Cancer Immunology, Immunotherapy

, Volume 63, Issue 9, pp 901–910 | Cite as

Preclinical evaluation of IL2-based immunocytokines supports their use in combination with dacarbazine, paclitaxel and TNF-based immunotherapy

  • Francesca Pretto
  • Giuliano Elia
  • Nadia Castioni
  • Dario NeriEmail author
Original Article


Antibody-cytokine fusion proteins (“immunocytokines”) represent a promising class of armed antibody products, which allow the selective delivery of potent pro-inflammatory payloads at the tumor site. The antibody-based selective delivery of interleukin-2 (IL2) is particularly attractive for the treatment of metastatic melanoma, an indication for which this cytokine received marketing approval from the US Food and drug administration. We used the K1735M2 immunocompetent syngeneic model of murine melanoma to study the therapeutic activity of F8–IL2, an immunocytokine based on the F8 antibody in diabody format, fused to human IL2. F8–IL2 was shown to selectively localize at the tumor site in vivo, following intravenous administration, and to mediate tumor growth retardation, which was potentiated by the combination with paclitaxel or dacarbazine. Combination treatment led to a substantially more effective tumor growth inhibition, compared to the cytotoxic drugs used as single agents, without additional toxicity. Analysis of the immune infiltrate revealed a significant accumulation of CD4+ T cells 24 h after the administration of the combination. The fusion proteins F8–IL2 and L19–IL2, specific to the alternatively spliced extra domain A and extra domain B of fibronectin respectively, were also studied in combination with tumor necrosis factor (TNF)-based immunocytokines. The combination treatment was superior to the action of the individual immunocytokines and was able to eradicate neoplastic lesions after a single intratumoral injection, a procedure that is being clinically used for the treatment of Stage IIIC melanoma. Collectively, these data reinforce the rationale for the use of IL2-based immunocytokines in combination with cytotoxic agents or TNF-based immunotherapy for the treatment of melanoma patients.


Immunocytokines Interleukin-2 Oncofetal fibronectin Vascular targeting 



Cytotoxic T-lymphocyte antigen 4






Extra domain A of fibronectin


Extra domain B of fibronectin


Food and drug administration


Injected dose




Natural killer cells




Optimal cutting temperature compound




Phosphate buffered saline




Standard error


Small immunoprotein


Tumor growth inhibition ratio


Tumor necrosis factor



The authors thank Kathrin Schwager for her help with biodistribution and immunofluorescence experiments. The study was supported by Swiss National Science Foundation, the Swiss Federal Institute of Technology (ETH), the European Union (FP7 ADAMANT and PRIAT Project) and the Kommission für Technologie und Innovation (KTI MedTech Award to Philochem).

Conflict of interest

Dario Neri is founder and shareholder of Philogen, a biotech company that owns the F8 antibody. Francesca Pretto and Giuliano Elia are Philochem employees. Nadia Castioni declares no conflict of interest.

Supplementary material

262_2014_1562_MOESM1_ESM.pdf (78.4 mb)
Supplementary material 1 (PDF 80271 kb)


  1. 1.
    Smith FO, Downey SG, Klapper JA et al (2008) Treatment of metastatic melanoma using interleukin-2 alone or in conjunction with vaccines. Clin Cancer Res 14(17):5610–5618. doi: 10.1158/1078-0432.CCR-08-0116 PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Ponce R (2008) Adverse consequences of immunostimulation. J Immunotoxicol 5(1):33–41. doi: 10.1080/15476910801897920 PubMedCrossRefGoogle Scholar
  3. 3.
    Baluna R, Sausville EA, Stone MJ, Stetler-Stevenson MA, Uhr JW, Vitetta ES (1996) Decreases in levels of serum fibronectin predict the severity of vascular leak syndrome in patients treated with ricin a chain-containing immunotoxins. Clin Cancer Res 2(10):1705–1712PubMedGoogle Scholar
  4. 4.
    Schwartzentruber DJ (2001) Guidelines for the safe administration of high-dose interleukin-2. J Immunother 24(4):287–293PubMedCrossRefGoogle Scholar
  5. 5.
    Kontermann RE (2012) Antibody-cytokine fusion proteins. Arch Biochem Biophys 526(2):194–205. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  6. 6.
    Pasche N, Neri D (2012) Immunocytokines: a novel class of potent armed antibodies. Drug Discov Today 17(11–12):583–590. doi: 10.1016/j.drudis.2012.01.007 PubMedCrossRefGoogle Scholar
  7. 7.
    Davis CB, Gillies SD (2003) Immunocytokines: amplification of anti-cancer immunity. Cancer Immunol Immunother 52(5):297–308. doi: 10.1007/s00262-002-0349-4 PubMedGoogle Scholar
  8. 8.
    Huston JS, McCartney J, Tai MS et al (1993) Medical applications of single-chain antibodies. Int Rev Immunol 10(2–3):195–217. doi: 10.3109/08830189309061696 PubMedCrossRefGoogle Scholar
  9. 9.
    Holliger P, Prospero T, Winter G (1993) “Diabodies”: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci U S A 90(14):6444–6448PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Gillies SD, Lan Y, Williams S et al (2005) An anti-CD20-IL-2 immunocytokine is highly efficacious in a SCID mouse model of established human B lymphoma. Blood 105(10):3972–3978. doi: 10.1182/blood-2004-09-3533 PubMedCrossRefGoogle Scholar
  11. 11.
    Ko YJ, Bubley GJ, Weber R et al (2004) Safety, pharmacokinetics, and biological pharmacodynamics of the immunocytokine EMD 273066 (huKS-IL2): results of a phase I trial in patients with prostate cancer. J Immunother 27(3):232–239PubMedCrossRefGoogle Scholar
  12. 12.
    Gillessen S, Gnad-Vogt US, Gallerani E et al (2013) A phase I dose-escalation study of the immunocytokine EMD 521873 (Selectikine) in patients with advanced solid tumours. Eur J Cancer 49(1):35–44. doi: 10.1016/j.ejca.2012.07.015 PubMedCrossRefGoogle Scholar
  13. 13.
    King DM, Albertini MR, Schalch H et al (2004) Phase I clinical trial of the immunocytokine EMD 273063 in melanoma patients. J Clin Oncol 22(22):4463–4473. doi: 10.1200/JCO.2004.11.035 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Carnemolla B, Borsi L, Balza E et al (2002) Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix. Blood 99(5):1659–1665PubMedCrossRefGoogle Scholar
  15. 15.
    Marlind J, Kaspar M, Trachsel E et al (2008) Antibody-mediated delivery of interleukin-2 to the stroma of breast cancer strongly enhances the potency of chemotherapy. Clin Cancer Res 14(20):6515–6524. doi: 10.1158/1078-0432.CCR-07-5041 PubMedCrossRefGoogle Scholar
  16. 16.
    Villa A, Trachsel E, Kaspar M et al (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(11):2405–2413. doi: 10.1002/ijc.23408 PubMedCrossRefGoogle Scholar
  17. 17.
    Tarli L, Balza E, Viti F et al (1999) A high-affinity human antibody that targets tumoral blood vessels. Blood 94(1):192–198PubMedGoogle Scholar
  18. 18.
    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(7):1814–1824. doi: 10.1158/0008-5472.CAN-11-1919 PubMedCrossRefGoogle Scholar
  19. 19.
    Pedretti M, Verpelli C, Marlind J et al (2010) Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma. Br J Cancer 103(6):827–836. doi: 10.1038/sj.bjc.6605832 PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    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(3):751–758. doi: 10.1038/jid.2012.376 PubMedCrossRefGoogle Scholar
  21. 21.
    Schliemann C, Palumbo A, Zuberbuhler K et al (2009) Complete eradication of human B-cell lymphoma xenografts using rituximab in combination with the immunocytokine L19–IL2. Blood 113(10):2275–2283. doi: 10.1182/blood-2008-05-160747 PubMedCrossRefGoogle Scholar
  22. 22.
    Halin C, Gafner V, Villani ME et al (2003) Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha. Cancer Res 63(12):3202–3210PubMedGoogle Scholar
  23. 23.
    Schwager K, Kaspar M, Bootz F et al (2009) Preclinical characterization of DEKAVIL (F8-IL10), a novel clinical-stage immunocytokine which inhibits the progression of collagen-induced arthritis. Arthritis Res Ther 11(5):R142. doi: 10.1186/ar2814 PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Schwager K, Villa A, Rosli C, Neri D, Rosli-Khabas M, Moser G (2011) A comparative immunofluorescence analysis of three clinical-stage antibodies in head and neck cancer. Head Neck Oncol 3:25. doi: 10.1186/1758-3284-3-25 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Johannsen M, Spitaleri G, Curigliano G et al (2010) The tumour-targeting human L19–IL2 immunocytokine: preclinical safety studies, phase I clinical trial in patients with solid tumours and expansion into patients with advanced renal cell carcinoma. Eur J Cancer 46(16):2926–2935. doi: 10.1016/j.ejca.2010.07.033 PubMedCrossRefGoogle Scholar
  26. 26.
    Eigentler TK, Weide B, de Braud F et al (2011) A dose-escalation and signal-generating study of the immunocytokine L19–IL2 in combination with dacarbazine for the therapy of patients with metastatic melanoma. Clin Cancer Res 17(24):7732–7742. doi: 10.1158/1078-0432.CCR-11-1203 PubMedCrossRefGoogle Scholar
  27. 27.
    Spitaleri G, Berardi R, Pierantoni C et al (2013) Phase I/II study of the tumour-targeting human monoclonal antibody-cytokine fusion protein L19–TNF in patients with advanced solid tumours. J Cancer Res Clin Oncol 139(3):447–455. doi: 10.1007/s00432-012-1327-7 PubMedGoogle Scholar
  28. 28.
    Papadia F, Basso V, Patuzzo R et al (2013) Isolated limb perfusion with the tumor-targeting human monoclonal antibody-cytokine fusion protein L19–TNF plus melphalan and mild hyperthermia in patients with locally advanced extremity melanoma. J Surg Oncol 107(2):173–179. doi: 10.1002/jso.23168 PubMedCrossRefGoogle Scholar
  29. 29.
    Weide B, Eigentler TK, Pflugfelder A et al (2011) Survival after intratumoral interleukin-2 treatment of 72 melanoma patients and response upon the first chemotherapy during follow-up. Cancer Immunol Immunother 60(4):487–493. doi: 10.1007/s00262-010-0957-3 PubMedCrossRefGoogle Scholar
  30. 30.
    Weide B, Derhovanessian E, Pflugfelder A et al (2010) High response rate after intratumoral treatment with interleukin-2: results from a phase 2 study in 51 patients with metastasized melanoma. Cancer 116(17):4139–4146. doi: 10.1002/cncr.25156 PubMedCrossRefGoogle Scholar
  31. 31.
    Weide B, Eigentler TK, Pflugfelder A et al (2014) Intralesional treatment of stage III metastatic melanoma patients with L19–IL2 results in sustained clinical and systemic immunologic responses. Cancer Immunol Res. doi: 10.1158/2326-6066.CIR-13-0206 PubMedGoogle Scholar
  32. 32.
    Kripke ML (1979) Speculations on the role of ultraviolet radiation in the development of malignant melanoma. J Natl Cancer Inst 63(3):541–548PubMedGoogle Scholar
  33. 33.
    Talmadge JE, Fidler IJ (1982) Enhanced metastatic potential of tumor cells harvested from spontaneous metastases of heterogeneous murine tumors. J Natl Cancer Inst 69(4):975–980PubMedGoogle Scholar
  34. 34.
    Frey K, Schliemann C, Schwager K, Giavazzi R, Johannsen M, Neri D (2010) The immunocytokine F8–IL2 improves the therapeutic performance of sunitinib in a mouse model of renal cell carcinoma. J Urol 184(6):2540–2548. doi: 10.1016/j.juro.2010.07.030 PubMedCrossRefGoogle Scholar
  35. 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(5):1206–1213. doi: 10.1038/bjc.2013.421 PubMedCrossRefGoogle Scholar
  36. 36.
    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(15):4092–4103. doi: 10.1158/1078-0432.CCR-12-0282 PubMedCrossRefGoogle Scholar
  37. 37.
    Schwager K, Bootz F, Imesch P, Kaspar M, Trachsel E, Neri D (2011) The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model. Hum Reprod 26(9):2344–2352. doi: 10.1093/humrep/der195 PubMedCrossRefGoogle Scholar
  38. 38.
    Borsi L, Balza E, Carnemolla B et al (2003) Selective targeted delivery of TNF alpha to tumor blood vessels. Blood 102(13):4384–4392. doi: 10.1182/blood-2003-04-1039 PubMedCrossRefGoogle Scholar
  39. 39.
    Starnes CO (1992) Coley’s toxins. Nature 360(6399):23. doi: 10.1038/360023b0 PubMedCrossRefGoogle Scholar
  40. 40.
    Pretto F, Neri D (2013) Pharmacotherapy of metastatic melanoma: emerging trends and opportunities for a cure. Pharmacol Ther 139(3):405–411. doi: 10.1016/j.pharmthera.2013.05.006 PubMedCrossRefGoogle Scholar
  41. 41.
    Gutbrodt KL, Schliemann C, Giovannoni L et al. (2013) Antibody-based delivery of interleukin-2 to neovasculature has potent activity against acute myeloid leukemia. Sci Transl Med 5 (201):201ra118. doi  10.1126/scitranslmed.3006221
  42. 42.
    Erba PA, Sollini M, Orciuolo E et al (2012) Radioimmunotherapy with radretumab in patients with relapsed hematologic malignancies. J Nucl Med 53(6):922–927. doi: 10.2967/jnumed.111.101006 PubMedCrossRefGoogle Scholar
  43. 43.
    Heuveling DA, de Bree R, Vugts DJ et al (2013) Phase 0 microdosing PET study using the human mini antibody F16SIP in head and neck cancer patients. J Nucl Med 54(3):397–401. doi: 10.2967/jnumed.112.111310 PubMedCrossRefGoogle Scholar
  44. 44.
    Santimaria M, Moscatelli G, Viale GL et al (2003) Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer. Clin Cancer Res 9(2):571–579PubMedGoogle Scholar
  45. 45.
    Sauer S, Erba PA, Petrini M et al (2009) Expression of the oncofetal ED-B-containing fibronectin isoform in hematologic tumors enables ED-B-targeted 131I-L19SIP radioimmunotherapy in hodgkin lymphoma patients. Blood 113(10):2265–2274. doi: 10.1182/blood-2008-06-160416 PubMedCrossRefGoogle Scholar
  46. 46.
    Frey K, Fiechter M, Schwager K et al (2011) Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions. Exp Dermatol 20(8):685–688. doi: 10.1111/j.1600-0625.2011.01314.x PubMedCrossRefGoogle Scholar
  47. 47.
    Halin C, Rondini S, Nilsson F et al (2002) Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature. Nat Biotechnol 20(3):264–269. doi: 10.1038/nbt0302-264 PubMedCrossRefGoogle Scholar
  48. 48.
    Kaspar M, Trachsel E, Neri D (2007) The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis. Cancer Res 67(10):4940–4948. doi: 10.1158/0008-5472.CAN-07-0283 PubMedCrossRefGoogle Scholar
  49. 49.
    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(2):467–477. doi: 10.1002/ijc.28359 PubMedCrossRefGoogle Scholar
  50. 50.
    Testori A, Faries MB, Thompson JF et al (2011) Local and intralesional therapy of in-transit melanoma metastases. J Surg Oncol 104(4):391–396. doi: 10.1002/jso.22029 PubMedCrossRefGoogle Scholar
  51. 51.
    Radny P, Caroli UM, Bauer J et al (2003) Phase II trial of intralesional therapy with interleukin-2 in soft-tissue melanoma metastases. Br J Cancer 89(9):1620–1626. doi: 10.1038/sj.bjc.6601320 PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Weide B, Zelba H, Derhovanessian E et al (2012) Functional T cells targeting NY-ESO-1 or Melan-A are predictive for survival of patients with distant melanoma metastasis. J Clin Oncol 30(15):1835–1841. doi: 10.1200/JCO.2011.40.2271 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Francesca Pretto
    • 1
  • Giuliano Elia
    • 1
  • Nadia Castioni
    • 2
  • Dario Neri
    • 2
    Email author
  1. 1.Philochem AGOtelfingenSwitzerland
  2. 2.Department of Chemistry and Applied BiosciencesSwiss Federal Institute of Technology (ETH)ZurichSwitzerland

Personalised recommendations