Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers.

Bi-, tri- and multispecific antibodies have enabled the development of targeted cancer immunotherapies redirecting immune effector cells to eliminate malignantly transformed cells. These antibodies allow for simultaneous binding of surface antigens on malignant cells and activating receptors on innate immune cells, such as natural killer (NK) cells, macrophages, and neutrophils. Significant progress with such antibodies has been achieved, particularly in hematological malignancies. Nevertheless, several major challenges remain, including increasing their immunotherapeutic efficacy in a greater proportion of patients, particularly in those harboring solid tumors, and overcoming dose-limiting toxicities and immunogenicity. Here, we discuss novel antibody-engineering developments designed to maximize the potential of NK cells by NK cell engagers mediating antibody-dependent cellular cytotoxicity (ADCC), thereby expanding the armamentarium for cancer immunotherapy.

Radiotherapy orchestrates natural killer cell dependent antitumor immune responses through CXCL8.

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

NBAS Variants Are Associated with Quantitative and Qualitative NK and B Cell Deficiency.

PURPOSE: Biallelic pathogenic NBAS variants manifest as a multisystem disorder with heterogeneous clinical phenotypes such as recurrent acute liver failure, growth retardation, and susceptibility to infections. This study explores how NBAS-associated disease affects cells of the innate and adaptive immune system. METHODS: Clinical and laboratory parameters were combined with functional multi-parametric immunophenotyping methods in fifteen NBAS-deficient patients to discover possible alterations in their immune system. RESULTS: Our study revealed reduced absolute numbers of mature CD56dim natural killer (NK) cells. Notably, the residual NK cell population in NBAS-deficient patients exerted a lower potential for activation and degranulation in response to K562 target cells, suggesting an NK cell-intrinsic role for NBAS in the release of cytotoxic granules. NBAS-deficient NK cell activation and degranulation was normalized upon pre-activation by IL-2 in vitro, suggesting that functional impairment was reversible. In addition, we observed a reduced number of naïve B cells in the peripheral blood associated with hypogammaglobulinemia. CONCLUSION: In summary, we demonstrate that pathogenic biallelic variants in NBAS are associated with dysfunctional NK cells as well as impaired adaptive humoral immunity.

Preclinical evaluation of AFM24, a novel CD16A-specific innate immune cell engager targeting EGFR-positive tumors.

Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG1-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with KD values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype.

PPARγ induces PD-L1 expression in MSS+ colorectal cancer cells.

Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor "programmed-cell-death-1" (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal -2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC.

Human innate immune cell crosstalk induces melanoma cell senescence.

Mononuclear phagocytes and NK cells constitute the first line of innate immune defense. How these cells interact and join forces against cancer is incompletely understood. Here, we observed an early accumulation of slan+ (6-sulfo LacNAc) non-classical monocytes (slanMo) in stage I melanoma, which was followed by an increase in NK cell numbers in stage III. Accordingly, culture supernatants of slanMo induced migration of primary human NK cells in vitro via the chemotactic cytokine IL-8 (CXCL8), suggesting a role for slanMo in NK cell recruitment into cancer tissues. High levels of TNF-α and IFN-γ were produced in co-cultures of TLR-ligand stimulated slanMo and NK cells, whereas much lower levels were contained in cultures of slanMo and NK cells alone. Moreover, TNF-α and IFN-γ concentrations in slanMo/NK cell co-cultures exceeded those in CD14+ monocyte/NK cell and slanMo/T cell co-cultures. Importantly, TNF-α and IFN-γ that was produced in TLR-ligand stimulated slanMo/NK cell co-cultures induced senescence in different melanoma cell lines, as indicated by reduced melanoma cell proliferation, increased senescence-associated ß-galactosidase expression, p21 upregulation, and induction of a senescence-associated secretory phenotype (SASP). Taken together, we identified a role for slanMo and NK cells in a collaborative innate immune defense against melanoma by generating a tumor senescence-inducing microenvironment. We conclude that enhancing the synergistic innate immune crosstalk of slanMo and NK cells could improve current immunotherapeutic approaches in melanoma.

Memory-Like NK Cells: Remembering a Previous Activation by Cytokines and NK Cell Receptors.

Natural Killer (NK) cells are cytotoxic innate lymphoid cells serving at the front line against infection and cancer. In inflammatory microenvironments, multiple soluble and contact-dependent signals modulate NK cell responsiveness. Besides their innate cytotoxic and immunostimulatory activity, it has been uncovered in recent years that NK cells constitute a heterogeneous and versatile cell subset. Persistent memory-like NK populations that mount a robust recall response were reported during viral infection, contact hypersensitivity reactions, and after stimulation by pro-inflammatory cytokines or activating receptor pathways. In this review, we highlight recent findings on the generation, functionality, and clinical applicability of memory-like NK cells and describe common features in comparison to other recent concepts of memory NK cells. Understanding of these features will facilitate the conception and design of novel NK cell-based immunotherapies.

CD16A Activation of NK Cells Promotes NK Cell Proliferation and Memory-Like Cytotoxicity against Cancer Cells.

CD16A is a potent cytotoxicity receptor on human natural killer (NK) cells, which can be exploited by therapeutic bispecific antibodies. So far, the effects of CD16A-mediated activation on NK cell effector functions beyond classical antibody-dependent cytotoxicity have remained poorly elucidated. Here, we investigated NK cell responses after exposure to therapeutic antibodies such as the tetravalent bispecific antibody AFM13 (CD30/CD16A), designed for the treatment of Hodgkin lymphoma and other CD30+ lymphomas. Our results reveal that CD16A engagement enhanced subsequent IL2- and IL15-driven NK cell proliferation and expansion. This effect involved the upregulation of CD25 (IL2Rα) and CD132 (γc) on NK cells, resulting in increased sensitivity to low-dose IL2 or to IL15. CD16A engagement initially induced NK cell cytotoxicity. The lower NK cell reactivity observed 1 day after CD16A engagement could be recovered by reculture in IL2 or IL15. After reculture in IL2 or IL15, these CD16A-experienced NK cells exerted more vigorous IFNγ production upon restimulation with tumor cells or cytokines. Importantly, after reculture, CD16A-experienced NK cells also exerted increased cytotoxicity toward different tumor targets, mainly through the activating NK cell receptor NKG2D. Our findings uncover a role for CD16A engagement in priming NK cell responses to restimulation by cytokines and tumor cells, indicative of a memory-like functionality. Our study suggests that combination of AFM13 with IL2 or IL15 may boost NK cell antitumor activity in patients by expanding tumor-reactive NK cells and enhancing NK cell reactivity, even upon repeated tumor encounters. Cancer Immunol Res; 6(5); 517-27. ©2018 AACR.

KIR downregulation by IL-12/15/18 unleashes human NK cells from KIR/HLA-I inhibition and enhances killing of tumor cells.

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition of human NK cells by HLA-I-expressing tumor cells. Here, we show that stimulation of NK cells with IL-12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re-induced already 3 days after re-culture in IL-2. Reduced KIR2DL2/L3 expression on IL-12/15/18-activated NK cells resulted in less inhibition upon antibody-mediated KIR engagement and increased CD16-dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL-12/15/18-stimulated NK cells against tumor cells expressing cognate HLA-I molecules. NK cells pre-activated with IL-12/15/18 were previously shown to exert potent anti-tumor activity and memory-like long-lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL-12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self-HLA-I due to decreased KIR expression, highlighting the potency of IL-12/15/18-activated NK cells for anti-tumor immunotherapy protocols.

MicroRNA-519a-3p mediates apoptosis resistance in breast cancer cells and their escape from recognition by natural killer cells.

Aggressive breast cancer is associated with poor patient outcome and characterized by the development of tumor cell variants that are able to escape from control of the immune system or are resistant to targeted therapies. The complex molecular mechanisms leading to immune escape and therapy resistance are incompletely understood. We have previously shown that high miR-519a-3p levels are associated with poor survival in breast cancer. Here, we demonstrate that miR-519a-3p confers resistance to apoptosis induced by TRAIL, FasL and granzyme B/perforin by interfering with apoptosis signaling in breast cancer cells. MiR-519a-3p diminished the expression of its direct target genes for TRAIL-R2 (TNFRSF10B) and for caspase-8 (CASP8) and its indirect target gene for caspase-7 (CASP7), resulting in reduced sensitivity and tumor cell apoptosis in response to apoptotic stimuli. Furthermore, miR-519a-3p impaired tumor cell killing by natural killer (NK) cells via downregulation of the NKG2D ligands ULBP2 and MICA on the surface of tumor cells that are crucial for the recognition of these tumor cells by NK cells. We determined that miR-519a-3p was overexpressed in more aggressive mutant TP53 breast cancer that was associated with poor survival. Furthermore, low levels of TRAIL-R2, caspase-7 and caspase-8 correlated with poor survival, suggesting that the inhibitory effect of miR-519a-3p on TRAIL-R2 and caspases may have direct clinical relevance in lowering patient's prognosis. In conclusion, we demonstrate that miR-519a-3p is a critical factor in mediating resistance toward cancer cell apoptosis and impairing tumor cell recognition by NK cells. This joint regulation of apoptosis and immune cell recognition through miR-519a-3p supports the hypothesis that miRNAs are key regulators of cancer cell fate, facilitating cancer progression and evasion from immunosurveillance at multiple and interconnected levels.

Tricking the balance: NK cells in anti-cancer immunity.

Natural Killer (NK) cells are classically considered innate immune effector cells involved in the first line of defense against infected and malignant cells. More recently, NK cells have emerged to acquire properties of adaptive immunity in response to certain viral infections such as expansion of specific NK cell subsets and long-lasting virus-specific responses to secondary challenges. NK cells distinguish healthy cells from abnormal cells by measuring the net input of activating and inhibitory signals perceived from target cells through NK cell surface receptors. Acquisition of activating ligands in combination with reduced expression of MHC class I molecules on virus-infected and cancer cells activates NK cell cytotoxicity and release of immunostimulatory cytokines like IFN-γ. In the cancer microenvironment however, NK cells become functionally impaired by inhibitory factors produced by immunosuppressive immune cells and cancer cells. Here we review recent progress on the role of NK cells in cancer immunity. We describe regulatory factors of the tumor microenvironment on NK cell function which determine cancer cell destruction or escape from immune recognition. Finally, recent strategies that focus on exploiting NK cell anti-cancer responses for immunotherapeutic approaches are outlined.

The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration.

The high-mobility group box 1 (HMGB1) protein has a central role in immunological antitumour defense. Here we show that natural killer cell-derived HMGB1 directly eliminates cancer cells by triggering metabolic cell death. HMGB1 allosterically inhibits the tetrameric pyruvate kinase isoform M2, thus blocking glucose-driven aerobic respiration. This results in a rapid metabolic shift forcing cells to rely solely on glycolysis for the maintenance of energy production. Cancer cells can acquire resistance to HMGB1 by increasing glycolysis using the dimeric form of PKM2, and employing glutaminolysis. Consistently, we observe an increase in the expression of a key enzyme of glutaminolysis, malic enzyme 1, in advanced colon cancer. Moreover, pharmaceutical inhibition of glutaminolysis sensitizes tumour cells to HMGB1 providing a basis for a therapeutic strategy for treating cancer.

Expression of the immune regulation antigen CD70 in osteosarcoma.

Osteosarcoma is the most frequent bone cancer in children and young adults. The outcome of patients with advanced disease is dismal. Exploitation of tumor-immune cell interactions may provide novel therapeutic approaches. CD70-CD27 interactions are important for the regulation of adaptive immunity. CD70 expression has been reported in some solid cancers and implicated in tumor escape from immunosurveillance. In this study, expression of CD70 and CD27 was analyzed in osteosarcoma cell lines and tumor specimens. CD70 protein was expressed on most osteosarcoma cell lines (5/7) and patient-derived primary osteosarcoma cultures (4/6) as measured by flow cytometry. In contrast, CD70 was detected on few Ewing sarcoma cell lines (5/15) and was virtually absent from neuroblastoma (1/7) and rhabdomyosarcoma cell lines (0/5). CD70(+) primary cultures were derived from CD70(+) osteosarcoma lesions. CD70 expression in osteosarcoma cryosections was heterogeneous, restricted to tumor cells and not attributed to infiltrating CD3(+) T cells as assessed by immunohistochemistry/immunofluorescence. CD70 was detected in primary (1/5) but also recurrent (2/4) and metastatic (1/3) tumors. CD27, the receptor for CD70, was neither detected on tumor cells nor on T cells in CD70(+) or CD70(-) tumors, suggesting that CD70 on tumor cells is not involved in CD27-dependent tumor-immune cell interactions in osteosarcoma. CD70 gene expression in diagnostic biopsies of osteosarcoma patients did not correlate with the occurrence of metastasis and survival (n = 70). Our data illustrate that CD70 is expressed in a subset of osteosarcoma patients. In patients with CD70(+) tumors, CD70 may represent a novel candidate for antibody-based targeted immunotherapy.

Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

BACKGROUND: In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. METHODS: Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. RESULTS: M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1ß. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. CONCLUSION: This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our observation that the induction of macrophage anti-tumor activity by L-MTP-PE required IFN-γ may be of relevance for the optimization of L-MTP-PE therapy in osteosarcoma patients.

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity.

Osteosarcoma and Ewing's sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-ß. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.

Adenovirus type 35, but not type 5, stimulates NK cell activation via plasmacytoid dendritic cells and TLR9 signaling.

In hematopoietic stem cell transplant (HSCT) recipients, disseminated adenoviral infections during the first two months after HSCT can lead to severe complications and fatal outcome. Since NK cells are usually the first lymphocytes to reconstitute after HSCT and have been implicated in the clearance of adenovirus-infected cells, it was investigated whether NK cells are activated by adenovirus in vitro. Exposure of PBMC to human adenovirus type 5 (HAdV5) or HAdV35 resulted in the up-regulation of the activation marker CD69 on NK cells and enhanced the cytolytic activity of NK cells. HAdV5-induced NK cell activation relied on the contribution of T cells as the depletion of T cells from PBMC abolished NK cell activation. In contrast, NK cell activation in response to HAdV35 occurred in the absence of T cells. Plasmacytoid dendritic cells (pDC) were necessary and sufficient to mediate NK cell activation. HAdV35 induced significantly more interferon-α (IFN-α) production by pDC than HAdV5. The increased IFN-α production and NK cell activation correlated with a higher infection efficiency of viruses with the type 35 fiber. The IFN-α response of pDC was enhanced by the presence of NK cells, suggesting a reciprocal interaction between pDC and NK cells. Incubation with a TLR9 antagonist impaired the IFN-α production by pDC as well as NK cell activation, implying that TLR9 signaling is critically involved in the IFN-α response of pDC and NK cell activation after HAdV35 exposure. In conclusion, two human adenovirus serotypes from two different species differ considerably in their capacity to stimulate pDC and NK cells.

Anti-EGFR antibody cetuximab enhances the cytolytic activity of natural killer cells toward osteosarcoma.

PURPOSE: Osteosarcoma and Ewing's sarcoma are the most common bone tumors in children and adolescents. Despite intensive chemotherapy, patients with advanced disease have a poor prognosis, illustrating the need for alternative therapies. Sarcoma cells are susceptible to the cytolytic activity of resting natural killer (NK) cells which can be improved by interleukin (IL)-15 stimulation. In this study, we explored whether the cytolytic function of resting NK cells can be augmented and specifically directed toward sarcoma cells by antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN: Epidermal growth factor receptor (EGFR) expression was examined on osteosarcoma and Ewing's sarcoma cell lines by flow cytometry and in osteosarcoma biopsy and resection specimens by immunohistochemistry. Cetuximab-mediated ADCC by NK cells from osteosarcoma patients and healthy controls was measured with 4-hour (51)Cr release assays. RESULTS: EGFR surface expression was shown on chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cells (12/12), most primary osteosarcoma cultures (4/5), and few Ewing's sarcoma cell lines (2/7). In the presence of cetuximab, the cytolytic activity of resting NK cells against all EGFR-expressing sarcoma cells was substantially increased and comparable with that of IL-15-activated NK cells. Surface EGFR expression on primary osteosarcoma cultures correlated with EGFR expression in the original tumor. The cytolytic activity of osteosarcoma patient-derived NK cells against autologous tumor cells was as efficient as that of NK cells from healthy donors. CONCLUSION: Our data show that the cytolytic potential of resting NK cells can be potentiated and directed toward osteosarcoma cells with cetuximab. Therefore, cetuximab-mediated immunotherapy may be considered a novel treatment modality in the management of advanced osteosarcoma.