scholarly journals Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines

2020 ◽  
Vol 8 (2) ◽  
pp. e000873 ◽  
Author(s):  
Ji-Eun Park ◽  
Seong-Eun Kim ◽  
Bhumsuk Keam ◽  
Ha-Ram Park ◽  
Soyeon Kim ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Nk Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Nk Cell ◽  
Cancer Cell Lines ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Healthy Donors ◽  
Nk Cell Cytotoxicity

BackgroundAlthough programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines.MethodsVarious cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by 51Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. FcγRIIIa (CD16a)-V158F genotyping was performed for healthy donors.ResultsWe demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing a FCGR3A high-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype.ConclusionThese results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

Quantification and Characterization of Peripheral Blood Natural Killer (NK) Cells Mediating Rituximab and Alemtuzumab Dependent Cytotoxicity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2808-2808
Author(s):  
Olaf Penack ◽  
Lars Fischer ◽  
Chiara Gentilini ◽  
Anne M. Asemissen ◽  
Carmen Scheibenbogen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Cell Activation ◽  
Nk Cell ◽  
Surface Expression ◽  
Leukemia Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Specific Lysis ◽  
Flow Cytometric

Abstract Background: Recent data suggest that NK cell mediated antibody dependent cellular cytotoxicity (ADCC) is a major mechanism of action of the anti-CD20 monoclonal antibody (mAb) rituximab and the anti-CD52 mAb alemtuzumab, which are frequently applied in patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. However, the exact mechanisms leading to NK cell activation are not completely understood and the cytotoxic subpopulation of peripheral blood NK cells mediating ADCC remains to be defined. In order to quantify and characterize the NK cells mediating ADCC, we used a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation. Methods: PBMCs from healthy individuals were coincubated at 37°C for 3 h with different human leukemia and lymphoma cell lines. In each tube, containing 200μl effector/target suspension (4x105 cells), 15μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. To assess antibody dependent cellular cytotoxicity (ADCC) saturating concentrations (10μg/ml) of rituximab or alemtuzumab were used. After the first 1 h 5μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3, NKG2D, CD69, CD94, NKp30, NK46) for flow cytometry. NK cell-mediated cytotoxicity (specific lysis) was analyzed by flow cytometric detection of propidium iodide uptake. Results: After coincubation with NK sensitive K562 cells up to 6% of CD56+ cells expressed CD107a, indicating that a subpopulation of NK cells releases cytotoxic granules after contact with these target cells. In contrast, coincubation with NK-resistant leukemia cells (ML2, EHEB, DAUDI, RAJI, AM0-1, YT-1) was not followed by an increased surface expression of CD107a. However, when rituximab was added to CD20+ lymphoma or leukemia cells (EHEB, DAUDI, RAJI) we observed that up to 15% of NK cells expressed CD107a after coincubation. In contrast no increased CD107a surface expression was observed when rituximab was added to the CD20− cell lines AMO-0 and YT-1, which excludes unspecific NK cell activation. When alemtuzumab was added to the CD52+ cell lines AMO-1, DAUDI, EHEB, RAJI and YT-1, surface expression of CD107a on NK cells was increased considerably. The majority of degranulating NK cells had the phenotype: CD56dim, CD69+, NKG2D+, NKp30−, NKp46− and CD94−. Furthermore we found that the CD107a assay can also visualize ADCC under clinical conditions as we observed increased numbers of NK cells degranulating in response to CD20+ lymphoma cell lines in patients with non-Hodgkin’s lymphoma treated with rituximab. The number of degranulating NK cells was closely related to the concentration of rituximab and the effector:target ratio, showing a maximum at a ratio of 1:1 and concentrations above 5μg/ml. CD107a surface expression and specific lysis demonstrated a strong positive correlation (r2 = 0.99), confirming that NK cell cytotoxicity can be assessed by this method. Conclusion: The CD107a assay represents a promising new method not only for assessment of natural cytotoxicity on a single cell level but also for determination of ADCC in vitro and in patients treated with mAb. In clinical practice, it may help to find optimal doses and time schedules for the treatment with different mAbs.

scholarly journals 688 Knockout of the inhibitory receptor TIGIT enhances anti-tumor response of ex vivo expanded NK cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A716-A716
Author(s):  
Tayler Croom-Perez ◽  
Md Faqrul Hasan ◽  
Thomas Dieffenthaller ◽  
Liza Robles-Carrillo ◽  
Jonathan Eloriaga ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Fine Tuning ◽  
Inhibitory Receptors ◽  
Wild Type

BackgroundNatural Killer (NK) cells are an important immune cell population crucial for the success of many immunotherapies due to their critical role in both the innate response and in priming an adaptive immune response. Recently, much focus has been on generating highly cytotoxic NK cells for use in adoptive cell therapy and combinatorial immune-oncology therapies. The robust cytotoxicity against cancer cells and NK cell activation relies on fine tuning of activating and inhibitory signals. NK cell inhibitory receptors are often upregulated upon stimulation and activation and can be a marker for exhaustion. One of the major NK inhibitory receptors, T-cell immunoglobulin and ITIM domain (TIGIT), is highly expressed in ex vivo expanded NK cells. In this study, we will investigate if knockout of TIGIT in ex vivo expanded NK cells will enhance their anti-tumor activity.MethodsCRISPR was used to make a targeted TIGIT knockout (KO) in ex vivo expanded NK cells. TIGIT KO NK cells were then compared to wild type NK cells to determine any changes in phenotypic markers. IFNγ, TNFα, and the degranulation marker CD107a expression were analyzed after co-culture with cancer cells. Cytotoxicity of TIGIT KO NK cells was compared to wild type NK cells against multiple different cancer cell spheroids using a kinetic live-cell imaging assay. Multiple NK cell:target cell ratios were analyzed over time to determine killing half-time and maximum killing. Data were fit to dose-response curves to determine cytotoxicity EC50 values.ResultsCRISPR was used to efficiently knockout TIGIT in ex vivo expanded NK cells and decreased expression levels to less than 5%. After co-culture with Raji cells expressing the TIGIT ligand PVR (CD155), TIGIT KO NK cells showed increased expression of IFNγ, TNFα and CD107a. TIGIT KO NK cells showed improved killing compared to wild type NK cells. TIGIT KO cells killed more target cells faster with significant decreases in half-killing time and more than a 2-fold decrease in EC50 cytotoxicity values in 3D spheroid cytotoxicity models against six different cancer cell lines. When NK cell:target cell ratios were low, the maximum cytotoxicity was also significantly higher in TIGIT KO cells.ConclusionsKnockout of the TIGIT gene in ex vivo expanded NK cells resulted in higher functioning NK cells with increased cytokine expression, degranulation, and cytotoxicity against multiple cancer cell lines. These TIGIT knockout NK cells with improved antitumor activity provide a promising universal effector population with the potential for enhanced therapeutic efficacy.AcknowledgementsWe thank FL DOH Grant #9JK04 for funding and MaxCyte for providing instrument for testing.

scholarly journals Antibody-Dependent Cellular Cytotoxicity Mediated by Cetuximab against Lung Cancer Cell Lines

2007 ◽  
Vol 13 (5) ◽  
pp. 1552-1561 ◽  
Author(s):  
Jun Kurai ◽  
Hiroki Chikumi ◽  
Kiyoshi Hashimoto ◽  
Kosuke Yamaguchi ◽  
Akira Yamasaki ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Lung Cancer Cell

scholarly journals 606 SAR444245 (THOR-707), an engineered non-alpha IL-2, enhances NK mediated antibody-dependent cellular cytotoxicity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A636-A636
Author(s):  
Julie-Ann Gavigan ◽  
Nicole Acuff ◽  
Christen Buetz ◽  
Michael Lampa ◽  
Chaomei Shi ◽  
...  
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Biology ◽  
Nk Cell ◽  
Cellular Cytotoxicity ◽  
Natural Amino Acid ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Dose Dependent

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.

470 Targeting Pan-Tumor Associated Antigen B7H3 via Combination of Tri-specific Killer Engager and Off-the-shelf NK Cell Therapy Enhances Specificity and Function Against a Broad Range of Solid Tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A500-A500
Author(s):  
Jeffrey Miller ◽  
Nicholas Zorko ◽  
Behiye Kodal ◽  
Zachary Davis ◽  
Alexander Lenvik ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Nk Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Repeated Measures ◽  
Prostate Cancer Cell ◽  
Nk Cell ◽  
Cancer Cell Lines ◽  
Prostate Cancer Cell Lines ◽  
Repeated Measures Anova

BackgroundB7H3 is a tumor associated antigen (TAA), found on numerous malignancies including prostate, lung, and breast cancers. High levels of B7H3 expression are correlated with late stage disease and poor prognosis. Furthermore, B7H3 is minimally expressed on normal tissue, making it an ideal TAA for broad cancer treatment strategy. We developed a tri-specific killer engager (TriKETM) consisting of a nanobody anti-CD16, IL-15, and nanobody anti-B7H3 joined by flexible linkers (camB7H3 TriKE) (figure 1A). The combination of B7H3 TriKE with an off-the-shelf NK cell therapy presents an appealing therapeutic strategy for the treatment of solid tumors with decreased risk of toxicity in allogeneic settings compared to T-cell derived products.MethodsAn anti-B7H3 nanobody was developed via biopanning and cloned into a TriKE vector. TriKE was produced in Expi293 cells and affinity purified using poly-His tag. NK cells were co-incubated with cell lines exhibiting a range of B7H3 expression and with 3nM of camelid B7H3 TriKE or control. We have previously derived NK cells expressing high affinity non-cleavable hnCD16, CD38 KO, and IL-15/IL-15R fusion from clonal master engineered iPSC lines. Engineered iNK cells were tested in conjunction with the TriKE. A repeated measures ANOVA was used for statistical comparisons as noted in figure legendsResultsEngineered iNK cells co-incubated with camB7H3 TriKE and C4-2 prostate cells significantly increased degranulation (CD107a) and cytokine production (IFN-gamma) compared to controls (figure 1B/C, P<0.05, n=3). camB7H3 TriKE directly bound C4-2 cells with an estimated EC50 of approximately 3nM. camB7H3 TriKE increased percentages of engineered iNK cells dividing robustly (3 or more times) compared to corresponding IL-15 doses at 3 nM (figure 1D, P<0.001, n=3). Furthermore, camB7H3 TriKE enhanced cytotoxic activity of engineered iNK cells against a variety of tumor cells in 2D and spheroid format independent of cytokine support (figure 1E-F). Engineered iNK cells incorporating an anti-B7H3 chimeric antigen receptor (CAR) is also being developed and will be discussed.Abstract 470 Figure 1A) Schematic of TriKE molecule demonstrating spatial relationship of anti-CD16 nanobody, IL-15, and anti-B7H3 nanobody with flexible linker regions. B) Percent of PB NK cells CD107a as a marker of NK degranulation. Unselected PBMCs were stimulated with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment with B7H3-expressing C4-2 prostate cancer cell lines Cells were stimulated for 5 hours and evaluated for degranulation (surface CD107a) by flow cytometry, gating on the NK (CD56+CD3-) population (displayed). Graphs display mean ± SEM. P<0.05 using repeated measures ANOVA. C) Percent of PB NK cells expressing intracellular interferon-gamma. Unselected PBMCs were stimulated with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment with B7H3-expressing C4-2 prostate cancer cell lines Cell were stimulated for 5 hours and evaluated for degranulation (surface CD107a) by flow cytometry, gating on the NK (CD56+CD3-) population (displayed). Graphs display mean ± SEM. P<0.05 using repeated measures ANOVA. D) Percent of PB NK cells dividing robustly (3 or more times) over a 7 day stimulation with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment. PBMCs were incubated with Cell-Trace Violet reagent prior to stimulation. Graphs display mean ± SEM. P<0.001 using repeated measures ANOVA. E) Representative 2D IncuCyte images of PC3 prostate cancer cell lines transduced with NucLight Red. Cells were co-incubated with iNK alone, iNK with 3nM IL-15 or iNK with 3 nM antiB7H3 TriKE. Images represent remaining NucLight Red transduced PC3 cells after 72 hours of co-incubation as noted above. F) Representative microspheriod IncuCyte images of PC3 prostate cancer lines transduced with NucLight Red. Cells were co-incubated with iNK cells alone, iNK with 3 nM IL-15 or iNK with 3 nM antiB7H3 TriKE. Images represent remaining NucLight Red transduced PC3 cells after 72 hours of co-incubation as noted aboveConclusionscamB7H3 TriKE dramatically increases function and activation on endogenous NK cells as well as engineered iNK cell, which can be adoptively transferred to patients with a broad range of cancers, including prostate cancer. TriKE activity was potent across a broad concentration spectrum and corresponded directly with B7H3 target expression. These studies represent the proof-of-concept of a novel pairing of off-the-shelf, engineered iNK cells with B7H3-directed pan-cancer engager molecules (TriKEs and CARs) to enhance specificity, persistence and anti-tumor function.

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