scholarly journals Single-cell integrative analysis of CAR-T cell activation reveals a predominantly TH1/TH2 mixed response independent of differentiation

2018 ◽  
Author(s):  
Iva Xhangolli ◽  
Burak Dura ◽  
GeeHee Lee ◽  
Dongjoo Kim ◽  
Yang Xiao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Objective Response ◽  
Effector Memory ◽  
Car T Cells ◽  
Mixed Response ◽  
Car T

We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19 4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4+ ‘helper’ and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines (e.g., IFNγ, TNFα, IL5, and IL13), as confirmed by the expression of master transcription factors TBX21 (T-bet) and GATA3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell and the regulatory T cell (Treg) activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. GM-CSF is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status although all major differentiation subsets such as naïve, central memory, effector memory and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GMCSF, supporting the notion that ‘polyfunctional’ CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights to the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

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 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.

scholarly journals The Enhanced Functionality of Low-Affinity CD19 CAR T Cells Is Associated with Activation Priming and Polyfunctional Cytokine Phenotype

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 52-53
Author(s):  
Ilaria M. Michelozzi ◽  
Eduardo Gomez-Castaneda ◽  
Ruben V.C. Pohle ◽  
Ferran Cardoso Rodriguez ◽  
Jahangir Sufi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Rna Seq ◽  
Car T Cells ◽  
Car T ◽  
Functional Phenotype

We have recently described a low-affinity second-generation anti-CD19 Chimeric Antigen Receptor (CAR) (CAT), characterized by faster antigen dissociation rate which showed enhanced expansion, cytotoxicity and anti-tumour efficacy compared with the high affinity (FMC63 based) CAR used in Tisagenlecleucel in pre-clinical models. Furthermore, CAT CAR T cells showed an excellent toxicity profile, enhanced in vivo expansion and long-term persistence in a Phase I clinical study (Ghorashian et al Nature Med 2019). However the molecular mechanisms behind the improved properties of CAT CAR T cells remain unknown. Herein, we performed a systematic in vitro characterization of the transcriptomic (bulk RNA-seq) and protein (CyTOF) changes occurring in CAR T cells expressing a low-affinity (CAT) vs high affinity (FMC63) anti-CD19 CARs following stimulation with CD19 expressing targets. Untransduced (UT) controls and T cells lentivirally transduced to express CAT or FMC63 CD19 CARs were compared both at baseline and following stimulation with CD19+ Acute Lymphoblastic Leukaemia cell line NALM6. In Principal Component Analysis for both RNA-seq and protein results, we found that the major variance across conditions was explained by CD19-mediated CAR T activation. Strikingly, unstimulated CAT CAR T cells showed an intermediate degree of activation between UT T cells and antigen stimulated CAR T cells. Indeed, when comparing RNA-seq results of unstimulated CAT vs FMC63, we found enhanced expression (FDR <0.1) of genes involved in cytotoxicity (GNLY, GZMK) and T cell activation (HLA-DRA and HLA-DPA1) (Figure 1a), confirmed at protein level by CyTOF. This "activation priming" observed in CAT CAR T cells was associated with and may be driven by residual CD19-expressing B-cells present in the manufacture product, preferentially inducing a T Central Memory (TCM) phenotype in CAT vs FMC63, in both CD4 and CD8 T cells. Such priming is likely to be instrumental to CAT CAR T cells more potent cytotoxic response upon NALM6 stimulation, when they displayed further increase in the expression of immune stimulatory cytokines (IFNG, CSF2), chemokines (CCL3L1, CCL4, CXCL8) and IFNg responsive genes (CIITA) by RNA-seq, as well as augmented T cell activation (CD25, NFAT1) and proliferation (pRB) markers by CyTOF. To identify the mechanisms underlying the stronger basal activation of CAT CAR T cells, we analysed cytokine expression at the single cell level by mass cytometry. Interestingly, rather than an increment in the expression of individual cytokines, we found that the distinctive feature of CAT CAR T cells was a shift toward a cytokine polyfunctional phenotype, with a marked increase in the proportion of cells co-expressing 3 or more cytokines (17.50% CAT vs 7.33% FMC63) (Figure 1b). Of note, cytokine polyfunctionality (expression of more than 1 cytokine/cell) in pre-infusion CAR T cell products has been associated to improved clinical efficacy. The functional phenotype observed in CAT CAR T cells was linked to the preferential activation of the p38 MAPK phospo-signalling, which is activated downstream of TCR CD3ζ chain (present in the CARs) but is also central to cytokine-dependent T cell activation in memory T cells. Interestingly, cytokine polyfunctional CAT CAR T cells were enriched in the CD3+CD19+ trogocytic (trog+) population, found at higher proportion in CAT vs FMC63 at 24h post antigen stimulation. Although trogocytosis has been associated to CAR T cell fratricide killing, trog+ CAT CAR T cells displayed higher levels of proliferation (pRB), activation (CD25, NFAT1) and cytotoxic (Granzyme B, Perforin B) markers, pointing at a stimulatory role of trogocytosis over fratricide killing, potentially due to the low-affinity CAR T cells distinctive property of better discriminating between low (trog+ CAR T cells) and high (tumour cells) target expression levels. In conclusion, we described the molecular mechanisms underlying the low affinity CAT CAR T cells functional phenotype. Our results show that the potent and long-term anti-tumour responses observed with CAT may be sustained by the establishment of CAR T cells self-reinforcing circuits activated through polyfunctional cytokine crosstalk. This work may inform the future design of versatile CAR T cells, capable of balancing safety, efficacy and long-term persistence. Disclosures Ghorashian: Amgen: Honoraria; UCLB: Patents & Royalties; Novartis: Honoraria. Pule:Autolus: Current Employment, Other: owns stock in and receives royalties, Patents & Royalties; UCLB: Patents & Royalties; Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL.

scholarly journals Enhancing clinical and immunological effects of anti-PD-1 with belapectin, a galectin-3 inhibitor

2021 ◽  
Vol 9 (4) ◽  
pp. e002371
Author(s):  
Brendan D Curti ◽  
Yoshinobu Koguchi ◽  
Rom S Leidner ◽  
Annah S Rolig ◽  
Elizabeth R Sturgill ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Dose Range ◽  
Objective Response ◽  
Eastern Cooperative Oncology Group ◽  
Effector Memory ◽  
Group Status ◽  
Galectin 3

BackgroundPD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC).MethodsWe performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404). Belapectin was administered at 2, 4, or 8 mg/kg IV 60 min before pembrolizumab (200 mg IV every 3 weeks for five cycles). Responding patients continued pembrolizumab monotherapy for up to 17 cycles. Main eligibility requirements were a functional Eastern Cooperative Oncology Group status of 0–2, measurable or assessable disease, and no active autoimmune disease. Prior T-cell checkpoint antibody therapy was permitted.ResultsObjective response was observed in 50% of MM (7/14) and and 33% of HNSCC (2/6) patients. Belapectin+pembrolizumab was associated with fewer immune-mediated adverse events than anticipated with pembrolizumab monotherapy. There were no dose-limiting toxicities for belapectin within the dose range investigated. Significantly increased effector memory T-cell activation and reduced monocytic myeloid-derived suppressor cells (M-MDSCs) were observed in responders compared with non-responders. Increased baseline expression of Gal-3+ tumor cells and PD-1+CD8+ T cells in the periphery correlated with response as did higher serum trough levels of pembrolizumab.ConclusionsBelapectin+pembrolizumab therapy has activity in MM and HNSCC. Increased Gal-3 expression, expansion of effector memory T cells, and decreased M-MDSCs correlated with clinical response. Further investigation is planned.

scholarly journals Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mohit Sachdeva ◽  
Brian W. Busser ◽  
Sonal Temburni ◽  
Billal Jahangiri ◽  
Anne-Sophie Gautron ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Immune Pathways ◽  
And Control

Abstract Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.

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 Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1690-1690
Author(s):  
Xiaomeng Hu ◽  
Mo Dao ◽  
Kathy White ◽  
Corie Gattis ◽  
Ryan Clarke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nk Cell ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T

Abstract Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction as well as the ability to generate a final product from healthy T cells. While TCR editing can effectively prevent graft-versus-host reactions, the significant host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells (N Engl J Med. 2021;384(7):673-674). We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced by this strategy. Since CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to delete b2m, CIITA, and TCR expression and lentiviral transduction was used to overexpress CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CAR T cells. Control T cells were unmanipulated except for lentiviral transduction used to overexpress the same CD19CAR and the deletion of the TCR. When transplanted into allogeneic humanized mice, hypoimmune CD19CAR T cells evade immune recognition by T cells even in previously sensitized animals as evidenced by a lack of T cell activation measured using ELISPOT analysis. In contrast, transplantation of non-hypoimmune-edited CD19CAR T cells generated from the same human donor resulted in a significant T cell activation (see figure: mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and non-edited CD19CAR T cells, respectively; p<0.0001 unpaired T-test). In addition to evading T cells, immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmune CAR T cells susceptible to macrophage and NK cell killing in vitro and in vivo, confirming the importance of CD47 overexpression to evade innate immune clearance. The hypoimmune CD19 CAR T cells retained their antitumor activity in both the Daudi and Nalm-6 B cell leukemia models, in vitro and in vivo. This indicated that the hypoimmune technology-i.e. isolated CD47 overexpression, deletion of b2m, CIITA, and TCR- did not show any effect on the cytotoxic potential of CD19 CAR T cells (see figure). These studies demonstrate that in vivo clearance of leukemic cells in NSG mice occurs across a range of tumor cell toCD19 CAR T cell ratios in a manner comparable to control, unedited CD19 CAR T cells (see figure). This result was validated using T cells from 3 different donors These findings show that, in these models, hypoimmune CD19 CAR T cells are functionally immune evasive in allogeneic humanized mouse recipients and have cytotoxic anti-tumor capacity. They suggest that hypoimmune CAR T cells could provide universal CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that hypoimmune CD19 CAR T cells can be used in sensitized patients and for re-dosing strategies. Figure 1 Figure 1. Disclosures Hu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dao: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. White: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Gattis: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Clarke: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Landry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Basco: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tham: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tucker: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Luo: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Bandoro: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Chu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Young: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dowdle: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Rebar: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Schrepfer: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Cite-Seq Profiling of T Cells in Multiple Myeloma Patients Undergoing BCMA Targeting CAR-T or Bites Immunotherapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Noemie Leblay ◽  
Ranjan Maity ◽  
Elie Barakat ◽  
Sylvia McCulloch ◽  
Peter Duggan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Single Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Surface Protein ◽  
T Cell Subsets ◽  
Cell Surface Protein ◽  
Car T

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) have demonstrated encouraging responses in heavily pre-treated multiple myeloma (MM) patients. However, the cellular and molecular predictors of clinical response are not fully understood as well as the mediators of acquired resistance remain elusive. Local immune suppression and T cell exhaustion are important mediators of responses therefore, it is plausible to speculate that a tolerant tumor microenvironment and the expansion of specific T cell populations may dictate clinical responses. In this study, we performed at the single cell level a broad immunophenotypic and transcriptomic characterization of the blood and bone marrow (BM) T cells of sensitive and resistant MM patients treated with adaptive T cell therapies. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in order to identify cellular mediators of resistance to these adoptive immune therapies. Serial blood samples and BM aspirates (n=12) were collected from patients treated with anti-BCMA CAR-T or BCMA-CD3 BiTEs at variable time points, prior and post initiation of therapy and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD3pos T cells. Unbiased mRNA profiling coupled with feature barcoding technology for cell surface protein (TotalSeq-B) of BM CD3pos T cells was then performed by using the chromium single cell (10x Genomics). Paired-end sequencing was performed on Illumina platform. Cell Ranger and Seurat pipeline were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting, cell surface protein expression and data analysis. CAR-T cells were identified by the expression of the chimeric CAR-T cell transcript. The parallel measurement of transcripts and cell surface protein phenotypes of CD3pos T cells using a panel of 19 immune surface markers underlined the T cell repertoire diversity and identified different T cell subsets among the CD8pos and CD4pos T cells. Notably, the cell surface protein information overlaid on the transcript-generated UMA allowed accurate identification of all main immune clusters, in particular for the CD45RA and CD45RO positive cells. Comparison of CITE-Seq features revealed that the T cells composition of the blood and BM niches differed significantly between sensitive and resistant patients. As such an enrichment of CD4pos T cells with a higher CD4:CD8 ratio was noted in responding patients. Phenotypic (CD45RA, CD45RO, CD95, CCR7, CD62L, CD28, CD27) and transcriptional signatures (TCF7, LEF1, GATA3, EOMES, TBX21, PRDM1) also identified a higher proportion of memory like T cells (Tscm, Tcm) in responding patients. In contrast, T cells of resistant patients were enriched with terminally exhausted (Tex) and senescent cells with loss of CD28, high GMZHand GMZB, CD57pos, CD69pos and CD160pos as well as upregulation of TBX21. Expression of T cell checkpoint inhibitors such as LAG3, TIGIT and PD1 was high in these Tex cells as well as in some Tem. Of note, ex vivo T cell activation studies with TIGIT blockade demonstrated T cell activation in an autologous MM and T cell co-culture system with enhanced MM cells death. An expanded cluster of regulatory T cells (Treg) FOXP3pos,CD25pos was also observed in two resistant patients. Of note, no loss of BCMA transcript or surface expression was noted in MM cells at the time of acquired resistance. Single cell transcriptome of primary MM cells and chromatin accessibility (ATAC-seq) analyses of T cells of these patients are ongoing to investigate the transcriptional programs and epigenetic factors underlying the immune escape. Combined single cell features profiling of the transcriptome and surface protein expression of T cells from MM patients receiving BCMA targeted CAR-T or BiTEs therapies revealed potential mediators of resistance. In particular, T cells composition (low CD4:CD8 ratio and reduced population of Tscm, Tcm) along with an enrichment of terminally exhausted T cells are the main features observed in resistant patients. Delineating these mechanisms will guide future T cells engineering studies to enhance the efficacy and response durability of adoptive immunotherapy in MM. Disclosures McCulloch: Amgen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Duggan:Jannsen: Consultancy; Amgen: Consultancy; Novartis: Honoraria; Celgene: Consultancy; Astra Zeneca: Consultancy. Jimenez-Zepeda:Janssen, Celgene, Amgen, Takeda: Honoraria. Bahlis:AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Karyopharm Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Neri:Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

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.

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