27743
2022
eng
13
article
1
--
2022-06-13
--
A TNFR2-Specific TNF fusion protein with improved in vivo activity
Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.
Methods
Single-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.
Results
STAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.
Conclusions
NewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.
Frontiers in Immunology
1664-3224
10.3389/fimmu.2022.888274
urn:nbn:de:bvb:20-opus-277436
2022-06-27T08:45:26+00:00
sword
swordwue
attachment; filename=deposit.zip
d3e003882d5028e773bf3d4c1f815bdb
Frontiers in Immunology (2022) 13:888274. DOI:10.3389/fimmu.2022.888274
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Juan Gamboa Vargas
Jennifer Wagner
Haroon Shaikh
Isabell Lang
Juliane Medler
Mohamed Anany
Tim Steinfatt
Josefina Peña Mosca
Stephanie Haack
Julia Dahlhoff
Maike Büttner-Herold
Carolin Graf
Estibaliz Arellano Viera
Hermann Einsele
Harald Wajant
Andreas Beilhack
eng
uncontrolled
agonist
eng
uncontrolled
GvHD
eng
uncontrolled
regulatory T cells
eng
uncontrolled
serum retention
eng
uncontrolled
TNF
eng
uncontrolled
TNFR2
Medizin und Gesundheit
open_access
Graduate School of Life Sciences
Abteilung für Molekulare Innere Medizin (in der Medizinischen Klinik und Poliklinik II)
Medizinische Klinik und Poliklinik II
Import
Förderzeitraum 2022
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/27743/DataSheet_1.pdf
https://opus.bibliothek.uni-wuerzburg.de/files/27743/fimmu-13-888274.pdf
17672
2018
eng
1060
9
article
1
2019-02-15
--
--
CD28 costimulation of T helper 1 cells enhances cytokine release in vivo
Compared to naive T cells, differentiated T cells are thought to be less dependent on CD28 costimulation for full activation. To revisit the role of CD28 costimulation in mouse T cell recall responses, we adoptively transferred in vitro generated OT-II T helper (Th) 1 cells into C57BL/6 mice (Thy1.2\(^{+}\)) and then either blocked CD28–ligand interactions with Fab fragments of the anti-CD28 monoclonal antibody (mAb) E18 or deleted CD28 expression using inducible CD28 knock-out OT-II mice as T cell donors. After injection of ovalbumin protein in adjuvant into the recipient mice we observed that systemic interferon (IFN)γ release strongly depended on CD28 costimulation of the Th1 cells, while secondary clonal expansion was not reduced in the absence of CD28 costimulation. For human memory CD4\(^{+}\) T cell responses we also noted that cytokine release was reduced upon inhibition of CD28 costimulation. Together, our data highlight the so far underestimated role of CD28 costimulation for the reactivation of fully differentiated CD4\(^{+}\) T cells.
Frontiers in Immunology
10.3389/fimmu.2018.01060
urn:nbn:de:bvb:20-opus-176726
Frontiers in Immunology 2018, Volume 9, Article 1060. DOI: 10.3389/fimmu.2018.01060
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Daniela Langenhorst
Stephanie Haack
Selina Göb
Anna Uri
Fred Lühder
Bernhard Vanhove
Thomas Hünig
Niklas Beyersdorf
eng
uncontrolled
CD4\(^{+}\) T helper cells
eng
uncontrolled
T helper 1 cells
eng
uncontrolled
antigenic recall
eng
uncontrolled
CD28 costimulation
eng
uncontrolled
cytokine secretion
eng
uncontrolled
mouse
eng
uncontrolled
human
Medizin und Gesundheit
open_access
Institut für Virologie und Immunbiologie
Förderzeitraum 2018
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17672/Langenhorst_Frontiers_in_Immunology.pdf
23902
2021
eng
738
741
3
51
article
1
--
--
--
Superagonistic CD28 stimulation induces IFN‐γ release from mouse T helper 1 cells in vitro and in vivo
Like human Th1 cells, mouse Th1 cells also secrete IFN‐γ upon stimulation with a superagonistic anti‐CD28 monoclonal antibody (CD28‐SA). Crosslinking of the CD28‐SA via FcR and CD40‐CD40L interactions greatly increased IFN‐γ release. Our data stress the utility of the mouse as a model organism for immune responses in humans.
European Journal of Immunology
10.1002/eji.202048803
urn:nbn:de:bvb:20-opus-239028
2021-06-01T16:19:00+00:00
sword
swordwue
attachment; filename=deposit.zip
b6fa53f479c7e459b0705d556e8d6954
European Journal of Immunology 2021, 51(3):738–741. DOI: 10.1002/eji.202048803
false
true
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Stephanie Haack
Sarah Baiker
Jan Schlegel
Markus Sauer
Tim Sparwasser
Daniela Langenhorst
Niklas Beyersdorf
eng
uncontrolled
CD28
eng
uncontrolled
Th1 cells
eng
uncontrolled
cytokine release
eng
uncontrolled
interferon γ
eng
uncontrolled
Superagonistic antibody
Medizin und Gesundheit
open_access
Institut für Virologie und Immunbiologie
Theodor-Boveri-Institut für Biowissenschaften
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/23902/EJI_EJI4933.pdf
23775
2021
eng
doctoralthesis
1
2021-05-11
--
2021-04-14
A novel mouse model for systemic cytokine release upon treatment with a superagonistic anti-CD28 antibody
Ein neues Mausmodell zur Untersuchung der Zytokinfreisetzung nach Behandlung mit einem superagonistischen anti-CD28 Antikörper
The adaptive immune system is known to provide highly specific and effective immunity against a broad variety of pathogens due to different effector cells. The most prominent are CD4+ T-cells which differentiate after activation into distinct subsets of effector and memory cells, amongst others T helper 1 (Th1) cells. We have recently shown that mouse as well as human Th1 cells depend on T cell receptor (TCR) signals concomitant with CD28 costimulation in order to secrete interferon (IFN) which is considered as their main effector function. Moreover, there is a class of anti-CD28 monoclonal antibodies that is able to induce T cell (re-)activation without concomitant TCR ligation. These so-called CD28-superagonists (CD28-SA) have been shown to preferentially activate and expand CD4+ Foxp3+ regulatory T (Treg) cells and thereby efficaciously conferring protection e.g. against autoimmune responses in rodents and non-human primates. Considering this beneficial effect, CD28-SA were thought to be of great impact for immunotherapeutic approaches and a humanized CD28-SA was subjected to clinical testing starting with a first-in-man trial in London in 2006. Unexpectedly, the volunteers experienced life-threatening side effects due to a cytokine release syndrome (CRS) that was unpredicted by the preclinical studies prior to the trial. Retrospectively, CD4+ memory T cells within the tissues were identified as source of pro-inflammatory cytokines released upon CD28-SA administration. This was not predicted by the preclinical testing indicating a need for more reliable and predictive animal models. Whether mouse CD4+ T cells are generally irresponsive to CD28-SA stimulation or rather the lack of a bona fide memory T cell compartment in cleanly housed specific-pathogen-free (SPF) mice is the reason why the rodent models failed to predict the risk for a CRS remained unclear. To provide SPF mice with a true pool of memory/effector T cells, we transferred in vitro differentiated TCR-transgenic OT-II Th1 cells into untreated recipient mice. Given that Treg cells suppress T cell activation after CD28- SA injection in vivo, recipients were either Treg-competent or Treg-deficient, wild type or DEREG mice, respectively. Subsequent CD28-SA administration resulted in induction of systemic pro-inflammatory cytokine release, dominated by IFN, that was observed to be much more pronounced and robust in Treg-deficient recipients. Employing a newly established in vitro system mirroring the in vivo responses to CD28-SA stimulation of Th1 cells revealed that antigen-presenting cells (APCs) amplify CD28-SAinduced IFN release by Th1 cells due to CD40/CD40L-interactions. Thus, these data are the first to show that mouse Th1 cells are indeed sensitive to CD28-SA stimulation in vivo and in vitro responding with strong IFN release accompanied by secretion of further pro-inflammatory cytokines, which is compatible with a CRS. In conclusion, this study will facilitate preclinical testing of immunomodulatory agents providing a mouse model constituting more “human-like” conditions allowing a higher degree of reliability and translationability.
Das adaptive Immunsystem ermöglicht mittels hocheffektiver, antigen-spezifischer Mechanismen und unterschiedlicher Effektorzellen den Schutz vor einer nahezu unbegrenzten Vielfalt von Pathogenen. Die Hauptakteure stellen hierbei CD4+ T-Zellen dar, welche nach Aktivierung distinkte Effektorpopulationen, unter anderem Th1 Zellen, bilden. Wir zeigten kürzlich, dass sowohl für Maus- als auch humane Th1-Zellen CD28-Kostimulation mit zeitgleicher T-Zellrezeptor (TZR)-Aktivierung essentiell für die Sekretion von Interferon (IFN), deren Haupteffektorfunktion, ist. Allerdings sind monoklonale anti-CD28 Anti-körper bekannt, die auch ohne TZR-Signal T-Zellen aktivieren können. Diese sogenannten CD28 Supera-gonisten (CD28-SA) aktivieren und expandieren vorrangig CD4+ Foxp3+ regulatorische T-Zellen (Treg) und vermitteln wirksamen Schutz vor z.B. Autoimmunreaktionen in Nagern und Primaten. Um diesen erfolgversprechenden Effekt für immuntherapeutische Ansätze nutzen zu können, wurde 2006 in Lon-don eine erste klinische Erprobung eines humanisierten CD28-SA begonnen. Unerwarteterweise zeigten sich bei den Probanden lebensbedrohliche Nebenwirkungen, die Ausdruck eines Zytokin-Ausschüttungs-Syndroms (Cytokine Release Syndrome, CRS) waren, welches durch die vorangegangenen präklinischen Studien nicht vorhersagbar war. Rückblickend konnte die Sekretion pro-inflammatorischer Zytokine auf CD4+ Gedächtnis-T-Zellen im Gewebe zurückgeführt werden, die so auf die Gabe des CD28-SA reagier-ten. Die unvorhersehbare Reaktion im Menschen zeigt deutlich, dass verlässlichere und prädiktivere Tiermodelle unverzichtbar sind. Ob Maus CD4+-T-Zellen möglicherweise nicht durch CD28-SA stimulier-bar sind oder dieser fehlgeleiteten Einschätzung über das mögliche Risiko eines CRS eher das Fehlen eines echten CD4+ Gedächtnis-T-Zellen-Kompartiments in sauber gehaltenen spezifischen-Pathogen-freien (SPF) Mäusen zugrunde liegt, ist bisher ungeklärt. Um in SPF-Mäusen ein Gedächtnis-T-Zell-Kompartiment zu etablieren, wurden in vitro-differenzierte Th1 Zellen, die TZR-transgenen OT-II-Mäusen entstammen, in unbehandelte Empfängermäuse transferiert. Da bekannt ist, dass Treg-Zellen die Aktivierung von T-Zellen nach Anwendung von CD28-SA in vivo supprimieren, wurden Treg-kompetente (wildtypische) oder -defiziente (DEREG) Empfänger verwendet. Die anschließend erfolgte Injektion von CD28-SA löste die systemische Sekretion pro-inflammatorischer Zytokine aus, wobei eine stark erhöhter IFN-Konzentration im Serum zu beobachten war, welche deutlich ausgeprägter und robuster bei den Treg-defizienten Empfängern ausfiel. Ein neu etabliertes in vitro-System, welches die in vivo Antwort der Th1-Zellen auf CD28-SA-Stimulation widerspiegelt, identifizierte Antigen-präsentierende Zellen (APZs) als essentiellen Faktor für die erhöhte IFN-Sekretion der Th1-Zellen nach CD28-SA-Stimulation in Abhängigkeit von CD40/CD40L-Interaktionen. Zusammenfassend zeigt diese Thesis zum ersten Mal, dass Maus Th1 Zellen sowohl in vivo als auch in vitro durch CD28 SA stimulierbar sind, wodurch eine starke IFN-Sekretion induziert wird, die von der gesteigerten Ausschüttung anderer pro-inflammatorischer Zytokine begleitet wird und in Abwesenheit von Treg einem CRS gleicht. Folglich kann diese Erkenntnis die präklinische Forschung bei der Erprobung neuer immuntherapeutischer Ansät-ze durch ein neues Mausmodell voranbringen, das dem menschlichen erfahreneren Immunsystem mehr als bisherige Modelle entspricht und somit verlässlichere Vorhersagen erlaubt und eine verbesserte Übertragbarkeit von Maus zu Mensch ermöglicht.
urn:nbn:de:bvb:20-opus-237757
10.25972/OPUS-23775
publish
X 129408
CC BY-SA: Creative-Commons-Lizenz: Namensnennung, Weitergabe unter gleichen Bedingungen 4.0 International
Stephanie Haack
deu
swd
CD28
eng
uncontrolled
CD28-SA
eng
uncontrolled
cytokine release syndrome
Medizin und Gesundheit
open_access
Graduate School of Life Sciences
Institut für Virologie und Immunbiologie
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/23775/Haack_Stephanie_CD28-SA.pdf
16574
2016
eng
2060
12
17
article
1
2018-07-26
--
--
Toll Like Receptor 2, 4, and 9 Signaling Promotes Autoregulative Tumor Cell Growth and VEGF/PDGF Expression in Human Pancreatic Cancer
Toll like receptor (TLR) signaling has been suggested to play an important role in the inflammatory microenvironment of solid tumors and through this inflammation-mediated tumor growth. Here, we studied the role of tumor cells in their process of self-maintaining TLR expression independent of inflammatory cells and cytokine milieu for autoregulative tumor growth signaling in pancreatic cancer. We analyzed the expression of TLR2, -4, and -9 in primary human cancers and their impact on tumor growth via induced activation in several established pancreatic cancers. TLR-stimulated pancreatic cancer cells were specifically investigated for activated signaling pathways of VEGF/PDGF and anti-apoptotic Bcl-xL expression as well as tumor cell growth. The primary pancreatic cancers and cell lines expressed TLR2, -4, and -9. TLR-specific stimulation resulted in activated MAP-kinase signaling, most likely via autoregulative stimulation of demonstrated TLR-induced VEGF and PDGF expression. Moreover, TLR activation prompted the expression of Bcl-xL and has been demonstrated for the first time to induce tumor cell proliferation in pancreatic cancer. These findings strongly suggest that pancreatic cancer cells use specific Toll like receptor signaling to promote tumor cell proliferation and emphasize the particular role of TLR2, -4, and -9 in this autoregulative process of tumor cell activation and proliferation in pancreatic cancer.
International Journal of Molecular Sciences
10.3390/ijms17122060
urn:nbn:de:bvb:20-opus-165743
International Journal of Molecular Sciences 2016, 17, 2060; doi:10.3390/ijms17122060
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Tanja Grimmig
Romana Moench
Jennifer Kreckel
Stephanie Haack
Felix Rueckert
Roberta Rehder
Sudipta Tripathi
Carmen Ribas
Anil Chandraker
Christoph T. Germer
Martin Gasser
Ana Maria Waaga-Gasser
eng
uncontrolled
tumor growth
eng
uncontrolled
TLR2
eng
uncontrolled
TLR4
eng
uncontrolled
TLR9
eng
uncontrolled
pancreatic cancer
eng
uncontrolled
inflammation
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Allgemein-, Viszeral-, Gefäß- und Kinderchirurgie (Chirurgische Klinik I)
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/16574/Grimmig_IJMS_2016.pdf