Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve

J Immunother Cancer. 2021 Feb;9(2):e001691. doi: 10.1136/jitc-2020-001691.


Background: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire.

Methods: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis.

Results: The MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the 'warm' MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the 'cool' 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model.

Conclusion: The hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

Trial registration: ClinicalTrials.gov NCT03493945.

Keywords: cytokines; immunomodulation; immunotherapy; translational medical research; vaccination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents, Immunological / administration & dosage*
  • Antineoplastic Agents, Immunological / pharmacology
  • Cell Line, Tumor
  • Colorectal Neoplasms / drug therapy*
  • Colorectal Neoplasms / immunology
  • Combined Modality Therapy
  • Docetaxel / administration & dosage*
  • Docetaxel / pharmacology
  • Female
  • Humans
  • Immune Checkpoint Inhibitors / administration & dosage*
  • Immune Checkpoint Inhibitors / pharmacology
  • Interleukin-15 / agonists
  • Mice
  • Receptors, OX40 / antagonists & inhibitors
  • Recombinant Fusion Proteins / administration & dosage*
  • Recombinant Fusion Proteins / pharmacology
  • Treatment Outcome
  • Triple Negative Breast Neoplasms / drug therapy*
  • Triple Negative Breast Neoplasms / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / antagonists & inhibitors
  • Vaccines, DNA / administration & dosage*
  • Vaccines, DNA / genetics
  • Vaccines, DNA / pharmacology
  • Xenograft Model Antitumor Assays


  • ALT-803
  • Antineoplastic Agents, Immunological
  • Immune Checkpoint Inhibitors
  • Interleukin-15
  • Receptors, OX40
  • Recombinant Fusion Proteins
  • Tumor Necrosis Factor Receptor Superfamily, Member 9
  • Vaccines, DNA
  • Docetaxel

Associated data

  • ClinicalTrials.gov/NCT03493945