搜索结果: 'gmp t cell activator'

Transition from the manual processes that are performed during the initial research and development (R&D) stage to automated processes for later and commercial stage cell therapy manufacturing can be challenging. It often requires significant effort,time,and costs – which hinders the therapy’s access to the clinic. To ease this transition,we have developed a novel and flexible manufacturing platform,Bioreactor with Expandable Culture Area (BECA),that aims to support both R&D and manufacturing to accelerate cell therapies from bench to bedside. This report introduces two models in this manufacturing platform: BECA-S for manual small-scale operation at R&D phase and BECA-Auto for functionally closed and automated scaled-out operation at manufacturing phase. We employed these two models to streamline transition of the T cell culture process from manual to automated and reported insignificant differences in the culture outcome between the two. Our work represents the first detailed development and demonstration of a standalone cell manufacturing platform that facilitates a seamless transition between manual and automated processing for autologous T cell therapy manufacturing. View Publication

With the approval of the antibody-drug conjugate enfortumab vedotin (EV),NECTIN4 has emerged as a bona fide therapeutic target in urothelial carcinoma (UC). Here,we report the development of a NECTIN4-directed chimeric antigen receptor (CAR) T cell,which exhibits reactivity across cells expressing a range of endogenous NECTIN4,with enhanced activity in high expressors. We demonstrate that the PPARγ pathway,critical for luminal differentiation,transcriptionally controls NECTIN4,and that the PPARγ agonist rosiglitazone primes and augments NECTIN4 expression,thereby increasing sensitivity to NECTIN4-CAR T cell-mediated killing. NECTIN4-CAR T cells have potent anti-tumor activity even against EV resistant cells,which largely retain NECTIN4 expression,including in a post-EV biopsy cohort. Our results elucidate a therapeutically actionable mechanism that UC cells use to control NECTIN4 expression and suggest therapeutic approaches that leverage PPARγ agonists for rational combinations with NECTIN4-targeting agents in UC,as well as future potential treatment options for EV-refractory patients. Subject terms: Bladder cancer,Cancer immunotherapy,Cancer therapeutic resistance,Oncology,Bladder cancer View Publication

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF),a cell surface C3/C5 convertase regulator,exerts control over T cell responses. Extending these results,we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1(-/-)) APCs produce significantly more IL-12,C5a,and IFN-gamma compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-gamma-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC,demonstrating a link between DAF,local complement activation,IL-12,and T cell-produced IFN-gamma. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-gamma-producing effector T cells. Overall,our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production,this in turn,contributes to directing T cell differentiation toward an IFN-gamma-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity. View Publication

Uveal melanoma (UM) is the most common intraocular cancer in adults,with metastatic disease (mUM) occurring in approximately half of the patients. Tebentafusp,an immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC),is a therapeutic shown to improve overall survival (OS) in HLA-A*02:01+ adult patients with mUM. Here we investigate the impact of tumor-associated macrophages (TAM) on ImmTAC activity. In vitro,M2 macrophages inhibit ImmTAC-mediated tumor-killing in a dose-dependent and contact-dependent manner. Accordingly,high baseline intratumoral TAM-to-T cell ratios correlate with shorter OS (HR = 2.09,95% CI,1.31–3.33,p = 0.002) in tebentafusp-treated mUM patients from a phase 2 trial. By contrast,IL-2 conditioning of T cells overcomes M2 macrophage-mediated suppression in vitro,while ImmTAC treatment leads to M2-to-M1 macrophage reprogramming both in vitro and in tebentafusp-treated mUM patients. Overall,we show that tebentafusp reshapes the tumor microenvironment to enhance anti-tumor T cell activity,whilst combining tebentafusp with IL-2 may enhance benefit in patients with high levels of TAM. ‘T cell engagers promote antitumor immunity,but how macrophage modulates this activity in tumor is still unclear. Here the authors show,using biopsies from patients with uveal melanoma and single cell analyses,that a T cell engager,tebentafusp,reprograms tumor-associated macrophages and ameliorates,in synergy with IL-2,immunosuppression to cancer. View Publication

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51),endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high,TH17-high,and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples,and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures,we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However,neutralization of IL-13 and IL-17 protected mice from eosinophilia,mucus hyperplasia,and airway hyperreactivity and abolished the neutrophilic inflammation,suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes. View Publication

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor,these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study,evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation,a non-JAK2-dependent signal,in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates,STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1,-3,-5a,and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT,JAK3/AP-1,and JAK3/MAPK pathways and their cellular consequences. Taken together,these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways. View Publication

The promise of inducing immunological tolerance through regulatory T cell (Treg) control of effector T cell function is crucial for developing future therapeutic strategies to treat allograft rejection as well as inflammatory autoimmune diseases. In the current study,we used murine allograft rejection as a model to identify microRNA (miRNA) regulation of Treg differentiation from na{\{i}}ve CD4 cells. We performed miRNA expression array in CD4+ T cells in the draining lymph node (dLN) of mice which received syngeneic or allogeneic grafts to determine the molecular mechanisms that hinder the expansion of Tregs. We identified an increase in miRNA cluster 297-669 (C2MC) after allogeneic transplantation in CD4+ T cells such that 10 of the 27 upregulated miRNAs were all from this cluster with one of its members mmu-miR-466a-3p (miR-466a-3p) targeting transforming growth factor beta 2 (TGF-$\beta$2) as identified through reporter luciferase assay. Transfection of miR-466a-3p in CD4+ T cells led to a decreased inducible FoxP3+ Treg generation while inhibiting miR-466a-3p expression through locked nucleic acid resulting in increased Tregs and a reduction in effector T cells. Furthermore in vivo inhibition of miR-466a-3p in an allogeneic skin-graft model attenuated T cell response against the graft through an increase in TGF-$\beta$2. TGF-$\beta$2 was as effective as TGF-$\beta$1 at both inducing Tregs and through adoptive transfer mitigating host effector T cell response against the allograft. Together the current study demonstrates for the first time a new role for miRNA-466a-3p and TGF-$\beta$2 in the regulation of Treg differentiation and thus offers novel avenues to control inflammatory disorders." View Publication

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4 + T cell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling,resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML,serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR T cell treatment on LSCs in vitro . Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore,promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML. View Publication

In clinical chimeric antigen receptor (CAR) T cell therapy,one of the strongest correlates of favorable patient responses is lower levels of differentiation in T cells from the peripheral blood mononuclear cell (PBMC) starting material or the CAR T cell product. T cells from older patients are inherently more differentiated,but we hypothesised that specific activation protocols could be used to limit CAR T cell differentiation during manufacturing,particularly in older patients. We used PBMCs from young (20–30 years old) and older (60+ years old) healthy donors to generate CAR T cells using two activation protocols: soluble anti‐(α) CD3 monoclonal antibody (mAb) vs immune complexes of αCD3 and αCD28 mAbs. Products were assessed for yield,function and differentiation,which was used as a measure of CAR T cell quality. T cells in PBMCs were assessed for CD28 expression and correlative analyses were performed. Older samples generated fewer,more differentiated CAR T cells than young samples,and the αCD3/CD28 mAb protocol exacerbated this,further reducing yield and quality. CD28 expression by T cells correlated with CAR T cell differentiation,but T cell differentiation in PBMC starting material was a stronger correlate of CAR T cell differentiation. Choice of activation protocol can substantially impact on the yield and quality of CAR T cells during manufacturing. This is a key consideration for older patients whose samples already generate a poorer yield and lower quality of CAR T cells. View Publication

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection,but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor,but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation,activation marker (CD44 and CD69) expression,and production of IFN-γ,IL-2,and granzyme B. Moreover,we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically,type I IFNs promote Ag presentation on MHC I molecules,CD86 and CD40 expression,and the production of IL-12 p70,IL-2,IL-6,and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation,although,in the context of LPS stimulation,this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection,which may be useful in the rational design of vaccines directed against pathogens and tumors. View Publication

Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU),a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen,and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses,and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response,nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells,and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity,and the expression of MC5r and A2Ar on human immune cells,suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients. View Publication

Here,we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation,a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT,inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells,demonstrates selectivity for Lck and FynT over ZAP-70,and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly,this compound selectively inhibits the induction of the IL-2 gene,but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function. View Publication