CD34+ progenitor cell target (72%). 57% of the PM patients achieved >2.0 × 106 CD34+ progenitor cells/kg body weight on day 1 of apheresis and nearby 70% of them underwent ASCT. Median time to engraftment was similar in PM and GM patients of the lymphoma cohort. Conclusions: Majority of PM patients with lymphoma were successfully mobilized and underwent ASCT. Most of them received PLX during the study.

Abstract Introduction: The combination of reduced-intensity conditioning (RIC) with in vivo T-cell depletion by alemtuzumab prior to hematopoietic stem cell transplantation (HSCT) has demonstrated efficient engraftment and reduced graft-versus-host disease (GVHD). However, this regimen is associated with slow lymphocyte recovery leading to a delayed anti-infectious and anti-malignant immunity. DLI can be used to improve immune reconstitution. Here we investigate on the impact of different DLI: prophylactic CD8-depleted DLI vs preemptive non-depleted DLI. Methods: 256 patients with different hematologic malignancies were planned for treatment with DLI after allogeneic HSCT following reduced intensity conditioning (Fludarabin, Melphalan, in vivo Alemtuzumab). All patients received PBSC. Donors were HLA-identical siblings or HLA-matched unrelated donors. The calcineurin-inhibitor used for GVHD-prophylaxis (Cyclospron A) was intended to be tapered until day 50. 134 patients should receive CD8deplDLI prophylactically after day +60 (Group A). 122 patients were planned for non-depleted DLI after day +100 in a preemptive setting. Trigger for preemptive DLI were mixed donor chimerism or MRD positivity. Both groups received DLI in escalating doses with an interval of 60 to 90 days. DLI application was stopped when GVHD occurred (Group A and B), or a full donor chimerism was achieved / MRD became negative (Group B). Both patient-groups did not differ in median age. The majority of patients either suffered from an acute leukemia / MDS (n=42%), lymphoma (n=28%), myeloma (n=17%), or myeloproliferative neoplasms (n=12%) and these diseases were equally distributed among the groups. All patients were treated at the university medical center in Mainz, Germany. Results: Of 134 patients (Group A) 41% received CD8deplDLI prophylactically (GVHD was the main cause for withholding DLI). In group B, 32% of 122 patients received preemptive non-depleted DLI. 2 Year-Overall survival (OS) significantly increased in all patients (both groups) after DLI (74% with DLI, 37% without DLI, p<0.0002). There was a trend for better OS in patients receiving preemptive DLI in Group B (72% vs 87%, p=0,235) compared to prophylactic CD8depl DLI (Group A). The relapse rate was reduced after DLI in both groups (24% after DLI, 37% without DLI p=0.05). Relapse occurred in the median 120 days later after DLI application. CD8depl prophylactic DLI did not induce less GVHD than CD3pos preemptive DLI (70,9% after CD8deplDLI vs 67,2% P=0.627) and GVHD was mainly limited (acute GVHD I-II°). Chronic GVHD occurred in 19.4% vs 23.1%. After 3 years OS (50% vs 54%), relapse rate (33% vs 26%) and non-relapse mortality (34% vs 37%) did not differ between group A and B. Analyzing lymphoma patients, OS did not differ in both groups (60% vs 58%), but group B with preemptive DLI showed a trend with lower relapse rates (31% vs 8%, p=0,298). Presence of any GVHD was protective in lymphoma-patients of both groups (p=0.007). Patients suffering from acute leukemia (AL) seem to benefit from prophylactic CD8 depleted DLI (group A): OS after 2 year was 56% in Group A vs 33% (p=0.261), progression free survival 54% vs 34%, NRM 27% vs 46% (p=0.532). The relapse rate did not differ between both groups (31% vs 31%). Discussion: In summary, the application of DLI (prophylactic CD8depl and preemptive CD3pos) after RIC in combination with in vivo Alemtuzumab is essential to improve OS. The differences in OS, NRM and disease control for AL and lymphoma patients concerning the different DLI modalities even in this retrospective analysis is remarkable. AL patients seems to benefit from CD8depl DLI in a prophylactic setting (OS, NRM, PFS), in lymphoma patients there seems to be an advantage of CD3pospreemptive DLI (OS, relapse rate). Due to small patient numbers our findings could not reach statistical significance. Our data strongly support a randomized trial, comparing prophylactic vs. preemptive / therapeutic DLI application for different disease groups in the context of T-cell depleted HSCT to assess the different potential to prevent form infections and relapse. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 4109 We have previously demonstrated that the application of CD8-depleted donor-lymphocyte infusions (DLI) is feasible after reduced-intensity conditioning and in vivo T-cell depletion by alemtuzumab. DLI overcome slow lymphocyte recovery associated with alemtuzumab-administration and improve anti-infectious immunity and reliably convert a decreasing T-cell chimerism (Meyer et al. Blood 2007 & BMT 2010). Here we provide clinical follow up data of 117 patients with different hematological diseases and a median observation time of 1 year (range, 1–86 months) post hematopoietic stem cell transplantation (HSCT). The majority of patients either suffered from an acute leukemia / MDS (n=54), lymphoma (n=32), myeloma (n=17), or myeloproliferative neoplasms (n=12). Two patients suffered from non-malignant diseases. The median age of the patients was 56 years (range, 19–71) and none of them qualified for a conventional conditioning regimen. 50 patients had undergone previous transplantations (autologous: n=47, allogeneic: n=3). Donors were matched siblings (n=20), matched unrelated donors (n=55), or unrelated donors with a single HLA mismatch (n=42). Between days 60 and 120 after HSCT, without calcineurin-inhibitors and in the absence of graft-versus-host disease (GVHD), 1×106 CD8-depleted DLI per kg bodyweight were administered. Up to three further DLI were given in escalating doses in 60 to 90 day intervals. Following this procedure, 45 patients received at least one dose of DLI. Among those patients who did not qualify for DLI, 50 patients had primary GVHD. In 22 patients DLI were not administered for other reasons (donor unavailable, infections, relapse). In 64% of DLI induced acute GVHD, which was the major reason for withholding the next DLI-dose step. The rate of acute GVHD > grade 2 was 30%. 10% suffered from extensive chronic GVHD. The 1 and 3 year overall survival was 63% and 43%, respectively. Survival significantly differed between the DLI and the non DLI group after 3 years (63% vs. 27%, p=0.002). Since this trial was not randomized, we also compared the DLI group to only those patients who did not receive DLI for other reasons than primary GVHD and found similar results (62% vs. 28%, p=0.01). As expected, the presence of GVHD at any time was associated with a reduced relapse rate in all patients (55.8% vs 30.8%, p=0.013). Although DLI was associated with a survival benefit, the relapse rate did not differ from that of the no-DLI cohort. AML/MDS patients represented the largest group of patients included in our study (n= 48). Among these, 41 patients achieved the time point for DLI administration, 16 of them received at least one dose of prophylactic DLI. 9 patients developed GVHD after DLI application. 20 patients had primary GVHD as major cause for not receiving DLI. The survival curves differed significantly between the DLI and non DLI group after 1 and 3 years (91.7% vs. 54%, and 82.5% vs 24% p=0.004). The estimated 5 year overall survival for all AML patients was 50.4%. There was no significant difference analyzing the relapse rate (20% vs 18.8%). In summary, the prophylactic application of CD8-depleted DLI in the absence of GVHD was associated with a survival benefit. However, we were not able to relate this benefit to a decreased relapse rate, and we assume a better control of infections. Our data strongly support a randomized trial, comparing prophylactic vs. preemptive / therapeutic DLI application in the context of T-cell depleted HSCT. Disclosures: Meyer: BMS: Membership on an entity's Board of Directors or advisory committees.

Reactivated varicella-zoster virus (VZV) infection causes herpes zoster and commonly occurs after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Because VZV-specific T cell immunity is essential to prevent virus reactivation, we developed an interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assay for the sensitive detection of VZV-reactive T cells at the single-cell level ex vivo. We used this assay to monitor the frequency of VZV-reactive T cells in 17 seropositive patients during the first year after T cell-depleted allo-HSCT. The patients did not receive anti-herpesvirus prophylaxis after stem cell engraftment. Independent of the magnitude of transferred donor immunity, VZV-reactive T cell numbers decreased to low levels (median, 2/mL; range, 0 to 35/mL) in peripheral blood early after transplantation. Only patients with subsequent zoster (n = 5) exhibited a dramatic boost in VZV-reactive T cells (median, 366/mL; range, 158 to 756/mL), which was induced by the reactivation event. The postzoster VZV-reactive T cell levels were similar to those seen in healthy virus carriers. In contrast, antiviral T cell levels remained low in patients without VZV disease. Our results demonstrate that VZV-specific T cell immunity recovered efficiently during zoster in T cell-depleted allo-HSCT recipients. It did not reconstitute spontaneously in nonzoster patients, even in the absence of antiviral prophylaxis. Prospective studies should investigate whether VZV vaccination can substitute for natural resensitization by virus disease.

Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C-mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.