Skip to main content

Advertisement

SpringerLink
Log in

A highly efficient modality to block the degradation of tryptophan for cancer immunotherapy: locked nucleic acid-modified antisense oligonucleotides to inhibit human indoleamine 2,3-dioxygenase 1/tryptophan 2,3-dioxygenase expression

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Tumors can utilize a diverse repertoire of immunosuppressive mechanisms to evade attack by the immune system. Despite promising success with blockade of immune checkpoints like PD-1 the majority of patients does not respond to current immunotherapies. The degradation of tryptophan into immunosuppressive kynurenine is an important immunosuppressive pathway. Recent attempts to target the key enzymes of this pathway—IDO1 and TDO2—have so far failed to show therapeutic benefit in the clinic, potentially caused by insufficient target engagement. We, therefore, sought to add an alternative, highly efficient approach to block the degradation of tryptophan by inhibiting the expression of IDO1 and TDO2 using locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs). We show that LNA-modified ASOs can profoundly inhibit the expression of IDO1 and TDO2 in cancer cells in vitro without using a transfection reagent with IC50 values in the sub-micromolar range. We furthermore measured kynurenine production by ASO-treated cancer cells in vitro and observed potently reduced kynurenine levels. Accordingly, inhibiting IDO1 expression in cancer cells in an in vitro system leads to increased proliferation of activated T cells in coculture. We furthermore show that combined treatment of cancer cells in vitro with IDO1-specific ASOs and small molecule inhibitors can reduce the production of kynurenine by cancer cells in a synergistic manner. In conclusion, we propose that a combination of LNA-modified ASOs and small molecule inhibitors should be considered as a strategy for efficient blockade of the degradation of tryptophan into kynurenine in cancer immunotherapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

AhR:

Aryl hydrocarbon receptor

ASO:

Antisense oligonucleotide

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GCN2:

General control nonderepressible 2 kinase

HPRT1:

Hypoxanthine phosphoribosyltransferase 1

IDO1:

Indoleamine 2,3-dioxygenase 1

LNA:

Locked nucleic acid

PGE2:

Prostaglandin E2

PTO:

Phosphorothioate

TAM:

Tumor associated macrophage

TDO2:

Tryptophan 2,3-dioxygenase

References

  1. Herbst RS, Baas P, Kim D-W et al (2016) Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet Lond Engl 387:1540–1550. https://doi.org/10.1016/S0140-6736(15)01281-7

    Article  CAS  Google Scholar 

  2. Reck M, Rodríguez-Abreu D, Robinson AG et al (2016) Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 375:1823–1833. https://doi.org/10.1056/NEJMoa1606774

    Article  CAS  PubMed  Google Scholar 

  3. Hodi FS, O’Day SJ, McDermott DF et al (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:711–723. https://doi.org/10.1056/NEJMoa1003466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Uyttenhove C, Pilotte L, Théate I et al (2003) Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9:1269–1274. https://doi.org/10.1038/nm934

    Article  CAS  PubMed  Google Scholar 

  5. Opitz CA, Litzenburger UM, Sahm F et al (2011) An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor. Nature 478:197–203. https://doi.org/10.1038/nature10491

    Article  CAS  PubMed  Google Scholar 

  6. Lee GK, Park HJ, Macleod M et al (2002) Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 107:452–460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Apetoh L, Quintana FJ, Pot C et al (2010) The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27. Nat Immunol 11:854–861. https://doi.org/10.1038/ni.1912

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu X, Shin N, Koblish HK et al (2010) Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood 115:3520–3530. https://doi.org/10.1182/blood-2009-09-246124

    Article  CAS  PubMed  Google Scholar 

  9. Crosignani S, Bingham P, Bottemanne P et al (2017) Discovery of a novel and selective indoleamine 2,3-dioxygenase (IDO-1) inhibitor 3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and its characterization as a potential clinical candidate. J Med Chem 60:9617–9629. https://doi.org/10.1021/acs.jmedchem.7b00974

    Article  CAS  PubMed  Google Scholar 

  10. Pei Z, Mendonca R, Gazzard L et al (2018) Aminoisoxazoles as potent inhibitors of tryptophan 2,3-dioxygenase 2 (TDO2). ACS Med Chem Lett 9:417–421. https://doi.org/10.1021/acsmedchemlett.7b00427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Beatty GL, O’Dwyer PJ, Clark J et al (2017) First-in-human phase 1 study of the oral inhibitor of indoleamine 2,3-dioxygenase-1 Epacadostat (INCB024360) in patients with advanced solid malignancies. Clin Cancer Res Off J Am Assoc Cancer Res 23:3269–3276. https://doi.org/10.1158/1078-0432.CCR-16-2272

    Article  CAS  Google Scholar 

  12. Mitchell TC, Hamid O, Smith DC et al (2018) Epacadostat plus pembrolizumab in patients with advanced solid tumors: phase I results from a multicenter, open-label phase I/II trial (ECHO-202/KEYNOTE-037). J Clin Oncol Off J Am Soc Clin Oncol. https://doi.org/10.1200/JCO.2018.78.9602(JCO2018789602)

    Article  Google Scholar 

  13. Perez RP, Riese MJ, Lewis KD et al (2017) Epacadostat plus nivolumab in patients with advanced solid tumors: preliminary phase I/II results of ECHO-204. J Clin Oncol 35:3003. https://doi.org/10.1200/JCO.2017.35.15_suppl.3003

    Article  Google Scholar 

  14. Long GV, Dummer R, Hamid O et al (2018) Epacadostat (E) plus pembrolizumab (P) versus pembrolizumab alone in patients (pts) with unresectable or metastatic melanoma: results of the phase 3 ECHO-301/KEYNOTE-252 study. J Clin Oncol 36:108. https://doi.org/10.1200/JCO.2018.36.15_suppl.108

    Article  Google Scholar 

  15. Eckstein F (1985) Nucleoside phosphorothioates. Annu Rev Biochem 54:367–402. https://doi.org/10.1146/annurev.bi.54.070185.002055

    Article  CAS  PubMed  Google Scholar 

  16. Stein CA, Hansen JB, Lai J et al (2010) Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res 38:e3–e3. https://doi.org/10.1093/nar/gkp841

    Article  CAS  PubMed  Google Scholar 

  17. Jaschinski F, Korhonen H, Janicot M (2015) Design and selection of antisense oligonucleotides targeting transforming growth factor beta (TGF-β) isoform mRNAs for the treatment of solid tumors. In: Walther W, Stein U (eds) Gene therapy of solid cancers: methods and protocols. Springer, New York, pp 137–151

    Chapter  Google Scholar 

  18. Kashyap AS, Thelemann T, Klar R et al (2019) Antisense oligonucleotide targeting CD39 improves anti-tumor T cell immunity. J Immunother Cancer. https://doi.org/10.1186/s40425-019-0545-9

    Article  PubMed  PubMed Central  Google Scholar 

  19. Beck B, Dörfel D, Lichtenegger FS et al (2011) Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission. J Transl Med 9:151. https://doi.org/10.1186/1479-5876-9-151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ianevski A, He L, Aittokallio T, Tang J (2017) SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinform Oxf Engl 33:2413–2415. https://doi.org/10.1093/bioinformatics/btx162

    Article  CAS  Google Scholar 

  21. R Core Team (2018) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  22. Loewe S (1953) The problem of synergism and antagonism of combined drugs. Arzneimittelforschung 3:285–290

    CAS  PubMed  Google Scholar 

  23. McDermott D, Lebbé C, Hodi FS et al (2014) Durable benefit and the potential for long-term survival with immunotherapy in advanced melanoma. Cancer Treat Rev 40:1056–1064. https://doi.org/10.1016/j.ctrv.2014.06.012

    Article  PubMed  Google Scholar 

  24. Robert C, Thomas L, Bondarenko I et al (2011) Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 364:2517–2526. https://doi.org/10.1056/NEJMoa1104621

    Article  CAS  PubMed  Google Scholar 

  25. Weber JS, D’Angelo SP, Minor D et al (2015) Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol 16:375–384. https://doi.org/10.1016/S1470-2045(15)70076-8

    Article  CAS  PubMed  Google Scholar 

  26. Robert C, Long GV, Brady B et al (2015) Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 372:320–330. https://doi.org/10.1056/NEJMoa1412082

    Article  CAS  PubMed  Google Scholar 

  27. Cady SG, Sono M (1991) 1-Methyl-dl-tryptophan, beta-(3-benzofuranyl)-dl-alanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]-dl-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase. Arch Biochem Biophys 291:326–333

    Article  CAS  PubMed  Google Scholar 

  28. Papadopoulos K, Eder P, Piha-Paul SA et al (2019) First-in-human phase I study of M4112, the first dual inhibitor of indoleamine 2,3-dioxygenase-1 and tryptophan 2,3-dioxygenase 2, in patients with advanced solid malignancies. In: AACR. Atlanta, p abstract no. CT011/6

  29. Théate I, van Baren N, Pilotte L et al (2015) Extensive profiling of the expression of the indoleamine 2,3-dioxygenase 1 protein in normal and tumoral human tissues. Cancer Immunol Res 3:161–172. https://doi.org/10.1158/2326-6066.CIR-14-0137

    Article  CAS  PubMed  Google Scholar 

  30. Zhai L, Ladomersky E, Lauing KL et al (2017) Infiltrating T cells increase IDO1 expression in glioblastoma and contribute to decreased patient survival. Clin Cancer Res 23:6650–6660. https://doi.org/10.1158/1078-0432.CCR-17-0120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kiyozumi Y, Baba Y, Okadome K et al (2018) IDO1 expression is associated with immune tolerance and poor prognosis in patients with surgically resected esophageal cancer. Ann Surg. https://doi.org/10.1097/SLA.0000000000002754

    Article  Google Scholar 

  32. Muller AJ, DuHadaway JB, Donover PS et al (2005) Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Nat Med 11:312–319. https://doi.org/10.1038/nm1196

    Article  CAS  PubMed  Google Scholar 

  33. Hou D-Y, Muller AJ, Sharma MD et al (2007) Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses. Cancer Res 67:792–801. https://doi.org/10.1158/0008-5472.CAN-06-2925

    Article  CAS  PubMed  Google Scholar 

  34. Spranger S, Koblish HK, Horton B et al (2014) Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment. J Immunother Cancer 2:3. https://doi.org/10.1186/2051-1426-2-3

    Article  PubMed  PubMed Central  Google Scholar 

  35. Geary RS, Norris D, Yu R, Bennett CF (2015) Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev 87:46–51. https://doi.org/10.1016/j.addr.2015.01.008

    Article  CAS  PubMed  Google Scholar 

  36. Yu J, Du W, Yan F et al (2013) Myeloid-derived suppressor cells suppress antitumor immune responses through IDO Expression and correlate with lymph node metastasis in patients with breast cancer. J Immunol 190:3783–3797. https://doi.org/10.4049/jimmunol.1201449

    Article  CAS  PubMed  Google Scholar 

  37. Muller AJ, Sharma MD, Chandler PR et al (2008) Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase. Proc Natl Acad Sci 105:17073–17078. https://doi.org/10.1073/pnas.0806173105

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work has been supported by a Grant from the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) (Grant number: 031B0459) and a research Grant provided to AZ from Secarna.

Author information

Authors and Affiliations

  1. Secarna Pharmaceuticals GmbH & Co. KG, Am Klopferspitz 19, 82152, Planegg, Germany

    Richard Klar, Sven Michel, Monika Schell, Lisa Hinterwimmer & Frank Jaschinski

  2. Cancer Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland

    Alfred Zippelius

  3. Medical Oncology, University Hospital Basel, Basel, Switzerland

    Alfred Zippelius

Authors
  1. Richard Klar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sven Michel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Monika Schell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lisa Hinterwimmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alfred Zippelius
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frank Jaschinski
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RK planned the study and experiments, analyzed data, performed statistical analysis and wrote the manuscript. SM performed bioinformatics analysis, performed statistical analysis of the data and wrote the manuscript. MS and LH performed experiments. AZ provided conceptual advice, interpreted data and wrote the manuscript. FJ planned the study, interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Richard Klar or Frank Jaschinski.

Ethics declarations

Conflict of interest

Richard Klar, Sven Michel, Monika Schell, Lisa Hinterwimmer and Frank Jaschinski are employed by Secarna. Richard Klar and Frank Jaschinski are holding patents for IDO1 and TDO2 ASOs, Sven Michel is holding a patent for TDO2 ASOs. Alfred Zippelius received research funding from Secarna Munich.

Ethical approval

Leukapheresis from healthy individuals was collected after informed consent following requirements of the local ethical board and principles of the Helsinki Declaration. The study was approved by the ethics commission of the Technical University of Munich (ethics commission reference: 329/16 S, study approval date: 9-19-2016).

Informed consent

Healthy donors consented in written form that their personal information was anonymized after leukapheresis and their PBMC were used to investigate the effect of antisense oligonucleotide-mediated knockdown of immunosuppressive factors in vitro.

Animal source

Not applicable.

Cell line authentication

EFO-21 (purchased from DSMZ), SKOV-3 (purchased from ATCC), MDA-MB-453 (purchased from ATCC) and A-172 (purchased from ATCC) cells were used in this study. Authentication of cell lines was not required as cells were purchased from professional vendors and master cell banks were generated shortly after taking the original vials into culture. Cells were kept in culture for a maximum time of 3 weeks to minimize the risk of contaminations.

Note on previous publication

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 449 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Klar, R., Michel, S., Schell, M. et al. A highly efficient modality to block the degradation of tryptophan for cancer immunotherapy: locked nucleic acid-modified antisense oligonucleotides to inhibit human indoleamine 2,3-dioxygenase 1/tryptophan 2,3-dioxygenase expression. Cancer Immunol Immunother 69, 57–67 (2020). https://doi.org/10.1007/s00262-019-02438-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-019-02438-1

Keywords

Search

Navigation