PRESS RELEASE
Cathepsin C Inhibitor Proved Effective in theTreatment of Vasculitis～Expectations for a Novel Therapeutic Agent for ANCA1*-Associated Vasculitis～Alivexis, Inc. and Hokkaido University today announced the publication of a joint research article
entitled “Cathepsin C inhibition reduces neutrophil serine protease activity and improves activatedneutrophil-mediated disorders,” published in Nature Communications on August 22, 2024.
The research, led by Assistant Professor Yuka Nishibata, Dr. Sakiko Masuda, and Professor Akihiro
Ishizu of the Graduate School of Health Sciences, Hokkaido University in collaboration with Alivexis,
has shown for the first time in an animal model that inhibiting cathepsin C (CatC) improves ANCAassociated vasculitis (AAV). AAV is an autoimmune disease mainly observed in the elderly, whichcauses rapid progression of kidney and lung damage. The published study targeted CatC, an enzyme
which controls the maturation of neutrophil serine protease (NSPs2*), since mature NSPs areessential for the formation of neutrophile extracellular traps (NETs3*), the underlying cause of AAV.
The team hypothesized that CatC inhibition would improve AAV symptoms, and to test this theory
the Hokkaido University research group developed a rat model of AAV to which they orally
administered MOD06051, a novel and highly specific CatC inhibitor developed by Alivexis. In animals
treated with MOD06051, both NET-forming neutrophils detected in peripheral blood and NETs
deposited in the kidneys were drastically reduced, and the clinical manifestations of kidney and lungdamage were also reduced. The results of this study suggest that CatC inhibition is an effective
therapeutic strategy for the treatment of various diseases caused by NETs, including AAV.
Key Scientific Findings


CatC inhibition improves kidney lesions and pulmonary hemorrhage in a rat model of AAV.

CatC inhibition suppressed NET formation, one of the major underlying mechanisms of AAV.

Expectations are high for the development of novel therapeutics for multiple diseases causedby NETs.

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Renal glomerular lesions in an animalmodel of AAV and ameliorative effect ofCatC inhibitor Neutrophils (green) positive
for the NET marker Cit-H34* (red) areobserved in the glomeruli of the diseasegroup, with ruptured glomerular capillaries(↓) resulting in blood components leaching
into the Bowman's sac. Cit-H3 is notobserved in the neutrophils infiltrating theglomeruli of the low-dose and high-dose
treatment groups, and the glomeruli maintainnormal morphology.
Bar, 50 µm【Background】
AAV is a systemic small vessel vasculitis that develops in the presence of the autoantibody against
neutrophil cytoplasmic antigen (ANCA). AAV is a disease mainly observed in the elderly and ischaracterized by rapidly progressing renal dysfunction and pulmonary bleeding. The Japan Ministryof Health, Labour and Welfare designates AAV as an intractable disease, with approximately 20,000
Japanese patients in 2020—a number which is increasing every year. Current treatment options for
AAV include immunosuppressive drugs such as corticosteroids and cyclophosphamide, as well as
molecular targeted drugs such as rituximab and avacopan. However, due to the number of nonresponders to these therapies and the cases of relapse after treatment, there is a strong need for
new treatment options. Furthermore, the existing therapeutics have side effects including decreasedimmunity, with treatment-related infections being a major issue that needs to be resolved.
NETs are DNA complexes coated in bactericidal enzymes originally expressed in the cytoplasm ofneutrophils which are released extracellularly by neutrophils in response to pathogenic
microorganisms. NETs released from neutrophils entangle the pathogens with their DNA andbombard them with enzymes to kill them. Although NETs play a significant role as a host defense
mechanism, in the absence of pathogens the enzymes released from NETs damage the wall of bloodvessels, leading to vasculitis (Fig. 1).
NSPs are a group of enzymes found in neutrophils, and include neutrophil elastase, proteinase 3,and cathepsin G. A common activation mechanism for all types of NSPs is the cleavage of two
peptides on the N-terminal side of immature (inactive) NSPs during neutrophil differentiation in thebone marrow, resulting in their conversion to the mature (active) form. The mature form of neutrophil
elastase plays an important role in the formation of NETs (Fig. 1).
CatC is an enzyme that acts during neutrophil differentiation in the bone marrow, cleaving the two
N-terminal peptides of immature (inactive) NSPs and converting them to the mature (active) form
(Fig. 1).

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【Methods】
Animal model of AAV: Twenty-four 4-week-old male Wistar Kyoto rats were immunized with humanderived MPO, a potential antigen for autoantibodies against neutrophils.
Administration of CatC inhibitor (MOD06051): The above rats were divided into 3 groups of 8 rats
each (disease group, low-dose treatment group, and high-dose treatment group). 0.3 mg/kg ofMOD06051 was orally administered twice daily in the low-dose treatment group and 3 mg/kg in thehigh-dose treatment group. The disease group received the same dose of solvent orally twice daily.
The administration period was 42 days from the day of model creation, and sampling was performed
from all individuals on the 42nd day.
Evaluation items: ANCA antibody titer in serum (enzyme-linked immunosorbent assay), NET-forming
neutrophils in blood (flow cytometry), NETs deposition in renal tissue (immunofluorescence), renal
tissue injury (PAS staining), and lung hemorrhage (HE staining) were evaluated.
【Results】
CatC inhibitory activity of MOD06051: MOD06051 selectively inhibited only CatC across ninecathepsin family enzymes in vitro and inhibited CatC enzyme activity in peripheral blood afteradministration to rats. It also inhibited the activity of NSPs in neutrophils differentiated from humanhematopoietic stem cells and in neutrophils extracted from rat bone marrow after repeated dosingof the compound, as well as inhibiting NET formation induced by various stimuli.
ANCA antibody titers in serum: Compared to normal rats (n=8) in the negative control group, thedisease control group showed significantly higher ANCA antibody titers than the negative controls;in the MOD06051-treated group, ANCA antibody titers were as high as in the disease control group,
regardless of compound dosage (Fig 2a).
NET-forming neutrophils in blood: Compared to the negative control group, the percentage ofperipheral neutrophils forming NETs was significantly higher in the disease control group than in thenegative controls, and the percentage was significantly and dose-dependently reduced to normal
levels in the MOD06051 treatment groups (Fig 2b).
NET deposition in renal tissue: Approximately 10% of kidney glomeruli-infiltrating neutrophils formed
NETs in the disease control group, while NET deposition in renal tissue was significantly and dosedependently reduced in the MOD06051 treatment groups (Fig 2c).
Renal tissue injury: Approximately 10% of glomeruli in the disease control group had lesions, while
the percentage of injured glomeruli was significantly reduced in the MOD06051 treatment groups(Fig 2d).
Pulmonary hemorrhage: MOD06051 dose-dependently reduced pulmonary hemorrhage, withstatistically significant improvement in the high-dose group compared to the disease control group(Fig 2e).
【Future ExpectationsProspects】
This study clearly showed that CatC inhibitors have the potential to be novel therapeutic candidates
for AAV. The fact that no effect on ANCA antibody titer was observed in this study means that theability to produce antibody, i.e., liquid immunity, was not affected by CatC inhibition. This finding
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indicates that, unlike existing therapeutic agents that suppress a wide range of immune functions,
CatC inhibitors potentially may avoid causing immunosuppressive adverse effects. In addition, since
it has been reported that mice genetically deficient in CatC have no loss of host defense-related
neutrophil functions, such as ROS release, adhesion, migration, or phagocytosis of neutrophils,
MOD06051 may improve disease symptoms while not effecting the neutrophil's host defensefunction. Further investigation, including clinical trial evaluation, is expected to confirm the safety
and efficacy of the compound in human diseases. In addition to AAV, NETs are also known tocontribute to the etiology of various other diseases such as sepsis, gout, diabetes, systemic lupus
erythematosus, and rheumatoid arthritis. Therefore, CatC inhibition is expected to become aneffective therapeutic strategy for the treatment of various NET-related diseases.
【Acknowledgements】
This research was supported by JSPS Grant-in-Aid for Scientific Research (21H0295802).
Publication Information
Title:Cathepsin C inhibition reduces neutrophil serine protease activity and improves activatedneutrophil-mediated disorders
Authors: Yuka Nishibata1, Suishin Arai1, Mai Taniguchi1, Issei Nakade1, Hodaka Ogawa1, Shota
Kitano1, Yumeka Hosoi1, Ayano Shindo1, Ryo Nishiyama1, Sakiko Masuda1, Daigo Nakazawa2, Utano
Tomaru3, Takafumi Shimizu4, William Sinko4, Tadashi Nagakura4, Yoh Terada4, Akihiro Ishizu1（1Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University,Sapporo, Japan, 2Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicineand Graduate School of Medicine, Hokkaido University, Sapporo, Japan, 3Department of Surgical
Pathology, Hokkaido University Hospital, Sapporo, Japan, 4Alivexis, Inc., Tokyo, Japan）
Journal: Nature Communications
ＤＯＩ:

10.1038/s41467-024-50747-6

Publication date: 10.00 BST/18.00 JST, August 22nd, 2024（online）
Contact Information for R&D
Faculty of Health Sciences, Hokkaido University, Professor
ＴＥＬ011-706-3385

ＦＡＸ011-706-4916

ＵＲＬ

https://www.p-i-labo-hs-hokudai.jp

Akihiro Ishizu

Mail aishizu@med.hokudai.ac.jp

Alivexis, Inc. Research and Development
ＴＥＬ03-6868-4547

MailＵＲＬ

https://alivexis.com

info@alivexis.com

Contact Information for Public Information
PR & Comms. Division, Office of Public Relations and Social Collaboration, Hokkaido University
ＴＥＬ011-706-2186

ＦＡＸ011-706-2092

Mail en-press@general.hokudai.ac.jp

Alivexis, Inc. Business Administration
ＴＥＬ03-6868-4547

Mail

info@alivexis.com
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Fig 1. Mechanism of action of cathepsin C inhibitors on tissue injury by neutrophils(a) ANCA antibody titer in serum.
(b) Percentage of NET-forming
neutrophils in peripheral blood. (c)PercentageofNET-forming

neutrophils in kidney glomeruliinfiltrating
Renal

neutrophils.

lesionsshown

(d)

as

thepercentage of injured glomeruli.
(e) Pulmonary hemorrhage shownas the number of foci of pulmonary
hemorrhage observed under themicroscope.
*p<0.05, **p<0.01, ***p<0.001,**** p<0.0001.

Fig

2.

EfficacyofCatC

Inhibitors in the Treatment ofAAV【Glossary】＊ 1 ANCA … Anti-Neutrophil Cytoplasmic Antibody is a pathogenic autoantibody that binds tomyeloperoxidase and proteinase 3 in the cytoplasm of neutrophils.
＊2 NSPs … Neutrophil Serine Proteases are a group of enzymes found in neutrophils, including
neutrophil elastase, proteinase 3, and cathepsin G, among others. Neurophil elastase
in its mature form plays a critical role in the formation of NETs.
＊3 NETs … Neutrophil Extracellular Traps are released from neutrophils as a defensive response
to pathogenic microorganisms in the body. In AAV, however, ANCA binding causes therelease of NETs from neutrophils, which in the absence of pathogenic microorganisms
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cause damage to the vascular endothelium, resulting in vascular inflammation.
＊4 Cit-H3 … Citrullinated Histone H3 is produced when histone H3, a nuclear protein around which
DNA coils, undergoes citrullination during NET formation, inducing DNA unwinding. The
presence of Cit-H3 is an indicator of NET formation.

Link to the Press Release from Hokkaido University: https://www.global.hokudai.ac.jp/blog/newcompound-shows-great-potential-for-patients-with-neutrophil-associated-inflammation/

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