T-cell exhaustion induced by continuous bispecific molecule exposure is ameliorated by treatment-free intervals.

T-cell-recruiting bispecific molecule therapy has yielded promising results in patients with hematologic malignancies; however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell-based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific molecule blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life-extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; P = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as a powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI-specific lysis on day 14: 34.9% vs 93.4%; n = 6; P < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.

Impaired function and delayed regeneration of dendritic cells in COVID-19.

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

SIRPα-αCD123 fusion antibodies targeting CD123 in conjunction with CD47 blockade enhance the clearance of AML-initiating cells.

BACKGROUND: Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance. METHODS: SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. RESULTS: SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123+ CD47+ AML cells even in the presence of CD47+ healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. CONCLUSIONS: SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.

COVID-19 in Patients Receiving CD20-depleting Immunochemotherapy for B-cell Lymphoma.

The clinical and immunological impact of B-cell depletion in the context of coronavirus disease 2019 (COVID-19) is unclear. We conducted a prospectively planned analysis of COVID-19 in patients who received B-cell depleting anti-CD20 antibodies and chemotherapy for B-cell lymphomas. The control cohort consisted of age- and sex-matched patients without lymphoma who were hospitalized because of COVID-19. We performed detailed clinical analyses, in-depth cellular and molecular immune profiling, and comprehensive virological studies in 12 patients with available biospecimens. B-cell depleted lymphoma patients had more severe and protracted clinical course (median hospitalization 88 versus 17 d). All patients actively receiving immunochemotherapy (n = 5) required ICU support including long-term mechanical ventilation. Neutrophil recovery following granulocyte colony stimulating factor stimulation coincided with hyperinflammation and clinical deterioration in 4 of the 5 patients. Immune cell profiling and gene expression analysis of peripheral blood mononuclear cells revealed early activation of monocytes/macrophages, neutrophils, and the complement system in B-cell depleted lymphoma patients, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration of COVID-19. Longitudinal immune cell profiling and functional in vitro assays showed SARS-CoV-2-specific CD8+ and CD4+ T-effector cell responses. Finally, we observed long-term detection of SARS-CoV-2 in respiratory specimens (median 84 versus 12 d) and an inability to mount lasting SARS-CoV-2 antibody responses in B-cell depleted lymphoma patients. In summary, we identified clinically relevant particularities of COVID-19 in lymphoma patients receiving B-cell depleting immunochemotherapies.