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Disinhibition of Early T cell Activation via CD5 Knock Out Enhances the Anti-Tumor Activity of Adoptive T Cell Therapies

ABSTRACT: The majority of patients treated with currently approved adoptive T-cell therapies (ACT) and, specifically, chimeric antigen receptor (CAR) T cells will eventually fail treatment. Furthermore, ACT has not been successful against solid cancers and several hematological malignancies, including T-cell lymphomas which have extremely poor prognosis. One of the main barriers to the effective activity of adoptively transferred T cells is their inhibition when they reach the tumor bed. In this study, we discover that CD5 inhibits CAR T activation and that the knockout (KO) of CD5 using CRISPR-Cas9 enhances the anti-tumor effect of CAR and TCR T cells in multiple hematological and solid cancer models. As a consequence, CD5 KO T cells display increased in vivo expansion and persistence. Despite this increased activity, no clear increased toxicity was observed in preclinical models. These findings support the development of CD5 KO adoptive T-cell therapies in early-phase clinical trials for relapsed and refractory cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248590 | GEO | 2024/07/18

REPOSITORIES: GEO

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Project description:Adoptive T cell therapies have been transformative in the treatment of a subset of hematological malignancies. However, many patients either fail to respond or relapse, and these therapies still have had much more limited success in solid tumors. A critical challenge for increasing patient response rates and achieving durable efficacy of adoptive T cell therapies is overcoming suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints. Targeted gene editing has the potential to enhance T cell therapeutic function and improve clinical responses. Here we perform multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to nominate genes that could be targeted to prevent T cell dysfunction. These CRISPR screens converged on RASA2 as a target to confer resistance to suppressive conditions found in tumor microenvironments. We identify RASA2 as a signaling checkpoint in human T cells that is downregulated upon acute TCR stimulation but increases gradually with chronic antigen exposure. Antigen titration showed that genetic ablation of RASA2 enhances mitogen activated protein kinase (MAPK) signaling and CAR-T cell cytolytic activity in response to low antigen levels. Repeated tumor antigen stimulation in vitro revealed that RASA2-deficient TCR-T and CAR-T cells have increased activation, cytokine production, and metabolic activity compared to control cells, and show a striking advantage in persistent cancer cell killing. RASA2 KO CAR-T cells showed a competitive fitness advantage over CAR-T control cells in the bone marrow of an in vivo leukemia model. Deletion of RASA2 in multiple preclinical models of TCR- and CAR-T cell therapies prolonged survival in animals xenografted with either liquid or solid tumors. Altogether, our findings from multiple genome-wide screens and preclinical studies highlight RASA2 as a promising new target to enhance both persistence and effector function in TCR-T and CAR-T cell therapies for cancer treatment.

Project description:CD5 is characterized as an inhibitory co-receptor with important regulatory role during T cell development. To study the molecular mechanism by which CD5 operates, we used quantitative mass spectrometry to analyze the components of the CD5 signaling machinery in primary T cells. In a first set of experiments, CD5-containing complexes were immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice. Thymocytes were treated with pervanadate to induce widespread activation of protein tyrosine kinases. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls, to discriminate CD5-binding molecules from the background of contaminant proteins. Eight biological replicates were prepared for both conditions, and samples were analyzed in duplicate LC-MS runs. This first set of experiment is presented in TableS1 of the paper, and is associated to the MaxQuant result file “Interactome WT” in the present PX dataset. We analyzed the CD5 interactome at different time of stimulation with pervanadate (1min and 10min) using thymocytes from wild-type (WT) C57BL/6 mice. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls. Three biological replicates were prepared for both conditions (WT and KO) and each time point (1min and 10min). This set of experiments is presented in TableS2 of the paper and is associated to the MaxQuant result file “Interactome at 1min and 10min” in the present PX dataset. We checked that the activation of CD5 (phosphorylation sites) and the formation of the signaling complex was comparable in different stimulatory conditions (comparison of WT thymocytes stimulated with either pervanadate or anti-CD3+anti-CD4 antibodies). We also analyzed the binding of CD5 interactors in thymocytes from a c-Cbl-/- mouse model (comparison of WT and c-Cbl-/-thymocytes stimulated with pervanadate). These experiments and the associated experimental design of the comparisons are summarized in TableS3 of the paper, and are based on the MaxQuant result file “Comparison of different stimulatory conditions” in this dataset. To check the phosphorylation status of the CD5 tyrosine residues at different time of stimulation, we analyzed samples immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice, following stimulation with anti-CD3+anti-CD4 antibodies for 1min, 3min and 10min. This set of experiment is presented in TableS4 of the paper and is associated to the MaxQuant result file “CD5 pY kinetics” in the present PX dataset. To determine the role of Y429 in the context of CD5 signaling, we expressed a wild-type (CD5tgWt) or a mutated form of CD5, containing a tyrosine to phenylalanine substitution at position 429 (CD5tgY429F) in transgenic mice. We analyzed the CD5 interactome by comparing samples immunoprecipitated from thymocytes of these 2 mice models (following pervanadate stimulation, 6 and 5 biological replicates respectively) with samples prepared in the same way from Cd5−/− mice as controls (6 biological replicates). The results are shown in TableS5 of the paper, and correspond to the MaxQuant result file “interactome CD5tgWt_CD5tgY429F”.

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Disinhibition of Early T cell Activation via CD5 Knock Out Enhances the Anti-Tumor Activity of Adoptive T Cell Therapies

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