P01.03 Targeting diaclyglycerol kinase alpha and zeta by self delivering RNAi to optimize tlymphocytes for adoptive therapy of solid tumors

BackgroundEvidence indicates that diacylglycerol kinases (DGK) are promising targets for the optimization of T cell activity, for example in the setting of adoptive cell therapy (ACT). The tumor microenvironment (TME) of human renal cell carcinoma (RCC) is an immunosuppressive setting where T and NK cell functionality is blocked. DGK–α is a negative regulator of TCR signaling, functioning by metabolizing diacylglycerol to phosphatidic acid and thereby limiting the activation of MAPK/ERK1/2 signaling pathway. DGK-α is found increased in tumor-infiltrating lymphocytes (TIL) from RCC patients and also in adoptively transferred T cells after infiltrating into the TME.1 We previously reported that inhibition of DGK-α restored functionality of unresponsive CD8 T cells and NK cells from RCC-TIL. Other studies demonstrated that knockdown or pharmacologic inhibition of DGK-α and DGK-ζ alone or together increased target cell killing and cytokine production, and protected T cells from inhibitory factors in the TME.2 However, there are no inhibitors for DGK-ζ and available DGK-α inhibitors have undesired pharmacokinetic/pharmacodynamic properties and are highly toxic precluding their clinical application. Here, we present data using a novel RNA interference (RNAi) technology that can specifically target each DGK isoform.Materials and MethodsINTASYL™ compounds incorporate drug-like properties into RNAi, resulting not only in enhanced cellular uptake in the presence of serum but also eliminating the need for further transfection reagents. Toxicity of compounds applied alone or in combination was assessed by 7-AAD flow cytometry analysis and WST assay. Silencing of mRNA and protein was analyzed by RT-qPCR and SimpleWestern. Downstream signaling pathways and T cell function were analyzed to demonstrate pharmacological efficacy.ResultsTwo DGK-ζ compounds and one DGK-α compound were analyzed using Jurkat T cells and primary human TCR-transduced T cells. No effects were seen on cell viability for the compounds applied alone or in combination. On-target knockdown was achieved in Jurkat T cells evidenced by RT-qPCR and SimpleWestern. Silencing of mRNA and protein occurred quickly after 24h, peaked between 48h and 72h and lasted at least for 96h. Stimulation under DGK-targeting INTASYL treatment resulted in enhanced levels of phosphorylated ERK1/2 and enhanced secretion of IL-2.ConclusionsINTASYL™ self-delivering RNAi compounds represent a promising approach to target intracellular immune checkpoints such as DGKs. The good toxicity profile allows for combined application of several compounds enabling targeting of multiple checkpoints, which likely is necessary to counteract the complex and heterogeneous inhibitory influences of the TME. The technology enables the anti-tumor activity of T and NK cells for immunotherapy, and can be used in ACT and direct therapeutic applications towards the TME.ReferencesMoon EK, Wang L-C, Dolfi DV, Wilson CB, Ranganathan R, Sun J, et al. Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 2014;20(16):4262–73.Jung I-Y, Kim Y-Y, Yu H-S, Lee M, Kim S, Lee J. CRISPR/Cas9-mediated knockout of DGK improves antitumor activities of human T cells. Cancer Res 2018;78(16):4692–703.Disclosure InformationA.S. Herbstritt: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals. M. Maxwell: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. D. Yan: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. B. Cuiffo: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. J. Cardia: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. S.P. Fricker: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. E. Noessner: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals.

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P01.08 Beyond PD-1: characterization of new checkpoints restricting function of cytotoxic lymphocytes infiltrating human carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.21 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A11.2-A12

Author(s):

AS Herbstritt ◽

PU Prinz ◽

M Maxwell ◽

M Kadiyala ◽

D Yan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cell ◽

Nk Cells ◽

Solid Tumors ◽

Principal Investigator ◽

Tumor Control ◽

Research Support ◽

Part Time ◽

Research Grant

BackgroundT and NK cells from human renal cell carcinoma (RCC) are functionally non-responsive. Analysis of the TCR signaling cascade required for effector function identified that proximal signaling molecules were activated whereas activation of downstream ERK was blocked. Further investigation showed increased diacylglycerol kinase alpha (DGK-α) levels in T and NK cells from the RCC tumor microenvironment (TME). These cells were refractory to stimulation showing no degranulation or IFN-γ production. Using a small molecule DGK–α inhibitor (R59022), the function of tumor-infiltrating lymphocytes was restored ex vivo. A correlation of high DGK-α and loss of function was also observed in an experimental mouse model of adoptive therapy where CAR T cells that had lost their activity after infiltrating into solid tumors were found to have increased DGK-α.1 Blockade of the Programmed cell death protein 1 (PD-1) with monoclonal antibodies is used in the clinic enabling some patients to achieve tumor control. However, not all patients respond. DGK-α activity is positioned downstream of PD-1 and should, if overactive, curb T cell function even if PD-1 inhibition is released. Thus, we hypothesize that dual inhibition of PD-1 and DGK–α might be required to fully unleash the T cell’s potential in the TME. Current DGK-α inhibitors are not suitable for clinical application. Therefore, we investigated alternative means using an RNA interference (RNAi) approach to target DGK-α alone as well as in combination with PD-1 in T and NK cells.Material and MethodsKnockdown is performed by RNAi using INTASYLTM compounds developed by Phio Pharmaceuticals. INTASYLTM compounds incorporate drug-like properties into the siRNA, resulting in enhanced uptake in the presence of serum with no need for further transfection reagents. Knockdown is analyzed by RT-qPCR and flow cytometry. Functional assays include cytotoxicity, degranulation and cytokine production in tumor mimicking environments.ResultsA tumor mimicking in vitro system was developed which allows for the demonstration of functional restoration or prevention of functional loss of cell activity. Using T cell/tumor cell co–cultures at high tumor cell density, functional suppression could be induced in T and NK cells comparable to those observed in the TME. Testing of DGK-α targeting INTASYLTM compounds, silencing of DGK-α was observed in human U2OS osteosarcoma cells. Using a fluorescently labeled compound, highly efficient transfection of human primary immune cells was seen. Combinations of PD-1 and DGK-α targeting compounds are being tested and evaluated for synergism in experimental models.ConclusionsStrong activity of specific T and NK cells is necessary for tumor control. Dual targeting of PD-1 and DGK-α may be required to fully enable T and NK cell reactivity in the TME. Current DGK-α inhibitors do not exhibit the desirable pharmacokinetic/pharmacodynamic (PK/PD) properties for clinical development. The tested self-delivering RNAi technology represents a promising approach to targeting intracellular immune checkpoints such as DGK-α.ReferenceMoon EK, Wang L-C, Dolfi DV, Wilson CB, Ranganathan R, Sun J, et al. Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 2014; 20(16):4262–73Disclosure InformationA.S. Herbstritt: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Phio Pharmaceuticals. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals. P.U. Prinz: None. M. Maxwell: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. M. Kadiyala: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. D. Yan: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. E. Noessner: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Phio Pharmaceuticals. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals.

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Galectin-9 expression defines exhausted T cells and impaired cytotoxic NK cells in patients with virus-associated solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001849 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001849

Author(s):

Isobel Okoye ◽

Lai Xu ◽

Melika Motamedi ◽

Pallavi Parashar ◽

John W Walker ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Solid Tumors ◽

Mononuclear Cells ◽

Cell Phenotype ◽

Peripheral Blood Mononuclear ◽

Effector Functions ◽

Cell Functions ◽

Cell Effector

BackgroundWe have previously reported that the upregulation of galectin-9 (Gal-9) on CD4+ and CD8+ T cells in HIV patients was associated with impaired T cell effector functions. Gal-9 is a ligand for T cell immunoglobulin and mucin domain-3, and its expression on T cells in cancer has not been investigated. Therefore, we aimed to investigate the expression level and effects of Gal-9 on T cell functions in patients with virus-associated solid tumors (VASTs).Methods40 patients with VASTs through a non-randomized and biomarker-driven phase II LATENT trial were investigated. Peripheral blood mononuclear cells and tumor biopsies were obtained and subjected to immunophenotyping. In this trial, the effects of oral valproate and avelumab (anti-PD-L1) was investigated in regards to the expression of Gal-9 on T cells.ResultsWe report the upregulation of Gal-9 expression by peripheral and tumor-infiltrating CD4+ and CD8+ T lymphocytes in patients with VASTs. Our results indicate that Gal-9 expression is associated with dysfunctional T cell effector functions in the periphery and tumor microenvironment (TME). Coexpression of Gal-9 with PD-1 or T cell immunoglobulin and ITIM domain (TIGIT) exhibited a synergistic inhibitory effect and enhanced an exhausted T cell phenotype. Besides, responding patients to treatment had lower Gal-9 mRNA expression in the TME. Translocation of Gal-9 from the cytosol to the cell membrane of T cells following stimulation suggests persistent T cell receptor (TCR) stimulation as a potential contributing factor in Gal-9 upregulation in patients with VASTs. Moreover, partial colocalization of Gal-9 with CD3 on T cells likely impacts the initiation of signal transduction via TCR as shown by the upregulation of ZAP70 in Gal-9+ T cells. Also, we found an expansion of Gal-9+ but not TIGIT+ NK cells in patients with VASTs; however, dichotomous to TIGIT+ NK cells, Gal-9+ NK cells exhibited impaired cytotoxic molecules but higher Interferon gamma (IFN-γ) expression.ConclusionOur data indicate that higher Gal-9-expressing CD8+ T cells were associated with poor prognosis following immunotherapy with anti-Programmed death-ligand 1 (PD-L1) (avelumab) in our patients’ cohort. Therefore, for the very first time to our knowledge, we report Gal-9 as a novel marker of T cell exhaustion and the potential target of immunotherapy in patients with VASTs.

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719 CD122-directed interleukin-2 complexes and αPD-L1 differentially require innate and adaptive immunity to treat local and metastatic bladder cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0719 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A761-A761

Author(s):

Ryan Reyes ◽

Yilun Deng ◽

Deyi Zhang ◽

Niannian Ji ◽

Neelam Mukherjee ◽

...

Keyword(s):

Bladder Cancer ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

San Antonio ◽

Interleukin 2 ◽

Γδ T Cells ◽

List Type ◽

Γδ T Cell

BackgroundαPD-L1 bladder cancer (BC) immunotherapy is effective in <30% of cases.1 To address the large αPD-L1-unresponsive subset of patients, we tested αIL-2/IL-2 complexes (IL-2c) that block IL-2 from binding high-affinity IL-2Rα (CD25) for preferential IL-2Rβ (CD122) binding.2 Immunosuppressive regulatory T cells capture IL-2 by CD25 whereas antitumor CD8+ T, γδ T, and NK cells use CD122. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses and that IL-2c effects could differ from, and thus complement αPD-L1.MethodsWe used PD-L1+ mouse BC cell lines MB49 and MBT-2, for orthotopic, intravesical (i.e., in bladder) and intravenous challenge studies of local versus lung metastatic BC.ResultsαPD-L1 or IL-2c alone reduced tumor burden and extended survival in local MB49 and MBT-2. Using in vivo cell depletions, we found that γδ T cells and NK cells, but strikingly not CD8+ T cells, were necessary for IL-2c efficacy in bladder. We confirmed γδ T cell requirements for IL-2c, but not αPD-L1 efficacy in γδ T cell-null TCRδKO mice. TCRβKO conventional T cell-null mice exhibited IL-2c, but not αPD-L1 responsiveness for orthotopic BC treatment. Neither agent alone treated lung metastatic MB49 or MBT-2 but the drug combination improved survival in both tumor models. Combination treatment effects in lungs were distinct from bladder, requiring CD8+ T and NK cells, but not γδ T cells.ConclusionsBC immunotherapy effects differ by anatomic compartment and use distinct mechanisms to treat primary and metastatic BC. CD122-directed IL-2 is a promising BC immunotherapy strategy, and IL-2c is a candidate mediator through innate immune effects. αPD-L1 could improve IL-2c efficacy by engagement of adaptive immune responses including to improve metastatic disease treatment efficacy.Ethics ApprovalAll procedures involving animals in this study were approved by the UT Health San Antonio Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with UT Health San Antonio Department of Laboratory Animal Resources standards.ReferencesShah AY, Gao J, Siefker-Radtke AO. Five new therapies or just one new treatment? A critical look at immune checkpoint inhibition in urothelial cancer: Future Medicine, 2017.Arenas-Ramirez N, Zou C, Popp S, et al. Improved cancer immunotherapy by a CD25-mimobody conferring selectivity to human interleukin-2. Science translational medicine 2016;8(367):367ra166-367ra166.

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Calcium-activated Potassium Channels Sustain Calcium Signaling in T Lymphocytes

Journal of Biological Chemistry ◽

10.1074/jbc.m011342200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12249-12256 ◽

Cited By ~ 130

Author(s):

Christopher M. Fanger ◽

Heiko Rauer ◽

Amber L. Neben ◽

Mark J. Miller ◽

Heike Rauer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocyte ◽

Lymphocyte Activation ◽

Vital Role ◽

Dominant Negative ◽

Jurkat T Cells ◽

Human T Cells ◽

Calcium Activated Potassium Channels ◽

Small Conductance

To maintain Ca2+entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca2+-activated K+(KCa) channels, hSKCa2in the human leukemic T cell line Jurkat and hIKCa1in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of KCachannels but not KVchannels reduce Ca2+entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native KCachannel in Jurkat T cells by overexpression of a truncated fragment of the clonedhSKCa2channel decreases Ca2+influx. Finally, introduction of the hIKCa1channel into Jurkat T cells maintains rapid Ca2+entry despite pharmacological inhibition of the native small conductance KCachannel. Thus, KCachannels play a vital role in T cell Ca2+signaling.

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P03.02 Suppression of T-cell proliferation and cytokine release by the adenosine axis are mediated by different mechanisms

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.42 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A22.2-A23

Author(s):

J Festag ◽

T Thelemann ◽

M Schell ◽

S Raith ◽

S Michel ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Degradation Products ◽

T Cell Proliferation ◽

Activated T Cells ◽

Human T Cells ◽

Part Time ◽

Ifn Γ ◽

Cd73 Expression

BackgroundThe so-called adenosine axis has emerged as a promising therapeutic target pathway as high adenosine levels in the tumor microenvironment contribute to the suppression of antitumor immune responses. The ectonucleotidases CD39 and CD73 act in concert to degrade extracellular immune-stimulating adenosine triphosphate (ATP) to immunosuppressive adenosine. According to the current model, subsequent suppression of effector immune cell function is caused by binding of adenosine to adenosine receptors like the A2a receptor (A2aR). The ectonucleotidases CD39 and CD73 as well as the A2aR have emerged as molecular targets within the adenosine axis with currently more than 20 clinical trials investigating antitumor effects of CD39-, CD73- or A2aR blockade. We aimed to perform a direct comparison of these targets with regard to their roles in regulating T-cell proliferation and IFN-γ secretion.Materials and MethodsCD39 and CD73 expression was suppressed using LNAplusTM antisense oligonucleotides (ASOs). ASOs were synthesized as gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA, leaving a central gap for recruitment of the RNA-degrading enzyme RNaseH I. Knockdown efficacy of ASOs on mRNA and protein level was investigated in primary human T cells. Furthermore, the effects of ATP, AMP and adenosine analogues on T–cell proliferation and IFN–γ secretion were investigated. A2aR was blocked using small molecule inhibitors that are currently under clinical investigation.ResultsTreatment of human T cells with LNA-modified ASOs specific for human CD39 and CD73 resulted in potent target knockdown in vitro without the use of a transfection reagent. T-cell proliferation was reduced after addition of ATP to activated T cells that was completely reverted by ASO-mediated suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogues inhibited IFN–γ secretion of activated T cells, however, they did not suppress T-cell proliferation. Blockade of the adenosine kinase was able to revert the anti-proliferative effect of ATP degradation products, arguing for downstream metabolites of adenosine, but not A2aR signaling, being responsible for the suppression of T-cell proliferation.ConclusionsCytokine secretion and proliferation of T cells might be differentially regulated by the adenosine axis. Adenosine might primarily affect cytokine secretion via A2aR signaling, whereas adenosine metabolites might especially impair proliferation of activated T cells independent from A2aR signaling. Therefore, inhibition of CD39 and/or CD73 holds exceptional advantages over A2aR blockade as both, A2aR dependent and A2aR independent effects of ATP degradation products are targeted simultaneously.Disclosure InformationJ. Festag: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. T. Thelemann: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. M. Schell: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. S. Raith: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. S. Michel: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. R. Klar: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. F. Jaschinski: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG.

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O1 Tumor lactic acidosis alters decisive T cell activities

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.6 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A3.2-A4

Author(s):

AJ Fischbeck ◽

AN Mendler ◽

M Balles ◽

J Schwarz ◽

R Zantl ◽

...

Keyword(s):

T Cells ◽

Lactic Acid ◽

T Cell ◽

Lactic Acidosis ◽

Tumor Cells ◽

Culture Medium ◽

Research Support ◽

Part Time ◽

Ifn Γ ◽

Regular Culture Medium

BackgroundAdoptive T cell therapy is a promising treatment strategy for tumor patients. However, when entering the tumor microenvironment (TME), T cells lose their effector function showing reduced degranulation and cytokine secretion. Besides T cell inhibition through checkpoint pathways (i.e. PD-1/L1, CTLA–4), suppressor cells (i.e. TAM, Treg) and cytokines (i.e. IL–10, TGF, VEGF), various metabolites of the TME also counteract antitumoral activities. Among the latter, lactate and extracellular acidosis are byproducts of the cancer metabolism and commonly observed in high concentrations in solid tumors. Previous experiments showed that tumor lactic acidosis selectively targets the signaling pathway including JNK/c-Jun and p38, resulting in inhibition of IFN-γ production. In contrast, granule exocytosis, which is regulated via the MEK1/ERK pathway, was moderately affected. Based on the contrasting effects on these two essential T cell effector activities, we investigated in more detail the effects of lactic acidosis on the killing process conducted by T cells.Material and MethodsTumor cells and cytotoxic T cells were co-cultured in lactic acid or regular culture medium and analyzed for effector function by flow cytometry and cell-mediated cytotoxicity assays. Additionally, ‘in-channel micropatterning’ in combination with artificial intelligence (AI) aided image analysis was used to visualize and analyze T cell cytotoxicity and mobility on a single cell level. Usage of collagen-matrices allowed the observation of T cell activity in a physiological three-dimensional environment. Cell metabolism was analyzed by Seahorse technology.ResultsIn the presence of lactic acid, IFN-γ production was strongly inhibited, while degranulation was only moderately reduced. Detailed analysis of the different processes involved in T cell cytotoxicity revealed that T cell recognition of tumor cells resulted in less secretion of cytotoxins (perforin, granzyme B and granzyme A). Lytic activity against tumor cells was strongly reduced at low T cell to tumor cell ratio (1:2). This deficiency could be compensated by increasing the T cell to tumor cell ratio (10:1). Using live cell imaging we investigated underlying mechanisms that might explain how higher T cell to target cell ratios might overcome lactic acid inhibition. T cells in lactic acid covered less distance, they moved for longer time periods and made less contacts with tumor cells in comparison to T cells cultured in regular culture medium.ConclusionsMicropatterning and AI based image analysis allows for detailed assessment of the processes involved in T cell-mediated killing such as mobility, speed, directionality and attachment on target cells. Lactic acidosis is hampering T cell killing activity by reducing the T cell’s capacity to find its target cell and attach to it. Repeated addition of T cells or neutralization of lactic acidosis in the TME are means to overcome these deficits and hold promise to improve the outcome of T cell-based immunotherapies.Disclosure InformationA.J. Fischbeck: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; IBIDI GmbH. A.N. Mendler: None. M. Balles: A. Employment (full or part-time); Significant; IBIDI GmbH. J. Schwarz: A. Employment (full or part-time); Significant; IBIDI GmbH. R. Zantl: A. Employment (full or part-time); Significant; IBIDI GmbH. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; IBIDI GmbH. E. Noessner: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; IBIDI GmbH.

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CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

Scientific Reports ◽

10.1038/srep20070 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 133

Author(s):

Shu Su ◽

Bian Hu ◽

Jie Shao ◽

Bin Shen ◽

Juan Du ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Checkpoint Inhibitors ◽

Gene Knockout ◽

Cell Activity ◽

Negative Regulator ◽

Activated T Cells ◽

Human T Cells ◽

New Strategy

Abstract Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

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788 A novel T- lymphocyte binding aptamer assembled into a bispecifc compound for the treatment of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.788 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A823-A823

Author(s):

Irit Carmi Levy ◽

Erez Lavi ◽

Neta Zilony Hanin ◽

Zohar Pode ◽

Karin Mizrahi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Tumor Cells ◽

Tumor Targeting ◽

Specific Antigen ◽

Therapeutic Agents ◽

List Type ◽

Bispecific T Cell Engager

BackgroundT-cell engagers are bispecific molecules directed against the CD3 complex on one end and a tumor specific antigen on the other end, allowing a physical link of T cell to a tumor cell, resulting in tumor killing and immune activation. Bispecific molecules harnessing and redirecting T-cells towards tumor cells are a promising therapeutic agents. Aptamers are single stranded oligonucleotides with binding and recognition propensities similar to those of antibodies. Aptamers have a number of advantages over bispecific antibodies including shorter generation time and low immunogenicity. Thus, aptamers capable of targeting T cells would have great potential for use as anti-cancer therapeuticsMethodsSystematic evolution of ligands by exponential enrichment (SELEX) methodology was employed in order to identify a novel CD3e binding aptamer. CD3 binding aptamer was subsequently linked into a bispecific T cell engager structure with a tumor-targeting aptameric arm. The tumor-targeting aptamer is developed by Aummune's proprietary tailored therapeutic platform.1 based on identifying functional aptamer sequences capable of specifically killing targeted tumor cells and sparing healthy tissue .Exemplary bispecific aptamers were tested for T cell stimulation by flow cytometry. In vivo antitumor activity was investigated in syngeneic and in xenograft tumor models.ResultsWe have successfully identified a novel CD3e –targeting aptamer with a Kd of 31nM. A bispecific T cell engager comprised of this aptamer and a tumor-targeting aptamer induced a potent stimulation of T cells in vitro, resulting in CD69 upregulation and IFNg secretion.Next, the CD3e targeting aptamer was hybridized to tumoricidal aptamers identified by Aummune's platform (VS12) to target either the human colon carcinoma HCT116 cells or (VS32) the murine triple negative breast cancer 4T1 cells. Both bispecific entities (CS6-VS12 and CS6-VS32) effectively lead to inhibition of tumor growth in vivo and increased survival in the corresponding models.ConclusionsOur data above provide a proof-of-concept for Aummune's Bispecific Aptamer efficacy and provide a framework for the clinical development of this novel tailored immune therapeutic agents. Indeed, we are currently in the process of developing a first-in-human clinical study in subjects with solid tumors.ReferenceMamet N, et al, Commun Biol 2020.

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827 Elucidating the roles of PIK3IP1/TrIP immune regulation on distinct T cell subsets in the context of cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0827 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A878-A878

Author(s):

Benjamin Murter ◽

Lawrence Kane

Keyword(s):

T Cells ◽

T Cell ◽

Immune Regulation ◽

Treg Cells ◽

Genome Project ◽

Negative Regulator ◽

T Cell Subsets ◽

Pi3k Signaling ◽

List Type ◽

The Impact

BackgroundThe signaling pathways involving phosphoinositide-3-kinases (PI3Ks) are highly conserved and tightly regulated to influence the activation, proliferation, and survival of all cell types. PI3K signaling plays a major role in T cell responses to antigen due to its position directly downstream of T cell receptor (TCR)/CD28 ligation. Our lab has recently shown that the cell surface protein TrIP (Transmembrane Inhibitor of PI3K, gene name: Pik3ip1) is capable of downregulating PI3K signaling in CD4+ T cells and can act as a negative regulator of T cell immune responses.1 This negative immune regulation was just recently reported to promote anti-tumor immunity, implicating TrIP as a potential immunotherapy target.2 Interestingly, although all effector subsets re-express TrIP to varying degrees, public expression data shows that Treg cells maintain higher TrIP message than other T-effector subsets.3MethodsUsing a conditional TrIP knockout mouse model developed in our lab, we have begun to interrogate how TrIP expression regulates the opposing activities of CD8+ T cells vs. Treg and how these affect the overall tumor immune landscape. With Treg-specific TrIP KO, we assessed the effects on syngeneic tumor growth in vivo, as well as analyzed primary and tumor-derived Treg phenotypes ex-vivo.ResultsThus far, we have found that TrIP knockout in the Treg compartment leads to no detectable differences in tumor burden. However, the lack of TrIP expression on Treg does have some effect on the effector phenotype of Treg cells isolated from the tumor.ConclusionsWe describe preliminary data on the role of TrIP in Treg function and phenotype and have begun to explore its effects on the tumor microenvironment. To build on this work we are currently developing TrIP over-expressing lentiviral constructs to compliment the knockout approaches described here. We have also now obtained mice with tamoxifen-inducible TrIP KO in Treg, so we will determine whether the timing of TrIP deletion affects the impact of TrIP deficiency.ReferencesUche UU, Piccirillo AR, Kataoka S, Grebinoski SJ, D’Cruz LM, Kane LP. PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt. J Exp Med 2018;215:3165–3179. doi:10.1084/jem.20172018.Chen Y, Wang J, Wang X, Li X, Song J, Fang J, et al. Pik3ip1 Is a Negative Immune Regulator that Inhibits Antitumor T-Cell Immunity. Clin Cancer Res 2019;25:6180–6194. doi:10.1158/1078-0432.CCR-18-4134.Heng TSP, Painter MW, Immunological Genome Project Consortium. The immunological genome project: networks of gene expression in immune cells. Nat Immunol 2008;9:1091–1094. doi:10.1038/ni1008-1091.

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