Antibody-based delivery of tumor necrosis factor (L19-TNFα) and interleukin-2 (L19-IL2) to tumor-associated blood vessels has potent immunological and anticancer activity in the syngeneic J558L BALB/c myeloma model

  • Hans D. Menssen
  • Ulf Harnack
  • Ulrike Erben
  • Dario Neri
  • Burkhard Hirsch
  • Horst Dürkop
Original Article – Cancer Research

Abstract

Purpose

To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice.

Methods

PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20 h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells.

Results

Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20 h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4− CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (− 50%), and an increase in PD-L1+ myeloma cells.

Conclusion

IL2, TNFα, or IL2 plus TNFα (final 20 h) increased the proportion of CD4− CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.

Keywords

J558L myeloma Targeted immunocytokines IL-2 TNF-α, immune modulation of tumor microenvironment PD-L1 PD-1 

Notes

Acknowledgements

We thank Paulina Kuczma, Ines Puschendorf, Katja Dörfel, and Edda von der Wall for thoroughly conducting the animal studies and for the immunohistological stainings.

Author contributions

HDM, DN, and HD designed the overall study concept and the animal studies. UH designed and performed the in vitro studies on human PBMCs. UE and BH performed the immunohistologic studies. HDM, UH, and HD wrote the manuscript with the help of UE and BH.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Statement of human rights

All procedures performed in studies involving human participants (blood samples obtained from human volunteers) were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Statement of animal welfare

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

References

  1. Agata Y et al (1996) Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 8:765–772CrossRefPubMedGoogle Scholar
  2. Balasa B et al (2015) Elotuzumab enhances natural killer cell activation and myeloma cell killing through interleukin‑2 and TNF‑α pathways. Cancer Immunol Immunother 64:61–73CrossRefPubMedGoogle Scholar
  3. Balza E et al (2006) Targeted delivery of tumor necrosis factor-alpha to tumor vessels induces a therapeutic T cell-mediated immune response that protects the host against syngeneic tumors of different histologic origin. Clin Cancer Res 12:2575–2582CrossRefPubMedGoogle Scholar
  4. Balza E et al (2010) Therapy-induced antitumor vaccination in neuroblastomas by the combined targeting of IL-2 and TNFalpha. Int J Cancer 127:101–110CrossRefPubMedGoogle Scholar
  5. Bootz F, Neri D (2010) Immunocytokines: a novel class of products for the treatment of chronic inflammation and autoimmune conditions. Drug Discov Today 21:180–189CrossRefGoogle Scholar
  6. Buda G et al (2016) Phase II study of the combination of IL-2 with zoledronic acid as maintenance therapy following autologous stem cell transplant in patients with multiple myeloma. Blood 128:56–97Google Scholar
  7. D’Agostino M, Boccadoro M, Smith EL (2017) Novel immunotherapies for multiple myeloma. Curr Hematol Malig Rep 12(4):344–357CrossRefPubMedGoogle Scholar
  8. Danielli R et al (2015) Intralesional administration of L19-IL2/L19-TNF in stage III or stage IVM1a melanoma patients: results of a phase II study. Cancer Immunol Immunother 64:999–1009CrossRefPubMedGoogle Scholar
  9. De Luca R et al (2017) Potency-matched dual cytokine-antibody fusion proteins for cancer therapy. Mol Cancer Ther 16(11):2442–2451CrossRefPubMedGoogle Scholar
  10. Eigentler TK 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 17:7732–7742CrossRefPubMedGoogle Scholar
  11. Erba PA et (2012) Radioimmunotherapy with radretumab in patients with relapsed hematologic malignancies. J Nucl Med 53:922–927CrossRefPubMedGoogle Scholar
  12. Filella X et al (1996) Cytokines (IL-6, TNF-alpha, IL-1 alpha) and soluble interleukin-2 receptor as serum tumor markers in multiple myeloma. Cancer Detect Prev 20:52–56PubMedGoogle Scholar
  13. Gavriatopoulou M et al (2017) Efficacy and safety of elotuzumab for the treatment of multiple myeloma. Exp Opin Drug Safety 16:237–245Google Scholar
  14. Gottlieb DJ et al (1990) Malignant plasma cells are sensitive to LAK cell lysis: pre-clinical and clinical studies of interleukin 2 in the treatment of multiple myeloma. Br J Haematol 75:499–505CrossRefPubMedGoogle Scholar
  15. 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 134:467–477CrossRefPubMedGoogle Scholar
  16. Hemmerle T et al (2013) The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity. Br J Cancer 109:1206–1213CrossRefPubMedPubMedCentralGoogle Scholar
  17. Henry JY et al (2013) Enhanced cross-priming of naïve CD38+ T cells by dendritic cells treated by the IMiDs immunomodulatory compounds lenalidomide and pomalidomide. Immunology 139:377–385CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hoving S et al (2006) Early destruction of tumor vasculature in tumor necrosis factor-alpha-based isolated limb perfusion is responsible for tumor response. Anticancer Drugs 17:949–959CrossRefPubMedGoogle Scholar
  19. Keir ME et al (2008) PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26:677–704CrossRefPubMedGoogle Scholar
  20. Klein C et al (2017) Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines. Oncoimmunology 6:e1277306CrossRefPubMedPubMedCentralGoogle Scholar
  21. Krejcik J et al (2016) Daratumumab depletes CD38+ immune regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma. Blood 128:384–394CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lejeune FJ, Lienard D, Matter M, Ruegg C (2006) Efficiency of recombinant human TNF in human cancer therapy. Cancer Immun 6:1–17Google Scholar
  23. Locher R et al (2014) Abundant in vitro expression of the oncofetal ED-B-containing fibronectin translates into selective pharmacodelivery of (131)I-L19SIP in a prostate cancer patient. J Cancer Res Clin Oncol 140:35–43CrossRefPubMedGoogle Scholar
  24. Lonial S et al (2015) Elotuzumab therapy for relapsed or refractory multiple myeloma. N Engl J Med 373:621–631CrossRefPubMedGoogle Scholar
  25. Mosely SI et al (2017) Rational selection of syngeneic preclinical tumro models for immunotherapeutic drug discovery. Cancer Immunol Res 5:29–41CrossRefPubMedGoogle Scholar
  26. Palumbo A et al (2016) Daratumumab, bortezomib, and dexamethasone for multiple myeloma. N Engl J Med 375:754–766CrossRefPubMedGoogle Scholar
  27. Papadia F 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 107:173–179CrossRefPubMedGoogle Scholar
  28. Peest D et al (1995) Low-dose recombinant interleukin-2 therapy in advanced multiple myeloma. Br J Haematol 89:328–337CrossRefPubMedGoogle Scholar
  29. Rapoport AP et al (2015) NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med 21:914–921CrossRefPubMedPubMedCentralGoogle Scholar
  30. Rodriguez-Otero P et al (2017) Is immunotherapy here to stay in multiple myeloma? Haematolgica 102:423–453CrossRefGoogle Scholar
  31. Santimaria M et al (2003) Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer. Clin Cancer Res 9:571–579PubMedGoogle Scholar
  32. Sauer S 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 113:2265–2274CrossRefPubMedGoogle Scholar
  33. Schliemann C et al (2009) Complete eradication of human B-cell lymphoma xenografts using rituximab in combination with the immunocytokine L19-IL2. Blood 113:2275–2283CrossRefPubMedGoogle Scholar
  34. 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 133:751–758CrossRefPubMedGoogle Scholar
  35. SEER Stat Fact Sheets (2017) SEER Stat Fact Sheets: Myeloma NCI surveillance, epidemiology, and end results program. SEER Stat Fact Sheets: Myeloma”. NCI (Retrieved 18 Aug 2017) Google Scholar
  36. Sehgal K et al (2015) Clinical and pharmacodynamics analysis of pomalidomide dosing strategies in myeloma: impact on immune activation and cereblon targets. Blood 125:4042–4051CrossRefPubMedPubMedCentralGoogle Scholar
  37. Sponaas AM et al (2016) PDL1expression on plasma and dendritic cells in myeloma bone marrow suggests benefit of targeted PD1-PDL1 therapy. PLoS One.  https://doi.org/10.1371/journal.pone.0139867 Google Scholar
  38. Szmania S et al (2015) Ex vivo-expanded natural killer cells demonstrate robust proliferation in vivo in high-risk relapsed multiple myeloma patients. J Immunother 38:24–36CrossRefPubMedPubMedCentralGoogle Scholar
  39. Vacca A et al (2015) A disturbance of the IL-2/IL2–2 receptor system parallels the activity of multiple myeloma. Clin Exp Immunol 84:429–434Google Scholar
  40. Wang L et al (2017) High level of soluble interleukin-2 receptor in serum predicts treatment resistance and poor progression-free survival in multiple myeloma. Ann Hematol.  https://doi.org/10.1007/s00277-017-3125-4 (Epub ahead of print)Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Hans D. Menssen
    • 1
  • Ulf Harnack
    • 2
  • Ulrike Erben
    • 3
  • Dario Neri
    • 4
  • Burkhard Hirsch
    • 5
    • 6
  • Horst Dürkop
    • 7
  1. 1.Division of Hematology and Oncology, Campus Benjamin Franklin, Department of MedicineCharité-Universitätsmedizin BerlinBerlinGermany
  2. 2.Division of Oncology and Hematology, Campus Mitte, Department of MedicineCharité-Universitätsmedizin BerlinBerlinGermany
  3. 3.Division of Gastroenterology, Infectious Diseases and Rheumatology, Medical DepartmentCampus Benjamin Franklin, Charité-Universitätsmedizin BerlinBerlinGermany
  4. 4.Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical SciencesETH ZurichZurichSwitzerland
  5. 5.Department of Pathology at Campus Benjamin Franklin, Campus Mitte, Institute of PathologyCharité-Universitätsmedizin BerlinBerlinGermany
  6. 6.Department of Medicine, Campus Mitte, Institute of PathologyCharité-Universitätsmedizin BerlinBerlinGermany
  7. 7.Pathodiagnostik BerlinBerlinGermany

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