• Increased GM-CSF, GM-CSF-Rα, and downstream pathway-associated protein levels in GCA biopsies were consistent with previously observed increased transcriptome signature11

• Expression of genes associated with GM-CSFR pathway and inflammatory cell infiltration was suppressed by mavrilimumab in cultured GCA arteries

• These data implicate the GM-CSF pathway in GCA pathophysiology and support confidence in rationale for targeting the GM-CSF pathway in GCA

• Giant Cell Arteritis (GCA) is a type of large vessel vasculitis that can cause pain and malaise and more severe complications including blindness and aortic aneurysms1

• A significant unmet medical need remains in GCA, as current treatment options are limited

• Relapse increases corticosteroid (CS) exposure and toxicity

• Tocilizumab blockade of IL-6 signaling significantly reduces relapses; however, ~45% of patients in the GiACTA trial did not achieve a sustained remission at 52 weeks following weekly or bi-weekly tocilizumab dosing2

• Macrophages/dendritic cells (DCs) and T helper (TH1/TH17) lymphocytes are major contributing cell types in GCA pathogenesis1

• Granulocyte-macrophage colony stimulating factor (GM-CSF) may contribute to GCA pathogenesis by promoting the actions of key cell types involved 3,4

• GM-CSF may drive DCs to program naïve CD4+ cells to TH1, TH17, TH follicular and THGM5 phenotypes characterized by the expression of IFNγ, IL-17, IL-21, and GM-CSF respectively

• GM-CSF produced primarily by CD4+ T helper TH1 and TH17 cells can stimulate conventional DCs and promote differentiation of monocyte-derived DCs

• GM-CSF may promote the proliferation and migration of vascular endothelial cells, thus contributing to angiogenesis

• GM-CSF can stimulate giant cell formation characteristic of the disease

• GM-CSF mRNA expression has been reported in GCA lesions6 and in peripheral blood mononuclear cells of symptomatic GCA patients7

• Mavrilimumab (KPL-301), a human monoclonal antibody targeting the GM-CSF receptor alpha subunit (GM-CSF-Rα), is currently under investigation as a treatment for GCA (Phase 2; NCT03827018), COVID-19 associated pneumonia and hyperinflammation (NCT04399980, NCT04397497), and relapsed or refractory large B-cell lymphoma, in an investigational combination with Yescarta (chimeric antigen receptor T [CAR-T] cell therapy)

GM-CSF Pathway is Implicated in Pathogenic Inflammatory Mechanism in Giant Cell ArteritisMaria C. Cid1, Sujatha Muralidharan2, Marc Corbera-Bellalta1, Georgina Espigol-Frigole1, Javier Marco-Hernandez1, Amanda Denuc3, Roberto Ríos-Garcés1, Nekane Terrades-Garcia1, John F. Paolini2, Annalisa D’Andrea2

1 Vasculitis Research Unit, Department of Autoimmune Diseases, Hospital Clínic, University of Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 2 Kiniksa Pharmaceuticals, Corp., Lexington, MA; 3HCB-IDIBAPS Biobank, Barcelona, Spain

BACKGROUND

METHODS

European League Against Rheumatism (EULAR) 2020 Congress | 3 June 2020 | E-Congress

RESULTS

OBJECTIVES

Immunofluorescence (IF) staining for protein expression • TABs (n=3 each) were fixed in 4% paraformaldehyde (PFA) in

phosphate-buffered saline (PBS), embedded in OCT, frozen at −80°C, sectioned at 10 microns thickness, and incubated at 4°C overnight with antibodies against GM-CSF and GM-CSFRα followed by secondary antibodies

• Slides were mounted with DAPI Fluoromount-G medium and examined using a laser scanning confocal Leica TCS SP5 microscope. Images were processed with Image J software

Immunohistochemical (IHC) staining for protein expression• FFPE TAB samples (n=1-3, sectioned at 5 microns thickness) were

used for IHC. Briefly, after antigen retrieval with citrate buffer (pH6, 20 min), samples were immunostained with antibodies to PU.1 or phosphoJAK2, developed with diaminobenzidine and counterstained with hematoxylin

• Slides were analyzed using the Leica Microsystems’ Bond-max™ automated immunostainer together with the Bond Polymer Refine Detection System (Leica Microsystems)

Ex vivo culture of temporal arteries• Control or GCA temporal arteries (n=8-11 each) were sectioned (0.8-

1 mm), embedded in Matrigel, and cultured in supplemented RPMI medium

• Each artery sample was cultured in the presence of Placebo [50 mM sodium acetate, 70 mM sodium chloride, 4% (w/v) trehalosedihydrate, 0.05% (w/v) polysorbate 80, pH 5.8] or mavrilimumab (20 mcg/ml) for 5 days. Supernatants were used for soluble factor ELISA, and cells were used for RNA isolation and expression of relevant genes analyzed by qRT-PCR using TaqmanTM assays. Data were normalized to housekeeping gene GUSb and expressed as relative expression units

Abstract 2689

• To explore localized expression of GM-CSF cytokine and receptor in different cell types in GCA arteries compared to control arteries at the protein level

• To examine the activation status of the GM-CSFR pathway associated signaling molecules in GCA arteries compared to control arteries

• To measure the effect of mavrilimumab on genes relevant to GCA pathophysiology in ex vivo GCA artery cultures

Figure 7: GM-CSF pathway in GCA pathophysiology

REFERENCES:1. Terrades-Garcia & Cid. Rheumatology, 2018; 57(2):51-62; 2. Stone et al. N Engl J Med, 2017; 377:317-328; 3. Lemaire et al. Journal of Leukocyte Biology, 1996; 60(4):509-18; 4. Wicks & Roberts. Nature Reviews. Rheumatology, 2016; 12(1):37-48; 5. Herndler-Brandstetter & Flavell. Cell Research, 2014; 24(12): 1379-80; 6. Weyand et al. Annals of Internal Medicine, 1994, 121(7):484-91; 7. Terrier et al. Arthritis and rheumatism, 2012; 64(6):2001-11; 8. Deng et al. Circulation, 2010; 121(7): 906–915; 9. Zielińska et al. Frontiers in Immunology, 2014; (7):592; 10. Burmester et al. Ann Rheum Dis. 2013 Sep 1;72(9):1445-52. 11. Cid et al. GM-CSF Pathway Signature Identified in Temporal Artery Biopsies of Patients With Giant Cell Arteritis. Poster presented Nov 2019 at American College of Rheumatology.

DISCLOSURES:This research was funded by Kiniksa Pharmaceuticals, Ltd.

M. Cid, presenting author, has received research grants from Kiniksa and consulting fees from Janssen and Abbvie; co-authors S. Muralidharan and J.F. Paolini are employees of Kiniksa Pharmaceuticals Corp.

CONCLUSIONS

Figure 5: Mavrilimumab shows trends in reduction of some TH1 and TH17 markers in ex vivo cultured arteries from patients with GCA

Figure 3: GM-CSF R-driven signaling pathways are activated in GCA lesions, and expression of regulated molecules is increased

• Expression of GM-CSF and GM-CSFRα was increased in GCA arteries compared to control arteries (Figure 1)

• GM-CSF protein was increased in the luminal endothelium, neovessels, vascular smooth muscle cells and immune cells such as macrophages and T cells in GCA TABs compared to control. GM-CSF-Rα protein was elevated in the luminal endothelium, neovessels and immune cells in GCA TABs compared to control TABs (Figure 2 and Table 1)

• Protein levels of phosphorylated JAK2 and nuclear localized PU.1 also appeared higher in GCA arteries indicating active GM-CSFR signaling pathway (Figure 3)

Figure 1: GMCSF and GM-CSFRα are upregulated in GCA lesions

Control GCA

GM-CSF

Control GCA

GM-CSFRα

Figure 2: Immune cells and endothelial cells present in GCA arteries express high levels of GM-CSF and GM-CSFRα

GM

-CSF

GM

-CSF

-Ra

GM-CSF CD31GM-CSF CD68 GM-CSF CD20GM-CSF CD3 GM-CSF SMA

GM-CSFRa CD31GM-CSFRa CD68 GM-CSFRa CD20GM-CSFRa CD3 GM-CSFRa SMA

Control GCA

GM

CSF

GM

CSF

Ra

GM

CSF

GM

CSF

Ra

Luminalendothelium - + +++ ++

Neovessels/ Adventitial

endothelium- - + +

SMC - - + +/-Neointimal

myofibroblasts - - ++ ++

Macrophages - - +++ ++B cells - - + +/-T cells - - ++ -

Table 1: GMCSF and GM-CSFRα are upregulated in GCA lesions

Cell nuclei (blue); PU.1 (brown)

100x 100x

PU.1

200x

Control GCA100x 100x

phospho-JAK2

Cell nuclei (blue); phospho-JAK2(brown)

GM-CSF

GM-CSFR

JAK/STAT PU.1

GCA ARTERY

Activated Macrophages/DCs

INFLAMMATORY DAMAGE, GIANT CELL FORMATION, VASCULAR REMODELING

• In ex vivo cultures of GCA arteries, mavrilimumab inhibited expression of CD83 and PU.1 at the mRNA level indicating blockade of GM-CSFR signaling pathway (Figure 4)

• There were trends for reduced expression of some inflammatory TH1/TH17 factors including TNFα, CXCL10 (IFNγ-stimulated chemokine), IL-6 and IL-1β at the mRNA or protein level with mavrilimumab in GCA artery cultures (Figure 5)

• Mavrilimumab induced decrease in mRNA expression of key cell surface markers including CD14 and CD16 (monocytes), CD3ε (T cells) and CD20 (B cells) in these GCA artery cultures indicating reduced infiltration of these inflammatory cell types into the artery (Figure 6)

GM-CSF

GM-CSFR

JAK/STAT PU.1

GCA ARTERY

Recruitment of monocytes and macrophages

IFNγCXCL10

TNFα

T-bet

TH1

T cell recruitment and activation

TH17 CS

IL-17A

IL-23AIL-6

RORγ

mRNA levels

Protein levels

Figure 6: Exposure to mavrilimumab reduces select infiltrating cell subsets in ex vivo cultured arteries from patients with GCA

40x

100x

40x

100x

40x 40x

100x 200x

Cell nuclei (blue); CD83 (brown)

CD83

40x 40x

Figure 4: Exposure to mavrilimumab inhibits GM-CSFR pathway associated molecules in ex vivo cultured arteries from patients with GCA

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