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Genome-wide CRISPR screen to identify genes involved in T cell dysfunction and novel targets for cancer immunotherapy

English title Genome-wide CRISPR screen to identify genes involved in T cell dysfunction and novel targets for cancer immunotherapy
Applicant Zippelius Alfred
Number 188576
Funding scheme Project funding (Div. I-III)
Research institution Abteilung für Onkologie Medizinische Universitätsklinik B Universitätsspital Basel
Institution of higher education University of Basel - BS
Main discipline Experimental Cancer Research
Start/End 01.03.2020 - 28.02.2023
Approved amount 495'715.00
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All Disciplines (2)

Discipline
Experimental Cancer Research
Immunology, Immunopathology

Keywords (7)

T cell engineering; checkpoint inhibition; t cell exhaustion; CRISPR/Cas9; T cells; cancer immunotherapy; cellular therapy

Lay Summary (German)

Lead
Gezielte Reaktivierung von Immunzellen für Krebs-Immuntherapie
Lay summary

Immunzellen im Tumor, wie beispielsweise T-Lymphozyten, können Krebszellen hemmen und schützen deshalb vor Tumorentwicklung. Allerdings werden diese tumorinfiltrierenden T-Lymphozyten bei vielen Patienten unterdrückt und fallen in eine Art „Erschöpfungszustand“. Dies ist verbunden mit einer Hochregulation von hemmenden Signalen, sog. Immun-Checkpoints wie PD-1, das zu einer Verschlechterung der Tumor gerichteten Effektor-Wirkung führt. Die Blockade von Immun-Checkpoints ist eine hocheffektive Therapie in der Onkologie, die zu einem dauerhaften Zurückdrängen des Tumors bei Patienten führen kann.  Allerdings ist eine Mehrheit der Patienten resistent gegen diese Medikamente. Eine der wichtigsten Aufgaben ist es daher, die Mechanismen der T-Zell Erschöpfung im Tumor besser zu verstehen, um somit die Therapie effektiver zu machen. Wir haben ein Modell entwickelt, das diesen „Erschöpfungszustand“ der T-Lymphozyten des Tumors nachstellt. T-Lymphozyten von gesunden Spendern werden dazu gebracht, einen Proteinkomplex zu bilden, der ein Tumorantigen erkennt. Nach wiederholter Stimulation mit einem spezifischen Tumorantigen können wir zeigen, dass auf verschiedenen Ebenen die T-Lymphozyten einen Zustand erreichen, der den T-Lymphozyten im Tumor gleicht. Mit unserem Model erreichen wir, eine grosse Zahl dieser T-Lymphozyten zu produzieren. Dies ermöglicht uns wissenschaftliche Untersuchungen durchführen zu können, um Signalwege und Zielmoleküle für eine gezielte Reaktivierung dieser «erschöpften» T-Lymphozyten zu identifizieren. Im Rahmen dieses Antrages wollen wir die sog. CRISPR-Cas9 Methode anwenden um das gesamte Genom systematisch nach potentiellen Zielproteinen zu untersuchen. Danach werden wir die Ergebnisse in Immunzellen von Patienten und Tumormodellen bestätigen. Unser Ziel ist es, dass ein verbessertes Verständnis der molekularen Zusammenhänge in den «erschöpften» T-Lymphozyten des Tumors in Zukunft dazu beiträgt, die Effizienz der Immuntherapie für Krebspatienten zu verbessern.

Direct link to Lay Summary Last update: 25.02.2020

Responsible applicant and co-applicants

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Project partner

Associated projects

Number Title Start Funding scheme
162575 Dissecting mechanisms of T cell dysfunction in patients with lung and ovarian cancer 01.05.2016 Project funding (Div. I-III)
170929 Paracrine delivery of therapeutic biologicals: developing a new technology for personalized cancer immunotherapy 01.01.2017 Sinergia

Abstract

Intra-tumoral T cell dysfunction, frequently referred to as T cell exhaustion, is considered a hallmark of cancer. Reinvigoration of T cell function by immune checkpoint inhibitors (ICI) can result in impressive clinical responses, but is effective only in a minority of cancer patients. The mechanisms involved in T cell dysfunction, and particularly its involvement in ICI therapy is still not fully understood. Moreover, the development of novel cancer immunotherapies urgently requires comprehensive understanding of molecular initiators and promotors of T cell dysfunction. Here, we have developed an ex vivo human model system to mimic tumor-specific T cell dysfunction in human cancers. To this end, circulating CD8 T cells from non-cancer individuals are transduced with a T cell receptor construct specifically recognizing a human tumor-associated antigen. Subsequently, these T cells are exposed to repetitive stimulation utilizing antigenic peptides with different affinities. We can demonstrate that this procedure stably renders CD8 T cells dysfunctional. The latter acquire a state which highly resembles CD8 T cells found in human tumors on a phenotypic and transcriptional level. Importantly, this model is capable to produce a high number of antigen-specific dysfunctional T cells sufficient to overcome restraints imposed by the limited availability of tumor-infiltrating T cells with unknown antigen specificity obtained from cancer patients. By taking advantage of this model, we aim to dissect individual genes or group of genes critical for the generation of T cell dysfunction and identify key genes and downstream pathways that could be targeted for T cell reinvigoration. To this end, we propose to perform an unbiased and comprehensive loss-of-function CRISPR screen with the following major research aims: (i) to discover genes involved in inducing and/or maintaining T cell dysfunction, and (ii) to mechanistically dissect how these genes are involved in T cell dysfunction. Experimentally, we will develop CRISPR-Cas9 mediated knockout systems scaled up to a genome-wide scale in antigen-specific CD8 T cells by co-transduction with a tumor antigen-specific T cell receptor and pooled single guide RNAs, combined with Cas9 protein electroporation. Initial experiments have already demonstrated the feasibility of the approach. After repetitive antigenic stimulation in compliance with our model system, the dysfunctional T cells will be restimulated and divided into functional/non-functional populations to systematically identify potential target genes that are critical in mediating T cell dysfunctionality. Validation of target gene knockouts will be accomplished in primary CD8 T cells and organotypic culture systems derived from cancer patients, utilizing proteomic and siRNA approaches. In addition, we will investigate the impact of target gene knockouts/inhibition using mouse tumor models of adoptive transfer and tumor-infiltrating lymphocytes (TIL) obtained from melanoma patients in the framework of a newly established clinical program for TIL transfer in our center. We expect that the findings advance our understanding of the molecular mechanisms that underpin T cell dysfunction in the context of cancer and in the future serve to optimize T cell-targeted immunotherapy for the benefit of patients.
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