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

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 A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR 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 A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-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. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Anti-mouse TNF, Clone MP6-XT22 "ICFC -Quality tested, Each lot of this antibody is quality control tested by intracellular immunofluorescentstaining with flow cytometric analysis. For flow cytometric staining using the µg size,the suggested use of this reagent is (0.25 µg per million cells in 100 µl volume. For flow cytometric staining using the µl size, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. It isrecommended that the reagent be titrated for optimal performance for eachapplication." Anti-mouse IFN-#, Clone XMG1.2, PE "ICFC -Quality tested, Each lot of this antibody is quality control tested by intracellular immunofluorescentstaining with flow cytometric analysis. For flow cytometric staining, the suggesteduse of this reagent is (0.25 µg per million cells in 100 µl volume. It is recommendedthat the reagent be titrated for optimal performance for each application." Anti-mouse CD45.2, Clone 104 Applications Reported: This 104 antibody has been reported for use in flow cytometric analysis. Applications Tested: This 104 antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Anti-mouse TIM-3, Clone RMT3-23, BV785 "FC-Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ( 0.25 µg per million cells in 100 µl volume. It is recommended that thereagent be titrated for optimal performance for each application." Anti-human NGFR, Clone ME20.4, PEcy7 "FC-Quality tested, e Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of wholeblood." Anti-human Gzm-B Clone GB11 AF700 Expression of granzyme B by peripheral blood CD8+ lymphocytes. Whole human blood was lysed with BD Pharm Lyse™ Lysing Buffer (Cat No. 555899) prior to staining with GB11. Whole lysed human blood was subsequently fixed, permeabilized and stained with mouse anti-human granzyme B antibody (Alexa Fluor® 700 GB11, Cat. No. 560213), gated on a positive CD8+ lymphocytes population. To demonstrate the specificity of this staining, the binding of Alexa Fluor® 700 GB11 was nature research | reporting summary April 2020 blocked by preincubation of the fixed/permeabilized cells with excess of an unlabelled GB11 antibody (10 µg, data not shown) prior to stainining.
Anti-human Ki-67 Clone B56 AF647 Flow cytometric analysis of Ki-67 expression by proliferating Jurkat and noncycling human peripheral blood mononuclear cells (PBMC). Jurkat and PBMC were fixed and permeabilized with 70% ice cold ethanol, washed, and stained with Alexa Fluor® 647 Mouse Anti-Ki-67 antibody (Cat. No. 561126) according to the BD Biosciences support protocol, Flow Cytometry Staining Protocol for Detection of Ki-67. The cells were then RNase A (Sigma Cat. No. R-5500) treated and counterstained with Propidium Iodide Staining Solution (Cat. No. 556463) to stain DNA . Flow cytometry was performed using a BD LSR™ II flow cytometry system.
Anti-mouse PD-1, Clone 29F.1A12, FITC "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of thisreagent is (1.0 µg per million cells in 100 µl volume. It is recommended that thereagent be titrated for optimal performance for each application. Anti-human/mouse IRF-4, clone 3E4, FITC "Applications Reported: This 3E4 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This 3E4 antibody has been tested by intracellular staining and flow cytometric analysis of stimulated normal human peripheral blood cells using the Foxp3/Transcription Factor Staining Buffer Set (cat 00-5523) and protocol. Please see Best Protocols Section (Staining intracellular Antigens for Flow Cytometry) for staining protocol (refer to Protocol B: One-step protocol for intracellular (nuclear) proteins). This can be used at less than or equal to 0.125 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay ofinterest." Anti-human CD3, clone UCHT1, Percpcy5.5 "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of wholeblood." Anti-FLAG, clone L5, PE "FC, ICFC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of thisr eagent is (0.125 µg per million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application." Anti-human CD4, clone SK3, BUV805 Flow cytometric analysis of CD4 expression on human peripheral blood lymphocytes. Human whole blood was stained with either BD Horizon™ BUV805 Mouse IgG1, % Isotype Control (Cat. No. 612897; dashed line histogram) or BD Horizon BUV805 Mouse Anti-Human CD4 antibody (Cat. No. 612887/612888; solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Anti-human CD8, clone SK1, BUV737 Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes. Human whole blood was stained with either BD Horizon™ BUV737 Mouse IgG1, % Isotype Control (Cat. No. 612758; dashed line histogram) or BD Horizon BUV737 Mouse Anti-Human CD8 antibody (Cat. No. 612754/612755; solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Anti-human CD69, clone FN50, Percpcy5.5 "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood." Anti-human TIM-3, clone F38-2E2, BV421 "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood." Mouse Anti-human IFN#, clone 45.B3, PEcy7 Expression of IFN-# by stimulated human peripheral blood lymphocytes. Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma) and calcium ionophore A23187 (500 ng/ml final concentration; Sigma) in the presence of GolgiStop™ Note that full information on the approval of the study protocol must also be provided in the manuscript.
Anti-human CD4, clone OKT4, BV785 "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood." Anti-human CD62L, clone DREG-56. APCcy7 "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of thisreagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of wholeblood." Anti-human PD-1, clone EH12.2H7, AF647 "FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining withflow cytometric analysis. For flow cytometric staining, the suggested use of thisreagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of wholeblood." Anti-human CD27, clone O323, BV711 FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood.
Anti-human CD45RA, clone HI100, BV421 FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. For immunohistochemistry, a concentration range of 5.0 -10.0 µg/ml is suggested. It is recommended that the reagent be titrated for optimal performance for each application.
Anti-human CD45RO, clone UCHL1, AF488 FC -Quality tested, Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. For immunohistochemistry, a concentration range of 5.0 -10 µg per ml is suggested. It is recommended that the reagent be titrated for optimal performance for each application.
Anti-human CD73, Clone AD2 FC-routinely tested. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 X 10e6 cells in a 100-µl experimental sample (a test).
The mouse colon adenocarcinoma MC38 cell line and sarcoma 24JK cell line were kindly provided by Dr. Jeff Schlom and Dr. Patrick Hwu, respectively (National Institute of Health, Maryland, USA). The mouse breast carcinoma cell line E0771 was a gift from Prof. Robin Anderson (Olivia Newton-John Cancer Centre, Victoria, Australia). OVCAR-3, MCF7 and MDA-MB-435 were obtained from the American Type Culture Collection.
Cell lines were not authenticated but were used within 10 passages of a master stock to ensure their accuracy All cell lines tested negative for mycoplasma contamination.
None of the cell lines used are listed on the ICLAC register.
C57BL/6J mice, NOD.Cg-Prkdc<scid>Il2rg<tm1Wjl> (NSG) and transgenic C57BL/6 Her2Tg mice were utilized where indicated. Studies were performed in female mice. Mice were used between 6-16 weeks of age with the exception of rechallenge experiments which necessitated the use of older mice.
No field collected samples were used in the study Ethics oversight was performed by the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee (AEEC).