Specific Lysis Research Articles

Branched Multipeptide-combined Adjuvants Potentially Improve the Antitumor Effects on Glioblastoma.

The promising immunotherapy effects of a multiple antigenic peptide on glioblastoma (GBM) in a previous study encourage the use of adjuvants to enhance its therapeutic efficacy. Among adjuvants, pan HLA-DR-binding epitope (PADRE) and anti-programmed cell death protein 1 (anti-PD1) have potentially been tested for cancer immunotherapy. Therefore, here we evaluated the ability of PADRE and anti-PD1 to enhance the function of the branched multipeptide against GBM. The potential utility of tumor-associated antigens (ErbB-2 and WT-1) targeting GBM with HLA-A24 was confirmed and a branched multipeptide was constructed from these antigens. The effects of the branched multipeptide and PADRE on immunophenotyping and polarized Th cytokine production in dendritic cells were clarified. The expression of PD1 on T cells and PDL1 on GBM cells was also investigated. The interferon-γ enzyme-linked immunospot and lactate dehydrogenase release assays were performed to determine the function of GBM peptide antigen-specific cytotoxic T cells against GBM cells. Overall, this study showed that both ErbB-2 and WT-1 are potential candidates for branched multipeptide construction. The branched multipeptide and PADRE enhanced the expression of major histocompatibility complex and co-stimulatory molecules and the production of polarized Th1 cytokines in dendritic cells. The increase in the number of interferon-γ+ effector T cells was consistent with the increase in the percentage specific lysis of GBM target cells by GBM peptide antigen-specific cytotoxic T cells in the presence of the branched multipeptide, PADRE, and anti-PD1. Our study suggests the combination of branched multipeptide and adjuvants such as PADRE and anti-PD1 can potentially enhance the effects of immunotherapy for GBM treatment.

Simultaneous BRAFV600E Protein and DNA Aberration Detection in Circulating Melanoma Cells Using an Integrated Multimolecular Sensor.

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.

Оценка местно-раздражающего, кожно- резорбтивного и аллергенного действия инсектицидного препарата ВЭИС-2

The main safety indicators of the new preparation VEIS-2, containing natural or chemically modified avermectins as an active ingredient, when exposed to the skin of mammals, intended to protect the downy feather raw material from keratophagous insects, have been studied. It has been established that the VEIS-2 compositions containing abamectin or avermectin B1a hemisuccinate as an active principle have a mild irritating effect on the conjunctival mucosa of the rabbit eye. With single and multiple applications on the skin, the above compositions do not cause such functional and structural changes in the skin as edema, erythema, cracks, desquamation. The presence of skin-resorptive and allergenic action in the compositions of the VEIS-2 preparation with abamectin or hemisuccinate avermectin B1a using the "test tube method" in rats, as well as using the reaction of specific lysis of leukocytes (RSLL), has not been established. It was shown that the VEIS-2 composition based on avermectin complex has a more pronounced local irritant effect on laboratory animals in terms of toxicity and allergenicity.

Antigen-Specific In Vivo Killing Assay.

The in vivo killing assay allows the quantification of the antigen-specific killing capacity of Cytotoxic CD8+ T Lymphocytes (CTLs) in mice. CTLs are indeed known for the lysis of cells expressing foreign or modified antigen peptides on their MHC class I molecules. Here we describe the detailed protocol used for the in vivo specific lysis of cells expressing the H-2 Kb immunodominant CD8+ T-cell epitope of the OVA protein: an 8 amino acid peptide corresponding to the 257-264 region of OVA (SIINFEKL).

Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL.

Improvements in Flow Cytometry-Based Cytotoxicity Assay.

The flow cytometry-based assay has been increasingly used to assess the cell-mediated cytotoxicity since the 1980s due to its advantages over the conventional radioactive 51 Cr release assay (CRA), such as higher sensitivity at the single-cell level and nonradioactivity. The basic principle of this assay is the usage of two dyes, one nontoxic dye for labeling targets or effector cells to distinguish one from another, one viability dye for discrimination of dead from live cells. Due to the problem of spontaneous release or leakage of the nontoxic dye, the concern about the cross-staining has not yet been clearly elucidated. In this study, carboxyfluorescein diacetate succinimidyl ester (CFSE) was utilized to label target cells and Hoechst 33258 was used as the viability dye. We confirmed that no cross-staining occurred between the effector and target cells after 4h of coculture. We also found that the cytotoxicity would be overestimated if effector cells instead of target cells were labeled due to the exclusion of viable targets in effector-target conjugates. Using EDTA at the end of culture or labeling targets can solve this problem. Furthermore, the gating strategy could be improved by plotting CFSE against forward scatter (FSC) to discriminate some early apoptotic events. Due to the loss of target cells lysed by effector cells, counting beads are normally preferable in this assay. Here, we found an alternative to the use of beads in standardizing the flow cytometry-based assay. Instead of using beads, sample acquisition in a fixed time was shown to have the same effect in specific lysis evaluation as the beads application but have a greater stability than the latter. With a good quality control, the acquisition time for each sample could be shortened to 15 s, thus making this work to be done efficiently, especially in the case of larger sample sizes. Collectively, the findings in this study can improve the flow cytometric cytotoxicity assay to be carried out in a more accurate, efficient, and cost-effective way. © 2020 International Society for Advancement of Cytometry.

A novel CD2 staining-based flow cytometric assay for assessment of natural killer cell cytotoxicity.

BackgroundAssessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non‐radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51Chromium‐release assay (CRA) and flow cytometry–based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs.MethodsHere, we report a rapid FCC for quantifying target cell death after co‐incubation with NK cells. In this assay, after 4 hours of NK cell‐target cell co‐incubation, fluorochrome‐conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V‐FITC were further used to detect target cell death in CD2‐negative population. In parallel, both CRA and FCC assay using CFSE/ 7‐AAD were performed to validate the reproducibility and replicability.ResultsWe observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI‐H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2‐based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7‐AAD.ConclusionsWe demonstrated that this CD2‐based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

Abstract 4229: Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform

Abstract Introduction: In the rapidly growing field of chimeric antigen receptor (CAR) engineered T cells new approaches aim to make CAR-T cell therapy safer and more effective. Recent, designs aim towards universal or modular CARs that do not directly recognize the target antigen itself, but instead facilitate contact to CAR-adaptor molecules, which in turn bind to the target antigen. Current CAR-adaptor molecules include human antibodies of the IgG1 isotype binding to CD16 or antibodies modified by peptide or hapten tags. We developed a modular CAR-T approach that recognizes the P329G mutation clinically used to silence the Fc effector function of therapeutic antibodies. These modular anti-P329G-CAR-T cells are only functional in the presence of antibodies that possess the P329G mutation. Methods: The anti-P329G interaction with P329G-containing Fc fragment was analyzed using surface plasmon resonance and co-crystallography. Lentivirus transfected anti-P329G CAR-T cells were characterized in vitro for their selectivity and potential to mediate antigen specific tumor cell lysis, cytokine secretion and proliferation. Immunological synapse formation was investigated using confocal microscopy. Results: Anti-P329G-CAR-T cells allow the precise recognition of the P329G mutation present in therapeutic IgG1 based adaptor-molecules. Crystal structure- and SPR-analysis revealed a 1:1 binding stoichiometry with low nanomolar affinity of the P329G-Fab fragment applied in the CAR for P329G-containing IgG1 antibodies. Potent tumor cell lysis was demonstrated for multiple tumor antigens e.g. CD20, HER2, FOLR1, EpCAM, FAP and others. For all tested antigens, a huIgG1 dose-dependent activation of anti-P329G-CAR-T cells as well as dose-dependent tumor cell lysis was observed. For selected antigens P329G-CAR-T activity was found comparable to the activity mediated by T cell bispecific antibodies recognizing the respective tumor antigen. Finally, the immunological synapse formed by P329-CAR-T cells was compared to the one formed by T cell bispecific antibodies in a 2+1 format. Conclusions: P329G-CAR-T cells mediate potent and specific tumor cell killing using various tumor targeted antibodies as adaptor molecules. Based on these data in vivo studies to investigate efficacy and safety of the approach are foreseen. Notably, this approach allows control of CAR-T activity and potential side effects by titrating the adaptor molecule, as well as the simultaneous targeting of more than one antigen at the same time with the goal to prevent tumor escape mechanisms. Combining the P329G-CAR with allogenic T-cells may provide a truly off-the-shelf P329G-CAR-T cell therapy approach. Citation Format: Diana Darowski, Christian Jost, Zeno Riester, Mohamed Benmebarek, Kay Stubenrauch, Anne Freimoser-Grundschober, Uwe Wessels, Jörg Benz, Ekkehard Mössner, Renier Myburgh, Floriana Cremasco, Mario Perro, Pablo Umana, Sebastian Kobold, Christian Klein. Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4229.

Abstract 4232: More than a bridging therapy: Targeting CD123 with rapidly switchable universal CAR-T cells for treatment of acute leukemia

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 or BCMA is tremendously effective in treating hematological malignancies. However, the application of CAR-T beyond certain B-cell malignancies such as ALL, MM, lymphoma and CLL remains challenging due to the lack of tumor-specific antigens and the limited ability to control acute and potential long term CAR-T reactivity in patients. Therefore, a rapidly switchable universal CAR-T platform (UniCAR) was developed. In this system, antigen-specificity is provided by soluble adaptors of short pharmacokinetic half-life, termed targeting modules (TM), which redirect T-cells engineered to express a universal CAR against tumors, allowing for excellent controllability of CAR-T reactivity while maintaining the high anti-tumor activity of CAR-T cells. Here we present results from late stage pre-clinical characterization of UniCAR-T targeting CD123 (UniCAR-T-CD123) for treatment of acute leukemia. Pre-clinical characterization confirmed specificity and safety of the approach. UniCAR-T proofed to be per se inert and safe in clinically relevant in vivo toxicity models. Activation of UniCAR-T occurs solely in the presence of the CD123-specific TM (TM123) and upon cross-linkage to CD123 expressing leukemic cells. Specific lysis of leukemic target cell lines and primary patient material is induced in a dose-dependent manner at pico-molar TM123 concentrations. Notably, induction of cytokine release occurs at higher TM doses than on-set of target cell lysis, opening a therapeutic window for clinical application. In vivo efficacy of UniCAR-T re-directed against CD123 was proven in CDX and PDX mouse models. Interestingly, anti-leukemic responses of switch-controllable UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro at comparable doses. In contrast to conventional CD123 CAR-T, CD123-specific toxicity of TM-activated UniCAR-T towards hematopoietic progenitors was reversible and could be abrogated by withdrawal of TM, allowing for normal development of human hematopoiesis in a xenograft model. In summary, in vitro and in vivo evidence suggests that UniCAR-T-CD123 could provide strong efficacy against CD123 expressing hematological malignancies while providing excellent control and ensuring recovery of normal hematopoiesis post treatment. A phase IA dose-finding study is ongoing. Citation Format: Simon Loff, Jan-Erik Meyer, Johannes Spehr, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Michael Pehl, Gerhard Ehninger, Armin Ehninger, Marc Cartellieri. More than a bridging therapy: Targeting CD123 with rapidly switchable universal CAR-T cells for treatment of acute leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4232.

Epigenetic modulation combined with PD-1/PD-L1 blockade enhances immunotherapy based on MAGE-A11 antigen-specific CD8+T cells against esophageal carcinoma.

Cancer testis antigens (CTAs) are promising targets for T cell-based immunotherapy and studies have shown that certain CT genes are epigenetically depressed in cancer cells through DNA demethylation. Melanoma-associated antigen A11 (MAGE-A11) is a CTA that is frequently expressed in esophageal cancer and is correlated with a poor esophageal cancer prognosis. Consequently, MAGE-A11 is a potential immunotherapy target. In this study, we evaluated MAGE-A11 expression in esophageal cancer cells and found that it was downregulated in several tumor cell lines, which restricted the effect of immunotherapy. Additionally, the specific recognition and lytic potential of cytotoxic T lymphocytes (CTLs) derived from the MAGE-A11 was determined. Specific CTLs could kill esophageal cancer cells expressing MAGE-A11 but rarely lysed MAGE-A11-negative tumor cells. Therefore, induction of MAGE-A11 expression is critical for CTLs recognition and lysis of esophageal cancer cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased MAGE-A11 expression in esophageal cancer cells and subsequently enhanced the cytotoxicity of MAGE-A11-specific CD8+T cells against cancer cell lines. Furthermore, we found that PD-L1 expression in esophageal cancer cells affected the antitumor function of CTLs. programmed death-1 (PD-1)/PD-L1 blockade could increase the specific CTL-induced lysis of HLA-A2+/MAGE-A11+ tumor cell lines treated with 5-aza-2'-deoxycytidine. These findings indicate that the treatment of tumor cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine augments MAGE-A11 expression in esophageal cancer cells. The combination of epigenetic modulation by 5-aza-2'-deoxycytidine and PD-1/PD-L1 blockade may be useful for T cell-based immunotherapy against esophageal cancer.

Feasibility of dendritic cell-based vaccine against glioblastoma by using cytoplasmic transduction peptide (CTP)-fused protein antigens combined with anti-PD1

ABSTRACT Recent clinical trials utilizing antigen-pulsed dendritic cells (DCs) have demonstrated increased survival of vaccinated cancer patients. Besides, the cytoplasmic transduction peptide (CTP) not only has an excellent transcellular efficiency but also shows a strong tendency to remain in the cytoplasm after transduction, without migrating into the nucleus. In this study, we investigated the effectiveness of immunotherapy against malignant gliomas using DCs pulsed with CTP-fused protein antigens combined with programmed cell death protein 1 blockade (anti-PD1). The expression of tumor associated antigen (WT1 and BIRC5) and PDL1 on glioblastoma (GBM) target cells was confirmed by western blot. The effect of CTP-fused protein antigens on mature DCs (VaxDCs) was determined. The immunophenotypes of VaxDCs pulsed with CTP-fused protein antigens was confirmed by flow cytometry and the cytokine production levels of T helper polarization were measured by enzyme-linked immunosorbent (ELISA) assay. The IFN-γ-enzyme linked immunospot and lactate dehydrogenase release assays were performed to estimate the cytotoxic activity of antigen-specific cytotoxic T lymphocytes (CTLs), stimulated by VaxDCs pulsed with CTP-fused protein antigens and anti-PD1, against malignant glioma cells expressing target antigens. VaxDCs pulsed with CTP-fused protein antigens showed enhanced expression of major histocompatibility complex (MHC) and co-stimulatory markers of DCs and resulted in Th1 cytokine polarization. The increase in the number of IFN-γ+ effector T cells paralleled with the enhanced percent specific lysis of GBM targets cells by antigen-specific CTLs. Our study suggested that using CTP-fused protein antigens for DC vaccine preparation along with PD1 blockade could be an effective immunotherapy strategy for GBM.

Human influenza-specific adaptive NK cells mediate potent HLA-E-restricted responses against antigenically distinct influenza strains

Abstract Novel influenza vaccine approaches to achieve cross-protection against a broad range of antigenically distinct strains are needed. Beyond their ability to eliminate infected cells without the need for prior sensitization, natural killer (NK) cells have been shown to mediate virus-specific recall responses. Notably, exposure to influenza antigens induces protective influenza-specific memory NK cells in mice, but evidence of influenza-specific memory NK cells in humans is still lacking. Using intracellular cytokine staining, we found that NK cells from 45% of adults can robustly respond to nucleoprotein (NP) from at least 2 heterosubtypic influenza strains completely independent of T cell help. To further determine if a subset of NK cells can specifically react to NP peptide pools from distinct strains, we clonally expanded individual NK cells (NKCL) and measured H1N1, H2N2 and H3N2 NP-specific killing by 33 NKCL from 6 donors. 63% of NKCL showed positive responses against the 2009 H1N1 pandemic strain (2%–57% specific lysis). Strikingly, half of H1N1 NP-specific NKCL also displayed cytotoxicity against either H2N2 or H3N2 (9%–100% specific lysis) or against both H2N2 and H3N2 strains (5%–11% specific lysis). Potent killing by NKCLs was associated with high cell surface expression of the activating NKG2C receptor, a ligand for HLA-E. Accordingly, we identified an HLA-E-restricted NP epitope that triggers activation of influenza-specific NK cells. Collectively, these data provide the first mechanistic evidence of influenza-specific memory NK cells in humans and suggest influenza-specific responses mediated by NK cells may have the potential to be harnessed to enhance influenza vaccine-induced cross-protection.

Therapeutic EBV-specific T cell cytotoxicity against allogeneic LCLs in vitro directly correlates to intracellular IFNγ/TNFα expression in response to EBV peptide stimulation

Background & Aim SNBTS has been involved in the manufacture of Epstein-Barr virus-specific T cells (EBV-VST) in treatment of post-transplant lymphroliferative disorder (PTLD) for over 20 years. Manufacture of cells for clinical use relies on comprehensive QC analysis to assess product safety, dose, identity, functionality and stability. We have developed an in vitro cytotoxicity assay of EBV-VST against EBV-infected lymphoblastoid cell line (LCL) targets to measure specific lysis compared to controls. Since the LCLs required for this assay may contain live virus, in the advancing regulatory setting this assay is not easily transferable to an accredited QC lab. The aim of this study was to investigate if VST cytotoxicity directly correlated to any other cytotoxic T cell functional assays such as cytokine production or expression of degranulation markers. Methods, Results & Conclusion Cryopreserved EBV-VST products were thawed, counted, and split for use in both a cytotoxicity assay and intracellular cytokine/ degranulation assay. For the cytotoxicity assay, HLA-matched and unmatched LCLs were labeled with PKH67, and then co-cultured alongside EBV-VSTs at increasing VST to LCL ratios against suitable controls. Plates were incubated for 5 hours, and cells subsequently labeled with Annexin V and DRAQ7 to assess target apoptotis and necrosis by flow cytometry. For the intracellular assay, EBV-VSTs were stimulated for 5 hours with EBV consensus peptides and then labeled with surface and intracellular markers in the following flow cytomeric panel: CD8, CD4, CD45RO, CD107a, Perforin, Granzyme B, IFNγ, TNFα, IL-2 and DRAQ7. Both assays were performed from the same start material at same time to ensure a direct comparison. The percentage of IFNγ/TNFα co-expressing cells in response to EBV peptide stimulation positively correlated with specific lysis of HLA-matched target LCLs in EBV-VST co-cultures (R2= 0.5602, p=0.008, n=12). No correlation of LCL specific lysis was seen with any of the other degranulation markers, and interestingly no correlation seen with other product-variable parameters (VST viability, CD8/CD4 ratio, number of HLA matches) suggesting these have less direct impact on overall killing efficiency. Flow cytometric analysis of IFNγ/TNFα expression in response to EBV peptide stimulation is an accurate surrogate marker of EBV-infected LCL killing by VSTs, and therefore presents a valid, safe and compliant alternative functional characterisation method to the conventional LCL cytotoxicity assay.

Application of individual HLA-binding prediction thresholds increases the detection of target cell surface-presented HPV16 E6- and E7-derived epitopes as a basis for therapeutic HPV vaccine design

Abstract For the development of a therapeutic vaccine against human papillomavirus (HPV), viral epitopes bona fide presented on the surface of HPV-transformed cells need to be identified. The constitutively expressed HPV oncoproteins E6 and E7 represent ideal targets for immunotherapeutic vaccination approaches. However, viral epitopes are low abundant and human leukocyte antigen (HLA)-restricted, posing a challenge for developing peptide-based vaccines. For the identification of novel epitopes, in silico HLA-binding prediction was used to preselect HPV16 E6- and E7-derived peptides for 7 major HLA-types representing 5 HLA supertypes. Based on performance evaluation of predictors, new individual binding affinity thresholds were applied. HLA-binding of synthesized predicted binders was validated in vitro in competitive cellular binding assays. Interferon-γ ELISpot assays performed with blood samples from HLA-characterized healthy donors identified memory T cell responses against HLA-binding HPV peptides. T cells of responding donors were further characterized for epitope-specific mediation of specific target cell lysis by CD8+ CTLs using a flow cytometry based cytotoxicity assay. Complementary, an immunopeptidomics approach was used to determine the surface presentation of epitopes on HPV16-transformed cancer cells. Targeted liquid chromatography-mass spectrometry was capable of detecting epitopes predicted within the new individual affinity thresholds but missed by common thresholds. Completing the identification of bona fide target cell surface-presented E6- and E7-derived T cell epitopes will provide a map of targets for the development of a therapeutic vaccine to treat HPV16-induced malignancies.

The liver controls regulatory T cell populations by enclysis, a CD4+ T cell engulfment process

Abstract Background and Aims Regulatory T cells (Treg) dampen inflammation and they are increased in the liver compared to blood, contributing to a tolerising environment. The swift neutralisation of Treg cells is important to enable immune effector functions to combat infection, but the mode of this is not understood. We noted that hepatocytes preferentially engulfed live Treg cells, and we set out to characterise the mechanism and outcome of this new phenomenon we termed “enclysis”. Method We measured enclysis, the enclosure and lysis of Treg cells, in vitro and ex vivo using perfused liver wedges, in the presence of inhibitors of known cell-in-cell processes. Reporter dyes for viability, mitochondrial activity and acidification were used to determine enclysis outcome. Cell-in-cell structures were documented by light, confocal, electron and multiphoton microscopy. FoxP3+ Treg and T-bet+ T helper cells were enumerated in donor and in end-stage disease livers of autoimmune and viral diseases. Results Hepatocytes preferentially engulfed live Treg cells in a process that differed in mechanism and kinetic to previously described cell-in-cell structures (efferocytosis, suicidal emperipolesis, entosis). Enclysis reflects the enclosure and specific lysis of live Treg cells inside hepatocytes, and beta-catenin-positive enclytic vesicles were connected to the endocytic pathway. Enclysis was increased in patients with autoimmune compared to viral hepatitis (p=0.0011), offering an explanation for the breach of tolerance in the liver. Conclusion Enclysis is the preferential engulfment and deletion of live Treg cells by hepatocytes and can be modulated pharmacologically, offering a new therapeutic target for liver immune regulation.

Novel car design reversing the PD-1 signal boosts the efficacy of car-T cells with low cytokine release and no off-target effect

Background & Aim Purpose The immunosuppressive tumor microenvironment (TIME) and cytokine release syndrome (CRS) hinder the progression of chimeric antigen receptor (CAR) T cell therapy. Strategies that block or reverse PD-1/PD-L1 inhibitory signals through CAR structural redesign to overcome TIME are extensively studied but still need be improved. Targeting the widely expressed PD-L1 without increasing the off-tumor effect while reduce cytokines release are critical challenges. Based on the above considerations, we engineered a novel bispecific CAR T cell type targeting CD19 and PD-L1 by incorporating the PD-1 extracellular domain next to the transmembrane domain (TMD) of CD19-targeting CAR domain, which we named EMD.PD-1 CAR. Methods, Results & Conclusion Experimental design In this study, we firstly engineered a novel CD19/PD-L1 bispecific CARs by incorporating the PD-1 domain into CD19 CAR and constructed second-generation CAR as control. Second, to assess the function of the novel anti-CD19 CAR T cells via antigen-specific stimulation, the antitumor functions, cytokine release, and off-target effects were studied in vitro and in vivo. We demonstrated that PD-L1 stimulation alone is not sufficient to activate EMD.PD-1 CAR T cells for the production of a antigen-specific immune response, including specific lysis, cytokines release, and proliferation. When challenged with CD19+PDL1+ tumor cells, EMD.PD-1 CARs showed a stronger specific immune response than 2nd CAR in vitro. Moreover, EMD.PD-1 CAR T cells exhibited only moderate specific cytokine release compared with 2nd CAR T cells when challenged with target tumor cells. In xenograft NOD/SCID/γc−/−(NSG) mouse model bearing CD19+PD-L1+ tumor cells, our results demonstrated that EMD.PD-1 CAR prolonged the overall survival and decreased the tumor burden compared with 2nd CAR. In addition, EMD.PD-1 CAR T cells showed a higher fraction of stem cell memory (TSCM) and central memory (TCM) T cell phenotype and lower PD-L1, TIM-3, CTLA-4, and intrinsic PD-1 expression in flow cytometry, CAR T cells demonstrated decreased differentiation and exhaustion marker expression. In conclusion, when faced with heterogeneous tumors with high expression of immunosuppressive signals, traditional second-generation CAR T cells are insufficient. The novel CAR, called EMD.PD-1 CAR with a low-avidity affinity to PD-L1 and moderate cytokine release can address this critical challenge.

Pancreatic and colon cancer stem cells escape NK cell effector function via PCNA–NKp44 interaction

Abstract Natural killer (NK) cells play an important role in the innate immune response against cancer. Unlike B or T lymphocytes, NK cells do not require prior sensitization and can immediately respond to target cells. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of cells. Cancer cells may escape NK-mediated responses by expressing inhibitory ligands. Proliferating cell nuclear antigen (PCNA), in association MHC I, forms the inhibitory ligand for NKp44. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. CSC can be identified by a variety of markers such as surface co-expression of CD44 and CD133. In both Panc-1 and HCT 116 cell lines, surface PCNA is associated with co-expression of CD44 and CD133. Triple positive (PCNA+CD44+CD133+) cells have increased CSC transcription factor (NANOG, SOX2, and Oct-4) expression compared to double positive (PCNA−CD44+CD133+) or negative cells. Additionally, blocking the PCNA–NKp44 interaction alters IFN-g secretion and increases specific lysis of cancer cells by NK cells. Taken together, these data suggest that surface co-expression of PCNA, CD44, and CD133 are markers of pancreatic and colon CSC and blocking the PCNA–NKp44 interaction may provide an immunotherapeutic approach to target pancreatic and colon CSC and prevent metastasis.

Efficient induction of cytotoxic T lymphocytes in hepatocellular carcinoma using the HLA-A2-restricted survivin peptide in vitro

Survivin is a newly identified tumour-associated antigen and has been demonstrated to be an excellent target for immunotherapy in several cancers, but its role in hepatocellular carcinoma treatment is still unknown. In this study, survivin-derived peptide-specific cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells of healthy donors were induced by multiple stimulations with HLA-A2- restricted survivin peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis of T2 cells pulsed with the peptide and HLA-A2+ hepatocellular carcinoma cells expressing survivin, while HLA-A2+ hepatocellular carcinoma cell lines that did not express survivin were not recognized by the CTLs. These results suggest that the survivin peptide epitope could be a potential target of specific immunotherapy for HLA-A2+ patients with hepatocellular carcinoma.

Design of a thrombin inhibitory staphylokinase based plasminogen activator with anti-reocclusion potential

In the quest of a therapeutically improved thrombolytic drug, we designed and expressed a Staphylokinase based, recombinant fusion protein with fibrin specific clot lysis and anti-thrombin activities derived from human thrombomodulin, a transmembrane glycoprotein with anticoagulant properties, known to form a complex with thrombin and then activating Protein C. The fusion construct was expressed in the yeast Pichia pastoris for cost effective and facile production. The purified construct had comparable plasminogen activation and clot lysis capability as compared to native SAK in that it showed plasmin dependent Plasminogen activation, but exhibited significantly lower fibrinogenolysis (an indicator of fibrin-specific action) even at much higher doses than SAK. In addition, its successful activation of the thrombin-mediated Protein C anticoagulant pathway was reflected in increased in vitro plasma clotting time, as well as inhibition of clot bound thrombin, which led to nearly 15–25% lesser re-formation of fibrin clots after initial successful clot lysis in a specially designed in vitro assay for re-occlusion. Thus, this novel chimeric protein could be of high therapeutic potential as a thrombolytic drug with enhanced fibrin specific clot dissolving properties along with lower bleeding and re-thrombosis complications- a dire need today, especially in the treatment of thrombotic strokes.