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NKG2D-Fc fusion protein promotes antitumor immunity through the depletion of immunosuppressive cells

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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

A major factor impeding the success of numerous therapeutic approaches in cancer is the immunosuppressive nature of the tumor microenvironment (TME). Hence, methods capable of reverting tumor immunosuppression through depletion or reprogramming of myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs) are of great clinical need. Here, we explore NKG2D-Fc as a modality to modulate antitumor immunity through the depletion of immunosuppressive MDSCs and Tregs in the TME. We have generated the NKG2D-Fc fusion protein and characterized its potential to mediate tumor control and overall survival in LL2 and MC38 murine models. Upon treatment of LL2 or MC38 tumor-bearing mice with NKG2D-Fc, we observe significant tumor control and enhanced survival compared to Fc control. When characterizing MDCSs and Tregs from tumor-bearing mice, we observe clear expression of NKG2D-ligand RAE1γ and subsequent binding of NKG2D-Fc fusion protein to both MDSCs and Tregs. Examining the immune profile of mice treated with NKG2D-Fc reveals significant depletion of MDSCs and Tregs in the TME, as well as an increase in NK cells likely due to the reversed suppressive TME. In conclusion, NKG2D-Fc induces antitumor immunity and tumor control through the depletion of MDSCs and Tregs, subsequently providing a niche for the infiltration and expansion of proinflammatory cells, such as NK cells. Strategies capable of modulating the immunosuppressive state in cancer are in high clinical demand. NKG2D-Fc is a simple, single tool capable of depleting both MDSCs and Tregs and should be further investigated as a therapeutic agent for the treatment of cancer.

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Availability of data and material

All data recorded for this study was performed at the Johns Hopkins Medical Institutes.

Abbreviations

ADCC:

Antibody-dependent cell-mediated cytotoxicity

ARG1:

Arginase 1

CTLA-4:

Cytotoxic T-lymphocyte-associated protein 4

ICB:

Immune checkpoint blockade

ICOS:

Inducible T cell costimulatory

IFNγ:

Interferon gamma

IL-10:

Interleukin-10

MDSCs:

Myeloid-derived suppressor cells

MHC:

Major histocompatibility complex

M-MDSCs:

Monocytic MDSCs

NO:

Nitric oxide

PD-L1:

Program death-ligand 1

PGE2:

Prostaglandin E2

PMN-MDSCs:

Granulocytic MDSCs

ROS:

Reactive oxidative species

scFv-NKG2D:

Single-chain variable fragment with the extracellular domain of NKG2D receptor

TAMs:

Tumor-associated macrophages

TGF-β:

Transforming growth factor beta

Tregs:

Regulatory T cells

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Acknowledgements

The authors would like to acknowledge Dr. T.-C. Wu for facilitating helpful science discussions and Mr. Christopher Carter Polston for providing administrative assistance. This study was supported by an NIH-supported research grant (R01CA233486).

Author information

Author notes

  1. Po-Hao Feng and Brandon Lam: Co-first authors.

Authors and Affiliations

  1. Department of Pathology, The Johns Hopkins University School of Medicine, 1550 Orleans St., CRB II – Room 307, Baltimore, MD, 21287, USA

    Po-Hao Feng, Brandon Lam, Ssu-Hsueh Tseng, Yu-Jui Kung, Emily Farmer, Max A. Cheng & Chien-Fu Hung

  2. Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan

    Po-Hao Feng

  3. Division of Pulmonary Medicine, Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan

    Po-Hao Feng

  4. Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    Chien-Fu Hung

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Contributions

PHF and BL equally contributed to the design of the study, the performance of the experiments, and the writing of the manuscript; SHT and YJK contributed to the performance of the experiments; EF and MC contributed to the writing and preparation of the manuscript; CH contributed to the design of the study.

Corresponding author

Correspondence to Chien-Fu Hung.

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Conflict of interest

The authors do not declare any competing interests.

Ethics approval and consent to participate

The housing and handling of mice follow guidelines established by Johns Hopkins Medical Institutions Animal Care and Use Committee and the National Institutes of Health. The Oncology Center Animal Facility has full-time veterinary support through the Department of Comparative Medicine. Animals are monitored daily for infection and other illnesses by trained animal technicians. The criteria include: infection, failure to thrive, perceived pain, respiratory distress, etc. Research support services include training classes and the capacity for full post-mortem analysis is available on request. Only trained laboratory personnel and animal technicians were allowed to handle laboratory animals. All individuals handling mice were registered to protocols at the Johns Hopkins Animal Care and Use Committee.

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Supplementary Fig.

 1. Anti-tumor effects of NKG2D-Fc at a lower dose. (A) MC38 tumor-bearing mice were treated with 30 μg of NKG2D-Fc or Fc control protein. Same kinetic was followed as in Fig. 1. Tumor growth and Kaplan–Meier survival curves were collected. Data are represented as mean ± SEM, with n = 5 in each group from one representative experiment (PNG 14 kb)

Supplementary Fig.

 2. Gating strategy for Tregs and MDSCs. (A) Representative flow dot plots from spleen depicting gating strategy used to identify MDSC and Tregs in PBMC, spleen, and tumor of mice (PNG 336 kb)

Supplementary Fig.

 3. RAE1 γ staining on circulating MDSCs and Tregs in naïve or tumor-bearing mice. (A) Representative histogram of RAE1 γ on MDSCs and (B) MFI represented as mean ± SEM. (C) Representative histogram of RAE1 γ on Tregs and (B) MFI represented as mean ± SEM. Naïve mice are shown as a grey histogram, and tumor-bearing mice as a purple histogram (PNG 98 kb)

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Feng, PH., Lam, B., Tseng, SH. et al. NKG2D-Fc fusion protein promotes antitumor immunity through the depletion of immunosuppressive cells. Cancer Immunol Immunother 69, 2147–2155 (2020). https://doi.org/10.1007/s00262-020-02615-7

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  • DOI: https://doi.org/10.1007/s00262-020-02615-7

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