Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

Fellermeier, Sina; Beha, Nadine; Meyer, Jan-Erik; Ring, Sarah; Bäder, S.; Kontermann, Roland E.; Müller, Dafne

doi:10.1080/2162402x.2016.1238540

Cited by 33 publications

(49 citation statements)

References 37 publications

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“…(GGGGS) 4 if involving 4-1BBL as one partner and (GGGGS) 3 in the other cases. Furthermore, duokines were also generated in a single-chain format (scDuokines) by connecting the single-chain versions of two TNFSF ligands via a 15 amino acid (GGGGS) 3 linker (Figure 1A), 20 thereby creating a bifunctional hexavalent single-chain molecule (Figure 1B). By incorporating two different TNFSF ligands, two modes of action (cis-acting and trans-acting) were realized.…”

Section: Resultsmentioning

confidence: 99%

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Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

et al. 2018

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Co-stimulatory signals induced by ligands of the tumor necrosis factor superfamily (TNFSF) play a central role in T cell activation and have emerged as a promising strategy in cancer immunotherapy. Here, we established a novel class of bifunctional co-stimulatory fusion proteins with the aim to boost T cell activation at the level of T cell – antigen-presenting cell (APC) interaction. These novel dual-acting cytokine fusion proteins were created by connecting two different homotrimeric TNFSF ligands to form homotrimeric bifunctional molecules (Duokines) or by connecting single-chain derivatives of two different homotrimeric TNFSF with a single, flexible linker (single-chain Duokines, scDuokines). By linking the TNFSF ligands 4-1BBL, OX40L and CD27L in all possible combinations, cis-acting Duokines were generated that act on the same or adjacent T cells, while combining CD40L with 4-1BBL, OX40L and CD27L resulted in trans-acting Duokines acting simultaneously on APCs and T cells. In vitro, co-stimulation of T cells was seen for cis- and trans-acting Duokines and scDuokines in an antigen-independent as well as antigen-specific setting. Trans-acting molecules furthermore activated B cells, which represent a subclass of APCs. In a pilot experiment using the syngeneic B16-FAP mouse tumor model scDuokines displayed antitumoral activity in vivo in combination with a primary T cell-activating bispecific antibody, evident from reduced number of lung metastasis compared to the antibody-only treated group. Our data show that the bifunctional, co-stimulatory duokines are capable to enhance T cell-mediated anti-tumor immune responses, suggesting that they can serve as a new class of immuno-stimulatory molecules for use in cancer immunotherapy strategies.

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Section: Resultsmentioning

confidence: 99%

“…in combination with T-cell retargeting bispecific antibodies. 16–20 However, although this approach incorporates a targeted delivery and accumulation in the tumor tissue, activation is restricted to a single receptor type.…”

Section: Introductionmentioning

confidence: 99%

Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

et al. 2018

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“…Our lab has had a long-standing interest in the characterization of the tumor-homing properties 48 and anti-cancer activity of antibody fusion proteins with members of the TNF superfamily 36,49 . F8-41BBL, F8-GITRL and F8-CD154 featured the TNF superfamily member as single-chain polypeptide in order to stabilize the homotrimeric structure 20 while the antibodies were used as recombinant diabody moieties 50 . A concentration-dependent NF-B reporter activity could be measured for all three antibodycytokine fusions, monitoring both luciferase activity and mCherry expression [Figure 3].…”

Section: Resultsmentioning

confidence: 99%

A universal reporter cell line for bioactivity evaluation of engineered cytokine products

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Pellegrino

Neri

2019

Preprint

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Engineered cytokine products represent a growing class of therapeutic proteins which need to be tested for biological activity at various stages of pharmaceutical development. In most cases, dedicated biological assays are established for different products, in a process that can be time-consuming and cumbersome. Here we describe the development and implementation of a universal cell-based reporter system for various classes of immunomodulatory proteins. The novel system capitalizes on the fact that the signaling of various types of pro-inflammatory agents (e.g., cytokines, chemokines, Toll-like receptor agonists) may involve transcriptional activation by NF-B. Using viral transduction, we generated stably-transformed cell lines of B or T lymphocyte origin and compared the new reporter cell lines with conventional bioassays. The experimental findings with various interleukins and with members of the TNF superfamily revealed that the newly-developed "universal" bioassay method yielded bioactivity data which were comparable to the ones obtained with dedicated conventional methods. The engineered cell lines with reporters for NF-B were tested with several antibody-cytokine fusions and may be generally useful for the characterization of novel immunomodulatory products. The newly developed methodology also revealed a mechanism for cytokine potentiation, based on the antibody-mediated clustering of TNF superfamily members on tumor-associated extracellular matrix components.

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“…Figure 1a] 57 , while its murine counterpart forms stable homodimers 58,59 . Stable trimeric structures can be engineered by connecting 4-1BBL monomeric domains with suitable polypeptide linkers 60 . Recombinant antibodies can be expressed as full IgG or as fragments, forming single-chain Fv (scFv) 61,62 or diabody 63 structures [Figure 1b and Supplementary Table 1] 2,64,65 .…”

Section: Introductionmentioning

confidence: 99%

An engineered 4-1BBL fusion protein with “activity-on-demand”

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Stringhini

Villa

et al. 2020

Preprint

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ABSTRACTEngineered cytokines are gaining importance for cancer therapy but those products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein (termed F8-4-1BBL), which does not exhibit cytokine activity in solution but regains biological activity upon antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively-spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent anti-tumor activity in various mouse models of cancer, without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity-on-demand” properties at the site of disease upon antigen binding and reduce toxicity to normal tissues.

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Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

Cited by 33 publications

References 37 publications

Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

A universal reporter cell line for bioactivity evaluation of engineered cytokine products

An engineered 4-1BBL fusion protein with “activity-on-demand”

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