scholarly journals P07.02 Regulation of CD19 CAR T- cell activation based on engineered Nuclear factor of activated T cells artificial transcription factors

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A23-A23
Author(s):  
D Lainšček ◽  
V Mikolič ◽  
Š Malenšek ◽  
A Verbič ◽  
R Jerala
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Activity ◽  
External Control ◽  
Car T Cells ◽  
Car T Cell ◽  
Artificial Transcription Factors ◽  
Car T

BackgroundCD19 CAR T- cells (Chimeric antigen receptor T cells that recognize CD19) present a therapeutic option for various malignant diseases based on their ability to specifically recognize the selected tumour surface markers, triggering immune cell activation and cytokine production that results in killing cancerous cell expressing specific surface markers recognized by the CAR. The main therapeutic effect of CAR is a specific T cell activation of adequate cell number with sequential destruction of tumorous cells in a safe therapeutic manner. In order to increase T cell activation, different activation domains were introduced into CAR. CAR T-cells are highly efficient in tumour cell destruction, but may cause serious side effects that can also result in patient death so their activity needs to be carefully controlled.1 Several attempts were made to influence the CAR T cell proliferation and their activation by adding T cell growth factors, such as IL-2, into patients, however this approach of increasing the number of activating T cells with no external control over their number can again lead to non-optimal therapeutic effects. Different improvements were made by designing synthetic receptors or small molecule-inducible systems etc., which influence regulated expansion and survival of CAR T cells.2Material and MethodsIn order to regulate CD19 CAR-T cell activity, different NFAT2 based artificial transcription factors were prepared. The full length NFAT2, one of the main players in T cell IL2 production, a key cytokine for T cell activation and proliferation was truncated by deletion of its own activation domain. Next, we joined via Gibson assembly tNFAT21-593 coding sequence with domains of different heterodimerization systems that interact upon adding the inductor of heterodimerization. The interaction counterparts were fused to a strong tripartite transcriptional activator domain VPR and/or strong repressor domain KRAB resulting in formation of an engineered NFAT artificial transcription (NFAT-TF) factors with external control. To determine the activity of NFAT-TF HEK293, Jurkat or human T cells were used.ResultsBased on luciferase assay, carried out on NFAT-TF transfected HEK293 cells we first established that upon adding the external inductor of heterodimerization, efficient gene regulation occurs, according to VPR or KRAB domain appropriate functions. Findings were then transferred to Jurkat cells that were electroporated with appropriate DNA constructs, coding for NFAT-TF and CD19 CAR. After Raji:Jurkat co-culture ELISA measurements revealed that IL2 production and therefore CD19 CAR-T cell activity can be controlled by the action of NFAT-TF. The same regulation over the activity and subsequent proliferation status was also observed in retrovirally transduced human T-cells.ConclusionWe developed a regulatory system for therapeutic effect of CD19 CAR-T cells, a unique mechanism to control T cell activation and proliferation based on the engineered NFAT2 artificial transcription factor.ReferencesBonifant CL, et al. Toxicity and management in CAR T-cell therapy. Mol Ther Oncolytics 2016;3:16011.Wu C-Y, et al. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science 2015;80:350.Disclosure InformationD. Lainšček: None. V. Mikolič: None. Š. Malenšek: None. A. Verbič: None. R. Jerala: None.

scholarly journals Mutation of the CD28 Costimulatory Domain Confers Decreased CAR T Cell Exhaustion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 966-966 ◽  
Author(s):  
Justin C. Boucher ◽  
Gongbo Li ◽  
Bishwas Shrestha ◽  
Maria Cabral ◽  
Dylan Morrissey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, a major obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation causing acute toxicities, but also lead to T cell 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. We hypothesized that by incorporating null mutations of the CD28 subdomains (YMNM, PRRP, or PYAP) 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 (Fig1A), IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytoxicity was enhanced with the PYAP only CAR T cells compared to non-mutated CARs (Fig1B). When we examined the PYAP (mut06) only mutant in an immune competent mouse model we found similar B cell aplasia and CAR T cell persistence compared to non-mutated CD28 CAR T cells. Additionally, PYAP only CAR T cells injected into mice had decreased (82% to 62%) expression of PD1 in the BM. Using a pre-clinical immunocompetent mouse tumor model we found the PYAP only CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells, with 100% survival of mice given PAYP only CAR T cells compared to 50% survival of mice given non-mutated CAR T cells (Fig1C). We next sought to determine what role CAR T cell exhaustion was playing using a Rag knockout mouse system. CAR T cells were given to Rag-/- mice and 1 week later mice were challenged with tumor. Studies in Rag-/- mice also showed PYAP only CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. We also found PYAP only CAR T cells had significantly less expression of PD1 compared to non-mutated CAR T cells (Fig1D). We then co-cultured CAR T cells with target cells expressing CD19 and PDL1 and found PYAP only CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This shows that PYAP only CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. Using Nur77, pAkt, and pmTOR to measure CAR signaling we found PYAP only CAR T cells had significantly reduced levels of Nur77 while still having higher expression then first generation CAR T cells. We then examined what affect the PYAP only CAR had on transcription factors. We found similar AP1 and NF-kB expression between PYAP only and non-mutated CD28 CAR T cells but a significant reduction of NFAT in the PYAP only mutants compared to non-mutated CD28 CAR T cells. This suggests reduced NFAT expression contributes to the PYAP only CAR's resistance to exhaustion. Finally, we made human CAR constructs of the PYAP only mutant. We found PYAP only human CAR T cells had increased cytoxicity and decreased exhaustion in vitro compared to non-mutated human CD28 CAR T cells. NFAT levels in human PYAP only CAR T cells were significantly reduced compared to non-mutated CAR T cells supporting our findings in mice. Our results demonstrate that CAR T cells with only a PYAP CD28 subdomain have better cytoxicity and decreased exhaustion compared to non-mutated CD28 CAR T cells. Our results suggest this is the result of decreased CAR and NFAT signaling. Additionally, we were able to validate these findings using human CAR constructs. This work allows for development of an enhanced 2nd and 3rd generation CAR T cell therapies for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities. Figure 1 Figure 1. Disclosures Davila: Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities

2021 ◽  
Vol 8 ◽  
Author(s):  
Li Du ◽  
Yaru Nai ◽  
Meiying Shen ◽  
Tingting Li ◽  
Jingjing Huang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transfection Efficiency ◽  
Cytotoxic Activities ◽  
Cell Preparation ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Adoptive immunotherapy using CAR-T cells is a promising curative treatment strategy for hematological malignancies. Current manufacture of clinical-grade CAR-T cells based on lentiviral/retrovirus transfection of T cells followed by anti-CD3/CD28 activation supplemented with IL-2 has been associated with low transfection efficiency and usually based on the use of terminally differentiated effector T cells. Thus, improving the quality and the quantity of CAR-T cells are essential for optimizing the CAR-T cell preparation. In our study, we focus on the role of IL-21 in the γc cytokine conditions for CAR-T cell preparation. We found for the first time that the addition of IL-21 in the CAR-T preparation improved T cell transfection efficiency through the reduction of IFN-γ expression 24–48 h after T cell activation. We also confirmed that IL-21 enhanced the enrichment and expansion of less differentiated CAR-T cells. Finally, we validated that IL-21 improved the CAR-T cell cytotoxicity, which was related to increased secretion of effector cytokines. Together, these findings can be used to optimize the CAR-T cell preparation.

scholarly journals A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization

2021 ◽  
Vol 12 ◽  
Author(s):  
Laura Grunewald ◽  
Tobias Lam ◽  
Lena Andersch ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Three Dimensional ◽  
Human Tumor ◽  
Analysis Tool ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

CAR-T Cells Are Serial Killers of Tumor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3088-3088
Author(s):  
Misty Jenkins ◽  
Alex Davenport ◽  
Ryan Cross ◽  
Carmen Yong ◽  
David S. Ritchie ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Kinetics Of ◽  
I Cells

Abstract Chimeric antigen receptor (CAR) T cells have shown clinical efficacy in refractory B cell malignancies. Despite these exciting clinical results, our fundamental understanding of CAR-T cell biology is limited, possibly limiting the broader application of CAR-T cells to additional haemopoetic and solid cancers. To date, the mechanism of CAR-T immunological synapse formation with tumor cells and kinetics of subsequent serial killing by CAR-T cells has not been explored. Here, we investigated the kinetics of CAR-T cell activation and cytotoxicity, including immune synapse formation, and kinetics of tumor cell killing, closely comparing to activation and cytotoxicity via the T cell receptor (TCR). To address this, we developed a mouse model in which the CD8+ T cells (termed CAR.OT-I cells) co-expressed two antigen receptors, the clonogenic OT-I TCR, and a second generation CAR comprising a scFV to human HER2, CD28 and CD3ζ signaling domains. Effector CAR.OT-I cells were activated via their antigen receptors using either SIINFEKL-pulsed or HER-2 expressing tumor cells, the interactions between the effector CAR.OT-I cells and tumor cells were then assessed by time lapse live microscopy. CAR.OT-I cell activation via the endogenous TCR or the CAR did not affect tumor killing kinetics, except the time taken from CAR.OT-I activation to detachment (from the dying tumor cell) was significantly slower when the endogenous TCR was engaged. Subsequently, we showed for the first time, that CAR.OT-I cells have serial killing capacity, which is important to consider when therapeutic numbers of CAR-T cells are likely to be outnumbered by tumor targets. Individual CAR.OT-I cells killed multiple tumor cells, whether activated via the endogenous TCR or the CAR. We further explored whether these findings have implications for killing of tumor cells using low effector:target cell ratio in short versus long-term killing assays, chromium release and xCELLigence killing assays respectively. We observed, no matter which antigen receptor was activated, the effector CAR.OT-I cells were equivalent killers of tumor cells in short term assays (4-8 hours). However, over a period of 50 hours, CAR.OT-I cells activated via the CAR killed tumor cells at a lower rate than when activated via the TCR. This was due to CAR.OT-I CAR expression down-regulation from 20-50 hours. This study highlights that fundamental differences occur in the way CAR-T cells kill tumor cells, depending on how the effector CAR-T cell is activated. Furthermore, the study provides important insights for CAR-T cell activation in vivo with implications for single- or dual-receptor-focused CAR-T cell therapy and improved clinical benefit. Disclosures No relevant conflicts of interest to declare.

scholarly journals G-CSF/Plerixafor Dual-Mobilized Donor Derived CD33CAR T-Cells As Potent and Effective AML Therapy in Pre-Clinical Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1716-1716
Author(s):  
Giacomo Canesin ◽  
Hillary Hoyt ◽  
Reid Williams ◽  
Mariana Silva ◽  
Melissa Chng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immune Cell ◽  
Ex Vivo ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract There are currently no known acute myeloid leukemia (AML) specific antigens. Genetic ablation of CD33 using CRISPR/Cas9 engineering of the hematopoietic stem cell (HSC) transplant (VOR33) represents a synthetic biology approach to generating a leukemia-specific antigen in the transplant recipient. VOR33 enables anti-CD33 CAR-T cell killing of AML cells while sparing normal myeloid lineage development and function, thereby potentially avoiding myelosuppression and increasing the therapeutic index of anti-CD33 CAR-T therapy. Mobilized leukapheresis product represents an attractive starting material for the generation of both a CD33 null HSC transplant and a complementary CD33CAR T-cell product. In this study, we sought to determine the impact of dual mobilization with Granulocyte-Colony Stimulating Factor (G-CSF) and plerixafor (mozobil) on immune cell composition, T cell phenotype, and the functionality of these T cells to control AML tumor growth upon chimeric antigen receptor (CAR) transduction. Mobilized (mob) peripheral blood mononuclear cells (PBMCs) were collected from healthy donors injected with G-CSF (10µg/kg/day, 5 consecutive days) and plerixafor (240µg/kg, on day 4 and 5). Non-mobilized (non-mob) PBMCs, collected from the same donors, were used as controls. Cells were analyzed by flow cytometry for immunophenotyping and T cell characterization including differentiation and bone marrow homing markers, as well as responses to T cell activation with anti-CD3 (OKT3) and IL-2. Non-/mob PBMC populations were also analyzed by single-cell next generation sequencing (CITEseq) using 127 immune cell phenotypic markers in combination with extensive transcriptome and T cell receptor repertoire analysis. In addition, lentiviral transduction of anti-CD33 CAR constructs enabled functional comparisons of mob- and non-mob-CAR T-cells in AML cell co-cultures as well as AML mouse models. Ex vivo immunophenotyping of PBMC from a total of over 30 healthy donor samples showed that mobilization decreases the overall percentage of CD3 + T cells but increases that of naïve T cells (CD45RA +/CCR7 +), at the expense of T effector-memory (CD45RA -/CCR7 -) and central-memory (CD45RA -/CCR7 +) populations. Bone marrow homing factors (e.g.: CXCR4) were increased in mob compared to non-mob T cell samples. As expected, higher percentages of monocytes (CD14 +) were detected in mob compared to non-mob donor samples, but this difference disappeared after culture under T cell activation conditions. T cell activation also led to similar increases in CD25, CD69 and CD137 expression, and a decrease in CD62L expression. Single cell sequencing analyses confirmed mobilization-induced increases in naïve T cells as well as changes in monocytes/macrophages, CD4 + T cells and NK cells percentages. Notably, functional in vitro cytotoxic assays demonstrated that mob-CD33-CAR T-cells are as effective as non-mob-CD33-CAR T-cells in killing CD33 + AML cells, with reduced 'bystander' activation of non-transduced T cells. Furthermore, results from in vivo AML mouse models indicate that mob-CD33-CAR T-cells are equally effective in clearing CD33 + tumors as non-mob-CD33-CAR T-cells. Our analysis showed phenotypical ex vivo differences between mob and non-mob PBMCs, which disappeared upon activation, indicating similar responses to T cell-specific stimulation. These findings are corroborated by similar in vitro cytotoxicity profiles of non-/mob-CAR T-cells. Non-transduced T cells in the mob-CAR T-cell population showed limited 'bystander' activation, indicating a potentially favorable clinical toxicity profile. Additional in vivo assessment of mob-CAR T-cell function shows effective tumor clearance, which supports further efforts towards their clinical use in combination with engineered HSCs for the treatment of AML patients. Disclosures Canesin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hoyt: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Williams: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Silva: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chng: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ung: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Qiu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Shin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Scherer: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Kassim: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

scholarly journals 206 The development of ‘chimeric CD3e fusion protein’ and ‘anti-CD3-based bispecific T cell activating element’ engineered T (CAB-T) cells for the treatment of solid malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A217-A217
Author(s):  
Andy Tsun ◽  
Zhiyuan Li ◽  
Zhenqing Zhang ◽  
Weifeng Huang ◽  
Shaogang Peng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
In Vivo Studies ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

BackgroundCancer immunotherapy has achieved unprecedented success in the complete remission of hematological tumors. However, serious or even fatal clinical side-effects have been associated with CAR-T therapies to solid tumors, which mainly include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome, etc. Furthermore, CAR-T therapies have not yet demonstrated significant clinical efficacy for the treatment of solid tumors. Here, we present a novel T cell therapeutic platform: a Chimeric CD3e fusion protein and anti-CD3-based bispecific T cell activating element (BiTA) engineered T (CAB-T) cells, which target tumor antigens via the secretion of BiTAs that act independently of MHC interactions. Upon BiTA secretion, CAB-T cells can simultaneously achieve anti-tumor cytotoxic effects from the CAB-T cells and simultaneously activate bystander T cells.MethodsCAB-T cells were generated by co-expressing a chimeric CD3e fusion protein and an anti-CD3-based bispecific T cell activating element. The chimeric CD3e contains the extracellular domain of CD3e, a CD8 transmembrane domain, 4-1BB costimulatory domain, CD3z T cell activation domain and a FLAG tag, while the BiTA element includes a tumor antigen targeting domain fused with an anti-CD3 scFv domain and a 6x His-tag. CAR-T cells were generated as a control. Cytokine release activity, T cell activation and exhaustion markers, T cell killing activity and T cell differentiation stages were analysed. We also tested their tumor growth inhibition activity, peripheral and tumor tissue distribution, and their safety-profiles in humanized mouse models.ResultsCAB-T cells have similar or better in vitro killing activity compared with their CAR-T counterparts, with lower levels of cytokine release (IL-2 and IFNγ). CAB-T cells also showed lower levels of exhaustion markers (PD-1, LAG-3 and TIM-3), and higher ratios of naive/Tscm and Tcm T cell populations, after co-culture with their target tumor cells (48h). In in vivo studies, CAIX CAB-T and HER2 CAB-T showed superior anti-tumor efficacy and tumor tissue infiltration activity over their corresponding CAR-T cells. For CLDN18.2 CAB-T cells, similar in vivo anti-tumor efficacy was observed compared to CAR-T after T cell infusion, but blood glucose reduction and animal mortality was observed in the mice administered with CAR-T cells.ConclusionsThe advantages of CAB-T in in vitro and in vivo studies may result from TCR signal activation of both the engineered CAB-T cells and the non-engineered bystander T cells via cross-bridging by the secreted BiTA molecules, thus offering superior anti-tumor efficacy with a potential better safety-profile compared to conventional CAR-T platforms.

scholarly journals Tonic Signaling Leads to Off-Target Activation of T Cells Engineered with Chimeric Antigen Receptors That Is Not Seen in T Cells Engineered with T Cell Antigen Coupler (TAC) Receptors

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Duane Moogk ◽  
Arya Afsahi ◽  
Vivian Lau ◽  
Anna Dvorkin-Gheva ◽  
Jonathan Bramson
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transcriptional Profiling ◽  
Binding Domain ◽  
Antigen Binding ◽  
Knock Out ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptors (CARs) are powerful tools that enable MHC-independent activation of T cells. Recent reports have indicated that constitutive, low-level (tonic) signaling by CARs can impair the utility of the engineered T cells. The single-chain antibody (scFv) binding domain was one of the features determined to promote tonic signaling. We have recently developed a novel chimeric receptor, known as the T cell antigen coupler (TAC), that is less prone to tonic signaling than second-generation CARs. The TAC consists of a scFv-based antigen binding domain, a CD3-binding domain that couples the TAC to endogenous T cell receptor (TCR), and a transmembrane and cytoplasmic coreceptor (CD4) domain. In contrast to CARs, this design enables TAC-T cells to signal through the endogenous TCR, which we propose provides a fidelity to natural T cell signal regulation. Interestingly, we have recently reported that CAR-T cells have a greater propensity for off-target activation than TAC-T cells, suggesting a safety advantage to TAC-T cells (Helsen et al., Nat. Comm., 2019). Further characterization of the differences between CAR- and TAC-T cell signal initiation and activation is required to understand how their design affects sensitivity, specificity and regulation of T cell activation. Examination of the activation requirements for BCMA-specific CAR-T cells and TAC-T cells confirmed that TAC-T cells are reliant upon the endogenous TCR for T cell activation whereas CAR-T cells are TCR-independent. TRAC knock-out CAR-T cells retained potent effector function at levels similar to CAR-T cells with intact TCR expression, whereas TRAC knock-out TAC T-cells showed significant impairment in effector function. Consistent with TCR-dependence, the immunological synapse produced by TAC-T cells displays all the hallmarks of a conventional immunological synapse, whereas CAR-T cells form unconventional synapses. Unlike TAC-T cells, immunological synapses formed by CAR-T cells display non-uniform central supramolecular activation clusters, disperse Lck distribution, a lack of an LFA-1 associated adhesion ring (Figure), as well as more disperse delivery of perforin to the cell interface. CAR-T cells also formed synapses faster than TAC-T cells. This suggests that while TAC T-cells are beholden to the requirement of organized, mature synapse formation, CAR T-cells can rapidly form less structurally organized synapses. Transcriptional profiling of CAR-T cells in the absence of antigen stimulation revealed a basal activation status associated with upregulation of Nur77, a transcription factor that is downstream of TCR activation. Transcriptional profiling of TAC-T cells failed to reveal evidence of TCR signaling in the absence of stimulation. Further evaluation of CAR- and TAC- T cells in the absence of stimulation revealed elevated levels of CD69, PD-1 and LAG-3 in CAR-T cells compared with TAC-T cells, as well as higher expression of IL-2, IFNγ, and TNF in CAR-T cells. Interestingly, the level of tonic signaling was dependent on the antigen-binding scFV, as otherwise identical BCMA-specific CAR- and TAC-T cells displayed different levels of CD69, PD-1 and LAG-3 depending on the identity of the BCMA-specific scFv. Despite different levels of basal activation, both CAR- and TAC-T cells displayed comparable activation kinetics as measured by upregulation of CD69 and Ki-67, as well as proliferation. However, the elevated level of basal activation rendered the CAR-T cells more easily activated by a cross-reactive off-target antigen that failed to stimulate TAC-T cells carrying the same binding domain. These data suggest that the TAC receptor offers a valuable alternate platform to CAR-T cells. The antigen-binding scFv domain has a direct impact on tonic signaling and basal activation in CAR-T cells. Conversely, TAC-T cells are less susceptible to basal activation and this works suggests that the TAC receptor can deploy scFv binding domains that are not suitable for CARs. This work was supported by Triumvira Immunologics and Genome Canada. Figure 1 Disclosures Bramson: McMaster University: Current Employment, Patents & Royalties; Triumvira Immunologics: Current Employment, Current equity holder in private company, Research Funding.

A Failure to Start: Aborted Activation of CAR T Cells in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 681-681
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Meng Wang ◽  
Stefan M Lundh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
Low Levels

Introduction: Chronic Lymphocytic Leukemia (CLL) is a CD19+ B-cell malignancy that accounts for approximately 25% of adult leukemia diagnoses in the developed world. While conventional therapies have some efficacy, there are few curative therapeutic options and many patients ultimately progress to relapsed or refractory disease. CD19-targeting chimeric antigen receptor (CAR) T cell therapy has provided some hope, but induces complete remission in only 26% of patients. This suboptimal response rate is believed to be due to T cell dysfunction and immune-suppression by CLL cells, the mechanisms of which are poorly understood. Results: To understand the causes of CAR T cell dysfunction in CLL we investigated the defects that CLL cells induced in normal donor CD19-targeting CAR T cells. CAR T cells were repeatedly stimulated at 5-day intervals with either primary CLL cells from patients or a CD19-expressing control cell line (aAPC). Repeat stimulation of CAR T cells with aAPCs resulted in 5.36 ± .94 population doublings after three stimulations, whereas CLL cells only evoked 2.39 ± .92 population doublings. We performed phenotyping, proliferation analysis, and cytokine analysis of stimulated CAR T cells. CLL-stimulated T cells appeared un-activated, with low levels of PD-1, LAG3, and TIM3, low levels of cytokine production, and a high proportion of non-cycling cells as measured by Ki67 staining. We first hypothesized that CLL cells induce an altered epigenetic program that prevents effector function and is stabilized by successive stimulations. To test this, we stimulated CAR T cells with CLL cells or aAPCs as indicated in Fig. 1A. CLL-stimulated CAR T cells failed to proliferate or produce cytokines, but subsequent stimulation with aAPCs rescued these functions (Fig. 1B). Further, CLL-stimulated CAR T cells did not differentiate, suggesting that CLL cells do not induce stable defects but rather insufficiently activate CAR T cells (Fig. 1C). These cells also appeared un-activated as indicated by low levels of PD-1 and Ki67. We then used flow cytometry to assess expression of costimulatory and inhibitory molecules on the primary CLL samples. We found that the levels of co-stimulatory and adhesion molecules, namely CD80/CD86 and CD54/CD58 respectively were found at low frequencies, and where present were expressed at low levels. This suggested that one mechanism behind the lack of CAR T cell effector responses may be that a lack of co-stimulation prevents proper CAR T cell targeting of these cells. Towards this, we incubated CLL cells with a murine fibroblast line expressing CD40 ligand for 24 hours with IL-4 to activate the CLL cells. We found that this activation significantly increased expression of CD80, CD86, CD54, and CD58 on the CLL cells. We then used these cells to stimulate CAR T cells in our re-stimulation assay and found that CAR T cells were able to proliferate in response to these activated CLLs (Fig. 1D). In addition, CAR T cells stimulated with activated CLL cells formed more cell-to-cell conjugates than those stimulated with un-activated CLL cells. These data suggest not only that insufficient activation of CAR T cells may be responsible for the poor response rates to CAR T cell therapy in CLL, but also implicate a need for additional co-stimulation in this CAR T cell setting. Another contributing factor may be immune suppression by CLL cells. To determine if CLL cells are immune-suppressive, we used a co-culture assay to stimulate CAR T cells with aAPCs and CLL cells mixed at known ratios. Interestingly, all mixed cultures proliferated similarly, suggesting that CLL cells did not prevent T cell activation in the presence of a strong activation signal. We also found that CLL cells are responsive to IL-2, as addition of this cytokine to culture media prolongs survival of CLL cells out to 10 days depending on the dose. This suggests that CLL cells express a functional IL-2 receptor and may be taking up IL-2 from the culture media, further impairing T cell activation. In support of this, supplementing IL-2 into our CLL/CAR T cell co-cultures rescued T cell proliferative capacity. Taken together, these data suggest that T cell dysfunction in CLL is the result of insufficient activation rather than true functional defects. Disclosures June: Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; Incyte: Research Funding.

Generating Chimeric Antigen Receptors Utilizing Novel Anti-CD3 Nanobeads

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5949-5949
Author(s):  
Liora M. Schultz ◽  
Debra K Czerwinski ◽  
Aihua Fu ◽  
Shoshana Levy ◽  
Ronald Levy
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antigen Receptors ◽  
Chimeric Antigen Receptors ◽  
Car T Cell ◽  
Car T ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract The processes of ex vivo transduction of T cells to express chimeric antigen receptors (CARs) and of CAR+ T cell expansion influence the phenotype, function and ultimate fate of the final CAR+T cell product infused into patients. CAR constructs, despite expression of endogenous activation signals, require exogenous T cell activation during CAR transduction to allow optimal lenti-viral or retroviral-mediated integration of the CAR gene of interest into T cells. Clinical CAR therapy trials utilize anti-CD3 antibody-mediated activation or combined CD3 and CD28 stimulation using CD3, CD28 specific magnetic beads. We introduce novel magnetic nanoparticle beads generated from iron oxide nanoparticles conjugated to streptavidin and bound to biotinylated T cell activating antibodies for the purpose of CAR transduction. The small size of these nanobeads confers the advantage of decreased steric hindrance and enhanced capability of bead surface antibodies to access T cell surface antigen for binding and stimulation. We achieve efficient CAR transduction using anti-CD3 nanobead-mediated T cell stimulation and demonstrate CD19 specific CAR-mediated cytotoxicity of CD19+ tumor using an annexin V and 7AAD cytotoxicity assay. Evaluation of T cell phenotype following anti-CD3 nanobead-mediated T cell activation demonstrates preferential activation of naïve T cells as compared to central and effector memory cells. Addition of anti-CD28 costimulation is not necessary to achieving or inhibiting this preferential naïve T cell activation. Naïve T cells exhibit greater replicative capacity and anti-tumor function as compared to both effector and central memory T cells for adoptive transfer. We anticipate that preferential generation of naïve T cell derived CAR+ T cells achieved by introducing anti-CD3 nanobead stimulation can further improve the outcomes of clinical trials using CAR therapy. Disclosures Fu: NVIGEN Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals P06.02 Enhancing CAR T cell persistence and memory through modulating mitochondrial function

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A42.1-A42
Author(s):  
A Hosseini Rad ◽  
G Min Yi Tan ◽  
A Poudel ◽  
A McLellan
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Viability ◽  
T Cell Activation ◽  
Mitochondrial Function ◽  
Luciferase Reporter ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundCAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF).Materials and MethodsOverexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells.ResultsOverexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells.ConclusionsOverexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells.Disclosure InformationA. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.

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