Killing Of Targets Research Articles

Pharmacokinetic and Pharmacodynamic Considerations of Antibiotics of Last Resort in Treating Gram-Negative Infections in Adult Critically Ill Patients.

We provide an overview of antimicrobials that are considered last resort for the treatment of resistant gram-negative infections in adult critically ill patients. The role in therapy, pharmacodynamic (PD) goals, and pharmacokinetic (PK) changes in critical illness for aminoglycosides, polymyxins, tigecycline, fosfomycin, and fluoroquinolones are summarized. Altered PK in septic patients in the intensive care unit (ICU) is observed with many of our agents of last resort. Based on the available literature, dosage adjustments may be required to optimize PK parameters and meet PD targets for most effective bacterial killing. Data is limited, studies are conducted in heterogeneous patient populations, and conclusions are frequently conflicting. Strategic dosing regimens such as high-dose extended interval dosing of aminoglycosides or loading doses with colistin and polymyxin B are examples of ways to optimize antibiotic PK in critically ill patients. Benefits of these strategies must be balanced with risks of increased toxicity. Patients with resistant gram-negative infections may present with septic shock in the ICU. Sepsis can significantly alter the PK of antibiotics and require dosage adjustments to attain optimal drug levels. An understanding of PK and PD properties of these agents of last resort will help to maximize therapeutic efficacy while minimizing toxic effects.

Pentocin MQ1: A Novel, Broad-Spectrum, Pore-Forming Bacteriocin From Lactobacillus pentosus CS2 With Quorum Sensing Regulatory Mechanism and Biopreservative Potential.

Micrococcus luteus, Listeria monocytogenes, and Bacillus cereus are major food-borne pathogenic and spoilage bacteria. Emergence of antibiotic resistance and consumer demand for foods containing less of chemical preservatives led to a search for natural antimicrobials. A study aimed at characterizing, investigating the mechanism of action and regulation of biosynthesis and evaluating the biopreservative potential of pentocin from Lactobacillus pentosus CS2 was conducted. Pentocin MQ1 is a novel bacteriocin isolated from L. pentosus CS2 of coconut shake origin. The purification strategy involved adsorption-desorption of bacteriocin followed by RP-HPLC. It has a molecular weight of 2110.672 Da as determined by MALDI-TOF mass spectrometry and a molar extinction value of 298.82 M−1 cm−1. Pentocin MQ1 is not plasmid-borne and its biosynthesis is regulated by a quorum sensing mechanism. It has a broad spectrum of antibacterial activity, exhibited high chemical, thermal and pH stability but proved sensitive to proteolytic enzymes. It is potent against M. luteus, B. cereus, and L. monocytogenes at micromolar concentrations. It is quick-acting and exhibited a bactericidal mode of action against its targets. Target killing was mediated by pore formation. We report for the first time membrane permeabilization as a mechanism of action of the pentocin from the study against Gram-positive bacteria. Pentocin MQ1 is a cell wall-associated bacteriocin. Application of pentocin MQ1 improved the microbiological quality and extended the shelf life of fresh banana. This is the first report on the biopreservation of banana using bacteriocin. These findings place pentocin MQ1 as a potential biopreservative for further evaluation in food and medical applications.

Abstract B009: Imprime PGG, a soluble yeast b-glucan PAMP, activates both innate and adaptive immune effector cells, resulting in enhanced antitumor responses that synergize with anti-PD-1 antibody therapy

Abstract Immune checkpoint therapy has made a splash in the cancer world by providing long-term durable responses in patients across multiple cancer indications. However, not all patients respond to checkpoint intervention as this therapeutic relies on an underlying recognition and activation of T cells against the tumor. Furthermore, recent evidence suggest that acquired resistance to CPIs may occur through beta-2 microglobulin mutations and loss of MHC class I-restricted T-cell responses, in which case other cytotoxic effector cells like natural killer (NK) cells could play a critical role. Therefore, combination therapies utilizing checkpoints and innate immune modulators such as PAMPs (pathogen-associated molecular patterns) are being evaluated to expand both the magnitude and duration of the response, as well as the responding patient population. Imprime PGG (Imprime) is a soluble, systemically delivered yeast 1,3/1,6 β-glucan PAMP capable of triggering innate immune cell function leading to a cascade of immune activation and enhanced antitumor killing. Imprime is currently being evaluated in combination with anti-PD1 therapy in multiple clinical trials. Here we employ a combination of experimental techniques, including both flow cytometry and multiplex immunofluorescence (IFC), to examine how Imprime modulates the antitumor response in the MC38 murine adenocarcinoma model. C57BL/6 mice were injected with MC38 cells subcutaneously and randomized into 4 treatment groups (vehicle, Imprime, anti-PD1, Imprime+anti-PD1) once tumors reached ~50 mm3. After 2 weeks of treatment, mice were evaluated for inhibition of tumor growth, presence and activation of tumor-infiltrating immune cells by IFC, and the induction of polyfunctional T cells by flow cytometry. Mice treated with the combination of Imprime and anti-PD1 had significantly smaller tumors than all other groups, including those treated with anti-PD1 alone. IFC-based analysis of these tumors showed that these mice had increased frequencies of CD8+GranzymeB+ cells, indicative of an enhanced antitumor T-cell response. TILs from these tumors also showed increased frequency of IL-2 production after stimulation ex vivo with a pool of known MC38 tumor antigens. Interestingly, there was also an increase in a CD3-GranzymeB+ population (also negative for B220, F4/80, Ly6C, Ly6G, MHCII) in the tumor of mice treated with the combination. We classified these as most likely to be NK cells. In peripheral blood, flow cytometric analysis of NK1.1+ NK cells also showed increased GranzymeB expression. To explore the impact of Imprime on NK-mediated tumor killing, we further evaluated in vivo killing of MHCI-deficient cancer cell targets after intravenous administration of Imprime. In these experiments Imprime was able to enhance the NK cytotoxic killing of the targets, providing further evidence that Imprime effects innate effector function. These data demonstrate a unique synergy between anti-PD1 and Imprime treatment. The enhanced cytotoxic T-cell response coupled with enhanced effector NK function represents an exciting novel mechanism that may provide enhanced therapeutic benefit to patients in our ongoing clinical trials. Citation Format: Steven Leonardo, Ross B. Fulton, Kathryn A. Fraser, Takashi O. Kangas, Keith B. Gorden, Ben Harrison, Adria LB Jonas, Anissa SH Chan, Yumi Yokayama, Nandita Bose, Jeremy R. Graff, Mark Uhlik. Imprime PGG, a soluble yeast b-glucan PAMP, activates both innate and adaptive immune effector cells, resulting in enhanced antitumor responses that synergize with anti-PD-1 antibody therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B009.

The research significance of concomitant use of CAR-CD138-NK and CAR-CD19-NK to target multiple myelomas

Multiple myeloma (MM) is a type of cancer characterized by abnormal proliferation of clonal cells; it is the very dangerous and highly prevalent disease. Although significant progress has been made in clinical research, especially with novel drugs such as bortezomib, lenalidomide, and carfilzomib, most of the patients with MM still suffer from often fetal relapses due to drug resistance. In this study, we aimed to develop immune cells that could specifically target and destroy MM cells. Chimeric antigen receptor–modified NK-92 (CAR-NK92) cells have been very effective against B-cell acute lymphoblastic leukemia (B-ALL); as MM shows high expression of CD138, we constructed CD138-directed CAR-NK-92MI cells (CAR-CD138). It 2is reported that there is a small subset of CD138–/CD19+ MM cells showing, to some extent, stem cell qualities. We therefore generated the CD19-directed CAR-NK-92MI cells (CAR-CD19) as well. These two CAR-NK cells showed strong in vitro biological activity in specifically killing target tumor cells. Thus, the concomitant use of these CAR-NK cells may achieve excellent results in vivo.

Google Maps Security Concerns

Google Maps and other such maps in GIS have a lot of significance in every one’s life for in modern world due to technological development as well as contemporary needs in travelling, business planning, agriculture, e-marketing supply chain management, census and planning and excessive use of mobile phones. Being a revolutionary technology, it attracts the users from its inception. It has been revolutionary in having an impact on one’s daily life by helping one explore geographical locations virtually anywhere on the whole planet. It has become a norm that people use Google Maps before or while commuting to a certain place as most of the people rely on it to provide the shortest or fastest route to a destination. Google Maps has had a profound impact not only one’s personal life, but has opened new avenues of marketing, business intelligence, urban planning, infrastructure strategy development, as well as traffic engineering. Hence, no one can deny the impact it has had on our society in a short period. However, Google Maps has security concerns associated with its use. This is because whenever a user is searching for a geographical location on Google Maps, there is no way to ascertain his/her intention. As result, whatever is requested by the user, it is provided without much security checks or personal specific logging history. Criminal minded people may use the technology to carry out unwarranted and uncalled-for activities such as terror attacks, exploitation of military assets, target killing, trailing a potential victim, kidnapping, demanding ransom money, and compromising national security etc. Numbers of such activities may be carried out using the guidance from “Google Maps” without getting noticed. This is largely due to that anonymous login is allowed into Google Maps. The paper highlights the main security issues that exist in the use of Google Maps and suggests the key areas to improve upon. In this research paper, we discuss the security concerns related to Google Maps utilization and try to identify the associated risks that may be worst in some situations due to that it is significant to assess the usage of this tremendous technology. We categorize the millions of Anonymous Google Maps users into two major categories (Anonymous Good Users and Anonymous Bad Users) based on Google Maps browsing and analyze the associated risks and potential threats and propose the way to minimize them. In future, if Google Maps adopts the proposed techniques and improves upon, then its utilization minimizes security concerns and makes the world much safe place.

Resistance to SL-401 in AML and BPDCN Is Associated with Loss of the Diphthamide Synthesis Pathway Enzyme DPH1 and Is Reversible By Azacitidine

SL-401 is a novel targeted therapy comprised of recombinant interleukin 3 (IL3) fused to a truncated diphtheria toxin (DT) payload. SL-401 delivers DT to cells expressing the IL3 receptor (CD123). After internalization, DT catalyzes ADP ribosylation of eukaryotic elongation factor 2 (eEF2), blocking protein synthesis and killing target cells. SL-401 is currently in clinical trials for CD123+ cancers, including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Other than CD123 expression, the determinants of response are largely unknown. Our goal was to study mechanisms of de novo and acquired resistance to inform combination strategies and enhance the efficacy of SL-401.In 16 AML and BPDCN patients enrolled in a phase 1-2 trial, we did not see decreased expression of CD123 by flow cytometry during or after SL-401. To study alternative resistance mechanisms, we generated 3-6 independent SL-401 resistant clones from each of 4 CD123+ AML (THP1, NOMO1, EOL1) or BPDCN (CAL1) cell lines. We treated cells with the LD95 (lethal dose to 95%), retreating upon recovery. All lines developed >2-3 log resistance to SL-401 within 28 days. All resistant clones maintained CD123 surface expression, consistent with what we observed in patients. Using confocal microscopy, we saw that a fluorescently tagged SL-401 was internalized equally in resistant and parental cells. SL-401 resistant cells were also resistant to full length DT, suggesting that the mechanism of resistance involved DT rather than IL3 binding/internalization.We performed whole transcriptome RNA-sequencing and whole exome sequencing (WES) on parental and SL-401 resistant cells. There were no recurrent acquired DNA mutations. However, in RNA-seq the most downregulated gene in 6 independent clones from 2 lines was DPH1 (FC -7.5, FDR<0.0001). DPH1 is the first enzyme in a cascade that converts histidine 715 on eEF2 to diphthamide, the direct target for ADP ribosylation by DT. Decreased expression of DPH1 was confirmed in the 6 resistant clones by qRT-PCR, and in 3 clones from an additional line. Across 33 cell lines and subclones there was an inverse linear correlation between DPH1 level and SL-401 IC50 (P=0.0005). To validate this finding in patients, we performed paired RNA-seq on CD45+CD123+ sorted blasts from 2 AMLs pre & post 2 cycles of SL-401. Both patients' AMLs had reduced DPH1 after exposure to SL-401 (mean -2.1 fold). To determine if loss of DPH1 was sufficient to confer de novo resistance to SL-401, we generated THP1, NOMO1, and CAL1 cells stably expressing the Cas9 nuclease and transduced them with 1 of 4 DPH1-targeting or 2 non-targeting CRISPR sgRNAs co-expressing GFP. Four days after infection at titers that achieve ~20% GFP+ cells, we treated with the LD95 of SL-401. We observed a survival advantage for DPH1 sgRNA-transduced cells (2.9 fold increase in GFP+ cells, P<0.0001) and decreased apoptosis measured by AnnexinV positivity, compared to GFP- cells in the same cultures. By contrast, there was no survival advantage for cells transduced with control sgRNAs. These data suggest that DPH1 loss is sufficient to mediate SL-401 resistance in AML and BPDCN.Pseudomonas toxin (PT) also targets eEF2 on diphthamide, and PT resistance in a B-ALL cell line has been associated with DPH1 loss via reversible promoter CpG DNA methylation (Hu, Leuk Res 2013). Therefore, we tested the DNA methyltransferase inhibitor azacitidine in combination with SL-401 and observed synergistic cytotoxicity, in naïve (combination index (CI) = 0.45; <1 indicates synergy) and SL-401 resistant (CI = 0.55) cells. Most strikingly, 4-week pulsatile treatment with non-lethal “epigenetic” doses of azacitidine (300 nM 2d on/2d off) fully reversed SL-401 resistance in 6 CAL1 and THP1 clones that were insensitive at baseline (Figure). Controls grown in vehicle or with weekly SL-401 challenge showed no reversion, suggesting that azacitidine had a specific sensitizing effect. Restoration of SL-401 sensitivity was accompanied by an increase in DPH1 expression compared to resistant clones.In summary, we found that DPH1 is a biomarker of SL-401 activity and acquired resistance, and resistance is reversible by azacitidine. Based on these data, we have initiated a multicenter phase 1 trial of the combination of SL-401 and azacitidine in patients with AML or MDS (NCT03113643), with correlative laboratory studies designed to explore these hypotheses. [Display omitted] DisclosuresvonEgypt:Stemline Therapeutics: Employment. Lindsay:Stemline Therapeutics: Employment. Brooks:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. Lane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

Lawsone derivatives target the Wnt/β-catenin signaling pathway in multidrug-resistant acute lymphoblastic leukemia cells

Multidrug resistance (MDR) represents a serious problem in cancer treatment. One strategy to overcome this obstacle is to identify agents that are selectively lethal to MDR cells. The aim of this study was to discover novel compounds against MDR leukemia and to determine the molecular mechanisms behind collateral sensitivity. A library of 1162 compounds was tested against parental, drug-sensitive CCRF-CEM cells using the resazurin assay. A total of 302 compounds showed reasonable activity (less than 50% cell viability). Eleven out of 30 lawsone derivatives revealed considerable collateral sensitivity in MDR P-glycoprotein (Pgp)-overexpressing CEM/ADR5000 cells. They reduced β-catenin activity in a Wnt/β-catenin reporter cell line. Their activities significantly correlated with apolar desolvation (R = 0.819). Compound (1) (3-hydroxy-1,4-dioxo-N-phenyl-naphthalene-2-carboxamide) was the most active compound and dose-dependently down-regulated protein expression of β-catenin, c-MYC, Pgp and Frizzled 7. By molecular docking, we predicted that compound (1) bound to the palmitoyl-binding groove of the cysteine-rich domain of Frizzled-7 and Frizzled-8. Compound (1) neither stimulated ATPase activity of Pgp nor reactive oxygen species generation, both of which have been previously described as possible mechanisms of collateral sensitivity. Instead, we found that Wnt/β-catenin signaling was selectively inhibited in CEM/ADR5000 cells, but not in CCRF-CEM cells. In conclusion, we found for the first time that the inhibition of Wnt/β-catenin signaling may represent a novel molecular mechanism of collateral sensitivity in MDR cells. Wnt/β-catenin signaling, therefore, represents a potential therapeutic target for the selective killing of Pgp-mediated MDR.

A single exercise bout augments the ex vivo expansion and anti-tumor activity of Vdelta2 T-cells: Implications for allogeneic adoptive transfer immunotherapy

Gamma-delta T-Cells are emerging candidates for allogeneic (donor to patient) adoptive transfer immunotherapy to treat both solid and ‘liquid’ tumors. However, the numbers of γ - δ T-cell in blood are relatively low and new, cost effective methods are sought to facilitate their mobilization and ex vivo expansion from healthy donors. The aim of this study was to determine if a single exercise bout would augment the ex vivo expansion and function of γ - δ T-cells using a clinical grade protocol designed to preferentially expand the Vdelta2 subtype. Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 12) before and immediately after 30-min of steady state cycling exercise were expanded with zoledronic acid and IL-2 for 14 days. Exercise doubled the rate of γ - δ T-cell expansion, even after adjusting for pre-expansion gamma-delta T-cells numbers. Exercise did not affect γ - δ T-cell phenotypic shifts following expansion. Using a series of standard allogeneic tumor target cells in a 4 h cytotoxicity assay, γ - δ T-cells expanded after exercise were better at killing target cells of leukemia (K562), lymphoma (721.221) and multiple myeloma origin (U266) (increase: 1.5-fold compared to pre-exercise). We conclude that a single exercise bout in healthy donors markedly augments the ex vivo expansion and anti-tumor activity of gamma-delta T-cells. Exercising donors prior to blood donation for adoptive T-cell therapy may elicit better clinical outcomes in cancer patients after transplant.

Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter.

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%-50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation.Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs.Results: CD8+BTLA- TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA- T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened "serial killing" capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation.Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151-64. ©2017 AACR.

Downmodulation of Effector Functions in NK Cells upon Toxoplasma gondii Infection.

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. The rapid transfer of T. gondii from infected dendritic cells to effector natural killer (NK) cells may contribute to the parasite's sequestration and shielding from immune recognition shortly after infection. However, subversion of NK cell functions, such as cytotoxicity or production of proinflammatory cytokines, such as gamma interferon (IFN-γ), upon parasite infection might also be beneficial to the parasite. In the present study, we investigated the effects of T. gondii infection on NK cells. In vitro, infected NK cells were found to be poor at killing target cells and had reduced levels of IFN-γ production. This could be attributed in part to the inability of infected cells to form conjugates with their target cells. However, even upon NK1.1 cross-linking of NK cells, the infected NK cells also exhibited poor degranulation and IFN-γ production. Similarly, NK cells infected in vivo were also poor at killing target cells and producing IFN-γ. Increased levels of transforming growth factor β production, as well as increased levels of expression of SHP-1 in the cytosol of infected NK cells upon infection, were observed in infected NK cells. However, the phosphorylation of STAT4 was not altered in infected NK cells, suggesting that transcriptional regulation mediates the reduced IFN-γ production, which was confirmed by quantitative PCR. These data suggest that infection of NK cells by T. gondii impairs NK cell recognition of target cells and cytokine release, two mechanisms that independently could enhance T. gondii survival.

Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children.

Pediatric patients with recurrent, refractory or advanced soft tissue sarcoma (STS) who are simultaneously showing signs of cumulative treatment toxicity are in need of novel therapies. In this preclinical analysis, we identified ErbB2 as a targetable antigen on STS cells and used cytokine-induced killer (CIK) cells transduced with the lentiviral 2nd-generation chimeric antigen receptor (CAR) vector pS-5.28.z-IEW to target ErbB2-positive tumors. Solely CIK cell subsets with the CD3+ T cell phenotype showed up to 85% cell surface expression of the respective CAR. A comparison of wildtype (WT), mock-vector and ErbB2-CAR-CIK cells showed, that engineered cells exhibited diminished in vitro expansion, retained WT CIK cell phenotype with higher percentages of differentiated effector memory/effector cells. Activating natural killer (NK) cell receptor NKG2D-restricted target cell recognition and killing of WT and ErbB2-CAR-CIK cells was maintained against ErbB2-negative tumors, while ErbB2-CAR-CIK cells demonstrated significantly increased cytotoxicity against ErbB2-positive targets, including primary tumors. ErbB2-CAR- but not WT CIK cells proliferated, infiltrated and efficiently lysed tumor cell monolayers as well as 3D tumor spheroids.Here, we demonstrate a potential cell therapeutic approach using ErbB2-CAR-CIK cells for the recognition and elimination of tumor cells expressing ErbB2, which we identified as a targetable antigen on high-risk STS cells.

Abstract 4513: Investigation of bystander effects of hypoxia activated prodrugs using three dimensional cell cultures

Abstract Background: Hypoxia in solid tumors acts as the major barrier to anti-cancer therapies and promotes tumor progression. Hypoxia-activated prodrugs (HAPs) are designed to not only eliminate these resistant cells, but also to exploit hypoxia as a basis of tumor selectivity. A key concept in exploiting hypoxia with HAPs is that active drug metabolite should be able to diffuse from hypoxic zones to maximize effect in surrounding aerobic tumor tissue. There is some evidence that these ‘bystander effects’ contribute to the antitumor activity of HAPs, but currently there is a lack of robust methods for their detection. Objectives: To investigate bystander effects from HAPs in a novel spheroid co-culture system using PR104A which releases a diffusible DNA crosslinking mustard and the tirapazamine analogue SN30000 which is converted to a short lived radical that is not expected to diffuse out of cells. Methods: A new method for measuring the bystander effect with multicellular spheroids under hypoxic conditions was developed using co-cultures of HCT116 colon cancer cells with high metabolic capacity for HAP metabolism (activators, transfected to overexpress cytochrome P450 reductase [POR]) and cells that cannot activate HAPs efficiently (targets, with POR knocked out by zinc finger nuclease gene targeting) along with differential antibiotic resistance and fluorescent protein markers to enable their quantitation in mixed cultures. Sensitivity of activators and targets to PR104A and SN30000 was determined in spheroid co-cultures by dissociation and clonogenic cell survival assay. Results were compared to output of a novel agent-based mathematical model (ABM) of spheroid co-cultures. Results: In monolayer cultures, activator cells showed markedly higher sensitivity to the HAPs than did targets. Tagging of activators and targets with fluorescent proteins allowed visualization of their spatial distribution in spheroid co-cultures. When spheroids were treated with PR104A, killing of targets markedly increased with increasing proportion of activators, confirming a bystander effect. However, increasing proportions of activators reduced cell killing of targets after SN30000 treatment, suggesting that rapid metabolism by activators inhibits its penetration, which was confirmed by flux experiments in multicellular layer co-cultures. ABM simulations of cell killing by PR104A and SN30000 in spheroids using our published transport parameters agreed with experimental results indicating diffusion of active metabolites of PR104A but not SN30000. Discussion: Using this novel spheroid co-culture model, a bystander effect of PR104A was demonstrated while SN30000 showed no bystander effect. This is the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the utility of the novel spheroid ABM in simulating active metabolite diffusion in 3D co-cultures. Citation Format: Cho Rong Hong, Gib Bogle, William R. Wilson, Kevin O. Hicks. Investigation of bystander effects of hypoxia activated prodrugs using three dimensional cell cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4513. doi:10.1158/1538-7445.AM2017-4513

Abstract 4618: TGF-B upregulates GSK3B and mediates NK dysfunction in cancer

Abstract Transforming growth factor-B (TGF-B) is an important regulator of tumorigenesis. Initially, it was discovered to have anti-cancer activities by promoting apoptosis and preventing hyperproliferation of cells. However, abundant presence of TGF-B in the tumor microenvironment is known to hinder cancer eradication by suppressing anti-tumor activities of immune cells. The molecular switches that regulate the role of TGF-B are not fully understood. One main target of TGF-B-mediated immune cell dysfunction are natural killer (NK) cells. NK cells are innate immune cells that lyse virally-infected and cancer cells. Recently, we have discovered that glycogen synthase kinase 3-beta (GSK3B) upregulation in NK cells leads to NK dysfunction. Another study has indicated that GSK3 phosphorylates SMAD3 to inhibit TGF-B-mediated growth dysfunction in AML12 cells. In addition, GSK3B inhibition has been linked to increased sensitivity of cancer cells to NK killing but the role of GSK3B in NK function is understudied. This study examines how the role that GSK3B has on TGF-B-mediated NK cell dysfunction. Methods: NK cells were isolated from the peripheral blood of healthy donors and expanded in vitro for 14 days. NK cells were either treated with or without 5ng/mL TGF-B for 72 hours, and GSK3B expression, and the expression of NK activating receptors was measured via flow cytometry. In addition, GSK3B expression is abrogated using a siRNA in NK cells. The NK cells were treated with or without TGF-B, and NK cell cytotoxicity is measured with a fluorometric cytotoxicity assay. Results: TGF-B pretreatment of NK cells led to 2-fold GSK3B expression in NK cells. TGF-B pretreatment led to decreased expression of NK activating receptors NKp46 and NKG2D. Also, TGF-B led to decreased expression of lymphocyte function-associated antigen-1 (LFA-1), an adhesion molecule necessary for NK cells to adhere to target cells prior to lysis of the target cells. TGF-B pretreatment of NK cells led to 33.3% reduced killing of both leukemic and solid tumor targets. Pharmacologic and genetic abrogation of GSK3 minimized TGF-B-mediated NK dysfunction as measured by calcein-AM based cytotoxicity assays. Importantly, inhibiting GSK3B activity with TGF-B treatment rescued NK cell cytotoxic function. Discussion: Our data suggests that TGF-B leads to upregulated GSK3B expression, and downregulation of NK activating receptors. In addition, TGF-B reduced NK killing of cancer cells. We believe that TGF-B-mediated NK dysfunction is facilitated by GSK3B upregulation. Further studies are underway to determine how TGF-B affects the expression of other NK receptors, and to elucidate the mechanism by which TGF-B directs GSK3B upregulation. Citation Format: Evelyn Ojo, Folashade Otegbeye, Stephen Moreton, David Wald. TGF-B upregulates GSK3B and mediates NK dysfunction in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2017-4618

Abstract 825: Cetixumab enhanced apoptosis effects of radiation on human prostate cancer cell line PC-3

Abstract Prostate cancer is a leading cause of cancer death in men in developed countries. Radiotherapy is the main treatment to prostate cancer. Dose escalation of irradiation with image guide rasdiotherapy and intensity modulated radiotherapy were used with more and more evidences in prostate cancer patients and bring in longer PSA-free survival time. Although the evolution of radiation techniques bring better outcomes, but modern modalities were not avaliable in non-western world. Radiation enhancement is still an improtant issus in prostate cancer in non-western world, especially in androgen independnt prostate cancer. The epidermal growth factor receptor (EGFR) network has rich targets for prostate cancer killing. The chimeric monoclonal antibody Cetuximab (IMC-A12, Erbitux®) binds to EGFR and prevents its intracellular signaling. Currently, it is approved for treatment of wild-type KRAS colon and head and neck cancer. The effect and mechanisms of Erbitux and radiotherapy are still not clear. In this study, we evaluated the effects of combining the EGFR inhibition and radiation on PC-3 prostate cancer cells. PC-3 cells were treated with various doses(1,2.5,5 and 10 ug/ml) of anti-EGFR antibody (CETUXIMAB) and irradiation(5, 7.5 Gy). The results revealed that 5 and 10 ug/ml of CETUXIMAB significnatly reduced the colony formation. The cell cycle analysis revealed that CETUXIMAB significnatly enhanced the radiation induced G2/M phase arrest and subG1 peak increasing. The Hoechst staining and DNA laddering analysis revealed radiation and CETUXIMAB combined use will induced more apoptotic bodies or DNA fragmentation than radiation alone use. By using a apaptosis protein array, we found that several apaptosis related protein (Catalase, cIAP-1, Clusterin, Fas, HIF-1α, HSP27, HSP60, HSP70, SMAC and Survivin) were significantly downregulated in radiation and CETUXIMAB combined group. Taken together, our results demonstrated that Erbitux enhanced apoptosis effects of radiation on PC-3. Citation Format: Chia-lun Chang, Szu-Yuan Wu. Cetixumab enhanced apoptosis effects of radiation on human prostate cancer cell line PC-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 825. doi:10.1158/1538-7445.AM2017-825

Abstract 3750: Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity

Abstract Introduction: Adoptive immune therapy has achieved great success in eradicating blood-borne cancers, prominently, the CD19 CAR T cells in B cell leukemia and lymphomas. However, CAR T cell therapy in solid tumors has been limited due to the scarcity of tumor antigens that are deemed safe for targeting. One strategy to overcome scarcity of tumor antigen is by tuning the affinity of CAR to limit T cell reaction with cells overexpressing target antigen while sparing cells with basal level expression. To rigorously test the idea of “affinity tuning”, we built variants of CARs possessing one million-fold difference in affinity spanning low nanomolar to high micromolar affinity to a target antigen, and examined the influence of CAR affinity on the rate of tumor killing and systemic toxicity. Methods: Antigen-binding domain of CAR was built from the inserted or I domain belonging to integrin LFA-1. Affinity of CAR expressed in T cells was confirmed by ICAM-1 binding by flow cytometry. For in vivo study, mice with systemic growth of ICAM-1 positive thyroid tumor were used, where tumors grew in lungs, liver, and bones. Tumor growth and killing were monitored by whole body luminescence imaging. Sera were collected for cytokine analysis. Body weight, cytokine profile, and overall behavior were used to assess the severity of systemic toxicity. Results: CAR T cells with a step-wise, one million-fold variation in affinity to ICAM-1 resulted in a rate of target killing in proportion to the increase in affinity and in ICAM-1 density. Owing to cross-reaction of human LFA-1 with murine ICAM-1, the influence of CAR affinity on efficacy and on-target, off-tumor toxicity was tested in mice bearing ICAM-1 positive human tumors. In vivo tumor elimination by CAR T cells was in contrast to in vitro affinity-dependent rate of target killing, demonstrating that micromolar affinity CAR T cells was superior to nanomolar affinity T cells in both tumor killing and safety aspects. Highest affinity CAR T cells led to uniform death of the host, caused by on-target, off-tumor toxicity, and high level cytokine release. Conclusion: Our study is the first comprehensive report examining the effect of CAR affinity on the rate of tumor killing, efficacy, and toxicity. In contrast to in vitro tumor killing effect, the increase in affinity of CAR beyond certain threshold was deleterious to T cell persistence and associated with more frequent tumor relapse. Our study highlights that CAR T cells approximating natural T cell receptor affinity is more efficacious in eliminating tumors with overexpressed antigens, and is safer by avoiding potential reaction with normal cells with basal expression of the same antigen. Citation Format: Enda Shevlin, Spencer Park, Yogindra Vedvyas, Marjan Zaman, Susan Park, Irene M. Min, Moonsoo M. Jin. Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3750. doi:10.1158/1538-7445.AM2017-3750

Apoptosis Induced via Gamma Delta T Cell Antigen Receptor "Blocking" Antibodies: A Cautionary Tale.

Mechanistic studies contribute greatly to our understanding of γδ T cell (γδTc) biology, aiding development of these cells as immunotherapeutic agents. The antibody blocking assay is an accepted method to determine the receptors involved in γδTc killing of tumor targets. Effectors and/or targets are preincubated with microgram quantities of monoclonal antibodies (mAb), often described by commercial sources to be useful for blocking assays. We and others have used such assays extensively in the past, correlating decreases in cytotoxicity against specific targets with involvement of the blocked receptor(s). However, we wondered whether other mechanisms might be at play beyond cytotoxicity inhibition. Indeed, administration of certain “blocking” mAb to the γδ T cell antigen receptor (γδTCR) induced γδTc death. Upon further investigation, we discovered that γδTc underwent apoptosis triggered by incubation with mAb to the γδTCR. This effect was specific, as no apoptosis was observed when αβ T cells (αβTc) were incubated with these mAb. Apoptosis was further potentiated by the presence of interleukin (IL)-2, often included in cytotoxicity assays; however, exogenous interleukin-2 (IL-2) did not contribute significantly to γδTc cytotoxicity against breast cancer cell lines. Here, we have investigated the usefulness of four mAb for use in blocking assays by assessing blocking properties in conjunction with their propensity to induce apoptosis in cultured primary human γδTc. We found that the 5A6.E9 clone was usually a better alternative to the commonly used B1 (or B1.1) and 11F2 clones; however, some variability in susceptibility to apoptosis induction was observed among donor cultures. Thus, viability assessment of primary effector cells treated with mAb alone should be undertaken in parallel with cytotoxicity assays employing blocking antibodies, to account for cytotoxicity reduction caused by effector cell death. Previous findings should be reassessed in this light.

Delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination

DNA rich in unmethylated CG motifs (CpGs) engage Toll-Like Receptor 9 (TLR-9) in endosomes and are well described stimulators of the innate and adaptive immune system. CpGs therefore can efficiently improve vaccines' immunogenicity. Packaging CpGs into nanoparticles, in particular into virus-like particles (VLPs), improves the pharmacological characteristics of CpGs as the protein shell protects them from DNAse activity and delivers the oligomers to the endosomal compartments of professional antigen presenting cells (APCs). The current consensus in packaging and delivering CpGs in VLP-based vaccines is that both adjuvants and antigens should be kept in close proximity (i.e. physically linked) to ensure delivery of antigens and adjuvants to the same APCs. In the current study, we harness the draining properties of the lymphatic system and show that also non-linked VLPs are efficiently co-delivered to the same APCs in lymph nodes. Specifically, we have shown that CpGs can be packaged in one VLP and mixed with another VLP displaying the antigen prior to administration in vivo. Both VLPs efficiently reached the same draining lymph node where they were taken up and processed by the same APCs, namely dendritic cells and macrophages. This resulted in induction of specific CTLs producing cytokines and killing target cells in vivo at levels seen when using VLPs containing both CpGs and chemically conjugated antigen. Thus, delivery of antigens and adjuvants in separate nanoparticles eliminates the need of physical conjugation and thus can be beneficial when designing precision medicine VLP-based vaccines or help to re-formulate existing VLP vaccines not naturally carrying immunostimulatory sequences.

Markers and function of human NK cells in normal and pathological conditions.

Natural killer (NK) cells, the most important effectors of the innate lymphoid cells (ILCs), play a fundamental role in tumor immune-surveillance, defense against viruses and, in general, in innate immune responses. NK cell activation is mediated by several activating receptors and co-receptors able to recognize ligands on virus-infected or tumor cells. To prevent healthy cells from auto-aggression, NK cells are provided with strong inhibitory receptors (KIRs and NKG2A) which recognize HLA class I molecules on target cells and, sensing their level of expression, allow killing of targets underexpressing HLA-class I. In vivo, NK cell-mediated anti-tumor function may be suppressed by tumor or tumor-associated cells via inhibitory soluble factors/cytokines or the engagement of the so called immune-check point molecules (e.g., PD1-PDL1). The study of these immune check-points is now offering new important opportunities for the therapy of cancer. In haemopoietic stem cell transplantation, alloreactive NK cells (i.e., those that express KIRs, which do not recognize HLA class I molecules on patient cells), derived from HSC of haploidentical donors, are able to kill leukemia blasts and patient's DC, thus preventing both tumor relapses and graft-versus-host disease. A clear correlation exists between size of the alloreactive NK cell population and clinical outcome. Thus, in view of the recent major advances in cancer therapy based on immuno-mediated mechanisms, the phenotypic analysis of cells and molecules involved in these mechanisms plays an increasingly major role. © 2017 International Clinical Cytometry Society.

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

ABSTRACTNatural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses.Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood.Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcϵRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcϵRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli.These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.