In Vitro Modulation of Cellular Immunity with Antioxidants in Patients with Thalassemia Major.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3775-3775
Author(s):  
Belkis Atasever ◽  
Serap Erdem Kuruca ◽  
Zeynep Karakas ◽  
Leyla Agaoglu
Keyword(s):  
T Cells ◽  
Free Radicals ◽  
Vitamin E ◽  
Vitamin C ◽  
Iron Overload ◽  
Healthy Volunteers ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Activity

Abstract Immunological disturbances have been reported in thalassemia and the possibility has been raised that these may be consequences of blood transfusion and iron overload. These disturbances are augmentation of the number of supressor T cells (CD8), decreased number and activity of helper T cells (CD4) and impaired activity of NK (natural killer) cells. Iron overload causes toxic tissue changes through the release of free radicals and induces oxidative stress. According to Fenton and Haber-Weiss reactions, iron plays a catalytic role occuring hydroxyl radicals (OH*) which are very reactive free radicals. Antioxidants, like vitamin E, vitamin C and selenium, may modulate oxidative damage. In the present study; firstly, normal lymphocytes mitogen responses and NK activity were investigated by colorimetric MTT test in 26 thalassemia patients and 10 healthy volunteers as control. Secondly, lymphocytes were incubated with vitamin E ( 150, 50, 15 mg/ml), vitamin C (200, 100, 20mg/ml) and selenium (10−5, 10−6, 10−7 M). Finally, lymphocytes mitogen responses and NK activity are investigated. The results were statistically analyzed comparing with controls and healthy volunteers. It was found decreased NK activity of thalassemia patients in comparison with healthy volunteers. The concentration of 10−7 M of selenium enhanced NK activity at the E:T (effector/target) ratio of 50:1 The concentration of 200 mg/ml of vitamin C enhanced NK activity at the E:T ratio of 10:1, 25:1 and 50:1. However, vitamin E decreased NK activity of both thalassemia patients and healhty volunteers. The concentration of 50 mg/ml vitamin E decreased NK activity at the E:T ratio of 5:1 in thalassemia patients and the concentration of 15 mg/ml of vitamin E decreased NK activity at the E:T ratio of 5:1 in healhty volunteers. It was not found any differences between thalassemia patients and healthy volunteers in lymphocytes mitogen responses. The concentration of 200 mg/ml of vitamin C decreased lymphocytes mitogen response against PHA. In conclusion, we suggest that vitamin C and selenium supplementation are required in patients with thalassemia for augmentation of NK cell activity.

Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice: natural killer cell activity in cultured spleen leukocytes concomitant with T-cell-dependent immune interferon production

1980 ◽  
Vol 30 (2) ◽  
pp. 473-483
Author(s):  
R M Welsh ◽  
W F Doe
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cytotoxic T Cells ◽  
Cell Activity ◽  
Lymphocytic Choriomeningitis ◽  
Type I ◽  
Spleen Cells

The characteristics and specificities of spleen and peritoneal cytotoxic cells generated during lymphocytic choriomeningitis virus (LCMV) infection of C3H/St mice were examined. Activated natural killer (NK) cell activity was identified in fresh leukocyte populations from the 2nd to 8th days postinfection, whereas virus-specific cytotoxic T-cell activity was detected from the 6th to 14th days. When leukocytes were cultured overnight at 37 degrees C before assay, T-cell activity was still observed, but nonspecific activated NK cell-like cytotoxicity was only detected on the 6th and to a lesser degree the 8th day postinfection. Overnight culture of leukocytes taken earlier in the infection eliminated their NK cell activity. Similar activities were seen with spleen cell, plastic-adherent peritoneal cell, and nonadherent peritoneal cell populations. The virus-specific cytotoxicity observed with adherent peritoneal cells was due to contamination with cytotoxic T cells, as shown by H-2-restricted cytotoxicity and sensitivity to anti-theta antibody and complement. The nonspecific cultured day 6 effector cell from either the spleen or peritoneum displayed killing specificities and other physical properties identical to those of activated NK cells, but had sensitivities to anti-theta antibody and complement intermediate between activated day 3 NK cells and cytotoxic T cells. Culture stable NK-like cells were not found in athymic nude mice, suggesting a T-cell-dependent mechanism. Whereas LCMV spleen homogenates contained 10-fold-higher levels of interferon at day 2 than at day 6 postinfection, substantially more (nearly 20-fold) interferon was made in cultures of day 6 cells than day 2 cells. Spleen interferon was predominantly type I, whereas the culture interferon was predominantly type II, as shown by acid lability studies. Significant levels of interferon were produced by nylon-wool-passed day 6 spleen cells, and virtually all interferon production was eliminated by treatment of either day 2 or day 6 cells with antibody to theta antigen and complement, suggesting that T cells produced the interferon in vitro. Furthermore, athymic nude mice had no culture-stable NK cells 6 days postinfection, and spleen cells from them failed to produce significant levels of interferon in vitro. Addition of interferon (type I, fibroblast) to cultured C3H spleen cells affect the already elevated levels of cytotoxicity in day 6 cultures, suggesting that the NK cells in the day 6 culture were already activated. Our results suggest that T cells responding to LCMV infection secrete interferon type II which causes the continued activation of NK cells in culture. The resulting population of activated NK cells therefore appears to be relatively stable in culture and to express more theta antigen because of this T-cell dependence. Although one could mistakenly or allospecific cytotoxic T cells or cytotoxic macrophages, more careful examination shows that they are most likely activated NK cells...

scholarly journals Marina crystal minerals (MCM) activate human dendritic cells to induce CD4+ and CD8+ T cell responses in vitro

2018 ◽  
Vol 32 ◽  
pp. 205873841879776 ◽  
Author(s):  
Mamdooh H Ghoneum ◽  
Takeshi Ogura ◽  
James K Gimzewski ◽  
Aya D Ghoneum ◽  
Michael C Henary ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Interferon Gamma ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Activity ◽  
Human Monocyte ◽  
Nutritional Supplement ◽  
Inducible Protein

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs’ maturation and function in vitro. Human monocyte–derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1β, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

In-Vitro Culture of Human CD80/IL2 Lentivirus Transduced Acute Myeloid Leukemia Cells (AML) Promote NK Cell Activation and Cytolytic Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2969-2969
Author(s):  
Wendy Ingram ◽  
Lucas Chan ◽  
Hayrettin Guven ◽  
Shahram Kordasti ◽  
Linda Barber ◽  
...  
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Activity ◽  
Cell Cytotoxicity ◽  
Release Assay ◽  
Nk Cell Cytotoxicity

Abstract Natural killer (NK) cells are increasingly recognized as an important component in the graft versus leukemia response following allogeneic hematopoietic stem cell transplantation. Immunotherapeutic strategies aim to promote NK cell activity however, the presence of regulatory T cells (Tregs) which inhibit effector immune responses pose a potential challenge to the efficacy of such regimens. We have previously shown that ‘in-vitro’ culture of AML cells transduced with a self-inactivating lentivirus (LV) encoding CD80 (B7.1) and IL2 enhance allogeneic (allo) and autologous (auto) T cell proliferation and cytotoxicity. The effect on NK cell activity and Tregs has not previously been studied and is of particular importance as IL2 stimulates NK cell and Treg activity. Peripheral blood mononuclear cells (PBMCs) from healthy donors (allo) or AML patients (auto) were cultured for 7 days ‘in-vitro’ with either unmodified or LV-CD80/IL2 AMLs. The number of NK cells (CD3−CD56+) and Tregs (CD3+CD4+CD25highFoxp3+) was examined by multi-color flow cytometry. We observe an increase in the number of NK cells (p<0.001) with an increase in the expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR following allo culture with LV-CD80/IL2 AML compared with unmodified AML. Autologous culture provides a weaker stimulus ‘in-vitro’ however, a higher number of NK cells (p=0.002) and a consistent increased expression of the activation receptors NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, as well as up regulation of the cytolytic marker CD107a was detected following auto stimulation with LV-CD80/IL2 AML compared with unmodified AML. Up regulation of CD107a was also observed in allo cultures stimulated with both unmodified and LV-CD80/IL2 AML cells. In contrast, a consistent increase in the number of Tregs was observed following allo (p=0.043) but not auto (p=0.515) LV-CD80/IL2 AML culture. Foxp3 may be unregulated on activated CD4+ T cells therefore the number of CD3+CD4+CD25highFoxp3+CD27+ Tregs was also examined. An increase in the number of CD27+ Tregs was observed following allo (p=0.017) but not auto (p=0.807) LV-CD80/IL2 AML cell culture. A standard 51Cr release assay was used to examine cytotoxicity against primary unmodified AMLs on days 0 and 7 following LV-CD80/IL2 AML cell culture. Tregs are capable of suppressing CD4+ and CD8+ T cell and NK cell cytotoxicity, therefore lysis of unmodified AMLs was initially examined using whole PBMCs as effectors. Even in the presence of Tregs an increase in lysis of allo unmodified AMLs was observed: 2.2% day 0, 4.6% following culture with unmodified AMLs; 20.4% following LV-CD80/IL2 AML cell culture. Importantly, an increase in lysis of auto AML was also detected: 0% day 0, 2.1% unmodified AML culture, 16% LV-CD80/IL2 AML culture. The ratio of Tregs to effector T cells is important for the suppressive function of Tregs. The number of Tregs in the cytotoxicity assays is likely to be lower than that required for a significant suppressive effect to be observed. We next examined the cytotoxicity of NK cells using K562 and unmodified AMLs as targets. NK cells were negatively isolated on days 0 and 7 following either unmodified AML or LV-CD80/IL2 AML cell culture and used as effectors in a 51Cr release assay. In keeping with the changes in NK cell activation receptor expression, we demonstrate a significant increase in NK cell cytotoxicity against both K562 and primary unmodified AMLs. Lysis of K562 increased from 46.7% on day 0 to 90.4% after LV-CD80/IL2 stimulation. Importantly, an increase in lysis of both allo and auto unmodified AMLs was detected following LV-CD80/IL2 AML cell culture. Lysis of allo AMLs increased from a median of 11.8% on day 0, 8.7% following culture with unmodified AML to 20.1% following LV-CD80/IL2 AML cell culture using a low effector: target ratio of just 5:1. Importantly, an increase in lysis of auto AML from 0.4% on day 0, 2.1% with unmodified AML cells to 21.5% following LV-CD80/IL2 AML stimulation was observed. LV-CD80/IL2 AML cells enhance NK cell activation and cytotoxicity against allo and auto unmodified AMLs. Furthermore, cytotoxicity is enhanced even in the presence of Tregs with an increase in Tregs only observed following allo culture. Vaccination of patients with LV-CD80/IL2 AML cells therefore represents a potential strategy to promote T and NK cell cytotoxicity and enhance anti-leukemia immune responses in patients with AML.

scholarly journals Natural killer cell activity in the peripheral blood of patients with Cushing's syndrome

1999 ◽  
pp. 299-306 ◽  
Author(s):  
RG Masera ◽  
A Staurenghi ◽  
ML Sartori ◽  
A Angeli
Keyword(s):  
Nk Cell ◽  
Cell Activity ◽  
Urinary Free Cortisol ◽  
Nk Activity ◽  
Plasma Acth ◽  
Ifn Gamma ◽  
Free Cortisol ◽  
Direct Cytotoxicity ◽  
Nk Cytotoxicity

BACKGROUND: Natural killer (NK) cells are CD3(-)CD16(+)CD56(+) bone-marrow-derived lymphocytes mediating first-line defence by direct cytotoxicity against various types of target cells without prior immunization. NK cell activity is positively regulated by immune interferon (IFN-gamma); among hormones, glucocorticoids are potent in vitro and in vivo inhibitors, whereas ACTH and beta-endorphin in many experimental circumstances enhance NK cytotoxicity. DESIGN: We measured NK cytotoxicity of peripheral blood mononuclear cells (PBMC) obtained at 0800h and 2000h from 26 patients with Cushing's syndrome (12 pituitary-dependent, 12 adrenal-dependent and two dependent on ectopic ACTH secretion). In vitro responsiveness to IFN-gamma or cortisol was also tested. METHODS: NK activity was measured in a 4-h direct cytotoxicity assay using K562 cells as targets. Plasma ACTH, serum and urinary free cortisol were concomitantly measured with commercially available kits. RESULTS: Spontaneous activity and responsiveness to IFN-gamma or cortisol were significantly greater in 15 age- and sex-matched controls than in Cushing's patients at 0800h. In pituitary-dependent Cushing's patients, plasma ACTH correlated positively with mean levels of spontaneous NK activity (r=0.64, P<0.05) and negatively with cortisol-dependent percentage inhibition (r=-0.69, P<0.02). In adrenal-dependent Cushing's patients, a negative correlation was observed between levels of spontaneous NK activity and urinary free cortisol (r=-0.67, P<0.02). CONCLUSIONS: Our data indicate that excess endogenous glucocorticoids affect spontaneous NK cell activity and responsiveness to exogenous IFN-gamma or cortisol. The differential patterns observed between pituitary-dependent and adrenal-dependent groups are compatible with a positive immunomodulatory role of pituitary pro-opiomelanocortin-derived peptides that effectively counterbalance, at least partially, glucocorticoid immunosuppression.

scholarly journals Targeting B7-H3 via chimeric antigen receptor T cells and bispecific killer cell engagers augments antitumor response of cytotoxic lymphocytes

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jie Liu ◽  
Shuo Yang ◽  
Bihui Cao ◽  
Guangyu Zhou ◽  
Fengjuan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Aerobic Glycolysis ◽  
Killer Cell ◽  
Cell Activity ◽  
Antitumor Efficacy ◽  
Tumor Tissues

Abstract Background B7-H3, an immune-checkpoint molecule and a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. Here, we aimed to systematically evaluate the value of B7-H3 as a target in NSCLC via T cells expressing B7-H3-specific chimeric antigen receptors (CARs) and bispecific killer cell engager (BiKE)-redirected natural killer (NK) cells. Methods We generated B7-H3 CAR and B7-H3/CD16 BiKE derived from an anti-B7-H3 antibody omburtamab that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Antitumor efficacy and induced-immune response of CAR and BiKE were evaluated in vitro and in vivo. The effects of B7-H3 on aerobic glycolysis in NSCLC cells were further investigated. Results B7-H3 CAR-T cells effectively inhibited NSCLC tumorigenesis in vitro and in vivo. B7-H3 redirection promoted highly specific T-cell infiltration into tumors. Additionally, NK cell activity could be specially triggered by B7-H3/CD16 BiKE through direct CD16 signaling, resulting in significant increase in NK cell activation and target cell death. BiKE improved antitumor efficacy mediated by NK cells in vitro and in vivo, regardless of the cell surface target antigen density on tumor tissues. Furthermore, we found that anti-B7-H3 blockade might alter tumor glucose metabolism via the reactive oxygen species-mediated pathway. Conclusions Together, our results suggest that B7-H3 may serve as a target for NSCLC therapy and support the further development of two therapeutic agents in the preclinical and clinical studies.

184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A198-A198
Author(s):  
Tingting Zhong ◽  
Xinghua Pang ◽  
Zhaoliang Huang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cells ◽  
Mixed Culture ◽  
Cell Activity ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Jurkat Cells ◽  
List Type ◽  
Dependent Manner ◽  
Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

scholarly journals Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15

2021 ◽  
Author(s):  
Shannon L. McArdel ◽  
Anne-Sophie Dugast ◽  
Maegan E. Hoover ◽  
Arjun Bollampalli ◽  
Enping Hong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Blood Cell ◽  
Nk Cells ◽  
Red Blood Cell ◽  
Safety Profile ◽  
Nk Cell ◽  
Cell Activity ◽  
In Vivo Studies

AbstractRecombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

scholarly journals 114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A126-A126
Author(s):  
John Goulding ◽  
Mochtar Pribadi ◽  
Robert Blum ◽  
Wen-I Yeh ◽  
Yijia Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Tumor Immunity ◽  
Immune Cell ◽  
Nk Cell ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

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