Because frail and patients ≥80 years with 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 endpoint was the overall response rate (ORR) after 6 cycles of treatment to test the null hypothesis of ORR ≤ 65%.53 patients were included into the trial and 34 patients are still on therapy. Adverse events (AEs) were the most frequent reason for early discontinuation (ten patients), whereas five 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 endpoint of this trial (p < 0.001). 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 (CTC° ≥ 3) events were reported in 63.5% of patients, there were no events of severe bleeding and atrial fibrillation was rare (two cases of CTC° 2 and 3). Five patients died, of which four deaths happened during or <28 days after treatment. Infections/COVID-19 were the cause of death in three cases. This is the first prospective trial in older and/or frail CLL patients demonstrating a high efficacy and safe treatment with acalabrutinib monotherapy. ClinicalTrials.gov ID: NCT04883749 EUDRA-CT: 2020-002142-17.

The significance of endogenous immune surveillance in acute lymphoblastic leukemia (ALL) remains controversial. Using clinical B-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. Tr1s then inhibit cytotoxic CD8+ T-cells, preventing effective leukemia clearance. Leukemic cells induce Tr1s 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 IL10R 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 towards Th1 states. Our findings uncover a mechanism that enables leukemic relapse and resolves existing controversies on the role of immune surveillance towards this cancer type. Therapeutic polarization of neoantigen-specific CD4+ T-cells away from Tr1 and towards Th1 states may improve contemporary immune therapies by reshaping the immune microenvironment towards states permissive for cytotoxic attack of residual leukemia.

ABSTRACTInnate 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 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 co-culture. Finally, in both human xenogeneic and murine allogeneic hematopoietic cell transplantation (HCT) 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 (GVHD) by inducing T cell senescence.

Current therapies, including autologous CAR-T 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 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 with CD19- and/or CD133-targeting CARs and investigate their anti-leukaemia activity against KMT2Ar-ALL in relevant in vitro and in vivo models. Compared to mono-specific counterparts and dual, bi-specific CAR-T, bi-specific CD19-CD133 CAR-iNKT have a more potent anti-leukemia activity, effectively targeting both CAR antigen-high and -low leukemia. Bi-specific CAR-iNKT eradicate medullary and, notably, leptomeningeal leukemia and induce sustained remissions without discernible hematologic toxicity. Mechanistically, the more potent anti-leukemia effect of CAR-iNKT over CAR-T cells is mediated by a pronounced CAR- and CAR antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT 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 two different types of leukemia-associated antigens i.e., CAR antigens and NKG2D ligands, CAR-iNKT 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 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-DLBCL), but their significance is less defined. We used RNA-seq immunoglobulin assembly to determine the frequency and CDR location of the acquired N-glycosylation sites (AGS) in two large independent DLBCL cohorts. Composition of the glycans occupying the AGS 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 AGS were observed in 41-46% of GCB-DLBCL but were rare in other DLBCL. Only CDR-located AGS of DLBCL with an FL signature were occupied by oligomannose-type glycans. These DLBCL were termed Mann-type DLBCL. Conversely, the AGS of the other DLBCL were either non-glycosylated 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-DLBCL cases, including those with an FL signature but without AGS, had the best outcomes. Mann-type DLBCL overexpressed gene-sets of cell growth, survival, and cycling, and underexpressed proinflammatory and apoptotic pathways, irrespective of concomitant MYC translocations. Acquisition of Mann-type glycans is a highly selective environmental pressure, identifying an aggressive GCB-DLBCL type with an origin related to FL. The detection of AGS 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 chronic-phase chronic myeloid leukemia (CP-CML) patients demonstrated somatic variants in blood cancer-related genes (CGVs) and rearrangements associated with the formation of the Philadelphia-chromosome (Ph-associated rearrangements) at diagnosis, collectively termed additional genetic abnormalities (AGA). AGAs had a negative impact on failure-free survival 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 (NGS) was performed on diagnostic samples of 315 patients consecutively enrolled in four clinical trials of frontline potent tyrosine kinase inhibitors (TKIs) in CP-CML. AGAs were present in 34% of patients at diagnosis, including 20% with CGVs and 18% with Ph-associated rearrangements (4% had both). While the negative impact of Ph-associated rearrangements was overcome by more potent inhibitors, patients with CGVs continued to have inferior outcomes. This was largely attributable to patients with ASXL1 variants, observed in 7% overall. Comparing patients with ASXL1 variants to ASXL1 wildtype: 12-month major molecular response 55% versus 83% (P=0.001); 2-year failure-free survival 61% versus 91% (P<0.001); and notably, development of treatment emergent BCR::ABL1 kinase domain mutations at 2 years, 35% versus 1% (P<0.001). In multivariable models, CGVs and ASXL1 variants were predictors of each of these outcomes. 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 (HDACi) 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 (R/R) CTCL and PTCL. Romidepsin was withdrawn from its PTCL indication following a negative randomized Phase IV study (Ro-CHOP) that showed no benefit over CHOP alone, further diminishing options for patients. Herein, we describe the development of a first-in-class polymer nanoparticle (PNP) of romidepsin using an innovative amphiphilic di-block 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 anti-tumor efficacy in multiple models including in vitro -TCL cell lines, ex vivo LGL leukemia patient samples, and murine TCL xenografts. Nanoromidepsin demonstrated greater accumulation in tumors and a statistically significant improvement in overall survival compared to romidepsin in murine xenograft models. These findings justify the clinical development of Nanoromidepsin in patients with T-cell malignancies.