scholarly journals Data-Driven Analysis of a Mechanistic Model of CAR T Cell Signaling Predicts Effects of Cell-to-cell Heterogeneity

2019 ◽  
Author(s):  
Colin G. Cess ◽  
Stacey D. Finley
Keyword(s):  
T Cell ◽  
Protein Expression ◽  
Cell Signaling ◽  
Mechanistic Model ◽  
Cell Cancer ◽  
Data Driven ◽  
Cell Heterogeneity ◽  
T Cell Signaling ◽  
Car T Cell ◽  
Car T

ABSTRACTDue to the variability of protein expression, cells of the same population can exhibit different responses to stimuli. It is important to understand this heterogeneity at the individual level, as population averages mask these underlying differences. Using computational modeling, we can interrogate a system much more precisely than by using experiments alone, in order to learn how the expression of each protein affects a biological system. Here, we examine a mechanistic model of CAR T cell signaling, which connects receptor-antigen binding to MAPK activation, to determine intracellular modulations that can increase cellular response. CAR T cell cancer therapy involves removing a patient’s T cells, modifying them to express engineered receptors that can bind to tumor-associated antigens to promote cell killing, and then injecting the cells back into the patient. This population of cells, like all cell populations, would have heterogeneous protein expression, which could affect the efficacy of treatment. Thus, it is important to examine the effects of cell-to-cell heterogeneity. We first generated a dataset of simulated cell responses via Monte Carlo simulations of the mechanistic model, where the initial protein concentrations were randomly sampled. We analyzed the dataset using partial least-squares modeling to determine the relationships between protein expression and ERK phosphorylation, the output of the mechanistic model. Using this data-driven analysis, we found that only the expressions of proteins relating directly to the receptor and the MAPK cascade, the beginning and end of the network, respectively, are relevant to the cells’ response. We also found, surprisingly, that increasing the amount of receptor present can actually inhibit the cell’s ability to respond due to increasing the strength of negative feedback from phosphatases. Overall, we have combined data-driven and mechanistic modeling to generate detailed insight into CAR T cell signaling.

scholarly journals Mutation of the CD28 Co-Stimulatory Domain Confers Enhanced CAR T Cell Function

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 248-248 ◽  
Author(s):  
Justin C. Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Maria Cabral ◽  
Dylan Morrissey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Signaling ◽  
Research Funding ◽  
Chromatin Accessibility ◽  
Target Cells ◽  
T Cell Signaling ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

CD70 as a novel target of CAR-T-cell therapy for gliomas.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 148-148 ◽  
Author(s):  
Linchun Jin ◽  
Haitao Ge ◽  
Changlin Yang ◽  
Yu Long ◽  
Yifan(Emily) Chang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Glioma Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Gene And Protein Expression ◽  
Car T ◽  
Human And Mouse

148 Background: Gliomas are the most common primary malignant brain tumor and are uniformly lethal. Cancer immunotherapy has the potential to target gliomas; however, its antitumor effects are restricted by limitations in clinically useful tumor specific targets. Chimeric antigen receptor modified T-cell (CAR-T) therapy is a highly promising option for cancer treatment, due to its combination of precision antibody recognition and T-cell tumor-specific killing. CD70 is an antigen expressed by limited subsets of normal lymphocytes and dendritic cells but is aberrantly overexpressed by glioma cells, which makes it an outstanding glioma-antigen target. Methods: The gene and protein expression of CD70 were evaluated to identify its potential as a glioma target. Human and mouse versions of CD70-specific CAR-T cells were generated, and human primary GBM lines as well as murine lines (GL-261, KR-158B) were used as human and mouse tumor targets, respectively. The antitumor effect of the human and mouse CD70-sepecific CARs were tested in vitro and in orthotopic xenograft and syngeneic murine models. Results: CD70 is only overexpressed by tumor cells in a subset of low-grade gliomas and GBM. The elevated gene and protein expression are associated with increased tumor grade and poor patient survival. Co-culturing CD70-specific CAR-T cells with CD70-positive glioma cells resulted in potent secretion of IFN-gamma and tumor-specific killing in a CD70-dependent manner. Irradiation enhances CD70 expression on glioma cells and thus increases CAR T-cell recognition. Adoptive transfer of the human and mouse CD70 CAR-T cells resulted in tumor regression of immunocompetent and immunodeficient mice, respectively. Conclusions: CD70 can be an excellent tumor target for gliomas, and CD70-specific CAR-T cells have potent antitumor activity against CD70-positive gliomas both in vitro and in vivo. Our study provides crucial preclinical evidence to support the future clinical application of CD70 CAR-T cells to treat gliomas.

scholarly journals 146 Evolving Mutliplexed shRNA to generate tailored CAR T cell therapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A154-A154
Author(s):  
Mikhail Steklov ◽  
Benjamin Lecalve ◽  
Jerome Marijse ◽  
Fanny Huberty ◽  
Nancy Ramelot ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Protein Expression ◽  
Clinical Development ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Vector Titer

BackgroundManipulating protein expression to generate cells with a specific desired phenotype is one of the central goals of engineered cell therapy. Short Hairpin RNA (shRNA) is a well-established approach to reduce protein expression through the targeted degradation of messenger RNA transcripts. However, the use of shRNA in the Chimeric Antigen Receptor (CAR) T cell therapy has been limited. We have recently shown that single shRNA incorporated into a CAR expression vector can knockdown expression of the target antigen when expressed on the CAR T itself to avoid fratricide or expression of some elements of the T cell receptor (TCR) to generate allogeneic CAR T cells. An attraction of the shRNA approach is to express multiple shRNA from the same vector that can regulate protein expression thereby optimzing CAR T cell phenotype.MethodsRetroviral vectors encoding a CAR targeting a well-studied antigen (generally BCMA) co-expressing a tag for cell enrichment and identification along with shRNA multiplexed were generated. The shRNA multiplexed were inserted within a microRNA (miR) framework to enable expression from a single PolII promoter (the retroviral LTR promoter). Functional assessment of target knockdown target in T cells along with retroviral titers was determinedResultsOur products in ongoing clinical development have employed a miR196a2 scaffold enabling the expression of the desired shRNA driven by the same promoter as that used for the CAR and other transgenes. Multiplexing the miR196a2 scaffold to express multiple shRNA (targeting CD247, beta 2 Microglobulin and CD95) was successful in terms of target knockdown but an obvious reduction in retroviral titer was observed. These titer reductions were variable between the duplex and triplex shRNA constructs examined but were uniformly low when considering clinical development. A proprietary scaffold was developed that coupled expression of duplexed and triplexed shRNA while also elevating vector titer by at least 2-3x.ConclusionsMultiplexing shRNA within a single vector format with scaffolds that ensure co-linked expression of the shRNA with therapeutic transgenes is a highly attractive approach to generate CAR T cells with bespoke, desired phenotypes. However, simply multiplexing shRNA using a currently clinical-used scaffold (miR196a2) resulted in reductions in vector titer. Engineering further proprietary scaffolds were produced that maintained shRNA expression but elevated retroviral titer to a level which does not preclude clinical development. These developments now provide the opportunity to develop second generation clinical candidates using shRNA multiplexed technology.

scholarly journals Transient “rest” induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

2020 ◽  
Author(s):  
Evan W. Weber ◽  
Rachel C. Lynn ◽  
Kevin R. Parker ◽  
Hima Anbunathan ◽  
John Lattin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Kinase Inhibitor ◽  
T Cell Signaling ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Epigenetic Remodeling ◽  
The Impact

SUMMARYT cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR-T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. “rest”) on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved anti-tumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.

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