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 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.

scholarly journals P09.05 Plasma CD27, a surrogate of intratumoral CD27-CD70 interaction, correlates with immunotherapy resistance in renal cancer

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A30.1-A30
Author(s):  
N Benhamouda ◽  
I Sam ◽  
N Epaillard ◽  
A Gey ◽  
A Saldmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Principal Investigator ◽  
Cell Phenotype ◽  
Metastatic Rcc ◽  
Research Grant

BackgroundCD70, a costimulatory molecule on antigen presenting cells, is known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). However, persistent interaction of CD27 and CD70 such as in chronic infection may exhaust the T cell pool and promote apoptosis. Surprisingly, our analysis based on TCGA database show that clear cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors. Despite the important clinical efficacy of immunotherapy by anti-PD-1 in RCC patients, the overall response to anti-PD1 remains modest. The relationship between the CD27-CD70 interaction in the RCC and the response to immunotherapy is still unclear.Materials and MethodsTo study the CD27 and CD70 expression in the tumor microenvironment (TME), FFPE tumor tissues from 25 RCC patients were analysed using multiplex in situ immunofluorescence. 10 fresh RCC tumor samples were collected to analyse the phenotype of CD27+ T cells by flow cytometry and 4 samples were proceeded for single-cell RNA-seq analysis. A cohort of metastatic RCC patients (n = 35) treated by anti-PD-1 were enrolled for the measurement of plasma sCD27 by ELISA and the survival analysis is also realized.ResultsIn the TME, we demonstrated that CD27+ T cells interact with CD70-expressing tumor cells. In fresh tumors from RCC patients, CD27+ T cells express higher levels of cleaved caspase 3 (a classical marker of apoptosis) than CD27- T cells. We confirmed the apoptotic signature (BAX, FASLG, BCL2L11, CYCS, FBXO32, LGALS1, PIK3R1, TERF1, TXNIP, CDKN2A) of CD27+ T cells by single-cell RNAseq analysis. CD27+T cells also had a tissue resident memory T cell phenotype with enriched gene expression of ITGAE, PRDM1, RBPJ and ZNF683. Moreover, CD27+T cells display an exhaustion phenotype with the expression of multiple inhibitory receptors gene signature (PDCD1, CTLA4, HAVCR2, LAG3, etc). Besides, intratumoral CD27-CD70 interaction significantly correlates with plasma sCD27 concentration in RCC (p = 0.0017). In metastatic RCC patients treated with anti-PD-1, higher levels of sCD27 predict poor overall survival (p = 0.037), while it did not correlate with inflammatory markers or clinical prognostic criteria.ConclusionsIn conclusion, we demonstrated that sCD27, a surrogate of T cell dysfunction in tumors likely induced by persistent interactions of CD27+T cells and CD70-expressing tumor cells, is a predictive biomarker of resistance to immunotherapy in mRCC. To our knowledge, this is the first report showing that a peripheral blood biomarker may reflect certain aspects of the tumor-host interaction in the tumor microenvironment. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be further extended to other types of tumors. CD70-CD27 interaction could thus be considered as a mechanism of tumor escape, but also a novel therapeutic target in cancers.Disclosure InformationN. Benhamouda: None. I. Sam: None. N. Epaillard: None. A. Gey: None. A. Saldmann: None. J. Pineau: None. M. Hasan: None. V. Verkarre: None. V. Libri: None. S. Mella: None. C. Granier: None. C. Broudin: None. P. Ravel: None. B. Jabla: None. N. Chaput: None. L. Albiges: None. Y. Vano: None. O. Adotevi: None. S. Oudard: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; SIRIC CARPEM, FONCER. E. Tartour: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Fondation ARC, INCA PLBio, Labex Immuno-Oncology, SIRIC CARPEM, FONCER, IDEX université de Paris, Inserm Transfert.

599 New checkpoints controlling function of cytotoxic lymphocytes infiltrating human carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A634-A634
Author(s):  
Anna Herbstritt ◽  
Elfriede Noessner ◽  
Petra Prinz ◽  
Mani Kadiyala ◽  
Melissa Maxwell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Growth ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Diacylglycerol Kinase ◽  
Tumor Control ◽  
T Cell Function

BackgroundAlthough present in high numbers, T and NK cells appear functionally impaired in the renal cell carcinoma (RCC) tumor milieu, as they cannot be stimulated to degranulation and IFN-γ production. This is in part due to altered regulation of signaling downstream of the T cell receptor (TCR). Increased diacylglycerol kinase alpha (DGK-α) has been observed in T and NK cells from the RCC tumor microenvironment (TME). Ex vivo inhibition of DGK-α by the commercially available inhibitor R59022 was able to restore responsiveness to stimulation.1 2 Inhibition of DGK-α is reported to also block tumor cell growth and survival.3 4 Many T cells from RCC additionally express the immune checkpoint Programmed cell Death-1 (PD-1). Interaction of PD-1 with PD-L1 on tumor cells blocks AKT signaling and inhibits T cell function. In the clinic, blocking the PD-1/PD-L1 interaction allows tumor control in some patients; however, the majority of patients do not respond long-term. Since DGK-α acts downstream of PD-1 it may, if overactive, curb T cell function despite PD-1/PD-L1 blockade. 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 investigate alternative means using RNA interference (RNAi) to target DGK-α alone as well as in combination with PD-1.MethodsKnockdown was achieved by RNAi using INTASYLTM compounds, developed by Phio Pharmaceuticals. These compounds incorporate drug-like properties into siRNA, resulting in enhanced uptake with no need for transfection reagents. Efficacy was analyzed on mRNA and protein level by rt-qPCR, flow cytometry and Western Blot. Functional assays include cytotoxicity and cytokine production in tumor-mimicking environments.ResultsUsing INTASYLTM compounds, silencing of DGK-α was observed in human U2OS osteosarcoma as well as K562 erythroleukemic cells. PD-1 knockdown was achieved in human T cells isolated from peripheral blood mononuclear cells (PBMC). Synergy of DGK-α and PD-1 knockdown is tested in tumor-mimicking in vitro systems using T cell/tumor cell co-cultures at high tumor cell density where T and NK cells become functional suppressed as observed in the TME.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. Self-delivering RNAi technology represents a promising approach to targeting intracellular immune checkpoints such as DGK-α, in addition to PD-1 inhibition.ReferencesPrinz PU, Mendler AN, Masouris I, Durner L, Oberneder R, Noessner E. High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention. J Immunol 2012 Jun 15;188(12):5990–6000. doi: 10.4049/jimmunol.1103028. Epub 2012 May 9. PMID: 22573804.Prinz PU, Mendler AN, Brech D, Masouris I, Oberneder R, Noessner E. NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention. Int J Cancer 2014 Oct 15;135(8):1832–41. doi: 10.1002/ijc.28837. Epub 2014 Mar 26. PMID: 24615391.Torres-Ayuso P, Daza-Martín M, Martín-Pérez J, Ávila-Flores A, Mérida I. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src. Oncotarget 2014 Oct 30;5(20):9710–26. doi: 10.18632/oncotarget.2344. PMID: 25339152; PMCID: PMC4259432.Yanagisawa K, Yasuda S, Kai M, Imai S, Yamada K, Yamashita T, Jimbow K, Kanoh H, Sakane F. Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-induced apoptosis of human melanoma cells through NF-kappaB activation. Biochim Biophys Acta 2007 Apr; 1771(4):462–74. doi: 10.1016/j.bbalip.2006.12.008. Epub 2007 Jan 8. PMID: 17276726.

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

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.

Therapeutic effect of local Interleukin-15 with radiotherapy in breast cancer.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 158-158
Author(s):  
Elena Garcia Martinez ◽  
Karsten A Pilones ◽  
Joseph Aryankalayil ◽  
Silvia Formenti ◽  
Sandra Demaria
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Therapeutic Effect ◽  
Tumor Immunity ◽  
Tumor Infiltrating Lymphocytes ◽  
Tumor Control ◽  
Complete Regression ◽  
Growth And Survival

158 Background: Interleukin (IL)-15 is a key regulator of T cell homeostasis with activity in cancer and a favorable toxicity profile compared to IL-2. IL-15 stimulates the proliferation and effector differentiation of CD8+T cells, and the proliferation and activation of natural killer (NK) cells. We observed IL-15 upregulation by gene arrays in radiotherapy (RT)-treated TSA mouse breast cancer, suggesting that it may play a role in RT-induced anti-tumor immunity. However, the upregulation was modest prompting us to test the hypothesis that administration of IL-15 may enhance in situ vaccination by RT. Methods: BALB/c mice with established poorly immunogenic TSA tumors were sham-treated, treated with tumor-targeted RT (8GyX3 days), IL-15 given peri-tumorally (2 ug/mouse/day for 10 days) starting on the first day of RT, and RT+IL-15, and monitored for tumor growth and survival. Tumor infiltrating lymphocytes (TIL) were analyzed by flow cytometry and immunostaining. In some experiments, Batf3-/-mice were used as tumor recipient. Results: IL-15 by itself was ineffective, but it significantly increased tumor control by RT (p=0.0007, RT versus RT+IL-15) leading to complete responses in 50% of the mice, most of them durable. Analysis of TILs showed significantly increased NK cells (CD45+ CD3- DX5+) in tumors treated with RT+IL-15 (p<0.0004 versus sham-treated; p<0.02 versus RT). NK cells were also more activated as indicated by expression of CD122 and CD137. Depletion of NK cells completely abrogated the therapeutic effect of the combination, while CD8 T cell depletion reduced tumor control and rate of complete regression. Interestingly, Batf3-/- mice, which lack CD103+ DCs, showed reduced response to RT+IL-15 compared to WT mice. Conclusions: Data suggest that local IL-15 with RT is an effective strategy to induce anti-tumor immunity to poorly immunogenic breast cancer. NK cells are critical mediators of the response, and may act by both killing tumor cells and promoting priming of CD8 T cells. Experiments are ongoing to determine the mechanisms of durable complete responses. <footer>Acknowledgments: IL-15 was provided by NCI BRB. Garcia-Martinez E was supported by GEICAM grant.</footer>

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 Combining De-Glycosylating Agents with CAR-T Cells for Targeting Solid Tumors and Reducing Toxicity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4544-4544
Author(s):  
Beatrice Greco ◽  
Katia Paolella ◽  
Barbara Camisa ◽  
Valeria Malacarne ◽  
Laura Falcone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Combined Approach ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Glycosylated Proteins

Abstract Background: The adoptive transfer of CAR-T cells have shown impressive results against B-cell malignancies, but still limited efficacy against solid tumors. The discovery of the key factors regulating the activity of CAR-T cells is required to improve their antitumor potency and modulate toxicities. Since solid tumors display a wide range of glycosylation alterations, including increased N-glycan branching, we hypothesized that peptidic epitopes may be masked by glycans from CAR-T cell targeting, especially in richly glycosylated proteins. Results: To investigate if sugar chains may be sterically hulking for CAR-T cell targeting, we generated N-glycosylation-defective pancreatic tumor cell lines. This aim has been achieved by knocking-out the expression of the glycosyltransferase Mgat5, a key enzyme involved in the process of N-glycan branching, using the CRISPR-Cas9 technology. As model antigens for CAR targeting, we focused on CD44v6 and CEACAM-5 (CEA) since they are both heavily glycosylated proteins over-expressed on a wide variety of solid tumors, including pancreatic adenocarcinoma. Strikingly, the impairment of N-glycosylation resulted in a dramatic increase of tumor targeting by both CD44v6 (4-fold, p<0,001) and CEA CAR-T cells (10-fold, p<0,001). This effect associated with improved CAR-T cell activation, suggesting more proficient antigen engagement. To exploit this mechanism in order to increase the efficacy of CAR-T cells against solid tumors, we sought to block tumor N-glycosylation with the clinical-grade glucose/mannose analogue 2-Deoxy-D-glucose (2DG). Similarly to genetically induced glycosylation blockade, treatment with 2DG also sensitized tumor cells to recognition by CAR-T cells, significantly increasing their elimination (CD44v6: 3-fold, p<0,01; CEA: 13-fold, p<0,001). Notably, 2DG alone proved to be ineffective as mono-therapy, suggesting a synergistic effect with CAR-T cells. To get more insights on this mechanism, we took advantage of previous studies reporting that 2DG interference with N-glycosylation can be reverted by the addition of exogenous mannose. Of notice, mannose did revert the synergy between 2DG and CAR-T cells (p<0,05), implying that blockade of N-glycosylation rather than glycolysis is the crucial mechanism involved. These findings were further confirmed by using the N-glycosylation inhibitor tunicamycin (CD44v6: 2,5-fold; CEA: 5-fold, p<0,01) and by Western blot, looking at the presence of de-glycosylated proteins on tumor cell surface after 2DG treatment. Next, we challenged the combined approach in a pancreatic adenocarcinoma xenograft mouse model. Accordingly with in vitro data, mice receiving CAR-T cells highly benefited from 2DG administration (5-fold less tumor at 7d, p<0,05), which conversely was unable to mediate any antitumor effect alone. Interestingly, improved antitumor activity was accompanied by a decrease in the frequency of CAR-T cells expressing one or more exhaustion and senescence markers, such as TIM-3, LAG-3, PD-1 and CD57 (SPICE software analysis, p=0,0105). Finally, thanks to metabolic deregulation (Warburg effect), 2DG is expected to selectively accumulate in cancer cells compared to healthy tissues, supporting the safety of the combined approach. Accordingly, we observed that the same doses of 2DG able to enhance tumor cell recognition by CAR-T cells failed to increase the elimination of healthy cells, such as keratinocytes. Conclusions: Our results indicate that i) the glycosylation status of tumor cells regulates the efficacy of CAR-T cells, especially when targeting highly glycosylated antigens, and ii) combining CAR-T cells with the de-glycosylation agent 2DG, which preferentially accumulates in tumor masses, may pave the way for a successful immunotherapy against solid tumors. Disclosures Bonini: Intellia Therapeutics: Research Funding. Bondanza:Novartis: Employment.

scholarly journals Enhanced Solid Tumor Recognition and T cell Stemness with SynNotch CAR Circuits

2021 ◽  
Author(s):  
Axel Hyrenius-Wittsten ◽  
Yang Su ◽  
Minhee Park ◽  
Julie M Garcia ◽  
Nathaniel Perry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Specific Antigen ◽  
Tumor Control ◽  
Car T Cells ◽  
Therapeutic Benefits ◽  
Car T ◽  
Tumor Recognition ◽  
Life Threatening

The lack of highly tumor-specific antigens limits the development of engineered T cell therapeutics because of life-threatening on-target/off-tumor toxicities. Here we identify ALPPL2 as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma. ALPPL2 can act as a sole target for chimeric antigen receptor (CAR) therapy or be combined with tumor-associated antigens such as MCAM or mesothelin in synthetic Notch (synNotch) CAR combinatorial antigen circuits. SynNotch CAR T cells display superior tumor control when compared to CAR T cells to the same antigens by prevention of CAR-mediated tonic signaling allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable target for multiple solid tumors and demonstrate the multi-faceted therapeutic benefits of synNotch CAR T cells.

scholarly journals iPSC-Derived NK Cells Synergize with T Cells and Anti-PD-1 Antibody to Mediate Durable Anti-Tumor Responses In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1933-1933
Author(s):  
Jeffrey S. Miller ◽  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Sajid Mahmood ◽  
Paul Rogers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Tumor Control ◽  
Tumor Spheroid ◽  
Advisory Committees ◽  
Tumor Reduction

Cancer immunotherapies have resulted in a paradigm shift in therapy. One of the most successful approaches has been administration of antibodies targeting immune checkpoint inhibitors (ICI), such as programmed death-1 (PD-1), that can reinvigorate functionally exhausted T cells. Unfortunately, durable tumor regression is limited to a minority of patients, and relapse remains a significant concern. Combining novel immunotherapies with ICI s a promising strategy to bolster antitumor responses and response rates. Natural killer (NK) cells mediate direct tumor cell lysis, effectively target MHC low or null transformed cells and are key regulators of T cell responses through the production of inflammatory cytokines and chemokines. In many cancers, NK cell numbers are low, and their functional responses are sub-optimal. The use of allogeneic peripheral blood NK cells for immunotherapy has shown significant clinical promise for the treatment of various cancers. However, sourcing NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs) that can be combined with ICI antibodies for multiple tumor types. To interrogate the ability of iPSC-derived NK cells to synergize with ICI therapy, we developed an in vitro 3D tumor spheroid system to model the combinatorial effects of activated CD3+ T cells, iPSC-derived NK (iNK) cells and ICI blockade for anti-tumor function in a more physiological context than the standard 2D cultures and in real time. Using SKOV-3 (an ovarian cancer line) spheroids as targets in a 160-hour killing assay, we found that iNK cells could mediate significant, but not complete destruction of tumor spheroids (46% tumor reduction). Twice as many activated CD3+ T cells by themselves also induced significant but incomplete tumor spheroid destruction (58% tumor reduction). Combined iNK and activated CD3+ T cells led to robust target cell destruction (71% tumor reduction). Importantly, adding anti-PD-1 antibody (mAb) to activated CD3+ T cells and iNK cells led to near complete elimination of tumor spheroid targets, with >99% tumor reduction. Cytokine and chemokine secretion analyses in co-cultures of activated CD3+ T cells and iNK cells revealed synergistic production of CCL3 and CCL4 for T cell recruitment, and TNF and IFN-γ to augment anti-tumor responses. To determine whether these striking 3D tumor spheroid results simulate the in vivo setting, we injected luciferase-expressing OVCAR8 (a human ovarian cancer line) cells into the peritoneal cavities of immunodeficient NSG mice. Following sublethal irradiation, we treated groups of mice with either anti-PD-1 mAb, activated CD3+ T cells or iNK cells alone or in various combinations. IL-2 was injected i.p. twice weekly for two weeks into all mice except those in the tumor alone group (Figure 1A). In mice that received iNK cells alone, significant tumor control was observed over the first 21 days but was not sustained. Tumor control was similar between groups of mice that received iNK cells and mice that received iNK cells and anti-PD-1 mAb. Mice treated with either anti-PD-1 mAb or activated CD3+ T cells alone exhibited similar rates of tumor growth relative to the untreated group. Modest tumor control was observed in the group of mice that received combined activated CD3+ T cells and anti-PD-1 mAb, though the effect did not reach statistical significance. Durable tumor control past day 21 was observed in groups of mice that received either combined activated CD3+ T cells and iNK cells or activated CD3+ T cells + iNK cells with anti-PD-1 mAb (Figure 1B). Importantly, 50% of mice in the activated CD3+ T cells + iNK cells + anti-PD-1 mAb group exhibited tumor bioluminescence readings well below other treatment group at day 35 (Figure 1C, D). Collectively, these data demonstrate that iNK cells can serve as an off-the-shelf source of high-quality NK cells and synergize with anti-PD-1 ICI therapy to enhance anti-tumor T cell responses in vitro and in vivo, providing a novel immunotherapeutic platform for tumors in which iNK cells, activated CD3+ T cells or anti-PD-1 mAb therapy alone is not sufficiently effective. The current program is under clinical investigation and can be found at clinicaltrials.gov NCT03841110. Disclosures Miller: Moderna: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bjordahl:Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moyar:Fate Therapeutics, Inc: Employment. Blazar:Abbvie Inc: Research Funding; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. OffLabel Disclosure: NK cells

scholarly journals Expression of MyD88/CD40 Drives In Vivo Activation and Proliferation of Chimeric Antigen Receptor-Modified T Cells That Can be Effectively Regulated By Inducible Caspase-9

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 851-851
Author(s):  
Aaron Foster ◽  
Peter Chang ◽  
Pei-Yi Lin ◽  
Jeannette Crisostomo ◽  
Aruna Mahendravada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Tumor Control ◽  
Tumor Cell Lines ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Introduction: Efficacy of chimeric antigen receptor (CAR)-modified T cells is dependent on their in vivo survival and expansion following infusion. The addition of accessory molecules (e.g., costimulatory and cytokine genes) may improve CAR-T proliferation and potency, but may also increase toxicity of these next generation CAR-T cell therapies, suggesting that the incorporation of a built in "safety switch" would balance safety and efficacy in a single, controllable therapy. Here, we demonstrate that cytosolic coexpression of a MyD88/CD40-derived fusion protein dramatically enhances CAR-T activation, cytokine production, and proliferation in vivo, resulting in improved antitumor efficacy. Importantly, CAR-T cell numbers, elevated cytokine levels, and observed CAR-T-related toxicity could be controlled by titratable rimiducid administration to reduce or eliminate CAR-T cells by activating the inducible caspase-9 (iC9) suicide gene. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with retrovirus encoding, iC9, a first generation CAR (with CD3ζ) targeting CD19, Her2 or PSCA, and a detached, fusion protein comprising signaling domains from MyD88 and CD40 (MC). For comparison, additional CARs were constructed without MC, with MyD88 or CD40 elements only, or with conventional CARs coexpressing CD28 within the CAR molecule (CAR.28.ζ). Transduced T cells were assessed in vitro for cytotoxicity, cytokine production and proliferation against tumor cell lines (CD19+: Daudi, Raji; Her2+: SK-BR-3; PSCA+: Capan-1, HPAC). In vivo antitumor efficacy of CAR-modified T cells was assessed using immunodeficient NSG mice engrafted with antigen-matched tumor cell lines (5x105 Raji, i.v.; 1x106 SK-BR-3, s.c; 2x106 HPAC, s.c.) followed by i.t. or i.v. injection of variable doses of T cells. Reduction or elimination of CAR-T cells was performed by i.p. injection of rimiducid (0 - 5 mg/kg). Tumor cell lines expressing luciferase or T cells co-transduced with luciferase-encoding vectors were used for bioluminescence imaging (BLI) to measure tumor growth or T cell expansion/elimination, respectively. Serum cytokine levels were assessed by blood draws and CAR-T cell frequency was measured by flow cytometry. Results: All CAR constructs were stably expressed in T cells (30-90%). CAR vectors coexpressing MC induced high IL-2 levels in vitro when exposed to target antigen+ tumor cells (CD19 = 4246 ± 52, Her2 = 2613 ± 1298, and PSCA = 3263 ± 1393 pg/ml per 1x105 T cells over 48 hrs) and corresponded to improved CAR-T cell proliferation and tumor elimination compared to control vectors. In NSG mice, MC costimulation resulted in >2,000-fold expansion of CD19-targeted CAR-T cells and complete tumor control for >100 days in 100% of mice engrafted with CD19+ Raji cells (p = 0.0002) following injection of 5x106 CAR-T cells, followed on day 7 with a single i.p. dose of rimiducid (5 mg/kg) to control toxicity. MC-enabled CAR-T cells were eliminated or partially reduced by rimiducid titrations, which corresponded to decreased cytokine (IL-6, IFN-γ, TNF-α) levels and restoration of health in animals showing signs of toxicity (e.g., ≥15% weight loss). For solid tumors, Her2-targeted, MC-enabled CAR-T cells showed a 150-fold in vivo expansion and compared favorably to first (Her2.ζ; p = 0.01) and second generation (Her2.28.ζ; p = 0.01) CARs, causing 100% elimination of SK-BR-3 tumors and enhanced survival for >60 days following i.t. injection (p = 0.0015). PSCA-targeted CARs expressing MC also drove complete and durable (>42 days) elimination of large (200 mm3) HPAC tumors in 100% of mice, after a single i.v. injection of 1x107 CAR-T cells followed on day 14 with a single 5 mg/kg i.p. rimiducid dose to reverse toxicity. Summary: Coexpression of MC, and the cell therapy safety switch "CaspaCIDe", in combination with a first generation CAR, together comprising the novel "CIDeCAR" platform technology, dramatically increases efficacy against a number of tumor targets by enhancing T cell engraftment and proliferation following infusion, while incorporating an effective, built-in safety mechanism. In three distinct tumor models, rimiducid administration promptly eliminated signs and symptoms of CAR toxicity without subsequent loss of tumor control. CIDeCAR technology may allow the development of safer and more effective CAR-T cell therapies for a range of difficult-to-treat liquid and solid tumors. Disclosures Foster: Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Lin:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Khalil:Bellicum Pharmaceuticals: Employment. Saha:Bellicum Pharmaceuticals: Employment. Shaw:Bellicum Pharmaceuticals: Employment. Morschl:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

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