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.

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 Direct T Cell Activation by Chimeric Single Chain Fv-Syk Promotes Syk-Cbl Association and Cbl Phosphorylation

1997 ◽  
Vol 272 (13) ◽  
pp. 8551-8557 ◽  
Author(s):  
Cheryl J. Fitzer-Attas ◽  
Daniel G. Schindler ◽  
Tova Waks ◽  
Zelig Eshhar
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Single Chain ◽  
Single Chain Fv

RIAM Regulates Actin Reorganization at the Immunological Synapse and Calcium Mobilization during T Cell Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1722-1722
Author(s):  
Esther M. Lafuente ◽  
Mathew Salanga ◽  
Yoshiko Iwamoto ◽  
Lequn Li ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Jurkat Cells ◽  
Jurkat T Cells ◽  
Actin Reorganization ◽  
Cytoskeletal Dynamics

Abstract Engagement of immune recognition subunits on lymphocytes results in cytoskeletal reorganization, polarization of polymerized actin, conjugate formation between T cells and APC, stabilization of the immunological synapse (IS), and initiation of signaling cascades leading to T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. We have identified RIAM, a novel adaptor molecule that interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. RIAM also has a mandatory role in regulating activation of integrins downstream of Rap1. The ability of a T cell to recognize and conjugate with an APC is dependent on cytoskeletal dynamics and “inside out” signaling leading to integrin-mediated adhesion. Activation of the b2 integrin LFA-1 leads to high avidity binding to ICAM proteins, which is crucial for stable T cell-APC conjugation. Because RIAM is involved both in cytoskeletal dynamics and integrin activation, we examined whether RIAM had a role in this process. Stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin and ZAP-70. Knockdown of RIAM in Jurkat T cells resulted in significant defects in conjugate formation with SEE loaded B cells. Thus RIAM is involved in actin reorganization in the IS and in the formation of T cell:APC conjugates. Because appropriate actin reorganization is required for T cell activation we next explored whether RIAM knockdown leads to defects in TCR-mediated signaling events. RIAM-knockdown Jurkat T cells displayed significantly impaired IL-2 production in response to SEE loaded Raji and in decreased IL-2 promoter driven transcriptional activity. Surprisingly, despite the significant decrease in IL-2 transcription, RIAM-knockdown Jurkat cells did not show altered TCR-induced phosphorylation of PLCg1, or activation of MAP kinases including MEK, Erk1/2 and JNK, compared to control cells. Moreover, AP-1 and NF-kB-mediated gene transcription induced by CD3-plus-CD28 was unaffected. In contrast, NFAT-driven transcription was significantly impaired. Assessment of intracellular calcium by flow cytometry indicated that RIAM-knockdown cells had significantly reduced TCR-mediated intracellular calcium signal. These results indicate that RIAM is an important component of a signaling pathway that regulates calcium mobilization and NFAT mediated gene transcription in T lymphocytes.

scholarly journals 702 Dual blockade of the PD-1 checkpoint pathway and the adenosinergic negative feedback signaling pathway with a PD-1/CD73 bispecific antibody for cancer immune therapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A744-A744
Author(s):  
Tingting Zhong ◽  
Zhaoliang Huang ◽  
Xinghua Pang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Negative Feedback ◽  
Immune Suppression ◽  
Specific Antibody ◽  
Cell Activation ◽  
Immune Therapy ◽  
B Cell Activation

BackgroundCD73 (ecto-5’-nucleotidase) is an ecto-nucleotidase that dephosphorylate AMP to form adenosine. Activation of adenosine signaling pathway in immune cells leads to the suppression of effector functions, down-regulate macrophage phagocytosis, inhibit pro-inflammatory cytokine release, as well as yield aberrantly differentiated dendritic cells producing pro-tumorigenic molecules.1 In the tumor microenvironment, adenosinergic negative feedback signaling facilitated immune suppression is considered an important mechanism for immune evasion of cancer cells.2 3 Combination of CD73 and anti-PD-1 antibody has shown promising activity in suppressing tumor growth. Hence, we developed AK119, an anti- human CD73 monoclonal antibody, and AK123,a bi-specific antibody targeting both PD-1 and CD73 for immune therapy of cancer.MethodsAK119 is a humanized antibody against CD73 and AK123 is a tetrameric bi-specific antibody targeting PD-1 and CD73. Binding assays of AK119 and AK123 to antigens, and antigen expressing cells were performed by using ELISA, Fortebio, and FACS assays. In-vitro assays to investigate the activity of AK119 and AK123 to inhibit CD73 enzymatic activity in modified CellTiter-Glo assay, to induce endocytosis of CD73, and to activate B cells were performed. Assay to evaluate AK123 activity on T cell activation were additionally performed. Moreover, the activities of AK119 and AK123 to mediate ADCC, CDC in CD73 expressing cells were also evaluated.ResultsAK119 and AK123 could bind to its respective soluble or membrane antigens expressing on PBMCs, MDA-MB-231, and U87-MG cells with high affinity. Results from cell-based assays indicated that AK119 and AK123 effectively inhibited nucleotidase enzyme activity of CD73, mediated endocytosis of CD73, and induced B cell activation by upregulating CD69 and CD83 expression on B cells, and showed more robust CD73 blocking and B cell activation activities compared to leading clinical candidate targeting CD73. AK123 could also block PD-1/PD-L1 interaction and enhance T cell activation.ConclusionsIn summary, AK119 and AK123 represent good preclinical biological properties, which support its further development as an anti-cancer immunotherapy or treating other diseases.ReferencesDeaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007; 204:1257–65.Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood. 1997; 90:1600–10.Novitskiy SV, Ryzhov S, Zaynagetdinov R, Goldstein AE, Huang Y, Tikhomirov OY, Blackburn MR, Biaggioni I,Carbone DP, Feoktistov I, et al. Adenosine receptors in regulation of dendritic cell differentiation and function. Blood 2008; 112:1822–31.

scholarly journals Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana Colado ◽  
Esteban Enrique Elías ◽  
Valeria Judith Sarapura Martínez ◽  
Gregorio Cordini ◽  
Pablo Morande ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Immunomodulatory Effects ◽  
Immunoglobulin Preparations

AbstractHypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

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.

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 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.

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