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599 New checkpoints controlling function of cytotoxic lymphocytes infiltrating human carcinoma
  1. Anna Herbstritt1,
  2. Elfriede Noessner1,
  3. Petra Prinz1,
  4. Mani Kadiyala2,
  5. Melissa Maxwell2,
  6. Dingxue Yan2,
  7. James Cardia2 and
  8. Simon Fricker2
  1. 1Helmholtz Zentrum München, Munich, Germany
  2. 2Phio Pharmaceuticals, Marlborough, MA, USA


Background Although 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.

Methods Knockdown 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.

Results Using 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.

Conclusions Strong 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.


  1. Prinz 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.

  2. 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.

  3. 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.

  4. 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.

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