Bispecific T-cell engagers (BiTE® molecules) have transformed the treatment of B-cell malignancies, yet clinical activity in AML has been modest. Resistance is driven in part by the genetic heterogeneity of AML, most notably TP53 mutations, present in 10-15% of de novo and up to 25% of therapy-related AML. Thus, we hypothesized that TP53 aberrations in AML contribute to cell-intrinsic and extrinsic resistance against T-cell-based immunotherapy. Cytotoxicity against TP53-deleted (DEL) primary AML cells and TP53-knockdown (KD) AML cell lines was reduced in co-cultures with T cells stimulated with the BiTE molecule AMG 330 (CD3×CD33). In addition, T-cell proliferation and proinflammatory cytokine secretion was impaired in co-cultures with TP53 KD cells. Transwell assays identified the secretome of TP53 KD AML cells as a key contributor to the immunosuppressive effects. Proteomic analysis revealed TGF-β1 in TP53 KD co-cultures as a mediator of T-cell suppression. RNA sequencing of T cells co-cultured with TP53 KD cells uncovered a transcriptional shift toward a senescent cell cycle profile. Our data collectively identify the immunosuppressive secretome of TP53-deficient AML as a key barrier to T-cell-engaging immunotherapies, underscoring an unmet clinical need for strategies able to restore T-cell function in TP53 KD AML.

T cell engagers (TCEs) have revolutionized the treatment of hematologic malignancies, with eight approved bispecific constructs and numerous bi- and trispecific TCEs currently in clinical trials. However, despite their clinical success, lack of response and high relapse rates remain major challenges. T cell exhaustion has emerged as an important mechanism of resistance to TCE therapy. Characterizing the T cell pool in patients prior to and during TCE therapy is warranted (1) to identify patients who will benefit most from TCE therapy and (2) to develop strategies to circumvent exhaustion during treatment to improve patient outcomes. We here review the current evidence of pre-existing and acquired T cell exhaustion during TCE therapy in patients with hematologic malignancies. We furthermore review state-of-the-art (pre)clinical strategies aimed at ameliorating exhaustion and reinvigorating T cell function.

This multicenter real-world study identifies critical determinants of outcome for tisagenlecleucel (tisa-cel) in treating post-HSCT relapse in 220 children/young adults with B-ALL from 31 European centers. Median follow-up was 30.0 months, with a 43.6% 2-year event-free survival (EFS), 67.2% overall-survival (OS), and 57.1% incidence of CAR-T failure. CAR-T for relapse after transplant from a matched sibling donor (MSD) compared to alternative donors was associated with lower 2-year-OS (MSD 59.1%, mismatched donor MMD 80.2%, matched family/unrelated donor MFD/MUD 68.3%, p = 0.046). Two-year incidence of CAR-T failure was highest for MSD (MSD 73.8%, MFD/MUD 49.7%, MMD 52.2%, p = 0.006). Patients who had relapsed early (< 6 months post HSCT) showed inferior 2-year-EFS (23.7%) and OS (47.2%) compared to patients with late relapse, ≥ 6 months after HSCT (EFS 49.8%, p = 0.001; OS 73.9%, p < 0.001). Early relapse was associated with a higher incidence of CAR-T failure and relapse after tisa-cel, particularly CD19+ relapses. Outcomes correlated with disease burden at lymphodepletion: 2-year-OS was 81.7% for MRD-, 69.2% for MRD+, and 55.2% for patients in non-remission (p = 0.003), with incidence of CAR-T failure highest in non-remission. Prior transplant from an MSD, early post-HSCT relapse, and disease burden at lymphodepletion identify patients at increased risk of CAR-T failure after HSCT.

Glofitamab, a bispecific antibody targeting CD20 and CD3, is approved for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) after at least two prior treatment lines, but real-world data is scarce. In this retrospective, multicenter, multinational study, we evaluated the outcomes of 70 patients with r/r DLBCL treated with glofitamab as part of the compassionate use patient program in the DACH region (Germany, Austria, Switzerland). The median number of prior treatment lines was four, with 71% of patients having received prior CAR-T therapy, and 71% being refractory to their last treatment. Cytokine release syndrome (CRS) was observed in 40% of patients (grade 3-4 in 2%), immune effector cell-associated neurotoxicity syndrome (ICANS) in 10% (grade 3 in 1%), and infections in 31% (grade 5 in 3%). The overall response rate was 47%, with 27% achieving complete responses (CR) and 20% partial responses (PR). The median progression-free survival (PFS) was 3.6 months, while the median overall survival (OS) was 5.7 months. Notably, 13 patients (19%) were in CR 6 months after initiation of glofitamab and exhibited durable responses. Elevated LDH is the most robust predictor of inferior outcome. Patients pretreated with bendamustine within 6 months prior glofitamab initiation exhibited significantly reduced PFS, suggesting that bendamustine may impair T-cell fitness and hence glofitamab efficacy. In summary, glofitamab demonstrates promising efficacy and a manageable safety profile in heavily pretreated r/r DLBCL patients in the real-world scenario and the optimal sequence of treatments should use T-cell-depleting agents before glofitamab with caution.

T-cell recruiting bispecific antibodies (BsAbs) are in clinical development for relapsed/refractory acute myeloid leukemia (AML). Despite promising results, early clinical trials have failed to demonstrate durable responses. We investigated whether activation of the innate immune system through stimulator of interferon genes (STING) can enhance target-cell killing by a BsAb targeting CD33 (CD33 BiTE® molecule, AMG 330). Indeed, we show that cytotoxicity against AML mediated by AMG 330 can be greatly enhanced when combined with the STING agonist 2',3'-cyclic GMP-AMP (cGAMP), or diABZI. We used in vitro cytotoxicity assays, immunoblotting, transcriptomic analyses, and extensive CRISPR-Cas9 knockout experiments to investigate the enhancing effect of a STING agonist on the cytotoxicity of AMG 330 against AML. Importantly, we validated our findings with primary AML cells, and in a xenograft AML model. Mechanistically, in addition to direct cytotoxic effects of STING activation on AML cells, activated T cells render AML cells more susceptible to STING activation through their effector cytokines interferon-gamma (IFNγ) and tumor necrosis factor (TNF), resulting in enhanced type I interferon production and induction of interferon-stimulated genes. This feeds back to the T cells, leading to a further increase in effector cytokines and an overall cytotoxic T-cell phenotype, contributing to the beneficial effect of cGAMP/diABZI in enhancing AMG 330-mediated lysis. We established a key role for IFNγ in AMG 330-mediated cytotoxicity against AML cells, and in rendering AML cells responsive to STING agonism. Here, we propose to improve the efficacy of CD33-targeting BsAbs by combining them with a STING agonist.

Immune deficits after CD19 chimeric antigen receptor (CAR) T-cell therapy can be long-lasting, predisposing patients to infections and non-relapse mortality. In B-cell non-Hodgkin lymphoma (B-NHL), the prognostic impact of immune reconstitution (IR) remains ill-defined, and detailed cross-product comparisons have not been performed to date. In this retrospective observational study, we longitudinally characterized lymphocyte subsets and immunoglobulin levels in 105 B-NHL patients to assess patterns of immune recovery arising after CD19 CAR-T. Three key IR criteria were defined as CD4+ T helper (TH) cells > 200/µL, any detectable B cells, and serum immunoglobulin G (IgG) levels >4 g/L. After a median follow-up of 24.6 months, 38% of patients displayed TH cells, 11% showed any B cells, and 41% had IgG recovery. Notable product-specific differences emerged, including deeper TH cell aplasia with CD28z- versus longer B-cell aplasia with 41BBz-based products. Patients with any IR recovery experienced extended progression-free survival (PFS) (median 20.8 vs. 1.7 months, p < 0.0001) and overall survival (OS) (34.9 vs. 4.0 months, p < 0.0001). While landmark analysis at 90 days confirmed improved PFS in patients with any recovery (34.9 vs. 8.6 months, p = 0.005), no significant OS difference was noted. Notably, 72% of patients with refractory disease never displayed recovery of any IR criteria. Early progressors showed diminished IR at the time of progression/relapse compared to patients with late progression/recurrence (after Day 90). Our results highlight the profound immune deficits observed after CD19 CAR-T and shed light on the intersection of IR and efficacy in B-NHL. Importantly, IR was impaired considerably postprogression, carrying significant implications for subsequent T-cell-engaging therapies and treatment sequencing.

Loss of anticancer NK cell function in AML patients is associated with fatal disease progression and remains poorly understood. Here, we demonstrate that AML-blasts isolated from patients rapidly inhibit NK cell function and escape NK cell-mediated killing. Transcriptome analysis of NK cells exposed to AML-blasts revealed increased CREM expression and transcriptional activity, indicating enhanced cAMP signalling, confirmed by uniform production of the cAMP-inducing prostanoid PGE2 by all AML-blast isolates from patients. Phosphoproteome analysis disclosed that PGE2 induced a blockade of LCK-ERK signalling that is crucial for NK cell activation, indicating a two-layered escape of AML-blasts with low expression of NK cell-activating ligands and inhibition of NK cell signalling. To evaluate the therapeutic potential to target PGE2 inhibition, we combined Fcg-receptor-mediated activation with the prevention of inhibitory PGE2-signalling. This rescued NK cell function and restored the killing of AML-blasts. Thus, we identify the PGE2-LCK signalling axis as the key barrier for NK cell activation in two-layered immune escape of AML-blasts that can be targeted for immune therapy to reconstitute anti-cancer NK cell immunity in AML patients.

The approval of venetoclax (VEN) in combination with hypomethylating agents (HMA) like Azacitidine (AZA) for newly diagnosed acute myeloid leukemia (AML) has significantly improved the outcomes of patients not fit for intensive chemotherapy. However, a considerable proportion of patients are primary refractory or relapse early with HMA/VEN, highlighting the need for novel combinatorial treatment options.Preclinical models have shown that blocking of the PD-1/PD-L1 pathway enhances antileukemic responses. However, in AML, single-agent efficacy of PD-1 inhibitors is limited. In preclinical findings, AZA upregulates PD-1 expression and the IFNγ signaling pathway. In relapsed/refractory (R/R) AML, AZA + Nivolumab demonstrated higher response rates and improved overall survival compared to historical outcomes (Daver et al 2019). We found that blocking the inhibitory immune checkpoint LAG-3 significantly increases anti-leukemic T-cell responses, in particular in combination with PD-1/PD-L1 blocking (Lichtenegger FS et al 2018). Here, we report interim results of a phase I/II trial designed to assess the safety and efficacy of AZA, nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) in R/R AML, and of AZA+VEN in combination with nivolumab and relatlimab in non-fit patients with newly diagnosed wild-type IDH/NPM1 AML. In the AARON trial (NCT04913922), AZA±VEN (applied per standard of care) is combined with nivolumab (480mg) and relatlimab (160mg) q 4 weeks. To date, 12 r/r AML patients (pts) have been treated. We observed no CR/PR (response rate 0%), but acceptable tolerability. Immune adverse events occurred in 25% of pts, but were well manageable, also post-SCT. The 11 non-fit frontline pts (median age 78 years, range 62-86) treated to date had a very high risk profile (adverse risk profile per ELN 2022 criteria in 100%). These pts received a median of 3 cycles (range 1-10), and in 5/11 pts (45%) treatment is ongoing. Best response was CR/CRi in 7/10 evaluable pts (response rate 70%), MLFS in 1/10 (10%) and blast response in 1/10 (10%). Only one pt had primary refractory disease. Immune-related adverse events were seen in 3/11 pts (27%, transaminitis, myocarditis and rash, all G3) and rapidly reversible with steroid treatment. 30-day mortality was 9% (septic shock unrelated to study treatment in 1 pt). In 3/11 pts, nivolumab/relatlimab treatment was discontinued, whereas AZA+VEN was continued. In conclusion, AZA/VEN + LAG-3/PD-1 inhibition was safe in adverse risk frontline AML patients not fit for intensive chemotherapy and showed promising efficacy. We continue to enroll non-fit frontline patients. The ongoing comprehensive immunomonitoring program aims to elucidate mechanisms of treatment response. Veit Buecklein, Giulia Magno, Christian Rausch, Maximilian Warm, Alica-Joana Emhardt, Adrian Gottschlich, Karsten Spiekermann, Christian Schmidt, Sabine Witt, Michael Unterhalt, Naval Daver, Marion Subklewe. AARON: An ongoing open-label phase I/II study of relatlimab (anti-LAG-3) with nivolumab (anti-PD-1) in combination with azacitidine ± venetoclax for the treatment of patients with relapsed/refractory and non-fit patients with newly diagnosed acute myeloid leukemia - Interim analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT225.

BACKGROUND:Despite revolutionary efficacy of CD19-CAR-T cell therapy (CAR-T) in aggressive B cell lymphoma, many patients still relapse mostly early. In early failure, distinct drugs support CAR-T which makes reliable and early prediction of imminent relapse/refractoriness critical. A complete metabolic remission (CR) on Fluor-18-Deoxyglucose (FDG) Positron-Emission-Computed Tomography (PET) 30 days after CAR-T (PET30) strongly predicts progression-free survival (PFS), but still fails in a relevant proportion of patients. We aimed to identify additional routine parameters in PET evaluation to enhance CAR-T response prediction. RESULTS:Thirty patients with aggressive B cell lymphoma treated with CAR-T were retrospectively analyzed. Pre-CAR-T, LDH was the strongest PFS-predictor also by multivariate analysis. Post-CAR-T, 10 out of 14 patients (71.4%) with PET30-CR remained in disease remission, while 12 out of 16 patients (75%) with incomplete metabolic remission (PET30-nCR) relapsed after CAR-T. 28.6% of patients with PET30-CR ultimately progressed. Change of liver FDG-uptake from baseline to day30 (Delta-Liver-SUVmean) was identified as an independent biomarker for response. PET30-nCR and a decrease of Delta-Liver-SUVmean were associated with a high risk of tumor progression (HR 4.79 and 3.99, respectively). The combination of PET30 and Delta-Liver-SUVmean identified patients at very low, at intermediate and at very high risk of relapse (PFS not reached, 7.5 months, 1.5 months, respectively). CONCLUSION:Additionally to PET30 metabolic remission, longitudinal metabolic changes in Delta-Liver-SUVmean predicted CAR-T efficiency. Our results may guide early intervention studies aiming to enhance CAR-T particularly in the very high-risk patients.

Mit Rituximab hat die Immuntherapie bereits vor Jahrzehnten Einzug in die Routinebehandlung von B-Zell-Lymphomen gehalten. Die Zulassung neuer bispezifischer T-Zell-rekrutierender Antikörper und der adoptiven Immuntherapie mit CAR-T-Zellen bringt tiefgreifende Änderungen der Therapiealgorithmen bei aggressiven und indolenten Lymphomen mit sich. Im Folgenden werden die wichtigsten aktuell zugelassenen T-Zell-rekrutierenden Behandlungsstrategien vorgestellt.

Management of relapsed/refractory Large B Cell Lymphoma (r/r LCBL) after CD19 Chimeric Antigen Receptor (CAR) T cells represents a major clinical challenge. Glofitamab and Epcoritamab, two CD3xCD20 bispecific T cell-recruiting antibodies, have recently been approved for third-line treatment. However, real-world experience with these bispecifics is limited, and predictive biomarkers for efficacy are lacking. Given that both bispecifics and CAR-T cells mediate their antineoplastic activity by redirecting the patient's T cells, we hypothesized that biomarkers associated with outcomes from CD19 CAR-T therapy would similarly predict outcomes post-CD20-directed bispecifics. To test this, we applied two established models from CAR-T cell therapy-CAR-HEMATOTOX (Rejeski et al, Blood 2021) and InflaMix (Raj et al, ASH Annual Meeting 2023) to our cohort of 63 patients treated with bispecifics. In this international, multicenter observational study, we analyzed outcomes of r/r LBCL patients treated with Glofitamab (n=55) or Epcoritamab (n=8) monotherapy. Baseline features were assessed on the first day of the first treatment cycle. Parameters were derived from standard laboratory analysis. For a subset of patients, cytokines were measured using the Legendplex flow-cytometry based multiplex immunoassay. Kaplan-Meier estimates of progression-free (PFS) and overall survival (OS) were compared using the log-rank test. Univariate and multivariate analyses of PFS were conducted using the Cox proportional hazards model. The CAR-HEMATOTOX model integrates five variables (ANC, hemoglobin, platelet count, C-reactive protein, and ferritin) determined at the time of lymphodepletion. The InflaMix model, based on up to 14 laboratory values assessed prior to CAR-T cell infusion, stratifies patients into inflammatory and non-inflammatory clusters. Both models discriminate between cohorts with significantly different clinical outcome. Median age was 67 years (range 35-86). Patients had received a median of 3 prior therapy lines (range 1-8), 46 patients (73%) underwent prior CAR-T cell therapy and 21 patients (33%) were refractory to their latest line of therapy. The objective response rate was 54%, with 16 complete remissions (25%) and 18 partial remissions (29%). With a median follow up of 4.1 months, median PFS was 3.5 months and median OS was 13.5 months. Cytokine release syndrome (CRS) occurred in 38.6% of patients (22/57), high grade CRS (grade ≥3) was reported in 3 cases. ICANS was noted in 5.3% of patients (3/57), with one patient experiencing high grade ICANS. In a univariate Cox regression analysis, the examined clinical factors (including sex, age, ECOG performance status, histology, Ann Arbor stage, presence of extranodal disease, number of prior lines of therapy and prior CAR-T exposure) were not significantly associated with PFS. Among the analyzed laboratory values, however, higher LDH (p<0.001), CRP (p=0.002), and ferritin levels (p=0.006) as well as low hemoglobin (p=0.021) were significantly associated with inferior PFS, highlighting inflammation as a key factor associated with response. Based on data availability, CAR-HEMATOTOX and InflaMix were applied to 45 and 55 patients, respectively. Patients presenting with a CAR-HEMATOTOX score ≥2 (24/45) showed inferior PFS (1.9 months vs not reached, p=0.005) and OS (3.9 vs 13.7 months, p=0.027). When applying InflaMix, 38.2% of the patients were assigned to the inflammatory cluster, showing a significantly shorter median PFS (1.7 vs 6.9 months, p<0.001) and OS (2.7 months vs not reached, p<0.001). In a multivariable Cox regression analysis accounting for LDH, age, prior CAR-T exposure and bispecific product, higher LDH and assignment to the inflammatory cluster by InflaMix remained independent risk factors for inferior PFS. Notably, InflaMix had the most significant effect on PFS (p=0.005). In summary, our multi-center international real-world data analysis demonstrates that the CAR-HEMATOTOX and the InflaMix models effectively identify LBCL patients with high risk of treatment failure, highlighting the importance of immune dysregulation in failure to bispecifics. Further validation of both models is ongoing. These scores may guide patient stratification into bispecifics monotherapy, combinatorial approaches and inclusion in clinical trials to further advance treatment approaches in LBCL.

ABSTRACT:Cytopenias represent the most common side effect of CAR T-cell therapy (CAR-T) and can predispose for severe infectious complications. Current grading systems, such as the Common Terminology Criteria for Adverse Events (CTCAE), neither reflect the unique quality of post-CAR-T neutrophil recovery, nor do they reflect the inherent risk of infections due to protracted neutropenia. For this reason, a novel EHA/EBMT consensus grading was recently developed for Immune Effector Cell-Associated HematoToxicity (ICAHT). In this multicenter, observational study, we applied the grading system to a large real-world cohort of 549 patients treated with BCMA- or CD19-directed CAR-T for refractory B-cell malignancies (112 multiple myeloma [MM], 334 large B-cell lymphoma [LBCL], 103 mantle cell lymphoma [MCL]) and examined the clinical sequelae of severe (≥3°) ICAHT. The ICAHT grading was strongly associated with the cumulative duration of severe neutropenia (r = 0.92, P < .0001), the presence of multilineage cytopenias, and the use of platelet and red blood cell transfusions. We noted an increased rate of severe ICAHT in patients with MCL vs those with LBCL and MM (28% vs 23% vs 15%). Severe ICAHT was associated with a higher rate of severe infections (49% vs 13%, P < .0001), increased nonrelapse mortality (14% vs 4%, P < .0001), and inferior survival outcomes (1-year progression-free survival: 35% vs 51%, 1-year overall survival: 52% vs 73%, both P < .0001). Importantly, the ICAHT grading demonstrated superior capacity to predict severe infections compared with the CTCAE grading (c-index 0.73 vs 0.55, P < .0001 vs nonsignificant). Taken together, these data highlight the clinical relevance of the novel grading system and support the reporting of ICAHT severity in clinical trials evaluating CAR-T therapies.

Introduction: Emerging evidence suggests that immune dysregulation drives resistance to cellular therapies. In particular, low CAR T-cell expansion and inferior patient outcomes have been associated with an inflamed immune milieu. Here, we investigated whether myeloid cells and serum proteomics are linked to T-cell dysfunction and treatment failure. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel, brexu-cel, or tisa-cel in the third- or later-line setting were retrospectively included (n=74). Aliquots of EDTA-anticoagulated peripheral blood and serum were collected. Immune checkpoint (IC) expression (PD-1, TIM-3, and LAG-3) was assessed through flow cytometry before CAR T-cell infusion (time of leukapheresis, day -5 and day 0) as a surrogate for T-cell exhaustion. More specifically, the abundance of different co-expression profiles of ICs was compared and defined as non-exhausted (PD-1-TIM-3-LAG-3-), progenitor-exhausted (PD-1+TIM-3+LAG-3-), and terminally-exhausted (PD-1+TIM-3+LAG-3+). Using the Olink® Immuno-Oncology panel, 92 immune-related proteins were measured at day 0. A next-generation single-cell proteogenomics approach (BD RhapsodyTM) was applied to analyze the transcriptome of myeloid cells before and after infusion (day -5 and day 7). Responding patients (R, complete or partial remission) were compared to non-responding patients (NR, stable or progressive disease) according to 3-month (PET-) CT scans. Results: At baseline, NR showed lower frequencies of non-exhausted (apheresis, p = 0.025; day -5, p = 0.0236; day 0, p = 0.0496) and higher frequencies of terminally-exhausted T-cells (-5, p = 0.0053) compared to R. Interestingly, these surrogates for pre-existing T-cell dysfunction in NR were linked to an inflammatory state. Patients with low levels of non-exhausted T-cells also had higher levels of CRP (p = 0.0059), Ferritin (p = 0.0333), and LDH (p = 0.0268) compared to patients with high levels of non-exhausted T-cells before infusion. Network analysis correlating the abundance of immune-modulatory proteins with baseline IC expression on T cells revealed numerous connections for NR but merely associations for R, suggesting an impact of the serum protein milieu on IC expression in NR. We dissected these networks in terms of the degree centrality differences for the different proteins (i.e., the variations in the number of connections among the nodes between NR and R). While we identified a higher degree centrality for co-stimulatory proteins such as IL-18, CD8A and ICOSLG in R, we noted higher degree centrality for immune-inhibitory and tumor-related proteins such as CXC3L1, TNFRSF21, VEGFA and IL-6 in NR. Accordingly, we linked a higher abundance of CD8A and a lower abundance of TNFRSF21 and IL-6 to the frequency of non-exhausted T-cells at baseline using a linear regression model. Finally, we asked whether the myeloid compartment at baseline drives these differences in the serum protein milieu observed between NR and R. We identified four phenotypic and transcriptomic different monocytic and dendritic cell populations. Interestingly, two classical monocyte-like populations (CD14+CD16+HLA-DRlo) showed divergent population dynamics in NR but not R when assessing pre- and post-infusion time points. Notably, gene set enrichment analysis revealed an upregulation of immunosuppressive pathways, such as genes regulated by NF-KB in response to TNF, in several myeloid populations in NR but not R (classical monocyte-like 2, p < 0.0001; non-classical monocyte-like, p < 0.0001; DC-like, p = 0.0024). Surprisingly, monocytic populations in R revealed an upregulation of genes with immune stimulatory functions linked to responses to interferon-alpha or -gamma proteins (classical monocyte-like 1 and 2, p = 0.0058 and p = 0.0003; non-classical monocyte-like, p = 0.0003). Conclusion: These data suggest that CAR-T non-responders exhibit pre-existing T-cell dysfunction resulting from a systemic inflamed proteomic and cellular environment. Notably, the profound alterations within the myeloid compartment highlight diverging immune-modulating functions in non-responding and responding B-NHL patients. These findings set the further scope for scientific investigations not only to mitigate immune dysregulation but also to target the innate immune compartment for enhanced CAR T-cell responses.

Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematological malignancy derived from plasmacytoid dendritic cells. The approval of Tagraxofusp (TAG) in 2018 has shifted treatment paradigms, offering a targeted option for induction therapy in BPDCN, especially for patients (pts) ineligible for an intensive chemotherapy. While TAG has shown promising results with a 75% overall response rate (ORR), the role of consolidation treatments in the TAG era remains to be determined. Historically, long-term disease control has been achieved through allogeneic stem cell transplantation (allo-SCT), but its optimal timing and patient selection in the context of TAG therapy remain unclear. There is a need for real-world data to better understand efficacy and outcomes of these treatment approaches in clinical routine. Methods We conducted a multicenter retrospective analysis including 32 pts treated with TAG as first-line treatment for BPDCN in 12 German/Austrian/Swiss tertiary centers between November 2019 and March 2024. Outcome was calculated from the day of first application of TAG until the respective event. Using a historical cohort of 40 BPDCN pts not treated with TAG, propensity score matching (nearest neighbor 1:1 matching with a caliper of 0.2 standard deviations, for age at diagnosis, number of therapy lines, use of auto-SCT as well as exact matching for allo-SCT as co-variates) was performed to compare outcomes and the role of consolidative allo-SCT between both groups. Results We identified 32 pts (84.4% male) with a median age of 71.5 years (range: 24-87) at diagnosis. ECOG performance score <2 was noted in 90.0% of cases. Disease manifestations included skin (93.8%), peripheral blood (84.4%), lymphadenopathy (56.3%), bone marrow (56.7%), hepatosplenomegaly (50.0%) and CNS (16.1%). The median time-to-TAG after BPDCN diagnosis was 20.5 days (range: 3-262), median number of TAG cycles was 2.5, and 74.2% of pts completed all planned administrations of the first cycle. Notable adverse events included tumor lysis syndrome (CTCAE≥3) in 9 pts (28.1%), and capillary leak syndrome in 8 pts (25.0%). During TAG treatment, 73.3% pts required albumin substitution and 75.9% steroid application. 15 pts (46.9%) underwent allo-SCT for consolidation, no auto-SCT was performed. Among allo-SCT recipients, 6/15 received myeloablative conditioning, 10/15 had TBI-based protocols, and 12/15 were transplanted from matched donors. ORR after TAG was 82.7% with a median response duration of 5.0 months (range 0.23-50.5). With a median follow-up of 11.1 months (range 2.1-50.5), the 1-year progression-free survival (PFS) and overall survival (OS) rates were 40.8% and 67.0%, respectively. The cumulative 1-year relapse incidence was 50.0%, while non-relapse mortality (NRM) was 9.5%. Multivariate regression analysis, including age and TAG response as covariates, revealed that consolidative allo-SCT was significantly associated with improved PFS rates with median PFS of 3.0 months (95% CI: 2.1-6.1) vs. not reached (95% CI: 22.7-NA, HR=0.06, 95% CI: 0.01-0.46, p=0.006). Propensity score matching identified 20 pair-matched pts from the historical non-TAG cohort, of which 8 with allo-SCT. While 1-year OS rates were comparable between the matched TAG (58.3%) and matched non-TAG cohort (58.7%, p=0.96), allo-SCT consolidation was associated with improved OS (HR=0.16, 95% CI: 0.05-0.50, p=0.002) irrespective of induction treatment. Conclusions Our real-world data highlight the efficacy of Tagraxofusp in BPDCN, with a response rate of >80%. Consolidation with allo-SCT significantly improved survival outcomes. Yet, survival was similar with prior TAG and conventional induction when consolidative allo-SCT was performed. These findings underscore the role of TAG as a bridge to allo-SCT and the importance of allo-SCT in maintaining durable remissions in the TAG era. Future studies are essential to optimize treatment strategies, identify prognostic factors, and improve long-term outcomes for BPDCN pts.

In acute myeloid leukemia (AML), the anti-leukemic potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and post-transplant donor lymphocyte infusion (DLI) hinges on the activity of T cells [1].Bispecific antibodies, including bispecific T-cellengager (BiTE ® ) molecules, redirect endogenous T cells against neoplastic cells for eradication by CD3-dependent T-cell activation.In B-lymphoid malignancies, high clinical efficacy has led to the approval of several T-cell-recruiting constructs [2, 3].In AML, several bispecific antibodies have been developed and have shown strong preclinical efficacy [4, 5].However, albeit early-phase I/II clinical trials in heavily pre-treated patients with R/R AML have yielded promising, dose-dependent results, sustained responses were not observed [6-8].We hypothesize that T-cell dysfunction contributes to BiTE resistance and a lack of long-term responses in AML.Evidence for the relevance of T-cell fitness to BiTE-mediated activity is derived from patients with B-cell precursor acute lymphoblastic leukemia in whom a predominance of T cells with an exhausted phenotype was associated with failure of blinatumomab treatment [9].Additionally, transcriptional profiles associated with T-cell dysfunction were found in nonresponding patients [10].Further evidence of an association between T-cell fitness and BiTE activity was found in a preclinical model of T-cell exhaustion after continuous BiTE exposure [11].So far, attempts to characterize T-cell phenotype and function in AML patients have yielded variable and sometimes contradictory results.Studies suggest that BM T cells in contrast to peripheral blood T cells better reflect the immune state and are the main mediators of BiTE-mediated cytotoxicity [12, 13].Hence, characterizing BM T cells at different time points during the course of the disease might help to guide the optimal clinical application of T-cell-based immunotherapies in AML.

Background: Substantial unmet medical need remains for patients with acute myeloid leukemia (AML) who often have minimal residual disease (MRD) at the end of induction therapy that drives disease recurrence. Emerging approaches to improve outcomes include application of novel targeted and immunotherapies to erradicate MRD in AML patients at high risk of relapse. CLN-049 is a humanized bispecific antibody, with dual binding specificities for FLT3 and CD3, on a human immunoglobulin G1 backbone with a silenced Fragment crystallizable domain. CLN-049 effectively redirects CD3+ T cells to kill FLT3-expressing AML cells within the blood and bone marrow to exert anti-leukemic effect. Study Design and Methods: This is a phase 1, open-label, multicenter, dose escalation and dose expansion study to evaluate the safety and tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of subcutaneously (SC) and intravenously (IV) administered CLN-049 in adult patients (≥ 18 years of age) with a diagnosis of AML in CR with positive MRD. Other key inclusion criteria include: patients who have exhausted or are ineligible to receive available treatment alternatives or are expected to receive alternative therapy (e.g. allo-HSCT) at a later date, and ECOG Performance Status 0-1. Key exclusion criteria include prior treatment with chimeric antigen receptor (CAR-T) cell therapy or other modified T cell therapy, FLT3-directed bispecific molecule, FLT3-targeted antibody, or allo-HSCT within 60 days of treatment. The study consists of an initial dose escalation phase commencing with accelerated titration in single-patient cohorts followed by a standard 3+3 dose escalation design exploring both SC and IV routes of administration to identify the maximum tolerated dose (MTD), or maximum administered dose if no MTD is defined. A subsequent dose expansion phase will further characterize the safety and preliminary efficacy of CLN-049 in this patient population at the dose, schedule, and preferred route of administration determined in dose escalation phase to allow determination of a RP2D. CLN-049 will be administered every 7 days in 21-day cycles until morphologic relapse, unacceptable toxicity, proceeding to alternative treatment (eg, allo-HSCT), or a maximum of 12 weeks of treatment. The initial administration of CLN-049, including potential step-up doses and the first target dose, will be administered on an inpatient basis. Subsequent doses may be given in the outpatient setting. Safety, PK, PD, and preliminary efficacy assessments will guide the dose selection and schedule for further evaluation. The primary endpoint is safety. Key secondary endpoints include the proportion of patients achieving MRD negativity, time to MRD negativity, duration of MRD negativity, time to disease progression (MRD or morphologic relapse), time to subsequent anti-leukemia therapy, progression-free survival, overall survival, PK, and anti-drug antibodies. Approximately 70 patients (50 in dose escalation and 20 in dose expansion) will be enrolled at 12 sites in Germany and Spain, including both community and academic centers. The study is currently enrolling in Germany and Spain (EU CT: 2023-506572-27-00).