CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism

Nelke, Johannes; Medler, Juliane; Weisenberger, Daniela; Beilhack, Andreas; Wajant, Harald

doi:10.1080/19420862.2020.1807721

Cited by 7 publications

(8 citation statements)

References 45 publications

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“…In accordance with the hypotheses that plasma membrane-associated presentation is the major factor determining agonistic activity of conventional αCD40- and α41BB antibodies, we and others recently reported anchoring-dependent agonism of αCD40- and α41BB antibody fusion proteins carrying an anchor domain specific for plasma membrane-exposed tumor marker proteins, such as CD20 12 , BCMA 13 , FAP 23 or mesothelin 24 . In this work, we exploit this mode of action to generate bifunctional αCD40- and α41BB antibody fusion proteins combining two synergistically acting activities, namely i) conditional FcγR-independent agonism and ii) PD1-PDL1 checkpoint inhibition.…”

Section: Discussionsupporting

confidence: 88%

“…This suggests that it is the sheer presentation in cell-bound form that is responsible for the high agonistic activity of FcγR-bound αCD40- and α41BB antibodies. In accordance with this idea, we recently found that antibody fusion proteins harboring an anchoring domain (AD), which recognizes a cell surface-exposed anchoring target (AT), display strong AT-dependent agonism resembling those of the FcγR-bound antibodies 12 , 13 . The much higher agonistic activity of FcγR- and AT-bound antibodies and antibody fusion proteins correspond well with the fact that the natural activators of CD40 and 41BB, the ligands CD40L and 41BBL, are also much more active as membrane-bound molecules than in soluble form.…”

Section: Introductionmentioning

confidence: 68%

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CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

Medler¹,

Kucka²,

Melo³

et al. 2022

Theranostics

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Background: A strategy to broaden the applicability of checkpoint inhibitors is the combined use with antibodies targeting the immune stimulatory receptors CD40 and 41BB. However, the use of anti-CD40 and anti-41BB antibodies as agonists is problematic in two ways. First, anti-CD40 and anti-41BB antibodies need plasma membrane-associated presentation by FcγR binding to exert robust agonism but this obviously limits their immune stimulatory efficacy by triggering ADCC, CDC or anti-inflammatory FcγRIIb activities. Second, off tumor activation of CD40 and 41BB may cause dose limiting systemic inflammation. Methods: To overcome the FcγR-dependency of anti-41BB and anti-CD40 antibodies, we genetically fused such antibodies with a PDL1-specific blocking scFv as anchoring domain to enable FcγR-independent plasma membrane-associated presentation of anti-CD40- and anti-41BB antibodies. By help of GpL-tagged variants of the resulting bispecific antibodies, binding to their molecular targets was evaluated by help of cellular binding studies. Membrane PDL1-restricted engagement of CD40 and 41BB but also inhibition of PDL1-induced PD1 activation were evaluated in coculture assays with PDL1-expressing tumor cell lines and 41BB, CD40 and PD1 responsible cell lines or T-cells. Results: The binding properties of the bispecific antibody fusion proteins remained largely unchanged compared to their parental molecules. Upon anchoring to membrane PDL1, the bispecific antibody fusion proteins activated CD40/41BB signaling as efficient as the parental anti-CD40/anti-41BB antibodies when bound to FcγRs or cells expressing membrane-bound CD40L/41BBL. PD1 inhibition remained intact and the anti-41BB fusion protein thus showed PDL1-restricted costimulation of T-cells activated in vitro with anti-CD3 or a BiTe. Conclusions: Targeting of anti-CD40 and anti-41BB fusion proteins to membrane PDL1 with a blocking PDL1 scFv links PD1-PDL1 checkpoint blockade intrinsically with engagement of CD40 or 41BB.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 68%

CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

Medler¹,

Kucka²,

Melo³

et al. 2022

Theranostics

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“…Plasma membrane binding-dependent agonism has, for example, been demonstrated by different groups for anti-TRAILR2 antibody fusion proteins with the ability to anchor to the plasma membrane with the help of a second antibody domain recognizing the cell surface antigens FAP, MCSP, and FolR1 ( Brunker et al, 2016 ; He et al, 2016 ; Shivange et al, 2018 ). Similarly, a 50- to >1,000-fold plasma membrane anchoring-dependent increase in their TNFR-stimulating potential has also been reported for various antibody fusion proteins targeting the category II TNFRs 4-1BB, CD27, CD40, CD95, Fn14, and TNFR2 ( Medler et al, 2019 ; Nelke et al, 2020 ).…”

Section: Tnf Receptor Activation Requirements: Consequences For the Dmentioning

confidence: 52%

Receptor Oligomerization and Its Relevance for Signaling by Receptors of the Tumor Necrosis Factor Receptor Superfamily

Kucka

Wajant

2021

Front. Cell Dev. Biol.

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With the exception of a few signaling incompetent decoy receptors, the receptors of the tumor necrosis factor receptor superfamily (TNFRSF) are signaling competent and engage in signaling pathways resulting in inflammation, proliferation, differentiation, and cell migration and also in cell death induction. TNFRSF receptors (TNFRs) become activated by ligands of the TNF superfamily (TNFSF). TNFSF ligands (TNFLs) occur as trimeric type II transmembrane proteins but often also as soluble ligand trimers released from the membrane-bound form by proteolysis. The signaling competent TNFRs are efficiently activated by the membrane-bound TNFLs. The latter recruit three TNFR molecules, but there is growing evidence that this is not sufficient to trigger all aspects of TNFR signaling; rather, the formed trimeric TNFL–TNFR complexes have to cluster secondarily in the cell-to-cell contact zone for full TNFR activation. With respect to their response to soluble ligand trimers, the signaling competent TNFRs can be subdivided into two groups. TNFRs of one group, designated as category I TNFRs, are robustly activated by soluble ligand trimers. The receptors of a second group (category II TNFRs), however, failed to become properly activated by soluble ligand trimers despite high affinity binding. The limited responsiveness of category II TNFRs to soluble TNFLs can be overcome by physical linkage of two or more soluble ligand trimers or, alternatively, by anchoring the soluble ligand molecules to the cell surface or extracellular matrix. This suggests that category II TNFRs have a limited ability to promote clustering of trimeric TNFL–TNFR complexes outside the context of cell–cell contacts. In this review, we will focus on three aspects on the relevance of receptor oligomerization for TNFR signaling: (i) the structural factors which promote clustering of free and liganded TNFRs, (ii) the signaling pathway specificity of the receptor oligomerization requirement, and (iii) the consequences for the design and development of TNFR agonists.

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“…In the past 10 years, extensive in vitro and in vivo studies have given comprehensive evidence that virtually every CD40-specific antibody elicits agonistic activity when bound to Fcγ receptors [ 68 , 123 , 127 , 128 ]. It is worth mentioning that the agonism of FcγR-interacting anti-CD40 antibodies is independent of FcγR downstream signaling [ 123 ] and can also be realized with FcγR-transfected non-immune cells e.g., [ 72 , 128 , 129 ]. Therefore, the sheer plasma membrane-associated mode of presentation of anti-CD40 antibody molecules appears to be sufficient to constitute the agonism of anti-CD40 antibody-FcγR complexes.…”

Section: Cd40 As Therapeutic Targetmentioning

confidence: 99%

FcγRs and Their Relevance for the Activity of Anti-CD40 Antibodies

Lang

Zaitseva

Wajant

2022

IJMS

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Inhibitory targeting of the CD40L-CD40 system is a promising therapeutic option in the field of organ transplantation and is also attractive in the treatment of autoimmune diseases. After early complex results with neutralizing CD40L antibodies, it turned out that lack of Fcγ receptor (FcγR)-binding is the crucial factor for the development of safe inhibitory antibodies targeting CD40L or CD40. Indeed, in recent years, blocking CD40 antibodies not interacting with FcγRs, has proven to be well tolerated in clinical studies and has shown initial clinical efficacy. Stimulation of CD40 is also of considerable therapeutic interest, especially in cancer immunotherapy. CD40 can be robustly activated by genetically engineered variants of soluble CD40L but also by anti-CD40 antibodies. However, the development of CD40L-based agonists is biotechnologically and pharmacokinetically challenging, and anti-CD40 antibodies typically display only strong agonism in complex with FcγRs or upon secondary crosslinking. The latter, however, typically results in poorly developable mixtures of molecule species of varying stoichiometry and FcγR-binding by anti-CD40 antibodies can elicit unwanted side effects such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of CD40 expressing immune cells. Here, we summarize and compare strategies to overcome the unwanted target cell-destroying activity of anti-CD40-FcγR complexes, especially the use of FcγR type-specific mutants and the FcγR-independent cell surface anchoring of bispecific anti-CD40 fusion proteins. Especially, we discuss the therapeutic potential of these strategies in view of the emerging evidence for the dose-limiting activities of systemic CD40 engagement.

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CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism

Cited by 7 publications

References 45 publications

CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

Receptor Oligomerization and Its Relevance for Signaling by Receptors of the Tumor Necrosis Factor Receptor Superfamily

FcγRs and Their Relevance for the Activity of Anti-CD40 Antibodies

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