CAR-Expressing Natural Killer Cells for Cancer Retargeting

Since the approval in 2017 and the outstanding success of Kymriah® and Yescarta®, the number of clinical trials investigating the safety and efficacy of chimeric antigen receptormodified autologous T cells has been constantly rising. Currently, more than 200 clinical trials are listed on clinicaltrial. gov. In contrast to CAR-T cells, natural killer (NK) cells can be used from allogeneic donors as an “off the shelf product” and provide alternative candidates for cancer retargeting. This review summarises preclinical results of CAR-engineered NK cells using both primary human NK cells and the cell line NK-92, and provides an overview about the first clinical CAR-NK cell studies targeting haematological malignancies and solid tumours, respectivel

Histone deacetylase inhibitors enhance expression of NKG2D ligands in Ewing sarcoma and sensitize for natural killer cell-mediated cytolysis

Background: Ewing sarcoma patients have a poor prognosis despite multimodal therapy. Integration of combination immunotherapeutic strategies into first-/second-line regimens represents promising treatment options, particularly for patients with intrinsic or acquired resistance to conventional therapies. We evaluated the susceptibility of Ewing sarcoma to natural killer cell-based combination immunotherapy, by assessing the capacity of histone deacetylase inhibitors to improve immune recognition and sensitize for natural killer cell cytotoxicity. Methods: Using flow cytometry, ELISA and immunohistochemistry, expression of natural killer cell receptor ligands was assessed in chemotherapy-sensitive/-resistant Ewing sarcoma cell lines, plasma and tumours. Natural killer cell cytotoxicity was evaluated in Chromium release assays. Using ATM/ATR inhibitor caffeine, the contribution of the DNA damage response pathway to histone deacetylase inhibitor-induced ligand expression was assessed. Results: Despite comparable expression of natural killer cell receptor ligands, chemotherapy-resistant Ewing sarcoma exhibited reduced susceptibility to resting natural killer cells. Interleukin-15-activation of natural killer cells overcame this reduced sensitivity. Histone deacetylase inhibitor-pretreatment induced NKG2D-ligand expression in an ATM/ATR-dependent manner and sensitized for NKG2D-dependent cytotoxicity (2/4 cell lines). NKG2D-ligands were expressed in vivo, regardless of chemotherapy-response and disease stage. Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls. Conclusion: Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

Figure S1. Feasibility of cryopreservation. (A) Tricistronic IDLV encoding for hGM-CSF, hIFN-α and CMV-pp65 protein used to generate SmyleDCpp65. (B) Scheme of SmyleDCpp65 generation. Monocytes were isolated by MACS selection, pre-conditioned with cytokines for 8 h, and transduced with IDLV-G2α2pp65 for 16 h. After transduction, cells were harvested and cryopreserved at 2x106 cells/mL/vial. Cells were analyzed immediately after thaw (AT) or cultured in medium without exogenous cytokines for 7 days. (C) Viability (7AADneg) and identity (CD14 + expression level) of cell product (AT). (D) Total IDLV copy numbers detected by RT-q-PCR in the transduced cell groups AT and after 7 days in culture. (E) pp65 expression in SmyleDCpp65 (CD14neg, CD11cbright) after 7 days of in vitro culture. (F) Viability, down regulation of monocyte marker (CD14), identity (CD11cbright and HLA-DR) and functional markers (CD86 and CD80) expressed in SmyleDCpp65 7 days after in vitro culture

IL-2 Stimulated but Not Unstimulated NK Cells Induce Selective Disappearance of Peripheral Blood Cells: Concomitant Results to a Phase I/II Study

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9–14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/−) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(−) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/−)CD69(+)NCR(high)CD62L(−) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy

Redirected Primary Human Chimeric Antigen Receptor Natural Killer Cells As an “Off-the-Shelf Immunotherapy” for Improvement in Cancer Treatment

Primary human natural killer (NK) cells recognize and subsequently eliminate virus infected cells, tumor cells, or other aberrant cells. However, cancer cells are able to develop tumor immune escape mechanisms to undermine this immune control. To overcome this obstacle, NK cells can be genetically modified to express chimeric antigen receptors (CARs) in order to improve specific recognition of cancer surface markers (e.g., CD19, CD20, and ErbB2). After target recognition, intracellular CAR domain signaling (CD3ζ, CD28, 4-1BB, and 2B4) leads to activation of PI3K or DNAX proteins (DAP10, DAP12) and finally to enhanced cytotoxicity, proliferation, and/or interferon γ release. This mini-review summarizes both the first preclinical trials with CAR-engineered primary human NK cells and the translational implications for “off-the-shelf immunotherapy” in cancer treatment. Signal transduction in NK cells as well as optimization of CAR signaling will be described, becoming more and more a focal point of interest in addition to redirected T cells. Finally, strategies to overcome off-target effects will be discussed in order to improve future clinical trials and to avoid attacking healthy tissues

CAR-Expressing Natural Killer Cells for Cancer Retargeting

Since the approval in 2017 and the outstanding success of Kymriah® and Yescarta®, the number of clinical trials investigating the safety and efficacy of chimeric antigen receptormodified autologous T cells has been constantly rising. Currently, more than 200 clinical trials are listed on clinicaltrial. gov. In contrast to CAR-T cells, natural killer (NK) cells can be used from allogeneic donors as an “off the shelf product” and provide alternative candidates for cancer retargeting. This review summarises preclinical results of CAR-engineered NK cells using both primary human NK cells and the cell line NK-92, and provides an overview about the first clinical CAR-NK cell studies targeting haematological malignancies and solid tumours, respectivel

CAR-Expressing Natural Killer Cells for Cancer Retargeting

Since the approval in 2017 and the outstanding success of Kymriah® and Yescarta®, the number of clinical trials investigating the safety and efficacy of chimeric antigen receptormodified autologous T cells has been constantly rising. Currently, more than 200 clinical trials are listed on clinicaltrial. gov. In contrast to CAR-T cells, natural killer (NK) cells can be used from allogeneic donors as an “off the shelf product” and provide alternative candidates for cancer retargeting. This review summarises preclinical results of CAR-engineered NK cells using both primary human NK cells and the cell line NK-92, and provides an overview about the first clinical CAR-NK cell studies targeting haematological malignancies and solid tumours, respectivel

Triplebody Mediates Increased Anti-Leukemic Reactivity of IL-2 Activated Donor Natural Killer (NK) Cells and Impairs Viability of Their CD33-Expressing NK Subset

Natural killer cells (NK) are essential for the elimination of resistant acute myeloid and acute lymphoblastic leukemia (AML and ALL) cells. NK cell-based immunotherapies have already successfully entered for clinical trials, but limitations due to immune escape mechanisms were identified. Therefore, we extended our established NK cell protocol by integration of the previously investigated powerful trispecific immunoligand ULBP2-aCD19-aCD33 [the so-called triplebodies (TBs)] to improve the anti-leukemic specificity of activated NK cells. IL-2-driven expansion led to strongly elevated natural killer group 2 member D (NKG2D) expressions on donor NK cells which promote the binding to ULBP2+ TBs. Similarly, CD33 expression on these NK cells could be detected. Dual-specific targeting and elimination were investigated against the B-cell precursor leukemia cell line BV-173 and patient blasts, which were positive for myeloid marker CD33 and B lymphoid marker CD19 exclusively presented on biphenotypic B/myeloid leukemia’s. Cytotoxicity assays demonstrated improved killing properties of NK cells pre-coated with TBs compared to untreated controls. Specific NKG2D blocking on those NK cells in response to TBs diminished this killing activity. On the contrary, the observed upregulation of surface CD33 on about 28.0% of the NK cells decreased their viability in response to TBs during cytotoxic interaction of effector and target cells. Similar side effects were also detected against CD33+ T- and CD19+ B-cells. Very preliminary proof of principle results showed promising effects using NK cells and TBs against primary leukemic cells. In summary, we demonstrated a promising strategy for redirecting primary human NK cells in response to TBs against leukemia, which may lead to a future progress in NK cell-based immunotherapies