CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy

Lauren Giuffrida; Kevin Sek; Melissa A Henderson; Junyun Lai; Amanda X Y Chen; Deborah Meyran; Kirsten L Todd; Emma V Petley; Sherly Mardiana; Christina Mølck; Gregory D Stewart; Benjamin J Solomon; Ian A Parish; Paul J Neeson; Simon J Harrison; Lev M Kats; Imran G House; Phillip K Darcy; Paul A Beavis

doi:10.1038/s41467-021-23331-5

CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy

Nat Commun. 2021 May 28;12(1):3236. doi: 10.1038/s41467-021-23331-5.

Authors

Lauren Giuffrida^#^{1

2}, Kevin Sek^#^{1

2}, Melissa A Henderson^{1

2}, Junyun Lai^{1

2}, Amanda X Y Chen^{1

2}, Deborah Meyran^{1

2}, Kirsten L Todd^{1

2}, Emma V Petley^{1

2}, Sherly Mardiana^{1

2}, Christina Mølck³, Gregory D Stewart⁴, Benjamin J Solomon², Ian A Parish^{1

2}, Paul J Neeson^{1

2}, Simon J Harrison^{2

5}, Lev M Kats^{2

3}, Imran G House^{6

7}, Phillip K Darcy^{8

9

10

11}, Paul A Beavis^{12

13}

Affiliations

¹ Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia.
² Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Vic, Australia.
³ Department of Pathology, University of Melbourne, Parkville, Vic, Australia.
⁴ Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic, Australia.
⁵ Clinical Haematology and Centre of Excellence for Cellular Immunotherapies, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Vic, Australia.
⁶ Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia. [email protected].
⁷ Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Vic, Australia. [email protected].
⁸ Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia. [email protected].
⁹ Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Vic, Australia. [email protected].
¹⁰ Department of Pathology, University of Melbourne, Parkville, Vic, Australia. [email protected].
¹¹ Department of Immunology, Monash University, Clayton, Vic, Australia. [email protected].
¹² Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia. [email protected].
¹³ Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Vic, Australia. [email protected].

^# Contributed equally.

Abstract

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A_2A receptor (A_2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A_2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A_2AR are superior to shRNA mediated knockdown or pharmacological blockade of A_2AR. Mechanistically, human A_2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A_2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A_2AR for the improvement of CAR T cell function in the clinic.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine / metabolism
Adenosine A2 Receptor Antagonists / pharmacology
Animals
CRISPR-Cas Systems / genetics
Cell Engineering / methods
Cell Line, Tumor / transplantation
Disease Models, Animal
Female
Gene Editing
Gene Expression Regulation, Neoplastic / immunology
Gene Knockdown Techniques
Gene Knockout Techniques
Humans
Immunotherapy, Adoptive / methods*
Lymphocytes, Tumor-Infiltrating / immunology
Mice
Mice, Transgenic
Neoplasms / genetics
Neoplasms / immunology
Neoplasms / therapy*
RNA, Small Interfering / metabolism
RNA-Seq
Receptor, Adenosine A2A / genetics*
Receptor, Adenosine A2A / metabolism
Receptor, ErbB-2 / genetics
Receptors, Chimeric Antigen / immunology
Receptors, Chimeric Antigen / metabolism
Signal Transduction / drug effects
Signal Transduction / genetics
Signal Transduction / immunology
T-Lymphocytes / immunology
T-Lymphocytes / metabolism
T-Lymphocytes / transplantation*
Tumor Escape / drug effects
Tumor Escape / genetics

Substances

ADORA2A protein, human
Adenosine A2 Receptor Antagonists
Adora2a protein, mouse
RNA, Small Interfering
Receptor, Adenosine A2A
Receptors, Chimeric Antigen
ERBB2 protein, human
Receptor, ErbB-2
Adenosine