CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC).

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

Harnessing NK Cell Checkpoint-Modulating Immunotherapies.

During the host immune response, the precise balance of the immune system, regulated by immune checkpoint, is required to avoid infection and cancer. These immune checkpoints are the mainstream regulator of the immune response and are crucial for self-tolerance. During the last decade, various new immune checkpoint molecules have been studied, providing an attractive path to evaluate their potential role as targets for effective therapeutic interventions. Checkpoint inhibitors have mainly been explored in T cells until now, but natural killer (NK) cells are a newly emerging target for the determination of checkpoint molecules. Simultaneously, an increasing number of therapeutic dimensions have been explored, including modulatory and inhibitory checkpoint molecules, either causing dysfunction or promoting effector functions. Furthermore, the combination of the immune checkpoint with other NK cell-based therapeutic strategies could also strengthen its efficacy as an antitumor therapy. In this review, we have undertaken a comprehensive review of the literature to date regarding underlying mechanisms of modulatory and inhibitory checkpoint molecules.

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 receptor-modified 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, respectively.

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1-2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.

Preclinical Assessment of Suitable Natural Killer Cell Sources for Chimeric Antigen Receptor Natural Killer-Based "Off-the-Shelf" Acute Myeloid Leukemia Immunotherapies.

The introduction of chimeric antigen receptors (CARs) to augment the anticancer activity of immune cells represents one of the major clinical advances in recent years. This work demonstrates that sorted CAR natural killer (NK) cells have improved antileukemia activity compared to control NK cells that lack a functional CAR. However, in terms of viability, effectiveness, risk of side effects, and clinical practicality and applicability, an important question is whether gene-modified NK cell lines represent better CAR effector cells than primary human donor CAR-NK (CAR-dNK) cells. Comparison of the functional activities of sorted CAR-NK cells generated using the NK-92 cell line with those generated from primary human dNK cells demonstrated that CAR-NK-92 cells had stronger cytotoxic activity against leukemia cells compared to CAR-dNK cells. CAR-NK-92 and CAR-dNK cells had similar CD107a surface expression upon co-incubation with leukemia cells. However, CAR-NK-92 cells secreted higher granzyme A and interleukin-17A levels, while CAR-dNK cells secreted more tumor necrosis factor alpha, interferon gamma, and granulysin. In addition, CAR-NK-92 cells revealed a significantly higher potential for adverse side effects against nonmalignant cells. In short, this work shows the feasibility for further development of CAR-NK strategies to treat leukemia.

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

[This corrects the article DOI: 10.3389/fimmu.2017.01100.].

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.

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.

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8+ T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.

Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors.

UNLABELLED: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to modify human NK cells, we compared different retroviral vector systems, retroviral pseudotypes, and transduction protocols. Using optimized transduction conditions, the highest transduction levels (up to 60%) were achieved with alpharetroviral vectors. Alpharetroviral-modified primary human NK cells exhibited no alteration in receptor expression and had similar degranulation activity as untransduced NK cells, thus demonstrating that alpharetroviral modification did not negatively affect NK cell cytotoxicity. Transduction of NK cells with an alpharetroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells, achieving nearly complete elimination of leukemia cells after 48 h. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications. KEY MESSAGES: Efficient modification of human NK cells using alpharetroviral vectors. Anti-CD19-CAR-NK cells exhibited improved cytotoxicity towards CD19(+) leukemia cells. Alpharetroviral vectors are promising tools for immunotherapy applications using NK cells.

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

BACKGROUND: Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development. METHODS: Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed. RESULTS: Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. CONCLUSION: These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.

Cytotoxicity and infiltration of human NK cells in in vivo-like tumor spheroids.

BACKGROUND: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation. METHODS: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation. RESULTS: The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity. CONCLUSION: Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.

Advantages and applications of CAR-expressing natural killer cells.

In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.

BACKGROUND: The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6. METHODS: To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ(+) T cells exceeded 0.03% of CD3(+) lymphocytes as determined by IFN-γ cytokine secretion assay. RESULTS: Starting with low concentration of IFN-γ(+) T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ(+)CD3(+) T cells (1.42 × 10(6), 0.05 × 10(6), and 1.15 × 10(6)) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8(+) than CD4(+) memory T cells, both of which were effectively enriched to a total of 81.0% CD8(+)IFN-γ(+) and 38.4% CD4(+)IFN-γ(+) T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes. CONCLUSIONS: The generation of antiviral CD4(+) and CD8(+) T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.

Interleukin-2-stimulated natural killer cells are less susceptible to mycophenolate mofetil than non-activated NK cells: possible consequences for immunotherapy.

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.

Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol.

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 10(8) CD56(+)CD3(-) cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56(+)CD3(-) NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

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.

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.