Cancer Immunology, Immunotherapy

, Volume 67, Issue 1, pp 47–60 | Cite as

Administration of low-dose combination anti-CTLA4, anti-CD137, and anti-OX40 into murine tumor or proximal to the tumor draining lymph node induces systemic tumor regression

  • Jonathan P. O. HebbEmail author
  • Adriane R. Mosley
  • Felipe Vences-Catalán
  • Narendiran Rajasekaran
  • Anna Rosén
  • Peter Ellmark
  • Dean W. FelsherEmail author
Original Article


The delivery of immunomodulators directly into the tumor potentially harnesses the existing antigen, tumor-specific infiltrating lymphocytes, and antigen presenting cells. This can confer specificity and generate a potent systemic anti-tumor immune response with lower doses and less toxicity compared to systemic administration, in effect an in situ vaccine. Here, we test this concept using the novel combination of immunomodulators anti-CTLA4, -CD137, and -OX40. The triple combination administered intratumorally at low doses to one tumor of a dual tumor mouse model had dramatic local and systemic anti-tumor efficacy in lymphoma (A20) and solid tumor (MC38) models, consistent with an abscopal effect. The minimal effective dose was 10 μg each. The effect was dependent on CD8 T-cells. Intratumoral administration resulted in superior local and distant tumor control compared to systemic routes, supporting the in situ vaccine concept. In a single tumor A20 model, injection close to the tDLN resulted in similar efficacy as intratumoral and significantly better than targeting a non-tDLN, supporting the role of the tDLN as a viable immunotherapy target in addition to the tumor itself. Distribution studies confirmed expected concentration of antibodies in tumor and tDLN, in keeping with the anti-tumor results. Overall intratumoral or peri-tDLN administration of the novel combination of anti-CTLA4, anti-CD137, and anti-OX40, all agents in the clinic or clinical trials, demonstrates potent systemic anti-tumor effects. This immunotherapeutic combination is promising for future clinical development via both these safe and highly efficacious routes of administration.


Combination immunotherapy Intratumoral Tumor draining lymph node CD137 OX40 CTLA4 





Enzyme-linked immunosorbent assay


Interferon gamma






Lymph node


Monoclonal antibody




Phosphate-buffered saline






Tumor draining lymph node


Tumor necrosis factor receptor



The authors would like to dedicate this study to the memory of Dr. Holbrook Kohrt, who conceptualized and inspired this work but sadly passed away before its completion. He was a great scientist, physician, and human being. He continues to inspire. Also thanks to Dr. Idit Sagiv-Barfi and Dr. Suparna Dutt for their advice and expertise during the course of the project.

Compliance with ethical standards

Conflict of interest

D. Felsher and J. Hebb received research funding from Alligator Biosciences. P. Ellmark and A. Rosén are employees of Alligator Biosciences. The remaining authors have no conflicts of interest to declare.


This project was funded by NIH grants R01 CA170378, U01 CA188383, and R01 CA184384 (D. W. Felsher) and Alligator Biosciences.

Supplementary material

262_2017_2059_MOESM1_ESM.docx (518 kb)
Supplementary material 1 (DOCX 518 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Jonathan P. O. Hebb
    • 1
    • 3
    Email author
  • Adriane R. Mosley
    • 1
  • Felipe Vences-Catalán
    • 1
  • Narendiran Rajasekaran
    • 1
    • 4
  • Anna Rosén
    • 5
  • Peter Ellmark
    • 5
    • 6
  • Dean W. Felsher
    • 1
    • 2
    Email author
  1. 1.Division of Oncology, Department of MedicineStanford University School of MedicineStanfordUSA
  2. 2.Department of PathologyStanford University School of MedicineStanfordUSA
  3. 3.Division of HematologyQEII Health Sciences Centre, Dalhousie UniversityHalifaxCanada
  4. 4.Department of Chemistry and BiochemistryNorthern Arizona UniversityFlagstaffUSA
  5. 5.Alligator BioscienceLundSweden
  6. 6.Department of ImmunotechnologyLund UniversityLundSweden

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