von Willebrand disease (VWD) type 1 is a bleeding disorder characterized by a quantitative deficiency of functional von Willebrand factor (VWF). We designed a novel bispecific nanobody, named KB-V13A12, that aims to increase endogenous VWF levels by bridging it to albumin. KB-V13A12 comprises 2 single-domain antibodies, 1 targeting VWF and 1 targeting albumin. VWF bound efficiently to the albumin/KB-V13A12 complex (2.0 ± 0.4 nM) in immunosorbent assays, and binding was stable at pH 5.6 and 7.4. VWF ristocetin activity and factor VIII binding remained unaffected in the presence of a 100- to 200-fold molar excess of KB-V13A12/albumin. Humanized VWD type 1 mice were used for in vivo analysis. A single subcutaneous dose of KB-V13A12 (5 mg/kg) was associated with a nanobody half-life of 3.0 ± 0.7 days, and dose-dependently increased VWF in VWD type 1 mice 1.4- to 2.1-fold for up to 14 days. Factor VIII activity was also increased during this period. The VWF propeptide/VWF antigen ratio (a marker for VWF clearance) was significantly reduced in the presence of KB-V13A12, suggesting that delayed clearance contributes to increased VWF levels. Clearance experiments in wild-type mice using recombinant VWF preincubated with KB-V13A12 indeed confirmed a prolonged survival, while this prolongation was absent in FcRn-deficient mice. Finally, treatment with KB-V13A12 resulted in a significantly improved bleeding tendency in VWD type 1 mice when using the saphenous vein puncture model. In conclusion, KB-V13A12 is a bispecific nanobody that efficiently increases functional levels of endogenous VWF, and could be a therapeutic option to treat VWD type 1.

Platelets in peripheral blood critically drive clot formation in health and disease. Previously considered to uniformly respond to vascular injury and inflammatory cues, recent studies have highlighted that circulating platelets exhibit marked heterogeneity, with distinct populations contributing differentially to hemostasis, thrombosis, and inflammation. In this review, we highlight platelet diversity as a consequence of origin (ie, megakaryocyte diversity), circulatory age (ie, young vs aged platelets), and, specifically, as both a sequela of and a contributing factor to cardiovascular and inflammatory diseases. This diversity includes reticulated platelets (RPs), newly released from the bone marrow, RNA-rich, and highly prothrombotic, vs aged platelets, which exhibit altered receptor expression and proinflammatory rather than hemostatic features. We further describe how platelet subsets actively shape disease progression. Hyperreactive RPs drive arterial thrombosis, whereas procoagulant platelets amplify fibrin formation in venous thromboembolism. In chronic inflammation, interactions of immune-responsive platelets with leukocyte subsets facilitate their recruitment and impact on polarization, but can also promote endothelial dysfunction and immune hyperactivation, perpetuating thromboinflammatory dysregulation. Moreover, platelet phenotypes are dynamically regulated by disease states, with systemic inflammation, altered shear forces, and metabolic stress influencing platelet turnover, activation thresholds, and functional specialization. Recognizing platelet heterogeneity in disease pathogenesis could provide new opportunities for precision medicine, potentially allowing stratification of thrombotic risk and differential tailoring of antiplatelet and anti-inflammatory therapies.

Because frail patients and patients aged ≥80 years with chronic lymphocytic leukemia (CLL) are still underrepresented in clinical trials, the CLL-Frail trial aimed to evaluate the efficacy and safety of acalabrutinib in these patients. The primary end point was the overall response rate (ORR) after 6 cycles of treatment to test the null hypothesis of ORR ≤65%. Fifty-three patients were included in the trial, and 34 patients are still on therapy. Adverse events (AEs) were the most frequent reason for early discontinuation (10 patients), whereas 5 patients stopped treatment because of death. Median age was 81 years, and 47.2% of patients were frail. The ORR for the 46 patients receiving ≥3 cycles of treatment was 93.5% (95% confidence interval, 82.1-98.6) meeting the primary end point of this trial (P < .001). The estimated 12-month progression-free and overall survival rates were 93.3% and 95.7%, respectively, after a median follow-up of 19 months. 53.5% of patients reported an improvement in their self-perceived frailty. Although all patients experienced AEs, and severe (Common Terminology Criteria of ≥3) events were reported in 63.5% of patients, there were no events of severe bleeding and atrial fibrillation was rare (2 cases of Common Terminology Criteria Grades 2 and 3). Five patients died, of which 4 deaths happened during or <28 days after treatment. Infections/COVID-19 were the cause of death in 3 cases. To our knowledge, this is the first prospective trial in older and/or frail patients with CLL demonstrating a high efficacy and safe treatment with acalabrutinib monotherapy. This trial was registered at www.ClinicalTrials.gov as #NCT04883749.

The significance of endogenous immune surveillance in acute lymphoblastic leukemia (ALL) remains controversial. Using clinical B-cell ALL samples and a novel mouse model, we show that neoantigen-specific CD4+ T cells are induced to adopt type 1 regulatory (Tr1) function in the leukemia microenvironment. Tr1 cells then inhibit cytotoxic CD8+ T cells, preventing effective leukemia clearance. Leukemic cells induce Tr1 cells by phenocopying hematopoietic stem cells, which normally are subject to effective surveillance by this CD4+ subset. This mechanism effectively redirects Tr1 cells from a role in preventing cancer to maladaptively promoting clinical relapse. In mouse models, addition of interleukin-10 receptor (IL-10R) blockade to cytotoxic therapy modestly affected Tr1 development but was insufficient to improve leukemia control. In contrast, combined therapy with a cytotoxic agent and anti-PDL1 blockade eradicated measurable residual disease. This correlates with polarization of the neoantigen-specific CD4+ T-cell population from Tr1 toward T helper 1 (Th1) states. Our findings uncover a mechanism that enables leukemic relapse and resolves existing controversies on the role of immune surveillance toward this cancer type. Therapeutic polarization of neoantigen-specific CD4+ T cells away from Tr1 and toward Th1 states may improve contemporary immune therapies by reshaping the immune microenvironment toward states permissive for cytotoxic attack of residual leukemia.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that regulate tissue homeostasis and immune responses. How ILCs modulate T cells, remains incompletely understood. To investigate the interaction between ILCs and T cells, we differentiated ILC2s and ILC3s from hematopoietic stem cells (HSCs). Both suppressed T-cell proliferation, enhanced cytokine production, and upregulated T-cell senescence-associated surface receptors (CD57, KLRG1, TIGIT, and TIM3). T cells exposed to ILCs also increased expression of senescence-related proteins, including p16, p21, p53, GATA4, and NF-κB. Mechanistically, ILCs produced interleukin-9 (IL-9), and IL-9 blockade prevented ILC-driven T-cell senescence. Conversely, addition of exogenous IL-9 to T cells recapitulated the effects of ILC coculture. Finally, in both human xenogeneic and murine allogeneic hematopoietic cell transplantation models, we observed ILC-mediated T-cell modulation in vivo, with evidence of T-cell senescence. In conclusion, HSC-derived ILCs from both humans and mice mitigate graft-versus-host disease by inducing T-cell senescence.

Current therapies, including autologous chimeric antigen receptor (CAR) T-cell immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL), a disease characterized by frequent central nervous system involvement, poor treatment response, early relapse, and lineage switching. More effective treatment strategies, including the availability of off-the-shelf immunotherapies, is particularly relevant in infants. PROM1/CD133 is a direct target of KMT2A-fusion oncoproteins and is expressed on leukemic cells. Allogeneic invariant natural killer T (iNKT) cells, "innately" more powerful effectors than T cells, can be deployed off-the-shelf without risk of acute graft-versus-host disease. Here, we equip iNKT cells with CD19- and/or CD133-targeting CARs, and investigate their antileukemia activity against KMT2Ar-ALL in relevant in vitro and in vivo models. Compared with monospecific counterparts and dual, bispecific CAR T cells, bispecific CD19-CD133 CAR-iNKT cells have a more potent antileukemia activity, effectively targeting both CAR antigen-high and -low leukemia. Bispecific CAR-iNKT cells eradicate medullary and, notably, leptomeningeal leukemia, and induce sustained remissions without discernible hematologic toxicity. Mechanistically, the more potent antileukemia effect of CAR-iNKT cells over CAR T cells is mediated by a pronounced CAR-dependent and CAR antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT cells, and its engagement by its corresponding ligands on KMT2Ar-ALL cells. This ensures effective leukemia targeting even with downregulation of CD133 or CD19. Thus, by engaging with 2 different types of leukemia-associated antigens, that is, CAR antigens and NKG2D ligands, CAR-iNKT cells provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.

The acquisition of N-glycosylation sites that are occupied by oligomannose-type glycans in the immunoglobulin complementarity-determining region (CDR) is an early, clonal, tumor-specific identifier of follicular lymphoma (FL). CDR-located N-glycosylation sites are also acquired in germinal center B-cell-like diffuse large B-cell lymphomas (GCB-DLBCLs), but their significance is less defined. We used RNA sequencing immunoglobulin assembly to determine frequency and CDR location of the acquired N-glycosylation sites (AGSs) in 2 independent DLBCL cohorts. Composition of the glycans occupying the AGSs was determined using liquid chromatography-mass spectrometry and correlated with cell of origin, FL signature (defined by EZB phenotype or BCL2 translocation), transcript profile, and clinical outcome. CDR-located AGSs were observed in 41% to 46% of GCB-DLBCLs but were rare in other DLBCLs. Only CDR-located AGSs of DLBCL with an FL signature were occupied by oligomannose-type glycans. These DLBCLs were termed Mann-type DLBCL. Conversely, the AGSs of the other DLBCLs were either nonglycosylated or occupied by complex-type glycans. Mann-type status was an independent marker of short progression-free survival and overall survival. In contrast, the other GCB-DLBCLs, including those with an FL signature but without AGSs, had the best outcomes. Mann-type DLBCLs overexpressed gene sets of cell growth, survival, and cycling, and underexpressed proinflammatory and apoptotic pathways, irrespective of the presence of concomitant MYC translocations. The acquisition of Mann-type glycans is a highly selective environmental pressure enabling the identification of an aggressive GCB-DLBCL type with origin related to FL. The detection of AGSs in the CDR of GCB-DLBCLs with an FL signature defines Mann-type DLBCLs, refines prognosis, and marks a precise tumor interaction to block early therapeutically.

Genomic profiling in patients with chronic-phase chronic myeloid leukemia (CP-CML) demonstrated somatic variants in blood cancer-related gene variants (CGVs) and rearrangements associated with the formation of the Philadelphia chromosome (Ph-associated rearrangements) at diagnosis, collectively termed additional genetic abnormalities (AGAs). AGAs had a negative impact on failure-free survival (FFS) and molecular response in imatinib-treated patients. We investigated whether treatment with more potent therapies could overcome the negative impact of AGAs at diagnosis. Targeted RNA-based next-generation sequencing was performed on diagnostic samples of 315 patients consecutively enrolled in 4 clinical trials of frontline potent tyrosine kinase inhibitors (TKIs) in CP-CML. AGAs were present in 34% of patients at diagnosis, including 20% harboring CGVs and 18% with Ph-associated rearrangements (4% had both). Although the negative impact of Ph-associated rearrangements was overcome by more potent inhibitors, patients with CGVs continued to experience inferior outcomes. This result was largely attributable to patients with ASXL1 variants, observed in 7% overall. Patients harboring ASXL1 variants also had inferior outcomes compared with those with wild-type ASXL1 in terms of 12-month major molecular response (55% vs 83%; P = .001), 2-year FFS (61% vs 91%; P < .001), and notably, the development of treatment-emergent BCR::ABL1 kinase domain mutations at 2 years (35% vs 1%; P < .001). In multivariable models, both CGVs and ASXL1 variants were predictors of each outcome. Treatment with frontline potent TKIs overcame the negative impact of Ph-associated rearrangements observed with frontline imatinib. However, inferior outcomes were still associated with the presence of CGVs. The acquisition of TKI-resistant BCR::ABL1 mutations was almost exclusively associated with mutated ASXL1 at diagnosis.

Histone deacetylase inhibitors (HDACis) are valued treatment options for patients with T-cell malignancies. Romidepsin is a selective class I HDACi initially approved for patients with relapsed or refractory cutaneous and peripheral T-cell lymphomas (PTCLs). Romidepsin was withdrawn from its PTCL indication following a negative randomized phase 4 study (romidepsin-CHOP [cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone]) that showed no benefit over CHOP alone, further diminishing options for patients. Herein, we describe the development of, to our knowledge, a first-in-class polymer nanoparticle (PNP) of romidepsin using an innovative amphiphilic diblock copolymer-based nanochemistry platform. Nanoromidepsin exhibited superior pharmacologic properties with improved tolerability and safety in murine models of T-cell lymphoma (TCL). The PNP also exhibited superior antitumor efficacy in multiple models, including in vitro TCL cell lines, ex vivo samples from patients with large granular lymphocyte (LGL) leukemia, and murine TCL xenografts. Nanoromidepsin demonstrated greater accumulation in tumors and a statistically significant improvement in overall survival compared with romidepsin in murine xenograft models. These findings justify the clinical development of nanoromidepsin in patients with T-cell malignancies.