TY - JOUR A1 - Vargas, Juan Gamboa A1 - Wagner, Jennifer A1 - Shaikh, Haroon A1 - Lang, Isabell A1 - Medler, Juliane A1 - Anany, Mohamed A1 - Steinfatt, Tim A1 - Mosca, Josefina Peña A1 - Haack, Stephanie A1 - Dahlhoff, Julia A1 - Büttner-Herold, Maike A1 - Graf, Carolin A1 - Viera, Estibaliz Arellano A1 - Einsele, Hermann A1 - Wajant, Harald A1 - Beilhack, Andreas T1 - A TNFR2-Specific TNF fusion protein with improved in vivo activity JF - Frontiers in Immunology N2 - 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. KW - agonist KW - GvHD KW - regulatory T cells KW - serum retention KW - TNF KW - TNFR2 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-277436 SN - 1664-3224 VL - 13 ER - TY - THES A1 - Aido, Ahmed T1 - Development of anti-TNF antibody-gold nanoparticles (anti-TNF-AuNPs) T1 - Entwicklung von Anti-TNF-Antikörper-Gold-Nanopartikeln N2 - Gold nanoparticles of diameter ca. 60 nm have been synthesized based on Turkevich and Frens protocols. We have demonstrated that the carboxyl-modified gold nanoparticles can be coupled covalently with antibodies (Ab) of interest using the EDC/NHS coupling procedure. Binding studies with Ab-grafted AuNPs and GpL fusion proteins proved that conjugation of AuNPs with antibodies enables immobilization of antibodies with preservation of a significant antigen binding capacity. More importantly, our findings showed that the conjugation of types of anti-TNF receptors antibodies such as anti-Fn14 antibodies (PDL192 and 5B6) (Aido et al., 2021), anti-CD40, anti-4-1BB and anti-TNFR2 with gold nanoparticles confers them with potent agonism. Thus, our results suggest that AuNPs can be utilized as a platform to immobilize anti-TNFR antibodies which, on the one hand, helps to enhance their agonistic activity in comparison to “free” inactive antibodies by mimicking the effect of cell-anchored antibodies or membrane-bound TNF ligands and, on the other hand, allows to develop new generations of drug delivery systems. These constructs are characterized with their biocompatibility and their tunable synthesis process. In a further work part, we combined the benefits of the established system of Ab-AuNPs with materials used widely in the modern biofabrication approaches such as the photo-crosslinked hydrogels, methacrylate-modified gelatin (GelMA), combined with embedded variants of human cell lines. The acquired results demonstrated clearly that the attaching of proteins like antibodies to gold nanoparticles might reduce their release rate from the crosslinked hydrogels upon the very low diffusion of gold nanoparticles from the solid constructs to the surrounding medium yielding long-term local functioning proteins-attached particles. Moreover, our finding suggests that hydrogel-embedded AuNP-immobilized antibodies, e.g. anti-TNFα-AuNPs or anti-IL1-AuNPs enable local inhibitory functions, To sum up, our results demonstrate that AuNPs can act as a platform to attach anti-TNFR antibodies to enhance their agonistic activity by resembling the output of cell-anchoring or membrane bounding. Gold nanoparticles are considered, thus, as promising tool to develop the next generation of drug delivery systems, which may contribute to cancer therapy. On top of that, the embedding of anti-inflammatory-AuNPs in the biofabricated hydrogel presents new innovative strategy of the treatment of autoinflammatory diseases. N2 - Gold-Nanopartikel mit einem Durchmesser von ca. 60 nm wurden auf Basis der Turkevich- und Frens-Protokolle synthetisiert. Bindungsstudien mit Ab-verankerten AuNPs und GpL-Fusionsproteinen haben gezeigt, dass die Konjugation von AuNPs mit Antikörpern die Immobilisierung von Antikörpern mit Erhaltung einer signifikanten Antigenbindungs-Kapazität ermöglicht. Noch wichtiger ist, dass unsere Ergebnisse zeigen, dass die Konjugation von Typen von Antikörpern gegen TNFRs wie anti-Fn14-Antikörper (PDL192 und 5B6), anti-CD40, anti-4-1BB und anti-TNFR2 mit Gold-Nanopartikeln ihnen eine starke agonistische Wirkung verleiht. Unsere Ergebnisse legen nahe, dass AuNPs als Plattform genutzt werden können, um Antikörper gegen TNFR zu immobilisieren, was einerseits dazu beiträgt, ihre agonistische Aktivität im Vergleich zu "freien" inaktiven Antikörpern zu erhöhen, indem sie die Wirkung von zellgebundenen Antikörpern oder membranverankerten TNF-Liganden nachahmen und andererseits die Entwicklung neuer Generationen von Wirkstoffabgabe Systemen ermöglicht. Diese Konstrukte zeichnen sich durch ihre Biokompatibilität und ihren einstellbaren Syntheseprozess aus. In einem weiteren Teil der Arbeit haben wir die Vorteile des etablierten Systems von Ab-AuNPs mit Materialien kombiniert, die in modernen Biofabrikationsansätzen weit verbreitet sind, nämlich Hydrogele, z.b. methacrylatmodifiziertes Gelatine (GelMA), kombiniert mit eingebetteten Varianten von menschlichen Zelllinien. Die erzielten Ergebnisse zeigten deutlich, dass die Anbindung von Proteinen wie Antikörpern an Gold-Nanopartikel ihre Freisetzung aus den vernetzten Hydrogelen reduzieren könnte, da die Diffusion von Gold-Nanopartikeln aus den festen Konstrukten in das umgebende Medium sehr gering ist und so langfristig Konstrukte mit lokalem Proteine load - erzeugt werden können. Darüber hinaus legt unser Befund nahe, dass in das Hydrogel eingebettete AuNP-immobilisierte Antikörper wie Anti-TNFα-AuNPs oder Anti-IL1-AuNPs eine lokal Immunsuppression erlauben. Diese können als vielversprechende Ansätze betrachtet werden, um verschiedene Arten von Autoimmunerkrankungen zu behandeln. Zusammenfassend zeigen unsere Ergebnisse, dass AuNPs als Plattform dienen können, um Anti-TNFR-Antikörper anzubinden und ihre agonistische Aktivität zu erhöhen. Goldnanopartikel werden daher als vielversprechendes Werkzeug zur Entwicklung der nächsten Generation von Wirkstofftransportsystemen angesehen, die zur Krebstherapie beitragen können. Darüber hinaus stellt die Einbettung von entzündungshemmenden-AuNPs in das biofabrizierte Hydrogel eine neue innovative Strategie für die Behandlung von autoinflammatorischen Erkrankungen dar. KW - AuNPs KW - TNF KW - Nanoparticles KW - Antibody KW - Gold Nanoparticles KW - Drug delivery system (DDS) KW - Nanopartikel Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349212 ER -