scholarly journals Immunotherapeutic Targeting of Mesothelin Positive Pediatric AML Using Bispecific T Cell Engaging Antibodies

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 5964
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Colin E. Correnti ◽  
Kristina Pilat ◽  
Ida Lin ◽  
Man Kid Chan ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Bone Marrow Cells ◽  
Intensive Therapy ◽  
Specific Antigen ◽  
Single Chain ◽  
Variable Regions ◽  
Pediatric Aml

Advances in the treatment of pediatric AML have been modest over the past four decades. Despite maximally intensive therapy, approximately 40% of patients will relapse. Novel targeted therapies are needed to improve outcomes. We identified mesothelin (MSLN), a well-validated target overexpressed in some adult malignancies, to be highly expressed on the leukemic cell surface in a subset of pediatric AML patients. The lack of expression on normal bone marrow cells makes MSLN a viable target for immunotherapies such as T-cell engaging bispecific antibodies (BsAbs) that combine two distinct antibody-variable regions into a single molecule targeting a cancer-specific antigen and the T-cell co-receptor CD3. Using antibody single-chain variable region (scFv) sequences derived from amatuximab-recognizing MSLN, and from either blinatumomab or AMG330 targeting CD3, we engineered and expressed two MSLN/CD3-targeting BsAbs: MSLNAMA-CD3L2K and MSLNAMA-CD3AMG, respectively. Both BsAbs promoted T-cell activation and reduced leukemic burden in MV4;11:MSLN xenografted mice, but not in those transplanted with MSLN-negative parental MV4;11 cells. MSLNAMA-CD3AMG induced complete remission in NTPL-146 and DF-5 patient-derived xenograft models. These data validate the in vivo efficacy and specificity of MSLN-targeting BsAbs. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients.

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 A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadine Aschmoneit ◽  
Sophia Steinlein ◽  
Lennart Kühl ◽  
Oliver Seifert ◽  
Roland E. Kontermann
Keyword(s):  
T Cell ◽  
Binding Site ◽  
Cancer Cell ◽  
Cancer Progression ◽  
T Cell Activation ◽  
Cell Activation ◽  
Egf Receptor ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Single Chain

AbstractHER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation. Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.

scholarly journals Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

2016 ◽  
Vol 27 (22) ◽  
pp. 3627-3636 ◽  
Author(s):  
Sophie V. Pageon ◽  
Philip R. Nicovich ◽  
Mahdie Mollazade ◽  
Thibault Tabarin ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Cell Receptor ◽  
Intact Cells ◽  
Analysis Strategy ◽  
Signaling Activity

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

2005 ◽  
Vol 55 (5) ◽  
pp. 503-514 ◽  
Author(s):  
Bernd Schlereth ◽  
Cornelia Quadt ◽  
Torsten Dreier ◽  
Peter Kufer ◽  
Grit Lorenczewski ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Single Chain ◽  
Single Chain Antibody

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.

scholarly journals T Cell Activation Machinery: Form and Function in Natural and Engineered Immune Receptors

2020 ◽  
Vol 21 (19) ◽  
pp. 7424
Author(s):  
Nicholas J. Chandler ◽  
Melissa J. Call ◽  
Matthew E. Call
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immune Cell ◽  
Single Chain ◽  
Design Work ◽  
Cell Therapies ◽  
Form And Function ◽  
Car T ◽  
And Function

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many different disease contexts. A rapidly growing body of design work is examining every modular component of traditional single-chain CARs as well as expanding out into many new and innovative engineered immunoreceptor designs that depart from this template. New approaches to immune cell and receptor engineering are being reported with rapidly increasing frequency, and many recent high-quality reviews (including one in this special issue) provide comprehensive coverage of the history and current state of the art in CAR-T and related cellular immunotherapies. In this review, we step back to examine our current understanding of the structure-function relationships in natural and engineered lymphocyte-activating receptors, with an eye towards evaluating how well the current-generation CAR designs recapitulate the most desirable features of their natural counterparts. We identify key areas that we believe are under-studied and therefore represent opportunities to further improve our grasp of form and function in natural and engineered receptors and to rationally design better therapeutics.

scholarly journals Induction of c-ets and c-fos gene expression upon antigenic stimulation of a T cell hybridoma with inducible cytolytic capacity.

1987 ◽  
Vol 166 (3) ◽  
pp. 810-815 ◽  
Author(s):  
Y Kaufmann ◽  
T Silverman ◽  
B Z Levi ◽  
K Ozato
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Specific Antigen ◽  
Functional Differentiation ◽  
Mrna Levels ◽  
Cellular Oncogenes ◽  
Differentiation Of Lymphocytes ◽  
Stimulation Of

Expression of cellular oncogenes was studied in a T cell hybridoma that undergoes cytolytic activation when stimulated by specific antigen or by anti-Thy-1 antibody. The activation occurs without induction of hybridoma proliferation, providing a model to examine oncogene expression during functional differentiation of lymphocytes. We found that c-fos and c-ets-1 mRNAs were transiently induced at high levels in the hybridoma 30 min and 4 h after stimulation, respectively. c-myc and c-ets-2 oncogenes were constitutively expressed in the hybridoma and their mRNA levels were unaffected during 4 h of stimulation, although c-myc expression was reduced in the later stage of stimulation. Inhibitors of T cell activation, cyclosporin A and anti-LFA-1 antibody, blocked the induction of c-fos and c-ets-1 mRNAs without reducing the levels of c-myc and c-ets-2. The results indicate that the functional activation of the CTL hybridoma is associated with induction of c-fos and c-ets-1 genes.

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