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Inefficient CAR-proximal signaling blunts antigen sensitivity

Nature Immunology volume 21, pages 848–856 (2020)Cite this article

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Abstract

Rational design of chimeric antigen receptors (CARs) with optimized anticancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative, single-molecule, live-cell imaging and found the sensitivity of CAR T cells toward antigen approximately 1,000-times reduced as compared to T cell antigen-receptor-mediated recognition of nominal peptide–major histocompatibility complexes. While CARs outperformed T cell antigen receptors with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-protein kinase ZAP-70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR designs, which presently limit the efficacy of CAR T cell therapies to target tumors with diminished antigen expression.

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Fig. 1: Protein-functionalized planar glass SLB system allows for highly resolved spatiotemporal imaging of CAR- or TCR-mediated antigen recognition.
Fig. 2: CMV-specific T cells detect antigen more efficiently than CAR T cells by 2–3 orders of magnitude.
Fig. 3: CAR T cells engage antigen efficiently yet require a high number of antigen-ligated CARs for ITAM phosphorylation.
Fig. 4: After synapse recruitment, ZAP-70 is activated less efficiently and is more stably antigen- receptor-associated in CAR T cells.

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Data availability

The data that support the findings of this study are available from the authors upon reasonable request; see the author contributions for specific datasets.

Code availability

The custom code employed for the analysis of calcium signaling can be made accessible at the reader’s request.

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Acknowledgements

This work was supported by the Austrian Science Fund through project no. P 25775-B2 (V.G., S.K., L.S. and J.H.), the Vienna Science and Technology Fund (no. LS14-031 to V.G. and J.H.), the Platform for Advanced Cellular Therapies (J.H., R.K.), the Doctoral Program Biomolecular Technology of Proteins supported by the Austrian Science Fund (no. W1224 to E.L. and R.K.) and the Innovative Training Network EN-ACTI2NG (European Network on Anti-Cancer Immuno-Therapy Improvement by modification of CAR and TCR Interactions and Nanoscale Geometry) supported by the European Commission. M.H. is a member of the Young Scholar Program (Junges Kolleg) and an extraordinary member of the Bavarian Academy of Sciences. This work was supported by a grant from German Cancer Aid (Max Eder Program, grant no. 70110313 to M.H.) and the German Research Foundation (project no. 324392634, TRR 221 to M.H.). We thank V. Mühlgrabner (Medical University of Vienna) for help with fluorescence-activated cell sorting; R. Platzer (Medical University of Vienna) for help with single-molecule tracking; O. Dushek (Oxford University) for sending us the T1-CAR constructs; and U. Jäger and N. Worel (Medical University of Vienna), M. Lehner (St. Anna Children’s Cancer Research) and A. Rehm (Max Delbrück Center for Molecular Medicine) for constructive criticism.

Author information

Author notes

  1. Lydia Scharf

    Present address: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

  2. Sebastian Königsberger

    Present address: Roche Diagnostics GmbH, Penzberg, Germany

  3. These authors contributed equally: Venugopal Gudipati, Julian Rydzek.

Authors and Affiliations

  1. Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria

    Venugopal Gudipati, Iago Doel-Perez, Lydia Scharf, Sebastian Königsberger, Hannes Stockinger & Johannes B. Huppa

  2. Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany

    Julian Rydzek, Vasco Dos Reis Gonçalves, Hermann Einsele & Michael Hudecek

  3. Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria

    Elisabeth Lobner & Renate Kunert

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Contributions

V.G., J.R., M.H. and J.B.H. conceived the project and wrote the manuscript. V.G. conducted and analyzed all imaging-related and biochemical experiments. J.R. and V.D.R.G. generated all T cell lines and conducted classical immunological assays. I.D-P. wrote the analysis software used for the single-cell calcium analysis. S.K. carried out the initial experiments. L.S. produced the HLA-A2 and HLA-A2(D227T/K228A) imaging probes. H.E. and H.S. contributed important ideas. E.L. and R.K. provided the CD19 cells for the SLB-related imaging experiments. E.L. contributed to the CD19-related imaging experiments.

Corresponding authors

Correspondence to Michael Hudecek or Johannes B. Huppa.

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The authors declare no competing interests.

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Peer review information Jamie D. K.Wilson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Biochemical and imaging-based analysis of SLB-embedded proteins as well as in vitro analysis of cytotoxicity, IFNγ secretion and proliferation of CMVsp- and ROR1-CAR-T cells.

a, 12.5% SDS PAGE analysis of the recombinant proteins employed for SLB functionalization. Data shown are representative of (n=3) independent experiments. b, The immobile fraction of SLB-anchored proteins was determined via Fluorescence Recovery After Photobleaching (FRAP). Fluorescence intensities were normalized with regard to initial intensity values and plotted versus time. Data shown are representative of (n=5) independent experiments. c, The diffraction-limited fluorescence signal of a single ROR1-AF555 molecule diffusing laterally in an SLB is marked with a green circle. The multi-step single molecule time trajectory is indicated as a green trace (scale bar: 1 µm). Data shown are representative of (n ≥ 2500) single molecule traces. Scale bar = 2 µm. d, SDS-PAGE of whole cell lysates performed under non-reducing/non-boiling and reducing/boiling conditions combined with immunoblot analysis carried out using an affinity-purified polyclonal rabbit anti-CD3ζ antibody confirms dimeric state of CAR-constructs featuring an IgG4-hinge domain, as employed in this study. Data shown are representative of (n=3) independent experiments. e, Schematic outline of CAR-constructs employed in this study. f, Assessment of CAR-T cell effector functions in cell-based in vitro assays. (top panel) Cytotoxicity of R12 4-1BB ζ-CAR-T cells, which were also specific for CMV, co-cultured with K562 or K562-ROR1 or K562-HLA-A2/CMV target cells (10:1 E:T ratio) over time. (middle panel) Proliferation of CFSE-labeled CMVsp-R12 4-1BB ζ-CAR-T cells co-cultured for 72 hours with K562-ROR1 or K562-HLA-A2/CMV target cells (4:1 ratio) or high IL-2 concentrations. As indicated, the decrease in fluorescence was reflective of the number of cell divisions. Data shown are representative of (n=3) independent experiments. (bottom panel) IFNγ secretion of CMVsp-R12 4-1BB ζ-CAR-T cells co-cultured for 24 hours with K562-ROR1 or K562-HLA-A2/CMV target cells (4:1 ratio) or in the presence of PMA and ionomycin. IFNγ concentration in the supernatant was analyzed by ELISA. Data (top and bottom panels) correspond to T cells derived from healthy donors (n=3) and are displayed as mean ± s.d. Data (middle panel) shown is representative of (n=3) independent experiments.

Extended Data Fig. 2 Epitopes targeted by ROR1-specific CARs, CD45 segregation patterns and the influence of the presence of B7-1 on CAR-T cell antigen sensitivity.

a, Schematic representation of ROR1-epitopes targeted by R12, R11 and 2A2 CARs with previously published binding constants 23,24. b, Antigen dose-response of R11 4-1BB ζ-s featuring a size-adjusted and a short linker as is indicated. Each data point comprises data from (n ≥ 230) cells. Data shown are representative of (n=3) independent experiments. c, Montage of representative immunofluorescence images (left panel) of a R12 4-1BB ζ-CAR-T cell confronted with an SLB presenting ROR1 (318 ROR1-AF555 μm−²), ICAM-1 and B7-1. The yellow line in the merged channel denotes the position of the fluorescence intensity line scan shown in the upper middle panel. The Pearson coefficient (upper right panel) was derived by plotting anti-CD45-antibody-AF647 intensities against ROR1-AF555 intensities within individually scanned pixels and contributed to the quantitation shown in lower middle panel. Distributions of Pearson coefficients (lower middle and right panels) are shown for indicated SLB densities of ROR-AF555 / HLA-A2/CMV-AF555 and anti-CD45 antibody-AF647within synaptic antigen clusters (lower panels; n ≥ 40 clusters per condition; n = 16 cells per panel). The negative Pearson coefficients indicate anti-correlated distribution of TCR/CAR and CD45. The degree of exclusion of CD45 from areas of bound antigen is indistinguishable for CAR-T cells engaging ROR1 and CMVsp T cells recognizing HLA-A2/CMV. Data shown are representative of (n=3) independent experiments; scale bar = 2 µm. d, Quantitation of the magnitude of the calcium signal (as measured using the ratiometric calcium sensor fura-2) of activated R12 CAR-T cells featuring a 4-1BB-, CD28- or no intracellular costimulatory module in response to 1.9 ROR1 molecules µm−2. Data are representative of (n=3) independent experiments with data points displaying the measured means (n ≥ 57 cells) ± s.d.. e, Relative surface expression levels (upper panels) of ROR1-CARs and CD28. Calcium response (lower panel) of T cells (n ≥ 841 cells per data point) modified with indicated CARs to increasing antigen densities in the presence or absence of B7-1. Data shown all panels are representative of independent experiments (n=3).

Extended Data Fig. 3 TCR/CAR expression levels and Lck-phosphorylation in the course of antigen recognition.

a, Comparison of TCR- and CAR-surface densities. (left panel) CMVsp-ROR1-CAR-T cells were labeled with either recombinant ROR-AF647 or AF647-conjugated anti-TCR mAb (clone IP26) to determine concentrations required for label saturation. (middle panel) Flow cytometric calibration of AF647 intensity values with the use of QuantumTM Alexa Flour® 647 MESF beads. Resulting MFIs were plotted against the number of equivalent AF647 fluorophores. (right panel) Quantitation of CARs or TCRs on the surface of CMVsp-ROR1-CAR-T cells based on label saturation (left panel) and flow cytometric calibration (middle panel). (left panel) Data shown is representative of independent experiments (n=3). (right and middle panel) Data corresponding to (n=4) donors is shown as mean ± s.d. b, Quantification of synaptic Lck activation (1st panel) and inhibition (2nd panel) as quantified via immunofluorescence in the course of antigen recognition by CMV-specific T cells as well as CAR-T cells as is indicated. Each data point shows mean ± s.e.m (n=30 cells). Plotted data are representative of independent experiments (n=3).

Extended Data Fig. 4 Relative ZAP-70 expression levels, synaptic architecture and the influence of ITAM numbers on CAR-T cell antigen sensitivity.

a, Levels of ectopically expressed ZAP-70-GFP as compared via immunoblot analysis to the expression of endogenous ZAP-70. Data shown are representative of independent experiments (n=3). b, Representative synapses of R12 4-1BB ζ-CAR-T cells and CMVsp-T cells contacting SLBs presenting ROR1-AF555 at a density of 11 molecules µm−2 or HLA-A2/CMV-AF555 at a density of 7 molecules µm−2, respectively (n=30 cells per condition). Data shown are representative of at independent experiments (n=5); scale bar = 5µm. c, Antigen dose-response of T cells modified with R12 4-1BB CARs featuring 2 x 1 ITAMs, 2 x 3 ITAMs or 2 x 6 ITAMs. Each data point comprises data from T cells (n ≥ 402). Data shown are representative of independent experiments (n=3).

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Gudipati, V., Rydzek, J., Doel-Perez, I. et al. Inefficient CAR-proximal signaling blunts antigen sensitivity. Nat Immunol 21, 848–856 (2020). https://doi.org/10.1038/s41590-020-0719-0

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