Inefficient ZAP70-Signaling Blunts Antigen Detection by CAR-T-Cells

Gudipati, Venugopal; Rydzek, Julian; Perez, Iago Doel; Scharf, Lydia; Königsberger, Sebastian; Einsele, Hermann; Stockinger, Hannes; Hudecek, Michael; Huppa, Johannes B.

doi:10.1101/720417

Cited by 5 publications

(4 citation statements)

References 61 publications

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“…Given that BLI is an optical technique that requires large amounts of protein binding for detection, the high amount of peptide immobilisation used to achieve this may mean that a large amount of intermolecular rebinding across different ITAMs is taking place leading to apparent long half-lives. Consistent with the present work, previous in vivo measurements of the ZAP70 half-life using fluorescence recovery after photobleaching have provided recovery timescales of ∼10 s (18,47,48).…”

Section: Discussionsupporting

confidence: 92%

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

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Depoil

Yang

et al. 2020

Preprint

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Protein-protein binding domains are critical in signalling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins have tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signalling, which is a critical requirement of noise filtering mechanisms such as kinetic proofreading. Here, we use modelling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases when tandem domains bind by a kinetic, but not a static, avidity mode. We use surface plasmon resonance to show that ZAP70, a tandem SH2 domain-containing kinase, binds kinetically to biphosphorylated peptides from the T cell antigen receptor (TCR) and that the unbinding rate can be accelerated by the phosphatase CD45. An important functional prediction of regulated unbinding is that the intracellular ZAP70/TCR half-life in T cells will be correlated to the extracellular TCR/antigen half-life and we show that this is the case in both cell lines and primary T cells. The work highlights that binding by kinetic avidity breaks the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discriminated mediated by kinetic proofreading. 1

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Section: Discussionsupporting

confidence: 92%

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Goyette

Depoil

Yang

et al. 2020

Preprint

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“…Because the H/T domain appeared to affect the interaction of the CAR T cell and tumor, we next imaged the CAR synapse, to test the hypothesis that differences in the H/T region affect receptor clustering and recruitment of ZAP70, which propagates CAR signaling (57). To do this, we generated CD19-4-1BBζ and CD19-CD28H/T-4-1BBζ mCherry fusion constructs that expressed similarly in primary human T cells (Supplementary Fig.…”

Section: The Cd28 H/t Domain Results In Faster Tumor-cell Killing and A More Efficient Immune Synapsementioning

confidence: 99%

Tuning the Antigen Density Requirement for CAR T-cell Activity

et al. 2020

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Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence.SignifiCAnCe: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigendensity tumors while maintaining their unique capacity for persistence.

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“…Another distinct feature of TCRs and BCRs is that they recognise surface anchored ligands whereas other receptors recognise ligands directly from solution (79). These features are also shared by synthetic CARs that, with available data, appear to exhibit baseline discrimination but they appear to have major defects in antigen recognition and signalling (80, 81). These observations may provide clues to the molecular mechanisms underlying discrimination.…”

Section: Discussionmentioning

confidence: 97%

The discriminatory power of the T cell receptor

Huhn

Wilson

Merwe

et al. 2020

Preprint

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T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity non-self pMHC antigens. The strength of this discrimination and the mechanisms that produce it remain controversial. Although a large number of mouse and human TCRs have now been characterised, they have not been used to precisely quantitate discrimination. Here, we systematically quantify the discrimination of TCRs using a discrimination power (α). Early influential studies on three mouse TCRs suggested that discrimination was nearly perfect (α ~ 9.0). In striking contrast, our analysis of published data on other mouse and human TCRs, and more recent data on the original mouse TCRs, produced significantly lower discrimination (α = 2.0). Although not perfect, the discriminatory power of TCR was greater than that of conventional receptors such as cytokine receptors, GPCRs, RTKs, and CARs (α ≤ 1). The revised discriminatory power of the TCR is readily explained by a kinetic proofreading mechanisms, and accounts for the ability of low affinity self-antigens to stimulate autoimmune and anti-tumour T cell responses.

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Inefficient ZAP70-Signaling Blunts Antigen Detection by CAR-T-Cells

Abstract: Rational design of chimeric antigen receptors (CARs) with optimized anti-cancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen-engagement triggers activation.

Cited by 5 publications

References 61 publications

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Tuning the Antigen Density Requirement for CAR T-cell Activity

The discriminatory power of the T cell receptor

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