Inefficient CAR-proximal signaling blunts antigen sensitivity
Authors: Iago Doel-Perez
Venugopal Gudipati
Michael Hudecek
Johannes B Huppa
Elisabeth Lobner
Hermann Einsele
Hannes Stockinger
Julian Rydzek
Lydia Scharf
Vasco Dos Reis Gonçalves
Sebastian Königsberger
Renate Kunert
Journal: Nature Immunology (2020)
Discipline: IMMUNOLOGY
Keywords: Chimeric antigen receptors; CAR T cells; Antigen recognition; Live-cell imaging; Immunological synapse
Affiliations: Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria., Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany., Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden., Roche Diagnostics GmbH, Penzberg, Germany., Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria., Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany. ., Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. .

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. 

Retracted: No

PubPeer comments:
 Dear Authors, We have discussed your paper u201cInefficient CAR-proximal signaling blunts antigen sensitivityu201d with great interest and excitement during our weekly literature meeting. However, as we have noticed a few things that we would have liked to see explained (or corrected) in future publications, we permit ourselves to present some suggestions for improvements: General comments: 1.tOne of the parameters that has not been controlled for in the presented experimental system, is the affinity of CAR vs TCR. It would be interesting to see how the CAR-TCR fusion (such as described here: u201cSynthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor responseu201d) would have behaved in the authorsu2019 system. Such a comparison should further confirm if it is only the intracellular part of CAR constructs that needs improvement in order to induce a more physiological T cell activation response. 2.tIt has recently been shown that Lck needs a part of the CD3u03b5 chain (other than ITAMs) to bind to and start the activation cascade (see: u201cNoncanonical binding of Lck to CD3u03b5 promotes TCR signaling and CAR functionu201d). It would be interesting to see if that sequence addition improves CAR signaling in the authorsu2019 experimental model. 3.tAre the images presented in Fig. 4e representative? If so, there is a peculiar difference between the behavior of CAR and TCR synapses. Why has ZAP70 not been recruited again to the middle of the synapse? Is it really an observation of ZAP70 turnover or is it de novo recruitment? Even if the architecture of the synapse is not physiological in this experimental system, a synapse will still form. Might it be that there is no ZAP70 turnover in the center of the synapse, but it can happen at the peripheries (and possibly at different co-receptors)? See: u201cA dynamic CD2-rich compartment at the outer edge of the immunological synapse boosts and integrates signalsu201d for interesting aspects of immunological synapse behavior that were also investigated with TIRF and supported lipid bilayers. 4.tShowing data as mean with SEM is not wrong, but it is not a really honest way of data presentation either. It would be better for the reader to judge results by seeing all the data points. Presenting medians with range would have been better. Specific comments: 1.tCould presence of endogenous ZAP70 influence the presented system? 2.tIn Fig. 1d, 3a, 4a, c and e the reader may get the impression that the cells are different between panels. As the cells are the same in all of these panels, it should be indicated that it is antigen on the SLBs that is the dependent variable (like in Fig. 2a). 3.tIt is not stated in the figure description what the stimulation time points (or length) of data presented are in the graphs plotted against u201cAntigen density on the lipid bilayeru201d. 4.tAdditional negative controls could also have been tested, such as irrelevant peptide loaded on HLA-A2 CMV, to test for T cell receptor unspecificity. 5.tIt seems that all the graphs in Fig. 2 are missing error bars (or range bars as suggested in general comments). 6.tIn Fig. 3b (bottom graph) it could have been indicated in the title of the Y-axis that the fold change is from before synapse formation. 7.tExtended data Fig. 1 could have noted the specific figures in which each construct has been used (for example in brackets next to the construct). It would make a perfect overview of the whole paper. 8.tExtended data Fig. 3 - In our experience anti-Lck pY394 and pY505 antibodies from Cell Signaling Technology, although perfect for Western Blotting, cannot be used for fluorescence measurements. We have tested these antibodies with various Lck mutants (including Phe505, which cannot be phosphorylated on that site) upon various stimulations with flow cytometry and the results were completely unspecific and incorrect (in distinct contrast to Western blotting results). As such, these antibodies should be used with caution for immunofluorescent staining. , 
 Hi, Thanks for your comments. Will be able to answer them in full but prefer to do that over Skype for reasons of deadlines and time. Best, Johannes ,