Marit Inngjerdingen

Natural killer (NK) cells are exploited in cellular therapies for cancer. While NK cell therapies are efficient against haematological cancers, it has been difficult to target solid tumours due to low tumour infiltration and a hostile tumour microenvironment. NK‐cell derived extracellular vesicles (NK‐EVs) target and kill cancer cells in vitro and represent an alternative treatment strategy for solid tumours. To exploit their potential, it is necessary to standardize NK‐EV production protocols. Here, we have performed a comparative analysis of EVs from the human NK‐92 cell line cultured in five serum‐free commercial media optimized for growth of human NK cells and one serum‐free medium for growth of lymphocytes. The effect of growing the NK‐92 cells in static cell cultures versus shaking flasks was compared. EVs were purified via ultracentrifugation followed by size‐exclusion chromatography. We found that there were no significant differences in EV yield from NK‐92 cells grown under static or dynamic conditions. However, we found clear differences between the different culture media in terms of EV purity as assessed by the enrichment of the CD63 and CD81 markers in the isolates that translated into their capacity to induce apoptosis of the colon cancer cell line HCT 116. These findings will be instructive for the design of future production protocols for therapeutic NK‐cell derived EVs.

There is a high medical need to develop new strategies for the treatment of patients with acute myeloid leukemia (AML) refractory to conventional therapy. In vitro, the combinations of the blast-modulatory response modifiers GM-CSF + Prostaglandin E1, (summarized as Kit M) have been shown to convert myeloid leukemic blasts into antigen-presenting dendritic cells of leukemic origin (DC_leu) that were able to (re-)activate the innate and adaptive immune system, direct it specifically against leukemic blasts, and induce memory cells. This study aimed to investigate the immune modulatory capacity and antileukemic efficacy of Kit M in vivo. Brown Norway rats suffering from AML were treated with Kit M (twofold application). Blasts and immune cells were monitored in peripheral blood (PB) and spleen. Upon the observation of promising immune modulatory effects in the treated animals, two patients with AML refractory to multiple lines of therapy were offered treatment with Kit M on an individualized basis. Safety, as well as immunological and clinical effects, were monitored. Samples obtained from a third patient in similar clinical conditions not receiving Kit M were used as controls for immune monitoring tests. Animal experiments: Drugs were well tolerated by the treated animals. After 9 days of treatment, DC_leu and memory-like T cells increased in the peripheral blood, whereas regulatory T cells, especially blasts, decreased in treated as compared to untreated control animals. Clinical courses: No severe side effects were observed. In patient 1482, PB blasts remained under the detection threshold during 27 days of treatment, thrombocytes were normalized, and (leukemia specific) immune effector cells of the adaptive and innate immune system increased up to 800-fold compared to the start of treatment. Patient 1601 responded with a 12% reduction in blasts in PB immediately after Kit M treatment. Several subtypes of (leukemia-specific) immune effector cells in PB increased up to four-fold during the 19 days of treatment. In contrast, immune-reactive cells decreased under mild chemotherapy in the PB of control patient 1511 with comparably refractory AML. Within the limitation of low numbers in both animal experiments and clinical applications, our data suggest that Kit M treatment of AML-diseased rats and patients is feasible and may induce leukemia-specific immune reactions and clinical improvement. A larger series and a prospective clinical trial will be required to confirm our observations. Beyond optimized doses and schedules of the applied compounds, the combination with other antileukemic strategies or the application of Kit M in less proliferative stages of the myeloid diseases need to be discussed. If effects are confirmed, the concept may add to the armamentarium of treatments for highly aggressive blood cancer.

Acute paediatric leukaemia is diagnosed and monitored via bone marrow aspirate assessment of blasts as a measure of minimal residual disease. Liquid biopsies in the form of blood samples could greatly reduce the need for invasive bone marrow aspirations, but there are currently no blood markers that match the sensitivity of bone marrow diagnostics. Circulating extracellular vesicles (EVs) represent candidate biomarkers that may reflect the blast burden in bone marrow, and several studies have reported on the utility of EVs as biomarkers for adult haematological malignancies. Increased levels of EVs have been reported for several haematological malignancies, and we similarly report here elevated EV concentrations in plasma from paediatric BCP‐ALL patients. Plasma EVs are very heterogeneous in terms of their cellular origin, thus identifying a cancer selective EV‐marker is challenging. Here, we undertook a reductionistic approach to identify protein markers selectively associated to plasma EVs derived from BCP‐ALL patients. The EV proteome of primary BCP‐ALL cell‐derived EVs were compared against EVs from healthy donor B cells and the BCP‐ALL cell line REH, and further against EVs isolated from plasma of healthy paediatric donors and paediatric BCP‐ALL patients. With this approach, we identified a signature of 6 proteins (CD317, CD38, IGF2BP1, PCNA, CSDE1, and GPR116) that were specifically identified in BCP‐ALL derived EVs only and not in healthy control EVs, and that could be exploited as diagnostic biomarkers.

NK cells are important for early tumor immune surveillance. In patients with hematological cancers, NK cells are generally functional deficient and display dysregulations in their receptor repertoires. Acute leukemia is the most common cancer in children, and we here performed a comparative phenotypic profiling of NK cells from pre-B cell acute lymphoblastic leukemia (BCP-ALL) patients to identify aberrant NK cell phenotypes. NK cell phenotypes, maturation, and function were analyzed in matched bone marrow and blood NK cells from BCP-ALL patients at diagnosis, during treatment, and at end of treatment and compared to age-matched pediatric controls. Expression of several markers were skewed in patients, but with large inter-individual variations. Undertaking a multi-parameter approach, we found that high expression levels of NKG2A was the single predominant marker distinguishing NK cells in BCP-ALL patients compared to healthy controls. Moreover, naïve CD57^-NKG2A NK cells dominated in BCP-ALL patients at diagnosis. Further, we found dysregulated expression of the activating receptor DNAM-1 in resident bone marrow CXCR6⁺ NK cells. CXCR6⁺ NK cells lacking DNAM-1 expressed NKG2A and had a tendency for lower degranulation activity. In conclusion, high expression of NKG2A dominates NK cell phenotypes from pediatric BCP-ALL patients, indicating that NKG2A could be targeted in therapies for this patient group.

Hematopoietic allogeneic stem cell transplantation (allo-SCT) is a curative option for patients with hematological malignancies. However, due to disparities in major and minor histocompatibility antigens between donor and recipient, severe inflammatory complications can occur, among which chronic graft-versus-host disease (cGVHD) can be life-threatening. A classical therapeutic approach to the prevention and treatment of cGVHD has been broad immunosuppression, but more recently adjuvant immunotherapies have been tested. This review summarizes and discusses immunomodulatory approaches with T cells, including chimeric antigen receptor (CAR) and regulatory T cells, with natural killer (NK) cells and innate lymphoid cells (ILCs), and finally with mesenchymal stromal cells (MSC) and extracellular vesicles thereof. Clinical studies and pre-clinical research results are presented likewise.

NK cell-based therapies have shown promise for hematological cancer forms, but their use against solid tumors is hampered by their poor ability to infiltrate the tumor. NK cells release extracellular vesicles (EVs) containing cytolytic proteins, indicating that NK-cell derived EVs may have therapeutic potential. In this study, we compared the tumor-targeting potential of EVs derived from either primary NK cells or the NK cell lines NK-92 and KHYG-1 cultured in IL-15 alone or in combination with IL-12 and IL-18. Primary NK cells were also stimulated through the activating receptor CD16. Tumor cell apoptosis was measured using a panel of human colon, melanoma, glioblastoma, prostate, breast, and ovarian tumor cell line spheroids. NK cells or NK-92 cells stimulated with IL-12, IL-15, and IL-18 generated EVs with higher efficiency than EVs from resting cells, although similar amounts of EVs were produced under both conditions. Proteomic analysis indicated similar distribution of cytolytic proteins in EVs from primary NK cells and NK-92, but lower levels in KHYG-1 EVs that translated into poor capacity for KHYG-1 EVs at targeting tumor cell lines. Further, we show that CD16-stimulated NK cells release low amounts of EVs devoid of cytolytic proteins. Importantly, EVs from cytokine-stimulated NK cells penetrate into the spheroid core, and tumor spheroid susceptibility to NK-cell derived EVs was linked to differential expression of the NKG2D ligands MICA/B, which was blocked with an anti-NKG2D antibody. We conclude that EVs from activated primary NK cells or NK-92 cells has the best potential to infiltrate and target solid tumors.