Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

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.     

Proteome characterization of human NK-92 cells identifies novel IFN-alpha and IL-15 target genes

Natural killer (NK) cells are important components of innate immune defense. NK cells kill virus-infected cells and secrete cytokines that are involved in activation of other immune cells. Macrophage-derived cytokines interferon-alpha (IFN-alpha) and interleukin-15 (IL-15) are in turn important activators of NK cells, but the receptors and intracellular pathways that are involved in NK cell functions are still incompletely known. Here we have used expression proteomics to find new IFN-alpha and IL-15 regulated proteins in human NK-92 cells, which have the characteristics of activated NK cells. Cells were stimulated with cytokines for 20 h, lysed, and soluble proteins were separated by two-dimensional electrophoresis, and differentially expressed protein spots were identified with mass spectrometry and database searches. A total of 57 protein spots were found to be reproducibly differentially expressed between control and cytokine stimulated gel pairs, 26 spots being more than 2-fold upregulated and 3 spots being at least 2-fold downregulated. The rest 28 spots showed minor, less than 2-fold changes in their expression levels after quantification. From the differentially expressed protein spots we identified 47 different proteins, most of which are new IFN-alpha and IL-15 target proteins. Interestingly, we show that e.g., adenylate kinase 2 is highly upregulated by IFN-alpha and IL-15 stimulation in NK-92 cells. The expression of selected genes with high expression level differences after cytokine stimulation were further studied at mRNA level. Northern blot analysis showed that the genes studied were induced by IFN-alpha, IL-15, and IL-2 already at 3 h time point, suggesting that they are primary target genes of these cytokines.     

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC.

     

NKP30-B7-H6 Interaction Aggravates Hepatocyte Damage through Up-Regulation of Interleukin-32 Expression in Hepatitis B Virus-Related Acute-On-Chronic Liver Failure

Background and Aims

Previous work conducted by our group has shown that the accumulation of hepatic natural killer (NK) cells and the up-regulation of natural cytotoxicity receptors (NKP30 and NKP46) on NK cells from patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) were correlated with disease progression in HBV-ACLF. The natural cytotoxicity receptors expressed on NK cells are believed to be probable candidates involved in the NK cell-mediated hepatocyte damage in HBV-ACLF. However, the underlying mechanisms remain to be elucidated. In the present study, we aimed to discover the role of NKP30-B7-H6 interaction in NK cells-mediated hepatocyte damage in HBV-ACLF.

Methods

Hepatic expressions of B7-H6 and interleukin-32 (IL-32) were examined by immunochemistry staining in samples from patients with HBV-ACLF or mild chronic hepatitis B (CHB). The cytotoxicity of NK-92 cell against target cells (Huh-7 and LO2) was evaluated by CCK8 assay. Expression of IL-32 in liver NK cell, T cells and NK-92 cell line was detected by the flow cytometric analysis. The effect of IL-32 on the apoptosis of Huh7 cells was evaluated using Annexin V/PI staining analysis.

Results

An enhancement of hepatic B7-H6 and IL-32 expression was associated with the severity of liver injury in HBV-ACLF. And there was a positive association between hepatic B7-H6 and IL-32 expression. Expressions of IL-32 in liver NK cells and T cells were increased in HBV-ACLF patients. In vitro NK-92 cells are highly capable of killing the high B7-H6 expressing Huh7 cells and B7-H6-tansfected hepatocyte line LO2 cells dependent on NKP30 and B7-H6 interaction. Furthermore, NK-92 cells exhibited elevated IL-32 expression when stimulated with anti-NKP30 antibodies or when co-cultured with Huh7 cells. IL-32 can induce the apoptosis of Huh7 cells in a dose-dependent manner.

Conclusion

Our results suggest that NKP30-B7-H6 interaction can aggravate hepatocyte damage, probably through up-regulation of IL-32 expression in HBV-ACLF.     

Development of mesothelin-specific CAR NK-92 cells for the treatment of gastric cancer

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR NK cell efficacy. It has been reported that mesothelin (MSLN) may be an ideal immunotherapy target for gastric cancer. However, the feasibility of using anti-MSLN CAR NK cells to treat gastric cancer remains to be studied.Methods: MSLN expression in primary human gastric cancer, normal tissues and cell lines were detected. MSLN and CD19 targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed, purified and verified. N87, MKN-28, AGS and Huh-7 cells expressing the GFP and luciferase genes were transduced. Cell- and patient-derived xenograft (PDX) were established via NSG mice. The ability of MSLN-CAR NK cells to kill MSLN-positive gastric cancer cells were evaluated in vitro and in vivo.Results: MSLN-CAR NK cells can specifically kill MSLN-positive gastric cancer cells (N87, MKN-28 and AGS), rather than MSLN negative cell (Huh-7), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretions were secreted in MSLN-CAR NK cells cocultured with N87, MKN-28 and AGS. Furthermore, MSLN-CAR NK cells can effectively eliminate gastric cancer cells in both subcutaneous and intraperitoneal tumor models. They could also significantly prolong the survival of intraperitoneally tumor-bearing mice. More importantly, the potent antitumor effect and considerable NK cell infiltration were observed in the patient-derived xenograft treated with MSLN-CAR NK cells, which further warranted the therapeutic effects of MSLN-CAR NK cells to treat gastric cancer.Conclusion: These results demonstrate that MSLN-CAR NK cells possess strong antitumor activity and represent a promising therapeutic approach to gastric cancer.     

Use of the anti-inflammatory cytokine interleukin-11 to reverse HIV-1gp120 repression of a natural killer cell line

Enhancing natural killer (NK) cell activation has been associated with protection from human immunodeficiency virus-1 (HIV-1) infections and slowed onset of immunodeficiency. However, soluble HIV-1 envelope protein, gp120, has been shown to impair NK cell cytokine secretion and cell-mediated cytotoxicity. Here we show that gp120 suppressed IFN-γ production and cytotoxic function of a human NK cell line NK-92MI. We furthermore demonstrated that an anti-inflammatory cytokine interleukin-11 can restore effector functions to repressed NK-92MI cells. These studies support the notion that IL-11 administration may reduce HIV-1-mediated immune activation and exhaustion while achieving elimination of virally-infected cells through restored NK cell function.     

Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.     

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CD19‐CAR engineered NK‐92 cells are sufficient to overcome NK cell resistance in B‐cell malignancies

Many B‐cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK‐92 cells, a continuously growing NK cell line, showing that anti‐CD19‐CAR (αCD19‐CAR) engineered NK‐92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK‐92 cells. The ‘first generation’ CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc‐tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK‐92 and transduced αCD19‐CAR NK‐92 cells towards CD19 negative targets was found. However, αCD19‐CAR NK‐92 cells specifically and efficiently lysed CD19 expressing B‐precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK‐92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK‐92 should be considered a treatment option for patients with lymphoid malignancies.     

IL-15-mediated induction of LFA-1 is a late step required for cytotoxic differentiation of human NK cells from CD34+Lin- bone marrow cells

Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56(+)CD3(-) NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34(+)Lin(-) bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.     

The NK-92 cell line—30 years later: its impact on natural killer cell research and treatment of cancer

The NK-92 cell line, established in 1992, mirrors all the characteristics of highly active blood natural killer (NK) cells but with much broader and greater cytotoxicity. The cell line was established from the blood cells of a patient with lymphoma and has been made widely available for research since it was deposited into the American Type Culture Collection in 1998. The worldwide distribution of NK-92 cells has led to a plethora of scientific discoveries that have greatly increased the understanding of NK-cell biology. NK-92 cells also have been developed for clinical use, overcoming the challenges of obtaining and expanding NK cells from donor or patient blood. More than 100 patients with cancer have now been treated all over the world with unmodified or genetically engineered NK-92 cells. Modified cells include high-affinity Fc-receptor expressing NK-92 cells (haNK^R) and various chimeric antigen receptor targeted haNK cells (t-haNK^TM). Infusions of either unmodified or modified NK-92 cells have been reported to be safe and efficacious, leading in some cases to disease remission even in patients who had failed multiple previous lines of therapy. It is the purpose of this review to distill the plethora of scientific data on NK-92 cells and its genetic variants, highlighting relevant experimental findings that have contributed to a better understanding of NK cell biology and summarize the therapeutic potential of these cells for treatment of cancer and infections.