Selective blockade of CD28 and not CTLA-4 with a single-chain Fv–α1-antitrypsin fusion antibody

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 564-570 ◽  
Author(s):  
Bernard Vanhove ◽  
Geneviève Laflamme ◽  
Flora Coulon ◽  
Marie Mougin ◽  
Patricia Vusio ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antibody Fragment ◽  
Single Chain ◽  
Selective Blockade ◽  
Mhc Molecules ◽  
Single Chain Fv

Abstract B7-1 and B7-2 are costimulatory molecules expressed on antigen-presenting cells. The CD28/B7 costimulation pathway is critical for T-cell activation, proliferation, and Th polarization. Blocking both cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and CD28 interactions with a CTLA-4/Ig fusion protein inhibits various immune-mediated processes in vivo, such as allograft rejection and autoimmunity. However, selective blockade of CD28 may represent a better strategy for immunosuppression than B7 blockade, because CTLA-4/B7 interactions have been shown to participate in the extinction of the T-cell receptor–mediated activation signal and to be required for the induction of immunologic tolerance. In addition, selective CD28 inhibition specifically decreases the activation of alloreactive and autoreactive T cells, but not the activation of T cells stimulated by exogenous antigens presented in the context of self major histocompatibility complex (MHC) molecules. CD28 blockade cannot be obtained with anti-CD28 dimeric antibodies, which cluster their target and promote T-cell costimulation, whereas monovalent Fab fragments can block CD28 and reduce alloreactivity. In this study, we report the construction of a monovalent single-chain Fv antibody fragment from a high-affinity antihuman CD28 antibody (CD28.3) that blocked adhesion of T cells to cells expressing the CD28 receptor CD80. Genetic fusion with the long-lived serum protein α1-antitrypsin led to an extended half-life without altering its binding characteristics. The anti-CD28 fusion molecule showed biologic activity as an immuno-suppressant by inhibiting T-cell activation and proliferation in a mixed lymphocyte reaction.

scholarly journals MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation

2011 ◽  
Vol 208 (5) ◽  
pp. 1041-1053 ◽  
Author(s):  
Sean O. Ryan ◽  
Jason A. Bonomo ◽  
Fan Zhao ◽  
Brian A. Cobb
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bacteroides Fragilis ◽  
Carbohydrate Antigen ◽  
Activated T Cells ◽  
Mhc Molecules

N-linked glycans are thought to protect class II major histocompatibility complex (MHC) molecules (MHCII) from proteolytic cleavage and assist in arranging proteins within the immune synapse, but were not thought to directly participate in antigen presentation. Here, we report that antigen-presenting cells (APCs) lacking native complex N-glycans showed reduced MHCII binding and presentation of the T cell activating glycoantigen (GlyAg) polysaccharide A from Bacteroides fragilis but not conventional peptides. APCs lacking native N-glycans also failed to mediate GlyAg-driven T cell activation but activated T cells normally with protein antigen. Mice treated with the mannosidase inhibitor kifunensine to prevent the formation of complex N-glycans were unable to expand GlyAg-specific T cells in vivo upon immunization, yet adoptive transfer of normally glycosylated APCs into these animals overcame this defect. Our findings reveal that MHCII N-glycosylation directly impacts binding and presentation of at least one class of T cell–dependent antigen.

scholarly journals P07.01 A modular and controllable T cell therapy platform for AML

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A22.2-A23
Author(s):  
M Benmebarek ◽  
B Loureiro Cadilha ◽  
M Herrmann ◽  
S Schmitt ◽  
S Lesch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Activation ◽  
Tumour Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). The mutational landscape, heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are equipped with synthetic agonistic receptors (SARs) that are selectively activated only in the presence of a target AML-associated antigen, and a cross-linking tandem single chain variable fragment (taFv) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific taFvs that target AML-associated antigens were designed and expressed in Expi293FTM cells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigens CD33 and CD123.ResultsAnti-CD33-EGFRvIII and anti-CD123 EGFRvIII taFv, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by taFv could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR-taFv combination could also mediate specific cytotoxicity against patient-derived AML blasts and leukemic stem cells whilst driving SAR T cell activation. In vivo, treatment with SAR-taFv combination could efficiently eradicate leukemia and enhance survival in an AML xenograft models. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility and modularity of said activation upon depletion of the T cell engaging molecule, both in vitro and in vivo.ConclusionsHere we apply the SAR-taFv platform in efforts to deliver specific and conditional activation of SAR-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its taFv facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B. Loureiro Cadilha: None. M. Herrmann: None. S. Schmitt: None. S. Lesch: None. S. Stoiber: None. A. Darwich: None. C. Augsberger: None. B. Brauchle: None. M. Schwerdtfeger: None. A. Gottschlich: None. A. Gottschlich Rataj: None. N.C. Fenn: None. C. Klein: None. M. Subklewe: None. S. Endres: None. K. Hopfner: None. S. Kobold: None.

scholarly journals Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Janett Göhring ◽  
Florian Kellner ◽  
Lukas Schrangl ◽  
René Platzer ◽  
Enrico Klotzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Antibody Fragment ◽  
Temporal Analysis ◽  
Single Chain ◽  
Single Chain Antibody

AbstractMechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

scholarly journals Temporal Analysis of T-Cell Receptor-Imposed Forces via Quantitative Single Molecule FRET Measurements

2020 ◽  
Author(s):  
Janett Göhring ◽  
Florian Kellner ◽  
Lukas Schrangl ◽  
René Platzer ◽  
Enrico Klotzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Antibody Fragment ◽  
Antigen Recognition ◽  
Single Chain ◽  
Single Chain Antibody

ABSTRACTMechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped with a TCR-reactive single chain antibody fragment, which was tethered to planar supported lipid bilayers (SLBs) and informs most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid SLBs instead, T-cells still exerted tensile forces yet with threefold reduced magnitude and only prior to but not upon activation. Our findings do not only provide first truly molecular information on TCR-imposed forces within the immunological synapse, they also recalibrate their significance in antigen recognition.

scholarly journals Bifunctional iRGD-anti-CD3 enhances antitumor potency of T cells by facilitating tumor infiltration and T-cell activation

2021 ◽  
Vol 9 (5) ◽  
pp. e001925
Author(s):  
Shujuan Zhou ◽  
Fanyan Meng ◽  
Shiyao Du ◽  
Hanqing Qian ◽  
Naiqing Ding ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Confocal Microscope ◽  
Mechanistic Studies ◽  
Tumor Spheroids ◽  
Antitumor Effects ◽  
Transport Pathway

BackgroundPoor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.MethodsT-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.ResultsWe generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.ConclusionAltogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.

NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Katia Urso ◽  
Arantzazu Alfranca ◽  
Sara Martínez-Martínez ◽  
Amelia Escolano ◽  
Inmaculada Ortega ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Gene Knockout ◽  
Activated T Cells ◽  
Knock Down ◽  
And Function

Abstract The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.

scholarly journals TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

scholarly journals 702 TJ-CD4B (ABL111), a Claudin18.2-targeted 4-1BB tumor engager induces potent tumor-dependent immune response without dose-limiting toxicity in preclinical studies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A730-A730
Author(s):  
Wenqing Jiang ◽  
Zhengyi Wang ◽  
Zhen Sheng ◽  
Jaeho Jung ◽  
Taylor Guo
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Gene Expression Analysis ◽  
Cell Activation ◽  
Clinical Development ◽  
Cynomolgus Monkeys ◽  
Anti Tumor Activity

Background4-1BB (CD137) is a co-stimulatory receptor that stimulates the function of multiple immune cells. Its ability to induce potent anti-tumor activity makes 4-1BB an attractive target for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic toxicities, we have developed a novel Claudin18.2 (CLDN18.2) x 4-1BB bispecific antibody, TJ-CD4B (ABL111) that stimulates 4-1BB pathway only when it engages with Claudin 18.2, a tumor-associated antigen specifically expressed in gastrointestinal cancers. TJ-CD4B (ABL111) is now being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04900818).MethodsTJ-CD4B (ABL111) was evaluated in vivo using the human 4-1BB knock-in mice bearing CLDN18.2 expressing MC38 tumor cells. Pharmacodynamic effects upon treatment were characterized in tumor tissue and blood. Immunophenotyping of the tumor microenvironment (TME) and peripheral blood was performed by flow cytometry. Soluble biomarkers were measured using Luminex-based multiplex assay. In-depth gene expression analysis was performed on primary human CD8+ T cells that were co-cultured with CLDN18.2 expressing cells in the presence of anti-CD3 using NanoString nCounter®. Pharmacokinetic (PK) and toxicity study were performed in cynomolgus monkeys.ResultsTJ-CD4B (ABL111) elicited complete tumor regression in 13 out of 18 MC38 tumor bearing mice given at a dose above 2 mg/kg. Dose-dependent anti-tumor activity was associated with enhanced T cell activation in TME and expansion of memory T cells in the peripheral blood. Increased CD8+ T cells number and proliferation were observed in both tumor nest and surrounding stroma while the level of soluble 4-1BB in the serum was also elevated in response to the treatment. In vitro gene expression analysis by Nanostring revealed TJ-CD4B(ABL111) effectively activated immune pathways characterized by IFN?-signaling and T cell inflammation. Preclinically, TJ-CD4B was well tolerated at the repeated doses up to 100 mg/kg/wk in cynomolgus monkeys without the adverse influence on the liver function which is generally affected by 4-1BB activation. Besides, no cytokine release or immune activation was observed in the periphery.ConclusionsTJ-CD4B (ABL111) is a novel CLDN18.2 dependent 4-1BB bispecific agonist antibody that induced T cell activation and memory response in tumor with CLDN18.2 expression, leading to a strong anti-tumor activity in vivo. TJ-CD4B did not induce systemic immune response nor hepatic toxicity due to the CLDN18.2 dependent 4-1BB stimulation. These data warrant the current clinical development in phase I trial to validate the safety properties and tumor specific responses.

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 Bispecific T-Cell Engagers Targeting Membrane-Bound IgE

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1568
Author(s):  
Aleksandra Rodak ◽  
Gerhard Stadlmayr ◽  
Katharina Stadlbauer ◽  
Dominic Lichtscheidl ◽  
Madhusudhan Reddy Bobbili ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Single Chain ◽  
Ige Antibodies ◽  
Serum Ige ◽  
Single Chain Fv ◽  
Membrane Bound

The increased incidence of allergies and asthma has sparked interest in IgE, the central player in the allergic response. Interaction with its high-affinity receptor FcεRI leads to sensitization and allergen presentation, extracellular membrane-proximal domain in membrane IgE can act as an antigen receptor on B cells, and the interaction with low-affinity IgE receptor CD23 additionally influences its homeostatic range. Therapeutic anti-IgE antibodies act by the inhibition of IgE functions by interfering with its receptor binding or by the obliteration of IgE-B cells, causing a reduction of serum IgE levels. Fusion proteins of antibody fragments that can act as bispecific T-cell engagers have proven very potent in eliciting cytotoxic T-lymphocyte-mediated killing. We have tested five anti-IgE Fc antibodies, recognizing different epitopes on the membrane-expressed IgE, for the ability to elicit specific T-cell activation when expressed as single-chain Fv fragments fused with anti-CD3ε single-chain antibody. All candidates could specifically stain the cell line, expressing the membrane-bound IgE-Fc and bind to CD3-positive Jurkat cells, and the specific activation of engineered CD3-overexpressing Jurkat cells and non-stimulated CD8-positive cells was demonstrated for 8D6- and ligelizumab-based bispecific antibodies. Thus, such anti-IgE antibodies have the potential to be developed into agents that reduce the serum IgE concentration by lowering the numbers of IgE-secreting cells.

Sign in / Sign up

Export Citation Format

Share Document