GFP Affects Human T Cell Activation and Cytokine Production following In Vitro Stimulation

BackgroundTCEs are effective in leukemias but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent CRS include step-up dosing and/or complex designs but are unsuccessful due to toxicity and/or enhanced immunogenicity. HER2-XPAT, or XTENylated Protease-Activated bispecific T-Cell Engager, is a prodrug TCE that exploits the protease activity present in tumors vs. healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core, two unstructured polypeptide masks (XTEN) sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a potent TCE with short T1/2, further improving the TI. HER2-XPAT, a tumor protease-activatable prodrug with wide safety margins, can co-opt T-cells regardless of antigenic specificity to induce T-cell killing of HER2+ tumors.MethodsPreclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytotoxicity in vitro, for anti-tumor efficacy in xenograft models, and for safety in NHPs.ResultsHER2-PAT demonstrated potent in vitro T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and cytokine production by hPBMCs. HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and no cytotoxicity against cardiomyocytes up to 1uM. In vivo, HER2-XPAT induced complete tumor regressions in BT-474 tumors with equimolar dosing to HER2-PAT, whereas HER2-NonClv had no efficacy, supporting requirement of protease cleavage for T-cell activity. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD). HER2-XPAT demonstrated early T-cell margination at 2 mg/kg but largely spared CRS, cytokine production, and tissue toxicity up to 42 mg/kg. PK profiles of HER2-XPAT and HER2-NonClv were comparable, consistent with ex vivo stability for cleavage when incubated in cancer pts plasma for 7 days at 37°C. HER2-PAT by continuous infusion induced lethal CRS and cytokine spikes at 0.3 mg/kg/d but was tolerated at 0.25 mg/kg/d, providing HER2-XPAT with >1300-fold protection in tolerability vs. HER2-PAT, >4 logs over cytotoxicity EC50s for HER2 cell lines, and a 20-fold safety margin over the dose required for pharmacodynamic activity.ConclusionsHER2-XPAT is a potent prodrug TCE with no CRS and a wide TI based on NHPs. With XTEN’s clinical data demonstrating low immunogenicity, the XPATs are a promising solution. IND studies are ongoing. Additional PK/PD, cytokines, safety, and efficacy data will be presented.

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Comparison of anti-arthritic properties of leflunomide with methotrexate and FK506: Effect on T cell activation-induced inflammatory cytokine production in vitro and rat adjuvant-induced arthritis

Inflammation Research ◽

10.1007/s00011-004-1294-9 ◽

2004 ◽

Vol 53 (10) ◽

pp. 544-550 ◽

Cited By ~ 21

Author(s):

K. Magari ◽

S. Miyata ◽

F. Nishigaki ◽

Y. Ohkubo ◽

S. Mutoh

Keyword(s):

T Cell ◽

T Cell Activation ◽

Inflammatory Cytokine ◽

Cell Activation ◽

Cytokine Production ◽

Inflammatory Cytokine Production ◽

Adjuvant Induced Arthritis

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107 Effects of IL-2 and IL-15 on the proliferative and antitumor capacities of allogeneic CD20 CAR-engineered γδ T cells in a 3D B cell lymphoma spheroid assay

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0107 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A119-A119

Author(s):

Lu Bai ◽

Kevin Nishimoto ◽

Mustafa Turkoz ◽

Marissa Herrman ◽

Jason Romero ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cytokine Production ◽

Γδ T Cells ◽

Γδ T Cell ◽

Car T

BackgroundAutologous chimeric antigen receptor (CAR) T cells have been shown to be efficacious for the treatment of B cell malignancies; however, widespread adoption and application of CAR T cell products still face a number of challenges. To overcome these challenges, Adicet Bio is developing an allogeneic γδ T cell-based CAR T cell platform, which capitalizes on the intrinsic abilities of Vδ1 γδ T cells to recognize and kill transformed cells in an MHC-unrestricted manner, to migrate to epithelial tissues, and to function in hypoxic conditions. To gain a better understanding of the requirements for optimal intratumoral CAR Vδ1 γδ T cell activation, proliferation, and differentiation, we developed a three-dimensional (3D) tumor spheroid assay, in which tumor cells acquire the structural organization of a solid tumor and establish a microenvironment that has oxygen and nutrient gradients. Moreover, through the addition of cytokines and/or tumor stromal cell types, the spheroid microenvironment can be modified to reflect hot or cold tumors. Here, we report on the use of a 3D CD20+ Raji lymphoma spheroid assay to evaluate the effects of IL-2 and IL-15, positive regulators of T cell homeostasis and differentiation, on the proliferative and antitumor capacities of CD20 CAR Vδ1 γδ T cells.MethodsMolecular, phenotypic, and functional profiling were performed to characterize the in vitro dynamics of the intraspheroid CD20 CAR Vδ1 γδ T cell response to target antigen in the presence of IL-2, IL-15, or no added cytokine.ResultsWhen compared to no added cytokine, the addition of IL-2 or IL-15 enhanced CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and cytokine production in a dose-dependent manner but were only able to alter the kinetics of Raji cell killing at low effector to target ratios. Notably, differential gene expression analysis using NanoString nCounter® Technology confirmed the positive effects of IL-2 or IL-15 on CAR-activated Vδ1 γδ T cells as evidenced by the upregulation of genes involved in activation, cell cycle, mitochondrial biogenesis, cytotoxicity, and cytokine production.ConclusionsTogether, these results not only show that the addition of IL-2 or IL-15 can potentiate CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation into antitumor effectors but also highlight the utility of the 3D spheroid assay as a high throughput in vitro method for assessing and predicting CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation in hot and cold tumors.

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Cobalt-chromium-molybdenum but not titanium-6aluminium-4vanadium alloy discs inhibit human t cell activation in vitro

BioMetals ◽

10.1007/bf00140600 ◽

1996 ◽

Vol 9 (4) ◽

pp. 321-326 ◽

Cited By ~ 14

Author(s):

Cristina Faleiro ◽

Inês Godinho ◽

Ulrich Reus ◽

Maria de Sousa

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cobalt Chromium ◽

Human T Cell ◽

Cobalt Chromium Molybdenum

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A high-affinity human TCR-like antibody detects celiac disease gluten peptide–MHC complexes and inhibits T cell activation

Science Immunology ◽

10.1126/sciimmunol.abg4925 ◽

2021 ◽

Vol 6 (62) ◽

pp. eabg4925

Author(s):

Rahel Frick ◽

Lene S. Høydahl ◽

Jan Petersen ◽

M. Fleur du Pré ◽

Shraddha Kumari ◽

...

Keyword(s):

Celiac Disease ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Therapeutic Potential ◽

Human Leukocyte ◽

Fab Fragment ◽

Biopsy Material ◽

Human T Cell

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)–like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.

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In Vitro Monitoring of Human T Cell Responses to Skin Sensitizing Chemicals—A Systematic Review

Cells ◽

10.3390/cells11010083 ◽

2021 ◽

Vol 11 (1) ◽

pp. 83

Author(s):

Marina Aparicio-Soto ◽

Caterina Curato ◽

Franziska Riedel ◽

Hermann-Josef Thierse ◽

Andreas Luch ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cross Reactivity ◽

T Cell Epitopes ◽

Cell Responses ◽

Human T Cell ◽

The Individual

Background: Chemical allergies are T cell-mediated diseases that often manifest in the skin as allergic contact dermatitis (ACD). To prevent ACD on a public health scale and avoid elicitation reactions at the individual patient level, predictive and diagnostic tests, respectively, are indispensable. Currently, there is no validated in vitro T cell assay available. The main bottlenecks concern the inefficient generation of T cell epitopes and the detection of rare antigen-specific T cells. Methods: Here, we systematically review original experimental research papers describing T cell activation to chemical skin sensitizers. We focus our search on studies published in the PubMed and Scopus databases on non-metallic allergens in the last 20 years. Results: We identified 37 papers, among them 32 (86%) describing antigen-specific human T cell activation to 31 different chemical allergens. The remaining studies measured the general effects of chemical allergens on T cell function (five studies, 14%). Most antigen-specific studies used peripheral blood mononuclear cells (PBMC) as antigen-presenting cells (APC, 75%) and interrogated the blood T cell pool (91%). Depending on the individual chemical properties, T cell epitopes were generated either by direct administration into the culture medium (72%), separate modification of autologous APC (29%) or by use of hapten-modified model proteins (13%). Read-outs were mainly based on proliferation (91%), often combined with cytokine secretion (53%). The analysis of T cell clones offers additional opportunities to elucidate the mechanisms of epitope formation and cross-reactivity (13%). The best researched allergen was p-phenylenediamine (PPD, 12 studies, 38%). For this and some other allergens, stronger immune responses were observed in some allergic patients (15/31 chemicals, 48%), illustrating the in vivo relevance of the identified T cells while detection limits remain challenging in many cases. Interpretation: Our results illustrate current hardships and possible solutions to monitoring T cell responses to individual chemical skin sensitizers. The provided data can guide the further development of T cell assays to unfold their full predictive and diagnostic potential, including cross-reactivity assessments.

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Differential effects of estradiol and progesterone on human T cell activation in vitro

European Journal of Immunology ◽

10.1002/eji.202049144 ◽

2021 ◽

Author(s):

Georgia Papapavlou ◽

Sandra Hellberg ◽

Johanna Raffetseder ◽

Jan Brynhildsen ◽

Mika Gustafsson ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Differential Effects ◽

Human T Cell

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Farnesyltransferase inhibitors inhibit T-cell cytokine production at the posttranscriptional level

Blood ◽

10.1182/blood-2006-06-031088 ◽

2007 ◽

Vol 110 (6) ◽

pp. 1982-1988 ◽

Cited By ~ 15

Author(s):

Reinhard E. Marks ◽

Allen W. Ho ◽

Christian Robbel ◽

Todd Kuna ◽

Seth Berk ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Intracellular Signaling ◽

Map Kinases ◽

Cell Activation ◽

Cytokine Production ◽

Immunosuppressive Agents ◽

Farnesyltransferase Inhibitors ◽

Human T Cell ◽

Posttranscriptional Level

Abstract Several cytoplasmic proteins, such as GTPases of the Ras family, containing a C-terminal CAAX motif are prenylated by farnesyltransferase to facilitate localization to cellular membranes where activation occurs. Farnesyltransferase inhibitors (FTIs) interfere with this farnesylation process, thereby preventing proper membrane localization and rendering the proteins unavailable for activation. Currently, FTIs are being explored as antineoplastic agents for the treatment of several malignancies. However, since farnesylated proteins like Ras are also involved in intracellular signaling in lymphocytes, FTIs might interfere with T-cell activation. Based on this hypothesis we examined the effect of several FTIs on cytokine production in response to anti-CD3 + anti-CD28 monoclonal antibodies or PMA + ionomycin. Murine Th1 and Th2 clones, stimulated in the presence of FTIs, showed a dose-dependent reduction of lineage-specific cytokine secretion (IFN-γ, IL-2, IL-4, IL-5). However, no inhibition of ERK or JNK MAP kinases was observed, nor was induction of cytokine mRNA affected. Rather, intracellular cytokine protein synthesis was blocked. Inhibition of human T-cell INF-γ production also was observed, correlating with reduced phosphorylation of p70S6K. These results indicate that FTIs inhibit T-cell activation at the posttranscriptional level and also suggest that they may have potential as novel immunosuppressive agents.

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