Cell Killing Assays Research Articles

Abstract 143: Stroke Impairs Endothelial Cells Through Downregulation of Programed Death Ligand-1 Expression

Introduction: Stroke triggers a robust inflammatory response, allowing leukocytes to cross the blood brain barrier. T cells infiltrate in an antigen-independent manner early after injury, leading to additional neuronal injury. The pro-inflammatory cytokine, interferon-gamma (IFNγ), upregulates programed death ligand-1 (PD-L1) expression. Engagement of PD-L1 and programed death-1 (PD-1) “calls off” the cytolytic activity of infiltrated T cells. The immunosuppressive activity of PD-L1 is modulated by ubiquitination and glycosylation. Glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces degradation of PD-L1. To date, the role of PD-L1 in endothelial cells (ECs) remains elusive. Hypothesis: PD-L1 in EC reduces neuroinflammation via protection of microvascular integrity. Methods: Human brain ECs, HBEC-5i, were subjected to IFNγ treatment in normal or oxygen-glucose deprivation (OGD) conditions. Western blot, Fluorescence-activated cell sorting analysis, PD-1 binding assays and an in vitro T cell killing assay were performed to investigate EC viability and PD-L1 function. Male mice (12 weeks) were subjected to a 60-minute middle cerebral artery occlusion (MCAO) and given either vehicle or GSK3β inhibitor VIII (4 mg/kg). We examined PD-L1 expression, blood vessel integrity and T cell activation in brain tissue using anti-PD-L1, p-GSK3β, CD31, and Granzyme B antibodies for immunohistochemistry (IHC). Results: IFNγ strongly induced PD-L1 expression in ECs. However, when ECs were exposed to OGD, IFNγ-mediated PD-L1 upregulation was compromised (from 48.9% to 31.4%). PD-L1 binding affinity of EC was also reduced after OGD (from 33.1% to 1.3%). Consistently, human (n=22) and mice (n=6) brain IHC staining found lower levels of PD-L1 and p-GSK3β in ECs and higher expression of Granzyme B in stroke samples compared to controls (p<0.05). MCAO mice treated with GSK3β inhibitor VIII showed higher PD-L1 expression and smaller infarcts. Conclusion: This study reveals a protective role of PD-L1 in ECs during early brain injury. Activation of GSK3β by stroke destabilized endothelial PD-L1. Thus, stabilizing PD-L1 or preventing downregulation of PD-L1 may maintain microvascular integrity and reduce neuroinflammation after stroke.

Host Resistance Assays.

The goal of immunotoxicity testing is to obtain data useful for immunotoxicity safety assessment. Guidance in the performance of immunotoxicity safety evaluations is provided in documents from the US EPA for chemicals and the ICH S8 document for pharmaceuticals. The ICH S8 document outlines a tiered approach that includes (1) standard toxicity studies with associated hematology, immune system organ weights, and histopathology data; (2) functional assays, such as cytotoxic T lymphocyte (CTL) assays, natural killer (NK) cell assays, respiratory burst, phagocytosis, and T-cell-dependent antibody response (TDAR) assays; and (3) host resistance assays. Host resistance assays are considered the gold standard in immunotoxicity testing and provide a critical overview of the extent to which innate, adaptive, and homeostatic regulatory immune functions are integrated to protect the host. Both comprehensive and targeted host resistance assays are available, each with distinct advantages. This chapter serves to provide a general overview of the various assays that may be used, as well as a summary of procedures.

Ambient Aqueous Synthesis of Ultrasmall Ni0.85Se Nanoparticles for Noninvasive Photoacoustic Imaging and Combined Photothermal-Chemotherapy of Cancer.

Large-size-induced long-term retention in the body has hampered the translational applications of many reported nanomedicines. Herein, we reported a multifunctional theranostic agent composed of ultrasmall poly(acrylic acid)-functionalized Ni0.85Se nanoparticles (PAA-Ni0.85Se NPs), which were successfully obtained through a facile ambient aqueous precipitation strategy. Without exhibiting any noticeable toxicity, the as-prepared PAA-Ni0.85Se NPs (average diameter of 6.40 ± 1.89 nm) showed considerable absorption in near-infrared (NIR) region and high photothermal conversion efficiency of 54.06%, which could induce remarkable photoacoustic signals for tumor imaging and heat for localized ablation of cancerous cells upon exposure to NIR light. Notably, the ultrasmall PAA-Ni0.85Se NPs, unlike conventional nanomaterials with larger sizes, showed reasonable body clearance within 8 h after intravenous injection. Furthermore, ascribed to protonation process of amino groups in DOX molecules and carboxyl groups in PAA molecules in an acidic microenvironment, the drug-loaded (doxorubicin hydrochloride, DOX·HCl) PAA-Ni0.85Se NPs (PAA-Ni0.85Se-DOX NPs) revealed promoted drug release at acidic pH, which could be useful for acidic tumor microenvironment responsive drug delivery. Evident from the results of cell-killing assay in vitro and tumor treatment study in vivo, PAA-Ni0.85Se-DOX NPs exhibited evident synergistic effects on killing 4T1 breast cancer cells. Thus, this study presents a multifunctional theranostic agent composed of ultrasmall PAA-Ni0.85Se NPs for potential cancer treatment without long-term toxicity concerns.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.

Abstract 978: Spatial-temporal delivery of OX40 agonist and PD-1 inhibitor using nanoparticles improves therapeutic efficacy of cancer immunotherapy

Abstract Background: Cancer immunotherapy is an exciting new approach to cancer treatment and there is strong interest in strategies to improve the long-term durable response rates of cancer immunotherapy. One approach is to combine checkpoint inhibitors such as aPD-1 with T cell activator such as OX40 agonist to further increase immune activation. We hypothesized that we can improve the therapeutic efficacy of this approach by temporally control the activation of OX40 and inhibition of PD-1 pathways. To accomplish this, we utilized nanoparticles that can deliver anti-OX40 and anti-PD1 antibodies simultaneously to T cells. Methods: Agonist antibody (anti-OX40) and antagonist antibody (anti-PD1) were conjugated to PLGA-PEG-Maleimide nanoparticles (AANPs) with precise ratio control and quantified by ELISA. Their specific binding to the target proteins was shown in vitro by flow cytometry. The tumor inhibition efficiency was assessed on mice bearing different tumor models. Two tumors were inoculated subcutaneously (105 B16F10 cells) or on fourth mammary fat pad (105 4T1 cells) on both flanks of mice. One side of tumor was irradiated once and AANPs were injected twice every 3 days. In vivo depletion experiments were tested on C56Bl6 mice and CD11b/c mice. Different populations of T cells in tumor and spleen were analyzed by flow cytometry and by fluorescent IHC staining. T cell killing assay and IFN-γ ELISpot were studied. Co-localization was demonstrated with fluorescent labeled antibodies and the corresponding AANPs. Results: AANPs showed a 30% cure rate, compared to 10% of free antibodies, 0% of anti-PD1 conjugated NPs, and 0% of anti-OX40 conjugated NPs in B16F10 melanoma model. We then re-challenged the cured mice with 2×105 B16F10 cells and none of the mice developed another tumor. In 4T1 breast cancer model, the survival rate on day 39 was 50% with AANPs treatment, compared to 22% in the mixture of anti-PD1 conjugated nanoparticles and anti-OX40 conjugated nanoparticles, and 0% of free antibodies. We demonstrated that AANPs led to a higher medium TCD8+/Treg ratio in tumors. The therapeutic effect was mediated by CD8+ T cells as elimination of these cells abrogated the therapeutic effects. In vitro study confirmed that AANPs were able to improve T cell stimulation compared to free antibodies by increasing IFN-γ excretion (2x). We further confirmed co-localization of antibodies with AANPs on tumor infiltration T cells in vivo. Our data demonstrated that spatial-temporal delivery of agonist and antagonist could improve T cell activation and cancer immunotherapy. Conclusions: Our data demonstrates that spatial-temporal delivery of agonist and antagonist can improve T cell activation and cancer immunotherapy. Citation Format: Yu Mi, Christof C. Smith, Feifei Yang, Jonathan Serody, Benjamin Vincent, Andrew Z. Wang. Spatial-temporal delivery of OX40 agonist and PD-1 inhibitor using nanoparticles improves therapeutic efficacy of cancer immunotherapy [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 978. doi:10.1158/1538-7445.AM2017-978

Abstract 3629: Engineering a novel asymmetric head-to-tail 2+1 T-cell bispecific (2+1 TCB) IgG antibody platform with superior T-cell killing compared to 1+1 asymmetric TCBs

Abstract T cell bispecific antibodies that recruit and engage T cells for tumor cell killing through binding to the T cell receptor (TCR) upon binding to a tumor antigen (TA) and subsequent crosslinking have attracted broad interest. Here, we describe a novel asymmetric head-to-tail 2+1 T cell bispecific antibody (2+1 TCB) platform characterized by the fusion of a flexible Fab fragment to the N-terminus of the CD3e Fab of a heterodimeric asymmetric bispecific TA-CD3e IgG1 antibody in head-to-tail geometry via a flexible linker. The resulting TCB is monovalent for CD3e (KD 70-100 nM) and binds bivalently with avidity to the TA on the target cell. Correct heavy chain pairing is enabled by knob-into-holes technology, correct light chain pairing by CrossMAb technology or using a common light chain. This enables production with standard processes in CHO cells. To exclude FcgR-mediated unspecific TCR and FcgR co-activation resulting in unspecific cytokine release, Fc- effector functions (ADCC, ADCP, CDC) are abolished by introduction of P329G LALA mutations while FcRn binding and IgG-like pharmacokinetic properties are retained as shown in mouse and Cynomolgus. For comparative profiling, the following TCBs were generated with specificity for the tumor antigens MCSP/CSPG4, FOLR1/FRalpha, CD19 and CD20: 2+1 TCBCD3-inside, 2+1 TCBCD3-outside, one-armed 1+1 TCBCD3-inside and a classical asymmetric 1+1 IgG TCB. In vitro Jurkat-NFAT, T cell killing, activation and proliferation assays show that both 2+1 TCB formats mediate superior potency of killing (for CSPG4, FOLR1, CD19, CD20) and superior absolute killing (for CSPG4, CD19) compared to the respective classical asymmetric 1+1 IgG TCB. Surprisingly, the 2+1 TCBCD3-inside format was found to be superior in potency compared to the 2+1 TCBCD3-outside format, although its binding affinity for CD3e is reduced. These data confirm that TCBs mediate extremely potent T cell killing with fM-pM EC50 values based on CD3e antibodies with affinities of only 70-100 nM. Notably, for CD19 both, 2+1 TCBCD3-inside and one-armed 1+1 TCBCD3-inside, mediate comparable potency and overall killing, and both were superior compared to the asymmetric 1+1 IgG TCB. These data underline the importance of the head-to-tail geometry with two Fabs on one arm attached to each other via a flexible G4S-linker. Finally, using 2+1 and 1+1 FOLR1 TCBs we demonstrate that bivalent binding allows better differentiation in killing of cells with high vs. low FOLR1 expression as compared to monovalent binding. Taken together, we demonstrate that the 2+1 TCBCD3-inside is the most potent, efficacious and versatile TCB design. Due to its orientation with the CD3e Fab inside, it allows the conversion of existing antibodies into potent TCBs without format restriction. Based on this platform, CEA CD3 TCB (RG7802, Phase I/Ib) and CD20 CD3 TCB (RG6026, Phase I) have entered clinical trials. Citation Format: Christian Klein, Christiane Neumann, Tanja Fauti, Tina Weinzierl, Anne Freimoser-Grundschober, Inja Waldhauer, Linda Fahrni, Sylvia Herter, Erwin van Puijenbroek, Sara Colombetti, Johannes Sam, Sabine Lang, Sherri Dudal, Wolfgang Schäfer, Jörg T. Regula, Samuel Moser, Oliver Ast, Ralf Hosse, Ekkehard Mössner, Peter Brünker, Marina Bacac, Pablo Umana. Engineering a novel asymmetric head-to-tail 2+1 T-cell bispecific (2+1 TCB) IgG antibody platform with superior T-cell killing compared to 1+1 asymmetric TCBs [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 3629. doi:10.1158/1538-7445.AM2017-3629

Antiviral activity of [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones against chikungunya virus targeting the viral capping nsP1

Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by Aedes mosquitoes. Since 2005, CHIKV has been spreading worldwide resulting in epidemics in Africa, the Indian Ocean islands, Asia and more recently in the Americas. CHIKV is thus considered as a global health concern. There is no specific vaccine or drug available for the treatment of this incapacitating viral infection. We previously identified 3-aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of CHIKV replication and proposed the viral capping enzyme nsP1 as a target. This work describes the synthesis of novel series of related compounds carrying at the aryl moiety a methylketone and related oximes combined with an ethyl or an ethyl-mimic at 5-position of the triazolopyrimidinone. These compounds have shown antiviral activity against different CHIKV isolates in the very low μM range based on both virus yield reduction and virus-induced cell-killing inhibition assays. Moreover, these antivirals inhibit the in vitro guanylylation of alphavirus nsP1, as determined by Western blot using an anti-cap antibody. Thus, the data obtained seem to indicate that the anti-CHIKV activity might be related to the inhibition of this crucial step in the viral RNA capping machinery.

Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work.FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5–20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

Combining impedence-based viability measurements and flow cytometric analyte quantitation to evaluate effector cell killing of T lymphocytes

Abstract Cancer immunotherapy is increasingly being evaluated in the lab, as well as the clinic as an approach for cancer treatment by harnessing and enhancing the immune system to attack cancer cells. Moving this research from the bench to the clinic is critically important, but we need reliable tools to be able to translate the in-vitro protocols and results for use in-vivo. Here, we have used a combination of impedence-based technology and flow cytometry to evaluate both the target and effector cells in an immune-cell based killing assay. The impedence based Real Time Cell Analysis technology, provides us with a continuous readout of target cell viability and flow cytometry allows us to measure responses using specific labels, such as effector cell viability and cytokine production in the supernatant of these assays. Here, we demonstrate cell killing assays measuring the cytotoxic effect of T-lymphocytes on a B-cancer cell line in conjunction with cytokine concentration measurements of IL-2, IL-4, IL-5, IL-10, TNF-a, IFN-g, IL-8, IL-1b, IL-6, IL-10, and IL-12p70 to simultaneously understand the T-lymphocyte response in these assays. Using both of these methods allows us to thoroughly evaluate immune cell killing efficacy and kinetics. The potency assays described in these studies have great potential for use in-vivo models and clinic.

Truncated Autoinducing Peptide Conjugates Selectively Recognize and Kill Staphylococcus aureus.

The accessory gene regulator (agr) of Staphylococcus aureus coordinates various pathogenic events and is recognized as a promising therapeutic target for virulence control. S.aureus utilizes autoinducing peptides (AIPs), cyclic-peptide signaling molecules, to mediate the agr system. Despite the high potency of synthetic AIP analogues in agr inhibition, the potential of AIP molecules as a delivery vehicle for antibacterial agents remains unexplored. Herein, we report that truncated AIP scaffolds can be fused with fluorophore and cytotoxic photosensitizer molecules without compromising their high agr inhibitory activity, binding affinity to the receptor AgrC, or cell specificity. Strikingly, a photosensitizer-AIP conjugate exhibited 16-fold greater efficacy in a S.aureus cell-killing assay than a nontargeting analogue. These findings highlight the potential of truncated AIP conjugates as useful chemical tools for in-depth biological studies and as effective anti-S.aureus agents.

A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay.

Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard 51 Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to 51 Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T-cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.

Effect of Cytotoxic T Lymphocytes Induced by Recombinant Adeno-Associated Virus on Different Hepatitis B Virus Genes

Background: Hepatitis B virus (HBV) infection is a significant global problem. The main therapies for chronic hepatitis B are nucleos(t)ide analogues (NAs) and (pegylated) interferon (IFN). However, the prognosis of chronic hepatitis B is still not so optimistic since the low serological conversion rate and the high recurrence rate after drug withdrawal. Objectives: This aim was to evaluate the killing activity of cytotoxic T lymphocytes (CTLs) induced by dendritic cells transduced recombinant adenovirus associated virus (rAAV) with different hepatitis B virus gene fragment (HBV-S, C, E, X). Methods: Peripheral blood mononuclear cells (PBMCs) were extracted from chronic hepatitis B patients , and the adhered cells were harvested to cultivate with recombinant adeno-associated virus with different hepatitis B virus (HBV) antigen gene fragment (rAAV-HBV-S, C, E, X) for 7 days by adding GM-CSF, IL-4 and TNF-α to generate mature dendritic cells (DCs). The DCs’ state were observed and their differentiation antigen molecules (CD) were detected by flow cytometry (FACS) to evaluate their maturation and function. DCs were co-cultured with prepared T lymphocytes to induce cytotoxic T lymphocytes (CTLs). The activity of CTLs induced by different DCs were compared by detection of lymphocyte CD molecules and cytokine levels. HepaG2.2.15 cells were target cells and HepaG2 cells as control. The specific killing activity of CTLs were compared by cell killing assay using 3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS). Results: The expression of phenotype CD14, CD80, CD83, CD86 from DCs transduced with rAAV-HBV-S, C, E, X and the secretion of cytokine IL-10, IL-12 were compared, respectively. CD80 in rAAV-HBV-S was the highest (P 0.05). The response of cytotoxic T-lymphocytes induced by different adeno-associated virus (AAV) vector delivery into dendritic cells had significant differences. In comparing the four antigens in terms of CD4+CD25+T cells, it was found that rAAV HBV-E group was the highest, and rAAV HBV-X group was the lowest (P 0.05). Conclusions: These data suggest that rAAV-HBV-S, C, E, X delivery into DC may be all useful for immunotherapeutic strategies against hepatitis B virus infection and that the HBV X antigen gene may be the most useful.

Immunotoxic effects of in vitro exposure of dolphin lymphocytes to Louisiana sweet crude oil and Corexit™.

The Deepwater Horizon oil spill was one of the worst environmental disasters on record in the United States. Response efforts to reduce the magnitude of the oil slick included the use of thousands of gallons of the chemical dispersant Corexit™ in surface and deep-water environments. The immunotoxicity of Louisiana sweet crude oil and the chemical dispersant Corexit was examined using lymphocyte proliferation (LP) and natural killer cell (NK) assays as measures of impact on the adaptive (LP) and innate (NK) immune response in bottlenose dolphins. Study results show that both high-energy media-accommodated fractions (MAF) and chemically enhanced MAF (CEMAF) mixtures modulate immune function. Following exposure to Louisiana sweet crude, both B- and T-cell proliferation of white blood cells was increased for all exposure concentrations, compared to control; however, this increase was only significant for the 50% and 100% treatments. In contrast, exposure of white blood cells to the CEMAF mixture significantly decreased both T- and B-cell proliferation in the 25%, 50% and 100% treatments. NK cell activity was enhanced significantly by CEMAF mixtures for the 50% and 100% treatments. The immunosuppression of LP at environmentally relevant concentrations of oil and dispersant suggests that marine mammals may be unable to mount an adequate defense against xenobiotic threats following exposure to oil and dispersant, leaving them more susceptible to disease. In contrast, NK cell activity was significantly enhanced, which may increase an organism's tumor or viral surveillance ability by mounting an enhanced immune response. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract 2299: ET1402L1-CART, a T cell therapy targeting the intracellular tumor antigen AFP, demonstrates potent antitumor activity in hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is the 5th most prevalent and 3rd most lethal cancer worldwide, due to limited treatment options. The lack of tumor-specific membrane targets is one of the key challenges currently facing immunotherapy for solid tumors, where on-target/off-tumor activity can lead to severe adverse responses. Most HCCs overexpress Alpha-Fetoprotein (AFP), a secreted fetal glycoprotein rarely expressed in adult tissues, making AFP an ideal target for immunotherapy. However, since AFP is only expressed intracellularly and secreted, it cannot be targeted with traditional antibodies against cell-surface antigens. To overcome this limitation, we exploited the fact that intracellular antigens, including AFP, are processed into peptides and presented by class I major histocompatibility complexes (MHCs) on the surface of tumor cells. Using an antibody discovery platform optimized for identifying candidates that bind peptide/MHC complexes, we developed ET1402L1, a fully-human scFv specifically targeting an immunogenic AFP peptide complexed with human leukocyte antigen (HLA)-A*02:01. ET1402L1 binds HLA-A*02:01/AFP with exquisite specificity and does not cross-react with naked HLA-A*02:01 molecules or with HLA-A*02:01 complexed with a panel of control peptides from endogenous human proteins. T cells engineered to express a second generation chimeric antigen receptor (CAR) constructed with ET1402L1 responded to HLA-A*02:01/AFP-expressing cells by degranulating and releasing IFN-γ, IL-2, IL-6, IL-8, IL-10, GM-CSF, and TNFα in an antigen-selective manner. In a cell killing assay, ET1402L1-CART selectively lysed HCC cells that expressed both HLA-A*02:01 and AFP, while sparing cells from multiple tissue types that were negative for either. In vivo, ET1402L1-CART demonstrated potent anti-tumor activity in multiple HCC xenograft models. While intravenous administration of ET1402L1-CART significantly inhibited the growth of established subcutaneous (s.c.) Hep G2 tumors (an HCC cell line positive for HLA-A*02:01 and AFP), intratumoral delivery resulted in rapid and prolonged tumor regression with complete regression observed in 75% of animals. These effects were recapitulated in a s.c. tumor model using another liver-derived HLA-A*02:01-positive cell line (SK-HEP-1) engineered to express the AFP peptide. Lastly, intraperitoneal injection of ET1402L1-CART led to regression of tumors in a disseminated abdominal HCC xenograft model. Our data demonstrates that CAR-T cell therapy targeting intracellular antigens via peptide/MHC complexes can effectively eradicate solid tumors in vivo. This approach expands the spectrum of antigens available for redirected T cell therapy against solid malignancies and provides a promising future therapy for HCC. A phase I clinical trial testing ET1402L1-CART in HCC patients is expected to begin in 2016. Citation Format: Hong Liu, Yiyang Xu, Jingyi Xiang, Li Long, Shon Green, Zhiyuan Yang, Jingwei Lu, Neal Cheng, Lucas H. Horan, Bin Liu, Su Yan, Pei Wang, Juan Diaz, Lian-xing Liu, Vivien Chan, Cheng Liu. ET1402L1-CART, a T cell therapy targeting the intracellular tumor antigen AFP, demonstrates potent antitumor activity in hepatocellular carcinoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2299.

Abstract 4698: BET protein inhibitors prevent TKI-induced drug resistance in solid tumors

Abstract Acquired drug resistance is one of the major challenges for clinical application of tyrosine kinase inhibitor (TKI) in cancer treatment. TKI-induced drug resistance could develop as a result of adaptive cell reprogramming that leads to pathway feedback activation in the same signaling pathway or parallel pathways or gatekeeper mutations abolishing drug binding. The findings of reversible drug response and lack of genetic mutations in TKI-resistant tumor cells suggest epigenetic basis during resistance development process. Targeting epigenetic modifiers and readers, in particular, the bromodomain and extra-terminal (BET) protein family, has shown promising efficacies against both primary and drug-resistant hematological malignancies via modulating transcription profile and suppressing the MYC oncogene. BGB-3619 is a novel, potent inhibitor of BET family proteins (BRD2, BRD3, BRD4 and BRDT) with IC50 ranging from 20 to 49 nM. It demonstrates superior anti-proliferation activity than iBET762 in multiple leukemia and lymphoma cell lines. In this study, we investigated the effect of BGB-3619 and iBET762 on solid tumor cells during and after TKI-resistance development. From cell killing assays, BET inhibitors were found to be much more potent to TKI-resistant cells (vemurafenib-resistant A375, HT29, SK-MEL-5, cells and erlotinib-resistant HCC827 cells) than their parental cells. Notably, long-term colony formation assays showed that combination of low dose BET inhibitors and TKI effectively suppressed the expansion of TKI-tolerated cell populations. The enhanced sensitivity of TKI-resistant cells to BET inhibitor was due to strong G1 phase arrest rather than induction of apoptosis. Furthermore, BET inhibitors blocked cell proliferation independent of c-Myc inhibition, suggesting they might affect a broad transcription network and provide favorable epigenetic state to induce cell cycle arrest. Taken together, these findings suggested potential therapeutic activity of BET inhibitor in TKI-resistant solid tumors and a strategy of combining BET inhibitors with TKIs to overcome resistance to targeted cancer therapies. Citation Format: Yuan Zhao, Yiyuan Wu, Hexiang Wang, Bo Ren, Ye Liu, Min Wei, Changyou Zhou, Lusong Luo. BET protein inhibitors prevent TKI-induced drug resistance in solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4698.

Abstract LB-062: Evaluation of drug conjugation sites in an anti-EGFR Centyrin

Abstract Centyrins are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with increased tissue penetration and simplified manufacturing. To identify optimal positions for chemical conjugation within the Centyrin scaffold, the crystal structure of an anti-EGFR Centyrin was solved and a library consisting of all possible single-cysteine variants was constructed and assessed for expression level in E. coli, efficiency of conjugation to maleimide derivatives, protein aggregation, thermostability, and EGFR binding. The best variants were conjugated to cytotoxic drugs and compared in a cell-killing assay. Three optimal positions were identified at which the Centyrin conjugated efficiently to maleimides; retained high expression, antigen binding, and biophysical properties; and showed increased cell-killing potency relative to the parent molecule. Citation Format: Rosa Cardoso, Shalom Goldberg, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven Jacobs, Gary Gilliland, Karyn O’Neil. Evaluation of drug conjugation sites in an anti-EGFR Centyrin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-062.

Short Term Hypoxia Synergizes with Interleukin 15 Priming in Driving Glycolytic Gene Transcription and Supports Human Natural Killer Cell Activities

Natural killer (NK) cells induce apoptosis in infected and transformed cells and are important producers of immunoregulatory cytokines. Therefore, they operate under low oxygen conditions (hypoxia) in inflammatory and tumor environments. In vitro studies of NK cells are, however, commonly performed in ambient air (normoxia). We used global gene expression profiling to evaluate changes in transcriptional pathways in primary human NK cells following short term culture under hypoxia compared with normoxia and in response to interleukin 15 (IL-15) priming using a 2 × 2 factorial design. The largest contrasts observed were priming dependences for associations between hypoxia and the hypoxia-inducible factor (Hif) 1 signaling and glycolysis pathways. RT-PCR confirmed positive synergistic hypoxia/IL-15 interactions for genes of key regulatory and metabolic enzymes. IL-15 primes NK cells for effector functions, which were recently demonstrated to depend on glycolytic switching. We did not, however, observe important increases in glycolytic flux through hypoxia and priming alone. Chemical Hif-1α inhibition suggested equal importance of this transcription factor for glycolysis and energy production under normoxia and hypoxia. Hypoxia promoted secretion of CC chemokines Ccl3/4/5 and macrophage migration inhibitory factor. Unexpectedly, hypoxia also stimulated migration of NK cells through the extracellular matrix and shifted amounts of susceptible leukemia target cells toward late apoptosis in a cell killing assay. We conclude that short term hypoxia supports these activities by positively interacting with NK cell priming at the level of glycolytic gene transcription. Hypoxic conditioning of NK cells may thus benefit their use in cell-based immunotherapy of cancer.

What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins

The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential.

Identification of the murine H-2D(b) and human HLA-A*0201 MHC class I-restricted HPV6 E7-specific cytotoxic T lymphocyte epitopes.

Recurrent respiratory papillomatosis is caused by human papillomavirus (HPV) infection, most commonly types 6 (HPV-6) and 11 (HPV-11). Due to failed host immune responses, HPV is unable to be cleared from the host, resulting in recurrent growth of HPV-related lesions that can obstruct the lumen of the airway within the upper aerodigestive tract. In our murine model, the HPV-6b and HPV-11 E7 antigens are not innately immunogenic. In order to enhance the host immune responses against the HPV E7 antigen, we linked calreticulin (CRT) to HPV-6b E7 and found that vaccinating C57BL/6 mice with the HPV-6b CRT/E7 DNA vaccine is able to induce a CD8+ T cell response that recognizes an H-2D(b)-restricted E7aa21-29 epitope. Additionally, vaccination of HLA-A*0201 transgenic mice with HPV-6b CRT/E7 DNA generated a CD8+ T cell response against the E7aa82-90 epitope that was not observed in the wild-type C57BL/6 mice, indicating this T cell response is restricted to HLA-A*0201. In vivo cytotoxic T cell killing assays demonstrated that the vaccine-induced CD8+ T cells are able to efficiently kill target cells. Interestingly, the H-2D(b)-restricted E7aa21-29 sequence and the HLA-A*0201-restricted E7aa82-90 sequence are conserved between HPV-6b and HPV-11 and may represent shared immunogenic epitopes. The identification of the HPV-6b/HPV-11 CD8+ T cell epitopes facilitates the evaluation of various immunomodulatory strategies in preclinical models. More importantly, the identified HLA-A*0201-restricted T cell epitope may serve as a peptide vaccination strategy, as well as facilitate the monitoring of vaccine-induced HPV-specific immunologic responses in future human clinical trials.

Abstract B087: Cyclic dinucleotide: A novel adjuvant for squamous cell carcinoma

Abstract Introduction and purpose of the study: Cyclic dinucleotides (CDNs), which signal through the STING (Stimulator of Interferon Gene) pathway, are a novel class of adjuvants that have been shown to induce anti-tumor responses in vivo. Preclinical studies will provide the necessary rationale for immunotherapy clinical trials for metastatic/recurrent HNSCC patients. In this study, we examined the role of CDNs in stimulating human dendritic cells and monocytes. We further examined the anti-tumor immune responses generated by natural and synthetic CDNs on SCCVII squamous cell tumors in a murine model. Experimental design and setup: Preclinical animal study and in vitro human sample experiments performed at an academic animal research facility. Cultured human dendritic cells (DCs) were stimulated with CDNs to examine cytokine response profiles and activity levels. We further attempted to model the effects of CDNs in a murine system. SCCFVII tumor bearing C3H/HeOUJ mice (n = 5 per group) were treated with three intratumoral injections of RR-CDG (synthetic cyclic-di-guanine), CDG (natural cyclic-di-guanine), R848 (TLR 7/8 agonist), or PBS (phosphate buffered saline, control). In addition, a group of RR-CDG treated mice was also treated with anti-CD8 T-cell depletion antibody to evaluate the role of CDNs in stimulating CD8+ T-cells. In vivo tumor growth rate was followed for 23 days, after which spleens were harvested and splenocytes were analyzed for cytokine profile using flow cytometry. In addition to measuring the anti-tumor immune response, the direct toxicity of the CDNs was measured by performing in vitro dose response live/dead cell viability assays. Findings or new outcome: The primary outcome measure was tumor volume. The secondary outcome measure was comparison of relative expression of Th1 vs. Th2 cytokine profiles from splenocytes in disparate murine treatment groups and from cultured human dendritic cells. In cultured human DCs, CDN stimulation resulted in higher levels of monocyte activation and increased IFNg and IL-12 levels when compared to PBS controls. In the murine model, RR-CDG and CDG treatment groups showed significantly decreased tumor size when compared to the R848, T-cell deficient RR-CDG, and PBS treatment groups (p<0.05). Splenocytes from the RR-CDG treated mice showed an enhanced Th1 response, as determined by increased expression of IFNγ, when compared to the PBS treatment group. In vitro tumor cell killing assay showed that there was no direct tumor cytotoxicity effect in response to CDNs. Conclusions: Synthetic and natural CDN treated mice had significantly reduced tumor volumes when compared to R848 (TLR7/8 agonist) and control treated mice. Mice treated with synthetic and natural CDNs also showed a predominant Th1 response when compared to mice treated with R848 (TLR7/8) agonists or controls. A pre-dominant Th1 cytokine response and increased monocyte activation in human cell studies demonstrate the translational nature of the animal studies and strongly support the future implementation of intratumoral injection of CDN into HNSCC patients. Citation Format: Shekhar K. Gadkaree, Rupashree Sen, Juan Fu, Clint Allen, Young J. Kim. Cyclic dinucleotide: A novel adjuvant for squamous cell carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B087.