scholarly journals O1 Tumor lactic acidosis alters decisive T cell activities

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.

scholarly journals Beyond the Lactate Paradox: How Lactate and Acidity Impact T Cell Therapies against Cancer

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 25
Author(s):  
Violet Y. Tu ◽  
Asma Ayari ◽  
Roddy S. O’Connor
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Biology ◽  
Redox Balance ◽  
Hematologic Malignancies ◽  
Activated T Cells ◽  
Unique Challenge ◽  
Cell Therapies

T cell therapies, including CAR T cells, have proven more effective in hematologic malignancies than solid tumors, where the local metabolic environment is distinctly immunosuppressive. In particular, the acidic and hypoxic features of the tumor microenvironment (TME) present a unique challenge for T cells. Local metabolism is an important consideration for activated T cells as they undergo bursts of migration, proliferation and differentiation in hostile soil. Tumor cells and activated T cells both produce lactic acid at high rates. The role of lactic acid in T cell biology is complex, as lactate is an often-neglected carbon source that can fuel TCA anaplerosis. Circulating lactate is also an important means to regulate redox balance. In hypoxic tumors, lactate is immune-suppressive. Here, we discuss how intrinsic- (T cells) as well as extrinsic (tumor cells and micro-environmental)-derived metabolic factors, including lactate, suppress the ability of antigen-specific T cells to eradicate tumors. Finally, we introduce recent discoveries that target the TME in order to potentiate T cell-based therapies against cancer.

scholarly journals Tumor Lactic Acidosis: Protecting Tumor by Inhibiting Cytotoxic Activity Through Motility Arrest and Bioenergetic Silencing

2020 ◽  
Vol 10 ◽  
Author(s):  
Angelika J. Fischbeck ◽  
Svenja Ruehland ◽  
Andreas Ettinger ◽  
Kerstin Paetzold ◽  
Ilias Masouris ◽  
...  
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
T Cell ◽  
Lactic Acidosis ◽  
Tumor Cell ◽  
Cytotoxic Activity ◽  
Adoptive T Cell Therapy ◽  
Normal Medium ◽  
Granule Exocytosis ◽  
Prolonged Contact

Adoptive T cell therapy (ACT) is highly effective in the treatment of hematologic malignancies, but shows limited success in solid tumors. Inactivation of T cells in the tumor milieu is a major hurdle to a wider application of ACT. Cytotoxicity is the most relevant activity for tumor eradication. Here, we document that cytotoxic T cells (CTL) in lactic acidosis exhibited strongly reduced tumor cell killing, which could be compensated partly by increasing the CTL to tumor cell ratio. Lactic acid intervened at multiple steps of the killing process. Lactic acid repressed the number of CTL that performed lytic granule exocytosis (degranulation) in tumor cell co-culture, and, additionally impaired the quality of the response, as judged by the reduced intensity of degranulation and lower secretion of cytotoxins (perforin, granzyme B, granzyme A). CTL in lactic acid switched to a low bioenergetic profile with an inability to metabolize glucose efficiently. They responded to anti-CD3 stimulation poorly with less extracellular acidification rate (ECAR). This might explain their repressed granule exocytosis activity. Using live cell imaging, we show that CTL in lactic acid have reduced motility, resulting in lower field coverage. Many CTL in lactic acidosis did not make contact with tumor cells; however, those which made contact, adhered to the tumor cell much longer than a CTL in normal medium. Reduced motility together with prolonged contact duration hinders serial killing, a defining feature of killing potency, but also locally confines cytotoxic activity, which helps to reduce the risk of collateral organ damage. These activities define lactic acid as a major signaling molecule able to orchestrate the spatial distribution of CTL inside inflamed tissue, such as cancer, as well as moderating their functional response. Lactic acid intervention and strategies to improve T cell metabolic fitness hold promise to improve the clinical efficacy of T cell–based cancer immunotherapy.

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

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.

scholarly journals The Injury Response to DNA Damage Promotes Anti-Tumor Immunity

2020 ◽  
Author(s):  
Ganapathy Sriram ◽  
Lauren Milling ◽  
Jung-Kuei Chen ◽  
Wuhbet Abraham ◽  
Erika D. Handly ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Injured Cell ◽  
Ifn Γ

ABSTRACTInhibition of immune checkpoints has shown promising results in the treatment of certain tumor types. However, the majority of cancers do not respond to immune checkpoint inhibition (ICI) treatment, indicating the need to identify additional modalities that enhance the response to immune checkpoint blockade. In this study, we identified a tumor-tailored approach using ex-vivo DNA damaging chemotherapy-treated tumor cells as a live injured cell adjuvant. Using an optimized ex vivo system for dendritic cell-mediated T-cell IFN-γ induction in response to DNA-damaged tumor cells, we identified specific dose-dependent treatments with etoposide and mitoxantrone that markedly enhance IFN-γ production by T-cells. Unexpectedly, the immune-enhancing effects of DNA damage failed to correlate with known markers of immunogenic cell death or with the extent of apoptosis or necroptosis. Furthermore, dead tumor cells alone were not sufficient to promote DC cross-presentation and induce IFN-γ in T-cells. Instead, the enhanced immunogenicity resided in the fraction of injured cells that remained alive, and required signaling through the RIPK1, NF-kB and p38MAPK pathways. Direct in vivo translation of these findings was accomplished by intra-tumoral injection of ex vivo etoposide-treated tumor cells as an injured cell adjuvant, in combination with systemic anti-PD1/CTLA4 antibodies. This resulted in increased intra-tumoral CD103+ dendritic cells and circulating tumor antigen-specific CD8+ T-cells, leading to enhanced anti-tumor immune responses and improved survival. The effect was abrogated in BATF3-deficient mice indicating that BATF3+ DCs are required for appropriate T-cell stimulation by live but injured DNA-damaged tumor cells. Notably, injection of the free DNA-damaging drug directly into the tumor failed to elicit such an enhanced anti-tumor response as a consequence of simultaneous damage to dendritic cells and T-cells. Finally, the DNA damage induced injured cell adjuvant and systemic ICI combination, but not ICI alone, induced complete tumor regression in a subset of mice who were then able to reject tumor re-challenge, indicating induction of a long-lasting anti-tumor immunological memory by the injured cell adjuvant treatment in vivo.

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

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.

IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 816-816 ◽  
Author(s):  
Mauro P. Avanzi ◽  
Dayenne G. van Leeuwen ◽  
Xinghuo Li ◽  
Kenneth Cheung ◽  
Hyebin Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cytokine Analysis ◽  
Car T ◽  
Ifn Γ

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Vaccination Strategies for Patients with B-CLL.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2106-2106
Author(s):  
Fatma V Okur ◽  
Eric Yvon ◽  
Gianpietro Dotti ◽  
George Carrum ◽  
Helen E. Heslop ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Th2 Cells ◽  
Ifn Γ ◽  
Adenoviral Vaccine

Abstract B-chronic lymphocytic leukemia (B-CLL) cells express tumor associated antigens that may generate a T cell mediated immune response, but present these antigens poorly. Moreover, patients with B-CLL often have poor immune function due to the disease or its treatment. We have shown that expression of transgenic CD40L increases the immunogenecity of human B-CLL cells ex vivo and in vivo, and that this effect can be potentiated by co-expression of transgenic IL2. Previous studies described outcomes when adenoviral vectors were used to obtain gene transfer, but because of the complexities and expense of manufacture of viral vectors, and their lingering safety concerns, we determined whether it was possible to use electroporation (with the MaxCyte device) as a physical means of transferring CD40L and IL2 plasmids to produce vaccines with similar biological properties in vitro and in vivo. Table 1 compares the phenotype of the vaccines using each vector. Table 1. Comparision of immunogenic characteristics and viability of the adenoviral and plasmid vaccines Type of Vaccine CD40L (%) CD80 (%) CD86 (%) IL-2 (pg/ml/10e6 cells) Viability (%) IL2 CD40L All the values are given as mean ± SE. * P&lt; 0.01, Paired Student’s t test. Adenoviral Pre 0.2 ± 0.01 2.6 ± 2.4 7.5 ± 3.9 Post 66.1 ± 5.5* 50.2 ± 7.8* 69.5 ±11* 253.5 ± 82.6 93.6 94.2 Plasmid Pre 1.3 ± 0.85 11.5 ± 6.2 19.7 ± 6.8 Post 55.5 ± 5.1* 19.2 ± 9.3 26.4 ± 9.7 4806.6 ±1398.9 84.4 88.4 Vaccines made by both approaches met the release criteria for CD40L and IL2 expression (CD40L ≥20% and IL-2 ≥ 150 pg/ml/1x10e6 cells ), but expression of IL2 was higher in the plasmid vaccines, expression of CD40L was equivalent in each and expression of the additional co-stimulatory molecules CD80 and CD86 (induced after CD40 activation by transgenic CD40L) was higher in the adenoviral vaccines. Fourteen patients were given adenoviral-vaccines and nine the plasmid transduced cells. Each of these patients received up to 18 s.c. injections of IL-2 secreting and CD40L expressing tumor cells. Both types of vaccine were well tolerated. Table 2 shows the results of culturing patient T cells with autologous B-CLL tumor cells. Table 2. Comparision of anti-B-CLL T cell responses induced by adenoviral and plasmid vaccines Type of Vaccine Pre-vaccine After 3rd vaccine After 6th vaccine All the values were are given as mean ± SE. *P&lt;0.05, Wilcoxon Signed Ranks test Adenoviral 307.3 ± 293.9 375 ± 306.8 656.8 ± 373.8 IFN-γ spots/10e6 T cells&#x2028; IL-5 spots/10e6 T cells 0 12.8 ± 7.9 5.8 ± 2.3 Plasmid 31.1 ± 14.8 38 ± 17.8 32.9 ± 19.5 IFN-γ spots/10e6 T cells&#x2028; IL-5 spots/10e6 T cells 4 ± 2.7 14 ± 10.2 203.9 ± 156.3* After 3 and 6 injections, both the adenoviral and plasmid vaccines had induced a rise in spot forming cells (SFC) for IL5, a cytokine associated with Th2 cells, but the rise was greatest in the recipients of the electroporated plasmid vaccine. By contrast, only the adenoviral vaccine induced a rise in SFC that produced IFN-γ, a cytokine associated with Th1 cells. Studies using MHC class I and II blocking antibodies showed that the IL5 and IFN-γ responses to both types of vaccine were mediated by HLA restricted T lymphocytes. The 1-year progression-free survival rates (PFS) for adenoviral vaccine group and plasmid vector group were 43% and 22% respectively. Figure 1 shows 1-year PFS rates for each group. Hence electroporation provides a more rapid and simpler means of preparing IL2/CD40L expressing B-CLL vaccines, but the cells express higher levels of IL2 and lower levels of “secondary” co-stimulator molecules than adenoviral vaccines, and produce an anti-tumor immune response of different polarity. Currently, we are evaluating electroporation of mRNA encoded CD40L which appears to augment upregulation of additional costimulatory molecules. Figure Figure

scholarly journals P06.14 Characterization of tumor-infiltrating T cells by highly multiplexed immunofluorescence imaging

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A47.2-A48
Author(s):  
E Criado-Moronati ◽  
A Gosselink ◽  
J Kollet ◽  
A Dzionek ◽  
B Heemskerk
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
T Cell Subsets ◽  
Imaging Technology ◽  
Part Time

BackgroundThe adoptive cell transfer (ACT) of tumor-infiltrating T lymphocytes (TILs) has shown remarkable results in patients with different cancer types. The antitumor effect of this therapy is mainly attributed to a small fraction of tumor-reactive T lymphocytes (TRLs) that recognize mutated peptides as well as overexpressed self-antigens. Therefore, the enrichment and expansion of TRLs constitutes a promising immunotherapy approach. However, the specific targeting of individual mutated antigens represents a daunting challenge for widespread therapeutic application. Alternatively, we hypothesize that TRLs could be identified and enriched by a surface marker (or combination thereof) in an antigen-independent manner as a result of the chronic antigen exposure and other factors present in the tumor microenvironment (TME).Materials and MethodsWe screened T cell activation and exhaustion markers, among others, on different tumor tissues using the MACSima™ Imaging Platform, an instrument for the highly multiplexed immunofluorescence imaging technology MICS (Multiparameter Imaging Cell Screen), enabling investigation of hundreds of markers on a single section. Moreover, flow cytometry and single-cell RNA sequencing analyses of T cells from tumor digests were performed to complement the characterization of TILs.ResultsThe MICS results highlighted the complexity of the TME, mainly composed of tumor cells, fibroblasts and endothelial vessels. In some cases, an extensive immune infiltrate consisted of T cells, plasma cells, some B cells and distinct myeloid cells was observed. Particularly, CD8 T cells from different tumor areas exhibited a tissue-resident memory phenotype with the expression of CD69, CD45RO or CD103. Activated/exhausted CD8 T cells were homogenously found across the imaged tumor areas. However, there was a tendency to find them in close proximity to tumor cells, especially for CD8 subsets expressing CD39 and other relevant markers, which may suggest the identification of tumor-reactive CD8 T cell populations. Flow cytometry data revealed the presence of similar T cell phenotypes in the patient´s TILs from tumor digests.ConclusionsThis imaging technology offers the possibility to study multiple parameters—including the localization—of relevant cells in the TME such as T cells. The phenotypic and functional characterization of different T cell subsets will allow the further investigation of their anti-tumor reactivity. Ultimately, the enrichment and expansion of the identified tumor-reactive T cell population hold great promises to improve the efficiency of T cell therapy against cancer.Disclosure InformationE. Criado-Moronati: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Gosselink: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. J. Kollet: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Dzionek: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. B. Heemskerk: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG.

scholarly journals 691 Identification of shared tumor epitopes from endogenous retroviruses inducing high avidity cytotoxic T cells for cancer immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A719-A719
Author(s):  
Paola Bonaventura ◽  
Vincent Alcazer ◽  
Virginie Mutez ◽  
Laurie Tonon ◽  
Juliette Martin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Endogenous Retroviruses ◽  
High Avidity ◽  
T Cell Epitopes ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

BackgroundHuman endogenous retroviruses (HERVs) are aberrantly expressed by tumor cells and may represent a source of T cell epitopesMethodsUsing TCGA pancancer RNAseq data (n=8,893 samples), we developed a bioinformatics-based method to select cancer-specific HERVs associated with a cytotoxic T cell response (“cyt-HERVs”) and identify shared T cell epitope candidates. T cells were primed with selected short and long peptide candidates from HLA-A2+ healthy donors. Peptide-specific dextramers were used to sort and expand specific CD8+ T cell clones and determine their TCR sequences and avidity. Cytotoxicity was assessed against HERV-expressing tumor cell lines and patient-derived organoids using Incucyte and Nanolive technologies (Flowchart, figure 1).ResultsIn a pancancer analysis, we identified 57 HML-2/HERV-K HLA-A*0201 epitope candidates from 27 distinct open reading frames. Six shared HLA-A2 strong binders 9-mer peptides, present on multiple HERVs located on different chromosomes, and with translational evidence found in mass spectrometry public datasets, were selected and synthetized. In vitro HLA binding assay confirmed peptide-HLA affinity. Priming assays showed the presence of specific CD8+ T cells leading to polyfunctional IFN-γ+ TNF-α+ T cell responses with upregulation of the degranulation marker CD107A upon co-culture with peptide-pulsed T2 cells. Synthetic long peptides containing the epitopes were used to confirm the correct processing by antigen-presenting cells. The functionality of the sorted T cell clones was confirmed using an Elispot assay (GrzB+ IFN-γ+). Their sequenced TCRs were predicted to stably interact with their respective MHC-peptide complexes in a 3D model. This was confirmed by measurement of the functional avidity, which was in the same order as CMV-specific T cell clones. HERV-specific CD8+ T cells induced specific cell death of HLA-A2+ cancer cell lines, associated with IFN-g production, in a HLA-A2 restricted manner. Finally, pre-existing HERV-specific CD8+ T cells were identified using dextramers among tumor infiltrating lymphocytes (TILs) from cancer patients. HERV-specific T cells co-cultured with patient derived organoids showed signs of activation with lysis of the organoid.ConclusionsOur bioinformatics-based approach allowed us to identify shared HERV-derived CD8+ T cell epitopes specifically expressed by tumor cells and inducing high avidity T cell clones able to kill tumor cells in a class I-restricted manner. The detection of TILs recognizing HERV peptides suggests natural presentation of these epitopes in the tumors. These HERV-derived epitopes may thus represent relevant targets for the development of new cancer vaccines or T cell-based therapies, especially in tumors with low mutational burden.Abstract 691 Figure 1Graphical flowchart of HERV antigen validation. Graphical representation of the flowchart used to identify and validate specific CD8+ T cells for shared tumor epitopes from endogenous retroviruses http://dx.doi.org/10.1136/jitc-2021-SITC2021.691

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

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A4.1-A4
Author(s):  
AS Herbstritt ◽  
M Maxwell ◽  
D Yan ◽  
B Cuiffo ◽  
J Cardia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Solid Tumors ◽  
Negative Regulator ◽  
List Type ◽  
Research Support ◽  
Jurkat T Cells ◽  
Human T Cells ◽  
Part Time

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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