Facile generation of biepitopic antibodies with intrinsic agonism for activating receptors in the tumor necrosis factor superfamily

Abstract

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.

Keywords: 4–1BB; CD137; FcγR; OX40; TNF; TNFR; agonist; antibody; biepitopic; mAb; receptor clustering.

Figure 1.. Overview of approach for isolating single-chain antibodies from human libraries with unique receptor (OX40) epitopes relative to a clinical-stage OX40 antibody.

A human single-chain (scFv) library displayed on yeast was enriched for binding to the human OX40 ectodomain via magnetic-activated cell sorting (MACS, one sort) and fluorescence-activated cell sorting (FACS, three sorts). Next, a clinical-stage OX40 IgG (11D4, also known as ivuxolimab) was used to block its OX40 epitope on the soluble OX40 ectodomain, and the library was further enriched against the OX40/IgG complex to identify scFvs with unique OX40 epitopes. Three unique scFvs (OX.F1, OX.F2, and OX.F3) were discovered using this approach. The figure was made using BioRender.

Figure 2.. Selected single-chain antibody possesses unique OX40 epitope relative to 11D4 IgG.

(A) The candidate single-chain antibody (OX.F2) was displayed on yeast and evaluated for binding to monovalent OX40 ectodomain when the 11D4 epitope is pre-blocked. (B) The wild-type 11D4 single-chain Fab (scFab) on yeast loses binding to OX40 when the ectodomain is pre-incubated with 11D4 IgG, as expected. (C) Selected OX40 scFv (OX.F2) shows strong binding to OX40 in the presence and absence of 11D4 IgG pre-blocking. The results are averages of three independent experiments and the error bars are standard deviations.

Figure 3.. Biepitopic OX40 antibody induces strong human CD4+ T cell activation in an FcγR-independent manner.

(A) Schematic illustration of the approach used to convert an existing clinical-stage antibody into a potent OX40 antibody with intrinsic agonist activity. (B) The biepitopic antibody induced strong human CD4+ T cell proliferation. Division index was calculated using the FlowJo Proliferation platform to evaluate proliferation at day 6. The results are averages of three independent experiments and the error bars are standard deviations. In (B), the biepitopic (IgG-scFv) antibodies displayed higher levels of proliferation than the monoepitopic antibodies and OX40 ligand at each concentration [p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****)].

Figure 4.. Generalization of the biepitopic antibody approach to additional OX40 clinical-stage antibodies results in potent human CD4+ T cell activation.

(A) Schematic of the process of competition-based analysis of antibody epitopes for clinical-stage OX40 agonists and antagonists, the resulting combination of IgGs with the OX.F2 scFv into biepitopic formats, and their evaluation for T cell activation. (B-C) Competition-based analysis of (B) 11D4 and (C) OX.F2 single-chain antibodies displayed on yeast binding to OX40 after pre-blocking OX40 with a clinical-stage OX40 antibody. (D) The biepitopic antibodies induced strong human CD4+ T cell proliferation. Division index was calculated using the FlowJo Proliferation platform to evaluate proliferation at day 6. The results are averages of three independent experiments and the error bars are standard deviations. 18D8-OX.F2 displayed higher levels of proliferation than telazorlimab-OX.F2 and OX40 ligand at each concentration, while pogalizumab-OX.F2 displayed higher levels of proliferation than telazorlimab-OX.F2 and OX40 ligand at 0.02 and 0.2 μg/mL [p-value <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****)].

Figure 5.. Generalization of the biepitopic antibody approach to an additional TNF receptor (CD137) results in potent human CD8+ T cell activation.

(A) A human scFv library was enriched against the human CD137 ectodomain via MACS (one sort) and FACS (three sorts). Next, the library was enriched for CD137 binding in the presence of a clinical-stage CD137 IgG (utomilumab, uto.; two sorts). (B) Schematic of the approach used to convert utomilumab into a biepitopic CD137 antibody with intrinsic agonist activity. (C) The isolated CD137 single-chain antibody (CD.K2) displayed on yeast bound CD137 ectodomain in the absence and presence of uto IgG. (D) The biepitopic antibody (utomilumab-CD.K2; uto-CD.K2) induced strong human CD8+ T cell proliferation, as judged by CD25 expression, relative to the monoepitopic antibodies and a control IgG. The results are averages of five independent experiments and the error bars are standard errors [p-value <0.01 (**) and <0.0001 (****)].