CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches

Argiriadi, M.A.; Benatuil, Lorenzo; Dubrovska, I.; Egan, David A.; Gao, Lei; Greischar, Amy; Hardman, Jennifer; Harlan, John E.; Iyer, Ramesh; Judge, Russell A.; Lake, Marc; Perron, Denise; Sadhukhan, Ramkrishna; Sielaff, Bernhard; Sousa, Silvino; Wang, Rui; McRae, Bradford L.

doi:10.1186/s12860-019-0213-4

Cited by 22 publications

(18 citation statements)

References 26 publications

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“…This epitope partially overlaps the CD40L binding site, but, is distinct from the binding sites of previously reported agonistic mAbs [39,42,43]. Furthermore, studies suggest that binding of this part of CD40 molecule may stabilize CD40 in an "inactive antiparallel state" and prevent downstream signaling [44].…”

Section: Discussionmentioning

confidence: 69%

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

2021

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Background CD40-CD40L is a key co-stimulatory pathway for B cell activation. As such, its blockade can inhibit pathogenic B cell responses in autoimmune diseases, such as Sjogren’s syndrome (SjS) and systemic lupus erythematosus (SLE). In this study, we examined the in vitro effects of KPL-404, a humanized anti-CD40 monoclonal antibody (Ab), on primary human B cells derived from either healthy donors (HD) or autoimmune patients and compared them to the effects of G28-5, a partially antagonistic anti-CD40 antibody. Methods PBMCs from HD or SjS and SLE patients were cultured in high-density cell cultures in the presence of IgG4 isotype control or anti-CD40 Abs KPL-404 or G28-5. Cells were stimulated with anti-CD3/CD28 cross-linking reagent ImmunoCult (IC) to induce CD40L-CD40-mediated B cell responses. B cell proliferation and activation, measured by dilution of proliferation tracker dye and the upregulation of CD69 and CD86, respectively, were assessed by flow cytometry. Anti-CD40 Ab cell-internalization was examined by imaging flow cytometry. Cytokine release in the PBMC cultures was quantified by bead-based multiplex assay. Results KPL-404 binds to CD40 expressed on different subsets of B cells without inducing cell depletion, or B cell proliferation and activation in in vitro culture. Under the same conditions, G28-5 promoted proliferation of and increased CD69 expression on otherwise unstimulated B cells. KPL-404 efficiently blocked the CD40L-CD40-mediated activation of B cells from HD at concentrations between 1 and 10 μg/ml. Treatment with KPL-404 alone did not promote cytokine production and blocked the production of IFNβ in healthy PBMC cultures. KPL-404 efficiently blocked CD40L-CD40-mediated activation of B cells from patients with SjS and SLE, without affecting their anti-IgM responses or affecting their cytokine production. Consistent with the differences of their effects on B cell responses, KPL-404 was not internalized by cells, whereas G28-5 showed partial internalization upon CD40 binding. Conclusions Anti-CD40 mAb KPL-404 showed purely antagonistic effects on B cells and total PBMCs. KPL-404 inhibited CD40L-CD40-mediated B cell activation in PBMC cultures from both healthy controls and autoimmune patients. These data support the therapeutic potential of CD40 targeting by KPL-404 Ab for inhibiting B cell responses in SjS and SLE.

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Section: Discussionmentioning

confidence: 69%

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

2021

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“…The data supports a mechanism where the best antagonist binds to the antiparallel dimer form of the receptor locking in the quiescent state [14,38]. In another example, an antagonist antibody to CD40 (TNFRSF5) has also been shown to bind to the antiparallel dimer form of the receptor [41]. The antibody bound as a single Fab domain making interactions to both CD40 monomers in the dimer.…”

Section: Tnfrsf Antiparallel Dimer Stabilization By Antibodies Effectmentioning

confidence: 53%

“…The antibody bound as a single Fab domain making interactions to both CD40 monomers in the dimer. It was further found that the antiparallel dimer stabilization was essential for antagonism as demonstrated by a closely related antibody that only interacted with a single CD40 monomer and had agonistic activity [41]. As we discuss next, the structure of the herpes virus entry mediator (HVEM) in complex with B and T lymphocyte attenuator (BTLA) represents another example of an antiparallel arrangement with BTLA assisting HVEM oligomerization, and playing a regulatory role in HVEM function.…”

Section: Tnfrsf Antiparallel Dimer Stabilization By Antibodies Effectmentioning

confidence: 95%

On the TRAIL of Better Therapies: Understanding TNFRSF Structure-Function

Vanamee

Faustman

2020

Cells

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Tumor necrosis factor (TNF) superfamily ligands show diverse biological functions, such as the induction of apoptotic cell death or cell survival and proliferation, making them excellent therapeutic targets for cancer and autoimmunity. We review the latest literature on TNF receptor superfamily signaling with a focus on structure-function. Using combinatorics, we argue that receptors that cluster on the cell surface and are activated by membrane-bound ligands need to arrange in a highly ordered manner, as the probability of random ligand and receptor arrangements matching up for receptor activation is very low. A growing body of evidence indicates that antiparallel receptor dimers that sequester the ligand binding site cluster on the cell surface, forming a hexagonal lattice. Upon ligand binding, this arrangement puts the activated receptors at the right distance to accommodate the downstream signaling partners. The data also suggest that the same geometry is utilized regardless of receptor type. The unified model provides important clues about TNF receptor signaling and should aid the design of better therapies for cancer and various immune mediated diseases.

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“…The importance of the IgG2 hinge on function has been recently confirmed by a study of the anti‐CD40 antibody agonist for cancer immunotherapy 20,22,24 . The structurally rigid IgG2 isoforms with the C127S mutation optimized agonism.…”

Section: Resultsmentioning

confidence: 88%

“…For years, the important antibody region for target specificity was the variable region, but here the focus is on antibody isotypes and isoforms. Antibody isotypes are known to have an impact on function 20–23 . Antibody shuffling between isoforms is defined as the process of creating variants through covalent intrachain interactions in the hinge made naturally in vivo and influenced by manufacturing conditions 22–29 .…”

Section: Resultsmentioning

confidence: 99%

Optimizing TNFR2 antagonism for immunotherapy with tumor microenvironment specificity

Yang

Trần

Torrey

et al. 2020

Journal of Leukocyte Biology

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Most approved cancer immunotherapies lack T‐regulatory (Treg) or tumor specificity. TNF receptor 2 (TNFR2) antibody antagonism is emerging as an attractive immunotherapy due to its tumor microenvironment (TME) specificity. Here we show that the human TNFR2 receptor is overexpressed on both human tumor cells and on human tumor‐residing Tregs, but negligibly expressed on beneficial T effectors (Teffs). Further, we found widespread, if variable, TNFR2 expression on 788 human tumor cell lines from diverse cancer tissues. These findings provided strong rationale for developing a targeted immunotherapy using a TNFR2 antibody antagonist. We designed a novel, human‐directed TNFR2 antibody antagonist and tested it for function using three cell‐based TME assays. The antagonist showed TME specificity by killing of TNFR2‐expressing tumor cells and Tregs, but sparing Teffs, which proliferated. However, the antagonist shuffled between five isoforms, only one of which showed the desirable function. We designed and tested several new chimeric human versions of the antagonist, finding that the IgG2 isotype functioned better than the IgG1 isotype. To further improve function, we introduced targeted mutations to its amino acid sequence to stabilize the natural variability of the IgG2 isotype's hinge. Altogether, our findings suggest that optimal TNFR2 antagonists are of the human IgG2 isotype, have hinge stabilization, and have wide separation of antibody arms to bind to newly synthesized TNFR2 on rapidly growing tumor cells. Antagonistic antibodies with these characteristics, when bound to TNFR2, can form a nonsignaling cell surface dimer that functions with high TME specificity.

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CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches

Cited by 22 publications

References 26 publications

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

On the TRAIL of Better Therapies: Understanding TNFRSF Structure-Function

Optimizing TNFR2 antagonism for immunotherapy with tumor microenvironment specificity

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