Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation

Abstract

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.

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Abbreviations

APCs:

Antigen-presenting cells

ATCC:

American Type Culture Collection

CD47 m:

CD47-translational blocking morpholino

CTLA4:

Cytotoxic T-lymphocyte-associated protein 4

MDSCs:

Myeloid-derived suppressor cells

NCI:

National Cancer Institute

NY-ESO-1:

Cancer/testis antigen 1B

PD-1:

Programmed death-1

RT:

Radiation therapy

SIRPα:

Signal-regulatory protein-α

TCGA:

The Cancer Genome Atlas

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Acknowledgements

We would like to thank the staff of NCI’s Pathology/Histotechnology Laboratory at Frederick for performing immunohistochemical staining for CD3.

Funding

This work was supported by the Intramural Research Program of the NIH/National Cancer Institute.

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Affiliations

Authors

Contributions

Anthony L. Schwartz, Pulak R. Nath, Michael Allgauer, Elizabeth C. Lessey-Morillon, Lisa A. Ridnour, John M. Sipes, and Yves M. Morillon performed experiments described herein. Zhiya Yu and Nicholas P. Restifo prepared ESO-specific human T-cell lines. Anthony L. Schwartz and David D. Roberts planned experiment design and wrote the paper.

Corresponding author

Correspondence to David D. Roberts.

Ethics declarations

Conflict of interest

Anthony L. Schwartz is the Chief Executive Officer and shareholder of Morphiex Biotherapeutics which holds licensing rights to the CD47 Morpholino. All other authors of this paper declare no conflicts of interest.

Ethical approval

Animal Studies: The B16 melanoma animal model was carried out under approved protocols (Protocol #LP-026, January 2016) following the guidelines of the National Cancer Institute’s Animal Care and Use Committee.

Informed consent

Human T-cell studies: TCGA data were obtained under informed consent as described (https://cancergenome.nih.gov/abouttcga/policies/informedconsent). No new human materials for T-cell experiments were obtained, thus did not require consent or IRB review.

Animal source

C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Cell line authentication

B16F10 (CRL-6475) mouse melanoma cell line was purchased from ATCC and were authenticated at the Frederick National Laboratory for Cancer Research (Frederick, MD). The origin of the T-cell lines was previously published, described and provided by the Surgery Branch, Center for Cancer Research, National Cancer Institute [33].

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Schwartz, A.L., Nath, P.R., Allgauer, M. et al. Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation. Cancer Immunol Immunother 68, 1805–1817 (2019). https://doi.org/10.1007/s00262-019-02397-7

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Keywords

  • CD47
  • Immunotherapy
  • Melanoma
  • Phosphorodiamidate morpholino oligomer
  • Thrombospondin-1
  • SIRP-α