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Synapse-tuned CARs enhance immune cell anti-tumor activity

Nature Biotechnology (2023)Cite this article

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Abstract

Chimeric antigen receptor (CAR) technologies have been clinically implemented for the treatment of hematological malignancies; however, solid tumors remain resilient to CAR therapeutics. Natural killer (NK) cells may provide an optimal class of immune cells for CAR-based approaches due to their inherent anti-tumor functionality. In this study, we sought to tune CAR immune synapses by adding an intracellular scaffolding protein binding site to the CAR. We employ a PDZ binding motif (PDZbm) that enables additional scaffolding crosslinks that enhance synapse formation and NK CAR cell polarization. Combined effects of this CAR design result in increased effector cell functionality in vitro and in vivo. Additionally, we used T cells and observed similar global enhancements in effector function. Synapse-tuned CAR immune cells exhibit amplified synaptic strength, number and abundance of secreted cytokines, enhanced killing of tumor cells and prolonged survival in numerous different tumor models, including solid tumors.

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Fig. 1: PDZbm scaffolding anchor enhances CAR NK cell synapse formation.
Fig. 2: CAR.PDZ NK cells exhibit enhanced avidity and calcium flux upon cancer cell recognition.
Fig. 3: CAR.PDZ NK cells have enhanced and distinct cytokine production.
Fig. 4: CAR.PDZ NK cells have enhanced cytolytic activity and invasive properties.
Fig. 5: CAR.PDZ NK cells control solid tumor growth and extend survival in vivo.
Fig. 6: CAR.PDZ T cells control tumor growth and extend survival in vivo.

Data availability

Data are available upon reasonable request. Source data are provided with this paper.

Code availability

Imaging analysis code is available on GitHub (https://github.com/Jorge-Ibanez-StJude/AutomatedImageAnalysis.git).

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Acknowledgements

We would like to acknowledge the technical support from H. Houke, C. O’Reilly and A. Chabot. This work was supported by the St. Jude Sumara Fellowship (P.C.), the American Lebanese Syrian Associated Charites (S.G.), the ChadTough Defeat DIPG Foundation (G.K.), National Institute of Neurological Disorders and Stroke grant R01NS121249 (G.K.), the Rally Foundation for Childhood Cancer Research (L.J.T.), the Garwood Postdoctoral Fellowship (J.I.V.) and National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P30 CA021765. Animal imaging was performed by the Center for In Vivo Imaging and Therapeutics, which is supported, in part, by NIH grants P01CA096832 and R50CA211481. Cellular images were acquired at the St. Jude Childrenʼs Research Hospital Cell & Tissue Imaging Center, which is supported by St. Jude and NCI P30 CA021765. Gene editing of cell lines was performed by the Center for Advanced Genome Engineering (CAGE), which is supported, in part, by NCI P30 CA021765. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

  1. Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Peter J. Chockley, Jorge Ibanez-Vega, Giedre Krenciute, Lindsay J. Talbot & Stephen Gottschalk

  2. Department of Surgery, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Lindsay J. Talbot

Authors
  1. Peter J. Chockley
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  4. Lindsay J. Talbot
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Contributions

P.J.C. conceptualized the study, performed experiments, analyzed data and wrote the manuscript. J.I. performed confocal microscopy and analysis. G.K. provided DIPG007 and DIPG7c model setup for immune cell studies and acquired funding. L.J.T. developed and performed the halo assay. S.G. acquired funding, supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Peter J. Chockley.

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Competing interests

S.G. and P.J.C. have patent applications in the fields of NK and T cell and/or gene therapy for cancer. S.G. has a research collaboration with TESSA Therapeutics, is a Data and Safety Monitoring Board member of Immatics and was on the scientific advisory board of Tidal. P.J.C. is a technical consultant for LUMICKS.

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Nature Biotechnology thanks Dongfang Liu, Weidong Han and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Primary NK cell transduction efficiency.

(a) Representative flow cytometry histogram plots detailing surface CAR expression. (b) Quantified flow cytometry data showing percent CAR positive NK cells of various donors. UN: n = 9, CAR Δ: n = 8, CAR: n = 9, CAR.PDZ: n = 6 donors, mean ± SEM shown. (c) Immunophenotype of NK cells via flow cytometry. n = 4 donors, mean ± SEM shown.

Source data

Extended Data Fig. 2 Scribble Polarization at 30 minutes.

(a) Confocal images as prepared in (Fig. 1) incubated for 30 minutes with NK cells in various groups quantified in (b). White bars indicate 10microns. Immunolabelling of Scribble in red, CD3ε in green, and filamentous actin (F-actin) in white. (b) Scribble polarization and accumulation at the immune synapse (IS). CAR Δ; n = 13, CAR; n = 7, CAR.PDZmut: n = 14, CAR.PDZ n = 9, One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. mean ± SEM shown of one donor.

Source data

Extended Data Fig. 3 WASp Polarization at 15 and 30 minutes.

Confocal images as prepared in (Fig. 1) incubated for 15 and 30 minutes with NK cells in various groups. Quantified WASp polarization and accumulation at the immune synapse (IS). CAR Δ; n = 34 and 25, CAR; n = 42 and 25, CAR.PDZ n = 19 and 39, for 15 and 30 minutes, respectively. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. Statistical difference delineated by q < 0.01 *, q < 0.0001 ****; mean ± SEM shown of one donor.

Source data

Extended Data Fig. 4 Live NK cell imaging reveals lysosomal condensing and enhanced synapse formation with increased calcium Flux.

Lysosomal coalescing from live cell imaging in Fig. 2g EphA2 targeting CARs UN; n = 27, CARΔ; n = 48, CAR; n = 44, CAR.PDZ; n = 48 cells. Peak lysosome signal was measured from the first calcium flux peak in each condition. One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; mean ± SEM shown of one donor.

Source data

Extended Data Fig. 5 LM7 Avidity assessment with EphA2-targeting CARs.

Normalized fold change of CAR NK cell binding compared to untransduced NK cells. Arrowed lines indicate the point of statistical difference at CAR.PDZ vs. CAR Δ at 268 pN, CAR.PDZ vs CAR at 343 pN, CAR.PDZ vs CAR.PDZmut at 363 pN which continued to 1000 pN indicated by dashed arrow lines. The only exception to this significance was from 650 to 738 pN for CAR.PDZ vs CAR.PDZmut. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; n = 1-3 donors, mean ± SEM shown.

Source data

Extended Data Fig. 6 B7-H3 CAR design with avidity, synapse, and calcium flux analyses.

(a) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-B7-H3 scFv): goldenrod, hinge and transmembrane domains (CD8αH/TM): grey, CD28 co-stimulatory domain: purple, CD3ζ activation domain: blue, PDZbm scaffolding anchor domain: red. (b) Example flow cytometry plot detailing B7-H3 CAR expression. (c) Normalized fold change of CAR NK cell binding compared to untransduced NK cells. Bracketed line indicates the scale of statistical difference at CAR.PDZ vs. CAR Δ and CAR from 194 to 646 pN for both comparisons except for 205-215 pN. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; q < 0.05 *, <0.001 ***, n = 3 donors, mean ± SEM shown. (d) Immune synapse area quantification of B7-H3 CAR and CAR.PDZ NK cells (n = 11 and 11 cells) Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.05 * at minute 5 and 12 with area under the curve analysis. (e) Calcium flux quantification of B7-H3 CAR and CAR.PDZ NK cells (n = 15 and 14 cells) with Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.05 * starting at minute 2; 1st peak AUC analysis with unpaired Student’s t-Test, mean ± SEM shown of one donor.

Source data

Extended Data Fig. 7 A549 and LM7 tumor rechallenge rejection.

(a) A549 tumor rechallenge timeline with identical initial cancer cell numbers. Indicated tumor volumes from palpable nodules overtime. (b) Intravital imaging of LM7 rechallenge with identical initial cancer cell numbers in complete responder mice. Color scale 1e6 to 1e7 of total photon flux(p/s). (c) Tumor flux values of weekly measurements.

Source data

Extended Data Fig. 8 CAR T cell phenotyping and cytokine production.

(a) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-B7-H3 scFv): goldenrod, hinge and transmembrane domains (CD8αH/TM): grey, CD28 co-stimulatory domain: purple, CD3ζ activation domain: blue, PDZbm scaffolding anchor domain: red. (b) Transduction efficiencies of various CAR constructs in T cells CAR Δ; n = 7, CAR; n = 8, CAR.PDZ n = 5 donors mean ± SEM shown. (c) CD4/8 T cell analysis post transduction at Day 5-6, n = 2 donors mean ± SEM shown. (d) Immunophenotype of CD4 CAR T cells Day 5-6 and longitudinally Day 12-13, n = 2 donors mean ± SEM shown. (e) Immunophenotype of CD8 CAR T cells Day 5-6 and longitudinally Day 12-13, n = 2 donors mean ± SEM shown. (f) B7-H3 CAR T cells were co-cultured with A549, LM7, and 143b cancer cells lines at a 2:1 ratio for 24 hours for n = 3 experiments. Supernatant was collected and multiplex cytokine assessment was performed. One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR mean ± SEM.

Source data

Extended Data Fig. 9 B7-H3 CAR T cell synapse and calcium flux analyses.

(a) Immune synapse area quantification of B7-H3 CAR and CAR.PDZ T cells (n = 12 and 9 cells) co-cultured with LM7 cells with Area Under the Curve analysis. (b) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cell (n = 16 and 22 cells) co-cultured with LM7 cells with Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.0001 **** at minute 4; 1st peak AUC analysis. mean ± SEM shown of one donor. (c) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cells (n = 42 and 15 cells) co-cultured with U87 cells. Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.0001 **** starting at minute 1; 1st peak AUC analysis with unpaired Student’s t-Test. mean ± SEM shown of one donor. (d) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cells (n = 22 and 31 cells) co-cultured with DIPG007 cells. Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.0001 **** starting at minute 1; 1st peak AUC analysis with unpaired Student’s t-Test. mean ± SEM shown of one donor.

Source data

Extended Data Fig. 10 B7-H3 expression on tumor cells.

143B, U87, LM7, DIPG7c, and DIPG007 tumor cells were analyzed for B7-H3 expression. Black histograms indicate Isotype controls and red histograms are immunolabeled cells. Each sample is 100% positive and the indicated gMFI values are delineated.

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Table of antibodies used.

Supplementary Video 1

Calcium flux movie.

Supplementary Video 2

Calcium flux movie.

Supplementary Video 3

Calcium flux movie.

Supplementary Video 4

Calcium flux movie.

Supplementary Video 5

Lysosome coalescing movie.

Supplementary Video 6

Lysosome coalescing movie.

Supplementary Video 7

Lysosome coalescing movie.

Supplementary Video 8

Lysosome coalescing movie.

Supplementary Video 9

Tumoroid killing movie.

Supplementary Video 10

Tumoroid killing movie.

Supplementary Video 11

Tumoroid killing movie.

Supplementary Video 12

Live synapse imaging movie.

Supplementary Video 13

Live synapse imaging movie.

Source data

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Chockley, P.J., Ibanez-Vega, J., Krenciute, G. et al. Synapse-tuned CARs enhance immune cell anti-tumor activity. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-022-01650-2

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