SUMMARY

• Guselkumab is a human immunoglobulin G (IgG)1λ monoclonal antibody (mAb) that binds selectively to the interleukin 23 (IL-23) protein with high specificity and affinity.¹
• Guselkumab, mirikizumab, risankizumab, and tildrakizumab are directed against the p19 subunit of IL-23 and have unique molecular attributes leading to important clinically relevant implications.^2,3,7 
• Guselkumab and risankizumab demonstrated binding to IL-23 and neutralized IL-23 signaling with similar high affinity and potency that was higher than that of tildrakizumab.^3,4
• Guselkumab and tildrakizumab, but not risankizumab, showed dose-dependent binding to human inflammatory monocytes in an Fc-dependent manner.^3-5
• Guselkumab, but not risankizumab, bound to the surface of inflammatory monocytes, correlating with cluster of differentiation 64 (CD64) expression, and simultaneously captured IL-23 endogenously secreted from the same cells. Guselkumab, but not risankizumab or mirikizumab, demonstrated binding to CD64.^2,7 
• Guselkumab, but not risankizumab, demonstrated binding to CD64 on the surface of macrophages and became internalized, leading to the intracellular uptake of IL-23 in a time-dependent manner. Additionally, following internalization, guselkumab colocalized with IL-23 in the endolysosomal compartments.^2,6
• Transcriptomic analysis demonstrated that expression of CD64 and IL-23 was significantly increased in inflamed gut biopsies of inflammatory bowel disease (IBD) patients vs non-inflamed control. Additionally, it was observed that CD64 was predominantly expressed by myeloid cells that also expressed IL-23p19.⁶
• Guselkumab, risankizumab, and mirikizumab exhibited similar potency in inhibiting IL-23 signaling induced by recombinant IL-23 in human peripheral blood mononuclear cells and exogenous native IL-23 in IL-23 bioassay cells. However, compared with risankizumab and mirikizumab, guselkumab demonstrated enhanced potency for neutralization of IL-23 signaling in an in vitro co-culture assay.⁷ 
• Guselkumab demonstrated binding to IL-23 in a monovalent manner and did not form polyvalent immune complexes with IL-23; therefore, it would not be expected to induce cytokine release.^2,8
• Compared to placebo, guselkumab decreased IL-23 pathway associated key inflammatory and effector cytokines in patients with Crohn's disease at Week 12 of a phase 2b study.It also significantly reduced cellular processes related to epithelial inflammation, inflammatory fibroblast activity, myeloid biology, interferon response, and IL-23 pathway processes.⁹

Company Core data sheet

About TREMFYA

Guselkumab is a human IgG1λ mAb that binds selectively to the IL-23 protein with high specificity and affinity through the antigen biding site. IL-23, a regulatory cytokine, affects the differentiation, expansion, and survival of T cell subsets, (e.g., Th17 cells and Tc17 cells) and innate immune cell subsets, which represent sources of effector cytokines, including IL-17A, IL-17F, and IL-22 that drive inflammatory disease. In humans, selective blockade of IL-23 was shown to normalize production of these cytokines.¹

Levels of IL-23 are elevated in the skin of patients with plaque psoriasis. In patients with ulcerative colitis, and Crohn’s Disease, levels of IL-23 are elevated in the colon tissue. In in vitro models, guselkumab was shown to inhibit the bioactivity of IL-23 by blocking its interaction with cell surface IL-23 receptor, disrupting IL-23-mediated signaling, activation, and cytokine cascades. Guselkumab exerts clinical effects in plaque psoriasis, psoriatic arthritis, ulcerative colitis, and Crohn's disease through blockade of the IL-23 cytokine pathway.¹

In vitro data

Molecular Differentiation In Vitro

• Guselkumab, risankizumab, mirikizumab, and tildrakizumab are mAbs directed against IL-23p19.^2,3,10

Unique Molecular Attributes

• Unique molecular attributes of guselkumab, risankizumab, and mirikizumab may have clinically relevant implications^7,11-13;see Figure: Molecular Attributes of Guselkumab, Risankizumab, and Mirikizumab.

Binding Affinity and Potency of Guselkumab and Risankizumab for IL-23

• Binding affinity was assessed using kinetic exclusion assay at room temperature (∼22°C) and surface plasmon resonance (SPR) at 37°C. Proximal signaling of IL-23 was assessed using a phosphorylated signal transducer and activator of transcription 3 (pSTAT3) assay in human peripheral blood mononuclear cells.⁴
• For the in vitro binding affinity and potency of guselkumab vs risankizumab for IL-23, see Figure: Binding Affinity and Potency of Antigen-Recognition Domains of Guselkumab and Risankizumab.

• As shown below in Figure: Binding Affinity and Potency of Guselkumab vs Risankizumab for IL-23 Inhibition, guselkumab and risankizumab bind IL-23 and neutralize IL-23 signaling with similar high affinity and potency that was higher than that of tildrakizumab.^3,4

Background on CD64/ Fc gamma receptor (FcγR)

• CD64⁺ cells are the predominant producers of IL-23 and are associated with disease activity.¹⁹ For a background on CD64⁺/Fc gamma receptor (FcγR), see Figure: Role of CD64⁺/FcγR in Disease Activity.

Binding to CD64⁺(FcγRs)

• Guselkumab and risankizumab were assessed for their ability to compete with a control D2-labeled IgG molecule for binding to FcγRs, as shown in Figure: Competitive Binding of Guselkumab and Risankizumab to FcγRs.
• Guselkumab demonstrated the strongest binding to cells transfected with CD64, as indicated by a decrease in Homogeneous Time Resolved Fluorescence (HTRF) for control antibody binding; risankizumab showed minimal binding to FcγRs.⁴

Binding to Human Inflammatory Monocytes

• CD14⁺ primary human monocytes were cultured in the presence of interferon gamma (IFNγ) leading to differentiation into monocytes with an inflammatory phenotype. Flow cytometry was used to assess IFNγ-induced upregulation of CD64 in CD14⁺ primary human monocytes and dose-dependent binding of guselkumab, risankizumab, modified guselkumab, and modified risankizumab.²⁶
• As shown in Figure: Binding of Guselkumab and Risankizumab to Human Inflammatory Monocytes, guselkumab and tildrakizumab, but not risankizumab, showed dosedependent binding to human inflammatory monocytes in an Fc-dependent manner.^3-5

Binding to Inflammatory Monocytes and Capture of Endogenously Secreted IL-23

• CD14⁺ primary human monocytes were cultured in the presence of granulocytemacrophage colony-stimulating factor and IFNγ leading to differentiation into monocytes with an inflammatory phenotype.²⁷
• To promote the production of endogenous IL-23, “inflammatory” monocytes were incubated with fluorescently labeled guselkumab or risankizumab and stimulated with toll-like receptor ligands (lipopolysaccharide and resiquimod).²⁷
• Flow cytometry was used to assess the binding of guselkumab and risankizumab to CD64⁺ inflammatory monocytes and capture of endogenously produced IL-23.
• As shown in Figure: Binding of Guselkumab and Risankizumab to inflammatory Monocytes and Capture of Endogenously Secreted IL-23, guselkumab, but not risankizumab, bound to the surface of inflammatory monocytes, correlating with CD64 expression, and simultaneously captured IL-23 endogenously secreted from the same cells.²

Binding to CD64⁺ Inflammatory Macrophages and Mediating Intracellular Uptake of IL-23 as Well as Enhancing Potency for Neutralizing IL-23 Signaling

• High-throughput spinning disk confocal microscopy was used to assess subcellular localization of Alexa Fluor™ 488 (AF488)-labeled mAbs and pHrodo Red-labeled IL-23.^2,29
• As shown in Figure: Quantitation of Binding of Guselkumab and Risankizumab to CD64⁺ Macrophages and Intracellular Uptake of IL-23, guselkumab, but not risankizumab, demonstrated binding to CD64 on the surface of macrophages and became internalized, leading to the intracellular uptake of IL-23 in a time-dependent manner.^2,29
• Additionally, following internalization, guselkumab colocalized with IL-23 in endolysosomal compartments.^2,6,29

• Guselkumab, risankizumab, and mirikizumab showed similar potency for inhibition of signaling induced by recombinant IL-23 in human peripheral blood mononuclear cells (assessed by pSTAT3 assay), and that induced by exogenous native IL-23 in IL-23 bioassay cells.⁷ 
• Guselkumab demonstrated enhanced potency compared with risankizumab, and mirikizumab for inhibition of IL-23 signaling. This was shown in the in vitro co-culture assay of IL-23-producing THP-1 cells (a CD64⁺ monocyte cell line activated to produce IL-23) and an IL-23 responsive cells.⁷ 

CD64 and IL-23 Expression in IBD Patient Gut Biopsies

• For bulk transcriptomic analyses, IBD ribonucleic acid sequencing (RNAseq) data along with read counts from rectal biopsies of patients with Crohn’s disease (115 inflamed samples, 251 non-inflamed), patients with ulcerative colitis (136 inflamed, 164 non-inflamed), and non-IBD controls (225 non-inflamed) were used. The data were converted to log₂ counts per million (CPM) using the edgeR package (Bioconductor) for plotting and statistical analysis.⁶
• As shown in Figure: Expression of CD64 and IL-23 in Gut Biopsies of IBD Patients vs Non-IBD Controls, the FcγR1A (CD64), IL23A (IL-23p19), and IL12B (p40 subunit of IL23) expression was significantly increased in inflamed vs non-inflamed IBD gut biopsies.⁶

• Additionally, it was observed that CD64 was predominantly expressed by myeloid cells that also expressed IL-23p19.

Key Proinflammatory and Effector Cytokines Levels

• At week 12, compared with placebo, guselkumab reduced RNA expression of IL-17A, IL-22, and IFNγ in the ileum and colon (P≤0.05).⁹
• Serum collagen degradation biomarker (C4M) and the C3M/PRO-C3 ratio showed an association with ileal narrowing and history of stricture at baseline (P≤0.001).⁹
• Intravenous TREMFYA induction reduced serum amyloid A (SAA), C-reactive protein (CRP), and IL-22 by week 4 (P≤0.01), with further reduction through week 12 in serum SAA, CRP, IL-6, IFNγ, IL-22, and IL-17A (P≤0.001).⁹
• Guselkumab also significantly reduced cellular processes associated with epithelial inflammation, inflammatory fibroblast and myeloid biology, interferon response, and IL‐23 pathway.⁹
• Regional molecular change was greatest in colon, followed by rectum and ileum.⁹

Cross-linking and Cytokine Production

• Guselkumab demonstrated binding to IL-23 in a monovalent manner, did not form polyvalent immune complexes with IL-23, and would not be expected to induce cytokine release;^2,8 see Figure: Interaction Between Guselkumab and IL-23.

• A human 41-plex cytokine bead assay was used to assess the production of 41 different cytokines.
• As shown in Figure: Assessment of Cytokine Production by Inflammatory Monocytes, the binding of guselkumab and risankizumab to CD64 did not induce production of IL-8, growth-regulated oncogene, IL-1 receptor antagonist, and tumor necrosis factor alpha or other cytokines evaluated.
• A goat anti-CD64 cross-linking antibody (positive control) induced cytokine production by inflammatory monocytes.

LITERATURE SEARCH

A literature search of MEDLINE^®, EMBASE^®, BIOSIS Previews^®, and DERWENT^® (and/or other resources, including internal/external databases) was conducted on 25 October 2024.