An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases

Abstract

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

Keywords: SIRPα; agonistic antibody; arthritis; autoimmune inflammation; colitis; monocyte; neutrophil.

Graphical abstract

Figure 1

Elevated SIRPα⁺ myeloid cells in inflammatory tissues and associated with treatment non-responsiveness (A) Transcriptional data of SIRPA expression in synovial biopsies from healthy control, OA, and RA patients (n = 69; each dot represents an individual patient biopsy). (B) Transcriptional data of SIRPA expression in colon biopsies from healthy control, CD, and UC patients (n = 254; each dot represents an individual patient biopsy). (C) Transcriptional heatmap of SIRPA and other neutrophil-/monocyte-associated genes in colonic biopsies from healthy control (HC), inflamed, and uninflamed UC/CD colonic biopsies. Data are presented as log2 relative expression. (D) Transcriptional heatmap of SIRPA and other neutrophil-/monocyte-associated genes in baseline colonic biopsies of patients who responded (R) or non-responded (NR) with infliximab or vedolizumab. Data are presented as log2 relative expression. (E) Immunohistochemical (IHC) staining of SIRPα in normal or RA synovium (top row) and normal or CD colons (bottom row). Scale bar, 100 μm (one representative image was selected from each group with 3–5 biopsy samples). Data were analyzed using a Kruskal-Wallis test with Dunn’s multiple comparisons test. p values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) were calculated using a one-way ANOVA and accounting for multiple testing.

Figure 2

The agonistic anti-SIRPα antibody limits neutrophil and inflammatory monocyte immune infiltration (A) Immunoblot analysis of phosphorylated SIRPα. RAW264.7 cells were stimulated with no treatment, isotype control, or agonistic anti-SIRPα for 5 min. (B) TNFα and G-CSF in supernatant from RAW 264.7 cells stimulated by mIgG2a-protein A beads immune complex (IC) in the presence of isotype control, agonistic anti-SIRPα, or CD47-Fc. (C) Flow cytometric quantification of mouse neutrophil (Ly6G⁺CD11b⁺) and monocyte (Ly6C⁺CD11b⁺) cell number from peritoneal lavage collected 6 h after zymosan injection. (D) IL-1β, TNFα, and G-CSF in peritoneal lavage collected 6 h after zymosan injection with indicated treatment. (E and F) Mice were treated with isotype control, agonistic anti-SIRPα, and control anti-SIRPα antibodies. Overnight after antibody treatment, neutrophil and monocyte cell number were quantified by flow cytometry quantification in peritoneal cavity 4 h post intraperitoneal injection of recombinant CXCL1. In (F), indicated groups of mice were pre-treated with blocking antibodies against LFA-1 and MAC-1 16 h before CXCL1 injection. (G) Mice were treated isotype control, agonistic anti-SIRPα, or control anti-SIRPα antibody. Overnight, after the treatment, migration of bone marrow neutrophils from indicated mouse groups in response to CXCL1 (2 ng/mL) was quantified by a transwell migration assay. Data are from one representative experiment of three independent experiments with at least three biological replicates per group. Each symbol represents one individual mouse. Bar graph is shown as mean ± standard error. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Figure 3

Anti-SIRPα agonistic antibody ameliorates experimental arthritis through reducing neutrophil and monocyte infiltration (A–G) Mice received indicated treatment agents following K/BxN serum transfer. (A) Daily arthritis score. (B and C) Representative joint histopathology image and paw/joint histological scores of mice 8 days post K/BxN serum transfer. Scale bar, 500 μm. (D) Area under the curve (AUC) of total clinical arthritis scores. (E and F) Flow cytometric quantification of neutrophils and monocytes (F) from knee joint (E) and spleen (F) on 8 days post K/BxN serum transfer. (G) RNA sequencing of total paw tissues (each from four individual mice) of naive, isotype control, and agonistic anti-SIRPα treated mice 7 days post K/BxN serum transfer. Heat maps showing differential expression of genes related to myeloid cells (left) and genes of cytokines and chemokines (right). (H and J) Clinical arthritis scores of naive or collagen-induced arthritis (CIA) mice with treatments of isotype control, agonistic anti-SIRPα, and CD47-Fc. (I) Representative joint histopathology of naive or CIA mice that received indicated treatment. Scale bar, 500 μm. Data are from one representative experiment of two independent experiments with at least three technical replicates per group. Each symbol represents one mouse. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

The agonistic anti-SIRPα ameliorates T cell transfer colitis through reducing neutrophils and inflammatory monocytes in tissues (A) Schema of the CD45RB^highCD4⁺ T cell transfer colitis therapeutic model. C.B17 SCID mice were transferred with unsorted splenocytes or sorted CD45RB^highCD4⁺ T cells. Six weeks post cell transfer, all mice were re-randomized and started with indicated treatments every other day. All mice were euthanized 12 weeks post cell transfer for tissue collection and histopathology. (B and C) Visual colon scores and histological scores of colons. (C) Hematoxylin and eosin staining of colon. Asterisks indicate increased lamina propria inflammation, epithelial hyperplasia, and areas of crypt loss. Scale bar, 400 μm. (D–F) IHC staining of colons was conducted per standard protocols on an autostainer. Sections were stained with antibodies for CD4, F4/80, and GR1(Ly6G/C). Scale bar, 200 μm; inset scale bar, 100 μm. Data are from one representative experiment of two independent experiments with at least three biological replicates per group. Each symbol represents one mouse. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, by ordinary one-way ANOVA with Tukey’s multiple comparisons test.