K562 Tumor Cells Research Articles

Linkage-Editing of Melibiosamine: Synthesis and Biological Evaluation of CH2- and CHF-Linked Analogs.

Melibiosamine (Gal-α(1,6)-GlcNH2), consisting of galactose and glucosamine linked by an α(1,6)-glycosidic bond, is an artificial disaccharide derivative that selectively inhibits the proliferation of K562 tumor cells relative to HUC-F2 normal cells. In this study, we employed a linkage-editing strategy to synthesize CH2- and CHF-linked melibiosamine analogs through chemo- and stereoselective hydrogenation of fluorovinyl-C-glycoside. (R)-CHF-Melibiosamine exhibited more potent antiproliferative activity than O-linked melibiosamine, while (S)-CHF-melibiosamine was less potent.

Abstract 1332: Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies

Abstract Invariant Natural Killer T (iNKT) cells are a unique subset of innate lymphocytes, constituting <1% of human T cells. iNKT cells express a semi-invariant TCR (iTCR) recognizing glycolipids presented by the monomorphic, MHC-like molecule CD1d. Previous studies indicate that due to distinctive TCR constitution and antigen recognition properties, iNKT cells do not induce acute graft-versus-host disease. Based on the CD4 and CD8 expression, mature human iNKT cells can be classified into CD4+CD8-, CD4-CD8- & CD4-CD8+ subsets with overlapping and distinct functions. The functional profiling of CAR19-iNKT cell subsets serves the dual purpose of ensuring their safety and efficacy as a therapeutic intervention. Here we characterized the phenotypic and functional profile of CD4+ and CD4- CAR19-iNKT cells. iNKT cells were isolated with >99% purity from healthy donors' peripheral blood and were transduced with a CD19 CAR-encoding 3rd generation lentivirus. After expansion, the CAR19-iNKT cells were cryopreserved and later analyzed post-thawing. Differential expression of CD27 and PD1 was noted in CAR+CD4+ and CAR+CD4- iNKT cells compared to other markers. For functional profiling, CAR+CD4+ iNKT cells were positively selected, and 24h cytotoxicity assays were performed using several tumor cell lines including SEM, Ramos and C1R-CD1d (+/- α-GalCer). The CAR+CD4- iNKT cells exhibited superior cytotoxicity to multiple tumor cell lines compared to that of CAR+CD4+ iNKT cells from two donors. However, both subsets lysed α-GalCer pulsed C1R-CD1d cells in a comparable manner indicating the potential of these subsets to recognize antigen through the iTCR. Subsequently, we assessed the proliferation ability of the CAR+CD4+ and CAR+CD4- subsets by exposing the cells to three rounds of stimulation, 24h apart, using SEM (CD19+CD1d-) or K562 (CD19-CD1d-) tumor cells with or without IL-15. Proliferation was assessed seven days following the first stimulation. The CAR+CD4+ subset of CAR19-iNKT cells demonstrated faster proliferation than CAR+CD4- cells in the presence of IL-15 after repeated exposure to tumor cells. Ongoing RNA seq analysis of CAR+CD4+/CAR+CD4- cells will further elucidate differences between these subsets. The outcomes of these studies have shown encouraging results, indicating the potential benefit of having diverse subsets among CAR19-iNKT cells for treating CD19+ cancers. The therapeutic potential of CAR+CD4+ iNKT cell subset could be enhanced to match that of CAR+CD4- subset with the use of α-GalCer. In summary, inclusion of both CD4+ and CD4- iNKT cells is critical for the functionality of allogeneic CAR-iNKT cell therapy. Extensive characterization is crucial for clinical translation, but additional research, including preclinical and clinical trials, is essential to determine the safety and effectiveness of CAR19-iNKT cell therapy in real-world applications. Citation Format: Kanagaraju Ponnusamy, Simon Poon, Nicole van der Weerden, Robson Dossa, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1332.

Human iPSC-Derived NK Cells with Knock-in of the BCL2 G101V Mutation Are Resistant to Venetoclax and Demonstrate Improved Anti-AML Activity In Vivo

Treatment options for acute myeloid leukemia (AML) patients who are unfit for intensive chemotherapy are limited. Both targeted therapy and cellular therapy have been investigated, typically as separate treatments. For patients with AML unfit for intensive induction chemotherapy, venetoclax, a BCL-2 inhibitor, containing regimens have significantly improved treatment outcomes. However, patients treated long-term with venetoclax can develop resistance. Based on the characterization of the BCL2 G101V mutation that mediates resistance to venetoclax, we investigated the ability of homozygous BCL2 G101V knock-in in induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells to promote resistance to venetoclax in these effector cells to enable concurrent treatment with venetoclax and the engineered iPSC-derived NK cells that we hypothesize will provide improved NK cell-mediated killing of AML. Our group has pioneered the production of genetically modified NK cells from human iPSCs. iPSCs provide an advantage of a stable platform for genome engineering and previous studies demonstrate we can engineer iPSCs to express or delete genes of interest to derive genetically modified iPSC-NK cells with improved anti-tumor activity. To generate iPSC-derived NK cells with resistance to venetoclax, we employed CRISPR-Cas9 technology to knock-in the BCL2 G101V mutation (BCL2 G101V) in iPSCs. iPSCs homozygous for BCL2 G101V were selected and differentiated to NK cells. Homozygous BCL2 G101V engineered iPSC-NK cells demonstrated resistance to venetoclax compared to wildtype (WT) iPSC-NK cells in vitro. BCL2 G101V iPSC-NK cells were 94-fold more resistant to venetoclax compared to wildtype iPSC NK cells with an EC50 of 6018 nM, above the serum levels of patients receiving venetoclax. Analysis of cell surface proteins demonstrated that both sets of iPSC-NK cells had a typical NK cell phenotype with no differences in expression of receptors analyzed. There was no difference seen in cytotoxicity against K562 tumor cells between BCL2 G101V iPSC-NK cells and WT-iPSC NK cells. Additional functional analyses demonstrated that activity of the BCL2 G101V iPSC NK cells was preserved upon exposure to venetoclax. Cytotoxic activity, as measured by CD107a expression on WT iPSC-NK cells stimulated by MOLM13 AML tumors cells, was reduced nearly 3-fold upon addition of venetoclax. In contrast, activation of BCL2 G101V iPSC NK cells was not significantly affected by addition of venetoclax. This resistance to venetoclax was confirmed in longer term 36-hour Incucyte cytotoxicity assays where BCL2 G101V iPSC-NK cells demonstrated more than 10-fold increase in anti-AML activity compared to the WT-iPSC-NK cells. We then conducted in vivo studies to test the WT and engineered NK cells with and without addition of venetoclax in an AML xenograft model using NSG mice (Figure Panels A-D). These studies used MOLM13 AML cells made resistant to venetoclax and demonstrated that without venetoclax treatment, both the WT and the BCL2 G101V iPSC NK cells mediate effective anti-AML killing. With addition of venetoclax, the WT NK cells were unable to effectively kill the AML cells while the BCL2 G101V iPSC cells mediated potent anti-AML activity and demonstrated significantly improved survival when the NK cells are given in combination with venetoclax. Specifically, in this system, the mice treated with WT iPSC-NK cells had a median survival of 22.8 days while the median survival of mice treated with the BCL2 G101V iPSC NK cells was not reached (p<0.01) Intriguingly, BCL2 G101V iPSC NK cells also demonstrated improved activity against MOLM13 cells resistant to venetoclax. Together our results demonstrate that iPSC-NK cells can be engineered to generate venetoclax-resistance for use in combination with concurrent venetoclax therapy to markedly improve treatment of AML. Furthermore, this work demonstrates that novel drug resistance mechanisms can be introduced via genome engineering into iPSC-derived NK cells as a new strategy to produce improved cell products for “off-the-shelf” therapy.

Generation of Singlet Oxygen by Laser Infrared Radiation 1273 nm and Evaluation of Cytotoxic Effect on Tumor Cell Lines

Increased reactivity makes singlet oxygen a cytotoxic agent. Infrared radiation at 1273 nm allows for direct photogeneration of singlet oxygen without the use of a photosensitizer. In this study, we demonstrated direct photogeneration of singlet oxygen in an aqueous environment using laser radiation at 1273 nm. Using chemical trapping methods, we determined the key parameters of generation, including the generation rate coefficient of singlet oxygen at an irradiance of 1 mW/cm2 and a concentration of dissolved triplet oxygen of 1 μM: ξ=(4.3±0.1) • 10-8 ((s•mW/cm2 )-1). We showed that irradiation of K562 and HeLa tumor cells with a focused laser beam at 1273 nm (diameter 100–1000 μm) leads to a significant cytotoxic effect in the irradiated area, not caused by thermal effects. We developed a mathematical model to calculate the photogenerated concentrations of singlet oxygen in an aqueous environment when irradiated with a 1273 nm laser. Based on experimental data, we calculated cumulative concentrations of singlet oxygen that result in cell death: 50% lethal dose for HeLa cells ~1000 μM, K562 cells ~3000 μM. Our research results confirm the potential application of 1273 nm laser radiation for direct photogeneration of singlet oxygen in tissues and the therapy of solid tumors

Cembranoids and their bioactivities in soft coral Sarcophyton glaucum

Chemical constituents in soft coral Sarcophyton glaucum were separated and purified by various chromatographic methods. Based on the spectral data, physicochemical properties, and comparison with the data reported in the literature, nine cembranoids, including a new cembranoid named sefsarcophinolide(1) together with eight known cembranoids, namely(+)-isosarcophine(2), sarcomilitatin D(3), sarcophytonolide J(4),(1S,3E,7E,13S)-11,12-epoxycembra-3,7,15-triene-13-ol(5), sarcophytonin B(6),(-)-eunicenone(7), lobophytin B(8), and arbolide C(9), were identified. As revealed by biological activity experiment results, compounds 2-6 had weak acetylcholinesterase inhibitory activity, and compound 5 displayed weak cytotoxicity against K562 tumor cell line.

Modakafusp Alfa (TAK-573), a Novel CD38-Targeting Attenuated Interferon-Alpha Immunocytokine, Kills MM Cells Via NK Cell Activation

Background Despite recent success of monoclonal antibodies (mAb) and T cell redirecting therapies, relapsed/refractory (RR) multiple myeloma (MM) patients remain in urgent need of new therapies with distinct mechanisms of action (MoA). Modakafusp alfa, previously known as TAK-573, is a novel immune-targeted attenuated cytokine specifically designed to deliver interferon-alpha to CD38-expressing cells via a CD38-targeting IgG4 mAb. Preliminary results of a first in human phase 1 trial with modakafusp alfa monotherapy (1.5mg/kg, every four weeks) in RRMM patients (90% anti-CD38 mAb refractory; 37% anti-BCMA agent exposed) indicate a promising 40% overall response with manageable toxicity (Kaufman et al. EHA 2022 abstract S181). Prior studies suggest a unique and multimodal MoA of modakafusp alfa, including anti-proliferative effects on tumor cells as well as stimulation of CD38-positive immune effector cells. Towards a thorough understanding of these immunomodulatory effects, specifically regarding the activation of innate immunity, we here studied the effects of modakafusp alfa on the anti-MM activity of NK cells, which highly express CD38. Aims We assessed the ex vivo activity of modakafusp alfa on NK cell activation, degranulation, and antibody-independent cytotoxicity. In addition, we investigated the impact of modakafusp alfa on NK cell-mediated, antibody-dependent cellular cytotoxicity (ADCC) against MM cells using daratumumab (anti-CD38) or elotuzumab (anti-SLAMF7). Methods NK cell activation was explored by flow cytometry after 24-hour incubation of peripheral blood mononuclear cells (PBMC) from healthy donors with modakafusp alfa or negative/specificity controls. These pre-incubated PBMCs were also used to examine NK cell degranulation after subsequent 4 hour stimulation with K562 or PMA/Ionomycin, or in a 16-hour cytotoxicity assay against K562. In addition, modakafusp alfa pre-incubated PBMCs or enriched NK cells were assessed for antibody-independent cytotoxicity or daratumumab/elotuzumab-mediated ADCC in 24-48 hour assays against MM cell lines RPMI-8226, MM.1S or UM9. Alternatively, modakafusp alfa was directly added to the assays without pre-incubation steps. Results We found that incubation of PBMCs with modakafusp alfa for 24 hours resulted in upregulation of activation markers CD38 and CD69 on the surface of NK cells. In addition, modakafusp alfa pre-incubation significantly improved the degranulation of NK cells after 4-hour stimulation with K562 or PMA/Ionomycin. Importantly, overnight pre-incubation of PBMCs or enriched NK cells with modakafusp alfa showed enhanced antibody-independent cytotoxicity against K562 tumor cells and MM cell lines RPMI-8226, MM.1S or UM9. Consequently, this also resulted in augmented killing of MM cells in the presence of daratumumab or elotuzumab. We observed similar effects when modakafusp alfa was directly added to the cytotoxicity assays without pre-incubation of immune effector cells. Conclusions Modakafusp alfa rapidly activates NK cells, and improves their degranulation and anti-MM effectivity. This important MoA also results in improved efficacy of daratumumab or elotuzumab in short-term cytotoxicity assays. Our findings warrant clinical investigation of combining modakafusp alfa with NK cell-exploiting immunotherapies.

Trained Immunity of IL-12-, IL-15-, and IL-18-Induced CD3+CD56+ NKT-Like Cells.

CD3+CD56+ natural killer T (NKT)-like cells have an immune function of T cells and NK cells, which play an important role in antitumor and antiviral immune responses. This study aims to establish a CD3+CD56+ NKT-like cell model by simulating the memory NK effect induced by cytokines IL-12, IL-15, and IL-18 (IL-12/15/18) and explore the formation mechanism. Our study found that the IL-12/15/18 preactivated CD3+CD56+ NKT-like cells exhibited enhanced IFN-γ production in response to restimulation with IL-12/15/18 for 6h on day 7. The intrinsic potential of these trained cells was significantly improved, showing an increase in IFN-γ, TNF-α, and cell proliferation potential. The IFN-γ release, granzyme B level, and proliferation ability significantly increased when stimulated by NK-cell-sensitive K562 tumor cells. Among these cytokines, the combination of IL-12/15/18 was particularly effective. After the preactivation of IL-12/15/18, some cell surface proteins related to function and differentiation, such as CD11b, CD62 L, NKp46, NKG2A, and CD127, showed an evident and consistent change trend. The CDK4/6 inhibitor can effectively weaken this effect, and the expression of cyclin D1, Rb protein phosphorylation, and E2F-1 decreased significantly. Our work revealed that cytokine IL-12/15/18 can induce CD3+CD56+ NKT-like cells to obtain enhanced training immunity, which was a memory-like phenomenon.

Abrogation of natural killer cell check point pathway LLT1/CD161 by novel anti LLT1 antibody ZM008 and its therapeutic applications in solid cancers.

e14503 Background: LLT1/CD161 interaction plays important roles in cancer immune surveillance, fine tuning of NK cells responsiveness and down regulating effector functions. LLT1 gene expression was confirmed on multiple tumors as well as on various infiltrating immune cells. Therefore, it is relevant to develop therapeutic monoclonal antibody against LLT1 that could potentiate ADCC, ADCP, CDC, bispecific, CAR-T/NK functions to eliminate tumor cells. In this work, we have described discovery of novel human anti-LLT1 mAb ZM008, NK cells activation and anti-tumor function of ZM008. A Phase 1 open label, multicenter dose escalation clinical trial is planned in patients with advanced solid tumors to evaluate the safety and tolerability of ZM008, as monotherapy and in combination with Pembrolizumab. Methods: LLT1 gene expression was analyzed using TCGA database for 33 cancer indications. 60 Prostate cancer FFPE samples from Mt. Sinai hospital biorepository, NY were tested. Multiple in vitro studies were conducted to demonstrate the mode of action of ZM008 and efficacy was determined in HuNOG-EXL animals with PC3 xenograft. Cynomolgus monkeys were dosed with ZM008 (QW, 4 weeks) to understand the safety, pharmacokinetics, and toxicokinetics. Results: TCGA analysis revealed differential LLT1 gene expression in advance stages of NSCLC, TNBC, HNSCC, PRAD and urothelial cancers and high correlation with immune checkpoint pathways. HALO® data analysis of PRAD FFPE samples have shown distinct and strong signal on tumor cells with ZM008. Cell based assays with ZM008 revealed activation of CD69, NKG2D, CD107 and IFNγ on human NK cells leading to significant cytotoxicity of PC3 (MHC+ve) and K562 (MHC-ve) tumor cells. In vivo tumor growth inhibition was achieved at 10 mg/kg ZM008 treatment in HuNOG-PC3 xenograft tumor model. Anti-tumor function of ZM008 was demonstrated by high infiltration of CD8+ T cells and CD56+ NK cells in the TME. Cynomolgus study suggests ZM008 was well tolerated at 10 mg/kg and upto 125 mg/kg dosing with no mortality or adverse reactions. Half-life of ZM008 was determined up to 259 hours. Conclusions: This study for the first time revealed intense staining of LLT1 protein in high Gleason score prostate cancer, demonstrating therapeutic potential of ZM008 in solid cancers not responsive to current I/O checkpoint therapies. In vitro and in vivo experiments have shown human NK cells activation resulting in significant tumor growth reduction. CD56+ NK and CD8+ T cells infiltrations indicate transformation of “Cold tumors” to “Hot tumors”. Targeting LLT1/CD161 interaction in TME could stimulate robust NK and T cell mediated anti-tumor checkpoint functions. The ZM008 drug product is safe and well tolerated in Cynomolgus monkey safety and toxicity studies paving the way for the open-label, multicenter, Phase 1 dose-escalation study with ZM008. .

Kalihioxepanes A—G: Seven Kalihinene Diterpenoids from Marine Sponge Acanthella cavernosa Collected off the South China Sea

Comprehensive SummarySeven new kalihinene diterpenoids, kalihioxepanes A—G (1—7) bearing a rare oxepane ring, were isolated from sponge Acanthella cavernosa collected from the South China Sea. The structures and absolute configurations were elucidated by comprehensive spectroscopic analysis, single crystal X‐ray diffraction, and quantum chemical calculation methods. Kalihioxepane A (1) displayed strong cytotoxicity against H69 and K562 tumor cells, while kalihioxepane B (2) showed moderate cytotoxicity against K562 cells.

CD95/Fas-ligation and resting NK or IL-2 activated NK (LAK) attack limit NK-92 efficacy.

Abstract Adoptively transferred NK-92 cells and genetically modified variants must undergo irradiation to prevent proliferation, since NK-92 cells are themselves tumors. This malignant quality also makes NK-92 cells susceptible to attack by patients’ NK cells. Here, we investigated the effect of gamma-irradiation and/or Fas-ligation on NK-92 killing against K562 tumor cells, as well as the effect of irradiation on the susceptibility of NK-92 cells to attack by primary NK cells. To evaluate killing, we used a 51Cr-release assay with multiple effector to target (E:T) K562 tumor cell ratios at 4- and 8-hour time points, 10 Gy irradiation and IgM antibody ligation of Fas. (K562 cells lack detectable Fas). NK-92 cells, maintained in 1000 U/ml of recombinant IL-2, retained high cytotoxic activity immediately after irradiation, but lost >50% activity one day after 10 Gy irradiation. NK-92 cells were highly resistant to death initiated by CD95-ligation, whether irradiated or not. However, the surviving NK-92 cells suffered loss of cytotoxicity following Fas-ligation. This loss of cytotoxicity was more pronounced when Fas-ligation and irradiation were combined. Irradiation also made NK-92 cells more sensitive to attack by resting NK cells and IL-2 activated NK (LAK) cells. These findings demonstrate that Fas-ligation and irradiation are detrimental to NK-92 functionality and support the recommendation that genetic removal of CD95/Fas may increase cytotoxicity by NK-92 cells and potentially, other cell lines used for adoptive immunotherapies. This research was supported in part by a UNR Foundation Award, an NIH P30 GM110767 for the Cytometry Center, a Nevada Undergraduate Research Award, and by the Nevada INBRE program (NIH GM103440) for research scholarship and supplies that helped support the project.

Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy

BackgroundThe NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells.MethodsTo evaluate serial killing, we used 51Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-h.ResultsNK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in 8 h. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost > 50% activity 1 day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, 1 day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells.ConclusionsIrradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Natural Phaeosphaeride A Derivatives Overcome Drug Resistance of Tumor Cells and Modulate Signaling Pathways.

In the present study, natural phaeosphaeride A (PPA) derivatives are synthesized. Anti-tumor studies are carried out on the PC3, K562, HCT-116, THP-1, MCF-7, A549, NCI-H929, Jurkat, and RPMI8226 tumor cell lines, and on the human embryonic kidney (HEK293) cell line. All the compounds synthesized turned out to have better efficacy than PPA towards the tumor cell lines listed. Among them, three compounds exhibited an ability to overcome the drug resistance of tumor cells associated with the overexpression of the P-glycoprotein by modulating the work of this transporter. Luminex xMAP technology was used to assess the effect of five synthesized compounds on the activation of intracellular kinase cascades in A431 cells. MILLIPLEX MAP Multi-Pathway Magnetic Bead 9-Plex was used, which allowed for the simultaneous detection of the following nine phosphorylated protein markers of the main intracellular signaling pathways: a universal transcription factor that controls the expression of immune-response genes, apoptosis and cell cycle NFκB (pS536); cAMP-dependent transcription factor (CREB (pS133); mitogen-activated kinase p38 (pT180/pY182); stress-activated protein kinase JNK (pT183/pY185); ribosomal SK; transcription factors STAT3 (pS727) and STAT5A/B (pY694/699); protein kinase B (Akt) (pS473); and kinase regulated by extracellular signals ERK1/2 (pT185/pY187). The effect of various concentrations of PPA derivatives on the cell culture was studied using xCelligence RTCA equipment. The compounds were found to modulate JNK, ERK1/2, and p38 signaling pathways. The set of activated kinase cascades suggests that oxidative stress is the main probable mechanism of the toxic action of PPA derivatives.

In vitro cytotoxicity against K562 tumor cell line, antibacterial, antioxidant, antifungal and catalytic activities of biosynthesized silver nanoparticles using Sophora pachycarpa extract

In the present study, we demonstrate the green synthesis of silver nanoparticles using Sophora pachycarpa extract ( S. pachycarpa; SPE ) as capping, reducing, and stabilizing agents. The biosynthesized silver nanoparticles ( SPE- AgNPs) were tested for catalytic, antibacterial, antifungal, antioxidant, and anti-cancer activities. The affecting parameters (the concentration of silver nitrate, the temperature of the reaction, and time of reaction) on the synthesis process were optimized. The biosynthesized SPE- AgNPs were studied by X-Ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDS) and Fourier-transform infrared spectroscopy (FT-IR). The FESEM and TEM results revealed spherical and oval-like morphology with sizes ranging from 30 to 40 nm. Photocatalytic performance experiments of SPE- AgNPs were determined by the rapid degradation of the eriochrome black T (EBT) and methylene blue (MB) under sunlight and UV irradiations. The results showed that SPE -AgNPs degraded more than 90% and 80% of both dyes under UV and sunlight irradiations, respectively. In addition, the SPE -AgNPs exhibited good antibacterial and antifungal properties against S. aureus , S. epidermidis , P. aeruginosa , E. coli , K. pneumoniae , E. faecalis , and C. albicans with MIC values of 6.25, 6.25, 0.78, 0.39, 0.78, 1.56 and 0.78 µg/ml. The green synthesized SPE -AgNPs were found to inhibit the activity of DPPH free radicals efficiently. Eventually, the SPE -AgNPs exhibited significant in vitro cytotoxicity against K562 tumor cell line (IC50 = 19.5 µg/ml). All these studies indicated that AgNPs synthesized using S. pachycarpa extract have applications in the environmental and biomedical fields.

Cytotoxic activity in basal and tumoral cell lines of the C0K3N3 protein from the snake venom Crotalus durissus collilineatus, variety crotamine negative

Snake venoms are natural sources of bioactive substances with therapeutic potential. In this work, we evaluated the cytotoxicity of the Crotalus durissus collilineatus, negative crotamine variety and the isolated fraction C0K3N3 in BALB C/3T3 and K562 cell lines. The results indicate that the C0K3N3 protein is more cytotoxic against the K562 tumor cell line than in the 3T3 baseline.

Synthesis and anticancer activity of Pt(II) complexes of spiro-5-substituted 2,4-dithiohydantoins

Despite the widely studied anticancer activity of metal complexes of various hydantoin derivatives, reports on biological properties of dithiohydantoin complexes are limited to only few. Main challenge is determination of their structure, since no crystallographic data are available for metal complexes of dithiohydantoins. Herein, we describe the synthesis and structure of Pt(II) complexes of two spiro-5-(2,4-dihiohydantoins) with large and potentially intercalating aromatic rings, namely 3′,4′-dihydro-2H,2′H,5H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dithione and spiro-(fluorene-9,4′-imidazolidine)-2′,5′-dithione. The anticancer activity of all compounds were tested against HL-60, BV-173, and K-562 tumor cell lines. Single-crystal X-ray analysis was performed for the spiro-(benzocyclohexane-4′-imidazolidine)-2′,5′-dithione, whereas all other compounds were characterized by integrated approach of spectroscopic (IR and solid-state NMR) and theoretical (DFT) studies. The analysis of the data suggested Pt2L4 composition of the complexes with bridging N^S coordination of four ligand molecules and two Pt(II) centers of square planar geometry. Polymeric structure in the solid state was also presumed. The complexes showed modest to moderate anticancer activity as compared to cisplatin and the chronic myeloid leukemia derived BV-173 proved to be the most responsive tumor model.

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast.

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a–c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82–97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7–30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6–18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions.

Detecting long-range interactions between migrating cells

Chemotaxis enables cells to systematically approach distant targets that emit a diffusible guiding substance. However, the visual observation of an encounter between a cell and a target does not necessarily indicate the presence of a chemotactic approach mechanism, as even a blindly migrating cell can come across a target by chance. To distinguish between the chemotactic approach and blind migration, we present an objective method that is based on the analysis of time-lapse recorded cell migration trajectories: For each movement step of a cell relative to the position of a potential target, we compute a p value that quantifies the likelihood of the movement direction under the null-hypothesis of blind migration. The resulting distribution of p values, pooled over all recorded cell trajectories, is then compared to an ensemble of reference distributions in which the positions of targets are randomized. First, we validate our method with simulated data, demonstrating that it reliably detects the presence or absence of remote cell-cell interactions. In a second step, we apply the method to data from three-dimensional collagen gels, interspersed with highly migratory natural killer (NK) cells that were derived from two different human donors. We find for one of the donors an attractive interaction between the NK cells, pointing to a cooperative behavior of these immune cells. When adding nearly stationary K562 tumor cells to the system, we find a repulsive interaction between K562 and NK cells for one of the donors. By contrast, we find attractive interactions between NK cells and an IL-15-secreting variant of K562 tumor cells. We therefore speculate that NK cells find wild-type tumor cells only by chance, but are programmed to leave a target quickly after a close encounter. We provide a freely available Python implementation of our p value method that can serve as a general tool for detecting long-range interactions in collective systems of self-driven agents.

Abstract 1555: Enhanced HER2-dependent immune activation by margetuximab, an investigational Fc-engineered anti-HER2 mAb, supports combination with checkpoint blockade

Abstract Background: Margetuximab (M) has demonstrated anti-tumor activity in patients with advanced HER2+ gastric cancer and PFS superiority to trastuzumab (T) in pre-treated metastatic HER2+ breast cancer patients (1). Similar to T, M inhibits HER2 signaling; additionally, M enhances engagement with the activating Fcγ receptor (FcR)IIIA, while diminishing interaction with the inhibitory FcRIIB. Given the role of FcRs in coupling innate and adaptive immune responses, clinical studies of M in combination with PD-1 or PD-1 plus LAG-3 blockade have been initiated (2,3). The combination of M with tebotelimab, an investigational bispecific DART® molecule blocking PD-1 and LAG-3, has demonstrated encouraging early evidence of anti-tumor activity in patients with advanced HER2+ malignancies, including those with PD-L1-negative tumors (3). Here we present data supporting the potential mechanisms underlying the anti-tumor activity elicited by M and tebotelimab combination therapy. Methods: PBMCs were challenged with HER2-expressing tumor cell lines in the presence of M or T. Immune modulation was assessed by analysis of immune cell phenotype, gene expression profiles, and cytokine secretome. Functional activity of immune cells following exposure to the combination of M with tebotelimab was evaluated. Results: M induced greater upregulation relative to T of both co-stimulatory (eg 4-1BB) and/or checkpoint molecules (eg PD-L1 and LAG-3) on NK cells and/or monocytes in PBMCs cocultured with HER2-expressing tumor cells. Compared with T, M also induced greater proinflammatory cytokine release, including IFNγ that in turn upregulates PD-L1 and MHC class I expression on co-cultured tumor cells. These effects were not observed with an Fc-null version of M or were inhibited by the addition of FcR-blocking Abs, demonstrating Fc:FcR dependency of M-induced immune activation. Interestingly, CD8 T cells, which lack FcR, also exhibited LAG-3 upregulation in the above cultures, suggesting the effects of M can also be mediated via a cascade mechanism. An IFNγ neutralizing mAb reduced tumor cell-associated PD-L1 and MHC class I upregulation, but not LAG-3 induction on T-cells. PBMCs, pre-treated with a combination of M and tebotelimab in the presence of HER2-expressing tumor cells, demonstrated enhanced cytolytic activity against both the canonical NK cell target, K562, or M-opsonized HER2-positive tumor cells. Conclusions: M, through an Fc-dependent mechanism, can upregulate checkpoint molecules on NK-cells, CD8 T cells and tumor cells, thereby sensitizing them to immune checkpoint blockade. In turn, PD-1 and LAG-3 blockade by tebotelimab enhances M-mediated NK cell cytolytic activity in vitro.

Effect of emoxipine on cytotoxicity of peripheral blood mononuclears under cultivation with cytarabine and cyclocytidine

Taking into account the special role of oxidative stress that increases during cancer chemotherapy, the effect of the antioxidant emoxipine on peripheral blood mononuclears was studied under conditions that simulate the cytotoxic effects of antimetabolites of a number of modified cytidine nucleosides in relation to the tumor cell line K562. Lymphoid cells were also a source for subsequent modelling of the immune response to the cancer. It was found that neither the modified nucleosides themselves nor their combination with emoxipine caused changes in IL-2-stimulated cytotoxicity of lymphoid cells in relation to K562 tumor cell line. A study of the expression of the CD107a marker showed a significant stimulating effect of 1 µmol/L of citarabine on the activation of subpopulations of T-lymphocytes (CD3+ ) and cytotoxic T-lymphocytes (CD3+ CD8+ ).

Overcoming the UCB HSCs \u2013Derived NK cells Dysfunction through Harnessing RAS/MAPK, IGF-1R and TGF-\u03b2 Signaling Pathways

BackgroundThe natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. This is due to the possibility to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC-derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-β signaling pathways using IL-15, IGF-1 and SIS3 respectively.MethodsThe CD34 + cells were isolated from human UCB mononuclear cells through magnetic activation cell sorting (MACS) with purity of (≥ 90%) and were subjected to differentiate into NK cells. After 21 days of induction with SFTG36 (SCF, FLt-3L, TPO, GM-CSF, IL-3 and IL-6), IS721 (IGF-1, SIS3, IL-7 and IL-21) and IL-15/Hsp70 media, NK cells phenotypes were studied and their cytotoxicity against K562 human erythroleukemia cells and SKOV3 ovarian carcinoma cells was analyzed.ResultsThe NK cells induced in SFTG36/IS721 medium were selected for activation due to their higher expression of CD56 + 16 + CD3 − (93.23% ± 0.75) and mean fluorescence intensity (MFI) of NKG2D + (168.66 ± 20.00) and also a higher fold expansion potential (11.893 ± 1.712) compared to the other groups. These cells once activated with IL-15, demonstrated a higher cytotoxicity against K562 (≥ 90%; P ≤ 0.001) and SKOV3 tumor cells (≥ 65%; P ≤ 0.001) compared to IL-15/Hsp70-activated NK cells.ConclusionsThe differentiation of ex vivo expanded CD34 + cells through manipulation of RAS/MAPK, IGF-1R and TGF-β signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.