Anti-citrullinated histone monoclonal antibody CIT-013, a dual action therapeutic for neutrophil extracellular trap-associated autoimmune diseases

Figures & data

Table 1. Amino acid sequence of non-citrullinated and citrullinated peptides.

Name	Amino acid position	Amino acid sequence	N-terminal modification	C-terminal modification
Histone H2A	1–12	SGRGKQGGKARA	Acetyl	Aminohexanoic acid–lysine–biotin
Cit-histone H2A	1–12	SGXGKQGGKARA	Acetyl	Aminohexanoic acid–lysine–biotin
Histone H3	1–14	ARTKQTARKSTGGK	Hydrogen	Aminohexanoic acid–lysine–biotin
Cit-histone H3	1–14	AXTKQTARKSTGGK	Hydrogen	Aminohexanoic acid–lysine–biotin
Histone H4	1–12	SGRGKGGKGLGK	Acetyl	Aminohexanoic acid–lysine–biotin
Cit-histone H4	1–12	SGXGKGGKGLGK	Acetyl	Aminohexanoic acid–lysine–biotin
Cit-tenascin	2182–2200	AEMKLXPSNFXNLEGXXKX	Hydrogen	Aminohexanoic acid–lysine–biotin
Cit-$\alpha$-enolase	4–21	LKIHAXEIFDSXGNPTVE	Hydrogen	Aminohexanoic acid–lysine–biotin
Cit-fibrinogen $\alpha$	36–48	GPXVVEXHQSACK	Hydrogen	Aminohexanoic acid–lysine–biotin
Cit-fibrinogen $\beta$	60–74	XPAPPPISGGGYXAX	Hydrogen	Aminohexanoic acid–lysine–biotin
Cit-vimentin	60–70	GVYATXSSAVRL	Hydrogen	Aminohexanoic acid–lysine–biotin
Cit-filaggrin	3692–3710	SHQESTXGRSRGRSGRSGS	Hydrogen	Aminohexanoic acid–lysine–biotin

X = Citrulline.

Table 2. CIT-013’s affinity for non-citrullinated and citrullinated N-terminal histone peptides.

Ligand	Kon (M s^−1)	Koff (s^−1)	KD (pM)
Histone H2A	1.5 × 10^6	7.61 × 10^–4	508
Histone H3	-	-	NB
Histone H4	-	-	NB
Cit-histone H2A	6.07 × 10^6	5.69 × 10^−5	9.37
Cit-histone H3	-	-	NB
Cit-histone H4	5.44 × 10^5	5.78 × 10^−5	106

NB = no binding detected.

Figure 1. Elevated CIT-013 epitope in RA synovial tissue with moderate to marked active synovitis.

Three representative images of H&E-stained synovial biopsies (left) from distinct RA patients with minimal active synovitis (a), moderate active synovitis (b), and moderate to marked active synovitis (c). For the detection of CIT-013’s epitope in RA synovial tissue (magenta staining in boxes on the right), samples were subsequently incubated with FITC-conjugated CIT-013, followed by a rabbit anti-FITC antibody and finally with an HRP-conjugated horse anti-rabbit antibody. Histological severity grade and incidence of CIT-013 staining were scored by a pathologist (Table 3). The scale bar for H&E staining is 2 mm, and the scale bar for CIT-013 staining is 200 µm.

Table 3. Histological severity grade of RA synovia and incidence of CIT-013 staining.

Biopsy	Histological severity grade	Key histological features	Incidence of CIT-013 staining	Histological localization of CIT-013 staining
1	No active synovitis	Quiescent synovium with a few scattered inflammatory cells within connective tissue	Occasional positive punctate cells	Infiltrating acute cells
2*	Minimal synovitis	Scattered lymphoid foci and scattered mixed inflammatory infiltrate, cartilage and bone admixed with the synovial matrix	Minimal diffuse staining, occasional positive punctate cells	Infiltrating acute cells
3	Minimal active synovitis	Focal areas of synovial hyperplasia with diffuse inflammatory infiltrates in the sub-lining	Minimal diffuse staining, occasional positive punctate cells	Infiltrating acute cells
4	Minimal reactive synovitis	Low-grade acute synovitis with a low-grade fibrogranular reaction around detritic foci, and low-grade acute inflammatory infiltrate around microvasculature	Occasional positive punctate cells	Infiltrating acute cells
5*	Moderate active synovitis	Lymphoid foci with microvascular sinusoid formation, hyperplastic synovial lining with zones of lymphoid, mononuclear and macrophage infiltration in sub-lining, peri-vascular acute inflammatory infiltrates, and diffuse stromal cell reaction	Moderate to marked multi-focal staining	Synovial lining, sub-lining, peri-vascular, peri-lymphoid, and demarcation of follicular zones
6*	Moderate to marked active immune synovitis	Marked lymphoid-rich (follicular) synovitis, multiple lymphoid foci with abundant microvascular sinusoid formation, markedly hyperplastic synovial lining with dense zones of lymphoid, mononuclear and macrophage infiltration in sub-lining, peri-vascular acute inflammatory infiltrates, and diffuse stromal cell reaction	Marked zonal staining	Synovial lining, sub-lining, peri-vascular, peri-lymphoid, and demarcation of follicular zones

*These biopsies are demonstrated in Figure 1.

Figure 2. CIT-013 does not bind blood cells and does not affect neutrophil anti-microbial activity other than NETosis.

(a) Binding of HiLyte^TM Fluor 488-conjugated isotype control Ab (HL488-cIgG) or HL488-CIT-013 to erythrocytes, thrombocytes, T cells, B cells, monocytes, and neutrophils was measured using flow cytometry. Neutrophils stimulated with A23187 for 120 min (netting neutrophils) were used as positive control. (b) Neutrophil phagocytosis of fluorescent pHrodo^TM Green S. aureus Bioparticles^TM Conjugates in the absence (No Ab) or presence of cIgG, CIT-013, or Cyto D (actin polymerization inhibitor as a positive control inhibitor of phagocytosis). (c) ROS production by neutrophils stimulated with Ig-opsonized heat-killed S. aureus in the absence or presence of cIgG, CIT-013, or DPI (NADPH oxidase inhibitor as a positive control inhibitor for ROS production) (n = 6). Statistics were performed on the area under the curve. (d) CD11b membrane degranulation marker on neutrophils stimulated with LPS in the absence or presence of cIgG, CIT-013, or Cyto D (positive control inhibitor of degranulation) at t = 180 min. *P<.05, ***P<.001, and ****P<.0001, two-tailed paired t test (a), two-tailed unpaired t test (b), and RM one-way ANOVA using Dunnett’s multiple comparisons test (c). RFU = relative fluorescence units; MFI = mean fluorescence intensity.

Figure 3. CIT-013 specifically inhibits the release of citrullination rich NETs.

A23187- and PMA-induced NET release was measured by a quantitative immunofluorescence live imaging NET assay using the impermeable DNA dye Sytox^TM Green. (a) Representative images of NET release in response to A23187 (red arrows) and PMA (yellow arrows) at different time points. Permeable neutrophils with intracellular chromatin are indicated with blue arrows. (b) Quantification of NET release over time (n = 5). Statistics were performed on t = 240 min. (c) Citrullinated nucleosome detection in NET harvest at t = 240 min. (d) A23187-induced NET release at t = 240 min in the absence (No Ab) or presence of cIgG or CIT-013. (e) Dose-dependent inhibition of A23187-induced NET release with CIT-013 (n = 3). Data were normalized to cIgG (set as 100% NET release). (f) PMA-induced NET release at t = 240 min in the absence or presence of cIgG or CIT-013. (g) Immune complex-induced NET release at t = 240 min in the presence of cIgG or CIT-013. A23187-induced NET release in the absence or presence of CIT-013 in combination with RF (h) or ACPA-rich pool of IgG from RA patients (i) at t = 60 and 180 min, respectively. Concentrations in (i) represent the level of ACPA present in the assay, not the level of IgGs. **P<.01, ***P<.001, and ****P<.0001, Repeated measures one-way ANOVA with Dunnett’s (b) or Tukey’s (d) multiple comparisons test, Friedman test with Dunn’s multiple comparisons test (c), two-tailed Wilcoxon matched-pairs signed rank test (g), and ordinary two-way ANOVA with Dunnett’s multiple comparisons test (h and i).

Figure 4. Bivalency is necessary for the NET-inhibitory capacity of CIT-013.

(a) Schematic design of a full-length CIT-013 antibody, a monovalent CIT-013 antibody, and a CIT-013 F(ab’)2 fragment. Monovalent CIT-013 was produced by co-expression of a light chain, a heavy chain containing a hole mutation (T366S:L368A:Y407V), and a truncated heavy chain containing a knob mutation (T366W). (b) Neutrophils were stimulated with A23187 in the presence of full-length CIT-013, monovalent CIT-013, or CIT-013 F(ab’)2 fragments (n = 3). Data were normalized to cIgG (set as 100% NET release). CL = constant light; VL = variable light; CH = constant heavy; VH = variable heavy; Fc = constant fragment.

Figure 5. CIT-013 blocks chromatin release from netting neutrophils.

(a) Representative image of a non-stimulated neutrophil characterized by its multi-lobed nucleus in the presence of HiLyte^TM Fluor 488-CIT-013 (HL488-CIT-013). (b) Representative images of an A23187-induced netting neutrophil in the presence of HL488-CIT-013. This neutrophil showed a re-organized rounded nucleus (blue arrow) in phase 1, nuclear membrane rupture and chromatin decondensation (red arrow) in phase 2, and plasma membrane rupture and chromatin release into the extracellular environment (yellow arrow) in phase 3a of the NETosis pathway. HL488-CIT-013 binding to netting neutrophils occurs in phase 3b followed by intracellular diffusion of HL488-CIT-013 beyond the rupture point (green arrow) in phase 3c. Quantification of the relative chromatin area of netting neutrophils in phase 1 (c), phase 2 (d), and phase 3b (e) of the NETosis pathway in the absence (No Ab) or presence of HL488-cIgG or HL488-CIT-013. The dotted line represents the relative chromatin area of non-stimulated neutrophils. ****P<.0001, Kruskal–Wallis test with Dunn’s multiple comparisons test. (f) Representative image of a netting neutrophil in the presence of HL488-cIgG. (g) Orthogonal representative projection of a netting neutrophil in phase 3b of the NETosis pathway confirming colocalization of CIT-013 with the site of plasma membrane rupture and chromatin escape. The scale bar in (a), (b), and (g) is 5 µm and in (f) is 10 µm.

Figure 6. Phagocytosis of CIT-013-opsonized NETs and netting neutrophils by macrophages.

Neutrophils were stimulated with A23187 in the absence (No Ab) or presence of cIgG (resulting in NETs) or CIT-013 (resulting in CIT-013-opsonized netting neutrophils) and co-cultured with macrophages. Pre-incubated macrophages with Cyto D were used as control for phagocytosis inhibition. (a) Orthogonal representative projection of a CIT-013-opsonized netting neutrophil that was internalized by a macrophage at t = 60 min. (b) Quantification of phagocytosis of netting neutrophils by macrophages at t = 60 min. (c) Quantification of phagocytosis of NET fragments in the presence of cIgG or CIT-013 in combination with Cyto D at t = 60 min. (d) Representative images of CIT-013-opsonized NET fragments that were internalized by a macrophage (white arrows) and accumulated in intracellular vesicles (yellow arrows) over time. (e) Quantification of phagocytosis of NET fragments in the presence of CIT-013 F(ab’)2 fragments or CIT-013 in combination with a mix of blocking antibodies against Fc$\gamma$RI, Fc$\gamma$RIIA, and Fc$\gamma$RIII (Fc$\gamma$R block) at t = 60 min. (f) Quantification of phagocytosis of NET fragments in the presence of CIT-013 in combination with RF at t = 60 min. *P<.05, **P<.01, and ****P<.0001, Friedman test with Dunn’s multiple comparisons test (b), RM one-way ANOVA with Dunnett’s multiple comparisons test (c and e), and ordinary two-way ANOVA with uncorrected Fisher’s LSD test (f). The scale bar in (a) and (d) is 10 µm.

Figure 7. Reduced inflammatory infiltrate and enhanced phagocytosis induced by m-ACHA in a neutrophilic airway inflammation mouse model.

CFA/HDM-sensitized Balb/c mice were challenged (i.n.) with HDM or vehicle. One hour before challenge, mice received Dex or vehicle p.o. (gray area) or received m-ACHA, mouse isotype control antibody (m-cIgG), or vehicle i.v. (white area). BALF and lung tissue were collected 24 hours after challenge. (a) m-ACHA epitope levels in BALF from vehicle and HDM challenged mice which did not receive any antibody treatment. (b) Representative fluorescence IHC images from lung tissue of vehicle and HDM challenged mice stained with DAPI (DNA in blue) and m-ACHA (yellow). The scale bar is 20 µm. (c) Semi-quantitative scores of perivascular neutrophilia in H&E-stained lung sections (2 sections per mouse). (d) Neutrophil numbers in BALF. (e) Anatomically matched and randomly selected fluorescence IHC images from lung tissue stained with anti-citrullinated histone H3 antibody (green), anti-MPO antibody (red), and DAPI. The scale bar is 20 µm. (f) Semi-quantitative scoring of NETs (admixing of citH3 and MPO) in the perivascular zone of immunofluorescent-stained lung sections. Autofluorescence signals were present in images (green channel), but NETs were considered extracellular based on the morphology or minimal distance of 3 nuclear radii from the nearest nucleus. (g) dsDNA levels in BALF. The total number of macrophages (h) and the percentage of phagocytic macrophages (i) in the perivascular zone. Graphs are depicted as median in (c and f) and as mean in (a, d, and g−i) of eight mice per group. Comparisons were made per administration route (p.o. or i.v.). *P <.05, **P <.01, ***P <.001, ****P <.0001, unpaired two-tailed t test (a), Kruskal-Wallis test with Dunn’s multiple comparison test (c and f−h), and ordinary one-way ANOVA with Holm-Šidák’s multiple comparisons test (d and i).

Data availability statement

All data are available under a material transfer agreement with Citryll B.V.