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

ABSTRACT: Antibodies that target a clinically relevant group of receptors within the tumor necrosis factor receptor superfamily (TNFRSF), including CD40 and CD95 (Fas/Apo-1), also require binding to Fc gamma receptors (FcγRs) to elicit a strong agonistic activity. This FcγR dependency largely relies on the mere cellular anchoring through the antibody's Fc domain and does not involve the engagement of FcγR signaling. The aim of this study was to elicit agonistic activity from αCD40 and αCD95 antibodies in a myeloma cell anchoring-controlled FcγR-independent manner. For this purpose, various antibody variants (IgG1, IgG1_N297A, Fab₂) against the TNFRSF members CD40 and CD95 were genetically fused to a single-chain-encoded B-cell activating factor (scBaff) trimer as a C-terminal myeloma-specific anchoring domain substituting for Fc domain-mediated FcγR binding. The antibody-scBaff fusion proteins were evaluated in binding studies and functional assays using tumor cell lines expressing one or more of the three receptors of Baff: BaffR, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA). Cellular binding studies showed that the binding properties of the different domains within the fusion proteins remained fully intact in the antibody-scBaff fusion proteins. In co-culture assays of CD40- and CD95-responsive cells with BaffR, BCMA or TACI expressing anchoring cells, the antibody fusion proteins displayed strong agonism while only minor receptor stimulation was observed in co-cultures with cells without expression of Baff-interacting receptors. Thus, our CD40 and CD95 antibody fusion proteins display myeloma cell-dependent activity and promise reduced systemic side effects compared to conventional CD40 and CD95 agonists.

SUBMITTER: Nelke J

PROVIDER: S-EPMC7531569 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

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