Skip to main content

Advertisement

SpringerLink
Log in

The antibody-mediated targeted delivery of interleukin-13 to syngeneic murine tumors mediates a potent anticancer activity

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

Abstract

We describe the expression and in vivo characterization of an antibody–cytokine fusion protein, based on murine Interleukin-13 (IL13) and the monoclonal antibody F8, specific to the alternatively spliced extra domain A of fibronectin, a marker of neo-angiogenesis. The IL13 moiety was fused at the C-terminal extremity of the F8 antibody in diabody format. The resulting F8-IL13 immunocytokine retained the full binding properties of the parental antibody and cytokine bioactivity. The fusion protein could be expressed in mammalian cells, purified to homogeneity and showed a preferential accumulation at the tumor site. When used as single agent at doses of 200 μg, F8-IL13 exhibited a strong inhibition of tumor growth rate in two models of cancer (F9 teratocarcinoma and Wehi-164), promoting an infiltration of various types of leukocytes into the neoplastic mass. This anticancer activity could be potentiated by combination with an immunocytokine based on the F8 antibody and murine IL12, leading to complete and long-lasting tumor eradications. Mice cured from Wehi-164 sarcomas acquired a durable protective antitumor immunity, and selective depletion of immune cells revealed that the antitumor activity was mainly mediated by cluster of differentiation 4-positive T cells. This study indicates that IL13 can be efficiently delivered to the tumor neo-vasculature and that it mediates a potent anticancer activity in the two models of cancer investigated in this study. The observed mechanism of action for F8-IL13 was surprising, since immunocytokines based on other payloads (e.g., IL2, IL4, IL12 and TNF) eradicate cancer by the combined contribution of natural killer cells and cluster of differentiation 8-positive T cells.

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

CD4+:

Cluster of differentiation 4-positive

CD8+:

Cluster of differentiation 8-positive

CHO:

Chinese hamster ovary

EDA:

Extra domain A

ELISA:

Enzyme-linked immunosorbent assay

i.v.:

Intravenous

IFN:

Interferon

ILx:

Interleukin-x

NK:

Natural killer

PAGE:

Polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

s.c.:

Subcutaneous

SDS:

Sodium dodecyl sulfate

SIP:

Signal peptide

Th:

T helper

TNF:

Tumor necrosis factor

References

  1. Jackaman C, Bundell CS, Kinnear BF, Smith AM, Filion P, van Hagen D, Robinson BW, Nelson DJ (2003) IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2. J Immunol 171:5051–5063. doi:10.4049/jimmunol.171.10.5051

    Article  CAS  PubMed  Google Scholar 

  2. Welander CE (1987) Overview of preclinical and clinical-studies of interferon Alfa-2b in combination with cytotoxic drugs. Invest New Drug 5:S47–S59. doi:10.1007/BF00207263

    CAS  Google Scholar 

  3. Halin C, Rondini S, Nilsson F, Berndt A, Kosmehl H, Zardi L, Neri D (2002) Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature. Nat Biotechnol 20:264–269. doi:10.1038/nbt0302-264

    Article  CAS  PubMed  Google Scholar 

  4. Hess C, Venetz D, Neri D (2014) Emerging classes of armed antibody therapeutics against cancer. Med Chem Commun 5:408–431. doi:10.1039/C3MD00360D

    Article  CAS  Google Scholar 

  5. Schwager K, Hemmerle T, Aebischer D, Neri D (2013) The immunocytokine L19-IL2 eradicates cancer when used in combination with CTLA-4 blockade or with L19-TNF. J Invest Dermatol 133:751–758. doi:10.1038/jid.2012.376

    Article  CAS  PubMed  Google Scholar 

  6. Hemmerle T, Neri D (2014) The antibody-based targeted delivery of interleukin-4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer. Int J Cancer 134:467–477. doi:10.1002/ijc.28359

    Article  PubMed  Google Scholar 

  7. Castellani P, Viale G, Dorcaratto A, Nicolo G, Kaczmarek J, Querze G, Zardi L (1994) The fibronectin isoform containing the ED-B oncofetal domain: a marker of angiogenesis. Int J Cancer 59:612–618. doi:10.1002/ijc.2910590507

    Article  CAS  PubMed  Google Scholar 

  8. Borsi L, Castellani P, Allemanni G, Neri D, Zardi L (1998) Preparation of phage antibodies to the ED-A domain of human fibronectin. Exp Cell Res 240:244–251. doi:10.1006/excr.1998.3946

    Article  CAS  PubMed  Google Scholar 

  9. Frey K, Fiechter M, Schwager K, Belloni B, Barysch MJ, Neri D, Dummer R (2011) Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions. Exp Dermatol 20:685–688. doi:10.1111/j.1600-0625.2011.01314.x

    Article  CAS  PubMed  Google Scholar 

  10. Moschetta M, Pretto F, Berndt A et al (2012) Paclitaxel enhances therapeutic efficacy of the F8-IL2 immunocytokine to EDA-fibronectin-positive metastatic human melanoma xenografts. Cancer Res 72:1814–1824. doi:10.1158/0008-5472.CAN-11-1919

    Article  CAS  PubMed  Google Scholar 

  11. Schliemann C, Wiedmer A, Pedretti M, Szczepanowski M, Klapper W, Neri D (2009) Three clinical-stage tumor targeting antibodies reveal differential expression of oncofetal fibronectin and tenascin-C isoforms in human lymphoma. Leuk Res 33:1718–1722. doi:10.1016/j.leukres.2009.06.025

    Article  CAS  PubMed  Google Scholar 

  12. Gutbrodt KL, Schliemann C, Giovannoni L, Frey K, Pabst T, Klapper W, Berdel WE, Neri D (2013) Antibody-based delivery of interleukin-2 to neovasculature has potent activity against acute myeloid leukemia. Sci Transl Med 5:201ra118. doi: 10.1126/scitranslmed.3006221

  13. Villa A, Trachsel E, Kaspar M, Schliemann C, Sommavilla R, Rybak JN, Rosli C, Borsi L, Neri D (2008) A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo. Int J Cancer 122:2405–2413. doi:10.1002/ijc.23408

    Article  CAS  PubMed  Google Scholar 

  14. Pasche N, Neri D (2012) Immunocytokines: a novel class of potent armed antibodies. Drug Discov Today 17:583–590. doi:10.1016/j.drudis.2012.01.007

    Article  CAS  PubMed  Google Scholar 

  15. Schrama D, Reisfeld RA, Becker JC (2006) Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov 5:147–159. doi:10.1038/nrd1957

    Article  CAS  PubMed  Google Scholar 

  16. Kontermann RE (2012) Antibody-cytokine fusion proteins. Arch Biochem Biophys 526:194–205. doi:10.1016/j.abb.2012.03.001

    Article  CAS  PubMed  Google Scholar 

  17. Pasche N, Wulhfard S, Pretto F, Carugati E, Neri D (2012) The antibody-based delivery of interleukin-12 to the tumor neovasculature eradicates murine models of cancer in combination with paclitaxel. Clin Cancer Res 18:4092–4103. doi:10.1158/1078-0432.CCR-12-0282

    Article  CAS  PubMed  Google Scholar 

  18. Gutbrodt KL, Casi G, Neri D (2014) Antibody-based delivery of IL2 and cytotoxics eradicates tumors in immunocompetent mice. Mol Cancer Ther 13:1772–1776. doi:10.1158/1535-7163.MCT-14-0105

    Article  CAS  PubMed  Google Scholar 

  19. de Vries JE (1998) The role of IL-13 and its receptor in allergy and inflammatory responses. J Allergy Clin Immunol 102:165–169. doi:10.1016/S0091-6749(98)70080-6

    Article  PubMed  Google Scholar 

  20. Hancock A, Armstrong L, Gama R, Millar A (1998) Production of interleukin 13 by alveolar macrophages from normal and fibrotic lung. Am J Respir Cell Mol Biol 18:60–65. doi:10.1165/ajrcmb.18.1.2627

    Article  CAS  PubMed  Google Scholar 

  21. de Waal MalefytR, Abrams JS, Zurawski SM et al (1995) Differential regulation of IL-13 and IL-4 production by human CD8+ and CD4+ Th0, Th1 and Th2 T cell clones and EBV-transformed B cells. Int Immunol 7:1405–1416. doi:10.1093/intimm/7.9.1405

    Article  Google Scholar 

  22. Zurawski G, de Vries JE (1994) Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today 15:19–26. doi:10.1016/0167-5699(94)90021-3

    Article  CAS  PubMed  Google Scholar 

  23. Minty A, Chalon P, Derocq JM et al (1993) Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature 362:248–250. doi:10.1038/362248a0

    Article  CAS  PubMed  Google Scholar 

  24. Blais Y, Gingras S, Haagensen DE, Labrie F, Simard J (1996) Interleukin-4 and interleukin-13 inhibit estrogen-induced breast cancer cell proliferation and stimulate GCDFP-15 expression in human breast cancer cells. Mol Cell Endocrinol 121:11–18. doi:10.1016/0303-7207(96)03843-9

    Article  CAS  PubMed  Google Scholar 

  25. Renard N, Duvert V, Banchereau J, Saeland S (1994) Interleukin-13 inhibits the proliferation of normal and leukemic human B-cell precursors. Blood 84:2253–2260

    CAS  PubMed  Google Scholar 

  26. Serve H, Oelmann E, Herweg A et al (1996) Inhibition of proliferation and clonal growth of human breast cancer cells by interleukin 13. Cancer Res 56:3583–3588

    CAS  PubMed  Google Scholar 

  27. Lebel-Binay S, Laguerre B, Quintin-Colonna F et al (1995) Experimental gene therapy of cancer using tumor cells engineered to secrete interleukin-13. Eur J Immunol 25:2340–2348. doi:10.1002/eji.1830250833

    Article  CAS  PubMed  Google Scholar 

  28. Porgador A, Bannerji R, Watanabe Y, Feldman M, Gilboa E, Eisenbach L (1993) Antimetastatic vaccination of tumor-bearing mice with two types of IFN-gamma gene-inserted tumor cells. J Immunol 150:1458–1470

    CAS  PubMed  Google Scholar 

  29. Hsi LC, Kundu S, Palomo J, Xu B, Ficco R, Vogelbaum MA, Cathcart MK (2011) Silencing IL-13Ralpha2 promotes glioblastoma cell death via endogenous signaling. Mol Cancer Ther 10:1149–1160. doi:10.1158/1535-7163.MCT-10-1064

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Gafner V, Trachsel E, Neri D (2006) An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties. Int J Cancer 119:2205–2212. doi:10.1002/ijc.22101

    Article  CAS  PubMed  Google Scholar 

  31. Pasche N, Woytschak J, Wulhfard S, Villa A, Frey K, Neri D (2011) Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7. J Biotechnol 154:84–92. doi:10.1016/j.jbiotec.2011.04.003

    Article  CAS  PubMed  Google Scholar 

  32. Frey K, Zivanovic A, Schwager K, Neri D (2011) Antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation. Integr Biol (Camb) 3:468–478. doi:10.1039/c0ib00099j

    Article  CAS  Google Scholar 

  33. Holliger P, Prospero T, Winter G (1993) “Diabodies”: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA 90:6444–6448. doi:10.1073/pnas.90.14.6444

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Sommavilla R, Pasche N, Trachsel E, Giovannoni L, Roesli C, Villa A, Neri D, Kaspar M (2010) Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12. Protein Eng Des Sel 23:653–661. doi:10.1093/protein/gzq038

    Article  CAS  PubMed  Google Scholar 

  35. Hemmerle T, Probst P, Giovannoni L, Green AJ, Meyer T, Neri D (2013) The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity. Br J Cancer 109:1206–1213. doi:10.1038/bjc.2013.421

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Hemmerle T, Doll F, Neri D (2014) Antibody-based delivery of IL4 to the neovasculature cures mice with arthritis. Proc Natl Acad Sci USA 111:12008–12012. doi:10.1073/pnas.1402783111

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Pretto F, Elia G, Castioni N, Neri D (2014) Preclinical evaluation of IL2-based immunocytokines supports their use in combination with dacarbazine, paclitaxel and TNF-based immunotherapy. Cancer Immunol Immunother 63:901–910. doi:10.1007/s00262-014-1562-7

    Article  CAS  PubMed  Google Scholar 

  38. Pedretti M, Verpelli C, Marlind J, Bertani G, Sala C, Neri D, Bello L (2010) Combination of temozolomide with immunocytokine F16-IL2 for the treatment of glioblastoma. Br J Cancer 103:827–836. doi:10.1038/sj.bjc.6605832

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Xie Y, Akpinarli A, Maris C, Hipkiss EL, Lane M, Kwon EK, Muranski P, Restifo NP, Antony PA (2010) Naive tumor-specific CD4(+) T cells differentiated in vivo eradicate established melanoma. J Exp Med 207:651–667. doi:10.1084/jem.20091921

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Quezada SA, Simpson TR, Peggs KS et al (2010) Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts. J Exp Med 207:637–650. doi:10.1084/jem.20091918

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Ebbinghaus C, Ronca R, Kaspar M, Grabulovski D, Berndt A, Kosmehl H, Zardi L, Neri D (2005) Engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy. Int J Cancer 116:304–313. doi:10.1002/ijc.20952

    Article  CAS  PubMed  Google Scholar 

  42. Hemmerle T, Neri D (2014) The dose-dependent tumor targeting of antibody-IFNgamma fusion proteins reveals an unexpected receptor-trapping mechanism in vivo. Cancer Immunol Res 2:559–567. doi:10.1158/2326-6066.CIR-13-0182

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Eidgenössische Technische Hochschule (ETH) Zürich, to the Swiss National Science Foundation, to The Commission for Technology and Innovation (CTI) Switzerland, to the European Union (FP7 Project PRIAT) and to Philochem AG for financial contribution.

Conflict of interest

Dario Neri is a cofounder and shareholder of Philogen SpA (Siena, Italy), the company that owns the F8 antibody. Other authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology Zurich (ETH Zürich), Vladimir-Prelog-Weg 1-5/10, 8093, Zurich, Switzerland

    Christian Hess & Dario Neri

Authors
  1. Christian Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dario Neri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dario Neri.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 818 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hess, C., Neri, D. The antibody-mediated targeted delivery of interleukin-13 to syngeneic murine tumors mediates a potent anticancer activity. Cancer Immunol Immunother 64, 635–644 (2015). https://doi.org/10.1007/s00262-015-1666-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-015-1666-8

Keywords

Search

Navigation